Pluripotent Stem Cell-derived Natural Killer Research Articles

SynNotch-programmed iPSC-derived NK cells usurp TIGIT and CD73 activities for glioblastoma therapy

Severe heterogeneity within glioblastoma has spurred the notion that disrupting the interplay between multiple elements on immunosuppression is at the core of meaningful anti-tumor responses. T cell immunoreceptor with Ig and ITIM domains (TIGIT) and its glioblastoma-associated antigen, CD155, form a highly immunosuppressive axis in glioblastoma and other solid tumors, yet targeting of TIGIT, a functionally heterogeneous receptor on tumor-infiltrating immune cells, has largely been ineffective as monotherapy, suggesting that disruption of its inhibitory network might be necessary for measurable responses. It is within this context that we show that the usurpation of the TIGIT − CD155 axis via engineered synNotch-mediated activation of induced pluripotent stem cell-derived natural killer (NK) cells promotes transcription factor-mediated activation of a downstream signaling cascade that results in the controlled, localized blockade of CD73 to disrupt purinergic activity otherwise resulting in the production and accumulation of immunosuppressive extracellular adenosine. Such “decoy” receptor engages CD155 binding to TIGIT, but tilts inhibitory TIGIT/CD155 interactions toward activation via downstream synNotch signaling. Usurping activities of TIGIT and CD73 promotes the function of adoptively transferred NK cells into intracranial patient-derived models of glioblastoma and enhances their natural cytolytic functions against this tumor to result in complete tumor eradication. In addition, targeting both receptors, in turn, reprograms the glioblastoma microenvironment via the recruitment of T cells and the downregulation of M2 macrophages. This study demonstrates that TIGIT/CD155 and CD73 are targetable receptor partners in glioblastoma. Our data show that synNotch-engineered pluripotent stem cell-derived NK cells are not only effective mediators of anti-glioblastoma responses within the setting of CD73 and TIGIT/CD155 co-targeting, but represent a powerful allogeneic treatment option for this tumor.

Engineered human pluripotent stem cell-derived natural killer cells with PD-L1 responsive immunological memory for enhanced immunotherapeutic efficacy

Adoptive chimeric antigen receptor (CAR)-engineered natural killer (NK) cells have shown promise in treating various cancers. However, limited immunological memory and access to sufficient numbers of allogenic donor cells have hindered their broader preclinical and clinical applications. Here, we first assess eight different CAR constructs that use an anti-PD-L1 nanobody and/or universal anti-fluorescein (FITC) single-chain variable fragment (scFv) to enhance antigen-specific proliferation and anti-tumor cytotoxicity of NK-92cells against heterogenous solid tumors. We next genetically engineer human pluripotent stem cells (hPSCs) with optimized CARs and differentiate them into functional dual CAR-NK cells. The tumor microenvironment responsive anti-PD-L1 CAR effectively promoted hPSC-NK cell proliferation and cytotoxicity through antigen-dependent activation of phosphorylated STAT3 (pSTAT3) and pSTAT5 signaling pathways via an intracellular truncated IL-2 receptor β-chain (ΔIL-2Rβ) and STAT3-binding tyrosine-X-X-glutamine (YXXQ) motif. Anti-tumor activities of PD-L1-induced memory-like hPSC-NK cells were further boosted by administering a FITC-folate bi-specific adapter that bridges between a programmable anti-FITC CAR and folate receptor alpha-expressing breast tumor cells. Collectively, our hPSC CAR-NK engineering platform is modular and could constitute a realistic strategy to manufacture off-the-shelf CAR-NK cells with immunological memory-like phenotype for targeted immunotherapy.

Advanced T and Natural Killer Cell Therapy for Glioblastoma.

Although immunotherapy has been broadly successful in the treatment of hematologic malignancies and a subset of solid tumors, its clinical outcomes for glioblastoma are still inadequate. The results could be due to neuroanatomical structures such as the bloodbrain- barrier, antigenic heterogeneity, and the highly immunosuppressive microenvironment of glioblastomas. The antitumor efficacy of endogenously activated effector cells induced by peptide or dendritic cell vaccines in particular has been insufficient to control tumors. Effector cells, such as T cells and natural killer (NK) cells can be expanded rapidly ex vivo and transferred to patients. The identification of neoantigens derived from tumor-specific mutations is expanding the list of tumor-specific antigens for glioblastoma. Moreover, recent advances in gene-editing technologies enable the effector cells to not only have multiple biological functionalities, such as cytokine production, multiple antigen recognition, and increased cell trafficking, but also relieve the immunosuppressive nature of the glioblastoma microenvironment by blocking immune inhibitory molecules, which together improve their cytotoxicity, persistence, and safety. Allogeneic chimeric antigen receptor (CAR) T cells edited to reduce graft-versushost disease and allorejection, or induced pluripotent stem cell-derived NK cells expressing CARs that use NK-specific signaling domain can be a good candidate for off-the-shelf products of glioblastoma immunotherapy. We here discuss current progress and future directions for T cell and NK cell therapy in glioblastoma.

Abstract 3151: A novel method to produce clinical scale induced pluripotent stem cell-derived natural killer (iPSC-NK) cells with improved anti-tumor activity for next-generation allogenic cell therapies

Abstract Natural killer (NK) cells are innate immune cells that play a key role in tumor immune surveillance. NK cells have self-tolerance to healthy cells and can kill allogeneic tumor cells- both hematological and solid malignancies. Recent advances to derive immune cells from human induced pluripotent stem cells (iPSCs) allow the production of NK cells that can be used for immunotherapy. Recent clinical trials suggest that high doses of NK cells (approx. 0.5-1 x 109 cells per dose) and multiple doses are both safe and likely necessary for clinical efficacy. Manufacturing large number of NK cells from a clonal master iPSC line provides a promising strategy to enable off-the-shelf, next generation allogenic cell therapies. To do this, we have developed a novel method to produce clinical scale iPSC-NK cells and improved the process to expand NK cells efficiently and consistently. Briefly, hematopoietic progenitor cells were induced using an improved spin embryoid body (EB) method and the hematopoietic progenitor cells were subsequently differentiated into mature NK cells without cell sorting step as well as stroma cells support. Using this novel protocol, differentiated NK cells expanded more than 3,000-fold to enable us to potentially produce 1 x 1012 iPSC-NK cells starting from 1 x 106 undifferentiated iPSC. This cell production scale can supply >200 doses of 109 cells per dose. Importantly, iPSC-NK cells produced using this method display the typical phenotype of NK cell activating receptor including NKG2D, NKp44, NKp46, and DNAM-1. These iPSC-NK cells demonstrate better anti-tumor activity (against AML) and higher levels of IFNγ and TNFα compared to primary peripheral blood-derived NK cells isolated from healthy donors. Additionally, our iPSC-NK cells demonstrated prolonged “serial killing” of AML cells up to six days. Moreover, this protocol has been tested in large scale using not only unmodified iPSC-NK cells, but also genetically engineered iPSC-NK cells including those with deletion of CISH in iPSC-derived NK cells us that demonstrate improved in vivo anti-tumor activity, in vivo persistence, metabolic fitness, polyfunctionality and resistance to cell exhaustion to create next-generation CAR-NK cell-based immunotherapies. Overall, this novel cell production strategy paves the way for clinical trials using higher doses of more potent iPSC-derived NK cells. Citation Format: Davide Bernareggi, Caryn Gonsalves, Max Schabla, Alejandra Garate-Carrilo, Eleanor O’Gorman, Nathan Chang, Luis Solchaga, Mohammad El-Kalay, William Sandborn, Dan S. Kaufman, Huang Zhu. A novel method to produce clinical scale induced pluripotent stem cell-derived natural killer (iPSC-NK) cells with improved anti-tumor activity for next-generation allogenic cell therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3151.

IPSC-Derived Natural Killer Cell Therapies - Expansion and Targeting.

Treatment of cancer with allogeneic natural killer (NK) cell therapies has seen rapid development, especially use against hematologic malignancies. Clinical trials of NK cell-based adoptive transfer to treat relapsed or refractory malignancies have used peripheral blood, umbilical cord blood and pluripotent stem cell-derived NK cells, with each approach undergoing continued clinical development. Improving the potency of these therapies relies on genetic modifications to improve tumor targeting and to enhance expansion and persistence of the NK cells. Induced pluripotent stem cell (iPSC)-derived NK cells allow for routine targeted introduction of genetic modifications and expansion of the resulting NK cells derived from a clonal starting cell population. In this review, we discuss and summarize recent important advances in the development of new iPSC-derived NK cell therapies, with a focus on improved targeting of cancer. We then discuss improvements in methods to expand iPSC-derived NK cells and how persistence of iPSC-NK cells can be enhanced. Finally, we describe how these advances may combine in future NK cell-based therapy products for the treatment of both hematologic malignancies and solid tumors.

Induced pluripotent stem cell-derived natural killer cells gene-modified to express chimeric antigen receptor-targeting solid tumors.

The use of allogeneic, pluripotent stem cell-derived immune cells for cancer immunotherapy has been the subject of recent research, including clinical trials. The use of pluripotent stem cells as the source for allogeneic immune cells facilitates stringent quality control of the final product, regarding efficacy, safety, and producibility. In this review, we have described the characteristics of natural killer (NK) cells from multiple cell sources, including pluripotent stem cells, the chimeric antigen receptor (CAR)-modification method and strategy for these NK cells, and the current and planned clinical trials of CAR-modified induced pluripotent stem cell-derived NK cells.

Metabolic Reprograming via Deletion of CISH in Human iPSC-Derived NK Cells Promotes InVivo Persistence and Enhances Anti-tumor Activity.

Cytokine-inducible SH2-containing protein (CIS; encoded by the gene CISH) is a key negative regulator of interleukin-15 (IL-15) signaling in natural killer (NK) cells. Here, we develop human CISH-knockout (CISH-/-) NK cells using an induced pluripotent stem cell-derived NK cell (iPSC-NK cell) platform. CISH-/- iPSC-NK cells demonstrate increased IL-15-mediated JAK-STAT signaling activity. Consequently, CISH-/- iPSC-NK cells exhibit improved expansion and increased cytotoxic activity against multiple tumor cell lines when maintained at low cytokine concentrations. CISH-/- iPSC-NK cells display significantly increased invivo persistence and inhibition of tumor progression in a leukemia xenograft model. Mechanistically, CISH-/- iPSC-NK cells display improved metabolic fitness characterized by increased basal glycolysis, glycolytic capacity, maximal mitochondrial respiration, ATP-linked respiration, and spare respiration capacity mediated by mammalian target of rapamycin (mTOR) signaling that directly contributes to enhanced NK cell function. Together, these studies demonstrate that CIS plays a key role to regulate human NK cell metabolic activity and thereby modulate anti-tumor activity.

Metabolic Reprograming Due to Deletion of <i>CISH</i> in Human Natural Killer Cells Promotes <i>in vivo</i> Persistence and Enhances Anti-Tumor Activity

Cytokine-inducible SH2-containing protein (CIS, encoded by the gene CISH) is a key negative regulator of IL-15 signaling in natural killer (NK) cells. Here, we developed human CISH knockout (CISH-/-) NK cells using an induced pluripotent stem cell-derived NK cell (iPSC-NK cell) platform. CISH-/- iPSC-derived NK cells demonstrate increased IL-15 mediated JAK-STAT pathway signaling activity. Consequently CISH-/- iPSC-NK cells exhibit improved expansion and increased cytotoxic activity against multiple tumor cell lines when maintained at low cytokine concentration. CISH-/- iPSC-NK cells display significantly increased in vivo persistence and inhibition of tumor progression in a leukemia xenograft model. Mechanistically, CISH-/- iPSC-NK cells display improved metabolic fitness characterized by a significant increase in basal glycolysis, glycolytic capacity, maximal mitochondrial respiration, ATP-linked respiration and spare capacity respiration which are mediated by mTOR signaling and directly contribute to enhanced NK cell function. In addition, we demonstrate that deletion of CISH in human NK cells increases single-cell polyfunctionality by secreting multiple effector cytokines, a profile has been proven to be positively associated with clinical outcome of CAR-T cells. Together, these studies demonstrate CIS plays a key role to regulate human NK cell metabolic activity and deletion of CIS leads to enhanced anti-tumor activity.

Deletion of CISH in Human Pluripotent Stem Cell-Derived Natural Killer Cells Enhances Anti-Tumor Activity Via Metabolic Reprogramming

Cellular metabolism is now recognized to play a key role in regulating function and differentiation of immune cells. Immune cells undergo dynamic metabolic shifts to support their activity. Recent studies have shown the importance of metabolism to regulate NK cell function under different conditions. IL-15 can regulate NK cell metabolism in a dose- and time- dependent manner. However, whether improving metabolic fitness of NK cells can lead to improved anti-tumor activity is largely unknown. Here, we deleted CIS (encoded by the CISH gene in human induced pluripotent stem cells (iPSC)-derived NK cells (CISH-/- iPSC-NK) using CRISPR/Cas9 technology. CIS is known to function as a critical negative regulator of IL-15 signaling and a potent inhibitory checkpoint in NK cell-mediated anti-tumor activity. Surprisingly, more than 3000 differentially expressed genes were identified in CISH-/- iPSC-NK cells through RNA sequencing analysis. Gene ontology (GO) enrichment analysis showed that the top 2 up-regulated signaling pathways were associated with leukocyte differentiation and activation. Notably, more than 50 genes that involved in positive regulation of lymphocyte activation including genes associated with NK cell effector functions were significantly up-regulated in CISH-/- iPSC-NK cells compared with unmodified iPSC-NK cells. As expected, CISH-/- iPSC-NK cells exhibited increased sensitivity to IL-15 stimulation. Specifically, IL-15 stimulated JAK1 tyrosine phosphorylation, STAT3 and STAT5 phosphorylation were all dramatically increased in CISH-/- iPSC-NK cells, compared with unmodified iPSC-NK cells. In addition, when incubated with low concentrations of IL-15 (5 ng/ml), a concentration that does not support the growth and functions of unmodified iPSC-NK cells, CISH-/- iPSC-NK cells maintained significantly better expansion with >10 fold expansion for CISH-/- iPSC-NK cells in 3 weeks, whereas most of WT iPSC-NK cells died under these conditions (p<0.001). These CISH-/- iPSC-NK cells exhibited improved cytotoxic against acute myeloid leukemia (AML) cell lines K562 and Molm-13. More importantly, in an AML (MOLM-13) xenograft model, CISH-/- iPSC-NK cells displayed significantly increased in vivo persistence thus lead to significantly better control of tumor progression. To further understand the underling mechanisms contributing to the increased functionality in CISH-/- iPSC-NK cells, we then analyzed the metabolic profiles of these cells using Agilent Seahorse assays. After treatment with low concentration IL-15 for 7 days, CISH-/- iPSC-NK cells displayed both increased glucose metabolism and increased mitochondrial respiration profiles when compared with unmodified iPSC-NK cells. Increased glucose metabolism include a significant increase in basal glycolysis (p=0.0003, 84.5 ± 1.0 vs 61.1 ± 1.8 mpH/min, n=6), and glycolytic capacity (p=0.0049, 15.2±0.4 vs 11.7±0.5 mpH/min, n=6). And increased mitochondrial respiration profile is characterized by an increase in the spare respiratory capacity (p=0.0009, 16.3±1.9 vs 6.9±0.5 pmol/min, n=6), maximal respiration (p<0.0001, 31.6±1.9 vs 15.7±0.8 pmol/min, n=6) and ATP-linked respiration (p<0.0001, 11.5 ± 0.4 vs 7.0±0.3 pmol/min, n=6). Interestingly, limiting the increase of glucose metabolism and mitochondrial respiration via utilization of an mTOR inhibitor (rapamycin) attenuated the increased functionality in CISH-/- iPSC-NK cells. The findings presented here show that deletion of CISH in iPSC-NK cells enhance anti-tumor activity, at least in part via metabolic reprogramming. These findings suggest that improving NK cell functions for cancer immunotherapy through metabolic reprogramming provides a promising therapeutic strategy. Disclosures Guan: OncoImmune: Equity Ownership. Kaufman:FATE Therapeutics: Consultancy, Research Funding.

Engineered human pluripotent stem cell-derived natural killer cells: the next frontier for cancer immunotherapy.

Adoptive immunotherapy using immune effector cells has revolutionized cancer treatments with approval of two autologous chimeric antigen receptor (CAR) T cell therapies by the US FDA. Clinical trials using natural killer (NK) cell-based adoptive immunotherapy have been shown to be safe and effective for treatment of multiple malignancies, especially acute myelogenous leukemia. However, most of these trails use primary NK cells isolated from peripheral or cord blood which can have donor-dependent variability and can be challenging to genetic engineer to improve antitumor functions, limiting the widespread use of this promising new therapy. NK cells can now be routinely produced from human pluripotent stem cells, both human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). These pluripotent stem cells are homogenous, easy to genetically modify on a clonal level and can be used as unlimited source of NK cells, making them ideal population to develop standardized, off-the-shelf adoptive NK cell therapy products. In this review, we discuss recent advances of obtaining and expanding hESC and iPSC-derived NK cells and novel genetic engineering strategies that are being applied to improve their antitumor functions.

Induction of human pluripotent stem cell-derived natural killer cells for immunotherapy under chemically defined conditions

Natural killer (NK) cells are innate lymphocytes and show cytotoxicity against tumor cells without prior antigen specific stimulation. Because of their innate properties, NK cells are being considered for immunotherapies against various malignancies or leukemia. Human pluripotent stem cells (hPSCs) are capable of inducing enough NK cells for allogeneic transplantation. However, current induction protocols require feeder cells or human or bovine serum for the differentiation and expansion of NK cells, which incurs potential risk for contamination and may cause lot dependency in the cells. To address these issues, here we established a differentiation protocol for developing functional NK cells from hPSCs under a completely chemically-defined condition. The resultant PSC-derived NK cells show comparable phenotypes to those produced under serum-containing condition, exerting strong killing potential against a leukemia cell line in vitro and resistance to tumor growth in vivo. Our protocol can be a useful tool for applying PSC-derived NK cells to future cellular cancer immunotherapies.

Off-the-shelf cell therapy with induced pluripotent stem cell-derived natural killer cells

Cell therapy is emerging as a very promising therapeutic modality against cancer, spearheaded by the clinical success of chimeric antigen receptor (CAR) modified T cells for B cell malignancies. Currently, FDA-approved CAR-T cell products are based on engineering of autologous T cells harvested from the patient, typically using a central manufacturing facility for gene editing before the product can be delivered to the clinic and infused to the patients. For a broader implementation of advanced cell therapy and to reduce costs, it would be advantageous to use allogeneic "universal" cell therapy products that can be stored in cell banks and provided upon request, in a manner analogous to biopharmaceutical drug products. In this review, we outline a roadmap for development of off-the-shelf cell therapy based on natural killer (NK) cells derived from induced pluripotent stem cells (iPSCs). We discuss strategies to engineer iPSC-derived NK (iPSC-NK) cells for enhanced functional potential, persistence, and homing.

CRISPR/Cas9 Genome-Editing System in Human Stem Cells: Current Status and Future Prospects.

Genome-editing involves the insertion, deletion, or replacement of DNA in the genome of a living organism using “molecular scissors.” Traditional genome editing with engineered nucleases for human stem cells is limited by its low efficiency, high cost, and poor specificity. The CRISPR system has recently emerged as a powerful gene manipulation technique with advantages of high editing efficiency and low cost. Although this technique offers huge potential for gene manipulation in various organisms ranging from prokaryotes to higher mammals, there remain many challenges in human stem cell research. In this review, we highlight the basic biology and application of the CRISPR/Cas9 system in current human stem cell research, discuss its advantages and challenges, and debate the future prospects for human stem cells in regenerative medicine.

Engineering Human Pluripotent Stem Cell-Derived Natural Killer Cells to Prevent CD16a Shedding for Enhanced Anti-Tumor Killing

Natural killer (NK) cells are lymphocytes able to recognize and lyse transformed cells without HLA-restriction and without prior sensitization, mediated through a repertoire of cell surface receptors. Solid tumors are typically more refractory to NK cell-mediated killing compared to hematologic malignancies. To improve NK cell-mediated killing of solid tumors, we have engineered NK cells to express a more stable form of CD16a. The IgG Fc receptor CD16a represents one of the most potent activators of NK cell anti-tumor activity. This receptor binds antibodies attached to target cells to initiate antibody-dependent cell-mediated cytotoxicity (ADCC). Importantly, CD16a undergoes a rapid down-regulation in expression by ADAM17 (A Disintegrin And Metalloprotease-17) mediated proteolytic cleavage upon cell activation. This loss of CD16a expression leads to reduced ADCC activity and decreased anti-tumor killing. Previous studies by our group identified cleavage sites in CD16a that are targeted by ADAM17 activity. Substitution of the serine at position 197 for a proline (S197P) in the middle of the cleavage region effectively blocks ADAM17-mediated CD16a cleavage. To evaluate the ability of this cleavage-resistant CD16a(S197P) to improve ADCC, we have used a system previously developed by our group to efficiently produce NK cells from both human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). These hESC/iPSC-derived NK cells have a phenotype and function similar to NK cells isolated from peripheral blood (PB-NK cells). However, unlike PB-NK cells, it is relatively easy to genetically-modify hESC/iPSC-derived NK cells by engineering at the stem cell level. Here, we use two strategies to generate iPSC-derived NK cells with stabilized CD16a expression. First, we used the Sleeping Beautytransposon system to stably express the cleavage-resistant CD16a(S197P) in iPSCs. Second, we have used the CRISPR/Cas9 system to delete the ADAM17 gene. This strategy allows us to compare specifically engineering stabilized CD16a expression to the broader effect of ADAM17-deletion which cleaves multiple cell-surface proteins.NK cells derived from the CD16a(S197P) iPSCs are resistant to CD16a shedding upon activation with IL-12 and IL-18 as 97.5% of iPSC-NK cells are CD16+ before stimulation and 95.2% of cells are CD16+ after stimulation. In contrast, PB-NK cells demonstrate a decrease from 85% CD16+ cells before stimulation to 67% CD16+ cells after stimulation. Next, we demonstrate improved ADCC and NK cell degranulation (measured by CD107a expression) in this engineered iPSC-derived NK cell compared to non-engineered cells upon addition of the anti-Her2 antibody (ab) Herceptin to ovarian cancer cells with differing levels of Her2 expression. Specifically, CD16a(S197P) iPSC-NK cells stimulated with the Her2-high SKOV3 ovarian carcinoma cells were 26.7% CD107a+ cells with addition of anti-Her2 and 10.8% CD107a+ cells without ab. In contrast, unmodified iPSC-NK cells were 9.6% vs. 6.9% CD107a+ with and without anti-Her2; and PB-NK cells were 12.4% vs. 9.4% CD107a+ with and without anti-Her2. However, when co-cultured with the Her2-low MA-148 ovarian carcinoma none of the three NK cell populations demonstrated increased CD107a+ cells with anti-Her2 treatment compared to no ab controls. Similar results were obtained when testing the ADCC of these cells using the Cr-51 release cytotoxicity assay. Killing of SKOV3 cells by CD16a(S197P) iPSC-NK cells was markedly increased upon addition of anti-Her2 ab from 32.2% cytoxicity with ab vs. 11% cytotoxicity without ab. In contrast, unmodified iPSC-NK cells had less killing of the SKOV3 cells (8.1% vs. 3.7% with or without anti-Her2 ab) and PB-NK cell killing of SKOV3 cells was 20.5% vs. 9.2% with or without anti-Her2 ab. Currently, we are comparing anti-tumor activity of the CD16a(S197P) iPSC-NK cells to the ADAM17-deleted iPSC-NK cells, both as resting NK cells and IL12+IL18-stimulated NK cells. Overall, this strategy will allow us to improve the ability of readily available anti-tumor antibodies to improve NK cell-mediated killing of solid tumors and use engineered iPSC-derived NK cells as an "off-the-shelf" approach for allogeneic cell-based immunotherapies. DisclosuresKaufman:Fate Therapeutics: Consultancy.

Human pluripotent stem cell-derived natural killer cells for anti-cancer therapy

Human pluripotent stem cell-derived natural killer cells for anti-cancer therapy

Induced Pluripotent Stem Cell-Derived Natural Killer Cells for Treatment of Ovarian Cancer

Natural killer (NK) cells can provide effective immunotherapy for ovarian cancer. Here, we evaluated the ability of NK cells isolated from peripheral blood (PB) and NK cells derived from induced pluripotent stem cell (iPSC) to mediate killing of ovarian cancer cells in a mouse xenograft model. A mouse xenograft model was used to evaluate the intraperitoneal delivery of three different NK cell populations: iPSC-derived NK cells, PB-NK cells that had been activated and expanded in long-term culture, and overnight activated PB-NK cells that were isolated through CD3/CD19 depletion of PB B and T cells. Bioluminescent imaging was used to monitor tumor burden of luciferase expressing tumor lines. Tumors were allowed to establish prior to administering NK cells via intraperitoneal injection. These studies demonstrate a single dose of any of the three NK cell populations significantly reduced tumor burden. When mice were given three doses of either iPSC-NK cells or expanded PB-NK cells, the median survival improved from 73 days in mice untreated to 98 and 97 days for treated mice, respectively. From these studies, we conclude iPSC-derived NK cells mediate antiovarian cancer killing at least as well as PB-NK cells, making these cells a viable resource for immunotherapy for ovarian cancer. Due to their ability to be easily differentiated into NK cells and their long-term expansion potential, iPSCs can be used to produce large numbers of well-defined NK cells that can be banked and used to treat a large number of patients including treatment with multiple doses if necessary.