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This supplementary file contains Supplemental Figures 1-6. Supplemental Figure 1 shows that 7-day ex vivo culture of NK cells with IL-15 and 5 μM CHIR99021 leads to an increase in the frequencies of NK cells with heterogeneous adaptive NK cell phenotypes (defined by expression of CD57, PLZF, SYK and FcεR1γ) relative to DMSO controls. Supplemental Figure 2 contains a detailed phenotypic characterization of receptor expression and cytotoxic granule component levels in sorted CD3-CD56dimCD57- and CD3-CD56dimCD57+ NK cells cultured for 7 days with IL-15 and either DMSO or 5 μM CHIR99021. Supplementary Figure 3 shows an analysis of NK cell phenotype, viability and proliferation after 7-day culture with IL-15 and either DMSO or CHIR99021 at several concentrations (1 μM, 3 μM and 5 μM). Supplemental Figure 4 shows an analysis of NK cell function (CD107a and IFN-γ) against K562 cells. NK cells were cultured for 7 days in IL-15 and either DMSO or 5 μM CHIR99021. Function was determined for individual CD3-CD56+ NK cell subsets gated on CD57 and NKG2C. Supplemental Figure 5 shows an analysis of NK cell function (CD107a and IFN-γ) against K562 cells. NK cells were cultured for 7 days in IL-15 and either DMSO or 5 μM CHIR99021. Function was determined for individual CD3-CD56+ NK cell subsets gated on CD57 and KIR. Supplemental Figure 6 shows the frequency of ex vivo expanded NK cells expressing CD57 and/or NKG2C 14 days after adoptive transfer into NSG mice.

Maturation of human natural killer (NK) cells as defined by accumulation of cell-surface expression of CD57 is associated with increased cytotoxic character and TNF and IFNγ production upon target-cell recognition. Notably, multiple studies point to a unique role for CD57+ NK cells in cancer immunosurveillance, yet there is scant information about how they mature. In this study, we show that pharmacologic inhibition of GSK3 kinase in peripheral blood NK cells expanded ex vivo with IL15 greatly enhances CD57 upregulation and late-stage maturation. GSK3 inhibition elevated the expression of several transcription factors associated with late-stage NK-cell maturation including T-BET, ZEB2, and BLIMP-1 without affecting viability or proliferation. When exposed to human cancer cells, NK cell expanded ex vivo in the presence of a GSK3 inhibitor exhibited significantly higher production of TNF and IFNγ, elevated natural cytotoxicity, and increased antibody-dependent cellular cytotoxicity. In an established mouse xenograft model of ovarian cancer, adoptive transfer of NK cells conditioned in the same way also displayed more robust and durable tumor control. Our findings show how GSK3 kinase inhibition can greatly enhance the mature character of NK cells most desired for effective cancer immunotherapy. Cancer Res; 77(20); 5664–75. ©2017 AACR.

Maturation of human natural killer (NK) cells as defined by accumulation of cell-surface expression of CD57 is associated with increased cytotoxic character and TNF and IFNγ production upon target-cell recognition. Notably, multiple studies point to a unique role for CD57+ NK cells in cancer immunosurveillance, yet there is scant information about how they mature. In this study, we show that pharmacologic inhibition of GSK3 kinase in peripheral blood NK cells expanded ex vivo with IL15 greatly enhances CD57 upregulation and late-stage maturation. GSK3 inhibition elevated the expression of several transcription factors associated with late-stage NK-cell maturation including T-BET, ZEB2, and BLIMP-1 without affecting viability or proliferation. When exposed to human cancer cells, NK cell expanded ex vivo in the presence of a GSK3 inhibitor exhibited significantly higher production of TNF and IFNγ, elevated natural cytotoxicity, and increased antibody-dependent cellular cytotoxicity. In an established mouse xenograft model of ovarian cancer, adoptive transfer of NK cells conditioned in the same way also displayed more robust and durable tumor control. Our findings show how GSK3 kinase inhibition can greatly enhance the mature character of NK cells most desired for effective cancer immunotherapy. Cancer Res; 77(20); 5664–75. ©2017 AACR.

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.

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

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

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.

Cellular immunotherapies are poised to transform the treatment of cancer and immunological disorders. In the most promising setting to date, genetic modification to the T lymphocytes in the form of chimeric antigen receptors (CAR) has dramatically increased therapeutic efficacy with reported initial complete remission rates in acute lymphoblastic leukemia ranging between 80-100%. However, pressing challenges remain to be solved to ensure that engineered T-cell immunotherapies can be cost-effectively and consistently manufactured, and safely and reliably delivered at the scale necessary to support wide patient base commercialization. Human induced pluripotent stem cell (hiPSC) derived T lymphocytes represent a unique, renewable source of genetically engineered T cells for "off-the-shelf" immunotherapy. Through the precise genetic engineering at the hiPSC stage, clonal and uniform populations of modified cell lines can be banked and reliably tapped into on demand to generate highly efficacious T cells for therapeutic applications. Although great progress has been made, several challenges need to be addressed including the ability to enhance effector function through genome-engineering of persistence, targeting, histocompatibility and controlled safety mechanisms at the hiPSC juncture while retaining the capacity to efficiently and reproducibly generate the intricate stages of lymphocyte development in an accurate and scalable process. 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 population of hiPSCs, our platform enables efficient multi-gene and multi-loci targeted engineering at a single cell level resulting in clonal population of pluripotent cell lines with desired genetic attributes. Here we will provide an update on our "off-the-shelf" T-cell immunotherapy preclinical program where engineered hiPSC lines are uniquely used as the renewable starting material. We will also highlight our novel differentiation platform to derive definitive hematopoietic progenitor cells termed hemogenic endothelium (HE); a well-defined, small molecule-driven, staged process that is currently being translated into cGMP (current good manufacturing practice) settings. The highly efficient differentiation system (on average >65% hiPSC to CD34 conversion) delivers approximately 100 CD34+ HE cells per each input hiPSC, representing a highly scalable process that is further expanded during lymphocyte differentiation and maturation. To validate that the iCD34+ HE is definitive in nature we demonstrate that during further hematopoietic differentiation the emerging CD43+ hematopoietic cells exhibit Notch dependency and high expression of key genes such as MYB and the HOXA cluster, found only in definitive hematopoietic progenitors. The hiPSC-derived HE exhibits multi-lineage potential and can be successfully cryopreserved and banked, serving as a highly-stable cell bank for subsequent therapeutic use. Through genetic modifications at the single cell hiPSC stage, we confer antigen-specificity via the expression of temporally inducible CARs as premature expression of CAR proteins during in vitro differentiation has been found to skew development towards innate-lymphoid like lineages. Utilizing our stage-specific hematopoietic differentiation platform we have identified the optimal developmental window to induce the expression of CAR proteins to maintain optimal differentiation towards functional effector lymphocytes. The hiPSC-derived engineered T lymphocytes are currently under preclinical investigation for in vitro and in vivo effector function including thymic rejuvenation, T cell repertoire repopulation, target specific recognition and enhanced killing potential. Preliminary data suggests that hiPSC-derived lymphocytes are functional and can home to their respective niche to support initial repopulation in vivo. Our study continues to support that naïve hiPSCs are an ideal renewal source for "off-the-shelf" hematopoietic cell-based immunotherapies and represent a potentially exponential advancement in adoptive T cell therapy. Disclosures Clarke: Fate Therapeutics: Employment. Groff:Fate Therapeutics: Employment. Sasaki:Fate Therapeutics: Employment. Bauer:Fate Therapeutics: Employment. Lee:Fate Therapeutics: Employment. Lan:Fate Therapeutics: Employment. Burrascano:Fate Therapeutics: Employment. Abujarour:Fate Therapeutics: Employment. Bonello:Fate Therapeutics: Employment. Robinson:Fate Therapeutics: Employment. Foster:Fate Therapeutics: Employment, Equity Ownership. Robbins:Fate Therapeutics: Employment, Equity Ownership. Wolchko:Fate Therapeutics: Employment. Shoemaker:Fate Therapeutics: Employment, Equity Ownership. Abbot:Fate Therapeutics: Employment. Valamehr:Fate Therapeutics, Inc: Employment.

Umbilical cord blood (UCB) offers many potential advantages as a source of hematopoietic stem cells (HSCs) for allogeneic transplantation, including ease of collection, rapid availability, flexibility of HLA-matching, lower rates of GvHD and potentially lower relapse rates. However, the low HSC content of UCB compared to other graft sources results in a prolonged time to engraftment, and higher rates of graft failure and early mortality. Pulse ex vivo exposure of HSCs to 16,16-dimethyl PGE2 (FT1050) has been demonstrated to enhance HSC engraftment potential, which could benefit clinical UCB transplant. FT1050 modulation promotes multiple mechanisms, including increased proliferation, reduced apoptosis, and improved migration and homing [North 2007&2009; Hoggatt 2009]. Improved HSC homing is mediated by induction of CXCR4 gene expression leading to increased cell surface CXCR4. Further optimization of the UCB modulation process demonstrated that incubation with 10µM FT1050 for 2 hrs at 37C resulted in a maximal biological response of the FT1050-UCB (ProHema®). A Phase 1 trial was performed to evaluate the safety of FT1050-UCB paired with an unmanipulated UCB unit in reduced-intensity double UCBT (dUCBT) [Cutler 2013]. We observed durable, multi-lineage engraftment of FT1050-UCB with acceptable safety. Earlier neutrophil engraftment was observed relative to historical controls (median 17.5 vs. 21 days (historical control), p=0.045), coupled with preferential engraftment of the FT1050-UCB unit in 10 of 12 subjects. A Phase 2 multi-center clinical trial of FT1050-UCB in adult patients undergoing dUCBT for hematologic malignancies was then initiated. Subjects are randomized 2:1 to FT1050-UCB-containing vs. standard dUCBT after high-dose conditioning. The primary endpoint is a categorical analysis of neutrophil engraftment using a pre-specified control median. Data on the initial 11 subjects, of which 8 were randomized to receive FT1050-UCB, continue to demonstrate acceptable safety with adverse events attributed to FT1050-UCB limited primarily to common infusion-related side effects. Of the 8 FT1050-UCB subjects, 1 died prior to neutrophil engraftment, with the remaining 7 subjects engrafting at a median of 28 days vs. 31 days for the 3 control subjects. With median overall follow-up of 16.1 months, 4 of 8 subjects on the FT1050-UCB arm are alive with a median survival not reached (> 11.0 months). 1 of 3 control subjects is alive with median survival of 6.0 months. During the clinical translation process, the media used during FT1050 modulation of UCB was identified as a key variable. Standard UCB washing media, consisting of a nutrient-free saline solution of low molecular weight dextran and human serum albumin (LMD/HSA), is used clinically to stabilize fragile cells post-thaw by reducing lysis. This media was used in the Phase 1 trial and to initiate Phase 2. Early during the Phase 2 trial, we identified a novel cell-stabilizing nutrient-rich formulation (NRM), containing glucose, amino acids and other HSC-supporting nutrients that promoted full FT1050 modulation of UCB and increased cell viability. The expression of key FT1050-pathway genes was significantly higher with NRM compared to intermediate levels observed with LMD/HSA. Modulation of human CD34+ (hCD34+) cells with FT1050 in NRM led to an 8-fold increase over LMD/HSA in induced CXCR4 gene expression (20-fold total), which translated to significantly increased surface CXCR4 protein. In vivo homing models demonstrated that UCB CD34+ cells modulated with FT1050 in NRM resulted in a 2.2-fold homing increase relative to vehicle (p < 0.001) compared to a 1.6-fold increase with LMD/HSA (p = 0.002), with a significant difference between the two media conditions (p = 0.04). A xenotransplantation study in NSG mice with hCD34+ cells modulated with FT1050 in either NRM or LMD/HSA demonstrated a 2-fold increase in circulating hCD45+ cells 12-weeks post-transplant with NRM (p = 0.007; unpaired t-test). These findings supported the incorporation of NRM into the FT1050-UCB manufacturing process in order to further improve its clinical engraftment potential. Enrollment of a 60-patient Phase 2 trial has been initiated that incorporates this manufacturing change. Disclosures Shoemaker: Fate Therapeutics: Employment, Equity Ownership. Rezner:Fate Therapeutics: Employment. Guerrettaz:Fate Therapeutics: Employment. Robbins:Fate Therapeutics: Employment. Medcalf:Fate Therapeutics: Employment. Wolchko:Fate Therapeutics: Employment, Equity Ownership. Ferraro:Fate Therapeutics: Employment. Multani:Fate Therapeutics: Employment.