Development of ZHPV16E7- granzyme B immunoaffitoxin: dual mechanisms targeting hpv16-positive cervical cancer through epithelial-mesenchymal transition inhibition and cell death
IntroductionCervical cancer, predominantly caused by high-risk human papillomavirus (HPV), remains a major global health challenge. HPV16 is the most prevalent type, and its oncoprotein E7 promotes epithelial-mesenchymal transition (EMT), a critical step in metastasis. Current therapies for HPV16-re...
Saved in:
| Main Authors: | , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Frontiers Media S.A.
2025-07-01
|
| Series: | Frontiers in Immunology |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/articles/10.3389/fimmu.2025.1616715/full |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1849429557245902848 |
|---|---|
| author | Xiaochun Tan Xiaochun Tan Jiani Yang Jiani Yang Yanheng Li Kairong Wan Sicong Feng Xishang Jing Zhenyun Xie Lifang Zhang Wenshu Li |
| author_facet | Xiaochun Tan Xiaochun Tan Jiani Yang Jiani Yang Yanheng Li Kairong Wan Sicong Feng Xishang Jing Zhenyun Xie Lifang Zhang Wenshu Li |
| author_sort | Xiaochun Tan |
| collection | DOAJ |
| description | IntroductionCervical cancer, predominantly caused by high-risk human papillomavirus (HPV), remains a major global health challenge. HPV16 is the most prevalent type, and its oncoprotein E7 promotes epithelial-mesenchymal transition (EMT), a critical step in metastasis. Current therapies for HPV16-related cancers are often insufficient, highlighting the need for targeted treatments. We engineered a novel immunotoxin, ZHPV16E7-GrB, by fusing an HPV16E7-specific affibody (ZHPV16E7) with the cytotoxic immune effector granzyme B (GrB). This construct was designed for precise targeting and therapeutic activity against HPV16-positive cervical cancer cells.MethodsZHPV16E7-GrB was engineered, expressed in E. coli, and purified. Binding specificity was assessed via ELISA and immunofluorescence using HPV16-positive (SiHa, CaSki), HPV18-positive (HeLa), and HPV-negative (C33A) cervical cancer cells. Functional assays evaluated cell viability (LDH release, luminescence), migration (Transwell), EMT markers (Western blot for E-cadherin, N-cadherin, Vimentin, Snail), apoptosis (TUNEL, flow cytometry, caspase activation), and pyroptosis (SYTOX Green uptake, cytokine release ELISA, GSDME cleavage). Caspase-3 knockdown and in vitro cleavage assays determined pyroptosis mechanisms.ResultsZHPV16E7-GrB exhibited strong binding specificity for HPV16E7. It significantly inhibited cell growth and suppressed EMT in HPV16-positive cells, evidenced by reduced migration and invasion, downregulation of Vimentin and Snail, increased E-cadherin, and decreased N-cadherin expression. Furthermore, ZHPV16E7-GrB induced apoptosis via caspase-3/caspase-8 activation and triggered pyroptosis through direct cleavage of gasdermin E (GSDME), independent of caspase-3, accompanied by membrane rupture and proinflammatory cytokine release. Crucially, ZHPV16E7-GrB demonstrated selective toxicity, effectively killing HPV16-positive cells while sparing non-HPV16-positive cells, minimizing off-target effects.DiscussionThis study highlights the dual mechanism of ZHPV16E7-GrB, inhibiting EMT and inducing cell death (apoptosis and pyroptosis). These findings demonstrate its significant promise as a targeted therapeutic agent for HPV16-associated cervical cancer, addressing critical unmet needs in current treatment strategies. |
| format | Article |
| id | doaj-art-eea29502f33448bca3b5acd8e4fbe237 |
| institution | Kabale University |
| issn | 1664-3224 |
| language | English |
| publishDate | 2025-07-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Immunology |
| spelling | doaj-art-eea29502f33448bca3b5acd8e4fbe2372025-08-20T03:28:20ZengFrontiers Media S.A.Frontiers in Immunology1664-32242025-07-011610.3389/fimmu.2025.16167151616715Development of ZHPV16E7- granzyme B immunoaffitoxin: dual mechanisms targeting hpv16-positive cervical cancer through epithelial-mesenchymal transition inhibition and cell deathXiaochun Tan0Xiaochun Tan1Jiani Yang2Jiani Yang3Yanheng Li4Kairong Wan5Sicong Feng6Xishang Jing7Zhenyun Xie8Lifang Zhang9Wenshu Li10Institute of Molecular Virology and Immunology, Department of Microbiology & Immunology, School of Basic Medical Sciences, Wenzhou Medical University, Wenzhou, ChinaDepartment of Laboratory Medicine, The First Hospital of Jiaxing, Affiliated Hospital of Jiaxing University, Jiaxing, ChinaInstitute of Molecular Virology and Immunology, Department of Microbiology & Immunology, School of Basic Medical Sciences, Wenzhou Medical University, Wenzhou, ChinaDepartment of Laboratory Medicine, Nanjing University of Chinese Medicine, Yancheng TCM Hospital, Yancheng, ChinaInstitute of Molecular Virology and Immunology, Department of Microbiology & Immunology, School of Basic Medical Sciences, Wenzhou Medical University, Wenzhou, ChinaInstitute of Molecular Virology and Immunology, Department of Microbiology & Immunology, School of Basic Medical Sciences, Wenzhou Medical University, Wenzhou, ChinaInstitute of Molecular Virology and Immunology, Department of Microbiology & Immunology, School of Basic Medical Sciences, Wenzhou Medical University, Wenzhou, ChinaInstitute of Molecular Virology and Immunology, Department of Microbiology & Immunology, School of Basic Medical Sciences, Wenzhou Medical University, Wenzhou, ChinaCommunication Department, Technical University of Valencia, Valencia, SpainInstitute of Molecular Virology and Immunology, Department of Microbiology & Immunology, School of Basic Medical Sciences, Wenzhou Medical University, Wenzhou, ChinaInstitute of Molecular Virology and Immunology, Department of Microbiology & Immunology, School of Basic Medical Sciences, Wenzhou Medical University, Wenzhou, ChinaIntroductionCervical cancer, predominantly caused by high-risk human papillomavirus (HPV), remains a major global health challenge. HPV16 is the most prevalent type, and its oncoprotein E7 promotes epithelial-mesenchymal transition (EMT), a critical step in metastasis. Current therapies for HPV16-related cancers are often insufficient, highlighting the need for targeted treatments. We engineered a novel immunotoxin, ZHPV16E7-GrB, by fusing an HPV16E7-specific affibody (ZHPV16E7) with the cytotoxic immune effector granzyme B (GrB). This construct was designed for precise targeting and therapeutic activity against HPV16-positive cervical cancer cells.MethodsZHPV16E7-GrB was engineered, expressed in E. coli, and purified. Binding specificity was assessed via ELISA and immunofluorescence using HPV16-positive (SiHa, CaSki), HPV18-positive (HeLa), and HPV-negative (C33A) cervical cancer cells. Functional assays evaluated cell viability (LDH release, luminescence), migration (Transwell), EMT markers (Western blot for E-cadherin, N-cadherin, Vimentin, Snail), apoptosis (TUNEL, flow cytometry, caspase activation), and pyroptosis (SYTOX Green uptake, cytokine release ELISA, GSDME cleavage). Caspase-3 knockdown and in vitro cleavage assays determined pyroptosis mechanisms.ResultsZHPV16E7-GrB exhibited strong binding specificity for HPV16E7. It significantly inhibited cell growth and suppressed EMT in HPV16-positive cells, evidenced by reduced migration and invasion, downregulation of Vimentin and Snail, increased E-cadherin, and decreased N-cadherin expression. Furthermore, ZHPV16E7-GrB induced apoptosis via caspase-3/caspase-8 activation and triggered pyroptosis through direct cleavage of gasdermin E (GSDME), independent of caspase-3, accompanied by membrane rupture and proinflammatory cytokine release. Crucially, ZHPV16E7-GrB demonstrated selective toxicity, effectively killing HPV16-positive cells while sparing non-HPV16-positive cells, minimizing off-target effects.DiscussionThis study highlights the dual mechanism of ZHPV16E7-GrB, inhibiting EMT and inducing cell death (apoptosis and pyroptosis). These findings demonstrate its significant promise as a targeted therapeutic agent for HPV16-associated cervical cancer, addressing critical unmet needs in current treatment strategies.https://www.frontiersin.org/articles/10.3389/fimmu.2025.1616715/fullHPV16E7affibodyGrBcervical cancerEMTcell death |
| spellingShingle | Xiaochun Tan Xiaochun Tan Jiani Yang Jiani Yang Yanheng Li Kairong Wan Sicong Feng Xishang Jing Zhenyun Xie Lifang Zhang Wenshu Li Development of ZHPV16E7- granzyme B immunoaffitoxin: dual mechanisms targeting hpv16-positive cervical cancer through epithelial-mesenchymal transition inhibition and cell death Frontiers in Immunology HPV16E7 affibody GrB cervical cancer EMT cell death |
| title | Development of ZHPV16E7- granzyme B immunoaffitoxin: dual mechanisms targeting hpv16-positive cervical cancer through epithelial-mesenchymal transition inhibition and cell death |
| title_full | Development of ZHPV16E7- granzyme B immunoaffitoxin: dual mechanisms targeting hpv16-positive cervical cancer through epithelial-mesenchymal transition inhibition and cell death |
| title_fullStr | Development of ZHPV16E7- granzyme B immunoaffitoxin: dual mechanisms targeting hpv16-positive cervical cancer through epithelial-mesenchymal transition inhibition and cell death |
| title_full_unstemmed | Development of ZHPV16E7- granzyme B immunoaffitoxin: dual mechanisms targeting hpv16-positive cervical cancer through epithelial-mesenchymal transition inhibition and cell death |
| title_short | Development of ZHPV16E7- granzyme B immunoaffitoxin: dual mechanisms targeting hpv16-positive cervical cancer through epithelial-mesenchymal transition inhibition and cell death |
| title_sort | development of zhpv16e7 granzyme b immunoaffitoxin dual mechanisms targeting hpv16 positive cervical cancer through epithelial mesenchymal transition inhibition and cell death |
| topic | HPV16E7 affibody GrB cervical cancer EMT cell death |
| url | https://www.frontiersin.org/articles/10.3389/fimmu.2025.1616715/full |
| work_keys_str_mv | AT xiaochuntan developmentofzhpv16e7granzymebimmunoaffitoxindualmechanismstargetinghpv16positivecervicalcancerthroughepithelialmesenchymaltransitioninhibitionandcelldeath AT xiaochuntan developmentofzhpv16e7granzymebimmunoaffitoxindualmechanismstargetinghpv16positivecervicalcancerthroughepithelialmesenchymaltransitioninhibitionandcelldeath AT jianiyang developmentofzhpv16e7granzymebimmunoaffitoxindualmechanismstargetinghpv16positivecervicalcancerthroughepithelialmesenchymaltransitioninhibitionandcelldeath AT jianiyang developmentofzhpv16e7granzymebimmunoaffitoxindualmechanismstargetinghpv16positivecervicalcancerthroughepithelialmesenchymaltransitioninhibitionandcelldeath AT yanhengli developmentofzhpv16e7granzymebimmunoaffitoxindualmechanismstargetinghpv16positivecervicalcancerthroughepithelialmesenchymaltransitioninhibitionandcelldeath AT kairongwan developmentofzhpv16e7granzymebimmunoaffitoxindualmechanismstargetinghpv16positivecervicalcancerthroughepithelialmesenchymaltransitioninhibitionandcelldeath AT sicongfeng developmentofzhpv16e7granzymebimmunoaffitoxindualmechanismstargetinghpv16positivecervicalcancerthroughepithelialmesenchymaltransitioninhibitionandcelldeath AT xishangjing developmentofzhpv16e7granzymebimmunoaffitoxindualmechanismstargetinghpv16positivecervicalcancerthroughepithelialmesenchymaltransitioninhibitionandcelldeath AT zhenyunxie developmentofzhpv16e7granzymebimmunoaffitoxindualmechanismstargetinghpv16positivecervicalcancerthroughepithelialmesenchymaltransitioninhibitionandcelldeath AT lifangzhang developmentofzhpv16e7granzymebimmunoaffitoxindualmechanismstargetinghpv16positivecervicalcancerthroughepithelialmesenchymaltransitioninhibitionandcelldeath AT wenshuli developmentofzhpv16e7granzymebimmunoaffitoxindualmechanismstargetinghpv16positivecervicalcancerthroughepithelialmesenchymaltransitioninhibitionandcelldeath |