Search

Multiple genetic or molecular alterations are known to be associated with cancer stem cell formation and cancer development. Targeting such alterations, therefore, may lead to cancer prevention. By crossing our previously established phosphatase and tensin homolog (Pten)-null acute T-lymphoblastic leukemia (T-ALL) model onto the recombination-activating gene 1-/- background, we show that the lack of variable, diversity and joining [V(D)J] recombination completely abolishes the Tcrα/δ-c-myc translocation and T-ALL development, regardless of β-catenin activation. We identify mammalian target of rapamycin (mTOR) as a regulator of β-selection. Rapamycin, an mTOR-specific inhibitor, alters nutrient sensing and blocks T-cell differentiation from CD4-CD8- to CD4+CD8+, the stage where the Tcrα/δ-c-myc translocation occurs. Long-term rapamycin treatment of preleukemic Pten-null mice prevents Tcrα/δ-c-myc translocation and leukemia stem cell (LSC) formation, and it halts T-ALL development. However, rapamycin alone fails to inhibit mTOR signaling in the c-KitmidCD3+Lin- population enriched for LSCs and eliminate these cells. Our results support the idea that preventing LSC formation and selectively targeting LSCs are promising approaches for antileukemia therapies. [ABSTRACT FROM AUTHOR]

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. 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 × 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/m2) and fludarabine (30 mg/m2) 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/m2) 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 5·0. The trial was registered with ClinicalTrials.gov , NCT04245722. 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. 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 1·8 × 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. Fate Therapeutics. [ABSTRACT FROM AUTHOR]

Cell therapy is emerging as a very promising therapeutic modality against cancer, spearheaded by the clinical success of chimeric antigen receptor (CAR) modified T cells for B cell malignancies. Currently, FDA-approved CAR-T cell products are based on engineering of autologous T cells harvested from the patient, typically using a central manufacturing facility for gene editing before the product can be delivered to the clinic and infused to the patients. For a broader implementation of advanced cell therapy and to reduce costs, it would be advantageous to use allogeneic "universal" cell therapy products that can be stored in cell banks and provided upon request, in a manner analogous to biopharmaceutical drug products. In this review, we outline a roadmap for development of off-the-shelf cell therapy based on natural killer (NK) cells derived from induced pluripotent stem cells (iPSCs). We discuss strategies to engineer iPSC-derived NK (iPSC-NK) cells for enhanced functional potential, persistence, and homing. [ABSTRACT FROM AUTHOR]

The tumor suppressor gene PTEN (phosphatase and tensin homologue deleted on chromosome 10) is frequently mutated or deleted in various human cancers. PTEN localizes predominantly to the cytoplasm and functions as a lipid phosphatase, thereby negatively regulating the phosphatidylinositol 3-kinase-AKT signaling pathway. PTEN can also localize to the nucleus, where it binds and regulates p53 protein level and transcription activity. However, the precise function of nuclear PTEN and the factors that control PTEN nuclear localization are still largely unknown. In this study, we identified oxidative stress as one of the physiological stimuli that regulate the accumulation of nuclear PTEN. Specifically, oxidative stress inhibits PTEN nuclear export, a process depending on phosphorylation of its amino acid residue Ser-380. Nuclear PTEN, independent of its phosphatase activity, leads to p53-mediated G1 growth arrest, cell death, and reduction of reactive oxygen species production. Using xenografts propagated from human prostate cancer cell lines, we reveal that nuclear PTEN is sufficient to reduce tumor progression in vivo in a p53-dependent manner. The data outlined in this study suggest a unique role of nuclear PTEN to arrest and protect cells upon oxidative damage and to regulate tumorigenesis. Since tumor cells are constantly exposed to oxidative stress, our study elucidates the cooperative roles of nuclear PTEN with p53 in tumor suppression. [ABSTRACT FROM AUTHOR]

Allogeneic cellular immunotherapies hold promise for broad clinical implementation but face limitations due to potential rejection of donor cells by the host immune system. Silencing of beta-2 microglobulin (B2M) expression is commonly employed to evade T cell-mediated rejection by the host, although the absence of B2M is expected to trigger missing-self responses by host natural killer (NK) cells. Here, we demonstrate that genetic deletion of the adhesion ligands CD54 and CD58 in B2M-deficient chimeric antigen receptor (CAR) T cells and multi-edited induced pluripotent stem cell (iPSC)-derived CAR NK cells reduces their susceptibility to rejection by host NK cells in vitro and in vivo. The absence of adhesion ligands limits rejection in a unidirectional manner in B2M-deficient and B2M-sufficient settings without affecting the antitumor functionality of the engineered donor cells. Thus, these data suggest that genetic ablation of adhesion ligands effectively alleviates rejection by host immune cells, facilitating the implementation of universal immunotherapy., Competing Interests: Declaration of interests K.-J.M. is a consultant and has research support from Fate Therapeutics. K.-J.M. has research support from Oncopeptides. K.-J.M. and Q.H. are consultants at Vycellix. All relationships have been approved by Oslo University Hospital, the University of Oslo, and the Karolinska Institute. R.M.M., E.V., Y.P., M.J., B.G., R.A., T.L., A.W., J.P.G., and B.V. are employees at Fate Therapeutics. Memorial Sloan Kettering has licensed IP (unrelated to this study) to Fate Therapeutics, and M.S. receives research support (for unrelated studies) from Fate Therapeutics., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Introduction: NK cells can mediate tumor cell killing by natural cytotoxicity and by antibody-dependent cell-mediated cytotoxicity (ADCC), an anti-tumor mechanism mediated through the IgG Fc receptor CD16A (FcγRIIIA). CD16A polymorphisms conferring increased affinity for IgG positively correlate with clinical outcomes during monoclonal antibody therapy for lymphoma, linking increased binding affinity with increased therapeutic potential via ADCC. We have previously reported on the FcγR fusion CD64/16A consisting of the extracellular region of CD64 (FcγRI), a high-affinity Fc receptor normally expressed by myeloid cells, and the transmembrane/cytoplasmic regions of CD16A, to create a highly potent and novel activating fusion receptor. Here, we evaluate the therapeutic potential of engineered induced pluripotent stem cell (iPSC)-derived NK (iNK) cells expressing CD64/16A as an "off-the-shelf", antibody-armed cellular therapy product with multi-antigen targeting potential., Methods: iNK cells were generated from iPSCs engineered to express CD64/16A and an interleukin (IL)-15/IL-15Rα fusion (IL-15RF) protein for cytokine independence. iNK cells and peripheral blood NK cells were expanded using irradiated K562-mbIL21-41BBL feeder cells to examine in in vitro and in vivo assays using the Raji lymphoma cell line. ADCC was evaluated in real-time by IncuCyte assays and using a xenograft mouse model with high circulating levels of human IgG., Results: Our data show that CD64/16A expressing iNK cells can mediate potent anti-tumor activity against human B cell lymphoma. In particular, (i) under suboptimal conditions, including low antibody concentrations and low effector-to-target ratios, iNK-CD64/16A cells mediate ADCC, (ii) iNK-CD64/16A cells can be pre-loaded with tumor-targeting antibodies (arming) to elicit ADCC, (iii) armed iNK-CD64/16A cells can be repurposed with additional antibodies to target new tumor antigens, and (iv) cryopreserved, armed iNK-CD64/16A are capable of sustained ADCC in a tumor xenograft model under saturating levels of human IgG., Discussion: iNK-CD64/16A cells allow for a flexible use of antibodies (antibody arming and antibody targeting), and an "off-the-shelf" platform for multi-antigen recognition to overcome limitations of adoptive cell therapies expressing fixed antigen receptors leading to cancer relapse due to antigen escape variants., Competing Interests: JW and BW are inventors on the patent application WO2019084388A1 Recombinant immune cells, methods of making, and methods of use. Human CD64/16A described in the patent application has been exclusively licensed to Fate Therapeutics. JSM is a paid consultant for Fate Therapeutics and JW, BW, and JSM receive research funds from Fate Therapeutics. Fate Therapeutics owns patent No. 10,626,372 Methods and compositions for inducing hematopoietic cell differentiation covering the iPSC derived NK cells. MH, RB, BH, SS, and BV are employees of Fate Therapeutics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Dixon, Snyder, Khaw, Hullsiek, Davis, Matson, Shirinbak, Hancock, Bjordahl, Hosking, Miller, Valamehr, Wu and Walcheck.)

Multiple myeloma (MM) is a plasma cell disease with a preferential bone marrow (BM) tropism. Enforced expression of tissue-specific chemokine receptors has been shown to successfully guide adoptively-transferred CAR NK cells towards the malignant milieu in solid cancers, but also to BM-resident AML and MM. For redirection towards BM-associated chemokine CXCL12, we armored BCMA CAR-NK-92 as well as primary NK cells with ectopic expression of either wildtype CXCR4 or a gain-of-function mutant CXCR4 R334X . Our data showed that BCMA CAR-NK-92 and -primary NK cells equipped with CXCR4 gained an improved ability to migrate towards CXCL12 in vitro . Beyond its classical role coordinating chemotaxis, CXCR4 has been shown to participate in T cell co-stimulation, which prompted us to examine the functionality of CXCR4-cotransduced BCMA-CAR NK cells. Ectopic CXCR4 expression enhanced the cytotoxic capacity of BCMA CAR-NK cells, as evidenced by the ability to eliminate BCMA-expressing target cell lines and primary MM cells in vitro and through accelerated cytolytic granule release. We show that CXCR4 co-modification prolonged BCMA CAR surface deposition, augmented ZAP-70 recruitment following CAR-engagement, and accelerated distal signal transduction kinetics. BCMA CAR sensitivity towards antigen was enhanced by virtue of an enhanced ZAP-70 recruitment to the immunological synapse, revealing an increased propensity of CARs to become triggered upon CXCR4 overexpression. Unexpectedly, co-stimulation via CXCR4 occurred in the absence of CXCL12 ligand-stimulation. Collectively, our findings imply that co-modification of CAR-NK cells with tissue-relevant chemokine receptors affect adoptive NK cell therapy beyond improved trafficking and retention within tumor sites., Competing Interests: AR and UH filed a patent application for the BCMA CAR used in this manuscript PCT/WO2017211900A1. AR and UH receive research funds from Fate Therapeutics San Diego, CA. JR, KM, JG, and BV are employees of Fate Therapeutics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Moles, Erdlei, Menzel, Massaro, Fiori, Bunse, Schrimpf, Gerlach, Gudipati, Reiser, Mathavan, Goodrich, Huppa, Krönke, Valamehr, Höpken and Rehm.)

Aim: We explored the generation of human induced pluripotent stem cells (iPSCs) solely through the transcriptional activation of endogenous genes by CRISPR activation (CRISPRa). Methods: Minimal number of human-specific guide RNAs targeting a limited set of loci were used with a unique cocktail of small molecules (CRISPRa-SM). Results: iPSC clones were efficiently generated by CRISPRa-SM, expressed general and naive iPSC markers and clustered with high-quality iPSCs generated using conventional reprogramming methods. iPSCs showed genomic stability and robust pluripotent potential as assessed by in vitro and in vivo . Conclusion: CRISPRa-SM-generated human iPSCs by direct and multiplexed loci activation facilitating a unique and potentially safer cellular reprogramming process to aid potential applications in cellular therapy and regenerative medicine., Competing Interests: R Abujarour, B Valamehr, M Pribadi, LK Fong, M Denholtz, A Gutierrez, M Haynes, TT Lee, are employees and shareholders of Fate Therapeutics, Inc. S Ding is a founder of Fate Therapeutics, Inc. S Ding has a patent related to this work and is licensed to Fate Therapeutics, Inc. The authors have no other competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript apart from those disclosed., (© 2024 Fate Therapeutics, Inc.)

Background: Antibody therapies can direct natural killer (NK) cells to tumor cells, tumor-associated cells, and suppressive immune cells to mediate antibody-dependent cell-mediated cytotoxicity (ADCC). This antigen-specific effector function of human NK cells is mediated by the IgG Fc receptor CD16A (FcγRIIIA). Preclinical and clinical studies indicate that increasing the binding affinity and avidity of CD16A for antibodies improves the therapeutic potential of ADCC. CD64 (FcγRI), expressed by myeloid cells but not NK cells, is the only high affinity IgG Fc receptor and is uniquely capable of stably binding to free monomeric IgG as a physiological function. We have reported on the generation of the FcγR fusion CD64/16A, consisting of the extracellular region of CD64 and the transmembrane and cytoplasmic regions from CD16A, retaining its signaling and cellular activity. Here, we generated induced pluripotent stem cell (iPSC)-derived NK (iNK) cells expressing CD64/16A as a potential adoptive NK cell therapy for increased ADCC potency., Methods: iPSCs were engineered to express CD64/16A as well as an interleukin (IL)-15/IL-15Rα fusion (IL-15RF) protein and differentiated into iNK cells. iNK cells and peripheral blood NK cells were expanded using irradiated K562-mbIL21-41BBL feeder cells and examined. NK cells, ovarian tumor cell lines, and therapeutic monoclonal antibodies were used to assess ADCC in vitro, performed by a DELFIA EuTDA assay or in real-time by IncuCyte assays, and in vivo. For the latter, we developed a xenograft mouse model with high circulating levels of human IgG for more physiological relevance., Results: We demonstrate that (1) iNK-CD64/16A cells after expansion or thaw from cryopreservation can be coupled to therapeutic antibodies, creating armed iNK cells; (2) antibody-armed iNK-CD64/16A cells can be redirected by added antibodies to target new tumor antigens, highlighting additional potential of these cells; (3) cytokine-autonomous activity by iNK-CD64/16A cells engineered to express IL-15RF; and that (4) antibody-armed iNK-CD64/16A cells thawed from cryopreservation are capable of sustained and robust ADCC in vitro and in vivo, as determined by using a modified tumor xenograft model with high levels of competing human IgG., Conclusions: iNK cells expressing CD64/16A provide an off-the-shelf multiantigen targeting platform to address tumor heterogeneity and mitigate antigen escape., Competing Interests: Competing interests: JW and BW are inventors on the patent application WO2019084388A1 (Recombinant immune cells, methods of making, and methods of use). Human CD64/16A described in the patent application has been exclusively licensed to Fate Therapeutics. JSM is a paid consultant for Fate Therapeutics and JW, BW and JSM receive research funds from Fate Therapeutics. Fate Therapeutics owns patent No. 10626372 (Methods and compositions for inducing hematopoietic cell differentiation) covering the iPSC derived NK cells. MH, RB, BH, SS, and BV are employees of Fate Therapeutics. All other authors declare no conflicts., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Allogeneic cell therapies hold promise for broad clinical implementation, but face limitations due to potential rejection by the recipient immune system. Silencing of beta-2-microglobulin ( B2M ) expression is commonly employed to evade T cell-mediated rejection, although absence of B2M triggers missing-self responses by recipient natural killer (NK) cells. Here, we demonstrate that deletion of the adhesion ligands CD54 and CD58 on targets cells robustly dampens NK cell reactivity across all sub-populations. Genetic deletion of CD54 and CD58 in B2M -deficient allogeneic chimeric antigen receptor (CAR) T and multi-edited induced pluripotent stem cell (iPSC)-derived NK cells reduces their susceptibility to rejection by NK cells in vitro and in vivo without affecting their anti-tumor effector potential. Thus, these data suggest that genetic ablation of adhesion ligands effectively alleviates rejection of allogeneic immune cells for immunotherapy.

Substantial numbers of B cell leukemia and lymphoma patients relapse due to antigen loss or heterogeneity after anti-CD19 chimeric antigen receptor (CAR) T cell therapy. To overcome antigen escape and address antigen heterogeneity, we engineered induced pluripotent stem cell-derived NK cells to express both an NK cell-optimized anti-CD19 CAR for direct targeting and a high affinity, non-cleavable CD16 to augment antibody-dependent cellular cytotoxicity. In addition, we introduced a membrane-bound IL-15/IL-15R fusion protein to promote in vivo persistence. These engineered cells, termed iDuo NK cells, displayed robust CAR-mediated cytotoxic activity that could be further enhanced with therapeutic antibodies targeting B cell malignancies. In multiple in vitro and xenogeneic adoptive transfer models, iDuo NK cells exhibited robust anti-lymphoma activity. Furthermore, iDuo NK cells effectively eliminated both CD19+ and CD19- lymphoma cells and displayed a unique propensity for targeting malignant cells over healthy cells that expressed CD19, features not achievable with anti-CAR19 T cells. iDuo NK cells combined with therapeutic antibodies represent a promising approach to prevent relapse due to antigen loss and tumor heterogeneity in patients with B cell malignancies., (© 2022 by The American Society of Hematology.)

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., (© 2022. The Author(s).)