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Background <p>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<sub>HPV16-18E7</sub>). 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.</p> Methods <p>Three forms of the affitoxin constructs (GrB-Z<sub>HPV16-18E7</sub>, Z<sub>HPV16E7</sub>-GrB-Z<sub>HPV18E7</sub>, and Z<sub>HPV16-18E7</sub>-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.</p> Results <p>Two bispecific affitoxins of Z<sub>HPV16E7</sub>-GrB-Z<sub>HPV18E7</sub> and Z<sub>HPV16-18E7</sub>-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.</p> Conclusions <p>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.</p>

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

  • 期刊论文

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  • 发表日期：

    2025年9月22日

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  • Kairong Wan,
  • Lijun Yu,
  • Sicong Feng,
  • Zhenyun Xie,
  • Yanheng Li,
  • Xisha Jing,
  • Junze Wu,
  • Lifang Zhang,
  • Wenshu Li

摘要

Background

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 ZHPV16-18E7). 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.

Methods

Three forms of the affitoxin constructs (GrB-ZHPV16-18E7, ZHPV16E7-GrB-ZHPV18E7, and ZHPV16-18E7-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.

Results

Two bispecific affitoxins of ZHPV16E7-GrB-ZHPV18E7 and ZHPV16-18E7-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.

Conclusions

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.