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Natural killer (NK) cells mediate the cytolysis of transformed cells and are currently used as an adoptive cellular therapy to treat cancer. Infection with human cytomegalovirus has been shown to expand a subset of "adaptive" NK cells expressing the activation receptor NKG2C that have preferred functional attributes distinct from conventional NK cells. Because NKG2C delivers a strong activating signal to NK cells, we hypothesized that NKG2C could specifically trigger NK-cell-mediated antitumor responses. To elicit a tumor-directed response from NKG2C+ NK cells, we created an anti-NKG2C/IL-15/anti-CD33 killer engager called NKG2C-KE that directs NKG2C+ cells to target CD33+ cells and tumor-associated antigen expressed by acute myelogenous leukemia cells. The NKG2C-KE induced specific degranulation, interferon-γ production, and proliferation of NKG2C-expressing NK cells from patients who reactivated cytomegalovirus after allogeneic transplantation. The NKG2C-KE was also tested in a more homogeneous system using induced pluripotent stem cell (iPSC)-derived NK (iNK) cells that have been engineered to express NKG2C at high levels. The NKG2C-KE triggered iNK-cell-mediated cytotoxicity against CD33+ cells and primary AML blasts. The NKG2C-KE-specific interaction with adaptive NK and NKG2C+ iNK cells represents a new immunotherapeutic paradigm that uniquely engages highly active NK cells to induce cytotoxicity against AML through redirected targeting.

Allogeneic natural killer (NK) cell adoptive transfer is a promising treatment for several cancers but is less effective for the treatment of multiple myeloma. In this study, we report on quadruple gene-engineered induced pluripotent stem cell (iPSC)-derived NK cells designed for mass production from a renewable source and for dual targeting against multiple myeloma through the introduction of an NK cell-optimized chimeric antigen receptor (CAR) specific for B cell maturation antigen (BCMA) and a high affinity, non-cleavable CD16 to augment antibody-dependent cellular cytotoxicity when combined with therapeutic anti-CD38 antibodies. Additionally, these cells express a membrane-bound interleukin-15 fusion molecule to enhance function and persistence along with knock out of CD38 to prevent antibody-mediated fratricide and enhance NK cell metabolic fitness. In various preclinical models, including xenogeneic adoptive transfer models, quadruple gene-engineered NK cells consistently demonstrate durable antitumor activity independent of exogenous cytokine support. Results presented here support clinical translation of this off-the-shelf strategy for effective treatment of multiple myeloma.

INTRODUCTION Natural Killer (NK) cells play pivotal roles in cancer immune surveillance and harbor potent anti-tumor activities. We have engineered an induced pluripotent stem cells (iPSCs) derived, NK cell (termed FT538) that express 1) a high-affinity non-cleavable CD16 Fc receptor to facilitate antibody engagement, 2) a membrane-bound IL-15/IL-15 receptor fusion to enhance NK activity and persistence and 3) a knock-out of CD38 to enhance NK metabolic fitness. FT538 is produced in a GMP-compliant manner as an off-the-shelf immunotherapy. Clinical experiences with FT538 have shown evidence of durable anti-tumor immunity after single administration in select lymphoma patients. METHODS We performed pre-clinical studies to characterize the tumoricidal activity of FT573 against glioblastomas. RESULTS Without specific antibody to engage CD16, FT538 exhibited potent in vitro activity against ~80% of a panel of isocitrate dehydrogenase wild-type patient-derived xenograft (PDX) glioblastoma lines, including MGG8. When directly injected into the murine brain, 50% of the FT538 persisted for > 2 weeks without addition of exogenous cytokine. Moreover, the mice exhibited no evidence of neurologic compromise, and brains harvested after injection show no signs of FT538 activation. In vivo intratumoral injection of FT538 into an orthotopically established MGG8 glioblastoma caused a ~20 fold increase in granzyme B release by FT538, with a comparable increase of cleaved-caspase 3 in the tumor. In independent experiments, > 90% of mice orthotopically implanted MGG8 survived beyond 100 days after intra-tumoral FT538 while all mice treated with mock injection died by 60 days. For glioblastoma PDX refractory to FT538, such as MGG4, tumoricidal activity of FT583 can be induced in vitro and in vivo by the addition of a tri-specific engager targeting B7H3, a surface protein highly expressed in glioblastomas. CONCLUSION Our results demonstrate great promise of FT538 as a glioblastoma therapy, with plans for translation into a first-in-human clinical trial.

Immune cell therapies derived from induced pluripotent stem cells (iPSC) provide a novel opportunity for the treatment of multiple cancer types. Assessment of the persistence and biodistribution of these product candidates requires specific and sensitive methods to detect engineered cells in both liquid and solid biopsies. Here, we present the development and validation of two complimentary nucleic acid-based detection assays for iPSC-derived natural killer (iNK) cell product candidates containing Fate’s proprietary high-affinity, non-cleavable CD16 transgene (hnCD16). The first assay is a droplet digital PCR (ddPCR) method to detect and quantify hnCD16 transgene copies present in a pool of genomic DNA (gDNA). The primers and probe were designed to recognize the optimized codons of hnCD16. Assay linearity and accuracy were assessed through titration studies using 0.024 to 1 ng of hnCD16-containing DNA spiked into different amounts of hnCD16-negative gDNA. Precision was determined through multiple assay runs by different operators on two instruments. The second assay is an in situ hybridization based method utilizing RNAscope࣪ technology to detect cells expressing hnCD16 in fixed tissue. Probes targeting hnCD16 were used to optimize signal specificity. Cells expressing hnCD16 and tissues from in vivo studies treated with iNK products served as positive controls. For the ddPCR assay, absolute limit of detection (aLoD) was determined to be 4.9 copies of hnCD16 per 20 µL reaction, regardless of total genomic mass input. Absolute limit of quantification (aLoQ) was 12 copies per 20 µL reaction with a %CV ≤30. Relative limit of quantification (rLoQ), assessing transgene to total DNA ratio, is affected by the background gDNA input and is less sensitive with lower input mass. rLoQ for total mass of 70 - 250 ng was 97 - 22 copies/µg gDNA (0.064% - 0.015%) with a %CV ≤30. The sensitivity of this input range allows evaluation of clinical samples with low cellularity. While ddPCR provides robust quantification of the hnCD16 transcript, the RNAscope࣪ assay informs localization of the iNK product. Specificity of the probe was established by confirming its lack of affinity for endogenous CD16 using a variety of human normal and tumor tissues and by staining hnCD16-positive fixed cell pellets and tissues from in vivo studies. In cell pellets, positive RNAscope࣪ signal correlated with the known ratio of transgene positive cells. In murine tissues previously confirmed to contain iNK cell product, the RNAscope࣪ positive staining correlated with NKG2A immunohistochemistry staining, confirming the presence of product NK cells. The combined use of both the ddPCR and RNAscope࣪ assays targeted to hnCD16 allows for detection and quantification of transgene-bearing iNK cells in a wide variety of patient samples including tumor biopsies. Both assays are being utilized for cell detection and quantification in our ongoing clinical trials. Citation Format: Cara E. Bickers, Judy L. Martin, Steven Castro, Jason Zhang, Thomas Dailey, Eric Sung, Suzanna Gasparian, Jason O'Rourke, Moyar Ge, Tom T. Lee, Janel Huffman, Jode Goodridge, Ryan Bjordahl, Bahram Valamehr, Peter M. Szabo, Lilly Wong, Sarah Cooley. Detection of genetically engineered iPSC-derived natural killer cells in blood and tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1138.

Chimeric antigen receptor (CAR) T-cell therapies have revolutionized the treatment of hematologic malignancies and have shown significant potential in solid tumor indications. However, logistical complexities associated with patient-specific CAR T-cell therapies often limit broad accessibility. Many of these challenges can be overcome with an allogeneic cellular product, but immune cell-mediated rejection of allogeneic cellular therapies remains a significant concern. Both allogeneic and autologous cell therapies currently rely on lymphodepleting conditioning to modulate the immune system and create greater access to homeostatic cytokines. However, protracted lympho-conditioning has been associated with poor immune reconstitution and increased susceptibility to opportunistic infections. Therefore, an ideal allogeneic cell therapy would be able to avoid immune rejection while reducing or eliminating the need for chemotherapeutic conditioning to deplete host lymphocytes. To address many of these challenges, we engineered our novel alloimmune defense receptor (ADR) that targets 41BB+ alloreactive immune cells while providing a CD3z signaling boost into our off-the-shelf iPSC derived NK cells expressing anti-CD19 CAR (CAR iNK). The ability of ADR+ CAR iNK cells to resist alloimmune rejection was tested by co-culturing ADR+ CAR iNK cells with allogeneic PBMCs from ten donors in a mixed lymphocyte reaction assay. Notably, ADR+ CAR iNK cells co-cultured with allogeneic PBMCs persisted to similar levels as the PBMC-free culture while ADR negative CAR iNK cells were eliminated when co-cultured with allogeneic PBMCs. Furthermore, all PBMC donors screened in ADR+ CAR iNK cell co-cultures showed ablation of reactive 41BB+ NK and T cells, with non-activated T cells remaining intact. CAR iNK cells +/- ADR were then compared in a Nalm6 disseminated in vivo model for anti-tumor efficacy. ADR+ CAR iNK cells and their ADR negative counterparts were found to equivalently control tumor. Building on this tumor model, we co-infused allogeneic T cells to mimic an immuno-competent setting. The data demonstrated that ADR negative CAR iNK cells were depleted and were unable to control tumor growth while significant levels of allogeneic T cells persisted. In contrast, ADR+ CAR iNK cells were able to resist allogeneic T cell attack, control tumor, and persist when compared to the ADR negative control. Collectively, our preliminary data suggest that ADR-armed CAR iNK cells withstand immune cell-mediated rejection with uncompromised effector function. We are actively developing models to confirm our initial finding that ADR+ effector cells also benefit from their engagement with alloreactive cells in immuno-competent settings to promote enhanced anti-tumor responses, proliferation, and persistence. Citation Format: Alan M. Williams, Ken Hayama, Yijia Pan, Brian Groff, Rina Mbofung, Lauren Fong, Nicholas Brookhouser, Berhan Mandefro, Ramzey Abujarour, Tom Lee, Quirin Hammer, Karl-Johan Malmberg, Maksim Mamonkin, Ryan Bjordahl, Jode Goodridge, Bahram Valamehr. A novel synthetic stealth receptor that redirects host immune cell alloreactivity and potentiates functional persistence of adoptively transferred off-the-shelf cell-based cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2828.

Introduction: B7-H3 (CD276) has gained significant clinical interest as a pan-tumor target antigen for development of various immuno-oncology agents. Due to its broad expression on a wide variety of solid tumors and minimal expression on normal tissues, B7-H3 is an ideal tumor antigen target. Additionally, high levels of B7-H3 are found on “immunologically cold” tumors such as glioblastoma multiforme, prostate cancer, head and neck cancer and soft tissue sarcomas, which typically have poor response to approved immune therapies. To effectively target B7-H3 with an off-the-shelf cellular therapy, we describe here the development of camB7-H3 CAR-NK cell utilizing our iPSC platform to engineer multiple modalities into a clonal iPSC line, which can serve as the starting cell source for mass production of off-the-shelf, iPSC-derived CAR-NK cells (CAR-iNK cells). Methods: A camelid nanobody specific for human B7-H3 (camB7-H3) was discovered using a phage display library and validated in functional assays. camB7-H3 CAR-iNK cells were designed to 1) express membrane-bound IL-15/IL-15 receptor fusion for enhanced persistence, 2) have a CD38 knockout to improve metabolic fitness, 3) express a high-affinity non-cleavable CD16 to maximize ADCC when combined with a therapeutic antibody, and 4) express an anti-camB7-H3 CAR optimized for NK cell signaling. As the initial preclinical study, camB7-H3 CAR-iNK cells were assessed using flow cytometry-based functional assays evaluating CD107a and IFNγ or xCelligence target killing assays against B7-H3 transgenic or naturally expressing tumor cell lines. Results: The camelid single domain B7-H3 initially tested in CAR-T exhibited B7-H3 specific binding and specific activity against several solid tumor cell lines (breast, ovarian, prostate, lung). We next produced camB7-H3 CAR-iNK cells and demonstrated superior B7-H3-specific target elimination compared to untransduced iNK cells. These results were seen across multiple tumor lines representing various solid tumor indications. Further enhancement of anti-tumor efficacy was seen when combined with therapeutic antibodies, including trastuzumab and cetuximab. Conclusions: We have successfully produced and validated the specificity and function of an engineered camB7-H3 CAR-iNK cell exhibiting robust target killing and on-target specificity. To our knowledge, this is the first camelid nanobody antigen recognition domain reported in a CAR-NK cell to be used as an off-the-shelf immunotherapy. The combining camB7-H3 CAR-NK with monoclonal antibodies targeting HER2 and EGFR as a dual targeting approach will add additional tumor specificity, further increase the efficacy of tumor cell elimination and prevent antigen escape. Additional camB7-H3 CAR-iNK cell preclinical studies are in process and will be discussed. Citation Format: Nicholas Zorko, Frank Cichocki, John Goulding, Bryan Hancock, Robert Blum, Mochtar Pribadi, Bjoern Gaertner, Tom Lee, Martin Felices, Ryan Bjordahl, Bahram Valamehr, Jeffrey S. Miller. FT573: Preclinical development of multiplexed-engineered iPSC-derived NK cells expressing a novel camelid nanobody chimeric antigen receptor (CAR) targeting pan-cancer antigen B7-H3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2761.

Resistance to CAR-T cell therapy through antigen loss and clonal heterogeneity is a major limiting factor in achieving durable responses, even in highly persistent cell therapies. Simultaneous targeting of multiple tumor antigens with a single therapeutic modality offers the potential to treat heterogeneous tumor populations, prevent antigen escape, and induce durable clinical remission. Here, we demonstrate the application of a unique dual-CAR approach simultaneously targeting two tumor associated antigens (TAA) for the treatment of multiple myeloma (MM) using an off-the-shelf induced pluripotent stem cell (iPSC) derived NK cell platform. The iPSC background has been functionally enhanced and can be combined with therapeutic antibodies. A CAR targeting B cell maturation antigen (BCMA), a well-defined TAA in MM, was designed utilizing a previously published high-affinity binding sequence shown to exhibit high selectivity to BCMA with enhanced recognition of low-BCMA expressing myeloma cells (Bluhm et al., Molec Ther 2018). This CAR was combined with a companion CAR targeting the pan-TAAs, MICA and MICB. The CAR binding sequence targets the conserved α3 domain of MICA/MICB, which we have previously shown to inhibit MICA/B shedding by blocking the putative cleavage site (Andrade et al., Science 2018). The designed anti-MICA/B-α3 CAR exhibits selective targeting potential against a broad range of tumor types. To determine the suitability of co-targeting BCMA and MICA/B in MM, we surveyed surface expression of BCMA and MICA/B on a variety of MM cancer cell lines and observed a complimentary pattern of co-expression compatible with a dual-CAR to broaden the targeting approach of malignant plasma cells. In a reductionist approach, dual CAR-iNK cells exhibited antigen-specific activation, degranulation and cytotoxicity against a Nalm6 target line constitutively expressing the BCMA and MICA/B. Similar trends were observed in a series of long-term cytotoxicity assays against several MM lines, consistent with antibody staining on target cells, illustrating that co-targeting of MICA/B and BCMA expands the breadth of coverage against MM. Known modulators of antigen expression were tested for their ability to provide further depth of response, and therapeutic antibodies such as anti-CD38 were tested in combination to exploit the non-cleavable CD16 and CD38KO edits in the iPSC backbone. These combinations were further tested in vivo against a disseminated model of multiple myeloma where BCMA/MICA dual CAR-iNK cells demonstrated superior tumor control relative to single-CAR controls, and TGI was augmented with the addition of Daratumamab. These data highlight the applicability of a multi-targeted approach in MM patients, whereby MM dual-CAR NK and/or T cells maintain responsiveness to malignant cells that shed or downregulate tumor antigens to evade treatment. Citation Format: Ketan Mathavan, John Reiser, Sajid Mahmood, Yijia Pan, Bryan Hancock, Robert Blum, Wen-I Yeh, Andrew Houk, Chia-Wei Chang, Tom Lee, Bobby Goulding, Jode Goodridge, Ryan Bjordahl, Bahram Valamehr, Uta Hoepken, Armin Rehm, Kai Wucherpfennig. Combining dual CAR iPSC-derived immune cells with antibody for multi-antigen targeting to overcome clonal resistance in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4190.

Background MHC class I related proteins A (MICA) and B (MICB) are induced by cellular stress and transformation, and their expression has been reported for many cancer types. NKG2D, an activating receptor expressed on natural killer (NK) and T cells, targets the membrane-distal domains of MICA/B, activating a potent cytotoxic response. However, advanced cancer cells frequently evade immune cell recognition by proteolytic shedding of the α1 and α2 domains of MICA/B, which can significantly reduce NKG2D function and the cytolytic activity. Methods Recent publications have shown that therapeutic antibodies targeting the membrane-proximal α3 domain inhibited MICA/B shedding, resulting in a substantial increase in the cell surface density of MICA/B and restoration of immune cell-mediated tumor immunity.1 We have developed a novel chimeric antigen receptor (CAR) targeting the conserved α3 domain of MICA/B (CAR-MICA/B). Additionally, utilizing our proprietary induced pluripotent stem cell (iPSC) product platform, we have developed multiplexed engineered, iPSC-derived CAR-MICA/B NK (iNK) cells for off-the-shelf cancer immunotherapy. Results A screen of CAR spacer and ScFv orientations in primary T cells delineated MICA-specific in vitro activation and cytotoxicity as well as in vivo tumor control against MICA+ cancer cells. The novel CAR-MICA/B design was used to compare efficacy against NKG2D CAR T cells, an alternative MICA/B targeting strategy. CAR-MICA/B T cells showed superior cytotoxicity against melanoma, breast cancer, renal cell carcinoma, and lung cancer lines in vitro compared to primary NKG2D CAR T cells (p Conclusions Ongoing work is focused on extending these preclinical studies to further support the clinical translation of an off-the-shelf, CAR-MICA/B iNK cell cancer immunotherapy with the potential to overcome solid tumor escape from NKG2D-mediated mechanisms of recognition and killing. Reference Ferrari de Andrade L, Tay RE, Pan D, Luoma AM, Ito Y, Badrinath S, Tsoucas D, Franz B, May KF Jr, Harvey CJ, Kobold S, Pyrdol JW, Yoon C, Yuan GC, Hodi FS, Dranoff G, Wucherpfennig KW. Antibody-mediated inhibition of MICA and MICB shedding promotes NK cell-driven tumor immunity. Science 2018 Mar 30;359(6383):1537–1542.

Background B7H3 is a tumor associated antigen (TAA), found on numerous malignancies including prostate, lung, and breast cancers. High levels of B7H3 expression are correlated with late stage disease and poor prognosis. Furthermore, B7H3 is minimally expressed on normal tissue, making it an ideal TAA for broad cancer treatment strategy. We developed a tri-specific killer engager (TriKETM) consisting of a nanobody anti-CD16, IL-15, and nanobody anti-B7H3 joined by flexible linkers (camB7H3 TriKE) (figure 1A). The combination of B7H3 TriKE with an off-the-shelf NK cell therapy presents an appealing therapeutic strategy for the treatment of solid tumors with decreased risk of toxicity in allogeneic settings compared to T-cell derived products. Methods An anti-B7H3 nanobody was developed via biopanning and cloned into a TriKE vector. TriKE was produced in Expi293 cells and affinity purified using poly-His tag. NK cells were co-incubated with cell lines exhibiting a range of B7H3 expression and with 3nM of camelid B7H3 TriKE or control. We have previously derived NK cells expressing high affinity non-cleavable hnCD16, CD38 KO, and IL-15/IL-15R fusion from clonal master engineered iPSC lines. Engineered iNK cells were tested in conjunction with the TriKE. A repeated measures ANOVA was used for statistical comparisons as noted in figure legends Results Engineered iNK cells co-incubated with camB7H3 TriKE and C4-2 prostate cells significantly increased degranulation (CD107a) and cytokine production (IFN-gamma) compared to controls (figure 1B/C, P Conclusions camB7H3 TriKE dramatically increases function and activation on endogenous NK cells as well as engineered iNK cell, which can be adoptively transferred to patients with a broad range of cancers, including prostate cancer. TriKE activity was potent across a broad concentration spectrum and corresponded directly with B7H3 target expression. These studies represent the proof-of-concept of a novel pairing of off-the-shelf, engineered iNK cells with B7H3-directed pan-cancer engager molecules (TriKEs and CARs) to enhance specificity, persistence and anti-tumor function.

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.

Antibody-dependent cellular cytotoxicity (ADCC) is a key effector mechanism of natural killer (NK) cells that is mediated by therapeutic monoclonal antibodies (mAbs). This process is facilitated by the Fc receptor CD16a on human NK cells. CD16a appears to be the only activating receptor on NK cells that is cleaved by the metalloprotease a disintegrin and metalloproteinase-17 upon stimulation. We previously demonstrated that a point mutation of CD16a prevents this activation-induced surface cleavage. This noncleavable CD16a variant is now further modified to include the high-affinity noncleavable variant of CD16a (hnCD16) and was engineered into human induced pluripotent stem cells (iPSCs) to create a renewable source for human induced pluripotent stem cell–derived NK (hnCD16-iNK) cells. Compared with unmodified iNK cells and peripheral blood–derived NK (PB-NK) cells, hnCD16-iNK cells proved to be highly resistant to activation-induced cleavage of CD16a. We found that hnCD16-iNK cells were functionally mature and exhibited enhanced ADCC against multiple tumor targets. In vivo xenograft studies using a human B-cell lymphoma demonstrated that treatment with hnCD16-iNK cells and anti-CD20 mAb led to significantly improved regression of B-cell lymphoma compared with treatment utilizing anti-CD20 mAb with PB-NK cells or unmodified iNK cells. hnCD16-iNK cells, combined with anti-HER2 mAb, also mediated improved survival in an ovarian cancer xenograft model. Together, these findings show that hnCD16-iNK cells combined with mAbs are highly effective against hematologic malignancies and solid tumors that are typically resistant to NK cell–mediated killing, demonstrating the feasibility of producing a standardized off-the-shelf engineered NK cell therapy with improved ADCC properties to treat malignancies that are otherwise refractory.

Natural killer (NK) cells are innate lymphocytes that target malignant cells via non-clonotypic receptors to induce natural cytotoxicity and that recognize tumor-bound antibodies to induce antibody-dependent cell-mediated cytotoxicity (ADCC). Human NK cells exclusively mediate ADCC through the IgG Fc receptor, CD16A, and studies have demonstrated that increasing the binding affinity between CD16A and therapeutic monoclonal antibodies (mAbs), mediated by the high-affinity 158V polymorphism, can augment clinical efficacy. Given the exquisite specificity and diverse antigen detection of anti-tumor mAbs, we sought to arm iPSC-derived NK (iNK) cells expressing a high-affinity recombinant FcγR with various mAbs as unique tumor-targeting strategy for various malignancies. As a member of the FcγR family, CD64 (FcγRI) possesses the highest affinity and can uniquely facilitate antibody preabsorption but it is normally expressed by myeloid cells. To leverage CD64 in NK cells, we developed a novel FcγR recombinant fusion comprising the extracellular region of CD64 with the transmembrane and intracellular regions of other NK cell activating receptors, including CD16A (CD64/16A) (figure 1A). The recombinant CD64/16A engineered into a clonal master induced pluripotent stem cell (iPSC) line for mass production of off-the-shelf iPSC-derived CD64/16A NK (iNK-CD64/16A) cells, can be armed with mAbs, including various combinations thereof to enable multi-antigen targeting and to address tumor heterogeneity (figures 1B and 2). To determine optimal binding and FcR saturation of iNK-CD64/16 cells, rituximab (anti-CD20 therapeutic mAb) was added in a two-hour preabsorbtion assay (figure 3A). Using an in vitro Delfia® ADCC assay, we show that iNK-CD64/16A cells mediated ADCC against Raji cells, a Burkitt Lymphoma cell line, when the iNKs were preabsorbed and armed with rituximab (figure 3B). Considering the high-affinity state of CD64, we examined the effects of free IgG on ADCC by iNK-CD64/16A cells. Using an IncuCyte® Live Cell Analysis, ADCC was evaluated in the presence of purified human IgG. Despite the high levels of excess IgG, iNK-CD64/16A cells mediated efficient ADCC when rituximab was either added to the assay (figure 4A) or preabsorbed to the cells (figure 4B), demonstrating that saturating levels of free IgG did not prevent ADCC in either setting. To determine the ability of preabsorbed and armed iNK-CD64/16 cells to retain rituximab and perform serial killing, we performed a sequential killing assay using an IncuCyte® Live Cell Analysis where preabsorbed iNK-CD64/16A cells were thawed and co-cultured with or without Raji cells for 48 hours, followed by a second round of co-culture. As shown in figure 5, iNK-CD64/16A cells armed with rituximab retain ADCC capacity and perform serial killing for an extended time. To establish that iNK-CD64/16A cells can be armed with assorted therapeutic mAbs to target other tumor-associated antigens, we next determined the ability of iNK-CD64/16A cells preabsorbed and armed with anti-HER2 mAb, trastuzumab, to target the adenocarcinoma ovarian cancer cell line SKOV-3. Indeed iNK-CD64/16A cells armed with preabsorbed trastuzumab were able to effectively kill SKOV-3 cells via in vitro ADCC by IncuCyte® Live Cell Analysis (figure 6). We next investigated in vivo ADCC using NSG mice implanted with 3x10 5 SKOV-3 cells expressing firefly luciferase intraperitoneally (IP). 10 million iNK-CD64/16A with or without preabsorbed trastuzumab were injected IP (figure 7A), and a significant reduction in tumor volume in animals treated with iNK-CD64/16A cells armed with trastuzumab compared to unarmed iNK-CD64/16A cells (figure 7B). Collectively, our data show that iNK-CD64/16A cells can be armed with various therapeutic mAbs through a unique preabsorption strategy to mediate a potent and durable ADCC activity. The versatility of mAb-armed iNK-CD64/16A cells is being further investigated in various preclinical models to further elucidate the potential of this approach to overcome antigen escape and address tumor heterogeneity. Figure 1 Figure 1. Disclosures Lee: Fate Therapeutics, Inc: Current Employment. Chu: Fate Therapeutics: Current Employment. Rogers: Fate Therapeutics: Current Employment. Bjordahl: Fate Therapeutics: Current Employment. Hosking: Fate Therapeutics: Current Employment. Shirinbak: Fate Therapeutics, Inc.: Current Employment. Miller: Fate Therapeutics, Inc: Consultancy, Patents & Royalties, Research Funding; GT Biopharma: Consultancy, Patents & Royalties, Research Funding; Vycellix: Consultancy; ONK Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Magenta: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Wugen: Membership on an entity's Board of Directors or advisory committees. Valamehr: Fate Therapeutics, Inc.: Current Employment. Walcheck: Fate Therapeutics: Research Funding.

Treatments for B-cell malignancies have improved over the past several decades with clinical application of the CD20-specific antibody rituximab and chimeric antigen receptor (CAR) T cells targeting CD19. Despite the success of these therapies, loss of CD20 after rituximab treatment has been reported in leukemia and lymphoma patients. Additionally, up to 50% of all patients receiving anti-CD19 CAR T-cell therapy relapse within the first year with many of those patients exhibiting CD19 loss. Thus, new therapeutic approaches are needed to address tumor antigen escape. Accordingly, we generated triple gene-modified iPSC-derived NK (iNK) cells, termed "iDuo" NK cells, tailored to facilitate multi-antigen targeting. The iPSC line was clonally engineered to express high-affinity, non-cleavable CD16a (hnCD16), an anti-CD19 CAR optimized for NK cell signaling, and a membrane-bound IL-15/IL-15R fusion (IL-15RF) molecule to enhance NK cell persistence (Fig. 1A). To model antigen escape, we generated CD19 knockout AHR77 lymphoma cells alongside wild type AHR77 cells (both CD20 +) as targets in cytotoxicity assays. Activated peripheral blood NK (PBNK) cells, non-transduced iNK cells, and iDuo NK cells were tested as effectors. Unlike PBNK cells or non-transduced iNK cells, iDuo NK cells efficiently eliminated wild type AHR77 cells with or without the addition of rituximab at all tested E:T ratios. Similarly, iDuo NK cells in combination with rituximab were uniquely able to efficiently eliminate CD19 KO AHR77 cells due to enhanced antibody-dependent cellular cytotoxicity (ADCC) driven by hnCD16 (Fig. 1B-E). Cytotoxicity mediated by iDuo NK cells was also evaluated using primary chronic lymphocytic leukemia (CLL) cells. Compared to expanded PBNK cells and non-transduced iNK cells, only iDuo NK cells (in the absence of rituximab) were able to kill primary CLL cells (Fig. 1F). Expression of IL-15RF by iDuo NK cells uniquely supports in vitro expansion without the need for cytokine supplementation. To determine whether IL-15RF supports in vivo persistence of iDuo NK cells, CD19 CAR iNK cells (lacking IL-15RF) and iDuo NK cells were injected into NSG mice without the addition of cytokines or CD19 antigen availability. iDuo NK cell numbers peaked within a week after injection and persisted at measurable levels for ~5 weeks, in marked contrast to CD19 CAR iNK cell numbers that were undetectable throughout (Fig. 1G). To evaluate the in vivo function of iDuo NK cells, NALM6 leukemia cells were engrafted into NSG mice. Groups of mice received tumor alone or were treated with 3 doses of thawed iDuo NK cells. iDuo NK cells alone were highly effective in this model as evidenced by complete survival of mice in the treatment group (Fig. 1H). To assess iDuo NK cells in a more aggressive model, Raji lymphoma cells were engrafted, and groups of mice received rituximab alone, iDuo NK cells alone, or iDuo NK cells plus rituximab. Mice given the combination of iDuo NK cells and rituximab provided extended survival compared to all other arms in the aggressive disseminated Raji lymphoma xenograft model (Fig. 1I). One disadvantage of anti-CD19 CAR T cells is their inability to discriminate between healthy and malignant B cells. Because NK cells express inhibitory receptors that enable "self" versus "non-self" discrimination, we reasoned that iDuo NK cells could have higher cytotoxicity against tumor cells relative to healthy B cells. To address this, we labeled Raji cells, CD19 + B cells from healthy donor peripheral blood mononuclear cells (PBMCs) and CD19 - PBMCs. Labeled populations of cells were co-cultured with iDuo NK cells, and specific killing was analyzed. As expected, iDuo NK cells did not target CD19 - PBMCs. Intriguingly, iDuo NK cells had much higher cytotoxic activity against Raji cells compared to primary CD19 + B cells, suggesting a preferential targeting of malignant B cells compared to healthy B cells. Together, these results demonstrate the potent multi-antigen targeting capability and in vivo antitumor function of iDuo NK cells. Further, these data suggest that iDuo NK cells may have an additional advantage over anti-CD19 CAR T cells by discriminating between healthy and malignant B cells. The first iDuo NK cell, FT596, is currently being tested in a Phase I clinical trial (NCT04245722) for the treatment of B-cell lymphoma. Figure 1 Figure 1. Disclosures Cichocki: Gamida Cell: Research Funding; Fate Therapeutics, Inc: Patents & Royalties, Research Funding. Bjordahl: Fate Therapeutics: Current Employment. Gaidarova: Fate Therapeutics, Inc: Current Employment. Abujarour: Fate Therapeutics, Inc.: Current Employment. Rogers: Fate Therapeutics, Inc: Current Employment. Huffman: Fate Therapeutics, Inc: Current Employment. Lee: Fate Therapeutics, Inc: Current Employment. Szabo: Fate Therapeutics, Inc: Current Employment. Wong: BMS: Current equity holder in publicly-traded company; Fate Therapeutics, Inc: Current Employment. Cooley: Fate Therapeutics, Inc: Current Employment. Valamehr: Fate Therapeutics, Inc.: Current Employment. Miller: Magenta: Membership on an entity's Board of Directors or advisory committees; ONK Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Vycellix: Consultancy; GT Biopharma: Consultancy, Patents & Royalties, Research Funding; Fate Therapeutics, Inc: Consultancy, Patents & Royalties, Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees; Wugen: Membership on an entity's Board of Directors or advisory committees.

Chimeric antigen receptor (CAR) T-cell therapies have revolutionized the treatment of hematologic malignancies, however, logistical complexities associated with patient-specific CAR T-cell therapies often limit broad accessibility to patients. Many of these challenges can be overcome with an allogeneic cellular product that is available off-the-shelf, and overcoming immune cell-mediated rejection of allogeneic cell therapy is an area of significant research. Conditioning chemotherapies, which are commonly administered with CAR T-cell therapy, suppress a patient's immune system and may create a suitable window of activity for allogeneic cell therapies to elicit clinical response. However, protracted lympho-conditioning has been associated with poor immune reconstitution and increased susceptibility to opportunistic infections. Deletion of human leukocyte antigen (HLA) surface expression is known to abrogate T-cell alloreactivity, but deletion of class I HLA must be combined with other immune-modulating strategies to avoid NK cell-mediated recognition. To this end, allogeneic models combining class I HLA deletion with NK cell inhibitory molecules, such as HLA-E and CD47, have been shown to abrogate NK cell reactivity in mouse models. However, since HLA-E is the canonical activator of NKG2C, a dominant activating receptor found on human NK cells, and since the ligand for CD47, SIRPα, is known to be expressed on macrophages and dendritic cells and not on human NK cells, the observed effects of these immune-modulating strategies may not translate into the patient-treatment setting. In assessment of their protective effects in a defined human system, we found that HLA-E or CD47 overexpression on class I HLA null human cells offer only partial protection in evading various human NK cell compartments. We found that class I HLA-null K562 cells engineered to over-express CD47 were ineffective in inhibiting NK cells (0 to 7% inhibition). Separately, K562 cells engineered to over-express HLA-E, while effective in inhibiting NKG2A+ NK cells (90.2% +/- 3.7% inhibition), were unable to completely inhibit CD56 dim NK cells (33.2% +/- 29.6% inhibition) and not only failed, but instead activated, NKG2C+ NK cells (167% +/- 69% activation). Our data highlight the limitations of engineered CD47 and HLA-E modalities in suppressing broad populations of NK cells in clinically relevant settings. We therefore evaluated expression of the alloimmune defense receptor (ADR) that uniquely targets alloreactive immune cells (Mo et al. Nat Biotechnol 2021). We have shown that the expression of ADR has the potential to evade host immune cells without the need for further genetic editing such as class I HLA deletion. To determine its applicability for off-the-shelf cell therapy, ADR expression was engineered into induced pluripotent stem cells (iPSCs) and iPSC-derived CAR-NK (CAR-iNK) cells were generated. CAR-iNK cells carrying the ADR modality (ADR+ CAR-iNK cells) showed normal patterns of differentiation (>99% CD56+ with co-expression of NK cell receptors such as NKG2D, NKp30 and NKp46), suggesting that ADR expression did not disrupt hematopoiesis or the expansion of iNK cells. Additionally, ADR+ and ADR-negative CAR-iNK cells produced similar cytotoxicity against tumor cells. We next tested the ability of ADR to provide resistance to alloimmune rejection by coculturing ADR+ CAR-iNK cells with allogeneic pBMCs in a mixed lymphocyte reaction (MLR) assay. Notably, ADR+ CAR-iNK cells maintained durable persistence throughout the entire duration of the MLR assay and suppressed the expansion of alloreactive T- and NK-cells in comparison to the control arm (Figure 1). Collectively, initial preclinical studies suggest that ADR-modified CAR-iNK cells resist host immune cell rejection, while eliciting a durable anti-tumor response. Our preliminary data show evidence toward a promising off-the-shelf solution for elimination of broad pools of alloreactive T- and NK- effector cells in the clinical setting without the need for lympho-depleting conditioning or genetic editing strategies. Figure 1 Figure 1. Disclosures Williams: Fate Therapeutics: Current Employment. Abujarour: Fate Therapeutics, Inc.: Current Employment. Lee: Fate Therapeutics, Inc.: Current Employment. Malmberg: Merck: Research Funding; Vycellix: Consultancy; Fate Therapeutics: Consultancy, Research Funding. Mamonkin: Beam Therapeutics: Other: Licensing payments; Fate Therapeutics: Other: Licensing payments; Allogene Therapeutics: Consultancy, Other: Licensing payments; Xenetic Biosciences: Consultancy, Membership on an entity's Board of Directors or advisory committees. Bjordahl: Fate Therapeutics: Current Employment. Valamehr: Fate Therapeutics, Inc.: Current Employment.

BackgroundChimeric antigen receptor (CAR)-T cell therapy has revolutionized cancer treatment, but it is associated with significant dose-limiting toxicities, restricted tumor targeting (limited by specific antigen expression), and, notably, a lack of multi-antigen targeting capability to mitigate tumor associated immune evasion and heterogeneity. Furthermore, dysfunctional starting material, product inconsistency, and small manufacturing lot size limits the application and on-demand availability of CAR-T cell therapy.MethodsTo overcome these considerable limitations, we have developed FT536, a first-of-kind, induced pluripotent stem cell (iPSC)-derived NK (iNK) cell with a novel CAR that ubiquitously targets cancer cells through canonical stress ligand recognition. We have previously reported FT536 recognizes the conserved α3 domain of the pan-tumor associated antigens MICA and MICB (MICA/B), and is derived from a renewable master iPSC line that contains multiplexed genetic edits to enhance effector cell functionality, persistence, and multi-antigen targeting capabilities via high affinity non cleavable CD16 (hnCD16) mediated antibody dependent cellular cytotoxicity (ADCC). Here we preview the nonclinical study for the investigational new drug (IND) application for FT536.ResultsUtilizing a manufacturing process analogous to pharmaceutical drug product development, we demonstrate FT536 can be consistently and uniformly produced with a greater than 4x10E7 fold cellular expansion per manufacturing campaign. Furthermore, FT536 can be cryopreserved at clinical scale to support off-the-shelf clinical application, with rapid product thaw and immediate patient infusion in an out-patient setting. Functional evaluation demonstrated that FT536 uniquely possesses potent and persistent antigen specific cytolytic activity against an array of solid and hematological tumor lines. Through its hnCD16 modality, FT536 can be utilized in combination with monoclonal antibodies to provide multi-antigen targeting capabilities and in conjunction with chemotherapeutics and/or radiation that augment surface MICA/B expression. In addition, directly thawed and infused FT536 demonstrated significant tumor growth inhibition in multiple solid and liquid in vivo xenograft models, in which tumor control was further enhanced in combination with a therapeutic antibody (figure 1). Finally, ongoing studies utilizing a lung adenocarcinoma model have highlighted the sustained persistence of FT536 in lung tissue up to 33 days following a single dose infusion without the need for exogenous cytokine support.Abstract 117 Figure 1FT536 provides statistically significant in vivo anti-tumor activity which is enhanced in combination with ADCC active monoclonal antibody therapy. (A-B) FT536 significantly reduced the number of lung and liver (not shown) metastases compared to CAR negative iNK control cells in a murine metastatic melanoma model using B16-F10 cells engineered to overexpress human MICA. (C-D) FT536 alone, and in combination with Herceptin, demonstrate significant tumor growth inhibition (TGI) compared to Herceptin alone in an orthotopic xenograft model of human lung adenocarcinoma.ConclusionsCollectively, these studies demonstrate that FT536 is a highly potent, multi-tumor targeting CAR-iNK cell product that is uniform in composition and can be effectively and safely used off-the-shelf for on-demand treatment of multiple solid and hematological malignancies. An IND submission is planned for 2021, with an initial Phase 1 clinical trial to follow.

BackgroundOvarian cancer is a leading cause of cancer-related deaths among women due to the development of therapeutic resistance. Natural killer (NK) cells are cytotoxic lymphocytes that can kill neoplastic cells without prior sensitization. A key anti-tumor function of human NK cells is antibody-dependent cell-mediated cytotoxicity (ADCC), mediated exclusively by the IgG Fcy (FcyR) receptor CD16A. The mechanism of action of several clinically successful antitumor therapeutic monoclonal antibodies (mAbs) involves ADCC; however, their therapeutic efficacy is reduced due to regulatory checkpoints of CD16A, which include its low IgG binding affinity and rapid downregulation upon NK cell activation by the membrane metalloprotease ADAM17.1–3 CD64, the highest affinity FcyR, is expressed on myeloid-derived cells and not lymphocytes and is also not cleaved by ADAM17. To enhance ADCC, we generated CD64/16A, a novel recombinant FcyR consisting of extracellular CD64 and intracellular and transmembrane CD16A to mediate high affinity IgG binding and potent cell signaling.4 5MethodsEngineered NK cells expressing CD64/16A were derived from human induced pluripotent stem-cells (iPSCs), referred to here as iNKs, which are clonal and clinically scalable NK cells. ADCC and natural cytotoxicity of three ovarian cancer cell lines were measured in vitro using Delfia EuTDA and IncuCyte cytotoxicity assays, and production of anti-tumor cytokines was determined via flow cytometry. Finally, tumor cell killing was assessed in vivo using a human xenograft mouse model of peritoneal metastatic ovarian cancer.ResultsOur data show that iNK-CD64/16A cells uniquely facilitate mAb absorption, robustly produce IFN-y and TNF-alpha, and kill several ovarian cancer cell lines in the presence of the therapeutic mAbs trastuzumab or cetuximab, which target HER2 or EGFR, respectively. We found that iNK-CD64/16A cells can capture soluble mAbs and retain antibody arming and ADCC efficacy after cryopreservation. Additionally, iNK-CD64/16A cells robustly mediate ADCC and reduce overall tumor burden of HER2+ tumor cells in vivo in the described metastatic ovarian cancer xenograft model.ConclusionsOur findings provide new insights into using high affinity Fc receptor-based adoptive NK cell therapies and lay the preclinical foundation necessary for developing an ‘off-the-shelf’ cellular therapy that can be combined with therapeutic tumor-targeting mAbs for the treatment of ovarian cancer. Importantly, iNK-CD64/16A cells can serve as a docking platform for therapeutic antibodies that can be switched and mixed for universal tumor antigen targeting to treat multiple malignancies.AcknowledgementsGrant supportNIH R01CA203348, HHMI Medical Research Fellows Program/Burroughs Wellcome FundReferencesJing Y, Ni Z, Wu J, et al. Identification of an ADAM17 cleavage region in human CD16 (FcgammaRIII) and the engineering of a non-cleavable version of the receptor in NK cells. PLoS One 2015;10(3):e0121788.Wu J, Mishra HK, Walcheck B. Role of ADAM17 as a regulatory checkpoint of CD16A in NK cells and as a potential target for cancer immunotherapy. J Leukoc Biol 2019;105(6):1297–303.Dixon KJ, Wu J, Walcheck B. Engineering Anti-Tumor Monoclonal Antibodies and Fc Receptors to Enhance ADCC by Human NK Cells. Cancers (Basel) 2021;13(2).Snyder KM, Hullsiek R, Mishra HK, et al. Expression of a Recombinant High Affinity IgG Fc Receptor by Engineered NK Cells as a Docking Platform for Therapeutic mAbs to Target Cancer Cells. Front Immunol 2018;9:2873.Walcheck B, Wu J. iNK-CD64/16A cells: a promising approach for ADCC? Expert Opin Biol Ther 2019;19(12):1229–32.Ethics ApprovalThis study was approved by the University of Minnesota’s Institutional Animal Care and Use Committee, protocol number 1902–36768A.

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.

Surface expression of the HLA-I related molecules MICA and MICB (MICA/B) in response to oncogenic and cellular stress acts as a natural anti-cancer immunosurveillance mechanism. The recognition of MICA/B by the activating immunoreceptor NKG2D, which is expressed by natural killer (NK) and T cell subsets, is responsible for the removal of many transformed and virally infected cells. However, tumors frequently evade NKG2D-mediated immunosurveillance by proteolytic shedding of MICA/B, which can inhibit NKG2D function and promote tumor immune escape. Recently, we demonstrated that monoclonal antibodies targeting the conserved, membrane-proximal α3 domain of MICA/B can prevent MICA/B shedding and enhance NK cell anti-tumor efficacy. With the goal of leveraging the ubiquity of MICA/B expression on malignant cells, we have developed a novel chimeric antigen receptor targeting the α3 domain of MICA/B (CAR-MICA/B) and are currently evaluating application of CAR-MICA/B in an off-the-shelf NK cell immunotherapy platform for both solid and hematopoietic tumor indications. Optimization of CAR-MICA/B design was performed by primary T cell transduction using a matrix of CAR spacers and ScFv heavy and light chain orientations. Six candidate CAR-MICA/B designs were screened in vitro against a panel of tumor cell lines and in vivo against the Nalm6 leukemia cell line engineered to express MICA (Nalm6-MICA). All tested constructs demonstrated MICA-specific in vitro activation and cytotoxicity as well as in vivo tumor control (Figure 1A). Additional studies utilizing the optimal CAR-MICA/B configuration demonstrated MICA/B-specific reactivity against a panel of solid and hematopoietic tumor cell lines in vitro, including melanoma, renal cell carcinoma, and lung cancer lines (Figure 1B). Further, CAR-MICA/B T cells were superior to NKG2D-CAR T cells in clearing A2058 melanoma cells in an in vivo xenograft metastasis model (Figure 1C). Although MICA/B expression has primarily been studied in the context of solid tumors, moderate MICA/B mRNA expression was identified in a number of hematopoietic tumor cell lines, including acute myeloid leukemia (AML) and multiple myeloma (MM) lines. Following the confirmation of surface MICA/B protein expression on a selection of MM and AML cell lines, we utilized MICA/B CAR primary T cells to further demonstrate MICA/B-specific activation and cytotoxicity and to confirm CAR-MICA/B targeting of hematological malignancies (Figure 1D). To further advance CAR-MICA/B development, we introduced the CAR-MICA/B construct into an induced pluripotent stem cell (iPSC) line designed for production of off-the-shelf natural killer (NK) cell immunotherapies. Using a panel of tumor cell lines expressing MICA/B, CAR-MICA/B iPSC-derived NK (iNK) cells displayed specific MICA reactivity, resulting in enhanced cytokine production, degranulation, and CAR-mediated cytotoxicity compared to CAR-negative iNK control cells (Figure 1E). In addition to MICA/B-specific cytotoxicity mediated by CAR, iNK cells also mediated innate cytotoxicity against cancer cells through endogenous NKG2D and other NK cell activating receptors, highlighting the multifaceted targeting capacity of CAR iNK cells. In order to isolate CAR-directed cytotoxicity from the iNK cells' innate anti-tumor capacity, an in vivo proof of concept study was performed using mouse B16-F10 melanoma cells engineered to express human MICA. In this model, iNK expressing CAR-MICA/B significantly reduced B16-F10-MICA liver and lung metastases from CAR-MICA/B iNK cells compared to CAR negative control cells, with reductions of the number of metastases by 87% in the lung (p Figure 1 Disclosures Bjordahl: Fate Therapeutics: Current Employment. Goulding:Fate Therapeutics: Current Employment. Blum:Fate Therapeutics: Current Employment. Chang:Fate Therapeutics: Current Employment. Wucherpfennig:Fate Therapeutics: Research Funding. Chu:Fate Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company; Roche Holding AG: Current equity holder in publicly-traded company. Chu:Fate Therapeutics, Inc: Current Employment. Gaidarova:Fate Therapeutics, Inc: Current Employment. Liu:Fate Therapeutics: Current Employment. Sikaroodi:Fate Therapeutics: Current Employment. Fong:Fate Therapeutics: Current Employment. Huffman:Fate Therapeutics: Current Employment. Lee:Fate Therapeutics, Inc.: Current Employment. Valamehr:Fate Therapeutics, Inc: Current Employment, Current equity holder in publicly-traded company.

Approximately 19,940 new cases of acute myeloid leukemia (AML) will be diagnosed in 2020 with an estimated 11,180 deaths, which has remained steady for 3 decades. We established that allogeneic natural killer (NK) cell infusions have an antileukemic effect and induce remission in 25-40% of relapsed/refractory AML patients. After hundreds of individual donor product infusions, we noted therapeutic limitations to include effector potency, cell numbers,persistence, specificity and exportability. To overcome these barriers, we developed a robust genetic editing and manufacturing platform for the uniform engineering and expansion of high-quality NK cells derived from induced pluripotent stem cells (iPSCs). This manufacturing system allows efficient production of iPSC-derived NK (iNK) cells engineered to enhance persistence and potency, and enables distribution of highly homogenous iNK cells to multiple clinical sites and multi-dosing to treat patients on-demand with advanced cancer. We hypothesized that iNK cells could be combined with the anti-CD38 monoclonal antibody daratumumab (dara) to directly target various tumors and harness their antibody-dependent cellular cytotoxicity (ADCC) potential. Previously, we reported on our ability to effectively target multiple myeloma with our FT538 program (the iNK defined below), now FDA approved for clinical trials. Here we discuss applying these same engineered iNK cells to specifically target and kill AML blasts induced to upregulate CD38 by retinoic acid exposure. We utilized iNK cells expressing a high-affinity, non-cleavable version of CD16 (the Fc receptor that mediates ADCC) termed hnCD16 alone or combined with CD38 knockout (KO) to prevent dara-mediated NK cell fratricide (Fig. 1A). CD38 KO iNK cells had intact calcium flux in response to ionomycin or CD16 crosslinking (Fig. 1B). Adding dara to peripheral blood NK cells or hnCD16 iNK cells triggered ADCC-mediated fratricide, while hnCD16/CD38 KO iNK cells that cannot be targeted by dara were unaffected (Fig 1C). CD38, expressed intracellularly and on the plasma membrane, functions as an NADase, degrading nicotinamide adenine dinucleotide (NAD+) into ADP-ribose and nicotinamide. Because NAD+ levels influence several key metabolic pathways, we assessed the impact of CD38 KO on iNK cell metabolism. We found that, relative to hnCD16 iNK cells, hnCD16/CD38 KO iNK cells had significantly higher intracellular NAD+, NADH, and ATP levels (Fig. 1D). Additionally, hnCD16/CD38 KO iNK cells exhibited elevated mitochondrial oxidative phosphorylation (Fig. 1E) and marked resistance to oxidative stress to hydrogen peroxide exposure (Fig. 1F). Together, these results demonstrate a significant enhancement of NK cell mitochondrial oxidative phosphorylation and redox homeostasis in iNK CD38 KO cells. To enhance hnCD16/CD38 KO iNK cells further, we incorporated a third modification consisting of an IL-15 receptor signaling complex (IL-15RF). This receptor complex is expressed on the cell surface and provides IL-15 signals required for NK cell survival and proliferation independent of exogenous cytokine. To determine the efficacy of hnCD16/CD38 KO/IL-15RF iNK cells combined with dara to target AML, we first assessed CD38 expression on THP-1 cells (an AML cell line) and primary AML blasts incubated with or without retinoic acid. We observed high CD38 expression on both THP-1 cells and primary AML cells further elevated with retinoic acid treatment (Fig. 1G). THP-1 and primary AML cells were labeled with CellTrace dye and used as targets for killing assays using hnCD16/CD38 KO/IL-15RF iNK cells. Compared to hnCD16/CD38 KO/IL-15RF iNK cells cultured with targets alone, adding dara led to higher target cell killing, especially after retinoic acid exposure (Fig. 1H). In 12-hour live imaging experiments testing iNK cell cytotoxicity against THP-1 cells, similar results were observed (Fig. 1I). Collectively, our results show that utilizing the iNK cell platform to uniformly express hnCD16 and IL15RF combined with complete CD38 KO is an effective strategy to promote effective ADCC against CD38+ cells in the absence of fratricide, and that CD38 KO reprograms NK cells for higher oxidative metabolic fitness for improved persistence and anti-tumor function. Furthermore, we have generated proof-of-concept data supporting triple gene-modified iNK cells combined with dara as a novel AML immunotherapy. Disclosures Cichocki: Fate Therapeutics, Inc: Consultancy, Patents & Royalties, Research Funding. Bjordahl:Fate Therapeutics: Current Employment. Bonello:Fate Therapeutics, Inc: Current Employment. Mahmood:Fate Therapeutics, Inc: Current Employment. Rogers:Fate Therapeutics, Inc: Current Employment. Ge:Fate Therapeutics, Inc: Current Employment. Lee:Fate Therapeutics, Inc.: Current Employment. Felices:GT Biopharma: Consultancy. Walcheck:Fate Therapeutics: Consultancy, Research Funding. Blazar:Fate Therapeutics Inc.: Research Funding; KidsFirst Fund: Research Funding; Magenta Therapeutics: Consultancy; Childrens' Cancer Research Fund: Research Funding; BlueRock Therapeutics: Research Funding; BlueRock Therapeuetic: Consultancy; Tmunity: Other: Co-founder. Valamehr:Fate Therapeutics, Inc: Current Employment, Current equity holder in publicly-traded company. Miller:GT Biopharma: Consultancy, Patents & Royalties, Research Funding; Onkimmune: Honoraria, Membership on an entity's Board of Directors or advisory committees; Nektar: Honoraria, Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics, Inc: Consultancy, Patents & Royalties, Research Funding; Vycellix: Consultancy. OffLabel Disclosure: FT538 is a genetically modified induced pluripotent stem cell derived NK cell product that can be combined with daratumumab to target CD38 on AML after retinoid acid induction.

Chimeric antigen receptor (CAR) directed therapies have been used successfully to treat a variety of hematological malignancies. With the advent of multi-modal engineering, adoptive cell therapy offers the opportunity to tackle increasingly complex disease settings such as multiple myeloma (MM), where targeting of single tumor associated antigen by CAR or monoclonal antibodies (mAb) is confounded by antigen loss and clonal heterogeneity. Further, expanding treatment options beyond primary T and NK cell based therapies has multiple advantages, including the use of induced pluripotent stem cells (iPSC) to derive effector cells using precision genetic engineering that can be uniformly manufactured at scale from a clonally-derived master cell bank (MCB).FT576 is a multiplex-edited, iPSC-derived CAR-NK (CAR-iNK) cell therapy designed for treatment of Multiple Myeloma. FT576 is engineered 1) to express a recombinant IL-15/IL-15 receptor signaling complex (IL-15RF) for enhanced persistence; 2) to express an enhanced high-affinity, non-cleavable CD16 (hnCD16) ; 3) to disrupt expression of CD38, allowing for enhanced ADCC without NK cell fratricide; and 4) to express a BCMA-targeted CAR with NK-cell optimized signaling.CAR-directed specificity of the FT576 cells for BCMA was demonstrated using a short-term cytotoxicity assay (90.8% cytotoxicity against BCMA+ vs 22.1% BCMA- cells, p Citation Format: Jode P. Goodridge, Ryan Bjordahl, Sajid Mahmood, John Reiser, Svetlana Gaidarova, Robert Blum, Frank Cichocki, Hui-yi Chu, Greg Bonello, Tom Lee, Brian Groff, Miguel Meza, Thomas Daley, Yu-waye Chu, Bruce Walcheck, Karl-Johan Malmberg, Jeffrey S. Miller, Armin Rehm, Bahram Valamehr. FT576 path to first-of-kind clinical trial: translation of a versatile multi-antigen specific off-the-shelf NK cell for treatment of multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1550.

Cancer immunotherapies have revolutionized cancer treatment by showing clinical efficacy across multiple cancer indications. However, tumor heterogeneity and evasion from host immune cell surveillance often limit the durability and efficacy of these strategies as monotherapies. Consequently, it is becoming common practice to combine existing anticancer treatments and novel immunotherapies to maximize clinical efficacy. The pan tumor-associated antigens MICA and MICB (MICA/B) are surface proteins induced by cellular stress, often associated with tumorigenesis, and are recognized by the NK cell activating receptor NKG2D. To evade immune recognition, cancer cells often proteolytically shed the membrane distal domains of MICA/B, leading to reduced NKG2D recognition. To combat this pervasive tumor escape mechanism and create a ubiquitous cancer targeting platform, we have developed a novel CAR-iPSC-derived NK (iNK) cell that targets the conserved α3 domain of MICA/B, rendering it resistant to inhibition by shed MICA/B. To enhance effector cell function, persistence and multi-antigen capacity, further genetic editing at the iPSC stage was conducted to equip the CAR-iNK cells with a unique IL-15/IL-15 receptor fusion, the knockout of CD38 and a novel high-affinity, non-cleavable CD16 (hnCD16) to enhance antibody-dependent cellular cytotoxicity (ADCC). In this study, we evaluated the function of multiplexed engineered MICA/B CAR iNK cells (termed FT536) in combination with monoclonal antibodies (mAbs), to elicit multi-antigen targeting, and radiation therapy, to augment surface MICA/B expression. FT536 showed superior in vitro cytotoxicity and in vivo tumor control against an array of MICA/B expressing tumor lines. Furthermore, ADCC, induced in combination with cetuximab or trastuzumab, enhanced the potency of FT536 against various solid tumor lines (p Citation Format: John Goulding, Robert Blum, Bryan Hancock, Moyar Ge, Brian Groff, Soheila Shirinbak, Joy Grant, Martin Hosking, Mochtar Pribadi, Yijia Pan, Hui-Yi Chui, Shohreh Sikaroodi, Lauren Fong, Janel Huffman, Wen-I Yeh, Chia-Wei Chang, Thomas Dailey, Miguel Meza, Cokey Nguyen, Lucas Ferrari de Andrade, Tom Lee, Ryan Bjordahl, Kai W. Wucherpfennig, Bahram Valamehr. FT536: Preclinical development of a novel off-the-shelf CAR-MICA/B NK cell immunotherapy combined with radiation and antibody treatments as a first-of-kind pan-cancer targeting strategy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1591.

Although chimeric antigen receptor (CAR) T cell therapies have demonstrated impressive efficacy in a number of hematological malignancies, successful application of CAR T cells in solid tumor indications has been challenging due to difficulty identifying and targeting suitable tumor associated antigens (TAA) without eliciting significant on-target, off-tumor toxicity. Furthermore, the challenges of tumor heterogeneity and antigen escape need to be addressed when treating bulky solid tumors. Successful development of CAR-targeted cellular therapies for solid tumor indications will consequently require careful selection of target antigen, effector cell, and cell engineering to balance on-tumor efficacy with potential for off-tumor toxicities. In contrast to CAR-T cell therapies, adoptive natural killer (NK) cell immunotherapies have been well tolerated, but conventional, donor-derived strategies have had limited efficacy. B7H3 is an immune checkpoint molecule of the B7 protein superfamily and an ideal TAA; B7H3 has broad expression in cancer and limited expression on healthy tissues. Clinically, upregulation of B7H3 is often associated with metastatic cancer and poor patient prognosis. To target B7H3 with a multifaceted effector cell population, we selected the use of NK cells to leverage their clinical safety profile and versatile innate tumor recognition and cytotoxicity features. Here we describe the optimization and characterization of a novel anti-B7H3 CAR and its application in an induced pluripotent stem cell-derived, off-the-shelf NK (iNK) cell immunotherapy. These anti-B7H3 CAR iNK cells utilize multiplexed genetic engineering to enhance persistence, effector function, and to enable multi-antigen targeting through combinations with monoclonal antibody therapy. Using a novel B7H3 binding domain, CAR constructs were first screened in primary T cells for the optimal spacer length by in vitro and in vivo cytotoxicity assays. Following optimized CAR selection, in vitro proof of concept studies demonstrated broad B7H3 CAR reactivity against an array of tumor cell line targets of both hematologic and solid organ cancers. B7H3 CAR-T cells were highly reactive against cell lines derived from ovarian, prostate, bone, and breast cancers as demonstrated by cytotoxicity and production of IFNγ and TNFα. iNK cells carrying the optimized CAR design were evaluated in vitro for B7H3 specificity using cytotoxicity assays and intracellular cytokine staining. In addition, B7H3 CAR iNK cells synergized with anti-EGFR and anti-HER2 monoclonal antibodies to produce antibody-dependent cellular cytotoxicity, leading to further enhancement of tumor targeting. In summary, these data demonstrate successful targeting of B7H3 using an off-the-shelf, engineered NK cell platform for the effective elimination of multiple tumor types. Citation Format: Ryan Bjordahl, John Goulding, Hui-Yi Chu, Frank Cichocki, Nicholas Zorko, Zachary Davis, Martin Felices, Alex Garcia, Svetlana Gaidarova, Wen-I Yeh, Tom T. Lee, Jeffrey S. Miller, Bahram Valamehr. Development of off-the-shelf B7H3 chimeric antigen receptor NK cell therapeutic with broad applicability across many solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1539.

The use of induced pluripotent stem cells (iPSCs) to derive immune effector cells offers distinct advantages for immune therapy over existing patient- or donor- derived platforms, not only in terms of scalable manufacturing and precision genetic engineering at the clonal level, but also in allowing the generation and combinational use of multiple effector cell types each with distinct characteristics. Taking cues from the natural propagation of innate to adaptive effector responses, here we describe the combined use of multi-engineered iPSC derived Natural Killer (iNK) and T (iT) cell platforms in order to exploit the unique properties of each cell type to achieve both depth and durability of response for hematological malignancies. As innate cells, NK cells are characterized by the capacity for spontaneous reactivity, either in response to cell surface antigen or downregulation of class I MHC, and the rapid kinetic under which NK cells operate make them an ideal candidate to achieve a depth of response that outpaces the limiting effects of cytokine support. FT596 was developed as a dual-targeted iNK cell platform engineered to express both a CD19-directed, NK cell-optimized (NKG2D-2B4-CD3ζ) chimeric antigen receptor (CAR) and a high-affinity, non-cleavable Fc receptor (hnCD16), enabling multi-targeting through combination with therapeutic antibodies. The activity of each receptor is further enhanced by the expression of an IL15-IL15Ra fusion receptor, which also allows the cells to expand in the absence of exogenous cytokine support and prolongs cell survival in vitro and in vivo. T cells are exquisitely specific and undergo rapid clonal expansion and differentiation in response to target antigen, and antigen driven persistence has been demonstrated as a key determinant in efficacy in primary CAR-T cell immune therapy. FT819 is an iT cell platform engineered to express a functionally optimized CD19-CAR (1XX) that has been genome edited into the T cell receptor (TCR) alpha constant (TRAC) locus to provide ideal CAR activity and to prevent TCR expression, thereby avoiding the complications of GVH reactivity in an allogeneic setting. In vivo, both FT596 and FT819 showed stable levels of tumor cell clearance against the CD19+ acute lymphoblastic leukemia cell line NALM6, comparable to that of primary CAR19 T cells (p* Citation Format: Jode P. Goodridge, John W. Reiser, Ryan Bjordahl, Milli Mandal, Chia-wei Chang, Raedun Clarck, Sajid Mahmood, Huang Zhu, Svetlana Gaidarova, Robert Blum, Frank Cichocki, Hui-ting Hsu, Greg Bonello, Tom Lee, Brian Groff, Karl-Johan Mamlberg, Bruce Walcheck, Jeffrey S. Miller, Dan Kaufman, Bahram Valamehr. Combinational strategy targeting B cell malignancy using iPSC engineered CAR-NK (FT596) and CAR-T cell (FT819) platforms with therapeutic antibody to achieve an effective deep and durable response [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2216.

Monoclonal antibody (mAb) treatment is an effective therapeutic strategy for many cancer types, though there remains meaningful opportunity to improve mAb efficacy by optimizing the interaction with natural killer (NK) cells to enhance antibody-dependent cellular cytotoxicity (ADCC). NK cells are an ideal effector cell for combined use with tumor-targeting mAbs, as NK cells effect both innate tumoricidal capacity and ADCC. CD38-targeting mAbs, such as daratumumab, are effective in treating multiple myeloma (MM) and achieve their efficacy through multiple mechanisms, including ADCC. However, because activated NK cells express high levels of CD38, daratumumab induces NK cell depletion through fratricide, potentially reducing treatment effectiveness. Adoptive NK cell immunotherapy therefore has the potential to augment daratumumab's ADCC activity if fratricide can be reduced or prevented. FT538 is an off-the-shelf adoptive NK cell immunotherapy product candidate designed for enhanced cellular persistence and ADCC while avoiding anti-CD38 mAb induced fratricide. It is derived from induced pluripotent stem cells (iPSC) engineered to lack CD38 expression, which we have previously shown to eliminate daratumumab-induced fratricide among iPSC-derived NK cells, resulting in enhanced long-term daratumumab-mediated ADCC. FT538 is engineered to express an IL-15 receptor alpha fusion protein (IL-15RF; IL-15 tethered to IL-15 receptor α) to enhance persistence and a high-affinity non-cleavable CD16 (hnCD16, FcRγIII) to increase ADCC. To support the clinical translation of FT538, and to enable the repeatable and scalable cell production to support off-the-shelf availability of a uniform NK cell product, a clinical-grade master pluripotent stem cell line was developed. The FT538 master pluripotent stem cell line was created by reprogramming donor fibroblasts into iPSCs using our non-integrating cellular reprogramming platform, and cells were further genetically edited by targeting IL-15RF and hnCD16 to the CD38 locus. Clonal iPSC lines were generated and screened for precise knock-in and knock-out edits at the CD38 locus and a lack of off-target genome integration (15% total success rate for CD38-/-IL-15RF+CD16+). Selected engineered iPSC clones were confirmed to be free of reprogramming transgenes and to maintain genomic stability. Engineered iPSC clones were additionally tested for their NK cell differentiation potential and function, and a single clone was selected to serve as the renewable starting material for cGMP manufacturing and clinical development. Upon differentiation and expansion FT538 demonstrated a mature NK cell phenotype with expression of NK cell receptors including NKp30, NKp46, NKG2D, KIR, NKG2A, and DNAM-1. The functional impact of CD38 knockout on FT538 NK cells was confirmed in an in vitro fratricide assay, where peripheral blood (PB)-NK cells exhibited fratricide at a frequency of 33% after 3 hr culture with increasing daratumumab concentrations. In contrast, FT538 cells were entirely resistant ( Disclosures Bjordahl: Fate Therapeutics, Inc.: Employment. Gaidarova:Fate Therapeutics, Inc: Employment. Cichocki:Fate Therapeutics, Inc: Research Funding. Bonello:Fate Therapeutics, Inc.: Employment. Robinson:Fate Therapeutics, Inc.: Employment. Ruller:Fate Therapeutics, Inc.: Employment. Pribadi:Fate Therapeutics, Inc.: Employment. Dinella:Fate Therapeutics, Inc.: Employment. Fong:Fate Therapeutics, Inc.: Employment. Huffman:Fate Therapeutics, Inc.: Employment. Chu:FATE THERAPEUTICS: Employment. Lee:Fate Therapeutics, Inc.: Employment. Abujarour:Fate Therapeutics, Inc.: Employment. Kaufman:FATE Therapeutics: Consultancy, Research Funding. Malmberg:Fate Therapeutics, Inc.: Consultancy, Research Funding; Vycellix: Consultancy, Membership on an entity's Board of Directors or advisory committees. Miller:CytoSen: Membership on an entity's Board of Directors or advisory committees; Moderna: Membership on an entity's Board of Directors or advisory committees; OnKImmune: Membership on an entity's Board of Directors or advisory committees; GT BioPharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Dr. Reddys Laboratory: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics, Inc: Consultancy, Research Funding. Valamehr:Fate Therapeutics, Inc: Employment.

Induced pluripotent stem cell (iPSC)-derived effector cells offer distinct advantages for immune therapy over existing patient- or donor- derived platforms, both in terms of scalable manufacturing from a renewable starting cellular material and precision genetic engineering that is performed at the single-cell level. iPSC derived natural killer (iNK) cells offer the further advantage of innate reactivity to stress ligands and MHC downregulation and the potential to recruit downstream adaptive responses. These unique features form the basis of our multi-antigen targeted chimeric antigen receptor (CAR) CAR-iNK cell product candidate, termed FT596, which is further combined with additional functionality to enhance effector function. FT596 is consistently manufactured from a master iPSC line engineered to uniformly express an NK cell-calibrated CD19-targeting CAR (CD19-CAR), an enhanced functioning high-affinity, non-cleavable CD16 (hnCD16) and a recombinant fusion of IL-15 and IL-15 receptor alpha (IL-15RF) for cytokine-autonomous persistence. The design of the CD19-CAR involved exploiting the intrinsic polyfunctionality of NK cells, which function by engaging multiple signaling pathways activated through combinations of distinct germline encoded receptors. Using this approach, the transmembrane region of activating receptor NKG2D, combined with the intracellular signaling domains of SLAM co-receptor 2B4 and CD3ζ, proved the most effective in triggering antigen specific functional responses in NK cells. Chimerization of an anti-CD19 scFv onto this NKG2D-2B4-CD3ζ signaling platform produced specific in vitro recognition of CD19+ B cell lymphoma cells in short-term and long-term NK cytotoxicity assays (>80% and The functionality of the CD19-CAR was further enhanced in combination with autonomous IL-15 signaling. Introduction of the IL-15RF enabled expansion of iNK cells without addition of soluble cytokine and greatly improved longevity and functional persistence of iNK cells both in vitro and in animal models. Moreover, iNK cells modified with IL-15RF showed enhanced functional maturation, including upregulated expression of effector molecules such as granzyme B. iNK cells with both CD19-CAR and IL-15RF resulted in enhanced CAR functionality in vitro, and mouse models for B cell malignancy demonstrated that treatment with iNK cells engineered with CD19-CAR and IL-15RF were curative against B cell lymphoma (p In combination with hnCD16, co-expression of CD19-CAR and IL15-RF culminates in iNK cells capable of dual-specificity through combinatorial use with monoclonal antibodies to tackle antigen escape. In long term killing assays, FT596 alone demonstrated equivalent levels of CD19 targeted anti-tumor activity as primary CD19-targeted CAR (CAR19) T cells when tested against CD19+ CD20+ B lymphoblast target cells and demonstrated enhanced levels of activity when used in combination with anti-CD20 (rituximab). When targeting CD19- CD20+ B lymphoblast target cells and used in combination with rituximab, only FT596 was able to effectively eliminate the CD19 antigen escaped target cell (64% vs 30% clearance of tumor cells at 36H vs rituximab alone). In vivo FT596 showed equivalent levels of tumor cell clearance as primary CAR19 T cells against the CD19+ acute lymphoblastic leukemia cell line NALM6 and CD19+CD20+ Burkitts lymphoma cell line RAJI, and enhanced clearance of RAJI tumor cells in combination with rituximab (p=0.0002). Furthermore, utilizing an allogenic human CD34 engrafted NSG mouse model, FT596 demonstrated improved survival and safety over primary CAR19 T cells, either as a monotherapy or as a combination therapy with rituximab versus RAJI tumor cells. Together, these studies demonstrate FT596 provides a multi-antigen targeting, potent and persistent engineered immune cell that is derived from a master iPSC line which utilizes the intrinsic versatility of NK cells to enable a highly effective combination therapy in a single, standardized, scalable, off-the-shelf platform and supports the rational for a first-of-kind Phase I Study as a monotherapy and in combination with CD20-targeted mAbs including rituximab in subjects with relapsed/refractory B-cell lymphoma and leukemia. Figure Disclosures Goodridge: FATE THERAPEUTICS: Employment. Mahmood:Fate Therapeutics, Inc: Employment. Gaidarova:Fate Therapeutics, Inc: Employment. Bjordahl:Fate Therapeutics, Inc.: Employment. Cichocki:Fate Therapeutics, Inc: Research Funding. Chu:FATE THERAPEUTICS: Employment. Bonello:Fate Therapeutics, Inc.: Employment. Lee:Fate Therapeutics, Inc.: Employment. Groff:FATE THERAPEUTICS: Employment. Meza:FATE THERAPEUTICS: Employment. Malmberg:Vycellix: Consultancy, Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics, Inc.: Consultancy, Research Funding. Miller:Moderna: Membership on an entity's Board of Directors or advisory committees; Dr. Reddys Laboratory: Membership on an entity's Board of Directors or advisory committees; CytoSen: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics, Inc: Consultancy, Research Funding; OnKImmune: Membership on an entity's Board of Directors or advisory committees; GT BioPharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Kaufman:FATE Therapeutics: Consultancy, Research Funding. Valamehr:Fate Therapeutics, Inc: Employment.

Cytolytic effector lymphocytes must contend with unfavorable microenvironments when infiltrating sites of infection or malignancy. Tumor cells typically have high levels of oxidative stress and produce reactive oxygen species (ROS) that suppress the cytotoxic functions of both natural killer (NK) cells and CD8+ T cells. Levels of activated granulocytes that release ROS are also elevated in cancer patients. Free radicals, such as ROS, cause detrimental cellular effects including protein oxidation, lipid peroxidation and DNA damage. Chronic viral infections, including CMV, are also associated with increased oxidative stress. We hypothesized that adaptive NK cells, which arise specifically in response to CMV, could have properties that allow these cells to persist and retain function in high oxidative stress environments. Adaptive NK cells are present in the peripheral blood of many otherwise healthy CMV seropositive individuals and expand in response to CMV reactivation in hematopoietic cell transplant (HCT) patients. Mounting evidence suggests that CMV peptides presented by HLA-E on infected cells can trigger the expansion of adaptive NK cells expressing the activating receptor NKG2C. The majority of NKG2C-positive adaptive NK cells co-express the terminal maturation marker CD57. Work by our group and others has shown that adaptive NK cells exhibit enhanced antibody-dependent cellular cytotoxicity (ADCC) and interferon (IFN)-γ production relative to canonical NK cells, appear to persist long-term and have metabolic attributes similar to memory CD8+ T cells. We also reported clinical correlations between adaptive NK cell numbers and reduced relapse risk in HCT patients with hematologic malignancies. Here, we show that CD38 expression is markedly reduced on adaptive NK cells from CMV seropositive individuals. This observation was first made from analyses of RNA-seq data comparing adaptive and canonical NK cells and was validated by flow cytometry (Figure 1A). CD38 is expressed both intracellularly and on the plasma membrane and functions as an NADase, degrading nicotinamide adenine dinucleotide (NAD+) into ADP-ribose and nicotinamide. NAD+ is a necessary cofactor for the sirturin family of protein deacetylases, which protect against oxidative stress. We hypothesized that CD38 downregulation in adaptive NK cells could be associated with more resistance to oxidative stress-induced cell death through increased NAD+ levels and sirturin activity. To determine whether there was a connection between CD38 expression and resistance to oxidative stress, we isolated NK cells from the peripheral blood of CMV seropositive donors and cultured them overnight with or without 15 mM H2O2 (hydrogen peroxide), known to induce oxidative stress and cell death. We found that NKG2C+ adaptive NK cells were markedly more resistant to oxidative stress-induced cell death compared to NKG2C-negative canonical NK cells as determined by annexin V and a fixable amine-reactive dye (LIVE/DEAD) that can permeate damaged membranes of dead cells and react with interior amines (Figure 1B). Similar results were observed in assays where NK cells from CMV seropositive donors were co-cultured with neutrophils pre-activated with phorbol 12-myristate 13-acetate (PMA) to induce the release of reactive oxygen species. To determine whether CD38 expression is directly associated with the NK cell response to oxidative stress, we generated induced pluripotent stem cell (iPSC) lacking CD38 through CRISPR/Cas9 gene editing that were differentiated into NK cells and tested for their ability to resist oxidative stress-induced death. Compared to control iPSC-derived NK (iNK) cells that express high levels of CD38 (Figure 1C), a substantially larger percentage of CD38 knockout iNK cells were viable when cultured overnight with H2O2 (Figure 1D). Our results have implications for adoptive immunotherapy to treat patients with cancer where a major goal is to manufacture cytotoxic cells that can persist and function in a tumor environment that contains high levels of oxidative radicals. We are exploring other cell stressors of high translational relevance such as freeze/thaw stress in adaptive and CD38 knockout cells that will be critical for cell therapy platforms. Disclosures Cichocki: Fate Therapeutics, Inc: Research Funding. Blazar:Kamon Pharmaceuticals, Inc: Membership on an entity's Board of Directors or advisory committees; Five Prime Therapeutics Inc: Co-Founder, Membership on an entity's Board of Directors or advisory committees; Regeneron Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Magenta Therapeutics and BlueRock Therapeuetics: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics, Inc.: Research Funding; RXi Pharmaceuticals: Research Funding; Alpine Immune Sciences, Inc.: Research Funding; Abbvie Inc: Research Funding; Leukemia and Lymphoma Society: Research Funding; Childrens' Cancer Research Fund: Research Funding; KidsFirst Fund: Research Funding; Tmunity: Other: Co-Founder; BlueRock Therapeutics: Membership on an entity's Board of Directors or advisory committees. Bjordahl:Fate Therapeutics, Inc.: Employment. Valamehr:Fate Therapeutics, Inc: Employment. Miller:Fate Therapeutics, Inc: Consultancy, Research Funding; CytoSen: Membership on an entity's Board of Directors or advisory committees; OnKImmune: Membership on an entity's Board of Directors or advisory committees; Dr. Reddys Laboratory: Membership on an entity's Board of Directors or advisory committees; Moderna: Membership on an entity's Board of Directors or advisory committees; GT BioPharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. OffLabel Disclosure: NK cells

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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