Natural killer cells expressing a mesothelin-targeted chimeric antigen receptor and interleukin-15 for the treatment of solid tumors

Abstract

Induced pluripotent stem cells (iPSCs) and iPSC-derived natural killer (iNK) cells co-expressing interleukin-15 (IL-15) and a chimeric antigen receptor (CAR) targeted to mesothelin are described. Nucleic acid molecules and vectors encoding a mesothelin-targeted CAR and IL-15, such as for transduction of iPSC and iNK cells, are also described. The CAR / IL-15-expressing iNK cells can be used for the treatment of solid tumors, such as mesothelin-positive solid tumors. Methods of producing hematopoietic progenitor cells and iNK cells from an iPSC line, such as the LiPSC-GR1.1 line, are also described.

Description

[0001]4239-111805-02 NATURAL KILLER CELLS EXPRESSING A MESOTHELIN-TARGETED CHIMERIC ANTIGEN RECEPTOR AND INTERLEUKIN-15 FOR THE TREATMENT OF SOLID TUMORS CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No.63 / 647,371, filed May 14, 2024, which is herein incorporated by reference in its entirety. FIELD This disclosure concerns natural killer (iNK) cells, differentiated from an induced pluripotent stem cell (iPSC) line, which co-express a mesothelin-specific chimeric antigen receptor (CAR) and interleukin-15 (IL-15). Use of the CAR / IL-15-expressing iNK cells for the treatment of mesothelin- positive solid tumors is also described. ACKNOWLEDGMENT OF GOVERNMENT SUPPORT This invention was made with government support under project number ZIA BC 010816 awarded by the National Institutes of Health. The government has certain rights in the invention. INCORPORATION OF ELECTRONIC SEQUENCE LISTING The electronic sequence listing, submitted herewith as an XML file named 4239-111805- 02.xml (31,415 bytes), created on May 5, 2025, is herein incorporated by reference in its entirety. BACKGROUND The mesothelin (MSLN) gene encodes a ~70 kDa precursor protein that is processed to a ~30 kDa N-terminal protein and a ~ 40 kDa C-terminal membrane-bound mature mesothelin (Hassan and Ho, Eur J Cancer 44:46-53, 2008). Mesothelin is present at relatively low levels in mesothelial cells of the pleura, peritoneum and pericardium of healthy individuals, but is highly expressed in malignant mesotheliomas (Chang et al., Cancer Res 52:181-186, 1992; Chang and Pastan, Proc Natl Acad Sci USA 93:136-140, 1996) and other solid tumors, such as lung cancer, stomach cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, thymic carcinoma, and colorectal cancer (Hassan et al., Clin. Cancer Res.10:3937-3942, 2004; Argani et al., Clin. Cancer Res.7:3862-3868, 2001; Hassan et al., Appl. Immunohistochem. Mol. Morphol.13:243-247, 2005; Li et al., Mol. Cancer Ther.7:286-296, 2008; Yu et al., J Cancer 1:141-1749, 2010; Tchou et al., Breast Cancer Res Treat 133(2):799-804, 2012; Thomas et al., Lung Cancer.101:104-110, 2016; Shiraishi et al., Oncotarget. 11(43): 3807–3817, 2020; U.S. Patent No.7,081,518). Due to its expression in a number of different types of cancer, and minimal expression in healthy tissues, mesothelin is a suitable immunotherapy target for the treatment of multiple solid tumors. 4239-111805-02 SUMMARY The present disclosure describes the generation of iNK cells from an induced pluripotent stem cell (iPSC) line using a method that includes differentiating the iPSCs into embryoid bodies (EBs) comprised of hematopoietic progenitor cells and further differentiating these progenitor cells into iNK cells. Also described are nucleic acid molecules and vectors that encode both a mesothelin-targeted chimeric antigen receptor (CAR) and human interleukin-15 (IL-15). The disclosed nucleic acid molecules and vectors can be used to transduce the iPSCs (prior to differentiation to iNK cells) to generate iPSCs that co-express the CAR and IL-15. The CAR / IL-15-expressing iPSCs are then differentiated into iNK cells, which can be used for the treatment of solid tumors, such as solid tumors positive for expression of mesothelin. Provided herein are nucleic acid molecules that include a nucleic acid sequence encoding a CAR that specifically binds mesothelin and a nucleic acid sequence encoding IL-15. In some aspects, the mesothelin-targeted CAR includes an antigen-binding domain having a variable heavy (VH) domain and a variable light (VL) domain that include the complementarity determining region (CDR) sequences of antibody hYP218. The CAR further includes a hinge region, a transmembrane domain, an intracellular co-stimulatory domain, and an intracellular signaling domain. In some examples, the CAR includes a CD8α hinge region, a NKG2D transmembrane domain, a 2B4 intracellular co- stimulatory domain and a CD3ζ intracellular signaling domain. In other examples, the CAR includes a CD8α hinge region, a CD8α transmembrane domain, a 4-1BB intracellular co-stimulatory domain and a CD3ζ intracellular signaling domain. Also provided are vectors that include a disclosed nucleic acid molecule. In some aspects, the vector is a transposon vector, such as a piggyBac transposon vector. Natural killer (NK) cells that include a nucleic acid molecule or vector disclosed herein are further provided. Also provided herein are NK cells that express a CAR that specifically binds mesothelin and IL-15, wherein the IL-15 is expressed from an exogenous nucleic acid molecule encoding IL-15. In some aspects, the mesothelin-targeted CAR includes an antigen-binding domain having a VH domain and a VL domain that include the CDR sequences of antibody hYP218. The CAR further includes a hinge region, a transmembrane domain, an intracellular co-stimulatory domain, and an intracellular signaling domain. In some examples, the CAR includes a CD8α hinge region, a NKG2D transmembrane domain, a 2B4 intracellular co-stimulatory domain and a CD3ζ intracellular signaling domain. In other examples, the CAR includes a CD8α hinge region, a CD8α transmembrane domain, a 4-1BB intracellular co-stimulatory domain and a CD3ζ intracellular signaling domain. In some aspects, the NK cell is derived from an iPSC line (such NK cells are referred to herein as “iNK” cells). In some examples, the iPSC line is LiPSC-GR1.1. 4239-111805-02 Compositions that include a pharmaceutically acceptable carrier and a nucleic acid molecule, vector or NK / iNK cell disclosed herein are also provided. Further provided are methods of treating a mesothelin-positive cancer in a subject. In some aspects, the method includes administering to the subject a therapeutically effective amount of a nucleic acid molecule, vector, NK / iNK cell or composition disclosed herein. Also provided herein is a method of differentiating iPSCs to hematopoietic progenitor cells. In some aspects, the method includes seeding iPSCs (such as trypsin-adapted iPSCs) in a culture vessel containing albumin polyvinylalcohol essential lipids (APEL) culture medium that includes human stem cell factor (SCF), human vascular endothelial growth factor (VEGF), human bone morphogenetic protein 4 (BMP-4) and Y-27632; and subjecting the seeded iPSCs to centrifugation and continuing to culture the iPSCs in the APEL culture medium to produce embryoid bodies (EBs) containing hematopoietic progenitor cells. In some aspects, the method further includes producing iNK cells from the EBs by culturing the EBs in NK cell differentiation medium that contains interleukin-3 (IL-3), IL-15, IL-7, SCF and fms-like tyrosine kinase 3 (FLT3) ligand, thereby producing iNK cells, such as CD45+CD56+ iNK cells. In some aspects, prior to differentiation of the iPSCs to hematopoietic progenitor cells, the iPSCs are transduced with a CAR / IL-15-encoding nucleic acid molecule or vector disclosed herein, and optionally enriching iPSCs that express the CAR. In some examples of these methods, the iPSCs are LiPSC-GR1.1 cells. The foregoing and other features of this disclosure will become more apparent from the following detailed description of several aspects which proceeds with reference to the accompanying figures. BRIEF DESCRIPTION OF THE DRAWINGS FIGS.1A-1F: Differentiation of iNKs from 6 human iPSC lines. (FIG.1A) Scheme of NK differentiation from iPSCs. EBs were generated using the spin method from single-cell dissociated human iPSCs seeded on day -6 and maintained in feeder- and serum-free conditions in a U-bottom 96 well plate. Six days later (after hematopoietic progenitor cells were generated), EBs were transferred into NK cell differentiation conditions on Day 0. NK cells were then harvested 4 weeks post differentiation. (FIG.1B) the expression of pluripotency markers SSEA-4 and TRA-1-60 was detected in the iPSC lines via flow cytometry. (FIG.1C) Left panel: the morphology of day 0 EBs and 4 weeks of NK differentiation from 6 different iPSC cell lines. The bars represent 1000 µm. Right panel: representative magnified pictures of differentiated iNKs 4 weeks post NK differentiation. The bars represent 200 µm. (FIG.1D) The iNK differentiation yield per EB from different iPSC lines (n = 2~3 independent differentiation experiments performed for each iPSC line). ND means no differentiation was observed in these lines. *p<0.05, ***p<0.001, analyzed using unpaired student t test. (FIG.1E) NK differentiation from the 3 iPSC lines LiPSC-GR1.1, NCRM5, NCRM6 4239-111805-02 was validated by staining for CD45 and CD56. (FIG.1F) The median fluorescence intensity (MFI) of CD56 was measured by flow cytometry on PBNKs (from 3 healthy donors) and iNKs (derived from LiPSC-GR1.1, NCRM5, and NCRM6). FIGS.2A-2G: Robust iNK differentiation yield from MSLN.CAR-IL-15 transduced LiPSC- GR1.1. (FIG.2A) MSLN.CAR-IL-15 piggyBac transposon vector construct encoding CAG promotor, MSLN.CAR (MSLN-targeted hYP218 scFv, CD8a hinge spacer, NKG2D transmembrane domain, 2B4 intracellular domain, and CD3ζ chain stimulatory domain), EGFRt, and human IL-15. GMCSFRss, GM-CSF receptor-α chain signal sequence directing cell surface expression. (FIG.2B) Genetic modification of LiPSC-GR1.1 cells with MSLN.CAR and MSLN.CAR-IL-15 piggyBac transposon systems. MSLN.CAR expression was examined by measuring EGFRt expression in transfected iPSCs. (FIG.2C) Human IL-15 production in the supernatant of iPSC culture. ****p<0.0001. (FIG.2D) NK differentiation yield per EB from MSLN.CAR-IL-15 modified iPSC and control iPSCs. ***p<0.001. (FIG.2E) iNK differentiation yield achieved with different concentration of IL-15 (10 ng / ml, 20 ng / ml, 40 ng / ml, 80 ng / ml) added to the mock LiPSC-GR1.1 iNK differentiation media. *p<0.05. (FIG.2F) NK differentiation and CAR expression in NK were validated though detecting CD45, CD56 and EGFRt expression in the cells. (FIG.2G) Cryopreserved differentiated iNKs were thawed and expanded for 7 days. The fold increase of expansion is shown. **p<0.01. The statistics were analyzed using unpaired student t test. FIGS.3A-3D: Characterization of freshly differentiated GR1.1-iNKs. GR1.1-iNKs were harvested 4 weeks post EB differentiation. (FIG.3A) Cell surface receptor expression in NK cells was detected via flow cytometry. CD56+CD3- PBNKs and CD56+GR1.1-iNKs cells were gated for the indicated biomarker analyses. The dark grey area represents biomarker staining. The light grey area represents isotype control. (FIGS.3B-3D) RNA-seq of PBNKs, mock iNKs and MSLN.CAR-IL-15 iNKs. Data are from one RNA-seq analysis with three biological replicates per group. (FIG.3B) Principal component analysis (PCA) of differentially expressed genes (DEGs). Each dot represents one sample. Each color represents a NK population. (FIG.3C) Bubble plot of GSEA analysis showing the highest-ranked top 10 KEGG and Hallmark gene sets significantly upregulated (p <0.05, adjusted p < 0.1) in mock iNK vs. PBNK (left panel), and MSLN.CAR-IL-15 iNKs v.s. mock iNK (right panel). The x-axis represents the enrichment score and the size of the bubble represents the number of genes in the gene set. (FIG.3D) The heatmap shows the gene expression (log2 (TPM+1)) of NK phenotypic markers in PBNKs, mock iNKs, and MSLN.CAR-IL-15 iNKs, including NK signature, activating receptors, inhibitory KIR, checkpoint, cytokine and receptors, chemokine receptors and cytolytic molecule related genes. FIGS.4A-4C: In vitro tumor killing function of MSLN.CAR-IL-15 GR1.1-iNKs. (FIG.4A) Cytotoxicity of mock iNKs, MSLN.CAR iNKs and MSLN.CAR-IL-15 iNKs against MSLN+KLM1- WT and MSLN- KLM1-KO cells. (FIG.4B) Cytotoxicity of all the iNKs against multiple MSLN+ 4239-111805-02 solid tumor cell lines. (FIG.4C) CD107a degranulation signature induced by the co-culture of iNKs with KLM-1 tumor cells. FIGS.5A-5D: The anti-tumor efficacy of MSLN.CAR-IL-15 GR1.1-iNKs in the NCI- meso63 mouse model. (FIG.5A) Schematic of tumor inoculation and iNK treatments in the NCI- Meso63 tumor model. (FIG.5B) Tumor growth monitored through BLI. P values were calculated using unpaired student t test. (FIG.5C) Tumor growth based on BLI measurements. The statistics were analyzed using unpaired student t test. (FIG.5D) Median overall survival of different groups is shown (p=0.07, Mock iNK vs Untreated; p<0.005, CAR-IL-15 iNK vs Mock iNK, analyzed using log-rank (Mantel-Cox) test). FIGS.6A-6H: Increased tumor infiltration of MSLN.CAR-IL-15 iNKs in NCI-meso63 tumors. (FIG.6A) Schematic of iNK treatments and tissue harvest in the NCI-Meso63 tumor model. (FIG.6B) Tumor growth monitored using BLI. (FIG.6C) Flow cytometric analysis of hCD45+mCD45- cells in the tumors on day 7 post iNK treatment (top panel). The hCD45+mCD45- cells were further gated to analyze hCD56 and hEGFRt expression (bottom panel). (FIG.6D) Percentage of hCD45+mCD45- cells among total live single cells isolated from spleens and tumors on day 7 (left and middle panels); percentage of hEGFRt+hCD56+cells among hCD45+mCD45- cells in spleens and tumors harvested from MSLN.CAR-IL-15 iNK treated mice on day 7 (right panel). (FIG. 6E) Multiplex immunofluorescence imaging of tumors harvested from mice treated with mock iNKs and MSLN.CAR-IL-15 iNKs on day 7 post iNK treatment. The bars represent 150 µm. (FIG.6F) hCD45+cell density in iNK-treated tumor. Three independent whole tissue images were analyzed. (FIG.6G) High magnification images showing Ki67+ hCD45+NK cells co-localized with hMSLN+tumor cells in mice treated with MSLN.CAR-IL-15 iNK. The bars represent 40 µm. (FIG.6H) The percentage of Ki67+cells among total hCD45+cells in iNK-treated tumor. Three independent whole tissue images were analyzed. P values were determined using unpaired student t test. FIGS.7A-7I: scRNAseq analysis of MSLN.CAR-IL-15 iNKs-treated NCI-meso63 tumors. (FIG.7A) Uniform manifold approximation and projection (UMAP) plots visualizing transcriptome- defined clusters of NK cells and tumor cells. (FIG.7B) Representative signature genes across NK and tumor cells. The size of the dots indicates the percentage of cells expressing the gene, while the color of the dots indicates average gene expression level. (FIG.7C) UMAP for total cells from untreated and treated groups. Cell types are annotated with the same color scheme as in FIG.7A. (FIG.7D) UMAP of identified aneuploid and diploid cells using copyKat analysis (top) and their fractions in each tumor clusters (bottom). (FIG.7E) The fractions of defined tumor subclusters among tumor cells in treated and untreated groups. (FIG.7F) The expression of representative tumor signature genes across tumor subclusters. (FIG.7G) MSLN expression in tumor subclusters in untreated and treated groups. (FIG.7H) The top upregulated and downregulated Hallmark pathways in treated versus untreated tumor subclusters (adjusted p < 0.25). The x-axis represents the enrichment scores and the 4239-111805-02 size of the bubble represents the number of genes in the gene set. (FIG.7I) The expression of HLA type I and HLA type II genes in untreated and iNK-treated tumor subclusters. FIGS.8A-8G: Single cell transcriptional profiles of NCI-meso63 tumor-infiltrating CAR-IL- 15 iNKs. (FIG.8A) The fractions of defined NK subclusters in NK cells in pre-infusion and post- infusion groups. (FIG.8B) The expression of representative signature genes across NK subclusters. (FIG.8C) The expression of NK associated biomarker genes (activation, inhibitory receptors, cytokines, chemokines and cytolytic related categories). (FIG.8D) The upregulated and downregulated Hallmark pathways in total post-infusion versus pre-infusion iNKs (adjusted p < 0.1). (FIGS.8E-8G) Heatmaps indicating the expression of selected gene sets in NK subtypes. FIG.9: Tri-lineage differentiation ability of the iPSC lines. The Scorecard assay confirms pluripotency and differentiation potential of the iPSC lines NCRM1, NCRM2, NCRM4, NCRM5, and NCRM6. Top panel showed Scorecard Values. Algorithm scores for the samples show up regulation or down regulation of the endoderm, mesoderm, ectoderm or pluripotent (self-renewal) markers relative to the reference set of nine undifferentiated pluripotent stem cell lines. Bottom panel is the heatmap of gene expression associated with self-renewal, mesendoderm, ectoderm, mesoderm and endoderm. Colors correlate to the fold change in expression of the indicated gene relative to the undifferentiated reference set. FIG.10: The representative pictures of iNK differentiation from genetically modified or mock iPSCs cell line LiPSC-GR1.1. Day 0 EBs and 4 weeks iNK differentiation from mock, MSLN.CAR-expressing, and MSLN.CAR-IL-15 expressing iPSC were shown. The bars represent 400 µm. FIGS.11A-11C: Differentially expressed genes analyses of RNA-seq data of different NK products. PBNKs were purified from 3 healthy donors. Mock iNKs and MSLN.CAR-IL-15 iNKs were harvested 4 weeks post iNK differentiation. (FIG.11A) Heatmap of hierarchical clustering of differentially expressed genes. (FIG.11B) Volcano plots of log2 (fold change) and –log10FDR of all tested genes between mock iNK and PBNK, as well as between MSLN.CAR-IL-15 iNKs and mock iNKs. Each dot in red represents an individual gene differentially expressed with log2FC >1, p < 0.05. (FIG.11C) The heatmap shows the gene expression (log2 (TPM+1)) of “adaptive” NK markers in PBNKs, mock iNKs, and MSLN.CAR-IL-15 iNKs. FIGS.12A-12D: In vivo efficacy of freshly differentiated GR1.1-iNK cells against human KLM-1 pancreatic tumors. (FIG.12A) Schematic of tumor inoculation and iNK treatments in KLM-1 tumor model. KLM-1 cells were i.p. injected 5 days before iNK treatment. The mice were then administrated three doses of freshly differentiated iNKs on day 0, day 7 and day 14. (FIG.12B) Tumor growth monitored through BLI. (FIG.12C) Summarized tumor growth curve. (FIG.12D) The median overall survival of different groups post iNK treatment (p<0.05, CAR-IL-15 iNK vs Mock iNK). 4239-111805-02 FIGS.13A-13C: In vivo efficacy of expanded frozen GR1.1-iNK cells against NCI-meso63 tumors. (FIG.13A) Tumor growth monitored through BLI. (FIG.13B) Summarized tumor growth curve. (FIG.13C) The median overall survival of different groups post iNK treatment (p<0.05, Mock iNK vs Untreated; p=0.01, CAR-IL-15 iNK vs Mock iNK). FIG.14: Surface biomarker expression of post-infusion CAR-IL-15 iNKs. Flow cytometry was performed to analyze the iNKs in tumors on day 7 post CAR-IL-15 iNK treatment. hCD56+cells were gated to analyze expression of the indicated biomarkers. The solid open lines represent biomarker staining. The grey areas represent isotype control. FIG.15: Schematic for production of MSLN.CAR / IL-15 GR1.1-iNK cells. LiPSC-GR1.1 was selected as the iPSC line for NK differentiation and genetic modification using the MSLN.CAR / IL-15 piggyBac transposon system disclosed herein. MSLN.CAR / IL-15 expressing iPSCs cells were enriched through FACS sorting for EGFR+ cells and expanded. The spin embryoid bodies (EBs) method was used to generate hematopoietic progenitor cells, which were further differentiated to iNK cells. The MSLN.CAR / IL-15 GR1.1-iNK product was validated through detecting NK phenotypic biomarkers and evaluating its specific killing ability against mesothelin positive solid tumors. FIGS.16A-16D: hYP218.CD8a.BB.Z.CAR / IL-15 modification also increased GR1.1-iNK cell differentiation yield and tumor killing ability. (FIG.16A) Schematic representation of the transgene including an EF1α promotor, the MSLN.CAR construct (which includes MSLN-targeted hYP218 scFv, CD8α hinge / spacer, CD8α transmembrane domain, 4-1BB intracellular co-stimulatory domain, and CD3ζ intracellular signaling domain), truncated human EGFR (EGFRt), and human IL- 15. (FIG.16B) The NK cell yield per EB from hYP218.CD8a.BB.Z.CAR / IL-15 modified and mock iPSCs. (FIG.16C) Representative flow cytometry analysis validating hCD56 and EGFRt expression in the differentiated iNKs from modified iPSCs. (FIG.16D) The cytotoxicity of hYP218.CD8a.BB.Z.CAR / IL-15 GR1.1-iNK cells against MSLN+ tumor cells. SEQUENCES The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and single letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. In the accompanying sequence listing: SEQ ID NO: 1 is the amino acid sequence of the hYP218 VH domain. SEQ ID NO: 2 is the amino acid sequence of the hYP218 VL domain. SEQ ID NOs: 3-8 are the amino acid sequences of the hYP218 CDRs according to Kabat. SEQ ID NOs: 9-14 are the amino acid sequences of the hYP218 CDRs according to IMGT. 4239-111805-02 SEQ ID NO: 15 is the amino acid sequence of a CD8α hinge region. SEQ ID NO: 16 is the amino acid sequence of a NKG2D transmembrane domain. SEQ ID NO: 17 is the amino acid sequence of a CD8α transmembrane domain. SEQ ID NO: 18 is the amino acid sequence of a 2B4 intracellular co-stimulatory domain. SEQ ID NO: 19 is the amino acid sequence of a 4-1BB intracellular co-stimulatory domain. SEQ ID NO: 20 is the amino acid sequence of a CD3ζ intracellular signaling domain. SEQ ID NO: 21 is the amino acid sequence of IL-15. SEQ ID NO: 22 is a nucleic acid sequence encoding hYP218.NKG2D.2B4.Z.CAR / IL-15. SEQ ID NO: 23 is a nucleic acid sequence encoding hYP218.CD8a.BB.Z.CAR / IL-15. SEQ ID NO: 24 is the amino acid sequence of hYP218.NKG2D.2B4.Z.CAR. SEQ ID NO: 25 is the amino acid sequence of hYP218.CD8a.BB.Z.CAR. DETAILED DESCRIPTION I. Introduction Chimeric antigen receptor (CAR) T cell therapy has been highly effective for treating blood cancers but has shown limited success against solid tumors (Albelda, Nat Rev Clin Oncol 21:47-66, 2024). CAR-engineered natural killer (NK) cells provide both CAR-dependent and independent antitumor effects with lower toxicity and offer scalable off-the-shelf production, making them a promising alternative cell therapy (Dagher and Posey, Nat Immunol 24:1994-2007, 2023; Wang et al., Cell Death Discov 10:40, 2024). Induced pluripotent stem cell (iPSC)-based NK (iNK) cell therapies have several advantages, such as a nearly limitless source, efficient and stable CAR expression with one-time genetic modification, and a relatively homogenous cell product with a more standardized production pipeline (Lin et al., Biomed Pharmacother 165:115123, 2023). However, CAR iNK manufacturing is a complex and challenging process that includes CAR engineering in iPSCs, differentiating iPSCs into functional iNKs and expanding these cells at a large scale (Maddineni et al., J Immunother Cancer 10:e004693, 2022). iPSCs derived from different donors exhibit genetic variability, which can influence the differentiation potential, functionality, and safety profile of the resulting iNKs (Cichocki et al., Blood 141:846-855, 2023). Thus, iPSC line diversity also poses challenges for standardizing cell manufacturing processes for iPSC-based therapies (Nath et al., Int J Mol Sci 24:16929, 2023). The Regenerative Medicine Program was launched by the U.S. National Institutes of Health to develop resources to catalyze therapeutic use of iPSCs. To date, this program has generated one clinical-grade cGMP iPSC line and 14 research-grade iPSC lines, all of which are accessible to the research community. Among them, six iPSC lines were generated from healthy donor-derived cord blood CD34+cells, including the cGMP-manufactured standardized line LiPSC- GR1.1, which has been well characterized (Baghbaderani et al., Stem Cell Reports 5:647-659, 2015; Baghbaderani et al., Stem Cell Rev Rep 12:394-420, 2016). Ideally, using well characterized cGMP- 4239-111805-02 manufactured human iPSC lines to produce CAR-iNK products may result in time and cost reduction in translation into the clinic due to elimination of the time-consuming and resource-intensive quality control measures needed for characterization of newly reprogrammed iPSCs. Mesothelin (MSLN) is a glycosylphosphatidylinositol-anchored membrane glycoprotein highly expressed in many solid tumors but with limited expression on normal tissues, making it an attractive target antigen for a cell therapy (Hassan et al., Clin Cancer Res 10:3937-3942, 2004). NK cells generally have a short lifespan in vivo, especially without support from cytokines like IL-2 or IL-15 (Zhang et al., Immunology 121:258-265, 2007), which play a critical role in NK cell development and homeostasis (Liu et al., J Clin Invest 123:4410-4422, 2013; Fehniger et al., J Clin Invest 106:117-124, 2000). IL-15 has been included in CAR-constructs for genetically modifying primary NK products to locally enhance NK cell survival, expansion, and anti-tumor activity (Ma et al., Trends Immunol 43:833-847, 2022). Most of the research in this area has focused on hematopoietic cancers, such as IL-15-expressing CD19-CAR NK therapies (Liu et al., Leukemia 32:520-531, 2018; Li et al., Sci Adv 9:eadd6997, 2023; Marin et al., Nat Med 30:772-784, 2024). Studies investigating IL-15-expressing CAR-NK cells in solid tumors are still relatively limited, and even fewer have explored the use of IL-15-CAR engineered iPSC-NK in this setting. Consequently, understanding of the molecular features of IL-15-expressing CAR iNK in the context of local solid tumors remains unclear. Disclosed herein is the development of an iPSC-derived natural killer (iNK) cell-based therapy in which the iNK cells co-express a mesothelin-targeted CAR (using the hYP218 scFv) and IL-15, for example from a transduced piggyBac transposon vector. The present disclosure also identifies the LiPSC-GR1.1 iPSC line as a superior iPSC line to produce hematopoietic progenitor cells and differentiated iNK cells. It is also disclosed herein that LiPSC-GR1.1 cells genetically modified by transduction with the MSLN.CAR / IL-15 piggyBac transposon vector robustly increased their differentiation to iNK cells and the differentiated iNK cells expressing MSLN.CAR and IL-15 exhibited potent anti-tumor killing in animal models of mesothelin-positive cancer. II. Abbreviations APEL albumin polyvinylalcohol essential lipids BMP-4 bone morphogenetic protein 4 CAR chimeric antigen receptor CDR complementarity determining region EB embryoid body E:T effector to target ratio EGFRt truncated epidermal growth factor receptor FLT3 fms-like tyrosine kinase 3 iNK iPSC-derived natural killer cells 4239-111805-02 iPSC induced pluripotent stem cell IL interleukin MSLN mesothelin NK natural killer PBNK peripheral blood natural killer cells SCF stem cell factor VEGF vascular endothelial growth factor VH variable heavy VL variable light III. Summary of Terms Unless otherwise noted, technical terms are used according to conventional usage. Definitions of many common terms in molecular biology may be found in Krebs et al. (eds.), Lewin’s genes XII, published by Jones & Bartlett Learning, 2017. As used herein, the singular forms “a,” “an,” and “the,” refer to both the singular as well as plural, unless the context clearly indicates otherwise. For example, the term “an antigen” includes singular or plural antigens and can be considered equivalent to the phrase “at least one antigen.” As used herein, the term “comprises” means “includes.” It is further to be understood that any and all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for descriptive purposes, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. To facilitate review of the various aspects, the following explanations of terms are provided: 2B4: A natural killer (NK) cell-surface receptor that is a member of the signaling lymphocyte activation molecule (SLAM)-related receptor family. 2B4 plays a role in stimulating NK cell cytotoxicity and cytokine production. 2B4 is also known as CD244. 2B4 sequences are publicly available, such as under NCBI Gene ID 51744. An exemplary 2B4 amino acid sequence is set forth herein as SEQ ID NO: 18. 4-1BB: A co-stimulatory molecule expressed by T cell receptor (TCR)-activated lymphocytes, and by other cells including natural killer cells. Ligation of 4-1BB induces a signaling cascade that results in cytokine production, expression of anti-apoptotic molecules and an enhanced immune response. 4-1BB is also known as TNF receptor superfamily member 9 (TNFRSF9) and CD137. 4-1BB sequences are publicly available, such as under NCBI Gene ID 3604. An exemplary amino acid sequence of 4-1BB is set forth herein as SEQ ID NO: 19. 4239-111805-02 Administration: To provide or give a subject an agent, such as iNK cells expressing a mesothelin-targeted CAR, by any effective route. Exemplary routes of administration include, but are not limited to, oral, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, intravenous, and intratumoral), sublingual, rectal, transdermal, intranasal, vaginal, and inhalation routes. Antibody: A polypeptide ligand comprising at least one variable region that recognizes and binds (such as specifically recognizes and specifically binds) an epitope of an antigen (such as mesothelin). Mammalian immunoglobulin molecules are composed of a heavy (H) chain and a light (L) chain, each of which has a variable region, termed the variable heavy (VH) domain and the variable light (VL) domain, respectively. Together, the VH domain and the VL domain are responsible for binding the antigen recognized by the antibody. There are five main heavy chain classes (or isotypes) of mammalian immunoglobulin, which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Antibody isotypes not found in mammals include IgX, IgY, IgW and IgNAR. IgY is the primary antibody produced by birds and reptiles and is functionally similar to mammalian IgG and IgE. IgW and IgNAR antibodies are produced by cartilaginous fish, while IgX antibodies are found in amphibians. Antibody variable regions contain "framework" regions and hypervariable regions, known as “complementarity determining regions” or “CDRs.” The CDRs are primarily responsible for binding to an epitope of an antigen. The framework regions of an antibody serve to position and align the CDRs in three-dimensional space. The amino acid sequence boundaries of a given CDR can be readily determined using any of a number of well-known numbering schemes, including those described by Kabat et al. (Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991; the “Kabat” numbering scheme), Chothia et al. (see Chothia and Lesk, J Mol Biol 196:901-917, 1987; Chothia et al., Nature 342:877, 1989; and Al-Lazikani et al., JMB 273,927-948, 1997; the “Chothia” numbering scheme), Kunik et al. (see Kunik et al., PLoS Comput Biol 8:e1002388, 2012; and Kunik et al., Nucleic Acids Res 40(Web Server issue):W521-524, 2012; “Paratome CDRs”) and the ImMunoGeneTics (IMGT) database (see, Lefranc, Nucleic Acids Res 29:207-9, 2001; the “IMGT” numbering scheme). The Kabat, Paratome and IMGT databases are maintained online. In addition, the AbRSA tool can be used to determine the CDR boundaries according to Kabat, IMGT or Chothia (online at aligncdr.labshare.cn / aligncdr / abrsa.php). A “single-domain antibody” refers to an antibody having a single domain (a variable domain) that is capable of specifically binding an antigen, or an epitope of an antigen, in the absence of an additional antibody domain. Single-domain antibodies include, for example, VH domain antibodies, VNAR antibodies, camelid VHH antibodies, and VL domain antibodies. VNAR antibodies are produced by cartilaginous fish, such as nurse sharks, wobbegong sharks, spiny dogfish and bamboo sharks. Camelid VHH antibodies are produced by several species including camel, llama, alpaca, dromedary, and guanaco, which produce heavy chain antibodies that are naturally devoid of light chains. 4239-111805-02 A “monoclonal antibody” is an antibody produced by a single clone of lymphocytes or by a cell into which the coding sequence of a single antibody has been transfected. Monoclonal antibodies are produced by methods known to those of skill in the art. Monoclonal antibodies include humanized monoclonal antibodies. A “chimeric antibody” has framework residues from one species, such as human, and CDRs (which generally confer antigen binding) from another species (such as mouse). A “humanized” antibody is an immunoglobulin including a human framework region and one or more CDRs from a non-human (for example a mouse, rabbit, rat, shark or synthetic) immunoglobulin. The non-human immunoglobulin providing the CDRs is termed a “donor,” and the human immunoglobulin providing the framework is termed an “acceptor.” In one aspect, all CDRs are from the donor immunoglobulin in a humanized immunoglobulin. Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, such as at least about 85-90%, such as about 95% or more identical. Hence, all parts of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to corresponding parts of natural human immunoglobulin sequences. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs. Humanized or other monoclonal antibodies can have additional conservative amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions. Binding affinity: Affinity of an antibody (or CAR) for an antigen (such as mesothelin). In one aspect, affinity is calculated by a modification of the Scatchard method described by Frankel et al., Mol. Immunol., 16:101-106, 1979. In another aspect, binding affinity is measured by an antigen / antibody dissociation rate. In another aspect, a binding affinity is measured by a competition radioimmunoassay. In another aspect, binding affinity is measured by ELISA. In other aspects, antibody affinity is measured by flow cytometry, surface plasmon reference, or biolayer interferometry (BLI). An antibody that “specifically binds” an antigen (such as mesothelin) is an antibody that binds the antigen with high affinity and does not significantly bind other unrelated antigens. In some examples, a CAR (such as a mesothelin-targeted CAR provided herein) specifically binds to a target (such as mesothelin) with a binding constant that is at least 103M-1greater, 104M-1greater or 105M-1greater than a binding constant for other molecules in a sample or subject. In some examples, a CAR has an equilibrium constant (KD) of 5 µM or less, such as 5,000 nM or less, 900 nM or less, 500 nM or less, 250 nM or less, 100 nM or less, 50 nM or less, 10 nM or less, 5 nM or less, or 1 nM or less. For example, a CAR binds to a target, such as mesothelin, with a binding affinity of at least about 1 x 10-6M, at least about 0.5 x 10-6M, at least about 1 x 10-7M, at least about 0.5 x 10-7M, at least about 1 x 10-8M, at least about 0.5 x 10-8M, at least about 1 x 10-9M, at least about 0.5 x 10-9M, or at least about 0.1 x 10-9. In certain aspects, a specific binding agent that binds to its target has a dissociation constant (Kd) of ≤1000 nM, ≤750 nM, 500 nM, ≤250 nM, ≤100 nM, ≤50 nM, ≤25 4239-111805-02 nM, ≤10 nM, ≤5 nM, ≤2.5 nM, ≤1 nM, ≤0.5 nM, ≤0.25 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10-6M or less, e.g., from 10-6M to 10-10M, e.g., from 10-10M to 10-12M). In some examples, binding affinity is measured using the Octet system (Creative Biolabs), which is based on BLI technology. In some examples, Kd is measured using surface plasmon resonance assays using a BIACORES-2000 or a BIACORES-3000 (BIAcore, Inc., Piscataway, N.J.). Breast cancer: A type of cancer that forms in tissues of the breast, usually the ducts (tubes that carry milk to the nipple) and lobules (glands that make milk). Triple negative breast cancer refers to a type of breast cancer in which the cancer cells do not express estrogen receptors, progesterone receptors or significant levels of HER2 / neu protein. Triple negative breast cancer is also called ER- negative PR-negative HER2 / neu-negative breast cancer. CD3ζ: A component of the T cell receptor (TCR) complex. The zeta chain plays an important role in coupling antigen recognition to several intracellular signal-transduction pathways. Low expression of the antigen results in impaired immune response. CD3ζ is also known as CD247. Sequences for CD3ζ are publicly available, such as under NCBI Gene ID 919. An exemplary amino acid sequence of the CD3ζ intracellular signaling domain is set forth herein as SEQ ID NO: 20. CD8 subunit alpha (CD8α): A transmembrane glycoprotein predominantly expressed by cytotoxic T lymphocytes, but can also be expressed by NK cells, cortical thymocytes and dendritic cells. CD8α mediates efficient cell-cell interactions within the immune system. The CD8 protein acts as a coreceptor with the T-cell receptor on T cells to recognize antigens displayed by an antigen presenting cell in the context of class I MHC molecules. The coreceptor functions as either a homodimer composed of two alpha chains or as a heterodimer composed of one alpha and one beta chain. Both alpha and beta chains share significant homology to immunoglobulin variable light chains. CD8α sequences are publicly available, such as under NCBI Gene ID 925. Exemplary amino acid sequences of CD8α hinge and CD8α transmembrane domains are set forth herein as SEQ ID NO: 15 and SEQ ID NO: 17, respectively. Chemotherapeutic agent: Any chemical agent with therapeutic usefulness in the treatment of diseases characterized by abnormal cell growth. Such diseases include tumors, neoplasms, and cancer as well as diseases characterized by hyperplastic growth. In one aspect, a chemotherapeutic agent is an agent of use in treating a mesothelin-expressing tumor. In one aspect, a chemotherapeutic agent is a radioactive compound. A skilled person can readily identify a chemotherapeutic agent of use (see for example, Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison's Principles of Internal Medicine, 14th edition; Perry et al., Chemotherapy, Ch.17 in Abeloff, Clinical Oncology 2nded., © 2000 Churchill Livingstone, Inc; Baltzer, L., Berkery, R. (eds.): Oncology Pocket Guide to Chemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995; Fischer, D.S., Knobf, M.F., Durivage, H.J. (eds): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 1993). Combination chemotherapy is the administration of more than one agent to treat cancer. One 4239-111805-02 example is the administration of a CAR that binds mesothelin, used in combination with a radioactive or chemical compound. In one example, a chemotherapeutic agent is a biologic, such as a therapeutic antibody (e.g., therapeutic monoclonal antibody), such as anti-PD1 or anti-PDL1 (e.g., pembrolizumab and nivolumab), anti-CTLA4 (e.g., ipilimumab), anti-EGFR (e.g., cetuximab), anti- VEGF (e.g., bevacizumab), or combinations thereof (e.g., anti-PD-1 and anti-CTLA-4). Chimeric antigen receptor (CAR): A chimeric molecule that includes an antigen-binding portion (such as a scFv) and a signaling domain, such as a signaling domain from a T cell receptor (for example, CD3ζ). Typically, CARs are comprised of an antigen-binding moiety, a hinge / spacer element, a transmembrane domain, and an endodomain. The endodomain typically includes a signaling chain having an immunoreceptor tyrosine-based activation motif (ITAM), such as CD3ζ or FcεRIγ. In some instances, the endodomain further includes the intracellular portion of at least one additional co-stimulatory domain, such as CD28, 4-1BB (CD137), ICOS, OX40 (CD134), CD27 and / or DAP10. In some examples, the CAR is multispecific (such as bispecific) or bicistronic. A multispecific CAR is a single CAR molecule comprised of at least two antigen-binding domains (such as scFvs) that each bind a different antigen or a different epitope on the same antigen (see, for example, US 2018 / 0230225). For example, a bispecific CAR refers to a single CAR molecule having two antigen-binding domains that each bind a different antigen. A bicistronic CAR refers to two complete CAR molecules, each containing an antigen-binding moiety that binds a different antigen. In some cases, a bicistronic CAR construct expresses two complete CAR molecules that are linked by a cleavage linker. T cells or NK cells expressing a bispecific or bicistronic CAR can bind cells that express both of the antigens to which the binding moieties are directed (see, for example, Qin et al., Blood 130:810, 2017; and WO / 2018 / 213337). Cholangiocarcinoma: A type of cancer that develops in cells that line the bile ducts in the liver. Colorectal cancer: Colorectal cancer starts in the colon or the rectum. These cancers can also be called colon cancer or rectal cancer, depending on where they start. Colon cancer and rectal cancer are often grouped together because they have many features in common. Complementarity determining region (CDR): Amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native Ig binding site. The light and heavy chains of an Ig each have three CDRs, designated LCDR1, LCDR2, LCDR3 and HCDR1, HCDR2 and HCDR3, respectively. Conservative variant: A protein containing conservative amino acid substitutions that do not substantially affect or decrease the activity or affinity of a protein, such as the affinity of an antibody or CAR to mesothelin. For example, a monoclonal antibody or CAR that specifically binds mesothelin can include at most about 1, at most about 2, at most about 5, and most about 10, or at most about 15 conservative substitutions and specifically bind mesothelin. The term “conservative 4239-111805-02 variant” also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid, provided that the antibody or CAR specifically binds mesothelin. Non-conservative substitutions are those that reduce an activity or binding to mesothelin. Conservative amino acid substitution tables providing functionally similar amino acids are well known. The following six groups are examples of amino acids that are considered to be conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). Degenerate variant: A polynucleotide encoding a polypeptide that includes a sequence that is degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included as long as the amino acid sequence of the polypeptide is unchanged. Embryoid body (EB): Three-dimensional aggregate formed by pluripotent stem cells, such as iPSCs or embryonic stem cells. Framework region: Amino acid sequences interposed between CDRs. Framework regions include variable light and variable heavy framework regions. The framework regions serve to hold the CDRs in an appropriate orientation for antigen binding. Fusion protein: A protein comprising at least a portion of two different (heterologous) proteins. Heterologous: Originating from a separate genetic source or species. Immune response: A response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus. In one aspect, the response is specific for a particular antigen (an “antigen- specific response”). In one aspect, an immune response is a T cell response, such as a CD4+response or a CD8+response. In another aspect, the response is a B cell response, and results in the production of specific antibodies. Induced pluripotent stem cell (iPSC): A type of pluripotent stem cell that is generated from a somatic cell. iPSCs are genetically reprogrammed to an embryonic stem cell-like status through forced expression of specific genes and factors. In some aspects herein, the iPSC is from the LiPSC- GR1.1 cell line, which is publicly available through the NIH Regenerative Medicine Program (RMP) (see commonfund.nih.gov / stemcells / lines; Baghbaderani et al., Stem Cell Rev and Rep 12:394-420, 2016; Baghbaderani et al., Stem Cell Reports 5:647-659, 2015). Interleukin-15 (IL-15): A cytokine that regulates T cell and natural killer cell activation and proliferation. IL-15 and IL-2 share many biological activities. They bind common hematopoietin 4239-111805-02 receptor subunits, and may compete for the same receptor, and thus negatively regulate each other's activity. The number of CD8+ memory cells is shown to be controlled by a balance between IL-15 and IL-2. IL-15 induces the activation of JAK kinases, as well as the phosphorylation and activation of transcription activators STAT3, STAT5, and STAT6. Sequences for human IL-15 are publicly available, such as under NCBI Gene ID 3600. An exemplary IL-15 amino acid sequence is set forth herein as SEQ ID NO: 21. Isolated: An “isolated” biological component, such as a nucleic acid, protein (including antibodies or CARs) or organelle, has been substantially separated or purified away from other biological components in the environment (such as a cell) in which the component occurs, e.g., other chromosomal and extra-chromosomal DNA and RNA, proteins and organelles. Nucleic acids and proteins that have been “isolated” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids and proteins. Linker: In some cases, a linker is a peptide within an antibody binding fragment (such as an Fv fragment) which serves to indirectly bond the variable heavy chain to the variable light chain. “Linker” can also refer to a peptide serving to link a targeting moiety, such as an antibody, to an effector molecule, such as a drug or a detectable label. In some aspects herein, the linker connects a VH domain to a VL domain of an scFv (such as an scFv targeting mesothelin). Lung cancer: Cancer that forms in tissues of the lung, usually in the cells lining air passages. The two main types are small cell lung cancer and non-small cell lung cancer (NSCLC). These types can be diagnosed using microscopy. Mesothelin: A 40 kDa cell-surface glycosylphosphatidylinositol (GPI)-linked glycoprotein. The human mesothelin protein is synthesized as a 70 kD precursor which is then proteolytically processed. The 30 kD amino terminus of mesothelin is secreted and is referred to as megakaryocyte potentiating factor (Yamaguchi et al., J. Biol. Chem.269:805808, 1994). The 40 kD carboxyl terminus remains bound to the membrane as mature mesothelin (Chang et al., Natl. Acad. Sci. USA 93:136140, 1996). Exemplary nucleic acid and amino acid sequences of mesothelin are as described in PCT Publication No. WO 97 / 25,068; U.S. Patent No.6,083,502; Chang and Pastan, Int. J. Cancer 57:90, 1994; Chang and Pastan, Proc. Natl. Acad. Sci USA 93:136, 1996; Brinkmann et al., Int. J. Cancer 71:638, 1997; and Chowdhury et al., Mol. Immunol.34:9, 1997. Amino acid sequences of human mesothelin are publicly available, such as under GenBank Accession No. AAH09272. Mesothelin also refers to mesothelin proteins or polypeptides which remain intracellular as well as secreted and / or isolated extracellular mesothelin protein. Mesothelioma: A type of neoplasm derived from the lining cells of the pleura and peritoneum which grows as a thick sheet covering the viscera, and is composed of spindle cells or fibrous tissue which may enclose gland-like spaces lined by cuboidal cells. Mesotheliomas often 4239-111805-02 originate in the tissue lining the lung, heart or abdomen. In some cases, mesotheliomas are caused by exposure to asbestos. Mesothelin-positive cancer: A cancer that expresses or overexpresses mesothelin. Examples of mesothelin-positive cancers include, but are not limited to, mesothelioma, lung cancer, stomach cancer, pancreatic cancer, cholangiocarcinoma, breast cancer (such as triple negative breast cancer), thymic carcinoma, colorectal cancer, and ovarian cancer. Natural killer (NK) cells: A type of cytotoxic lymphocyte that plays an important role in the innate immune system. NK cells target and kill virus-infected cells and tumor cells. NK cells typically express CD56 and CD45. An iNK cell is an NK cell differentiated from an iPSC cell. Neoplasia, malignancy, cancer or tumor: A neoplasm is an abnormal growth of tissue or cells that results from excessive cell division. Neoplastic growth can produce a tumor. The amount of a tumor in an individual is the “tumor burden” which can be measured as the number, volume, or weight of the tumor. A tumor that does not metastasize is referred to as “benign.” A tumor that invades the surrounding tissue and / or can metastasize is referred to as “malignant.” NKG2D: A transmembrane protein of the NKG2 family that is characterized by a type II membrane orientation (has an extracellular C terminus) and the presence of a C-type lectin domain. It binds to a diverse family of ligands that include MHC class I chain-related A and B proteins and UL- 16 binding proteins, where ligand-receptor interactions can result in the activation of NK and T cells. The surface expression of these ligands is important for the recognition of stressed cells by the immune system, and thus this protein and its ligands are therapeutic targets for the treatment of immune diseases and cancers. NKG2D is also known as killer cell lectin like receptor K1 (KLKR1). NKG2D sequences are publicly available such as under NCBI Gene ID 22914. An exemplary NKG2D amino acid sequence is set forth herein as SEQ ID NO: 16. Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame. Ovarian cancer: Cancer that forms in tissues of the ovary (one of a pair of female reproductive glands in which the ova, or eggs, are formed). Most ovarian cancers are either ovarian epithelial carcinomas (cancer that begins in the cells on the surface of the ovary) or malignant germ cell tumors (cancer that begins in egg cells). Pancreatic cancer: A disease in which malignant (cancer) cells are found in the tissues of the pancreas. Also called exocrine cancer. Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers of use are conventional. Remington: The Science and Practice of Pharmacy, The University of the Sciences in 4239-111805-02 Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, PA, 21stEdition (2005), describes compositions and formulations suitable for pharmaceutical delivery of the CAR-expressing cells and other compositions disclosed herein. In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (such as powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate. Preventing, treating or ameliorating a disease: “Preventing” a disease refers to inhibiting the full development of a disease. “Treating” refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop, such as a reduction in tumor burden or a decrease in the number of size of metastases. “Ameliorating” refers to the reduction in the number or severity of signs or symptoms of a disease, such as cancer. Recombinant: A recombinant nucleic acid or protein is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques. Sequence identity: The similarity between amino acid or nucleic acid sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologs or variants of a polypeptide or nucleic acid molecule will possess a relatively high degree of sequence identity when aligned using standard methods. Methods of alignment of sequences for comparison are well-known. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math.2:482, 1981; Needleman and Wunsch, J. Mol. Biol.48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444, 1988; Higgins and Sharp, Gene 73:237, 1988; Higgins and Sharp, CABIOS 5:151, 1989; Corpet et al., Nucleic Acids Research 16:10881, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444, 1988. Altschul et al., Nature Genet.6:119, 1994, presents a detailed consideration of sequence alignment methods and homology calculations. The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol.215:403, 1990) is available from several sources, including the National Center for Biotechnology Information 4239-111805-02 (NCBI, Bethesda, MD) and on the internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. A description of how to determine sequence identity using this program is available on the NCBI website on the internet. Homologs and variants of a VL or a VH of an antibody that specifically binds mesothelin, or a fragment thereof are typically characterized by possession of at least about 75%, for example at least about 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity counted over the full length alignment with the amino acid sequence of the antibody using the NCBI Blast 2.0, gapped blastp set to default parameters. For comparisons of amino acid sequences of greater than about 30 amino acids, the Blast 2 sequences function is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1). When aligning short peptides (fewer than around 30 amino acids), the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties). Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. When less than the entire sequence is being compared for sequence identity, homologs and variants will typically possess at least 80% sequence identity over short windows of 10-20 amino acids, and may possess sequence identities of at least 85% or at least 90% or 95% depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are available at the NCBI website on the internet. A skilled person will appreciate that these sequence identity ranges are provided for guidance only; it is entirely possible that strongly significant homologs could be obtained that fall outside of the ranges provided. Stomach cancer: Cancer that forms in tissues lining the stomach. Also called gastric cancer. Subject: Living multi-cellular vertebrate organisms, a category that includes both human and veterinary subjects, including human and non-human mammals. In some aspects, a subject is a human with a mesothelin-positive cancer. Therapeutically effective amount: A quantity of a specific substance sufficient to achieve a desired effect in a subject being treated. For instance, this can be the amount of a CAR necessary to inhibit or suppress growth of a tumor. In one aspect, a therapeutically effective amount is the amount necessary to eliminate, reduce the size, or prevent metastasis of a tumor (such as a mesothelin-positive cancer), such as reduce a tumor size and / or volume by at least 10%, at least 20%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, or even 100%, and / or reduce the number and / or size / volume of metastases by at least 10%, at least 20%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, or even 100%, for example as compared to a size / volume / number prior to treatment. In one aspect, a therapeutically effective amount is the amount necessary to increase the survival time of a subject with a tumor (such as a mesothelin-positive cancer), such as increase survival time by at least 10%, at least 20%, at least 50%, at least 75%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, or at least 500%, for example as compared to 4239-111805-02 a survival time compared to a subject with no treatment or a different treatment. In one aspect, a therapeutically effective amount is the amount necessary to increase the survival time of a subject with a tumor (such as a mesothelin-positive cancer), such as increase survival time by at least 3 months, at least 6 months, at least 9 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, at least 48 months, or at least 60 months, for example as compared a survival time compared to a subject with no treatment or a different treatment. In some aspects, combinations of these affects are achieved. When administered to a subject, a dosage will generally be used that will achieve target tissue concentrations (for example, in tumors) that has been shown to achieve a desired in vitro effect. Thymic carcinoma: Cancer that forms in the cells that cover the outside surface of the thymus. The thymus is a small organ that lies in the upper chest above the heart and under the breastbone. Transduced: A virus or vector “transduces” a cell when it transfers nucleic acid into the cell. Transposon: A mobile genetic element that can move from one location in a genome to another location in the genome. The term “transposon” also includes polynucleotides that are capable of being excised from a donor polynucleotide (such as a vector) and integrating into a target site of a nucleic acid (such as genomic DNA). Vector: A nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell. A vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector may also include one or more selectable marker genes and other genetic elements known in the art. In some aspects, the vector is a virus vector, such as a lentivirus vector, an adeno-associated virus (AAV) vector, or an adenovirus vector. In other aspects, the vector is a transposon vector, such as a piggyBac vector. IV. Mesothelin-Targeted Chimeric Antigen Receptor / IL-15 Constructs Described herein are mesothelin-targeted CAR constructs that include an antigen-binding domain from antibody hYP218, which binds a membrane-proximal region of the mesothelin protein (WO 2014 / 031476). The CAR molecules further include a hinge region, a transmembrane domain, an intracellular co-stimulatory domain and an intracellular signaling domain. The disclosed constructs further include IL-15, a cytokine that promotes differentiation, activation and proliferation of natural killer (NK) cells and can promote NK cell persistence in vivo. In some aspects, the CAR / IL-15 constructs further include a truncated EGFR to facilitate detection and enrichment of CAR-expressing cells. iPSCs, such as LiPSC-GR1.1 cells, that co-express the disclosed mesothelin-targeted CAR and IL-15 are described. NK cells, such as iPSC-derived NK cells, that co-express the disclosed mesothelin-targeted CAR and IL-15 are also described. The NK / iNK cells can be used, for example, to treat solid tumors, such as mesothelin-positive tumors. 4239-111805-02 A. Antibody and CAR Sequences Provided below are the amino acid sequences of the variable heavy (VH) and variable light (VL) domain of humanized antibody YP218 (hYP218). The CDR residues according to Kabat are underlined and the CDR residues according to IMGT are shown in bold font. The sequence of each CDR is also listed in Table 1. hYP218 VH domain (SEQ ID NO: 1) HMEVQLVESGGGLVQPGGSLRLSCAASGFDLGFYFYACWVRQAPGKGLEWVSCIYTAGSG STYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSTANTRSTYYLNLWGQGT LVTVSS hYP218 VL domain (SEQ ID NO: 2) DIQMTQSPSSLSASVGDRVTITCQASQRISSYLSWYQQKPGKVPKLLIYGASTLASGVPSRFS GSGSGTDFTLTISSLQPEDVATYYCQSYAYFDSNNWHAFGGGTKVEIKATS Table 1. CDR sequences of hYP218 CDR Kabat SEQ ID IMGT SEQ ID NO: NO: In some aspects of the disclosed CAR constructs, the CAR includes a CD8α hinge region, a NKG2D or a CD8α transmembrane domain, a 2B4 or a 4-1BB intracellular co-stimulatory domain, and a CD3ζ intracellular signaling domain. Provided below are exemplary amino acid sequences of each of these CAR components. CD8α hinge region (SEQ ID NO: 15) TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD NKG2D transmembrane domain (SEQ ID NO: 16) PFFFCCFIAVAMGIRFIIMVT 4239-111805-02 CD8α transmembrane domain (SEQ ID NO: 17) IYIWAPLAGTCGVLLLSLVIT 2B4 intracellular co-stimulatory domain (SEQ ID NO: 18) WRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAPTSQEPA YTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFDVYS 4-1BB intracellular co-stimulatory domain (SEQ ID NO: 19) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL CD3ζ intracellular signaling domain (SEQ ID NO: 20) RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYN ELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR IL-15 (SEQ ID NO: 21) AIAMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIED LIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNG NVTESGCKECEELEEKNIKEFLQSFVHIVQMFI Provided below are exemplary nucleic acid sequences of two CAR / IL-15 constructs disclosed herein. The construct hYP218.NKG2D.2B4.Z.CAR / IL-15 includes the coding sequences for the hYP218 scFv, a CD8α hinge region, an NKG2D transmembrane domain, a 2B4 intracellular co- stimulatory domain, and a CD3ζ intracellular signaling domain as the CAR component. Construct hYP218.CD8a.BB.Z.CAR / IL-15 includes the coding sequences for the hYP218 scFv, a CD8α hinge region, a CD8α transmembrane domain, a 4-1BB intracellular co-stimulatory domain, and a CD3ζ intracellular signaling domain as the CAR component. Both nucleic acid constructs further include a coding sequence for EGFRt and a coding sequence for IL-15. The coding sequences for the CAR, the EGFRt, and the IL-15 are each separated by a coding sequence for a T2A site (see FIG.2A and FIG. 16A). hYP218.NKG2D.2B4.Z.CAR / IL-15 (SEQ ID NO: 22) atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctgatcccacatatggaagtacaactggtagagtcaggaggc ggcctcgtacaaccaggtgggtcccttcggctttcatgtgccgcttctggtttcgatctcggtttctacttctacgcttgttgggtacgccaagcccctg gaaaagggttggagtgggtgtcatgtatttacaccgccggatctgggtctacgtattatgcctcctgggccaaaggaaggttcactattagccgcgat aactccaagaacacgctctacttgcaaatgaatagccttagggcagaggacacagcagtatactactgcgcgcggagtacggcgaacaccaggtc cacatactatctcaacctctgggggcaaggaaccctcgtgaccgttagctctggtggaggcggttcaggaggtggtggaagcggtggagggggt agtgatattcaaatgacgcagtcaccgtcaagcctctcagcctcagttggagaccgagtgaccattacctgtcaagcgtcccagagaatcagtagct acttgtcctggtatcagcagaagcccggaaaagttcctaagttgctgatttatggggcatctacattggcttctggggtgccatccagattttcaggtag cggatccgggaccgatttcaccttgaccataagttcattgcagcccgaggatgtggcaacctattattgccagtcctacgcctattttgactcaaataat tggcacgcttttggaggtgggacgaaagtcgagattaaggcgactagtaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatc gcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgacc cattttttttctgctgcttcatcgctgtagccatgggaatccgtttcattattatggtaacatggaggagaaagaggaaggagaagcagtcagagacca 4239-111805-02 gtcccaaggaatttttgacaatttacgaagatgtcaaggatctgaaaaccaggagaaatcacgagcaggagcagacttttcctggaggggggagc accatctactctatgatccagtcccagtcttctgctcccacgtcacaagaacctgcatatacattatattcattaattcagccttccaggaagtctggatcc aggaagaggaaccacagcccttccttcaatagcactatctatgaagtgattggaaagagtcaacctaaagcccagaaccctgctcgattgagccgc aaagagctggagaactttgatgtttattccagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataa cgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaaga accctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggc aaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgcgagggca gaggaagtcttctaacatgcggtgacgtggaggagaatcccggccctatgcttctcctggtgacaagccttctgctctgtgagttaccacacccagc attcctcctgatcccacgcaaagtgtgtaacggaataggtattggtgaatttaaagactcactctccataaatgctacgaatattaaacacttcaaaaact gcacctccatcagtggcgatctccacatcctgccggtggcatttaggggtgactccttcacacatactcctcctctggatccacaggaactggatattc tgaaaaccgtaaaggaaatcacagggtttttgctgattcaggcttggcctgaaaacaggacggacctccatgcctttgagaacctagaaatcatacg cggcaggaccaagcaacatggtcagttttctcttgcagtcgtcagcctgaacataacatccttgggattacgctccctcaaggagataagtgatgga gatgtgataatttcaggaaacaaaaatttgtgctatgcaaatacaataaactggaaaaaactgtttgggacctccggtcagaaaaccaaaattataagc aacagaggtgaaaacagctgcaaggccacaggccaggtctgccatgccttgtgctcccccgagggctgctggggcccggagcccagggactg cgtctcttgccggaatgtcagccgaggcagggaatgcgtggacaagtgcaaccttctggagggtgagccaagggagtttgtggagaactctgagt gcatacagtgccacccagagtgcctgcctcaggccatgaacatcacctgcacaggacggggaccagacaactgtatccagtgtgcccactacatt gacggcccccactgcgtcaagacctgcccggcaggagtcatgggagaaaacaacaccctggtctggaagtacgcagacgccggccatgtgtgc cacctgtgccatccaaactgcacctacggatgcactgggccaggtcttgaaggctgtccaacgaatgggcctaagatcccgtccatcgccactggg atggtgggggccctcctcttgctgctggtggtggccctggggatcggcctcttcatggagggcagaggaagtcttctaacatgcggtgacgtggag gagaatcccggccctgcgatcgccatgagaatttcgaaaccacatttgagaagtatttccatccagtgctacttgtgtttacttctaaacagtcattttcta actgaagctggcattcatgtcttcattttgggctgtttcagtgcagggcttcctaaaacagaagccaactgggtgaatgtaataagtgatttgaaaaaaa ttgaagatcttattcaatctatgcatattgatgctactttatatacggaaagtgatgttcaccccagttgcaaagtaacagcaatgaagtgctttctcttgga gttacaagttatttcacttgagtccggagatgcaagtattcatgatacagtagaaaatctgatcatcctagcaaacaacagtttgtcttctaatgggaatg taacagaatctggatgcaaagaatgtgaggaactggaggaaaaaaatattaaagaatttttgcagagttttgtacatattgtccaaatgttcatc hYP218.CD8a.BB.Z.CAR / IL-15 (SEQ ID NO: 23) atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctgatcccacatatggaagtacaactggtagagtcaggaggc ggcctcgtacaaccaggtgggtcccttcggctttcatgtgccgcttctggtttcgatctcggtttctacttctacgcttgttgggtacgccaagcccctg gaaaagggttggagtgggtgtcatgtatttacaccgccggatctgggtctacgtattatgcctcctgggccaaaggaaggttcactattagccgcgat aactccaagaacacgctctacttgcaaatgaatagccttagggcagaggacacagcagtatactactgcgcgcggagtacggcgaacaccaggtc cacatactatctcaacctctgggggcaaggaaccctcgtgaccgttagctctggtggaggcggttcaggaggtggtggaagcggtggagggggt agtgatattcaaatgacgcagtcaccgtcaagcctctcagcctcagttggagaccgagtgaccattacctgtcaagcgtcccagagaatcagtagct acttgtcctggtatcagcagaagcccggaaaagttcctaagttgctgatttatggggcatctacattggcttctggggtgccatccagattttcaggtag cggatccgggaccgatttcaccttgaccataagttcattgcagcccgaggatgtggcaacctattattgccagtcctacgcctattttgactcaaataat tggcacgcttttggaggtgggacgaaagtcgagattaaggcgactagtaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatc gcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgacat ctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccaaacggggcagaaagaaactcctgtatatattcaaac aaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtg aagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgat gttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaa agataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagta cagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgcgagggcagaggaagtcttctaacatgcggtgacgtggagg agaatcccggccctatgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctgatcccacgcaaagtgtgtaacgga ataggtattggtgaatttaaagactcactctccataaatgctacgaatattaaacacttcaaaaactgcacctccatcagtggcgatctccacatcctgc cggtggcatttaggggtgactccttcacacatactcctcctctggatccacaggaactggatattctgaaaaccgtaaaggaaatcacagggtttttgc tgattcaggcttggcctgaaaacaggacggacctccatgcctttgagaacctagaaatcatacgcggcaggaccaagcaacatggtcagttttctctt gcagtcgtcagcctgaacataacatccttgggattacgctccctcaaggagataagtgatggagatgtgataatttcaggaaacaaaaatttgtgctat gcaaatacaataaactggaaaaaactgtttgggacctccggtcagaaaaccaaaattataagcaacagaggtgaaaacagctgcaaggccacagg ccaggtctgccatgccttgtgctcccccgagggctgctggggcccggagcccagggactgcgtctcttgccggaatgtcagccgaggcagggaa tgcgtggacaagtgcaaccttctggagggtgagccaagggagtttgtggagaactctgagtgcatacagtgccacccagagtgcctgcctcaggc catgaacatcacctgcacaggacggggaccagacaactgtatccagtgtgcccactacattgacggcccccactgcgtcaagacctgcccggca ggagtcatgggagaaaacaacaccctggtctggaagtacgcagacgccggccatgtgtgccacctgtgccatccaaactgcacctacggatgca ctgggccaggtcttgaaggctgtccaacgaatgggcctaagatcccgtccatcgccactgggatggtgggggccctcctcttgctgctggtggtgg ccctggggatcggcctcttcatggagggcagaggaagtcttctaacatgcggtgacgtggaggagaatcccggccctgcgatcgccatgagaatt 4239-111805-02 tcgaaaccacatttgagaagtatttccatccagtgctacttgtgtttacttctaaacagtcattttctaactgaagctggcattcatgtcttcattttgggctgt ttcagtgcagggcttcctaaaacagaagccaactgggtgaatgtaataagtgatttgaaaaaaattgaagatcttattcaatctatgcatattgatgctac tttatatacggaaagtgatgttcaccccagttgcaaagtaacagcaatgaagtgctttctcttggagttacaagttatttcacttgagtccggagatgcaa gtattcatgatacagtagaaaatctgatcatcctagcaaacaacagtttgtcttctaatgggaatgtaacagaatctggatgcaaagaatgtgaggaac tggaggaaaaaaatattaaagaatttttgcagagttttgtacatattgtccaaatgttcatc In the amino acid sequences below, the first and second underlined regions indicate the CAR and IL-15 components of the construct, respectively. hYP218.NKG2D.2B4.Z.CAR (SEQ ID NO: 24) MLLLVTSLLLCELPHPAFLLIPHMEVQLVESGGGLVQPGGSLRLSCAASGFDLGFYFYACWV RQAPGKGLEWVSCIYTAGSGSTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA RSTANTRSTYYLNLWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTIT CQASQRISSYLSWYQQKPGKVPKLLIYGASTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATY YCQSYAYFDSNNWHAFGGGTKVEIKATSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAV HTRGLDFACDPFFFCCFIAVAMGIRFIIMVTWRRKRKEKQSETSPKEFLTIYEDVKDLKTRRN HEQEQTFPGGGSTIYSMIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKS QPKAQNPARLSRKELENFDVYSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRR GRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKD TYDALHMQALPPREGRGSLLTCGDVEENPGPMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIG EFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQA WPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYAN TINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGREC VDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAG VMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLV VALGIGLFMEGRGSLLTCGDVEENPGPAIAMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFI LGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVI SLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFI CAR = residues 23-575 of SEQ ID NO: 24 IL-15 = residues 969-1130 of SEQ ID NO: 24 hYP218.CD8a.BB.Z.CAR (SEQ ID NO: 25) MLLLVTSLLLCELPHPAFLLIPHMEVQLVESGGGLVQPGGSLRLSCAASGFDLGFYFYACWV RQAPGKGLEWVSCIYTAGSGSTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA RSTANTRSTYYLNLWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTI TCQASQRISSYLSWYQQKPGKVPKLLIYGASTLASGVPSRFSGSGSGTDFTLTISSLQPEDVAT YYCQSYAYFDSNNWHAFGGGTKVEIKATSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGA VHTRGLDFACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP PREGRGSLLTCGDVEENPGPMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNI KHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFE NLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSG QKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPR EFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWK YADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFMEGR GSLLTCGDVEENPGPAIAMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTE ANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDT VENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFI 4239-111805-02 CAR = residues 23-497 of SEQ ID NO: 25 IL-15 = residues 891-1052 of SEQ ID NO: 25 B. Nucleic Acid Molecules Encoding a Mesothelin-Targeted CAR and IL-15 Provided herein are nucleic acid molecules that include a nucleic acid sequence encoding a chimeric antigen receptor (CAR) that specifically binds mesothelin and a nucleic acid sequence encoding interleukin-15 (IL-15), such as human IL-15. In some aspects, the CAR includes an antigen-binding domain having a variable heavy (VH) domain and a variable light (VL) domain, wherein the VH domain includes the complementarity determining region 1 (CDR1), CDR2 and CDR3 sequences of the VH domain of antibody hYP218 (SEQ ID NO: 1) and the VL domain includes the CDR1, CDR2 and CDR3 sequences of the VL domain of antibody hYP218 (SEQ ID NO: 2). In some aspects, the CAR further includes a hinge region; a transmembrane domain; an intracellular co-stimulatory domain; and / or an intracellular signaling domain. In some aspects of the encoded CAR, the amino acid sequences of the VH domain CDR1, CDR2 and CDR3 respectively include SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, or respectively include SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11. In some aspects of the encoded CAR, the amino acid sequences of the VL domain CDR1, CDR2 and CDR3 respectively include SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, or respectively include SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14. In some aspects of the encoded CAR, the amino acid sequence of the VH domain is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 1 and includes the CDR1, CDR2 and CDR3 sequence of SEQ ID NO: 1; and / or the amino acid sequence of the VL domain is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 2 and includes the CDR1, CDR2 and CDR3 sequence of SEQ ID NO: 2. In some examples, the amino acid sequence of the VH domain includes or consists of SEQ ID NO: 1 and / or the amino acid sequence of the VL domain includes or consists of SEQ ID NO: 2. In some aspects of the encoded CAR, the hinge region is a CD8α hinge region; the transmembrane domain is a NKG2D or a CD8α transmembrane domain; the intracellular co- stimulatory domain is a 2B4 or a 4-1BB intracellular co-stimulatory domain; and / or the intracellular signaling domain is a CD3ζ intracellular signaling domain. In some examples, the hinge region is a CD8α hinge region; the transmembrane domain is a NKG2D transmembrane domain; the intracellular co-stimulatory domain is a 2B4 intracellular co-stimulatory domain; and the intracellular signaling domain is a CD3ζ intracellular signaling domain. In other examples, the hinge region is a CD8α hinge region; the transmembrane domain is a CD8α transmembrane domain; the intracellular co- 4239-111805-02 stimulatory domain is a 4-1BB intracellular co-stimulatory domain; and the intracellular signaling domain is a CD3ζ intracellular signaling domain. In some aspects of the encoded CAR, the amino acid sequence of the CD8α hinge region is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 15. In some aspects, the amino acid sequence of the CD8α hinge region includes or consists of SEQ ID NO: 15. In some aspects of the encoded CAR, the amino acid sequence of the NKG2D transmembrane domain is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 16. In some aspects, the amino acid sequence of the NKG2D transmembrane domain includes or consists of SEQ ID NO: 16. In some aspects of the encoded CAR, the amino acid sequence of the CD8α transmembrane domain is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 17. In some aspects, the amino acid sequence of the CD8α transmembrane domain includes or consists of SEQ ID NO: 17. In some aspects of the encoded CAR, the amino acid sequence of the 2B4 intracellular co- stimulatory domain is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 18. In some aspects, the amino acid sequence of the 2B4 intracellular co-stimulatory domain includes or consists of SEQ ID NO: 18. In some aspects of the encoded CAR, the amino acid sequence of the 4-1BB intracellular co- stimulatory domain is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 19. In some aspects, the amino acid sequence of the 4-1BB intracellular co-stimulatory domain includes or consists of SEQ ID NO: 19. In some aspects of the encoded CAR, the amino acid sequence of the CD3ζ intracellular signaling domain is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 20. In some aspects, the amino acid sequence of the CD3ζ intracellular signaling domain includes or consists of SEQ ID NO: 20. In some aspects of the nucleic acid molecule, the amino acid sequence of IL-15 is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 21. In some aspects, the amino acid sequence of IL-15 includes or consists of SEQ ID NO: 21. In some aspects, the nucleic sequence of the nucleic acid molecule is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 22 or SEQ ID NO: 23, or a degenerate variant thereof. In some aspects, the nucleic sequence of the nucleic acid molecule includes or consists of SEQ ID NO: 22 or SEQ ID NO: 23, or a degenerate variant thereof. 4239-111805-02 In some aspects, the nucleic acid molecule is operably linked to a promoter, such as an inducible, constitutive, or tissue-specific promoter. Also provided are vectors that include a nucleic acid molecule disclosed herein. In some aspects, the vector is a transposon vector. In specific aspects, the vector is a piggyBac transposon vector. In other aspects, the vector is a viral vector, such as a lentivirus vector, an adeno-associated virus vector, or an adenovirus vector. Further provided are natural killer (NK) cells that include a nucleic acid molecule or vector disclosed herein. In some aspects, the NK cells are induced NK (iNK) cells, such as cells induced from an induced pluripotent stem cell (iPSC) line. C. iPSCs Co-Expressing a Mesothelin-Targeted CAR and IL-15 Provided herein are iPSCs that co-express (i) a chimeric antigen receptor (CAR) that specifically binds mesothelin, and (ii) interleukin (IL)-15, wherein the IL-15 is expressed from an exogenous nucleic acid molecule encoding IL-15. In some aspects, the CAR includes an antigen- binding domain having a variable heavy (VH) domain and a variable light (VL) domain, wherein the VH domain includes the complementarity determining region 1 (CDR1), CDR2 and CDR3 sequences of the VH domain of antibody hYP218 (SEQ ID NO: 1) and the VL domain includes the CDR1, CDR2 and CDR3 sequences of the VL domain of antibody hYP218 (SEQ ID NO: 2). In some aspects, the CAR further includes a hinge region; a transmembrane domain; an intracellular co- stimulatory domain; and / or an intracellular signaling domain. In some aspects of the CAR, the amino acid sequences of the VH domain CDR1, CDR2 and CDR3 respectively include SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, or respectively include SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11. In some aspects of the CAR, the amino acid sequences of the VL domain CDR1, CDR2 and CDR3 respectively include SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, or respectively include SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14. In some aspects of the CAR, the amino acid sequence of the VH domain is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 1 and includes the CDR1, CDR2 and CDR3 sequence of SEQ ID NO: 1; and / or the amino acid sequence of the VL domain is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 2 and includes the CDR1, CDR2 and CDR3 sequence of SEQ ID NO: 2. In some examples, the amino acid sequence of the VH domain includes or consists of SEQ ID NO: 1 and / or the amino acid sequence of the VL domain includes or consists of SEQ ID NO: 2. In some aspects of the CAR, the hinge region is a CD8α hinge region; the transmembrane domain is a NKG2D or a CD8α transmembrane domain; the intracellular co-stimulatory domain is a 4239-111805-02 2B4 or a 4-1BB intracellular co-stimulatory domain; and / or the intracellular signaling domain is a CD3ζ intracellular signaling domain. In some examples, the hinge region is a CD8α hinge region; the transmembrane domain is a NKG2D transmembrane domain; the intracellular co-stimulatory domain is a 2B4 intracellular co-stimulatory domain; and the intracellular signaling domain is a CD3ζ intracellular signaling domain. In other examples, the hinge region is a CD8α hinge region; the transmembrane domain is a CD8α transmembrane domain; the intracellular co-stimulatory domain is a 4-1BB intracellular co-stimulatory domain; and the intracellular signaling domain is a CD3ζ intracellular signaling domain. In some aspects of the CAR, the amino acid sequence of the CD8α hinge region is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 15. In some aspects, the amino acid sequence of the CD8α hinge region includes or consists of SEQ ID NO: 15. In some aspects of the CAR, the amino acid sequence of the NKG2D transmembrane domain is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 16. In some aspects, the amino acid sequence of the NKG2D transmembrane domain includes or consists of SEQ ID NO: 16. In some aspects of the CAR, the amino acid sequence of the CD8α transmembrane domain is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 17. In some aspects, the amino acid sequence of the CD8α transmembrane domain includes or consists of SEQ ID NO: 17. In some aspects of the CAR, the amino acid sequence of the 2B4 intracellular co-stimulatory domain is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 18. In some aspects, the amino acid sequence of the 2B4 intracellular co-stimulatory domain includes or consists of SEQ ID NO: 18. In some aspects of the CAR, the amino acid sequence of the 4-1BB intracellular co- stimulatory domain is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 19. In some aspects, the amino acid sequence of the 4-1BB intracellular co-stimulatory domain includes or consists of SEQ ID NO: 19. In some aspects of the CAR, the amino acid sequence of the CD3ζ intracellular signaling domain is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 20. In some aspects, the amino acid sequence of the CD3ζ intracellular signaling domain includes or consists of SEQ ID NO: 20. In some aspects of the iPSCs, the amino acid sequence of the CAR is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to residues 23-575 of SEQ ID NO: 24. In some aspects, the amino acid sequence of the CAR includes or consists of residues 23-575 of SEQ ID NO: 24. In other aspects, the amino acid sequence of the CAR is at least 4239-111805-02 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to residues 23-497 of SEQ ID NO: 25. In some aspects, the amino acid sequence of the CAR includes or consists of residues 23-497 of SEQ ID NO: 25. In some aspects of the iPSCs, the amino acid sequence of IL-15 is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 21. In some aspects, the amino acid sequence of IL-15 includes or consists of SEQ ID NO: 21. In some aspects, the iPSC is an iPSC from the LiPSC-GR1.1 line (see commonfund.nih.gov / stemcells / lines; Baghbaderani et al., Stem Cell Rev and Rep 12:394-420, 2016; Baghbaderani et al., Stem Cell Reports 5:647-659, 2015). In other aspects, the iPSC is an iPSC from the NCRM5 or NCRM6 iPSC line (commonfund.nih.gov / stemcells / lines). D. Natural Killer Cells Co-Expressing a Mesothelin-Targeted CAR and IL-15 Provided herein NK cells that co-express (i) a chimeric antigen receptor (CAR) that specifically binds mesothelin, and (ii) interleukin (IL)-15, wherein the IL-15 is expressed from an exogenous nucleic acid molecule encoding IL-15. In some aspects, the CAR includes an antigen- binding domain having a variable heavy (VH) domain and a variable light (VL) domain, wherein the VH domain includes the complementarity determining region 1 (CDR1), CDR2 and CDR3 sequences of the VH domain of antibody hYP218 (SEQ ID NO: 1) and the VL domain includes the CDR1, CDR2 and CDR3 sequences of the VL domain of antibody hYP218 (SEQ ID NO: 2). In some aspects, the CAR further includes a hinge region; a transmembrane domain; an intracellular co- stimulatory domain; and / or an intracellular signaling domain. In some aspects of the CAR, the amino acid sequences of the VH domain CDR1, CDR2 and CDR3 respectively include SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, or respectively include SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11. In some aspects of the CAR, the amino acid sequences of the VL domain CDR1, CDR2 and CDR3 respectively include SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, or respectively include SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14. In some aspects of the CAR, the amino acid sequence of the VH domain is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 1 and includes the CDR1, CDR2 and CDR3 sequence of SEQ ID NO: 1; and / or the amino acid sequence of the VL domain is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 2 and includes the CDR1, CDR2 and CDR3 sequence of SEQ ID NO: 2. In some examples, the amino acid sequence of the VH domain includes or consists of SEQ ID NO: 1 and / or the amino acid sequence of the VL domain includes or consists of SEQ ID NO: 2. In some aspects of the CAR, the hinge region is a CD8α hinge region; the transmembrane domain is a NKG2D or a CD8α transmembrane domain; the intracellular co-stimulatory domain is a 4239-111805-02 2B4 or a 4-1BB intracellular co-stimulatory domain; and / or the intracellular signaling domain is a CD3ζ intracellular signaling domain. In some examples, the hinge region is a CD8α hinge region; the transmembrane domain is a NKG2D transmembrane domain; the intracellular co-stimulatory domain is a 2B4 intracellular co-stimulatory domain; and the intracellular signaling domain is a CD3ζ intracellular signaling domain. In other examples, the hinge region is a CD8α hinge region; the transmembrane domain is a CD8α transmembrane domain; the intracellular co-stimulatory domain is a 4-1BB intracellular co-stimulatory domain; and the intracellular signaling domain is a CD3ζ intracellular signaling domain. In some aspects of the CAR, the amino acid sequence of the CD8α hinge region is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 15. In some examples, the amino acid sequence of the CD8α hinge region includes or consists of SEQ ID NO: 15. In some aspects of the CAR, the amino acid sequence of the NKG2D transmembrane domain is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 16. In some examples, the amino acid sequence of the NKG2D transmembrane domain includes or consists of SEQ ID NO: 16. In some aspects of the CAR, the amino acid sequence of the CD8α transmembrane domain is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 17. In some examples, the amino acid sequence of the CD8α transmembrane domain includes or consists of SEQ ID NO: 17. In some aspects of the CAR, the amino acid sequence of the 2B4 intracellular co-stimulatory domain is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 18. In some examples, the amino acid sequence of the 2B4 intracellular co-stimulatory domain includes or consists of SEQ ID NO: 18. In some aspects of the CAR, the amino acid sequence of the 4-1BB intracellular co- stimulatory domain is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 19. In some examples, the amino acid sequence of the 4- 1BB intracellular co-stimulatory domain includes or consists of SEQ ID NO: 19. In some aspects of the CAR, the amino acid sequence of the CD3ζ intracellular signaling domain is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 20. In some examples, the amino acid sequence of the CD3ζ intracellular signaling domain includes or consists of SEQ ID NO: 20. In some aspects of the NK cells, the amino acid sequence of the CAR is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to residues 23-575 of SEQ ID NO: 24. In some examples, the amino acid sequence of the CAR includes or consists of residues 23-575 of SEQ ID NO: 24. In other aspects, the amino acid sequence of the CAR is at least 4239-111805-02 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to residues 23-497 of SEQ ID NO: 25. In some examples, the amino acid sequence of the CAR includes or consists of residues 23-497 of SEQ ID NO: 25. In some aspects of the NK cells, the amino acid sequence of IL-15 is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 21. In some examples, the amino acid sequence of IL-15 includes or consists of SEQ ID NO: 21. In some aspects, the NK cell is derived from an induced pluripotent stem cell (iPSC) line. In some examples, the iPSC line is LiPSC-GR1.1 (see commonfund.nih.gov / stemcells / lines; Baghbaderani et al., Stem Cell Rev and Rep 12:394-420, 2016; Baghbaderani et al., Stem Cell Reports 5:647-659, 2015). In other examples, the iPSC line is the NCRM5 or NCRM6 iPSC line (commonfund.nih.gov / stemcells / lines). The iNK cells can be produced from an iPSC line using any known method, such as the methods described in section VII and Examples 1-3. V. Compositions Also provided herein are compositions that include an NK cell, such as an iNK cell, co- expressing a mesothelin-targeted CAR and IL-15 as disclosed herein, and a pharmaceutically acceptable carrier. The compositions can be prepared in unit dosage forms for administration to a subject. The amount and timing of administration are at the discretion of the treating clinician to achieve the desired outcome. The NK / iNK cell composition can be formulated for systemic or local (such as intra-tumor) administration. In some aspects, the NK / iNK cell composition is formulated for parenteral administration, such as intravenous administration. The compositions for administration can include a solution of NK / iNK cells in a pharmaceutically acceptable carrier, such as an aqueous carrier. A variety of aqueous carriers can be used, for example, buffered saline and the like. These solutions are sterile and generally free of undesirable matter. These compositions can be sterilized by conventional sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration or quantity of NK / iNK cells in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the subject’s needs. The compositions that include NK / iNK cells can be formulated in unit dosage form suitable for individual administration of precise dosages. In addition, the compositions can be administered in a single dose or in a multiple dose schedule. A multiple dose schedule is one in which a primary course of treatment may be with more than one separate dose, for instance 1-10 doses, such as 1, 2, 3, 4239-111805-02 4, 5, 6, 7, 8, 9 or 10 doses, followed by other doses given at subsequent time intervals as needed to maintain or reinforce the action of the compositions. Treatment can involve daily or multi-daily doses of compound(s) over a period of a few days to months, or even years. Thus, the dosage regime will also, at least in part, be determined based on the particular needs of the subject to be treated and will be dependent upon the judgment of the administering practitioner. Exemplary dosages of the NK / iNK cell compositions or additional agents can range from about 1×105to about 1×109NK cells / kg, such as from about 5×105to about 5×108NK cells / kg. In some examples, the dosage is at least about 5×105NK cells / kg, at least about 6×105NK cells / kg, at least about 7×105NK cells / kg, at least about 8×105NK cells / kg, at least about 9×105NK cells / kg, at least about 1×106NK cells / kg, at least about 2×106NK cells / kg, at least about 3×106NK cells / kg, at least about 4×106NK cells / kg, at least about 5×106NK cells / kg, at least about 6×106NK cells / kg, at least about 7×106NK cells / kg is at least about 8×106NK cells / kg, at least about 9×106NK cells / kg, at least about 1×107NK cells / kg, at least about 2×107NK cells / kg, at least about 3×107NK cells / kg, at least about 4×107NK cells / kg, at least about 5×107NK cells / kg, at least about 6×107NK cells / kg, at least about 7×107NK cells / kg, at least about 8×107NK cells / kg, at least about 1×108NK cells / kg, at least about 2×108NK cells / kg, at least about 3×108NK cells / kg, at least about 4×108NK cells / kg, or at least about 5×108NK cells / kg. In particular examples, the subject is administered an NK / iNK cell or composition thereof, or additional agent(s), on a multiple daily dosing schedule, such as at least two consecutive days, 10 consecutive days, and so forth, for example for a period of weeks, months, or years. In one example, the subject is administered the NK / iNK cell composition and / or additional agent(s) for a period of at least 30 days, such as at least 2 months, at least 4 months, at least 6 months, at least 12 months, at least 24 months, or at least 36 months. In some aspects, the NK / iNK cell composition is administered intravenously, intraperitoneally, intratumorally, intrapleurally, subcutaneously or by another mode daily or multiple times per week for a period of time, followed by a period of no treatment, then the cycle is repeated. In some aspects, the NK / iNK cell composition is administered intraperitoneally. In some aspects, the initial period of treatment (e.g., administration of the therapeutic agent daily or multiple times per week) is for 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks or 12 weeks. In a related aspect, the period of no treatment lasts for 3 days, 1 week, 2 weeks, 3 weeks or 4 weeks. In certain aspects, the dosing regimen of the therapeutic agent is daily for 3 days followed by 3 days off; or daily or multiple times per week for 1 week followed by 3 days or 1 week off; or daily or multiple times per week for 2 weeks followed by 1 or 2 weeks off; or daily or multiple times per week for 3 weeks followed by 1, 2 or 3 weeks off; or daily or multiple times per week for 4, 5, 6, 7, 8, 9, 10, 11 or 12 weeks followed by 1, 2, 3 or 4 weeks off. The compositions disclosed herein can also be administered by other routes, including via inhalation, oral, or topical. In some examples, the composition is administered via fine-needle. 4239-111805-02 The NK / iNK cell compositions may be provided in sterile solutions of known concentration. The NK / iNK cell solution is then added to an infusion bag containing 0.9% sodium chloride, USP, and in some cases administered at a dosage of from 5×105to about 1×108NK cells / kg of body weight. NK / iNK cell compositions can be administered by slow infusion, rather than in an intravenous push or bolus. In one example, a higher loading dose is administered, with subsequent, maintenance doses being administered at a lower level. Controlled release parenteral formulations can be made as implants, oily injections, or as particulate systems. For a broad overview of protein delivery systems see, Banga, A.J., Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Technomic Publishing Company, Inc., Lancaster, PA, (1995). Particulate systems include, for example, microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Microcapsules contain the therapeutic composition as a central core. In microspheres, the therapeutic composition is dispersed throughout the particle. Particles, microspheres, and microcapsules smaller than about 1 µm are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively. Capillaries have a diameter of approximately 5 µm so that only nanoparticles are administered intravenously. Microparticles are typically around 100 µm in diameter and are administered subcutaneously or intramuscularly. See, for example, Kreuter, J., Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, NY, pp.219-342 (1994); and Tice & Tabibi, Treatise on Controlled Drug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, NY, pp.315-339, (1992). Polymers can be used for ion-controlled release of the NK / iNK cell compositions disclosed herein. Various degradable and nondegradable polymeric matrices for use in controlled drug delivery are known (e.g., see Langer, Accounts Chem. Res.26:537-542, 1993). For example, the block copolymer, polaxamer 407, exists as a viscous yet mobile liquid at low temperatures but forms a semisolid gel at body temperature. Alternatively, hydroxyapatite has been used as a microcarrier for controlled release of proteins (Ijntema et al., Int. J. Pharm.112:215-224, 1994). In yet another aspect, liposomes are used for controlled release (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA (1993)). Numerous additional systems for controlled delivery of therapeutic compositions are known (see U.S. Patent Nos.5,055,303; 5,188,837; 4,235,871; 4,501,728; 4,837,028; 4,957,735; 5,019,369; 5,055,303; 5,514,670; 5,413,797; 5,268,164; 5,004,697; 4,902,505; 5,506,206; 5,271,961; 5,254,342 and 5,534,496). VI. Methods of Treatment Further provided herein are methods of treating a mesothelin-positive cancer in a subject. In some aspects, the method includes administering to the subject a therapeutically effective amount of a MSLN.CAR / IL-15 expressing NK cell or composition disclosed herein. In some examples, the 4239-111805-02 mesothelin-positive cancer is mesothelioma, lung cancer, stomach cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, thymic carcinoma, or colorectal cancer. The NK / iNK cell compositions disclosed herein can be administered to slow or inhibit the growth of tumor cells, inhibit the metastasis of tumor cells, and / or increase the survival of a subject having a tumor, such as a mesothelin-positive tumor, such as a mesothelin-positive solid tumor. In these applications, a therapeutically effective amount of a composition is administered to a subject in an amount sufficient to inhibit growth, replication or metastasis of cancer cells, increase the survival of a subject having a tumor, and / or to inhibit a sign or a symptom of the cancer. Suitable subjects may include those diagnosed with a cancer that expresses mesothelin, such as, but not limited to mesothelioma, lung cancer, stomach cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, thymic carcinoma, or colorectal cancer. Provided herein is a method of treating a mesothelin-positive cancer in a subject by administering to the subject a therapeutically effective amount of an NK / iNK cell co-expressing a mesothelin-targeted CAR and IL-15, or composition thereof, as disclosed herein. In some examples, such a method reduces the size and / or volume of a mesothelin-positive cancer by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, at least 90%, at least 95%, or even 100% reduction the cancer (such as a reduction of 10-95%, 10-80%, or 20-50%), for example as compared to the size and / or volume of a mesothelin-positive cancer prior to treatment with a therapeutically effective amount of an NK / iNK cell co-expressing a mesothelin-targeted CAR and IL-15 provided herein, or as compared to a treatment that does not include administration of an NK / iNK cell co- expressing a mesothelin-targeted CAR and IL-15 provided herein. Also provided herein is a method of reducing tumor growth or metastasis of a mesothelin-positive cancer in a subject by administering to the subject a therapeutically effective amount of an NK / iNK cell co-expressing a mesothelin- targeted CAR and IL-15, or composition thereof, as disclosed herein. In some examples, such a method reduces the size, volume, and / or number of a mesothelin-positive cancer metastases by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, at least 90%, at least 95%, or even 100% reduction the metastases (such as a reduction of 10-95%, 10-80%, or 20-50%), for example as compared to the size, volume, and / or number of a mesothelin-positive cancer metastases prior to treatment with a therapeutically effective amount of an NK / iNK cell co-expressing a mesothelin-targeted CAR and IL-15 provided herein, or as compared to a treatment that does not include administration of an NK / iNK cell co-expressing a mesothelin-targeted CAR and IL-15 provided herein. In some aspects, the mesothelin-positive cancer is mesothelioma, lung cancer, stomach cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, thymic carcinoma, or colorectal cancer. In some aspects, the mesothelin-positive cancer is mesothelioma. In some aspects, the mesothelin-positive cancer is pancreatic cancer. In some aspects, the mesothelin- positive cancer is ovarian cancer. In some aspects, the mesothelin-positive cancer is a gastric cancer, such as a stomach cancer. 4239-111805-02 A therapeutically effective amount of an NK / iNK cell composition disclosed herein can depend upon the severity of the disease, the type of disease, and the general state of the patient’s health. A therapeutically effective amount of the NK / iNK cell composition is that which provides either subjective relief of a symptom(s) or an objectively identifiable improvement as noted by the clinician or other qualified observer. Administration of the NK / iNK cell compositions disclosed herein can also be accompanied by administration of other anti-cancer agents or therapeutic treatments (such as surgical resection of a tumor). Any suitable anti-cancer agent can be administered in combination with the NK / iNK cell compositions disclosed herein, such as administered prior to, concurrently with, or following administration of the NK / iNK cell composition. Exemplary anti-cancer agents include, but are not limited to, chemotherapeutic agents, such as, for example, mitotic inhibitors, alkylating agents, anti- metabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, anti-survival agents, biological response modifiers, anti-hormones (e.g. anti- androgens) and anti-angiogenesis agents. Other anti-cancer treatments include radiation therapy and other antibodies that specifically target cancer cells. Non-limiting examples of alkylating agents include nitrogen mustards (such as mechlorethamine, cyclophosphamide, melphalan, uracil mustard or chlorambucil), alkyl sulfonates (such as busulfan), nitrosoureas (such as carmustine, lomustine, semustine, streptozocin, or dacarbazine). Non-limiting examples of antimetabolites include folic acid analogs (such as methotrexate), pyrimidine analogs (such as 5-FU or cytarabine), and purine analogs, such as mercaptopurine or thioguanine. Non-limiting examples of natural products include vinca alkaloids (such as vinblastine, vincristine, or vindesine), epipodophyllotoxins (such as etoposide or teniposide), antibiotics (such as dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, or mitomycin C), and enzymes (such as L-asparaginase). Non-limiting examples of miscellaneous agents include platinum coordination complexes (such as cis-diamine-dichloroplatinum II also known as cisplatin), substituted ureas (such as hydroxyurea), methyl hydrazine derivatives (such as procarbazine), and adrenocrotical suppressants (such as mitotane and aminoglutethimide). Non-limiting examples of hormones and antagonists include adrenocorticosteroids (such as prednisone), progestins (such as hydroxyprogesterone caproate, medroxyprogesterone acetate, and magestrol acetate), estrogens (such as diethylstilbestrol and ethinyl estradiol), antiestrogens (such as tamoxifen), and androgens (such as testerone proprionate and fluoxymesterone). Examples of the most commonly used chemotherapy drugs include Adriamycin, Alkeran, Ara-C, BiCNU, Busulfan, CCNU, Carboplatinum, Cisplatinum, Cytoxan, Daunorubicin, DTIC, 5-FU, Fludarabine, Hydrea, Idarubicin, Ifosfamide, Methotrexate, Mithramycin, Mitomycin, Mitoxantrone, Nitrogen Mustard, 4239-111805-02 Taxol (or other taxanes, such as docetaxel), Velban, Vincristine, VP-16, while some more newer drugs include Gemcitabine (Gemzar), Herceptin, Irinotecan (Camptosar, CPT-11), Leustatin, Navelbine, Rituxan STI-571, Taxotere, Topotecan (Hycamtin), Xeloda (Capecitabine), Zevelin and calcitriol. Non-limiting examples of immunomodulators that can be used include AS-101 (Wyeth- Ayerst Labs.), bropirimine (Upjohn), gamma interferon (Genentech), GM-CSF (granulocyte macrophage colony stimulating factor; Genetics Institute), IL-2 (Cetus or Hoffman-LaRoche), human immune globulin (Cutter Biological), IMREG (from Imreg of New Orleans, La.), SK&F 106528, and TNF (tumor necrosis factor; Genentech). Another common treatment for some types of cancer is surgical treatment, for example surgical resection of the cancer or a portion of it. Another example of a treatment is radiotherapy, for example administration of radioactive material or energy (such as external beam therapy) to the tumor site to help eradicate the tumor or shrink it prior to surgical resection. VII. Methods of Producing iPSC-Derived Natural Killer Cells (iNK cells) Also provided herein are methods of producing hematopoietic stem cells from induced pluripotent stem cells (iPSCs), and methods of differentiating the hematopoietic stem cells to iNK cells (see, e.g., FIG.15). In some aspects, the method of differentiating iPSCs to hematopoietic progenitor cells includes seeding iPSCs in a culture vessel containing albumin polyvinylalcohol essential lipids (APEL) culture medium that includes human stem cell factor (SCF), human vascular endothelial growth factor (VEGF), human bone morphogenetic protein 4 (BMP-4) and / or a Rho kinase inhibitor (such as Y-27632); and subjecting the seeded iPSCs to centrifugation and continuing to culture the iPSCs in the APEL culture medium to produce embryoid bodies (EBs) containing hematopoietic progenitor cells. In some aspects, the hematopoietic progenitor cells are further differentiated to produce iNK cells. In some examples, differentiating the hematopoietic progenitor cells to iNK cells includes culturing the EBs in NK cell differentiation medium. In particular examples, the NK cell differentiation medium contains interleukin-3 (IL-3), IL-15, IL-7, SCF and fms-like tyrosine kinase 3 (FLT3) ligand. In some aspects of these methods, the iPSCs seeded into the culture vessel are trypsin-adapted iPSCs. For example, the trypsin-adapted iPSCs can be TrypLE-adapted iPSCs. In some aspects of the disclosed methods, the culture vessel is as tissue culture vessel, such as a 96-well plate, such as a 96-well round-bottom plate. However, a skilled person can select an appropriate alternative culture vessel for carrying out the disclosed methods. In some aspects, the EBs are cultured in the APEL culture medium for about 2 to about 14 days, such as about four, about five, about six, about seven, about eight, about nine, about ten, about 11, about 12, about 13, or about 14 days. 4239-111805-02 In some aspects, the APEL culture medium includes about 1-100 ng / ml SCF, about 1-100 ng / ml VEGF, about 1-100 ng / ml BMP-4, and / or about 0.1-100 μM Y-27632. In some examples, the APEL culture medium includes about 10 ng / ml, about 20 ng / ml, about 40 ng / ml, about 50 ng / ml, about 60 ng / ml, about 70 ng / ml, about 80 ng / ml, about 90 ng / ml or about 100 ng / ml SCF. In specific examples, the APEL culture medium includes about 40 ng / ml SCF. In some examples, the APEL culture medium includes about 10 ng / ml, about 20 ng / ml, about 40 ng / ml, about 50 ng / ml, about 60 ng / ml, about 70 ng / ml, about 80 ng / ml, about 90 ng / ml or about 100 ng / ml VEGF. In specific examples, the APEL culture medium includes about 20 ng / ml VEGF. In some examples, the APEL culture medium includes about 10 ng / ml, about 20 ng / ml, about 40 ng / ml, about 50 ng / ml, about 60 ng / ml, about 70 ng / ml, about 80 ng / ml, about 90 ng / ml or about 100 ng / ml BMP-4. In specific examples, the APEL culture medium includes about 20 ng / ml BMP-4. In some examples, the APEL culture medium includes about 0.1 μM, about 0.5 μM, about 1.0 μM, about 2.0 μM, about 5.0 μM, about 10 μM, about 25 μM, about 50 μM, about 75 μM, or about 100 μM Y-27632. In specific examples, the APEL culture medium includes about 10 μM Y-27632. In some aspects of the disclosed methods, the hematopoietic differentiation method is EB formation method with APEL culture medium. However, alternative hematopoietic differentiation methods using non-EB method and other hematopoietic differentiation medium system can be used for carrying out the disclosed methods. In some aspects, the EBs are cultured in the NK cell differentiation medium for about two to about seven weeks, such as about two, about three, about four, about five, about six, or about seven weeks. In some examples, the EBs are cultured in the NK cell differentiation medium for at least two weeks, at least three weeks, or at least four weeks. In some aspects, the NK cell differentiation medium includes about 1-100 ng / ml IL-3, about 1-100 ng / ml IL-15, about 1-100 ng / ml IL-7, about 1-100 ng / ml SCF and / or about 1-100 ng / ml FLT3 ligand. In some examples, the NK cell differentiation medium includes about 1 ng / ml, about 2.5 ng / ml, about 5.0 ng / ml, about 7.5 ng / ml, about 10 ng / ml, about 20 ng / ml, about 40 ng / ml, about 50 ng / ml, about 60 ng / ml, about 70 ng / ml, about 80 ng / ml, about 90 ng / ml or about 100 ng / ml IL-3. In specific examples, the NK cell differentiation medium includes about 5 ng / ml IL-3. In some examples, NK cell differentiation medium includes about 1 ng / ml, about 2.5 ng / ml, about 5.0 ng / ml, about 7.5 ng / ml, about 10 ng / ml, about 20 ng / ml, about 40 ng / ml, about 50 ng / ml, about 60 ng / ml, about 70 ng / ml, about 80 ng / ml, about 90 ng / ml or about 100 ng / ml IL-15. In specific examples, the NK cell differentiation medium includes about 10 ng / ml IL-15. In some examples, the NK cell differentiation medium includes about 10 ng / ml, about 20 ng / ml, about 40 ng / ml, about 50 ng / ml, about 60 ng / ml, about 70 ng / ml, about 80 ng / ml, about 90 ng / ml or about 100 ng / ml IL-7. In specific examples, the NK cell differentiation medium includes about 20 ng / ml IL-7. In some examples, the NK cell differentiation medium includes about 10 ng / ml, about 20 ng / ml, about 40 ng / ml, about 50 ng / ml, about 60 ng / ml, about 70 ng / ml, about 80 ng / ml, about 90 ng / ml or about 100 ng / ml SCF. In 4239-111805-02 specific examples, the NK cell differentiation medium includes about 20 ng / ml SCF. In some examples, NK cell differentiation medium includes about 1 ng / ml, about 2.5 ng / ml, about 5.0 ng / ml, about 7.5 ng / ml, about 10 ng / ml, about 20 ng / ml, about 40 ng / ml, about 50 ng / ml, about 60 ng / ml, about 70 ng / ml, about 80 ng / ml, about 90 ng / ml or about 100 ng / ml FLT3 ligand. In specific examples, the NK cell differentiation medium includes about 10 ng / ml FLT3 ligand. In some examples, IL-3 is present in the NK differentiation medium for only the first 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days of culture. In specific examples, IL-3 is present in the NK differentiation medium for only the first week of culture. In some aspects of the disclosed methods, the NK differentiation medium includes IL-3, IL- 15, IL-7, SCF and / or FLT3 ligand. However, an alternative NK differentiation method or medium system can be used for carrying out the disclosed methods. In some aspects, the iNK cells produced by the disclosed methods are CD45+CD56+. In some aspects of the disclosed methods, prior to differentiation of the iPSCs to hematopoietic cell progenitors, the iPSCs are transduced with a nucleic acid molecule or vector encoding a mesothelin-targeted CAR and IL-15, such as a nucleic acid or vector disclosed herein. In some examples of these methods, the method further includes the step of enriching iPSCs that express the CAR encoded by the transduced nucleic acid molecule or vector. In particular examples, the enriched iPSCs are also express IL-15 encoded by the transduced nucleic acid molecule or vector. In some aspects of the methods, the iPSCs are LiPSC-GR1.1 cells. In other aspects, the iPSCs are from a different established iPSC line, such as an iPSC line available through the NIH Regenerative Medicine Program (see commonfund.nih.gov / stemcells / lines; Baghbaderani et al., Stem Cell Rev and Rep 12:394-420, 2016; Baghbaderani et al., Stem Cell Reports 5:647-659, 2015). Also provided herein are methods of generating hematopoietic progenitor cells by differentiating LiPSC-GR1.1 cells to hematopoietic progenitor cells. Methods of generating hematopoietic progenitor cells from pluripotent stem cells, including iPSC lines, are known and a skilled person can select an appropriate differentiation method, such as the EB method described in Example 1, or any method described in Zheng et al. (Cell Regeneration 12:31, 2023). EXAMPLES The following examples are provided to illustrate particular features of certain aspects of the disclosure, but the scope of the claims should not be limited to those features exemplified. Example 1: Materials and Methods This example describes the materials and experimental procedures for the studies described in Examples 2-11. 4239-111805-02 Study Design The goals of this study included the development of a robustly differentiated mesothelin targeting CAR-IL-15 iNK product using the cGMP-manufactured iPSC line LiPSC-GR1.1, to phenotypically characterize MSLN.CAR-IL-15 GR1.1-iNK, and to investigate the anti-tumor efficacy and the mechanism of action of MSLN.CAR-IL-15 GR1.1-iNKs in MSLN positive solid tumors. Six iPSC lines were tested for iNK differentiation using the spin EB method. LiPSC-GR1.1 was identified as a superior iPSC line for iNK differentiation and selected for genetic modification using a MSLN.CAR-IL-15 expressing piggyBac transposon / transposase system. LiPSC-GR1.1 iPSCs stably expressing MSLN.CAR-IL-15 were differentiated to GR1.1-iNK cells. NK cell receptor expression levels on GR1.1-iNKs were then determined by flow cytometry and the transcriptome of GR1.1-iNKs compared to PBNKs isolated from healthy donors was analyzed employing bulk RNA-seq. The anti- tumor efficacy of MSLN.CAR-IL-15 GR1.1-iNK against mesothelin positive solid tumors was evaluated using in vitro and mouse tumor models. In addition, the infiltration of MSLN.CAR-IL-15 GR1.1-iNK in solid tumor tissues was investigated by flow cytometry and multiplex immunofluorescence. Lastly, to better understand tumor responses to MSLN.CAR-IL-15 GR1.1-iNK, as well as NK signatures associated with tumor homing and anti-tumor effects, single-cell RNAseq analysis was performed on the total cells isolated from MSLN.CAR-IL-15 GR1.1-iNK-treated tumor tissues, untreated tumor tissues, as well as pre-infusion MSLN.CAR-IL-15 GR1.1-iNK product. Mice, tumor cell lines, iPSC lines, and reagents NOD / SCID / γc− / − (NSG) mice were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). All mice were maintained in a dedicated pathogen-free environment following National Institutes of Health guidelines. Animal procedures reported in this study were conducted under an animal study protocol (#TGOB-001) approved by the Animal Care and Use Committee of the National Cancer Institute and in accordance with federal regulatory requirements and standards. Established tumor cell lines KLM-1 MSLN-WT / KO (received from Dr. Christine Alewine, CCR, NCI, NIH, Bethesda, MD), OVCAR-8 (received from Dr. Hisataka Kobayashi, CCR, NCI, NIH, Bethesda, MD), N87 (obtained from ATCC; American Type Culture Collection, Manassas, VA), and the EBV-transformed B cell line SMI-LCL (received from Dr. Richard W. Childs, Cellular and Molecular Therapeutics Branch, National Heart Lung and Blood Institute, NIH, Bethesda, MD) (Childs and Berg, Hematology Am Soc Hematol Educ Program 2013:234-246, 2013), were maintained in RPMI1640 medium with 10% FBS, 2 mmol / L l-glutamine, and 100 U penicillin– streptomycin. Early-passage mesothelioma cell lines (NCI-Meso21, NCI-Meso29 and NCI-Meso63) were maintained in RPMI1640 medium with 20% FBS, 2 mmol / L l-glutamine, and 100 U penicillin– streptomycin. The mesothelioma cell lines were established from ascites or pleural fluid obtained from patients with mesothelioma treated at the NCI (Bethesda, MD) under an Institutional Review 4239-111805-02 Board (IRB)–approved protocol (ClinicalTrials.gov NCT 01950572). The methods for establishment of primary culture cell lines have been described previously (Zhang et al., Clin Cancer Res 23:1564- 1574, 2017). KLM-1 MSLN-WT / KO, OVCAR-8, NCI-Meso29 and NCI-Meso63 were stable luciferase-expressing cell lines (Tomar et al., Mol Cancer Ther 21:1195-1206, 2022). The iPSC line LiPSC-GR1.1 was generated at Lonza Walkersville, Inc., as previously described (Baghbaderani et al., Stem Cell Reports 5:647-659, 2015; Baghbaderani et al., Stem Cell Rev Rep 12:394-420, 2016), and distributed through RUCDR Infinite Biologics at Rutgers University (stemcells.nindsgenetics.org). The iPSC lines NCRM1, NCRM2, NCRM4, NCRM5, and NCRM6 were obtained from the iPSC core of the National Heart, Lung, and Blood Institute (NHLBI). These lines are available to the research community. All of the iPS cell lines were generated from healthy donor derived cord blood CD34+cells and cryopreserved in CryoStor CS10 (STEMCELL technologies). Recombinant human cytokines used in NK differentiation and culture included vascular endothelial growth factor (VEGF) and bone morphogenetic protein 4 (BMP-4) (both obtained from R&D Systems Inc), as well as stem cell factor (SCF), IL-3, IL-15, IL-7, flt3 ligand, IL- 2, and IL-21 (all obtained from PeproTech). The antibodies used for flow cytometry and immunofluorescence included mouse anti-human CD56-BV785 (BioLegend, Cat#362549); rabbit anti-human EGFR-APC (Novus Biologicals, Cat#NBP2-52671APC); mouse anti-human DNAM-1-PerCP / Cy5.5 (BioLegend, Cat#338313); mouse anti-human NKp46-PE / Cy7 (BioLegend, Cat#331915); mouse anti-human NKG2D-FITC (BioLegend, Cat#320820); mouse anti-human TRAIL-PE / Cy7 (BioLegend, Cat#308216); mouse anti-human Fas-L-PE (BioLegend, Cat#306407); mouse anti-human CD16-PerCP / Cy5.5 (BioLegend, Cat#360712); mouse anti-human NKp44-PerCP / Cy5.5 (BioLegend, Cat#325113); mouse anti-human CD94-PE / Cy7 (BioLegend, Cat#305515); mouse anti-human NKG2A-PE (R & D Systems, Cat#FAB1059P-025); mouse anti-human CD158 (KIR2DL1 / S1 / S3 / S5)-FITC (BioLegend, Cat#339504); mouse anti-human CD107a-PerCP / Cy5.5 (BioLegend, Cat#328616); mouse anti-human CD45-PE-Cy7 (BioLegend, Cat#982310); rat anti-mouse CD45-PerCP / Cy5.5 (BioLegend, Cat#103132); mouse anti-human CD3-BV510 (BioLegend, Cat#317331); mouse anti-human CD158d (KIR2DL4)-APC (BioLegend, Cat#347007); mouse anti-human CD159c (NKG2C)-BV421 (BioLegend, Cat#375013); mouse anti-human CD337 (NKp30)-BV421 (BioLegend, Cat#325227); mouse anti-human CD45-AF532 (Thermo Fisher Scientific, Cat#58-0459-41); mouse anti-human CD56-PE-CF594 (BD Biosciences, Cat#562289); mouse anti-human Ki67-AF700 (BD Biosciences, Cat#561277); mouse anti-human Mesothelin (Rockland, Cat#200-301-A88); donkey anti-mouse IgG- AF790 (Thermo Fisher Scientific, Cat#A11371). CAR constructs cloning Lentiviral packaging plasmid psPAX2 expressing CD8TM.BBICD.Z.CAR (composed of MSLN-targeted hYP218 scFv, CD8a hinge spacer, CD8 transmembrane domain (TM), 4239-111805-02 4-1BB costimulatory intracellular domain (ICD), and stimulatory domain (SD) CD3ζ chain), as well as a truncated human EGFR polypeptide (EGFRt) was created previously (Tomar et al., Mol Cancer Ther 21:1195-1206, 2022). The fragment of IL-15 plus T2A between EGFRt and IL-15 was synthesized as a gene fragment (by GeneWiz) and subcloned into the psPAX2 vector through seamless cloning using NEBuilder® HiFi DNA Assembly Cloning Kit (New England biolabs) according to the manufacturer’s instructions. CD8-TM and 4-1BB-ICD were then replaced with gene fragment of NKG2D-TM and 2B4-ICD (synthesized by Genescript) through seamless cloning to generate plasmid psPAX2 expressing NKG2DTM / 2B4ICD.Z.CAR as well as NKG2DTM / 2B4ICD.Z.CAR-IL15. The PiggyBac transposon vector PBCAG-eGFP with a CAG promoter was a gift from Joseph Loturco (Addgene plasmid # 40973) (Chen and LoTurco, J Neurosci Methods 207:172-180, 2012). To create CAR expressing PBCAG plasmids, the expression cassette encoding NKG2DTM / 2B4ICD.Z.CAR-EGFRt or NKG2DTM / 2B4ICD.Z.CAR-EGFRt-IL-15 was amplified from the template plasmid psPAX2 through PCR and then subcloned into the XmaI and NotI site of PBCAG vector through seamless cloning. Cell culture of human iPSCs iPS cell lines were maintained feeder-free in complete Essential 8 Flex medium (Thermo Fisher Scientific) in 6-well plates coated with truncated vitronectin recombinant human protein (Thermo Fisher Scientific, A14700) at a concentration 0.5 µg / cm2. They were routinely passaged as small clumps using 0.5 mM EDTA in PBS at a split ratio of 1:6 to 1:10 every 3 to 4 days after reaching 60%–80% confluence. After EDTA treatment, hiPSCs were transferred to new vitronectin- coated plates in fresh medium supplemented with ROCK inhibitor (ROCKi) Y-27632 (10 μM, R&D Systems Inc). Next day, the medium was changed to hiPSC medium without ROCKi. NK cell differentiation from human iPSCs The derivation of NK cells from iPSCs has been previously described (Zhu and Kaufman, Methods Mol Biol 2048:107-119, 2019). Briefly, 8000 TrypLE-adapted iPSCs were seeded in 96-well round-bottom plates with APEL medium containing 40 ng / ml SCF, 20 ng / ml VEGF, 20 ng / ml BMP- 4, and Y-27632 (10 μM). After 6 days of hematopoietic differentiation, spin embryoid bodies (EBs) were washed once with NK differentiation medium then transferred into 6-well plates with NK differentiation medium containing 5 ng / mL IL-3 (first week only), 10 ng / mL IL-15, 20 ng / mL IL-7, 20 ng / mL SCF, and 10 ng / mL flt3 ligand for 4 weeks. Half-media changes were performed twice a week. NK cells were harvested 4 weeks post NK differentiation. For the IL-15 concentration test, different concentrations of IL-15 (10 ng / mL, 20 ng / ml, 40 ng / ml, 80 ng / ml) were used during the 4 weeks of NK differentiation. Three independent experiments were performed. 4239-111805-02 Stable CAR constructs expression in LiPSC-GR1.1 Line LiPSC-GR1.1 cells were dissociated as single cell suspensions using TrypLE, then washed with fresh E8 medium supplemented with ROCKi.0.2 million iPSCs were harvested through centrifugation, resuspended in 20 µl P3 primary solution containing 1 µg CAR expressing PBCAG transposon vector plus 0.25 µg Super PiggyBac Transposase (Cat#PB210PA-1, System Bioscience), and transferred into nucleocuvette strips. The nucleofection was performed using Lonza 4D- Nucleofector with pulse setting CA-137 according to the manufacturer’s guidelines. The iPSCs nucleofected without PBCAG transposon were used as mock control. CAR expressing iPSCs were enriched through FACS sorting EGFR+iPSCs during day 7-9 post nucleofection. IL-15 ELISA iPSCs were seeded at 3×105cells / well in 6-well plate and cultured for 72 hours without medium change. Then the supernatants of the iPSC culture were collected. IL-15 was analyzed using the ELISA MAX™ Deluxe Set Human (BioLegend) according to the manufacturer’s guidelines. Three independent experiments were performed. iNK cryopreservation and expansion Freshly differentiated iNKs were harvested 4 weeks post differentiation and cryopreserved in CryoStor CS10 at 1.0×107cells / ml. For expansion testing, iNKs were initially thawed and cultured in complete Xpander media supplemented with 10% heat-inactivated human AB serum, 100 U / ml IL-2, 100 ng / ml IL-21 and irradiated (100 Gy) SMI-LCL feeder cells (7.5:1 feeder-to-NK cell ratio) with starting cell concentration at 1.0×106cells / ml. Expanded iNK were harvested on day 7 post expansion for the following studies. PBNK isolation Human PBMCs from healthy donors were obtained from the NIH Clinical Center Department of Transfusion Medicine under NIH IRB-approved and consented healthy donor program. PBMCs were isolated by density gradient centrifugation on Histopaque 1077 (Sigma). Briefly, 15 ml of the collected blood sample was diluted in an equal volume of PBS and layered over onto an equal volume of Histopaque 1077 (Sigma). Gradients were centrifuged at 400 × g for 30 minutes at room temperature. The PBMC interface was carefully removed by pipetting and washed twice with PBS by centrifugation at 300 × g for 10 minutes. For all cell isolations, cell number was determined using a Countess Automated Cell Counter (Invitrogen). Primary NK cells were purified from PBMCs using human NK Cell Isolation Kit (Miltenyi Biotec). The NK purity was confirmed as 80%~95% by flow cytometry. Three independent experiments were performed. 4239-111805-02 In vitro NK cell cytotoxicity assays NK cell cytotoxicity was determined in vitro in a coculture of NKs and tumor cells by measuring direct killing of mesothelin-expressing tumor cells by the NK cells. Briefly, 100 μL of complete RPMI media containing 5,000 tumor cells were seeded in a 96-well U-bottom plate.4 hours after seeding the tumor cells, 100 μL of media containing NKs were added into the co-culture across a range of effector-to-target (E:T) ratios. The luciferase signal from the remaining live tumor cells was determined 24 hours post coculture using the Luciferase Assay System (Promega, catalog No. E1501). Percent killing was calculated using the formula: % killing = 100 × [1- relative light units (RLUs) from coculture wells / RLU from target-alone wells]. Two or three independent experiments were performed. Mouse tumor model study To evaluate iNK cell function against NCI-Meso63 tumors in vivo, 5- to 6-week-old female NSG mice (Jackson Laboratories) were intraperitoneally (i.p.) injected with 3×106luciferase- expressing NCI-Meso63 tumor cells on day -7. Bioluminescent imaging (BLI) (PerkinElmer, IVIS Lumina III In vivo Imaging System) was performed on day -1. The mice were then conditioned with 225 cGy radiation and divided into groups with similar starting BLI values. The two doses of 8×106iNK cells (harvested either on day 28 post NK differentiation or 1 week post expansion from frozen iNK stock) were i.p. injected into tumor-bearing mice on day 0 and day 7. Tumor growth was monitored weekly via BLI. To evaluate iNK cell function against KLM-1 tumor cells in vivo, NSG mice were i.p. injected with 5×105luciferase-expressing KLM-1 tumor cells on day -5. BLI imaging was performed on day -1. The mice were then conditioned with 225 cGy radiation and randomly divided into groups with similar starting BLI values. Two doses of 1×107and 1 dose of 5×106iNK cells harvested during day 28 to day 35 post NK differentiation were i.p. injected into tumor-bearing mice on day 0 and day 7 and tumor growth monitored weekly via BLI (n = 5~7 mice per group for all the tumor model efficacy study). To investigate the tumor response and iNKs tumor infiltration in the NCI-Meso63 tumor model, mice were injected with 6×106NCI-meso63 tumor cells on day -16 and treated with 6×106iNK cells (product of 1 week expansion from frozen iNK stock) on day 0. BLI was performed to monitor the tumor growth on day -1 and day 6. All tissues and blood were harvested on day 7 post iNK treatment. The tumors were harvested for flow cytometry, immunofluorescence, and scRNAseq analysis. The spleens were harvested for flow cytometry analysis (n =3 mice per group). Two or three independent experiments were performed. Bulk mRNAseq Total RNA was isolated from freshly isolated PBNKs (from healthy donors) and freshly differentiated iNK cells were harvested 4 weeks post NK differentiation using TRIzol reagent according to the manufacturer’s guide (Invitrogen). Quantity and quality of RNA were analyzed 4239-111805-02 using Agilent TapeStation. mRNA-Seq samples were pooled and sequenced on NextSeq 2000 P2 using Illumina stranded mRNA ligation kit having 77 to 88 million pass filter reads with more than 92% of bases above the quality score of Q30. Reads of the samples were trimmed for adapters quality bases using Cutadapt before alignment with the reference genome (hg38) and the annotated transcripts using STAR. The average mapping rate of all samples was 95%. Unique alignment was above 90%. There were 3.33 to 4.82% unmapped reads. The mapping statistics were calculated using Picard software. The samples had 0.47% ribosomal bases. Percent coding bases were between 42-51%. Percent UTR bases were 31-33%, and mRNA bases were between 74-83% for all samples. Library complexity was measured in terms of unique fragments in the mapped reads using Picard’s MarkDuplicate utility. The samples had 70-75% non-duplicate reads. In addition, the gene expression quantification analysis was performed for all samples using STAR / RSEM tools. The normalization of the RNA counts and differentially expressed genes (DEGs) analysis were performed using the Limma Voom R package. Transcripts with significantly differential expression (p<0.05, log2 fold change >1) were used for subsequent pre-ranked gene set enrichment analysis (GSEA) using EasyGSEA tool (Cheng et al., Nucleic Acids Res 49:W207-W215, 2021) for Hallmark and KEGG gene sets scoring. Single cell isolation from mouse tissues From NCI-meso63 tumor bearing mice, spleens were harvested in cold PBS, gently mashed, and filtered through a two-chamber sterile filter bag (Fisher Scientific), and the resulting single-cell suspension was collected from the other side of the bag. The mouse peritoneal tumors were harvested in cold serum-free RPMI-1640 medium and total cells were isolated using tumor dissociation kit (Miltenyi Biotec) according to manufacturer’s instructions. Briefly, tumor tissues were digested in serum-free RPMI-1640 medium supplemented with human tumor dissociation kit enzymes (Miltenyi Biotec) at 37°C under continuous rotation for 90 minutes. Dissociated single cell suspensions were then filtered through a 70 μm mesh filter and pelleted at 300 × g for 5 minutes. For flow cytometry analysis, all of the single cells isolated from the spleens and tumors were washed with FACS buffer (PBS + 0.5% FBS, + 1 mM EDTA). For scRNAseq study, the single cells isolated from the tumors were washed with PBS+0.04% BSA. The single cells prepared were then used for the following study. Flow cytometry For human iPSCs, iPSCs were dissociated into a single-cell suspension with TrypLE, harvested, washed, then resuspended in E8 medium containing Essential 8 Flex medium, and incubated with fluorescent conjugated antibodies at 4°C for 30 minutes. For iNK cells, cells were harvested from the cultured supernatant, washed, then resuspended in PBS, and incubated with fluorescent conjugated antibodies at 4°C for 30 minutes. For the cells harvested from NK / tumor coculture or isolated from peritoneal tumors and spleens, cells were first stained with Zombie Aqua 4239-111805-02 Fixable Viability Kit (Biolegend, Cat#423102) at room temperature for 30 minutes, then incubated with 1% FBS at 4°C for 15 minutes to block non-specific binding sites before incubating with fluorescent conjugated antibodies at 4°C for 30 min. For CD107a degranulation assay, NK cells were incubated with or without KLM-1 cells at 2:1 effector to target ratios in the presence of anti-CD107a antibody for 1 hour, followed by adding GolgiStop (BD Biosciences, Cat#554724) for additional 2 hours incubation. At the completion of incubation, cells were washed with PBS, then first stained with Zombie Aqua Fixable Viability Kit at room temperature for 30 minutes, then incubated with 1% FBS at 4°C for 15 minutes to block non-specific binding sites before incubating with fluorescent conjugated antibodies at 4°C for 30 minutes. All flow cytometry detection was performed on CytoFLEX LX and analyzed using FlowJo software. Two or three independent experiments were performed. Multiplex immunofluorescence Fresh isolated tumor tissues were fixed in BD Cytofix / Cytoperm solution (BD Bioscience, Cat#: 554722) diluted 1:4 in PBS for 2 days at 4°C, washed in PBS three times and maintained in PBS for 10 hours at 4°C. Then, the tissue blocks were transferred into PBS containing 30% sucrose for 2 days until the tissue blocks sank to the bottom of the 30% sucrose solutions. The tissues were then embedded in Scigen Tissue-Plus™ O.C.T. Compound (Fisher Scientific, Cat# 23-730-571) and stored in -80°C before use.12-micron thick cross sections of the tumor tissue blocks were cut by using a Leica CM1950 cryostat. O.C.T. compound of tissue sections was removed by washing the tissue sections with PBS for 30 minutes before being blocked in 1% BSA, 0.1 M pH 7.4 Tris buffer containing 0.3% Triton X-100 for 30 minutes at room temperature. A microwave-assisted immune- fluorescent staining protocol was adapted for mesothelin detection by using a mouse anti-human mesothelin antibody amplified by a fluorescent dye directly conjugated donkey anti-mouse secondary antibody (Radtke et al., Proc Natl Acad Sci USA 117:33455-33465, 2020). The sections were then blocked in 1% BSA and 1% mouse serum in 0.1 M pH 7.4 Tris buffer containing 0.3% Triton X-100, stained with the following fluorescent dye directly conjugated primary antibodies: CD45-AF532, CD56-PE / CF594, Ki67-AF700 together with nuclear dye Helix NP-NIR (BioLegend, Cat# 425301). Then, tissue sections were washed in 0.1 M pH 7.4 Tris buffer and mounted with SlowFade Gold Antifade Mounting solution (Thermo Fisher Scientific Cat#S36937) using #1.5 cover glass (VWR, Cat#: 48393-241). Digital scan of stained tumor sections was performed using an inverted Leica TCS SP8 X confocal system equipped with an 80 MHz pulsed white light laser, 4 Gallium-Arsenide (GaAs) Hybrid Detectors (HyDs) and 1 multialkali photomultiplier tube (PMT) with spectral detection capability. A 40X (NA 1.3) oil emersion objective lens was used for scanning the sections with a pixel size of 568.74X568.74 mm2and pixel dwell time of 1.2 microseconds. Digital images acquired by the Leica confocal system were tile stitched and then processed to correct signal spillovers from nearby channels and autofluorescence signal within the tumor sections by using the 4239-111805-02 manual unmixing method of the Automatic Dye Separation function within the Leica Application Suite X (LAS X) software package (version 4.4.0.24861). The output images (.lif files, Leica file format) were converted into Imaris version 5.5 files by Imaris software (Version 9.5.0, Bitplane) and imported into Imaris. Images of individual channels were passing through a Gaussian filter to remove random noise before pseudo-color assignment and visualization. In all image processing steps, image size and aspect ratio were maintained identical by keeping the pixel size unchanged. Two or three independent experiments were performed. Single-cell RNA sequencing Eight single cell samples were prepared for scRNA-seq, including 2 samples of pre-infusion product, 3 samples of total cells isolated from the untreated tumors (individual mouse as one sample) and 3 samples of total cells isolated from the MSLN.CAR-IL-15 iNK-treated tumors (4 mice pooled as one sample). Dead cells and debris were removed using Dead Cell Removal Kit (Miltenyi Biotec, 130-090-101) following the manufacturer’s instructions. Single cell suspensions were washed once with ice-cold PBS with 0.04% BSA by centrifugation at 350g and gently resuspended in fresh buffer. The cellular samples were loaded in the lane according to the 10X Genomics Chromium Next GEM Single Cell 5’ v2 (Dual Index) User Guide with a single capture lane per sample. The recovery was 6,000 cells per lane. Cell partitioning completed with a uniform emulsion consistency, and the reverse transcription PCR was run. All subsequent steps of library preparation and quality control were performed as described in the 10x Genomics user guide. Then the sequencing was performed on the NovaSeq 6000 system with the NovaSeq S2 Reagent kit v1.5 (100 cycles) following the cycling parameters: 26 bp (read1), 10 bp (index 1), 10 bp (index2), 90 bp (read2). scRNA-seq data processing Base calling was performed using RTA 3.4.4, demultiplexing was performed using cellranger v8.0.0 (Bcl2fastq 2.20.0), and alignment was performed using cellranger v8.0.0 (STAR 2.7.2a). Sequenced reads were aligned to a custom human GRCh38 and mouse GRCm39 reference sequence made by adding hYP218-scFv sequence to the refdata-gex-GRCh38_and_GRCm39-2024-A reference. UMI-adjusted aligned reads were used to generate a single cell barcode and gene expression matrix for downstream analysis. The preprocessed gene expression matrix generated by the CellRanger (10x Genomics) pipeline was imported into Seurat (v5.1.0). Putative droplet doublets were detected using scDblFinder (v1.16.0) with default parameters. Doublet cells were removed from each sample separately. Samples were merged. As a quality-control step, all cells expressing <200 genes were removed, as well as cells that contained <500 unique molecular identifiers (UMIs) and >20% mitochondrial counts. The filtered gene expression matrix was normalized using the NormalizeData function with default parameters. 3000 variable genes were identified using the FindVariableFeatures function with the vst method. 4239-111805-02 Principal component analysis (PCA) was applied with the RunPCA function to reduce dimensionality after regressing for the number of UMIs (counts) and percentage mitochondrial genes with the ScaleData function. The 15 most informative principal components (PCs) were used for clustering and uniform manifold approximation and projection for dimension reduction (UMAP). Shared nearest neighbors (SNN) were computed with the FindNeighbors function, and cells were then clustered using the FindClusters function with resolution set to 0.8. A UMAP was generated with the RunUMAP function. After the first-round of unsupervised clustering, the percentages of human and mouse genes was calculated in a cell in each cell cluster. The mouse cell clusters were removed from downstream analysis which focused on human cells. Genes of the mouse genome were also removed from human cells. Then a second-round of unsupervised clustering was performed on human cells. The second-round clustering procedure was performed the same as the first-round clustering with 0.7 resolution and dimensionality reduction with top 20 PCs for visualization. Human NKs and tumor clusters were annotated using curated in-house signatures. The cells annotated as B cells (LCL NK feeder cells) were removed from the analysis. R (v4.3.1) and RStudio (2023.09.1 Build 494, "Desert Sunflower" Release) were used for analysis. DEGs were performed using the FindMarkers function with Wilcoxon Rank Sum test, with the logfc.threshold, min.pct, and only.pos parameter set to log2(1.5), 0.1, F, respectively. DEGs were filtered with cutoff either pct.1 or min pct.2 >0.25 and adj P<0.05 for GSEA. Gene set variation analysis implemented in the fgsea R package (v1.28.0) was used for gene set enrichment analysis with 10,000 permutations performed. The Hallmark gene sets were retrieved from the msigdbr R package (v7.5.1). The input genes were calculated by FindMarkers and sorted by the avg_log2FC, and enriched pathways with less than 3 genes in the leadingEdge or adjusted P > 0.25 were filtered out. To recognize malignant cell subsets, copyKAT (v1.1.0) was used to determine human normal cells and malignant cells with the hg20 genome and other parameters set as default (Gao et al., Nat Biotechnol 39:599-608, 2021). The identified aneuploid cells were extracted as malignant cells. Trilineage Differentiation and Scorecard Assay Directed three germ layer differentiation was performed using StemMACS™ Trilineage Differentiation Kit (Miltenyi Biotec, 130-115-660) according to manufacturer's instructions. The cells were harvested, and RNA was extracted using the PureLink™ RNA Mini Kit (Thermo Fisher Scientific, 12183018A). DNase-treated RNA was prepared according to the High-capacity cDNA Reverse Transcription kit with RNase Inhibitor (Thermo Fisher Scientific, 4374966). The differentiation potential was assessed by quantitative PCR using the hPSC Scorecard Assay (Thermo Fisher Scientific, A16179) according to the manufacturer’s instructions. The scores representing gene expression associated with self-renewal, ectoderm, mesoderm or endoderm tissues were calculated according to algorithms determined by the manufacturer relative to a reference standard. 4239-111805-02 Statistical analysis Unpaired Student t test was used to compare the differences between groups for in vitro and in vivo studies. Mouse survival differences were compared using log-rank (Mantel-Cox) test. Statistical analysis was performed using GraphPad Prism 6.0. P < 0.05 was considered statistically significant. Example 2: LiPSC-GR1.1 identified as a superior human iPSC line for NK cell differentiation The well characterized LiPSC-GR1.1 line (Baghbaderani et al., Stem Cell Reports 5:647-659, 2015; Baghbaderani et al., Stem Cell Rev Rep 12:394-420, 2016) and 5 NCRM iPSC lines with normal karyotype were selected for NK differentiation study. The NCRM iPSC lines exhibited similar SSEA-4+TRA-1-60+pluripotency phenotype (FIG.1B) and were validated for their tri-lineage differentiation potential through scorecard assay (FIG.9). Using a standardized, simple, and efficient “spin-EB” iPSC-NK differentiation protocol (FIG.1A) (Zhu and Kaufman, Methods Mol Biol 2048:107-119, 2019), LiPSC-GR1.1 were compared to the 5 NCRM iPSC lines for their iNK differentiation potential.8000 TrypLE-adapted iPSCs were seeded in each well of 96-well round- bottom plates in APEL culture medium containing ROCK inhibitor, SCF, VEGF, and BMP-4 and spun to form embryoid bodies (EB). All of the cell lines initially aggregated and formed EBs on day 6 post cell seeding (FIG.1C). After day 6, EBs were then directly transferred into 6-well plates with NK cell differentiation medium for 4 weeks. During week 2 to week 4, substantial hematopoietic cell differentiation from EBs derived from LiPSC-GR1.1 and NCRM5 was observed. The EBs of these two lines began to disappear, with more free-floating, multicellular aggregates observed during the third week of NK differentiation. A low number of floating hematopoietic cells was observed with the EBs derived from the NCRM6 line. The EBs derived from NCRM1, NCRM2, and NCRM4 failed to further differentiate. The average iNK differentiation yield was 1.3×105per EB, 8.8×104per EB, and 9.0×103per EB from iPSC lines LiPSC-GR1.1, NCRM5, and NCRM6 respectively (FIG.1D). After 4 weeks of NK differentiation, > 98% of cells harvested from the supernatant of LiPSC-GR1.1 and NCRM5, as well as > 90% of cells harvested from the supernatant of NCRM6 were CD45+CD56+NK cells (FIG.1E) with higher CD56 expression as compared to the NKs derived from peripheral blood (PBNKs) (FIG.1F). Example 3: Robust differentiation of NK cells from MSLN.CAR-IL-15-engineered LiPSC- GR1.1 cells To target MSLN expressing solid tumors, the DNA construct MSLN.CAR that encodes the hYP218 scFv, followed by a CD8a hinge spacer, the NKG2D transmembrane domain (TM), the 2B4 intracellular domain, and the CD3ζ intracellular signaling domain, along with an independently translated truncated human EGFR polypeptide (EGFRt) sequence for tracking purpose, was subcloned 4239-111805-02 into a piggyBac transposon vector. IL-15 was also included in the construct to support NK persistence in vivo (FIG.2A) (9). LiPSC-GR1.1 cells were genetically engineered with the piggyBac transposon system carrying MSLN.CAR or MSLN.CAR-IL-15, and the iPSCs stably expressing the components of the constructs were enriched by FACS sorting of EGFRt+iPSCs. CAR expression by the expanded CAR or CAR-IL15-engineered LiPSC-GR1.1 cells was validated using flow cytometry (FIG.2B). IL- 15 production was detected only in the supernatant of the cultures of CAR-IL-15-engineered iPSCs using ELISA (FIG.2C). NK cell differentiation potential of these iPSCs was then tested. A robust yield of iNKs was attained from MSLN.CAR-IL-15 iPSCs (~1.6×106per EB) compared to limited iNK yield from mock iPSCs (~2.3×105per EB) or MSLN.CAR-engineered iPSCs (~ 4.5×105per EB) at 4 weeks post differentiation initiation (FIG.2D, FIG.10). In contrast, only a slightly increased NK cell yield was observed from the LiPSC-GR1.1 cells when the IL-15 concentration was increased to 20 ng / ml and 40 ng / ml in the differentiation medium (FIG.2E), suggesting that genetically modifying iPSCs to constantly express endogenous IL-15 ensures more robust and sustained signaling for hematopoietic differentiation within the mesoderm. NK markers and CAR expression by these differentiated iNKs were next examined using flow cytometry. The percentage of CD45+CD56+NKs in culture supernatants was >96% in all 3 groups (FIG.2F). CAR expression was 66.4% in MSLN.CAR iNKs and 85.8% in MSLN.CAR-IL-15 iNKs. In addition to the significant increase in differentiation yield, the recovery and expansion capacity of differentiated MSLN.CAR-IL-15 iNK cells post cryopreservation was also superior compared to both mock iNK cells and MSLN.CAR iNK cells (FIG.2G), suggesting that IL-15 expression could also benefit the viability and functionality of iNK cells after preservation. Example 4: Phenotypic and transcriptome profiling of MSLN.CAR-IL-15 engineered GR1.1- iNKs Flow cytometry was used to detect NK biomarkers on iNKs freshly harvested at 4 weeks post initiation of differentiation, as compared to PBNK control. High levels of NKG2D, DNAM-1, NKp46, NKp44, CD94, Fas-L, TRAIL, an intermediate level of NKG2A and low levels of CD16 and CD158 (KIR2DL1 / S1 / S3 / S5) were observed on mock iNK, MSLN.CAR iNKs and MSLN.CAR-IL- 15 iNKs (FIG.3A). To compare transcriptional profiling of freshly differentiated iNKs to mature PBNKs, RNAseq was performed on the RNA extracted from healthy donors PBNKs, mock iNKs, and MSLN.CAR-IL-15 modified iNKs. Principal-component analysis (PCA) (FIG.3B) and unsupervised hierarchical clustering of gene expression (FIG.11A) showed that three NK populations formed three distinct clusters. PBNKs were strongly separated from mock iNKs and MSLN.CAR-IL-15 iNK along the PC1 axis; mock iNKs and MSLN.CAR-IL-15 iNKs shared similar PC1 value but were clearly separated along the PC2 axis. The differentially expressed genes (DEGs) and the corresponding gene- enriched pathways were then analyzed. Overall, 4724 DEGs (2373 up-regulated and 2351 down- 4239-111805-02 regulated) were identified when comparing mock iNK versus PBNKs; 1371 DEGs (649 up-regulated and 722 down-regulated) were identified when comparing MSLN.CAR-IL-15 iNKs versus mock iNKs (FIG.11B). KEGG and hallmark pathway gene set enrichment analysis (GSEA) of the DEGs showed a significant increase of pathways including cell division, DNA replication, p53 signaling pathway, AGE-RAGE signaling pathway, and cholesterol homeostasis in iNKs as compared to PBNKs (FIG.3C). Compared to mock iNKs, MSLN.CAR-IL-15 iNKs showed increased expression of genes associated with cell division, DNA replication, NF-κB signaling, TRP channel calcium signaling, and Wnt signaling pathways. The expression of NK activity-associated genes was then profiled in different categories (FIG.3D). Although GR1.1-iNKs showed similar expression of many NK markers in common with PBNKs, they exhibited distinct features. The genes that were increased in GR1.1-iNKs included NCAM1 (CD56), NCR2 (NKp44), NCR3 (NKp30), KLRC1 (NKG2A), CD96, HAVCR2 (Tim3), CD276 (B7-H3), TNF, TNFSF14 (LIGHT), CCR1, CCR5, CCR6, CCR8, CXCR3 and TNFSF10 (TRAIL), while the genes that were decreased in GR1.1-iNKs included FCGR3A (CD16A), B3GAT1 (CD57) KLRF1 (NKp80), CD160, TIGIT, LAG3, CXCR1, CXCR2, CXCR4, and CX3CR1. MSLN.CAR-IL-15 iNKs expressed a similar level of most markers and cytokines except for higher IL15, and lower CD200R1 and CEACAM1, as compared to mock iNK. Additionally, high level expression of some “adaptive NK” feature genes IL32, S100A4, and ITGA1 (CD49A) were observed in GR1.1-iNKs (FIG.11C) (Woan et al., Cell Stem Cell 28:2062-2075, 2021). Example 5: Enhanced cytotoxicity of MSLN.CAR-IL-15 iNKs against human solid tumor cells To evaluate the tumor cell killing ability of the GR1.1-iNKs, cytotoxicity assays were performed by coculturing different modified GR1.1-iNKs with target cancer cell lines for 24 hours. MSLN.CAR-IL-15 iNK displayed the highest killing ability against all of the tumor cells tested (pancreatic cancer KLM-1, mesothelioma NCI-meso29, NCI-meso21, NCI-meso63, ovarian cancer OVCAR8, and gastric carcinoma cell line NCI-N87), as compared to mock iNK and MSLN.CAR iNK (FIGS.4A-4B). MSLN.CAR iNK and MSLN.CAR-IL-15 iNK killed more MSLN+KLM1-WT than MSLN- KLM1-KO target cells, suggesting MSLN recognition by CAR increased specific killing (FIG.4A). On the other hand, MSLN.CAR-IL-15 iNK showed higher killing ability than mock iNK and MSLN.CAR iNK when cocultured with MSLN- KLM1-KO target cells, indicating that chronic stimulation by IL-15 led to NK activation that promoted cytotoxicity independent of the CAR targeting specificity (FIG.4A). CD107a expression (granule release) of iNKs was then tested. Without target stimulation, slightly higher CD107a expression was observed in MSLN.CAR-IL-15 iNKs than in mock iNK and MSLN.CAR iNK when stimulated with KLM-1 tumor cells. MSLN.CAR iNK and MSLN.CAR-IL-15 iNK demonstrated higher CD107a expression, while mock iNK had limited CD107a expression (FIG.4C). These results demonstrate that MSLN.CAR-IL15 modification 4239-111805-02 of the LiPSC-GR1.1 cells not only significantly increased the iNK differentiation yield but also dramatically enhanced the killing function of the differentiated iNK against various tumor cells. Example 6: Tumor regression and improved survival in MSLN.CAR-IL15 iNK-treated MSLN+tumor-bearing mice To evaluate the anti-tumor efficacy of MSLN.CAR-IL15 iNKs in vivo, the patient mesothelioma-derived xenograft (PDX) NCI-meso63 mouse model established previously (Jiang et al., Sci Transl Med 12:eaaz7252, 2020) was used. The luciferase expressing NCI-meso63 tumor cells were injected i.p.7 days before iNK treatment. The mice were then administrated two doses of freshly differentiated iNKs on day 0 and day 7 and monitored weekly using bioluminescent imaging (BLI) (FIG.5A). Tumor growth was dramatically decreased in mice receiving MSLN.CAR-IL-15 iNKs as compared to mice receiving mock iNKs, although the mice receiving mock iNKs did show some anti- tumor effects when compared to the untreated animals due to CAR independent activity of NK cells (FIGS.5B-5C). The median overall survival was 48 days without treatment, 57 days with mock iNK treatment, and 79 days with MSLN.CAR-IL-15 iNK treatment (FIG.5D). In addition, the iNK were tested in a more aggressive pancreatic tumor KLM-1 mouse model (FIG.12A). Only MSLN.CAR-IL- 15 iNK treatment showed significant anti-tumor effects and prolonged survival, while mock iNK treatment had a minimal effect in this tumor model (FIGS.12B-12D). In both mouse models tested, no toxicity was observed in the mice treated with CAR-IL-15 GR1.1-iNKs. Considering the logistical advantages of cryopreservation for large-scale production and distribution of an off-the shelf iNK product, the efficacy of iNK expanded from cryopreserved differentiated iNK stocks was also evaluated using the NCI-meso63 tumor model (FIGS.13A-13C). These expanded MSLN.CAR-IL-15 iNKs also exhibited enhanced tumor killing and improved overall survival of these mice compared to the expanded mock iNK treatment. The anti-tumor activity was similar to the anti-tumor effects achieved with freshly differentiated MSLN.CAR-IL-15 iNKs. Given the advantages of scalability and logistical ease, the expanded iNKs from frozen stocks were used for the following mechanistic mouse model studies. Example 7: Increased infiltration of MSLN.CAR-IL-15 iNKs in NCI-meso63 tumors Treatment of solid tumors is partially constrained by the limited tumor homing and persistence of NK cells. To investigate the solid tumor infiltration ability of iNKs, the experimental design was adjusted by increasing NCI-meso63 tumor cell number, treating the tumor with a lower number of iNKs, and doing the iNK transfer on day 16 post tumor inoculation when the tumor was substantially larger than in the preceding experiment (FIG.6A). With only one low dose iNK treatment, no significant tumor reduction was observed on day 6 post iNK treatment (FIG.6B). Tumor tissue was harvested on day 7 after iNK treatment and a substantial increase of hCD45+hCD56+mCD45- cells was observed in the MSLN.CAR-IL-15 iNK-treated tumors compared 4239-111805-02 to tumors from mock iNK-treated mice and CAR iNK-treated mice (FIGS.6C-6D). Approximately 80% of these hCD45+hCD56+cells expressed the MSLN.CAR. There was a small number of hCD45+mCD45- cells in the spleens of mice given MSLN.CAR-IL-15 iNKs and this small number was higher than what was observed in spleens from mock iNK-treated mice (FIG.6D). Notably, the percentage of CAR+iNKs was much higher in tumor than in spleen. Next, iNK tumor infiltration in situ was visualized using multiplex immunofluorescent staining for MSLN (tumor marker), hCD45 / hCD56 (NK marker) and Ki67 (proliferation marker). hCD45+and hCD56+cells were significantly increased in the MSLN.CAR-IL-15 iNK-treated MSLN+tumor as compared to tumors from mice receiving mock iNKs (FIGS.6E-6F). hCD45+cells colocalized with MSLN+tumor cells suggesting possible direct interaction (FIG.6G). Some of these hCD45+cells co-expressed Ki67 (FIGS.6G-6H), indicating recent or ongoing proliferation of MSLN.CAR-IL-15 iNKs within the tumors. These findings suggest that the MSLN.CAR-IL-15 modification significantly improved iNKs tumor infiltration and persistence. Example 8: Single-cell transcriptional profiling of MSLN.CAR-IL-15 iNK treated NCI-meso63 tumors NCI-Meso63 is a highly metastatic PDX-derived mesothelioma cell line established without single-cell cloning which retained its heterogeneity (Jiang et al., Sci Transl Med 12:eaaz7252, 2020). To characterize the transcriptional state of the NCI-Meso63 tumors during therapy with MSLN.CAR- IL-15 iNKs, as well as that of tumor infiltrating MSLN.CAR-IL-15 iNKs, scRNAseq was performed on total cells isolated from untreated tumors (3 samples), iNK-treated tumors (3 samples) (same harvest on day 7 post MSLN.CAR-IL-15 GR1.1-iNK treatment as shown in FIG.6A), as well as MSLN.CAR-IL-15 GR1.1-iNK pre-infusion product (2 samples) (FIG.7A). To achieve unbiased comparison among groups, scRNAseq data from the three groups were pooled for downstream analyses. After quality control filtering, a total of 23287 human cells across 8 samples were collected. Through unsupervised clustering, 7 tumor clusters (11501 cells) and 7 NK clusters (11786 cells) were classified (FIG.7A) based on the respective canonical marker genes of mesothelioma (MSLN, KRT8 and KRT18) and NK cells (PTPRC, NCAM1, NKG7 and hYP218 (CAR)) (FIG.7B), as well as top marker genes (up to 100) expressed in each cluster. Example 9: Upregulation of MHC molecules and reduction of target MSLN in TGF-β-rich tumors post MSLN.CAR-IL-15 iNKs treatment The features of tumor subclusters was next investigated. Consistent with the flow cytometry and immunofluorescence findings, both tumor clusters and NK clusters were observed as the major populations (57% NKs vs.43% tumor cells) in the MSLN.CAR-IL-15 GR1.1-iNK-treated tumors as compared to the untreated tumors (FIG.7C). Using copyKAT copy number variant analysis (Gao et al., Nat Biotechnol 39:599-608, 2021), T2, T3, T4, and T6 were identified as the malignant tumor 4239-111805-02 clusters (FIG.7D) with high expression of MSLN (FIG.7B). Further studies focused on these clusters for the downstream analysis. The proportion of T3, which expressed the highest level of MSLN among all of the tumor subclusters, was significantly reduced after MSLN.CAR-IL-15 GR1.1-iNK treatment (FIG.7E). Instead, clusters T2 and T6 emerged as the dominant populations after treatment. The four malignant tumor clusters highly expressed mesenchymal genes and TGFB1, along with various genes linked to cell growth, metabolism, inflammation, and stress responses, highlighting tumor heterogeneity and the critical role of TGF-β in creating an immunosuppressive microenvironment. In addition, significant reduction of MSLN expression was observed in all four clusters in the treated group (FIG.7G), indicating effective targeted specific killing mediated by MSLN.CAR-IL-15 iNK. Further analysis of Hallmark pathways enrichment revealed that allograft rejection, interferon gamma response, interferon alpha response pathways were enriched in all the tumor clusters, while TNFA signaling via NFκB and epithelial mesenchymal transition pathways were distinctly enriched in different subclusters (FIG.7H). The expression of both HLA class I and HLA class II related genes was significantly upregulated in tumor clusters post iNK treatment (FIG.7I), which contributed to the allograft rejection pathway enrichment score, suggesting that tumor cells may regulate iNKs activity through the MHC molecules (He and Tian, Cell Mol Immunol 14:321-330, 2017; Niehrs and Altfeld, Front Cell Infect Microbiol 10:55, 2020). Example 10: Robust activation, increased cytotoxicity and cycling ability of tumor-infiltrating MSLN.CAR-IL-15 iNK cells Lastly, an additional study sought to elucidate the alterations in the transcriptional profile of the iNKs after they encounter the tumor micro-environment. The pre-infusion iNKs were mainly composed of 32.1% NK2, 32.7% NK3, 21.9% NK4, and 12.6% NK6, as compared to the tumor- infiltrating (post-infusion) iNKs primarily composed of 75.4%, NK1, 15% NK5 and 8.5% NK7 (FIG. 8A). The hYP218-scFv sequence (CAR) expression was observed higher in tumor-infiltrating NK1, NK5, NK7 compared to pre-infusion NK clusters, suggesting that the high expression of CAR may be essential for directing iNKs into the tumor (FIG.7B). Distinct characteristics of pre-infusion and post- infusion iNKs based on the expression of cluster-defining marker genes was observed (FIG.8B). In the pre-infusion NK subclusters, NK2 consisted of highly proliferative iNKs in culture, marked by high expression of cell division genes like MKI67 and AURKB; NK3 represented activated NK cells, with high expression of IFNG and NK signaling genes such as CLNK and FYB1; The NK4 cluster showed elevated expression of inhibitory receptors KLRC1 / KLRD1 (NKG2A / CD94) and TNFRSF4 (OX40); NK6 displayed high levels of mitochondrial genes, indicating cell stress and cellular damage. In the post-infusion NK cells, NK1 was the dominant subcluster, expressing high levels of cytotoxic effector genes (CST7, SRGN, GZMA), indicating its key role in tumor cell killing. The NK5 cluster was characterized by high expression of cell division genes, including STMN1, RRM2, and ZWINT, along with the tumor-infiltrating NK marker RGS1 (Tang et al., Cell 186:4235-4251, 2023), 4239-111805-02 suggesting they may correspond to the Ki67+NK population observed in immunofluorescence imaging. Additionally, NK5 co-expressed marker genes of both the NK1 and NK2 clusters, which are associated with proliferation and cytotoxicity, highlighting its strong cytolytic activity and ability to persist in the tumor environment. NK7, marked by high expression of SEMA4D (CD100), PRKCH (PKCθ) and CBLB, exhibited the lowest expression of NK function-related genes among the post- infusion clusters. Notably, RGS1, CBLB, and FYN were expressed at higher levels, while IFNG was expressed at lower levels across all tumor-infiltrating NK clusters compared to the pre-infusion NK clusters. Next, the expression of NK-associated biomarkers in the NK subclusters was evaluated (FIG. 8C). Tumor-infiltrating clusters NK1 and NK5 showed high expression of NK activating receptors, including NKG2D-DAP10 (KLRK1 / HCST), NKG2C (KLRC2), CD94 (KLRD1), and NKp30 (NCR3), suggesting they were robustly activated. Among the inhibitory KIR and immunosuppressive checkpoint genes, high expression of KIR2DL4, intermediate levels of NKG2A (KLRC1), and notable reduction of CD96 in the tumor-infiltrating iNKs were observed, along with negligible expression of other checkpoint markers. The protein expression of cell surface biomarkers of NKG2D, NKG2C, CD94, NKp30, NKG2A, and KIR2DL4 in tumor infiltrating CAR-IL-15 iNKs were validated through flow cytometry (FIG.14). The chemokine and receptor profiles of tumor-infiltrating NK1 and NK5 showed increased expression of CCL5, CCL4, XCL1, XCL2, and CXCR3, suggesting they may be involved in NK tumor homing and activation (Kohli et al., Cancer Gene Ther 29:10-21, 2022; Wendel et al., Cancer Res 68:8437-8445, 2008). In contrast, CCR1and CCR5 were decreased in tumor-infiltrating NKs, which promote NK liver homing (Wald et al., J Immunol 176:4716-4729, 2006; Levy et al., Cancers (Basel) 13:872, 2021). Regarding cytotoxicity-related genes, GZMB and SYTL3 were upregulated in the NK1 cluster, while PFR1 (perforin) and TNFSF10 (TRAIL) were downregulated in tumor-infiltrating iNKs as compared to pre-infusion iNKs. Example 11: Reduction of CISH, TGFBR2, and BATF in IL-15 producing tumor-infiltrating iNK cells to adapt to the hypoxic, TGF-β-rich tumor microenvironment The pathway enrichment analysis revealed the pathways significantly enriched in tumor- infiltrating NKs versus pre-infusion NKs, including TNFα signaling via NFκB, hypoxia, interferon gamma response, interferon alpha response, apoptosis, heme metabolism, IL2-STAT5 signaling, estrogen response and p53 pathway (FIG.8D). In contrast, the cell division-related pathway E2F target and G2M checkpoint were downregulated in the tumor-infiltrating NK cells compared to the highly proliferative pre-infusion NK product. These findings indicate that tumor-infiltrating NK cells were actively engaged in complex biological processes to mount an effective immune response against the tumor and tuning their own function and metabolism to adapt to the tumor microenvironment. The expression of different gene sets was then analyzed to understand the biological processes across different NK subclusters. 4239-111805-02 Significant upregulation of IL-15 signaling genes was observed in tumor-infiltrating NK clusters, including IL15, IL2RB, IL2RG, STAT5B, and JAK1 (FIG.8E). Notably, these genes exhibited higher expression levels in the NK1 subcluster compared to NK5. Unexpectedly, JAK3 was downregulated in post-infusion NK cells compared to pre-infusion iNK cells. In addition, the negative regulators of IL-15 signaling CISH was dramatically reduced while SOCS1 was markedly elevated in post-infusion iNKs as compared to pre-infusion iNKs. In the tumor microenvironment characterized by high levels of TGF-β, NK1 and NK5 clusters exhibited low expression of TGFBR2 (with particularly low levels in the NK5 cluster), and simultaneously upregulated negative regulators of TGF-β signaling SKI, SKIL, and TGIF1, indicating that these NK clusters may be adapting to mitigate the suppressive effects of TGF-β. In contrast, the cluster NK7 with impaired NK function showed high expression of genes involved in TGF-β signaling. It was also noted that AP-1 family transcription factors were enriched in post-infusion NK clusters except for gene BATF, which exhibited lower expression in post-infusion NKs compared to pre-infusion NKs. A series of genes related to cell cycle arrest, DNA repair, cell survival processes were enriched in post-infusion NK1 and NK5 clusters at similar levels (FIG.8F), contributing to NK response to hypoxia and collectively enabling NK cells to thrive in challenging tumor microenvironments. Interestingly, the pro-apoptotic genes BCL2L11, BTG1, and DUSP22 were expressed at lower levels in NK5 compared to NK1. As compared to pre-infusion NK cells, post- infusion NK cells showed upregulation of genes involved in nutrient uptake and mitophagy, lower levels of oxidative phosphorylation (OXPHOS), and similar levels of glycolysis (FIG.8G). Enhanced nutrient uptake in tumor infiltrating NK suggests that NK cells are adapting to their environment by increasing the availability of amino acids and other substrates, which can support cell survival and function despite other metabolic impairments (Santosa et al., Nat Immunol 24:1685-1697, 2023). Upregulation of mitophagy may help remove dysfunctional mitochondria, thereby maintaining mitochondrial health and function, even when overall oxidative metabolism is compromised (O'Sullivan et al., Immunity 43:331-342, 2015). This may help NK cells maintain their effector functions. High expression of METRNL was also observed in post-infusion NK1 and NK5, which may be involved in disrupting mitochondrial function in NK cells (Jackson et al., Immunity 57:1864-1877, 2024). Additionally, NK5 displayed higher levels of OXPHOS and glycolysis compared to NK1, aligning with the increased proliferation observed in NK5. This metabolic fitness advantage in NK5 may enable these NK cells to meet the metabolic demands necessary for both cytotoxicity and proliferation, thereby enhancing their sustained functionality in the hypoxia-stressed tumor microenvironment. Discussion In this study, LiPSC-GR1.1 was identified as a superior iPSC cell line for producing iNK product using a standardized simple differentiation method. Through genetic modification of LiPSC- 4239-111805-02 GR1.1 with an MSLN targeting CAR-IL-15 construct, robust iNK differentiation yield was achieved. The characteristics and functionality of these MSLN. CAR-IL-15 iNKs were also demonstrated through comprehensive in vitro and in vivo experiments, including validating CAR expression, profiling iNK cell biomarkers, and demonstrated the tumor-killing efficacy of these engineered iNKs against MSLN positive solid tumors. In addition, a significant infiltration of MSLN.CAR-IL-15 engineered NK cells into MSLN positive tumor was observed, demonstrating their tumor-homing ability and proliferation within the tumor microenvironment. Finally, studies detailed the single cell transcriptional profiles of iNK-treated and untreated tumors, as well as of tumor-infiltrating MSLN.CAR-IL-15 iNKs, which uncovered the early responses of MSLN positive tumor cells to iNKs mediated killing and provided a comprehensive analysis of characteristics of MSLN.CAR-IL-15 iNKs adapting to solid tumors context. iPSC-NK differentiation is a prolonged and intricate process (Saetersmoen et al., Semin Immunopathol 41:59-68, 2019). To address the challenges of large-scale production, enrichment and expansion of CD34+ hematopoietic progenitor cells before iNK differentiation is frequently employed, with further post-differentiation NK cell expansion using feeder cells being a necessary step (Cichocki et al., Sci Transl Med 12:872, 2020). The spin-EBs method to generate iPSC-NK in feeder-free conditions without CD34+enrichment significantly simplifies the operational workflow (Zhu and Kaufman, Methods Mol Biol 2048:107-119, 2019). The drawback is that only certain iPSC lines can be efficiently differentiated into NK cells using this method and the iNK yield is relatively low. The present disclosure identified LiPSC-GR1.1 as a superior source for producing iNK using this simple method. By introducing IL-15 in LiPSC-GR1.1, the iPSC-NK differentiation yield was increased by nearly tenfold, which could significantly shorten the expansion and simplify manufacturing process. IL-15 plays a multifaceted role in the regulation of NK cells. While it is essential for NK cell development and homeostasis, prolonged exposure to IL-15 can result in NK cell exhaustion (Felices et al., JCI Insight 3:e96219, 2018). Of note, the dosing experiment indicated that higher IL-15 concentrations did not necessarily result in better differentiation outcomes. In fact, it was observed that increasing IL-15 to a high level (e.g., 80 ng / ml) had a detrimental impact on NK cell differentiation. Therefore, picking iPSC colonies highly expressing IL-15 for NK production may negatively affect subsequent NK cell differentiation. It was also demonstrated that freshly differentiated MSLN.CAR-IL-15 GR1.1-iNK cells can be cryopreserved as a stock and subsequently expanded after thawing to an even larger scale without loss of functionality. This approach offers a standardized method for streamlined iNK production using the LiPSC-GR1.1 cell line. Additionally, this protocol can be adapted for various CAR genetic modifications or strategies to enhance CAR-NK therapy. In summary, the examples herein describe an approach for robustly differentiating iNK from the LiPSC-GR1.1 iPSC line genetically modified to stably express MSLN.CAR-IL-15. The present disclosure demonstrates the substantial anti-tumor effects of these iNK against solid tumors, conducts 4239-111805-02 a deep characterization of their phenotypic and transcriptional state, and reports how the interplay between tumor cells and MSLN.CAR-IL-15 iNK affects both populations during therapy. More importantly, this work supports the clinical translation of MSLN.CAR-IL-15-engineered GR1.1-iNK for the treatment of patients with advanced treatment refractory solid tumors that highly express mesothelin. Example 12: Alternative hYP218 CAR construct A second hYP218-based CAR / IL-15 construct was generated, which included the hYP218 scFv, a CD8α hinge region, a CD8α transmembrane domain, a 4-1BB co-stimulatory domain and a CD3ζ signaling domain, along with coding sequences for EGFRt and IL-15 (hYP218.CD8a.BB.Z.CAR / IL-15), as shown in FIG.16A. The hYP218.CD8a.BB.Z.CAR / IL-15 construct was tested in LiPSC-GR1.1 cells to evaluate its effect on iNK cell differentiation. The results showed a similarly increased yield of CD56+iNK cell differentiation than the iNK cell yield from mock GR1.1 iPSCs (FIGS.16B-16C), with high EGFRt (CAR) expression (FIG.16C) and enhanced tumor killing ability (FIG.16D). It will be apparent that the precise details of the methods or compositions described may be varied or modified without departing from the spirit of the described aspects of the disclosure. We claim all such modifications and variations that fall within the scope and spirit of the claims below.

Claims

4239-111805-02 CLAIMS 1. A nucleic acid molecule, comprising: (i) a nucleic acid sequence encoding a chimeric antigen receptor (CAR) that specifically binds mesothelin, wherein the CAR comprises: an antigen-binding domain comprising a variable heavy (VH) domain and a variable light (VL) domain, wherein the VH domain comprises the complementarity determining region 1 (CDR1), CDR2 and CDR3 sequences of SEQ ID NO: 1 and the VL domain comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 2; a hinge region; a transmembrane domain; an intracellular co-stimulatory domain; and an intracellular signaling domain; and (ii) a nucleic acid sequence encoding interleukin (IL)-15.

2. The nucleic acid molecule of claim 1, wherein: the amino acid sequences of the VH domain CDR1, CDR2 and CDR3 respectively comprise SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 and the amino acid sequences of the VL domain CDR1, CDR2 and CDR3 respectively comprise SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8; or the amino acid sequences of the VH domain CDR1, CDR2 and CDR3 respectively comprise SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11 and the amino acid sequences of the VL domain CDR1, CDR2 and CDR3 respectively comprise SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO:

14.

3. The nucleic acid molecule of claim 1 or claim 2, wherein: the amino acid sequence of the VH domain is at least 95% identical to SEQ ID NO: 1 and comprises the CDR1, CDR2 and CDR3 sequence of SEQ ID NO: 1; and / or the amino acid sequence of the VL domain is at least 95% identical to SEQ ID NO: 2 and comprises the CDR1, CDR2 and CDR3 sequence of SEQ ID NO:

2.

4. The nucleic acid molecule of any one of claims 1-3, wherein: the amino acid sequence of the VH domain comprises or consists of SEQ ID NO: 1; and / or the amino acid sequence of the VL domain comprises or consists of SEQ ID NO:

2.

5. The nucleic acid molecule of any one of claims 1-4, wherein: the hinge region is a CD8α hinge region; the transmembrane domain is a NKG2D or CD8α transmembrane domain;4239-111805-02 the intracellular co-stimulatory domain is a 2B4 or 4-1BB intracellular co-stimulatory domain; and / or the intracellular signaling domain is a CD3ζ intracellular signaling domain.

6. The nucleic acid molecule of claim 5, wherein: the amino acid sequence of the CD8α hinge region comprises SEQ ID NO: 15; the amino acid sequence of the NKG2D transmembrane domain comprises SEQ ID NO: 16 or the amino acid sequence of the CD8α transmembrane domain comprises SEQ ID NO: 17; the amino acid sequence of the 2B4 intracellular co-stimulatory domain comprises SEQ ID NO: 18, or the amino acid sequence of the 4-1BB intracellular co-stimulatory domain comprises SEQ ID NO: 19; and / or the amino acid sequence of the CD3ζ intracellular signaling domain comprises SEQ ID NO:

20.

7. The nucleic acid molecule of any one of claims 1-6, wherein the amino acid sequence of IL-15 comprises or consists of SEQ ID NO:

21.

8. The nucleic acid molecule of any one of claims 1-7, comprising a nucleic acid sequence at least 95% identical to SEQ ID NO: 22 or SEQ ID NO: 23, or a degenerate variant thereof.

9. The nucleic acid molecule of any one of claims 1-8, comprising or consisting of SEQ ID NO: 22 or SEQ ID NO: 23, or a degenerate variant thereof.

10. The nucleic acid molecule of any one of claims 1-9, operably linked to a promoter.

11. A vector comprising the nucleic acid molecule of any one of claims 1-10.

12. The vector of claim 11, wherein the vector is a piggyBac transposon vector.

13. A natural killer (NK) cell comprising the nucleic acid molecule or vector of any one of claims 1-12.

14. A natural killer (NK) cell expressing: (i) a chimeric antigen receptor (CAR) that specifically binds mesothelin, wherein the CAR comprises:4239-111805-02 an antigen-binding domain comprising a variable heavy (VH) domain and a variable light (VL) domain, wherein the VH domain comprises the complementarity determining region 1 (CDR1), CDR2 and CDR3 sequences of SEQ ID NO: 1 and the VL domain comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 2; a hinge region; a transmembrane domain; an intracellular co-stimulatory domain; and an intracellular signaling domain; and (ii) interleukin (IL)-15, wherein the IL-15 is expressed from an exogenous nucleic acid molecule encoding IL-15.

15. The NK cell of claim 14, wherein: the amino acid sequences of the VH domain CDR1, CDR2 and CDR3 respectively comprise SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 and the amino acid sequences of the VL domain CDR1, CDR2 and CDR3 respectively comprise SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8; or the amino acid sequences of the VH domain CDR1, CDR2 and CDR3 respectively comprise SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11 and the amino acid sequences of the VL domain CDR1, CDR2 and CDR3 respectively comprise SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14 16. The NK cell of claim 14 or claim 15, wherein: the amino acid sequence of the VH domain is at least 95% identical to SEQ ID NO: 1 and comprises the CDR1, CDR2 and CDR3 sequence of SEQ ID NO: 1; and / or the amino acid sequence of the VL domain is at least 95% identical to SEQ ID NO: 2 and comprises the CDR1, CDR2 and CDR3 sequence of SEQ ID NO:

2.

17. The NK cell of any one of claims 14-16, wherein: the amino acid sequence of the VH domain comprises or consists of SEQ ID NO: 1; and / or the amino acid sequence of the VL domain comprises or consists of SEQ ID NO:

2.

18. The NK cell of any one of claims 14-17, wherein: the hinge region is a CD8α hinge region; the transmembrane domain is a NKG2D or CD8α transmembrane domain; the intracellular co-stimulatory domain is a 2B4 or 4-1BB intracellular co-stimulatory domain; and / or the intracellular signaling domain is a CD3ζ intracellular signaling domain.4239-111805-02 19. The NK cell of claim 18, wherein: the amino acid sequence of the CD8α hinge region comprises SEQ ID NO: 15; the amino acid sequence of the NKG2D transmembrane domain comprises SEQ ID NO: 16 or the amino acid sequence of the CD8α transmembrane domain comprises SEQ ID NO: 17; the amino acid sequence of the 2B4 intracellular co-stimulatory domain comprises SEQ ID NO: 18, or the amino acid sequence of the 4-1BB intracellular co-stimulatory domain comprises SEQ ID NO: 19; and / or the amino acid sequence of the CD3ζ intracellular signaling domain comprises SEQ ID NO:

20.

20. The NK cell of any one of claims 14-19, wherein the amino acid sequence of IL-15 comprises or consists of SEQ ID NO:

21.

21. The NK cell of any one of claims 14-20, wherein the amino acid sequence of the CAR comprises or consists of residues 23-575 of SEQ ID NO: 24 or residues 23-497 of SEQ ID NO:

25.

22. The NK cell of any one of claims 13-21, wherein the NK cell is derived from an induced pluripotent stem cell (iPSC) line.

23. The NK cell of claim 22, wherein the iPSC line is LiPSC-GR1.

1.

24. A composition, comprising the NK cell of any one of claims 13-23 and a pharmaceutically acceptable carrier.

25. A method of treating a mesothelin-positive cancer in a subject, comprising administering to the subject a therapeutically effective amount of the NK cell of any one of claims 13- 23 or the composition of claim 24.

26. The method of claim 25, wherein the mesothelin-positive cancer is mesothelioma, lung cancer, stomach cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, thymic carcinoma, or colorectal cancer.

27. A method of differentiating induced pluripotent stem cells (iPSCs) to hematopoietic progenitor cells, comprising:4239-111805-02 (i) seeding iPSCs in a tissue culture vessel containing albumin polyvinylalcohol essential lipids (APEL) culture medium comprising human stem cell factor (SCF), human vascular endothelial growth factor (VEGF), human bone morphogenetic protein 4 (BMP-4) and Y-27632; and (ii) subjecting the seeded iPSCs to centrifugation and continuing to culture the iPSCs in the APEL culture medium of step (i) to produce embryoid bodies (EBs) containing hematopoietic progenitor cells.

28. The method of claim 27, further comprising producing iPSC-derived natural killer (iNK) cells from the EBs, comprising: (iii) culturing the EBs in NK cell differentiation medium comprising interleukin-3 (IL-3), IL-15, IL-7, SCF and fms-like tyrosine kinase 3 (FLT3) ligand, thereby producing iNK cells.

29. The method of claim 27 or claim 28, wherein: the iPSCs of step (i) are trypsin-adapted iPSCs; the tissue culture vessel is a 96-well round-bottom plate; the EBs are cultured in the APEL culture medium for about four, about five, about six, about seven or about eight days; the EBs are cultured in the NK cell differentiation medium for at least two weeks, at least three weeks, or at least four weeks; the APEL culture medium comprises about 1-100 ng / ml SCF, about 1-100 ng / ml VEGF, about 1-100 ng / ml BMP-4, and about 0.1-100 μM Y-27632; the NK cell differentiation medium comprises about 1-100 ng / ml IL-3, about 1-100 ng / ml IL- 15, about 1-100 ng / ml IL-7, about 1-100 ng / ml SCF, and / or about 1-100 ng / ml FLT3 ligand; IL-3 is present in the NK differentiation medium for only the first week of culture; and / or the iNK cells are CD45+CD56+.

30. The method of any one of claims 27-29, wherein prior to step (i), the iPSCs are transduced with a nucleic acid molecule or vector comprising: (1) a nucleic acid sequence encoding a chimeric antigen receptor (CAR) that specifically binds mesothelin, wherein the CAR comprises: an antigen-binding domain comprising a variable heavy (VH) domain and a variable light (VL) domain, wherein the VH domain comprises the complementarity determining region 1 (CDR1), CDR2 and CDR3 sequences of SEQ ID NO: 1 and the VL domain comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 2; a hinge region; a transmembrane domain; an intracellular co-stimulatory domain; and4239-111805-02 an intracellular signaling domain; and (2) a nucleic acid sequence encoding interleukin (IL)-15.

31. The method of claim 30, wherein prior to step (i), the method further comprises enriching iPSCs expressing the chimeric antigen receptor (CAR) encoded by the transduced nucleic acid molecule or vector.

32. The method of claim 30 or claim 31, wherein the iPSCs express IL-15 encoded by the transduced nucleic acid molecule or vector.

33. The method of any one of claims 27-32, wherein the iPSCs are LiPSC-GR1.1 cells.

34. A method of generating hematopoietic progenitor cells, comprising differentiating LiPSC-GR1.1 cells to hematopoietic progenitor cells.

35. Use of the LiPSC-GR1.1 cell line to produce hematopoietic progenitor cells.

36. Use of the NK cell of any one of claims 13-23 or the composition of claim 24 for treating a mesothelin-positive cancer in a subject.

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