PROJECT SUMMARY Significant progress in recent years on the development of oncolytic virotherapy has led to the FDA approval of a type I herpes simplex virus (HSV-1) based oncolytic virus (Talimogene Laherparepvec or T-VEC) for the treatment of advanced melanoma. However, it has become increasingly clear that there is a need to further improve the current version of OV for a better clinical benefit. Our lab has constructed a HSV-2 based oncolytic virus - FusOn-H2, the first of its kind, by a novel mechanism. Our recent studies showed that arming FusOn- H2 with a chimeric NK engager (C-NK-E) that can engage the infiltrated natural killer (NK) cells with tumor cells could significantly enhance the effectiveness of this virotherapy. Moreover, we observed that tumor destruction by the joint effect of the direct oncolysis and the engaged NK cells led to a measurable elicitation of neoantigen-specific antitumor immunity. Based on these exciting findings, we propose to develop a three- pronged strategy to enhance the antitumor efficacy of FusOn-H2 via combinatorial immuno-oncolytic virotherapy (IOV). The antitumor activities from this strategic three-pronged IOV come in waves in a sequential order. The first wave comes immediately and it derives primarily from the virus-mediated direct oncolysis. The second wave comes from the NK-mediated tumor cell killing enabled jointly by the virotherapy-trigged NK cell infiltration and the released C-NK-E from the armed virus. The third wave is the outcome of a series of chain events that ultimately result in induction and homing of neoantigen-specific T cells. Our major hypothesis is that this three-pronged strategy will act in a consequential and concerted way to significantly enhance the therapeutic efficacy of this IOV. We have designed three specific aims to test our major hypothesis. In Aim 1, we will dissect the mechanism of C-NK-E-armed virus in generating neoantigen-specific immunity and to design additional strategies to further potentiate this effect. Neoantigens are attractive targets for cancer immunotherapy, but there lacks a simple and effective way of delivering them. In principle, OV offers a simple means to release these neoantigens in individual patients, and our proposed strategy will ensure their efficient and timely presentation to the host’s immune system without the need for additional procedures. In Aim 2, we will investigate if combining C-NK-Es that engage two different activation receptors can further potentiate the NK cell killing and neoantigen-specific T cell immunity. In Aim 3, we will demonstrate that the three-pronged IOV can produce an efficient therapeutic effect against metastatic diseases by the systemic delivery route. We have recently developed novel strategies to overcome key obstacles for systemic delivery - the rapid clearance by the macrophage system and the neutralizing antibodies. We hypothesize that the combination of the high potency of the three-pronged IOV with the ...