Design and construction of genetic construct expressing anti-EGFR VIII immunotoxincontains exotoxin A of Pseudomonas aeruginosa for glioblastoma cancer

Cancer is one of the most common causes of human mortality in today's societies, and extensive efforts are being made to combat it. EGFRVIII is a tumor-specific antigen that is not observed in normal tissues, making it possible to target cancerous tissues without damaging normal tissues. ANTI-EGFRVIII-based immunotoxins can lead to the death of cancer cells that express an excessive amount of the EGFRVIII receptor by directing toxic components to these cells.

The objective of the present study is to develop a new human immunotoxin against EGFRVIII (huscFv-PE38) by genetically fusing a single-chain human anti-EGFRVIII antibody with a truncated form of the Pseudomonas aeruginosa exotoxin A (PE) (PE38KDEL).

The PE-38 exotoxin A fragment was amplified using PCR and attached to pET22b-antiEGFRVIII huscFv. The reaction was confirmed by PCR and enzymatic digestion. The immunotoxin was expressed in E. coli BL21 (plysS) and then purified using Ni-NTA chromatography. After that, the purified immunotoxin reaction was evaluated using ELISA methods.

The PCR and restriction digestion experiments confirmed the accuracy and integrity of the designed immunotoxin structure. Purification of the expressed immunotoxin using Ni-NTA chromatography column resulted in a highly pure, non-recombined immunotoxin with a molecular weight of 60 kDa shown on SDS-PAGE gel.

The results of this study demonstrate that the designed immunotoxin was successfully cloned, expressed, and purified, and can be considered as a promising candidate for the inhibition of EGFRVIII-positive cancer cells.