Work

A novel bispecific affitoxin simultaneously targeting E7 of HPV16/18 types: superior anti-tumor activity and EMT reversal in HPV-driven cervical cancer therapy

Year: 2025

Type: article

Abstract: Sustained infection with high-risk HPV of the 16 and 18 types is accounted for nearly 75% of cervical cancer (CC), but now there is an absence of agents aimed at eradicating HPV infections. Notwithstanding, affibody-based affitoxins may represent a breakthrough in tumor-targeted therapy. Our previous work has constructed a bispecific affibody simultaneously targeting the early oncogenic proteins E7 of HPV16 and 18 types (named as Z HPV16-18E7 ). In the present study, Granzyme B (GrB, cytotoxic effector) was introduced to construct three bispecific affitoxins for the enhanced therapy against HPV-infected CC cells. Three forms of the affitoxin constructs (GrB-Z HPV16-18E7 , Z HPV16E7 -GrB-Z HPV18E7 , and Z HPV16-18E7 -GrB) were designed and prepared, and their binding to the target protein and cells were confirmed by SPR analysis and a confocal immunofluorescence assay. The inhibition of cell viability by a CellTiter-Lumi™ luminescence assay, the induction of apoptosis by a fluorometric TUNEL method, and the reversal of EMT by wound healing and transwell assays, for the affitoxins against the target cells were evaluated. The in vivo inhibition in tumor-bearing mice along with the acute toxicity, pharmacokinetics, and stability of the affitoxins were tested. Two bispecific affitoxins of Z HPV16E7 -GrB-Z HPV18E7 and Z HPV16-18E7 -GrB were successfully prepared. They can bind to the target protein and cells, inhibited cell viability as well. Compared to the bispecific affibody (without GrB), the bispecific affitoxins induced a more pronounced apoptosis characterized by the release of active caspase-3 and may inhibit cell migration by reversing the epithelial-mesenchymal transition (EMT) pathway. In vivo, the bispecific affitoxin exhibited significant tumor-targeting accumulation in tumor-bearing mice. Compared to the bispecific affibody, the bispecific affitoxin showed a more significant inhibition of the growth of the xenograft tumor in mice, with no acute toxic reactions and a certain degree of stability. This work has developed a bispecific affitoxin that simultaneously targets HPV16- and HPV18-type CC cells, with a significant dual-functional advantage, combining the targeted inhibitory of the affibody with the cytotoxicity of the toxin molecule. Our research offers a novel design and approach for targeted therapy in HPV-driven cervical cancer.

Source: Journal of Translational Medicine

DOI: 10.1186/s12967-025-06971-9