Background FT596 is an induced pluripotent stem-cell (iPSC)-derived chimeric antigen receptor (CAR) natural killer (NK) cell therapy with three antitumour modalities: a CD19 CAR; a high-affinity, non-cleavable CD16 Fc receptor; and interleukin-15-interleukin-15 receptor fusion. In this study, we aimed to determine the recommended phase 2 dose (RP2D) and evaluate the safety and tolerability of FT596 as monotherapy and in combination with rituximab. We also aimed to evaluate the antitumour activity and characterise the pharmacokinetics of FT596 as monotherapy and in combination with rituximab. Methods In this phase 1, first-in-human trial, we evaluated FT596 in patients with relapsed or refractory B-cell lymphoma at nine sites in the USA. Patients who had received at least one previous systemic therapy and had no curative treatment options were eligible for inclusion. FT596 was administered after conditioning chemotherapy without rituximab (regimen A) or combined with rituximab (regimen B). The study consisted of a dose-escalation phase using a 3 + 3 design, with dose escalation commencing at 3 x 107 viable cells as a single dose on day 1 and done independently for individual regimens. A treatment cycle consisted of conditioning chemotherapy with cyclophosphamide (500 mg/m(2)) and fludarabine (30 mg/m(2)) intravenously on days -5 to -3, followed by FT596 administered at various doses and schedules, without (regimen A) or with (regimen B) a single dose of rituximab (375 mg/m(2)) intravenously on day -4. Supportive care was determined by the treating investigator. Patients were observed for dose-limiting adverse events for 28 days. Patients who tolerated therapy and derived clinical benefit could receive subsequent cycles of study treatment, with modification of conditioning chemotherapy dose if clinically indicated. The dose-expansion phase evaluated additional patients at selected doses and dosing schedules that had been found to be tolerable. The primary endpoints of the study were the incidence and nature of dose-limiting toxicities within each dose-escalation cohort to determine the maximum tolerated dose or maximum assessed dose to establish the RP2D and the incidence, nature, and severity of adverse events, with severity determined according to National Cancer Institute Common Toxicity Criteria and Adverse Events version 50. The trial was registered with ClinicalTrials.gov, NCT04245722. Findings Between March 19, 2020, and Jan 12, 2023, 86 patients with B-cell lymphoma received FT596 on regimen A (n=18) or regimen B (n=68). 22 (26%) of 86 patients were female and 72 (84%) of 86 patients were White. Patients had received a median of four previous lines of therapy (range 1-11) and 33 (38%) of 86 patients had received previous CAR T-cell therapy. The maximum tolerated dose was not reached. Cytokine release syndrome was reported in one (6%) of 18 patients (maximum grade 1) on regimen A and nine (13%) of 68 patients on regimen B (six with maximum grade 1 and three with grade 2). Neurotoxicity was not observed. Interpretation FT596 was well tolerated as monotherapy or with rituximab and induced deep and durable responses in patients with indolent and aggressive lymphomas and the RP2D was preliminarily identified to be 18 x 109 cells for three doses per cycle. This study supports that cell therapy using iPSC-derived, gene-modified NK cells is a potent platform for cancer treatment and suggests that such a platform might address limitations of currently available immune cell therapies, including manufacturing time, heterogeneity, access, and cost. Funding Fate Therapeutics. Copyright (c) 2025 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license.

Introduction: Allogeneic natural killer (NK) cell therapies have been well tolerated with documented anti-tumor activity in patients with relapsed/refractory (R/R) multiple myeloma (MM) (Lupo et al. 2019). However, limited availability of suitable donors, relatively short in vivo persistence, and manufacturing constraints that affect the ability to administer >1 dose of cells sufficient to achieve clinical activity are major barriers to maximizing the clinical benefit of allogeneic NK cell therapy. Induced pluripotent stem cell (iPSC)-derived immune effector cells offer distinct advantages over existing patient- and donor-derived therapeutic approaches, notably the use of a clonal master engineered iPSC line as a renewable source for the mass production of multiplexed-engineered immune cells of uniform composition for off-the-shelf availability, repeated dosing, and broad patient access. FT538 is an off-the-shelf, multiplexed-engineered NK cell therapy generated from a clonal master engineered iPSC line. FT538 incorporates 3 functional modalities for enhanced innate immunity: (1) high-affinity 158V, non-cleavable CD16 Fc receptor for augmented antibody-dependent cellular cytotoxicity (ADCC); (2) IL-15/IL-15 receptor fusion that promotes NK cell persistence; and (3) CD38 knockout to mitigate NK cell fratricide by CD38-directed monoclonal antibodies and to promote higher rates of glycolysis with improved metabolic fitness and resistance to oxidative stress within the tumor microenvironment. In preclinical studies, FT538 displays increased persistence without the need for exogenous cytokine support and, when combined with daratumumab against MM targets, demonstrates avoidance of daratumumab-mediated fratricide and significantly enhanced ADCC in vitro in a serial stimulation cytotoxicity assay compared with peripheral blood NK cells. The combination of FT538 and daratumumab led to highly effective tumor control compared with daratumumab alone in an in vivo MM xenograft model (Bjordahl et al. 2019). Methods: This is a multicenter, Phase I clinical trial of FT538 as monotherapy in R/R acute myelogenous leukemia (Regimen A) or in combination with daratumumab in R/R MM (Regimen B) (ClinicalTrials.gov: NCT04614636). In Regimen B, conditioning chemotherapy (fludarabine 30 mg/m2 and cyclophosphamide 300 mg/m2) followed by 3 once-weekly doses of FT538 (Days 1, 8, 15 of a 28-d cycle), ranging from 100 million cells/dose up to 1.5 billion cells/dose, are being evaluated using a standard 3 + 3 dose-escalation design. Daratumumab is first administered on Day -11 and given per prescribing information. A second cycle of therapy (daratumumab, conditioning chemotherapy, FT538) may be administered with FDA approval in subjects who have derived clinical benefit from their first cycle of therapy. The primary objectives are to evaluate safety and tolerability and to determine the recommended Phase II dose. Additional key objectives include anti-tumor activity, pharmacokinetics, and anti-product immunogenicity. Results: As of a data cutoff date of 05 Jul 2022, 6 patients with R/R MM were treated and evaluable in the first 2 dose cohorts (100 and 300 million cells/dose) of Regimen B. No dose-limiting toxicities, and no events of any grade of cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), or graft-versus-host disease (GvHD), were observed. No B- or T-cell mediated anti-product responses were observed. Conclusions: Administration of up to 3 doses of FT538 cells at 100 or 300 million cells/dose in combination with daratumumab is safe and well tolerated without CRS, neurotoxicity, or GvHD. Interim clinical data, including safety and tolerability and initial anti-tumor activity from the ongoing Phase I dose-escalation study of FT538 in combination with daratumumab in R/R MM, will be presented at the conference. References: Bjordahl R, Gaidarova S, Woan K, et al. FT538: Preclinical development of an off-the-shelf adoptive NK cell immunotherapy with targeted disruption of CD38 to prevent anti-CD38 antibody-mediated fratricide and enhance ADCC in multiple myeloma when combined with daratumumab. Blood. 2019;134 (Supplement_1):133. Lupo KB, Matosevic S. Natural killer cells as allogeneic effectors in adoptive cancer immunotherapy. Cancers (Basel). 2019;11(6):769.

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.

Introduction: Despite the recent FDA approval of autologous chimeric antigen receptor (CAR) T-cell therapies targeting B-cell maturation antigen (BCMA), multiple myeloma (MM) remains incurable and relapsed/refractory (R/R) disease remains an area of high unmet medical need. Additionally, patient access to autologous CAR T-cell therapies is currently limited due to manufacturing constraints, the need for bridging therapy, and potentially life-threatening toxicities including cytokine release syndrome and neurologic toxicities (Munshi et al. 2021). Off-the-shelf natural killer (NK) cell therapies may offer an improved therapeutic profile and broader patient access than autologous CAR T-cell therapies. FT576 is a first-of-kind, multiplexed-engineered BCMA CAR NK cell therapy generated from a clonal master engineered induced pluripotent stem cell line, which can be used as a renewable source for the mass production of off-the-shelf NK cells of uniform composition. FT576 is engineered with 4 modalities to combine multifaceted innate immunity with multi-antigen-targeting capability: (1) high-affinity 158V, non-cleavable CD16 (hnCD16) Fc receptor for augmented antibody-dependent cellular cytotoxicity; (2) IL-15/IL-15 receptor fusion that promotes NK cell persistence; (3) CD38 knockout to mitigate NK cell fratricide by CD38-directed monoclonal antibodies (mAbs) and that provides improved metabolic fitness and resistance to oxidative stress within the tumor microenvironment; and (4) a BCMA-directed CAR to target plasma cells. These modalities are designed to enhance the potency and persistence of FT576 and to enable multi-antigen targeting when combined with tumor-targeting mAbs. In preclinical studies, FT576 combined with the anti-CD38 mAb daratumumab demonstrated highly effective tumor control compared to either treatment alone or to primary CAR T cells in a disseminated MM xenograft model (Goodridge et al. 2021), suggestive that limitations in MM treatment confounded by clonal heterogeneity and antigen loss can be overcome with a dual antigen-targeting approach. Methods: This is a Phase I trial of FT576 in patients with R/R MM. The primary objectives are to assess safety and tolerability and to determine the recommended Phase II dose of FT576, given as single or multiple doses, as monotherapy and in combination with daratumumab in R/R MM. Key secondary objectives include anti-tumor activity and pharmacokinetics. The dose-escalation stage of the trial has the following 4 arms: single-dose FT576 monotherapy on Day 1 (Regimen A); multi-dose FT576 monotherapy on Days 1 and 15 (Regimen A1); single-dose FT576 + daratumumab on Day 1 (Regimen B); and multi-dose FT576 + daratumumab on Days 1 and 15 (Regimen B1). Dose levels of FT576 starting from 100 million cells/dose are being evaluated using a modified toxicity probability interval dose-escalation design. Daratumumab is administered per approved dose and schedule. Outpatient conditioning chemotherapy consists of 3 consecutive days of fludarabine and cyclophosphamide administered prior to the first dose of FT576. Results: As of a data cutoff date of 18 Jul 2022, 9 patients with R/R MM were treated and evaluable for safety and efficacy, in the first 2 dose levels of Regimen A (n = 6) and in the first dose level of Regimen B (n = 3). No dose-limiting toxicities, and no events of any grade of cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome, or graft-versus-host disease (GvHD), were observed. Conclusions: Administration of a single dose of FT576 at 100 or 300 million cells/dose alone or in combination with daratumumab is safe and well tolerated thus far without CRS, neurotoxicity, or GvHD. Interim clinical data, including safety and tolerability and initial anti-tumor activity, from the ongoing Phase I dose-escalation study of FT576 will be presented at the conference. References: Goodridge JP, Bjordahl R, Mahmood S, et al. FT576: Multi-specific off-the-shelf CAR-NK cell therapy engineered for enhanced persistence, avoidance of self-fratricide and optimized mAB combination therapy to prevent antigenic escape and elicit a deep and durable response in multiple myeloma. Blood. 2020;136 (Supplement 1):4-5. Munshi NC, Anderson LD Jr, Shah N, et al. Idecabtagene vicleucel in relapsed and refractory multiple myeloma. N Engl J Med. 2021;384(8):705-716.

7541 Background: FT516 is an investigational, NK cell cancer immunotherapy derived from a clonal master iPSC line. FT516 is engineered with a novel hnCD16 Fc receptor, demonstrated preclinically to maximize antibody-dependent cellular cytotoxicity (Zhu et al. Blood 2020). FT516 can be mass produced and made available off-the-shelf for broad pt access and multi-dose administration. Methods: This is a Phase I trial of FT516 combined with rituximab (R) in pts with R/R BCL. Treatment consists of 2 cycles, each with 3 days lympho-conditioning (fludarabine 30 mg/m2 and cyclophosphamide 500 mg/m2) and 1 dose of R followed by 3 weekly infusions of FT516 (planned doses 30-900 million/dose) with IL-2 (6 MIU after each FT516 dose). The primary objective is to identify the incidence of dose-limiting toxicity (DLT)/dose cohort and the recommended Phase II dose using a standard 3+3 design. Additional objectives include safety, tolerability, preliminary activity, pharmacokinetics, and immunogenicity. Results: Six pts (5 DLBCL, 1 FL, median age 65.5 y) have completed (5) or discontinued (1) study treatment after the DLT period (data cutoff 9 Dec 2020): 2 received 30 million cells/dose, 3 received 90 million cells/dose, and 1 received 300 million cells/dose. All pts received > 1 prior R-containing regimen, and median number of prior therapies was 3 (range 2-6), including CAR-T in 3 pts. FT516 was primarily administered in the outpatient setting. No FT516-related Grade ≥3 adverse events (AEs) or serious AEs, and no events of cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), or graft-versus-host disease (GvHD) of any grade were reported. DLT (Grade 4 neutrophil count decreased, not recovered to baseline by D29) was reported in the first pt at 30 million cells/dose and R dosing of 375 mg/m2 weekly x 4/cycle, resulting in modification of R dosing to once/cycle; no DLTs were observed with modified R dosing. Most common all grade AEs in ≥3 pts: fatigue (4 pts) and decreased appetite, nausea, neutrophil count decreased, and headache (3 pts each). Grade ≥3 AEs in ≥2 pts: neutrophil count decreased (3 pts) and febrile neutropenia and platelet count decreased (2 pts each); none considered related to FT516. Host anti-product B- or T-cell immunogenicity was not observed. Three of 4 pts treated at ≥90 million cells/dose achieved objective response (2 complete responses [CRs] and 1 partial response). Conclusions: Administration of up to 6 doses of FT516 cells, including up to 300 million cells/dose, appears to be safe and tolerable, without CRS, ICANS, or GvHD. Activity was observed, including CRs, in heavily pretreated pts. Dose escalation is ongoing. Updated clinical and translational data will be presented. Clinical trial information: NCT04023071.

Background: Allogeneic natural killer (NK) cell therapies have documented anti-tumor activity in patients with relapsed/refractory (R/R) hematologic malignancies, including B-cell lymphoma (BCL), and may offer an improved safety profile characterized by the absence of cytokine release syndrome (CRS) and neurologic toxicity compared with T-cell therapies (Liu et al. 2020). However, limited availability of suitable donors, relatively short in vivo persistence, and manufacturing constraints limiting the ability to consistently deliver multiple doses remain barriers to maximizing the clinical benefit of NK cell therapy.

Background: The use of a clonal master engineered induced pluripotent stem cell (iPSC) line as a renewable source for the mass production of immune effector cells offers distinct advantages over existing patient (pt)- and donor-derived cell-based cancer immunotherapy approaches, including off-the-shelf availability for broad pt access and multi-dose administration. FT596 is an iPSC-derived, off-the-shelf, CD19-directed chimeric antigen receptor (CAR) natural killer (NK) cell therapy capable of multi-antigen targeting in combination with monoclonal antibody (mAb) therapies. FT596 has three anti-tumor modalities: (1) a proprietary CD19-targeting CAR; (2) a novel high-affinity, non-cleavable CD16 Fc receptor that enables tumor targeting and enhanced antibody-dependent cell cytotoxicity in combination with a therapeutic mAb; and (3) IL-15/IL-15 receptor fusion promoting cytokine-autonomous persistence. Preclinical in vivo models of leukemia and lymphoma demonstrate potent CAR-mediated efficacy of FT596 against CD19+ tumor cells and activity against both CD19+ and CD19- tumor cells when combined with the anti-CD20 agent rituximab (Goodridge et al. 2019).

Haematopoietic stem and progenitor cells (HSPCs) have been the focus of developmental and regenerative studies, yet our understanding of the signalling events regulating their specification remains incomplete. We demonstrate that supt16h , a component of the Facilitates chromatin transcription (FACT) complex, is required for HSPC formation. Zebrafish supt16h mutants express reduced levels of Notch-signalling components, genes essential for HSPC development, due to abrogated transcription. Whereas global chromatin accessibility in supt16h mutants is not substantially altered, we observe a specific increase in p53 accessibility, causing an accumulation of p53. We further demonstrate that p53 influences expression of the Polycomb-group protein PHC1, which functions as a transcriptional repressor of Notch genes. Suppression of phc1 or its upstream regulator, p53 , rescues the loss of both Notch and HSPC phenotypes in supt16h mutants. Our results highlight a relationship between supt16h , p53 and phc1 to specify HSPCs via modulation of Notch signalling.

Snail2 is a zinc-finger transcription factor best known to repress expression of genes encoding cell adherence proteins to facilitate induction of the epithelial-to-mesenchymal transition. While this role has been best documented in the developmental migration of the neural crest and mesoderm, here we expand on previously reported preliminary findings that morpholino knock-down of snai2 impairs the generation of hematopoietic stem cells (HSCs) during zebrafish development. We demonstrate that snai2 morphants fail to initiate HSC specification and show defects in the somitic niche of migrating HSC precursors. These defects include a reduction in sclerotome markers as well as in the Notch ligands dlc and dld, which are known to be essential components of HSC specification. Accordingly, enforced expression of the Notch1-intracellular domain was capable of rescuing HSC specification in snai2 morphants. To parallel our approach, we obtained two mutant alleles of snai2. In contrast to the morphants, homozygous mutant embryos displayed no defects in HSC specification or in sclerotome development, and mutant fish survive into adulthood. However, when these homozygous mutants were injected with snai2 morpholino, HSCs were improperly specified. In summary, our morpholino data support a role for Snai2 in HSC development, whereas our mutant data suggest that Snai2 is dispensable for this process. Together, these findings further support the need for careful consideration of both morpholino and mutant phenotypes in studies of gene function.

Hematopoietic stem cells (HSCs) are a unique population of adult stem cells that continually replenish all types of terminally differentiated blood cells. This unique ability makes HSCs exploitable for treatment of hematopoietic disorders such as anemia and leukemia; however, these transplants are limited by a paucity of matched bone marrow donors. Furthermore, transplants have limited success rates due to immune rejection. Therefore, one major aim of regenerative medicine is to generate an alternative source of HSCs in vitro. Unfortunately, differentiation of therapeutically viable HSCs from pluripotent precursors is not yet possible. To this end, improvement of in vitro techniques necessitates a better understanding of the complex spatial and temporal cues of HSC development in vivo. The genetic control of hematopoiesis is widely conserved across vertebrate phyla. Therefore, the zebrafish is an ideal model for examining HSC emergence as they develop externally and are translucent, allowing for real-time imaging of HSCs and the cells that form their niche. During embryogenesis, HSCs arise from the hemogenic endothelium (HE), a unique population of aortic endothelial cells. For this process to occur, multiple tissues must interact with and provide essential specification signals to the developing HE. Interestingly, a transcriptome analysis of stromal cell lines derived from the murine embryonic HSC niche revealed expression of the zinc-finger transcription factor snai2 (Slug) to be enriched in the cell lines that better supported maintenance and differentiation of HSCs and hematopoietic progenitors. While Snai2 is best known for its role in neural crest specification and migration through its induction of the epithelial-to-mesenchymal transition, we have shown in zebrafish that it is also required for HSC specification by means of morpholino injection. However, it is still unclear where, when, and how this transcription factor plays its critical role. Here we present ongoing data to further elucidate the temporal and spatial requirement of Snai2 for HSC specification, as well as the development of a Snai2 mutant zebrafish generated with the Crispr/Cas9 system.