2565 Background: Engineered cell therapies expressing a chimeric antigen receptor (CAR) in human T cells have demonstrated significant promise for the treatment of hematological malignancies. However, translating CAR T cell therapies to solid tumors remains challenging due to two limitations: (1) specificity, as few antigens are truly unique to solid tumors, leading to off-target toxicities; and (2) suppression, as solid tumors establish immunosuppressive microenvironments that limit T cell survival and cytotoxicity. To address these challenges and improve the safety and potency of solid tumor cell therapies, we engineered cells with synthetic circuits designed to amplify hypoxia-induced signaling through feedback and synthetic transcriptional control, enabling augmented and tunable responses within both modestly and profoundly hypoxic environments. Methods: We evaluated circuit architectures in HEK293FT cells in which the HBS promoter drove either native HIF transcription factors or synthetic transcription factors and promoters controlling a fluorescent reporter. Circuits were stably integrated using PiggyBac transposon vectors, and cells were cultured under normoxic (21% O₂), modestly hypoxic (5% O₂), and profoundly hypoxic (1% O₂) conditions. Fluorescent reporter expression was quantified by flow cytometry over three days, and candidate circuits exhibiting high-output, low-background expression under hypoxia were identified. These candidate circuits were subsequently introduced into primary human T cells using transposon vectors. CAR expression in engineered T cells was measured by flow cytometry after culture for three days in modest or profound hypoxia, and cytotoxic activity was assessed in co-culture assays with BT-474 breast cancer and SKOV3 ovarian cancer cells. Results: We identified novel circuit architectures that produced rapid induction with minimal background under hypoxia and defined design rules governing transgene amplification. Circuit topologies incorporating synthetic transcription factor–mediated feedback exhibited markedly amplified expression relative to native HIF-based circuits. CAR-expressing T cells demonstrated cytotoxic activity compared with unmodified T cells in co-culture assays. Conclusions: We present a therapeutic platform that enables hypoxia-dependent sensing and high-output control of therapeutic gene expression within solid tumor microenvironments. This approach has the potential to improve the safety and efficacy of engineered cell therapies for solid tumors and to provide mechanistic insights that inform the development of hypoxia-responsive cell therapies.
Schreiber et al. (Wed,) studied this question.