Chimeric antigen receptor (CAR) T-cell therapy continues to struggle in reaching its full potential in solid tumors, largely due to the tumor microenvironment (TME) where various small molecules induce immunosuppressive signaling in CAR T-cells. Two key molecules, adenosine and lysoPS, are overly abundant in the TME and activate their respective G-protein coupled receptors (GPCRs) A2AR and GPR174. This hyper-activation triggers excessive cAMP production, ultimately leading to CAR T treatment failure. In this thesis, we develop G-protein-based strategies to mitigate the inhibitory effects of the TME on CAR T function.

First, we employed a minimal Gs (mGs) variant to block GPCR signaling. Wild-type (WT) mGs constructs effectively lowered cAMP levels under non-stimulated conditions but were unable to reduce cAMP levels in cells experiencing high A2AR stimulation. Through in silico design, we identified point mutations that enhanced the cAMP-blocking capabilities of mGs. We then combined the top three mutations to create the mGs_3x variant, which successfully reduced cAMP levels in A2AR-activated HEK and Jurkat cells.

Second, we engineered Gi-Gs (Gis) and Gq-Gs (Gqs) chimeric proteins to rewire the inhibitory Gs signaling into a beneficial pathway. Our Gis and Gqs chimeras lowered cAMP levels similarly to mGs_WT and were able to induce Gi and Gq signaling, respectively. Further in silico redesign of the G-protein-A2AR binding interface led to the identification of the Gis_F3 variant, which contains 24 point mutations and reduced cAMP levels in stimulated JK cells by 10-fold.

In summary, through the use of our mGs, Gis, and Gqs constructs, we effectively lowered cAMP production and rewired Gs signaling. We also successfully developed mGs and Gis variants that robustly blocked cAMP production upon A2AR stimulation in HEK and JK cells. Overall, our G-protein-based strategy shows promise in targeting the GPCR-Gs-cAMP pathway, thereby reducing TME-induced CAR T-cell immunosuppression.