The long-term goal of this project is to develop an effective immunotherapy for patients with metastatic and/or recurrent OS osteosarcoma (OS), whose prognosis remains poor despite aggressive multimodality therapy. Immunotherapy with chimeric antigen receptor (CAR)-expressing T cells has the potential to improve outcomes for patients with OS since CAR T-cell killing does not rely on the cytotoxic mechanism of conventional therapies. While CAR T cells have shown promise for the immunotherapy of B-cell malignancies in early phase clinical studies, T-cell therapy for solid tumors such as OS faces formidable challenges. For example, antitumor activity of T cells relies on significant in vivo expansion of adoptively transferred T cells, which is difficult to achieve at solid tumor sites. To overcome this obstacle, we have developed Engager (ENG) T cells, which secrete bispecific engager molecules consisting of single chain variable fragments specific for CD3 and a tumor antigen. These cells have the unique ability to redirect bystander T cells to tumors, amplifying antitumor effects. Indeed, our preliminary results indicate that ENG T cells targeting EphA2, a tumor-associated antigen expressed in OS, redirect bystander T cells to EphA2-positive tumor cells, and have potent antitumor activity in xenograft models. However, treated tumors ultimately progressed, indicating limited ENG T-cell activation at solid tumor sites. To overcome this limitation we now have generated a so-called costimulatory CAR with a CD28.OX40 endodomain targeting HER2 (HER2-CoCAR), a tumor-associated antigen also expressed in OS. Indeed our preliminary results indicate that EphA2-ENG/HER2-CoCAR T cells have superior effector function in comparison to EphA2-ENG T cells when stimulated with EphA2+/HER2+ target cells as judged by enhanced IL2 production and T-cell proliferation. Targeting two antigens should also reduce potential ‘on target/off cancer’ toxicities since full T-cell activation is restricted to tumor sites in which both tumor-associated antigens are expressed. Thus, we now hypothesize that EphA2-ENG T cells expressing a HER2-specific CoCAR with a CD28.OX40 endodomain (EphA2-ENG/HER2-CoCAR T cells) will have improved effector function compared to EphA2-ENG T cells resulting in enhanced anti-OS activity. This hypothesis will be evaluated in two interrelated research aims. Aim 1 compares the effector function of EphA2-ENG and EphA2-ENG/HER2-CoCAR T cells in vitro using standard immunological assays, and Aim 2 compares the expansion, persistence, and anti-OS activity of EphA2-ENG- and EphA2-ENG/HER2-CoCAR T cells in vivo using the LM7 OS xenograft model.