DEVELOPMENT OF ANTI-EGFR MONOCLONAL ANTIBODY: A BIOSIMILAR PROTOTYPE

INTRODUCTION AND OBJECTIVES: Approximately 51% of Brazilian annual public health budget is dedicated to purchase only 5% of medicines, the biopharmaceuticals. The demand for biopharmaceuticals is also driven by the growing prevalence of chronic diseases, such as cancer. Many monoclonal antibodies will have their patent expired by 2030, enabling the production of biosimilars, biological products with the same efficacy and safety as an approved commercial reference. Therefore, there is an urgent need to develop national technology for monoclonal antibody (mAb) production. This study aims to express and purify an anti-epidermal growth factor receptor (EGFR) mAB, as well as its structural and binding characterization. MATERIAL AND METHODS: The genes for both heavy chain (HC) and light chain (LC) of the antibody were commercially synthesized and inserted in the pcDNA 3.1 plasmid. The plasmids were cloned in E.Coli top 10 strain using electroporation and extracted using a purification kit. The cell line used to express the mAB was Expi293F. The cell culture was incubated with the DNA containing the HC and LC genes, and lipofectamine, for 5 days at 37 ºC, with 8% of CO₂ and orbital agitation of 125 rpm. The supernatant was submitted to an affinity purification using a HiTrap column in the ÄKTA start system. The purified mAb was then analyzed by electrophoresis using a SDS/PAGE gel and Western Blot technique (WB). A thermal stability test was conducted, and an enzyme-linked immunosorbent assay (ELISA). Finally, DU145 cells, a prostate cancer cell line, were incubated with the biosimilar prototype, the commercial mAb, and the epidermal growth factor, to test its binding capacity to the cells, and the competition between the anti-EGFR and the EGF. The cells were analyzed using flow cytometry technique. RESULTS AND CONCLUSION: The cloning, extraction and transfection of the genes were successful as well as the purification. A protein peak was identified in the chromatogram indicating the presence of the mAb, which was quantified at 500 ug/mL. Bands were visible in the same height as the commercial mAb in the SDS/PAGE and WB analysis. The biosimilar prototype showed higher binding affinity than the reference and the same thermal stability. The flow cytometry results indicated that there is a competition between the EGF and the anti-EGF expressed, as the binding between the EGF and its receptor went down by 30%. In conclusion, the gene construction strategy of the expression system demonstrated satisfactory expression level. The biosimilar mAb and the commercial product showed a similar profile of binding to the EGFR protein. The present research is ongoing for further mAb characterization and optimization of protein yield.