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 peripheral blood 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 3 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 anti-ovarian 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.

Background aims There is an urgent need for novel therapeutic strategies for relapsed ovarian cancer. Dramatic clinical antitumor effects have been observed with interleukin (IL)-2 activated natural killer (NK) cells; however, intravenous delivery of NK cells in patients with ovarian cancer has not been successful in ameliorating disease. We investigated in vivo engraftment of intraperitoneally (IP) delivered NK cells in an ovarian cancer xenograft model to determine if delivery mode can affect tumor cell killing and circumvent lack of NK cell expansion. Methods An ovarian cancer xenograft mouse model was established to evaluate efficacy of IP-delivered NK cells. Tumor burden was monitored by bioluminescent imaging of luciferase-expressing ovarian cancer cells. NK cell persistence, tumor burden and NK cell trafficking were evaluated. Transplanted NK cells were evaluated by flow cytometry and cytotoxicity assays. Results IP delivery of human NK cells plus cytokines led to high levels of circulating NK and was effective in clearing intraperitoneal ovarian cancer burden in xenografted mice. NK cells remained within the peritoneal cavity 54 days after injection and had markers of maturation. Additionally, surviving NK cells were able to kill ovarian cancer cells at a rate similar to pre-infusion levels, supporting that in vivo functionality of human NK cells can be maintained after IP infusion. Conclusions IP delivery of NK cells leads to stable engraftment and antitumor response in an ovarian cancer xenograft model. These data support further pre-clinical and clinical evaluation of IP delivery of allogeneic NK cells in ovarian cancer.

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a valuable tool for understanding ovarian cancer, their use has many limitations. These include the lack of heterogeneity and the plethora of genetic alterations associated with extended in vitro passaging. Here we describe a method that allows for rapid establishment of primary ovarian cancer cells form solid clinical specimens collected at the time of surgery. The method consists of subjecting clinical specimens to enzymatic digestion for 30 min. The isolated cell suspension is allowed to grow and can be used for downstream application including drug screening. The advantage of primary ovarian cancer cell lines over established ovarian cancer cell lines is that they are representative of the original specific clinical specimens they are derived from and can be derived from different sites whether primary or metastatic ovarian cancer.

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Natural killer (NK) cells are a key part in the innate immune system and have the ability to recognize diverse types of tumors and virally-infected targets. NK cells represent an attractive cell population for adoptive immunotherapy due to their ability to kill target cells in a human leukocyte antigen (HLA) non-restricted manner and without prior sensitization. Clinical studies using IL-2 activated NK cells demonstrate significant anti-tumor effects when adoptively transferred into patients with refractory leukemia (mainly AML). However, there has been a more limited response observed in clinical trials for the treatment of ovarian cancer and other solid malignancies. Chimeric antigen receptors (CARs) consist of an antigen-specific single chain antibody variable fragment fused to intracellular signaling domains derived from receptors involved in lymphocyte activation. CARs targeting various tumor-associated antigens have been developed and tested via expression in primary T cells with promising clinical results. However, engineering these T cells must be done on a patient-specific basis, thus limiting the number of patients who can be treated. In order to produce a potential targeted, “off-the-shelf” product suitable to treat patients with diverse tumors or chronic infections, we have generated human pluripotent stem cells with stable CAR expression. Previous studies by our group demonstrate that human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) provide an accessible, genetically tractable, and homogenous starting cell population to develop NK cells. We use a combined approach using “Spin-EB”- mediated differentiation of hESCs/iPSCs, followed by co-culture with artificial antigen presenting cells (aAPCs) that express mbIL-21. Using this strategy, we can generate 109 NK cells from a population of approximately 106 undifferentiated hESCs or iPSCs. This GMP compatible method is fully defined, without xenogeneic stromal cells or serum. Here, we have expressed both an anti-CD19 (targeting B cell malignancies) and an anti-mesothelin CAR (targeting ovarian cancer cells and other adenocarcinomas) in both hESCs and iPSCs. Using the Sleeping Beauty transposon system, both hESCs and iPSCs have been genetically engineered to express 3rd generation CARs, which express a single chain antibody fragment recognizing either CD19 or mesothelin, a CD8α hinge region, the transmembrane protein CD28, a co-stimulatory protein 4-1BB, and the activating domain CD3ζ. NK cells derived from hESCs/iPSCs with or without CAR expression are phenotypically similar to NK cells isolated from peripheral blood. These NK cells are CD56+, CD94+/CD117-, Nkp44+, Nkp46+, NKG2A+, NKG2D+, and KIR+. In 51Cr release assays against tumor targets expressing either CD19 or mesothelin, NK cells expressing the corresponding CAR show an enhanced killing ability. In cell lines lacking CD19 or mesothelin expression, the engineered cell lines exhibit equal activity compared to their non-engineered counterparts. Specifically, at a 10:1 effector:target ratio, anti-CD19 CAR+ iPSC-NK cells kill 58% of Lax7R cells (a CD19+ ALL cell line) compared to just 5% cell killing by CAR- iPSC-NK cells. Anti-CD19 CAR+ iPSC-NK cells also killed 2 other CD19+ ALL cell lines (018Z and Raji) better than CAR- iPSC-NK cells killing 63% vs 18% and 61% vs 8%, respectively. Similar results are seen against the mesothelin+ ovarian tumor line A1847. Here, anti-mesothelin CAR+ iPSC-NK cells kill 39% vs 14% for CAR- iPSC-NK cells. Currently, CAR-expressing NK cells derived from hESCs and iPSCs are being tested in vivo against both mesothelin+ ovarian tumor lines and CD19+ leukemia cells. Together, these studies demonstrate engineering hESCs and iPSCs with tumor-specific receptors provides a novel strategy to produce targeted NK cells suitable for immune therapies against refractory malignancies. Disclosures: No relevant conflicts of interest to declare.