Development of a Novel Antibody for Cancer Therapy Targeting Intracellular RAS Mutant Proteins
Abstract – In the modern pharmaceutical industry, monoclonal antibodies are often used as therapeutic agents. However, they are restricted to cell surface antigens due to their inability to penetrate the outer cell membrane and maintain normal function in the reducing environment. Previous research conducted to engineer a cell-penetrating peptide-tagged single-chain variable fragment that binds to mutated HRAS (G12V) (CPP-scFv(Ras)) was proven successful through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blotting and binding assay. To further develop CPP-scFv(Ras) antibody as cancer therapy, this study utilized the next-generation sequencing technique (RNA sequencing) to investigate the down-regulation of the Ras signal pathway genes after CPP-scFv(Ras) treatment. To analyze the transcriptomic effect of CPP-scFv(Ras) treatment on cancer cells, we used the bladder cancer cell line (T24) because this cell line contains HRas (G12V) mutation. Simultaneously data from RNA sequencing were analyzed to study the effect of CPP-scFv(Ras) on other gene regulations to isolate possible side effects of using CPP-scFv(Ras) antibodies in a clinical setting. Cancer cell viability assays were conducted prior to RNA sequencing to identify the optimal concentration of CPP-scFv(Ras) antibody concentration for inducing cancer cell death. After analyzing the total of 18096 human gene transcriptomes from the cancer cells, CPP-scFv(Ras) treatment proved effective in the downregulation of Ras pathways. This result indicates that our novel designed antibody can be applied for personalized cancer therapy for most cancer caused by Ras mutation.