Engaging Immune Cells within the Tumor Microenvironment to Enhance Efficacy of Oncolytic Virotherapy



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Oncolytic viruses are particularly attractive for many of the current cancer-immunotherapeutic modalities in their capacity to simultaneously cause direct lysis of tumor cells and indirect augmentation of host anti-tumor immunity. Despite widespread interest in the direct anti-cancer activity of oncolytic viruses, only limited attention has been paid to the interaction between viral therapy and the tumor microenvironment. In Chapter 2 of this dissertation, we studied the impact of FusOn-H3 (HSV-2 oncolytic virus) on the tumor microenvironment (TME), using single-cell RNA sequencing (scRNAseq) to investigate the infiltration and functionality within global populations or at the single-cell level. Our data show that FusOn-H3 can induce significant infiltration of both innate and adaptive immune cells. Detailed analysis by scRNAseq revealed the influx of T cells, B cells, NK cells, and Neutrophils into the TME, contributing to the conversion of cold tumors into hot ones.
In Chapter 3 of this dissertation, we harnessed the immune modulation potential of oncolytic viruses by engaging the immune cells within the TME, using chimeric engagers for improved anti-tumor efficacy. To accomplish this, we have generated two chimeric proteins, the Bispecific and Trispecific engagers (BiCEP and TriCEP), the genes encoding for which were delivered by an HSV amplicon system, packaged with the oncolytic virus as a helper virus. Oncolytic virotherapy can recruit NKG2D bearing NK or T cells to the tumor site, and the simultaneous release of these chimeric engagers by the co-delivered amplicons mediate cytotoxicity against tumor cells by crosslinking them with the immune cells via the overexpressed EGFR. Indeed, our data demonstrate that HSV-1 amplicon encoding Bi- and TriCEP combined with the oncolytic virus (Synco-2D) lead to durable remissions and protective anti-tumor immunity in an immunocompetent mouse model. In Chapter 4 of this dissertation, we characterized the transcriptional changes in the tumor cells upon oncolytic viral infection using scRNA sequencing. Analysis of viral and host transcriptomes in the same single cell enabled us to resolve the cellular heterogeneity of infected tumor cells as compared with the bystanders/uninfected cells. These findings give us an understanding of changes in the immune cell landscape and viral-host interactions in complex tumor microenvironment and open new avenues for virotherapy.



Oncolytic viruses, Immunotherapy, Virotherapy, FusOn-H3, single-cell RNA sequencing