@article { author = {Falahatgar, Dianoush and Farajnia, Safar and Zarghami, Nosratollah and Tanomand, Asghar and Ahdi Khosroshahi, Shiva and Akbari, Bahman and Farajnia, Hadi}, title = {Expression and Evaluation of HuscFv Antibody -PE40 Immunotoxin for Target Therapy of EGFR-Overexpressing Cancers}, journal = {Iranian Journal of Biotechnology}, volume = {16}, number = {4}, pages = {241-247}, year = {2018}, publisher = {National Institute of Genetic Engineering and Biotechnology of Iran}, issn = {1728-3043}, eissn = {2322-2921}, doi = {10.21859/ijb.1743}, abstract = {Background: Epidermal growth factor receptor (EGFR) plays an important role in the progression and tumorigenesis of the various cancers. In this regards, anti-EGFR antibodies are valuable approved therapeutics for the EGFR over-expressing cancers. However, the occurrence of mutations in the EGFR and/or KRAS genes; a common phenomenon which is seen in many cancers, lead to the resistance to the EGFR-directed antibodies. EGFR based immunotoxins are capable of overcoming this limitation by directing the toxin moieties to the cancer cells resulting in cell death.Objectives: In the present study, a novel immunotoxin consisting of the truncated Pseudomonas exotoxin A (PE-40) and anti-EGFR huscFv was developed and evaluated for the induction of cell death in EGFR positive A431tumoral cells.Materials and Methods: PE-40 fragment of the exotoxin A was amplified by using PCR and ligated to pET22b-huscFv. The reaction was confirmed by PCR and restriction digestion. The immunotoxin was expressed in E. coli BL21 (plysS) and then was purified by Ni-NTA affinity column. Subsequently, the toxicity of the purified immunotoxin was evaluated on EGFR over-expressing epidermoid carcinoma of skin, A431 cell line.Results: PCR and restriction digestion experiments have verified the integrity of the immunotoxin construct. Purification by affinity column resulted in a highly purified recombinant immunotoxin. MTT assay revealed the growth inhibitory effect of the huscFv-PE40 immunotoxin on EGFR-over-expressing A431 cells with an IC50 value of 250 ng.mL-1.Conclusion: In conclusion, the results indicated that the immunotoxin developed in this study has a high toxicity on the EGFR-over-expressing tumor cells and could be considered as a promising candidate for the treatment of the EGFR positive cancers.}, keywords = {Cancer target therapy,EGFR,HuscFv,Immunotoxin,Pseudomonas exotoxin A}, url = {https://www.ijbiotech.com/article_80166.html}, eprint = {https://www.ijbiotech.com/article_80166_0a42c9d1c6ce2d9e2265e3614c638707.pdf} }