Abstract 6542: CAL1 vaccinia virus as oncolytic agent and potential use of cell-based platform to enhance its therapeutic effects
Authors: Nguyen Duong H. , Herrmann Thomas , Alamillo Ashley , Neuharth Forrest , Gomez Alberto , Minev Ivelina , Härtl Barbara , Schneider Laura , Minev Boris , Draganov Dobrin , Santidrian Antonio F. 
Published: September 2020

Abstract
Background: Oncolytic virotherapy is a promising immuno-oncology approach that has not realized its potential due to rapid elimination by humoral immunity mediated by complement and neutralizing antibodies. We propose to use an adipose-derived mesenchymal stemcell-based platform,where the virus can be protected and amplified and potentiated inside the stem cells in order to minimize the clearance by anti-viral immunity. ACAM2000, the smallpox vaccine currently licensed in the U.S., is a clonal derivative of Dryvax® with reduced virulence and a well-documented safety profile in humans. This vaccinia virus strain can potentially be used as an oncolytic virus for cancer treatment. In this study, we evaluate the ability of ACAM2000 to (1) selectively kill cancer cells, (2) to be genetically modified without affecting its natural tumor selectivity, and to (3) determine if a stem cell-based platform can protect the virus from inactivation and potentiate its anti-tumor effects.
Methods: ACAM2000 was amplified in CV1 cells and named CAL1. CAL1 was tested for its ability to replicate and selectively kill various human prostate cancer cell lines in vitro and in vivo. Additionally, CAL1 was loaded into adipose-derived mesenchymal stem cells to generate a new therapeutic agent called SuperNova1 (SNV1). Both CAL1 and SNV1 were tested for their ability to kill cancer cells in the presence of active complement and neutralizing antibodies in cell culture as well as in mice. Furthermore, CAL1 was used as the backbone to generate derivative CAL2 viruses using CRISPR/Cas9 technology to insert the gene encoding the fluorescent protein TurboFP into the intergenic locus between ORF-157 and ORF-158 of CAL1 without disrupting any existing CAL1 ORFs.
Results: We showed that in vitro CAL1 preferentially infected, amplified in and lysed tumor cells and was also able to cause tumor regression in vivo without signs of toxicity. Furthermore, we demonstrated that the backbone of CAL1 can be used to engineer recombinant viruses, CAL2, that carry therapeutic genes without additionally attenuating the ability of the virus to amplify or kill tumor cells. SNV1 significantly enhanced protection of CAL1 virus from clearance by the immune system, leading to higher therapeutic efficacy. Furthermore, SNV1 provided instantly active viral particles for immediate infection and simultaneous release of therapeutic proteins in the injected tumors.
Conclusions: CAL1 could be used as an oncolytic agent. We show here that a major advantage of using a cell-based platform to deliver and potentiate oncolytic vaccinia virus is the prevention of viral inactivation by the humoral immune system resulting in enhanced oncolytic viral therapy.
Citation Format: Duong H. Nguyen, Thomas Herrmann, Ashley Alamillo, Forrest Neuharth, Alberto Gomez, Ivelina Minev, Barbara Härtl, Laura Schneider, Boris Minev, Dobrin Draganov, Antonio F. Santidrian. CAL1 vaccinia virus as oncolytic agent and potential use of cell-based platform to enhance its therapeutic effects [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6542.