PROJECT SUMMARY While the clinical outcome of multiple myeloma (MM) patients has shown remarkable improvements over the past two decades, MM remains incurable for most patients. Thus, more efficacious therapies and novel strategies are urgently needed. Oncolytic (cancer destroying) vaccinia virus (VV) is an appealing addition to the current cancer therapies due to its preference for infecting and killing tumor cells and a potential for activating the immune system component called T cells that can travel to distant sites and kill any tumor cells they find, even those not infected by the virus. At present the activation of tumor-specific T cells is suboptimal. More importantly, a desirable systemic intravenous administration of the oncolytic virus is not feasible due to the strong neutralizing antibody response against it. Our goal is to engineer a VV that will efficiently stimulate the anti- tumor immunity by directly engaging and activating resident T cells surrounding tumor cells so that they will kill tumor cells and stop new tumors from growing. To activate T cells surrounding MM we constructed a new molecule called a T-cell engager that couples T cells to MM and increases T-cell activation and tumor killing. We believe that this optimized VV producing the new T-cell engager molecule will be more effective in killing MM, compared to other oncolytic viruses. Additionally, we will modify viral glycoproteins to mask VV from antibody recognition and neutralization, making it suitable for systemic delivery. The central hypothesis of this study is that recombinant BCMA-TEA-VVNEV will: 1) escape neutralization by human anti-VV Abs in vitro and in vivo; 2) direct endogenous T cells to kill BCMA+ MMs that are not infected with virus (by-stander killing), enhancing its oncolytic activity; and 3) promote T-cell activation, resulting in the cytokines release and creation of a pro-inflammatory microenvironment, inhibiting tumor growth. Aim 1 is to generate and test the quality of BCMA-TEA-VVNEV in vitro. BCMA-TEA-VVNEV will be produced in our laboratory located at JLABS@TMC. The purity and stability will be tested. In addition, the Ab neutralization, anti-tumor effect (e.g. direct and bystander killing), replicative capacity, and T-cell activation of human BCMA-TEA-VVNEV will be tested using transformed cell cultures and standard immune assays. Aim 2 is to evaluate the anti-tumor efficacy of BCMA- TEA-VVNEV in vivo. BCMA-TEA-VVNEV and control VVs will be administered to tumor bearing mice and virus replication, anti-tumor immune responses, and antitumor efficacy will be compared. Aim 3 will evaluate the safety of BCMA-TEA-VVNEV in mouse models. BCMA-TEA-VVNEV and control VVs will be administrated to tumor bearing mice and safety of BCMA-TEA-VVNEV will be assessed with biodistribution (tissue histology and in vivo viral replication) and mouse survival. Our therapy is expected to have a major impact on the treatment of MM patients with relapsed disease, which are often incurable with current treatment strategies.