Abstract 588: Preclinical evaluation of novel chimeric vesiculovirus mediated expression of proteolytic enzymes targeting two major stromal components of pancreatic ductal adenocarcinoma

Abstract The vesiculovirus genus has recently garnered interest as an oncolytic virotherapy for pancreatic ductal adenocarcinoma (PDAC). A new synthetic chimeric vesiculovirus, VMG, demonstrated efficacy and safety against PDAC both in vitro and in vivo. The therapeutic effectiveness of virotherapies against PDAC is often hindered by immunosuppressive factors in the tumor environment, as well as the presence of hyaluronic acid (HA) and collagen (CN), which increase tumor stiffness and interstitial fluid pressure, leading to reduced drug/oncolytic virus and immune cell penetration into tumor. To address this, we engineered VMG viruses incorporating enzymes PH20, collagenase (CNase), and novel proteolytic enzyme SMC, the latter exhibits both PH20 and CNase-like activity. We characterized these modified viruses for their cytotoxicity and performance in vitro. Various PDAC cell lines displayed varied sensitivity to VMG vectors, yet they showed consistent oncolytic effects relative to the parent VMG-GFP virus. Our results revealed no compromise in viral replication or apoptosis-inducing ability after transgene insertion, which is evident as infectious viral progenies were produced to approximately similar level of magnitude of around 108 TCID50/ml in all the cell line tested. Additionally, the VMG-SMC and VMG-hPH20-CNase variants demonstrated notable enzyme activity. To enhance clinical relevance, we utilized an ex vivo live tumor platform, which indicated around 20% tumor cell death using VMG vectors compared to that of the control, signifying tumor viability and virus replicability 72 hours post-infection. Fluorescent imagery highlighted the viruses' ability to spread and express functional enzymes within tumors. In summary, VMG vectors expressing stroma-degrading proteolytic enzymes can effectively express PH20, CNase and SMC enzymes that degrade stromal barrier of HA and CN without interrupting the replicability, performance, and oncolytic characteristics of the VMG vectors. Future research should prioritize the safety of these vectors and their potential to augment neoantigen expression and immune cell infiltration in vivo, which are the hallmarks of antitumor efficacy. Citation Format: Khandoker Usran Ferdous, Mulu Z. Tesfay, Camila C. Simoes, Aleksandra Cios, Bolni Marius Nagalo. Preclinical evaluation of novel chimeric vesiculovirus mediated expression of proteolytic enzymes targeting two major stromal components of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 588.