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Natural Killer (NK) cells are cytotoxic lymphocytes capable of immune surveillance and represent an excellent source of cells for cancer immunotherapy for numerous reasons: 1) they mediate direct killing of transformed cells with reduced or absent MHC expression, 2) they can carryout antibody-dependent cell-mediated cytotoxicity (ADCC) on cells bound by appropriate antibodies via CD16, 3) they are readily available and easy to isolate from peripheral blood, 4) they can be expanded to clinically relevant numbers in vitro. Moreover, as NK cells do not cause graft versus host disease, they are inherently an off-the-shelf cellular product, precluding the need to use a patient's own NK cells to treat their cancer. In light of these attributes, NK cells have been used in many clinical trials to treat a number of cancer types; however, the results have not been as successful as other cellular based immunotherapies, such as CAR-T. In light of this, many groups have taken approaches to augment NK cell function, such as high dose IL15, CARs and Bi- or Tri-specific killer engagers. A synergistic or even alternative approach to these technologies is the use of CRISPR/Cas9-based genome editing to disrupt or manipulate the function of NK genes to improve their utility as an immunotherapeutic agent. In order to enhance the immunotherapeutic efficacy of NK cells we have implemented the CRISPR/Cas9 system to edit genes and deliver CARs. To this end, we have developed methods for high efficiency nucleic acid delivery to NK cells using electroporation. First, primary human NK cells are immunomagnetically isolated from peripheral blood mononuclear cells (PBMCs) of healthy donors. Purified NK cells are then activated and expanded using artificial antigen presenting cells (aAPCs) expressing membrane bound IL21 and 41BB for 7 days and subsequently electroporated (Figure 1A). Using this approach with EGFP encoding mRNA, we achieve high rates of transfection (>90%) and high viability (>90%) (Figure 1B). We next developed gRNAs targeting PD1, CISH, and ADAM17. PD1 is a negative regulator of NK cell function and its cognate receptor, PD-L1, is upregulated in a number of cancers. ADAM17 mediates CD16 cleavage on NK cells to negatively regulate their ability to perform ADCC. CISH is a recently described negative regulator of NK cell activation and integrates cytokine signals, including IL-15. We consistently achieved high rates (up to 90%) of gene inactivation in primary human NK cells across multiple donors (Figure 1C). Importantly, these gene edits do not affect expansion potential and are stable over several rounds of expansion (Figure 1D, E). Moreover, ADAM17 KO NK cells are highly resistant to CD16 cleavage upon activation (Figure 2A-E) and PD1 KO NK cells demonstrate significantly enhanced function against PD-L1 expressing cancer cell lines in vitro and in vivo (Figure 2F-J). These data demonstrate that high efficiency gene editing of NK cells can significantly enhance their function while maintaining in vitro expansion. In an effort to engineer NK cell specificity for cancer immunotherapy, we recently developed CAR molecules designed for use in NK cells (Li et al., 2018, Cell Stem Cell 23, 1-12). To this end, we engineered and tested 10 mesothelin CAR molecules with NK specific transmembrane domains (CD16, NKp44, NKp46, or NKG2D) and intracellular signaling domains (2B4, DAP10, DAP12, CD3ζ, and/or CD137). Utilizing several cancer models, we identified an architecture that significantly enhanced NK activation compared to T-CAR architectures (CAR4: scFv-NKG2D-2B4-CD3ζ). Moreover, NK-CAR4 cells demonstrated increased in vivo expansion, improved activity, and reduced toxicity compared to CAR-T cell therapy. In our studies to develop novel NK CARs, CARs were delivered to iPSC derived test NK cells (iNKs) using the PiggyBac transposon system. In order to deliver NK-CAR4 to peripheral blood NK cells we developed methods for high frequency, site specific integration. To this end, we utilized CRISPR/Cas9 combined with non-integrating recombinant Adeno-Associated Virus (rAAV) DNA donor for homologous recombination. Using an EGFP reporter we were able to optimize this process and deliver EGFP reporter to the AAVS1 safe harbor site with efficiencies >80% in NK cells. We are now utilizing our optimized gene editing approaches to generate multiplex edited CAR-NK cells and results from these studies will be presented. Disclosures Webber: BEAM Therapeutics: Consultancy; B-MoGen Biotechnologies: Employment, Equity Ownership. Felices:GT Biopharma: Research Funding. Moriarity:BEAM Therapeutics: Consultancy; B-MoGen Biotechnologies: Employment, Equity Ownership.

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.

Abstract 3611 Adoptive transfer of haploidentical natural killer (NK) cells can induce remissions in patients with refractory myeloid leukemia (AML). However, NK cells do not expand and persist in all patients despite lymphodepleting chemotherapy. In trials of adoptive NK cell therapy in solid tumors or lymphoma, host regulatory T cells (Treg) often expand in response to IL-2 given to stimulate donor NK cell expansion. Although murine studies report that Tregs inhibit NK cells, the influence of human Treg on NK cell proliferation and function is not well characterized. We studied the effect of allogeneic Tregs that were derived from human umbilical cord blood (UCB) as described by our group. Resting CFSE labelled NK cells or Teff were purified from healthy donors, and mixed with UCB Treg at various ratios. Unstimulated NK cells did not proliferate and thus IL-2 or IL-15 were added to the media at concentrations of 0.1, 0.25 and 0.5 ng/ml. In the absence of Treg, both cytokines induced equal NK cell proliferation at 5 days as measured by CFSE dilution in a concentration dependent manner. CFSE dilution was inhibited by Treg at a 1:1 ratio, especially at low cytokine concentrations. There were marked differences between the two cytokine conditions. Following IL-15 induced stimulation, the reduction in NK cell proliferation by Treg ranged from 1–35% (at different concentrations tested), whereas the inhibition of IL-2 stimulated NK cell proliferation ranged from 65–85%. Treg inhibition of NK cell proliferation could be measured at ratios as low as 1:8 in the presence of IL-2, but not IL-15. This inhibitory effect was partially explained by competition from CD25+ Tregs for IL-2. We measured Treg utilization of IL-2 by incubating NK cells with or without Treg in 0.5 ng/ml IL-2 for 4 days. The level of IL-2 with NK cells alone was 40 pg/ml vs. 17 pg/ml with Treg (compared to 330 pg/ml in IL-2-supplemented media without cells). Based on this data, we have incorporated host Treg depletion to enhance NK expansion after adoptive transfer to treat patients with refractory AML. As murine data from Blazar's group shows that CTL therapy is enhanced by Treg depletion, we added one dose of denileukin diftitox (ONTAK®, Eisai Inc) at 12 mg/kg to our lymphodepleting preparative regimen of fludarabine 25 mg/m2 × 5 days, cyclophosphamide 60 mg/kg × 2 days for 12 AML patients. Haploidentical NK cells (CD3- and CD19-depleted PBMCs and overnight activated with IL-2 1000 U/ml) were infused on Day 0, followed by 6 doses subcutaneous IL-2 (9 million units) given every other day to promote in vivo NK cell expansion. Eleven of 12 patients were evaluable, having received at least 4 of 6 planned doses of IL-2. Blood and marrow were collected 7 and 14 days after infusion to assess NK cell and Treg expansion, as well as leukemia clearance. Of the 10 patients with interpretable day 7 chimerism data, 9 had detectable donor DNA (median 68% donor DNA). At day 14, 4 of the 12 patients (33%) had successfully expanded NK cells in vivo, with absolute donor derived NK cell counts of 480, 530, 1470 and 12390 cells/μL blood, improving on our previous 10% rate of in vivo NK cell expansion which was observed with the same regimen, without Treg depletion. In the 4 patients who expanded NK cells in vivo, there were no detectable Treg (defined as a CD25+CD4+FoxP3+ lymphocyte population) at either day 7 or day 14. In contrast, the presence of a bona fide Treg population at either day 7 [range 9.5–53%] or day 14 [27–71%] correlated with a lack of in vivo NK cell expansion at day 14. Clinically, 8 of the 11 evaluable subjects cleared leukemia (72%), 7 of whom recovered neutrophils (63% CRp) and 6 of whom went on to best donor transplant (45%). In summary, we demonstrate in vitro and in vivo suppression of NK cell proliferation by IL-2 stimulated Treg. This effect is not seen in vitro with IL-15. We have shown that the absence of host Treg correlates with in vivo NK cells expansion. Although an increased rate of donor NK expansion was observed with a single dose of denileukin diftitox, it did not completely overcome the IL-2 induced host Treg expansion. Future trials testing additional doses of denileukin difitox or other methods of Treg depletion, as well as the use of IL-15 are planned. Disclosures: No relevant conflicts of interest to declare.