BIO-PROTAC: Application of Cell Penetrating Scfv to Treat Undruggable KRAS Mutant Cancer

Activation of mutations in RAS has been found in high ratio in human cancers, inducing downstream critical effectors corresponding tumorigenesis. In recent years, RAS-RAF-ERK1/2 pathway is known as a crucial target for the anti-cancer drug development because of high prevalence of ERK activation in human cancers. The attempts in direct targeting to activated RAS, which is GTP bound, has been somewhat successful in the treatment of certain type of cancer, however, still beset by the resistance. Our aim is to develop inhibitor that cell-penetrating scFv (single-chain variable fragment) which targets mutated KRAS (mKBscFv; mutated KRAS binding scFv). The Fv fragment is the smallest unit composed of the variable region of heavy chain and light chain in an immunoglobulin molecule, that has a function of antigen-binding activity. The scFv can specifically bind to an antigen, thereby reducing side effects of non-specific binding seen in chemical drugs. However, due to the lack of cell penetrating ability of scFv, a delivery carrier is often required. In this study, the mKBscFv was measured binding affinity toward wild type KRAS and mutated KRAS using surface plasmon resonance (SPR). The scFv has higher affinity to mutated KRAS than wild type KRAS. A cell-penetrating peptide (CPP) was developed for drug delivery and was expressed at the C-terminus of mKBscFv for transferring cells. The mKBscFv-CPP was tagged with His tag to express in CHO cells, which was purified by FPLC. The purity of mKBscFv-CPP was confirmed by SDS PAGE and immunoblotting. The mKBscFv-CPP treated KRAS mutated cancer cells decreased cell viability in a concentration-dependent manner, and decreased expression of mutated GTP-bound KRAS. In addition to the bioactivity of the mKBscFv, it has further advantage as a proteolysis-targeting chimera (PROTAC). The plasmid DNA was prepared by adding a VHL-expressing sequence to the C-terminus of mKBscFv (mKBscFv-Vh). This gene was transfected into KRAS mutated cancer cells to express the protein, and as a result mutated GTP-bound KRAS was reduced. A CPP peptide domain was added to the C-terminus of this gene to express it as a protein in CHO cells. The mutated GTP-bound KRAS was reduced by mKBscFv-Vh-CPP in KRAS mutated cacner cells. Taken together, mKBscFv that binds to mutated KRAS was developed, and it can be expected to become a new protein anticancer drug effective against mutated KRAS-mediated cancer cells by fusing cell penetrating peptide and PROTAC technology. Citation Format: Gookjin Yoon, Beom Soo Jo, Dong Woo Lee, Jinwook Yang, Min-Ho Park, Sanghui Seok, Jue Yeon Lee, Chong Pyoung Chung, Yoon Shin Park, Yoon Jeong Park. BIO-PROTAC: Application of cell penetrating scFv to treat undruggable KRAS mutant cancer [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B039.