Characteristics of Oncolytic Vesicular Stomatitis Virus Displaying Tumor-Targeting Ligands

Abstract

bWe sought proof of principle that tumor-targeting ligands can be displayed on the surface of vesicular stomatitis virus (VSV) by engineering its glycoprotein. Here, we successfully rescued VSVs displaying tumor vasculature-targeting ligands. By using a rational approach, we investigated various feasible insertion sites on the G protein of VSV (VSV-G) for display of tumor vasculature-targeting ligands, cyclic RGD (cRGD) and echistatin. We found seven sites on VSV-G that tolerated insertion of the 9-residue cRGD peptide, two of which could tolerate insertion of the 49-amino acid echistatin domain. All of the ligand-displaying viruses replicated as well as the parental virus. In vitro studies demonstrated that the VSVechistatin viruses specifically bound to targeted integrins. Since the low-density lipoprotein receptor (LDLR) was recently identified as a major receptor for VSV, we investigated the entry of ligand-displaying viruses after masking LDLR. The experiment showed that the modified viruses can enter the cell independently of LDLR, whereas entry of unmodified virus is significantly blocked by a specific monoclonal antibody against LDLR. Both parental and ligand-displaying viruses displayed equal oncolytic efficacies in a syngeneic mouse myeloma model. We further demonstrated that single-chain antibody fragments against tumor-specific antigens can be inserted at the N terminus of the G protein and that corresponding replication-competent VSVs can be rescued efficiently. Overall, we demonstrated that functional tumor-targeting ligands can be displayed on replication-competent VSVs without perturbing viral growth and oncolytic efficacy. This study provides a rational foundation for the future development of fully retargeted oncolytic VSVs. V esicular stomatitis virus (VSV) is an enveloped, negativestrand RNA virus that belongs to the Vesiculovirus genus of the Rhabdoviridae family. VSV has the ability to infect and kill cancer cells while sparing normal cells (1-4). Exploitation of this oncolytic property provides a promising alternative approach for the treatment of cancer. For disseminated cancer, virotherapy should ideally be administered systemically (2, 5-7), but this route of delivery brings its own set of problems. The major concerns for VSV virotherapy are neurotoxicity, antibody neutralization, and sequestration in off-target organs, especially the liver and spleen. Many attempts have been made to address these drawbacks. To reduce the neurotoxicity, the matrix protein of VSV was mutated (8, 9), and microRNA targets (10) or picornaviral internal ribosome entry sites (11) were engineered into the VSV genome. Serum neutralization has been avoided by PEGylating the virus (12, 13) or loading onto antigen-specific T cells (14), which ultimately improved virotherapy outcomes.To circumvent all of the above hurdles in a single step, pseudotyping VSV with other viral envelope glycoproteins was considered a potentially feasible approach. Recent studies demonstrated that VSV neurotoxicity can be circumvented by...

Experimental Evolution Generates Novel Oncolytic Vesicular Stomatitis Viruses with Improved Replication in Virus-Resistant Pancreatic Cancer Cells

“…8A). LDLR and LDLR family members have been shown to serve as receptors for VSV (3)(4)(5)(6). As VSV-G is responsible for VSV attachment to host cells and we observed an improved attachment of SUIT-2-passaged viruses to SUIT-2 cells (Fig.…”

“…VSV is able to infect and replicate in a wide range of cell types (2) due to the use of ubiquitously expressed cell surface molecules. The low-density lipoprotein receptor (LDLR) and other members of the LDLR family have been shown to serve as VSV receptors (3)(4)(5)(6), and additional studies showed that other cell surface molecules, such as phosphatidylserine (7)(8)(9), sialoglycolipids (10), and heparan sulfate (11) could also play a role in VSV attachment to host cells.…”

Recent advances in vesicular stomatitis virus-based oncolytic virotherapy: a 5-year update

“…The 49-amino acid echistatin domain could be inserted at two sites on VSV-G. VSV-echistatin was able to specifically target integrin avb3 in vitro and showed equal oncolytic efficacy in a mouse myeloma model [37]. The same study also showed that larger polypeptide ligands like single-chain antibodies with specificity to tumour-associated receptors EGFR and HER2 could be inserted at the N terminus of the G protein to target cancer cells more accurately [37].…”

Ruxolitinib and Polycation Combination Treatment Overcomes Multiple Mechanisms of Resistance of Pancreatic Cancer Cells to Oncolytic Vesicular Stomatitis Virus

“…Recently, LDLR was proposed to be one of the receptors for VSV (16,35,36). As a high level of variation in LDLR expression between different cell lines of pancreatic origin was shown (37), we hypothesized that HPAF-II cells could have a defect in LDLR expression, which could explain the ineffective VSV attachment.…”

“…In general, our results suggest that HPAF-II cells are highly resistant to VSV because they are not only nonpermissive to VSV replication due to their constitutive antiviral state but also nonsusceptible to VSV due to impaired virus attachment, which is type I IFN independent. As LDLR has been shown to be one of the receptors for VSV (16,35,36), our study examined if HPAF-II cells have a defect in LDLR expression or functionality. Our data show that HPAF-II cells have lower LDLR expression levels.…”