Establishing a potent and highly tumour selective immunooncolytic virotherapy

Abstract

Tumours grow by evading surveillance through immunosuppressive microenvironments. These microenvironments upregulate immune checkpoints, which inhibit T-cell activation. Targeting these checkpoints such as CTLA-4, PD-1/PDL-1, or LAG-3 with immune checkpoint inhibitors has been shown to enhance the host anti-tumour response and improve long-term survival in some patients. However, some tumours may be resistant to this therapy, and systemic administration of inhibitors can cause significant systemic toxicities. Oncolytic viruses (OV) have significant potential to induce immunogenic cell death (ICD), turning immunologically 'cold' tumours 'hot'. This potential can be enhanced when combined with immunotherapies, such as immune checkpoint inhibitors (ICI). Combining OV and ICI into single agents capable of targeting tumour cells following intravenous delivery can result in tumour-selective expression of ICI within the tumour microenvironment, encouraging 'ontarget' and minimising 'off-target' activity. We have developed a refined tumour-selective OV, Ad5NULL-A20, which has proven efficacious in peritoneal ovarian cancer and incorporated an antibody fragment targeted against different ICIs controlled by a CMV promoter and containing a CD33 secretion signal. In vitro, these viruses transduce αvβ6-positive tumour cells, expressing and secreting scFv-Fc. The purified anti-LAG-3 scFv-Fc was biologically active, blocking the interaction between MHC-II and LAG3 in engineered cells. The scFv-Fcbound LAG-3, and had equal or better affinity than an anti-LAG-3 control antibody, validating the approach as a new precision virotherapy. Despite the promising results in vitro, we have encountered some challenges when testing the scFv-Fc in a more complex system, such as co-culture assays using PBMCs where T-reg numbers and levels of activation were limiting. We developed a relevant 4T1 mouse cancer model based on previous work to test the virus in vivo. Results were inconclusive and require further investigation as discussed in this thesis. Overall, this project demonstrates the efficacy of both Ad5NULL-A20 as a platform to deliver immunovirotherapies and the anti-LAG-3 scFv-Fc, in vitro. However, more experiments are needed to demonstrate its efficacy in vivo