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.

Type 1 diabetes (T1D) is characterized by the loss of immune self-tolerance, resulting in an aberrant immune responses against self-tissue. A few therapeutics have been partially successful in reverting or slowing down T1D progression in patients, and the infusion of autologous hematopoietic stem cells (HSCs) is emerging as an option to be explored. In this study, we proposed to pharmacologically enhance by ex vivo modulation with small molecules the immunoregulatory and trafficking properties of HSCs to provide a safer and more efficacious treatment option for patients with T1D and other autoimmune disorders. A high-throughput targeted RNA sequencing screening strategy was used to identify a combination of small molecules (16,16-dimethyl PGE2 and dexamethasone), which significantly upregulate key genes involved in trafficking (e.g., CXCR4) and immunoregulation (e.g., programmed death ligand 1). The pharmacologically enhanced, ex vivo-modulated HSCs (regulatory HSCs [HSC.Regs]) have strong trafficking properties to sites of inflammation in a mouse model of T1D, reverted autoimmune diabetes in NOD mice, and delayed experimental multiple sclerosis and rheumatoid arthritis in preclinical models. Mechanistically, HSC.Regs reduced lymphocytic infiltration of pancreatic β cells and inhibited the activity of autoreactive T cells. Moreover, when tested in clinically relevant in vitro autoimmune assays, HSC.Regs abrogated the autoimmune response. Ex vivo pharmacological modulation enhances the immunoregulatory and trafficking properties of HSCs, thus generating HSC.Regs, which mitigated autoimmune diabetes and other autoimmune disorders.

Despite the success of chimeric antigen receptor (CAR)-T cell therapy in various hematologic malignancies, obstacles to an effective therapeutic outcome are highly dependent on the tumor type being targeted and the immune microenvironment that the CAR-T cells encounter. For example, the presence of suppressive cells and soluble factors in the tumor microenvironment (TME) can prevent continued antitumor function of CAR-T cells. Toward this end, we explored multiple genetic editing options, including IL15-based edits, for improving the persistence and activation state of CAR-T cells in the TME. CAR-T cells engineered to express one of five different molecular barcoded constructs were developed and compared, including two versions of an IL-15 signaling complex (IL15RF), constitutively active IL-7 receptor (ca-IL7R), IL-21 signaling complex (IL21RF), and CD16 transgenes. The use of molecular tags allowed us to track CAR-T cell subpopulations in a complex pool with great resolution via next-generation sequencing (NGS) technology. Subsequent in vitro functional testing was performed to assess CAR-T expansion and function in response to serial stimulation with tumor cells bearing cognate antigen. Results showed that after four rounds of stimulation, cytotoxicity was enhanced in CAR-T cells engineered with the ca-IL7R and IL15RF transgene edits (1.5-fold increase in target cell lysis compared to control). Furthermore, an increased proportion of IL-2 producing cells was seen in CAR-T cells expressing the ca-IL7R and IL15RF-based edits (2-fold increase compared to control). In the initial proof of concept study, the best expansion after eight rounds of stimulation was seen in CAR-Ts engineered with IL15RF-based edits. Furthermore, using NGS to screen for the unique molecular barcodes in the CAR-T cell pool, we confirmed the enrichment of CAR-T cells with IL15RF-based edits over multiple rounds of stimulation. Single cell RNAseq was also performed after four and eight rounds of stimulation, where multiple clusters of CAR-T cells were identified and traced back to performance in vitro. Analysis of single cell clusters without IL15RF-based edits exhibited an increase in expression of the checkpoint receptor CTLA4 (p = 4.2E-2) and transcription factor GATA3 (p = 6.9E-5), while clusters with IL15RF-based edits had increased expression of effector molecules GZMB (p = 3.6E-2) and GZMH (p = 2.9E-8), T cell memory related markers CD62L (p = 5.2E-3) and CD27 (p = 2.2E-6), as well as increased expression of the cell proliferation marker Ki-67 (p = 3.3E-12). Because the presence and expansion of T cells in the tumor can be a good prognostic indicator for response to therapy, we used the pool of barcoded CAR-T cells and tested for enrichment/infiltration in a subcutaneous solid tumor implanted in NSG mice. Importantly, enrichment for CAR-T cells with IL15RF-based edits was observed using an NGS readout for the molecular barcodes present in the tumors. Analysis of the data from spatial transcriptomics on tumor sections, and single cell RNAseq of dissociated tumor samples, further informed our understanding of how CAR-T cells with IL15-based edits performed better in the TME (4-fold increase compared to control). The strategy of using molecular barcoded constructs for evaluating clonal populations of engineered CAR-T cells in a pool is shown here to be feasible and that it can be applied as a precise method to concurrently screen many distinct engineered modalities to improve effector cell function, homing and residence in various solid tumor settings. Disclosures Peralta: Fate Therapeutics, Inc.: Current Employment. Robbins:Fate Therapeutics, Inc.: Current Employment. Carron:Fate Therapeutics, Inc.: Current Employment. Denholtz:Fate Therapeutics, Inc: Current Employment. Navarrete:Fate Therapeutics, Inc.: Current Employment. Lu:Fate Therapeutics, Inc.: Current Employment. Yao:Fate Therapeutics, Inc.: Current Employment. Hanok:Fate Therapeutics, Inc.: Current Employment. Sui:Fate Therapeutics, Inc.: Current Employment. Gentile:Fate Therapeutics, Inc.: Current Employment. Sung:Fate Therapeutics, Inc.: Current Employment. ORourke:Fate Therapeutics, Inc.: Current Employment. Lee:Fate Therapeutics, Inc.: Current Employment. Shoemaker:Fate Therapeutics, Inc.: Current Employment. Nguyen:Fate Therapeutics, Inc.: Current Employment. Valamehr:Fate Therapeutics, Inc: Current Employment, Current equity holder in publicly-traded company.

Background: ProTmune is an allogeneic Hematopoietic Progenitor Cell, Apheresis (HPC,A) product that is intended to reduce the incidence and severity of acute graft-versus-host disease (GvHD) and maintain graft-versus-leukemia (GvL) activity. ProTmune consists of pharmacologically modulated HPC,A cells. The manufacture of ProTmune is designed to integrate into current hematopoietic cell transplantation (HCT) procedures that are performed at the point of care in clinical cell processing facilities (CPFs). While several groups accredit these facilities (FACT, AABB), most CPFs are not currently required to comply with the full extent of current good manufacturing practices (cGMP). Here we describe results of Phase 1 manufacture of ProTmune and outline a comprehensive quality system designed to ensure its safety, purity and potency.

Acute graft-versus-host disease (aGvHD) is a serious complication of allogeneic hematopoietic cell transplantation (allo-HCT), occurring in up to half of all patients and remains the leading cause of morbidity and non-relapse mortality. ProTmune was developed as a next-generation graft by modulating the properties of mobilized peripheral blood with a combination of two small molecules, designed to simultaneously reduce the incidence and severity of aGvHD, while maintaining immune protection against infections and relapse. Mechanistically, we have demonstrated that modulated T cells have: (i) dampened T cell receptor (TCR) and (ii) inflammatory cytokine receptor signaling that manifests in reduced overall activation and expansion. Alloreactive T-cell responses typically initiate during the first 48-96 hours after allo-HCT, driven by coordinated signals from the TCR, co-stimulation, and inflammatory cytokines that lead lead to unwanted expansion, proliferation and differentiation of T effector cells that migrate and attack aGvHD target organs. ProTmune modulated T cells, when immediately activated by allo-antigen, TCR crosslinking, or cognate antigen, expand poorly in vitro compared to unmodulated T cells. We next assessed the relative strength of TCR signaling within naïve T cells. Both ERK phosphorylation and CD25 expression, two immediate outcomes of TCR signaling, were dramatically abrogated within ProTmune modulated T cells. The duration of this effect was evaluated by transferring modulated and unmodulated naïve T cells into congenic mice. Relative TCR signal strength was reassessed 14 days later, and, unlike the input T cells, no deficits in pERK, CD25, and TNF expression were observed, demonstrating that ProTmune transiently restricts the relative strength of TCR signaling, thereby limiting their activation and eventual expansion. The JAK-STAT signaling pathway mediates inflammatory cytokine signaling and has been implicated in aGVHD pathogenesis; we therefore assessed whether ProTmune would impact these pathways in human donor T cells. Both Stat1 and Stat3 phosphorylation was significantly limited within ProTmune modulated T cells compared to unmodulated T cells, suggesting that ProTmune modulation also limits the sensitivity of T cells to activating inflammatory cytokines. In summary, these data suggest that T cells within ProTmune modulated grafts are transiently less responsive to TCR signals and inflammatory cytokines, resulting in reduced overall activation and expansion immediately following infusion. These observations provide a rationale for the simultaneously reduced incidence and severity of aGvHD and intact GvL responses previously observed in preclinical animal models. ProTmune is currently undergoing clinical testing in PROTECT, an ongoing Phase 1-2 clinical trial in adult patients with hematologic malignancies.

Abstract 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<0.0001) in the CD57+ NK cell population, and total CD3-CD56+ NK cells were highly enriched (90.9% ± 2.2) relative to pre-culture CD3/CD19 depletion (23.3% ± 2.5) (p<0.0001). We used a high-resolution imaging approach to analyze in vitro NK cell-mediated killing of the ovarian tumor cell line SKOV-3 and the lung carcinoma line A549. We demonstrate that NK cells from CD3/CD19-depleted peripheral blood products cultured for 7 days with the GSK3β inhibitor exhibit significantly more rapid killing kinetics and overall tumor killing relative to NK cells cultured for 7 days with IL-15 and the vehicle control. Superior tumor control of NK cells cultured with IL-15 and the GSK3β inhibitor was also observed against SKOV-3 tumor cells in a murine xenogeneic adoptive transfer model that included IL-2 injections. 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

Abstract 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. <10% CD16+ PB-derived NK cell, upon target cell-mediated activation), and demonstrated enhanced antibody binding compared to PB-derived NK cells expressing the low-affinity variant. In addition to increased expression of the cytolytic molecules perforin and granzyme B and enhanced direct cytotoxicity against tumor targets, hnCD16-NK cells displayed superior ADCC capacity and cytokine production in response to CD16 stimulation. Importantly, manufacture of hnCD16-NK cells was proven to be highly scalable, delivering up to 107 fold expansion over a 35 day period. The maintained proliferative capacity can be in part associated with longer telomere length seen in hnCD16-NK cells. Furthermore, deletion of classical human leukocyte antigen molecules and ectopic expression of immunosuppressive proteins engineered at the iPSC level provided the ability of hnCD16-NK cells to potentially overcome the host histocompatibility barrier and to improve persistence in the allogeneic setting. In conclusion, the preclinical data presented herein highlight the therapeutic value of hnCD16-iNK cells as an ideal ADCC-mediated “off-the-shelf” NK cell-based immunotherapeutic product with augmented persistence, anti-tumor capacity, manufacturing reliability and preclinical efficacy. 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

Abstract 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.

Since the initial identification of natural killer (NK) cells, considerable effort has been made to harness their inherent anti-tumor capacities for the treatment of cancer. Despite recent clinical advancements utilizing the adoptive transfer of allogeneic NK cells, particularly with haplo-identical NK cells for acute myeloid leukemia, significant opportunities remain to further augment the anti-tumor activity of NK cells. Clinical trials have shown the persistence of adoptively transferred NK cells to be generally correlated with improved patient outcome. Natural killer cell persistence can be enhanced through several strategies, including by the administration of long-lived sub-populations such as adaptive-memory NK cells or of NK cells that are genetically modified to prolong survival or attenuate rejection by the host immune system. However, conventional sources of primary allogeneic NK cells have proven to be highly variable and difficult to genetically modify.

T cell-based immunotherapies are at the forefront of an emerging wave of medical discovery focused on harnessing the power of the immune system to treat cancer and other immune disorders. The most dramatic clinical outcome in this new era of cancer treatment has been seen in clinical trials evaluating autologous chimeric antigen receptor (CAR) therapy for the treatment of refractory B cell acute lymphoblastic leukemia where complete responses have reproducibly occurred in 80-90% of patients. While patient-specific CAR therapy hold great promise as a potentially curative therapeutic option, several obstacles hamper its range of application including the challenge of controlling the T cell product consistency, the inherent variability of manipulating a heterogenous cellular population and the inability to create the quantity of CAR T cells needed to support a wide patient base. Human induced pluripotent stem cells (hiPSC)-derived T cells represent a practical and sustainable source of highly defined lymphocytes for cancer immunotherapy. We previously demonstrated a cellular reprogramming platform supporting the efficient derivation of naive state, self-renewing, clonal hiPSC lines that can be effectively engineered with multi-gene and multi-loci targeting strategies. In addition, guided by a small molecule stage-specific differentiation protocol to derive definitive hemogenic endothelium, we have previously developed a highly efficient and scalable hiPSC differentiation system that delivers a consistent and reproducible source of cellular material for further hematopoietic specification. We have also previously demonstrated that hiPSCs reprogrammed from T cells encoding a rearranged endogenous αβTCR (TiPSCs) can generate effector T lymphocytes that, when engineered with a CD19-specific CAR, confer antigen-specific cytotoxic activity against CD19-expressing tumors in vivo. However, the derived T cells appeared skewed both phenotypically and functionally towards an innate γδ T cell subset expressing the CD8αα co-receptor, and were unable to induce complete tumor regression as seen with primary CDαβ CAR T cells. Therefore, to capitalize on the therapeutic potential of TiPSC-derived T cells, it is essential to generate CD8αβ-expressing CTLs. Through specific modulation of the Notch and T cell receptor (TCR) signaling pathways, we have now developed a directed TiPSC differentiation protocol that recapitulates the developmental stages of T cell commitment and have generated CD4 - CD8αβ + single positive T lymphocytes as defined by phenotype and gene expression profile analysis. In vitro assays validate the functionality of these TiPSC-derived T lymphocytes as demonstrated by proliferation in response to CD3/CD28 stimulation (>90% entry into cell cycle), upregulation of activation markers CD25 and CD69 (>50%, >90% respectively), production of the T cell effector cytokines IL2, IFNγ and TNFα (>70%, >30%, >90% respectively) and the release of the cytolytic proteins Granzyme B and Perforin upon target cell engagement. Utilizing our directed TiPSC differentiation platform, a single TiPSC clone produces a clonal and expandable CD4 - CD8αβ + CTL population, wherein greater than 5x10 6 CD4 - CD8αβ + T cells are derived from the single input TiPSC. Furthermore, we demonstrate that CAR-TiPSC can also differentiate efficiently into CD4 - CD8αβ + T cells using our directed differentiation protocol and these CD4 - CD8αβ + CAR T cells target tumor in an antigen specific manner, 70% lysis of CD19 + Raji vs. 5% CD19 - Raji, 77% lysis of CD19 + NALM6 vs. 10% CD19 - NALM6. Further in vitro and in vivo studies are ongoing and will be discussed. In summary, the advances presented here demonstrate the combination of engineered hiPSC clones, T cell differentiation and CAR-T technologies to produce a renewable source of off-the-shelf engineered T cells for cancer immunotherapies. Disclosures Clarke: Fate Therapeutics Inc.: Employment, Equity Ownership. Chang: Fate Therapeutics Inc.: Employment, Equity Ownership. Sasaki: Fate Therapeutics Inc.: Employment, Equity Ownership. Lee: Fate Therapeutics Inc.: Employment, Equity Ownership. Lan: Fate Therapeutics Inc.: Employment, Equity Ownership. Valamehr: Fate Therapeutics: Employment, Equity Ownership.

Abstract 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.

Abstract 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

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 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.

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.

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.

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<0.0001) in the CD57+ NK cell population. NK cells were highly enriched (90.9% ± 2.2) relative to the pre-cultured population post CD3/CD19 depletion (23.3% ± 2.5) (p<0.0001) Sorting experiments where purified NK cell subsets were cultured with monocytes obtained from the same donor and either vehicle or GSK3β inhibitor clearly demonstrated that the GSK3β inhibitor enhanced NK cell maturation. Importantly, NK cells expanded in the presence of the GSK3β inhibitor exhibited enhanced interferon (IFN)-γ production relative to the vehicle control in response to leukemia cells in vitro (2.15-fold ± 0.60, n=7, p=0.0002)and were superior in their ability to control tumor growth out to at least one month in a xenogeneic adoptive transfer model. To determine whether inhibition of GSK3β acted directly or indirectly on NK cells to drive terminal differentiation, purified CD56+ NK cells were cultured with or without monocytes in the presence or absence of the GSK3β inhibitor.The presence of differentiated monocytes was required for effective terminal NK cell differentiation, suggesting a monocyte-dependent, indirect effect of GSK3β (Figure 1A). Monocytes cultured with the GSK3β inhibitor exhibited markedly higher surface expression of an array of markers associated with monocyte maturation including HLA-DR, IL-15Rα, CD80, CD83 and CCR7 (Figure 1B). Thus, our data demonstrate that efficient NK cell differentiation is dependent upon the maturation state of the co-cultured monocytes.We have scaled our process to manufacture a GMP product (referred to as FATE-NK100) for clinical use. Using a representative apheresis product from a CMV seropositive donor containing 19.8 x 108 CD57+ NK cells and 1.7 x 108 CD57+NKG2C+ adaptive NK cells, we achieved 6.4-fold NK cell expansion resulting in a final GMP-grade product containing 142.2 x 108 CD57+ NK cells and 15.8 x 108 CD57+NKG2C+ adaptive NK cells.The cytotoxicity of activity of these ex vivo expanded adaptive NK cells in response to tumor targets is superior that of CD3/CD19-depleted haploidentical NK cells activated overnight with either IL-2 or IL-15 in the NK products 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.

While allogeneic hematopoietic cell transplantation (allo-HCT) is a potentially curative option for many hematologic malignancies, complications such as graft-versus-host disease (GVHD) result in significant morbidity and mortality. Conventional approaches to manage GVHD, such as prophylaxis with immunosuppressive agents or T-cell depletion strategies, are limited by increases in graft failure, viral-associated lymphoproliferative disorders, and disease relapse. Here we present a novel strategy to reduce the rates of GVHD by programming mobilized peripheral blood ex vivo with a cocktail of small molecules prior to allo-HCT. An established xenogeneic mouse model was used to examine the potential of this cell programming strategy to reduce rates of GVHD. Sub-lethally irradiated NOD-scid IL2rγnull (NSG) mice were transplanted with human peripheral blood mononuclear cells (PBMC) pulse treated ex vivo with either vehicle or a cocktail of two small molecules (FT1050+FT4145). Recipients of pharmacologically programmed PBMCs had significantly lower GVHD scores, decreased levels of circulating IFN-ɣ and enhanced survival relative to recipients of vehicle PBMCs (p<0.0001, Mantel-Cox log rank). In addition to xenograft-GVHD studies, we explored the impact of this cell programming strategy in a murine model of GVL. Lethally irradiated BALB/c (H-2Kd) recipient mice were transplanted with either control or FT1050+FT4145 programmed C57BL/6 (H-2Kb) CD8+ T cells and T cell-depleted bone marrow. Prior to allo-HCT, recipients were injected with 2x104 luciferase-expressing A20 lymphoma cells (A20-luc). Bioluminescence imaging was used to monitor the tumor burden over a 28 day period post-HCT. Donor cells programmed with this small molecule cocktail significantly improved survival (p<0.001) while retaining GVL effects against the A20 lymphoma cells. Combined, these studies demonstrate that pharmacologic programming of hematopoietic cells with FT1050+FT4145 prior to allo-HCT may offer an innovative therapeutic approach to reduce rates of GVHD without compromising GVL activity.

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.

Ex vivo exposure of hematopoietic stem cells (HSCs) to 16,16-dimethyl PGE2 (FT1050) has been shown to enhance HSC engraftment potential in preclinical models. Several FT1050-dependent mechanisms may contribute to improved engraftment including increased proliferation, reduced apoptosis, and improved homing by upregulation of CXCR4. CXCR4 is the cell surface receptor for SDF-1, a chemokine that directs migration to the bone marrow niche. We have initiated clinical trials to evaluate whether FT1050 modulation improves outcomes for patients undergoing umbilical cord blood (UCB) transplantation.