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WO2025240496A1 - Cellules tueuses naturelles exprimant un récepteur antigénique chimérique ciblant la mésothéline et l'interleukine-15 pour le traitement de tumeurs solides - Google Patents

Cellules tueuses naturelles exprimant un récepteur antigénique chimérique ciblant la mésothéline et l'interleukine-15 pour le traitement de tumeurs solides

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Publication number
WO2025240496A1
WO2025240496A1 PCT/US2025/029169 US2025029169W WO2025240496A1 WO 2025240496 A1 WO2025240496 A1 WO 2025240496A1 US 2025029169 W US2025029169 W US 2025029169W WO 2025240496 A1 WO2025240496 A1 WO 2025240496A1
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seq
domain
car
cells
amino acid
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Inventor
Qun JIANG
Raffit HASSAN
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US Department of Health and Human Services
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US Department of Health and Human Services
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/35—Cytokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/15—Natural-killer [NK] cells; Natural-killer T [NKT] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4254—Adhesion molecules, e.g. NRCAM, EpCAM or cadherins
    • A61K40/4255—Mesothelin [MSLN]

Definitions

  • 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.
  • mesothelin 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.
  • EBs embryoid bodies
  • nucleic acid molecules and vectors that encode both a mesothelin-targeted chimeric antigen receptor (CAR) and human interleukin-15 (IL-15).
  • CAR mesothelin-targeted chimeric antigen receptor
  • IL-15 human interleukin-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.
  • nucleic acid molecules that include a nucleic acid sequence encoding a CAR that specifically binds mesothelin and a nucleic acid sequence encoding IL-15.
  • 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.
  • the CAR includes a CD8 ⁇ hinge region, a NKG2D transmembrane domain, a 2B4 intracellular co- stimulatory domain and a CD3 ⁇ intracellular signaling domain.
  • the CAR includes a CD8 ⁇ hinge region, a CD8 ⁇ transmembrane domain, a 4-1BB intracellular co-stimulatory domain and a CD3 ⁇ intracellular signaling domain.
  • vectors that include a disclosed nucleic acid molecule.
  • 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.
  • 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.
  • 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.
  • the CAR includes a CD8 ⁇ hinge region, a NKG2D transmembrane domain, a 2B4 intracellular co-stimulatory domain and a CD3 ⁇ intracellular signaling domain.
  • the CAR includes a CD8 ⁇ hinge region, a CD8 ⁇ transmembrane domain, a 4-1BB intracellular co-stimulatory domain and a CD3 ⁇ intracellular signaling domain.
  • the NK cell is derived from an iPSC line (such NK cells are referred to herein as “iNK” cells).
  • 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.
  • 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.
  • 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.
  • APEL albumin polyvinylalcohol essential lipids
  • SCF human stem cell factor
  • VEGF human vascular endothelial growth factor
  • BMP-4 human bone morphogenetic protein 4
  • 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.
  • IL-3 interleukin-3
  • IL-7 interleukin-7
  • SCF fms-like tyrosine kinase 3
  • FLT3 fms-like tyrosine kinase 3
  • FIGS.1A-1F Differentiation of iNKs from 6 human iPSC lines.
  • FIGS.1A-1F 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.
  • 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 ⁇ m.
  • Right panel representative magnified pictures of differentiated iNKs 4 weeks post NK differentiation. The bars represent 200 ⁇ m.
  • 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 CD3 ⁇ chain stimulatory domain), EGFRt, and human IL-15.
  • GMCSFRss GM-CSF receptor- ⁇ 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.
  • 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.
  • 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.
  • 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 ⁇ m.
  • FIG.6F hCD45 + cell density in iNK-treated tumor.
  • 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.
  • 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).
  • FIG.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 ⁇ m.
  • 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.
  • FIGS.13A-13C 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.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.
  • EBs spin embryoid bodies
  • 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 EF1 ⁇ promotor, the MSLN.CAR construct (which includes MSLN-targeted hYP218 scFv, CD8 ⁇ hinge/spacer, CD8 ⁇ transmembrane domain, 4-1BB intracellular co-stimulatory domain, and CD3 ⁇ 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.
  • 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 CD8 ⁇ 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 CD8 ⁇ 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 CD3 ⁇ 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).
  • 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).
  • 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).
  • 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).
  • MSLN Mesothelin
  • 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).
  • 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.
  • 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.
  • 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.
  • an antigen includes singular or plural antigens and can be considered equivalent to the phrase “at least one antigen.”
  • 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.
  • 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.
  • TCR T cell receptor
  • 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.
  • VH domain and the VL domain are responsible for binding the antigen recognized by the antibody.
  • 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.
  • 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 V H H 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.”
  • 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.
  • 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.
  • 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 is an antibody that binds the antigen with high affinity and does not significantly bind other unrelated antigens.
  • 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 10 3 M -1 greater, 10 4 M -1 greater or 10 5 M -1 greater than a binding constant for other molecules in a sample or subject.
  • a CAR has an equilibrium constant (KD) of 5 ⁇ M 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.
  • a CAR binds to a target, such as mesothelin, with a binding affinity of at least about 1 x 10 -6 M, at least about 0.5 x 10 -6 M, at least about 1 x 10 -7 M, at least about 0.5 x 10 -7 M, at least about 1 x 10 -8 M, at least about 0.5 x 10 -8 M, at least about 1 x 10 -9 M, at least about 0.5 x 10 -9 M, or at least about 0.1 x 10 -9 .
  • 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 -6 M or less, e.g., from 10 -6 M to 10 -10 M, e.g., from 10 -10 M to 10 -12 M).
  • Kd dissociation constant
  • binding affinity is measured using the Octet system (Creative Biolabs), which is based on BLI technology.
  • 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.
  • CD3 ⁇ A component of the T cell receptor (TCR) complex.
  • TCR T cell receptor
  • 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.
  • CD3 ⁇ is also known as CD247.
  • Sequences for CD3 ⁇ are publicly available, such as under NCBI Gene ID 919.
  • An exemplary amino acid sequence of the CD3 ⁇ intracellular signaling domain is set forth herein as SEQ ID NO: 20.
  • CD8 subunit alpha A transmembrane glycoprotein predominantly expressed by cytotoxic T lymphocytes, but can also be expressed by NK cells, cortical thymocytes and dendritic cells. CD8 ⁇ 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.
  • CD8 ⁇ sequences are publicly available, such as under NCBI Gene ID 925.
  • 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.
  • a chemotherapeutic agent is an agent of use in treating a mesothelin-expressing tumor.
  • a chemotherapeutic agent is a radioactive compound.
  • 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 2 nd ed., ⁇ 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.
  • 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.
  • 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).
  • a therapeutic antibody e.g., therapeutic monoclonal antibody
  • 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
  • combinations thereof e.g
  • Chimeric antigen receptor 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, CD3 ⁇ ).
  • 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 CD3 ⁇ or Fc ⁇ RI ⁇ .
  • ITAM immunoreceptor tyrosine-based activation motif
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • Embryoid body 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.
  • the response is specific for a particular antigen (an “antigen- specific response”).
  • an immune response is a T cell response, such as a CD4 + response or a CD8 + response.
  • 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.
  • 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).
  • RMP NIH Regenerative Medicine Program
  • IL-15 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • 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).
  • 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, 21 st Edition (2005), describes compositions and formulations suitable for pharmaceutical delivery of the CAR-expressing cells and other compositions disclosed herein.
  • 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.
  • pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like
  • 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.
  • 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 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.
  • 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.
  • 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).
  • 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.
  • Sequence identity is 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.
  • 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.
  • a tumor such as a mesothelin-positive cancer
  • 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.
  • a tumor such as a mesothelin-positive cancer
  • 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.
  • a dosage 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.
  • the vector is a virus vector, such as a lentivirus vector, an adeno-associated virus (AAV) vector, or an adenovirus vector.
  • 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.
  • 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.
  • VH variable heavy
  • VL variable light domain of humanized antibody YP218
  • hYP218 VH domain (SEQ ID NO: 1) HMEVQLVESGGGLVQPGGSLRLSCAASGFDLGFYFYACWVRQAPGKGLEWVSCIYTAGSG STYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSTANTRSTYYLNLWGQGT LVTVSS hYP218 VL domain (SEQ ID NO: 2) DIQMTQSPSSLSASVGDRVTITCQASQRISSYLSWYQQKPGKVPKLLIYGASTLASGVPSRFS GSGSGTDFTLTISSLQPEDVATYYCQSYAYFDSNNWHAFGGGTKVEIKATS Table 1.
  • the CAR includes a CD8 ⁇ hinge region, a NKG2D or a CD8 ⁇ transmembrane domain, a 2B4 or a 4-1BB intracellular co-stimulatory domain, and a CD3 ⁇ intracellular signaling domain.
  • a CD8 ⁇ hinge region a CD8 ⁇ transmembrane domain
  • a 2B4 or a 4-1BB intracellular co-stimulatory domain a CD3 ⁇ intracellular signaling domain.
  • CD3 ⁇ intracellular signaling domain Provided below are exemplary amino acid sequences of each of these CAR components.
  • CD8 ⁇ hinge region (SEQ ID NO: 15) TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD NKG2D transmembrane domain (SEQ ID NO: 16) PFFFCCFIAVAMGIRFIIMVT 4239-111805-02 CD8 ⁇ transmembrane domain (SEQ ID NO: 17) IYIWAPLAGTCGVLLLSLVIT 2B4 intracellular co-stimulatory domain (SEQ ID NO: 18) WRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQEQTFPGGGSTIYSMIQSQSSAPTSQEPA YTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFDVYS 4-1BB intracellular co-stimulatory domain (SEQ ID NO: 19) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL CD3 ⁇ intracellular signaling domain (SEQ ID NO: 20) RV
  • the construct hYP218.NKG2D.2B4.Z.CAR/IL-15 includes the coding sequences for the hYP218 scFv, a CD8 ⁇ hinge region, an NKG2D transmembrane domain, a 2B4 intracellular co- stimulatory domain, and a CD3 ⁇ intracellular signaling domain as the CAR component.
  • Construct hYP218.CD8a.BB.Z.CAR/IL-15 includes the coding sequences for the hYP218 scFv, a CD8 ⁇ hinge region, a CD8 ⁇ transmembrane domain, a 4-1BB intracellular co-stimulatory domain, and a CD3 ⁇ 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 (SEQ ID NO: 24) MLLLVTSLLLCELPHPAFLLIPHMEVQLVESGGGLVQPGGSLRLSCAASGFDLGFYFYACWV RQAPGKGLEWVSCIYTAGSGSTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA RSTANTRSTYYLNLWGQGTLVTVSSGGGGSGGGGSGGSDIQMTQSPSSLSASVGDRVTIT CQASQRISSYLSWYQQKPGKVPKLLIYGASTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATY YCQSYAYFDSNNWHAFGGGTKVEIKATSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAV HTRGLDFACDPFFFCCFIAVAMGIRFIIMVTWRRKRKEKQSETSPKEFLTIYEDVKDLKTRRN HEQEQTFPGGGSTIYSMIQS
  • nucleic Acid Molecules Encoding a Mesothelin-Targeted CAR and IL-15
  • 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.
  • CAR chimeric antigen receptor
  • IL-15 interleukin-15
  • 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).
  • the CAR further includes a hinge region; a transmembrane domain; an intracellular co-stimulatory domain; and/or an intracellular signaling domain.
  • 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.
  • 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.
  • 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.
  • 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.
  • the hinge region is a CD8 ⁇ hinge region; the transmembrane domain is a NKG2D or a CD8 ⁇ 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 CD3 ⁇ intracellular signaling domain.
  • the hinge region is a CD8 ⁇ 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 CD3 ⁇ intracellular signaling domain.
  • the hinge region is a CD8 ⁇ hinge region; the transmembrane domain is a CD8 ⁇ 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 CD3 ⁇ intracellular signaling domain.
  • the amino acid sequence of the CD8 ⁇ 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 CD8 ⁇ 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.
  • the amino acid sequence of the CD8 ⁇ 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.
  • the amino acid sequence of the CD8 ⁇ transmembrane domain includes or consists of SEQ ID NO: 17.
  • 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.
  • the amino acid sequence of the 2B4 intracellular co-stimulatory domain includes or consists of SEQ ID NO: 18.
  • 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.
  • the amino acid sequence of the 4-1BB intracellular co-stimulatory domain includes or consists of SEQ ID NO: 19.
  • the amino acid sequence of the CD3 ⁇ 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.
  • the amino acid sequence of the CD3 ⁇ intracellular signaling domain includes or consists of SEQ ID NO: 20.
  • 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.
  • the amino acid sequence of IL-15 includes or consists of SEQ ID NO: 21.
  • 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.
  • 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
  • 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.
  • the vector is a transposon vector.
  • the vector is a piggyBac transposon vector.
  • the vector is a viral vector, such as a lentivirus vector, an adeno-associated virus vector, or an adenovirus vector.
  • NK natural killer
  • the NK cells are induced NK (iNK) cells, such as cells induced from an induced pluripotent stem cell (iPSC) line.
  • iPSCs Co-Expressing a Mesothelin-Targeted CAR and IL-15
  • 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.
  • CAR chimeric antigen receptor
  • IL interleukin
  • 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).
  • the CAR further includes a hinge region; a transmembrane domain; an intracellular co- stimulatory domain; and/or an intracellular signaling domain.
  • 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.
  • 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.
  • 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.
  • 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.
  • the hinge region is a CD8 ⁇ hinge region; the transmembrane domain is a NKG2D or a CD8 ⁇ 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 CD3 ⁇ intracellular signaling domain.
  • the hinge region is a CD8 ⁇ 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 CD3 ⁇ intracellular signaling domain.
  • the hinge region is a CD8 ⁇ hinge region; the transmembrane domain is a CD8 ⁇ transmembrane domain; the intracellular co-stimulatory domain is a 4-1BB intracellular co-stimulatory domain; and the intracellular signaling domain is a CD3 ⁇ intracellular signaling domain.
  • the amino acid sequence of the CD8 ⁇ 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 CD8 ⁇ 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.
  • the amino acid sequence of the CD8 ⁇ 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.
  • the amino acid sequence of the CD8 ⁇ transmembrane domain includes or consists of SEQ ID NO: 17.
  • 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.
  • the amino acid sequence of the 2B4 intracellular co-stimulatory domain includes or consists of SEQ ID NO: 18.
  • 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.
  • the amino acid sequence of the 4-1BB intracellular co-stimulatory domain includes or consists of SEQ ID NO: 19.
  • the amino acid sequence of the CD3 ⁇ 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.
  • the amino acid sequence of the CD3 ⁇ intracellular signaling domain includes or consists of SEQ ID NO: 20.
  • 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.
  • 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.
  • the amino acid sequence of IL-15 includes or consists of SEQ ID NO: 21.
  • 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).
  • the iPSC is an iPSC from the NCRM5 or NCRM6 iPSC line (commonfund.nih.gov/stemcells/lines). D.
  • 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.
  • CAR chimeric antigen receptor
  • IL interleukin
  • 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).
  • the CAR further includes a hinge region; a transmembrane domain; an intracellular co- stimulatory domain; and/or an intracellular signaling domain.
  • 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.
  • 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.
  • 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.
  • 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.
  • the hinge region is a CD8 ⁇ hinge region; the transmembrane domain is a NKG2D or a CD8 ⁇ 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 CD3 ⁇ intracellular signaling domain.
  • the hinge region is a CD8 ⁇ 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 CD3 ⁇ intracellular signaling domain.
  • the hinge region is a CD8 ⁇ hinge region; the transmembrane domain is a CD8 ⁇ transmembrane domain; the intracellular co-stimulatory domain is a 4-1BB intracellular co-stimulatory domain; and the intracellular signaling domain is a CD3 ⁇ intracellular signaling domain.
  • the amino acid sequence of the CD8 ⁇ 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 CD8 ⁇ 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.
  • the amino acid sequence of the CD8 ⁇ 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.
  • the amino acid sequence of the CD8 ⁇ transmembrane domain includes or consists of SEQ ID NO: 17.
  • 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.
  • the amino acid sequence of the 2B4 intracellular co-stimulatory domain includes or consists of SEQ ID NO: 18.
  • 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.
  • the amino acid sequence of the 4- 1BB intracellular co-stimulatory domain includes or consists of SEQ ID NO: 19.
  • the amino acid sequence of the CD3 ⁇ 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 CD3 ⁇ 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.
  • 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.
  • the NK cell is derived from an induced pluripotent stem cell (iPSC) line.
  • 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).
  • 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 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.
  • 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.
  • 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.
  • compositions that include NK/iNK cells can be formulated in unit dosage form suitable for individual administration of precise dosages.
  • 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.
  • 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 ⁇ 10 5 to about 1 ⁇ 10 9 NK cells/kg, such as from about 5 ⁇ 10 5 to about 5 ⁇ 10 8 NK cells/kg.
  • the dosage is at least about 5 ⁇ 10 5 NK cells/kg, at least about 6 ⁇ 10 5 NK cells /kg, at least about 7 ⁇ 10 5 NK cells/kg, at least about 8 ⁇ 10 5 NK cells /kg, at least about 9 ⁇ 10 5 NK cells /kg, at least about 1 ⁇ 10 6 NK cells /kg, at least about 2 ⁇ 10 6 NK cells/kg, at least about 3 ⁇ 10 6 NK cells/kg, at least about 4 ⁇ 10 6 NK cells/kg, at least about 5 ⁇ 10 6 NK cells /kg, at least about 6 ⁇ 10 6 NK cells/kg, at least about 7 ⁇ 10 6 NK cells/kg is at least about 8 ⁇ 10 6 NK cells/kg, at least about 9 ⁇ 10 6 NK cells/kg, at least about 1 ⁇ 10 7 NK cells/kg, at least about 2 ⁇ 10 7 NK cells/kg, at least about 3 ⁇ 10 7 NK cells/kg, at least about 4 ⁇ 10 7 NK cells/kg, at least about 5 ⁇ 10 7 NK cells/kg
  • 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.
  • 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.
  • 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.
  • the NK/iNK cell composition is administered intraperitoneally.
  • the initial period of treatment e.g., administration of the therapeutic agent daily or multiple times per week
  • the period of no treatment lasts for 3 days, 1 week, 2 weeks, 3 weeks or 4 weeks.
  • 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.
  • 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 ⁇ 10 5 to about 1 ⁇ 10 8 NK 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.
  • 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 ⁇ m are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively.
  • Capillaries have a diameter of approximately 5 ⁇ m so that only nanoparticles are administered intravenously. Microparticles are typically around 100 ⁇ m in diameter and are administered subcutaneously or intramuscularly. See, for example, Kreuter, J., Colloidal Drug Delivery Systems, J.
  • the block copolymer, polaxamer 407 exists as a viscous yet mobile liquid at low temperatures but forms a semisolid gel at body temperature.
  • hydroxyapatite has been used as a microcarrier for controlled release of proteins (Ijntema et al., Int. J. Pharm.112:215-224, 1994).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the mesothelin-positive cancer is mesothelioma, lung cancer, stomach cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, thymic carcinoma, or colorectal cancer.
  • the mesothelin-positive cancer is mesothelioma.
  • the mesothelin-positive cancer is pancreatic cancer.
  • the mesothelin- positive cancer is ovarian cancer.
  • the mesothelin-positive cancer is a gastric cancer, such as a stomach cancer.
  • 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.
  • 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.
  • 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).
  • 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
  • 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).
  • 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
  • enzymes such as L-asparaginase
  • 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).
  • platinum coordination complexes such as cis-diamine-dichloroplatinum II also known as cisplatin
  • substituted ureas such as hydroxyurea
  • methyl hydrazine derivatives such as procarbazine
  • adrenocrotical suppressants such as mitotane and aminoglutethimide
  • 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).
  • 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
  • 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 Jersey, 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.
  • 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.
  • iPSC-Derived Natural Killer 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).
  • 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.
  • APEL albumin polyvinylalcohol essential lipids
  • SCF human stem cell factor
  • VEGF human vascular endothelial growth factor
  • BMP-4 human bone morphogenetic protein 4
  • Rho kinase inhibitor such as Y-27632
  • the hematopoietic progenitor cells are further differentiated to produce iNK cells.
  • differentiating the hematopoietic progenitor cells to iNK cells includes culturing the EBs in NK cell differentiation medium.
  • the NK cell differentiation medium contains interleukin-3 (IL-3), IL-15, IL-7, SCF and fms-like tyrosine kinase 3 (FLT3) ligand.
  • the iPSCs seeded into the culture vessel are trypsin-adapted iPSCs.
  • the trypsin-adapted iPSCs can be TrypLE-adapted iPSCs.
  • the culture vessel is as tissue culture vessel, such as a 96-well plate, such as a 96-well round-bottom plate.
  • tissue culture vessel such as a 96-well plate, such as a 96-well round-bottom plate.
  • a skilled person can select an appropriate alternative culture vessel for carrying out the disclosed methods.
  • 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
  • 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 ⁇ M Y-27632.
  • 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.
  • 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.
  • the APEL culture medium includes about 0.1 ⁇ M, about 0.5 ⁇ M, about 1.0 ⁇ M, about 2.0 ⁇ M, about 5.0 ⁇ M, about 10 ⁇ M, about 25 ⁇ M, about 50 ⁇ M, about 75 ⁇ M, or about 100 ⁇ M Y-27632. In specific examples, the APEL culture medium includes about 10 ⁇ M Y-27632.
  • 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.
  • 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.
  • 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.
  • the NK cell differentiation medium includes about 5 ng/ml IL-3.
  • 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.
  • the NK cell differentiation medium includes about 10 ng/ml IL-15.
  • 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.
  • 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.
  • the NK cell differentiation medium includes about 20 ng/ml SCF.
  • 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.
  • the NK cell differentiation medium includes about 10 ng/ml FLT3 ligand.
  • 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.
  • the NK differentiation medium includes IL-3, IL- 15, IL-7, SCF and/or FLT3 ligand.
  • an alternative NK differentiation method or medium system can be used for carrying out the disclosed methods.
  • the iNK cells produced by the disclosed methods are CD45+CD56+.
  • the iPSCs prior to differentiation of the iPSCs to hematopoietic cell progenitors, 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.
  • the method further includes the step of enriching iPSCs that express the CAR encoded by the transduced nucleic acid molecule or vector.
  • the enriched iPSCs are also express IL-15 encoded by the transduced nucleic acid molecule or vector.
  • the iPSCs are LiPSC-GR1.1 cells.
  • 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.
  • Example 1 Materials and Methods This example describes the materials and experimental procedures for the studies described in Examples 2-11.
  • 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.
  • MSLN.CAR-IL-15 GR1.1-iNK 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.
  • MSLN.CAR-IL-15 GR1.1-iNK 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/ ⁇ c ⁇ / ⁇ (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.
  • 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.
  • 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).
  • VEGF vascular endothelial growth factor
  • BMP-4 bone morphogenetic protein 4
  • SCF stem cell factor
  • IL-3 IL-15
  • IL-7 flt3 ligand
  • IL- 2 flt3 ligand
  • IL-21 all obtained from PeproTech.
  • mice 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 (BioLegen
  • CAR constructs cloning Lentiviral packaging plasmid psPAX2 expressing CD8 TM .BB ICD .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) CD3 ⁇ 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 NKG2D TM /2B4 ICD .Z.CAR as well as NKG2D TM /2B4 ICD .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).
  • the expression cassette encoding NKG2D TM /2B4 ICD .Z.CAR-EGFRt or NKG2D TM /2B4 ICD .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.
  • iPSCs 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 ⁇ g/cm 2 . 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 ⁇ M, R&D Systems Inc).
  • ROCK inhibitor ROCK inhibitor
  • 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 ⁇ M).
  • 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.
  • 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.
  • 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 ⁇ l P3 primary solution containing 1 ⁇ g CAR expressing PBCAG transposon vector plus 0.25 ⁇ g 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.
  • 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 ⁇ 10 5 cells/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 MAXTM 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 ⁇ 10 7 cells/ml.
  • 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 ⁇ 10 6 cells/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).
  • 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.
  • % killing 100 ⁇ [1- relative light units (RLUs) from coculture wells/RLU from target-alone wells]. Two or three independent experiments were performed.
  • mice were i.p. injected with 5 ⁇ 10 5 luciferase-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 ⁇ 10 7 and 1 dose of 5 ⁇ 10 6 iNK cells harvested during day 28 to day 35 post NK differentiation were i.p.
  • mice were injected with 6 ⁇ 10 6 NCI-meso63 tumor cells on day -16 and treated with 6 ⁇ 10 6 iNK 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.
  • 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.
  • GSEA gene set enrichment analysis
  • EasyGSEA tool Choeng et al., Nucleic Acids Res 49:W207-W215, 2021
  • 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 ⁇ m 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).
  • FACS buffer PBS + 0.5% FBS, + 1 mM EDTA
  • 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.
  • 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.
  • NK/tumor coculture or isolated from peritoneal tumors and spleens 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.
  • FBS 1% FBS
  • 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.
  • 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-PlusTM 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).
  • 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.
  • GaAs Gallium-Arsenide
  • HyDs Hybrid Detectors
  • PMT multialkali photomultiplier tube
  • a 40X (NA 1.3) oil emersion objective lens was used for scanning the sections with a pixel size of 568.74X568.74 mm 2 and 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.
  • 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.
  • PCA Principal component analysis
  • the cells annotated as B 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.
  • 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 StemMACSTM Trilineage Differentiation Kit (Miltenyi Biotec, 130-115-660) according to manufacturer's instructions. The cells were harvested, and RNA was extracted using the PureLinkTM 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.
  • 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).
  • 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).
  • EBs were then directly transferred into 6-well plates with NK cell differentiation medium for 4 weeks.
  • 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.
  • NCRM1, NCRM2, and NCRM4 failed to further differentiate.
  • the average iNK differentiation yield was 1.3 ⁇ 10 5 per EB, 8.8 ⁇ 10 4 per EB, and 9.0 ⁇ 10 3 per EB from iPSC lines LiPSC-GR1.1, NCRM5, and NCRM6 respectively (FIG.1D).
  • > 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
  • MSLN.CAR that encodes the hYP218 scFv, followed by a CD8a hinge spacer, the NKG2D transmembrane domain (TM), the 2B4 intracellular domain, and the CD3 ⁇ 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.
  • iNKs A robust yield of iNKs was attained from MSLN.CAR-IL-15 iPSCs ( ⁇ 1.6 ⁇ 10 6 per EB) compared to limited iNK yield from mock iPSCs ( ⁇ 2.3 ⁇ 10 5 per EB) or MSLN.CAR-engineered iPSCs ( ⁇ 4.5 ⁇ 10 5 per EB) at 4 weeks post differentiation initiation (FIG.2D, FIG.10).
  • 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).
  • 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).
  • 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.
  • PCA Principal-component analysis
  • FIG.11A unsupervised hierarchical clustering of gene expression
  • 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).
  • MSLN.CAR-IL-15 iNKs showed increased expression of genes associated with cell division, DNA replication, NF- ⁇ B signaling, TRP channel calcium signaling, and Wnt signaling pathways.
  • the expression of NK activity-associated genes was then profiled in different categories (FIG.3D).
  • 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
  • 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).
  • 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.
  • MSLN.CAR-IL-15 iNKs 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).
  • Example 6 Tumor regression and improved survival in MSLN.CAR-IL15 iNK-treated MSLN + tumor-bearing mice
  • PDX mesothelioma-derived xenograft
  • 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).
  • BBI bioluminescent imaging
  • 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).
  • hCD45 + hCD56 + mCD45- cells were 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).
  • 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).
  • 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).
  • 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.
  • FIG.7A 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.
  • MSLN, KRT8 and KRT18 mesothelioma
  • NK cells PPRC, NCAM1, NKG7 and hYP218 (CAR)
  • 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).
  • 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.
  • 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- ⁇ in creating an immunosuppressive microenvironment.
  • 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.
  • 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).
  • 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).
  • NK2 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.
  • NK1 was the dominant subcluster, expressing high levels of cytotoxic effector genes (CST7, SRGN, GZMA), indicating its key role in tumor cell killing.
  • CST7, SRGN, GZMA cytotoxic effector genes
  • 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.
  • 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 (PKC ⁇ ) and CBLB, exhibited the lowest expression of NK function-related genes among the post- infusion clusters.
  • 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.
  • FIG. 8C the expression of NK-associated biomarkers in the NK subclusters was evaluated (FIG. 8C).
  • 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.
  • NK activating receptors including NKG2D-DAP10 (KLRK1/HCST), NKG2C (KLRC2), CD94 (KLRD1), and NKp30 (NCR3), suggesting they were robustly activated.
  • KLRK1/HCST NKG2D-DAP10
  • KLRC2C NKG2C
  • CD94 KLRD1
  • NCR3 NKp30
  • 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).
  • 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- ⁇ -rich tumor microenvironment
  • the pathway enrichment analysis revealed the pathways significantly enriched in tumor- infiltrating NKs versus pre-infusion NKs, including TNF ⁇ signaling via NF ⁇ B, hypoxia, interferon gamma response, interferon alpha response, apoptosis, heme metabolism, IL2-STAT5 signaling, estrogen response and p53 pathway (FIG.8D).
  • NK1 subcluster compared to NK5.
  • JAK3 was downregulated in post-infusion NK cells compared to pre-infusion iNK cells.
  • 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.
  • NK1 and NK5 clusters exhibited low expression of TGFBR2 (with particularly low levels in the NK5 cluster), and simultaneously upregulated negative regulators of TGF- ⁇ signaling SKI, SKIL, and TGIF1, indicating that these NK clusters may be adapting to mitigate the suppressive effects of TGF- ⁇ .
  • the cluster NK7 with impaired NK function showed high expression of genes involved in TGF- ⁇ 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.
  • NK1 and NK5 clusters 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.
  • the pro-apoptotic genes BCL2L11, BTG1, and DUSP22 were expressed at lower levels in NK5 compared to NK1.
  • 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).
  • 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.
  • 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.
  • LiPSC- 4239-111805-02 GR1.1 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.
  • 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.
  • 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.
  • a high level e.g. 80 ng/ml
  • 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 CD8 ⁇ hinge region, a CD8 ⁇ transmembrane domain, a 4-1BB co-stimulatory domain and a CD3 ⁇ 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.

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Abstract

L'invention concerne des cellules souches pluripotentes induites (iPSC) et des cellules tueuses naturelles dérivées d'iPSC (iNK) co-exprimant l'interleukine-15 (IL-15) et un récepteur antigénique chimérique (CAR) ciblé sur la mésothéline. L'invention concerne également des molécules d'acide nucléique et des vecteurs codant pour un CAR ciblant la mésothéline et l'IL-15, par exemple pour la transduction de cellules iPSC et iNK. Les cellules iNK exprimant CAR/IL-15 peuvent être utilisées pour le traitement de tumeurs solides, telles que des tumeurs solides positives à la mésothéline. L'invention concerne en outre des procédés de production de cellules progénitrices hématopoïétiques et de cellules iNK à partir d'une lignée iPSC, telle que la lignée LiPSC-GR1.1.
PCT/US2025/029169 2024-05-14 2025-05-13 Cellules tueuses naturelles exprimant un récepteur antigénique chimérique ciblant la mésothéline et l'interleukine-15 pour le traitement de tumeurs solides Pending WO2025240496A1 (fr)

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