S. Ghovvati, Mohammad Reza Nassiri, Hossein Borghaei and Gregory P. Adams
Department of Biotechnology, University of Guilan, Rasht, Iran; Fox Chase Cancer Center, Philadelphia, Pennsylvania, USA
Nowadays, ErbB2 (HER2) is become a validated target for antibody-based cancer immunotherapy. ErbB2 receptor tyrosine kinase is a member of the epidermal growth factor receptor family of transmembrane receptors that is over expressed on surface of a wide range of tumor cells where it is often play critical roles in the development of malignancy. Recently, antibody targeting of exogenous RNases to tumor surface antigens has emerged as a potentially promising approach for cancer therapy. While RNases of different species have been employed, we believe that the Human Pancreatic-RNase (HP-RNase) is greater potential as a component of a targeted fusion protein. The aim of this study was to engineer and construct a panel of fully-human antibody-RNase fusion protein that could be less immunogenic and more tolerant as a suitable agent for HER2 positive breast cancer therapy to modulate the cell killing effects of immunotoxins. Hence, we engineered, designed and expressed a fusion antibody containing two anti-HER2 single chain variables Fragment (scFv), a human IgG1 Fc domain and two HP-RNase (scFv-Fc-HPR). We also created variants containing different combinations of linker segments in an attempt to increase the escape rate of the HP-RNase from the endosomal compartment after receptor mediated internalization and improve subsequent localization in cytoplasm or nucleus. These fusion proteins were constructed using the pSEC2A vector, expressed transiently in HEK-293T cells and purified using IMAC directed against a poly-his tag. Fusion antibodies binding to HER2-ECD were characterized in parallel with HER2 positive cell lines using flow-cytometry. The flow-cytometry results demonstrated convincingly that the fusion antibodies bound to HER2-positive breast cancer cells similar to their parental scFv (Trastuzumab) and they have no detectable fluorescence intensities on HER2 negative breast cancer cells lacking HER2 amplification. In vitro dose response cytotoxicity assay results indicated that the engineered antibodies were capable of mediating significantly greater growth inhibition than what observed with the Trastuzumab on HER2 over expressing breast cancer cell lines and Herceptin resistant breast cancer cell line. Furthermore, they have no detectable impacts on the viability of the HER2-negative tumor cell lines. Owing to this fusion immunoconjugate antibodies have acquired RNase-based cytotoxicity in addition to the inhibitory growth effects of the Trastuzumab. In conclusion, current study results indicate that engineered antibodies could be a promising novel fully human anti-cancer therapeutics and well-tolerated agents for cancer therapy.
Keywords: Antibody engineering, Breast cancer, Cytotoxicity, Cancer, ErbB2, Flow-cytometry, Immunotherapy.