Evaluation of Oncolytic Viral Therapy Combined with or without Artificial Immunoadjuvant Cell Therapy in Immunocompetent Mouse Tumor Models
Authors: Ung Weng-Chit , Futami Muneyoshi , Sato Kota , Tojo Arinobu 
Published: October 2019

Abstract
Oncolytic viruses have a great potential to provide clinical improvement for cancer patients who do not respond to conventional therapies. The mechanisms of action include direct infection, immune-mediated cell death, and vascular collapse. We previously reported that a miRNA-regulated, thymidine kinase-deleted vaccinia virus (MDVV) specifically infects multiple myeloma (MM) cells and showed that the intravenous injection of MDVV significantly prolonged survival of severe combined immunodeficiency mice harboring a subcutaneous human RPMI8226 MM tumor (Blood 2014:124:2082, Mol Ther Oncolytics 2017;6:57). Although it was highly effective, there remained to be confirmed whether the MDVV would be similarly effective in the immunocompetent setting. To address this question, we performed several in vivo infection using immunocompetent C57BL/6 (B6) models.
A vaccinia virus (VV) strain LC16, which was derived from the Lister strain, was widely used in 1970s for the smallpox eradication program with no severe adverse effect. Because its safety is robust, we used the LC16 for the backbone of the MDVV. To improve its safety and tumor specificity, we deleted viral thymidine kinase gene, and introduced miRNA (let-7a)-target sequences into the critical viral B5R gene. Because let-7a is abundant in normal differentiated tissues, and the endogenous let-7a down-regulates viral B5R gene, the MDVV becomes far more tumor specific (Mol Ther Oncolytics 2017;6:57).
To observe whether the MDVV protects immunocompetent mice from MM, we generated a mouse MM model by transplanting 1x10^6 Vk*Myc tumor cells into syngeneic B6 mice. After confirming M-proteins in the serum, 1x10^7 pfu of MDVV was injected intravenously. Contrary to our expectation, we could not observe a clearly infected region, nor a prolongation of survival of these mice; probably due to difference in infectivity between species. Although mouse derived cell lines are generally insensitive to vaccinia virus as compared with human cell lines, a mouse lung carcinoma cell line 3LL showed a relatively high infectivity to VV in our screening in vitro infection experiment (Figure), therefore we used the B6/3LL subcutaneous tumor model instead of B6/Vk*Myc model.
The B6/3LL mice were injected with mock or 1x10^7 pfu MDVV intravenously when the subcutaneous tumor became more than 5 mm in diameter. Although viral signal (determined by in vivo imaging using luciferase) disappeared 3 days after infection, the therapeutic benefit had remained, and prolongation of survival was observed Because natural killer (NK) cells were thought to play in the clearance of VV in the B6 mice, we attempted to extend the duration of infection by depleting NK cells using anti-asialo GM1 antibody, which, we thought, enhances viral oncolytic activity. As expected, we observed a persistent viral signal for up to 2 weeks in anti-asialo GM1 antibody treated B6/3LL mice. However, the anti-tumor effect seen with anti-asialo GM1 antibody was rather milder than that of without anti-asialo GM1 antibody, indicating the importance of NK activities in anti-tumor immunity.
Based on this finding, we, in reverse, tried to stimulate the anti-tumor immunity using artificial immunoadjuvant cells co-expressing CD1d and tumor antigen/GM-CSF fusion protein. The CD1d is one of non-classical MHC molecules and is involved in the presentation of lipid antigens. When α-GlaCer is loaded on the CD1d, it activates natural killer T (NKT) cells through the interaction with invariant T cell receptor on NKT cells. When activated, NKT cells produce Th1 and Th2 cytokines, and activate NK cells. GM-CSF stimulates antigen-presenting cells (APCs), and fusion proteins of tumor antigen and GM-CSF are more efficiently taken up by APCs than tumor antigen alone (Nature 1993, 362:755). We generated the artificial immunoadjuvant cells by transducing 3LL tumor cells with lentiviral vectors expressing CD1d and ovalbumin (OVA)/GM-CSF fusion protein. After loading with α-GlaCer, the immunoadjuvant cells were irradiated with 50Gy, and injected intravenously into B6/3LL-OVA mice. We observed a significant reduction in tumor mass and prolongation of survival as compared with mock-injected mice. Currently, we are exploring the optimal setting of combination with MDVV virotherapy and immunoadjuvant cell therapy. In this meeting, we will present these results.

Disclosures
No relevant conflicts of interest to declare.