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.

Targeting Human Papillomavirus-Associated Cancer by Oncoprotein-Specific Recombinant Antibodies

“…The result was confirmed using two different HPV16-positive tumour cells, namely C3 and TC-1 cells, and also by employing higher amounts of TC-1 cells to mimic tumours that are more aggressive. Through histology and immunohistochemistry, we showed that the antitumour activity is based on the induction of tumour cell death by apoptosis [72].…”

“…We then investigated the I7nuc antitumour activity in mouse models for HPV tumours based on the injection of HPV16-positive tumour cells in C57BL6 mice. The scFv capability to either prevent cancer development from scFv-expressing tumour cells, or to hinder cancer progression by delivery to already established tumours, was evaluated [71,72] (Figure 4). We observed a clear impairment of tumour growth in all mice injected with TC-1 tumour cells expressing I7nuc by retroviral transduction before inoculation into mice, with 60% of them completely protected from tumour onset for the 4 months of observation time [71].…”

“…In addition, the scFv43M2SD intracellular expression was able to inhibit significantly and specifically the proliferation of different HPV16-positive cell lines [85]. The scFv43M2 was then tested in vivo in mouse HPV tumour models, demonstrating the ability to counteract tumour progression both when administered to tumour cells before their injection into mice and when administered to already implanted tumours [72,85] (Figure 4).…”

“…As such, it can be exploited in a wide range of applications, particularly in immunotherapy [96]. One of the studies reported here used EP to achieve efficient expression of therapeutic scFvs injected as DNA plasmids in HPV-driven tumours [72]. Nevertheless, the methodology could even be used to deliver scFvs as proteins or mRNAs.…”

Therapeutic Potential of Intrabodies for Cancer Immunotherapy: Current Status and Future Directions

“…A scFv intrabody targeting VEGFR2/KDR was selected from an immune phage display library of mice [ 63 ]. Recently, intrabodies targeting human papillomavirus 16 E6 and E7 oncoproteins for the treatment of established HPV-associated tumors were generated from a synthetic human antibody phage display library [ 64 ]. One scFv against 16 E7 was targeted to the ER and interferes with the binding of E7 to retinoblastoma tumor suppressor (pRb).…”