Selective sensitization of cancer cells to elimination by cytotoxic lymphocytes

Abstract

The present disclosure provides cancer cells that have been specifically sensitized to enhance their elimination by cytotoxic lymphocytes. The sensitized cancer cells, in some examples, overexpress one or more newly identified cytotoxic-lymphocyte sensitizing proteins, where the overexpression is achieved with, for example, a guide-based gene activation system, or transformation of the cancer cells with an open reading frame encoding the cytotoxic-lymphocyte sensitizing protein. The disclosure further provides cytotoxic lymphocytes engineered to overexpress antitumor enhancing ligands that act in a paracrine signaling mechanism to induce increased cytotoxic activity for the engineered lymphocytes. Also provided are methods for producing the sensitized cancer cells and engineered cytotoxic lymphocytes, and methods for treating or preventing a cancer.

Description

PATENT Attorney Docket No.110221-1499146-011610WO Client Ref. No. CZB-310S-PC / S24-186 SELECTIVE SENSITIZATION OF CANCER CELLS TO ELIMINATION BY CYTOTOXIC LYMPHOCYTES CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Application No. 63 / 651,817 filed May 24, 2024, the full disclosure of which is incorporated by reference in its entirety for all purposes. BACKGROUND

[0002] CD8+T cells can selectively target and eliminate cancer cells. However, cancer cells can evade this form of cytotoxicity in a variety of ways. Some of these are complex and are mediated by the tumor microenvironment and other cell types, while others are more direct. For example, cell autonomous resistance mechanisms can protect malignant cells from targeted T cell elimination, resulting in resistance to a broad range of immunotherapies. High throughput gene knockout and gene inhibition screens have been instrumental in identifying key drivers of such mechanisms. However, these screens often uncover gene activities that are essential but not sufficient for response. Therefore, a need exists for the identification and manipulation of additional regulators of the cancer-immune interface. By describing a catalog of immune evasion mechanisms and an array of new targets, and by demonstrating how synthetic gene activation and / or gene knockout can diminish immune evasion and open novel avenues for agonist-based and RNA-based interventions, the present disclosure addresses this need and provides associated and other advantages. BRIEF SUMMARY

[0003] This summary provides a high-level overview of various aspects of the disclosure and introduces some of the concepts that are described and illustrated in the present document and the 79685919V.1accompanying figures. The summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used in isolation to determine the scope of the claimed subject matter. Covered embodiments of the disclosure are defined by the claims, not this summary. The subject matter should be understood by reference to appropriate portions of the entire specification, any or all figures, and each claim. Some of the exemplary embodiments of the present disclosure are discussed below.

[0004] In one aspect, the disclosure provides a method for sensitizing a cancer cell to cytotoxic lymphocytes. The method includes introducing into the cancer cell a polynucleotide including or encoding a guide RNA targeting a gene encoding a cytotoxic-lymphocyte sensitizing protein. The cytotoxic-lymphocyte sensitizing protein is a protein that increases elimination of the cancer cell upon recognition of the cancer cell by a cytotoxic lymphocyte targeting the cancer cell.

[0005] In another aspect, the disclosure provides an engineered cytotoxic lymphocyte genetically engineered to overexpress an antitumor enhancing ligand. The antitumor enhancing ligand is a ligand that binds to a corresponding binding partner expressed by the engineered cytotoxic lymphocyte. The engineered cytotoxic lymphocyte induces increased elimination of a cancer cell as compared to the elimination of the cancer cell induced by a corresponding cytotoxic lymphocyte not overexpressing the antitumor enhancing ligand.

[0006] In another aspect, the disclosure provides a method for producing an engineered cytotoxic lymphocyte. The method includes providing a cytotoxic lymphocyte. The method further includes introducing into the cytotoxic lymphocyte a polynucleotide encoding an antitumor enhancing ligand, thereby producing the engineered cytotoxic lymphocyte. The antitumor enhancing ligand is a ligand that binds to a corresponding binding partner expressed by the engineered cytotoxic lymphocyte. The engineered cytotoxic lymphocyte induces increased elimination of a cancer cell as compared to the elimination of the cancer cell induced by a corresponding cytotoxic lymphocyte not overexpressing the antitumor enhancing ligand.

[0007] In another aspect, the disclosure provides another method for producing an engineered cytotoxic lymphocyte. The method includes providing a cytotoxic lymphocyte that includes a gene encoding a tumor enhancing ligand. The method further includes introducing into the cytotoxic lymphocyte a polynucleotide including or encoding a guide RNA targeting the gene, thereby producing the engineered cytotoxic lymphocyte. The antitumor enhancing ligand is a ligand that 79685919V.1binds to a corresponding binding partner expressed by the engineered cytotoxic lymphocyte. The engineered cytotoxic lymphocyte induces increased elimination of a cancer cell as compared to the elimination of the cancer cell induced by a corresponding cytotoxic lymphocyte not overexpressing the antitumor enhancing ligand.

[0008] In another aspect, the disclosure provides a method for treating a cancer in a subject. The method includes administering to the subject a polynucleotide including or encoding a guide RNA. The guide RNA targets a gene encoding a cytotoxic-lymphocyte sensitizing protein that increases elimination of a cancer cell of the cancer upon recognition of the cancer cell by a cytotoxic lymphocyte targeting the cancer cell.

[0009] In another aspect, the disclosure provides another method for treating a cancer in a subject. The method includes administering to the subject a polynucleotide encoding a cytotoxic- lymphocyte sensitizing protein. The cytotoxic-lymphocyte sensitizing protein is a protein that increases elimination of a cancer cell upon recognition of the cancer cell by a cytotoxic lymphocyte targeting the cancer cell.

[0010] In another aspect, the disclosure provides another method for treating a cancer in a subject. The method includes administering to the subject any of the engineered cytotoxic lymphocytes disclosed herein.

[0011] In another aspect, the disclosure provides another method for sensitizing a cancer cell to cytotoxic lymphocytes. The method includes introducing into the cancer cell a polynucleotide targeting a gene of the cancer cell. A decrease or inhibition of expression of the targeted gene resulting from the introduction of the polynucleotide increases elimination of the cancer cell upon recognition of the cancer cell by a cytotoxic lymphocyte targeting the cancer cell. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIG.1 presents a schematic illustration of a mechanism of action for sensitizing apoptosis genes with synthetic lethality in the presence of granzymes in accordance with provided embodiments. The illustrated technique involves selectively targeting cancer cell resistance to programmed cell death (PCD) through activation of the RNA expression of PCD proenzymes and proteins (e.g., CASP3, and BID) in combination with exposure to targeting CD8 T cells. The PCD 79685919V.1proteins are in an inactive form at baseline. In the presence of targeting CD8 T cells, an immune synapse is formed between the T cells and cancer cells, mediating the selective release of granzymes from the T cells and the intake of these granzymes by the cancer cells. The granzymes then activate the PCD proteins (e.g., CASP3, BID), resulting in selective elimination of the cancer cells.

[0013] FIG. 2 presents a schematic illustration of a mechanism of action of sensitizing ligands, i.e., antitumor enhancing ligands, in accordance with provided embodiments.

[0014] FIG. 3 presents a schematic illustration of a CRISPR activation screen for identification of RNA-based interventions that sensitize cancer cells to targeted T cell mediated elimination. Cancer cells stably expressing CRISPR / dCas9 were transduced to express a library of CRISPR activation single guide RNAs (sgRNAs). Transduction was performed at low multiplicity of infection (MOI) to obtain one gene activation per cell. Cancer cells were then cultured in co-culture with targeting TCR-engineered CD8 T cells (1), co-culture with non-targeting CD8 T cells (2), or monoculture (3). sgRNA sequencing reads from the cell populations post-selection were analyzed to identify perturbations that sensitize the cancer cells to targeted T cell mediated elimination.

[0015] FIG. 4 presents a graph showing that cancer cells are selectively eliminated by TCR engineered CD8 T cells. The graph plots the percentage of surviving cancer cells (melanoma A375) in co-culture with targeting TCR engineered CD8 T cells (light bars) vs. co-culture with non-targeting CD8 T cells (dark bars). The results are shown across a range of effector to target (E:T) ratios, where the effector cells are the CD8 T cells, and the target cells are the cancer cells. 0:1 E:T denotes the matching monoculture.

[0016] FIG. 5 presents a graph showing that a CRISPR activation screen in melanoma cells identified dozens of genes that (de)sensitize cancer cells to targeted T cell mediated elimination. Each of the 2,921 genes activated in the screen is represented by a point in the graph having a position that denotes the significance (y axis) and effect size (x axis) of the gene activation on the susceptibility of the cancer cell to targeted CD8 T cell mediated elimination. Negative and positive fold change (FC) values represent sensitizing and desensitizing perturbations, respectively.

[0017] FIG. 6 presents a series of graphs showing validation of top CRISPR activation sensitizing hits. Top sensitizing hits (grey bars) from the screen were validated by generating an 79685919V.1array of syngeneic cancer cells, each with a single gene activation via a single CRISPR activation sgRNA. The survival (y-axis) of the resulting cancer cells in co-culture with targeting TCR- specific CD8 T cells was compared to that of control cells (NTC, white bars), showing substantial sensitization across a range of E:T ratios.

[0018] FIG. 7 presents a graph showing validation of top CRISPR activation sensitizing hits using Open Reading Frames (ORFs). Top sensitizing hits (grey bars) from the screen were validated by generating an array of syngeneic cancer cells, each with a single gene activation using transduction with Open Reading Frame (ORF) constructs. The survival (y-axis) of the resulting cancer cells in co-culture with targeting TCR-specific CD8 T cells was compared to that of control cells (Empty backbone, white bar), as well as cancer cells transduced with the ORF of the resistance gene CD274 (encoding for PDL1, black bar). All sensitizing hits also showed substantial sensitization when activated via ORFs.

[0019] FIG. 8 presents a pair of graphs showing that CDCP1 is required for robust NK cell mediated elimination of breast cancer cells. The CDCP1 knockout (KO) was identified as a top NK resistance hit in breast cancer. Follow-up validations demonstrated that CDCP1 KO indeed leads to substantial resistance to NK mediated cytotoxicity, when compared to either control cells (NTC, black bars) or well-known resistance knockouts (ICAM1 and JAK1, represented by two KO sgRNAs, grey bars)0.01.

[0020] FIG. 9 presents a series of graphs showing spatial effects on gene activation signature data for CD44, VAV1, and MYC.

[0021] FIG. 10 presents a graph showing the effects of perturbations of VAV1, MYC, CD44, WNT3A, TSPYL2, PDPN, CD274, TCF7L2, or CASP3 on up-regulation and down-regulation of other genes in the cell transcriptome.

[0022] FIG. 11 presents a Western blot of pro-caspase-3 and cleaved caspase-3 protein levels following CASP3 overexpression.

[0023] FIG. 12 presents a graph showing that overexpression of top sensitizing hits does not impact A375 cancer cell proliferation in monoculture. 79685919V.1

[0024] FIG. 13 present a pair of graphs plotting A375 cancer cell viability following 24-hourtreatment with IFN or TNF, where the A375 cells overexpress different sensitizing hits.

[0025] FIG. 14 presents graph plotting data showing that pan-caspase inhibitor (Z-VAD-FMK) is blocking TCR-specific cytotoxicity in A375 cancer cells, also upon CASP3 overexpression.

[0026] FIG.15 presents a schematic illustration of additional testing of top sensitizing hits via a model that is based on the presentation and TCR-based recognition of a viral antigen. ORF-based overexpression of BID, CASP3, and SAFB in HPV+cervical cancer cells (CaSki) resulted in enhanced sensitivity to TCR-dependent cytotoxicity in coculture with E7 TCR T cells.

[0027] FIG. 16 presents a pair of graphs plotting data from the testing of FIG. 15, showing that overexpression of BID and CASP3 does not impact CasKi cell viability and proliferation in monoculture.

[0028] FIG. 17 presents schematic illustrations of a model of CASP3 RNA-based synthetic lethality with TCR-dependent cytotoxicity.

[0029] FIG. 18 presents a graph plotting data showing the toxicity of the FDA approved BH3 mimetics and Bcl2 inhibitor Venetoclax to A375 and primary CD8 T cells.

[0030] FIG. 19 presents a pair of graphs plotting data showing that Venetoclax is equally toxic to control and CASP3OEA375 cells (left) and decreases the efficacy of TCR-dependent cytotoxicity (right).

[0031] FIG. 20 presents a graph plotting data showing stable ORF-based overexpression of CASP3 in primary CD8 T cells

[0032] FIG. 21 presents a graph plotting data showing similar proliferation of the control and CASP3-overexpressing CD8 T cells of FIG.20.

[0033] FIG.22 presents a graph plotting data showing similar cytotoxicity levels for the control and CASP3-overexpressing CD8 T cells of FIG.20.

[0034] FIG. 23 presents a schematic illustration of CASP3 RNA delivery via cationic lipid / polymer-based nanoparticles in A375 cancer cell coculture with NY-ESO1 T cells. 79685919V.1

[0035] FIG. 24 presents a graph plotting data showing CASP3 overexpression following the RNA delivery via cationic lipid / polymer-based nanoparticles to the A375 cancer cell coculture of FIG.23.

[0036] FIG.25 presents a graph plotting data showing enhanced cancer cell death following the RNA delivery via cationic lipid / polymer-based nanoparticles to the A375 cancer cell coculture of FIG.23.

[0037] FIG.26 presents a graph plotting data showing that top CRISPR activation hits sensitize breast cancer cells to NK-92-mediated cytotoxicity in single-guide validation assays. The data in the graph are representative of up to three independent experiments and are presented as mean ± SEM from three to nine replicate wells. Statistical analysis was performed using one-way ANOVAwith Dunnett's multiple comparisons; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0038] FIG. 27 presents a graph plotting the percent cytotoxicity of breast cancer cells (MDA- MB-231) with CRISPR activation of SLAMF1 or non-targeting control (NTC) following 24 h, 48 h, and 72 h co-culture with NK-92 cells at varying effector-to-target (E:T) ratios. Data are representative of up to three independent experiments and are presented as mean ± SEM from three to nine replicate wells. Statistical analysis was performed using one-way ANOVA with Dunnett’smultiple comparisons; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0039] FIG.28 presents a graph plotting the percentage of surviving breast cancer cells (MDA- MB-231) with CRISPR activation of SLAMF1 or non-targeting control (NTC) following 24 h and 48 h co-culture with NK-92 cells at varying effector-to-target (E:T) ratios. Data are presented as mean ± SEM from four replicates, each pooled from duplicate wells. Statistical analysis wasperformed using Sidak’s multiple comparisons; *p < 0.05, **p < 0.01, ***p < 0.001,****p < 0.0001.

[0040] FIG. 29 presents a graph plotting the percent cytotoxicity of breast cancer cells (MDA- MB-231) with CRISPR activation of SLAMF1 or non-targeting control (NTC) following 48 h co- culture with IL-2-expressing NK-92 cells at varying effector-to-target (E:T) ratios. Data are presented as mean ± SEM from four replicate wells. Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons; *p < 0.05, **p < 0.01, ***p < 0.001,****p < 0.0001.79685919V.1

[0041] FIG. 30 presents a graph plotting data showing that SLAMF1-mediated cytotoxicity requires SLAMF1 expression on both NK and cancer cells. Data are presented as mean ± SEM and are representative of three to four replicate wells. Statistical analysis was performed usingtwo-way ANOVA with Sidak’s multiple comparisons; *p < 0.05, **p < 0.01, ***p < 0.001,****p < 0.0001.

[0042] FIG.31 presents a graph plotting the percent cytotoxicity of SLAMF1-activated and NTC breast cancer cells (MDA-MB-231) in co-culture with wild-type or anti-EpCAM CAR-NK-92 cells co-cultured for 6 hours at a 1:1 E:T ratio. Data represent mean ± SEM from three replicate wells. Statistical significance was determined using two-way ANOVA with Sidak’s multiplecomparisons; *p < 0.05, **p < 0.01, ***p < 0.001,< 0.0001.

[0043] FIG.32 presents a graph plotting the percent cytotoxicity of SLAMF1-activated and NTC breast cancer cells (MDA-MB-231) in co-culture with wild-type or anti-EpCAM CAR-NK-92 cells co-cultured for 20 hours at varying E:T ratios. Data represent mean ± SEM from three replicate wells. Statistical significance was determined using two-way ANOVA with Tukey’smultiple comparisons; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0044] FIG. 33 presents a graph plotting data showing that top CRISPR knockout hits sensitize breast cancer cells to NK-92-mediated cytotoxicity in syngeneic gene knockout (KO) experiments. The data in the graph are representative of up to three independent experiments and are presented as mean ± SEM from four to twelve replicate wells. Statistical analysis was performed using one-way ANOVA with Dunnett's multiple comparisons; *p < 0.05, **p < 0.01, ***p < 0.001,****p < 0.0001.

[0045] FIG. 34 presents a graph plotting the percent cytotoxicity of MDA-MB-231 gene knockout (KO) cell lines following 48-hour co-culture with NK-92 cells at varying E:T ratios. Data are representative of two independent experiments and are presented as mean ± SEM from six to eight replicate wells. Statistical significance was determined using two-way ANOVA withSidak’s multiple comparisons; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0046] FIG.35 presents a graph plotting data from flow cytometry analysis of cancer cell death in MDA-MB-231 KO cell lines under monoculture (0:1) and co-culture (1:1 E:T ratio with NK- 92 cells) conditions. Data are representative of up to five independent experiments and are 79685919V.1presented as mean ± SEM from a minimum of two replicate wells. Statistical significance was determined using two-way ANOVA with Dunnett’s multiple comparisons.

[0047] FIG. 36 presents a graph plotting the percent cytotoxicity of IKBKB knockout (KO) MDA-MB-231 cell lines, generated using the top two sgRNAs, following 48- and 72-hour co- culture with NK-92 cells at varying E:T ratios. Data represent mean ± SEM from five to six replicate wells. Statistical significance was determined using two-way ANOVA with Sidak’smultiple comparisons; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0048] FIG. 37 presents a graph plotting the IFN- levels in co-culture supernatants from NK-92 cells and IKBKB KO or NTC MDA-MB-231 cells, measured by ELISA at varying E:T ratios and time points. Data represent mean ± SEM from four replicate wells. Statistical significance wasdetermined using two-way ANOVA with Dunnett’s multiple comparisons; *p < 0.05, **p < 0.01,***p < 0.001, ****p < 0.0001.

[0049] FIG. 38 presents a graph plotting the percent cytotoxicity of MDA-MB-231 IKBKB knockout (KO) or NY-ESO-1-expressing IKBKB KO cell lines following 48-hour co-culture with either wild-type (WT) or NY-ESO-1 TCR-engineered primary T cells from a donor designated Donor17. Data represent mean ± SEM from triplicate wells. Statistical significance wasdetermined using two-way ANOVA with Tukey’s multiple comparisons; *p < 0.05, **p < 0.01,***p < 0.001, ****p < 0.0001.

[0050] FIG. 39 presents a graph plotting the percent cytotoxicity of MDA-MB-231 IKBKB knockout (KO) or NY-ESO-1-expressing IKBKB KO cell lines following 48-hour co-culture with either wild-type (WT) or NY-ESO-1 TCR-engineered primary T cells from a donor designated Donor18. Data represent mean ± SEM from triplicate wells. Statistical significance wasdetermined using two-way ANOVA with Tukey’s multiple comparisons; *p < 0.05, **p < 0.01,***p < 0.001, ****p < 0.0001.

[0051] FIG. 40 presents a graph plotting the percent cytotoxicity of MDA-MB-231 IKBKB knockout (KO) or NY-ESO-1-expressing IKBKB KO cell lines following 48-hour co-culture with either wild-type (WT) or NY-ESO-1 TCR-engineered primary T cells from a donor designated Donor19. Data represent mean ± SEM from triplicate wells. Statistical significance was 79685919V.1determined using two-way ANOVA with Tukey’s multiple comparisons; *p < 0.05, **p < 0.01,***p < 0.001, ****p < 0.0001.

[0052] FIG.41 presents a graph plotting percent cytotoxicity values for MDA-MB-231 KO lines following 48- and 72-hour co-culture with primary NK cells from multiple donors at a 1:1 effector- to-target (E:T) ratio. Experiments were performed separately for each donor, with at least two replicate wells per donor. Data represent mean ± SEM. Statistical significance was determinedusing two-way ANOVA with Sidak’s multiple comparisons; *p < 0.05, **p < 0.01, ***p < 0.001,****p < 0.0001.

[0053] FIG. 42 presents a graph plotting the average NK cytotoxicity level per donor (y-axis) for NTC and IKBKB KO MDA-MB-231 (x-axis) after 48 hours of co-culture at 1:1 E:T ratio. The datapoints per donor are connected with a dotted line. Statistical significance was determined usingmixed-effect models; ****p < 0.0001.

[0054] FIG. 43 presents a graph plotting the average NK cytotoxicity level per donor (y-axis) for NTC and IKBKB KO MDA-MB-231 (x-axis) after 72 hours of co-culture at 1:1 E:T ratio. The datapoints per donor are connected with a dotted line. Statistical significance was determined usingmixed-effect models; ****p < 0.0001.

[0055] FIG.44 presents a graph plotting percent cytotoxicity values for MDA-MB-231 IKBKB KO and NTC lines following 24- and 48-hour co-culture with either wild-type NK-92 or anti- EpCAM CAR-NK-92 cells at a 1:1 E:T ratio. Data are representative of two independent experiments and are presented as mean ± SEM from four to eight replicate wells. Statistical significance was determined using two-way ANOVA with Tukey’s multiple comparisons;*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.DETAILED DESCRIPTION I. INTRODUCTION

[0056] The present disclosure generally relates to materials and methods for selectively sensitizing cancer cells to, for example, cytotoxic lymphocyte mediated killing, by fixing deficits in programmed cell death systems of the cancer cells. Programmed cell death, also known as 79685919V.1apoptosis, is a crucial component of cancer therapy due to its role in maintaining cellular homeostasis. In normal physiological conditions, apoptosis serves as a defense mechanism to eliminate cells that are damaged, aged, or under stress. However, malignant cells often develop mechanisms to evade apoptosis, leading to uncontrolled proliferation and tumor growth. By triggering apoptosis in these cells, cancer therapies can effectively halt tumor progression and reduce tumor burden. Moreover, inducing apoptosis in cancer cells can also enhance the efficacy of other therapeutic strategies, such as chemotherapy and radiation, by sensitizing resistant cancer cells to these treatments.

[0057] It has proven difficult, however, to induce the programmed cell death of cancer cells safely and effectively using existing techniques. Pro-apoptotic compounds have been explored in oncology and some are FDA approved or are undergoing clinical trials (e.g., BH3 mimetics, BCL- 2 and MCL-1 inhibitors). These compounds, though, typically lack specificity and are therefore more toxic, since they activate cell death in a general fashion not limited to the malignant cells that a cancer therapy aims to eliminate.

[0058] The materials and methods provided by the present disclosure address these difficulties by, in some embodiments, overexpressing particular classes of proteins, i.e., cytotoxic-lymphocyte sensitizing proteins, that do not themselves activate cell death, but instead respond to signals that, in combination with the overexpressed proteins, initiate programmed cell death (FIG. 1). In other embodiments, the provided materials and methods include activating and / or modifying cytotoxic lymphocyte ligands, i.e., tumor enhancing ligands, that act in an autocrine manner to stimulate the cytotoxic lymphocytes and improve their ability to eliminate cancer cells (FIG. 2). In still other embodiments, the provided materials and methods include inhibiting and / or silencing expression of one or more particular genes. The disclosed procedures can involve, for example, RNA-based interventions delivered to a site of malignant or pre-malignant cancer cells to selectively sensitize them to cytotoxic lymphocyte (e.g., T cell and / or NK cell) mediated killing. Clinically applicable methods for RNA-based interventions and delivery are available and are further developing and improving. For example, the RNA delivery can be achieved with viral-like particles carrying linear or circular RNA. Additional or alternative approaches can use cytotoxic lymphocytes that have been themselves engineered to release the RNA molecules for transfer to the cancer cells. Beneficially, the provided approaches can be synergistically combined with therapies using CAR 79685919V.1T cells and other types of engineered T cells, overcoming the resistance of cancer cells to T cell mediated killing and enhancing T cells through other pathways. II. DEFINITIONS

[0059] Unless specifically indicated otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this disclosure belongs. In addition, any method or material similar or equivalent to a method or material described herein can be used in the practice of the present disclosure. For purposes of the present disclosure, the following terms are defined.

[0060] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a phospholipid” optionally includes a combination of two or more phospholipids, and the like.

[0061] As used herein, the terms “about” and “approximately,” when used to modify an amount specified in a numeric value or range, indicate that the numeric value as well as reasonable deviations from the value known to the skilled person in the art, for example ± 20%, ± 10%, or ± 5%, are within the intended meaning of the recited value.

[0062] As used herein, the term “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0063] As used herein, the terms “including,” “comprising,” “having,” “containing,” and variations thereof, are inclusive and open-ended and do not exclude additional, unrecited elements or method steps beyond those explicitly recited. As used herein, the phrase “consisting of” is closed and excludes any element, step, or ingredient not explicitly specified. As used herein, the phrase “consisting essentially of” limits the scope of the described feature to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the disclosed feature.

[0064] As used herein, the term “cytotoxic-lymphocyte sensitizing protein” refers to a protein, or a subunit or a fragment thereof, that, when expressed by a cancer cell, enhances the sensitivity of the cancer cell to elimination, e.g., apoptosis, upon recognition of the cancer cell by a cytotoxic 79685919V.1lymphocyte targeting the cancer cell, e.g., a cytotoxic lymphocyte recognizing an antigen of the cancer cell.

[0065] As used herein, the term “antitumor enhancing ligand” refers to a ligand, that, when binding to a corresponding binding partner expressed by a cytotoxic lymphocyte, enhances the ability of the cytotoxic lymphocyte to induce elimination, e.g., apoptosis, of a cancer cell by the cytotoxic lymphocyte.

[0066] As used herein, the terms “protein, “peptide,” and “polypeptide” refer to polymers comprised of covalently linked natural or chemically modified amino acid residues.

[0067] As used herein, the terms “polynucleotide” and “nucleic acid molecule” refer to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. A nucleic acid sequence may comprise combinations of deoxyribonucleic acids and ribonucleic acids. Such deoxyribonucleic acids and ribonucleic acids include both naturally occurring molecules and synthetic analogs. The polynucleotides of the disclosure also encompass all forms of sequences including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and- loop structures, circular structures, and the like.

[0068] The terms “variant,” and “fragment,” refer to a polynucleotide related to a wild-type polynucleotide, for example, either by nucleic acid sequence, structure (e.g., secondary and / or tertiary), activity (e.g., enzymatic activity) and / or function. Variants and fragments of a polynucleotide can include one or more nucleic acid variations (e.g., mutations, insertions, and deletions), truncations, modifications, or combinations thereof compared to a wild-type polynucleotide. A variant or fragment can include at least 50%, e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the sequence, structure, activity, and / or function of the corresponding wild-type polynucleotide. 79685919V.1

[0069] As used herein, the terms “proenzyme” and “zymogen” refer to a biologically inactive substance that is metabolized into an active enzyme. A proenzyme typically requires a biochemical change, such as a hydrolysis reaction revealing the active site, or a configuration change to reveal the active site, for the proenzyme to be transformed into an active enzyme. The term “proenzyme” is thus used to describe an inactive precursor of an enzyme, where the enzyme precursor has the potential to become active under certain conditions and / or in the presence of certain other compounds.

[0070] As used herein, the term “overexpression,” in the context of a protein described herein, refers to an increase in the amount and / or activity of the protein, e.g., the amount and / or activity of the protein in a cell or in a population of cells. The overexpression of the protein can be the result of an increased copy number of a gene encoding the protein, an increased transcription of a gene encoding the protein, an increased translation of RNA encoding the protein, and / or a post- translational modification to the protein. In some examples, an overexpression of a protein results from a modification to a cell that expressed the protein prior to the modification, where the modification increases the amount and / or activity of the protein to a higher level than would be in cell absent the modification. In other examples, an overexpression of a protein results from a modification to a cell that did not express the protein prior to the modification, where the modification introduces expression of the protein to the cell.

[0071] As used herein, the term “introducing,” in the context of a polynucleotide described herein, refers to presenting a nucleic acid sequence to a host cell in such a manner that the sequence gains access to the interior of the cell. Methods for introducing nucleic acid sequences into cells are known in the art and include, but are not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods. “Stable transformation” is intended to mean that the nucleotide construct introduced into a host cell integrates into the genome of the host cell and / or is capable of being inherited by the progeny of the transformed host cell. “Transient transformation” is intended to mean that a polynucleotide is introduced into the host cell and does not integrate into the genome and / or is not inherited by the progeny of the transformed host cell.

[0072] As used herein, the term “gene” refers to a segment of DNA encoding a polypeptide chain. A gene may include regions preceding (i.e., leader regions) and following (i.e., trailer regions) a 79685919V.1coding region, as well as intervening sequences (i.e., introns) between individual coding segments (i.e., exons).

[0073] As used herein, the terms “treat,” “treating,” and “treatment” refer to a procedure resulting in any indicia of success in the elimination or amelioration of an injury, pathology, condition, or symptom (e.g., pain), including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the symptom, injury, pathology or condition more tolerable to the patient; decreasing the frequency or duration of the symptom or condition; or, in some situations, preventing the onset of one or more symptoms. The treatment or amelioration of symptoms can be based on any objective or subjective parameter; including, e.g., the result of a physical examination or laboratory test.

[0074] As used herein, the term “subject” refers to a vertebrate, and preferably to a mammal. Mammalian subjects for which the provided composition is suitable include, but are not limited to, mice, rats, simians, humans, farm animals, sport animals, and pets. In some embodiments, the subject is human. In some embodiments, the subject is male. In some embodiments, the subject is female. In some embodiments, the subject is an adult. In some embodiments, the subject is an adolescent. In some embodiments, the subject is a child. In some embodiments, the subject is above 10 years of age, e.g., above 20 years of age, above 30 years of age, above 40 years of age, above 50 years of age, above 60 years of age, above 70 years of age, or above 80 years of age. In some embodiments, the subject is less than 80 years of age, e.g., less than 70 years of age, less than 60 years of age, less than 50 years of age, less than 40 years of age, less than 30 years of age, less than 20 years of age, or less than 10 years of age.

[0075] As used herein, the term “administering” refers to oral administration, administration as a suppository, topical contact, parenteral, intravenous, intraperitoneal, intramuscular, intralesional, intranasal or subcutaneous administration, intrathecal administration, or the implantation of a slow-release device e.g., a mini-osmotic pump, to the subject.

[0076] As used herein, the term “therapeutically effective amount” refers to an amount or dose that produces therapeutic effects for which it is administered. The exact amount or dose will depend on the purpose of the treatment, and will be readily ascertainable by one skilled in the art using known techniques. 79685919V.1III. SENSITIZED CANCER CELLS

[0077] In some aspects, the present disclosure provide various methods for sensitizing cancer cells to cytotoxic lymphocytes. In some embodiments, the provided methods generally involve overexpressing a cytotoxic-lymphocyte sensitizing protein that increases elimination of the cancer cell upon recognition of the cancer cell by a cytotoxic lymphocyte targeting the cancer cell. The overexpressing of the cytotoxic-lymphocyte sensitizing protein can include activating the transcription of a gene in the cancer cell encoding the cytotoxic-lymphocyte sensitizing protein, and / or can include introducing such a gene into the cancer cell. Preferably, the overexpressed cytotoxic-lymphocyte sensitizing protein is an inactive protein, e.g., a proenzyme. Thus, unlike comparative approaches to induce programmed cell death systems of cancer cells (e.g., through the use of BCL-2 inhibitors and / or BH3 mimetics) the overexpression of the cytotoxic-lymphocyte sensitizing protein by itself will not elicit a programmed cell death response. However, when cancer cells sensitized using the provided methods share an environment with CD8+T cells, granzymes released by the T cells will interact with the overexpress cytotoxic-lymphocyte sensitizing proteins in these cancer cells, thereby inducing programmed cell death only in the cancer cells. In other embodiments, the provided methods involve decreasing or inhibiting (i.e., silencing) expression of a target gene of the cancer cell by introducing a polynucleotide to the cancer cell. As with the gene activation embodiments described herein, the provided gene silencing or gene knockout embodiments also advantageously sensitize cancer cells to cytotoxic lymphocytes.

[0078] Cancer cells generally evolve to evade programmed cell death. Accordingly, a massive upregulation of anti-apoptotic genes can be observed in cancer cells even after a short exposure to CD8+T cells. Therefore, the ability to fix resulting deficits in programmed cell death in a manner that is specific to the cancer cells can provide a significant advantage for effective and safe therapies. This form of synthetic lethality is under-recognized in the field and provides important improvements for sensitizing tumors to immune responses, both irrespective of additional therapies and in the context of existing immunotherapies and other cell therapies.

[0079] Some provided methods for sensitizing a cancer cell to cytotoxic lymphocytes involve overexpressing at least one cytotoxic-lymphocyte sensitizing protein in the cancer cell by using a guide-based gene activation system, e.g., a system relying on a guide RNA. For example, the 79685919V.1methods can include introducing a polynucleotide into the cancer cell, where the polynucleotide includes a guide RNA that targets a gene encoding the cytotoxic-lymphocyte sensitizing protein. Alternatively or additionally, the methods can include introducing into the cancer cell a polynucleotide that encodes a guide RNA targeting the gene encoding the cytotoxic-lymphocyte sensitizing protein.

[0080] For guide-based gene activation methods, the cancer cell will generally include other necessary molecular components of a guide-base gene activation system, e.g., a CRISPRa system. In some examples, the method includes introducing to the cancer cell one or more molecular components of a guide-based gene activation system. The molecular components of the guide- based gene activation system can include, for example, CRISPR / dCas9, CRISPR / cas9, CRISPRon, CRISPR / dCas9-SAM, CRISPR / dCas9-VPR, cas12, cas13, other cas or cas-like proteins, or any combination thereof. In some embodiments, the method includes co-delivering the guide RNA to the cancer cell together with one more CRISPRa gene activation system molecular components, e.g., CRISPR / dCas9-VPR. In some embodiments, the gene activation system molecular components are co-delivered to the cancer cell at the protein level, e.g., with the guide RNA incorporated into a CRISPR complex, i.e., as a ribonucleotide complex. In some embodiments, the gene activation system molecular components are co-delivered to the cancer level at the nucleic acid level, e.g., as one or more polynucleotides that together encode the gene activation system molecular components and encode or include the guide RNA.

[0081] Some provided methods for sensitizing a cancer cell to cytotoxic lymphocytes involve overexpressing at least one cytotoxic-lymphocyte sensitizing protein in the cancer cell by using an open reading frame, e.g., a polynucleotide encoding the cytotoxic-lymphocyte sensitizing protein. For example, the methods can include introducing into the cancer cell a polynucleotide, where the polynucleotide encodes the cytotoxic-lymphocyte sensitizing protein. In some embodiments, the cancer cell is sensitized by introducing at least two polynucleotides into the cancer cell, where at least one of the at least two polynucleotides includes an open reading frame encoding a cytotoxic- lymphocyte sensitizing protein, and where at least one of the at least two polynucleotides includes or encodes a guide RNA targeting the open reading frame, i.e., the gene. In some embodiments, the cancer cell is sensitized by introducing into the cancer cell a polynucleotide that both includes 79685919V.1an open reading frame encoding a cytotoxic-lymphocyte sensitizing protein, and also includes or encodes a guide RNA targeting the open reading frame.

[0082] In some embodiments, the polynucleotide encoding or including the guide RNA, and / or the polynucleotide encoding the cytotoxic-lymphocyte sensitizing protein includes or consists of RNA. For example, the method can include introducing mRNA to the cancer cell, where the mRNA encodes the cytotoxic-lymphocyte sensitizing protein, the guide RNA, or both. In some embodiments, the polynucleotide encoding or including the guide RNA, and / or the polynucleotide encoding the cytotoxic-lymphocyte sensitizing protein includes or consists of DNA. For example, the method can include introducing DNA to the cancer cell, where the DNA encodes the cytotoxic- lymphocyte sensitizing protein, the guide RNA, or both. In some embodiments, the DNA delivered to the cancer cell becomes integrated into the genome of the cancer cell. In some examples of the provided method, the cytotoxic-lymphocyte sensitizing protein is introduced or delivered to the cancer cell in protein form, rather than in the form of a polynucleotide encoding the protein.

[0083] In some examples, the cytotoxic-lymphocyte sensitizing protein is an endogenous protein of the cancer cells. In these cases, overexpressing the endogenous cytotoxic-lymphocyte sensitizing protein can include activating an endogenous gene encoding the endogenous cytotoxic- lymphocyte sensitizing protein by introducing to the cancer cell a guide RNA targeting the endogenous gene. Additionally or alternatively, overexpressing the endogenous cytotoxic- lymphocyte sensitizing protein can include increasing the copy number of the endogenous gene encoding the endogenous cytotoxic-lymphocyte sensitizing protein by introducing to the cancer cell an open reading frame encoding the cytotoxic-lymphocyte sensitizing protein. Other suitable methods for overexpressing the endogenous cytotoxic-lymphocyte sensitizing protein can include enhancing the transcriptional or translation rate of the endogenous cytotoxic-lymphocyte sensitizing protein.

[0084] In some examples, the cytotoxic-lymphocyte sensitizing protein is exogenous to the cancer cells. In these cases, overexpressing the exogenous cytotoxic-lymphocyte sensitizing protein can include introducing to the cancer cell an open reading frame encoding the cytotoxic- lymphocyte sensitizing protein. Overexpressing the exogenous cytotoxic-lymphocyte sensitizing protein can further include activating the introduced open reading frame encoding the exogenous 79685919V.1cytotoxic-lymphocyte sensitizing protein by also introducing to the cancer cell a guide RNA targeting the open reading frame.

[0085] The introduction of polynucleotides (e.g., a polynucleotide including or encoding a guide RNA, a polynucleotide encoding a cytotoxic-lymphocyte sensitizing protein, and / or a polynucleotide encoding a molecular component of a guide-based gene activation system) to the cancer cell can involve, for example, transduction with one or more viruses containing the polynucleotides. Additionally or alternatively, the introduction of the polynucleotides to the cancer cell can involve transduction with one or more virus-like particles containing the polynucleotides. In other examples, the introduction of a polynucleotide to the cancer cell involves contacting the cancer cell with an immune cell that contains the polynucleotide and induces transfer of the polynucleotide from the immune cell to the cancer cell.

[0086] In some examples, the cytotoxic-lymphocyte sensitizing protein is an inactive protein. For example, the cytotoxic-lymphocyte sensitizing protein can be a proenzyme. In some preferred embodiments, the cytotoxic-lymphocyte sensitizing protein is a proenzyme or other inactive protein, where the cytotoxic-lymphocyte sensitizing protein is transformed to an active protein, e.g., an active enzyme, upon recognition of the cancer cell by the cytotoxic lymphocyte targeting the cancer cell. For example, the cytotoxic-lymphocyte sensitizing protein can be a proenzyme that is transformed into an active enzyme after a cytotoxic lymphocyte targeting the cancer cell releases granzymes that catalyze a reaction transforming the cytotoxic-lymphocyte sensitizing protein.

[0087] In some examples, the cytotoxic-lymphocyte sensitizing protein includes or consists of one or more B-cell lymphoma 2 (BCL-2) family proteins or subunits or fragments thereof. For example, the cytotoxic-lymphocyte sensitizing protein can include or consist of BCL-2 homology domain 3 interacting domain death agonist (BID), BCL-2 antagonist / killer 1 (BAK1), BCL-2 interacting killer (BIK), phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1), or a variant or combination thereof.

[0088] In some examples, the cytotoxic-lymphocyte sensitizing protein includes or consists of one or more cysteine-aspartic acid protease (caspase) family proteins or subunits or fragments thereof. For example, the cytotoxic-lymphocyte sensitizing protein can include or consist of caspase-3 (CASP3), caspase-8 (CASP8), or a variant or combination thereof. 79685919V.1

[0089] In some examples, the cytotoxic-lymphocyte sensitizing protein includes or consists of one or more transcription factors, transcriptional regulators, or subunits or fragments thereof. For example, the cytotoxic-lymphocyte sensitizing protein can include or consist of an ETS family transcription factor or a subunit or fragment thereof, e.g., ETS proto-oncogene 1 transcription factor (ETS1), ETS variant transcription factor 4 (ETV4), or a variant or combination thereof. In some examples, the cytotoxic-lymphocyte sensitizing protein includes or consists of a GATA family transcription factor or a subunit or fragment thereof, e.g., GATA-binding factor 4 (GATA4), GATA-binding factor 6 (GATA6), tricho-rhino-phalangeal syndrome Type 1 (TRPS1), or a variant or combination thereof. In some examples, the cytotoxic-lymphocyte sensitizing protein includes or consists of a paired box (PAX) family transcription factor or a subunit or fragment thereof, e.g., PAX3, PAX8, or a variant or combination thereof. In some examples, the cytotoxic-lymphocyte sensitizing protein includes or consists of a helix-loop-helix transcription factor, a helix-loop-helix transcriptional regulator, or a subunit or fragment thereof, e.g., DNA binding 1 (ID1), hairy / enhancer-of-split related with YRPW motif-like protein (HEYL), microphthalmia-associated transcription factor (MITF), MYC proto-oncogene BHLH transcription factor (MYC), or a variant or combination thereof. In some examples, the cytotoxic- lymphocyte sensitizing protein includes or consists of paired-like homeobox 2B (PHOX2B), chromodomain-helicase-DNA-binding protein 8 (CHD8), histone deacetylase 4 (HDAC4), histone deacetylase 7 (HDAC7), hypermethylated in cancer 1 protein (HIC1), high mobility group box 1 protein (HMGB1), interferon regulatory factor 1 (IRF1), nuclear receptor 4A3 (NR4A3), nuclear protein 1 (NUPR1), peroxisome proliferator-activated receptor gamma coactivator 1-beta (PPARGC1B), PR domain containing 16 (PRDM16), protein arginine N-methyltransferase 2 (PRMT2), homeobox protein prophet of PIT-1 (PROP1), prothymosin alpha (PTMA), transcription factor RelB (RELB), SET nuclear proto-oncogene (SET), homeobox protein SIX4 (SIX4), TATA-box binding protein associated factor 15 (TAF15), transcription factor AP-2 alpha (TFAP2A), DNA topoisomerase 1 (TOP1), Wilms tumor protein (WT1), or a variant or combination thereof.

[0090] In some examples, the cytotoxic-lymphocyte sensitizing protein includes or consists of one or more tumor necrosis factor receptor superfamily (TNFRSF) proteins or subunits or fragments thereof. For example, the cytotoxic-lymphocyte sensitizing protein can include or consist of the Fas cell surface death receptor (FAS), TNFRSF member 1A (TNFRSF1A), TNFRSF 79685919V.1member 1B (TNFRSF1B), tumor necrosis factor ligand superfamily member 9 (TNFSF9), or a variant or combination thereof.

[0091] In some examples, the cytotoxic-lymphocyte sensitizing protein includes or consists of one or more transmembrane ligands or subunits or fragments thereof. For example, the cytotoxic- lymphocyte sensitizing protein can include or consist of CUB domain-containing protein 1 (CDCP1), delta-like canonical Notch ligand 4 (DLL4), or a variant or combination thereof.

[0092] In some examples, the cytotoxic-lymphocyte sensitizing protein includes or consists of one or more immunoglobulin superfamily proteins or subunits or fragments thereof. For example, the cytotoxic-lymphocyte sensitizing protein can include or consist of CD47, CD58, or a variant or combination thereof.

[0093] In some examples, the cytotoxic-lymphocyte sensitizing protein includes or consists of one or more collagen subunits or fragments thereof. For example, the cytotoxic-lymphocyte sensitizing protein can include or consist of collagen alpha-1(V) chain (COL5A1), collagen alpha- 2(IV) chain (COL4A2), or a variant or combination thereof.

[0094] In some examples, the cytotoxic-lymphocyte sensitizing protein includes or consists of one or more interferon family signaling proteins or subunits or fragments thereof. For example, the cytotoxic-lymphocyte sensitizing protein can include or consist of interferon beta 1 (IFNB1), interferon gamma (IFNG), or a variant or combination thereof.

[0095] In some examples, the cytotoxic-lymphocyte sensitizing protein includes or consists of one or more mitosis regulatory proteins or subunits or fragments thereof. For example, the cytotoxic-lymphocyte sensitizing protein can include or consist of cyclin B1 (CCNB1), centrosomal protein of 70 kDa (CEP70), close homolog of L1 (CHL1), kinesin family member 14 (KIF14), or a variant or combination thereof.

[0096] In some examples, the cytotoxic-lymphocyte sensitizing protein includes or consists of one or more protein phosphatases or subunits or fragments thereof. For example, the cytotoxic- lymphocyte sensitizing protein can include or consist of dual specificity phosphatase 1 (DUSP1), protein phosphatase 1J (PPM1J), serine / threonine-protein phosphatase 2A regulatory subunit B'' subunit gamma (PPP2R3C), or a variant or combination thereof. 79685919V.1

[0097] In some examples, the cytotoxic-lymphocyte sensitizing protein includes or consists of nipped-B-like protein (NIPBL), C-C motif chemokine 22 (CCL22), CLK4-associating serine / arginine rich protein (CLASPR), 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNP), coronin-6 (CORO6), crystatin-F (CST7), cancer / testis antigen 1A (CTAG1A), eyes absent homolog 2 (EYA2), family with sequence similarity 161 centrosomal protein B (FAM161B), family with sequence similarity 46 member D (FAM46D), alpha-(1,3)-fucosyltransferase (FUT9), glutamate [NMDA] receptor subunit epsilon-2 (GRIN2B), immediate early response gene 5-like protein (IER5L), insulin receptor substrate 4 (IRS4), KHDRBS protein 1 (KHDRBS1), alpha-1,3- mannosyl-glycoprotein 4-beta-N-acetylglucosaminyltransferase A (MGAT4A), metallophosphoesterase domain-containing protein 2 (MPPED2), neuraminidase 3 (NEU3), nucleophosmin 1 (NPM1), proliferation and apoptosis adaptor protein 15 (PEA15), Pleckstrin homology domain interacting protein (PHIP), calcium-dependent phospholipase A2 (PLA2G5), proline-rich protein 5 (PRR5), retrotransposon Gag-like 5 (RTL5), ribonucleotide-diphosphate reductase subunit M2B (RRM2B), scaffold attachment factor B (SAFB), secreted frizzled related protein 2 (SFRP2), serine and arginine rich splicing factor 3 (SRSF3), tenascin C (TSC), TSPY- like 2 (TSPYL2), taxilin gamma (TXLNG), vimentin (VIM), or a variant or combination thereof.

[0098] In some embodiments, the provided methods include introducing a polynucleotide to a cancer cell, where the polynucleotide decreases or inhibits the expression of a particular gene (i.e., a target gene), and the silencing, knockout, or inhibition of the gene sensitizes the cancer cell to cytotoxic lymphocytes. In some embodiments, the polynucleotide may be, for example, a DNA oligonucleotide or an RNA oligonucleotide. In other embodiments, the oligonucleotide may be used in a CRISPR / Cas system.

[0099] In some embodiments, the oligonucleotide may be an siRNA or shRNA or antisense RNA. In some embodiments, the oligonucleotide may be an antisense oligonucleotide that mediates an RNase H-dependent cleavage of the mRNA transcript of the target gene. In other embodiments, the oligonucleotide may be a miRNA. In yet other embodiments, the oligonucleotide may be used in a CRISPR / Cas system.

[0100] In some embodiments, the mRNA transcript of the target gene may be targeted for cleavage and degradation. Different portions of the mRNA transcript may be targeted to decrease or inhibit the expression of the target gene. In some embodiments, a DNA oligonucleotide may be 79685919V.1used to target the mRNA transcript and form a DNA:RNA duplex with the mRNA transcript. The duplex may then be recognized and the mRNA cleaved by specific proteins in the cell. In other embodiments, an RNA oligonucleotide may be used to target the mRNA transcript of the target gene.

[0101] In some cases, the delivery of the inhibitory polynucleotide may result in a knockdown of the target gene to an extent that is at least about 25%, 50%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0102] In some aspects, the inhibitory polynucleotide (e.g., ASO or siRNA) comprises one or more modified nucleotides to improve certain properties of the nucleic acids, such as binding affinity, stability, and / or nuclease resistance. Accordingly, in some embodiments, the nucleic acid introduced to the cancer cell can include at least one nucleotide that is modified. In some aspects, the modified nucleotide comprises a sugar modification, a nucleic acid base modification, and / or a phosphate backbone modification. Modifications that are useful for optimizing inhibitory nucleic acids are described, e.g., in Freier & Altmann (1997), Nucl. Acid Res., 25, 4429; Uhlmann (2000), Curr. Opinion Drug Develop., 3(2), 293; and Deleavey and Damha (2012), Chem. Biol., 19: 937, and U.S. Patent Nos. 5,684,143, 5,858,988, and 6,291,438, as well as Filippova et al. (2019), Biochimie., 167:49.

[0103] Suitable antisense RNA molecules, siRNA, miRNA, shRNA can be produced by standard methods of oligonucleotide synthesis or by ordering such molecules from a contract research organization or supplier by providing the polynucleotide sequence, e.g., gene, being targeted.

[0104] Depending on whether transient or stable expression is desired one can select an appropriate delivery vector. Examples of delivery vectors that may be used with the present disclosure are viral vectors, plasmids, exosomes, liposomes, bacterial vectors, or nanoparticles. Exemplary viral vectors include adenovirus vector, adeno-associated viral vector (AAV), retrovirus vector, lentivirus vector.

[0105] siRNA and shRNA are involved in the RNA interference (RNAi) pathway where they can induce degradation of a target RNA. Methods for constructing siRNAs useful for inhibiting target RNAs are known to those of skill in the art, see e.g., Fire et al. (1998), Nature, 391:806; 79685919V.1Elbashir et al. (2001), Nature, 411:494; Brummelkamp (2002), Science, 296:550; Wittrup and Lieberman (2015), Nature Rev. Genet., 16:543–552; and Vickers et al. (2003), J. Biol. Chem., 278:7108. siRNAs comprise a sense strand and a complementary antisense strand annealed together by standard Watson Crick base pairing interactions. The sense strand may comprise a nucleic acid sequence that is identical to a target sequence contained within a target RNA, and the antisense strand may comprise a nucleic acid sequence that is complementary to a target sequence contained within the target RNA. In cells, the sense strand is degraded by RISC and the antisense strand directs RISC to an mRNA that has a complementary sequence. A protein called Ago2 in the RISC then cleaves the mRNA, or in some cases, represses translation of the mRNA, leading to its destruction and an eventual reduction in the protein encoded by the mRNA. Thus, the siRNA leads to targeted gene silencing.

[0106] A short hairpin RNA or small hairpin RNA (shRNA) is an artificial RNA molecule that is converted to siRNA and, thus, can be used to silence target gene expression via the same (RNAi) pathway described above. See, e.g., Fire et. al., Nature 391:806, 1998; Elbashir et. al., Nature 411:494, 2001; Chakraborty et al. Mol. Ther. Nucleic Acids 8:132, 2017; Bouard et al., and Br. J. Pharmacol.157:153, 2009. In the case of the shRNA, the sense and antisense strand are covalently linked by a single-stranded loop region, and the shRNA is converted into a siRNA by a cleavage event mediated by the enzyme Dicer. The loop region may be between 2 and 12 nucleotides in length. In some cases, the loop region is from 4 to 10 nucleotides in length. Details on the structure of shRNAs can be found, for example, in Paddison et al. (2002), Genes Dev., 16(8):948; Brummelkamp (2002), Science, 296:550; and Yu et al. (2002), Proc. Natl. Acad. Sci. USA, 99:6047.

[0107] In some embodiments, the siRNA or shRNA is 15-100, e.g., 15-50, 16-30, or 19-25 nucleotides in length. In some embodiments, the siRNA or shRNA is 21 nucleotides in length. In some embodiments, the siRNA or shRNA comprises at least 15 contiguous nucleotides identical or complimentary to the target gene. In some aspects, the siRNA or shRNA comprises a sense strand and an antisense strand, where the antisense strand includes a region that is identical or substantially identical to a target sequence of the target gene and the sense strand includes a region that is complementary or substantially complementary to a region of the antisense strand. 79685919V.1

[0108] In some embodiments, the siRNA or shRNA comprises an overhang on the sense strandand / or the antisense strand. The overhang may be at the 5 end and / or the 3 end of either of thestrands. The overhang can have any nucleotide sequence and may be 1-10 nucleotides in length, e.g., 2-6, e.g., 2-4 nucleotides in length.

[0109] RNase H-dependent antisense oligonucleotides (ASOs) are single-stranded, chemically modified oligonucleotides that bind to complementary sequences in target mRNAs and reduce gene expression both by RNase H-mediated cleavage of the target RNA and by inhibition of translation by steric blockade of ribosomes.

[0110] RNase H is an endonuclease enzyme that catalyzes the cleavage of RNA in an RNA:DNA duplex. The most well studied endogenous function for this enzyme is the removal of Okazaki fragments (small RNAs) used to prime the DNA duplication during cell division. In some embodiments, to target the mRNA transcript of the target gene for degradation, a nucleic acid (e.g., DNA oligonucleotide) capable of hybridizing to a portion of the mRNA may be administered to the subject. Once inside the cell, the DNA oligonucleotide base pairs with its targeted mRNA transcript. RNase H may bind to the resulting duplex and cleave the mRNA transcript at one or more places. The DNA oligonucleotide may further bind to other mRNA transcripts to target them for RNase H degradation. Thus, the expression of the target gene may be greatly reduced in a cancer cell of a subject.

[0111] A DNA oligonucleotide capable of hybridizing to an mRNA transcript of a target gene may contain, e.g., between 12 and 30 nucleotides (e.g., 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30 nucleotides). In some embodiments, the DNA oligonucleotide may be 100% identical to a portion of a target gene. In other embodiments, the ASO may be less than 100% identical (e.g., 95%, 90%, 85%, 80%, 75%, or 70% complementarity) to a portion of a target gene, but can still form a stableRNA:DNA duplex for the RNase H to cleave the mRNA transcript. The ASO may bind to the 5UTR or the 3 UTR of the mRNA transcript of the target gene.

[0112] A microRNA (miRNA) is a small non-coding RNA molecule that functions in RNA silencing and post-transcriptional regulation of gene expression. miRNAs base pair with complementary sequences within the mRNA transcript. As a result, the mRNA transcript may be silenced by one or more of the mechanisms such as cleavage of the mRNA strand, destabilization of the mRNA through shortening of its poly(A) tail, and decreased translation efficiency of the 79685919V.1mRNA transcript into proteins by ribosomes. In some embodiments, miRNAs resemble the siRNAs of the shRNA pathway, except that miRNAs derive from regions of RNA transcripts that fold back on themselves to form short hairpins, which are also called pri-miRNA. Once transcribed as pri-miRNA, the hairpins are cleaved out of the primary transcript in the nucleus by an enzyme called Drosha. The hairpins, or pre-miRNA, are then exported from the nucleus into the cytosol. In the cytosol, the loop of the hairpin is cleaved off by an enzyme called Dicer. The resultingproduct is now a double strand RNA with overhangs at the 3 end, which is then incorporated intoRISC. Once in the RISC, the second strand is discarded and the miRNA that is now in the RISC is a mature miRNA, which binds to mRNAs that have complementary sequences.

[0113] A difference between miRNAs and siRNAs from the shRNA pathway is that base pairingwith miRNAs comes from the 5 end of the miRNA, which is also referred to as the seed sequence.Since the seed sequence is short, each miRNA may target many more mRNA transcripts. In some embodiments, an miRNA targeting the target gene may be used in methods described herein. Suitable miRNAs are described in e.g., Lowery et al. (2016), Stem Cells Int.; 2016:7290686. Exemplary miRNA can also be found e.g., in the “miRTarBase” database available at mirtarbase.cuhk.edu.cn / php / idex.php, which includes experimentally validated microRNA-target interactions.

[0114] In some embodiments, the knocking out or knocking down of the target gene is performed using a gene editing system such as the CRISPR / Cas system. See Sanders and Joung, Nature Biotechnol. 32:347, 2014, Huang et al., J. Cell. Physiol. 10:1, 2017 and Mitsunobu et al., Trends Biotechnol. 17:30132, 2017. The “CRISPR / Cas” system refers to a widespread class of bacterial systems for defense against foreign nucleic acid. CRISPR / Cas systems include type I, II, and III sub-types. Any CRISPR / Cas system that is capable of altering a target polynucleotide sequence in a cell can be used in methods described here. Wild-type type II CRISPR / Cas systems use the RNA-mediated nuclease, for example, Cas9, in complex with guide and activating RNA to recognize and cleave foreign nucleic acid. In nature, many CRISPR systems include transactivating crisp RNA (tracrRNA), which binds the Cas endonuclease, and crisp RNA (crRNA), which binds to the DNA target sequence. Some CRISPR systems (e.g., CRISPR Cas12a / Cpf1) require only crRNA. In research and biomedical applications it is more typical to use a chimeric single guide RNA (“sgRNA”), which is a crRNA-tracrRNA fusion that binds both 79685919V.1the Cas endonuclease and the DNA target sequence (e.g., target gene sequence). It will be understood that, except where apparent from context, reference to a “gRNA” includes any suitable guide RNA with appropriate binding specificity (e.g., a sgRNA, crRNA, or other RNA that binds to a target gene). The most commonly used sgRNAs comprise a nucleic acid sequence approximately 20 nucleotides in length. Methods for designing sgRNAs that target a specified target sequence are well known in the art. See e.g., Doench et al. (2016), Nat. Biotechnol.34:184; Horlbeck et al. (2016), eLife.5, e19760 (2016); Cui et al., Interdiscip. Sci.2018, 10:455–465; and Kiani et al. (2015), Nat. Methods.2015, 12:1051.

[0115] Aspects of the invention relate to a nucleic acid that is a guide RNA (gRNA) that targets a polynucleotide encoding a target gene. In some aspects, introduction of the gRNA in a cancer cell expressing the target gene inhibits expression of the target gene. In some embodiments, the gRNA is of 20 nucleotides in length. In some cases, the guide RNA is an sgRNA.

[0116] In some aspects, the invention relates to a CRISPR / Cas system, where the system comprises a Cas protein and a guide RNA (e.g., an sgRNA) as described above. The sgRNA and Cas can be expressed from the same or different vectors of the system. Cas proteins and their amino acid sequence are well known in the art. The Cas protein used in the methods described herein can be a naturally occurring Cas protein or a functional derivative thereof. A “functional derivative” includes, but is not limited to, fragments of a native sequence and derivatives of a native sequence polypeptide and its fragments, provided that they have a biological activity in common with the corresponding native sequence polypeptide. A biological activity contemplated herein is the ability of the functional derivative to hydrolyze a DNA substrate (e.g., a target gene) into fragments. The term “derivative” encompasses both amino acid sequence variants of polypeptide, covalent modifications, and fusions thereof. Suitable derivatives of a Cas protein or a fragment thereof include but are not limited to mutants, fusions, or covalent modifications of Cas protein. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof. An exemplary Cas9 protein is the Streptococcus pyogenes Cas9 protein. The amino acid sequence of S. pyogenes Cas9 protein may 79685919V.1be found in the SwissProt database under accession number Q99ZW2. Additional Cas9 proteins and homologs thereof are described in, e.g., Chylinksi, et al., RNA Biol. 2013 May 1; 10(5): 726; Nat. Rev. Microbiol. 2011 June; 9(6): 467; Hou, et al., Proc. Natl. Acad. Sci. USA. 2013 Sep 24;110(39):15644; Sampson et al., Nature. 2013 May 9;497(7448):254; and Jinek, et al., Science. 2012 Aug 17;337(6096):816.

[0117] In some cases, the gRNA binds to a target sequence that is contiguous with a protospacer adjacent motif (PAM) recognized by the Cas protein. For example, Cas9 generally requires the PAM motif NGG for activity. Thus, in some systems, certain target sequences will be preferred based on the proximity of the target sequence to a PAM. However, some Cas proteins, including variants of Cas9, have flexible PAM requirements (see e.g., Legut et al., 2020, Cell Reports 30:2859; Gleditzsch et al., 2019, RNA Biol.2019 Apr; 16(4): 504) and other Cas proteins are PAM- independent (e.g., Cas14a1). Exemplary PAMs are described, e.g., in Zhao et al. (2017), Int. J. Biol. Sci.; 13(12):1470-1478.

[0118] The sensitized cancer cell can be a cell of any cancer, including a cell of a solid tumor or a liquid tumor. In some embodiments, the cancer cell is a melanoma cell. In some embodiments, the cancer cell is a breast cancer cell. IV. ENGINEERED CYTOTOXIC LYMPHOCYTES

[0119] In other aspects, the present disclosure provides various engineered cytotoxic lymphocytes and various methods for their production. The engineered cytotoxic lymphocytes generally overexpress an antitumor enhancing ligand that increases the elimination of a cancer cell by the engineered cytotoxic lymphocyte. The overexpressing of the antitumor enhancing ligand can include activating the transcription of a gene encoding the antitumor enhancing ligand in the cytotoxic lymphocyte, and / or can include introducing such a gene into the cytotoxic lymphocyte. The antitumor enhancing ligand binds to a corresponding binding partner, e.g., a receptor, of the engineered cytotoxic lymphocyte. Therefore, overexpression of the tumor enhancing ligand by the engineered cytotoxic lymphocyte can result in enhanced autocrine signaling that induces stem-like and other properties for improved cytotoxic lymphocyte function. The disclosed materials and methods thus provide an advantageous cell engineering approach for improved cytotoxic lymphocyte-based therapies addressing cancer. 79685919V.1

[0120] Some provided methods for engineering a cytotoxic lymphocyte involve overexpressing at least one antitumor enhancing ligand binding in the cytotoxic lymphocyte by using a guide- based gene activation system, e.g., a system relying on a guide RNA. For example, the methods can include introducing into the cytotoxic lymphocyte a polynucleotide, where the polynucleotide includes a guide RNA that targets a gene encoding the antitumor enhancing ligand. Alternatively or additionally, the methods can include introducing into the cytotoxic lymphocyte a polynucleotide that encodes a guide RNA targeting the gene encoding the antitumor enhancing ligand.

[0121] For guide-based gene activation methods, the cytotoxic lymphocyte will generally include other necessary molecular components of a guide-base gene activation system, e.g., a CRISPRa system. In some examples, the method includes introducing to the cytotoxic lymphocyte one or more molecular components of a guide-based gene activation system. The molecular components of the guide-based gene activation system can include, for example, CRISPR / dCas9, CRISPR / cas9, CRISPRon, CRISPR / dCas9-SAM, CRISPR / dCas9-VPR, cas12, cas13, other cas or cas-like proteins, or any combination thereof. In some embodiments, the method includes co- delivering the guide RNA to the cytotoxic lymphocyte together with one more CRISPRa gene activation system molecular components, e.g., CRISPR / dCas9-VPR. In some embodiments, the gene activation system molecular components are co-delivered to the cytotoxic lymphocyte at the protein level, e.g., with the guide RNA incorporated into a CRISPR complex, i.e., as a ribonucleotide complex. In some embodiments, the gene activation system molecular components are co-delivered to the cytotoxic lymphocyte at the nucleic acid level, e.g., as one or more polynucleotides that together encode the gene activation system molecular components and encode or include the guide RNA.

[0122] Some provided methods for engineering a cytotoxic lymphocyte involve overexpressing at least one antitumor enhancing ligand in the cytotoxic lymphocyte by using an open reading frame, e.g., a polynucleotide encoding the antitumor enhancing ligand. For example, the methods can include introducing into the cytotoxic lymphocyte a polynucleotide, where the polynucleotide encodes the antitumor enhancing ligand. In some embodiments, the cytotoxic lymphocyte is sensitized by introducing at least two polynucleotides into the cancer cell, where at least one of the at least two polynucleotides includes an open reading frame encoding an antitumor enhancing 79685919V.1ligand, and where at least one of the at least two polynucleotides includes or encodes a guide RNA targeting the open reading frame, i.e., the gene. In some embodiments, the cytotoxic lymphocyte is sensitized by introducing into the cytotoxic lymphocyte a polynucleotide that both includes an open reading frame encoding an antitumor enhancing ligand, and also includes or encodes a guide RNA targeting the open reading frame.

[0123] In some embodiments, the polynucleotide encoding or including the guide RNA, and / or the polynucleotide encoding the antitumor enhancing ligand includes or consists of RNA. For example, the method can include introducing mRNA to the cytotoxic lymphocyte, where the mRNA encodes the antitumor enhancing ligand, the guide RNA, or both. In some embodiments, the polynucleotide encoding or including the guide RNA, and / or the polynucleotide encoding the antitumor enhancing ligand includes or consists of DNA. For example, the method can include introducing DNA to the cytotoxic lymphocyte, where the DNA encodes the antitumor enhancing ligand, the guide RNA, or both. In some embodiments, the DNA delivered to the cytotoxic lymphocyte becomes integrated into the genome of the cytotoxic lymphocyte. In some examples of the provided method, the antitumor enhancing ligand is introduced or delivered to the cytotoxic lymphocyte in protein form, rather than in the form of a polynucleotide encoding the ligand.

[0124] In some examples, the antitumor enhancing ligand is an endogenous protein of the cytotoxic lymphocyte. In these cases, overexpressing the endogenous antitumor enhancing ligand can include activating an endogenous gene encoding the endogenous antitumor enhancing ligand by introducing to the cytotoxic lymphocyte a guide RNA targeting the endogenous gene. Additionally or alternatively, overexpressing the endogenous antitumor enhancing ligand can include increasing the copy number of the endogenous gene encoding the endogenous antitumor enhancing ligand by introducing to the cytotoxic lymphocyte an open reading frame encoding the antitumor enhancing ligand. Other suitable methods for overexpressing the endogenous antitumor enhancing ligand can include enhancing the transcriptional or translation rate of the endogenous antitumor enhancing ligand.

[0125] In some examples, the antitumor enhancing ligand is exogenous to the cytotoxic lymphocyte. In these cases, overexpressing the exogenous antitumor enhancing ligand can include introducing to the cytotoxic lymphocyte an open reading frame encoding the antitumor enhancing ligand. Overexpressing the exogenous antitumor enhancing ligand can further include activating 79685919V.1the introduced open reading frame encoding the exogenous antitumor enhancing ligand by also introducing to the cytotoxic lymphocyte a guide RNA targeting the open reading frame.

[0126] In some embodiments, the binding partner of the antitumor enhancing ligand, e.g. the receptor that binds to the ligand, is an endogenous protein of the cytotoxic lymphocyte. In some embodiments, the method further includes overexpressing the endogenous binding partner by activating an endogenous gene encoding the endogenous binding partner through introduction of a guide RNA to the cytotoxic lymphocyte, where the guide RNA targets the endogenous gene. Additionally or alternatively, overexpressing the endogenous binding partner can include increasing the copy number of the endogenous gene encoding the endogenous binding partner by introducing to the cytotoxic lymphocyte an open reading frame encoding the binding partner. Other suitable methods for overexpressing the endogenous binding partner can include enhancing the transcriptional or translation rate of the endogenous binding partner.

[0127] In some examples, the binding partner of the antitumor enhancing ligand is exogenous to the cytotoxic lymphocyte. In some embodiments, the method further includes overexpressing the exogenous binding partner by introducing to the cytotoxic lymphocyte an open reading frame encoding the binding partner. Overexpressing the exogenous binding partner can further include activating the introduced open reading frame encoding the exogenous binding partner by also introducing to the cytotoxic lymphocyte a guide RNA targeting the open reading frame.

[0128] The introduction of polynucleotides (e.g., a polynucleotide including or encoding a guide RNA, a polynucleotide encoding an antitumor enhancing ligand, and / or a polynucleotide encoding a molecular component of a guide-based gene activation system) to the cytotoxic lymphocyte can involve, for example, transduction with one or more viruses containing the polynucleotides. Additionally or alternatively, the introduction of the polynucleotides to the cytotoxic lymphocytes can involve transduction with one or more virus-like particles containing the polynucleotides. In other examples, the introduction of a polynucleotide to the cytotoxic lymphocyte involves contacting the cytotoxic lymphocyte with an immune cell that contains the polynucleotide and induces transfer of the polynucleotide from the immune cell to the cytotoxic lymphocyte.

[0129] In some examples, the antitumor enhancing ligand includes or consists of one or more Wnt family proteins or subunits or fragments thereof. For example, the antitumor enhancing ligand can include or consist of Wnt family member 1 (WNT1), Wnt family member 3 (WNT3), Wnt 79685919V.1family member 3A (WNT3A), Wnt family member 10B (WNT10B), or a variant or combination thereof. In some embodiments, the antitumor enhancing ligand includes or consists of CD58, CDCP1, CD47, or a variant or combination thereof. In some examples, the corresponding binding partner to the antitumor enhancing ligand includes or consists of one or more frizzled family transmembrane receptors, CD2, CD6, signal-regulatory protein gamma (SIRPG), signal- regulatory protein alpha (SIRPA), or a variant thereof.

[0130] In some embodiments, the engineered cytotoxic lymphocyte is a T cell, e.g., a CD8+T cell. In other embodiments, the engineered cytotoxic lymphocyte is an NK cell. The engineered cytotoxic lymphocyte can express a chimeric antigen receptor (CAR) and / or an engineered T-cell receptor (TCR). In some embodiments, the cytotoxic lymphocyte has an antigen-specific receptor that targets a particular cancer cell type, e.g., any of the sensitized cancer cells described in Section III. V. CANCER TREATMENT OR PREVENTION

[0131] In other aspects, the present disclosure provides various methods for treating or preventing a cancer in a subject. In some embodiments, the methods include sensitizing cancer cells of the subject using any of the methods described in Section III. For example, the methods can include administering to the subject a polynucleotide that includes or encodes a guide RNA targeting a gene encoding a cytotoxic-lymphocyte sensitizing protein. Additionally or alternatively, the methods can include administering to the subject a polynucleotide that encodes a cytotoxic-lymphocyte sensitizing protein. In some embodiments, the methods include administering to the subject any of the engineered cytotoxic lymphocytes described in Section IV. Further, the methods can include producing engineered cytotoxic lymphocytes using any of the techniques described in Section IV before administering the engineered cytotoxic lymphocytes to the subject.

[0132] Non-limiting examples of cancers that can be treated with the provided methods include Acanthoma, Acinic cell carcinoma, Acoustic neuroma, Acral lentiginous melanoma, Acrospiroma, Acute eosinophilic leukemia, Acute lymphoblastic leukemia, Acute megakaryoblastic leukemia, Acute monocytic leukemia, Acute myeloblastic leukemia with maturation, Acute myeloid dendritic cell leukemia, Acute myeloid leukemia, Acute promyelocytic leukemia, Adamantinoma, 79685919V.1Adenocarcinoma, Adenoid cystic carcinoma, Adenoma, Adenomatoid odontogenic tumor, Adrenocortical carcinoma, Adult T-cell leukemia, Aggressive NK-cell leukemia, AIDS-Related Cancers, AIDS-related lymphoma, Alveolar soft part sarcoma, Ameloblastic fibroma, Anal cancer, Anaplastic large cell lymphoma, Anaplastic thyroid cancer, Angioimmunoblastic T-cell lymphoma, Angiomyolipoma, Angiosarcoma, Appendix cancer, Astrocytoma, Atypical teratoid rhabdoid tumor, Basal cell carcinoma, Basal-like carcinoma, B-cell leukemia, B-cell lymphoma, Bellini duct carcinoma, Biliary tract cancer, Bladder cancer, Blastoma, Bone Cancer, Bone tumor, Brain Stem Glioma, Brain Tumor, Breast Cancer, Brenner tumor, Bronchial Tumor, Bronchioloalveolar carcinoma, Brown tumor, Burkitt's lymphoma, Cancer of Unknown Primary Site, Carcinoid Tumor, Carcinoma, Carcinoma in situ, Carcinoma of the penis, Carcinoma of Unknown Primary Site, Carcinosarcoma, Castleman's Disease, Central Nervous System Embryonal Tumor, Cerebellar Astrocytoma, Cerebral Astrocytoma, Cervical Cancer, Cholangiocarcinoma, Chondroma, Chondrosarcoma, Chordoma, Choriocarcinoma, Choroid plexus papilloma, Chronic Lymphocytic Leukemia, Chronic monocytic leukemia, Chronic myelogenous leukemia, Chronic Myeloproliferative Disorder, Chronic neutrophilic leukemia, Clear-cell tumor, Colon Cancer, Colorectal cancer, Craniopharyngioma, Cutaneous T-cell lymphoma, Degos disease, Dermatofibrosarcoma protuberans, Dermoid cyst, Desmoplastic small round cell tumor, Diffuse large B cell lymphoma, Dysembryoplastic neuroepithelial tumor, Embryonal carcinoma, Endodermal sinus tumor, Endometrial cancer, Endometrial Uterine Cancer, Endometrioid tumor, Enteropathy-associated T-cell lymphoma, Ependymoblastoma, Ependymoma, Epithelioid sarcoma, Erythroleukemia, Esophageal cancer, Esthesioneuroblastoma, Ewing Family of Tumor, Ewing Family Sarcoma, Ewing's sarcoma, Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Extramammary Paget's disease, Fallopian tube cancer, Fetus in fetu, Fibroma, Fibrosarcoma, Follicular lymphoma, Follicular thyroid cancer, Gallbladder Cancer, Gallbladder cancer, Ganglioglioma, Ganglioneuroma, Gastric Cancer, Gastric lymphoma, Gastrointestinal cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Stromal Tumor, Gastrointestinal stromal tumor, Germ cell tumor, Germinoma, Gestational choriocarcinoma, Gestational Trophoblastic Tumor, Giant cell tumor of bone, Glioblastoma multiforme, Glioma, Gliomatosis cerebri, Glomus tumor, Glucagonoma, Gonadoblastoma, Granulosa cell tumor, Hairy Cell Leukemia, Hairy cell leukemia, Head and Neck Cancer, Head and neck cancer, Heart cancer, Hemangioblastoma, 79685919V.1Hemangiopericytoma, Hemangiosarcoma, Hematological malignancy, Hepatocellular carcinoma, Hepatosplenic T-cell lymphoma, Hereditary breast-ovarian cancer syndrome, Hodgkin Lymphoma, Hodgkin's lymphoma, Hypopharyngeal Cancer, Hypothalamic Glioma, Inflammatory breast cancer, Intraocular Melanoma, Islet cell carcinoma, Islet Cell Tumor, Juvenile myelomonocytic leukemia, Kaposi Sarcoma, Kaposi's sarcoma, Kidney Cancer, Klatskin tumor, Krukenberg tumor, Laryngeal Cancer, Laryngeal cancer, Lentigo maligna melanoma, Leukemia, Leukemia, Lip and Oral Cavity Cancer, Liposarcoma, Lung cancer, Luteoma, Lymphangioma, Lymphangiosarcoma, Lymphoepithelioma, Lymphoid leukemia, Lymphoma, Macroglobulinemia, Malignant Fibrous Histiocytoma, Malignant fibrous histiocytoma, Malignant Fibrous Histiocytoma of Bone, Malignant Glioma, Malignant Mesothelioma, Malignant peripheral nerve sheath tumor, Malignant rhabdoid tumor, Malignant triton tumor, MALT lymphoma, Mantle cell lymphoma, Mast cell leukemia, Mediastinal germ cell tumor, Mediastinal tumor, Medullary thyroid cancer, Medulloblastoma, Medulloblastoma, Medulloepithelioma, Melanoma, Melanoma, Meningioma, Merkel Cell Carcinoma, Mesothelioma, Mesothelioma, Metastatic Squamous Neck Cancer with Occult Primary, Metastatic urothelial carcinoma, Mixed Mullerian tumor, Monocytic leukemia, Mouth Cancer, Mucinous tumor, Multiple Endocrine Neoplasia Syndrome, Multiple Myeloma, Multiple myeloma, Mycosis Fungoides, Mycosis fungoides, Myelodysplastic Disease, Myelodysplastic Syndromes, Myeloid leukemia, Myeloid sarcoma, Myeloproliferative Disease, Myxoma, Nasal Cavity Cancer, Nasopharyngeal Cancer, Nasopharyngeal carcinoma, Neoplasm, Neurinoma, Neuroblastoma, Neuroblastoma, Neurofibroma, Neuroma, Nodular melanoma, Non- Hodgkin Lymphoma, Non-Hodgkin lymphoma, Nonmelanoma Skin Cancer, Non-Small Cell Lung Cancer, Ocular oncology, Oligoastrocytoma, Oligodendroglioma, Oncocytoma, Optic nerve sheath meningioma, Oral Cancer, Oral cancer, Oropharyngeal Cancer, Osteosarcoma, Osteosarcoma, Ovarian Cancer, Ovarian cancer, Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian Low Malignant Potential Tumor, Paget's disease of the breast, Pancoast tumor, Pancreatic Cancer, Pancreatic cancer, Papillary thyroid cancer, Papillomatosis, Paraganglioma, Paranasal Sinus Cancer, Parathyroid Cancer, Penile Cancer, Perivascular epithelioid cell tumor, Pharyngeal Cancer, Pheochromocytoma, Pineal Parenchymal Tumor of Intermediate Differentiation, Pineoblastoma, Pituicytoma, Pituitary adenoma, Pituitary tumor, Plasma Cell Neoplasm, Pleuropulmonary blastoma, Polyembryoma, Precursor T-lymphoblastic lymphoma, Primary central nervous system lymphoma, Primary effusion lymphoma, Primary Hepatocellular 79685919V.1Cancer, Primary Liver Cancer, Primary peritoneal cancer, Primitive neuroectodermal tumor, Prostate cancer, Pseudomyxoma peritonei, Rectal Cancer, Renal cell carcinoma, Respiratory Tract Carcinoma Involving the NUT Gene on Chromosome 15, Retinoblastoma, Rhabdomyoma, Rhabdomyosarcoma, Richter's transformation, Sacrococcygeal teratoma, Salivary Gland Cancer, Sarcoma, Schwannomatosis, Sebaceous gland carcinoma, Secondary neoplasm, Seminoma, Serous tumor, Sertoli-Leydig cell tumor, Sex cord-stromal tumor, Sezary Syndrome, Signet ring cell carcinoma, Skin Cancer, Small blue round cell tumor, Small cell carcinoma, Small Cell Lung Cancer, Small cell lymphoma, Small intestine cancer, Soft tissue sarcoma, Somatostatinoma, Soot wart, Spinal Cord Tumor, Spinal tumor, Splenic marginal zone lymphoma, Squamous cell carcinoma, Stomach cancer, Superficial spreading melanoma, Supratentorial Primitive Neuroectodermal Tumor, Surface epithelial-stromal tumor, Synovial sarcoma, T-cell acute lymphoblastic leukemia, T-cell large granular lymphocyte leukemia, T-cell leukemia, T-cell lymphoma, T-cell prolymphocytic leukemia, Teratoma, Terminal lymphatic cancer, Testicular cancer, Thecoma, Throat Cancer, Thymic Carcinoma, Thymoma, Thyroid cancer, Transitional Cell Cancer of Renal Pelvis and Ureter, Transitional cell carcinoma, Urachal cancer, Urethral cancer, Urogenital neoplasm, Uterine sarcoma, Uveal melanoma, Vaginal Cancer, Verner Morrison syndrome, Verrucous carcinoma, Visual Pathway Glioma, Vulvar Cancer, Waldenstrom's macroglobulinemia, Warthin's tumor, Wilms' tumor, and combinations thereof.

[0133] The treated or prevented cancer can be a solid cancerous tumor or a liquid cancerous tumor. The solid cancerous tumor can be, for example, breast cancer tumor or a tubo-ovarian cancer tumor. The liquid cancerous tumor can be, for example, a melanoma. A tumor treated with the methods disclosed herein can result in stabilized tumor growth (e.g., one or more tumors do not increase more than 1%, 5%, 10%, 15%, or 20% in size, and / or do not metastasize). In some embodiments, a tumor is stabilized for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more weeks. In some embodiments, a tumor is stabilized for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more months. In some embodiments, a tumor is stabilized for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more years. In some embodiments, the size of a tumor or the number of tumor cells is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more. In some embodiments, the tumor is completely eliminated, or reduced below a level of detection. In some embodiments, a subject remains tumor free (e.g. in remission) for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more weeks following 79685919V.1treatment. In some embodiments, a subject remains tumor free for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more months following treatment. In some embodiments, a subject remains tumor free for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more years after treatment.

[0134] One skilled in the art will also appreciate that the provided materials be co-administered with other therapeutic agents for the treatment of cancer. Suitable anti-cancer agents for combination therapy include, without limitation, cytotoxins and agents such as antimetabolites, alkylating agents, anthracyclines, antibiotics, antimitotic agents, procarbazine, hydroxyurea, asparaginase, corticosteroids, interferons, radiopharmaceuticals, peptides with anti-tumor activitysuch as TNF- , pharmaceutically acceptable salts thereof; derivatives thereof, prodrugs thereof,and combinations thereof. For example, the provided engineered immune cells can be administered to a patient before, during, or after administration of an anti-cancer agent or combination of anti- cancer agents either before, during, or after chemotherapy.

[0135] In some embodiments, the provided treatment method results in stable disease, partial remission, or complete remission in the subject (e.g., the methods described herein provide a treatment that kills or otherwise slows the growth or progression of cancer cells and leads to stable disease or to partial or complete remission of the cancer in the subject). In some embodiments, the provided treatment results in a reduction in metastases of the cancer in the subject (e.g., the methods described herein provide a treatment that reduces metastases of the cancer in the subject). In some embodiments, the provided treatment results in a reduction in volume, size, or growth of a tumor in the subject (e.g., the methods described herein provide a treatment that reduces the volume, size, or growth of a tumor in the subject). In some embodiments, the provided treatment results in an increased responsiveness of the cancer to a subsequently administered anti-cancer agent (e.g., the methods described herein provide a treatment that increases responsiveness of the cancer to a subsequently administered anti-cancer agent).

[0136] In some embodiments, the provided method further includes obtaining a test sample from the subject. The test sample can include, for example, a blood sample, a tissue sample, a urine sample, a saliva sample, a cerebrospinal fluid sample, or a combination thereof. In some embodiments, the provided method further includes determining the level of one or more biomarkers in the obtained test sample. Determining the presence or level of biomarkers(s) can be 79685919V.1used to, as non-limiting examples, determine response to treatment or to select an appropriate composition for the prevention or treatment of the disease.

[0137] In some embodiments, the provided method further includes comparing the determined level of the one of more biomarkers in the obtained test sample to the level of the one or more biomarkers in a reference sample. The reference sample can be obtained, for example, from the subject, with the reference sample being obtained prior to the obtaining of the test sample, e.g., prior to the administering to the subject of the therapeutically effective amount of the provided materials. In this way, the reference sample can provide information about baseline levels of the biomarkers in the sample before the treatment, and the test sample can provide information about levels of the biomarkers after the treatment.

[0138] Alternatively, the reference sample can be obtained, for example, from a different subject, e.g., a subject in which the treatment is not provided according to the provided methods. In this way, the reference sample can provide information about baseline levels of the biomarkers without treatment, and the test sample can provide information about levels of the biomarkers with treatment. The reference sample can also be obtained, for example, from a population of subjects, e.g., subjects in which the treatment is not provided according to the provided method. In this way, the reference sample can provide population-averaged information about baseline levels of the biomarkers without treatment, and the test sample can provide information about levels of the biomarkers with treatment.

[0139] The reference sample can also be obtained from an individual or a population of individuals after treatment is provided according to the provided methods, and can serve as, for example, a positive control sample. In some embodiments, the reference sample is obtained from normal tissue. In some embodiments, the reference sample is obtained from abnormal tissue.

[0140] Depending on the biomarker, an increase or decrease relative to a normal control or reference sample can be indicative of the presence of a disease, or response to treatment for a disease. In some embodiments, an increased level of a biomarker in a test sample, and hence the presence of a disease, e.g., a cancer, increased risk of the disease, or response to treatment is determined when the biomarker levels are at least, 1.1-fold, e.g., at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9- fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at 79685919V.1least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, or at least 20-fold higher in comparison to a negative control. In other embodiments, a decreased level of a biomarker in the test sample, and hence the presence of the disease, increased risk of the disease, or response to treatment is determined when the biomarker levels are at least 1.1-fold, e.g., at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5- fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, or at least 20-fold lower in comparison to a negative control.

[0141] The biomarker levels can be detected using any method known in the art, including the use of antibodies specific for the biomarkers. Exemplary methods include, without limitation, polymerase chain reaction (PCR), Western Blot, dot blot, ELISA, radioimmunoassay (RIA), immunoprecipitation, immunofluorescence, FACS analysis, electrochemiluminescence, and multiplex bead assays, e.g., using Luminex or fluorescent microbeads. In some instances, nucleic acid sequencing is employed. In some embodiments, the detection of levels of biomarkers such as metabolites can include use of mass spectrometry (e.g., liquid chromatography linked to mass spectrometry (LC-MS)) and / or receptor (e.g., GPCR) reporting, for example as described in Example 3.

[0142] In certain embodiments, the presence of decreased or increased levels of one or more biomarkers is indicated by a detectable signal, e.g., a blot, fluorescence, chemiluminescence, color, or radioactivity in an immunoassay or PCR reaction, e.g., quantitative PCR. This detectable signal can be compared to the signal from a reference sample or to a threshold value.

[0143] In some embodiments, the results of the biomarker level determinations are recorded in a tangible medium. For example, the results of diagnostic assays, e.g., the observation of the presence or decreased or increased presence of one or more biomarkers, and the diagnosis of whether or not there is an increased risk or the presence of a disease, e.g., a cancer, or whether or not a subject is responding to treatment can be recorded, for example, on paper or on electronic media, e.g., audio tape, a computer disk, a CD-ROM, or a flash drive. 79685919V.1

[0144] In some embodiments, the provided method further includes the step of providing to the subject a diagnosis and / or the results of treatment. VI. EXEMPLARY EMBODIMENTS

[0145] The following embodiments are contemplated. All combinations of features and embodiments are contemplated.

[0146] Embodiment 1: A method for sensitizing a cancer cell to cytotoxic lymphocytes, wherein the cancer cell comprises a gene encoding a cytotoxic-lymphocyte sensitizing protein that increases elimination of the cancer cell upon recognition of the cancer cell by a cytotoxic lymphocyte targeting the cancer cell, the method comprising introducing into the cancer cell a polynucleotide comprising or encoding a guide RNA targeting the gene.

[0147] Embodiment 2: An embodiment of embodiment 1, wherein the method further comprises introducing into the cancer cell a CRISPR-based activation system.

[0148] Embodiment 3: A method for sensitizing a cancer cell to cytotoxic lymphocytes, the method comprising: introducing into the cancer cell a polynucleotide encoding a cytotoxic- lymphocyte sensitizing protein that increases elimination of the cancer cell upon recognition of the cancer cell by a cytotoxic lymphocyte targeting the cancer cell.

[0149] Embodiment 4: An embodiment of any one of embodiments 1-3, wherein the polynucleotide comprises RNA.

[0150] Embodiment 5: An embodiment of any one of embodiments 1-4, wherein the cytotoxic- lymphocyte sensitizing protein is an endogenous protein of the cancer cell.

[0151] Embodiment 6: An embodiment of any one of embodiments 1-5, wherein the cytotoxic- lymphocyte sensitizing protein is an inactive protein.

[0152] Embodiment 7: An embodiment of embodiment 6, wherein the inactive enzyme is a proenzyme.

[0153] Embodiment 8: An embodiment of embodiment 6 or 7, wherein the inactive protein transforms to an active protein upon recognition of the cancer cell by the cytotoxic lymphocyte targeting the cancer cell. 79685919V.1

[0154] Embodiment 9: An embodiment of embodiment 8, wherein a granzyme released by the cytotoxic lymphocyte induces transformation of the inactive protein to the active protein.

[0155] Embodiment 10: An embodiment of any one of embodiments 1-9, wherein the cytotoxic- lymphocyte sensitizing protein comprises a B-cell lymphoma 2 (BCL-2) family protein or a subunit or fragment thereof.

[0156] Embodiment 11: An embodiment of embodiment 10, wherein the BCL-2 family protein or the subunit or fragment thereof comprises BCL-2 homology domain 3 interacting domain death agonist (BID), BCL-2 antagonist / killer 1 (BAK1), BCL-2 interacting killer (BIK), phorbol-12- myristate-13-acetate-induced protein 1 (PMAIP1), or a variant or combination thereof.

[0157] Embodiment 12: An embodiment of any one of embodiments 1-11, wherein the cytotoxic-lymphocyte sensitizing protein comprises a cysteine-aspartic acid protease (caspase) family protein or a subunit or fragment thereof.

[0158] Embodiment 13: An embodiment of embodiment 12, wherein the caspase family protein or the subunit or fragment thereof comprises caspase-3 (CASP3), caspase-8 (CASP8), or a variant or combination thereof.

[0159] Embodiment 14: An embodiment of any one of embodiments 1-13, wherein the cytotoxic-lymphocyte sensitizing protein comprises a transcription factor, a transcriptional regulator, or a subunit or fragment thereof.

[0160] Embodiment 15: An embodiment of embodiment 14, wherein the transcription factor or the subunit or fragment thereof comprises an ETS family transcription factor or a subunit or fragment thereof.

[0161] Embodiment 16: An embodiment of embodiment 15, wherein the ETS family transcription factor or the subunit or fragment thereof comprises ETS proto-oncogene 1 transcription factor (ETS1), ETS variant transcription factor 4 (ETV4), or a variant or combination thereof.

[0162] Embodiment 17: An embodiment of any one of embodiments 14-16, wherein the transcription factor or the subunit or fragment thereof comprises a GATA family transcription factor or a subunit or fragment thereof. 79685919V.1

[0163] Embodiment 18: An embodiment of embodiment 17, wherein the GATA family transcription factor or the subunit or fragment thereof comprises GATA-binding factor 4 (GATA4), GATA-binding factor 6 (GATA6), tricho-rhino-phalangeal syndrome Type 1 (TRPS1), or a variant or combination thereof.

[0164] Embodiment 19: An embodiment of any one of embodiments 14-18, wherein the transcription factor or the subunit or fragment thereof comprises a paired box (PAX) family transcription factor or a subunit or fragment thereof.

[0165] Embodiment 20: An embodiment of embodiment 19, wherein the PAX family transcription factor or the subunit or fragment thereof comprises PAX3, PAX8, or a variant or combination thereof.

[0166] Embodiment 21: An embodiment of any one of embodiments 14-20, wherein the transcription factor or the subunit or fragment thereof comprises a helix-loop-helix transcription factor, a helix-loop-helix transcriptional regulator, or a subunit or fragment thereof.

[0167] Embodiment 22: An embodiment of embodiment 21, wherein the helix-loop-helix transcription factor, the helix-loop-helix transcriptional regulator, or the subunit or fragment thereof comprises inhibitor of DNA binding 1 (ID1), hairy / enhancer-of-split related with YRPW motif-like protein (HEYL), microphthalmia-associated transcription factor (MITF), MYC proto- oncogene BHLH transcription factor (MYC), or a variant or combination thereof.

[0168] Embodiment 23: An embodiment of any one of embodiments 14-22, wherein the transcription factor or the subunit or fragment thereof comprises paired-like homeobox 2B (PHOX2B), chromodomain-helicase-DNA-binding protein 8 (CHD8), histone deacetylase 4 (HDAC4), histone deacetylase 7 (HDAC7), hypermethylated in cancer 1 protein (HIC1), high mobility group box 1 protein (HMGB1), interferon regulatory factor 1 (IRF1), nuclear receptor 4A3 (NR4A3), nuclear protein 1 (NUPR1), peroxisome proliferator-activated receptor gamma coactivator 1-beta (PPARGC1B), PR domain containing 16 (PRDM16), protein arginine N- methyltransferase 2 (PRMT2), homeobox protein prophet of PIT-1 (PROP1), prothymosin alpha (PTMA), transcription factor RelB (RELB), SET nuclear proto-oncogene (SET), homeobox protein SIX4 (SIX4), TATA-box binding protein associated factor 15 (TAF15), transcription 79685919V.1factor AP-2 alpha (TFAP2A), DNA topoisomerase 1 (TOP1), Wilms tumor protein (WT1), or a variant or combination thereof.

[0169] Embodiment 24: An embodiment of any one of embodiments 1-23, wherein the cytotoxic-lymphocyte sensitizing protein comprises a tumor necrosis factor receptor superfamily (TNFRSF) protein or a subunit or fragment thereof.

[0170] Embodiment 25: An embodiment of embodiment 24, wherein the TNFRSF protein or the subunit or fragment thereof comprises the Fas cell surface death receptor (FAS), TNFRSF member 1A (TNFRSF1A), TNFRSF member 1B (TNFRSF1B), tumor necrosis factor ligand superfamily member 9 (TNFSF9), or a variant or combination thereof.

[0171] Embodiment 26: An embodiment of any one of embodiments 1-25, wherein the cytotoxic-lymphocyte sensitizing protein comprises a transmembrane ligand or a subunit or fragment thereof.

[0172] Embodiment 27: An embodiment of embodiment 26, wherein the transmembrane ligand or the subunit or fragment thereof comprises CUB domain-containing protein 1 (CDCP1), delta- like canonical Notch ligand 4 (DLL4), or a variant or combination thereof.

[0173] Embodiment 28: An embodiment of any one of embodiments 1-27, wherein the cytotoxic-lymphocyte sensitizing protein comprises an immunoglobulin superfamily protein or a subunit or fragment thereof.

[0174] Embodiment 29: An embodiment of embodiment 28, wherein the immunoglobulin superfamily protein or the subunit or fragment thereof comprises CD47, CD58, or a variant or combination thereof.

[0175] Embodiment 30: An embodiment of any one of embodiments 1-29, wherein the cytotoxic-lymphocyte sensitizing protein comprises a collagen subunit.

[0176] Embodiment 31: An embodiment of embodiment 30, wherein the collagen subunit comprises collagen alpha-1(V) chain (COL5A1), collagen alpha-2(IV) chain (COL4A2), or a variant or combination thereof. 79685919V.1

[0177] Embodiment 32: An embodiment of any one of embodiments 1-31, wherein the cytotoxic-lymphocyte sensitizing protein comprises an interferon family signaling protein or a subunit or fragment thereof.

[0178] Embodiment 33: An embodiment of embodiment 32, wherein the interferon family signaling protein or the subunit or fragment thereof comprises interferon beta 1 (IFNB1), interferon gamma (IFNG), or a variant or combination thereof.

[0179] Embodiment 34: An embodiment of any one of embodiments 1-33, wherein the cytotoxic-lymphocyte sensitizing protein comprises a mitosis regulatory protein or a subunit or fragment thereof.

[0180] Embodiment 35: An embodiment of embodiment 34, wherein the mitosis regulatory protein or the subunit or fragment thereof comprises cyclin B1 (CCNB1), centrosomal protein of 70 kDa (CEP70), close homolog of L1 (CHL1), kinesin family member 14 (KIF14), or a variant or combination thereof.

[0181] Embodiment 36: An embodiment of any one of embodiments 1-35, wherein the cytotoxic-lymphocyte sensitizing protein comprises a protein phosphatase or a subunit or fragment thereof.

[0182] Embodiment 37: An embodiment of embodiment 36, wherein the protein phosphatase or the subunit or fragment thereof comprises dual specificity phosphatase 1 (DUSP1), protein phosphatase 1J (PPM1J), serine / threonine-protein phosphatase 2A regulatory subunit B'' subunit gamma (PPP2R3C), or a variant or combination thereof.

[0183] Embodiment 38: An embodiment of any one of embodiments 1-37, wherein the cytotoxic-lymphocyte sensitizing protein comprises signaling lymphocytic activation molecule family member 1 (SLAMF1), sphingosine 1-phosphate transporter (SPNS2), mucin 21 (MUC21), translocation associated membrane protein 2 (TRAM2), cluster of differentiation 80 (CD80), family with sequence similarity 9 member C (FAM9C), solute carrier family 7 member 3 (SLC7A3), ATPase family AAA domain containing 1 (ATAD1), nipped-B-like protein (NIPBL), C-C motif chemokine 22 (CCL22), CLK4-associating serine / arginine rich protein (CLASPR), 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNP), coronin-6 (CORO6), crystatin-F (CST7), cancer / testis antigen 1A (CTAG1A), eyes absent homolog 2 (EYA2), family with sequence 79685919V.1similarity 161 centrosomal protein B (FAM161B), family with sequence similarity 46 member D (FAM46D), alpha-(1,3)-fucosyltransferase (FUT9), glutamate [NMDA] receptor subunit epsilon- 2 (GRIN2B), immediate early response gene 5-like protein (IER5L), insulin receptor substrate 4 (IRS4), KHDRBS protein 1 (KHDRBS1), alpha-1,3-mannosyl-glycoprotein 4-beta-N- acetylglucosaminyltransferase A (MGAT4A), metallophosphoesterase domain-containing protein 2 (MPPED2), neuraminidase 3 (NEU3), nucleophosmin 1 (NPM1), proliferation and apoptosis adaptor protein 15 (PEA15), Pleckstrin homology domain interacting protein (PHIP), calcium- dependent phospholipase A2 (PLA2G5), proline-rich protein 5 (PRR5), retrotransposon Gag-like 5 (RTL5), ribonucleotide-diphosphate reductase subunit M2B (RRM2B), scaffold attachment factor B (SAFB), secreted frizzled related protein 2 (SFRP2), serine and arginine rich splicing factor 3 (SRSF3), tenascin C (TSC), TSPY-like 2 (TSPYL2), taxilin gamma (TXLNG), vimentin (VIM), or a variant or combination thereof.

[0184] Embodiment 39: An embodiment of any one of embodiments 1-38, wherein the introducing of the polynucleotide to the cancer cell comprises contacting the cancer cell with a virus, a virus-like particle, or an immune cell, wherein the virus, the virus-like particle, or the immune cell comprises the polynucleotide.

[0185] Embodiment 40: An embodiment of any one of embodiments 1-39, wherein the cancer cell comprises a melanoma cell or a breast cancer cell.

[0186] Embodiment 41: An engineered cytotoxic lymphocyte genetically engineered to overexpress an antitumor enhancing ligand, the antitumor enhancing ligand binding to a corresponding binding partner expressed by the engineered cytotoxic lymphocyte, wherein the engineered cytotoxic lymphocyte induces increased elimination of a cancer cell as compared to the elimination of the cancer cell induced by a corresponding cytotoxic lymphocyte not overexpressing the antitumor enhancing ligand.

[0187] Embodiment 42: An embodiment of embodiment 41, wherein the engineered cytotoxic lymphocyte is further genetically engineered to overexpress the corresponding binding partner

[0188] Embodiment 43: An embodiment of embodiment 41 or 42, wherein the antitumor enhancing ligand comprises a Wnt family protein or a subunit or fragment thereof. 79685919V.1

[0189] Embodiment 44: An embodiment of embodiments 43, wherein the Wnt family protein or the subunit or fragment thereof comprises Wnt family member 1 (WNT1), Wnt family member 3 (WNT3), Wnt family member 3A (WNT3A), Wnt family member 10B (WNT10B), or a variant or combination thereof.

[0190] Embodiment 45: An embodiment of any one of embodiments 41-44, wherein the antitumor enhancing ligand comprises CD58, CDCP1, CD47, or a variant or combination thereof.

[0191] Embodiment 46: An embodiment of any one of embodiments 41-45, wherein the cytotoxic lymphocyte is a T cell or a natural killer (NK) cell.

[0192] Embodiment 47: An embodiment of any one of embodiments 41-46, wherein the engineered cytotoxic lymphocyte expresses a chimeric antigen receptor (CAR).

[0193] Embodiment 48: An embodiment of any one of embodiments 41-47, wherein the engineered cytotoxic lymphocyte expresses an engineered T-cell receptor (TCR).

[0194] Embodiment 49: An embodiment of any one of embodiments 41-48, wherein the engineered cytotoxic lymphocyte comprises a CRISPR-based activation system effecting overexpression of the antitumor enhancing ligand by the engineered cytotoxic lymphocyte.

[0195] Embodiment 50: An embodiment of any one of embodiments 41-49, wherein the engineered cytotoxic lymphocyte comprises an exogenous gene encoding the antitumor enhancing ligand.

[0196] Embodiment 51: A method for producing an engineered cytotoxic lymphocyte, the method comprising: providing a cytotoxic lymphocyte; and introducing into the cytotoxic lymphocyte a polynucleotide encoding an antitumor enhancing ligand, thereby producing the engineered cytotoxic lymphocyte; wherein the antitumor enhancing ligand binds to a corresponding binding partner expressed by the engineered cytotoxic lymphocyte; and wherein the engineered cytotoxic lymphocyte induces increased elimination of a cancer cell as compared to elimination of the cancer cell induced by a corresponding cytotoxic lymphocyte not overexpressing the antitumor enhancing ligand.

[0197] Embodiment 52: An embodiment of embodiment 51, wherein the method further comprises engineering the cytotoxic lymphocyte to overexpress the corresponding binding partner. 79685919V.1

[0198] Embodiment 53: A method for producing an engineered cytotoxic lymphocyte, the method comprising: providing a cytotoxic lymphocyte comprising a gene encoding an antitumor enhancing ligand; and introducing into the cytotoxic lymphocyte a polynucleotide comprising or encoding a guide RNA targeting the gene, thereby producing the engineered cytotoxic lymphocyte; wherein the antitumor enhancing ligand binds to a corresponding binding partner of the engineered cytotoxic lymphocyte; and wherein the engineered cytotoxic lymphocyte induces increased elimination of a cancer cell as compared to elimination of the cancer cell induced by a corresponding cytotoxic lymphocyte not overexpressing the antitumor enhancing ligand.

[0199] Embodiment 54: An embodiment of embodiment 53, wherein the engineered cytotoxic lymphocyte comprises a CRISPR-based activation system effecting overexpression of the antitumor enhancing ligand by the engineered cytotoxic lymphocyte

[0200] Embodiment 55: An embodiment of any one of embodiments 51-54, wherein the polynucleotide comprises RNA.

[0201] Embodiment 56: An embodiment of any one of embodiments 51-55, wherein the antitumor enhancing ligand is an endogenous protein of the cytotoxic lymphocyte.

[0202] Embodiment 57: An embodiment of any one of embodiments 51-55, wherein the antitumor enhancing ligand is exogenous to the cytotoxic lymphocyte.

[0203] Embodiment 58: An embodiment of any one of embodiments 51-57, wherein the antitumor enhancing ligand comprises a Wnt family protein or a subunit or fragment thereof.

[0204] Embodiment 59: An embodiment of embodiment 58, wherein the Wnt family protein or the subunit or fragment thereof comprises WNT1, WNT3, WNT3A, WNT10B, or a variant or combination thereof.

[0205] Embodiment 60: An embodiment of any one of embodiments 51-59, wherein the antitumor enhancing ligand are CD58, CDCP1, CD47, or a variant or combination thereof.

[0206] Embodiment 61: An embodiment of any one of embodiments 51-60, wherein introducing the polynucleotide comprises transduction of the polynucleotide into the cytotoxic lymphocyte using a virus or a virus-like particle, the virus or virus-like particle comprising the polynucleotide. 79685919V.1

[0207] Embodiment 62: An embodiment of any one of embodiments 51-61, wherein the cytotoxic lymphocyte is a T cell or a natural killer (NK) cell.

[0208] Embodiment 63: An embodiment of any one of embodiments 51-62, wherein the engineered cytotoxic lymphocyte expresses a chimeric antigen receptor (CAR).

[0209] Embodiment 64: An embodiment of any one of embodiments 51-63, wherein the engineered cytotoxic lymphocyte expresses an engineered T-cell receptor (TCR).

[0210] Embodiment 65: A method for treating a cancer in a subject, the method comprising: administering to the subject a polynucleotide comprising or encoding a guide RNA, the guide RNA targeting a gene encoding a cytotoxic-lymphocyte sensitizing protein that increases elimination of a cancer cell of the cancer upon recognition of the cancer cell by a cytotoxic lymphocyte targeting the cancer cell.

[0211] Embodiment 66: A method for treating a cancer in a subject, the method comprising: administering to the subject a polynucleotide encoding a cytotoxic-lymphocyte sensitizing protein that increases elimination of a cancer cell of the cancer upon recognition of the cancer cell by a cytotoxic lymphocyte targeting the cancer cell.

[0212] Embodiment 67: An embodiment of embodiment 65 or 66, wherein the polynucleotide comprises RNA.

[0213] Embodiment 68: An embodiment of any one of embodiments 65-67, wherein the cytotoxic-lymphocyte sensitizing protein is an endogenous protein of the cancer cell.

[0214] Embodiment 69: An embodiment of any one of embodiments 65-68, wherein the cytotoxic-lymphocyte sensitizing protein is an inactive protein.

[0215] Embodiment 70: An embodiment of embodiment 69, wherein the inactive protein is a proenzyme.

[0216] Embodiment 71: An embodiment of embodiment 69 or 70, wherein the inactive protein transforms to an active protein upon recognition of the cancer cell by the cytotoxic lymphocyte targeting the cancer cell. 79685919V.1

[0217] Embodiment 72: An embodiment of embodiment 71, wherein a granzyme released by the cytotoxic lymphocyte induces transformation of the inactive protein to the active protein.

[0218] Embodiment 73: An embodiment of any one of embodiments 65-72, wherein the cytotoxic-lymphocyte sensitizing protein comprises a BCL-2 family protein or a subunit or fragment thereof.

[0219] Embodiment 74: An embodiment of embodiment 73, wherein the BCL-2 family protein or the subunit or fragment thereof comprises BID, BAK1, BIK, PMAIP1, or a variant or combination thereof.

[0220] Embodiment 75: An embodiment of any one of embodiments 65-74, wherein the cytotoxic-lymphocyte sensitizing protein comprises a caspase family protein or a subunit or fragment thereof.

[0221] Embodiment 76: An embodiment of embodiment 75, wherein the caspase family protein or the subunit or fragment thereof comprises CASP3, CASP8, or a variant or combination thereof.

[0222] Embodiment 77: An embodiment of any one of embodiments 65-76, wherein the cytotoxic-lymphocyte sensitizing protein comprises a transcription factor, a transcriptional regulator, or a subunit or fragment thereof.

[0223] Embodiment 78: An embodiment of embodiment 77, wherein the transcription factor or the subunit or fragment thereof comprises an ETS family transcription factor or a subunit or fragment thereof.

[0224] Embodiment 79: An embodiment of embodiment 78, wherein the ETS family transcription factor or the subunit or fragment thereof comprises ETS1, ETV4, or a variant or combination thereof.

[0225] Embodiment 80: An embodiment of any one of embodiments 77-79, wherein the transcription factor or the subunit or fragment thereof comprises a GATA family transcription factor or a subunit or fragment thereof.

[0226] Embodiment 81: An embodiment of embodiment 80, wherein the GATA family transcription factor or the subunit or fragment thereof comprises GATA4, GATA6, TRPS1, or a variant or combination thereof. 79685919V.1

[0227] Embodiment 82: An embodiment of any one of embodiments 77-81, wherein the transcription factor or the subunit or fragment thereof comprises a PAX family transcription factor or a subunit or fragment thereof.

[0228] Embodiment 83: An embodiment of embodiment 82, wherein the PAX family transcription factor or the subunit or fragment thereof comprises PAX3, PAX8, or a variant or combination thereof.

[0229] Embodiment 84: An embodiment of any one of embodiments 77-83, wherein the transcription factor or the subunit or fragment thereof comprises a helix-loop-helix transcription factor, a helix-loop-helix transcriptional regulator, or a subunit or fragment thereof.

[0230] Embodiment 85: An embodiment of embodiment 84, wherein the helix-loop-helix transcription factor, the helix-loop-helix transcriptional regulator, or the subunit or fragment thereof comprises ID1, HEYL, MITF, MYC, or a variant or combination thereof.

[0231] Embodiment 86: An embodiment of any one of embodiments 77-85, wherein the transcription factor or the subunit or fragment thereof comprises PHOX2B, CHD8, HDAC4, HDAC7, HIC1, HMGB1, IRF1, NR4A3, NUPR1, PPARGC1B, PRDM16, PRMT2, PROP1, PTMA, RELB, SET, SIX4, TAF15, TFAP2A, TOP1, WT1, or a variant or combination thereof.

[0232] Embodiment 87: An embodiment of any one of embodiments 65-86, wherein the cytotoxic-lymphocyte sensitizing protein comprises a TNFRSF protein or a subunit or fragment thereof.

[0233] Embodiment 88: An embodiment of embodiment 87, wherein the TNFRSF protein or the subunit or fragment thereof comprises FAS, TNFRSF1A, TNFRSF1B, TNFSF9, or a variant or combination thereof.

[0234] Embodiment 89: An embodiment of any one of embodiments 65-88, wherein the cytotoxic-lymphocyte sensitizing protein comprises a transmembrane ligand or a subunit or fragment thereof.

[0235] Embodiment 90: An embodiment of embodiment 89, wherein the transmembrane ligand or the subunit or fragment thereof comprises CDCP1, DLL4, or a variant or combination thereof. 79685919V.1

[0236] Embodiment 91: An embodiment of any one of embodiments 65-90, wherein the cytotoxic-lymphocyte sensitizing protein comprises an immunoglobulin superfamily protein or a subunit or fragment thereof.

[0237] Embodiment 92: An embodiment of embodiment 91, wherein the immunoglobulin superfamily protein or the subunit or fragment thereof comprises CD47, CD58, or a variant or combination thereof.

[0238] Embodiment 93: An embodiment of any one of embodiments 65-92, wherein the cytotoxic-lymphocyte sensitizing protein comprises a collagen subunit.

[0239] Embodiment 94: An embodiment of embodiment 93, wherein the collagen subunit comprises COL5A1, COL4A2, or a variant or combination thereof.

[0240] Embodiment 95: An embodiment of any one of embodiments 65-94, wherein the cytotoxic-lymphocyte sensitizing protein comprises an interferon family signaling protein or a subunit or fragment thereof.

[0241] Embodiment 96: An embodiment of embodiment 95, wherein the interferon family signaling protein or the subunit or fragment thereof comprises IFNB1, IFNG, or a variant or combination thereof.

[0242] Embodiment 97: An embodiment of any one of embodiments 65-96, wherein the cytotoxic-lymphocyte sensitizing protein comprises a mitosis regulatory protein or a subunit or fragment thereof.

[0243] Embodiment 98: An embodiment of embodiment 97, wherein the mitosis regulatory protein or the subunit or fragment thereof comprises CCNB1, CEP70, CHL1, KIF14, or a variant or combination thereof.

[0244] Embodiment 99: An embodiment of any one of embodiments 65-98, wherein the cytotoxic-lymphocyte sensitizing protein comprises a protein phosphatase or a subunit or fragment thereof.

[0245] Embodiment 100: An embodiment of embodiment 99, wherein the protein phosphatase or the subunit or fragment thereof comprises DUSP1, PPM1J, PPP2R3C, or a variant or combination thereof. 79685919V.1

[0246] Embodiment 101: An embodiment of any one of embodiments 65-100, wherein the cytotoxic-lymphocyte sensitizing protein comprises SLAMF1, SPNS2, MUC21, TRAM2, CD80, FAM9C, SLC7A3, ATAD1, NIPBL, CCL22, CLASPR, CNP, CORO6, CST7, CTAG1A, EYA2, FAM161B, FAM46D, FUT9, GRIN2B, IER5L, IRS4, KHDRBS1, MGAT4A, MPPED2, NEU3, NPM1, PEA15, PHIP, PLA2G5, PRR5, RTL5, RRM2B, SAFB, SFRP2, SRSF3, TSC, TSPYL2, TXLNG, VIM, or a variant or combination thereof.

[0247] Embodiment 102: A method for treating a cancer in a subject, the method comprising: administering to the subject the engineered cytotoxic lymphocyte of any one of embodiments 41- 50.

[0248] Embodiment 103: A method for sensitizing a cancer cell to cytotoxic lymphocytes, the method comprising introducing into the cancer cell a polynucleotide configured to reduce or eliminate expression of a target gene by the cancer cell. wherein the reduction or the elimination of the target gene expression increases elimination of the cancer cell upon recognition of the cancer cell by a cytotoxic lymphocyte targeting the cancer cell.

[0249] Embodiment 104: An embodiment of embodiment 103, wherein the target gene encodes inhibitor of nuclear factor kappa B kinase subunit beta (IKBKB), presenilin enhancer gamma- secretase subunit (PSENEN), secretoglobin family 1D member 1 (SCGB1D1), glycogen synthase kinase 3 beta (GSK3B), ring finger protein 20 (RNF20), tuberous sclerosis complex-1 (TSC1), or ubiquitin conjugating enzyme E2 F (UBE2F). EXAMPLES

[0250] The present disclosure will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes only, and are not intended to limit the disclosure in any manner. Example 1. Identification of regulators of resistance and sensitivity to targeted CD8+T cell mediated cytotoxicity using CRISPR activation screens

[0251] To identify genes whose activation in malignant cells sensitizes or desensitizes cells to targeted T cell mediated cytotoxicity, a CRISPR activation (CRISPRa) screen was conducted in melanoma cells under different T cell mediated selection pressures. (FIG. 3) To distinguish 79685919V.1between targeted T cell receptor (TCR) dependent and non-specific (TCR-independent) T cell cytotoxicity human primary CD8+T cells were engineered to express a TCR that recognizes a specific antigen (the cancer testis antigen NY-ESO1) and the A375 melanoma cell line that homogenously expresses the antigen genes (i.e., CTAG1A and CTAG1B) and protein. An important goal of the screen was to distinguish between specific and non-specific immune effects because of the significant benefits of not merely sensitizing cancer or other cells to non-specific immune responses, e.g., responses related to cytokines.

[0252] A comparison of the TCR engineered and wildtype T cells confirmed TCR-dependent cytotoxicity in 2D co-culture with the melanoma A375 cells (FIG. 4) and, interestingly, also demonstrated TCR-dependent infiltration in 3D co-culture with A375 spheroids. Using this model, in vitro 2D co-culture screens were conducted to map the impact of 2,919 genes on melanoma cell susceptibility to targeted TCR-dependent cytotoxicity (FIG. 5). Key genes were identified and further examined via high content single cell and spatial screens in 3D models and in vivo.

[0253] The in vitro screen focused on cell surface proteins, epigenetic and transcriptional regulators, as well as other genes previously identified as strongly linked to immune checkpoint blockade (ICB) resistance and response in melanoma patients based on single cell data (L. Jerby- Arnon et al., Cell 175, (2018): 984). In brief, A375 cells were transduced to stably express CRISPR dCas9-VPR and single genes were activated in a pooled fashion using a single guide RNA (sgRNA) library encompassing 2,921 genes (10 sgRNAs per gene and 280 control gRNAs). The melanoma cells were grown in monoculture and challenged with up to 3 rounds of selection in co- culture [0.25:1 effector to target (E:T) ratio for 24 hours] with either the TCR-engineered or wildtype T cells. Exemplary results are shown in FIG.6.

[0254] Stringent statistical cutoffs across technical and biological replicates (Benjamini- Hochberg false discovery rate (BH FDR) < 0.05, MAGeCK (W. Li et al., Genome Biology 15, (2014): 554; W. Li et al., Genome Biol. 16, (2015): 281)) were used to identify 38 resistance and 90 sensitizing hits that were depleted or enriched specifically in the context of TCR-dependent cytotoxicity. As expected, with each co-culture round there was an increase in the number of significant hits identified, and CTAG1A, which encodes for the NY-ESO1 antigen, was among the top sensitizing hits (BH FDR = 2.32×10-6, MAGeCK). Comparing these findings to a collection of five published immune evasion CRISPR screens in melanoma and other cancer types (K. A. 79685919V.1Lawson et al., Nature 586, (2020): 120; S. J. Patel et al., Nature 548, (2017): 537; R. T. Manguso et al., Nature 547, (2017): 413; G. K. Griffin et al., Nature 595, (2021): 309; J. Joung et al., Nat. Commun.13, (2022): 1606), 9 of the resistance genes (P = 2.68×10-4, hypergeometric test, BCL2, BCL2L1, CD44, EP300, FADD, ITGAV, JUN, MAN2B2, MCL1) and 11 of the sensitizing genes (P = 1.42×10-3, hypergeometric test, e.g., CASP3, CASP8, CHD8, CLASRP, ETS1, FAS, IRF1, PPP2R3C, SAFB) were previously reported to sensitize or confer resistance upon knockout, respectively. While statistically significant and testifying to the validity of the screen, this is only a small fraction (< 16%) of the hits identified here, demonstrating the distinction between inhibition- and activation-based assessment of gene function in this context.

[0255] The hits highlight key cellular processes and mechanisms involved in the programmed response to targeted TCR-dependent cytotoxicity. Resistance genes are enriched for genes involved in negative regulation of cell death (BH FDR = 2.58×10-5, hypergeometric test, BCL2A1, MCL1, BCL2L1, BCL2), and transcriptional regulation (BH FDR = 7.54×10-5, hypergeometric test, e.g., EP300, ETV6, FOSL1, GFI1, JUN, KLF4), and include surface glycoproteins (PDPN, CD44, TNC), genes involved in protein glycosylation (B3GNT7, SDC2), insulin signaling (e.g., TCF7L2, GRB10), and mesoderm development (SNAI1, PAX2, TBX3). Sensitizing hits are enriched for pro- apoptosis and programmed cell death genes (BH FDR = 2.37×10-4, hypergeometric test, e.g., BAK1, BID, CASP3, CASP8, FAS), transcription and epigenetic regulators (BH FDR < 7.23×10-6, hypergeometric test, e.g., SAFB, NIPBL, ETS1, PHOX2B, TRPS1, ID1), and genes involved in developmental processes and organ morphogenesis (BH FDR = 1.32×10-3, e.g., COL5A1, DLL4, ETV4, GATA4, GATA6, NIPBL, PAX3, PAX8, WT1). The sensitizing genes may be under shared transcriptional regulation, with enrichment for regulation by specific transcription factors, including NFAT (Nuclear Factor of Activated T cell) and AP2-gamma (BH FDR < 0.05). Of note, AP2-gamma is also known to repress CD44 expression (A. R. Cyr et al., Oncogene 34, (2015): 436), which was identified here as a resistance gene.

[0256] The resistance and sensitizing hits are involved in 79 and 99 ligand-receptor interactions, respectively, providing leads to identify underlying autocrine and paracrine signaling and pinpoint the limiting factors of these circuits. For example, TNFRSF1A and TNFRSF1B are two sensitizing receptors identified in the screen, where both bind to TNF. As TNF is not a hit in the screen, it is likely that CD8+T cells secrete sufficiently high levels of TNF in co-culture such that the TNF 79685919V.1receptor expression by the malignant cells becomes a limiting factor. Sensitizing ligands include the 4-1BB ligand (4-1BBL, also known as TNFSF9) that provides costimulatory signals to CD8+T cells via 4-1BB (A. T. C. Cheuk, G. J. Mufti & B. Guinn, Cancer Gene Ther.11, (2004): 215), interferons (IFNB1 and IFNG), and multiple Wnt signaling ligands (WNT1, WNT3, WNT3A, and WNT10B). However, the sensitizing hits are not enriched for IFN signaling response genes or other Wnt signaling genes (BH FDR > 0.1, hypergeometric test), and, although IFN and Wnt genes bind to a total of 46 different receptors, none of these were found as hits except for SFPR2 (Secreted frizzled-related protein 2), which act as soluble modulators of Wnt signaling and was found as a sensitizing hit. This suggests that ligand scarcity is a limiting factor in IFN and Wnt signaling or that the mechanism of action involves paracrine signaling with nearby CD8+T cells. Indeed, IFN is a key regulator of T cell effector function and Wnt signaling in known to have profound effect on T cell differentiation and stemness (X. Li et al., Front. Immunol.10, (2019): 2293; G. Escobar, D. Mangani & A. C. Anderson, Sci. Immunol.5, (2020); X. Zhao, Q. Shan & H.-H. Xue, Nat. Rev. Immunol., (2021): doi:10.1038 / s41577-021-00563-6). Moreover, in accordance with the findings presented here, Wnt signaling has been shown to promote the generation of self-renewing multipotent CD8+memory stem cells with substantially better antitumor capacities compared to central and effector memory T cell subsets (L. Gattinoni et al., Nat. Med.15, (2009): 808).

[0257] Further stratifying the (de)sensitizing hits based on baseline fitness effects in the absence of antigen-specific T cells, 115 and 33 positive and negative fitness hits were identified, which, as expected, were enriched for tumor suppressors and oncogenes, respectively (P = 5.61×10-6, 1.73×10-2, hypergeometric test). However, 18 of the 33 positive fitness genes (including MYC, WNT1, and WNT3A) completely lost their selective advantage in the context of TCR+co-cultures, with 11 of them being sensitizing hits. In accordance with this, 14 of the sensitizing hits are known oncogenes (P = 1.6×10-3, hypergeometric test), demonstrating that targeted TCR-dependent cytotoxicity significantly impacts the malignant cell fitness landscape such that certain oncogenes and pro-proliferation genes exhibit a selective disadvantage upon active adaptive immune selection. Additionally, 68 sensitizing hits (including central apoptosis regulates as CASP3, BID, BAK) show no other positive or negative effect on fitness at baseline, indicating that their deleterious effect is highly specific and synthetically lethal in combination with a targeted T cell response. Special emphasis was placed on this group of sensitizing perturbations as these are less likely to induce cell proliferation or be deleterious to non-malignant cells that do not present 79685919V.1immunogenic (neo)antigens, hence making these candidates more suitable for pre-clinical and clinical applications. Example 2. Transcriptional programs regulating susceptibility to targeted TCR-dependent cytotoxicity revealed by Perturb-seq

[0258] Both resistance and sensitizing hits were highly enriched for transcription factors and transcriptional regulators (BH FDR < 7.54×10-5, hypergeometric test). To elucidate their function, a follow-up Perturb-seq screen was carried out in the form of a CRISPRa screen with single cell RNA-seq (scRNA-seq) readouts (A. Dixit et al., Cell 167, (2016): 1853). A total of 61 genes were selected, including top hits from the screen along with positive and negative controls, to be activated in the cancer cells in a pooled manner via a library of 276 sgRNAs (4 different sgRNAs per gene and 32 NTC sgRNAs). The cancer cells were cultured alone (monoculture) or with TCR-engineered T cells (24-hour co-culture at 1:1 E:T), and subsequently profiled via 5 droplet-basedscRNA-seq in conjunction with direct capture and sequencing of the sgRNA protospacer. The Perturb-seq data includes 29,498 high-quality scRNA profiles from the co-culture (12,192 cells) and monoculture (17,306 cells) conditions, with 468 cells per target on average.

[0259] Analyzing the Perturb-seq data first involved identifying the main sources of variation that exist irrespective of the genetic perturbations. Within each condition, cell cycle is a major source of variation. The overall expression of a cell cycle signature previously derived based on melanoma single cell RNA-seq study followed a bimodal distribution, and thus each cell was annotated as either positive or negative for active cell cycle. A moderate drop in the number of cycling cells was observed in the co-culture (74% cycling) vs. monoculture (82% cycling).

[0260] To characterize the transcriptional response to T cells, differentially expressed genes in the co-culture vs. monoculture were identified, considering only control cells (i.e., carrying non- targeting control guides). A total of 408 genes were significantly overexpressed in the coculture vs. monoculture conditions. Gene set enrichment analyses showed that these genes are enriched for expected gene ontologies, including immune response, response to type I interferon, inflammatory response, cellular response to oxidative stress, cell adhesion (P < 10-17, hypergeometric test), and other immune related pathways. More specifically, among these genes are genes involved in antigen presentation MHC-I and MHC-II genes), cell adhesion (ICAM1), the JAK-STAT pathway and interferon response genes (IRF1 / 2 / 7 / 9, ISG15 / 20, JAK2, 79685919V.1STAT1 / 2 / 3), cytokines (CXCL1 / 11 / 9 and CXCL10; the latter expressed exclusively in coculture), and double strand RNA editing (ADAR). The 177 genes that are underexpressed in the co-culture vs. monoculture conditions were enriched for other sets of processes, including the epithelial mesenchymal transition, p53 pathway, amino acid metabolic process, and estrogen responses (P < 10-5, hypergeometric test).

[0261] Further examination of the genes overexpressed in co-culture vs. monoculture demonstrated that exposure to T cells primes the cancer cells to the immune attack, but within a short time frame of 24 hours already elicits resistance mechanisms at the transcriptional level, activating the expression of anti-apoptotic genes. The genes overexpressed in co-culture vs. monoculture are enriched for genes associated with positive regulation of cell death, but also include multiple key anti-apoptotic genes (e.g., BCL2A1, MCL1, BCL3, BIRC2, BIRC3). Based on the collection of immune sensitizing and resistance genes that complied here, the overexpressed genes are enriched for both sensitizing (e.g., B2M, CASP8, DTX3L, FAS, IL15, IRF1, JAK2, STAT1 / 2, TAP1 / 2, TAPBP, TNC, TNFSF9) and resistance genes (e.g., ADAR, BCL2A1, BIRC2, CD274, JUN, JUNB, MCL1, SOCS1). Genes that were underexpressed in co-culture vs. monoculture do not show an association with program cell death or the CRISPR hits. Collectively these findings further support the need and potential of activating the expression of BID, CASP3, and other of the pro-apoptotic sensitizing hits identified in the screen to balance these anti- apoptotic effects, while in contrast the JAK-STAT pathway is already activated in this setting.

[0262] Further studies sought to identify the transcriptional effects of each perturbation. Per condition and for each of the target genes activated in the screen, a gene activation (GA) signature was identified, which consisted of all the genes significantly over or under-expressed in the perturbed cells (carrying the CRISPR activation guides of the pertaining target gene) compared to the control cells (carrying the non-targeting control guides). The GA signature identified based on the monoculture vs. co-culture datasets were distinct at the gene level, but where highly concordant in their overall gene expression patterns across the cells. More specifically, the expression of a GA signature was defined as the overall expression of the upregulated genes minus the overall expression of the downregulated genes. The overall expression of the GA signatures derived from the monoculture dataset was significantly higher in the cells subject to the pertaining gene 79685919V.1activation vs. the control cells, both when considering the cells in monoculture and in co-culture. Similar results were obtained when examining the co-culture GA signatures.

[0263] Before further examining the GA signatures, three tests were applied to examine the validity of the data, analytical approach, and resulting signatures. First, unlike CRISPR KO Perturb-seq screens where the detection of the guide is often not sufficient to conclude that the gene KO indeed took place, results here confirmed on-target gene activation. With 88% of the guides there is a significant up-regulation of the target expression in the cells where the guide was detected compared to the control cells. Second, the consistency across guides was examined via a leave-one-guide-out (LOGO) cross validation procedure. In this setting the GA signature was identified for each one of the target genes using only 2 out of the 3 target’s guides, and then examined to determine whether the expression of the resulting signature (defined as the overall expression of the up regulated genes minus the overall expression of the down regulated genes) was significantly higher in the cells with the test guide compared to those with control guides. In 82% of the cases this was indeed the case (P < 0.05, t-test). Third, while the guide detection was only used to conclude that a cell was subject to the pertaining guide activation, it was found that even when the target was removed from the GA signatures, these are still overexpressed in cells with high vs. low expression of the target gene, both when considering only the cells with the relevant gene target guides, and when considering only the control cells. The GA signatures were then used to group the hits into functional groups based on their transcriptional readouts.

[0264] Collectively, these findings suggest that sensitizing and resistance genes are tightly co- regulated at the transcriptional level, and thus perturbations that alter the cell transcriptome, including both exposure to T cells as well as genetic perturbations, result in coordinated activation of both types of functional genes. Thus, an effective approach can involve sensitizing cancer cells to T cell mediated killing through non-transcriptional perturbations, including the activation of BID, BAK, and other similar pro-apoptotic genes that were found as hits in the screen. More specifically, gene activation of BID and CASP3 appears to form a synthetic lethal interaction with granzymes secreted by CD8+T cells. Pro-apoptotic drugs as Venetoclax that inhibit BCL-2 are highly lethal, not only to the cancer cells, but also to the CD8+T cells, while activating BID and CASP3 at the RNA level does not lead to toxicities as these gene encode for inactive proteins that 79685919V.1will be activated only with a secondary signal. Such secondary signal can be provided by CD8+T cells upon granzyme secretion. Example 3. In situ Perturb-seq for genetic screens with spatial transcriptomics and proteomics readouts

[0265] CRISPR screens with single cell sequencing readouts, such as those conducted here, provide a powerful approach to decipher gene functions (A. Dixit et al., Cell 167, (2016): 1853; E. P. Mimitou et al., Nat. Method 16, (2019): 409; D. A. Jaitin, D. A et al., Cell 167, (2016): 1883; T. M. Norman et al., Science 365, 786; B. Adamson et al., Cell 167, (2016): 1867; C. J. Frangieh et al., Nat. Genet. 53, (2021): 332). However, these methods are limited in capturing key aspects of intercellular circuits or other inherently spatial processes (e.g., immune recruitment) as the spatial information is lost. Methods that preserve spatial information and detect genetic perturbations in situ have begun to fill this gap, but have thus far only measured a few dozen genes / proteins (M. Dhainaut et al., Cell, (2022): doi:10.1016 / j.cell.2022.02.015). To overcome this barrier, in situ Perturb-seq was developed. In situ Perturb-seq detects genetic perturbations in situ in combination with image-based spatial transcriptomics (up to 6,000 genes) and proteomics (64 proteins) at subcellular resolution. In addition to providing the spatial context, in situ Perturb-seq provides a cost-effective design for sequencing hundreds of thousands of cells. Importantly, perturbations can be conducted in the same cell type or across different cell types (e.g., one set of genes modified in malignant cells and another set of genes modified in CD8 T cells) to dissect intercellular circuits, as demonstrated here.

[0266] In brief, in situ Perturb-seq is compatible with both sgRNAs and ORFs that were modified to include a transcribed barcode detectable with in situ probes. Probes are either padlock probes for in situ sequencing via rolling circle amplification (compatible with Xenium (R. Ke et al., Nat. Methods 10, (2013): 857)) or error-robust probes for multiplexed fluorescence in situ hybridization (compatible with CosMx (S. He et al., Nat. Biotechnol. 40, (2022): 1794)). For efficient barcoding of sgRNAs (that bypasses barcode-sgRNA recombination (L. M. Sack, T. Davoli, Q. Xu, M. Z. Li & S. J. Elledge, G3 Genes Genomes Genet. 6, (2016): 2781; L. M. Sack et al., Cell 173, (2018): 499)) the CROP-seq design was adopted (P. Datlinger et al., Nat. Methods 14, (2017): 297), whereby double integration of the sgRNA cassette results in sgRNA expression 79685919V.1both via the RNA polymerase III from a human U6 promoter and through RNA polymerase II that transcribes both the barcode and the sgRNA.

[0267] In situ Perturb-seq was first tested and validated in genetically modified cancer spheroids, carrying either CRISPR / dCas9-VPR system in combination with barcoded sgRNAs or barcoded ORFs. Spheroids were generated with melanoma A375 cells such that each spheroid contained a single sgRNA or ORF, and was then co-cultured with wild-type or engineered CD8+T cells for 24 hours, collected, embedded, and sectioned for use with the CosMx spatial imaging platform (S. He et al., Nat. Biotechnol. 40, (2022): 1794). Both ORFs and sgRNAs were detected with high accuracy along with high quality spatial single cell transcriptomes. Following validation, in situ Perturb-seq was used to investigate how the (de)sensitizing genetic perturbations in malignant cells impact not only the malignant cell fitness and transcriptome, but also T cell recruitment and T cell states, both in vitro in 3D models and in vivo in the intact tissue.

[0268] Barcoded ORFs were used to transduce A375 melanoma cells to overexpress one of the top hits (WNT3A, MYC, TSPYL2, CASP3, PDPN, CD44, TCF7L2), CD274 (encoding for PD-L1) as a positive control, VAV1, or an empty control ORF with barcode only. Xenograft tumors containing the pool of genetically modified melanoma cells were engrafted, collected, and profiled via CosMx37 for detection of 1,000 genes at single molecule resolution along with in situ detection of the 10 ORFs. Data was analyzed based on image segmentation (N. F. Greenwald et al., Nat. Biotechnol.40, (2022): 555) to convert pixel level RNA detection to a cell by gene counts matrix.

[0269] In total, the in situ Perturb-seq data includes 950,988 cells, 337,075 of which are cancer cells carrying one of the 10 ORFs. Cells where an ORF barcode was detected showed the overexpression of the target gene based on the transcriptional readouts. Cancer cells harboring a specific perturbation were often spatially clustered together as a clone, creating “perturbational zonation” within the tumor and thus mitigating cross-perturbation confounding effects even in such a pooled setting. This also demonstrates that in vivo screens can suffer from spatial confounding effects, as cancer cells with a particular perturbation may just happen to be in a certain area within the tumor compared to the control cells and differently express certain genes due to microenvironmental effects that are independent of the genetic perturbation itself. One of the advantages of in situ Perturb-seq is that the spatial information is recorded and can be incorporated in the statistical model to account and control for random spatial effects. To test this, the GA 79685919V.1signatures per target gene were identified with and without controlling for spatial location. Indeed, only the genes that are supported by the spatially aware statistical model show robust and generalizable perturbation driven differential expression when tested on unseen data from unseen tumors (FIG. 9).

[0270] A large variation was observed in the transcriptional footprint of the different perturbations, with certain perturbations having a substantial effect on the cell transcriptome (FIG. 10). In these cases, the GA signatures identified with in situ Perturb-seq showed a significant overlap to those identified in the Perturb-seq screen conducted with CRISPRa (p < 10-3, hypergeometric test), demonstrating a high level of consistency across platforms (in situ Perturb- seq vs. Perturb-seq) even with different gene overexpression methods (ORF vs. CRISPRa) and models (in vivo vs. in vitro). Taking into consideration both types of data provides strong evidence that the perturbation is leading to the transcriptional shifts. For example, the WNT3A GA signature supported by both datasets shows enrichment for overexpression of genes involved in stem cell proliferations (e.g., BMP7, BMP4, FGFR1, and WNT5A; p = 8.3×10-6), and that the MYC GA signature is enriched for overexpression of MYC target genes (e.g., HSP90AB1, PTGES3, PCNA, RPL34; p = 1.64×10-4, hypergeometric test). In contrast, overexpression of CASP3 had no significant impact on the cell transcriptome other than the overexpression of the CASP3 gene itself (FIG. 10), indicating that, similar to BID, CASP3 is directly sensitizing cancer cells to TCR- dependent T cell cytotoxicity. Example 4. TCR-dependent cytotoxicity of CASP3 overexpression

[0271] Based on the CRISPRa screen and single gene validations, overexpression of BID and CASP3 selectively sensitizes cancer cells to TCR-dependent cytotoxicity. Additionally, activation of BID and CASP3 expression has minimal impact on the cell transcriptome based on Perturb-seq and in situ Perturb-seq screens, respectively, indicating that the sensitizing effects are directly mediated by BID and CASP3. Focusing on CASP3 further experiments investigated why overexpression of this pro-apoptotic gene was not lethal or detrimental to cell health at baseline. Examining caspase-3 at the protein level reveals that A375 cells carrying the CASP3 ORF (denoted as CASP3OE) have substantially higher levels of the conformationally inactive pro-caspase 3 protein, but provides no evidence of the cleaved active caspase-3 (FIG. 11) and thus this RNA- based intervention does not impact cells in the absence of a cell death trigger. In accordance with 79685919V.1caspase-3 being a substrate of granzyme B and an important mediator of T cell cytotoxicity in target cells (K. Tuomela, A. R. Ambrose & D. M. Davis, Front. Immunol.13, (2022): 867098; D. Martinvalet, Oxid. Med. Cell Longev. 2019, (2019): 9165214; I. S. Goping et al., Immunity 18, (2003): 355; S. P. Cullen & S. J. Martin, Cell Death & Differentiation 15, (2008): 251), 4 hours after coculture with NY-ESO-1 TCR T cells the CASP3OEA375 cells show a marked increase in cleaved active caspase-3 heterotetramer (FIG.11). In contrast, pro-caspase-3 levels were lower in the control A375 both in monoculture and coculture and there was no evidence of cleaved caspase- 3 in the control A375 cells despite the exposure to targeting T cells (FIG.11). In accordance with this, CASP3 overexpression did not impact cell viability and proliferation in A375 cells (FIG.12), but did significantly increase TCR-dependent T cell cytotoxicity (FIG. 7). Further demonstrating the specificity to TCR-dependent cytotoxicity, CASP3 overexpression does not sensitize cancer cells to IFNg or TNF (FIG. 13). Pan-caspase inhibition via Z-VAD-FNK compound restored resistance to TCR-dependent T cell cytotoxicity in CASP3OEA375 cells and severely desensitized control A375 cells to TCR-dependent T cell cytotoxicity (FIG.14).

[0272] To examine the generalizability of these findings, additional experiments tested a different cancer type with a different TCR that recognizes a viral antigen (FIG. 15). Using the cervical cancer cell line (CaSki) that is positive for the Human Papilloma Virus (HPV) and expresses the immunogenic HPV16- E7 protein along with primary CD8 T cells transduced to express a TCR targeting the E7 protein (N. B. Nagarsheth et al., Nat. Med.27, (2021): 419), it was found that overexpression of the top hits BID, CASP3, and SAFB significantly sensitizes CaSki cells to TCR-dependent T cell cytotoxicity, while WNT3A and TSPYL2 do not (FIG.16, left). Once again, CASP3 overexpression did not impact cell viability and proliferation also in the CasKi cells (FIG. 16, right).

[0273] Based on these findings, it was hypothesized that increasing the levels of CASP3 transcripts could form a new approach to selectively sensitize cancer cells to TCR-dependent cytotoxicity in a manner that will be more effective and less toxic to non-cancer cells compared to conventional pro-apoptotic drugs (FIG.17). This approach was thus tested in direct comparison to BH3 mimetics Venetoclax (the only FDA approved BH3 mimetic). Venetoclax has been shown to be highly effective in hematological malignancies, primarily chronic lymphocytic leukemia and small lymphocytic lymphoma, with ongoing clinical trials for use against solid tumors (I. Ploumaki 79685919V.1et al., Clin. Transl. Oncol. 25, (2023): 1554; B. A. Carneiro & W. S. El-Deiry, Nat. Rev. Clin. Oncol. 17, (2020): 395). Treating A375 and T cells with Venetoclax in monoculture shows that the drug is even more toxic to the T cells than to the melanoma cells (FIG. 18). Moreover, Venetoclax addition in coculture decreases, rather than increases, cancer cell killing by T cells (FIG.19, right). This demonstrates the intrinsic resistance that can exist in cancer cells to this line of treatments and the possibility of weakening the anti-tumor immune response due to the toxicities to T cells. Moreover, CASP3OEcells did not show greater sensitivity to Venetoclax compared to control A375 cells (FIG.19, left), further demonstrating that CASP3 overexpression is selectively sensitizing cancer cells to TCR-dependent cytotoxicity. Examining the impact of CASP3 overexpression in non-malignant cells shows that, in contrast to the Bcl2 inhibitor, overexpression of CASP3 in primary CD8 T cells does not interfere with T cell viability, expansion (FIGS.20 and 21), and cytotoxicity to target cancer cells (FIG. 22), such that CASP3 RNA delivery to coculture of wildtype A375 and targeting T cells via a cationic lipid / polymer- based nanoparticle significantly enhanced TCR-dependent cytotoxicity (FIGS.23-25). Example 5. Methods

[0274] The following methods were used in performing the studies described in Examples 1-4. Plasmids

[0275] Individual sgRNAs were cloned into pMCB306 (GFP), pMCB307 (BFP), or pMCB320 (mCherry) at the BstXI and BlpI restriction sites. The resulting plasmids were transformed into DH5a competent E. Coli (Zymo) then extracted using the GeneJET Plasmid Miniprep Kit. Plasmid sequences were confirmed through sanger sequencing (MCLAB). Cell Culture

[0276] The melanoma cell line A375 was obtained from ATCC (American Type Culture Collection) and cultured in DMEM high glucose (Sigma-Aldrich, D6429) supplemented with 10% fetal bovine serum (FBS, Life Technologies, A3840102) and 1x Pen / Strep (Cytiva, SV30010). The LENTI-XTMHEK293T cell line was obtained from Takara Bio (Cat #632180) and cultured in DMEM high glucose supplemented with 10% FBS. Peripheral blood mononuclear cells (PBMCs) were obtained from buffy coats by Ficoll-Paque (Cytiva, 17544202) in SepMate Tubes (Stemcell Technologies, Cat #85450) following the manufacturer’s protocol. CD8+T Cells were isolated 79685919V.1from PBMCs using immunomagnetic negative selection (Stemcell Technologies, Cat #17953) and cultured in RPMI-1640 medium with GlutaMAX and HEPES (Gibco, 72400-047), supplemented with 10% FBS, 1% human serum type AB (Sigma-Aldrich, H4522), 5 mM MEM Non-Essential Amino Acids (Corning, 25-025-Cl), 5 mM Sodium Pyruvate (Corning, 25-000-Cl), 50 μM B- mercaptoethanol (Sigma-Aldrich, M6250), and 30 units recombinant IL-2 (PeproTech, 10779- 568). All cells were regularly tested for mycoplasma and confirmed negative (PromoCell Inc., PK- CA91-1024). Lentivirus Production

[0277] The melanoma cell line A375 was obtained from ATCC (American Type Culture Collection) and cultured in DMEM high glucose (Sigma-Aldrich, D6429) supplemented with 10% fetal bovine serum (FBS, Life Technologies, A3840102) and 1x Pen / Strep (Cytiva, SV30010). The LENTI-XTMHEK293T cell line was obtained from Takara Bio (Cat #632180) and cultured in DMEM high glucose supplemented with 10% FBS. Peripheral blood mononuclear cells (PBMCs) were obtained from buffy coats by Ficoll-Paque (Cytiva, 17544202) in SepMate Tubes (Stemcell Technologies, Cat #85450) following the manufacturer’s protocol. CD8+T Cells were isolated from PBMCs using immunomagnetic negative selection (Stemcell Technologies, Cat #17953) and cultured in RPMI-1640 medium with GlutaMAX and HEPES (Gibco, 72400-047), supplemented with 10% FBS, 1% human serum type AB (Sigma-Aldrich, H4522), 5 mM MEM Non-Essential Amino Acids (Corning, 25-025-Cl), 5 mM Sodium Pyruvate (Corning, 25-000-Cl), 50 μM B- mercaptoethanol (Sigma-Aldrich, M6250), and 30 units recombinant IL-2 (PeproTech, 10779- 568). All cells were regularly tested for mycoplasma and confirmed negative (PromoCell Inc., PK- CA91-1024). Lentivirus Titration

[0278] To titrate lentivirus, 2×106cells were seeded in 6 wells of a 12-well plate in growth media plus 8 μg / mL polybrene. Immediately after, 0 μL, 2.5 μL, 5 μL, 10 μL, 20 μL, or 40 μL of lentivirus was added to the wells and mixed thoroughly. Twenty-four hours after transduction, cells for each condition were trypsinized and seeded into a 96-well plate at a concentration of 5×104cells / well in 2 wells with selection antibiotic, and in 2 wells without selection antibiotic. When the no-virus condition with selection antibiotic contained no viable cells, cell viability was 79685919V.1measured with PrestoBlue (Invitrogen, Cat# A13261) following the manufacturer protocol. The titer was calculated using the formula: Titer (TU / μL) = (N × P × D) / V where N = cell number in each well used for infection, P = proportion of live cells compared to the no-antibiotic condition, D = dilution fold of the virus, V = virus volume used for infection in each well, in μL, and TU = transduction unit. TCR Engineering

[0279] Lentiviral plasmid containing the TCR recognizing the HLA-A*02-restricted melanoma antigen NY-ESO-1 (NY-ESO-1:157-165 epitope) was used. This construct contains an HA-tag on the alpha chain and a PC-tag on the beta chain of the TCR. One day after stimulation with CD3 / CD28 dynabeads, 2×106T cells were transduced with 300 μL of concentrated lentivirus and expanded for 3 days. Cells were then labeled with anti-HA and anti-PC antibodies and sorted for double-positive cells. The sorted cells were expanded and used for experiments or frozen for later use. To confirm the engineered TCR was expressed on the T cell surface, peptide-MHC tetramers were used as described in the following Flow Cytometry protocol. To confirm the engineered TCR was functional, differential cytotoxicity was quantified in co-culture with A375 cells where CTAG1B (encoding for NYESO1) was either activated via CRISPRa or knocked out, compared to matching control A375 cells. Flow Cytometry

[0280] To assess the purity of CD8+T cell populations, cells were labeled with fluorescent- conjugated anti-CD3, anti-CD4, and anti-CD8 antibodies. Two methods were employed to confirm the expression of the NY-ESO-1 TCR on the T cell surface. First, CD8+T cells were labeled with primary anti-HA and anti-PC antibodies, followed by anti-mouse AF-488 (Cell Signaling Technology, 4408S) and anti-rabbit AF-647 (Cell Signaling Technology, 4414S) secondary antibodies. This labels the HA and PC tags that are present on the NY-ESO-1 TCR. Second, in an independent experiment, T cells were labeled with an APC-labeled tetramer that binds to a TCR that specifically recognizes human HLA-A*02:01 conjugated with the NY-ESO-1 antigen SLLMWITQC (NIH Tetramer Facility). Flow cytometry was performed on a Sony SH800 79685919V.1(Stanford CZ Biohub), BD FACSAria Fusion, BD FACSAria II, or BD Influx (Stanford Shared FACS Facility) flow cytometer. In vitro 2D Co-culture Assays

[0281] For the co-culture assays, 1×104cells were seeded in 100 μL growth media per well in a 96-well plate. Subsequently, 4 hours after seeding, CD8+T cells were added in 100 μL of T cell media at the appropriate E:T ratio and co-cultured for the desired amount of time. T cells were washed away with PBS and cancer cell viability was measured with PrestoBlue using the Infinite M1000 plate reader (Tecan). Western Blot

[0282] Total protein lysate was extracted from cells using Pierce RIPA buffer (Thermo Fisher Scientific, 89900) supplemented with Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific, 87785). Protein concentration was measured using the Qubit Protein Assay Kit (Thermo Fisher Scientific, Q33211). 20 μg of lysate was mixed with Laemmli buffer, boiled at 95 °C for 5 min, then either stored at -80 °C for future use or immediately used in gel electrophoresis. Samples were run through an SDS-PAGE gel and then wet-transferred to a PVDF membrane. Membranes were blocked with EveryBlot Blocking Buffer (Bio-Rad, 12010020) at RT for 5 min, and then incubated with primary antibody in Tris-buffered saline with 0.1% Tween (TBST) at 4 °C overnight. The following day, membranes were washed and incubated with a secondary antibody in TBST at RT for 1 hour. The bands were detected by incubating the membrane with Clarity Western ECL Substrate and exposing it to a digital imager. Quantitative PCR

[0283] Total RNA was extracted using the Aurum Total RNA Mini Kit (Bio-Rad, 7326820) following the manufacturer’s protocol. qRT-PCR was performed using the iTaq Universal SYBR Green One-Step Kit (Bio-Rad, 1725151) on a Bio-Rad CFX96 Touch Real-Time PCR Detection System. Relative RNA expression was determined by normalizing to GAPDH expression. CRISPRa Screens

[0284] A375 cell line stably expressing dCas9-VPR cell line was established as follows. A375 cells were transduced with a lentiviral construct containing dCas9-VPR-t2a-GFP at a multiplicity 79685919V.1of infection (MOI) of ~0.2. GFP high or low cells were sorted as single cells into two 96-well plates and allowed to proliferate for 1 month. The activity of dCas9-VPR in clones was assessed by transducing cells with a CD2 sgRNA and examining CD2 expression by qPCR and flow cytometry. A clone was selected for future experiments based on high induction of CD2 expression as well as similar growth characteristics to WT A375 cells.

[0285] Pooled lentivirus sgRNA library (Addgene catalog number 101926) targeting a total of 2,921 genes, with a total of 31,324 sgRNAs (10 sgRNAs per gene and 280 control gRNAs) was transduced into dCas9-VPR-expressing A375 cells at an MOI of < 0.3. Cells were selected with puromycin (1 μg / mL) for 3 days, then allowed to recover and expand for another 7 days before freezing at 20×106cells per vial.

[0286] CRISPRa screens were conducted with A375 cells under two co-culture conditions: (1) co-culture with WT TCR CD8+T cells (i.e., non-targeting T cells), and (2) co-culture with NY- ESO-1 TCR CD8+T cells (i.e., targeting T cells). In all cases an E:T ratio of 0.25:1 was used. Prior to adding CD8+T cells, 20×106library cells are seeded into T-225 flasks for 4 hours. In the first screen A375 cells were co-cultured with either targeting or non-targeting T cells for 24 hours before T cells were removed by washing twice with PBS. In the second screen A375 cells were subject to 3 rounds of co-culture with either targeting or non-targeting T cells at a 0.25:1 E:T ratio, with 4 days of recovery in between each round. A coverage of 500x of the library is maintained throughout the duration of the screens. All screens were performed in triplicate.

[0287] Genomic DNA was extracted with the DNA Blood Maxi Kit (Qiagen, cat# 51194) according to the manufacturer protocol. The library was prepared for sequencing as described previously (Deans et al., 2016). PCRs were run on the VeritiPro 96-well Thermocycler (Applied Biosystems). Sequencing was done through the Stanford Chan-Zuckerberg Biohub using the Illumina NextSeq 550 with a library read coverage of > 200X per sample. Perturb-seq Screen

[0288] The Perturb-seq sgRNA library was designed to include top hits from the CRISPRa screens (FDR < 0.05, Fisher combined test) with a focus on novel genes and mechanisms of interest. The library included a total of 276 CRISPRa sgRNA: 4 sgRNAs per gene targeting a total of 61 genes, and 20 additional non-targeting sgRNAs. The library was cloned into the pMCB320 79685919V.1backbone and transduced via lentivirus into A375 dCas9-VPR cells at an MOI < 0.3. Transduced cells were seeded at 5×105cells per well in 6-well plates, then either co-cultured with antigen- specific CD8+T cells at an E:T ratio of 0.5:1 or as mono-culture for 24 hours. Cells were harvested at ~1000 cell / μL then loaded onto the Chromium chip with 10,000 cells per condition. DNA libraries were prepared following the protocol for the Chromium Next GEM Single Cell 5' Reagent Kits v2 (Dual Index) with Feature Barcode technology for CRISPR Screening. Libraries were sequenced with the NextSeq 550 at a 10:1 ratio of gene expression to CRISPR libraries. Spatial 3D in vitro CRISPRa Screen

[0289] A375 cells expressing dCas9-VPR were transduced with individual sgRNA or ORF plasmids containing 50 base pair (bp) RNA barcodes. To generate spheroids, cancer cells were trypsinized and resuspended at 200,000 cells / mL in growth media supplemented with 2.5% Matrigel (Corning, 356231), then seeded into 96-well round-bottom ultra-low attachment plates (Corning, 7007). Plates were spun for 5 min at 400 g. After 24 hours, CD8+T cells were added. For sectioning, spheroids were harvested, washed with PBS, then fixed for 1 hour at room temperature with 4% PFA (Alfa Aesar, J61899). After fixation, spheroids were transferred to OCT molds and mixed with Histogel (Epredia, HG-4000-012). Once the Histogel solidified, spheroid blocks were sent to the Stanford Human Pathology / Histology Service Center for FFPE processing and sectioning. Spatial transcriptomics was performed using the CosMx Spatial Molecular Imager following the manufacturer’s protocol (MAN-10159-02). Example 6. Identification of target genes using CRISPR activation and CRISPR knockout screens in breast cancer cells

[0290] Top CRISPR activation hits were shown to sensitize breast cancer cells to NK-92- mediated cytotoxicity in single-guide validation assays. Results from these assays are shown in the graph of FIG. 26, which plots percent cytotoxicity values for MDA-MB-231 gene activation cell lines in dCas9-expressing cells following 48-hour co-culture with NK-92 cells at a 1:1 effector-to-target (E:T) ratio. Cytotoxicity was assessed using the PrestoBlue cell viability assay. The bar graph of FIG. 26 displays the percentage of cancer cell death for each activation line relative to the non-targeting control (NTC). 79685919V.1

[0291] Activation of SLAMF1 was shown to sensitize breast cancer cells to NK cell-mediated cytotoxicity. The experimental results presented by FIG.27 show the percent cytotoxicity of breast cancer cells (MDA-MB-231) with CRISPR activation of SLAMF1 or non-targeting control (NTC) following 24-, 48-, and 72-hour co-culture with NK-92 cells at varying effector-to-target (E:T) ratios. Cytotoxicity was measured using the PrestoBlue cell viability assay. FIG.28 shows results from a flow cytometry analysis of a comparative killing assay comparing SLAMF1-activated and NTC breast cancer cells (MDA-MB-231), differentially labeled with Cytopainter Red or Cytopainter Green. To control for potential dye bias, fluorescent labels were reversed betweenreplicates. Cancer cells were co-cultured with NK-92 cells and gated on live BFP populations toquantify the relative survival of each labeled population. The results presented by FIG. 29 show the percent cytotoxicity of SLAMF1-activated and NTC breast cancer cells following 48-hour co- culture with IL-2-expressing NK-92 cells at varying E:T ratios, measured by PrestoBlue assay.

[0292] Cytotoxicity mediated by SLAMF1 was shown to require SLAMF1 expression on both NK and cancer cells. The experimental results presented by FIG.30 show the percent cytotoxicity of SLAMF1-activated and NTC breast cancer cells (MDA-MB-231) after 24-hour co-culture with either NTC or SLAMF1 knockout (KO) NK-92 cells at a 2:1 E:T ratio. Cytotoxicity was measured via the PrestoBlue cell viability assay. The FIG. 30 bar graph displays the percentage of cancer cell death in co-culture with NTC or SLAMF1 KO NK-92 cells, each generated using one of three distinct sgRNAs.

[0293] Further experiments demonstrated that SLAMF1 activation further enhances CAR-NK cytotoxicity, and that SLAMF1 sensitizes breast cancer cells to CAR-NK92 cells in short co- culture incubations. Results from these experiments are presented by FIGS.31 and 32, which each plot percent cytotoxicity of SLAMF1-activated and NTC breast cancer cells (MDA-MB-231) in co-culture with wild-type or anti-EpCAM CAR-NK-92 cells. Cells were co-cultured for 6 hours at a 1:1 E:T ratio (FIG.31) or for 20 hours at varying E:T ratios (FIG.32). The bar graphs of FIGS. 31 and 32 show cytotoxicity relative to wild-type NK-92.

[0294] Top CRISPR knockout hits were also shown to sensitize breast cancer cells to NK-92- mediated cytotoxicity in syngeneic gene knockout (KO) experiments. Results from these experiments are shown in the graph of FIG.33, which plots percent cytotoxicity values for MDA- MB-231 gene knockout cell lines following 48-hour or 72-hour co-culture with NK-92 cells at a 79685919V.11:1 effector-to-target (E:T) ratio. Cytotoxicity was assessed using the PrestoBlue cell viability assay. The bar graph of FIG. 33 displays the percentage of cancer cell death for each KO line relative to non-targeting control (NTC) at both time points.

[0295] Knockout of IKBKB was shown to sensitize MDA-MB-231 cells to NK-92-mediated cytotoxicity. The bar graph of FIG. 34 provides percent cytotoxicity values for MDA-MB-231 gene knockout (KO) cell lines following 48-hour co-culture with NK-92 cells at varying E:T ratios. Cytotoxicity was assessed using the PrestoBlue viability assay and is shown relative to the corresponding NTC. The bar graph of FIG. 35 provides results from a flow cytometry analysis of cancer cell death in MDA-MB-231 KO cell lines under monoculture (0:1) and co-culture (1:1 E:Tratio with NK-92 cells) conditions. Dead target cells were identified as CD56 Sytox Blue .

[0296] The use of IKBKB knockout to sensitize breast cancer cells to NK-92-mediated killingalso results in an increase in IFN- production. The bar graph of FIG. 36 provides percentcytotoxicity values for IKBKB knockout (KO) MDA-MB-231 cell lines, generated using the top two sgRNAs, following 48-hour and 72-hour co-culture with NK-92 cells at varying E:T ratios. Cytotoxicity was assessed using the PrestoBlue viability assay and is shown relative to thecorresponding NTC. The bar graph of FIG. 37 provides IFN- concentrations measured in co-culture supernatants from NK-92 cells and IKBKB KO or NTC MDA-MB-231 cells, measured by ELISA at varying E:T ratios and time points.

[0297] Additional experiments demonstrated that IKBKB knockout sensitizes breast cancer cells to targeted antigen-TCR-dependent T cell mediated killing. These experiments measured the percent cytotoxicity of MDA-MB-231 IKBKB knockout (KO) or NY-ESO-1-expressing IKBKB KO cell lines following 48-hour co-culture with either wild-type (WT) or NY-ESO-1 TCR- engineered primary T cells from three different donors (designated Donor17, Donor18, and Donor19). Cytotoxicity was assessed using the PrestoBlue cell viability assay. The bar graphs of FIGS. 38-40 show the percentage of cancer cell death for each IKBKB KO line relative to the corresponding non-targeting control (NTC).

[0298] The knockout of IKBKB also sensitizes cancer cells to primary NK cells. This effect was confirmed in a study showing that IKBKB knockout sensitizes while CDCP1 knockout confers resistance to primary NK cell–mediated cytotoxicity in MDA-MB-231 cells across multiple donors. The bar graph of FIG. 41 provides percent cytotoxicity of MDA-MB-231 KO lines 79685919V.1following 48-hour and 72-hour co-culture with primary NK cells from multiple donors at a 1:1 effector-to-target (E:T) ratio. Cytotoxicity was assessed using the PrestoBlue cell viability assay. The data in FIG. 41 show the percentage of cancer cell death relative to the corresponding non- targeting control (NTC). The graphs of FIGS. 42 and 43 show the average NK cytotoxicity level per donor for NTC and IKBKB KO MDA-MB-231 after 48 hours (FIG. 42) and 72 hours (FIG. 43) of co-culture at 1:1 E:T ratio.

[0299] Additionally, IKBKB knockout has been shown to sensitize breast cancer cells to NK- 92 and CAR-NK-92 cells, thereby enhancing killing by EpCAM-CAR NK-92 cells. The bar graph of FIG. 44 provides percent cytotoxicity values for MDA-MB-231 IKBKB KO and NTC lines following 24-hour and 48-hour co-culture with either wild-type NK-92 or anti-EpCAM CAR-NK- 92 cells at a 1:1 E:T ratio. Cytotoxicity was assessed using the PrestoBlue cell viability assay. Example 7. Methods

[0300] The following methods were used in performing the studies described in Example 6. CRISPR Knockout and Activation Screens

[0301] CRISPR knockout (CRISPRko) and activation (CRISPRa) screens were performed using pooled sgRNA libraries transduced into MDA-MB-231 cells. For CRISPRko, MDA-MB-231 cells constitutively expressing Cas9 (Genecopoeia, SL515) were transduced with the Cellecta Human Genome-Wide Knockout Library, Module I (Cellecta, KOHGW-M1-P) at a multiplicity ofinfection (MOI) < 0.3 and selected with puromycin (1 μg / mL). Library-transduced cells weremaintained at > 1,000× coverage unless otherwise noted. Cells were cultured under three conditions: (1) monoculture, (2) 1:1 co-culture with NK-92 cells, and (3) 2.5:1 co-culture with NK-92 cells. All conditions were maintained for 72 hours, after which cells were washed twicewith 1× DPBS to remove NK-92 cells, snap frozen, and stored at -80 °C for genomic DNA (gDNA)extraction. Conditions (1) and (2) were re-cultured and harvested again on day 6 post-recovery, and on day 9 following an additional 72-hour culture.

[0302] For CRISPRa, MDA-MB-231 cells were first sequentially transduced with lentiviruses encoding dCas9-VP64 (Addgene, 61425) and MS2-p65-HSF1 (Addgene, 89308), followed byselection with blasticidin (12.5 μg / mL) and hygromycin B (500 μg / mL), respectively. These cellswere transduced with the Wright Human Membrane Protein Activation Library (Addgene, 79685919V.1113345) at MOI < 0.3 and selected with puromycin (1 μg / mL). Cells were cultured under twoconditions: (1) monoculture and (2) 1:1 co-culture with NK-92 cells. After 72 hours, cells werewashed twice with 1× DPBS, snap frozen, and stored at -80 °C. Cells were harvested again on day9 post-recovery, and again on day 27 following a second 72-hour co-culture. Genomic DNA was extracted using the QIAamp DNA Maxi Blood Kit (Qiagen, 51194), and sgRNA sequences were PCR-amplified for next-generation sequencing.

[0303] CRISPRko and CRISPRa libraries were sequenced on the NextSeq 550 High (single- end) and NextSeq 2000 P3 (single-end) platforms, respectively, at the Chan Zuckerberg Biohub. Guide enrichment or depletion was analyzed with MAGeCK to identify genes affecting NK-92– mediated cytotoxicity. Lentivirus Production

[0304] Lentivirus was produced as previously described (Y.-M. Kim, R. V. Akana, O. Laveroni & L. Jerby, bioRxiv, (2025): 03.21.644686; C. Y. Yeh et al., Nat. Immunol. 25, (2024): 1943).Lenti-X 293T cells were transfected at 80–90% confluency with transfer vector (14 μg), psPAX2(10 μg; Addgene, 12260), and pMD2.G (4.33 μg; Addgene, 12259) using TransIT-Lenti reagent(MirusBio, 6603) in Opti-MEM. After 6 hours, the medium was replaced with cOpti-MEMcontaining 1× ViralBoost (Alstem Bio, VB100). Supernatants were collected at 48 h, filtered(0.45 μm), concentrated using Lenti-X Concentrator (Takara, 631232), aliquoted, and stored at-80 °C.In vitro NK cell Cytotoxicity Assay and IFN- Secretion Quantification

[0305] MDA-MB-231 cells were seeded in black-walled, clear-bottom 96-well plates (Greiner, 655090). One day after seeding, NK cells were added at varying effector-to-target (E:T) ratios and co-cultured for different durations, as indicated in the figures and figure legends. At the end ofeach co-culture period, supernatants were collected and analyzed for human IFN- using an ELISAkit (BioLegend, 430104) according to the manufacturer’s instructions. Remaining cells were gently washed with PBS up to two times, followed by addition of PrestoBlue Cell ViabilityReagent (Thermo Fisher, A13262) and incubation for up to 2 hours at 37 °C. Fluorescence wasmeasured at excitation / emission 560 / 590 nm using a Tecan Infinite M1000 plate reader. Percentcytotoxicity was calculated relative to target-only controls. 79685919V.1

[0306] For the competitive cytotoxicity assay, SLAMF1-activated and non-targeting control (NTC) MDA-MB-231 cells were stained with Cytopainter Green (Abcam, ab176735) or Cytopainter Red (Abcam, ab176736) at room temperature for 30 minutes. After staining, cells were washed, counted, and mixed at a 1:1 ratio. The mixed cancer cell population was then co- cultured with NK-92 cells at the indicated effector-to-target (E:T) ratio and incubation time. After co-culture, cells were harvested and analyzed by flow cytometry. Gating was performed on liveBFP populations to identify transduced target cells, and the relative abundance of CytopainterGreen versus Red cells was quantified. To control for dye-specific effects, fluorescent labelswere swapped between SLAMF1-activated and NTC cells in biological replicates. Flow Cytometry and Cell Sorting

[0307] Cells were harvested and washed with FACS buffer (PBS + 2% FBS). Human TruStain FcX (Fc receptor blocker) (Biolegend, 422302, 1:50) was used to minimize nonspecific binding of antibodies. For cytotoxicity analysis, co-cultured cells were stained with anti-human CD56 (Miltenyi Biotech, 130-113-305) to label NK-92 cells and Sytox Blue (Thermo Fisher, S34857) toidentify dead cells. Dead target cells were gated as CD56 Sytox . For surface marker analysis andsorting, cells were blocked with Fc receptor blocker and stained with appropriate antibodies Cells were then stained for surface or intracellular markers at 4 ºC in the dark for 20 min unless stated otherwise. Anti-human SLAM (Biolegend, 306308) antibodies were used. Flow cytometry was performed using Sony SH800S. Cell sorting was conducted using the Sony SH800S Cell Sorter. All flow cytometry data were analyzed using FlowJo version 10.10.0. Generation of Single-guide CRISPR Knockout or Activation Cell Lines

[0308] MDA-MB-231 cells stably expressing Cas9 or dCas9 were generated as described previously, and Cas9-expressing NK-92 cells were obtained as a gift. Briefly, NK-92 cells were transduced with lentivirus encoding GFP-tagged Cas9 at a multiplicity of infection (MOI) < 0.3,and GFP cells were sorted by flow cytometry. Protospacer sequences for non-targeting control(NTC) and target sgRNAs were obtained from either the Human Genome Knockout Library Module I (Cellecta, KOHGW-M1-P) or the Wright Human Membrane Protein Activation Library (Addgene, 113345), and synthesized by Integrated DNA Technologies (IDT). For CRISPR knockout experiments, oligos were annealed and cloned into the lentiGuide-Puro backbone (Addgene, 52963) digested with FastDigest BsmBI (Thermo Scientific, FD0454). For CRISPR 79685919V.1activation, oligos were cloned into the pKVL2-U6gRNA_SAM (BbsI)-PGKpuroBFP-W vector (Addgene, 112925) digested with FastDigest BpiI. Lentivirus was produced by transfecting Lenti- X 293T cells with individual sgRNA constructs, and the resulting viral supernatants were used to transduce Cas9- or dCas9-expressing MDA-MB-231 cells or Cas9-expressing NK-92 cells at MOI < 0.3. Transduced cells were selected with 1 μg / mL puromycin for 5–7 days prior to use in downstream assays. Primary NK and CD8 T Cell Isolation and NY-ESO-1 TCR Engineering

[0309] Primary human NK and CD8 T cells were isolated from whole blood buffy coatsobtained from the Stanford Blood Center, as previously described (Y.-M. Kim, R. V. Akana, O. Laveroni & L. Jerby, bioRxiv, (2025): 03.21.644686; C. Y. Yeh et al., Nat. Immunol. 25, (2024): 1943). Briefly, PBMCs were enriched using Ficoll-Paque Premium Medium (Cytiva, 17-5442-02)and SepMate tubes (STEMCELL Technologies, 85450). CD8 T cells were isolated using theEasySep Human CD8 T Cell Isolation Kit (STEMCELL, 17953), activated with Dynabeads(Gibco, 11131D), and transduced with a lentiviral vector encoding the NY-ESO-1–specific TCR (1G4). Transduced T cells were stained with PC-Rabbit (Cell Signaling Technology, 68083S) and HA-Mouse (Cell Signaling Technology, 2367S) primary antibodies (1:50), followed by Alexa Fluor 647 anti-rabbit (CST, 4414S) and Alexa Fluor 488 anti-mouse (CST, 4408S) secondary antibodies (1:500), and sorted based on HA- and PC- expression. Primary NK cells were isolated using the EasySep Human NK Cell Isolation Kit (STEMCELL, 17955) and cultured in EL837 serum-free NK medium (EliteCell, ELM-1000) supplemented with EliteGro-Adv, 5% heat-inactivated human AB serum, 200 U / mL IL-2, and 100 ng / mL IL-18.Generation of IL-2-expressing NK-92 Cells

[0310] The IL-2 ORF was synthesized (Twist Bioscience) and cloned into the pLV-EF1a-IRES- Puro vector (Addgene, 85132) using BamHI-HF and EcoRI-HF digestion followed by Gibson assembly. Lentivirus was produced from the IL-2 construct and used to transduce NK-92 cells,which were subsequently selected with 1 μg / mL puromycin.

[0311] Although the foregoing disclosure has been described in some detail by way of illustration and example for purpose of clarity of understanding, one of skill in the art will appreciate that certain changes and modifications within the spirit and scope of the disclosure may 79685919V.1be practiced, e.g., within the scope of the appended claims. It should also be understood that aspects of the disclosure and portions of various recited embodiments and features can be combined or interchanged either in whole or in part. In the foregoing descriptions of the various embodiments, those embodiments which refer to another embodiment may be appropriately combined with other embodiments as will be appreciated by one of skill in the art. Furthermore, those of ordinary skill in the art will appreciate that the foregoing description is by way of example only and is not intended to limit the disclosure. In addition, each reference provided herein is incorporated by reference in its entirety for all purposes to the same extent as if each reference was individually incorporated by reference. 79685919V.1

Claims

1. WHAT IS CLAIMED IS:

1. A method for sensitizing a cancer cell to cytotoxic lymphocytes, wherein the cancer cell comprises a gene encoding a cytotoxic-lymphocyte sensitizing protein that increases elimination of the cancer cell upon recognition of the cancer cell by a cytotoxic lymphocyte targeting the cancer cell, the method comprising introducing into the cancer cell a polynucleotide comprising or encoding a guide RNA targeting the gene.

2. The method of claim 1, wherein the method further comprises introducing into the cancer cell a CRISPR-based activation system.

3. A method for sensitizing a cancer cell to cytotoxic lymphocytes, the method comprising: introducing into the cancer cell a polynucleotide encoding a cytotoxic-lymphocyte sensitizing protein that increases elimination of the cancer cell upon recognition of the cancer cell by a cytotoxic lymphocyte targeting the cancer cell.

4. The method of claim 1, wherein the polynucleotide comprises RNA.

5. The method of claim 1, wherein the cytotoxic-lymphocyte sensitizing protein is an endogenous protein of the cancer cell.

6. The method of claim 1, wherein the cytotoxic-lymphocyte sensitizing protein is an inactive protein.

7. The method of claim 6, wherein the inactive enzyme is a proenzyme.

8. The method of claim 6, wherein the inactive protein transforms to an active protein upon recognition of the cancer cell by the cytotoxic lymphocyte targeting the cancer cell.

9. The method of claim 8, wherein a granzyme released by the cytotoxic lymphocyte induces transformation of the inactive protein to the active protein. 79685919V.

110. The method of claim 1, wherein the cytotoxic-lymphocyte sensitizing protein comprises a B-cell lymphoma 2 (BCL-2) family protein or a subunit or fragment thereof.

11. The method of claim 10, wherein the BCL-2 family protein or the subunit or fragment thereof comprises BCL-2 homology domain 3 interacting domain death agonist (BID), BCL-2 antagonist / killer 1 (BAK1), BCL-2 interacting killer (BIK), phorbol-12-myristate-13- acetate-induced protein 1 (PMAIP1), or a variant or combination thereof.

12. The method of claim 1, wherein the cytotoxic-lymphocyte sensitizing protein comprises a cysteine-aspartic acid protease (caspase) family protein or a subunit or fragment thereof.

13. The method of claim 12, wherein the caspase family protein or the subunit or fragment thereof comprises caspase-3 (CASP3), caspase-8 (CASP8), or a variant or combination thereof.

14. The method of claim 1, wherein the cytotoxic-lymphocyte sensitizing protein comprises a transcription factor, a transcriptional regulator, or a subunit or fragment thereof.

15. The method of claim 14, wherein the transcription factor or the subunit or fragment thereof comprises an ETS family transcription factor or a subunit or fragment thereof.

16. The method of claim 15, wherein the ETS family transcription factor or the subunit or fragment thereof comprises ETS proto-oncogene 1 transcription factor (ETS1), ETS variant transcription factor 4 (ETV4), or a variant or combination thereof.

17. The method of claim 14, wherein the transcription factor or the subunit or fragment thereof comprises a GATA family transcription factor or a subunit or fragment thereof.

18. The method of claim 17, wherein the GATA family transcription factor or the subunit or fragment thereof comprises GATA-binding factor 4 (GATA4), GATA-binding factor 6 (GATA6), tricho-rhino-phalangeal syndrome Type 1 (TRPS1), or a variant or combination thereof. 79685919V.

119. The method of claim 14, wherein the transcription factor or the subunit or fragment thereof comprises a paired box (PAX) family transcription factor or a subunit or fragment thereof.

20. The method of claim 19, wherein the PAX family transcription factor or the subunit or fragment thereof comprises PAX3, PAX8, or a variant or combination thereof.

21. The method of claim 14, wherein the transcription factor or the subunit or fragment thereof comprises a helix-loop-helix transcription factor, a helix-loop-helix transcriptional regulator, or a subunit or fragment thereof.

22. The method of claim 21, wherein the helix-loop-helix transcription factor, the helix-loop-helix transcriptional regulator, or the subunit or fragment thereof comprises inhibitor of DNA binding 1 (ID1), hairy / enhancer-of-split related with YRPW motif-like protein (HEYL), microphthalmia-associated transcription factor (MITF), MYC proto-oncogene BHLH transcription factor (MYC), or a variant or combination thereof.

23. The method of claim 14, wherein the transcription factor or the subunit or fragment thereof comprises paired-like homeobox 2B (PHOX2B), chromodomain-helicase-DNA- binding protein 8 (CHD8), histone deacetylase 4 (HDAC4), histone deacetylase 7 (HDAC7), hypermethylated in cancer 1 protein (HIC1), high mobility group box 1 protein (HMGB1), interferon regulatory factor 1 (IRF1), nuclear receptor 4A3 (NR4A3), nuclear protein 1 (NUPR1), peroxisome proliferator-activated receptor gamma coactivator 1-beta (PPARGC1B), PR domain containing 16 (PRDM16), protein arginine N-methyltransferase 2 (PRMT2), homeobox protein prophet of PIT-1 (PROP1), prothymosin alpha (PTMA), transcription factor RelB (RELB), SET nuclear proto-oncogene (SET), homeobox protein SIX4 (SIX4), TATA-box binding protein associated factor 15 (TAF15), transcription factor AP-2 alpha (TFAP2A), DNA topoisomerase 1 (TOP1), Wilms tumor protein (WT1), or a variant or combination thereof.

24. The method of claim 1, wherein the cytotoxic-lymphocyte sensitizing protein comprises a tumor necrosis factor receptor superfamily (TNFRSF) protein or a subunit or fragment thereof. 79685919V.

125. The method of claim 24, wherein the TNFRSF protein or the subunit or fragment thereof comprises the Fas cell surface death receptor (FAS), TNFRSF member 1A (TNFRSF1A), TNFRSF member 1B (TNFRSF1B), tumor necrosis factor ligand superfamily member 9 (TNFSF9), or a variant or combination thereof.

26. The method of claim 1, wherein the cytotoxic-lymphocyte sensitizing protein comprises a transmembrane ligand or a subunit or fragment thereof.

27. The method of claim 26, wherein the transmembrane ligand or the subunit or fragment thereof comprises CUB domain-containing protein 1 (CDCP1), delta-like canonical Notch ligand 4 (DLL4), or a variant or combination thereof.

28. The method of claim 1, wherein the cytotoxic-lymphocyte sensitizing protein comprises an immunoglobulin superfamily protein or a subunit or fragment thereof.

29. The method of claim 28, wherein the immunoglobulin superfamily protein or the subunit or fragment thereof comprises CD47, CD58, or a variant or combination thereof.

30. The method of claim 1, wherein the cytotoxic-lymphocyte sensitizing protein comprises a collagen subunit.

31. The method of claim 30, wherein the collagen subunit comprises collagen alpha-1(V) chain (COL5A1), collagen alpha-2(IV) chain (COL4A2), or a variant or combination thereof.

32. The method of claim 1, wherein the cytotoxic-lymphocyte sensitizing protein comprises an interferon family signaling protein or a subunit or fragment thereof.

33. The method of claim 32, wherein the interferon family signaling protein or the subunit or fragment thereof comprises interferon beta 1 (IFNB1), interferon gamma (IFNG), or a variant or combination thereof.

34. The method of claim 1, wherein the cytotoxic-lymphocyte sensitizing protein comprises a mitosis regulatory protein or a subunit or fragment thereof. 79685919V.

135. The method of claim 34, wherein the mitosis regulatory protein or the subunit or fragment thereof comprises cyclin B1 (CCNB1), centrosomal protein of 70 kDa (CEP70), close homolog of L1 (CHL1), kinesin family member 14 (KIF14), or a variant or combination thereof.

36. The method of claim 1, wherein the cytotoxic-lymphocyte sensitizing protein comprises a protein phosphatase or a subunit or fragment thereof.

37. The method of claim 36, wherein the protein phosphatase or the subunit or fragment thereof comprises dual specificity phosphatase 1 (DUSP1), protein phosphatase 1J (PPM1J), serine / threonine-protein phosphatase 2A regulatory subunit B'' subunit gamma (PPP2R3C), or a variant or combination thereof.

38. The method of claim 1, wherein the cytotoxic-lymphocyte sensitizing protein comprises signaling lymphocytic activation molecule family member 1 (SLAMF1), sphingosine 1-phosphate transporter (SPNS2), mucin 21 (MUC21), translocation associated membrane protein 2 (TRAM2), cluster of differentiation 80 (CD80), family with sequence similarity 9 member C (FAM9C), solute carrier family 7 member 3 (SLC7A3), ATPase family AAA domain containing 1 (ATAD1), nipped-B-like protein (NIPBL), C-C motif chemokine 22 (CCL22), CLK4-associating serine / arginine rich protein (CLASPR), 2',3'-cyclic-nucleotide 3'- phosphodiesterase (CNP), coronin-6 (CORO6), crystatin-F (CST7), cancer / testis antigen 1A (CTAG1A), eyes absent homolog 2 (EYA2), family with sequence similarity 161 centrosomal protein B (FAM161B), family with sequence similarity 46 member D (FAM46D), alpha-(1,3)- fucosyltransferase (FUT9), glutamate [NMDA] receptor subunit epsilon-2 (GRIN2B), immediate early response gene 5-like protein (IER5L), insulin receptor substrate 4 (IRS4), KHDRBS protein 1 (KHDRBS1), alpha-1,3-mannosyl-glycoprotein 4-beta-N-acetylglucosaminyltransferase A (MGAT4A), metallophosphoesterase domain-containing protein 2 (MPPED2), neuraminidase 3 (NEU3), nucleophosmin 1 (NPM1), proliferation and apoptosis adaptor protein 15 (PEA15), Pleckstrin homology domain interacting protein (PHIP), calcium-dependent phospholipase A2 (PLA2G5), proline-rich protein 5 (PRR5), retrotransposon Gag-like 5 (RTL5), ribonucleotide- diphosphate reductase subunit M2B (RRM2B), scaffold attachment factor B (SAFB), secreted frizzled related protein 2 (SFRP2), serine and arginine rich splicing factor 3 (SRSF3), tenascin C 79685919V.1(TSC), TSPY-like 2 (TSPYL2), taxilin gamma (TXLNG), vimentin (VIM), or a variant or combination thereof.

39. The method of claim 1, wherein the introducing of the polynucleotide to the cancer cell comprises contacting the cancer cell with a virus, a virus-like particle, or an immune cell, wherein the virus, the virus-like particle, or the immune cell comprises the polynucleotide.

40. The method of claim 1, wherein the cancer cell comprises a melanoma cell or a breast cancer cell.

41. An engineered cytotoxic lymphocyte genetically engineered to overexpress an antitumor enhancing ligand, the antitumor enhancing ligand binding to a corresponding binding partner expressed by the engineered cytotoxic lymphocyte, wherein the engineered cytotoxic lymphocyte induces increased elimination of a cancer cell as compared to the elimination of the cancer cell induced by a corresponding cytotoxic lymphocyte not overexpressing the antitumor enhancing ligand.

42. The engineered cytotoxic lymphocyte of claim 41, wherein the engineered cytotoxic lymphocyte is further genetically engineered to overexpress the corresponding binding partner.

43. The engineered cytotoxic lymphocyte of claim 41, wherein the antitumor enhancing ligand comprises a Wnt family protein or a subunit or fragment thereof.

44. The engineered cytotoxic lymphocyte of claim 43, wherein the Wnt family protein or the subunit or fragment thereof comprises Wnt family member 1 (WNT1), Wnt family member 3 (WNT3), Wnt family member 3A (WNT3A), Wnt family member 10B (WNT10B), or a variant or combination thereof.

45. The engineered cytotoxic lymphocyte of claim 41, wherein the antitumor enhancing ligand comprises CD58, CDCP1, CD47, or a variant or combination thereof.

46. The engineered cytotoxic lymphocyte of claim 41, wherein the cytotoxic lymphocyte is a T cell or a natural killer (NK) cell. 79685919V.

147. The engineered cytotoxic lymphocyte of claim 41, wherein the engineered cytotoxic lymphocyte expresses a chimeric antigen receptor (CAR).

48. The engineered cytotoxic lymphocyte of claim 41, wherein the engineered cytotoxic lymphocyte expresses an engineered T-cell receptor (TCR).

49. The engineered cytotoxic lymphocyte of claim 41, wherein the engineered cytotoxic lymphocyte comprises a CRISPR-based activation system effecting overexpression of the antitumor enhancing ligand by the engineered cytotoxic lymphocyte.

50. The engineered cytotoxic lymphocyte of claim 41, wherein the engineered cytotoxic lymphocyte comprises an exogenous gene encoding the antitumor enhancing ligand.

51. A method for producing an engineered cytotoxic lymphocyte, the method comprising: providing a cytotoxic lymphocyte; and introducing into the cytotoxic lymphocyte a polynucleotide encoding an antitumor enhancing ligand, thereby producing the engineered cytotoxic lymphocyte; wherein the antitumor enhancing ligand binds to a corresponding binding partner expressed by the engineered cytotoxic lymphocyte; and wherein the engineered cytotoxic lymphocyte induces increased elimination of a cancer cell as compared to elimination of the cancer cell induced by a corresponding cytotoxic lymphocyte not overexpressing the antitumor enhancing ligand.

52. The method of claim 51, wherein the method further comprises engineering the cytotoxic lymphocyte to overexpress the corresponding binding partner.

53. A method for producing an engineered cytotoxic lymphocyte, the method comprising: providing a cytotoxic lymphocyte comprising a gene encoding an antitumor enhancing ligand; and introducing into the cytotoxic lymphocyte a polynucleotide comprising or encoding a guide RNA targeting the gene, thereby producing the engineered cytotoxic lymphocyte; 79685919V.1wherein the antitumor enhancing ligand binds to a corresponding binding partner of the engineered cytotoxic lymphocyte; and wherein the engineered cytotoxic lymphocyte induces increased elimination of a cancer cell as compared to elimination of the cancer cell induced by a corresponding cytotoxic lymphocyte not overexpressing the antitumor enhancing ligand.

54. The method of claim 53, wherein the engineered cytotoxic lymphocyte comprises a CRISPR-based activation system effecting overexpression of the antitumor enhancing ligand by the engineered cytotoxic lymphocyte.

55. The method of claims 51, wherein the polynucleotide comprises RNA.

56. The method of claims 51, wherein the antitumor enhancing ligand is an endogenous protein of the cytotoxic lymphocyte.

57. The method of claims 51, wherein the antitumor enhancing ligand is exogenous to the cytotoxic lymphocyte.

58. The method of claims 51, wherein the antitumor enhancing ligand comprises a Wnt family protein or a subunit or fragment thereof.

59. The method of claim 58, wherein the Wnt family protein or the subunit or fragment thereof comprises WNT1, WNT3, WNT3A, WNT10B, or a variant or combination thereof.

60. The method of claims 51, wherein the antitumor enhancing ligand are CD58, CDCP1, CD47, or a variant or combination thereof.

61. The method of claims 51, wherein introducing the polynucleotide comprises transduction of the polynucleotide into the cytotoxic lymphocyte using a virus or a virus-like particle, the virus or virus-like particle comprising the polynucleotide.

62. The method of claims 51, wherein the cytotoxic lymphocyte is a T cell or a natural killer (NK) cell. 79685919V.

163. The method of claims 51, wherein the engineered cytotoxic lymphocyte expresses a chimeric antigen receptor (CAR).

64. The method of claims 51, wherein the engineered cytotoxic lymphocyte expresses an engineered T-cell receptor (TCR).

65. A method for treating a cancer in a subject, the method comprising: administering to the subject a polynucleotide comprising or encoding a guide RNA, the guide RNA targeting a gene encoding a cytotoxic-lymphocyte sensitizing protein that increases elimination of a cancer cell of the cancer upon recognition of the cancer cell by a cytotoxic lymphocyte targeting the cancer cell.

66. A method for treating a cancer in a subject, the method comprising: administering to the subject a polynucleotide encoding a cytotoxic-lymphocyte sensitizing protein that increases elimination of a cancer cell of the cancer upon recognition of the cancer cell by a cytotoxic lymphocyte targeting the cancer cell.

67. The method of claim 65, wherein the polynucleotide comprises RNA.

68. The method of claim 65, wherein the cytotoxic-lymphocyte sensitizing protein is an endogenous protein of the cancer cell.

69. The method of claim 65, wherein the cytotoxic-lymphocyte sensitizing protein is an inactive protein.

70. The method of claim 69, wherein the inactive protein is a proenzyme.

71. The method of claim 69, wherein the inactive protein transforms to an active protein upon recognition of the cancer cell by the cytotoxic lymphocyte targeting the cancer cell.

72. The method of claim 71, wherein a granzyme released by the cytotoxic lymphocyte induces transformation of the inactive protein to the active protein.

73. The method of claim 65, wherein the cytotoxic-lymphocyte sensitizing protein comprises a BCL-2 family protein or a subunit or fragment thereof. 79685919V.

174. The method of claim 73, wherein the BCL-2 family protein or the subunit or fragment thereof comprises BID, BAK1, BIK, PMAIP1, or a variant or combination thereof.

75. The method of claim 65, wherein the cytotoxic-lymphocyte sensitizing protein comprises a caspase family protein or a subunit or fragment thereof.

76. The method of claim 75, wherein the caspase family protein or the subunit or fragment thereof comprises CASP3, CASP8, or a variant or combination thereof.

77. The method of claim 65, wherein the cytotoxic-lymphocyte sensitizing protein comprises a transcription factor, a transcriptional regulator, or a subunit or fragment thereof.

78. The method of claim 77, wherein the transcription factor or the subunit or fragment thereof comprises an ETS family transcription factor or a subunit or fragment thereof.

79. The method of claim 78, wherein the ETS family transcription factor or the subunit or fragment thereof comprises ETS1, ETV4, or a variant or combination thereof.

80. The method of claim 77, wherein the transcription factor or the subunit or fragment thereof comprises a GATA family transcription factor or a subunit or fragment thereof.

81. The method of claim 80, wherein the GATA family transcription factor or the subunit or fragment thereof comprises GATA4, GATA6, TRPS1, or a variant or combination thereof.

82. The method of claim 77, wherein the transcription factor or the subunit or fragment thereof comprises a PAX family transcription factor or a subunit or fragment thereof.

83. The method of claim 82, wherein the PAX family transcription factor or the subunit or fragment thereof comprises PAX3, PAX8, or a variant or combination thereof.

84. The method of claim 77, wherein the transcription factor or the subunit or fragment thereof comprises a helix-loop-helix transcription factor, a helix-loop-helix transcriptional regulator, or a subunit or fragment thereof. 79685919V.

185. The method of claim 84, wherein the helix-loop-helix transcription factor, the helix-loop-helix transcriptional regulator, or the subunit or fragment thereof comprises ID1, HEYL, MITF, MYC, or a variant or combination thereof.

86. The method of claim 77, wherein the transcription factor or the subunit or fragment thereof comprises PHOX2B, CHD8, HDAC4, HDAC7, HIC1, HMGB1, IRF1, NR4A3, NUPR1, PPARGC1B, PRDM16, PRMT2, PROP1, PTMA, RELB, SET, SIX4, TAF15, TFAP2A, TOP1, WT1, or a variant or combination thereof.

87. The method of claim 65, wherein the cytotoxic-lymphocyte sensitizing protein comprises a TNFRSF protein or a subunit or fragment thereof.

88. The method of claim 87, wherein the TNFRSF protein or the subunit or fragment thereof comprises FAS, TNFRSF1A, TNFRSF1B, TNFSF9, or a variant or combination thereof.

89. The method of claim 65, wherein the cytotoxic-lymphocyte sensitizing protein comprises a transmembrane ligand or a subunit or fragment thereof.

90. The method of claim 89, wherein the transmembrane ligand or the subunit or fragment thereof comprises CDCP1, DLL4, or a variant or combination thereof.

91. The method of claim 65, wherein the cytotoxic-lymphocyte sensitizing protein comprises an immunoglobulin superfamily protein or a subunit or fragment thereof.

92. The method of claim 91, wherein the immunoglobulin superfamily protein or the subunit or fragment thereof comprises CD47, CD58, or a variant or combination thereof.

93. The method of claim 65, wherein the cytotoxic-lymphocyte sensitizing protein comprises a collagen subunit.

94. The method of claim 93, wherein the collagen subunit comprises COL5A1, COL4A2, or a variant or combination thereof. 79685919V.

195. The method of claim 65, wherein the cytotoxic-lymphocyte sensitizing protein comprises an interferon family signaling protein or a subunit or fragment thereof.

96. The method of claim 95, wherein the interferon family signaling protein or the subunit or fragment thereof comprises IFNB1, IFNG, or a variant or combination thereof.

97. The method of claim 65, wherein the cytotoxic-lymphocyte sensitizing protein comprises a mitosis regulatory protein or a subunit or fragment thereof.

98. The method of claim 97, wherein the mitosis regulatory protein or the subunit or fragment thereof comprises CCNB1, CEP70, CHL1, KIF14, or a variant or combination thereof.

99. The method of claim 65, wherein the cytotoxic-lymphocyte sensitizing protein comprises a protein phosphatase or a subunit or fragment thereof.

100. The method of claim 99, wherein the protein phosphatase or the subunit or fragment thereof comprises DUSP1, PPM1J, PPP2R3C, or a variant or combination thereof.

101. The method of claim 65, wherein the cytotoxic-lymphocyte sensitizing protein comprises SLAMF1, SPNS2, MUC21, TRAM2, CD80, FAM9C, SLC7A3, ATAD1, NIPBL, CCL22, CLASPR, CNP, CORO6, CST7, CTAG1A, EYA2, FAM161B, FAM46D, FUT9, GRIN2B, IER5L, IRS4, KHDRBS1, MGAT4A, MPPED2, NEU3, NPM1, PEA15, PHIP, PLA2G5, PRR5, RTL5, RRM2B, SAFB, SFRP2, SRSF3, TSC, TSPYL2, TXLNG, VIM, or a variant or combination thereof.

102. A method for treating a cancer in a subject, the method comprising: administering to the subject the engineered cytotoxic lymphocyte of claim 41.

103. A method for sensitizing a cancer cell to cytotoxic lymphocytes, the method comprising introducing into the cancer cell a polynucleotide configured to reduce or eliminate expression of a target gene by the cancer cell. wherein the reduction or the elimination of the target gene expression increases elimination of the cancer cell upon recognition of the cancer cell by a cytotoxic lymphocyte targeting the cancer cell. 79685919V.1104. The method of claim 103, wherein the target gene encodes inhibitor of nuclear factor kappa B kinase subunit beta (IKBKB), presenilin enhancer gamma-secretase subunit (PSENEN), secretoglobin family 1D member 1 (SCGB1D1), glycogen synthase kinase 3 beta (GSK3B), ring finger protein 20 (RNF20), tuberous sclerosis complex-1 (TSC1), or ubiquitin conjugating enzyme E2 F (UBE2F). 79685919V.1

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