WO2025158400A1 - Compositions and methods of natural killer cell hyperboosts for enhancement of nk cell therapy - Google Patents

Abstract

Genetically modified Natural Killer (NK) cells and compositions and methods thereof for treating cancer are provided. In some forms, the modified NK cells have up-regulated expression of SGSM2, OR7A10, APLN, PDP1, or CYB5B genes. NK and CAR-NK cells that overexpress SGSM2, OR7A10, APLN, PDP1, or CYB5B genes enable enhanced Adoptive Cell Therapy (ACT). Also provided are compositions and methods for genomic CRISPRa-based gain-of- function screening of NK cells. Exemplary methods provide a library of sgRNAs specific for NK cell genes to implement CRISPRa-based genomic editing of populations of NK cells. Methods of administering cytokines IL15 and/or IL21 to enhance CAR-NK based ACT for treating cancer are also described.

Description

COMPOSITIONS AND METHODS OF NATURAL KILLER CELL HYPERBOOSTS FOR ENHANCEMENT OF NK CELL THERAPY

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of and priority to U.S. Application No. 63/624,561, filed January 24, 2024, which is incorporated herein by reference in its entirety.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing XML submitted as a file named “YU_8835_PCT_ST26.xml,” created on January 24, 2025, and having a size of 5,258 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.834(c)(1).

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under HT9425-23- 1-0472 and W81XWH-20-1-0072 awarded by the U.S. Department of Defense. The government has certain rights in the invention.

FIELD OF THE INVENTION

The invention is generally related to the field of screening technology for identifying gain-of-function genetic modifications for enhancing natural killer (NK) cell activity, and more particularly to compositions and methods for genetic engineering in live NK cells to enhance chimeric antigen receptor (CAR)-NK cell anti-cancer therapy against solid tumors.

BACKGROUND OF THE INVENTION

Natural killer (NK) cells are cytotoxic lymphocytes with a potent ability to kill both tumors and virally infected cells, bypassing major histocompatibility complex restriction and prior sensitization ¹’ ^{2, 3, 4}. NK cells recognize germline-encoded ligands associated with oncogenic transformation ⁵’ ⁶’ ⁷ and can therefore kill cancer cells with low mutational burden or lack neoantigen presentation ⁸’ ⁹’ ¹⁰. In addition, preclinical studies have shown that adoptive chimeric antigen receptor (CAR)-NK cell transfer is comparatively safe, has a low risk of graft- versus-host disease (GVHD) or cytokine release syndrome (CRS), and is highly feasible for “off-the-shelf’ manufacturing ^{111 12}. Collectively, these promising features have led to a rapid surge of interest and efforts aimed at harnessing NK cells as a safe and effective treatment approach for immunotherapy of solid tumors ¹³. As of November 2023, there are more than 2,000 clinical trials involving NK cells and 110 with CAR-NK therapy (ClinicalTrials.gov). Recent clinical trials have demonstrated favorable outcomes in the treatment of hematological malignancies ¹⁴, however, current forms of NK cell-based immunotherapy candidates face a number of obstacles, for example, the paucity, lower proliferative capacity, and particularly decreased effectiveness, persistence or tumor infiltration (Cozar, et al., Cancer Discov 11, 34-44 (2021); and Ge, et al., Immunopharmacol Immunotoxicol 42, 187-198 (2020)). In the context of solid cancers, the efficacy to date of CAR NK cell therapy has been variable due to tumor- evolved mechanisms that inhibit local immune cell activity. NK cells encode the same collection of -20,000 protein coding genes in their genome, many of which might play important roles in regulating or limiting the anti-tumor function of NK cells.

Various approaches have been actively utilized to address these hurdles, including ex vivo activation, expansion, and genetic modifications such as cytokine engineering and gene perturbation (Chu, J. et al. Journal of Translational Medicine 20, 240 (2022)) ¹⁹.

While a small number of genes, such as the CISH ²⁰, have been implicated as important regulators of NK cells’ anti-tumor capabilities, the understanding of the genomic landscape governing CAR-NK function remains largely unexplored. Certain studies have screened how genes in cancer cells mediate their susceptibility to NK-mediated cytotoxicity ^{21, 22, 23}. Notably, a recent preprint reported a CRISPR knockout (KO) screen on primary mouse NK cells²⁴. While using gene knockout for NK function enhancement is promising, it is dependent on CRISPR- mediated gene editing, which involves more complex manufacturing in cell therapy²⁵.

There is an urgent need for genome-scale gain-of-function screening in human NK cells, to guide the identification of new genes that can be manipulated to enhance NK function, such as activation, proliferation, repression of inhibitory signals or exhaustion, persistence, or tumor infiltration.

Therefore, it is an object of the invention to provide systems and methods of genomescale screening for genes that, when over-expressed, enhance anti-tumor activity of NK cells in vivo.

It is another object of the invention to provide systems for therapeutic cell engineering of NK cells.

It is another object of the invention to provide gene-edited human NK cells having enhanced tumor penetration and anti-tumor activity as compared to non-engineered NK cells.

SUMMARY OF THE INVENTION

Compositions and methods for highly efficient screening of genetically engineered NK cells are provided. The disclosed compositions and methods are especially applicable to development of enhanced chimeric antigen receptor engineered NK cell therapy (CAR-NK).

Genetically modified Natural Killer (NK) cells are described. Typically, the NK cells are modified to up-regulate expression of at least one gene selected from the group SGSM2, OR7A10, APLN, PDP1, GABBR1, PRR14L, TIAM1, KRT82, PLA2G1B, REM2, HIST1H2BN, CYB5B, LRRC23, NXPE3, MEGF11, FKBP5, PPFIA2, LRRC23, PEAR1, REM2, TIAM2, HPRT1, MMACHC, and ZBTB20, as compared to a non-genetically modified control NK cell. In some forms, NK cells are modified to up-regulate expression of at least one cytokine selected from Interleukin- 15 (IL-15) and Interleukin-21 (IL-21). Generally, the modification enhances an anti-cancer efficacy of the NK cell as compared to a non-genetically modified control NK cell. Therefore, in certain forms, NK cells are modified to up-regulate expression of at least one gene selected from the group including SGSM2, OR7A10, APLN, PDP1, GABBR1, PRR14L, TIAM1, KRT82, PLA2G1B, REM2, HIST1H2BN, CYB5B, LRRC23, NXPE3, MEGF11, FKBP5, PPFIA2, LRRC23, PEAR1, REM2, TIAM2, HPRT1, MMACHC, and ZBTB20, as compared to a non- genetically modified control NK cell, and/or to overexpress Interleukin- 15 (IL- 15), or Interleukin-21 (IL-21), or both IL-15 and IL-21. In some forms, the modification causes increased or enhanced expression, activation, presentation, and/or function of one or more protein(s) encoded by the gene(s).

In particular forms, the modification causes increased or enhanced expression of one or more of the genes SGSM2, OR7A10, APLN, PDP1 and CYB5B and/or the full-length protein(s) encoded by the gene(s) SGSM2, OR7A10, APLN, PDP1 and CYB5B, as compared to a non- genetically modified NK cell. Therefore, in some forms, the modification includes recombinant expression of the SGSM2 gene, and/or increased or enhanced expression of the full-length protein encoded by the SGSM2 gene. In other forms, the modification includes recombinant expression of the OR7A10 gene, and/or increased or enhanced the full-length protein encoded by the OR7A10 gene. In other forms, the modification includes recombinant expression of the APLN gene and/or increased or enhanced expression of the APLN gene and/or the full-length protein encoded by the APLN gene. In other forms, the modification includes recombinant expression of the PDP1 gene and/or increased or enhanced expression of the PDP1 gene and/or the full-length protein encoded by the PDP1 gene. In other forms, the modification includes recombinant expression of the CYB5B gene and/or increased or enhanced expression of the CYB5B gene and/or the full-length protein encoded by the CYB5B gene.

In some forms, the genetically modified NK cell expresses 11-15 and 11-21 in addition to enhanced or up-regulated expression of the SGSM2 gene, and/or the OR7A10 gene, and/or the APLN gene, and/or the PDP1 gene, and/or the CYB5B gene. In other forms, the genetically modified NK cell further includes at least one additional genetic modification. For example, in some forms, the NK cell expresses or encodes a Chimeric Antigen Receptor (CAR). A typical CAR targets a cancer antigen. In an exemplary form, the cancer antigen is a neoantigen derived from a subject. Exemplary cancer antigens include ENPP3, 4- IBB, 5T4, adenocarcinoma antigen, alpha fetoprotein, BAFF, B lymphoma cell, C242 antigen, CA 125, carbonic anhydrase 9 (CA IX), C-MET, CCR4, CD 152, CD 19, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44 v6, CD51, CD52, CD56, CD74, CD80, CEA, CNTO888, CTLA 4, DR5, EGFR, EpCAM, CD3, FAP, fibronectin extra domain B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HER2/neu, HGF, human scatter factor receptor kinase, IGF 1 receptor, IGF I, IgGl, LI CAM, IL 13, IL 6, insulin-like growth factor I receptor, integrin a5pi, integrin avP3, MORAb 009, MS4A1, MUC1, mucin CanAg, N glycolylneuraminic acid, NPC 1C, PDGF R a, PDL192, phosphatidylserine, prostatic carcinoma cells, RANKL, RON, ROR1, SCH 900105, SDC1, SLAMF7, TAG 72, tenascin C, TGF beta 2, TGF , TRAIL Rl, TRAIL R2, tumor antigen CTAA16.88, VEGF A, VEGFR 1, VEGFR2, and vimentin.

In some forms, the NK is derived from a subject diagnosed as having, or who is identified as being at increased risk of having a disease or disorder. For example in some forms, the subject is diagnosed as having, or is identified as being at increased risk of having cancer. In other forms, the NK is derived from a healthy subject prior to the genetic modification. In some forms, the modification causes increased or enhanced expression, activation, presentation, and/or function of one or more cytokine(s), as compared to a non-genetically modified control NK cell. For example, in some forms, at least one of IL-15 and IL-21 is recombinantly expressed in the cell; in certain forms, IL- 15 is recombinantly expressed in the cell; in other forms, IL-21 is recombinantly expressed in the cell; in further forms, IL- 15 and IL-21 are both recombinantly expressed in the cell. Populations of genetically modified NK cells, derived by expanding any of the described genetically modified NK cells, are also provided. Typically, the population of genetically modified NK cells is homogenous, however heterogenous populations, including merged or mixtures of cells expanded from differently-modified NK cells are also described. A pharmaceutical composition including the described population(s) of NK cells, and a pharmaceutically acceptable buffer, carrier, diluent or excipient for administration in vivo are also described. In some forms, the pharmaceutical composition includes at least one cytokine, such as IL- 15, or IL-21, or both IL- 15 and IL-21.

Methods of treating a subject having a disease, disorder, or condition including administering to the subject an effective amount of a pharmaceutical composition including a population of genetically modified NK cells, modified to up-regulate expression of at least one gene selected from the group including SGSM2, OR7A10, APLN, PDP1, GABBR1, PRR14L, TIAM1, KRT82, PLA2G1B, REM2, HIST1H2BN, CYB5B, LRRC23, NXPE3, MEGF11, FKBP5, PPFIA2, LRRC23, PEAR1, REM2, TIAM2, HPRT1, MMACHC, or ZBTB20, as compared to a non-genetically modified control NK cell, and/or that are modified to up-regulate expression of at least one of IL- 15 and IL-21 are also described. In some forms, a method of treating a subject having a disease, disorder, or condition associated with an elevated expression or specific expression of an antigen, includes administering to the subject an effective amount of the population of genetically modified NK cells, modified to up-regulate expression of at least one gene selected from the group including SGSM2, OR7A10, APLN, PDP1, GABBR1, PRR14L, TIAM1, KRT82, PLA2G1B, REM2, HIST1H2BN, CYB5B, LRRC23, NXPE3, MEGF11, FKBP5, PPFIA2, LRRC23, PEAR1, REM2, TIAM2, HPRT1, MMACHC, or ZBTB20, as compared to a non-genetically modified control NK cell, and/or that are modified to up-regulate expression of at least one of IL- 15 and IL-21, wherein the NK cells further include a CAR that targets the antigen. In some forms, the genetically modified NK cells include increased or enhanced expression of one or more of the genes and/or the full-length protein(s) encoded by the gene(s) SGSM2, OR7A10, APLN, PDP1 and CYB5B as compared to a non-genetically modified control NK cell, whereby the modification enhances the anti-cancer efficacy of the NK cell as compared to a non-genetically modified control NK cell. In some forms, the NK cell is genetically modified to express or encode a Chimeric Antigen Receptor (CAR), such as a CAR that targets an antigen expressed by the cancer. Exemplary cancers that can be treated by the described methods include leukemia, vascular cancer such as multiple myeloma, adenocarcinomas and bone, bladder, brain, breast, cervical, ovarian, colorectal, esophageal, kidney, liver, lung, nasopharyngeal, pancreatic, prostate, skin, stomach, and uterine cancer. In certain forms, the cancer is breast cancer, lung cancer, colorectal cancer, ovarian cancer, or skin cancer. In an exemplary method, the NK cell is genetically modified to express at least one recombinant cytokine selected from the group including IL- 15, and IL-21. In some forms, the NK cells are derived from the subject prior to genetic modification. Therefore, in some forms, the methods include obtaining NK cells from the subject, and modifying the NK cells, prior to readministering the modified NK cells to the subject. In certain forms, the methods further include administering to the subject cytokine IL-15, or cytokine IL-21, or both cytokines IL-15 and IL- 21. In some forms, the administration includes injection of the composition of cells into or directly adjacent to a tumor, or into the blood stream, or into the brain or into a ventricle of the heart of the subject. The methods can also include administering to the subject one or more additional therapeutic agents and/or procedures. An exemplary additional therapeutic agent and/or procedure is selected from a chemotherapeutic agent, an antimicrobial agent, an immune checkpoint inhibitor, a PD-I inhibitor, a CTLA-4 inhibitor, radiation treatment and surgery.

Methods of performing gain of function (GOF) screening of a Natural Killer (NK) cell are also described. Typically, the methods include (i) transducing an NK cell with one or more CRISPRa single-guide RNA(s) (sgRNAs), (ii) causing the NK cell to be genetically modified by CRISPRa-mediated genome editing of a gene targeted by the sgRNA; and (iii) screening the NK cell for tumor cell killing. An exemplary sgRNA includes (i) a guide sequence; and (ii)a tracrRNA, including a nucleic acid sequence selected from a library. For example, in some forms, the library includes all or part of a human genomic reference sequence library. Generally, the sgRNA is included within a vector, such as a lentiviral vector. In some forms, the vector further includes an expression cassette for the sgRNA. In some forms, the expression cassette further includes a nucleic acid construct configured to express or encode a chimeric antigen receptor (CAR). In some methods, steps (i)-(iii) are carried out using a plurality of NK cells, and each of the plurality of NK cells is contacted by one or more sgRNAs including one or more sequences of a library of sequences. In some methods, the plurality of NK cells is collectively contacted by a multiplicity of sgRNAs, whereby an sgRNA of the multiplicity of sgRNAs includes a single sequence from the library, and whereby an NK cell of the plurality of NK cells contacted by an sgRNA over-expresses a single gene, relative to a control NK cell that is not contacted by the sgRNA. In some forms, the step of screening the NK cell for tumor cell killing is carried out in vitro. In other forms, the step of screening the NK cell for tumor cell killing is carried out in vivo. For example, in some forms the in vivo screening is carried out using a tumor-bearing animal model, for example, whereby the screening includes selecting genetically modified NK cells from animals with enhanced survival/reduced tumor burden as compared to control animals that did not receive the same genetically modified NK cells. In some forms, the methods further include characterizing the mutant NK cell(s) by single cell transcriptome analysis, and/or by sequence analysis, to identify mutated genes. In some forms, the methods include repeating steps (i)-(iii) using a selected pool of sgRNAs for one or more additional rounds. Genetically modified NK cell created according to the described methods are also provided.

A pharmaceutical composition including (i) a population of genetically modified NK cells derived by expanding a genetically modified NK cell modified to up-regulate expression of at least one gene selected from the group including SGSM2, OR7A10, APLN, PDP1, GABBR1, PRR14L, TIAM1, KRT82, PLA2G1B, REM2, HIST1H2BN, CYB5B, LRRC23, NXPE3, MEGF11, FKBP5, PPFIA2, LRRC23, PEAR1, REM2, TIAM2, HPRT1, MMACHC, and ZBTB20, as compared to a non-genetically modified control NK cell, and/or modified to up-regulate expression of at least one of IL- 15 and IL-21; and (ii) a pharmaceutically acceptable excipient for administration in vivo is also provided.

A genetically modified Natural Killer (NK) cell including a mutation that causes up- regulated or enhanced expression of one or more genes selected from the group including SGSM2, OR7A10, APLN, PDP1, and CYB5B and/or the functional protein encoded by one or more genes selected from the group including SGSM2, OR7A10, APLN, PDP1 and CYB5B in the cell as compared to a non-genetically modified NK cell, wherein the mutation enhances the anti- cancer efficacy of the genetically modified NK cell as compared to a non-genetically modified NK cell, and wherein the genetically modified NK cell expresses or encodes a Chimeric Antigen Receptor (CAR) that targets a cancer antigen is also provided. In some forms, the cancer antigen is selected from ENPP3, 4-1BB, 5T4, adenocarcinoma antigen, alpha fetoprotein, BAFF, B lymphoma cell, C242 antigen, CA 125, carbonic anhydrase 9 (CA IX), C-MET, CCR4, CD 152, CD 19, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44 v6, CD51, CD52, CD56, CD74, CD80, CEA, CNTO888, CTLA 4, DR5, EGFR, EpCAM, CD3, FAP, fibronectin extra domain B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HER2/neu, HGF, human scatter factor receptor kinase, IGF 1 receptor, IGF I, IgGl, LI CAM, IL 13, IL 6, insulin-like growth factor I receptor, integrin a5pi, integrin avP3, MORAb 009, MS4A1, MUC1, mucin CanAg, N glycolylneuraminic acid, NPC 1C, PDGF R a, PDL192, phosphatidylserine, prostatic carcinoma cells, RANKL, RON, ROR1, SCH 900105, SDC1, SLAMF7, TAG 72, tenascin C, TGF beta 2, TGF , TRAIL Rl, TRAIL R2, tumor antigen CTAA16.88, VEGF A, VEGFR 1, VEGFR2, and vimentin. In some forms, the genetically modified NK has increased tumor penetration and/or increased anti-tumor cytotoxicity as compared to a non-genetically modified NK cell.

A genetically modified Natural Killer (NK) cell, wherein expression of the SGSM2 gene is up-regulated and/or enhanced in the cell, as compared to a non-genetically modified control NK cell, whereby the modification increases or enhances expression, activation, presentation, and/or function of one or more protein(s) encoded by the SGSM2 gene(s) and enhances the anticancer efficacy of the NK cell as compared to a non-genetically modified control NK cell, and whereby the modified NK cell expresses or encodes a Chimeric Antigen Receptor (CAR) that targets a cancer antigen.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the disclosed method and compositions and together with the description, explain the principles of the disclosed method and compositions.

Figures 1A-1O illustrate the genome-scale in vivo CRIPSR activation screen that identifies SGSM2 and OR7A10 that enhance CAR-NK anti-tumor efficacy. FIG. 1A is a schematic of the genome-scale in vivo CRIPSR activation screen for CAR-NK anti-tumor efficacy in HT29 tumor model. FIG. IB is a graph of tumor growth curve of HT29 tumorbearing mice following different treatments, including: PBS control (♦; n = 4 mice), treated with CAR-NK92 (•; n = 4 mice) or Library-CAR-NK92 (-A-; n = 12 mice) cells. Arrow indicates the time of adoptive transfer of CAR-NK92 cells. Data are shown as mean ± SEM. Two-way ANOVA was used to assess statistical significance with multiple testing comparisons; The statistical significance levels are indicated in the plots. FIG. 1C is a scatter plot graph of screen analysis results, presented by gene enrichment score and significance (-loglO adjusted p value) (SAMBA analysis). Genes are colored by the dot density, and a dashed line is shown for significance threshold (adjusted p < 0.01); a rug plot graph presented above the x axis to show the distribution of NTC sgRNA scores (grayscale) with the 90^th percentile score (10% FDR) indicated by the arrow. FIGS. 1D-1G is a set of graphs showing RT-qPCR for overexpression of each of SGSM2 (FIG. ID), OR7A10 (FIG. IE), APLN (FIG. IF), and PDP1 (FIG. 1G) genes, respectively, after lentiviral transduction (n = 4 biological replicates for SGSM2, OR7A10, and PDP1; n = 3 biological replicates for APLN). FIGS. 1H-1M are histograms showing co-culture assays of each of SGSM2 (A); OR7A10 (▼ ); APLN( ); PDP1(»); CFB5B-OE(«) CAR-NK92 cells and Vector! •)/NTC('O) controls, with HT29-GL (HT29 with GFP and luciferase reporters) cancer cells, with two E:T ratios (E:T=1:1 and E:T=l:10), and at each of two time points (12 and 24 hrs), respectively. Individual replicate data points were shown (n = 3 biological replicates). FIG. 1J is a histogram showing co-culture assay of each of SGSM2 ( A ); OR7A10 ( );

APLN( ); PDP1(»); CFB5B-OE(«) CAR-NK92 cells and Vector( •)/NTC(O) controls, with SKOV3-GL (SKOV3 with GFP and luciferase reporters) cancer cells, respectively, with E:T=1:1 at 8 hrs. Individual replicate data points were shown (n = 3 biological replicates). FIGS. 1K-1L are histograms showing co-culture assays of SGSM2 ( A ); OR7A10 ( ); APLN( ); PDP1(»); CYB5B-OE(u) CAR-NK92 cells and Vector(«)/NTC(O) controls, with MCF7-PL (MCF7 with puromycin and luciferase reporters) cancer cells, respectively, with two E:T ratios (E:T=1:1 and E:T=l:10), and at two time points (12 and 24 hrs). Individual replicate data points were shown (n = 3 biological replicates). FIG. IM is a histogram showing a co-culture assay of SGSM2 ( A ); OR7A10 (▼ ); APLN( ); PDP1(»); CFB5B-OE(«) CAR-NK92 cells and Vector(«)/NTC(O) controls, with A375-PL (A375 with puromycin and luciferase reporters) cancer cells, respectively, with E:T=1:1 at 8 hrs. Individual replicate data points were shown (n = 3 biological replicates). FIG. IN is a histogram showing a co-culture assay of each of SGSM2 ( A ); OR7A10 (▼ ); APLN( ); PDP1(»); CFB5B-OE(«) CAR-NK92 cells and Vector(«)/NTC(O) controls, with H1299-PL (H1299 with puromycin and luciferase reporters) cancer cells, respectively, with E:T=l:10 at two time points (8 and 24 hrs). Individual replicate data points were shown (n = 3 biological replicates). FIG. IO is a histogram showing a co-culture assay of each of SGSM2 (A ); OR7A10 (▼ ); APLN( ); PDP1(»); CFB5B-OE(«) CAR-NK92 cells and Vector(#)/NTC(O) controls, with MDA-MB-231-PL (MDA-MB-231 with puromycin and luciferase reporters) cancer cells, respectively, with E:T=1:1 at two time points (8 and 24 hrs). Individual replicate data points were shown (n = 3 biological replicates). Figures 2A-2F show overexpressing SGSM2 or OR7A10 in CAR-NK92 cells enhances in vivo anti-tumor efficacy and tumor infiltration. FIG.2A is a schematic of in vivo tumor cytotoxicity validation of SGSM2/OR7A10-OE a-HER2-CAR-NK92-hIL2 cells. FIG.2B is a graph showing tumor growth curve of HT29 tumor-bearing mice following different treatments: PBS control n = 5 mice), adoptive transfer of sgNTC n = 6 mice), sgSGSM2 (“A”; n = 6 mice) or sgOR7A10 n = 6 mice) a-HER2-CAR-NK92-hIL2 cells. Arrows indicate the time of adoptive transfer of CAR-NK92 cells, show functional genetic screens in four in vivo tumor models identifies candidate genes that enhance NK cell tumor infiltration. FIGS.2C-2F is a set of spider curve graphs of the tumor growth curve of HT29 tumor-bearing mice depicted in FIG.2B, for the tumor growth in individual mice, plotted by group. In all bar blots, data are shown as mean ± SEM. The statistical significance levels are indicated in the plots by Two-way ANOVA (b and e) or unpaired t test (f). ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Figure 3A is a schematic of in vivo tumor cytotoxicity validation of SGSM2/OR7A10- OE a-HER2-CAR-NK92-hIL2 cells. Figure 3B is a graph of the tumor growth curve of HT29 tumor-bearing mice following different treatments: PBS control n = 5 mice), adoptive transfer mice), SGSM2-OE (“A”; n = 8 mice) mice) a-HER2-CAR-NK92-hIL2 cells with mCherry or GFP. Arrows indicate the time of adoptive transfer of CAR-NK92 cells. Figures 3C-3E are bar graphs depicting quantification of tumor infiltrating CAR-NK cells in mice treated with NTC, SGSM2-OE, or OR7A10-OE a- HER2-CAR-NK92 cells. Representative bar plots are presented for percent CD56+ cell (FIG.3C),, GFP+ cells (FIG.3D), and mCherry+ cells (FIG.3E); comparison between groups. Experiments were performed with n = 8 tumors per group. In all bar blots, data are shown as mean ± SEM. The statistical significance levels are indicated in the plots by Two-way ANOVA (b and e) or unpaired t test (f). ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Figure 4A is a schematic of NTC, SGSM2-OE, or OR7A10-OE a-HER2-CAR-NK92- GFP-hIL2 co-cultured with HT29 cells and following bulk RNA sequencing. Figures 4B-4C are a set of bar graphs, showing RT-qPCR of SGSM2 in SGSM2-OE (FIG.4B) and OR7A10 in OR7A10-OE a-HER2-CAR-NK92-GFP-hIL2 cells (FIG.4C), respectively, (n = 3 biological replicates). Data are shown as mean ± SEM. The statistical significance levels are indicated in the plots by unpaired t test.

Figures 5A-5L illustrate SGSM2 or OR7A10 overexpression enhances proliferation, activation and effector function of CAR-NK cells. FIGS.5A-5B are a set of graphs depicting the flow analysis of proliferation of Vector/NTC/SGSM2-OE/OR7A10-OE a-HER2-CAR-NK92 - *cells using Ki-67 (FIG. 5A) and cell Trace dye (FIG. 5B) staining Quantifications (n = 3 biological replicates for Ki-67 and n=4 biological replicates for cell trace dye) (right). FIG.5C is a graph depicting the Flow analysis of degranulation (CD107a) of Vector/NTC/SGSM2- OE/OR7A10-OE a-HER2-CAR-NK92 cells upon MCF7-HER2-PL (MCF7 with HER2 overexpression, puromycin, and luciferase reporters) cancer cell stimulation at E:T=1:2. (left) Representative flow plots, (right) Quantifications, (n = 3 biological replicates). FIGS.5D-5G are a set of graphs depicting Flow analysis of effector cytokine production of Vector/NTC/SGSM2- OE/OR7A10-OE a-HER2-CAR-NK92 cells upon HT29 cancer cell stimulation with E:T=1:1 at 24 hrs. IFNg (FIG. 5D), TNF-a (FIG. 5E), GZMB (FIG. 5F), and Perforin (FIG. 5G). Each with Representative flow plots and quantifications, (n = 4 biological replicates). FIG.5H-5K are a set of bar graphs depicting flow analysis of key activating marker expression of Vector/NTC/SGSM2-OE/OR7A10-OE a-HER2-CAR-NK92 cells upon HT29 cancer cell stimulation at E:T=1:1. 4-1BB (FIG. 5H), CD69 (FIG. 51), NKG2D (FIG. 5J), and CD25 (FIG. 5K with Representative quantifications, (n = 4 biological replicates). FIG.5L is a histogram graph depicting Quantifications of Flow analysis of ERK1/2 phosphorylation (pT202/pY204) of Vector/NTC/SGSM2-OE/OR7A10-OE a-HER2-CAR-NK92 cells upon HT29 cancer cell stimulation at E:T=1:1. (n = 4 biological replicates). In all bar blots, data are shown as mean ± SEM. The statistical significance levels are indicated in the plots by Two-way ANOVA (b) or unpaired t test (a, c, and d). ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Figures 6A-6E illustrate genome-scale in vivo identification of boosters that enhance CAR-NK anti-tumor efficacy. FIG.6A is a heatmap of the correlation between CRISPRa screen samples. Sample correlation is presented as the Spearman rho values. FIG.6B is a line plot graph of the empirical cumulative distribution functions (CDFs) of CRISPRa screen samples. CDF lines are shown for the individual and grouped samples in the left and right samples, respectively. FIGs.6C and 6D are a set of line graphs of the multidimensional scaling of data from CRISPRa screen samples for individual samples (FIG.6C) and sample groups (FIG.6D), respectively. The samples were clustered by k-means, and a convex hull was drawn around the clustered samples. FIG.6E is a Scatter plot graph comparing the gRNA counts between tumor samples and cell control samples of the in vivo CRISPRa screen data. The position of each point represents the sample-averaged log2 counts-per-million values. Point color depicts the point density on the plot, and labels are shown for the gene name of the gRNAs that have the greatest residual variation from the loess trend line in blue.

Figures 7A-7F illustrate overexpression of SGSM2 or OR7A10 in CAR-NK92 cells enhances in vitro anti-tumor efficacy. FIGS.7A-7D are bar graphs showing RT-qPCR for baseline mRNA level of high-ranked hit genes from the screen (n = 4 biological replicates for SGSM2, OR7A10, and PDPP. n = 3 biological replicates for APLN). Higher CT values indicate the candidates’ mRNA levels as lower than house-keeping gene GADPH in each of NK92 cells (FIGS.7A-7B) or primary human NK cells (FIGS.7C-7D) cells, respectively. FIG.7B is a set of graphs showing flow analysis of HER2 expression (FIG.7E) and MHC-I expression (FIG.7F), respectively, on each of A375, HT29, H1299, MCF7, SK0V3, and MDA-MB-231 cancer cells.

Figure 8 is a heatmap graph of sample correlation of transcriptomic data from unstimulated and stimulated NTC, SGSM2-0E, or OR7A10-OE CAR-NK92 cells, showing the correlation between bulk mRNA-sequencing data from different CAR-NK92 cells. Sample correlation is presented as the Spearman rho values.

Figures 9A-9D illustrate differential expression analysis of transcriptomic data from unstimulated and stimulated NTC, SGSM2-0E, or OR7A10-OE CAR-NK92 cells. FIGS.9A-9B are volcano plots of the differential expression (DE) analysis of SGSM2-0E (FIGS.9A-9B) or OR7A10-OE (FIGS.9C-9D) vs control (NTC) CAR-NK92 cells, showing each of unstimulated (FIGS.9A, 9C), and at 6 hours-post-stimulation (FIGS. 9B, 9D), respectively. DE genes (DEG) had q < 0.01 and an absolute log2 fold-change > 1.

Figure 10 illustrates that SGSM2 or OR7A10 overexpression enhances proliferation, activation and effector function of CAR-NK cells. FIG.10 is a graph of cell number quantification of Vector/NTC/SGSM2-OE/OR7A10-OE a-HER2-CAR-NK92 cells at day 5. (n = 4 biological replicates).

Figure 11 is a graph of quantification of flow analysis of degranulation (CD 107a) of Vector/NTC/SGSM2-OE/OR7A10-OE a-HER2-CAR-NK92 cells upon HT29 cancer cell stimulation at E:T=1:2; (left) (n = 3 biological replicates). For all bar blots, data are shown as mean ± SEM. The statistical significance levels are indicated in the plots by Two-way ANOVA (b) or unpaired t test (a), ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Figures 12A-12B are Flow cytometry graphs, showing scatter over signal for each of unstained, WT NK 92 and Positive selection for CAR+FITC-Flag, respectively (FIG.12A); and showing scatter over signal for control and ENPP3 expressing HEK-293T cells; and a graph quantitating these data (FIG.12B).

Figure 13A is a graph of Incucyte Caspase3/7 Dye based cell cytotoxicity results, showing object Count/well over time (h) for ENPP3-HEK293 killing by control (WT; “•”) or engineered ENPP3-CARNK92 cells (“•”); with cytokine IL15 (“▼”); or cytokine IL 21 (“♦”), or both cytokine IL15 and cytokine IL 21 (“•”) expression, respectively. FIGS.13B-13E are graphs of mCherry ACHN serial killing assay, showing enumeration of mCherry-ACHN tumor killing (object Count/well over time (h)) by control (WT; “•”) or engineered CARNK92 cells (“♦”); with cytokine IL15 (“A”); or cytokine mbIL15 (“■”), or both cytokine IL15 and cytokine IL 21 (“>”) expression, respectively, for each of rounds 1-4 (FIGS.13B-13E), respectively.

Figures 14A-14C are a set of graphs illustrating Estimation of each of IL-6 (FIG.14A) IFN-gamma (IFN-y) (FIG.14B), and TNF-alpha (TNF-a) (FIG.14C), in culture supernatant after ENPP3-HEK293T tumor killing by engineered CARNK92 cells.

Figure 15 is a graph of object Count/well over time (h) for IL15 and/or IL21 cytokines, illustrating the effect of IL 15 and/or IL21 alone on tumor cell proliferation.

Figures 16A-16C are graphs of tumor cell (ACHN at 1:1 E:T ratio) killing in the presence of NK92 cells with/without IL- 15 and/or IL-21, showing object Count/well over time (h) for IL- 15 and/or IL-21 cytokines, illustrating the effect of IL- 15 when administered at each of 2.5 ng/ml (“•”), 5 ng/ml (“■”), 10 ng/ml (“A”), or 15 ng/ml (“▼”), as compared with -ve control (“★ ”)(FIG.16A); IL-21 when administered at each of 5 ng/ml (“♦”), 10 ng/ml (“•”), 20 ng/ml (“■”), or 30 ng/ml (“A”), as compared with -ve control (“★ ”) (FIG.16B); or both IL- 15 and IL-21, when administered at each of 2.5 ng/ml IL-15/10 ng/ml IL-21 (“▼”); 5 ng/ml IL- 15/10 ng/ml IL-21 (“♦”); 10 ng/ml IL-15/10 ng/ml IL-21 (“•”) (FIG.16C), as compared with - ve control

Figure 17 is a Flowchart depicting the methodology used to generate a listing of top 10 candidate genes for gain of function.

DETAILED DESCRIPTION OF THE INVENTION

The disclosed method and compositions can be understood more readily by reference to the following detailed description of embodiments and the Examples included therein and to the Figures and their previous and following description.

Natural killer (NK) cells are an innate immune cell type that serves at the first level of defense against pathogens and cancer. NK cells have clinical potential, however, their effector function, persistence, and tumor infiltration naturally hinder the successful implementation of NK cell therapy against cancer. To unbiasedly reveal the functional genetic landscape underlying important NK cell characteristics against cancer, compositions and methods for Gain of Function (GOF) mapping of tumor infiltrating NK cells by in vitro CRISPRa-based genetic modification, in vivo screening for anti-tumor function, and single cell sequencing are described. Selected genes can be expressed co-cistronically in the same vector as a CAR when making gain-of- function recombinant CAR-NK cells, literally leaving the chemistry, manufacturing and control (CMC) process for generating these cells unchanged. Also provided are cytokine combinations and dosages that enhance NK function. I. Definitions

The term “transposon” or “transposable element” means a nucleic acid sequence, such as a chromosomal segment, that can undergo “transposition”, i.e., to change its position within a genome, especially a segment of DNA encoding one or more genes that can be translocated within a host cell, sometimes creating or reversing mutations and altering the cell's genetic identity and genome size. Exemplary transpositions include introduction of one or more components of plasmid DNA into chromosomal DNA in the absence of a complementary sequence in the host DNA.

The term “transposase” means an enzyme that binds to the end of a transposon and catalyzes its movement, e.g., into a genome at a specific point part, by a cut and paste mechanism or a replicative transposition mechanism.

“Introduce” in the context of genome modification refers to bringing in to contact. For example, to introduce a gene editing composition to a cell is to provide contact between the cell and the composition. The term encompasses penetration of the contacted composition to the interior of the cell by any suitable means, e.g., via transfection, electroporation, transduction, gene gun, nanoparticle delivery, etc.

The term “operably linked” or “operationally linked” refers to functional linkage between a regulatory sequence (e.g., promoter, enhancer, silencer, polyadenylation signal, 5’ or 3’ untranslated region (UTR), splice acceptor, IRES, triple helix, 2A self-cleaving peptides such as F2A, E2A, P2A and T2A) and a heterologous nucleic acid sequence permitting them to function in their intended manner e.g., resulting in expression of the latter). The term encompasses positioning of a regulatory region (sequence), a sequence to be transcribed, and/or a sequence to be translated in a nucleic acid so as to influence transcription or translation of such a sequence. The regulatory sequence can be positioned at any suitable distance from the sequence being regulated (e.g., 1 nucleotide - 10,000 nucleotides). For example, to bring a coding sequence under the control of a promoter, the translation initiation site of the translational reading frame of the polypeptide is typically positioned between one and about fifty nucleotides downstream of the promoter. A promoter can, however, be positioned as much as about 5,000 nucleotides upstream of the translation initiation site or about 2,000 nucleotides upstream of the transcription start site. A promoter typically includes at least a core (basal) promoter.

The term “complementary” refers to the degree of anti-parallel alignment between two nucleic acid strands. Complete complementarity requires that each nucleotide be across from its opposite. No complementarity requires that each nucleotide is not across from its opposite. The degree of complementarity determines the stability of the sequences to be together or anneal/hybridize. Furthermore various DNA repair functions as well as regulatory functions are based on base pair complementarity.

As used herein, a DNA or RNA nucleotide sequence as recited refers to a polynucleotide molecule including the indicated bases in a 5' to 3' direction, from left to right.

The term “CRISPR/Cas” or “clustered regularly interspaced short palindromic Repeats” or “CRISPR” refers to DNA loci containing short repetitions of base sequences followed by short segments of spacer DNA from previous exposures to a virus or plasmid. Bacteria and archaea have evolved adaptive immune defenses termed CRISPR/CRISPR associated (Cas) systems that use short RNA to direct degradation of foreign nucleic acids. In bacteria, the CRISPR system provides acquired immunity against invading foreign DNA via RNA-guided DNA cleavage. The “CRISPR/Cas” system or “CRISPR/Cas-mediated gene editing” refers to a CRISPR/Cas system that has been modified for genome editing/engineering. For a type II CRISPR/Cas system, it typically includes a “guide” RNA (gRNA) and a non-specific CRISPR- associated endonuclease (Cas9). “Guide RNA (gRNA)” is used interchangeably herein with “short guide RNA (sgRNA)” or “single guide RNA” (sgRNA). The sgRNA is a short synthetic RNA composed of a “scaffold” sequence necessary for Cas9-hinding and a user-defined, ~20 nucleotide “spacer” or “targeting” sequence which defines the genomic target to be modified. The genomic target of Cas9 can be modified by changing the targeting sequence present in the sgRNA.

The term “cleavage” refers to the breakage of covalent bonds, such as in the backbone of a nucleic acid molecule or the hydrolysis of peptide bonds. Cleavage can be initiated by a variety of methods, including, but not limited to, enzymatic or chemical hydrolysis of a phosphodiester bond. Both single- stranded cleavage and double-stranded cleavage are possible. Double-stranded cleavage can occur as a result of two distinct single-stranded cleavage events. DNA cleavage can result in the production of either blunt ends or staggered ends. In certain embodiments, fusion polypeptides can be used for targeting cleaved double stranded DNA.

The term “knockdown” refers to a decrease in gene expression of one or more genes. The term “knockout”, or “KO” refers to the ablation of gene expression of one or more genes.

“Endogenous” refers to any material from or produced inside an organism, cell, tissue or system. “Exogenous” refers to any material introduced from or produced outside an organism, cell, tissue or system.

The term “antigen” as used herein is defined as a molecule capable of being bound by an antibody or T-cell receptor. An antigen can additionally be capable of provoking an immune response. This immune response can involve either antibody production, or the activation of specific immunologically competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen.

Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which includes a nucleotide sequence or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an “antigen” as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full-length nucleotide sequence of a gene. It is readily apparent that the disclosed compositions and methods includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid. In the context of cancer, “antigen" refers to an antigenic substance that is produced in a tumor cell, which can therefore trigger an immune response in the host. These cancer antigens can be useful as markers for identifying a tumor cell, which could be a potential candidate/target during treatment or therapy. There are several types of cancer or tumor antigens. There are tumor specific antigens (TSA) which are present only on tumor cells and not on healthy cells, as well as tumor associated antigens (TAA) which are present in tumor cells and on some normal cells. In some forms, the chimeric antigen receptors are specific for tumor specific antigens. In some forms, the chimeric antigen receptors are specific for tumor associated antigens. In some forms, the chimeric antigen receptors are specific both for one or more tumor specific antigens and one or more tumor associated antigens.

“Bi-specific chimeric antigen receptor” refers to a CAR that includes two domains, wherein the first domain is specific for a first ligand/antigen/target, and wherein the second domain is specific for a second ligand/antigen/target. In some forms, the ligand is a B-cell specific protein, a tumor-specific ligand/antigen/target, a tumor associated ligand/antigen/target, or combinations thereof. A bispecific CAR is specific to two different antigens. A multi-specific or multivalent CAR is specific to more than one different antigen, e.g., 2, 3, 4, 5, or more. In some forms, a multi-specific or multivalent CAR targets and/or binds three or more different antigens.

“Encoding” or “encode” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

As used herein, the term “locus” is the specific physical location of a DNA sequence (e.g., of a gene) on a chromosome. It is understood that a locus of interest can not only qualify a nucleic acid sequence that exists in the main body of genetic material (i.e., in a chromosome) of a cell but also a portion of genetic material that can exist independently to said main body of genetic material such as plasmids, episomes, virus, transposons or in organelles such as mitochondria as non-limiting examples.

“Isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell. An “isolated nucleic acid” refers to a nucleic acid segment or fragment which has been separated from sequences which flank it in a naturally occurring state, e.g., a DNA fragment which has been removed from the sequences which are normally adjacent to the fragment, i.e., the sequences adjacent to the fragment in a genome in which it naturally occurs. The term also applies to nucleic acids which have been substantially purified from other components which naturally accompany the nucleic acid, e.g., RNA or DNA or proteins, which naturally accompany it in the cell. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (i.e., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes: a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence, complementary DNA (cDNA), linear or circular oligomers or polymers of natural and/or modified monomers or linkages, including deoxyribonucleosides, ribonucleosides, substituted and alpha-anomeric forms thereof, peptide nucleic acids (PNA), locked nucleic acids (LNA), phosphorothioate, methylphosphonate, and the like.

In the context of cells, the term “isolated” also refers to a cell altered or removed from its natural state. That is, the cell is in an environment different from that in which the cell naturally occurs, e.g., separated from its natural milieu such as by concentrating to a concentration at which it is not found in nature. “Isolated cell” is meant to include cells that are within samples that are substantially enriched for the cell of interest and/or in which the cell of interest is partially or substantially purified.

As used herein, “transformed,” “transduced,” and “transfected” encompass the introduction of a nucleic acid or other material into a cell by one of a number of techniques known in the art.

A “vector” is a composition of matter which includes an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Examples of vectors include but are not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” encompasses an autonomously replicating plasmid or a virus. The term is also construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus (AAV) vectors, retroviral vectors, and the like.

“Tumor burden” or “tumor load” as used herein, refers to the number of cancer cells, the size or mass of a tumor, or the total amount of tumor/cancer in a particular region of a subject. Methods of determining tumor burden for different contexts are known in the art, and the appropriate method can be selected by the skilled person. For example, in some forms tumor burden can be assessed using guidelines provided in the Response Evaluation Criteria in Solid Tumors (RECIST).

As used herein, “subject” includes, but is not limited to, animals, plants, parasites and any other organism or entity. The subject can be a vertebrate, more specifically a mammal (e.g., a human, horse, pig, rabbit, dog, sheep, goat, non-human primate, cow, cat, guinea pig or rodent), a fish, a bird or a reptile or an amphibian. The subject can be an invertebrate, more specifically an arthropod (e.g., insects and crustaceans). The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. A patient refers to a subject afflicted with a disease or disorder. The term “patient” includes human and veterinary subjects. In some forms, the subject can be any organism in which the disclosed method can be used to genetically modify the organism or cells of the organism.

The term “inhibit” or other forms of the word such as “inhibiting” or “inhibition” means to decrease, hinder or restrain a particular characteristic such as an activity, response, condition, disease, or other biological parameter. It is understood that this is typically in relation to some standard or expected value, i.e., it is relative, but that it is not always necessary for the standard or relative value to be referred to. “Inhibits” can also mean to hinder or restrain the synthesis, expression or function of a protein relative to a standard or control. Inhibition can include, but is not limited to, the complete ablation of the activity, response, condition, or disease. “Inhibits” can also include, for example, a 10% reduction in the activity, response, condition, disease, or other biological parameter as compared to the native or control level. Thus, the reduction can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,

28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,

54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,

80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, or any amount of reduction in between as compared to native or control levels. For example, “inhibits expression” means hindering, interfering with or restraining the expression and/or activity of the gene/gene product pathway relative to a standard or a control.

“Treatment” or “treating” means to administer a composition to a subject or a system with an undesired condition (e.g., cancer). The condition can include one or more symptoms of a disease, pathological state, or disorder. Treatment includes medical management of a subject with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological state, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological state, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological state, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological state, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological state, or disorder. It is understood that treatment, while intended to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder, need not actually result in the cure, amelioration, stabilization or prevention. The effects of treatment can be measured or assessed as described herein and as known in the art as is suitable for the disease, pathological condition, or disorder involved. Such measurements and assessments can be made in qualitative and/or quantitative terms. Thus, for example, characteristics or features of a disease, pathological condition, or disorder and/or symptoms of a disease, pathological condition, or disorder can be reduced to any effect or to any amount.

“Prevention” or “preventing” means to administer a composition to a subject or a system at risk for an undesired condition (e.g., cancer). The condition can include one or more symptoms of a disease, pathological state, or disorder. The condition can also be a predisposition to the disease, pathological state, or disorder. The effect of the administration of the composition to the subject can be the cessation of a particular symptom of a condition, a reduction or prevention of the symptoms of a condition, a reduction in the severity of the condition, the complete ablation of the condition, a stabilization or delay of the development or progression of a particular event or characteristic, or reduction of the chances that a particular event or characteristic will occur.

As used herein, the terms “effective amount” or “therapeutically effective amount” means a quantity sufficient to alleviate or ameliorate one or more symptoms of a disorder, disease, or condition being treated, or to otherwise provide a desired pharmacologic and/or physiological effect. Such amelioration only requires a reduction or alteration, not necessarily elimination. The precise quantity will vary according to a variety of factors such as subjectdependent variables (e.g., age, immune system health, weight, etc.), the disease or disorder being treated, as well as the route of administration, and the pharmacokinetics and pharmacodynamics of the agent being administered.

By “pharmaceutically acceptable” is meant a material that is not biologically or otherwise undesirable, i.e., the material can be administered to a subject along with the selected compound without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.

As used herein, the terms “variant” or “active variant” refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide, but retains essential properties (e.g., functional or biological activity). A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and/or deletions). A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polypeptide may be naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. Modifications and changes can be made in the structure of the polypeptides of the disclosure and still obtain a molecule having similar characteristics as the polypeptide e.g., a conservative amino acid substitution). For example, certain amino acids can be substituted for other amino acids in a sequence without appreciable loss of activity. Because it is the interactive capacity and nature of a polypeptide that defines that polypeptide’s biological or functional activity, certain amino acid sequence substitutions can be made in a polypeptide sequence and nevertheless obtain a polypeptide with like properties (e.g., functional or biological activity).

Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.

Use of the term “about” is intended to describe values either above or below the stated value in a range of approx. +/- 10%; in other forms the values can range in value either above or below the stated value in a range of approx. +/- 5%; in other forms the values can range in value either above or below the stated value in a range of approx. +/- 2%; in other forms the values can range in value either above or below the stated value in a range of approx. +/- 1%. The preceding ranges are intended to be made clear by context, and no further limitation is implied.

II. Compositions

Compositions of genetically modified NK cells that have enhanced anti-tumor activity are described.

Populations of genetically modified NK cells that have enhanced anti-tumor activity, and compositions thereof for the treatment of cancer in vivo are also provided.

A. Modified Natural Killer Cells

Genetically-modified NK cells are provided. In some forms, the genetically modified NK cells are for use in cell-based immunotherapy applications. Typically, the NK cells are modified by knock-down or knock-out of one or more of the genes or gene expression products of the NK cell.

Natural killer (NK) cells are lymphocytes with important effector functions in innate immunity (Vivier, et al., Nat Immunol 9, 503-510 (2008)) that do not require sensitization or specific antigens to initiate an effective immune response (Ben-Shmuel, et al., Frontiers in immunology 11, 275-275 (2020)). NK Cells are lymphocytes in the same family as T and B cells, coming from a common progenitor. However, as cells of the innate immune system, NK cells are classified as group I Innate Lymphocytes (ILCs) and respond quickly to a wide variety of pathological challenges. NK cells are best known for killing virally infected cells and detecting and controlling early signs of cancer. Effector populations of NK cells are able to lyse adjacent cells based on the expression of oncogenic transformation-associated surface markers (Shimasaki, et al., Nat Rev Drug Discov 19, 200-218 (2020)). In addition, regulatory NK populations can influence the functions of DCs (Peterson, et al., Frontiers in Immunology 11 (2021)., Fernandez, et al., Nature Medicine 5, 405-411 (1999).), monocytes, T cells, and B cells via cytokine production or through direct cell-cell contact in a receptor-ligand interaction- dependent manner (Abel, et al., Front Immunol 9, 1869 (2018), Zwirner, et al., Front Immunol 8, 25 (2017)). As well as protecting against disease, specialized NK cells are also found in the placenta and may play an important role in pregnancy. In humans, CD56, CD161, CD16, CD94 or CD 57 represent prototypic markers of NK cells.

NK cells were first noticed for their ability to kill tumor cells without any priming or prior activation (in contrast to cytotoxic T cells, which need priming by antigen presenting cells). They are named for this ‘natural’ killing. Additionally, NK cells secrete cytokines such as IFNy and TNFa, which act on other immune cells like Macrophage and Dendritic cells to enhance the immune response. While on patrol NK cells constantly contact other cells. Whether or not the NK cell kills these cells depends on a balance of signals from activating receptors and inhibitory receptors on the NK cell surface. Activating receptors recognize molecules that are expressed on the surface of cancer cells and infected cells, and ‘switch on’ the NK cell. Inhibitory receptors act as a check on NK cell killing. Most normal healthy cells express MHC I receptors which mark these cells as ‘self’. Inhibitory receptors on the surface of the NK cell recognize cognate MHC I, and this ‘switches off’ the NK cell, preventing it from killing. Cancer cells and infected cells often lose their MHC I, leaving them vulnerable to NK cell killing. Once the decision is made to kill, the NK cell releases cytotoxic granules containing perforin and granzymes, which leads to lysis of the target cell.

Compositions of genetically modified NK cells including a chimeric antigen receptor and overexpressing one or more genes are provided. The compositions are useful for NK-based cell therapy, such as Adoptive Cell Therapy (ACT). NK-based cell therapy is a promising emerging branch of cancer immunotherapies. NK cell therapy leverages the advantages of rapid cytotoxic anti-tumor immune responses, TCR-independence, enhanced safety, simplicity in generating off- the-shelf allogeneic products, reduced off-target immune responses (Zhang, et al., Immunology 121, 258-265 (2007)), and reduced production of molecules associated with cytokine release syndrome (CRS) relative to other cell types (Chou, et al., Bone Marrow Transplant 54, 780-784 (2019), Hunter, et al., J Natl Cancer Inst 111, 646-654 (2019), Xie, et al., EBioMedicine 59, 102975 (2020)).

In preferred forms, the NK cell to be modified is a human cell. In some forms, the cell is from an established cell line, or a primary cell. The term “primary cell,” refers to cells and cell cultures derived from a subject and allowed to grow in vitro for a limited number of passages, i.e. splitting, of the culture.

In some forms, the genetically modified cell is modified by a gain-of-function CRISPRa screen to enhance the transcription and/or expression of at least one or more of the genes or gene expression products of the NK cell according to the described methods. An exemplary genetically modified Natural Killer (NK) cell includes a modification that increases the expression of at least one gene selected from SGSM2, OR7A10, APLN, PDP1, GABBR1, PRR14L, TIAM1, KRT82, PLA2G1B, REM2, HIST1H2BN, CYB5B, LRRC23, NXPE3, MEGF11, FKBP5, PPFIA2, LRRC23, PEAR1, REM2, TIAM2, HPRT1, MMACHC, ZBTB20, IL- 15, and IL-21, as compared to a non-genetically modified control NK cell. Typically, the modification enhances the anti-cancer efficacy of the NK cell as compared to a non-genetically modified control NK cell. For example, in some forms, the modification causes increased or enhanced expression, activation, presentation, and/or function of one or more protein(s) encoded by the gene(s). Preferably, the modification causes increased or enhanced expression of one or more of the genes and/or the full-length protein(s) encoded by the gene(s) SGSM2, OR7A10, APLN, and PDP1.

Any of the gain of function NK cells can also be modified to expressor encode a Chimeric Antigen Receptor (CAR) that, e.g., targets a cancer antigen.

An exemplary genetically modified Natural Killer (NK) cell is modified to increase the expression of the SGSM2 gene, as compared to a non-genetically modified control NK cell, whereby the modification increases or enhances expression, activation, presentation, and/or function of one or more protein(s) encoded by the SGSM2gene(s) and enhances the anti-cancer efficacy of the NK cell as compared to a non-genetically modified control NK cell, and whereby the modified NK cell expresses or encodes a Chimeric Antigen Receptor (CAR) that targets a cancer antigen.

Populations of genetically modified NK cells, derived by expansion of a genetically modified NK cell, and compositions thereof are also provided.

1. Up-Regulated/ Over-expressed Genes

Genetically modified Natural Killer (NK) cells, including a gain of expression, or increased transcription, or increased translation of at least one gene as compared to control nonmodified (e.g., wild-type) NK cell, are provided. As described in the Examples, it has been established that increased expression of certain genes enhances the anti-tumor efficacy of NK cells. The disclosed compositions of modified Natural Killer (NK) cells that over-express one or more endogenous genes are effective for enhanced NK-based anti-cancer therapy.

Typically, the gene that is over-expressed in the genetically modified NK cell is an endogenous gene, for example, that forms one or more components of the wild-type NK cell genome, and/or transcriptome, and/or proteome.

In some forms, the modified cell over-expresses a gene that alters the phenotype of the NK cell, for example, to improve, enhance or increase one or more anti-tumor functions of the NK cell. Exemplary anti-tumor functions that can be modified include tumor penetration, tumor cytotoxicity and cell proliferation.

As used herein, over expression, increased expression and increased transcription refer to modifying gene expression to initiate, increase or otherwise up-regulate the gene or gene product expression or bioactivity in the modified NK cell relative to a corresponding control, such as a non-modified (e.g., wild-type) NK cell. Thus, for example, encompassed are genetic modifications including deletions, substitutions, insertions, and combinations thereof, to promoters and/or coding regions and/or other regulatory elements, of a gene that initiate, increase or otherwise up-regulate the gene or gene product. In some forms, the modification initiates, increases or enhances the amount of a functional protein in the modified NK cell as compared to a corresponding control, such as a non-modified (e.g., wild-type) NK cell. Typically, the modified NK cell has an increased amount of wildtype protein. In some forms, the modification enhances cellular anti-cancer functions by reducing the relative amount of a nonfunctional protein or protein with reduced bioactivity, for example, truncated or mutated protein, relative to functional wildtype protein.

As demonstrated in the examples, it has been established that initiation or increase in the expression of one or more of the genes SGSM2, OR7A10, APLN, PDP1, GABBR1, PRR14L, TIAM1, KRT82, PLA2G1B, REM2, HIST1H2BN, CYB5B, LRRC23, NXPE3, MEGF11, FKBP5, PPFIA2, LRRC23, PEAR1, REM2, TIAM2, HPRT1, MMACHC, ZBTB20, IL15, and IL21, may enhance tumor penetration and/or tumor killing/tumor reduction by the genetically modified NK cells, as compared to a corresponding control, such as a non-modified (e.g., wild-type) NK cell.

Therefore, in some forms, NK cells that are genetically modified to initiate or increase the expression of one or more genes selected from SGSM2, OR7A10, APLN, PDP1, GABBR1, PRR14L, TIAM1, KRT82, PLA2G1B, REM2, HIST1H2BN, CYB5B, LRRC23, NXPE3, MEGF11, FKBP5, PPFIA2, LRRC23, PEAR1, REM2, TIAM2, HPRT1, MMACHC, ZBTB20, IL15 and IL2I are provided. In preferred forms, the genetic modification(s) to the NK cells increase expression and/or bioactivity of the full-length protein(s) encoded by genes SGSM2, OR7A10, APLN, PDP1, GABBR1, PRR14L, TIAM1, KRT82, PLA2G1B, REM2, HIST1H2BN, CYB5B, LRRC23, NXPE3, MEGF11, FKBP5, PPFIA2, LRRC23, PEAR1, REM2, TIAM2, HPRT1, MMACHC, ZBTB20, IL15 and IL21. In preferred forms, the gene(s) that is over-expressed in the modified NK cell(s) is one or more selected from SGSM2, OR7A10, APLN, and PDP1. In most preferred forms, the gene(s) that is over-expressed in the modified NK cell(s) is SGSM2, and/or OR7A10. In some forms, the gene that is over-expressed in the modified NK cell(s) is SGSM2. In some forms, the gene that is over-expressed in the modified NK cell(s) is OR7A10. Therefore, genetically modified NK cells including over-expression and/or increased function of one or more of the genes SGSM2, OR7A10, APLN, PDP1, GABBR1, PRR14L, TIAM1, KRT82, PLA2G1B, REM2, HIST1H2BN, CYB5B, LRRC23, NXPE3, , MEGF11, FKBP5, PPFIA2, LRRC23, PEAR1, REM2, TIAM2, HPRT1, MMACHC, ZBTB20, IL15 and/or IL21 are provided. Preferably, expression and/or bioactivity of the protein(s) encoded by one or more of SGSM2, OR7A10, APLN, PDP1, GABBR1, PRR14L, TIAM1, KRT82, PLA2G1B, REM2, HIST1H2BN, CYB5B, LRRC23, NXPE3, MEGF11, FKBP5, PPFIA2, LRRC23, PEAR1, REM2, TIAM2, HPRT1, MMACHC, ZBTB20, IL15 and/or IL21 is increased.

In some forms, the genetic modification(s) to the NK cells increase expression and/or bioactivity of the full-length protein(s) encoded by SGSM2. In some forms, the genetic modification(s) to the NK cells increase expression and/or bioactivity of the full-length protein(s) encoded by OR7A10. In some forms, the genetic modification(s) to the NK cells increase expression and/or bioactivity of the full-length protein(s) encoded by APLN. In some forms, the genetic modification(s) to the NK cells increase expression and/or bioactivity of the full-length protein(s) encoded by PDPL In some forms, the genetic modification(s) to the NK cells increase expression and/or bioactivity of the full-length protein(s) encoded by GABBRI. In some forms, the genetic modification(s) to the NK cells increase expression and/or bioactivity of the full-length protein(s) encoded by PRR14L. In some forms, the genetic modification(s) to the NK cells increase expression and/or bioactivity of the full-length protein(s) encoded by TIAML In some forms, the genetic modification(s) to the NK cells increase expression and/or bioactivity of the full-length protein(s) encoded by KRT82. In some forms, the genetic modification(s) to the NK cells increase expression and/or bioactivity of the full-length protein(s) encoded by PLA2G1B. In some forms, the genetic modification(s) to the NK cells increase expression and/or bioactivity of the full-length protein(s) encoded by REM2. In some forms, the genetic modification(s) to the NK cells increase expression and/or bioactivity of the full-length protein(s) encoded by HIST1H2BN. In some forms, the genetic modification(s) to the NK cells increase expression and/or bioactivity of the full-length protein(s) encoded by CYB5B. In some forms, the genetic modification(s) to the NK cells increase expression and/or bioactivity of the full-length protein(s) encoded by LRRC23. In some forms, the genetic modification(s) to the NK cells increase expression and/or bioactivity of the full-length protein(s) encoded by NXPE3. In some forms, the genetic modification(s) to the NK cells increase expression and/or bioactivity of the full-length protein(s) encoded by CYB5B. In some forms, the genetic modification(s) to the NK cells increase expression and/or bioactivity of the full-length protein(s) encoded by MEGF11. In some forms, the genetic modification(s) to the NK cells increase expression and/or bioactivity of the full-length protein(s) encoded by FKBP5. In some forms, the genetic modification(s) to the NK cells increase expression and/or bioactivity of the full-length protein(s) encoded by PPFIA2. In some forms, the genetic modification(s) to the NK cells increase expression and/or bioactivity of the full-length protein(s) encoded by LRRC23. In some forms, the genetic modification(s) to the NK cells increase expression and/or bioactivity of the full-length protein(s) encoded by PEARL In some forms, the genetic modification(s) to the NK cells increase expression and/or bioactivity of the full-length protein(s) encoded by REM2. In some forms, the genetic modification(s) to the NK cells increase expression and/or bioactivity of the full-length protein(s) encoded by TIAM2. In some forms, the genetic modification(s) to the NK cells increase expression and/or bioactivity of the full-length protein(s) encoded by HPRT1. In some forms, the genetic modification(s) to the NK cells increase expression and/or bioactivity of the full-length protein(s) encoded by MMACHC. In some forms, the genetic modification(s) to the NK cells increase expression and/or bioactivity of the full-length protein(s) encoded by and/or ZBTB20. a. SGSM2

In some forms, the genetically modified NK cells over-express and/or have enhanced function of a sphingomyelin synthase 2 (SGSM2) gene or gene product.

As described in the Examples, it has been established that overexpression or increased function of the SGSM2 gene particularly enhances the anti-tumor efficacy of NK cells. Therefore, in some forms genetically modified NK cells overexpress or have increased function of one or more SGSM2 genes or its gene expression product, the sphingomyelin synthase 2 enzyme relative to a control NK cell.

The SGSM2 gene is also known as CDL or SMS2 (NCBI Gene ID accession number: 166929) located in humans on Chromosome 4q25, (see NCBI Reference No: NC_000004.12, positions 107824563 - 107915047. Sphingomyelin, a major component of cell and Golgi membranes, is made by the transfer of phosphocholine from phosphatidylcholine onto ceramide, with diacylglycerol as a side product. The protein encoded by this gene is an enzyme that catalyzes this reaction primarily at the cell membrane. The synthesis is reversible, and this enzyme can catalyze the reaction in either direction. The encoded protein is required for cell growth. Three transcript variants encoding the same protein have been found for this gene. There is evidence for more variants, but the full-length nature of their transcripts has not been determined.

It has been shown that overexpression of the human Sphingomyelin synthase protein in mouse causes increased non-HDL-sphingomyelin and non-HDL cholesterol levels, decreased HDL-sphingomyelin and HDL-cholesterol levels and increases the atherogenic potential of non- HDL lipoprotein particles. Therefore, in some forms, the genetically modified NK cells have one or more of increased non-HDL-sphingomyelin and non-HDL cholesterol levels, decreased HDL- sphingomyelin and HDL-cholesterol levels, as compared to a corresponding control, such as a non-modified (e.g., wild-type) NK cell. In some forms, the genetically modified NK cell includes recombinant expression of the SGSM2 gene, for example, whereby the recombinant expression causes increased or enhanced expression of the SGSM2 gene and/or the full-length protein encoded by the SGSM2 gene.

In some forms, the gene expression product of the human SGSM2 gene that is overexpressed is the 365 amino acid SGSM2 enzyme having UNIPROT accession ID No. Q8NHU3 and having an amino acid sequence of:

MDI IETAKLEEHLENQPSDPTNTYARPAEPVEEENKNGNGKPKSLSSGLRKGTKKYPDYIQIAM PTESRNKFPLEWWKTGIAFI YAVFNLVLTTVMITWHERVPPKELSPPLPDKFFDYIDRVKWAF SVSEINGI ILVGLWITQWLFLRYKS IVGRRFCFI IGTLYLYRCITMYVTTLPVPGMHFQCAPKL NGDSQAKVQRILRLISGGGLSITGSHILCGDFLFSGHTVTLTLTYLFIKEYSPRHFWWYHLICW LLSAAGI ICILVAHEHYTIDVI IAYYITTRLFWWYHSMANEKNLKVSSQTNFLSRAWWFPIFYF FEKNVQGS IPCCFSWPLSWPPGCFKSSCKKYSRVQKIGEDNEKST (SEQ ID NOG).

Therefore, in some forms, the genetically modified human NK cells over-express and/or up-regulate a gene that expresses a protein having the amino acid sequence of SEQ ID NOG, as compared to a wild-type NK cell. In some forms, the genetically modified human NK cells have increased or induced expression or bioactivity of a protein having the amino acid sequence of SEQ ID NOG. b. OR7A10

In some forms, the genetically modified NK cells over-express and/or have enhanced function of a Olfactory receptor 7A10 (0R7A10) gene or gene product.

As described in the Examples, it has been established that overexpression or increased function of the OR7A10 gene particularly enhances the anti-tumor efficacy of NK cells. Therefore, in some forms genetically modified NK cells overexpress or have increased function of one or more OR7A10 genes or its gene expression product, the Olfactory receptor 7A10 protein relative to a control NK cell.

The OR7A10 gene is also known as OST027, and olfactory receptor OR19-18 (NCBI Gene ID accession number: 390892) located in humans on Chromosome 19pl3.12, (see NCBI Reference No: NC_000019.10 (14840466-14848922, complement Olfactory receptors interact with odorant molecules in the nose, to initiate a neuronal response that triggers the perception of a smell. The olfactory receptor proteins are members of a large family of G-protein-coupled receptors (GPCR) arising from single coding-exon genes. Olfactory receptors share a 7- transmembrane domain structure with many neurotransmitter and hormone receptors and are responsible for the recognition and G protein-mediated transduction of odorant signals. The olfactory receptor gene family is the largest in the genome.

Therefore, in some forms, the genetically modified NK cells have an increased expression of an olfactory receptor, as compared to a corresponding control, such as a nonmodified (e.g., wild-type) NK cell. In some forms, the genetically modified NK cell includes recombinant expression of the OR7A10 gene, for example, whereby the recombinant expression causes increased or enhanced expression of the OR7A10 gene and/or the full-length protein encoded by the OR7A10 gene.

In some forms, the gene expression product of the human OR7A10 gene that is overexpressed is the 309 amino acid OR7A10 protein having UNIPROT accession ID No. 076100 and having an amino acid sequence of: MKSWNNTI ILEFLLLGI SEEPELQAFLFGLFLSMYLVTVLGNLLI I LATI SDSHLHTPMYFFLS NLSFVD ICFVSTTVPKMLVNIQTHNKVITYAGCI TQMCFFLLFVGLDNFLLTVMAYDRFVAICH PLHYMVIMNPQLCGLLVLASWIMSVLNSMLQSLMVLPLPFCTHMEIPHFFCEINQVVHLACSDT FLNDIVMYFAVALLGGGPLTGILYSYSKIVSS IRAI SSAQGKYKAFSTCASHLSWSLFYGTCL GVYLSSAATHNSHTGAAASVMYTWTPMLNPF IYSLRNKHIKGAMKTFFRGKQ (SEQ ID NO:4).

Therefore, in some forms, the genetically modified human NK cells over-express and/or up-regulate a gene that expresses a protein having the amino acid sequence of SEQ ID NO:4, as compared to a wild-type NK cell. In some forms, the genetically modified human NK cells have increased or induced expression or bioactivity of a protein having the amino acid sequence of SEQ ID NO:4.

2. Chimeric Antigen Receptor (CAR)-NK Cells

Typically, the genetically modified cell is an NK cell that expresses or includes a chimeric antigen receptor (CAR), i.e., a CAR-NK Cell. Genetically modified NK cells, such as genetically modified CAR NK cells that overexpress one or more genes, such as SGSM2, OR7A10, APLN, PDP1, GABBR1, PRR14L, TIAM1, KRT82, PLA2G1B, REM2, HIST1H2BN, CYB5B, LRRC23, NXPE3, MEGF11, FKBP5, PPFIA2, LRRC23, PEAR1, REM2, TIAM2, HPRT1, MMACHC, ZBTB20, IL15 and/or IL21 are described.

Immunotherapy using NK cells genetically engineered to express a chimeric antigen receptor (CAR) is rapidly emerging as a promising new treatment for hematological and non-hematological malignancies. The development of CAR-NK cells has increased the therapeutic potential of CAR-reprogramming by adding a reduced risk for alloreactivity and Graft- vs-Host Disease, potentially allowing for CAR-NK to be mass produced in a more cost- effective manner than CAR-T cells. NK cell-based immunotherapies require effective anti-tumor function, exhaustion, durable immune responses (persistence), and tumor infiltration. This requires rational engineering of substantially enhanced NK cells, particularly by modification of endogenous genes.

The term “Chimeric antigen receptor” or “CAR” refers to an engineered receptor that is expressed on a NK cell or any other effector cell type capable of cell-mediated cytotoxicity. In some forms a CAR includes an extracellular domain having an antigen binding domain that is specific for a ligand or receptor. In some forms a CAR also includes a transmembrane domain, and a costimulatory signaling domain. In some forms a CAR includes a hinge. In some forms, the antigen binding domain is specific for EGFRvlll. In some forms the costimulatory signaling domain is a 4-1BB signaling domain. In some forms a CAR further includes a CD3 zeta signaling domain. A CAR-NK cell is a NK cell engineered to express a CAR.

CARs are engineered receptors that possess both antigen-binding and cell-activating functions. Based on the location of the CAR in the membrane of the cell, the CAR can be divided into three main distinct domains, including an extracellular antigen-binding domain, followed by a space region, a transmembrane domain, and the intracellular signaling domain. The antigen-binding domain, most commonly derived from variable regions of immunoglobulins, typically contains VH and VL chains that are joined up by a linker to form the so-called “scFv.” The segment interposing between the antigen-binding domain (e.g., scFv) and the transmembrane domain is a “spacer domain.” The spacer domain can include the constant IgGl hinge-CH2-CH3 Fc domain. In some cases, the spacer domain and the transmembrane domain are derived from CD 8. The intracellular signaling domains mediating T cell activation can include a CD3^ co-receptor signaling domain derived from C-region of the TCR a and chains and one or more costimulatory domains.

In some forms, the antigen-binding domain of a CAR is derived from an antibody. The term antibody herein refers to natural or synthetic polypeptides that bind a target antigen. The term includes polyclonal and monoclonal antibodies, including intact antibodies and functional (e.g., antigen-binding) antibody fragments, including Fab fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rlgG) fragments, single chain antibody fragments, including single chain variable fragments (scFv), and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments.

The term encompasses genetically engineered and/or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di scFv, tandem tri scFv. The term also encompasses intact or full-length antibodies, including antibodies of any class or subclass, including IgG and sub classes thereof, IgM, IgE, IgA, and IgD. The antigen-binding domain of a CAR can contain complementary determining regions (CDR) of an antibody, variable regions of an antibody, and/or antigen binding fragments thereof. For example, the antigen-binding domain for a CD 19 CAR can be derived from a human monoclonal antibody to CD 19, such as those described in U.S. Patent 7,109,304, for use in accordance with the disclosed compositions and methods. In some forms, the antigen-binding domain can include an F(ab')2, Fab', Fab, Fv or scFv.

In some forms, the CAR includes one or more spacer domain(s) (also referred to as hinge domain) that is located between the extracellular antigen-binding domain and the transmembrane domain. A spacer domain is an amino acid segment that is generally found between two domains of a protein and may allow for flexibility of the protein and movement of one or both of the domains relative to one another. Any amino acid sequence that provides such flexibility and movement of the extracellular antigen-binding domain relative to the transmembrane domain can be used. The spacer domain can be a spacer or hinge domain of a naturally occurring protein. In some forms, the hinge domain is derived from CD8a, such as, a portion of the hinge domain of CD8a, e.g., a fragment containing at least 5 (e.g., 5, 10, 15, 20, 25, 30, 35, or 40) consecutive amino acids of the hinge domain of CD8a. Hinge domains of antibodies, such as an IgG, IgA, IgM, IgE, or IgD antibodies can also be used. In some forms, the hinge domain is the hinge domain that joins the constant CHI and CH2 domains of an antibody. Non-naturally occurring peptides may also be used as spacer domains. For example, the spacer domain can be a peptide linker, such as a (GxS)n linker, wherein x and n, independently can be an integer of 3 or more, including 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more.

In some forms, the CAR includes a transmembrane domain that can be directly or indirectly fused to the antigen-binding domain. The transmembrane domain may be derived either from a natural or a synthetic source. As used herein, a “transmembrane domain” refers to any protein structure that is thermodynamically stable in a cell membrane, preferably a eukaryotic cell membrane. In some forms, the transmembrane domain of the CAR includes a transmembrane domain of an alpha, beta or zeta chain of a T-cell receptor, CD8, CD4, CD28, CD 137, CD80, CD86, CD 152 or PD1, or a portion thereof. Transmembrane domains can also contain at least a portion of a synthetic, non-naturally occurring protein segment. In some forms, the transmembrane domain is a synthetic, non-naturally occurring alpha helix or beta sheet. In some forms, the protein segment is at least about 15 amino acids, e.g., at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acids. Examples of synthetic transmembrane domains are known in the art, for example in U.S. Patent No. 7,052,906 and PCT Publication No. WO 2000/032776. The intracellular signaling domain is responsible for activation of at least one of the normal effector functions of the immune effector cell expressing the CAR. The term effector function refers to a specialized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines. In some forms, an intracellular signaling domain includes the zeta chain of the T cell receptor or any of its homologs (e.g., CD3 eta, CD3 delta, CD3 gamma or CD3 epsilon), MB1 chain, B29, Fc RIII, Fc RI and combinations of signaling molecules such as CD3^ and CD28, 4-1BB, 0X40 and combination thereof, as well as other similar molecules and fragments. Intracellular signaling portions of other members of the families of activating proteins can be used, such as FcyRIII and FcaRI.

Many immune effector cells require co-stimulation, in addition to stimulation of an antigen-specific signal, to promote cell proliferation, differentiation and survival, as well as to activate effector functions of the cell. Therefore, in some forms, the CAR includes at least one co-stimulatory signaling domain. The term co-stimulatory signaling domain, refers to at least a portion of a protein that mediates signal transduction within a cell to induce an immune response such as an effector function. The co-stimulatory signaling domain can be a cytoplasmic signaling domain from a co-stimulatory protein, which transduces a signal and modulates responses mediated by immune cells, such as T cells, NK cells, macrophages, neutrophils, or eosinophils. In some forms, the co-stimulatory signaling domain is derived from a co-stimulatory molecule selected from CD27, CD28, CD137, 0X40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, EIGHT, NKG2C, B7-H3, ligands of CD83 and combinations thereof.

CARs can be used in order to generate immuno-responsive cells, such as NK cells, specific for selected targets, such as malignant cells, with a wide variety of receptor chimera constructs having been described (see U.S. Patent No. 11,207,350 and PCT Publications WO 2016123333 Al and WO 2016201300 Al). Alternative CAR constructs can be characterized as belonging to successive generations. First-generation CARs typically include a single-chain variable fragment of an antibody specific for an antigen, for example including a VL linked to a VH of a specific antibody, linked by a flexible linker, for example by a CD8a hinge domain and a CD 8a transmembrane domain, to the transmembrane and intracellular signaling domains of either CD3^ or FcRy (scFv-CD3^ or scFv- FcRy; see U.S. Patent No. 7,741,465; U.S. Patent No. 5,912,172; U.S. Patent No. 5,906,936). Second-generation CARs incorporate the intracellular domains of one or more costimulatory molecules, such as CD28, 0X40 (CD134), or 4-1BB (CD137) within the endo-domain (for example scFv-CD28/OX40/4-lBB-CD3^; see U.S. Patent Nos.8, 911,993; 8,916,381; 8,975,071; 9,101,584; 9,102,760; 9,102,761). Third-generation CARs include a combination of costimulatory endodomains, such a CD3^-chain, CD97, GDI la-CD18, CD2, ICOS, CD27, CD154, CDS, 0X40, 4-1BB, or CD28 signaling domains (for example scFv-CD28-4-lBB-CD3^ or scFv-CD28-OX40-CD3^; see U.S. Patent No.8,906,682; U.S. Patent No.8,399,645; U.S. Pat. No. 5,686,281; PCT Publication No. WO2014134165; PCT Publication No. WO2012079000). Alternatively, co-stimulation can be orchestrated by expressing CARs in antigen-specific T cells, chosen so as to be activated and expanded following engagement of their native aPTCR, for example by antigen on professional antigen- presenting cells, with attendant co-stimulation. Any of the first, second, or third generation CARs described above can be used in accordance with the disclosed compositions and methods.

In some forms, the gene of interest within a transposon encodes a CAR targeting one or more antigens specific for cancer, an inflammatory disease, a neuronal disorder, HIV/AIDS, diabetes, a cardiovascular disease, an infectious disease, an autoimmune disease, or combinations thereof. One of skill in the art, based on general knowledge in the field and/or routine experimentation would be able to determine the appropriate antigen to be targeted by a CAR for a specific disease, disorder or condition. a. Cancer-specific CARs

In some forms, the genetically-modified CAR-NK cells include a CAR component that targets a cancer antigen. Exemplary antigens specific for cancer that could be targeted by the CAR include, but are not limited to, ENPP3, 4-1BB, 5T4, adenocarcinoma antigen, alpha-fetoprotein, BAFF, B -lymphoma cell, C242 antigen, CA-125, carbonic anhydrase 9 (CA-IX), C-MET, CCR4, CD 152, CD 19, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44 v6, CD51, CD52, CD56, CD74, CD80, CEA, CNTO888, CTLA-4, DR5, EGFR, EpCAM, CD3, FAP, fibronectin extra domain-B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HER2/neu, HGF, human scatter factor receptor kinase, IGF-1 receptor, IGF-I, IgGl, Ll-CAM, IL-13, IL-6, insulin-like growth factor I receptor, integrin a5pi, integrin avP3, MGRAb-009, MS4A1, MUC1, mucin CanAg, N-glycolylneuraminic acid, NPC-1C, PDGF-R a, PDL192, phosphatidylserine, prostatic carcinoma cells, RANKL, RON, ROR1, SCH 900105, SDC1, SLAMF7, TAG-72, tenascin C, TGF beta 2, TGF-p, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2, vimentin, and combinations thereof.

In preferred forms, the CAR targets CD19, CD22, or both CD19 and CD22.

Exemplary antigens specific for an inflammatory disease that could be targeted by the CAR include, but are not limited to, AOC3 (VAP-1), CAM-3001, CCL11 (eotaxin-1), CD 125, CD 147 (basigin), CD 154 (CD40L), CD2, CD20, CD23 (IgE receptor), CD25 (a chain of IL-2 receptor), CD3, CD4, CD5, IFN-a, IFN-y, IgE, IgE Fc region, IL-1, IL-12, IL-23, IL-13, IL-17, IL-17A, IL-22, IL-4, IL-5, IL-5, IL-6, IL-6 receptor, integrin a4, integrin a4p7, Lama glama, LFA-1 (CD I la), MEDI-528, myostatin, OX-40, rhuMAb P7, scleroscin, SOST, TGF beta 1, TNF-a, VEGF-A, and combinations thereof.

Exemplary antigens specific for a neuronal disorder that could be targeted by the CAR include, but are not limited to, beta amyloid, MABT5102A, and combinations thereof. Exemplary antigens specific for diabetes that could be targeted by the CAR include, but are not limited to, L-I p, CD3, and combinations thereof.

Exemplary antigens specific for a cardiovascular disease that could be targeted by the CAR include, but are not limited to, C5, cardiac myosin, CD41 (integrin alpha-lib), fibrin II, beta chain, ITGB2 (CD 18), sphingosine- 1 -phosphate, and combinations thereof. Exemplary antigens specific for an infectious disease that could be targeted by the CAR include, but are not limited to, anthrax toxin, CCR5, CD4, clumping factor A, cytomegalovirus, cytomegalovirus glycoprotein B, endotoxin, Escherichia coli, hepatitis B surface antigen, hepatitis B virus, HIV-1, Hsp90, Influenza A hemagglutinin, lipoteichoic acid, Pseudomonas aeruginosa, rabies virus glycoprotein, respiratory syncytial virus, TNF-a, and combinations thereof.

In preferred forms, the CAR targets one or more antigens selected from an antigen listed in Table 1.

Table 1. Non-limiting examples of CAR targets

3. Cytokines IL-15 and/or IL-21

In some forms, the genetically modified cells (i.e., NK cells or CAR-NK cells) express one or more exogenous genes, i.e., to upregulate the expression of one or more gene products that are not typically expressed, or ae expressed at lower level by a control (i.e., wild-type NK cell). Preferably, the over-expression products enhance or increase the anti-tumor activity of the genetically modified cells relative to control cells (i.e., wild-type NK cell).

In some forms, the NK cells include one or more genetic modifications to express one or more cytokines to a greater extent than a control (i.e., wild- type NK cell). In some forms, the genetically modified NK cells include one or more genetic modifications to express interleukin 15 (IL-15) to a greater extent than a control (i.e., wild-type NK cell). In some forms, the genetically modified NK cells include one or more genetic modifications to express interleukin 21 (IL-21) to a greater extent than a control (i.e., wild-type NK cell). In some forms, the genetically modified NK cells include one or more genetic modifications to express both IL- 15 and IL-21 to a greater extent than a control (i.e., wild-type NK cell).

Exogenously expressed cytokines can be secreted as soluble molecules, or can be tethered or otherwise associated with the cell, such as via attachment to or within the cell membrane. Typically the amount and location of the expressed cytokine(s) in the genetically modified NK cell is sufficient to enhance the anti-tumor efficacy of the modified NK cell, relative to a genetically equivalent NK cell that lacks expression of the cytokine(s).

In some forms, the genetically modified NK cells that express IL- 15 and/or IL-21 include no additional genetic modifications as compared to a control (i.e., wild-type NK cell). Therefore, in some forms, the genetically modified NK cells are genetically identical to a control (i.e., wildtype NK cell), apart from the expression or up-regulation of IL- 15 and/or IL-21. In other forms, the genetically modified NK cells are engineered to express IL- 15 and/or IL-21 in addition to expression of a Chimeric Antigen Receptor (CAR). In further forms, the genetically modified NK cells are engineered to express IL- 15 and/or IL-21 in addition to the over-expression or upregulation of one or more genes selected from SGSM2, OR7A10, APLN, PDP1, GABBR1, PRR14L, TIAM1, KRT82, PLA2G1B, REM2, HIST1H2BN, CYB5B, LRRC23, NXPE3, MEGF11, FKBP5, PPFIA2, LRRC23, PEAR1, REM2, TIAM2, HPRT1, MMACHC, ZBTB20, IL15 and/or IL21. In further forms, the genetically modified NK cells are engineered to express IL- 15 and/or IL-21 in addition to the over-expression or up-regulation of one or more genes selected from SGSM2, OR7A10, APLN, PDP1, GABBR1, PRR14L, TIAM1, KRT82, PLA2G1B, REM2, HIST1H2BN, CYB5B, LRRC23, NXPE3, MEGF11, FKBP5, PPFIA2, LRRC23, PEAR1, REM2, TIAM2, HPRT1, MMACHC and ZBTB20, in addition to expression of a Chimeric Antigen Receptor (CAR), such as a CAR that targets a cancer antigen. In an exemplary from, genetically modified NK cells are engineered to express IL- 15 and IL-21 in addition to over-expression or up-regulation of one or more genes selected from SGSM2 or OR7A10, and expression of a CAR that targets a cancer antigen.

4. Other Genetic Modifications

In some forms, the described genetically modified cells (i.e., NK cells or CAR-NK cells) that over-express one or more genes relative to a control (i.e., wild-type NK cell) and/or express one or more cytokines, and that exhibit enhanced anti-tumor activity relative to a control (i.e., wild- type NK cell), include one or more additional genetic modifications.

Exemplary additional modifications include expression of one or more heterologous genes, deletion or reduced expression of one or more autologous genes, and altered gene expression profiles. In some forms, the cells include one or more genetic modifications to express a protein that is not normally expressed by a control (i.e., wild-type NK cell).

5. Sources of NK cells

In preferred forms, the NK cells to be genetically modified are obtained from a human subject. For example, in some forms, the cells are autologous cells, i.e., cells obtained from a subject prior to genetic modification and re-introduction to the same subject following modification. In other forms, the cells are heterologous cells, i.e., cells obtained from a different subject than the intended recipient. In some forms, the cells are frozen prior to or after genetic modification. Methods and compositions for freezing and thawing viable eukaryotic cells are known in the art. In some forms, the cells are autologous immune cells, such as T cells or progenitor cells/stem cells. a. Autologous human NK cells

In some forms, the NK cells are obtained from a human subject, prior to modification and reintroduction to the same human subject for use as cell therapy. In some forms, NK cells are obtained from a healthy subject. In other forms, cells are obtained from a subject identified as having or at risk of having a disease or disorder, such as cancer and/or an auto-immune disease. In some forms, prior to expansion and/or genetic modification, NK cells are obtained from a diseased or healthy subject. NK cells can be obtained from a number of samples, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In some forms, NK cells are obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as FICOLL™ separation. In one preferred form, NK cells from the circulating blood of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. The cells collected by apheresis can be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In some forms, the cells are washed with phosphate buffered saline (PBS). In some forms, the wash solution lacks calcium and can lack magnesium or can lack many if not all divalent cations. After washing, the cells can be resuspended in a variety of biocompatible buffers, such as, for example, Ca2+-free, Mg2+-free PBS, PLASMALYTE A, or other saline solution with or without buffer. Alternatively, the undesirable components of the apheresis sample are removed and the cells directly resuspended in culture media.

In some forms, the NK cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient or by counterflow centrifugal elutriation. In specific forms, a specific subpopulation of NK cells, such as CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, is further isolated by positive or negative selection techniques. For example, in some forms, T cells are isolated by incubation with anti-CD3/anti-CD28 (i.e., 3x28) - conjugated beads, such as DYNABEADS® M-450 CD3/CD28 T, for a time period sufficient for positive selection of the desired T cells. b. Introduction of Modifications

Any of the genetic modifications presented herein can be introduced into, and expressed by the cells, using any suitable means. Expression can be via a genomic or extrachromosomal transgene, by modifying an endogenous gene to have increased expression, or a combination thereof. Different genes (e.g., gain of function gene and CAR) can introduced in the same or different expression cassettes.

For example, an isolated nucleic acid encoding a gene(s) can be introduced as a recombinant DNA that is incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, lentivirus, adenovirus, or herpes virus), or into the genomic DNA of the cell. In some embodiments, an endogenous gene(s) is modified by a gene editing composition such as CRISPR/Cas to increase expression thereof. Such modification can be to the promoter and/or other endogenous expression control sequences to increase expression of the endogenous target gene. The gene editing compositions can also be delivered by a vector.

In some forms the vector is a viral vector. In general, viral vectors are genetically engineered viruses carrying modified viral DNA or RNA that has been rendered non-infectious, but still contains viral promoters and transgenes, thus allowing for translation of the transgene through a viral promoter. Because viral vectors are frequently lacking infectious sequences, they require helper viruses or packaging lines for large-scale transfection. Examples of viral vectors that can be used include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, pox virus vectors, enteric virus vectors, Venezuelan Equine Encephalitis virus vectors, Semliki Forest Virus vectors, Tobacco Mosaic Virus vectors, lentiviral vectors, arenavirus viral vectors, replication-deficient arenavirus viral vectors or replication-competent arenavirus viral vectors, bi-segmented or tri-segmented arenavirus, infectious arenavirus viral vectors, nucleic acids which include an arenavirus genomic segment wherein one open reading frame of the genomic segment is deleted or functionally inactivated (and replaced by a nucleic acid encoding a disclosed polypeptide or another therapeutic polypeptide as described herein), arenavirus such as lymphocytic choriomeningitidis virus (LCMV), e.g., clone 13 strain or MP strain, and arenavirus such as Junin virus e.g., Candid #1 strain, etc.

In some forms, the viral vector is an adenovirus vector, e.g., a recombinant adenovirus vector. A recombinant adenovirus vector can for instance be derived from a human adenovirus (HAdV, or AdHu), or a simian adenovirus such as chimpanzee or gorilla adenovirus (ChAd, AdCh, or SAdV) or rhesus adenovirus (rhAd). Preferably, an adenovirus vector is a recombinant human adenovirus vector, for instance a recombinant human adenovirus serotype 26, or any one of recombinant human adenovirus serotype 5, 4, 35, 7, 48, etc. In other forms, an adenovirus vector is a rhAd vector, e.g. rhAd51, rhAd52 or rhAd53. In some forms, a recombinant viral vector is prepared using methods known in the art in view of the present disclosure. For example, in view of the degeneracy of the genetic code, several nucleic acid sequences can be designed that encode the same polypeptide. In some forms, a polynucleotide encoding a disclosed polypeptide is codon-optimized to ensure proper expression in the host cell (e.g., bacterial or mammalian cells). Codon-optimization is a technology widely applied in the art, and methods for obtaining codon-optimized polynucleotides will be well known to those skilled in the art in view of the present disclosure.

In some forms, the vectors, e.g., a DNA plasmid or a viral vector (particularly an adenoviral vector), include any regulatory elements to establish conventional function(s) of the vector, including but not limited to replication and expression of a disclosed polypeptide encoded by the polynucleotide sequence of the vector.

In some forms, the vector is adeno-associated viral vector (AAV). AAV vector used in the compositions and methods can be a naturally occurring serotype of AAV or an artificial variant. In preferred forms, the serotype of the AAV vector is AAV6 or AAV9.

In some forms, the vector for inclusion in the gene editing compositions or for providing elements of the gene editing compositions e.g., transposon) is a viral vector such as a vesicular stomatitis (VSV) vector, a Bocavirus vector, such as a human bocavirus 1 (HBoVl) vector, a Herpes simplex virus (HSV) vector, or an adenovirus vector (AdV).

In some forms, the viral vector is a Herpes simplex virus (HSV) vector. Herpes simplex viruses (HSV) are large, enveloped dsDNA viruses characteristic of their lytic and latent nature of infection, which result in life-long latent infection of neurons and allows for long-term transgene expression. Deletion of HSV genes has generated expression vectors with low toxicity and an excellent packaging capacity of >30 kb foreign DNA. In some forms, the viral vector is a Vesicular stomatitis virus (VSV) vector. Vesicular stomatitis virus is a non-segmented, negative- stranded RNA virus that belongs to the family Rhabdoviridae, genus Vesiculovirus. VSV infects a broad range of animals, including cattle, horses, and swine. The genome of the virus codes for five major proteins, glycoprotein (G), matrix protein (M), nucleoprotein (N), large protein (L), and phosphoprotein (P). The G protein mediates both viral binding and host cell fusion with the endosomal membrane following endocytosis. The L and P proteins are subunits of the viral RNA-dependent RNA polymerase. The simple structure and rapid high-titer growth of VSV in mammalian and many other cells has made recombinant VSV a useful tool in the fields of cellular and molecular biology and virology.

In some forms, the viral vector is a human Bocavirus vector (HBoV). Exemplary human bocavirus vectors include human bocaviruses 1-4 (HBoVl -4), As well as Gorilla BoV. In other forms, the viral vector is an adenovirus vector. In some forms, the vector is a chimeric vector, such as a vector that is based on a chimeric virus formed from a combination of one or more components from two or more different viral vectors. An exemplary chimeric viral vector is a chimeric bocavirus/adeno-associated virus vector. Therefore, in some forms, the vector is a chimeric HBoVl/AAV2 vector (e.g., rAAV2/HBoVl chimeras).

In some forms, the vector is an AAV vector that can transduce diverse cell types with minimal cellular toxicity, leading to highly efficient and stable genomic modifications.

An exemplary method for introducing a disclosed gene into a cell includes introducing to the cell a viral vector including a transposon encoding the gene and a sequence that encodes one or more transposase enzymes configured to specifically mediate targeted integration of the transposon into the cellular genome.

Also disclosed are systems for introducing a disclosed polypeptide into a cell, where the system includes a viral vector including a transposon encoding the disclosed polypeptide and a sequence that encodes one or more transposase enzymes configured to specifically mediate targeted integration of the transposon into the cellular genome.

In some forms, the expression vector also includes one or more additional functional elements, for example, for genetic modification of the host cell by removal or silencing of one or more of the host genes.

In some forms, the vector provides combinations of simultaneous multiplexed knockout and knock-in genomic modifications in the host cell. In some forms, the compositions include an RNA-guided endonuclease and one or more AAV vectors containing a sequence (e.g., a crRNA) that encodes one or more crRNAs that collectively direct the endonuclease to one or more target genes. Optionally, at least one of the AAV vectors contains or further contains one or more HDR templates. The crRNA array can encode two or more crRNAs each of which direct the endonuclease to a different target gene. In some forms, the method can involve introducing two AAV vectors. In the foregoing method, the one or more HDR templates include (a) a sequence that encodes a reporter gene and/or a disclosed polypeptide, and (b) one or more sequences homologous to one or more target sites. The HDR template can further include a promoter and/or polyadenylation signal operationally linked to each reporter gene, disclosed polypeptide, or combination thereof.

In some forms, the RNA-guided endonuclease is capable of disruption of the target genes and/or the one or more HDR templates can mediate targeted integration of the reporter gene, the disclosed polypeptide, or combinations thereof at the target sites. A target site can be within the locus of the disrupted gene or at a locus different from the disrupted gene. B. Formulations of Genetically Modified NK Cells

In some forms, the NK Cells modified according to the described compositions and systems are formulated into pharmaceutical compositions for administration in vivo. For example, in some forms, pharmaceutical compositions include a plurality of genetically modified NK cells, such as genetically modified CAR NK cells that overexpress one or more genes, such as SGSM2, OR7A10, APLN, PDP1, GABBR1, PRR14L, TIAM1, KRT82, PLA2G1B, REM2, HIST1H2BN, CYB5B, LRRC23, NXPE3, MEGF11, FKBP5, PPFIA2, LRRC23, PEAR1, REM2, TIAM2, HPRT1, MMACHC, ZBTB20, IL15 and/or IL21, optionally combined with excipients and/or other reagents suitable for administration to a subject in the form of a “living drug” or therapeutic agent. In some forms, a plurality of NK cells (that may or may not express a chimeric antigen receptor) genetically modified according to the methods are combined with excipients and/or other reagents suitable for administration to a subject to provide a NK cell therapy for a subject in need thereof.

In some forms, compositions containing NK and/or CAR NK cells include between about 10⁴ and about 10⁹ cells per kg body weight of the intended recipient (i.e., between 7x 10⁵ and 7xlO¹⁰ cells for an average adult), preferably 10⁵ to 10⁷ cells/kg body weight, including all integer values within those ranges. Pharmaceutical compositions containing a genetically modified NK cell, or a population of genetically modified NK cells are provided. In some forms, the pharmaceutical compositions include one or more of a pharmaceutically acceptable buffer, carrier, diluent, or excipients. In some forms, the pharmaceutical compositions include a specific number or population of cells, for example, expanded by culturing and expanding an isolated genetically modified NK cell e.g., CAR NK cell that overexpresses one or more genes, such as SGSM2, OR7A10, APLN, PDP1, GABBR1, PRR14L, TIAM1, KRT82, PLA2G1B, REM2, HIST1H2BN, CYB5B, LRRC23, NXPE3, MEGF11, FKBP5, PPFIA2, LRRC23, PEAR1, REM2, TIAM2, HPRT1, MMACHC, ZBTB20, IL15 and/or IL21). e.g., a homogenous population. Therefore, in some forms, pharmaceutical compositions include a homogenous population of modified NK cells. In other forms, the pharmaceutical compositions include populations of cells that contain variable or different genetically modified NK cells, e.g., a heterogeneous population. In some forms, the pharmaceutical compositions include CAR-NK cells that are bispecific or multi-specific. In some forms, the NK cells have been isolated from a diseased or healthy subject prior to genetic modification. Introduction of gene editing compositions (e.g., lentiviral-sgRNA vectors) to the NK cell can be performed ex vivo.

1. Cytokines

In some forms, compositions of NK Cells modified according to the described compositions and systems are formulated into pharmaceutical compositions that include one or more additional cytokines. For example, in some forms, a pharmaceutical composition including a population of genetically modified CAR NK cells that overexpress one or more genes, such as SGSM2, OR7A10, APLN, PDP1, GABBR1, PRR14L, TIAM1, KRT82, PLA2G1B, REM2, HIST1H2BN, LRRC23, NXPE3, CYB5B, MEGF11, FKBP5, PPFIA2, LRRC23, PEAR1, REM2, TIAM2, HPRT1, MMACHC, ZBTB20, IL15 and/or /L2/ includes an effective amount of at least one cytokine selected from IL- 15 and IL-21. In some forms, a pharmaceutical composition including a population of genetically modified CAR NK cells that overexpress one or more genes, such as SGSM2, OR7A10, APLN, PDP1, GABBR1, PRR14L, TIAM1, KRT82, PLA2G1B, REM2, HIST1H2BN, CYB5B, LRRC23, NXPE3, MEGF11, FKBP5, PPFIA2, LRRC23, PEAR1, REM2, TIAM2, HPRT1, MMACHC, ZBTB20, IL15 and/or IL21 includes an effective amount of IL-15 and IL-21. In some forms, IL-15 and/or IL-21 is present in a composition including a population of genetically modified CAR NK cells in an amount between about 0.01 mg and 10,000 mg, inclusive.

Cytokines can be deployed using a delivery system. The delivery vehicles can be, for example, polymeric particles, inorganic particles, silica particles, liposomes, micelles, multilamellar vesicles, etc. Delivery vehicles can be microparticles or nanoparticles. Nanoparticles are often utilized for inter-tissue application, penetration of cells, and certain routes of administration. a. Interleukin 15 (IL-15)

In some forms, the described compositions of genetically modified NK Cells are formulated as a composition in combination with Interleukin- 15 (IL- 15). As used herein, the gene(s) encoding human IL- 15 are termed IL15.

IL- 15 is an importantcytokine that plays a pivotal role in enhancing the efficacy of Chimeric Antigen Receptor (CAR) natural killer (NK) cell therapies, that holds significant importance in CAR NK cell immunotherapy due to its unique ability to promote the proliferation, activation, and survival of NK cells.

IL- 15 acts as a powerful stimulant, bolstering the cytotoxic potential of CAR NK cells, which are engineered to target specific tumor antigens (Li, et al. (2023) "Loss of metabolic fitness drives tumor resistance after CAR-NK cell therapy and can be overcome by cytokine engineering." Sci Adv 9(30): eadd6997; Laskowski, et al. (2022). "Natural killer cells in antitumor adoptive cell immunotherapy." Nat Rev Cancer 22(10): 557-575). It may be that, by providing a robust and sustained activation signal, IL- 15 helps CAR NK cells persist in the hostile tumor microenvironment, thereby increasing their capacity to seek out and destroy cancerous cells. Therefore, it is envisioned that IL- 15 not only amplifies the immediate anti- tumor response, but also endows CAR NK cells with a memory-like phenotype, enabling them to mount rapid and potent reactions upon re-encountering the target antigen.

Therefore, compositions including a population of genetically modified CAR NK cells that overexpress one or more genes, such as SGSM2, OR7A10, APLN, PDP1, GABBR1, PRR14L, TIAM1, KRT82, PLA2G1B, REM2, HIST1H2BN, LRRC23, NXPE3, CYB5B, MEGF11, FKBP5, PPFIA2, LRRC23, PEAR1, REM2, TIAM2, HPRT1, MMACHC, ZBTB20, IL15 and/or IL2I include IL-15. For example, in some forms, IL-15 is present in a composition including a population of genetically modified CAR NK cells in an amount between about 1.0 ng and 10,000 mg, inclusive. In some forms, the amount of IL- 15 is between about 1.0 mg and about 10 mg, for example, about 1 mg, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg of IL-15. As demonstrated in the Examples, an amount of IL-21 sufficient to induce enhance anti-cancer activity can equivalent to an in vitro concentration of between about 1 and about 100 ng/ml, inclusive, such as between about 2.5 ng/ml and about 15 ng/ml, inclusive, or between about 5 ng/ml and about 10 ng/ml, inclusive. In some forms, the amount of IL-21 within a formulation is equivalent to an in vitro concentration of about 2.5 ng/ml. In an exemplary form, IL15 is present in an amount of approximately 2.5 ng/ml, and IL21 is present in an amount of approximately 10 ng/ml.

Purified IL- 15 is commercially available from multiple sources. b. Interleukin 21 (IL-21)

In some forms, the described compositions of genetically modified NK Cells are formulated as a composition in combination with Interleukin-21 (IL-21). As used herein, the gene(s) encoding human Interleukin 21 are termed IL21.

Interleukin-21 (IL-21) plays a crucial role in augmenting the effectiveness of Chimeric Antigen Receptor (CAR) modified natural killer (NK) cells. It may be that IL-21 acts as a potent stimulator, enhancing the cytotoxic potential of the described genetically modified NK cells and CAR NK cells engineered to target specific tumor antigens. It is envisioned that IL-21 fosters robust proliferation and activation of NK cells, allowing for a more aggressive and sustained assault on cancer cells. Additionally, IL-21 promotes the development of memory-like properties in CAR NK cells, enabling them to mount swift and potent responses upon re-exposure to the target antigen.

Therefore, compositions including a population of genetically modified CAR NK cells that overexpress one or more genes, such as SGSM2, OR7A10, APLN, PDP1, GABBR1, PRR14L, TIAM1, KRT82, PLA2G1B, REM2, HIST1H2BN, CYB5B, LRRC23, NXPE3, MEGF11, FKBP5, PPFIA2, LRRC23, PEAR1, REM2, TIAM2, HPRT1, MMACHC, ZBTB20, IL15 and/or IL2I include IL-21. For example, in some forms, IL-21 is present in a composition including a population of genetically modified CAR NK cells in an amount between about 1.0 ng and 10,000 mg, inclusive. In some forms, the amount of IL-21 is between about 1.0 mg and about 10 mg, for example, about 1 mg, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg of IL-21. As demonstrated in the Examples, an amount of IL-21 sufficient to induce enhance anti-cancer activity can be equivalent to an in vitro concentration of between about 1 and about 100 ng/ml, inclusive, such as between about 5 ng/ml and about 30 ng/ml, inclusive. In some forms, the amount of IL-21 within a formulation is equivalent to an in vitro concentration of about 10 ng/ml.

Purified IL-21 is commercially available from multiple sources. c. Exemplary Formulations

In some forms, a formulation including NK cells that express one or more CARs also includes IL- 15 and IL-21. For example, in some forms, a formulation of CAR NK cells includes IL-21 at a concentration of about 10 ng/ml IL-21, and IL-15 at a concentration of about 2.5 ng/ml.

In some forms, a formulation of genetically engineered NK cells that over-express or up- regulate one or more genes including SGSM2, OR7A10, APLN, PDP1, GABBR1, PRR14L, TIAM1, KRT82, PLA2G1B, REM2, HIST1H2BN, CYB5B, LRRC23, NXPE3, MEGF11, FKBP5, PPFIA2, LRRC23, PEAR1, REM2, TIAM2, HPRT1, MMACHC, ZBTB20, IL15 and/or IL21 relative to a wild-type NK cell also includes IL- 15 and IL-21. For example, in some forms, a formulation of the described genetically engineered NK cells that over-express or up-regulate one or more genes including SGSM2, OR7A10, APLN, PDP1, GABBR1, PRR14L, TIAM1, KRT82, PLA2G1B, REM2, HIST1H2BN, CYB5B, LRRC23, NXPE3, MEGF11, FKBP5, PPFIA2, LRRC23, PEAR1, REM2, TIAM2, HPRT1, MMACHC, ZBTB20, IL15 and/or IL21 relative to a wild-type NK cell includes IL-21 at a concentration of about 10 ng/ml IL-21, and IL- 15 at a concentration of about 2.5 ng/ml.

2. Additional Active Agents

In some forms, the described genetically modified NK or CAR NK cells are formulated in combination with one or more additional active agents. Typically, only additional therapeutic agents that do not negatively impact the viability or efficacy of the genetically modified NK or CAR NK cells are formulated together with the cells.

Exemplary additional active agents include therapeutic, prophylactic, nutraceutical and diagnostic agents. Exemplary therapeutic agents include anti-cancer and anti-autoimmune agents. a. Anti-cancer agents

In some forms, the described genetically modified NK or CAR NK cells are formulated in combination with one or more additional anti-cancer agents. Any conventional therapeutic agents effective against cancer can be formulated together with the described genetically modified NK or CAR NK cells.

Numerous antineoplastic drugs can be used in combination with the disclosed pharmaceutical compositions. In some forms, the additional therapeutic agent is a chemotherapeutic or antineoplastic drug. The majority of chemotherapeutic drugs can be divided into alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, monoclonal antibodies, and other anti-tumor agents. b. Therapeutic agents against Autoimmune diseases

In some forms, the described genetically modified NK or CAR NK cells are formulated in combination with one or more additional agents active against Autoimmune diseases. Any conventional therapeutic agents effective against autoimmune diseases can be formulated together with the described genetically modified NK or CAR NK cells.

Exemplary aganets include immunosuppressive agents, such as steroids or cytostatic drugs, analgesics, non-steroidal anti-inflammatory drugs, glucocorticoids, immunosuppressive and immunomodulatory agents, such as methotrexate, leflunomide, hydroxychloroquine, and sulfasalazine, TNF a inhibitors, belimumab and rituximab, T cell co stimulation blocker, antiinterleukin 6 (IL-6) monoclonal antibody (mAh), anti IL-1 mAh, protein kinase inhibitors, anti TNFa mAh, anti CD19 mAh, anti CD20 mAh, anti CD22 mAh, and anti IL6R mAh, or other mAbs that target multiple B cell subtypes, and other aberrant cells in autoimmune diseases.

3. Pharmaceutical Excipients

The term “Pharmaceutically acceptable carrier” describes a pharmaceutically acceptable material, composition, or vehicle that is involved in carrying or transporting a compound of interest from one tissue, organ, or portion of the body to another tissue, organ, or portion of the body. For example, in some forms the carrier is a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or a combination thereof. Each component of the carrier must be “pharmaceutically acceptable” in that it must be compatible with the other ingredients of the formulation. It must also be suitable for use in contact with any tissues or organs with which it may come in contact, meaning that it must not carry a risk of toxicity, irritation, allergic response, immunogenicity, or any other complication that excessively outweighs its therapeutic benefits.

In some forms, pharmaceutical compositions include buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The pharmaceutical compositions can be formulated for delivery via any route of administration. The term “Route of administration” can refer to any administration pathway known in the art, including but not limited to aerosol, nasal, oral, intravenous, intramuscular, intraperitoneal, inhalation, transmucosal, transdermal, parenteral, implantable pump, continuous infusion, topical application, capsules and/or injections. The pharmaceutical compositions are preferably formulated for intravenous administration.

Typically, the disclosed pharmaceutical compositions are administered in a manner appropriate to a disease to be treated (or prevented). The quantity and frequency of administration is typically determined by such factors as the condition of the patient, and the type and severity of the patient's disease, although appropriate dosages can be determined by clinical trials.

The disclosed pharmaceutical compositions can be delivered in a therapeutically effective amount. The precise therapeutically effective amount is that amount of the composition that will yield the most effective results in terms of efficacy of treatment in a given subject. This amount will vary depending upon a variety of factors, including but not limited to the characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication), the nature of the pharmaceutically acceptable carrier or carriers in the formulation, and the route of administration. One skilled in the clinical and pharmacological arts will be able to determine a therapeutically effective amount through routine experimentation, for instance, by monitoring a subject's response to administration of a compound and adjusting the dosage accordingly. For additional guidance, see Remington: The Science and Practice of Pharmacy (Gennaro ed. 20th edition, Williams & Wilkins PA, USA) (2000).

III. Methods of Screening/Producing Genetically Modified NK Cells

Methods for screening genes associated with NK cells to identify candidate targets that enhance the anti-cancer efficacy of NK cells have been developed. Screening systems that enable the step-wise selection of Natural Killer (NK) cells recombinantly over-expressing one or more selected genes are provided. Methods for providing genetically modified NK cells based on data obtained from the screening methods are also provided. Methods of formulating pharmaceutical formulations including genetically modified NK cells for treating diseases and disorders are also provided. Methods of treating diseases and disorders in a subject in need thereof by administering formulations of genetically modified NK cells to the subject are also described. An exemplary method of performing a Gain-of-Function screening of a Natural Killer (NK) cell includes one or more steps of:

(i) transducing an NK cell with one or more CRISPRa single-guide RNA(s) (sgRNAs),

(ii) causing the NK cell to be genetically modified by CRISPRa-mediated genome editing of a gene targeted by the sgRNA; and

(iii) screening the NK cell for tumor cell killing.

In some forms, an sgRNA includes: (i) a guide sequence; and (ii) a tracrRNA sequence including a nucleic acid sequence selected from a library, such as all or part of a human genomic reference sequence library. In some forms, the sgRNA is included within a vector, such as a lentiviral vector. Typically, the vector further includes an expression cassette for the sgRNA. In some forms, the expression cassette further includes a nucleic acid construct configured to express or encode a chimeric antigen receptor (CAR). Generally, the methods including steps (i)- (iii) are carried out using a plurality of NK cells, for example, whereby each of the plurality of NK cells is contacted by one or more sgRNAs including one or more of the sequences of the library. Therefore, in some forms, the methods collectively contact a plurality of NK cells with a multiplicity of sgRNAs, whereby a single sgRNA of the multiplicity of sgRNAs includes a single sequence from the library, and whereby an NK cell of the plurality of NK cells that are contacted by an sgRNA over-expresses a single gene, relative to a control NK cell that is not contacted by the sgRNA.

Typically, the methods also include one or more steps of the screening genetically modified NK cells for tumor cell killing. Methods for screening of NK cell killing are known in the art. As described in the Examples, in some forms, screening for tumor cell killing is carried out in vitro, for example, using a cancer cell line or cells derived from a tumor sample ex vivo. In some forms, the screening for tumor cell killing is carried out in vivo, for example, by directly injecting a population of modified NK cells into a test animal, such as tumor-bearing animal model. An exemplary tumor-bearing animal model is an HT29 tumor-bearing mouse. In an exemplary method, screening includes selecting genetically modified NK cells from animals with enhanced survival/reduced tumor burden. The screening methods can include a control. For example, in some forms, the anti-cancer effect of the described NK cells in a tumor-bearing animal model is compared to control animals that did not receive the same genetically modified NK cells.

In some forms, the methods include one or more steps of characterizing the genetically modified NK cell(s). For example, in some forms, the methods characterize a genetically modified NK cell by single cell transcriptome analysis. In some forms, the methods further include characterizing the genetically modified NK cell(s) by sequence analysis to identify one or more modified genes.

In some forms, the screening methods are repeated once, or more than once, using the same NK cells, or a subset of the initially screened NK cells. Therefore, in some forms, the methods repeat all or part of steps (i)-(iii) once or more than once to include one or more additional “rounds” of the screening methods, for example, using a selected pool of sgRNAs for the one or more additional rounds. Genetically modified NK cells created according to the screening methods, and populations thereof generated by expansion of the genetically modified NK cells, are also provided. Therefore, in some forms, the methods include one or more steps of selecting and/or isolating one or more genetically modified NK cells identified as having one or more desired properties, relative to one or more other genetically modified NK cells and/or control cells (e.g., wild-type, non-modified NK cells). Exemplary desired properties include one or more function of enhanced, increased and/or prolonged tumor cell killing; enhanced, increased and/or prolonged solid tumor infiltration; enhanced and/or increased proliferation; enhanced, increased and/or prolonged activation; enhanced, increased and/or prolonged effector cytokine production; and enhanced and/or increased cancer cell cytotoxicity.

Methods for isolating one or more cells from a population of cells are known in the art. In some forms, the step of selecting genetically modified NK cells identified as having one or more desired properties includes the steps of (i) determining the sequence of the genetically modified NK cells identified as having one or more desired properties; and (ii) identifying one or more perturbed (e.g., upregulated and/or over expressed genes within the cell as compared to one or more other genetically modified NK cells and/or control cells (e.g., wild-type, non-modified NK cells). In some forms, the methods repeat the genetic modification in one or more further cells. In some forms, the methods expand or proliferate the genetically modified NK cells identified as having one or more desired properties.

A. Gain-of-Function Modification of NK cells

A high-throughput Gain-of-Function (GOF) gene perturbation screen for highly efficient identification and engineering of therapeutic NK cells has been established. In some forms, the screen identifies genes whose perturbation enhances the anti-tumor properties of NK cells. The methods employ a library of sgRNAs designed to target genomic components of NK cells to over-express the genes through CRISPR-based gene editing. 1. Single Guide RNA (sgRNA) Libraries

Single guide RNA (sgRNA, gRNA) libraries including a plurality of nucleic acids are provided.

In some forms, an sgRNA library has a size of between about 30,000 and about 100,000 different sgRNAs represented. In some forms, an sgRNA library has a size of between about 40,000 and about 90,000 different sgRNAs represented. In some forms, an sgRNA library has a size of between about 40,000 and about 80,000 different sgRNAs represented. In some forms, an sgRNA library has a size of between about 50,000 and about 70,000 different sgRNAs represented. In some forms, an sgRNA library has a size of between about 55,000 and about 70,000 different sgRNAs represented. Although an sgRNA library including between about 55,000 and about 70,000 different sgRNAs has a certain inevitable library representation loss, a library of this size can still consistently be used to recover a substantial fraction of the library without selection pressure. Typically, an sgRNA library having between about 55,000 and about 70,000 different sgRNAs can identify meaningful hits with strong selection and genetic modification phenotypes, despite partial library loss, even though the screen is not saturated, based on library representation. Therefore, in some forms, a high-density CRISPR library having between about 55,000 and about 70,000 different sgRNAs includes extensive sgRNA redundancy (i.e., >10 sgRNA / gene) to target collections of genes belonging to certain classes or annotated pathways, in a relatively unbiased manner. Exemplary molecular pathways that are targeted include all surface proteins, all kinases / phosphatases, all transcription factors, all KEGG enzymes, and combinations of two or more of these. Therefore, this library size represents a “sweet spot” of target range and in vivo coverage.

Typically, the libraries include a multiplicity of different sgRNAs within a single pool or group of pools. Typically, the libraries include at least one copy of each sgRNA represented within the library. In some forms, the libraries include multiple copies of each sgRNA. In particular forms, the numbers of copies of each sgRNA within in library are equal or are similar. In other forms, the numbers of copies of each sgRNA are not equal. For example, in some forms, sgRNA libraries are enriched for one or more of the multiplicity of sgRNAs within the library.

In some forms, the sgRNA library includes between about 1 and about 100 or more sgRNA sequences. In some forms, the library includes about 1,000, or more than 1,000 sgRNA sequences, up to 10,000 sgRNA sequences. In some forms, the library includes about 10,000 or more sgRNA sequences. In further forms, the library includes about 20,000 or more sgRNA sequences. In further forms, the library includes about 30,000 or more than 30,000, up to 50,000 sgRNA sequences. In further forms, the library includes about 40,000 sequences or more than 40,000, up to 50,000 sgRNA sequences. In further forms, the library includes about 50,000 sequences or more than 50,000, up to 60,000 sgRNA sequences. In yet further forms, the library includes about 40,000 sequences or more than 60,000, up to 70,000 sgRNA sequences.

Typically, the library includes a sufficient number of sgRNA sequences to enable coverage of a target gene set, whilst enabling complete (100%) or a high-degree (i.e., greater than 50%) representation in a screen. In some forms, of all the distinct species of sgRNAs present in a library, a screen according to the described methods includes at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, up to 100% representation of all the distinct species of sgRNAs present in a library.

2. crRNA/sgRNA Sequences

The structure and function of sgRNAs is known in the art. Each sgRNA is made up of two parts:

(i) a crispr RNA (crRNA), a 17-20 nucleotide sequence complementary to the target DNA; and

(ii) a Trans-activating CRISPR RNA (tracr RNA), which serves as a binding scaffold for the Cas nuclease.

The tracrRNA pairs with complementary repeat sequences within the pre-crRNA primary transcript and forms an RNA duplex, pre-crRNA:tracrRNA, which is recognized and cleaved by RNase III in the presence of Cas9 protein. The crRNA includes 17-20 contiguous nucleic acids that specifically bind to one gene with thin the NK cell genome.

In some forms, the different species of sgRNAs in a library are combined to have a level of redundancy with respect to coverage of one or more target genes, such as a group of genes associated with one or more molecular pathway or cellular function. In some forms, the different species of sgRNAs in a library include at least 1% redundancy, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or more than 10%, such as 15% or 20% redundancy in the coverage of one or more target genes, such as a group of genes associated with one or more molecular pathway or cellular function.

3. Vectors

In certain forms, the library includes each sgRNA encoded or encompassed within one or more vectors. For example, in some forms, the sgRNA library is packaged into a vector. Any vector known to one of ordinary skill in the art can be used. In some forms, the vector is a viral vector, including but not limited to lenti viral vectors.

In some forms, suitable viral vectors include, without limitation, vectors derived from bacteriophages, baculoviruses, retroviruses (such as lentiviruses), adenoviruses, poxviruses, and Epstein-Barr viruses. In some forms, the viral vector is derived from a DNA virus (e.g., dsDNA or ssDNA virus) or an RNA virus (e.g., an ssRNA virus). Numerous vectors and expression systems are commercially available from commercial vendors including Addgene, Novagen (Madison, WI), Clontech (Palo Alto, CA), Stratagene (La Jolla, CA), and Invitrogen/Life Technologies (Carlsbad, CA).

In some forms, the viral vector is a lentivirus vector. In an exemplary form, Lentivirus is produced by HEK293T cells, the supernatant was collected and precipitated using Lenti-X Concentrator (Takara). In an exemplary form, Lentiviral pellets are resuspended with NK92 complete culture media, then aliquoted and stored at -80°C. In an exemplary method, when the viral vector is a lentiviral vector, cells are transduced with lentivirus at l-2e6 cells / ml in a 12- well plate, which is pre-coated with Retronectin (Takara) in PBS, overnight at 4°C. In some forms, spin-infection is performed (e.g., at 32°C at 900 x g for 90 min).

In some forms, the vector is a viral vector such as a vesicular stomatitis (VSV) vector, a Bocavirus vector, such as a human bocavirus 1 (HBoV 1) vector, a Herpes simplex virus (HSV) vector, or an adenovirus vector (AdV).

In some forms, the viral vector is a Herpes simplex virus (HSV) vector. Herpes simplex viruses (HSV) are large, enveloped dsDNA viruses characteristic of their lytic and latent nature of infection, which result in life-long latent infection of neurons and allows for long-term transgene expression. Deletion of HSV genes has generated expression vectors with low toxicity and an excellent packaging capacity of >30 kb foreign DNA.In some forms, the viral vector is a Vesicular stomatitis virus (VSV) vector. Vesicular stomatitis virus is a non-segmented, negative- stranded RNA virus that belongs to the family Rhabdoviridae, genus Vesiculovirus. VSV infects a broad range of animals, including cattle, horses, and swine. The genome of the virus codes for five major proteins, glycoprotein (G), matrix protein (M), nucleoprotein (N), large protein (L), and phosphoprotein (P). The G protein mediates both viral binding and host cell fusion with the endosomal membrane following endocytosis. The L and P proteins are subunits of the viral RNA-dependent RNA polymerase. The simple structure and rapid high-titer growth of VSV in mammalian and many other cells has made recombinant VSV a useful tool in the fields of cellular and molecular biology and virology.

In some forms, the viral vector is a human Bocavirus vector (HBoV). Exemplary human bocavirus vectors include human bocaviruses 1-4 (HBoVl-4), As well as Gorilla BoV.

In other forms, the viral vector is an adenovirus vector. In some forms, the vector is a chimeric vector, such as a vector that is based on a chimeric virus formed from a combination of one or more components from two or more different viral vectors. An exemplary chimeric viral vector is a chimeric bocavirus/adeno-associated virus vector. Therefore, in some forms, the vector is a chimeric HBoVl/AAV2 vector (e.g., rAAV2/HBoVl chimeras). In some forms, the sgRNA library is a vector-sgRNA library than includes a multiplicity of vectors each encapsulating or otherwise associated with one or more sgRNA.

In some forms, the library includes a plurality of vectors, wherein each vector includes an expression cassette for an sgRNA including a nucleotide sequence.

Vectors including the described sgRNA libraries are described for gene editing and high- throughput screen in NK cells. Therefore, gene editing compositions for use in methods of modifying the genome of a cell are disclosed.

In exemplary forms, an sgRNA library is encapsulated or associated with a vector configured for CRISPR-based gene editing in target NK cells. One skilled in the art would understand that any system suitable for delivery of CRSIPR gene-editing compositions can be used for delivering the described sgRNA libraries to target cells.

In an exemplary form each CRISPR vector within the library includes one or more of an antibiotic resistance sequence, two ITRs, two sleeping beauty (SB) IR/DR repeats, a RNA pol- III promoter (e.g., U6), an sgRNA from the library (spacer and tracrRNA backbone), a promoter (EFS), a Thyl.l selection marker, an SB lOOx transposase, and a short poly A region.

4. CRISPR Components for Gene Editing

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is an acronym for DNA loci that contain multiple, short, direct repetitions of base sequences. The prokaryotic CRISPR/Cas system has been adapted for use as gene editing (silencing, enhancing or changing specific genes) for use in eukaryotes (see, for example, Cong, Science, 15 :339(6121): 819- 823 (2013) and Jinek, et al., Science, 337(6096):816-21 (2012)). Methods of preparing compositions for use in genome editing using the CRISPR/Cas systems are described in detail in WO 2013/176772 and WO 2014/018423, which are specifically incorporated by reference herein in their entireties.

As used herein, the term “Cas” generally refers to an effector protein of a CRISPR Cas system or complex. The term “Cas” may be used interchangeably with the terms “CRISPR” protein, “CRISPR Cas protein,” “CRISPR effector,” CRISPR Cas effector,” “CRISPR enzyme,” “CRISPR Cas enzyme” and the like, unless otherwise apparent. In general, “CRISPR system” refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g., tracrRNA or an active partial tracrRNA), a tracr-mate sequence (encompassing a “direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as a “spacer” in the context of an endogenous CRISPR system), or other sequences and transcripts from a CRISPR locus. One or more tracr mate sequences operably linked to a guide sequence (e.g., direct repeat-spacer-direct repeat) can also be referred to as pre-crRNA (pre-CRISPR RNA) before processing or crRNA after processing by a nuclease. Typically, the described vectors include an sgRNA from the described libraries, together with a Crispr-Cas effector protein. a. sgRNA structures

In some forms, the described sgRNA libraries include a tracrRNA and crRNA that are linked and form a chimeric crRNA-tracrRNA hybrid where a mature crRNA is fused to a partial tracrRNA via a synthetic stem loop to mimic the natural crRNA:tracrRNA duplex as described in Cong, Science, 15:339(6121):819— 823 (2013) and Jinek, et al., Science, 337(6096):816-21 (2012)). A single fused crRNA-tracrRNA construct can also be referred to as a guide RNA or gRNA (or single-guide RNA (sgRNA)). Within an sgRNA, the crRNA portion can be identified as the ‘target sequence’ and the tracrRNA is often referred to as the ‘scaffold’. b. Crispr-Cas effector protein

The Crispr-Cas effector protein may be without limitation a type II, type V, or type VI Cas effector protein.

Non-limiting examples of Crispr-Cas effector proteins include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, homologues thereof, or modified versions thereof. In some forms, the unmodified CRISPR enzyme has DNA cleavage activity. Preferably, the Crispr-Cas effector protein is mutated with respect to a corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence. c. Cas9

In some forms, the Type II CRISPR enzyme is a Cas9 enzyme such as disclosed in International Patent Application Publication No. WO/2014/093595. In some forms, the Cas9 enzyme is S. pneumoniae, S. pyogenes or S. thermophilus Cas9, and may include mutated Cas9 derived from these organisms. The enzyme may be a Cas9 homolog or ortholog. Additional orthologs include, for example, Cas9 enzymes from Corynebacter diptheriae, Eubacterium ventriosum, Streptococcus pasteurianus, Lactobacillus farciminis, Sphaeroachaeta globus, Azospirillum B510, Gluconacetobacter diazo trophicus, Neisseria cinereal, Roseburia intestinalis, Parvibaculum lavamentivorans, Staphylococcus aureus, Nitratifractor salsuginis DSM 16511, Camplyobacter lari CF89 12, and Streptococcus thermophilus LMD 9.

In some forms, the Cas9 effector protein and orthologs thereof may be modified for enhanced function. For example, improved target specificity of a CRISPR Cas9 system may be accomplished by approaches that include, but are not limited to, designing and preparing guide RNAs having optimal activity, selecting Cas9 enzymes of a specific length, truncating the Cas9 enzyme making it smaller in length than the corresponding wild-type Cas9 enzyme by truncating the nucleic acid molecules coding therefor and generating chimeric Cas9 enzymes wherein different parts of the enzyme are swapped or exchanged between different orthologs to arrive at chimeric enzymes having tailored specificity.

The methods combine the lentivirus-encoding sgRNAs for overexpression of specific genes, and adoptively transfer them into tumor bearing test animals for immediate functional assessment in vivo. Therefore, the methods introduce user-defined genetic modifications into NK cells in a controllable and highly efficient manner to target one or more genes.

The gene or genes that are targeted can be transcriptionally and/or translationally transduced and/or upregulated/over-expressed by other targeting methods. Other targeting methods include, but are not limited to, Cas9 coupled with transcriptional activators, receptor agonists, and the like.

5. Exemplary Targets for Gene Editing

In some forms, the methods include genetically modifying an NK cell to express or overexpress at least one gene selected from SGSM2, OR7A10, APLN. PDP1, GABBR1, PRR14L, TIAM1, KRT82, PLA2G1B, REM2, HIST1H2BN, CYB5B, LRRC23, NXPE3, MEGF11, FKBP5, PPFIA2, LRRC23, PEAR1, REM2, TIAM2, HPRT1, MMACHC, ZBTB20, IL15 and/or IL21. In some forms, the methods include stimulating a NK cell by contacting the NK cell with a therapeutically effective amount of an agonist of at least one gene selected from SGSM2, OR7A10, APLN, PDP1, GABBR1, PRR14L, TIAM1, KRT82, PLA2G1B, REM2, HIST1H2BN, CYB5B, LRRC23, NXPE3, MEGF11, FKBP5, PPFIA2, LRRC23, PEAR1, REM2, TIAM2, HPRT1, MMACHC, ZBTB20, IL15 and/or IL21. In certain forms the CRISPR system includes a Cas9, and at least one sgRNA complementary to SGSM2, OR7A10, APLN, PDP1, GABBR1, PRR14L, TIAM1, KRT82, PLA2G1B, REM2, HIST1H2BN, CYB5B, LRRC23, NXPE3, MEGF11, FKBP5, PPFIA2, LRRC23, PEAR1, REM2, TIAM2, HPRT1, MMACHC, ZBTB20, IL15 and/or IL2I.

In some forms, the methods perform genome editing and screening of a NK cell for a genetic modification in vitro. An exemplary method includes contacting the NK cell with Cas9 and a lentiviral vector library. In some forms, the lentiviral vector library includes a plurality of vectors, whereby each vector includes an expression cassette for an sgRNA and a chimeric antigen receptor (CAR). According to the methods, the NK cell undergoes genome editing and is then screened for a genetic modification in vitro. In other forms, the methods edit the genome and screen NK cells for a genetic modification in vivo. For example, in some forms, the methods contact an NK cell with Cas9 and a lentiviral vector library including a plurality of vectors, each of which includes an expression cassette for an sgRNA and a chimeric antigen receptor (CAR).

In some forms, the methods contact an NK cell with Cas9 and a lentiviral vector library including a plurality of vectors, wherein each vector includes an expression cassette for an sgRNA and a chimeric antigen receptor (CAR).

In some forms, the methods modify the NK cell in vitro and administer the NK cell to a subject and the NK cell is screened for a mutation in vivo. In certain forms the sgRNA mediates efficient transcriptional activation at an endogenous genomic loci to express at least one gene selected from SGSM2, OR7A10, APLN, PDP1, GABBR1, PRR14L, TIAM1, KRT82, PLA2G1B, REM2, HIST1H2BN, CYB5B, LRRC23, NXPE3, MEGF11, FKBP5, PPFIA2, LRRC23, PEAR1, REM2, TIAM2, HPRT1, MMACHC, ZBTB20, IL15 and/or IL21.

In some forms, the methods include one or more steps to screen and/or genetically modify a NK cell that is a CAR-NK cell. In some forms, the methods modify a NK cell in vitro to express a CAR and also to up-regulate or over express one or more genes, relative to a control (e.g., a wild-type NK cell). In some forms, the methods include one or more further steps to administer the CAR NK cell to a subject and the CAR NK cell is screened for a mutation in vivo.

In certain forms, methods for genetic modification of NK cells include administering into the cells an sgRNA to mediate efficient transcriptional activation at an endogenous genomic loci to express at least one gene selected from SGSM2, OR7A10, APLN. PDP1, GABBR1, PRR14L, TIAM1, KRT82, PLA2G1B, REM2, HIST1H2BN, LRRC23, NXPE3, CYB5B, MEGF11, FKBP5, PPFIA2, LRRC23, PEAR1, REM2, TIAM2, HPRT1, MMACHC, ZBTB20, IL15 and/or IL21, together with a construct encoding a CAR. Therefore, in some forms, the methods engineer a NK cell to express a CAR and also to over-express or up-regulate expression of one or more genes relative to a control. Typically, the engineered NK cells and/or CAR NK cells that overexpress or up-regulate expression of one or more genes relative to a control have increased anticancer efficacy relative to a control.

B. Selecting Anti-Cancer NK Cells

Typically, following methods for GOF modification of NK cells, the methods characterize modified NK cells having desired phenotypes.

In an exemplary form, the methods characterize the anti-cancer efficacy/function of modified NK cells by injection into a tumor-bearing animal model and identify animals with enhanced survival/reduced tumor burden. For example, in some forms, the methods isolate target NK cells from the tumor, and characterize the most abundant mutants within the isolated NK cells, e.g., using single cell transcriptome analysis. The methods optionally sequence the pool of selected Gain of Function (GOF) mutants and repeat the screen steps using only best sgRNAs for one or more additional rounds.

As described in the Examples, methods for CRISPRa GOF screening successfully identified modified NK cells that enhance the anti-cancer activity of NK cells. In some forms, the methods include one or more steps for identifying and isolating or selecting genetically modified NK cells having one or more desirable characteristics. The desirable characteristics can be phenotypic, such as reduced exhaustion, enhanced tumor penetration, reduced apoptosis, enhanced tumor killing, etc. All the characteristics of genetically modified NK cells can be compared to a control NK cell or population of control NK cells. Methods for assessing phenotypic characteristics of cells are known to those skilled in the art. In an exemplary form, the methods identify genetically modified NK cells having enhanced tumor killing efficacy.

In some forms, the methods include one or more steps for genetically characterizing the genetically modified NK cells that are identified as having one or more desirable characteristics. Identifying the genetically modified NK cells can include any method commonly known to one of ordinary skill in the art including but not limited to methods of nucleotide sequencing, sgRNA PCR, and/or flow cytometry. Nucleotide sequencing or “sequencing”, as it is commonly known in the art, can be performed by standard methods commonly known to one of ordinary skill in the art. In certain forms, sequencing is performed via next-generation sequencing. Nextgeneration sequencing (NGS), also known as high-throughput sequencing, describes a number of different modem sequencing technologies that allow sequencing of DNA and RNA much more quickly and cheaply than Sanger sequencing.

In some forms, the methods screen for anti-cancer activity using cancer cells in vitro, ex vivo or in vivo. In an exemplary form, the methods screen the NK cells for anti-tumor activity using in vivo using an animal model. An exemplary animal model is a syngeneic mouse model of human colorectal cancer, HT29 tumor-bearing mice. Methods for establishing mouse models of diseases such as cancer are known in the art.

C. Exemplary Methods

In an exemplary method, CRISPRa GOF screening is performed using naive NK cells isolated from a subject. An exemplary method includes one or more steps as follows: i. Provide a genome wide library of sgRNAs, each sgRNA of the library configured to to mediate efficient transcriptional activation of a single, distinct gene in NK cells; ii. Package the library into a lentiviral vector to form a lentiviral sgRNA library; iii. Transduce the vectors into the target NK cell population; and iv. characterize modified NK cells having desired phenotypes. In an exemplary method, between IxlO⁶ and 5xl0⁷ Cas9+ NK cells are transduced with a lentiviral sgRNA library. For example, in some forms, the methods include first generating NK cells (e.g., NK92 cells) that constitutively express an a-HER2-CAR 27, along with two CRISPR activation system components: dCAS9-VP64 and MS2-P65-HSF1, then transduced the a-HER2- CAR-NK92 cells with a genome-scale CRISPRa single-guide RNA (sgRNA) library, and adoptively transferring them into HT29 tumor-bearing mice via subcutaneous injection or via intravenous (iv) administration (tail vein injection). The methods typically include one or more controls, e.g., by injecting wild-type/non-modified NK cells. In some forms, the methods investigate the resulting effects upon the tumors within the model animals, for example, by investigation of tumor tissues removed up to 24 days post tumor inoculation. The NK cell screen is different from T cell or other screen due to the distinct biology, culture condition, gene editing and the nature of the NK cell type. An exemplary method for screening a pool of GOF NK cells for anti-cancer efficacy is set forth in Figure 1.

IV. Methods of Treatment

Methods of treatment including administering the genetically modified NK cells as therapeutic agents to a subject in need thereof are described. In some forms, the methods include Adoptive Cell Therapy (ACT) of a subject in need thereof. Typically, the methods include ACT employing the genetically modified NK cells prepared according to the described methods for screening and genetic manipulation of NK cells.

An exemplary method involves treating a subject e.g., a human) having a disease, disorder, or condition by administering to the subject an effective amount of a pharmaceutical composition including genetically modified NK cells prepared according to the described methods and compositions.

In some forms, the methods treat a subject having a disease, disorder, or condition associated with an elevated expression or specific expression of an antigen by administering to the subject an effective amount of a pharmaceutical composition including NK cells modified according to the disclosed methods. In some forms, the methods treat a subject having a disease, disorder, or condition associated with an elevated expression or specific expression of an antigen by administering to the subject an effective amount of a pharmaceutical composition including genetically modified NK cells modified to exhibit one or more characteristics that enhance the therapeutic activities of the NK cells in the context of the disease or disorder that is to be treated. Typically, the NK cells are modified to up-regulate and/or over-express at least one gene selected from SGSM2, OR7A10, APLN, PDP1, GABBR1, PRR14L, TIAM1, KRT82, PLA2G1B, REM2, HIST1H2BN, CYB5B, LRRC23, NXPE3, MEGF11, FKBP5, PPFIA2, LRRC23, PEAR1, REM2, TIAM2, HPRT1, MMACHC, ZBTB20, IL15 and/or IL21, as compared to a non- genetically modified control NK cell.

Methods of treating a subject having a disease, disorder, or condition including administering to the subject an effective amount of a pharmaceutical composition including live, viable NK cells engineered to enhance therapeutic efficacy are provided. In exemplary forms, the methods treat a subject having cancer by administering to the subject an effective amount of a pharmaceutical composition including live, viable NK cells engineered to up-regulate and/or over-express at least one gene selected from SGSM2, OR7A10, APLN, PDP1, GABBR1, PRR14L, TIAM1, KRT82, PLA2G1B, REM2, HIST1H2BN, CYB5B, LRRC23, NXPE3, MEGF11, FKBP5, PPFIA2, LRRC23, PEAR1, REM2, TIAM2, HPRT1, MMACHC, ZBTB20, IL15 and/or IL21, as compared to a non-genetically modified control NK cell. In exemplary forms, the methods treat a subject having cancer by administering to the subject an effective amount of a pharmaceutical composition including live, viable NK cells engineered to up-regulate and/or over-express SGSM2, and/or OR7A10.

In some forms, the methods treat a subject having a disease, disorder, or condition associated with an elevated expression or specific expression of an antigen. In some forms, the methods include administering to the subject an effective amount of the described genetically modified NK cells, further modified to express a CAR that targets the antigen. Therefore, in some forms, the methods treat a subject having cancer by administering to the subject an effective amount of a pharmaceutical composition including live, viable CAR-NK cells engineered to up-regulate and/or over-express at least one gene selected from SGSM2, OR7A10, APLN, PDP1, GABBR1, PRR14L, TIAM1, KRT82, PLA2G1B, REM2, HIST1H2BN, CYB5B, LRRC23, NXPE3, MEGF11, FKBP5, PPFIA2, LRRC23, PEAR1, REM2, TIAM2, HPRT1, MMACHC, ZBTB20, IL15 and/or IL21, as compared to a non-genetically modified control NK cell, or CAR NK cell.

Therefore, in some forms, the methods treat a subject having cancer by administering to the subject an effective amount of a pharmaceutical composition including live, viable CAR-NK cells engineered to up-regulate and/or over-express at least one gene selected from SGSM2, OR7A10, APLN, PDP1, GABBR1, PRR14L, TIAM1, KRT82, PLA2G1B, REM2, HIST1H2BN, CYB5B, LRRC23, NXPE3, MEGF11, FKBP5, PPFIA2, LRRC23, PEAR1, REM2, TIAM2, HPRT1, MMACHC, ZBTB20, IL15 and/or IL21, as compared to a non-genetically modified control NK cell, or CAR NK cell. In some forms, the methods treat a subject having cancer by administering to the subject an effective amount of a pharmaceutical composition including live, viable CAR NK cells engineered to up-regulate and/or over-express SGSM2 or OR7A10, and/or having enhanced or increased function of the SGSM2 or OR7A10 gene product(s). In an exemplary form, the methods treat a subject having cancer by administering to the subject an effective amount of a pharmaceutical composition including live, viable CAR NK cells engineered to up-regulate and/or over-express the SGSM2 gene, and/or having enhanced or increased function of the SGSM2 gene product(s). In a further exemplary form, the methods treat a subject having cancer by administering to the subject an effective amount of a pharmaceutical composition including live, viable CAR NK cells engineered to up-regulate and/or over-express the OR7A10 gene, and/or having enhanced or increased function of the OR7A10 gene product(s). The NK cell can have been isolated from the subject having the disease, disorder, or condition, or from a healthy donor, prior to genetic modification.

Unfavorable efficacy, safety issues, exhaustion, limited infiltration, and poor persistence are among the current major obstacles that hinder the clinical success of CAR therapy. Results presented below that IL- 15 and/or IL-21, particularly is certain doses, can reduce NK exhaustion and/or enhance cancer cell killing upon repeated exposures. Thus, in some embodiments, the disclosed compositions are more effective over time. In some embodiments, fewer administrations are needed relative to wildtype or CAR therapy without the addition one or more of the modifications or adjuncts provided herein, such as increase expression or presence of IL- 15 and/or IL-21.

A. Diseases to be treated

Methods of treating a subject having a disease or disorder are provided. Typically, the methods administer the genetically modified NK cells and/or CAR NK cells to the subject in an amount effective to treat and/or prevent the disease, or disorder.

The subject to be treated can have a disease, disorder, or condition such as but not limited to, cancer, an immune system disorder such autoimmune disease, an inflammatory disease, a neuronal disorder, HIV/AIDS, diabetes, a cardiovascular disease, an infectious disease, or combinations thereof, or can be identified as being at increased risk of developing the disease or disorder. The disease, disorder, or condition can be associated with an elevated expression or specific expression of an antigen. In some forms, the methods treat or prevent cancer and/or autoimmune disease in a subject in need thereof.

1. Cancer

In some forms, the methods treat or prevent cancer in a subject, or reduce, ameliorate or otherwise prevent one or more symptoms of cancer in a subject.

Cancer is a disease of genetic instability, allowing a cancer cell to acquire the hallmarks proposed by Hanahan and Weinberg, including (i) self-sufficiency in growth signals; (ii) insensitivity to anti-growth signals; (iii) evading apoptosis; (iv) sustained angiogenesis; (v) tissue invasion and metastasis; (vi) limitless replicative potential; (vii) reprogramming of energy metabolism; and (viii) evading immune destruction Cell., 144:646-674, (2011)).

Tumors, which can be treated in accordance with the disclosed methods, are classified according to the embryonic origin of the tissue from which the tumor is derived. In some forms, the tumor is a carcinoma. Carcinomas are tumors arising from endodermal or ectodermal tissues such as skin or the epithelial lining of internal organs and glands. In some forms, the tumor is a sarcoma. Sarcomas, which arise less frequently, are derived from mesodermal connective tissues such as bone, fat, and cartilage. In some forms, the tumor is a leukemia, or a lymphoma. The leukemias and lymphomas are malignant tumors of hematopoietic cells of the bone marrow. Leukemias proliferate as single cells, whereas lymphomas tend to grow as tumor masses. Malignant tumors may show up at numerous organs or tissues of the body to establish a cancer.

Methods of treating a subject having cancer, including administering to the subject an effective amount of a pharmaceutical composition including live, viable NK cells and/or CAR NK cells engineered or otherwise treated as provided herein are provided. In exemplary forms, the methods treat a subject having cancer by administering to the subject an effective amount of a pharmaceutical composition including live, viable NK cells and/or CAR NK cells engineered to engineered to up-regulate and/or over-express the SGSM2, OR7A10, APLN, PDP1, GABBR1, PRR14L, TIAM1, KRT82, PLA2G1B, REM2, HIST1H2BN, CYB5B, LRRC23, NXPE3, MEGF11, FKBP5, PPFIA2, LRRC23, PEAR1, REM2, TIAM2, HPRT1, MMACHC, ZBTB20, IL15 and/or IL21, gene or a combination thereof, as compared to a non-genetically modified control NK cell or CAR NK cell. The NK cell can have been isolated from the subject having the cancer prior to genetic modification (i.e., can be an autologous NK cell), or can be isolated from a healthy donor (i.e., can be a heterologous NK cell) prior to genetic modification.

A non-limiting list of cancers for which the CAR of the disclosed methods and compositions can target a specific or an associated antigen is provided in Table 2. Table 2: Listing of cancers

The disclosed compositions and methods of treatment thereof are generally suited for treatment of carcinomas, sarcomas, lymphomas and leukemias. Generally, the described compositions and methods are useful for treating, or alleviating subjects having benign or malignant tumors by delaying or inhibiting the growth/proliferation or viability of tumor cells in a subject, reducing the number, growth or size of tumors, inhibiting or reducing metastasis of the tumor, and/or inhibiting or reducing symptoms associated with tumor development or growth. In some forms, the disclosed compositions are used in a method of treating one or more of the cancers provided in Table 2.

The types of cancer that can be treated with the provided compositions and methods include, but are not limited to, cancers such as vascular cancer such as multiple myeloma, adenocarcinomas and sarcomas, of bone, bladder, brain, breast, cervical, colorectal, esophageal, kidney, liver, lung, naso-pharangeal, pancreatic, prostate, skin, stomach, and uterine. In some forms, the compositions are used to treat multiple cancer types concurrently. In some forms, the compositions are used to treat metastases or tumors at multiple locations. The described genetically modified NK cells and CAR-NK cells have enhanced anti-tumor efficacy, as compared with wild- type NK cells. The genetically modified NK cells are effective for killing tumor cells in vivo and in vitro. Exemplary tumor cells include, but are not limited to, tumor cells of cancers, including leukemias including, but not limited to, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemias such as myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia leukemias and myelodysplastic syndrome, chronic leukemias such as, but not limited to, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia; polycythemia vera; lymphomas such as, but not limited to, Hodgkin’s disease, non-Hodgkin’s disease; multiple myelomas such as, but not limited to, smoldering multiple myeloma, non-secretory myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma and extramedullary plasmacytoma; Waldenstrom’s macroglobulinemia; monoclonal gammopathy of undetermined significance; benign monoclonal gammopathy; heavy chain disease; bone and connective tissue sarcomas such as, but not limited to, bone sarcoma, osteosarcoma, chondrosarcoma, Ewing’s sarcoma, malignant giant cell tumor, fibrosarcoma of bone, chordoma, periosteal sarcoma, soft-tissue sarcomas, angiosarcoma (hemangiosarcoma), fibrosarcoma, Kaposi’s sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, neurilemmoma, rhabdomyosarcoma, synovial sarcoma; brain tumors including, but not limited to, glioma, astrocytoma, brain stem glioma, ependymoma, oligodendroglioma, nonglial tumor, acoustic neurinoma, craniopharyngioma, medulloblastoma, meningioma, pineocytoma, pineoblastoma, primary brain lymphoma; breast cancer including, but not limited to, adenocarcinoma, lobular (small cell) carcinoma, intraductal carcinoma, medullary breast cancer, mucinous breast cancer, tubular breast cancer, papillary breast cancer, Paget’s disease, and inflammatory breast cancer; adrenal cancer, including, but not limited to, pheochromocytom and adrenocortical carcinoma; thyroid cancer such as but not limited to papillary or follicular thyroid cancer, medullary thyroid cancer and anaplastic thyroid cancer; pancreatic cancer, including, but not limited to, insulinoma, gastrinoma, glucagonoma, vipoma, somatostatin-secreting tumor, and carcinoid or islet cell tumor; pituitary cancers including, but not limited to, Cushing’s disease, prolactin-secreting tumor, acromegaly, and diabetes insipius; eye cancers including, but not limited to, ocular melanoma such as iris melanoma, choroidal melanoma, and ciliary body melanoma, and retinoblastoma; vaginal cancers, including, but not limited to, squamous cell carcinoma, adenocarcinoma, and melanoma; vulvar cancer, including, but not limited to, squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and Paget’s disease; cervical cancers including, but not limited to, squamous cell carcinoma, and adenocarcinoma; uterine cancers including, but not limited to, endometrial carcinoma and uterine sarcoma; ovarian cancers including, but not limited to, ovarian epithelial carcinoma, borderline tumor, germ cell tumor, and stromal tumor; esophageal cancers including, but not limited to, squamous cancer, adenocarcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma, adeno-squamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small cell) carcinoma; stomach cancers including, but not limited to, adenocarcinoma, fungating (polypoid), ulcerating, superficial spreading, diffusely spreading, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; colon cancers; rectal cancers; liver cancers including, but not limited to, hepatocellular carcinoma and hepatoblastoma, gallbladder cancers including, but not limited to, adenocarcinoma; cholangio- carcinomas including, but not limited to, papillary, nodular, and diffuse; lung cancers including, but not limited to, non-small cell lung cancer, squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large-cell carcinoma and small-cell lung cancer; testicular cancers including, but not limited to, germinal tumor, seminoma, anaplastic, classic (typical), spermatocytic, nonseminoma, embryonal carcinoma, teratoma carcinoma, choriocarcinoma (yolk-sac tumor), prostate cancers including, but not limited to, adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; penal cancers; oral cancers including, but not limited to, squamous cell carcinoma; basal cancers; salivary gland cancers including, but not limited to, adenocarcinoma, mucoepidermoid carcinoma, and adenoid-cystic carcinoma; pharynx cancers including, but not limited to, squamous cell cancer, and verrucous; skin cancers including, but not limited to, basal cell carcinoma, squamous cell carcinoma and melanoma, superficial spreading melanoma, nodular melanoma, lentigo malignant melanoma, acral lentiginous melanoma; kidney cancers including, but not limited to, renal cell cancer, adenocarcinoma, hypernephroma, fibrosarcoma, transitional cell cancer (renal pelvis and/ or uterer); Wilms’ tumor; bladder cancers including, but not limited to, transitional cell carcinoma, squamous cell cancer, adenocarcinoma, and carcinosarcoma. For a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia and Murphy et al., 1997, Informed Decisions: The Complete Book of Cancer Diagnosis, Treatment, and Recovery, Viking Penguin, Penguin Books U.S.A., Inc., United States of America). 2. Other Disease or Disorders

In some forms, the methods administer the described genetically modified NK cells and/or CAR-NK cells to treat one or more non-cancer disease or disorder in a subject in need thereof. For example, in some forms the methods treat one or more genetic disease or disorders in a subject, such as a hereditary genetic disease or disorder, or a somatic genetic disease or disorder in a subject. In some forms, the methods administer genetically modified NK cells and/or CAR-NK cells to a subject to treat or prevent an autoimmune disease or disorder in the subject.

Any of the methods can include treating a subject having an underlying disease or disorder. For example, in some forms, the methods treat a disease or disorder, such as a cancer or auto-immune disease in a patient having another disease or disorder, such as diabetes, a bacterial infection (e.g., Tuberculosis), viral infection (e.g., Hepatitis, HIV, HPV infection, etc.), or a drug-associated disease or disorder. In some forms, the methods treat an immunocompromised subject. In some forms, the methods treat a subject having a disease of the kidney, liver, heart, lung, brain, bladder, reproductive system, bowel/intestines, stomach, bones or skin.

B. Effective Amounts

The effective amount, or therapeutically effective amount of a pharmaceutical composition including modified cells, such as therapeutic NK cells and/or CAR NK cells, is generally a dosage sufficient to treat, inhibit, or alleviate one or more symptoms of a disease or disorder, such as a cancer or autoimmune disease, or to otherwise provide a desired pharmacologic and/or physiologic effect, for example, reducing, inhibiting, or reversing one or more of the underlying pathophysiological mechanisms underlying a disease or disorder, such as cancer or autoimmune disease in a subject in need thereof.

In some forms, when administration of the pharmaceutical compositions including modified cells, such as therapeutic NK cells and/or CAR NK cells, elicits an anti-cancer response, the amount administered can be expressed as the amount effective to achieve a desired anti-cancer effect in the recipient. For example, in some forms, the amount of the pharmaceutical compositions including the described genetically modified NK cells and/or CAR NK cells, is effective to inhibit the viability or proliferation of cancer cells in the recipient. In some forms, the amount of the pharmaceutical composition including modified cells, such as therapeutic NK cells and/or CAR NK cells, is effective to reduce the tumor burden in the recipient, or reduce the total number of cancer cells, and combinations thereof. In other forms, the amount of the pharmaceutical compositions including modified cells, such as therapeutic NK cells and/or CAR NK cells, is effective to reduce one or more symptoms or signs of cancer in a cancer patient, or signs of an autoimmune disease in a patient having an autoimmune disease or disorder. Signs of cancer can include cancer markers. Cancer markers, and methods for the detection thereof, are known in the art. An exemplary marker is the amount or presence of Prostate-specific membrane antigen (PSMA) detected in the blood of a subject as indicative of whether the subject has, or is at increased risk of prostate cancer.

The effective amount of the pharmaceutical compositions including modified cells, such as therapeutic NK cells and/or CAR NK cells, that is required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the disorder being treated, and its mode of administration. Thus, it is not possible to specify an exact amount for every pharmaceutical composition. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein. For example, effective dosages and schedules for administering the pharmaceutical compositions including therapeutic NK cells and/or CAR NK cells can be determined empirically, and making such determinations is within the skill in the art. In some forms, the dosage ranges for the administration of the compositions including the described genetically-modified therapeutic NK cells and/or CAR NK cells are those large enough to effect reduction in cancer cell proliferation or viability, or to reduce tumor burden for example.

Generally, the dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. In some forms, the dosage will vary with the age, condition, and sex of the patient, route of administration, whether other drugs are included in the regimen, and the type, stage, and location of the disease to be treated. Typically, the dosage is adjusted by an individual physician in the event of any counterindications. It will also be appreciated that the effective dosage of the composition including therapeutic NK cells and/or CAR NK cells used for treatment can increase or decrease over the course of a particular treatment. Changes in dosage can result and become apparent from the results of diagnostic assays.

Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the subject or patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages can vary depending on the relative potency of individual pharmaceutical compositions and can generally be estimated based on ECsos found to be effective in in vitro and in vivo animal models.

It can generally be stated that a pharmaceutical composition containing NK cells and/or CAR NK cells described herein can be administered at a dosage of 10⁴ to 10⁹ cells/kg body weight, preferably IO⁵ to 10⁷ cells/kg body weight, including all integer values within those ranges. In some forms, patients can be treated by infusing a disclosed pharmaceutical composition containing CAR expressing cells (e.g., genetically modified engineered to up- regulate and/or over-express at least one gene selected from SGSM2, OR7A10, APLN, PDP1, GABBR1, PRR14L, TIAM1, KRT82, PLA2G1B, REM2, HIST1H2BN, CYB5B, LRRC23, NXPE3, MEGF11, FKBP5, PPFIA2, LRRC23, PEAR1, REM2, TIAM2, HPRT1, MMACHC, ZBTB20, IL- 15, and IL21 as compared to a control, such as a non-genetically modified control NK cell or CAR NK cell) in the range of about 10⁴ to 10¹² or more cells per square meter of body surface (cells/m).

The infusion can be repeated as often and as many times as the patient can tolerate until the desired response is achieved. Compositions of NK cells and/or CAR NK cells can also be administered once or multiple times at these dosages. The cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly. In some forms, the unit dosage is in a unit dosage form for intravenous injection. In some forms, the unit dosage is in a unit dosage form for oral administration. In some forms, the unit dosage is in a unit dosage form for inhalation. In some forms, the unit dosage is in a unit dosage form for intra-tumoral injection.

Treatment can be continued for an amount of time sufficient to achieve one or more desired therapeutic goals, for example, a reduction of the amount of cancer cells relative to the start of treatment, or complete absence of cancer cells in the recipient. Treatment can be continued for a desired period of time, and the progression of treatment can be monitored using any means known for monitoring the progression of anti-cancer treatment in a patient. In some forms, administration is carried out every day of treatment, or every week, or every fraction of a week. In some forms, treatment regimens are carried out over the course of up to two, three, four or five days, weeks, or months, or for up to 6 months, or for more than 6 months, for example, up to one year, two years, three years, or up to five years.

The efficacy of administration of a particular dose of the pharmaceutical compositions including modified cells, such as therapeutic genetically-modified NK cells, according to the methods described herein can be determined by evaluating the aspects of the medical history, signs, symptoms, and objective laboratory tests that are known to be useful in evaluating the status of a subject in need for the treatment of cancer or other diseases and/or conditions. These signs, symptoms, and objective laboratory tests will vary, depending upon the particular disease or condition being treated or prevented, as will be known to any clinician who treats such patients or a researcher conducting experimentation in this field. For example, if, based on a comparison with an appropriate control group and/or knowledge of the normal progression of the disease in the general population or the particular individual: (1) a subject’s physical condition is shown to be improved (e.g., a tumor has partially or fully regressed), (2) the progression of the disease or condition is shown to be stabilized, or slowed, or reversed, or (3) the need for other medications for treating the disease or condition is lessened or obviated, then a particular treatment regimen will be considered efficacious. In some forms, efficacy is assessed as a measure of the reduction in tumor volume and/or tumor mass at a specific time point (e.g., 1-5 days, weeks, or months) following treatment.

C. Modes of Administration

Any of the disclosed genetically modified cells (e.g., genetically modified NK or CAR NK cells engineered to up-regulate and/or over-express at least one gene selected from SGSM2, OR7A10, APLN, PDP1, GABBR1, PRR14L, TIAM1, KRT82, PLA2G1B, REM2, HIST1H2BN, CYB5B, LRRC23, NXPE3, MEGF11, FKBP5, PPFIA2, LRRC23, PEAR1, REM2, TIAM2, HPRT1, MMACHC, ZBTB20 IL15, and IL21 as compared to a control, such as a non-genetically modified control NK cell or CAR NK cell) can be used therapeutically in combination with a pharmaceutically acceptable carrier.

The compositions described herein can be conveniently formulated into pharmaceutical compositions composed of one or more of the compounds in association with a pharmaceutically acceptable carrier. See, e.g., Remington's Pharmaceutical Sciences, latest edition, by E.W. Martin Mack Pub. Co., Easton, PA, which discloses typical carriers and conventional methods of preparing pharmaceutical compositions that can be used in conjunction with the preparation of formulations of the therapeutics described herein and which is incorporated by reference herein. These most typically would be standard carriers for administration of compositions to humans. In one aspect, for humans and non-humans, these include solutions such as sterile water, saline, and buffered solutions at physiological pH. Other therapeutics can be administered according to standard procedures used by those skilled in the art.

The pharmaceutical compositions including modified cells, such as genetically modified NK cells and/or CAR NK cells, described herein can include, but are not limited to, carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the therapeutic(s) of choice.

Pharmaceutical compositions containing one or more modified cells, such as therapeutic T cells, and optionally one or more additional therapeutic agents can be administered to the subject in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Thus, for example, a pharmaceutical composition including modified cells, such as therapeutic NK cells and/or CAR NK cells, can be administered as an intravenous infusion, or directly injected into a specific site, for example, into or surrounding a tumor. Moreover, a pharmaceutical composition can be administered to a subject as an ophthalmic solution and/or ointment to the surface of the eye, vaginally, rectally, intranasally, orally, by inhalation, or parenterally, for example, by intradermal, subcutaneous, intramuscular, intraperitoneal, intrarectal, intraarterial, intralymphatic, intravenous, intrathecal and intratracheal routes. In some forms, the compositions are administered directly into a tumor or tissue, e.g., stereotactically.

Parenteral administration, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. A more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Patent No. 3,610,795, which is incorporated by reference herein. Suitable parenteral administration routes include intravascular administration (e.g., intravenous bolus injection, intravenous infusion, intra-arterial bolus injection, intra-arterial infusion and catheter instillation into the vasculature); peri- and intratissue injection e.g., intraocular injection, intra-retinal injection, or sub-retinal injection); subcutaneous injection or deposition including subcutaneous infusion (such as by osmotic pumps); direct application by a catheter or other placement device (e.g., an implant including a porous, non-porous, or gelatinous material).

Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions which can also contain buffers, diluents and other suitable additives. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives can also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

Administration of the pharmaceutical compositions containing one or more genetically modified cells (e.g., genetically modified NK or CAR NK cells engineered to up-regulate and/or over-express at least one gene selected from SGSM2, OR7A10, APLN, PDP1, GABBR1, PRR14L, TIAM1, KRT82, PLA2G1B, REM2, HIST1H2BN, CYB5B, LRRC23, NXPE3, MEGF11, FKBP5, PPFIA2, LRRC23, PEAR1, REM2, TIAM2, HPRT1, MMACHC, ZBTB20 IL-15, and IL- 61 21, as compared to a control, such as a non-genetically modified control NK cell or CAR NK cell) can be localized (i.e., to a particular region, physiological system, tissue, organ, or cell type) or systemic.

It is to be understood that the disclosed method and compositions are not limited to specific synthetic methods, specific analytical techniques, or to particular reagents unless otherwise specified, and, as such, can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular forms only and is not intended to be limiting.

D. Combination therapy

Any of the disclosed pharmaceutical compositions including modified cells, such as therapeutic NK cells (e.g., genetically modified NK or CAR NK cells engineered to up-regulate and/or over-express at least one gene selected from SGSM2, OR7A10, APLN, PDP1, GABBR1, PRR14L, TIAM1, KRT82, PLA2G1B, REM2, HIST1H2BN, CYB5B, LRRC23, NXPE3, MEGF11, FKBP5, PPFIA2, LRRC23, PEAR1, REM2, TIAM2, HPRT1, MMACHC, ZBTB20, IL-15, and IL- 21 as compared to a control, such as a non-genetically modified control NK cell or CAR NK cell), can be used alone, or in combination with other therapeutic agents or treatment modalities, for example, chemotherapy or stem-cell transplantation. As used herein, “combination” or “combined” refer to either concomitant, simultaneous, or sequential administration of the therapeutics.

In some embodiments, IL- 15 and/or IL-21 are administered to the subject in combination with an effective amount of disclosed modified NK cells.

In some forms, two or more pharmaceutical compositions and/or other therapeutic agents are administered separately through the same route of administration. In other forms, two or more pharmaceutical compositions and other therapeutic agents are administered separately through different routes of administration. The combinations can be administered either concomitantly (e.g., as an admixture), separately but simultaneously (e.g., via separate intravenous lines into the same subject; one agent is given orally while the other agent is given by infusion or injection, etc.,), or sequentially (e.g., one agent is given first followed by the second).

Examples of preferred additional therapeutic agents include other conventional therapies known in the art for treating the desired disease, disorder or condition. In some forms, the therapeutic agent is one or more other targeted therapies (e.g., a targeted cancer therapy) and/or immune-checkpoint blockage agents e.g., anti-CTLA-4, anti-PDl, and/or anti-PDLl agents such as antibodies).

The compositions and methods described herein may be used as a first therapy, second therapy, third therapy, or combination therapy with other types of therapies known in the art, such as chemotherapy, surgery, radiation, gene therapy, immunotherapy, bone marrow transplantation, stem cell transplantation, targeted therapy, cryotherapy, ultrasound therapy, photodynamic therapy, radio-frequency ablation or the like, in an adjuvant setting or a neoadjuvant setting.

The disclosed pharmaceutical compositions and/or other therapeutic agents, procedures or modalities can be administered during periods of active disease, or during a period of remission or less active disease. The pharmaceutical compositions can be administered before the additional treatment, concurrently with the treatment, post-treatment, or during remission of the disease or disorder. When administered in combination, the disclosed pharmaceutical compositions and the additional therapeutic agents (e.g., second or third agent), or all, can be administered in an amount or dose that is higher, lower or the same than the amount or dosage of each agent used individually, e.g., as a monotherapy. In certain forms, the administered amount or dosage of the disclosed pharmaceutical composition, the additional therapeutic agent (e.g., second or third agent), or all, is lower e.g., at least 20%, at least 30%, at least 40%, or at least 50%) than the amount or dosage of each agent used individually, e.g., as a monotherapy (e.g., required to achieve the same therapeutic effect).

1. Cytokines IL-15 and/or IL-21

In some forms, the methods including administering genetically modified NK or CAR NK cells e.g., genetically modified NK or CAR NK cells engineered to up-regulate and/or overexpress at least one gene selected from SGSM2, OR7A10, APLN, PDP1, GABBR1, PRR14L, TIAM1, KRT82, PLA2G1B, REM2, HIST1H2BN, CYB5B, LRRC23, NXPE3, MEGF11, FKBP5, PPFIA2, LRRC23, PEAR1, REM2, TIAM2, HPRT1, MMACHC, ZBTB20, IL15 and/or IL21 , as compared to a control, such as a non-genetically modified control NK cell or CAR NK cell in combination with one or more additional cytokines, such as Interleukin 15 (IL- 15) and/or Interleukin-21 (IL-21) to a subject.

In some forms, the methods include one or more steps of administering at least one cytokine to the subject in addition to the described genetically-modified NK cells. For example, in some forms, the methods include one or more steps of administering IL- 15 to the subject in addition to the described genetically-modified NK cells. In other forms, the methods include one or more steps of administering IL-21 to the subject in addition to the described genetically- modified NK cells. In some forms, the methods include one or more steps of administering IL- 15 and IL-21 to the subject in addition to the described genetically-modified NK cells. When the methods administer IL-15 and/or IL-21 to the subject, the administration can be together in one composition, or separately, as two or more different compositions, for example, administered to the subject as two or more different composition, with each administration being simultaneous or separated by a period of time of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 minutes, hours, days or weeks.

In some forms, the methods include administering a formulation including engineered NK cells that over-express or up-regulate one or more genes including SGSM2, OR7A10, APLN, PDP1, GABBR1, PRR14L, TIAM1, KRT82, PLA2G1B, REM2, HIST1H2BN, CYB5B, LRRC23, NXPE3, MEGF11, FKBP5, PPFIA2, LRRC23, PEAR1, REM2, TIAM2, HPRT1, MMACHC, ZBTB20, IL15 and/or IL21, relative to a wild-type NK cell, and/or which express a CAR, in combination with one or more compositions including an effective amount of IL- 15 and/or IL-21 to increase the anti-tumor activity of the NK or CAR-NK cells. For example, in some forms, a formulation of the described genetically engineered NK cells that express a CAR and/or overexpress or up-regulate one or more genes including SGSM2, OR7A10, APLN, PDP1, GABBR1, PRR14L, TIAM1, KRT82, PLA2G1B, REM2, HIST1H2BN, CYB5B, LRRC23, NXPE3, MEGF11, FKBP5, PPFIA2, LRRC23, PEAR1, REM2, TIAM2, HPRT1, MMACHC, ZBTB20, IL15 and/or IL2I relative to a wild- type NK cell is administered in combination with one or more formulations that include IL-21 at a concentration of about 10 ng/ml IL-21, and IL- 15 at a concentration of about 2.5 ng/ml. In some forms, the formulations are administered to a subject together. In other forms, the formulations are administered to a subject at different times, for example, separated by one or more minutes, hours, days, or weeks.

2. Additional anti-cancer agents

In some forms, the methods administer genetically modified NK or CAR NK cells e.g., genetically modified NK or CAR NK cells engineered to up-regulate and/or over-express at least one gene selected from SGSM2, OR7A10, APLN, PDPI, GABBR1, PRR14L, TIAM1, KRT82, PLA2G1B, REM2, HIST1H2BN, CYB5B, LRRC23, NXPE3, MEGF11, FKBP5, PPFIA2, LRRC23, PEAR1, REM2, TIAM2, HPRT1, MMACHC, ZBTB20, IL15 and/or IL21 , as compared to a control, such as a non-genetically modified control NK cell or CAR NK cell in combination with one or more additional anti-cancer agents to a subject.

In the context of cancer, targeted therapies are therapeutic agents that block the growth and spread of cancer by interfering with specific molecules ("molecular targets") that are involved in the growth, progression, and spread of cancer. Many different targeted therapies have been approved for use in cancer treatment. These therapies include hormone therapies, signal transduction inhibitors, gene expression modulators, apoptosis inducers, angiogenesis inhibitors, immunotherapies, and toxin delivery molecules. Numerous antineoplastic drugs can be used in combination with the disclosed pharmaceutical compositions. In some forms, the additional therapeutic agent is a chemotherapeutic or antineoplastic drug. The majority of chemotherapeutic drugs can be divided into alkylating agents, antimetabolites, anthracy clines, plant alkaloids, topoisomerase inhibitors, monoclonal antibodies, and other anti-tumor agents.

3. Additional therapeutic agents against Autoimmune diseases

In some forms, the methods administer genetically modified NK or CAR NK cells e.g., genetically modified NK or CAR NK cells engineered to up-regulate and/or over-express at least one gene selected from SGSM2, OR7A10, APLN, PDP1, GABBR1, PRR14L, TIAM1, KRT82, PLA2G1B, REM2, HIST1H2BN, CYB5B, LRRC23, NXPE3, MEGF11, FKBP5, PPFIA2, LRRC23, PEAR1, REM2, TIAM2, HPRT1, MMACHC, ZBTB20, IL15 and/or IL21, as compared to a control, such as a non-genetically modified control NK cell or CAR NK cell in combination with one or more conventional therapies for autoimmune diseases to the subject.

Exemplary therapies for autoimmune diseases include immunosuppressive agents, such as steroids or cytostatic drugs, analgesics, non-steroidal anti-inflammatory drugs, glucocorticoids, immunosuppressive and immunomodulatory agents, such as methotrexate, leflunomide, hydroxychloroquine, and sulfasalazine. In some forms, the methods administer one or more disease-modifying antirheumatic drugs (DMARDs). In some forms, the methods administer one or more biologic agents for localized treatment (i.e., agents that do not affect the entire immune system), such as TNF-a inhibitors, belimumab and rituximab depleting B cells, T-cell co-stimulation blocker, anti-interleukin 6 (IL-6), anti-IL-1, and protein kinase inhibitors. In other forms, the methods also administer one or more monoclonal antibodies (mAbs), such as anti-TNFa, anti-CD19, anti-CD20, anti-CD22, and anti-IL6R, or other mAbs that target multiple B cell subtypes, and other aberrant cells in autoimmune diseases.

V. Kits

Compositions, reagents, and other materials for cellular genomic engineering and/or therapy can be packaged together in any suitable combination as a kit useful for performing, or aiding in the performance of, the methods. It is useful if the components in a kit are designed and adapted for use together in the method. For example, kits with one or more compositions for administration to a subject, may include a pre-measured dosage of the composition in a sterile needle, ampule, tube, container, or other suitable vessel. The kits may include instructions for dosages and dosing regimens.

Provided are kits containing an sgRNA library, for example, including a multiplicity of RNAs having a spacer and tracrRNA backbone, the tracrRNA including one or more sequences engineered to mediate efficient transcriptional activation of at least one gene selected from SGSM2, OR7A10, APLN, PDP1, GABBR1, PRR14L, TIAM1, KRT82, PLA2G1B, REM2, HIST1H2BN, CYB5B, LRRC23, NXPE3, MEGF11, FKBP5, PPFIA2, LRRC23, PEAR1, REM2, TIAM2, HPRT1, MMACHC, ZBTB20, IL15 and/or IL21. Optionally the kits include one or more CRISPR NK cell vectors for efficient gene editing and high-throughput screening in NK cells, and instructional material for use thereof.

In preferred forms, the kit includes a plurality of vectors, where each vector independently contains a single sgRNA having a spacer and tracrRNA backbone. In some forms, the kit contains a population of NK cells (e.g., naive NK cells or CAR NK cells). The instructional material can include a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the compositions and methods of the kit. For example, the instructional material may provide instructions for methods using the kit components, such as performing transfections, transductions, infections, and conducting screens.

In some forms, the kit includes a transposon including a CAR that is specific for an antigen that is selected from a cancer antigen selected from 4 IBB, 5T4, adenocarcinoma antigen, alpha fetoprotein, BAFF, B lymphoma cell, C242 antigen, CA 125, carbonic anhydrase 9 (CA IX), C MET, CCR4, CD 152, CD 19, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44 v6, CD51, CD52, CD56, CD74, CD80, CEA, CNTO888, CTLA 4, DR5, EGFR, EpCAM, CD3, FAP, fibronectin extra domain B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HER2/neu, HGF, human scatter factor receptor kinase, IGF 1 receptor, IGF I, IgGl, LI CAM, IL 13, IL 6, insulin-like growth factor I receptor, integrin a5pi, integrin avP3, MORAb 009, MS4A1, MUC1, mucin CanAg, N glycolylneuraminic acid, NPC 1C, PDGF R a, PDL192, phosphatidylserine, prostatic carcinoma cells, RANKL, RON, ROR1, SCH 900105, SDC1, SLAMF7, TAG 72, tenascin C, TGF beta 2, TGF p, TRAIL Rl, TRAIL R2, tumor antigen CTAA16.88, VEGF A, VEGFR 1, VEGFR2, and vimentin; in some forms, the CAR is bispecific or multivalent; in some forms, the CAR is anti HER2.

The disclosed compositions and methods can be further understood through the following numbered paragraphs.

1. A genetically modified Natural Killer (NK) cell, wherein

(i) at least one gene selected from the group including SGSM2, OR7A10, APLN, PDP1, GABBR1, PRR14L, TIAM1, KRT82, PLA2G1B, REM2, HIST1H2BN, CYB5B, LRRC23, NXPE3, MEGF11, FKBP5, PPFIA2, LRRC23, PEAR1, REM2, TIAM2, HPRT1, MMACHC, and ZBTB20 is up-regulated or over-expressed in the cell, as compared to a non-genetically modified control NK cell; and/or

(ii) at least one cytokine selected from Interleukin- 15 (IL- 15) and Interleukin-21 (IL- 21) is up-regulated or over-expressed by the cell. 2. The genetically modified Natural Killer (NK) cell of paragraph 1, wherein the modification enhances the anti-cancer efficacy of the NK cell as compared to a non-genetically modified control NK cell.

3. The genetically modified NK cell of paragraph 1 or 2, wherein the modification causes increased or enhanced expression, activation, presentation, and/or function of one or more protein(s) encoded by the gene(s).

4. The genetically modified NK cell of paragraph 1 or 2, wherein the modification causes increased or enhanced expression of one or more of the genes SGSM2, OR7A10, APLN, PDP1 and CYB5B and/or the full-length protein(s) encoded by the gene(s) SGSM2, OR7A10, APLN, PDP1, and CYB5B.

5. The genetically modified NK cell of any one of paragraphs 1-4, wherein the modification includes recombinant expression of the SGSM2 gene.

6. The genetically modified NK cell of paragraph 5, wherein the recombinant expression causes increased or enhanced expression of the SGSM2 gene and/or the full-length protein encoded by the SGSM2 gene.

7. The genetically modified NK cell of any one of paragraphs 1-4, wherein the modification includes recombinant expression of the OR7A10 gene.

8. The genetically modified NK cell of paragraph 7, wherein the recombinant expression causes increased or enhanced expression of the OR7A10 gene and/or the full-length protein encoded by the OR7A10 gene.

9. The genetically modified NK cell of any one of paragraphs 1-4, wherein the modification includes recombinant expression of the APLN gene.

10. The genetically modified NK cell of paragraph 9, wherein the recombinant expression causes increased or enhanced expression of the APLN gene and/or the full-length protein encoded by the APLN gene.

11. The genetically modified NK cell of any one of paragraphs 1-4, wherein the modification includes recombinant expression of the PDP1 gene.

12. The genetically modified NK cell of paragraph 11, wherein the recombinant expression causes increased or enhanced expression of the PDP1 gene and/or the full-length protein encoded by the PDP1 gene.

13. The genetically modified NK cell of any one of paragraphs 1-4, wherein the modification includes recombinant expression of the CYB5B gene.

14. The genetically modified NK cell of paragraph 13, wherein the recombinant expression causes increased or enhanced expression of the CYB5B gene and/or the full-length protein encoded by the CYB5B gene. 15. The genetically modified NK cell of any one of paragraphs 1-14, wherein the genetically modified NK cell expresses 11-15 and 11-21 in addition to enhanced or up-regulated expression of the SGSM2 gene, and/or the OR7A10 gene, and/or the APLN gene, and/or the PDP1 gene, and/or the CYB5B gene.

16. The genetically modified NK cell of any one of paragraphs 1-15, further including at least one additional genetic modification.

17. The genetically modified NK cell of any one of paragraphs 1-16, wherein the NK cell expresses or encodes a Chimeric Antigen Receptor (CAR).

18. The genetically modified NK cell of paragraph 17, wherein the CAR targets a cancer antigen.

19. The genetically modified NK cell of paragraph 18, wherein the cancer antigen is a neoantigen derived from a subject.

20. The genetically modified NK cell of paragraph 19, wherein the cancer antigen is selected from the group including ENPP3, 4-1BB, 5T4, adenocarcinoma antigen, alpha fetoprotein, BAFF, B lymphoma cell, C242 antigen, CA 125, carbonic anhydrase 9 (CA IX), C- MET, CCR4, CD 152, CD 19, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44 v6, CD51, CD52, CD56, CD74, CD80, CEA, CNTO888, CTLA 4, DR5, EGFR, EpCAM, CD3, FAP, fibronectin extra domain B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HER2/neu, HGF, human scatter factor receptor kinase, IGF 1 receptor, IGF I, IgGl, LI CAM, IL 13, IL 6, insulin-like growth factor I receptor, integrin a5pi, integrin avP3, MORAb 009, MS4A1, MUC1, mucin CanAg, N glycolylneuraminic acid, NPC 1C, PDGF R a, PDL192, phosphatidylserine, prostatic carcinoma cells, RANKL, RON, ROR1, SCH 900105, SDC1, SLAMF7, TAG 72, tenascin C, TGF beta 2, TGF p, TRAIL Rl, TRAIL R2, tumor antigen CTAA16.88, VEGF A, VEGFR 1, VEGFR2, and vimentin.

21. The genetically modified NK cell of any one of paragraphs 1-20, wherein the NK is derived from a subject diagnosed as having, or who is identified as being at increased risk of having a disease or disorder.

22. The genetically modified NK cell of paragraph 21, wherein the subject is diagnosed as having, or is identified as being at increased risk of having cancer.

23. The genetically modified NK cell of any one of paragraphs 1-20, wherein the NK is derived from a healthy subject prior to the genetic modification.

24. The genetically modified NK cell of any one of paragraphs 1-23, wherein the modification causes increased or enhanced expression, activation, presentation, and/or function of one or more cytokine(s), as compared to a non-genetically modified control NK cell. 25. The genetically modified NK cell of paragraph 24, wherein at least one of IL- 15 and IL-21 is recombinantly expressed in the cell.

26. The genetically modified NK cell of paragraph 24 or 25, wherein IL-15 is recombinantly expressed in the cell.

27. The genetically modified NK cell of paragraph 24 or 25, wherein IL-21 is recombinantly expressed in the cell.

28. The genetically modified NK cell of paragraph 24 or 25, wherein IL-15 and IL-21 are both recombinantly expressed in the cell.

29. A population of NK cells derived by expanding the genetically modified NK cell of any one of paragraphs 1-28.

30. A pharmaceutical composition including the population of NK cells of paragraph 29 and a pharmaceutically acceptable buffer, carrier, diluent or excipient for administration in vivo.

31. The pharmaceutical composition of paragraph 30, further including at least one cytokine.

32. The pharmaceutical composition of paragraph 31, wherein the cytokine includes IL- 15.

33. The pharmaceutical composition of paragraph 31, wherein the cytokine includes IL- 21.

34. The pharmaceutical composition of paragraph 31, wherein the cytokine includes 1L- 15 and IL-21.

35. A method of treating a subject having a disease, disorder, or condition including administering to the subject an effective amount of the pharmaceutical composition of any one of paragraphs 30-34.

36. A method of treating a subject having a disease, disorder, or condition associated with an elevated expression or specific expression of an antigen, the method including administering to the subject an effective amount of the population of genetically modified NK cells of paragraph 29, wherein the NK cells include a CAR that targets the antigen.

37. A method of treating cancer in a subject in need thereof, including administering to the subject an effective amount of a population of genetically modified NK cells, wherein at least one gene selected from the group including SGSM2, OR7A10, APLN, PDP1, GABBR1, PRR14L, TIAM1, KRT82, PLA2G1B, REM2, HIST1H2BN, CYB5B, LRRC23, NXPE3, MEGF11, FKBP5, PPFIA2, LRRC23, PEAR1, REM2, TIAM2, HPRT1, MMACHC, and ZBTB20, is up-regulated or over-expressed in the NK cell, as compared to a non-genetically modified control NK cell; and/or at least one cytokine selected from Interleukin- 15 (11-15) and Interleukin-21 (11-21) is expressed by the cell.

38. The method of paragraph 37, wherein the genetically modified NK cells include increased or enhanced expression of one or more of the genes and/or the full-length protein(s) encoded by the gene(s) SGSM2, OR7A10, APLN, PDP1 and CYB5B as compared to a non- genetically modified control NK cell, and wherein the modification enhances the anti-cancer efficacy of the NK cell as compared to a non-genetically modified control NK cell.

39. The method of paragraph 37 or 38, wherein the genetically modified NK cells include recombinant expression of the SGSM2 gene and/or the functional protein encoded by the SGSM2 gene.

40. The method of paragraph 37 or 38, wherein the genetically modified NK cells include recombinant expression of the OR7A10 gene and/or the functional protein encoded by the OR7A10 gene.

41. The method of any one of paragraphs 37 to 40, wherein the NK cell is genetically modified to express or encode a Chimeric Antigen Receptor (CAR).

42. The method of paragraph 41, wherein the CAR targets an antigen expressed by the cancer.

43. The method of any one of paragraphs 37 to 42, wherein the cancer is selected from the group including leukemia, vascular cancer such as multiple myeloma, adenocarcinomas and bone, bladder, brain, breast, cervical, ovarian, colorectal, esophageal, kidney, liver, lung, nasopharangeal, pancreatic, prostate, skin, stomach, and uterine cancer.

44. The method of any one of paragraphs 37 to 43, wherein the cancer is breast cancer, lung cancer, colorectal cancer, ovarian cancer, or skin cancer.

45. The method of any one of paragraphs 37 to 44, wherein the NK cell is genetically modified to express at least one recombinant cytokine selected from the group including IL- 15, and IL-21.

46. The method of any one of paragraphs 37 to 45, wherein the NK cells are derived from the subject prior to genetic modification.

47. The method of any one of paragraphs 37 to 46, further including administering to the subject cytokine IL- 15, or cytokine IL-21, or both cytokine IL- 15 and cytokine IL-21.

48. The method of any one of paragraphs 37 to 47, wherein the administration includes injection of the composition of cells into or directly adjacent to a tumor, or into the blood stream, or into the brain or into a ventricle of the heart of the subject. 49. The method of any one of paragraphs 37 to 48, further including administering to the subject one or more additional therapeutic agents and/or procedures.

50. The method of paragraph 49, wherein the additional therapeutic agent and/or procedure is selected from the group including a chemotherapeutic agent, an antimicrobial agent, an immune checkpoint inhibitor, a PD-I inhibitor, a CTLA-4 inhibitor, radiation treatment and surgery.

51. A method of performing gain of function screening of a Natural Killer (NK) cell, the method including:

(i) transducing an NK cell with one or more CRISPRa single-guide RNA(s) (sgRNAs),

(ii) causing the NK cell to be genetically modified by CRISPRa-mediated genome editing of a gene targeted by the sgRNA; and

(iii) screening the NK cell for tumor cell killing.

52. The method of paragraph 51, wherein one sgRNA includes

(i) a guide sequence; and

(ii) a tracrRNA including a nucleic acid sequence selected from a library.

53. The method of paragraph 52, wherein the library includes all or part of a human genomic reference sequence library.

54. The method of any one of paragraphs 51-53, wherein the sgRNA is included within a vector.

55. The method of paragraph 54, wherein the vector is a lentiviral vector.

56. The method of paragraph 54 or 55, wherein the vector further includes an expression cassette for the sgRNA.

57. The method of paragraph 56, wherein the expression cassette further includes a nucleic acid construct configured to express or encode a chimeric antigen receptor (CAR).

58. The method of any one of paragraphs 51-57, wherein steps (i)-(iii) are carried out using a plurality of NK cells, and wherein each of the plurality of NK cells is contacted by one or more sgRNAs including one or more sequences of a library of sequences.

59. The method of paragraph 58, wherein the plurality of NK cells is collectively contacted by a multiplicity of sgRNAs, wherein an sgRNA of the multiplicity of sgRNAs includes a single sequence from the library, and wherein an NK cell of the plurality of NK cells contacted by an sgRNA over-expresses a single gene, relative to a control NK cell that is not contacted by the sgRNA. 60. The method of any one of paragraphs 51-59, wherein screening the NK cell for tumor cell killing is carried out in vitro.

61. The method of any one of paragraphs 51-59, wherein screening the NK cell for tumor cell killing is carried out in vivo.

62. The method of paragraph 61, wherein the in vivo screening is carried out using a tumor-bearing animal model, and wherein the screening includes selecting genetically modified NK cells from animals with enhanced survival/reduced tumor burden as compared to control animals that did not receive the same genetically modified NK cells.

63. The method of any one of paragraphs 51-62, further including characterizing the mutant NK cell(s) by single cell transcriptome analysis.

64. The method of any one of paragraphs 51-63, further including characterizing the mutant NK cell(s) by sequence analysis to identify mutated genes.

65. The method of any one of paragraphs 51-64, further including repeating steps (i)-(iii) using a selected pool of sgRNAs for one or more additional rounds.

66. A genetically modified NK cell created according to the method of any one of paragraphs 51-65.

67. A pharmaceutical composition including

(i) a population of genetically modified NK cells derived by expanding the genetically modified NK cell of paragraph 66; and

(ii) a pharmaceutically acceptable excipient for administration in vivo.

68. A genetically modified Natural Killer (NK) cell including a mutation that causes up- regulated or enhanced expression of one or more genes selected from the group including SGSM2, OR7A10, APLN, PDP1, and CYB5B and/or the functional protein encoded by one or more genes selected from the group including SGSM2, OR7A10, APLN, PDP1 and CYB5B in the cell as compared to a non-genetically modified NK cell, wherein the mutation enhances the anticancer efficacy of the genetically modified NK cell as compared to a non-genetically modified NK cell, and wherein the genetically modified NK cell expresses or encodes a Chimeric Antigen Receptor (CAR) that targets a cancer antigen.

69. The genetically modified NK cell of paragraph 68, wherein the cancer antigen is selected from the group including ENPP3, 4-1BB, 5T4, adenocarcinoma antigen, alpha fetoprotein, BAFF, B lymphoma cell, C242 antigen, CA 125, carbonic anhydrase 9 (CA IX), C- MET, CCR4, CD 152, CD 19, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44 v6, CD51, CD52, CD56, CD74, CD80, CEA, CNTO888, CTLA 4, DR5, EGFR, EpCAM, CD3, FAP, fibronectin extra domain B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HER2/neu, HGF, human scatter factor receptor kinase, IGF 1 receptor, IGF I, IgGl, El CAM, IE 13, IL 6, insulin-like growth factor I receptor, integrin a5pi, integrin avP3, MORAb 009, MS4A1, MUC1, mucin CanAg, N glycolylneuraminic acid, NPC 1C, PDGF R a, PDL192, phosphatidylserine, prostatic carcinoma cells, RANKL, RON, ROR1, SCH 900105, SDC1, SLAMF7, TAG 72, tenascin C, TGF beta 2, TGF p, TRAIL Rl, TRAIL R2, tumor antigen CTAA16.88, VEGF A, VEGFR 1, VEGFR2, and vimentin.

70. The genetically modified NK cell of paragraph 68 or 69, wherein the genetically modified NK has increased tumor penetration and/or increased anti-tumor cytotoxicity as compared to a non-genetically modified NK cell.

71. A genetically modified Natural Killer (NK) cell, wherein the SGSM2 gene is up- regulated and/or over-expressed in the cell, as compared to a non-genetically modified control NK cell, wherein the modification increases or enhances expression, activation, presentation, and/or function of one or more protein(s) encoded by the SGSM2 gene(s) and enhances the anticancer efficacy of the NK cell as compared to a non-genetically modified control NK cell, and wherein the modified NK cell expresses or encodes a Chimeric Antigen Receptor (CAR) that targets a cancer antigen.

The disclosure will be further understood by reference to the following examples.

EXAMPLES

Example 1: Genome-scale in vivo CRISPRa screen identified gene boosters that enhance CAR-NK anti-tumor efficacy

An in vivo genome-scale CAR-NK CRISPR activation (CRISPRa) screen has been developed to directly identify genes that, when overexpressed, could enhance the in vivo antitumor function of CAR-NK cells. To systematically identify genes that can be harnessed to engineer potent CAR-NK cells against solid tumor, an unbiased CRISPR activation (CRISPRa) screen was developed in human CAR-NK cells in vivo. This screen reveals several high-rank genes previously unlinked to NK or CAR-NK cells, whose gain-of-function (GOF) can augment the anti-tumor function of CAR-NK cells (hyper-boosters), as validated individually in CAR-NK cells against multiple types of cancer.

Methods

To systematically identify the genes that can enhance the in vivo anti-tumor efficacy of CAR-NK cells, a genome-scale in vivo GOF CAR-NK CRISPRa screen was developed, as depicted in FIG. 1A. Systematic interrogation of gene function requires the ability to modify gene expression in a robust and generalizable manner. Structure-guided engineering of a CRISPR-Cas9 complex to mediate efficient transcriptional activation at endogenous genomic loci was implemented (Konermann, et al. Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature 517, 583-588 (2015)). The engineered Cas9 activation complexes were implemented to investigate single-guide RNA (sgRNA) targeting for effective transcriptional activation, to upregulate activation of genes. A library including 70,290 guides targeting all human RefSeq coding isoforms was used to screen for genes that, upon activation, impart enhanced NK ant-tumor function.

Mouse models

Prior to all cancer-related experiments, each mouse was determined to be in good general health (BAR: bright, alert, and responsive). Female and male mice, aged 8-12 weeks, were used for all experiments. The NOD-scid IL2R-gamma-null (NSG) mice are used for this study. Each mouse strain was purchased from 28 JAX and bred in-house for in vivo tumor model experiments. All animal work was performed under the guidelines of Yale University Institutional Animal Care and Use Committee (IACUC) with approved protocols (Chen 2018- 20068; 2021-20068).

Cell lines

NK-92 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). NK-92 and CAR-NK92 cells were cultured in MEM-a (no nucleosides), supplemented with 2 mM L-glutamine, 0.2 mM myo-inositol, 0.02 mM folic acid, 0.1 rnM 2- mercaptoethanol, 200 lU/ml human recombinant IL-2 (Biolegend), 10% FBS, 10% horse serum, and 1% penicillin/streptomycin (Gibco, Life Technologies, America). 293T human embryonic kidney (HEK) cells, HT29, MCF7, MDA-MB-231, SKOV3, A-375, and NCLH1299 were bought from ATCC and cultured in DMEM, supplemented with 10% FBS and 1% penicillin/streptomycin (D10 media). K-562 cells were bought from ATCC and cultured in RPMI medium (Gibco), supplemented with 10% FBS and 1% penicillin/streptomycin. HT29 and SKOV3 cells were infected with lenti virus with GFP-Lucif erase (pXD024 plasmid, GL) for the in vitro tumor killing assays. MCF7, MDA-MB-231, SKOV3, A-375, and H1299 cells were infected with lentivirus with Puromycin-Luciferase (pXD023 plasmid, PL) for the in vitro tumor killing assays. MCF7-HER2-PL cells were generated by infection with HER2-Blasticidin lentiviral vectors. Human PBMC was purchased from Stemcell Technologies.

Construct design

All lentiviral plasmids were designed using the 3^rd generation lentiviral backbone (Addgene, #75112). Different fragments were assembled using both Gibson assembly and conventional restriction cloning methods. Lentivirus production

Lentivirus was produced using low-passage HEK293T cells. One day before transfection, HEK293T cells were seeded in 15 cm-dish at 50-60 % confluency. Before transfection, DIO media was replaced with 20 mL fresh pre-warmed DIO media. For each plate, 20 pg transfer plasmid, 15 pg psPAX2 (Addgene), and 10 pg pMD2.G (Addgene) were diluted into 700 pL of DMEM. 135 pL LipoD293 (SignaGen) was diluted into 700 pL of DMEM. The diluted LipoD293 was immediately added to the diluted DNA solution all at once. After pipetting up and down for 3 times, the mixture was incubated for 10 min at room temperature and then added dropwise to the cells. After 12 hours of transfection, the media was replaced with 20 ml pre-warmed D10 media. Viral supernatant was collected at 48 hours post-transfection, filtered using 0.45 pm filters (Fisher/VWR) to remove cell debris, and then concentrated using Lenti-X Concentrator (Takara). Lenti viral pellets were resuspended with NK92 complete culture media, then aliquoted and stored at -80 °C.

Lenti- O-HER2-CAR-NK92 cell transduction

NK92 cells were transduced with lentivirus at l-2e6 cells / ml in a 12-well plate, which was pre-coated with Retronectin (Takara) in PBS, overnight at 4°C. The spin-infection was performed at 32 °C at 1 ,000 g for 45 min. The CAR-positive NK92 cells were selected with 3 pg/mL puromycin for 3 days and measured at day 7 after transduction. Then the CAR-NK92 cells were used for different assays.

Genome-scale in vivo Gain-of-function CAR-NK screen

The (X-HER2-CAR-NK92 cell were transduced with dCas9-VP64-Blast and MS2-P65- HSFl-Hygro at MOI < 1 and selected with Hygromycin and Blastcidin for 5-7 days. After complete selection, 1.8e8 CAR-NK92 cells were transduced with the sgRNA library at MOI = 0.2 (0.2 infectivity * 1.8e8 cells/70,290 sgRNAs > 500-fold coverage). After 7 days of selection with Zeocin, > 4e7 Library-infected CAR-NK92 cells were maintained (4e7 cells/70,290 sgRNAs > 500-fold coverage). Eight-to-twelve-week-old female NSG mice were inoculated with le6 HT29-GL cells through subcutaneous injection. On day 8, library-infected CAR-NK92 cells were adoptively transferred into tumor-bearing mice via intravenous injection. On 13-day post-transfer, mice were euthanized, and tumors were isolated.

Tissue processing and genomic DNA extraction

Genomic DNA was extracted from tumors and pre-injected cell pellets using the methods from a previously study⁴². Briefly, each sample was put in a 15 mL Falcon tube and had 6 mL NK Lysis Buffer (50 mM Tris, 50 mM EDTA, 1% SDS, pH adjusted to 8.0) and 30 pL of 20 mg/mL Proteinase K (Qiagen) added, and then incubated at 55 °C overnight. After tissue digestion, 30 pL of 10 mg/mL RNase A (Qiagen) was added to the lysed sample, which was then inverted 20 times and incubated at 37 °C for 30 min. Digested tissues were cooled on ice before adding 2 mL cold 7.5 M ammonium acetate (Sigma) to precipitate proteins. Samples were vortexed at high speed for 10 s and then centrifuged at 4,000 g at 4 °C for 15 min. After the spin, the supernatant was moved to a new 15 mL Falcon tube, then 6 mL 100% isopropanol was added, and samples were inverted 50 times and centrifuged at 4,000 g at 4 °C for 10 min. Genomic DNA pellets were visible as a small white pallet in each tube. The supernatant was discarded, 6 mL 70% ethanol was added, the tubes were inverted 10 times, and then centrifuged at 4,000 g at 4 °C for 5 min. The supernatant was discarded by pouring, and the remaining ethanol was removed using a pipette. Genomic DNA was air dried for 30-60 min, and then resuspended in 0.5-1 mL nuclease-free water overnight at room temperature. The next day, the gDNA solution was vortexed briefly and transferred to Eppendorf tubes. The gDNA concentration was measured using a Nanodrop (Thermo Scientific). For pre-injection cells, 100- 200 pL QuickExtract solution (Epicenter) was directly added to cells and incubated at 65 °C for 30 min until the cell pellets were completely dissolved followed by a 5 minute incubation at 95 °C to denature proteins found in QuickExtract. sgRNA library readout by deep sequencing

The sgRNA library readout was performed using a two-steps PCR strategy, where the first PCR includes enough genomic DNA to preserve full library complexity (5 pg per reaction * 20 reactions = 100 ug gDNA per sample, 100 pg gDNA / 6.6 pg gDNA/cell / 70,290 sgRNAs > 200-fold coverage), and the second PCR adds appropriate sequencing adapters to the products from the first round PCR (2 pL per reaction, 6 reactions per sample).

For PCR#1, a region containing sgRNA cassette was amplified using primers specific to the CRISPR library vector:

Forward: 5’-GTGCGCCAATTCTGCAGACAAATGG-3’ (SEQ ID NO:1)

Reverse: 5’-CAGGATCCAAAAAAAGCACCGACTC-3’ (SEQ ID NO:2)

PCR was performed using Phusion Flash High Fidelity Master Mix (ThermoFisher). For PCR#1, the thermocycling parameters were: 98 °C for 2 min, 25 cycles of (98 °C for 1 s, 62 °C for 5 s, 72 °C for 30 s), and 72 °C for 2 minutes. The final PCR products for each biological sample were pooled and used for amplification with barcoded second PCR primers. Second PCR products were pooled and gel purified from a 2% E-gel EX (Life Technologies) using the QiaQuick Gel Extraction kit (Qiagen). The purified library was then quantified with a gel-based method using the Low-Range Quantitative Ladder (Life Technologies) and dsDNA High- Sensitivity Qubit (Life Technologies). Libraries were sequenced with 5-20% PhiX using an Illumina NovaSeq 4000 sequencer at the Yale Center for Genomic Analysis (YCGA).

Screen data analysis sgRNA counts were quantified from all samples by trimming, aligning, and quantifying sequencing reads. Briefly, raw fastq files were trimmed down to the sgRNA spacer sequence with Cutadapt v3.4, using a 10% error rate and filtering reads < 15nt long. Spacer sequences were aligned using Bowtie vl.3.0⁴³, using the following settings: -vO, -ml -best. The reads were quantified and analyzed using SAMBA vl.l R package (https://github.com/Prenauer/SAMBA), which performed quasi-likelihood F tests (formula: ~ screen) and sgRNA-score aggregation into a gene score for comparison.

Results

Genome-scale in vivo CRISPRa screen identified gene boosters that enhance CAR-NK anti-tumor efficacy

To systematically identify the genes that can enhance the in vivo anti-tumor efficacy of CAR-NK cells, a genome-scale in vivo GOF CAR-NK CRISPRa screen was performed (FIG. 1A). HT29, a human colorectal cancer cell line was used in a subcutaneous tumor model and the screen performed in NK92, a human NK cell line that has been used in CAR-NK studies and advanced to the clinical trial stage ²⁶. Firstly, NK92 cells were generated that constitutively express an a-HER2-CAR ²⁷ , along with two CRISPR activation system components: dCAS9- VP64 and MS2-P65-HSF1 ²⁸. The a-HER2-CAR-NK92 cells were then transduced with a genome-scale CRISPRa single-guide RNA (sgRNA) library and adoptively transferred into HT29 tumor-bearing mice (FIG. 1A). The treatment of non-transduced CAR-NK92 cells demonstrated no significant therapeutic effect compared to the PBS control, reflecting the resistance of solid tumors to current CAR-NK therapy without additional genetic modifications (FIG. IB). The library-transduced CAR-NK92 cells significantly attenuated tumor growth compared to both non-transduced CAR-NK92 cell and PBS controls (FIG. IB), implying that certain sgRNAs in the library might have enhanced the anti-tumor efficacy of CAR-NK92 cells. Subsequently, genomic DNA was extracted from both the tumors in the cohort treated with library-transduced CAR-NK92 and the pre-injection cells for screen readout using nextgeneration sequencing (NGS), as described in the Methods.

Analysis of screen data showed that there was a strong difference between the screen and control CAR-NK92 samples, based on the separate clustering of samples in a correlation analysis heatmap and multidimensional scaling plots (FIGS. 6A-6B). There is a clear separation between tumor and pre-injected cell samples’ library representation in cumulative distribution function (CDF) plots (FIGS. 6C-6D), indicative of strong positive selection. The tumor samples have a small dominant fraction of sgRNAs (FIG. 6C-6D). Using the SAMBA analysis method, 66 genes were identified whose GOF perturbations were significantly enriched in tumorinfiltrating CAR-NK92 cells relative to pre-injection cells (FDR adjusted p value (q) < 0.01) (FIG. 1C; FIG. 6E). Several top candidate genes have been previously shown to be important for cell proliferation and migration, such as APLN²⁹ and PDP1 ^{30, 31} , although not in NK cells previously

Example 2: Genes SGSM2 and OR7A10 are key regulators that enhance CAR-NK function

Two G-protein pathway components, SGSM2 and OR7A10, were identified and subsequently validated as key regulators that serve as potent CAR-NK functional boosters. For HER2-targeting CAR-NK cells that face complete resistance against HER2 -positive solid tumors, the top two hyperboosters (SGSM2 and OR7A10, both of which are G-protein pathway components), when overexpressed, significantly boost the in vivo anti-tumor efficacy of these HER2 CAR-NKs

Methods

RT-qPCR

For overexpression of target genes, at least one week after sgRNAs lentivirus transduction and zeocin selection, CAR-NK92 cells were collected for RNA preparation. All RNA preparations were performed using RNasy Plus Mini Kit (Qiagen). Total mRNA was reverse transcribed into cDNA by using SuperScript IV Reverse Transcriptase (Thermo Fisher). Gene expression was quantified using Taqman Fast Universal PCR Master Mix (Thermo Fisher) and Taqman probes (Invitrogen). RNA expression level was normalized to GADPH (human). Relative mRNA expression was determined via the DD Ct method.

CAR-NK92 cytotoxicity assay

To detect the cytotoxic capabilities of SGSM2/OR7A10/APLN/PDP1/CYB5B-OE CAR- NK92 cells and controls, cancer cells were seeded in a 96-well plate at 5e4 cells/well, then different Effector (NK92 cells): Target (cancer cells) ratio (E: T ratio) co-cultures were setup. Cytolysis was measured by adding 150 pg/mL D-Luciferin (PerkinElmer) using a multi-channel pipette. After 15 minutes incubation, the luciferase bioluminescence was determined using PerkinElmer plate reader. The luminescence units recorded were normalized to the cancer cells only group, referred to as LUcanceronly. Tumor killing percentage calculation formula is below:

% Cytotoxicity = 100 100 Results

Top-ranked genes identified from the GOF screen enhance in vitro cytotoxicity of CAR-NK cells

To individually investigate whether the increased expression of high-rank candidate genes (hits) could enhance the in vitro cytotoxicity of CAR-NK92 cells the CRISPRa-mediated overexpression of SGSM2, OR7A10, APLN, and PDP1, four highest-scoring genes identified in the screen, were validated in CAR-NK92 cells using qRT-PCR (FIG. 1D-1G). It was noted that all of these genes have low mRNA expression levels compared with the housekeeping gene GAPDH in both NK92 and human primary NK cells, leaving sufficient room for GOF CAR-NK engineering (FIG. 7A). in vitro co-culture assays were then performed and it was found that overexpression of SGSM2, OR7A10, APLN, and PDP1 significantly enhances in vitro cytotoxicity against HT29 cancer cells, the one used in a screen (FIGS. 1H, II).

It was then further tested whether the effect of these genes in CAR-NK92 are universal across different cancer cell lines with varying levels of HER2 and major histocompatibility complex class I (MHC-I) expression, including MCF7 and MDA-MB-231 breast cancer cells, H1299 lung cancer cells, SK0V3 ovarian cancer cells, and A375 melanoma cells (FIGS. 1J-1O, 7B). Against SK0V3 cells, overexpression of all five top hits in CAR-NK92 cells showed increased cytotoxicity (FIG. 1 J). Against MCF7 cells, SGSM2, OR7A10, APLN, and PDP1 overexpressed CAR-NK92 cells showed increased cytotoxicity, while CYB5B overexpressed CAR-NK92 cells showed a marginal effect as compared to NTC control CAR-NK92 cells (FIGS. 1K-1L). Against A375 cells, overexpression of all five top hits in CAR-NK92 cells showed increased cytotoxicity (FIG. IM). Against H1299 cells, SGSM2-, OR7A10-, and APLN-overexpressed (OE) CAR-NK92 cells showed increased cytotoxicity (FIG. IN). Against MDA-MB-231 cells, SGSM2-overexpressed (OE) CAR-NK92 cells showed increased cytotoxicity (FIG. IO). Collectively, among the five target genes, SGSM2-0E and OR7A10-OE CAR-NK92 cells exhibit the highest killing capability and most robust phenotypes across the vast majority of the cancer cell lines tested (Table 3). These two genes were chosen for downstream studies and in vivo testing as CAR-NK hyper-boosters. Table 3: Summary of results from co-culture assays for the top five CAR-NK hyperboosters candidates tested.

Example 3: Overexpression of SGSM2 or OR7A10 significantly increases both the in vitro and in vivo anti-tumor efficacy of CAR-NK cells

Engineered CAR-NK cells over-expressing SGSM2 or OR7A10 have improved tumor infiltration, increased proliferation, enhanced degranulation, elevated secretion of effector cytokines, and increased activation of CAR-NK cells. Both SGSM2’s and OR7A10’s GOF leads to enhanced tumor infiltration, proliferation, activation, cytokine production and cytotoxicity of CAR-NK cells. These features are supported by the increased calcium influx, elevated intracellular signaling, and alterations of the gene expression programs.

Methods

In vivo animal experiments

NOD-scid IL2R-gamma-null (NSG) mice were purchased from JAX and bred in-house. Eight-to-twelve-week-old male mice were inoculated with 2e6 HT29 cells through subcutaneous injection. After 19 days, NTC, SGSM2-OE, and OR7A10-QE a-HER2-CAR-NK92-hIL2 were injected intravenously into tumor-bearing mice. In the following days, CAR-NK92 cells were treated once a week and sequentially for 3 weeks. Treatment dose and time-point were labeled in the appropriate figures. Tumor volumes were measured by caliper and calculated with the following formula: vol = JT/6 * length * width * height. All mice were sacrificed once they reached the endpoint, based on lACUC-approved protocols.

In vivo tumor infiltration assay

Eight-to-twelve-week-old female NSG mice were inoculated with 2e6 HT29 cells through subcutaneous injection. On day 26, tumor-bearing mice were randomly assigned to one of four groups and treated intravenously with PBS, 2e7 NTC, SGSM2-0E, or OR7A10-OE anti-HER2- CAR-NK92-mCherry-hIL2 cells. On day 28, these mice received a second intravenous dose of 2e7 NTC, SGSM2-OE, or OR7AIO-OE anti-HER2-CAR-NK92-GFP-hIL2. The mice were euthanized on day 29, and tumor samples were collected for flow cytometry analyses. All mice were sacrificed on day 29 post-inoculation.

Isolation of tumor infiltrating NK cells

Mice were euthanized at indicated time point. Tumors were collected and immediately placed in ice-cold 2% FBS PBS. Tumors were minced into 1- to 3-mm size pieces using a scalper and then digested using Collagenase IV at 37 °C in the shaker with speed of 1000 rpm for one hour. Tumor suspensions were filtered through a 100-pm cell strainer to remove large bulk masses. Red blood cells were lysed by incubating the samples with 1 mL of ACK Lysis Buffer (Lonza) per tumor sample for one minute at room temperature. The lysed samples were then diluted with 10 ml 2% FBS PBS and filtered through a 40- pm filter. The resulting single-cell suspensions derived from the tumors were used for flow cytometry staining.

FACS analysis of tumor infiltrating NK cells

Single tumor cell suspensions were prepared using the Collagenase IV digestion with the method described above. Tumor cells were blocked using FcR Blocking Reagent, mouse (Miltenyi), following the manufacturer’s instructions. NK cells at a density of 10⁷ ml^-1 were stained with dimethyl sulfoxide-dissolved live/ dead staining dye, and BV510 conjugated anti- CD56 antibody in MACS buffer (PBS + 0.5% BSA+2 mM EDTA) and incubated on ice for 30 min. Stained cells were washed three times before being analyzed on a BD FACSAria. Stained cells were washed three times and resuspended in 200 pL MACS with 30 pL Precision Count Beads ™ (Biolegend) before being analyzed on a BD FACSAria.

Bulk mRNA sequencing (mRNA-seq) library preparation

The mRNA library preparations were performed using a NEBNext® Ultra™ RNA Library Prep Kit, and samples were multiplexed using barcoded primers provided by NEBNext® Multiplex Oligos for Illumina® (Index Primers Set 2). NTC, SGSM2-OE, and OR7AIO-OE anti- HER2-CAR-NK92-GL-hIL2 cells were stimulated with HT29 cancer cells at a 1:1 E:T ratio for 0, 6, 24 hours. CAR-NK92-GFP cells were sorted, had RNA extracted, and underwent mRNA- seq library preparations. Libraries were sequenced using a Novaseq 4000 (Illumina).

RNA-seq data analysis

For RNA-seq data preprocessing, raw fastq files were aligned to the human genome (GRCh38 Gencode v44) using STAR aligner v2.7.11⁴⁴. The aligned data were quantified into gene counts using RSEM vl.3.3⁴⁵ with default parameters. The data were then processed and analyzed with the edgeR package for R using a standard pipeline⁴⁶. Specifically, gene expression data were filtered (filterByExpr function), normalized by Trimmed Mean of M-values (TMM) method, had dispersion calculated (default), were fitted to generalized linear model (formula: ~ Mutant), and analyzed by likelihood ratio test. Differential expression was analyzed separately for each time-point: 0, 6 and 24 hrs post-stimulation.

Pathway network analyses were performed using differentially expressed genes (DEG) (q < 0.01 & absolute log2 fold-change > 2) that were pooled from each time-point analysis. First, a time-specific network was constructed from a matrix of the pooled DEGs, featuring the log2 fold-changes (LFC) from each DE analysis time-point (0, 6 and 24 hrs post-stimulation). The resulting LFC matrix (time vs DEG), vertex data were calculated via non-linear UMAP method (uwot R package v.0.1.14) to produce two dimensions/gene. Next, network weights were calculated as the Euclidean distances between all genes based on the UMAP embedding; distances were transformed (z-score of 1/square-root of distance) to ensure higher weights for closer points; and the resulting weights were filtered for the top 50%. Gene modules were then calculated by weighted Leiden clustering (cluster_leiden function of igraph R package vl.5.1) using a resolution of 0.5 and 4 iterations. Finally, pathway analyses were performed separately on the gene modules using Overrepresentation Analyses (fora function of fgsea R package vl.22.0) and Reactome pathway terms from MSig database v7.4 (including terms with size > 2, size < 1000, DEG overlap > 2, and < 50% of DEG overlap due to a set of paralogous genes (example: H2AC1-20 cannot make up >=50% of ontology overlap)). The gene modules were represented by the most significant filtered pathway term. Transcription factor (TF) regulator analyses were performed on DEG (q < 0.01 & absolute log2 fold-change > 0.5) using the decoupleR R package v2.2.2⁴⁷ and reference data for directional interactions between TF / target-genes from the DoRothEa database (human data; A- C interaction confidence)⁴⁸. Specifically, the analysis was performed using the Gene Set Enrichment Analysis method (run_fgsea function of decoupleR R package v2.2.2) with DEG ordered by likelihood ratio.

Cell proliferation assay le6 cells were collected and resuspended in 1 ml PBS. 1 pL CellTrace™ Violet dye (1:1000 dilution) was added to cell suspension. Cells were incubated at 37 °C for 5 min, and then were washed three times using 10% FBS-RPMI medium to remove any excess dye. Then the cells were pelleted, resuspended in fresh, pre-warmed complete culture medium, and incubated in the incubator. Five days later, cells were counted and analyzed by flow cytometry.

CD107a degranulation assay

To detect the degranulation of SGSM2-OE and OR7AIO-OE CAR-NK92 cells, cancer cell lines MCF-7-HER2-PL or HT29 cells were seeded in a 96-well plate at le5 cells/well. CAR- NK92 cells were added at an E: T ratio of 1:2 and stimulated for 2 hours, 4 hours, and 6 hours. One hour before collection, media was supplemented with 2 nM monensin and anti-CD107a-PE antibody (BioLegend) (1:1000 dilution). At the end of each co-culture, CAR-NK92 were washed with PBS and stained with anti-CD56-BV510 for 30 min on ice. Cells were analyzed using a BD FACS Aria.

Flow cytometry for surface activation markers

After co-culture with HT29 cancer cells at E:T=1:1 for 24 hrs, CAR-NK92 cells were collected and washed once using MACS buffer (0.5% BSA and 2 mM EDTA in PBS) before staining. CAR-NK92 cells were stained on ice for 30 min after adding antibodies (1:200 dilution), and then washed twice with 1 mL cold MACS buffer. All samples were run on a BD FACSAria cytometer, and analysis was performed using FlowJo software (Threestar, Ashland, OR).

Flow cytometry for intracellular cytokine production

CAR-NK92 cells were co-cultured with HT29 cancer cells at E:T=1:1. 6 hrs before collection (18 hrs after stimulation), 5 mg/mL Brefeldin A was added to the co-culture media. After 24 hrs, cells were collected, washed, and stained for membrane protein. Cells were fixed and permeabilized using BD Cytofix/Cytoperm Fixation/Permeabilization Kit (Thermo Fisher), and then specific antibodies were added. All samples were run on a BD FACSAria cytometer, and analysis was performed using FlowJo software (Threestar, Ashland, OR).

Calcium flux assay le7 CAR-NK92 cells were stained with 5 pM Cal520, AM (AAT Bioquest) in cRPMI medium with 0.04% Pluronic F-127 (Thermo) at 37 °C for 30 minutes. The cells were then washed once with Hank’s balanced salt solution (HBSS) and incubated with 10 pg/ml soluble HER2-Biotin protein (Aero Biosystems) on ice for 30 min. After binding with HER2 protein, the cells were washed twice with HBSS and resuspended in 1 mL HBSS. The cells were then incubated at 37 °C for 10 min and flow recorded for baseline FITC fluorescence at 37 °C. The streptavidin was added to a final concentration of 10 pg/ml, and the cells were continuously recorded for FITC signal changes for 10 min. p-ERKl/2 assay le7/mL CAR-NK92 cells underwent serum starvation overnight to minimize background phosphorylation. le7/mL HT29 cells were added to the wells to stimulate CAR-NK92 cells at E:T=1:1 for 5 minutes. Post stimulation, cells were rapidly fixed using prewarmed Fix Buffer I (BD Biosciences) for 10 min at 37 °C. Fixed cells were permeabilized with cold Phosflow Perm Buffer III (BD Biosciences) for 30 min on ice. Then cells were stained with PE p-ERKl/2 (pT202/pY204) (Biolegend) and flow recorded.

Sample size determination

Sample size was determined according to the lab's prior work or from published studies of similar scope within the appropriate fields. Replication

The number of biological replicates (typically n >= 3) are indicated in the figure legends. Key findings (non-NGS) were replicated in at least two independent experiments. NGS experiments were performed with biological replications, as indicated in the manuscript.

Randomization and blinding statements

Regular in vitro experiments were not randomized or blinded. Mouse experiments were randomized by using littermates and blinded using generic cage barcodes and ear tags where applicable. High-throughput experiments and analyses were blinded by barcoded metadata.

Standard statistical analysis

Standard statistical analyses were performed using common statistical methods with GraphPad Prism, Excel, and R. Different levels of statistical significance were accessed based on specific p values and type I error cutoffs (0.05, 0.01, 0.001, 0.0001). Further details of statistical tests were provided in figure legends and/or supplemental information.

Data Collection summary

Flow cytometry data was collected by a BD FACSAria.

All deep sequencing data were collected using Illumina Sequencers at Yale Center for Genome Analysis (YCGA). Co-culture killing assay data were collected with PE Envision Plate Reader.

Data analysis summary

Flow cytometry data were analyzed by FlowJo v.10.7. All simple statistical analyses were done with Prism 9. All NGS analyses were performed using custom codes.

Results

SGSM2-OE or OR7A10-OE CAR-NK92 cells demonstrate enhanced in vivo anti-tumor efficacy and tumor infiltration

To validate whether SGSM2 or OR7A10 overexpression could enhance the therapeutic efficacy of CAR-NK cells in in vivo settings, a-HER2-CAR-NK92-hIE2 cells, transduced with lentivirus encoding sgRNAs for overexpression of specific genes were established, and adoptively transferred into mice bearing HT29 tumors (FIG. 2A). It was observed that while sgNTC-transduced CAR-NK92 cells exhibited no significant in vivo efficacy against HT29 tumors, suggesting that these solid tumors are resistant to CAR-NK92 cells without genetic modification. In sharp contrast, SGSM2-OE or OR7AIO-OE CAR-NK92 cells showed robust anti-tumor activity compared to both sgNTC-transduced CAR-NK92 cell and PBS control groups (FIGS. 2B-2F).

It was next investigated whether the GOF of SGSM2 or OR7A10 could enhance CAR- NK92 cells’ tumor infiltration in vivo. a-HER2-CAR-NK92-hIE2 cells were labeled with either GFP or mCherry, transduced with lentivirus encoding NTC-, SGSM2- or OR7A10- sgRNAs, and adoptively transferred into mice bearing sizable HT29 tumors on day 26 and 28, respectively (FIG. 3A). The tumor growth curve data again showed that SGSM2-0E or OR7AIO-OE exhibited heightened efficacy of adoptive CAR-NK therapy (FIG. 3B). The tumor samples were collected on day 29 and analyze tumor-infiltrating NK cells by flow cytometry, and a significant increase was observed in the total number of tumor-infiltrating NK92 cells (marked by CD56), as well as GFP⁺ and mCherry⁺ tumor-infiltrating NK92 cells, in both the SGSM2-0E and OR7A10-OE group compared to the sgNTC group (FIG. 3C-3E). These findings demonstrated that SGSM2-0E or OR7AIO-OE can promote CAR-NK92 cells’ tumor infiltration and antitumor efficacy, further supporting them as CAR-NK hyperboosters.

Transcriptional time-course analyses show that SGSM2-0E and OR7A10-OE remodel multiple CAR-NK92 transcriptional programs

To unbiasedly map the gene expression alterations driven by SGSM2 and OR7A10 overexpression, bulk mRNA-seq was performed in SGSM2-0E, OR7A10-OE, and control (sgNTC-transduced) a-HER2-CAR-NK92-hIL2 before and after HT29 co-culture stimulation for 6 and 24 hours (hrs) (FIG. 4A). The overexpression of SGSM2 and OR7A10 was first validated in CAR-NK92 cells in these mRNA-seq samples by qPCR (FIGs. 4B-4C). Sample correlation analyses of the mRNA-seq data showed that there is a strong difference between stimulated and unstimulated cells, as well as between different CAR-NK92 genetic modification groups (FIG. 8). The differentially expressed (DE) genes were then identified using the edgeR pipeline (as described in the Methods section)

The SGSM2-0E in CAR-NK92 cells revealed 204, 537, and 204 upregulated genes; along with 637, 487, and 1788 downregulated genes at 0, 6, and 24 hrs, respectively (absolute log2 foldchange > 1 & q < 0.01) (FIGS. 9A-9D volcano plots). Notable significantly upregulated genes upon SGSM2-OE include LRRN3, BEST, GPR183, ZC2HC1B, IRF4 and IFNG. Highly significant downregulated genes include SYK, PRUNE2, KIR2DL4, NR4A2. Pathway analysis of SGSM2-OE showed that the upregulated genes are enriched in the pathways of Rho GTPase cycle and extracellular matrix (ECM) organization 6 hrs after stimulation, and then G alpha I signaling once stimulated. Immunoregulatory interaction pathway is significantly enriched at all timepoints, yet this pathway is most upregulated in unstimulated cells. Upstream regulator analysis revealed the most significantly enriched upstream regulators of SGSM2-OE- driven DE genes in CAR-NK92 cells include FOXK2, WT1 and REL in the un-activated state; SOX10 at 6h post activation; and REL and KLF13 at 24hr post activation.

The OR7A10-OE in CAR-NK92 cells revealed 257, 183, and 124 upregulated genes; along with 711, 608, and 1828 downregulated genes at 0, 6, and 24 hrs, respectively (absolute log2 foldchange > 1 & q < 0.01) (FIGS. 9A-9D, volcano plots). Notable significantly upregulated genes upon OR7A10-OE include FTH1, LRRN3, C3, TNFRSF9/4-1BB and IFNG. Highly significant downregulated genes include SYK, PIPOX, RXRA, TEE1, GNEY, as well as CISH, a gene whose knockout has been previously showed to enhance NK function ²⁰. Pathway analysis of OR7A10-OE showed that the upregulated genes are enriched in the pathways of cell junction organization, biological oxidations, and Rho GTPase cycle prominently in unstimulated cells. At 6 hrs of stimulation, the most upregulated pathways shift to GPCR signaling, HDACs deacetylate histones, and immunoregulatory interactions between lymphoid and non-lymphoid cells. Notably, immunoregulatory interaction pathway genes are upregulated at 6 and 24 hrs post stimulation in OR7A10-OE but not SGSM2-0E CAR-NK92 cells. Upstream regulator analysis revealed the most significantly enriched upstream regulators of OR7A10-OE-driven DE genes in CAR-NK92 cells include SOX9, NR1H2, REL, NR1H3 and KLF4 in the un-activated state; IRF3, TFDP1, RFX5 and NFKB1 at 6h post activation; and REL again at 24hr post activation.

SGSM2 and OR7A10 overexpression enhances multiple features of CAR-NK function The immunological features of SGSM2-0E or OR7A10-OE a-HER2-CAR-NK92-hIL2 cells were characterized. To assess the proliferation of a-HER2-CAR-NK92-hIL2 cells, flow cytometry analyses were conducted using Ki-67 and cell trace dye, as well as cell number quantification (FIGS. 5A, 5B, 5C, FIG. 10).

It was found that, in comparison to the vector and sgNTC control groups, both SGSM2-OE and OR7A10-OE CAR-NK92 cells have higher levels of Ki-67 and lower levels of cell trace dye (indicative of higher proliferation) (FIGS. 5A-5C). Raw cell number count also validated that there were more of SGSM2-OE or OR7A10-OE a-HER2-CAR-NK92-hIL2 cells compared to controls starting from equal number of cells grown over five days (FIG. 10). It was found that both SGSM2-OE and OR7A10-OE CAR-NK92 cells exhibit increased degranulation (surface CD107a) when co-cultured with cognate HER2-OE-MCF7 cells and HT29 cells (FIG. 5C; FIG. 11). Both SGSM2-OE and OR7A10-OE HER2-CAR-NK92-hIL2 cells compared to controls produce strikingly higher levels of IFNa, when co-cultured with HT29 cells (FIG. 5D-5G), which is consistent with the bulk RNA-seq result. It was observed that other major effector cytokines such as TN Fa, granzyme B, and perforin were also produced at higher levels by both SGSM2-OE and OR7A10-OE HER2-CAR-NK92-hIL2 cells compared to controls (FIG. 5D-5G).

The CAR-NK cell activation ability of SGSM2-OE or OR7A10-OE a-HER2-CAR- NK92-hIL2 cells was then compared. NK activation was analyzed using CD69, the IL-2 receptor alpha CD2532, the NK activating receptor NKG2D, and the costimulatory receptor 4-1BB (CD 137; TNFRSF9) and elevated levels of all these markers was observed in both SGSM2-OE and OR7A10-OE HER2-CAR-NK92-hIL2 cells compared to controls (FIG. 5H-5K). To monitor real-time calcium flux upon antigen stimulation, the Cal-520-labeled CAR-NK92 cells were incubated with biotin-labeled HER2 antigen and then cross-linked by streptavidin. Consistently, it was observed that both SGSM2-0E and OR7A10-OE HER2-CAR-NK92-ML2 cells mediated stronger and more durable calcium flux compared to controls (FIG. 5L). Furthermore, it was observed that both SGSM2-0E and OR7A10-OE HER2-CAR-NK92-hIL2 cells exhibited stronger extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation (pT202/pY204) (no simulation [1.83%], vector [8.88%], NTC [9.36%], SGSM2 [12.5%], OR7A10 [23.7%]) (FIGs. 5A-5L;), one of the major activation signals of NK cells resulting in granule polarization and exocytosis ⁷’ ^{33, 34}. These data together showed that SGSM2 and OR7A10 overexpression enhances multiple features of CAR-NK function including proliferation, activation and effector function.

Discussion

These data together suggest that in vivo GOF screening is an effective strategy to identify genes that can be harnessed to enhance NK cell function and reveal two cancer type agnostic hyper-boosters for CAR-NK therapy against solid tumors.

Clinical and preclinical studies have shown that the infusion of allogeneic NK cells can successfully traverse human leukocyte antigen (HLA) barriers, thus mitigating the graft-vs-host reactions, which present an inherent challenge in allogeneic T cell transfer^{3, 17, 35, 36}. In addition, NK cell therapy leverages the advantages of rapid cytotoxic anti-tumor immune responses, TCR/CAR-independence, enhanced safety, reduced off-target immune responses, and reduced production of molecules associated with cytokine release syndrome (CRS). Despite these promising attributes, NK cell-based immunotherapies still have many obstacles to overcome against solid tumors. The next generation of NK cell products will be engineered to enhance activating signals and suppress inhibitory signals, and promote their homing, infiltration into tumors, proliferation and persistence. This requires rational engineering of substantially enhanced CAR-NK cells, particularly by modification of endogenous genes.

To systematically identify genes that can function as NK boosters and thereby CAR-NK therapy, a genome-scale in vivo gain-of-function CAR-NK screen was conducted using an HT29 tumor model. Five candidates, SGSM2, OR7A10, APEN, PDP1, and CYB5B, were identified that can enhance the in vitro cytotoxicity of CAR-NK cells against various types of cancer cells. Among these targets, SGSM2 and OR7A10 showed highest cytotoxicity and have not been studies in NK cells before. SGSM2 has previously been known as a GTPase- activating protein (GAP) modulating small G protein (RAP and RAB)-mediated signaling pathways ³⁷ ’ ³⁸. Notably, Ras-associated protein 1(RAP1), which is shown as substrates of SGSM2 ³⁹, facilitates NK cell polarization, cytokine and chemokine production, phosphorylation of B-Raf, C-Raf, and ERK1/2 ⁴⁽⁴⁴¹. OR7A10 is a G-protein coupled receptor annotated as an olfactory receptor that might be capable of transducing signals. While these genes are normally not expressed or lowly expressed in NK cells, the data revealed that SGSM2 or OR7A10 overexpression/GOF can significantly increase the cytotoxicity, degranulation, proliferation, effector cytokine secretion, and tumor infiltration of CAR-NK cells. Importantly, SGSM2 or OR7A10 GOF renders CAR-NK cells to overcome solid tumor resistance and showed robust in vivo efficacy where the baseline CAR-NK has no in vivo activity.

In summary, the human genome was scanned for genes that can be used to enhance antitumor function of CAR-NK cells directly in vivo, and several of such hyper-boosters have been identified that were previously undocumented. SGSM2 and OR7A10 were validated, and characterized using various in vitro and in vivo assays, establishing them as two potent and broadly applicable functional boosters that can be engineered to enhance anti-tumor immunity of NK based cell therapy.

Example 4: Interleukins 15 and 21 Enhance CAR-NK Function in a Dose-Dependent manner

Interleukin- 15 (IL- 15) is an important cytokine that plays a pivotal role in enhancing the efficacy of Chimeric Antigen Receptor (CAR) natural killer (NK) cell therapies (Daher, et al., Targeting a cytokine checkpoint enhances the fitness of armored cord blood CAR-NK cells." Blood 137(5): 624-636 (2021)). Engineering of IL15 has been shown to be associated with systemic toxicities and its continuous treatment is known to exhaust NK cells (Misra, et al., (2021) "Activation of ADAM17 by IL-15 Limits Human NK Cell Proliferation." Front Immunol 12: 711621; Felices, et al. (2018). "Continuous treatment with IL-15 exhausts human NK cells via a metabolic defect." JCI Insight 3(3))

Interleukin-21 (IL-21) holds significant promise in the realm of CAR NK cell therapy (McMichael, et al., (2017). "IL-21 Enhances Natural Killer Cell Response to Cetuximab-Coated Pancreatic Tumor Cells." Clin Cancer Res 23(2): 489-502) IL-21 acts as a potent stimulator, enhancing the cytotoxic potential of CAR NK cells engineered to target specific tumor antigens. IL- 15 and IL-21 secreting as well as membrane bound IL- 15 and/or IL-21 NK92 cells were engineered to investigated these properties in greater detail.

Methods

NK cells expressing IL21 cytokine and/or IL 15 cytokine were engineered, and their effect on ENPP3 tumor antigen targeted tumor killing by CAR-NK92 cells was tested on at least two tumor models. The serial killing dynamics and cytokine secretion profiles of the engineered NK cells were also investigated. ENPP3 CAR-NK92 Engineering cells

Materials:

NK92 scFV 50 (60 pl) 2X MACS; mbIL-15 (Vector Builder: SFFV-CD8SP-IL15- CD8hinge-CD8TMD-T2A-puro); IL-15 (Vector Builder: SFFV-T2A-IL2SP-IL15) IL-21 (Vector Builder: SFFV-T2A-IL2SP-IL21); IL-15+IL-21 (Vector Builder: SFFV-T2A- IL2SP-IL 15-P2 A-IL2SP-IL21 ).

Spinoculation Protocol:

1. Day 1: Coat non-tissue treated plates with retronectin 3.5-5.0 pg/cm² for 2 hours RT or 4 °C overnight, using approx. 40 pg/mL of retronectin in PBS, 250 pl per well in 24 well dish;

2. Day 2: Block with 2% BSA for 30 minutes RT, wash with PBS;

3. Incubate viral particles in respective cell growth medias and desired MOI;

4. Centrifuge at 2,000xg for 1 hour at 32 °C, then pre-warm the centrifuge at least 30 minutes beforehand to get to temp;

5. Incubate plate + LV in 37 °C incubator for 1 hour;

6. Add NK7NK92 cells with 1.5 pM BX795 to wells, centrifuge at l,000xg for 10 min, 32 °C , plate 5e5 cells/well in 24 well plate or 2e6 cell/well in 6 well plate;

7. Day 3: Spin down, and replace media to remove extra viruses; and

8. Day 5: Check CAR % using FLAG-FITC antibody and 7AAD.

Cell/cytokine Assay

Materials:

NK92 Growth Media (RPMI +12.5% FBS +12.5% HS +200 mM L-glutamine + 0.2 mM Myo-Inositol + 0.02 mM Folic Acid + 0.1 mM 2-Mercaptoethanol +1% P/S); HEK ENPP3 1 pl - DMEM+10% FBS (Figures 12A-12B); Recombinant IL-2 100 IU/mL’ Caspase 3/7 green dye (1:1000 or 5 pM).

Assay protocol:

Day 0: Count target cells and seed at 5K cells/well in 100 pl of target cell growth media listed above in triplicate. Culture them in 37 °C overnight to allow cells to adhere;

Day 1: Count effector cells and seed them at 5K, 15K, 25K cells/100 pl in effector growth media + 200 IU/mL IL-2 (100 IU/mL when combined with target media) + 1:500 dilution of caspase 3/7 to effector media (1:1000 when added with target media). Run for 24-72 hours. Use 4X optical laser, Green lens, read whole well every 2 hours in 96-well plate for 3 days;

Day 4: Spin down assay plate 300xg, 5 minutes. Carefully remove 100 pl of media and store in 96-well plate + seal in = 80 °C for future cytokine analysis. Figure 13A shows Incucyte Caspase3/7 Dye based cell cytotoxicity results showing the ENPP3-HEK293 killing by engineered ENPP3-CARNK92 cells with or without cytokine/s expression. mCherry ACHN serial killing assay

Materials:

ACHN_mCherry_Luc - MEM + 10% FBS; NK92 Growth Media (RPMI + 12.5% FBS + 12.5% HS + 200 mM L-glutamine + 0.2 mM Myo-Inositol + 0.02 mM Folic Acid + 0.1 mM 2- Mercaptoethanol + 1% P/S); Recombinant IL-2 100 lU/mL.

Assay Protocol:

Day 0: Count target cells and seed at 5K cells/well in 100 pl of target cell growth media listed above in triplicate. Culture them in 37 °C overnight to allow cells to adhere;

Day 1: Count effector cells and seed them at 5K cells/100 pl in effector growth media + 200 lU/mL IL-2 (100 lU/mL when combined with target media). Use 4X optical laser, Red and Green lens, read whole well every 2 hours in 96-well plate for 3 days;

Day 3 (1 day before next round): Seed target cells 5K cells/well in target cell media;

Day 4: Spin down assay plate 300 g, 5 minutes. Carefully remove 100 pl of media and store in 96-well plate + seal in -80 °C for future cytokine analysis. For the remaining 100 pl, resuspend the cells by pipetting 3-4 times and transfer all media to new plate containing target cells (no external cytokines added); Repeat seeding 1 day before, spin and save supernatant, and plate remaining cells to new plate for next round stimulations.

Figures 13B-13E show enumeration of mCherry-ACHN tumor killing by engineered NK92 cells after each of rounds 1-4, respectively.

Cytokine measurement in tumor killing Assay

Cytokine measurement was done in supernatant culture of HEK293T cells after 72 hours by using a commercial kit (Legendplex; Biolegend) by flow cytometry.

Figures 14A-14C show estimation of each of IFN-gamma, IL-6 and TNF-alpha in culture supernatant after ENPP3-HEK293T tumor killing by engineered CARNK92 cells.

Cytokine mediated killing

To rule out whether the tumor cells were getting killed by cytokine alone and not through the CAR NK92 mediated lysis, an experiment was executed to compare the growth rate of tumor alone with the presence of different cytokines. Figure 15 depicts the effect of IL15 and IL21 cytokines both alone and in combination on the tumor cell proliferation. No effect of cytokines on the tumor growth rate was observed, indicating that the cytokines are improving the killing efficiencies of CAR-NK cells. Role of Exogenous Cytokines on CAR-NK92 mediated killing

Whether the addition of exogenous cytokine/s will improve the killing efficiencies of CAR-NK cells was investigated. Figures 16A-16C depicts the results of the assay where it was found that the lower concentration of IL15 (i.e., 2.5 ng/ml depicted in blue with circle) had improved killing efficiency than all the higher concentration of IL15 (i.e., 5, 10 and 15 ng/ml depicted in Square Red, Triangle Green, and inverted purple triangle respectively). However, the best IL21 concentration was 10 ng/ml. Considering the in vivo utility of IL 15 it was postulated that the 2.5 ng/ml of IL 15 along with 10 ng/ml of IL21 would be a winning combination in tumor bearing mice.

Results

The results indicated that the combination of cytokines IL 15 and IL21 provide a superior effector function in serial killing as well as cytokine secretion profile for this specific CAR. Example 5: Hyper-boost Assay Analysis

The hyper-boost lead from the data generated through the in vivo directed evolution in tumor bearing mice was carried out by following the method depicted in the flow diagram of Figure 17. Basically, the method included:

• NGS Count data were scaled by the TMM (Trimmed Mean of M-values” normalization) method from the edgeR package

• Scaled data will be further filtered by a feature with read counts greater than 3 for at least 4 tumor samples will be kept for the following analysis

• Two orthogonal approaches:

(1) Open source Mageck Package to identify target genes from significantly enriched guide RNAs

(2) customized scripts using the Elastic Net model (GLMNET) to identify features to classify tumor vs cell line samples

(3) 10 overlapped genes from the top 20 genes of each result will be the final candidates

The top 10 genes which are evaluated as a hyper-boost for gain of CAR-NK cell function include:

1. GABBRl.NM_021903

2. PRR14L.NM_173566

3. TIAMl.NM_003253

4. KRT82.NM_033033

5. PLA2G1B.NM_OOO928

6. REM2.NM_173527

7. HIST1H2BN.NM_OO352O 8. CYB5B.NM_030579

9. LRRC23.NM_006992

10. NXPE3.NM_001134456

These analyses used the same CRISPR screen data set demonstrated in Example 1 but implemented two different analytical approaches (Mageck and GLMNET). This approach was adopted, since it was postulated that these two different analysis algorithms may rank genes differently and thereby more rigorously identify the same or overlapping high-scoring genes. To capture the variation of different approaches and include more potential candidate genes, the topscoring genes identified from the two different approaches were overlapped, to identify the top 10 gene candidates from the combined algorithms. One of these 10 genes (GABBR1, PRR14L, TIAM1, KRT82, PLA2G1B, REM2, HIST1H2BN, CYB5B, LRRC23, and NXPE3), CYB5B, was also identified according to the methodology set forth in Example 1 (as described above).

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It is understood that the disclosed method and compositions are not limited to the particular methodology, protocols, and reagents described as these can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present disclosure which will be limited only by the appended claims.

Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed method and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a nucleic acid sequence is disclosed and discussed and a number of modifications that can be made to a number of molecules including the nucleic acid sequence are discussed, each and every combination and permutation of the nucleic acid sequence and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited, each is individually and collectively contemplated. Thus, is this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Further, each of the materials, compositions, components, etc. contemplated and disclosed as above can also be specifically and independently included or excluded from any group, subgroup, list, set, etc. of such materials. These concepts apply to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed.

It must be noted that as used herein and in the appended claims, the singular forms "a ", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "a nucleic acid sequence" includes a plurality of such nucleic acids, reference to "the nucleic acids" is a reference to one or more nucleic acid and equivalents thereof known to those skilled in the art, and so forth.

“Optional” or “optionally” means that the subsequently described event, circumstance, or material may or may not occur or be present, and that the description includes instances where the event, circumstance, or material occurs or is present and instances where it does not occur or is not present.

Unless the context clearly indicates otherwise, use of the word “can” indicates an option or capability of the object or condition referred to. Generally, use of “can” in this way is meant to positively state the option or capability while also leaving open that the option or capability could be absent in other forms or embodiments of the object or condition referred to. Unless the context clearly indicates otherwise, use of the word “may” indicate an option or capability of the object or condition referred to. Generally, use of “may” in this way is meant to positively state the option or capability while also leaving open that the option or capability could be absent in other forms or embodiments of the object or condition referred to. Unless the context clearly indicates otherwise, use of “may” herein does not refer to an unknown or doubtful feature of an object or condition. Ranges can be expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, also specifically contemplated, and considered disclosed is the range from the one particular value and/or to the other particular value unless the context specifically indicates otherwise. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the value forms another, specifically contemplated embodiment that should be considered disclosed unless the context specifically indicates otherwise. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint unless the context specifically indicates otherwise. All of the individual values and sub-ranges of values contained within an explicitly disclosed range are also specifically contemplated and should be considered disclosed unless the context specifically indicates otherwise. Finally, all ranges refer both to the recited range as a range and as a collection of individual numbers from and including the first endpoint to and including the second endpoint. In the latter case, any of the individual numbers can be selected as one form of the quantity, value, or feature to which the range refers. In this way, a range describes a set of numbers or values from and including the first endpoint to and including the second endpoint from which a single member of the set (i.e. a single number) can be selected as the quantity, value, or feature to which the range refers. The foregoing applies regardless of whether in particular cases some or all of these embodiments are explicitly disclosed.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each claim of this application.

Throughout this specification the word “comprise,” or variations such as “comprises” or “comprising,” will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed method and compositions belong. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present method and compositions, the particularly useful methods, devices, and materials are as described.

Publications cited herein and the material for which they are cited are hereby specifically incorporated by reference. Nothing herein is to be construed as an admission that the present disclosure is not entitled to antedate such disclosure by virtue of prior disclosure. No admission is made that any reference constitutes prior art. The discussion of references states what their authors assert, and applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of publications are referred to herein, such reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

Although the description of materials, compositions, components, steps, techniques, etc. can include numerous options and alternatives, this should not be construed as, and is not an admission that, such options and alternatives are equivalent to each other or, in particular, are obvious alternatives. Thus, for example, a list of different gene targets does not indicate that the listed gene targets are obvious one to the other, nor is it an admission of equivalence or obviousness.

Every component disclosed herein is intended to be and should be considered to be specifically disclosed herein. Further, every subgroup that can be identified within this disclosure is intended to be and should be considered to be specifically disclosed herein. As a result, it is specifically contemplated that any component, or subgroup of components can be either specifically included for or excluded from use or included in or excluded from a list of components.

Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the method and compositions described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

CLAIMS We claim:

1. A genetically modified Natural Killer (NK) cell, wherein

(i) at least one gene selected from the group consisting of SGSM2, OR7A10, APLN, PDP1, GABBR1, PRR14L, TIAM1, KRT82, PLA2G1B, REM2, HIST1H2BN, CYB5B, LRRC23, NXPE3, MEGF11, FKBP5, PPFIA2, LRRC23, PEAR1, REM2, TIAM2, HPRT1, MMACHC, and ZBTB20 is up-regulated or over-expressed in the cell, as compared to a non-genetically modified control NK cell; and/or

(ii) at least one cytokine selected from Interleukin- 15 (IL- 15) and Interleukin-21 (IL- 21) is up-regulated or over-expressed by the cell.

2. The genetically modified Natural Killer (NK) cell of claim 1, wherein the modification enhances the anti-cancer efficacy of the NK cell as compared to a non-genetically modified control NK cell.

3. The genetically modified NK cell of claim 1 or 2, wherein the modification causes increased or enhanced expression, activation, presentation, and/or function of one or more protein(s) encoded by the gene(s).

4. The genetically modified NK cell of claim 1 or 2, wherein the modification causes increased or enhanced expression of one or more of the genes SGSM2, OR7A10, APLN, PDP1 and CYB5B and/or the full-length protein(s) encoded by the gene(s) SGSM2, OR7A10, APLN, PDP1, and CYB5B.

5. The genetically modified NK cell of any one of claims 1-4, wherein the modification comprises recombinant expression of the SGSM2 gene.

6. The genetically modified NK cell of claim 5, wherein the recombinant expression causes increased or enhanced expression of the SGSM2 gene and/or the full-length protein encoded by the SGSM2 gene.

7. The genetically modified NK cell of any one of claims 1-4, wherein the modification comprises recombinant expression of the OR7A10 gene.

8. The genetically modified NK cell of claim 7, wherein the recombinant expression causes increased or enhanced expression of the OR7A10 gene and/or the full-length protein encoded by the OR7A10 gene.

9. The genetically modified NK cell of any one of claims 1-4, wherein the modification comprises recombinant expression of the APLN gene.

10. The genetically modified NK cell of claim 9, wherein the recombinant expression causes increased or enhanced expression of the APLN gene and/or the full-length protein encoded by the APLN gene.

11. The genetically modified NK cell of any one of claims 1-4, wherein the modification comprises recombinant expression of the PDP1 gene.

12. The genetically modified NK cell of claim 11, wherein the recombinant expression causes increased or enhanced expression of the PDP1 gene and/or the full-length protein encoded by the PDP1 gene.

13. The genetically modified NK cell of any one of claims 1-4, wherein the modification comprises recombinant expression of the CYB5B gene.

14. The genetically modified NK cell of claim 13, wherein the recombinant expression causes increased or enhanced expression of the CYB5B gene and/or the full-length protein encoded by the CYB5B gene.

15. The genetically modified NK cell of any one of claims 1-14, wherein the genetically modified NK cell expresses 11-15 and 11-21 in addition to enhanced or up-regulated expression of the SGSM2 gene, and/or the OR7A10 gene, and/or the APLN gene, and/or the PDP1 gene, and/or the CYB5B gene.

16. The genetically modified NK cell of any one of claims 1-15, further comprising at least one additional genetic modification.

17. The genetically modified NK cell of any one of claims 1-16, wherein the NK cell expresses or encodes a Chimeric Antigen Receptor (CAR).

18. The genetically modified NK cell of claim 17, wherein the CAR targets a cancer antigen.

19. The genetically modified NK cell of claim 18, wherein the cancer antigen is a neoantigen derived from a subject.

20. The genetically modified NK cell of claim 19, wherein the cancer antigen is selected from the group consisting of ENPP3, 4-1BB, 5T4, adenocarcinoma antigen, alpha fetoprotein, BAFF, B lymphoma cell, C242 antigen, CA 125, carbonic anhydrase 9 (CA IX), C-MET, CCR4, CD 152, CD 19, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44 v6, CD51, CD52, CD56, CD74, CD80, CEA, CNTO888, CTLA 4, DR5, EGFR, EpCAM, CD3, FAP, fibronectin extra domain B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HER2/neu, HGF, human scatter factor receptor kinase, IGF 1 receptor, IGF I, IgGl, LI CAM, IL 13, IL 6, insulin-like growth factor I receptor, integrin a5pi, integrin avP3, MORAb 009, MS4A1, MUC1, mucin CanAg, N glycolylneuraminic acid, NPC 1C, PDGF R a, PDL192, phosphatidylserine, prostatic carcinoma cells, RANKL, RON, ROR1, SCH 900105, SDC1, SLAMF7, TAG 72, tenascin C, TGF beta 2, TGF p, TRAIL Rl, TRAIL R2, tumor antigen CTAA16.88, VEGF A, VEGFR 1, VEGFR2, and vimentin.

21. The genetically modified NK cell of any one of claims 1-20, wherein the NK is derived from a subject diagnosed as having, or who is identified as being at increased risk of having a disease or disorder.

22. The genetically modified NK cell of claim 21, wherein the subject is diagnosed as having, or is identified as being at increased risk of having cancer.

23. The genetically modified NK cell of any one of claims 1-20, wherein the NK is derived from a healthy subject prior to the genetic modification.

24. The genetically modified NK cell of any one of claims 1-23, wherein the modification causes increased or enhanced expression, activation, presentation, and/or function of one or more cytokine(s), as compared to a non-genetically modified control NK cell.

25. The genetically modified NK cell of claim 24, wherein at least one of IL- 15 and IL- 21 is recombinantly expressed in the cell.

26. The genetically modified NK cell of claim 24 or 25, wherein IL-15 is recombinantly expressed in the cell.

27. The genetically modified NK cell of claim 24 or 25, wherein IL-21 is recombinantly expressed in the cell.

28. The genetically modified NK cell of claim 24 or 25, wherein IL-15 and IL-21 are both recombinantly expressed in the cell.

29. A population of NK cells derived by expanding the genetically modified NK cell of any one of claims 1-28.

30. A pharmaceutical composition comprising the population of NK cells of claim 29 and a pharmaceutically acceptable buffer, carrier, diluent or excipient for administration in vivo.

31. The pharmaceutical composition of claim 30, further comprising at least one cytokine.

32. The pharmaceutical composition of claim 31, wherein the cytokine comprises IL-15.

33. The pharmaceutical composition of claim 31, wherein the cytokine comprises IL-21.

34. The pharmaceutical composition of claim 31, wherein the cytokine comprises IL- 15 and IL-21.

35. A method of treating a subject having a disease, disorder, or condition comprising administering to the subject an effective amount of the pharmaceutical composition of any one of claims 30-34.

36. A method of treating a subject having a disease, disorder, or condition associated with an elevated expression or specific expression of an antigen, the method comprising administering to the subject an effective amount of the population of genetically modified NK cells of claim 29, wherein the NK cells comprise a CAR that targets the antigen.

37. A method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of a population of genetically modified NK cells, wherein at least one gene selected from the group consisting of SGSM2, OR7A10, APLN, PDP1, GABBR1, PRR14L, TIAM1, KRT82, PLA2G1B, REM2, HIST1H2BN, CYB5B, LRRC23, NXPE3, MEGF11, FKBP5, PPFIA2, LRRC23, PEAR1, REM2, TIAM2, HPRT1, MMACHC, and ZBTB20, is up-regulated or over-expressed in the NK cell, as compared to a non-genetically modified control NK cell; and/or at least one cytokine selected from Interleukin- 15 (11-15) and Interleukin-21 (11-21) is expressed by the cell.

38. The method of claim 37, wherein the genetically modified NK cells comprise increased or enhanced expression of one or more of the genes and/or the full-length protein(s) encoded by the gene(s) SGSM2, OR7A10, APLN, PDP1 and CYB5B as compared to a non- genetically modified control NK cell, and wherein the modification enhances the anti-cancer efficacy of the NK cell as compared to a non-genetically modified control NK cell.

39. The method of claim 37 or 38, wherein the genetically modified NK cells comprise recombinant expression of the SGSM2 gene and/or the functional protein encoded by the SGSM2 gene.

40. The method of claim 37 or 38, wherein the genetically modified NK cells comprise recombinant expression of the OR7A10 gene and/or the functional protein encoded by the OR7A10 gene.

41. The method of any one of claims 37 to 40, wherein the NK cell is genetically modified to express or encode a Chimeric Antigen Receptor (CAR).

42. The method of claim 41, wherein the CAR targets an antigen expressed by the cancer.

43. The method of any one of claims 37 to 42, wherein the cancer is selected from the group consisting of leukemia, vascular cancer such as multiple myeloma, adenocarcinomas and bone, bladder, brain, breast, cervical, ovarian, colorectal, esophageal, kidney, liver, lung, nasopharangeal, pancreatic, prostate, skin, stomach, and uterine cancer.

44. The method of any one of claims 37 to 43, wherein the cancer is breast cancer, lung cancer, colorectal cancer, ovarian cancer, or skin cancer.

45. The method of any one of claims 37 to 44, wherein the NK cell is genetically modified to express at least one recombinant cytokine selected from the group consisting of IL- 15, and IL-21.

46. The method of any one of claims 37 to 45, wherein the NK cells are derived from the subject prior to genetic modification.

47. The method of any one of claims 37 to 46, further comprising administering to the subject cytokine IL- 15, or cytokine IL-21, or both cytokine IL- 15 and cytokine IL-21.

48. The method of any one of claims 37 to 47, wherein the administration comprises injection of the composition of cells into or directly adjacent to a tumor, or into the blood stream, or into the brain or into a ventricle of the heart of the subject.

49. The method of any one of claims 37 to 48, further comprising administering to the subject one or more additional therapeutic agents and/or procedures.

50. The method of claim 49, wherein the additional therapeutic agent and/or procedure is selected from the group consisting of a chemotherapeutic agent, an antimicrobial agent, an immune checkpoint inhibitor, a PD-I inhibitor, a CTLA-4 inhibitor, radiation treatment and surgery.

51. A method of performing gain of function screening of a Natural Killer (NK) cell, the method comprising:

(i) transducing an NK cell with one or more CRISPRa single-guide RNA(s) (sgRNAs),

(ii) causing the NK cell to be genetically modified by CRISPRa-mediated genome editing of a gene targeted by the sgRNA; and

(iii) screening the NK cell for tumor cell killing.

52. The method of claim 51, wherein one sgRNA comprises

(i) a guide sequence; and

(ii) a tracrRNA comprising a nucleic acid sequence selected from a library.

53. The method of claim 52, wherein the library comprises all or part of a human genomic reference sequence library.

54. The method of any one of claims 51-53, wherein the sgRNA is comprised within a vector.

55. The method of claim 54, wherein the vector is a lentiviral vector.

56. The method of claim 54 or 55, wherein the vector further comprises an expression cassette for the sgRNA.

57. The method of claim 56, wherein the expression cassette further comprises a nucleic acid construct configured to express or encode a chimeric antigen receptor (CAR).

58. The method of any one of claims 51-57, wherein steps (i)-(iii) are carried out using a plurality of NK cells, and wherein each of the plurality of NK cells is contacted by one or more sgRNAs comprising one or more sequences of a library of sequences.

59. The method of claim 58, wherein the plurality of NK cells is collectively contacted by a multiplicity of sgRNAs, wherein an sgRNA of the multiplicity of sgRNAs comprises a single sequence from the library, and wherein an NK cell of the plurality of NK cells contacted by an sgRNA over-expresses a single gene, relative to a control NK cell that is not contacted by the sgRNA.

60. The method of any one of claims 51-59, wherein screening the NK cell for tumor cell killing is carried out in vitro.

61. The method of any one of claims 51-59, wherein screening the NK cell for tumor cell killing is carried out in vivo.

62. The method of claim 61, wherein the in vivo screening is carried out using a tumorbearing animal model, and wherein the screening comprises selecting genetically modified NK cells from animals with enhanced survival/reduced tumor burden as compared to control animals that did not receive the same genetically modified NK cells.

63. The method of any one of claims 51-62, further comprising characterizing the mutant NK cell(s) by single cell transcriptome analysis.

64. The method of any one of claims 51-63, further comprising characterizing the mutant NK cell(s) by sequence analysis to identify mutated genes.

65. The method of any one of claims 51-64, further comprising repeating steps (i)-(iii) using a selected pool of sgRNAs for one or more additional rounds.

66. A genetically modified NK cell created according to the method of any one of claims 51-65.

67. A pharmaceutical composition comprising

(i) a population of genetically modified NK cells derived by expanding the genetically modified NK cell of claim 66; and

(ii) a pharmaceutically acceptable excipient for administration in vivo.

68. A genetically modified Natural Killer (NK) cell comprising a mutation that causes up-regulated or enhanced expression of one or more genes selected from the group consisting of SGSM2, OR7A10, APLN, PDP1, and CYB5B and/or the functional protein encoded by one or more genes selected from the group consisting of SGSM2, OR7A10, APLN, PDP1 and CYB5B in the cell as compared to a non-genetically modified NK cell, wherein the mutation enhances the anti-cancer efficacy of the genetically modified NK cell as compared to a non-genetically modified NK cell, and wherein the genetically modified NK cell expresses or encodes a Chimeric Antigen Receptor (CAR) that targets a cancer antigen.

69. The genetically modified NK cell of claim 68, wherein the cancer antigen is selected from the group consisting of ENPP3, 4-1BB, 5T4, adenocarcinoma antigen, alpha fetoprotein, BAFF, B lymphoma cell, C242 antigen, CA 125, carbonic anhydrase 9 (CA IX), C-MET, CCR4, CD 152, CD 19, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44 v6, CD51, CD52, CD56, CD74, CD80, CEA, CNTO888, CTLA 4, DR5, EGFR, EpCAM, CD3, FAP, fibronectin extra domain B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HER2/neu, HGF, human scatter factor receptor kinase, IGF 1 receptor, IGF I, IgGl, LI CAM, IL 13, IL 6, insulin-like growth factor I receptor, integrin a5pi, integrin avP3, MORAb 009, MS4A1, MUC1, mucin CanAg, N glycolylneuraminic acid, NPC 1C, PDGF R a, PDL192, phosphatidylserine, prostatic carcinoma cells, RANKL, RON, ROR1, SCH 900105, SDC1, SLAMF7, TAG 72, tenascin C, TGF beta 2, TGF p, TRAIL Rl, TRAIL R2, tumor antigen CTAA16.88, VEGF A, VEGFR 1, VEGFR2, and vimentin.

70. The genetically modified NK cell of claim 68 or 69, wherein the genetically modified NK has increased tumor penetration and/or increased anti-tumor cytotoxicity as compared to a non-genetically modified NK cell.

71. A genetically modified Natural Killer (NK) cell, wherein the SGSM2 gene is up- regulated and/or over-expressed in the cell, as compared to a non-genetically modified control NK cell, wherein the modification increases or enhances expression, activation, presentation, and/or function of one or more protein(s) encoded by the SGSM2 gene(s) and enhances the anticancer efficacy of the NK cell as compared to a non-genetically modified control NK cell, and wherein the modified NK cell expresses or encodes a Chimeric Antigen Receptor (CAR) that targets a cancer antigen.