Protein-based pan-RAS inhibitor induces tumor regression in female mice via IFNγ and CD8+ T cell-dependent tumor necrosis

Abstract Mutations in K/H/N-RAS occur in approximately 30% of human cancers, yet most RAS mutants remain undruggable in clinical settings. Here, we describe a protein-based pan-RAS inhibitor, RRSP-RBD, that combines a RAS/Rap1A-specific endopeptidase (RRSP) with a RAS-binding domain (RBD). This engineered fusion protein localizes to RAS on the plasma membrane, where it cleaves RAS, disrupts RAS-effector interactions, and effectively inhibits downstream RAS signaling. To achieve intracellular delivery of RRSP-RBD in vivo, we engineer two cell-permeable variants. The diphtheria toxin-based version (RRSP-RBD-DTB) demonstrates femtomolar anti-tumor potency and induces tumor regression in a xenograft mouse model. The cell-permeable peptide-based version (RRSP-RBD-TAT) exhibits robust anti-tumor activity in syngeneic models without inducing irreversible toxicity in normal tissues. Interestingly, anti-tumor efficacy of RRSP-RBD-TAT critically depends on the tumor microenvironment, requiring infiltration by IFNγ + CD8 + T cells to mediate tumor regression. Pharmacokinetic and toxicity evaluations indicate that RRSP-RBD is tolerated under the conditions tested and support further investigation as a protein-based pan-RAS inhibitor (all mouse studies were performed in female mice).