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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.

The development of immunotherapeutic monoclonal antibodies targeting checkpoint inhibitory receptors, such as programmed cell death 1 (PD-1), or their ligands, such as PD-L1, has transformed the oncology landscape. However, durable tumor regression is limited to a minority of patients. Therefore, combining immunotherapies with those targeting checkpoint inhibitory receptors is a promising strategy to bolster antitumor responses and improve response rates. Natural killer (NK) cells have the potential to augment checkpoint inhibition therapies, such as PD-L1/PD-1 blockade, because NK cells mediate both direct tumor lysis and T cell activation and recruitment. However, sourcing donor-derived NK cells for adoptive cell therapy has been limited by both cell number and quality. Thus, we developed a robust and efficient manufacturing system for the differentiation and expansion of high-quality NK cells derived from induced pluripotent stem cells (iPSCs). iPSC-derived NK (iNK) cells produced inflammatory cytokines and exerted strong cytotoxicity against an array of hematologic and solid tumors. Furthermore, we showed that iNK cells recruit T cells and cooperate with T cells and anti-PD-1 antibody, further enhancing inflammatory cytokine production and tumor lysis. Because the iNK cell derivation process uses a renewable starting material and enables the manufacturing of large numbers of doses from a single manufacture, iNK cells represent an "off-the-shelf" source of cells for immunotherapy with the capacity to target tumors and engage the adaptive arm of the immune system to make a "cold" tumor "hot" by promoting the influx of activated T cells to augment checkpoint inhibitor therapies.

FT819 is a first-of-kind, allogeneic, off-the-shelf CAR T-cell therapy derived from a clonal master induced pluripotent stem cell (iPSC) line precisely engineered to insert a novel 1XX anti-CD19 chimeric antigen receptor (CAR) under the regulation of the T-cell receptor alpha constant (TRAC) locus for optimized control of anti-tumor activity and to completely delete T-cell receptor (TCR) expression to eliminate the potential of graft-versus-host disease (GvHD). Unlike conventional allogeneic CAR T-cell therapies which require repeatedly sourcing of T cells from various donors as the starting material, the use of a clonal master engineered iPSC line serves as a renewable starting cell source and ensures routine mass production of a uniformly engineered, homogenous CAR T-cell product for broad patient access. T cell-derived iPSCs were generated using a proprietary non-integrating cellular reprogramming system and genetically modified to integrate a novel anti-CD19 1XX CAR into both alleles of the TRAC gene. After single cell subcloning, each engineered iPSC clone was screened for multiple critical quality attributes including pluripotency, identity, genomic stability, cassette integration, on/off-target integration, T-cell differentiation propensity, and CAR T-cell function. Accordingly, the ideal single cell-derived engineered iPSC clone was selected as the clonal master iPSC line for FT819 and was converted into a master cell bank (MCB). The iPSC MCB serves as a renewable source for the routine GMP manufacture of FT819 drug product. The FT819 production process consists of three stages: 1) generation of CD34-expressing hematopoietic progenitor cells from iPSCs (>90% CD34+ cells post enrichment); 2) lineage-specification to T cells followed by T-cell expansion (>5e5 fold expansion); and 3) fill/finish and cryopreservation of the drug product. As an example, in an initial small-scale manufacturing campaign, a total of 2.5 × 10 10 FT819 CAR T-cells were generated and filled and finished starting from one vial of the MCB. The FT819 drug product was tested on safety, identity, purity, and potency. The final product was comprised of CD45+CD7+ lymphocytes (>99%), with homogeneous CAR expression (>99% CAR+) and lacking expression of TCRαβ (not detected) on the cell surface. Importantly, there were no residual iPSCs detected in the FT819 drug product. The FT819 drug product exhibited potent and consistent effector function against NALM6 leukemia cells. The FT819 drug product is currently being used in a landmark Phase I study (NCT04629729), the first-ever iPSC-derived T-cell therapy to undergo clinical investigation, for the treatment of patients with relapsed/refractory B-cell lymphoma, chronic lymphocytic leukemia and precursor B-cell acute lymphoblastic leukemia. In summary, FT819 is a first-of-kind, off-the-shelf, CAR T-cell therapy uniquely derived from a clonal multiplexed-engineered master iPSC line. The novel manufacturing paradigm enables mass production of a uniformly engineered, homogenous cell therapy product that is available on-demand for broad patient access. A multi-center Phase 1 study of FT819 is currently ongoing for the treatment of B-cell malignancies. Key Words: cancer immunotherapy, cell therapy, CAR-T, CD19, allogeneic, induced pluripotent stem cell, iPSC, clonal master iPSC line, engineered, off-the-shelf, cGMP, production, manufacturing, FT819 Disclosures Yuan: Fate Therapeutics, Inc.: Current Employment. Clarke: Fate Therapeutics, Inc.: Current Employment. Lai: Fate Therapeutics, Inc.: Current Employment. Chang: Fate Therapeutics, Inc.: Current Employment. Yang: Fate Therapeutics, Inc.: Current Employment. Hsia: Fate Therapeutics, Inc.: Current Employment. Abujarour: Fate Therapeutics, Inc.: Current Employment. Lee: Fate Therapeutics, Inc.: Current Employment. van der Stegen: Fate Therapeutics, Inc.: Current Employment. Shaked: Fate Therapeutics, Inc.: Current Employment. Jalloh: Fate Therapeutics, Inc.: Current Employment. Moreno: Fate Therapeutics, Inc.: Current Employment. ORourke: Fate Therapeutics, Inc.: Current Employment. Sung: Fate Therapeutics, Inc.: Current Employment. Gutierrez: Fate Therapeutics, Inc.: Current Employment. Rezner: Fate Therapeutics, Inc.: Current Employment. Eberhart: Fate Therapeutics, Inc.: Current Employment. Magdaleno: Fate Therapeutics, Inc.: Current Employment. Farnan: Fate Therapeutics, Inc.: Current Employment. Plavsic: Fate Therapeutics, Inc.: Current Employment. Bressi: Fate Therapeutics, Inc.: Current Employment. Rivière: Centre for Commercialization of Cancer Immunotherapy: Other: Provision of Services; Fate Therapeutics: Other: Provision of Services, Patents & Royalties; The Georgia Tech Research Corporation (GTRC): Other: Provision of Services (uncompensated); FloDesign Sonics: Other: Provision of Services; Juno Therapeutics: Patents & Royalties. Valamehr: Fate Therapeutics, Inc.: Current Employment.

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.

FT538 is an investigational, off-the-shelf, multiplexed engineered natural killer (NK) cell cancer immunotherapy that is derived from a clonal master induced pluripotent stem cell (iPSC) line engineered with three functional components to enhance innate immunity: a novel high-affinity, non-cleavable CD16 (hnCD16) Fc receptor; an IL-15/IL-15 receptor fusion (IL-15RF); and the elimination of CD38 expression. The use of a clonal master engineered iPSC line as a starting cell source enables routine mass production of FT538 and supports off-the-shelf product availability to reach many patients. The clonal master iPSC line for the manufacture of FT538 was made by reprogramming and engineering donor-consented human fibroblasts to induce pluripotency using a proprietary non-integrating system and to integrate a bicistronic cassette containing hnCD16 and IL-15RF into the CD38 locus, which resulted in complete disruption of the CD38 gene. The engineered iPSC population was then sorted to isolate single clones, and each engineered iPSC clone was screened for multiple critical quality attributes including pluripotency, identity, genomic stability, cassette integration, and off-target effects of engineering. Accordingly, a single engineered iPSC clone was selected as the FT538 clonal master iPSC line. The clonal master engineered iPSC line serves as a renewable source for the routine cGMP mass production of FT538 drug product. Routine cGMP manufacture of FT538 drug product consists of three stages: 1) differentiation of the clonal master engineered iPSC line to CD34-expressing hematopoietic progenitor cells; 2) further differentiation to and expansion of NK cells; and 3) fill/finish and cryopreservation of the FT538 drug product. A cGMP manufacturing campaign yielded sufficient CD34-expressing hematopoietic progenitor cells to support at least 15 batches of NK cell differentiation, the first batch of which was used to produce a total of 3 × 1011 FT538 NK cells filled into over 300 units of cryopreserved drug product. The cGMP campaign had a theoretical yield of 4.5 x 1012 FT538 NK cells. The FT538 drug product was characterized and found to be comprised of uniformly engineered CD56+ NK cells with homogeneous expression of hnCD16 and lacking expression of CD38. Importantly, there were no residual iPSCs detected in the FT538 drug product. Additionally, the FT538 drug product exhibited potent effector function in a candidate potency assay measuring IFN-γ release in response to RPMI 8226 target cells in the presence of daratumumab. An Investigational New Drug application for FT538 has been cleared by the U.S. Food and Drug Administration (FDA) for the conduct of a multicenter, multi-dose Phase I clinical trial for the treatment of patients with relapsed/refractory (r/r) acute myelogenous leukemia (AML) and multiple myeloma (MM). The dose-escalation utilizes a 3+3 design to identify the maximum tolerated dose of three doses of FT538 on Days 1, 8, and 15 as a monotherapy in r/r AML (Regimen A) and in combination with daratumumab (Regimen B) or elotuzumab (Regimen C) in r/r MM. The trial will test up to five FT538 dose levels ranging from 50 million to 1.5 billion cells, and up to 105 patients will be enrolled. The trial is expected to begin patient enrollment in 2020. Disclosures Rezner: Fate Therapeutics, Inc: Current Employment, Current equity holder in publicly-traded company. Solchaga:Fate Therapeutics, Inc: Current Employment. Reyes:Fate Therapeutics, Inc: Current Employment. Lin:Fate Therapeutics, Inc: Current Employment. Abujarour:Fate Therapeutics, Inc: Current Employment. Lee:Fate Therapeutics, Inc.: Current Employment. Valamehr:Fate Therapeutics, Inc: Current Employment, Current equity holder in publicly-traded company. Wang:Fate Therapeutics, Inc: Current Employment.

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.

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.

Early environmental events have profound influences on a wide range of adult behavior. In the current study, we assessed the influence of maternal stress during gestation on psychostimulant and neurochemical responsiveness to cocaine, cocaine self-administration, and reinstatement of cocaine-seeking in adult offspring. Pregnant, female Sprague–Dawley rats were subjected to either no treatment or to restraint stress three times per day for the last 7 days of gestation and cocaine-related behavior was assessed in offspring at 10 weeks of age. Relative to controls, a noncontingent cocaine injection elevated locomotor activity as well as nucleus accumbens levels of extracellular dopamine and glutamate to a greater extent in both cocaine-naïve and cocaine-experienced prenatal stress (PNS) rats and elevated prefrontal cortex dopamine in cocaine-experienced PNS rats. To assess the impact of PNS on cocaine addiction-related behavior, rats were trained to lever press for intravenous (i.v.) infusions of cocaine (0.25, 0.5, or 1 mg/kg/infusion), with each infusion paired with a light + tone-conditioned stimulus. Lever-pressing was extinguished and cocaine-seeking reinstated by re-exposure to the conditioned cues or by intraperitoneal cocaine-priming injections (5 or 10 mg/kg). PNS elevated active lever responding both during extinction and cocaine-primed reinstatement, but not during self-administration or conditioned-cued reinstatement. PNS also did not alter intake during self-administration. These findings demonstrate that PNS produces enduring nervous system alterations that increase the psychomotor stimulant, motivational, and neurochemical responsiveness to noncontingent cocaine. Thus, early environmental factors contribute to an individual's initial responsiveness to cocaine and propensity to relapse to cocaine-seeking.

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

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.

Adoptive cellular therapies using engineered chimeric antigen receptor T cells (CAR-T cells) are rapidly emerging as a highly effective treatment option for a variety of life-threatening hematological malignancies. Small molecule-mediated modulation of T cell differentiation during the in vitro CAR-T manufacturing process has great potential as a method to optimize the therapeutic potential of cellular immunotherapies. In animal models, T cells with a central or stem memory (TCM/SCM) phenotype display enhanced in vivoefficacy and persistence relative to other T cell subpopulations. We sought to identify small molecules that promote skewing towards a TCM/SCM phenotype during the CAR-T manufacturing process, with associated enhanced viability, expansion and metabolic profiles of the engineered cells. To this end, we developed a high-throughput functional screening platform with primary human T cells using a combination of high-content immunophenotyping and gene expression-based readouts to analyze cells following a high-throughput T cell culture platform that represents a scaled-down model of clinical CAR-T cell production. Multicolor flow cytometry was used to measure expansion, cell viability and the expression levels of cell surface proteins that define TCM cells (e.g., CCR7, CD62L and CD27) and markers of T cell exhaustion (e.g., PD1, LAG3, and TIM3). In parallel, a portion of each sample was evaluated using high content RNA-Seq based gene expression analysis of ~100 genes representing key biological pathways of interest. A variety of known positive and negative control compounds were incorporated into the high-throughput screens to validate the functional assays and to assess the robustness of the 384-well-based screening. The ability to simultaneously correlate small molecule-induced changes in protein and gene expression levels with impacts on cell proliferation and viability of various T cell subsets, enabled us to identify multiple classes of small molecules that favorably enhance the therapeutic properties of CAR-T cells. Consistent with results previously presented by Perkins et al. (ASH, 2015), we identified multiple PI3K inhibitors that could modify expansion of T cells while retaining a TCM/SCM phenotype. In addition, we identified small molecules, and small molecule combinations, that have not been described previously in the literature that could improve CAR-T biology. Several of the top hits from the screens have been evaluated across multiple in vitro (e.g., expansion, viability, CAR expression, serial restimulation/killing, metabolic profiling, and evaluation of exhaustion markers) and in vivo (e.g., mouse tumor models for persistence and killing) assays. Results from the initial screening hits have enabled us to further refine the optimal target profile of a pharmacologically-enhanced CAR-T cell. In addition, we are extending this screening approach to identify small molecules that enhance the trafficking and persistence of CAR-T cells for treating solid tumors. In conclusion, the approach described here identifies unique small molecule modulators that can modify CAR-T cells during in vitro expansion, such that improved profiles can be tracked and selected from screening through in vitro and in vivo functional assays. Disclosures Rosen: Fate Therapeutics: Employment, Equity Ownership. Rezner:Fate Therapeutics, Inc: Employment, Equity Ownership. Robbins:Fate Therapeutics: Employment, Equity Ownership. Hardy:Fate Therapeutics: Employment, Equity Ownership. Peralta:Fate Therapeutics: Employment, Equity Ownership. Maine:Fate Therapeutics: Employment, Equity Ownership. Sabouri:Fate Therapeutics: Employment, Equity Ownership. Reynal:Fate Therapeutics: Employment. Truong:Fate Therapeutics: Employment, Equity Ownership. Moreno:Fate Therapeutics, Inc.: Employment, Equity Ownership. Foster:Fate Therapeutics: Employment, Equity Ownership. Borchelt:Fate Therapeutics: Employment, Equity Ownership. Meza:Fate Therapeutics: Employment, Equity Ownership. Thompson:Juno Therapeutics: Employment, Equity Ownership. Fontenot:Juno Therapeutics: Employment, Equity Ownership. Larson:Juno Therapeutics: Employment, Equity Ownership. Mujacic:Juno Therapeutics: Employment, Equity Ownership. Shoemaker:Fate Therapeutics: Employment, Equity Ownership.

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.

Human induced pluripotent stem cells (hiPSCs) are a unique population of cells that can serve as an unlimited source for "off-the-shelf" cellular immunotherapeutics. Similar to master cell lines used in the manufacture of monoclonal antibodies, engineered hiPSC lines have the potential to serve as a renewable cell source for the consistent manufacture of homogeneous populations of effector cells for the treatment of thousands of patients. However, the creation of an effective master line is largely dependent on the ability to genetically edit hiPSCs in a precise, efficient and clonal manner. Furthermore, the genetically edited hiPSCs must maintain their inherent ability to continuously self-renew while retaining ability to express engineered modalities upon directed differentiation to the cell type of choice. We have previously reported the use of stage-specific media compositions to enable the footprint-free generation and long-term maintenance of single cell naïve hiPSCs with enhanced clonogenicity, an attribute critical for the derivation of engineered single cell-derived lines. Here we demonstrate the use of our naïve hiPSC platform to precisely introduce, in a site-specific manner, multiple genes into multiple safe harbor loci. By combining our single-cell naïve hiPSC platform with different nuclease-independent and -dependent strategies, we are able to generate large numbers of precisely engineered iPSC clones. The single cell-derived hiPSC clones were subsequently screened in a multiplexed fashion for successful multi-parameter engineering, maintained pluripotency and propensity for differentiation with lack of undesired phenotypes and genomic alterations. Using this approach, we derived individual clones containing a uniform population (>99%) of multi-engineered modalities consisting of tumor targeting, a controllable safety switch and a tracking marker. Moreover, we show that engineered modalities are expressed in undifferentiated and differentiated hiPSCs, including being expressed in >95% of both CD34 positive hematopoietic progenitor cells and CD56 positive natural killer cells. Furthermore, we have generated hiPSC clones with dual suicide genes (inducible Caspase 9 (iCasp9) and Herpes simplex virus thymidine kinase (HSV-TK)) targeted into two independent safe harbor loci, in both mono- and bi-allelic manner. The dual-targeted hiPSC clones were confirmed to have specific insertions into the predicted sites and were screened to exclude random insertions. Concurrent activation of both suicide genes led to complete elimination of engineered hiPSCs and no treatment-refractory cells were observed unlike the case when only one suicide gene was activated. In addition to robust targeted insertions, we were able to generate small insertions and deletions in up to 70% of naïve hiPSCs without selection and homozygous knockout of gene of interest in 100% of cells after selection. Finally, we will discuss efforts to temporally synchronize ectopic gene expression through endogenously regulated promoters by simultaneous endogenous gene disruption and transgene insertion. Overall, we show our naïve hiPSC platform is an ideal renewable source to efficiently generate, genetically engineer, isolate and bank clonally-derived homogenous population of pluripotent cells. These highly-stable pluripotent clonal banks can be repeatedly tapped to facilitate the consistent production of homogenous populations of potent, persistent, scalable and safer off-the-shelf cellular immunotherapeutics. Disclosures Abujarour: Fate Therapeutics: Employment. Lan:Fate Therapeutics: Employment. Lee:Fate Therapeutics: Employment. Bonello:Fate Therapeutics: Employment. Meza:Fate Therapeutics: Employment, Equity Ownership. Robinson:Fate Therapeutics: Employment. Clarke:Fate Therapeutics: Employment. Truong:Fate Therapeutics: Employment, Equity Ownership. Robbins:Fate Therapeutics: Employment, Equity Ownership. Rezner:Fate Therapeutics, Inc: Employment, Equity Ownership. Abbot:Fate Therapeutics: Employment. Shoemaker:Fate Therapeutics: Employment, Equity Ownership. Valamehr:Fate Therapeutics, Inc: Employment.

Summary Cell banking, disease modeling, and cell therapy applications have placed increasing demands on hiPSC technology. Specifically, the high-throughput derivation of footprint-free hiPSCs and their expansion in systems that allow scaled production remains technically challenging. Here, we describe a platform for the rapid, parallel generation, selection, and expansion of hiPSCs using small molecule pathway inhibitors in stage-specific media compositions. The platform supported efficient and expedited episomal reprogramming using just OCT4/SOX2/SV40LT combination (0.5%–4.0%, between days 12 and 16) in a completely feeder-free environment. The resulting hiPSCs are transgene-free, readily cultured, and expanded as single cells while maintaining a homogeneous and genomically stable pluripotent population. hiPSCs generated or maintained in the media compositions described exhibit properties associated with the ground state of pluripotency. The simplicity and robustness of the system allow for the high-throughput generation and rapid expansion of a uniform hiPSC product that is applicable to industrial and clinical-grade use., Graphical Abstract, Highlights • FRM supports highly efficient minimal factor episomal reprogramming • Clonal derivation of hiPSCs through high-resolution single-cell sorting • FMM supports long-term maintenance of genomically stable, transgene-free hiPSCs • Maintenance of homogeneous pluripotent culture resembling the ground state, Cell therapy and banking applications have placed increasing demands on hiPSC technology. Here, Flynn and colleagues demonstrate a simple multiplex selection and maintenance platform utilizing stage-specific small molecule media additives and flow cytometry sorting to derive minimal factor episomal hiPSCs that are genomically stable and resemble ground state pluripotent stem cells. This platform may serve as a path to generate clinically relevant hiPSCs.

Umbilical cord blood (UCB) is a valuable source of hematopoietic stem cells (HSCs) for use in allogeneic transplantation. Key advantages of UCB are rapid availability and less stringent requirements for HLA matching. However, UCB contains an inherently limited HSC count, which is associated with delayed time to engraftment, high graft failure rates, and early mortality. 16,16-Dimethyl prostaglandin E2 (dmPGE2) was previously identified to be a critical regulator of HSC homeostasis, and we hypothesized that brief ex vivo modulation with dmPGE2 could improve patient outcomes by increasing the "effective dose" of HSCs. Molecular profiling approaches were used to determine the optimal ex vivo modulation conditions (temperature, time, concentration, and media) for use in the clinical setting. A phase 1 trial was performed to evaluate the safety and therapeutic potential of ex vivo modulation of a single UCB unit using dmPGE2 before reduced-intensity, double UCB transplantation. Results from this study demonstrated clear safety with durable, multilineage engraftment of dmPGE2-treated UCB units. We observed encouraging trends in efficacy, with accelerated neutrophil recovery (17.5 vs 21 days, P = .045), coupled with preferential, long-term engraftment of the dmPGE2-treated UCB unit in 10 of 12 treated participants.

hiPSC derivation and selection remains inefficient; with selection of high quality clones dependent on extensive characterization which is not amenable to high-throughput (HTP) approaches. We recently described the use of a cocktail of small molecules to enhance hiPSC survival and stability in single cell culture and the use of flow cytometry cell sorting in the HTP-derivation of hiPSCs. Here we report an enhanced protocol for the isolation of bona fide hiPSCs in FACS-based selection using an optimized combination of cell surface markers including CD30. Depletion of CD30(+) cells from reprogramming cultures almost completely abolished the NANOG and OCT4 positive sub-population, suggesting it is a pivotal marker of pluripotent cells. Combining CD30 to SSEA4 and TRA-1-81 in FACS greatly enhanced specificity and efficiency of hiPSC selection and derivation. The current method allows for the efficient and automated, prospective isolation of high-quality hiPSC from the reprogramming cell milieu.

As the field of human induced pluripotent stem cell (hiPSC) research continues to advance, and as the clinical investigation of genetically-engineered hiPSC-derived cellular therapeutics begins to emerge, safety concerns relating to the administration of genetically-altered cells must be addressed and mitigated. A number of strategies including recombinant peptides, monoclonal antibodies, small molecule-modulated enzyme activity and gene-specific modifications have been explored to facilitate the selective elimination of aberrant cells. Of these, the application of genetically-encoded inducible "suicide" systems that can be rapidly activated by a specific non-toxic chemical inducer represents a very attractive, targeted approach for eliminating administered cells without damaging surrounding cells and tissues. Most previous studies have employed viral vectors and short promoters to stably introduce suicide-genes, including herpes complex virus thymidine kinase or inducible caspase-9 (iCasp9), into human cells. The use of viral vectors can lead to random integration events which can disrupt or activate disease-related genes, potentially causing deleterious effects. In addition, many artificial promoters and genome regions are prone to epigenetic silencing in both pluripotent and differentiated states, resulting in cells becoming unresponsive to suicide gene induction. Thus, it is of great importance to identify optimal integration sites and promoters in order to maintain functional suicide gene responses and facilitate the development of genetically-engineered cellular therapeutics. To effectively select and test suicide systems under the control of various promoters in combination with different safe harbor loci integration strategies, we took advantage of our proprietary hiPSC platform, which enables single cell passaging and high-throughput, 96-well plate-based flow cytometry sorting. In a single hiPSC per well manner, we utilized both nuclease-independent and nuclease-dependent strategies to efficiently and precisely integrate various suicide gene expression cassettes in AAVS1 or ROSA26 safe harbor loci. Several integration vectors, each containing a suicide gene expression cassette downstream of various exogenous and endogenous promoters, including endogenous AAVS1 or ROSA26, cytomegalovirus, elongation factor 1α, phosphoglycerate kinase, hybrid CMV enhancer/chicken β-actin and ubiquitin C promoters, were tested to systematically analyze and compare the activity of different suicide systems in both hiPSCs and hiPSC-derived differentiated cells. To conduct high-throughput analyses of these integration and expression strategies, we selected an iCasp9 suicide gene platform where rapid caspase-9 mediated cell death can be induced by small molecule chemical inducers of dimerization such as AP1903. Several endogenous promoters were found to drive persistent expression of iCasp9 during clonal expansion of hiPSCs, but the expression level was determined to be too low to effectively respond to AP1903 treatment. Expression of iCasp9 under the control of various exogenous promoters was lost during prolonged clonal expansion of hiPSCs, and failed to drive AP1903-induced cell death. However, one promoter maintained high levels of iCasp9 expression during the long-term clonal expansion of hiPSCs. Furthermore, these iCasp9-integrated clonal lines underwent rapid cell death in the presence of AP1903, and no residual cell survival was observed when cultures were allowed to recover in the absence of the dimerizing molecule. To test whether epigenetic landscape alterations would abrogate suicide gene-mediated response, hiPSC clones were differentiated into three somatic lineages in vitro and were found to be completely subject to AP1903-induced cell death. Clones were also specifically differentiated towards hematopoietic cells to demonstrate complete induction of cell death by AP1903 treatment. When injected into NSG mice to form teratomas, similar cell death-mediated response was observed in vivo. Notably, one hiPSC clone contained certain rare cells and did escape induced cell death, and this clone and these rare cells were characterized to assess the molecular mechanisms of escape. Our study describes novel findings toward designing optimal safety systems for integration into hiPSC-derived cellular therapies. Disclosures Huang: Fate Therapeutics Inc: Employment. Lan:Fate Therapeutics Inc: Employment. Parone:Fate Therapeutics Inc: Employment. Clarke:Fate Therapeutics Inc: Employment. Abujarour:Fate Therapeutics Inc: Employment. Robinson:Fate Therapeutics Inc: Employment. Meza:Fate Therapeutics Inc: Employment. Lee:Fate Therapeutics Inc: Employment. Shoemaker:Fate Therapeutics Inc: Employment. Valamehr:Fate Therapeutics Inc: Employment.

Allogeneic hematopoietic stem cell transplant (HSCT) represents a potential curative treatment for a number of life-threatening blood malignancies. The utility of this treatment regimen, however, is limited by a number of serious complications including graft versus host disease, which occurs in approximately half of all transplant patients. Standard-of-care for treating acute GvHD has remained unchanged for several decades and consists of high doses of steroids, which are only effective in approximately 35 percent of the cases. Therefore, the reduction of GvHD represents a large unmet medical need, and new approaches are needed to effectively attenuate GvHD. Here we present a fundamentally novel strategy for potentially reducing GVHD - by modulating donor mobilized peripheral blood cells with small molecules prior to HSCT, a programmed mobilized peripheral blood (mPB) allogeneic graft, with reduced T-cell alloreactivity, can be administered as the hematopoietic cell source for HSCT. To this end, we applied our screening platform to identify a combination of small molecule modulators (FT1050, FT4145) that promote the activation of genes implicated in cell cycle, immune tolerance and anti-viral properties of T cells, as well as in the survival, proliferation and engraftment potential of CD34+ cells. Genome-wide expression analysis of the T-cell compartment of mobilized peripheral blood following treatment with FT1050+FT4145 revealed the induction of genes involved in cell cycle (e.g., CCND1, CCNE1), immune tolerance (e.g., ALDH, AREG) and anti-viral properties (e.g., EFNB2). To further assess the therapeutic impact of ex vivo programming with FT1050 and FT4145, a number of T cell assays to assess T cell phenotype and function were conducted on mPB. Overall, ex vivo programming of mPB resulted in reduced allogeneic T cell responses and was accompanied by reduced capacity of modulated T cells to produce Interferon Gamma (IFN-ɣ). Concomitantly, the ability of the modulated T cells to make Interleukin 4 (IL-4) and 10 (IL-10) was enhanced, suggesting a polarization of these cells towards a less inflammatory functional state. This was further evidenced by increased surface expression of an immune-inhibitory molecule, PD1, and reduced expression of the activation markers 41BB and ICOS. We next examined the potential beneficial role of ex vivo programming with FT1050+FT4145 in a major histocompatibility complex (MHC) mis-matched HSCT mouse model. Briefly, lethally irradiated BALB/c mice received bone marrow and splenocytes from C57BL/6 donor mice pulse treated with vehicle or FT1050+FT4145. Significantly less GvHD, as determined by survival, weight loss, GVHD score (diarrhea, inactivity, hunched posture, ruffled fur, eye lesion, snout swelling/skin integrity), cytokine production and histopathology of GvHD target organs was observed in recipients receiving FT1050+FT4145 treated cells as compared to those receiving vehicle treated cells. In addition, we observed increased levels of donor T regulatory cells (Tregs) in secondary lymphoid organs concomitant with decreased levels of circulating IFN-ɣ in recipients receiving FT1050+FT4145 treated cells. Based on the attenuation of alloreactive T-cell responses in these preclinical studies, we believe our findings provide a compelling scientific basis to support the clinical evaluation of ex vivo programmed mobilized peripheral blood in patients undergoing HSCT for the treatment of hematologic malignancies. Disclosures Levin: Fate Therapeutics, Inc: Employment, Equity Ownership. Shoemaker:Fate Therapeutics Inc: Employment.

Human induced pluripotent stem cell (hiPSC) technology enables the generation of a potentially unlimited source of therapeutically viable hematopoietic cells for the treatment of numerous hematological and non-hematological malignancies, and represents a highly promising approach for overcoming many of the challenges and limitations of patient-derived cancer immunotherapies. To advance the promise of hiPSC technology as an "off-the-shelf" source of hematopoietic cellular therapeutics, it is essential to be able to efficiently and reproducibly generate not only hematopoietic stem cells (HSCs) but also immune effector populations, including the diverse subsets of T and NK lymphoid cells, through a robust and scalable process. The in vitro derivation of HSCs and lymphocytes is complicated by the existence of at least two temporally and spatially distinct waves of blood cell formation during embryonic development: primitive and definitive. Primitive hematopoiesis initiates in the extraembryonic yolk sac and generates a transient and restricted hematopoietic repertoire consisting mainly of primitive erythroid and myeloid cells. Nascent HSCs only emerge later during the definitive wave from a specialized endothelial progenitor within the arterial vasculature termed hemogenic endothelium (HE). HE undergoes an endothelial-to-hematopoietic transition to give rise to HSCs, which then ultimately migrate to the bone marrow where they sustain multi-lineage hematopoiesis, including T and NK lymphoid cells, throughout adult life. Therefore the generation of HSCs and the formation of lymphoid effector cells from hiPSCs is dependent upon our ability to accurately recapitulate the intricate stages of early embryonic hematopoietic development towards the definitive program. While a limited number of studies have described the directed differentiation of hiPSCs to definitive HE in vitro, a major hurdle in utilizing hiPSCs for therapeutic purposes has been the requirement to initially co-culture such cells with murine-derived stromal cells in the presence of ill-defined serum-containing media in order to maintain pluripotency and induce differentiation. In addition, these protocols have employed an intermediate strategy consisting of embryoid body (EB) formation, which is difficult to scale and hindered by lack of reproducibility. We have previously demonstrated that our proprietary platform for robust and rapid derivation of clonal hiPSC lines, which utilizes small molecule reprograming and single cell selection strategies, generates cells with properties indicative of the naïve, or ground state of pluripotency. In addition to maintaining a homogeneous population of hiPSCs, our platform enables the genetic engineering of such pluripotent cells, at a single cell level, in both nuclease-dependent and -independent strategies. Here we describe a novel method for the generation of definitive HE from naïve hiPSCs in a scalable manner, void of an EB intermediate, under serum/feeder-free conditions. This platform represents a well-defined, small molecule-driven, staged protocol that can readily be translated to meet current good manufacturing practice (cGMP) requirements for the development of "off-the-shelf" hematopoietic cell-based immunotherapies. The derived HE population is definitive in nature as determine by Notch dependency and exhibits multi-lineage potential, as demonstrated through the formation of both T and NK lymphoid cells. HE generated by this protocol can be successfully cryopreserved and banked, serving as a highly-stable feedstock for subsequent derivation of various cell types for therapeutic use, including for T and NK cell-based immunotherapies. We have demonstrated that our proprietary, clinically-adaptable method for the large-scale production of definitive HE can efficiently give rise to a variety of lymphoid cell subsets. These derived lymphocytes, including NK cells, have been extensively characterized in vitro and in vivo, and we have demonstrated functionality through cytokine release and cellular cytotoxicity. Furthermore, through genetic modifications at the single cell hiPSC stage, tumor antigen-targeting and inducible caspase-mediated safety systems have been introduced into safe harbor loci to improve the specificity and safety profiles of hiPSC-derived T and NK cells for cancer immunotherapy applications. Disclosures Clarke: Fate Therapeutics Inc: Employment. Abujarour:Fate Therapeutics Inc: Employment. Robinson:Fate Therapeutics Inc: Employment. Huang:Fate Therapeutics Inc: Employment. Shoemaker:Fate Therapeutics Inc: Employment. Valamehr:Fate Therapeutics Inc: Employment.

16,16 dimethyl prostaglandin E2 (FT1050) was previously identified to be a critical regulator of hematopoetic stem cell (HSC) homeostasis and we hypothesized that a brief ex vivo modulation could improve patient outcomes in umbilical cord blood (UCB) transplantation by increasing the “effective dose” of HSCs. Using preclinical models, we have demonstrated that pulse treating human HSCs with FT1050 significantly enhances the homing and engraftment of donor cells to the bone marrow niche, in part by upregulating CXCR4. To date, preclinical and clinical translation of this therapeutic paradigm has focused on UCB. In an initial clinical evaluation, ex vivo modulation of UCB with FT1050 (2 hours, on site, just prior to infusion) was found to accelerate neutrophil recovery (median 17.5 vs 21 days, p = 0.045) in adult patients undergoing double UCB transplantation (Cutler, 2013). In this Phase 1b study, we also observed low rates of viral reactivation (17% incidence of CMV viremia vs. 42-67% reported in the literature) and GvHD, suggesting that FT1050 may also be having a therapeutically relevant effect on the T-cell compartment of the treated UCB units. To explore the potential therapeutic application of ex vivo modulation of other allogeneic HSC sources, we conducted a series of in vitro studies characterizing the effect of FT1050 on HSCs and T-cells derived from mobilized peripheral blood (mPB). CD34+ cells isolated from mPB or UCB were treated with 10μM FT1050 for 2 hours at 37ºC. We showed that FT1050 binds to the EP2 and EP4 receptors on the cell surface, activating adenylate cyclase which results in a rapid increase in intracellular cAMP levels. Results of a competitive antibody binding assay demonstrated robust and comparable increases in cAMP levels in CD34+ cells from both UCB and mPB. Genome-wide expression analysis of FT1050 treated CD34+ cells showed a highly similar expression response in cells isolated from mPB and UCB, including a 20-fold increase in the key HSC homing receptor CXCR4. We also observed similar FT1050-dependent increases in the surface CXCR4 protein levels and significant improvements in transwell migration rates to gradients of SDF-1. Genome-wide expression analysis within the T-cell compartments from both UCB and mPB (e.g., CD8, CD4, NK, Tregs) revealed a highly similar set of FT1050 induced genes which are involved in cell cycle (e.g., CCND1, CCNE1), tolerance (e.g., DUSP5, FLT1) and anti-viral response (e.g., CD56, EFNB2). The results of this preclinical characterization study provides the basis upon which to explore the future use of FT1050 modulated mPB in a clinical setting. Disclosures Robbins: Fate Therapeutics: Employment, Equity Ownership. Le:Fate Therapeutics: Employment, Equity Ownership. Troung:Fate Therapeutics: Employment, Equity Ownership. Foster:Fate Therapeutics: Employment, Equity Ownership. Rezner:Fate Therapeutics: Employment, Equity Ownership. Mitchell:Fate Therapeutics: Employment, Equity Ownership. Guerrettaz:Fate Therapeutics: Employment, Equity Ownership. Multani:Fate Therapeutics: Employment, Equity Ownership. Shoemaker:Fate Therapeutics: Employment, Equity Ownership.

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.