The Intracellular Delivery Of Anti-HPV16 E7 scFvs Through Engineered Extracellular Vesicles Inhibits The Proliferation Of HPV-Infected Cells

Flavia Ferrantelli,1,* Claudia Arenaccio,1,* Francesco Manfredi,1 Eleonora Olivetta,1 Chiara Chiozzini,1 Patrizia Leone,1 Zulema Percario,2 Alessandro Ascione,1 Michela Flego,1 Paola Di Bonito,3 Luisa Accardi,3 Maurizio Federico1 1National Center for Global Health, Istituto Superiore Di Sanità (ISS), Rome, Italy; 2Department of Science, Roma Tre University, Rome, Italy; 3Department of Infectious Diseases, Istituto Superiore Di Sanità (ISS), Rome, Italy*These authors contributed equally to this workCorrespondence: Maurizio FedericoNational Center for Global Health, Istituto Superiore Di Sanità, Viale Regina Elena, 299, Rome 00161, ItalyTel +39-06-4990-3605Fax +39-06-49903210Email maurizio.federico@iss.itPurpose: Single-chain variable fragments (scFvs) are one of the smallest antigen-binding units having the invaluable advantage to be expressed by a unique short open reading frame (ORF). Despite their reduced size, spontaneous cell entry of scFvs remains inefficient, hence precluding the possibility to target intracellular antigens. Here, we describe an original strategy to deliver scFvs inside target cells through engineered extracellular vesicles (EVs). This approach relies on the properties of a Human Immunodeficiency Virus (HIV)-1 Nef mutant protein referred to as Nefmut. It is a previously characterized Nef allele lacking basically all functions of wt Nef, yet strongly accumulating in the EV lumen also when fused at its C-terminus with a foreign protein. To gain the proof-of-principle for the efficacy of the proposed strategy, the tumor-promoting Human Papilloma Virus (HPV)16-E7 protein was considered as a scFv-specific intracellular target. The oncogenic effect of HPV16-E7 relies on its binding to the tumor suppressor pRb protein leading to a dysregulated cell duplication. Interfering with this interaction means impairing the HPV16-E7-induced cell proliferation.Methods: The Nefmut gene was fused in frame at its 3ʹ-terminus with the ORF coding for a previously characterized anti-HPV16-E7 scFv. Interaction between the Nefmut-fused anti-HPV16-E7 scFv and the HPV16-E7 protein was tested by both confocal microscope and co-immunoprecipitation analyses on co-transfected cells. The in cis anti-proliferative effect of the Nefmut/anti-HPV16-E7 scFv was assayed by transfecting HPV16-infected cells. The anti-proliferative effect of EVs engineered with Nefmut/anti-HPV16-E7 scFv on HPV16-E7-expressing cells was evaluated in two ways: i) through challenge with purified EVs by a Real-Time Cell Analysis system and ii) in transwell co-cultures by an MTS-based assay.Results: The Nefmut/anti-HPV16-E7 scFv chimeric product is efficiently uploaded in EVs, binds HPV16-E7, and inhibits the proliferation of HPV16-E7-expressing cells. Most important, challenge with cell-free EVs incorporating the Nefmut/anti-HPV16-E7 scFv led to the inhibition of proliferation of HPV16-E7-expressing cells. The proliferation of these cells was hindered also when they were co-cultured in transwells with cells producing EVs uploading Nefmut/anti-HPV16-E7 scFv.Conclusion: Our data represent the proof-of-concept for the possibility to target intracellular antigens through EV-mediated delivery of scFvs. This finding could be relevant to design novel methods of intracellular therapeutic interventions.Keywords: single-chain variable fragments, Human Papilloma Virus E7 protein, exosomes, human immunodeficiency virus Nef protein, scFv intracellular delivery