Extracellular Vesicles-Mimetic Encapsulation Improves Oncolytic Viro-Immunotherapy in Tumors With Low Coxsackie and Adenovirus Receptor.

Yonghui Zhang,Hailin Zhang,Junhua Wu,Junyi Wu,Jiwu Wei

doi:10.3389/fbioe.2020.574007

Abstract

The oncolytic adenovirus (Adv) exhibited poor infection efficiency in tumor cells with low coxsackie and adenovirus receptor (CAR) on the cell surface, which limits the therapeutic efficacy of the Adv-mediated cancer gene therapy. In addition, the abundant adenovirus neutralizing antibodies also abrogate the viral infection of cancer cells. Therefore, novel strategies are required to overcome these two major hurdles to improve the Adv-mediated cancer virotherapy. We constructed a recombinant adenovirus expressing the extracellular domain of PD1 (Ad5-P). The 293T cells expressing VSV-G protein on the cell surface (293T-VSV-G) were infected with Ad5-P. Then Ad5-P infected 293T-VSV-G cells were harvested and squeezed stepwisely through a serial of polycarbonate membranes. Next, the extracellular vesicles-mimetic (EVM) encapsulated Ad5-P (EVM/VSV-G Ad5-P) were collected by density gradient centrifugation. In cell lines with low CAR expression, EVM/VSV-G Ad5-P showed a significantly improved infection efficiency, oncolytic ability, and soluble PD-1 production. In passively immunized mice with Ad5 neutralizing antibody, EVM/VSV-G Ad5-P successfully escaped from antibodies, and the soluble PD-1expression of Ad5-P was significantly prolonged. Finally, EVM/VSV-G Ad5-P treatment significantly improved the antitumor immune responses and prolonged survival of mice with HCC ascites. The EVM/VSV-G Ad5-P not only bypasses the limitation of low CAR expression in tumor cells to improve the viral entry, but also significantly protects the virus from the neutralization antibodies. The EVM encapsulation technology can be successfully used for loading of non-enveloped viruses to generate the extracellular vesicle-mimetic encapsulated viral particles. Our results provide a novel strategy in OVs manufacture to improve the efficacy of tumor oncolytic virotherapy.

Highlights

In the last decade, remarkable achievements in tumor immunotherapy have been reported
To overcome the limited entry in low-coxsackie and adenovirus receptor (CAR) cells, we sought to encapsulate the Adenovirus serotype 5 (Ad5) viral particles into vesicle mimetics, we propagated extracellular vesicles-mimetic (EVM) Ad5 in 293T cells expressing vesicular stomatitis Indiana virus G protein (VSV-G) (293TVSV-G, Supplementary Figure S1), a ligand of LDL receptor commonly expressed by most tumor cells
The non-replicative adenoviruses expressing GFP protein (Ad5-GFP) were encapsulated in EVM/VSV-G, and the particles were analyzed by transmission electron microscopy (TEM)

Summary

Introduction

Remarkable achievements in tumor immunotherapy have been reported. Infection with Ad5 depends on the level of CAR expression on the cell surface, and previous studies have shown that CAR expression is downregulated during the growth of primary tumor cells, which limits Ad5 entry into tumor cells and its antitumor effect (Philipson et al, 1968; Miller et al, 1998; Li et al, 1999; Nigatu et al, 2013). Adenovirus treatment elicits the production of neutralizing antibodies and triggers antiviral immunity, resulting in virus clearance, which limits the subsequent application of adenoviruses (Sumida et al, 2004). Strategies aiming to resolve these limitations will substantially increase the antitumor effect and applications of oncolytic adenoviruses

Methods

Results

Conclusion