The Inhibitory Effect of an HPV16E7-Specific Affibody Conjugated to Granzyme B (GrB) on Cervical Cancer Cells by Bifunctional Activity

Abstract

Infection with high-risk human papillomavirus 16 (HPV16) alone is responsible for over 50% of cervical cancer (CC) cases, and the HPV early oncogenic protein E7 participates in inducing epithelial–mesenchymal transition (EMT), leading to malignant transformation. The lack of effective target drugs against CC has aroused considerable concern about targeted CC treatments based on the clearance of HPV-infected cells. A novel specific affibody targeting HPV16E7 (Z_HPV16E7) was conjugated to GrB (an immune-mediated killing effector) to construct an immunoaffitoxin (Z_HPV16E7-GrB), which was proven to have significant target affinity for and growth inhibitory effects against HPV16-positive CC cells both in vivo and in vitro. In the present study, the bifunctional inhibitory activities of Z_HPV16E7-GrB, i.e., whether EMT is blocked or retarded after targeting of E7 by Z_HPV16E7-GrB and whether the cytotoxicity induced by Z_HPV16E7-GrB manifests as not only apoptosis but also pyroptosis, was further investigated. Our results showed that after targeting E7, Z_HPV16E7-GrB significantly decreased cell viability and promoted LDH release in HPV16-positive SiHa and CaSki CC cells, and this inhibitory effect was achieved by blocking EMT, as characterized by the decreases in Vimentin and Snail expression and the increase in E-cadherin expression. On the other hand, Z_HPV16E7-GrB induced obvious apoptosis and pyroptosis in cells by directly cleaving the pyroptotic executor protein GSDME through a caspase-3-independent pathway. In addition, Z_HPV16E7-GrB did not cause acute toxic reactions in vivo. Our research demonstrated that Z_HPV16E7-GrB has an improved cytotoxic advantage mediated by accurate delivery based on the Z_HPV16E7 affibody.