While
engineered chimeric antigen receptor (CAR) T cells have shown
promise in detecting and eradicating cancer cells within patients,
it remains difficult to identify a set of truly cancer-specific CAR-targeting
cell surface antigens to prevent potentially fatal on-target off-tumor
toxicity against other healthy tissues within the body. To help address
this issue, we present a novel tamoxifen-gated photoactivatable split-Cre
recombinase optogenetic system, called TamPA-Cre, that features high
spatiotemporal control to limit CAR T cell activity to the tumor site.
We created and optimized a novel genetic AND gate switch by integrating
the features of tamoxifen-dependent nuclear localization and blue-light-inducible
heterodimerization of Magnet protein domains (nMag, pMag) into split
Cre recombinase. By fusing the cytosol-localizing mutant estrogen
receptor ligand binding domain (ERT2) to the N-terminal half of split
Cre(2–59aa)-nMag, the TamPA-Cre protein ERT2-CreN-nMag is physically
separated from its nuclear-localized binding partner, NLS-pMag-CreC(60–343aa).
Without tamoxifen to drive nuclear localization of ERT2-CreN-nMag,
the typically high background of the photoactivation system was significantly
suppressed. Upon blue light stimulation following tamoxifen treatment,
the TamPA-Cre system exhibits sensitivity to low intensity, short
durations of blue light exposure to induce robust Cre-loxP recombination
efficiency. We finally demonstrate that this TamPA-Cre system can
be applied to specifically control localized CAR expression and subsequently
T cell activation. As such, we posit that CAR T cell activity can
be confined to a solid tumor site by applying an external stimulus,
with high precision of control in both space and time, such as light.
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