Intrabodies targeting human papillomavirus 16 E6 and E7 oncoproteins for therapy of established HPV-associated tumors

Abstract Background The oncogenic activity of the high risk human papillomavirus type 16 (HPV16) is fully dependent on the E6 and E7 viral oncoproteins produced during viral infection. The oncoproteins interfere with cellular homeostasis by promoting proliferation, inhibiting apoptosis and blocking epithelial differentiation, driving the infected cells towards neoplastic progression. The causal relationship between expression of E6/E7 and cellular transformation allows inhibiting the oncogenic process by hindering the activity of the two oncoproteins. We previously developed and characterized some antibodies in single-chain format (scFvs) against the HPV16 E6 and E7 proteins, and demonstrated both in vitro and in vivo their antitumor activity consisting of protective efficacy against tumor progression of HPV16-positive cells. Methods Envisioning clinical application of the best characterized anti-HPV16 E6 and –HPV16 E7 scFvs, we verified their activity in the therapeutic setting, on already implanted tumors. Recombinant plasmids expressing the anti-HPV16 E6 scFvI7 with nuclear targeting sequence, or the anti-HPV16 E7 scFv43M2 with endoplasmic reticulum targeting sequence were delivered by injection followed by electroporation to three different preclinical models using C57/BL6 mice, and their effect on tumor growth was investigated. In the first model, the HPV16+ TC-1 Luc cells were used to implant tumors in mice, and tumor growth was measured by luciferase activity; in the second model, a fourfold number of TC-1 cells was used to obtain more aggressively growing tumors; in the third model, the HPV16+ C3 cells where used to rise tumors in mice. To highlight the scFv possible mechanism of action, H&E and caspase-3 staining of tumor section were performed. Results We showed that both the anti-HPV16 E6 and HPV16 E7 scFvs tested were efficacious in delaying tumor progression in the three experimental models and that their antitumor activity seems to rely on driving tumor cells towards the apoptotic pathway. Conclusion Based on our study, two scFvs have been identified that could represent a safe and effective treatment for the therapy of HPV16-associated lesions. The mechanism underlying the scFv effectiveness appears to be leading cells towards death by apoptosis. Furthermore, the validity of electroporation, a methodology allowed for human treatment, to deliver scFvs to tumors was confirmed.

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MicroRNA 203 Expression in Keratinocytes Is Dependent on Regulation of p53 Levels by E6

Journal of Virology ◽

10.1128/jvi.00703-10 ◽

2010 ◽

Vol 84 (20) ◽

pp. 10644-10652 ◽

Cited By ~ 53

Author(s):

Declan J. McKenna ◽

Simon S. McDade ◽

Daksha Patel ◽

Dennis J. McCance

Keyword(s):

Dna Damage ◽

Human Papillomavirus ◽

Cell Biology ◽

Viral Oncoprotein ◽

Hpv16 E6 ◽

Human Papillomavirus Type 16 ◽

Proliferation And Differentiation ◽

Short Hairpin ◽

E6 And E7 ◽

Short Hairpin Rnas

ABSTRACT A screen of microRNA (miRNA) expression following differentiation in human foreskin keratinocytes (HFKs) identified changes in several miRNAs, including miRNA 203 (miR-203), which has previously been shown to play an important role in epithelial cell biology by regulating p63 levels. We investigated how expression of human papillomavirus type 16 (HPV16) oncoproteins E6 and E7 affected miR-203 expression during proliferation and differentiation of HFKs. We demonstrated that miR-203 expression is reduced in HFKs where p53 function is compromised, either by the viral oncoprotein E6 or by knockout of p53 using short hairpin RNAs (p53i). We show that the induction of miR-203 observed during calcium-induced differentiation of HFKs is significantly reduced in HFKs expressing E6 and in p53i HFKs. Induction of miR-203 in response to DNA damage is also reduced in the absence of p53. We report that proliferation of HFKs is dependent on the level of miR-203 expression and that overexpression of miR-203 can reduce overproliferation in E6/E7-expressing and p53i HFKs. In summary, these results indicate that expression of miR-203 is dependent on p53, which may explain how expression of HPV16 E6 can disrupt the balance between proliferation and differentiation, as well as the response to DNA damage, in keratinocytes.

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Intra-tumor injection of lentiviral-vector delivered shRNA targeting human papillomavirus E6 and E7 oncogenes reduces tumor growth in a xenograft cervical cancer model in mice

Journal of Solid Tumors ◽

10.5430/jst.v2n4p4 ◽

2012 ◽

Vol 2 (4) ◽

Cited By ~ 4

Author(s):

Jiezhong Chen ◽

Nigel McMillan ◽

Wenyi Gu

Keyword(s):

Cervical Cancer ◽

Human Papillomavirus ◽

Tumor Growth ◽

Lentiviral Vector ◽

Cancer Model ◽

E6 And E7

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Cooperative role of c-MYC and E6/E7 from two molecular variants of human papillomavirus type 16 upon proliferation and in vitro transformation of primary human keratinocytes

Revista de Medicina ◽

10.11606/issn.1679-9836.v98i1p53-59 ◽

2019 ◽

Vol 98 (1) ◽

pp. 52-58 ◽

Cited By ~ 1

Author(s):

Jimena Hochmann ◽

Silvaneide Ferreira ◽

João Sobrinho ◽

Laura Sichero

Keyword(s):

Human Papillomavirus ◽

Cellular Transformation ◽

Soft Agar ◽

Nuclear Antigen ◽

Hpv 16 ◽

Human Papillomavirus Type 16 ◽

In Vitro Transformation ◽

Molecular Variants ◽

E6 And E7

The roles of E6 and E7 oncoproteins of Human Papillomavirus type 16 (HPV-16) in the progression of immortalized epithelial cells to invasive tumors are not fully understood. Here, we establish a novel link between E6 and E7 of two molecular variants of HPV-16 (AA and E-350G), and c-MYC, regarding the cooperation in promoting malignant transformation of primary human foreskin keratinocytes (PHK). We aimed to study the synergistic effects of E6/E7 and c-MYC upon proliferation, and the in vitro transformation potential of PHK. We evaluated cellular proliferation through the expression of the Proliferating Cell Nuclear Antigen (PCNA) protein and colony formation abilities using soft agar and low attachment plates. We observed that E-350G-c-MYC PHKs exhibited discrete higher PCNA levels and formed significantly more colonies in both soft-agar and when growth in low-adhesion culture plates. Overall, we concluded that the E-350G variant co-transfected with c-MYC might promote malignant cellular transformation with a better efficiency than the AA-c-MYC counterpart. The enhanced oncogenic properties exhibited by the E-350G-c-MYC variant offer insights into mechanisms that may operate in human cervical neoplasia, given the higher frequency of its occurrence in the progression of high-grade precursor lesions to invasive carcinomas.

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E6^E7, a Novel Splice Isoform Protein of Human Papillomavirus 16, Stabilizes Viral E6 and E7 Oncoproteins via HSP90 and GRP78

mBio ◽

10.1128/mbio.02068-14 ◽

2015 ◽

Vol 6 (1) ◽

Cited By ~ 38

Author(s):

Masahiko Ajiro ◽

Zhi-Ming Zheng

Keyword(s):

Cervical Cancer ◽

Human Papillomavirus ◽

Critical Role ◽

Splice Isoforms ◽

Hpv16 E6 ◽

Human Papillomavirus 16 ◽

Hpv18 E6 ◽

Splice Isoform ◽

E6 And E7

ABSTRACTTranscripts of human papillomavirus 16 (HPV16) E6 and E7 oncogenes undergo alternative RNA splicing to produce multiple splice isoforms. However, the importance of these splice isoforms is poorly understood. Here we report a critical role of E6^E7, a novel isoform containing the 41 N-terminal amino acid (aa) residues of E6 and the 38 C-terminal aa residues of E7, in the regulation of E6 and E7 stability. Through mass spectrometric analysis, we identified that HSP90 and GRP78, which are frequently upregulated in cervical cancer tissues, are two E6^E7-interacting proteins responsible for the stability and function of E6^E7, E6, and E7. Although GRP78 and HSP90 do not bind each other, GRP78, but not HSP90, interacts with E6 and E7. E6^E7 protein, in addition to self-binding, interacts with E6 and E7 in the presence of GRP78 and HSP90, leading to the stabilization of E6 and E7 by prolonging the half-life of each protein. Knocking down E6^E7 expression in HPV16-positive CaSki cells by a splice junction-specific small interfering RNA (siRNA) destabilizes E6 and E7 and prevents cell growth. The same is true for the cells with a GRP78 knockdown or in the presence of an HSP90 inhibitor. Moreover, mapping and alignment analyses for splicing elements in 36 alpha-HPVs (α-HPVs) suggest the possible expression of E6^E7 mostly by other oncogenic or possibly oncogenic α-HPVs (HPV18, -30, -31, -39, -42, -45, -56, -59, -70, and -73). HPV18 E6^E7 is detectable in HPV18-positive HeLa cells and HPV18-infected raft tissues. All together, our data indicate that viral E6^E7 and cellular GRP78 or HSP90 might be novel targets for cervical cancer therapy.IMPORTANCEHPV16 is the most prevalent HPV genotype, being responsible for 60% of invasive cervical cancer cases worldwide. What makes HPV16 so potent in the development of cervical cancer remains a mystery. We discovered in this study that, besides producing two well-known oncoproteins, E6 and E7, seen in other high-risk HPVs, HPV16 produces E6^E7, a novel splice isoform of E6 and E7. E6^E7, in addition to self-interacting, binds cellular chaperone proteins, HSP90 and GRP78, and viral E6 and E7 to increase the steady-state levels and half-lives of viral oncoproteins, leading to cell proliferation. The splicingciselements in the regulation of HPV16 E6^E7 production are highly conserved in 11 oncogenic or possibly oncogenic HPVs, and we confirmed the production of HPV18 E6^E7 in HPV18-infected cells. This study provides new insight into the mechanism of splicing, the interplay between different products of the polycistronic viral message, and the role of the host chaperones as they function.

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Identification of High-Risk Human Papillomavirus DNA, p16 and E6/E7 Oncoproteins in Laryngeal and Hypopharyngeal Squamous Cell Carcinomas

Viruses ◽

10.3390/v13061008 ◽

2021 ◽

Vol 13 (6) ◽

pp. 1008

Author(s):

Andrejs Lifsics ◽

Valerija Groma ◽

Maksims Cistjakovs ◽

Sandra Skuja ◽

Renars Deksnis ◽

...

Keyword(s):

Human Papillomavirus ◽

High Risk ◽

Squamous Cell ◽

Surrogate Marker ◽

Hpv Infection ◽

Hpv16 E6 ◽

Hpv Dna ◽

Molecular Virology ◽

Hpv E6 ◽

E6 And E7

Human papillomavirus (HPV) was proven to play a significant role in cancer development in the oropharynx. However, its role in the development of laryngeal (LSCC) and hypopharyngeal squamous cell carcinoma (HPSCC) remains to be clarified. High-risk HPV (HR-HPV) viral proteins E6 and E7 are considered to be pertinent to HPV-related carcinogenesis. Hence, our aim was to estimate LSCC and HPSCC for HR-HPV DNA, p16, and E6/E7 oncoprotein status by using molecular virology and immunohistochemistry methods. The prevalence of HPV16 infection was 22/41 (53.7%) and 20/31 (64.5%) for LSCC and HPSCC, accordingly. The majority of HPV16+ tumor samples were stage III or IV. In most samples, the presence of either HPV16 E6 or HPV16 E7 viral protein in dysplastic or tumor cells was confirmed using immunohistochemistry. Our results suggest a high prevalence of HPV16 as a primary HR-HPV type in LSCC and HPSCC. The lack of HPV E6/E7 oncoproteins in some tumor samples may suggest either the absence of viral integration or the presence of other mechanisms of tumorigenesis. The utilization of p16 IHC as a surrogate marker of HR-HPV infection is impractical in LSCC and HPSCC.

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Immunodiagnosis and Immunotherapeutics Based on Human Papillomavirus for HPV-Induced Cancers

Frontiers in Immunology ◽

10.3389/fimmu.2020.586796 ◽

2021 ◽

Vol 11 ◽

Author(s):

Zhen Dong ◽

Renjian Hu ◽

Yan Du ◽

Li Tan ◽

Lin Li ◽

...

Keyword(s):

Human Papillomavirus ◽

Early Diagnosis ◽

Hpv Infection ◽

Research Progress ◽

Immunological Methods ◽

Malignant Neoplasms ◽

Single Chain ◽

Induced Tumors ◽

Antibody Mimics ◽

E6 And E7

Infection with human papillomavirus (HPV) is one of the main causes of malignant neoplasms, especially cervical, anogenital, and oropharyngeal cancers. Although we have developed preventive vaccines that can protect from HPV infection, there are still many new cases of HPV-related cancers worldwide. Early diagnosis and therapy are therefore important for the treatment of these diseases. As HPVs are the major contributors to these cancers, it is reasonable to develop reagents, kits, or devices to detect and eliminate HPVs for early diagnosis and therapeutics. Immunological methods are precise strategies that are promising for the accurate detection and blockade of HPVs. During the last decades, the mechanism of how HPVs induce neoplasms has been extensively elucidated, and several oncogenic HPV early proteins, including E5, E6, and E7, have been shown to be positively related to the oncogenesis and malignancy of HPV-induced cancers. These oncoproteins are promising biomarkers for diagnosis and as targets for the therapeutics of HPV-related cancers. Importantly, many specific monoclonal antibodies (mAbs), or newly designed antibody mimics, as well as new immunological kits, devices, and reagents have been developed for both the immunodiagnosis and immunotherapeutics of HPV-induced cancers. In the current review, we summarize the research progress in the immunodiagnosis and immunotherapeutics based on HPV for HPV-induced cancers. In particular, we depict the most promising serological methods for the detection of HPV infection and several therapeutical immunotherapeutics based on HPV, using immunological tools, including native mAbs, radio-labelled mAbs, affitoxins (affibody-linked toxins), intracellular single-chain antibodies (scFvs), nanobodies, therapeutical vaccines, and T-cell-based therapies. Our review aims to provide new clues for researchers to develop novel strategies and methods for the diagnosis and treatment of HPV-induced tumors.

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HPV16-E6 but not E7 inhibits the antitumor activity of LKB1 in lung cancer cells by down regulating the expression of KIF7

10.21203/rs.3.rs-19692/v2 ◽

2020 ◽

Author(s):

Yue Hu ◽

Ming-Zhe Wu ◽

Na-Jin Gu ◽

Xue-Shan Qiu ◽

En-Hua Wang ◽

...

Keyword(s):

Lung Cancer ◽

Antitumor Activity ◽

Cancer Cells ◽

Genetic Manipulation ◽

Lung Cancer Cells ◽

Hpv16 E6 ◽

E6 And E7 ◽

E7 Proteins ◽

First Time ◽

New Evidence

Abstract Background: The E6 and E7 proteins in HPV16 are the main oncogenes in the occurrence of lung cancer. In recent studies, we had found that E6 and E7 downregulated the expression of LKB1 in lung cancer cells. However, it is not clear how E6 and E7 regulate LKB1 in lung cancer cells. Methods: The double directional genetic manipulation and Nuclear plasma separation technology were performed to explore the molecular mechanism of E6 and E7 inhibiting the antitumor activity of LKB1 in well-established lung cancer cell lines. Results: E6 but not E7 significantly downregulated the expression of tumor suppressor KIF7 at protein level, and the inhibition of KIF7 further reduced the expression of LKB1 both in the nuclei and in the cytoplasm, whereas reduced the expression of p-LKB1 in the cytoplasm only. Therefore, we suggested that HPV16 E6 but not E7 downregulates the antitumor activity of LKB1 by down regulating the expression of p-LKB1 in the cytoplasm only. Conclusion: We demonstrated for the first time that E6 but not E7 inhibits the antitumor activity of LKB1 in lung cancer cells by downregulating the expression of KIF7. Our findings provided new evidence to support the important role of KIF7 in the pathogenesis of lung cancer and suggested new therapeutic targets.

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HPV16-E6 but not E7 inhibits the antitumor activity of LKB1 in lung cancer cells by down regulating the expression of KIF7

10.21203/rs.3.rs-19692/v1 ◽

2020 ◽

Author(s):

Yue Hu ◽

Ming-Zhe Wu ◽

Na-Jin Gu ◽

Xue-Shan Qiu ◽

En-Hua Wang ◽

...

Keyword(s):

Lung Cancer ◽

Antitumor Activity ◽

Cancer Cells ◽

Genetic Manipulation ◽

Lung Cancer Cells ◽

Hpv16 E6 ◽

E6 And E7 ◽

E7 Proteins ◽

First Time ◽

New Evidence

Abstract Background: The E6 and E7 proteins in HPV16 are the main oncogenes in the occurrence of lung cancer. In recent studies, we had found that E6 and E7 downregulated the expression of LKB1 in lung cancer cells. However, it is not clear how E6 and E7 regulate LKB1 in lung cancer cells. Methods: The double directional genetic manipulation and Nuclear plasma separation technology were performed to explore the molecular mechanism of E6 and E7 inhibiting the antitumor activity of LKB1 in well-established lung cancer cell lines. Results: E6 but not E7 significantly downregulated the expression of tumor suppressor KIF7 at protein level, and the inhibition of KIF7 further reduced the expression of LKB1 both in the nuclei and in the cytoplasm, whereas reduced the expression of p-LKB1 in the cytoplasm only. Therefore, we suggested that HPV16 E6 but not E7 downregulates the antitumor activity of LKB1 by down regulating the expression of p-LKB1 in the cytoplasm only. Conclusion: We demonstrated for the first time that E6 but not E7 inhibits the antitumor activity of LKB1 in lung cancer cells by down regulating the expression of KIF7. Our findings provided new evidence to support the important role of KIF7 in the pathogenesis of lung cancer and suggested new therapeutic targets.

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Epitope Mapping and Computational Analysis of Anti-HPV16 E6 and E7 Antibodies in Single-Chain Format for Clinical Development as Antitumor Drugs

Cancers ◽

10.3390/cancers12071803 ◽

2020 ◽

Vol 12 (7) ◽

pp. 1803 ◽

Cited By ~ 1

Author(s):

Carla Amici ◽

Maria Gabriella Donà ◽

Barbara Chirullo ◽

Paola Di Bonito ◽

Luisa Accardi

Keyword(s):

Epitope Mapping ◽

Binding Capacity ◽

Clinical Stage ◽

Public Health Problem ◽

Molecular Engineering ◽

Antigen Binding ◽

Clinical Use ◽

Single Chain ◽

Hpv16 E6 ◽

E6 And E7

Human Papillomavirus 16-associated cancer, affecting primarily the uterine cervix but, increasingly, other body districts, including the head–neck area, will long be a public health problem, despite there being a vaccine. Since the virus oncogenic activity is fully ascribed to the viral E6 and E7 oncoproteins, one of the therapeutic approaches for HPV16 cancer is based on specific antibodies in single-chain format targeting the E6/E7 activity. We analyzed the Complementarity Determining Regions, repositories of antigen-binding activity, of four anti-HPV16 E6 and -HPV16 E7 scFvs, to highlight possible conformity to biophysical properties, recognized to be advantageous for therapeutic use. By epitope mapping, using E7 mutants with amino acid deletions or variations, we investigated differences among the anti-16E7 scFvs in terms of antigen-binding capacity. We also performed computational analyses to determine whether length, total net charge, surface hydrophobicity, polarity and charge distribution conformed well to those of the antibodies that had already reached clinical use, through the application of developability guidelines derived from recent literature on clinical-stage antibodies, and the Therapeutic Antibodies Profiler software. Overall, our findings show that the scFvs investigated may represent valid candidates to be developed as therapeutic molecules for clinical use, and highlight characteristics that could be improved by molecular engineering.

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