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Cancer immunotherapies have resulted in a paradigm shift in therapy. One of the most successful approaches has been administration of antibodies targeting immune checkpoint inhibitors (ICI), such as programmed death-1 (PD-1), that can reinvigorate functionally exhausted T cells. Unfortunately, durable tumor regression is limited to a minority of patients, and relapse remains a significant concern. Combining novel immunotherapies with ICI s a promising strategy to bolster antitumor responses and response rates. Natural killer (NK) cells mediate direct tumor cell lysis, effectively target MHC low or null transformed cells and are key regulators of T cell responses through the production of inflammatory cytokines and chemokines. In many cancers, NK cell numbers are low, and their functional responses are sub-optimal. The use of allogeneic peripheral blood NK cells for immunotherapy has shown significant clinical promise for the treatment of various cancers. However, sourcing NK cells for adoptive cell therapy has been limited by both cell number and quality. Thus, we developed a robust manufacturing system for the differentiation and expansion of high-quality NK cells derived from induced pluripotent stem cells (iPSCs) that can be combined with ICI antibodies for multiple tumor types. To interrogate the ability of iPSC-derived NK cells to synergize with ICI therapy, we developed an in vitro 3D tumor spheroid system to model the combinatorial effects of activated CD3+ T cells, iPSC-derived NK (iNK) cells and ICI blockade for anti-tumor function in a more physiological context than the standard 2D cultures and in real time. Using SKOV-3 (an ovarian cancer line) spheroids as targets in a 160-hour killing assay, we found that iNK cells could mediate significant, but not complete destruction of tumor spheroids (46% tumor reduction). Twice as many activated CD3+ T cells by themselves also induced significant but incomplete tumor spheroid destruction (58% tumor reduction). Combined iNK and activated CD3+ T cells led to robust target cell destruction (71% tumor reduction). Importantly, adding anti-PD-1 antibody (mAb) to activated CD3+ T cells and iNK cells led to near complete elimination of tumor spheroid targets, with >99% tumor reduction. Cytokine and chemokine secretion analyses in co-cultures of activated CD3+ T cells and iNK cells revealed synergistic production of CCL3 and CCL4 for T cell recruitment, and TNF and IFN-γ to augment anti-tumor responses. To determine whether these striking 3D tumor spheroid results simulate the in vivo setting, we injected luciferase-expressing OVCAR8 (a human ovarian cancer line) cells into the peritoneal cavities of immunodeficient NSG mice. Following sublethal irradiation, we treated groups of mice with either anti-PD-1 mAb, activated CD3+ T cells or iNK cells alone or in various combinations. IL-2 was injected i.p. twice weekly for two weeks into all mice except those in the tumor alone group (Figure 1A). In mice that received iNK cells alone, significant tumor control was observed over the first 21 days but was not sustained. Tumor control was similar between groups of mice that received iNK cells and mice that received iNK cells and anti-PD-1 mAb. Mice treated with either anti-PD-1 mAb or activated CD3+ T cells alone exhibited similar rates of tumor growth relative to the untreated group. Modest tumor control was observed in the group of mice that received combined activated CD3+ T cells and anti-PD-1 mAb, though the effect did not reach statistical significance. Durable tumor control past day 21 was observed in groups of mice that received either combined activated CD3+ T cells and iNK cells or activated CD3+ T cells + iNK cells with anti-PD-1 mAb (Figure 1B). Importantly, 50% of mice in the activated CD3+ T cells + iNK cells + anti-PD-1 mAb group exhibited tumor bioluminescence readings well below other treatment group at day 35 (Figure 1C, D). Collectively, these data demonstrate that iNK cells can serve as an off-the-shelf source of high-quality NK cells and synergize with anti-PD-1 ICI therapy to enhance anti-tumor T cell responses in vitro and in vivo, providing a novel immunotherapeutic platform for tumors in which iNK cells, activated CD3+ T cells or anti-PD-1 mAb therapy alone is not sufficiently effective. The current program is under clinical investigation and can be found at clinicaltrials.gov NCT03841110. Disclosures Miller: Moderna: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics, Inc: Consultancy, Research Funding; CytoSen: Membership on an entity's Board of Directors or advisory committees; OnKImmune: Membership on an entity's Board of Directors or advisory committees; Dr. Reddys Laboratory: Membership on an entity's Board of Directors or advisory committees; GT BioPharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Bjordahl:Fate Therapeutics, Inc.: Employment. Gaidarova:Fate Therapeutics, Inc: Employment. Mahmood:Fate Therapeutics, Inc: Employment. Rogers:Fate Therapeutics, Inc: Employment. Moyar:Fate Therapeutics, Inc: Employment. Blazar:Abbvie Inc: Research Funding; KidsFirst Fund: Research Funding; Childrens' Cancer Research Fund: Research Funding; Leukemia and Lymphoma Society: Research Funding; Regeneron Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Magenta Therapeutics and BlueRock Therapeuetics: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics, Inc.: Research Funding; Tmunity: Other: Co-Founder; BlueRock Therapeutics: Membership on an entity's Board of Directors or advisory committees; RXi Pharmaceuticals: Research Funding; Alpine Immune Sciences, Inc.: Research Funding; Kamon Pharmaceuticals, Inc: Membership on an entity's Board of Directors or advisory committees; Five Prime Therapeutics Inc: Co-Founder, Membership on an entity's Board of Directors or advisory committees. Kaufman:FATE Therapeutics: Consultancy, Research Funding. Valamehr:Fate Therapeutics, Inc: Employment. Cichocki:Fate Therapeutics, Inc: Research Funding. OffLabel Disclosure: NK cells

Multiple myeloma (MM) is a B cell neoplasm that originates from the malignant transformation of plasma cells, with treatment strategies that include chemotherapeutic agents and immunomodulatory drugs. Recently, significant effort has been applied to the development of monoclonal antibody (mAb) and chimeric antigen receptor (CAR) T cell therapies for the treatment of advanced MM. Anti-CD38 mAb therapy is at the forefront of these efforts, with clearly demonstrated clinical benefit and availability of a FDA-approved mAb in daratumumab. Antibody-dependent cellular cytotoxicity (ADCC) is a key mechanism of action of CD38-targeted mAbs; however, high CD38 expression on natural killer (NK) cells results in fratricide, which depletes the NK cells necessary for ADCC. In addition to CD38, targeting of other MM-associated cell-surface proteins has been explored. Of these antigens, the TNF-superfamily member BCMA is among the most researched and is under development by multiple groups as a CAR target. Several clinical trials in MM have shown promising initial results targeting BCMA with CAR T cells, however there remains significant opportunity to improve both relapse rates and treatment of relapsed patients. Collectively, clinical data would suggest that combinatorial targeting of both CD38 and BCMA may improve clinical efficacy compared with targeting either antigen alone. We have developed a multiple-target, adoptive NK cell immunotherapy approach for the treatment of MM. The strategy utilizes our off-the-shelf NK cell platform with four engineered attributes: 1) an anti-BCMA CAR for direct MM targeting, 2) high affinity non-cleavable CD16 (hnCD16) for enhanced ADCC in combination with anti-CD38 mAbs, 3) CD38 deletion for resistance to anti-CD38 mAb induced NK cell depletion, and 4) IL-15/IL-15 receptor α fusion protein (IL-15RF; IL-15 fused to IL-15Rα) for enhanced NK cell persistence. The anti-BCMA CAR consists of a unique single chain variable fragment (scFv) targeting domain with a BCMA binding affinity in the low nanomolar range, providing high functional avidity and efficacy in disease settings where BCMA antigen density is low. Our approach utilizes NK cells derived from a genetically engineered, clonally-derived master pluripotent stem cell line with uniform expression of anti-BCMA CAR, IL-15RF, hnCD16, and CD38 bi-allelic knockout. The engineered master pluripotent stem cell line serves as the starting material for consistent and repeatable manufacture of off-the-shelf NK cells that contain all described attributes in a homogenous manner (termed FT576) and that can be produced at a scale to support multi-dose treatment strategies and on-demand dose availability. In preclinical studies, FT576 NK cells exhibited uniform expression of CD16, CAR, and IL15-RF and did not express CD38 (99% cytotoxicity in a 3D-spheroid culture model. Preclinical studies are ongoing to support the advancement of FT576 as the first-of-kind cellular therapeutic for the combination of anti-BCMA CAR and mAb-directed targeting of MM. Disclosures Bjordahl: Fate Therapeutics, Inc.: Employment. Gaidarova:Fate Therapeutics, Inc: Employment. Goodridge:FATE THERAPEUTICS: Employment. Mahmood:Fate Therapeutics, Inc: Employment. Bonello:Fate Therapeutics, Inc.: Employment. Robinson:Fate Therapeutics, Inc.: Employment. Ruller:Fate Therapeutics, Inc.: Employment. Pribadi:Fate Therapeutics, Inc.: Employment. Lee:Fate Therapeutics, Inc.: Employment. Abujarour:Fate Therapeutics, Inc.: Employment. Dinella:Fate Therapeutics, Inc.: Employment. Huffman:Fate Therapeutics, Inc.: Employment. Chu:FATE THERAPEUTICS: Employment. Valamehr:Fate Therapeutics, Inc: Employment.

Monoclonal antibody (mAb) treatment is an effective therapeutic strategy for many cancer types, with significant opportunity to optimize natural killer (NK) cell and mAb interaction to improve antibody-dependent cellular cytotoxicity (ADCC). NK cells are critical mediators of ADCC, where they recognize and kill malignant cells coated with antibody through the Fc receptor CD16. However, NK cell function is often impaired in cancer patients, which limits the induction of ADCC in mAb therapy. To enhance ADCC in combination with commercialized mAb therapies, we have developed FT516; a novel, off-the-shelf NK cell immunotherapeutic engineered to uniformly express a high-affinity, non-cleavable version of CD16 (hnCD16). FT516 is manufactured from a renewable master induced pluripotent stem cell (iPSC) line with the potential to generate hundreds to thousands of doses of allogeneic NK cells uniformly expressing hnCD16 (hnCD16 iNK cells) per manufacturing run. In an in vivo xenograft model of disseminated lymphoma, FT516 reduced tumor burden below the limit of detection at day 28 after transplant when delivered in combination with rituximab, which was significantly more potent than peripheral blood NK cells (p = 0.03). This was attributed to enhanced CD16-mediated activation of FT516, as observed through improved calcium flux and enhanced activation of signaling pathways such as ERK (p = 0.016), LAT (p = 0.0007), and ZAP70 (p = 0.0003), leading to enhanced ADCC and cytokine production. FT516 also maintained tumor cell specificity, with preferential targeting of K562 leukemia cells when presented with a mixture of K562 and normal PBMC targets (p < 0.0001). We also explored strategies to further engineer therapeutic function to enhance FT516 efficacy. Combined expression of hnCD16 with an IL-15/IL-15ra fusion construct enhanced the persistence of iNK cells and allowed survival of up to 8 weeks in vivo without exogenous cytokine (p < 0.0001), with correlation to improved efficacy. In vitro modeling of FT516 with daratumumab demonstrated ADCC against multiple myeloma (MM) targets. However, as reported, daratumumab induced NK cell fratricide through binding of CD38 on NK cells. To rescue daratumumab-mediated fratricide, we specifically deleted CD38 at the iPSC level and demonstrated that fratricide was undetectable in hnCD16 CD38-/- iNK cells ( Citation Format: Ryan Bjordahl, Huang Zhu, Paul Rogers, Svetlana Gaidarova, Moyar Q. Ge, Robert Blum, Frank Cichocki, Jode Goodridge, Helen Chu, Greg Bonello, Tom Lee, Brian Groff, Ramzey Abujarour, Bruce Walcheck, Jeffrey Miller, Dan Kaufman, Bahram Valamehr. FT516, an off-the-shelf engineered NK cell therapeutic product for universal anti-tumor targeting strategy in combination with monoclonal antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3191.

The unprecedented success of chimeric antigen receptor (CAR) and monoclonal antibody (mAb) -based immune-therapies has provided a clear indication that a system as complex as human immunity can be harnessed, even enhanced, toward a growing number of hematological cancers. Here we describe pre-clinical progress to develop a multi-functional induced pluripotent stem cell (iPSC)-derived natural killer (iNK) cell platform that combines engineered longevity with CAR and mAb-based modalities to leverage the intrinsic polyfunctionality of NK cells. As frontrunners of immune surveillance, NK cells employ a diverse array of germline encoded receptors in distinct combinations, which engage multiple signaling pathways to deliver potent effector responses that can be directed toward tumor cells, drive rapid proliferation, and pave the way for recruitment of adaptive immunity. Specific engagement of multiple signaling pathways was achieved in iNK cells through design of an NK cell-centric CAR combining the transmembrane domain of activating receptor NKG2D with intracellular signaling domains of 2B4 and CD3ζ. Recombining an anti-CD19 scFv onto this signaling platform, CAR modified iNK cells produced specific in vitro recognition of CD19+ B cell lymphoma cells in short term and long term cytotoxicity assays (84% vs 40% clearance of tumor cells at 60H, p99% vs 90% clearance of tumor cells) and CD19- targets (>99% vs 50% clearance of tumor cells by iNK with anti-CD19 CAR alone, p Citation Format: Jode Goodridge, Sajid Mahmood, Huang Zhu, Svetlana Gaidarova, Robert Blum, Ryan Bjordahl, Frank Cichocki, Hui-Yi Chu, Greg Bonello, Tom Lee, Brian Groff, Karl-Johan Malmberg, Bruce Walcheck, Jeffrey Miller, Dan Kaufman, Bahram Valamehr. Preclinical development of first-of-kind dual-targeted off-the-shelf CAR-NK cell product with engineered persistence for an effective treatment of B cell malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3207.

Adoptive transfer of haplo-donor NK cells can achieve remission in patients with relapsed or refractory acute myeloid leukemia (AML). This strategy is limited by NK cell heterogeneity, lack of specificity, and single dosed strategies. To overcome these barriers, we genetically modified induced pluripotent stem cells (iPSCs) to stably over-express NKG2C alone or along with DAP12. CD94/NKG2C is a heterodimeric receptor that binds to HLA-E and associates with DAP12, a protein containing an immunoreceptor tyrosine-based activating motif. Efficient expression of CD94/NKG2C on the cell surface requires the presence of DAP12, and charged amino acids in the transmembrane domains of DAP12 and NKG2C mediate this interaction. Physiologic NKG2C expression is limited to subsets of NK and CD8+ T cells that expand in response to cytomegalovirus (CMV) infection. Engineered iPSCs were differentiated into CD34+ hematopoietic progenitor cells and subsequently differentiated into CD56+ NK cells (iNK cells) using a stepwise differentiation and expansion strategy. Surface NKG2C expression was low on unmodified iNK cells (10.7%) with higher levels of NKG2C on the surface of iNK cells derived from the NKG2C only line (65.1%). These results demonstrate that transgenic NKG2C can pair with endogenous CD94 and DAP12 to form stable structures on the cell surface. Importantly, surface NKG2C expression was highest on iNK cells derived from the NKG2C/DAP12 line (84.1%) with higher MFI, demonstrating the importance of engineering a line with both components (Figure 1A). To validate function via engagement of NKG2C on iNK cells, we used a redirected antibody dependent cellular cytotoxicity (ADCC) assay where P815 cells (which bind the Fc portion of antibodies) were coated with a monoclonal anti-NKG2C agonist antibody at a range of concentrations (0.01-10 mg/ml) and co-cultured with unmodified iNK cells, NKG2C iNK cells or NKG2C/DAP12 iNK cells. We found that at all concentrations tested, NKG2C/DAP12 iNK cells demonstrated the strongest functional response as measured by the frequencies of surface CD107a expression, interferon (IFN)-g production and tumor necrosis factor (TNF) production. Intermediate functional responses were observed for NKG2C iNK cells (Figure 1B). Having demonstrated stable expression and intact signaling of NKG2C/DAP12 in iNK cells, we next created a Tri-specific Killer Engager (TriKE) molecule with single chain Fv fragments specific for NKG2C (on iNK cells) and CD33 (on AML cells), as well as IL-15 (to support iNK cell survival and proliferation). This NKG2C/CD33/IL-15 TriKE was tested in functional assays where NKG2C iNK cells or NKG2C/DAP12 iNK cells were used as effectors against CD33+ THP1 cells (an AML cell line shown to be refractory to NK cell cytotoxicity). Without the addition of NKG2C/CD33/IL-15 TriKE, the frequencies of both NKG2C iNK cells and NKG2C/DAP12 iNK cells producing IFN-g was relatively low (< 10%). Addition of NKG2C/IL-15/CD33 TriKE in the assay markedly increased the frequency of IFN-g production by both NKG2C iNK cells (19.1%) and NKG2C/DAP12 iNK cells (25.8%) (Figure 1C). Finally, we directly tested the ability of the NKG2C/IL-15/CD33 TriKE molecule to trigger target killing by engineered iNK cells monitored over a 24-hour period. THP1 target cells were dye-labeled and plated in wells with either NKG2C iNK cells or NKG2C/DAP12 iNK cells at a 5:1 effector-to-target ratio with or without NKG2C/CD33/IL-15 TriKE. We found that in the absence of TriKE, there was no killing of THP1 targets. In contrast, both NKG2C iNK cells and NKG2C/DAP12 iNK cells mediated robust THP1 killing with the addition of NKG2C/CD33/IL-15 TriKE, with NKG2C/DAP12 iNK cells exhibiting the strongest response (Figure 1D). Our results provide a proof-of-concept that iNK cells engineered to express NKG2C/DAP12 can be used in combination with a novel NKG2C/IL-15/CD33 TriKE molecule to effectively target cancer cells. Engaging NK cells through NKG2C, restricted to adaptive NK cells or genetically modified iPSC will be more specific than CD16, which will bind to CD16A on NK cells but also have off-target binding to CD16B on neutrophils. Because of the enormous expansion and engineering capacity of the iNK cell platform, we are in a unique position to create an "off-the-shelf" NK cellular therapy that is targeted, specific and efficacious. Disclosures Cichocki: Fate Therapeutics Inc.: Consultancy, Research Funding. Felices:GT Biopharma: Research Funding. Chu:Fate Therapeutics Inc.: Employment. Ge:Fate Therapeutics Inc.: Employment. Bjordahl:Fate Therapeutics Inc.: Employment. Kaufman:Fate Therapeutics: Consultancy, Research Funding. Malmberg:Fate Therapeutics Inc.: Consultancy, Research Funding. Valamehr:Fate Therapeutics Inc.: Employment.

Daratumumab targets the cell surface protein CD38 and is the only FDA approved monoclonal antibody that has demonstrated single agent efficacy in relapsed refractory myeloma. CD38 is broadly expressed in the immune system, and its high expression on multiple myeloma cells allows for effective targeting by daratumumab. Daratumumab induces myeloma cell death through multiple mechanisms, including complement-dependent cytotoxicity, antibody-dependent cellular phagocytosis, and perhaps most importantly, antibody-dependent cellular cytotoxicity (ADCC). ADCC is mediated by binding of the antibody Fc region to the CD16 Fc receptor expressed on natural killer (NK) cells. Engagement of CD16 induces NK cell activation and target cell cytolysis. However, because CD38 is also expressed on the surface of NK cells, daratumumab treatment can induce NK cell fratricide, which likely impairs the effectiveness of ADCC-mediated targeting and elimination of myeloma. In addition, NK cell function is often suppressed or absent in patients with myeloma, as a result of the tumor itself or from its therapy, further reducing the effectiveness of daratumumab. Collectively, preclinical and clinical observations suggest a potential therapeutic benefit of maintaining NK cell numbers and function in patients to support daratumumab-mediated ADCC and augment the treatment of multiple myeloma. We have developed an off-the-shelf NK cell immunotherapy derived from genetically engineered, induced pluripotent stem cells (iPSC) for enhanced ADCC in combination with daratumumab. iPSCs were engineered to express a high-affinity, non-cleavable CD16 construct (hnCD16) in combination with complete bi-allelic disruption of the CD38 gene (hnCD16 CD38-/-), and the engineered iPSCs were subsequently differentiated into NK (iNK) cells. We hypothesized that CD38-deficient iNK cells would exhibit improved survival by avoiding daratumumab-induced NK cell fratricide, while expression of the hnCD16 transgene would enhance ADCC against myeloma cells in combination with daratumumab. Genetic modification was confirmed in hnCD16 CD38-/- iNK cells by flow cytometry, demonstrating abrogation of CD38 expression (Fig. 1A) and constitutive high expression of CD16 (Fig. 1B). Additionally, hnCD16 iNK cells and hnCD16 CD38-/- iNK cells expressed similar levels of SLAMF7/CD319 (the target of elotuzumab) and NKG2A (Fig. 1C and D). No significant difference in iNK cell differentiation, expansion, maturation, activation, or ability to mediate natural cytotoxicity was observed. In contrast to previous reports, we observed no effect of CD38-deficiency on CD16-mediated calcium flux between hnCD16 iNK cells and hnCD16 CD38-/- iNK cells (Figure 1E). In vitro culture of NK cells in the presence of daratumumab led to NK cell fratricide for both peripheral blood-derived NK cells and hnCD16 iNK cells (Fig. 1F). Daratumumab-induced NK cell fratricide was dependent upon expression of both CD16 and CD38, as unmodified iNK with low CD16 levels (~20% of cells) showed reduced cell death in the presence of daratumumab, which was entirely absent in hnCD16 CD38-/- iNK cells (Fig. 1F). This data was confirmed by extended culture of NK cells with RPMI-8226 tumor spheroids in the presence or absence of daratumumab. The number of hnCD16 iNK cells and peripheral blood NK cells were significantly reduced compared to hnCD16 CD38-/- iNK cells (p>0.005, Fig. 1 G). Importantly, hnCD16 CD38-/- iNK cells were better able to mediate ADCC towards MM1.S multiple myeloma cells compared to hnCD16 iNK cells (Fig. 1H). Taken together, these data support our hypothesis that targeted knock out of CD38 on NK cells alleviates daratumumab-induced NK cell fratricide that occurs through the crosslinking of CD16 and CD38 on neighboring NK cells, leading to augmented anti-myeloma immunity. These data provide a translatable, proof of concept study demonstrating precision genetic engineering of iPSC to generate off-the-shelf NK cell immunotherapy to enhance daratumumab mediated ADCC in multiple myeloma. We propose a strategy of off-the-shelf hnCD16 CD38-/- iNK infusion in combination with daratumumab to overcome NK cell depletion effects of CD38 targeted agents and to improve myeloma patient outcomes. Figure 1. Figure 1. Disclosures Bjordahl: Fate Therapeutics Inc.: Employment. Cichocki:Fate Therapeutics Inc.: Consultancy, Research Funding. Gaidarova:Fate Therapeutics Inc: Employment. Pride:Fate Therapeutics Inc.: Employment. Kaufman:Fate Therapeutics: Consultancy, Research Funding. Malmberg:Fate Therapeutics Inc.: Consultancy, Research Funding. Valamehr:Fate Therapeutics Inc.: Employment.

T lymphocytes that express an anti-CD19 chimeric antigen receptor (CAR) to redirect target specificity exhibit remarkable remissions in B cell malignancies. However, these cells are typically produced in a patient-specific manner that is relatively inefficient and expensive. Additionally, CAR-T cell treatment can lead to severe adverse events (SAEs) such as cytokine release syndrome (CRS) and neurotoxicity, as well as graft versus host disease (GvHD) when given in allogeneic setting. To circumvent these safety issues while maintaining multi-faceted anti-tumor activity, we developed an off-the-shelf natural killer (NK) cell consisting of a novel CAR combined with other effector mechanisms to enhance targeted cytotoxicity. NK cells are potent anti-tumor effector cells that play an important role in innate and adaptive immunity. Multiple clinical studies have demonstrated that adoptive transfer of allogeneic NK cells can induce durable remissions in patients with cancers that have relapsed or are refractory to standard therapies without detection of SAEs such as CRS. However, NK cells are challenging to genetically engineer, and are dependent on cytokine support for persistence and exhibit donor-to-donor variability factors that make it difficult to create a consistent clinical product from NK cells. Here we report the use of human induced pluripotent stem cells (iPSCs) to produce a renewable source of precisely engineered NK cells. This iPSC platform was utilized to evaluate a combination of CARs comprised of distinct NK-cell specific signaling and transmembrane domains with an autonomous protein to create a persistent and targeted NK cell immunotherapy. The selected NK cell optimized CAR (NK-CAR) backbone contains an NKG2D transmembrane domain, a 2B4 co-stimulatory domain, and a CD3ζ signaling domain to mediate a potent NK cell activating signal. To provide directed anti-tumor activity, anti-CD19 scFv was added to the NK-CAR backbone, engineered at the iPSC stage and subsequently differentiated on-demand to produce a uniform population of CAR-expressing NK cells. In addition, an IL-15RF fusion transgene was introduced to provide self-stimulating signals to support NK cell function and persistence. The IL-15RF construct was created by fusing IL-15Rα to IL-15 at the C-terminus through a flexible linker. As a third modality, a metalloprotease ADAM17-resistant version of the high-affinity CD16a (hnCD16) 158V variant was introduced at the iPSC stage to augment antibody-dependent cellular-cytotoxicity (ADCC) when used in combination with monoclonal antibodies. The selected iPSC clone exhibits stable expression of all three modalities and represents a renewable source of starting material for the reproducible generation of NK cells consisting of NK-CAR, IL-15RF and hnCD16 with product purity that is greater than 95% CD45+CD56+ with a product expansion greater than one million-fold over the course of the manufacturing process. In preclinical studies, these multi-functional engineered NK cells demonstrated enhanced directed cytotoxicity against CD19+ tumor targets when compared to non-engineered NK cells or iPSC-derived NK cells engineered with other CAR constructs. Additionally, the multi-functional engineered iPSC-CAR-NK cells significantly reduced tumor burden in a xenograft model of B acute lymphoblastic leukemia (p Disclosures Kaufman: Fate Therapeutics: Consultancy, Research Funding. Bjordahl:Fate Therapeutics Inc.: Employment. Mahmood:Fate Therapeutics Inc.: Employment. Bonello:Fate Therapeutics Inc.: Employment. Lee:Fate Therapeutics Inc.: Employment. Cichocki:Fate Therapeutics Inc.: Consultancy, Research Funding. Valamehr:Fate Therapeutics Inc.: Employment.

Natural killer (NK) cells are potent antitumor effectors that play an important role in innate and adaptive immunity. Despite recent clinical advances in the therapeutic use of NK cells, significant opportunities remain to harness their full potential in adoptive immunotherapy. For example, achieving consistent manufacturing of cancer immunotherapies using patient- and donor-sourced cells remains a significant challenge to delivering therapies to all patients who may benefit. There is also the need to improve the efficacy and persistence of adoptively transferred lymphocytes to promote favorable patient outcomes. We have previously demonstrated that induced pluripotent stem cells (iPSCs) can be genetically edited at the single-cell level and expanded to create clonal master engineered pluripotent cell lines, representing a renewable and reliable starting material for the creation of off-the-shelf (OTS) adoptive NK cell therapy. Using this unique platform, we evaluated the combination of NK cell-specific chimeric antigen receptors (CARs) with an autonomous protein to create a highly effective, persistent, and targeted NK cell therapy. The NK cell optimized CAR (NK-CAR) backbone contains the NKG2D transmembrane domain and the 2B4 co-stimulatory and the CD3ζ signaling domains to mediate a strong increase in NK cell signaling. To provide directed antitumor activity, anti-mesothelin (meso) and anti-CD19 scFvs were added to the NK-CAR backbone, and then engineered into the iPSC and subsequently differentiated to NK cells expressing NK-CARmeso or NK-CAR19. Using an ovarian cancer xenograft model in the initial study, a single dose of NK-CARmeso NK cells markedly inhibited tumor growth and mediated enhanced survival (84 days) compared to various controls, including NK cells harboring a 3rd-generation T-cell CAR construct with CD28 and 4-1BB costimulatory domains (p < 0.002). We next engineered an IL-15RF fusion protein to provide self-stimulating signals to support NK cell function and persistence. The IL-15RF construct was created by fusing mature IL-15Rα to IL-15 at the C-terminus through a flexible linker. The design mimics the trans-presentation of IL-15 bound to IL-15Rα that is presented to IL-15Rβ/γC dimer to initiate signaling. While both iPSC-derived NK cells (iNKs) and iNKs bearing IL-15RF expanded in vitro in a similar manner in the presence of soluble IL-15 and IL-2 (2040- and 3615-fold expansion in 14 days, respectively), only the iNKs bearing IL-15RF significantly proliferated in the absence of cytokines (10- vs. 701-fold expansion in 14 days, respectively). We are currently investigating the combination of NK-CAR and IL-15RF to determine the potential synergy between the two modalities to create cytokine-autonomous, OTS NK cell cancer immunotherapies, including those that are CAR-targeted against solid and liquid tumors. Citation Format: Tom T. Lee, Ye E. Li, Ryan Bjordahl, Robert Blum, Sajid Mahmood, Huang Zhu, Gregory B. Bonello, Bahram Valamehr, Dan S. Kaufman. Cytokine-autonomous, CAR-directed, off-the-shelf natural killer cells derived from a clonal engineered master pluripotent cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3574.

The development of PD1/PDL1 targeting checkpoint inhibitors (CI) has transformed the oncology landscape, providing long term remissions in multiple indications. However, many tumor subtypes are resistant to checkpoint blockade therapy, and relapse remains a significant concern. Novel therapeutic approaches with the ability to overcome CI resistance are needed, and there is significant opportunity for therapies capable of additively or synergistically enhancing T-cell activation and recruitment when combined with CI. Adoptive transfer of NK cells from healthy donors has the potential to recruit T cells to the tumor microenvironment and augment T-cell activation at the tumor site. NK cells have both direct anti-tumor activity and the capacity to secrete inflammatory cytokines and chemokines upon activation, enabling the cells to play a unique and critical role in regulating anti-tumor T cell activity. We sought to determine whether FT500, an off-the-shelf NK cell product derived from a clonal master pluripotent cell line, could synergize with CI to relieve local immunosuppression and enhance T-cell activation and recruitment to the tumor site. FT500 is universally negative for cell surface PD1, and expression of PDL1 on tumor lines had no discernable effect on FT500 cytotoxicity. Similarly, addition of PDL1 blocking antibody had no effect on FT500 cytotoxicity or degranulation, suggesting that FT500 is inherently resistant to PDL1-PD1 mediated inhibition. Additionally, activation of FT500 induced the secretion of soluble factors capable of enhancing T-cell activation, as evidenced by increased upregulation of CD69. We hypothesized that FT500 might also enhance CI by promoting recruitment of T cells to the tumor site. Using conventional in vitro transwell migration assays, we found that FT500 produced soluble factors that promoted the migration of activated T cells. Additional profiling confirmed FT500 production of a range of chemokines, including CCL3, CCL4, CXCL10 and CCL22. Furthermore, using an in vivo recruitment model, FT500 was able to recruit T cells out of the circulation and into the peritoneal cavity. Similarly, utilizing a three-dimensional tumor spheroid model in vitro, infiltration of T cells into tumor spheroids was significantly enhanced when combined with FT500, suggesting that FT500 can enhance tumor infiltration of T cells. Our data suggest that FT500 is a potent producer of chemokines and can facilitate the recruitment of T cells to the tumor site. In addition to its direct cytotoxic potential, FT500 is also able to enhance T-cell activation, suggesting an ability to synergize with CI to reduce tumor burden. Together, our data provide evidence supporting the combination of FT500, an off-the-shelf NK cell cancer immunotherapy, with CI to overcome checkpoint blockade resistance. Citation Format: Ryan Bjordahl, Sajid Mahmood, Svetlana Gaidarova, Ramzey Abujarour, Raedun Clarke, Laurel Stokely, Paul Rogers, Moyar Ge, Megan Robinson, Betsy Rezner, Tom Tong Lee, Bahram Valamehr. FT500, an off-the-shelf NK cell cancer immunotherapy derived from a master pluripotent cell line, enhances T-cell activation and recruitment to overcome checkpoint blockade resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3576.

Both peptidoglycan and muropeptides potently modulate inflammatory and innate immune responses. The secreted Listeria monocytogenes p60 autolysin digests peptidoglycan and promotes bacterial infection in vivo. Here, we report that p60 contributes to bacterial subversion of NK cell activation and innate IFN-gamma production. L. monocytogenes deficient for p60 (Deltap60) competed well for expansion in mice doubly deficient for IFNAR1 and IFN-gammaR1 or singly deficient for IFN-gammaR1, but not in wild-type, IFNAR1(-/-), or TLR2(-/-) mice. The restored competitiveness of p60-deficient bacteria suggested a specific role for p60 in bacterial subversion of IFN-gamma-mediated immune responses, since in vivo expansion of three other mutant L. monocytogenes strains (DeltaActA, DeltaNamA, and DeltaPlcB) was not complemented in IFN-gammaR1(-/-) mice. Bacterial expression of p60 was not required to induce socs1, socs3, and il10 expression in infected mouse bone marrow macrophages but did correlate with enhanced production of IL-6, IL-12p70, and most strikingly IFN-gamma. The primary source of p60-dependent innate IFN-gamma was NK cells, whereas bacterial p60 expression did not significantly alter innate IFN-gamma production by T cells. The mechanism for p60-dependent NK cell stimulation was also indirect, given that treatment with purified p60 protein failed to directly activate NK cells for IFN-gamma production. These data suggest that p60 may act on infected cells to indirectly enhance NK cell activation and increase innate IFN-gamma production, which presumably promotes early bacterial expansion through its immunoregulatory effects on bystander cells. Thus, the simultaneous induction of IFN-gamma production and factors that inhibit IFN-gamma signaling may be a common strategy for misdirection of early antibacterial immunity.

Natural killer (NK) cells represent a lineage of immune cells capable of direct cytotoxicity against tumor cells and are a critical source of key inflammatory cytokines such as interferon (IFN)-γ and tumor necrosis factor (TNF). NK cell function is often impaired in the setting of cancer, reducing the effectiveness of the endogenous immune system. The unique biological attributes of NK cells, including multifaceted effector function, tumor cell recognition independent of antigen presentation and target cell selectivity independent of HLA-matching, has enabled NK cells from a donor to be adoptively transferred to a patient for the treatment of cancer. This safe and effective administration of donor NK cells to patients validates their potential for broad use as part of an off-the-shelf cancer immunotherapy strategy, including in combination with monoclonal antibody and checkpoint inhibitor therapies. We have previously shown that human induced pluripotent stem cells (hiPSC) can be clonally-selected, cryopreserved and banked, and that these master pluripotent cell lines (MPCLs) can be used to renewably generate large clonal populations of NK cells. The use of MPCLs represents a highly-promising, off-the-shelf approach to cell-based cancer immunotherapy, with the potential to overcome many of the challenges and limitations of patient-sourced and donor-derived cell therapies. However, to clinically and commercially enable this off-the-shelf strategy, it is essential to efficiently and reproducibly differentiate MPCLs to fully-functional NK cells using a robust and scalable process that meets regulatory requirements. Here we describe a novel paradigm for the manufacture of hiPSC-derived NK (iNK) cells consisting of a well-defined, small molecule-driven, staged protocol that enables clinical translation and is compatible with current good manufacturing practice (cGMP) requirements. The manufacturing protocol is currently being transferred from the laboratories of Fate Therapeutics to Molecular and Cellular Therapeutics at the University of Minnesota, which is a state-of-the-art GMP/GTP compliant, full-service developer and manufacturer of cell- and tissue-based products. iNK cell therapy manufacture consists of four unique steps including: 1) the derivation and master cell banking of a clonal pluripotent cell line (> 95% SSEA4+/TRA181+ hiPSCs); 2) differentiation of the clonal pluripotent cell line towards hematopoietic progenitor cells (enriched for > 80% CD34+ cells); 3) differentiation and expansion of iNKs (Figure 1A, approximately 1,000-fold expansion in 14 days); and 4) freeze and thaw of drug product, comprised of a sufficiently pure homogenous population of iNKs (Figure 1B, > 95% CD45+, > 90% CD56+, minimal CD3+ T cells). Importantly, testing at both the molecular and culture stages demonstrate that no hiPSCs exist in the final drug product (limit of detection 1 hiPSC in 1.25 million iNK cells). This novel manufacturing paradigm supports the generation of significant numbers of iNK cells: approximately 1 million-fold cell expansion is achieved in less than 50 days, such that a very small population of hiPSCs can readily produce 1x1012 iNK cells. We estimate that this represents hundreds of doses of drug product per each manufacturing run (Figure 1A). The iNK cells display markedly augmented effector function relative to ex vivo expanded primary peripheral blood or cord blood NK cells with respect to cytokine release (IFN-γ and TNF) and cellular cytotoxicity against various leukemic and solid tumor-derived target cells including K562, Raji, A549 and SKOV3 (Figure 1C). To enable centralized manufacturing, we established a freeze and thaw strategy that supports greater than 85% viability with a recovery of greater than 80% iNK cells at twenty-four hours post-thaw. Because the freeze process uses an infusible medium formulation, we demonstrated in vitro and in vivo that the iNK cells maintain their efficacy post-thaw and can be immediately infused into patients. The manufacturing data presented herein support the filing of an Investigational New Drug application for an off-the-shelf iNK cell therapy product to treat advanced hematologic and solid tumor malignancies alone or in combination with monoclonal antibody and checkpoint inhibitor therapies. Disclosures Bjordahl: Fate Therapeutics: Employment, Equity Ownership. Gaidarova: Fate Therapeutics Inc.: Employment, Equity Ownership. Rogers: Fate Therapeutics Inc.: Employment, Equity Ownership. Clarke: Fate Therapeutics Inc.: Employment, Equity Ownership. Groff: Fate Therapeutics Inc.: Employment. Moreno: Fate Therapeutics Inc.: Employment. Abujarour: Fate Therapeutics Inc.: Employment. Robinson: Fate Therapeutics Inc.: Employment. Bonello: Fate Therapeutics Inc.: Employment. Lee: Fate Therapeutics Inc.: Employment, Equity Ownership. Lan: Fate Therapeutics Inc.: Employment, Equity Ownership. Rezner: Fate Therapeutics, Inc.: Employment. Abbot: Fate Therapeutics Inc.: Employment. Wolchko: Fate Therapeutics Inc.: Employment. Kaufman: Fate Therapeutics: Consultancy, Research Funding. Valamehr: Fate Therapeutics: Employment, Equity Ownership. Miller: Oxis Biotech: Consultancy; Celegene: Consultancy; Fate Therapeutics: Consultancy, Research Funding.

Encouraging clinical outcomes in autologous cellular immunotherapy have garnered hope and excitement. However, limitations of patient-derived cancer immunotherapies remain to be addressed to deliver reliable and efficacious therapies with broader applicability. Induced pluripotent stem cells (iPSCs) are a unique, renewable source for the continuous generation of cellular therapeutics and represent a highly promising approach for overcoming many of the limitations of autologous therapy. To advance the promise of iPSC technology as an “off-the-shelf” (OTS) source of cellular therapeutics, several considerations need to be addressed. Ensuring the persistence of allogeneic OTS therapies after adoptive cell transfer across histocompatibility barriers is a key requirement. Establishing a master cell line from genetically engineered clonal iPSC lines with the capacity to continuously generate homogenous populations of highly functional effector cells will also be necessary. Here we demonstrate a comprehensive approach for the generation of immune tolerant effector cells derived from a genetically engineered iPSC master cell line. We successfully combined deletion of classical human leukocyte antigen molecules with expression of immunosuppressive proteins to generate clonal iPSC lines with the ability to escape immune rejection. Utilizing in vitro quantitative live cell analysis we show that OTS-iPSCs elicit a significantly decreased cytotoxic response from both peripheral blood (PB)-NK cells (47.9 vs. 91.4% survival at 3:1 E:T ratio) and PB-T cells (>2.7-fold greater number of OTS-iPSC derived cells remaining at 88 hrs). Additionally, mixed lymphocyte reactions employing unfractionated PB mononuclear cells resulted in significantly decreased activation and proliferation of CD8+ T cells (63.4 vs. 29.6%), CD4+ T cells (70.9 vs. 17.3%) and NK cells (46.8 vs. 11.6%). In preclinical mouse models we demonstrate that OTS-iPSCs exhibit improved persistence in vivo. Bilateral engraftments were established in non-conditioned, fully immune-competent recipient mice using luciferized wildtype and OTS-iPSCs. Daily bioluminescence imaging revealed a significant increase in persistence of OTS-iPSCs during the 48-196 hour post injection window (>5.5 fold greater luminescence at 96 hrs). Using our potent chemically-defined stage-specific monolayer hematopoietic differentiation platform, we demonstrate that OTS-iPSC derived CD34 expressing hematopoietic cells are reproducibly scaled and readily give rise to functional lymphocytes carrying the engineered targeted modality in a homogenous manner (95 +/- 5%). The outlined preclinical data illustrate that iPSCs are an ideal renewable source for OTS hematopoietic cell-based immunotherapies and represent a potentially exponential advancement in adoptive immunotherapy. Citation Format: Raedun L. Clarke, Matthieu Bauer, Ryan Bjordahl, Jeffrey Sasaki, Brian Groff, Svetlana Gaidarova, Tom Tong Lee, Weijie Lan, Michelle Burrascano, Ramzey Abujarour, Greg Bonello, Megan Robinson, Stewart Abbot, Scott Wolchko, Daniel Shoemaker, Bob Valamehr. Overcoming host histocompatibility barrier to create a renewable source of off-the-shelf effector lymphocytes for adoptive immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 609. doi:10.1158/1538-7445.AM2017-609

The unique attributes of a combinatorial tumor recognition system, diminished off-tumor cytotoxicity, and multifaceted effector function make natural killer (NK) cells a prime candidate for a universal approach to cancer immunotherapy. In addition, NK cells are the principal mediator of antibody-directed cellular cytotoxicity (ADCC). However, NK cell function is often impaired in the setting of cancer, reducing the effectiveness of the endogenous immune system and the therapeutic efficacy of monoclonal antibodies. To address the need for advanced and combinatorial cancer therapies, we developed a unique and effective strategy to create a renewable source of engineered “off-the-shelf” NK cells with augmented function, including enhanced ADCC and persistence. Key challenges associated with genetic editing, limited expansion, persistence and variability of peripheral blood (PB)-derived NK cells were overcome by utilizing our induced pluripotent stem cell (iPSC) technology as the unlimited starting material for the reproducible and consistent derivation of engineered NK cells. Through targeted transgene integration, we produced a clonal iPSC master cell line to continuously produce NK cells engineered to uniformly express a high affinity, non-cleavable version of CD16 (hnCD16-NK). In directed differentiation, the hnCD16-NK cells displayed homogeneous expression of CD16 (>95%) and a mature CD56+ NK cell phenotype, as exhibited by expression of KIR, NCRs, DNAM-1, and NKG2D. In contrast to endogenous CD16 expression, the engineered hnCD16 molecule was shown to be cleavage resistant upon NK cell activation (>95% CD16+ hnCD16-NK vs. Citation Format: Ryan Bjordahl, Frank Cichocki, Raedun Clarke, Svetlana Gaidarova, Brian Groff, Paul Rogers, Stacey Moreno, Ramzey Abujarour, Greg Bonello, Tom Lee, Weijie Lan, Matthieu Bauer, Dave Robbins, Betsy Rezner, Sarah Cooley, Bruce Walcheck, Stewart Abbot, Bruce Blazar, Scott Wolchko, Daniel Shoemaker, Jeffrey S. Miller, Bahram Valamehr. Renewable and genetically engineered natural killer cells for off-the-shelf adoptive cellular immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3755. doi:10.1158/1538-7445.AM2017-3755

Natural killer (NK) cells are innate lymphoid cells that mediate immune responses against pathogens and cancer. Human NK cells are distinguished by the surface phenotype CD3-CD56+, and maturation of CD56dim NK cells is associated with acquisition of CD57. Rather than being an immunosenescence marker, CD57 acquisition represents a shift toward greater effector function, including increased CD16 signaling (Fc receptor responsible for triggering antibody-dependent cellular cytotoxicity), more potent cytotoxicity and enhanced inflammatory cytokine production after target cell engagement. The main challenge in enriching for CD57+ NK cells using current ex vivo expansion protocols is that interleukin (IL)-15, the cytokine that drives NK cell proliferation and is critical for NK cell survival, preferentially expands less mature NK subsets that fail to terminally differentiate in culture. Our group has developed a novel NK cell expansion method that overcomes this barrier. Peripheral blood mononuclear cells from are depleted of CD3+ T cells and CD19+ B cells and cultured for 7 days with IL-15 and a small molecule inhibitor of glycogen synthase kinase 3-beta (GSK3β), a multifunctional kinase downstream of the PI(3)K pathway. Compared to vehicle control, addition of the GSK3β inhibitor led to a substantial increase (2.2-fold ± 0.19, n=23, p We have scaled our process to manufacture a GMP product (referred to as FATE-NK100) for clinical use. Using an apheresis product from a donor containing 21.5 x 108 CD57+ NK cells, we achieved 6.4-fold NK cell expansion resulting in a final GMP-grade product containing 158 x 108 CD57+ NK cells. The cytotoxicity of these ex vivo expanded NK cells in response to SKOV-3 cells is superior to that of CD3/CD19-depleted haploidentical NK cells activated overnight with either IL-2 or IL-15 (representing the NK products used in current clinical trials). These data have been presented to the FDA in preparation for a clinical trial of FATE-NK100 in lymphodepleted patients with advanced AML anticipated for Q1 2017. Citation Format: Frank Cichocki, Barham Valamehr, Ryan Bjordahl, Bin Zhang, Dhifaf Sarhan, Sarah Cooley, Bruce Blazar, Betsy Rezner, Paul Rogers, Chad Green, Stewart Abbot, Daniel Shoemaker, Scott Wolchko, Jeffrey S. Miller. FATE-NK100: A novel NK cell-mediated cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3752. doi:10.1158/1538-7445.AM2017-3752

The roles of many non-oncogenic mutations in cancer may influence tumor growth, survival, or how tumors interact with their surroundings. Here we characterize the functional relevance of missense mutations within the gene encoding the tumor necrosis receptor family member HVEM (TNFRSF14), a locus frequently targeted within human lymphoma and other cancers. We find that point mutations identified in human lymphoma were localized to the extracellular domain and specifically target ligand binding, resulting in preferential loss of CD160 and BTLA interactions compared to LIGHT (TNFSF14). Missense mutations were associated with alterations in cytotoxic effector cell signatures within tumor biopsies, while deletion mutations were associated with changes in myeloid cell signatures. Finally, we find that mutated HVEM proteins retained the capacity to inhibit T cell signaling through BTLA, while reducing costimulation of cytolysis in NK cells through CD160. Together, these data provide evidence for how immune selective pressures may drive mutation of TNFRSF14 resulting in greater tumor fitness.

Natural killer (NK) cells are innate lymphoid cells that mediate immune responses against pathogens and cancer. Human NK cells are distinguished by the surface phenotype CD3-CD56+ and differential expression of the CD56 surface antigen defines subsets. CD56bright NK cells are presumed to be precursors of CD56dim NK cells, and terminal maturation of CD56dim NK cells is associated with acquisition of CD57. Rather than being an immunosenescence marker CD57 acquisition represents a shift toward greater effector function, including increased CD16 signaling (Fc receptor responsible for triggering antibody-dependent cellular cytotoxicity), enhanced cytotoxicity and decreased responsiveness to interleukin (IL)-12 and IL-18 stimulation. Cytomegalovirus (CMV) infection is uniquely associated with expansion of CD57+ NK cells expressing the activating receptor NKG2C.We have reported that in vivo expanded of CD57+NKG2C+ NK cells (referred to as adaptive NK cells) persist for over one year and are directly associated with reduced leukemia relapse after reduced intensity hematopoietic cell transplantation. Ex vivo expansion to enrich the subset of cells with the adaptive NK cell phenotype represents a new strategy to obtain high numbers of NK cells with enhanced effector function for use in adoptive transfer to treat cancer patients. The main challenge in enriching for CD57+ NK cells using current ex vivo expansion protocols is that IL-15, the cytokine that drives NK cell proliferation and is critical for NK cell survival preferentially expands less mature NK subsets that fail to terminally differentiate in culture. Our group has developed a novel NK cell expansion method that overcomes this barrier. Peripheral blood mononuclear cells from CMV seropositive donors are depleted of CD3+ T cells and CD19+ B cells and cultured for 7-9 days with IL-15 and a small molecule inhibitor of glycogen synthase kinase 3-beta (GSK3β), a multifunctional kinase downstream of the PI(3)K pathway. Compared to vehicle control, addition of the GSK3β inhibitor led to a substantial increase (2.2-fold ± 0.19, n=23, p Disclosures Cichocki: Fate Therapeutics, Inc: Research Funding. Valamehr:Fate Therapeutics, Inc: Employment. Cooley:Fate Therapeutics: Research Funding. Bjordahl:Fate Therapeutics, Inc: Employment. Rezner:Fate Therapeutics, Inc: Employment, Equity Ownership. Rogers:Fate Therapeutics, Inc: Employment. Green:Fate Therapeutics, Inc: Employment. McKenna:Fate Therapeutics, Inc: Research Funding. Shoemaker:Fate Therapeutics: Employment, Equity Ownership. Wolchko:Fate Therapeutics: Employment. Miller:Fate Therapeutics: Consultancy, Research Funding; Oxis Biotech: Consultancy, Other: SAB.

Encouraging clinical outcomes in autologous cellular immunotherapy have garnered hope and excitement. However, considerable challenges and limitations of patient-derived cancer immunotherapies remain and need to be addressed in order to consistently deliver reliable and efficacious therapies with broadened applicability. Human induced pluripotent stem cells (hiPSCs) are a unique, renewable source for the continuous generation of cellular therapeutics for the treatment of hematological and non-hematological malignancies, and represent a highly promising approach for overcoming many of the limitations of autologous therapy. To advance the promise of hiPSC technology as an "off-the-shelf" source of cellular therapeutics, several considerations need to be addressed. Enabling cell transfer across histocompatibility barriers to permit persistence and therapeutic efficacy in an allogeneic setting is a key requirement. In addition to improving persistence, the ability to overcome histocompatibility barriers may facilitate multi-dosing regimens which may be a requirement in more advanced and complicated disease settings. Genetic incompatibilities between donor and recipient among the classical human leukocyte antigen (HLA) molecules is the leading cause of alloresponse by the host immune system and is currently mitigated by immunosuppressive strategies. Unfortunately, this treatment strategy is not only a stressful event for the patient but also damages the endogenous immune system, compromising the patient's ability to continue to fight the disease and opportunistic infections. Genetic editing of the HLA genes to generate histocompatible universal cell products is a viable opportunity that is currently being investigated. In addition to selective editing of unique genes to avoid a T cell mediated alloresponse, additional considerations such as natural killer (NK) cell-mediated rejection will need to be addressed. We have previously demonstrated that our proprietary reprogramming platform supports efficient and rapid derivation of clonal hiPSC lines with properties indicative of the naïve state of pluripotency. In addition to maintaining a homogeneous renewable population of hiPSCs, our platform is amenable to precise multi-gene and multi-loci targeted engineering at the single cell level, in both nuclease -dependent and -independent strategies. Furthermore, we have shown through small molecule-guided differentiation protocols, these highly-stable pluripotent cell lines can be banked and repeatedly tapped to consistently produce homogenous populations of immune cells with enhanced effector properties. Here we demonstrate a multi-faceted and comprehensive approach for the generation of immune tolerant hiPSCs and hiPSC-derived immune effector cells. We successfully combined deletion of classical HLA molecules with enforced expression of robust immunosuppressive proteins, including non-classical HLA molecules, to generate clonal hiPSC lines with the ability to escape immune rejection for "off-the-shelf" (OTS-hiPSCs) cellular immunotherapy. Utilizing in vitro real-time quantitative live cell analysis we determined that OTS-hiPSCs elicit a significantly decreased cytotoxic response from both activated peripheral blood (PB)-NK cells and primed PB-T cells compared to wildtype controls. Furthermore we demonstrate that OTS-hiPSCs exhibit improved persistence in xenograft studies in vivo. Bilateral teratomas were formed in a non-conditioned, fully immune-competent recipient mice using luciferized wildtype and OTS-hiPSCs. Daily bioluminescence imaging over a period of 7 days revealed a significant increase (>50 fold difference) in persistence of OTS-hiPSCs compared to wildtype hiPSCs during the 60-144 hour post injection window. Lastly we demonstrate that OTS-hiPSCs can successfully differentiate into functional effector lymphocytes using our potent chemically-defined monolayer hematopoietic differentiation platform. Our current studies focus on the functional characterization of OTS-hiPSC-derived effector lymphocytes in humanized mouse models and generating increased potency of OTS-hiPSC-derived effector lymphocytes through precise genetic engineering of antigen targeting and costimulatory proteins to create and optimized source of "off-the-shelf" cell-based immunotherapies. Disclosures Bauer: Fate Therapeutics: Employment. Clarke:Fate Therapeutics: Employment. Sasaki:Fate Therapeutics: Employment. Groff:Fate Therapeutics: Employment. Lee:Fate Therapeutics: Employment. Lan:Fate Therapeutics: Employment. Abujarour:Fate Therapeutics: Employment. Bonello:Fate Therapeutics: Employment. Burrascano:Fate Therapeutics: Employment. Robinson:Fate Therapeutics: Employment. Bjordahl:Fate Therapeutics, Inc: Employment. Gaidarova:Fate Therapeutics: Employment. Abbot:Fate Therapeutics: Employment. Wolchko:Fate Therapeutics: Employment. Shoemaker:Fate Therapeutics: Employment, Equity Ownership. Valamehr:Fate Therapeutics, Inc: Employment.

Natural Killer (NK) cells play a crucial role in immunosurveillance and form a first line of defense against cancer. In comparison to other lymphocytes, NK cells are unique in their capability to elicit tumoricidal responses without the need for antigen presentation or prior sensitization. Clinical data from bone marrow transplant and allogeneic NK immunotherapy suggest that MHC mismatch is advantageous in promoting graft-versus-leukemia without eliciting graft-versus-host, providing evidence that NK cells hold promisa as an allogeneic, universal immunotherapeutic. Further, the anti-tumor effect of many monoclonal antibodies is mediated through binding of the low-affinity Fc receptor CD16 on NK cells, which induces tumor cell killing through antibody-dependent cellular cytotoxicity (ADCC). Thus, NK cells represent a unique source of effector cells that can be combined with monoclonal antibodies, bispecific engagers or chimeric antigen receptors to direct tumor specificity and enhance cytotoxicity. Despite the significant potential of NK cell therapy, current clinical practices are limited by the need for large numbers of healthy NK cells, lack of in vivo persistence, and a burdensome manufacturing strategy that requires donor cell extraction, modulation, expansion and re-introduction per each patient. The ability to generate universally histocompatible and genetically-enhanced NK cells from continuously renewable human induced pluripotent stem cell (hiPSC) lines offers the potential to develop a true "off-the-shelf" cellular immunotherapy. While NK differentiation from hiPSC has been demonstrated, the clonal derivation of engineered hiPSCs to improve effector function has been challenging and the scalability and robustness of the differentiation method has been limited by skewed development towards primitive hematopoiesis and the cumbersome use of embryoid bodies. Here we highlight our "off-the-shelf" NK cell therapy preclinical program by demonstrating robust and highly scalable generation of functionally mature, genetically targeted and universally histocompatible NK cells. This program utilizes our previously described naïve hiPSC platform where we uniquely create clonal lines of precisely engineered, renewable hiPSCs and drive definitive hematopoiesis in a highly scalable manner. Because hiPSC differentiation is lineage directed, minimal cellular contamination is seen, including the lack of T and B cells, in the final product. Through precise genetic engineering of naïve hiPSC lines, we have engineered HLA-class I deficient NK cells uniformly expressing a high affinity, non-cleavable version of the Fc receptor CD16 (NcHaCD16-NK). The hiPSC-derived NcHaCD16-NKs display markers of maturity, including CD16, KIR, NCRs, and CD94. When compared to conventional cord blood and peripheral blood sourced NK cells, NcHaCD16-NKs exhibit superior cytotoxicity and production of effector cytokines in response to both solid and liquid tumor cell challenge in vitro. NcHaCD16-NKs exhibit augmented cytokine response following Fc-mediated stimulation, demonstrating function competence of the engineered CD16 construct. Because surface expression of CD16 is resistant to activation-induced shedding, NcHaCD16-NKs continuously maintain enhanced ADCC while retaining the capacity for general cytotoxicity. Importantly, the hiPSC-derived hematopoietic cells can be successfully cryopreserved and banked, serving as a highly-stable cell bank for subsequent therapeutic use. Preliminary data also shows NcHaCD16-NKs elicit preferred specificity for cancer stem cells as defined by expression of ALDH1 and surface markers such as CD24. In conclusion, the outlined preclinical data demonstrate the potential therapeutic utility of NK cells developed via precision genetic engineering of a renewable, scalable hiPSC platform, and highlights the therapeutic value of NcHaCD16-NKs as an ideal ADCC-mediated "off-the-shelf" NK cell-based immunotherapeutic product with augmented persistence, anti-tumor capacity and preclinical efficacy. Disclosures Bjordahl: Fate Therapeutics, Inc: Employment. Clarke:Fate Therapeutics: Employment. Gaidarova:Fate Therapeutics: Employment. Groff:Fate Therapeutics: Employment. Rogers:Fate Therapeutics, Inc: Employment. Moreno:Fate Therapeutics, Inc.: Employment, Equity Ownership. Abujarour:Fate Therapeutics, Inc.: Employment. Bonello:Fate Therapeutics, Inc.: Employment. Lee:Fate Therapeutics: Employment. Lan:Fate Therapeutics: Employment. Burrascano:Fate Therapeutics: Employment. Bauer:Fate Therapeutics: Employment. Robinson:Fate Therapeutics: Employment. Sasaki:Fate Therapeutics, Inc.: Employment. Kim:Fate Therapeutics, Inc.: Employment. Robbins:Fate Therapeutics: Employment, Equity Ownership. Rezner:Fate Therapeutics, Inc: Employment, Equity Ownership. Abbot:Fate Therapeutics: Employment. Wolchko:Fate Therapeutics: Employment. Shoemaker:Fate Therapeutics: Employment, Equity Ownership. Valamehr:Fate Therapeutics, Inc: Employment.

The T cell response to B cell lymphomas differs from the majority of solid tumors in that the malignant cells themselves are derived from B lymphocytes, key players in immune response. B cell lymphomas are therefore well situated to manipulate their surrounding microenvironment to enhance tumor growth and minimize anti-tumor T cell responses. We analyzed the effect of T cells on the growth of a transplantable B cell lymphoma and found that iNKT cells suppressed the anti-tumor CD8(+) T cell response. Lymphoma cells transplanted into syngeneic wild type (WT) mice or Jalpha18(-/-) mice that specifically lack iNKT cells grew initially at the same rate, but only the mice lacking iNKT cells were able to reject the lymphoma. This effect was due to the enhanced activity of tumor-specific CD8(+) T cells in the absence of iNKT cells, and could be partially reversed by reconstitution of iNKT cells in Jalpha 18(-/-) mice. Treatment of tumor-bearing WT mice with alpha -galactosyl ceramide, an activating ligand for iNKT cells, reduced the number of tumor-specific CD8(+) T cells. In contrast, lymphoma growth in CD1d1(-/-) mice that lack both iNKT and type II NKT cells was similar to that in WT mice, suggesting that type II NKT cells are required for full activation of the anti-tumor immune response. This study reveals a tumor-promoting role for iNKT cells and suggests their capacity to inhibit the CD8(+) T cell response to B cell lymphoma by opposing the effects of type II NKT cells.

Tregs play a key role in immune system suppression during autoimmunity and tumor development. Tregs are classified as CD4+CD25+FoxP3+, and exist as either nTregs originating from the thymus, or iTregs derived from CD4+ effector T cells in the periphery. Reduction in Treg numbers leads to autoimmunity; this is clearly shown in scurfy mice where FoxP3 has been deleted, Treg numbers are severely reduced, and mice succumb to autoimmunity early in life. In contrast, Tregs play a debilitating role in cancer biology when the Treg numbers are elevated, and the immune response is dampened allowing cancer cells to evade the immune system. Thus, the correct balance of Tregs is essential for maintaining immune homeostasis. FoxP3 has been widely used as a marker of Tregs, and many antibodies are available on the market that distinguish the Treg population in human or mouse cells. We developed a novel FoxP3 antibody and Fix and Perm buffer system that is comparable to current clones, but recognizes both mouse and human FoxP3 equally well, eliminating the need for separate antibodies. The ms/hu FoxP3 antibody was generated using our Rabbit Recombinant antibody technology, which allows for selecting high specificity, high affinity antibodies, lot to lot consistency and detection of both mouse and human nTregs and iTregs. This allows researchers to extend their mouse models into clinical studies without a change in the antibody clone eliminating a variable and streamlining the process.