Project Summary/Abstract Osteosarcoma (OS) is the most common primary bone malignancy affecting long bones in children and the third most frequent in adults in the United States. The survival rates over the last 20 years are unchanged. OS is also the most common malignant bone tumor (80%) in dogs. Treatment options for OS are limited. Immunotherapy is a promising new approach that has yet to be fully explored in OS. Chimeric antigen receptor (CAR) T cells are designed to express recombinant receptors to target specific tumor cell surface antigens and promote T cell- mediated cancer cell death. However, one of the biggest challenges in CAR T cell therapy is the immunosuppressive tumor micro-environment (TME). Another immunotherapy strategy is the use of immune checkpoint inhibitors, such as monoclonal antibodies (mAb) against PD-1. Anti-PD-1 mAbs have shown significant efficiency in treating multiple tumors but cause adverse effects due to systemic delivery of the mAb. To avoid systemic delivery, oncolytic viruses can be designed to produce immune checkpoint inhibitors directly in TME to boost the immune system and enable T cells to kill tumor cells. Conditionally replicative adenoviruses (CRAds) replicate only in tumor cells, lyse them, and stimulate anti-tumor immunity in TME and at distant sites of disease, causing a bystander effect. However, the limited number of effector T cells against cancer antigens limits the efficacy of this approach. Therefore, we propose a combination therapy to synergize these strategies to combat tumor cells. We will combine CAR T cell therapy, oncolytic virus, and PD-1/PD-L1 inhibitor to target osteosarcoma tumor cells. We have developed an armed oncolytic adenovirus to produce anti-PD-1 Ab in the TME. We will create two more armed oncolytic viruses that will produce secreted single domain antibodies (sdAb) to PD1 and PDL1. We will evaluate CRAd and CAR T cell (against B7-H3) induced tumor cells lysis. The conditionally replicated oncolytic viruses will produce anti-PD1 and anti-PDL1 sdAb in TME, reducing the adverse effects of systemic administration, and enhancing anti-tumor immunity. CAR T cells against B7-H3 will initiate tumor cell killing in an activated TME. Ultimately, beyond the scope of this proposal, this approach will be tested in spontaneous OS in canine patients, before translation to pediatric OS patients.

Project Narrative A combination cancer immunotherapy utilizing CAR T cells and an oncolytic virus armed with immune checkpoint inhibitors will be used to target osteosarcoma cells. Each treatment will be assessed individually and in combination for their ability to stimulate the immune system and kill osteosarcoma cells, in 2D and 3D cell culture. These activities will be assessed by measurement of cell responses, including cytokines and chemokines secreted to induce immune responses in the tumor microenvironment.