Engineered protein circuits for cancer therapy

Many targeted therapies indirectly suppress cancer cells by inhibiting oncogenic signaling pathways such as Ras1-4. This renders them susceptible to resistance and limits their long-term clinical efficacy4-10. Engineered protein circuits11-25 have been envisioned as an alternative to pharmacological inhibition that directly rewires oncogenic activity to cell death. However, it has remained unclear whether engineered protein circuits can potently and safely treat cancers. Here, we show that Ras-targeting circuits can accurately discriminate between cancer and non-cancer cells, circumvent intrinsic and acquired resistance mechanisms that limit pharmacological inhibitors, and suppress cancer in vivo. These circuits combine three modules: a protease-based sensor that responds to a broad spectrum of clinically relevant Ras mutations, an optional protease amplifier, and protease-triggered cell death effectors. These effectors can flexibly trigger either non-inflammatory apoptosis or immunogenic pyroptosis, which has been shown to extend therapeutic effects beyond transfected cells26,27. The resulting sense-kill circuits can be safely, efficiently, and transiently delivered to cells as mRNA in lipid nanoparticles (LNPs). The circuits exhibited potent efficacy against Ras-mutant human cancer cell lines with minimal off-target killing of wild-type Ras cells. In immunocompetent mice bearing aggressive, multifocal Ras-driven liver tumors, systemically-delivered mRNA-LNP circuits significantly reduced tumor burden. Further, therapeutic circuits provided more potent killing of Ras-mutant cancer cells than the Ras inhibitors Sotorasib and RMC-79777,28-30, and exhibited increased sensitivity to Sotorasib-resistant cells in vitro. These results establish a potent, specific, and programmable mechanism for treating cancer and other human diseases.