Solid tumors are characterized by cell clusters experiencing chronic and intermittent hypoxia due to haphazard growth. Importantly, highly migratory tumorigenic stem cells are localized within hypoxic niches, shielded by hypoxia-induced metabolites that inhibit tumor-infiltrating T cells, restricting their survival, cytotoxicity, and beneficial immunomodulatory cytokine responses. To eliminate bulky solid tumors including stem cells, T cells must overcome physiological changes distinctive for the tumor microenvironment, such as hypoxia, depleted nutrient levels, and low extracellular pH. To exploit the hypoxic tumor microenivornment, we introduce a new approach that exploits hypoxia as a condition for T-cell activation. We demonstrate a chimeric antigen receptor (CAR), which is specific for CD19 and capable of activating T cells through chimeric CD28 and/or CD3-zeta signaling endodomain, that can be conditionally expresed in a strictly oxygen-sensitive manner. Using the Sleeping Beauty transposon/transposase system, we can achieve stable, persistent CAR transgene expression without the expense and complexity of retroviral production. Cell surface expression of our CAR is high at 1% O and not detectable at 20% O (Figure). Anticipating the circulatory nature of T cells in vivo, our oxygen-sensitive CAR is designed to deactivate upon exiting from hypoxic tumor microenvironment to minimize deleterious off-target effects. When supplemented with one or more survival factors and/or homing receptors, this approach to CAR expression has the advantage of

  • transforming hypoxia from an adverse factor to a triggering mechanism,

  • efficiency, and

  • high levels of transgene expression.

Figure:

Flow cytometry expression of CAR under normoxia and hypoxia.

Figure:

Flow cytometry expression of CAR under normoxia and hypoxia.

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Disclosures: No relevant conflicts of interest to declare.

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