Expression and Evaluation of HuscFv Antibody -PE40 Immunotoxin for Target Therapy of EGFR-Overexpressing Cancers

Document Type: Research Paper

Authors

1 Department of Medical Biotechnology, Faculty of Advanced Medical Science, Tabriz University of Medical Sciences, Tabriz, Iran.

2 Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran.

3 Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.

4 Maragheh University of Medical Sciences, Maragheh, Iran.

5 Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.

6 Department of Medical Biotechnology, Faculty of Medicine, Kermanshah University of Medical Sciences, Kermanshah, Iran.

7 Tuberculosis and Lung Disease Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.

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.

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