Abstract 569: BIO-PROTAC: New treatment modality to treat undruggable KRAS mutants by application of cell penetrating scFv
Authors: Yoon Gookjin , Jo Beom Soo , Lee Dong Woo , Yang Jinwook , Park Min-Ho , Seok Sanghui , Lee Jue-Yeon , Chung Chong Pyung , Park Yoon Jeong 
Published: April 2023

Activation of mutations in RAS has been found in high ratio in human cancers, inducing downstream critical effectors corresponding tumorigenesis. 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. It has been well established that the tumor engagement is quite related with the interaction between mutated RAS protein and RAF kinase in ERK cascade. Our aim is to develop inhibitor that cell-penetrating scFv (single chain fragment variable) which targets mutated KRAS (mKBscFv). 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, a cell-penetrating synthetic peptide (CPP) was developed for drug delivery carrier and was expressed at the C-terminus of mKBscFv for transferred cells. In addition, the CPP peptide has a function of inhibiting the interaction of mutated RAS and RAF. The mKBscFv-CPP specifically binding to mutated KRAS and tagged with His tag to express in CHO cells, which was purified by FPLC. The purity of mKBscFv-CPP was confirmed by SDS PAGE. In the SPR results, not only mKBscFv-CPP showed high binding ability to mutated KRAS, but also in cell viability test, it affected the proliferation of KRAS mutated cells. The muatated GTP-bound KRAS was reduced by mKBscFv in KRAS mutated cancer cells. 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 cell penetrating synthetic 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 cancer 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 Pyung Chung, Yoon Jeong Park. BIO-PROTAC: New treatment modality to treat undruggable KRAS mutants by application of cell penetrating scFv [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 569.