Previously, we described VSV-GP, a modified version of the vesicular stomatitis virus, as a non-neurotoxic oncolytic virus that is effective for the treatment of malignant glioblastoma and ovarian cancer. Here, we evaluate the therapeutic efficacy of VSV-GP for malignant melanoma. All of the human, mouse, and canine melanoma cell lines that were tested, alongside most primary human melanoma cultures, were infected by VSV-GP and efficiently killed. Additionally, we found that VSV-GP prolonged the survival of mice in both a xenograft and a syngeneic mouse model. However, only a few mice survived with long-term tumor remission. When we analyzed the factors that might limit VSV-GP’s efficacy, we found that vector-neutralizing antibodies did not play a role in this context, as even after eight subsequent immunizations and an observation time of 42 weeks, no vector-neutralizing antibodies were induced in VSV-GP immunized mice. In contrast, the type I IFN response might have contributed to the reduced efficacy of the therapy, as both of the cell lines that were used for the mouse models were able to mount a protective IFN response. Nevertheless, early treatment with VSV-GP also reduced the number and size of lung metastases in a syngeneic B16 mouse model. In summary, VSV-GP is a potent candidate for the treatment of malignant melanoma; however, factors limiting the efficacy of the virus need to be further explored.

The L-protein of vesicular stomatitis virus (VSV) is a single-chain multi-domain RNA-dependent RNA polymerase. Previously reported attempts of intramolecular insertions of fluorescent proteins into the L-protein resulted in temperature-sensitive and highly attenuated polymerase activity. Here, we describe a novel insertion site that was selected based on in silico prediction. Of five preselected locations, insertion of the fluorescent protein mCherry in the VSV polymerase between amino acids 1620 and 1621 preserved polymerase function even after extended passaging and showed only mild attenuation compared to wildtype VSV polymerase. High magnification fluorescence imaging revealed a corpuscular cytosolic pattern for the L-protein. To confirm that the insertion site tolerates inclusion of proteins others than mCherry, we cloned mWasabi into the same position in L, generating a VSV-LmWasabi, which was also functional. We also generated a functional dual-color-dual-insertion VSV construct with intramolecularly labeled P and L-proteins. Together, our data present an approach to tag VSV polymerase intramolecularly without perturbing enzymatic activity. This L fusion protein might enable future tracing studies to monitor intracellular location of the VSV transcription and replication machinery in real-time life-imaging studies.

Introduction: Oncolytic viruses (OVs), which selectively replicate in and destroy tumor cells, form a highly promising class of cancer therapeutics with a strong immune stimulatory potential. VSV-GP, a chimeric Vesicular Stomatitis Virus (VSV) pseudotyped with the glycoprotein of the lymphocytic choriomeningitis virus (LCMV) can selectively replicate and express therapeutic cargos in the tumor microenvironment. Lymphotoxin alpha (LTα) - a member of the TNF superfamily - may present as a potent therapeutic cargo based on its immune stimulatory and cell death modulatory characteristics.In this study, we explore the immune promoting and tumor cell death inducing properties of VSV-GP-LTα, a next generation variant with an NF-κB facilitated mode of action. We further investigate clinically relevant combination therapies with “second mitochondria-derived activator of caspases” mimetic compounds (SMCs). Experimental Procedures: Viral fitness, cargo expression and test substance quality were assessed using growth kinetic, ELISA, RT-qPCR and titration assays. Therapeutic efficacy and safety of VSV-GP-LTα, with or without SMC co-treatment, were assessed in vivo in syngeneic C57BL/6 mice bearing subcutaneous LLC1-IFNAR-/- or B16F10-OVA tumors. To shed further light into the VSV-GP-LTα treatment effects, we applied immunohistochemistry, flow cytometry, multiplex ELISA and Nanostring® assays. Data Summary: VSV-GP-LTα induced stronger anti-tumor effects and increased overall survival compared to VSV-GP. It was also better tolerated than VSV-GP armed with TNF-α, a therapeutic cargo from the same class. Histological and gene expression analysis confirmed widespread cell killing within the tumor and activation of innate and adaptive immune response markers. However, further characterization of the adaptive immune compartment did not show significant changes induced by the expressed LTα. Building on the observed therapeutic strengths of VSV-GP-LTα, we further explored a treatment combination with SMC. This resulted in a therapeutic synergy with improved treatment-induced tumor cell death, especially in a less permissive tumor setting. Conclusion: Together, here we show that arming VSV-GP with LTα resulted in a strong inflammation of the tumor microenvironment and enhanced levels of tumor cell death induction. This effect could be further enhanced by the co-treatment with SMC. Citation Format: Bart Spiesschaert, Philipp Müller, Katharina Angerer, Krishna Das, Tobias Nolden, Carles Urbiola, Judy Ng, Fabian Heinemann, Birgit Stierstorfer, Patrik Erlmann, Guido Wollmann. Lymphotoxin-alpha-armed oncolytic VSV-GP synergizes with SMAC mimetics to induce enhanced tumor cell death and regression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4193.