Recombinant cell penetrating peptides and intrabodies targeting membrane-bound mutated KRAS antigens

Abstract

One of the drivers for early carcinogenesis involves specific somatic point substitution mutations in the KRAS gene which damages its ability to conduct signal transduction. Although antibodies can be used for the targeting of KRAS antigen, their localization along the cell’s inner membrane serves as a barrier against the accessibility of the antibodies. This study describes the evaluation of two internalization strategies, namely the endocytosis-based cell penetrating peptide (CPP) approach and the adenoviral-based intrabody (IB) approach, for the delivery of an anti-mutant KRAS single-chain variable fragment (scFv) into the cell. Splicing by overhang extension polymerase chain reaction (SOE-PCR) was used for the fusion of scFv with an enhanced green fluorescence protein (eGFP) and Antennapedia-PTD (Antp), a cell penetrating signal peptide. The fused construct (Antp-scFv-eGFP) at a concentration of 0.085 mg/ml was expressed in E. coli (BL21), while recombinant adenoviral particles containing the scFv-eGFP gene were harvested from HEK 293 cells. Both SW480 and HeLa cells were treated with Antp-scFv-eGFP and recombinant adenoviral particles, and their eGFP localization and intensity were compared to determine their scFv binding efficiencies. The IB approach was shown to exhibit a 3-fold higher fluorescence signal intensity compared to the CPP approach. This proof-of-concept study demonstrated that both antigens for either screening, diagnostic approaches can be potentially adopted when targeting various intracellular or therapeutic purposes.