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Chimeric antigen receptor (CAR) T-cell therapy offers promise to improve outcomes for pediatric patients with recurrent/refractory osteosarcoma (OS), a ‘poor-prognosis’ cancer. However, early-phase clinical studies have shown limited activity despite potent antitumor activity in preclinical xenograft models, highlighting the need to develop better preclinical models. B7-H3 has emerged as a promising immunotherapeutic target for pediatric solid tumors including OS, and we and other investigators are actively exploring B7-H3-CAR T cell therapies in early-phase clinical studies. Our goal was to develop a murine immunocompetent OS model (F331) for the realistic pre-clinical evaluation of B7-H3-CAR T cells and to explore additional genetic modifications to improve their effector function. We generated murine B7-H3-CAR T cells expressing a 2nd generation B7-H3-CD28.ζ CAR by retroviral transduction. In vitro, B7-H3-CAR T cells recognized B7-H3-positive F331 cells as judged by IFNγ production and cytolytic activity in contrast to B7-H3-negative tumor cells; Control (ctrl) CAR T-cells recognizing an irrelevant antigen (SP6) did not recognize or kill B7-H3-positive tumor cells confirming specificity. Since lung is the primary OS metastasis site, we focused on the intravenous (i.v.) disseminated lung F331 OS metastasis model. In this model, a single i.v. infusion of 5x106 CD8+ B7-H3-CAR T cells demonstrated significant antitumor activity compared to CD8+ ctrl CAR T cells, resulting in a significant survival advantage (median: 70 vs 38 days respectively). Unfortunately, tumors invariably recurred. We found limited B7-H3-CAR T cell expansion and persistence, even though recurring tumors still expressed B7-H3, excluding tumor antigen escape mechanism. We therefore embarked on a comprehensive screen to knock out (KO) known negative regulators of T-cell function, starting with Regnase-1 (Reg-1). Reg-1 is known to have RNase activity and to regulate activation of immune cells. KO of Reg-1 in CD8+ B7-H3-CAR T cells improved their expansion post-infusion in spleen and lungs as judged by flow cytometric analysis without systemic toxicities based on mice body weight assessment. This resulted in a significant improvement in overall survival in comparison to control KO B7-H3-CAR T cells at a cell dose (1x106) at which ctrl-KO CD8+ B7-H3-CAR T cells were ineffective. Reg1-KO ctrl CAR T cells had no therapeutic benefit, excluding nonspecific effects. In summary, we have established an immune-competent OS model to evaluate the effector function of B7-H3-CAR T cells and have demonstrated the advantage given by 2nd genetic modifications to enhance their antitumor activity. We are currently using this model to define mechanisms of immune resistance with the goal of further enhancing OS-redirected CAR T-cell therapy. Citation Format: Adeleye O. Adeshakin, Peipei Zhou, Jean-Yves Métais, Phuong Nguyen, Scott Perry, Heather Sheppard, Xiang Sun, Trevor Cunningham, Hao Shi, Jessica Wagner, Jason T. Yustein, Christopher DeRenzo, Giedre Krenciute, Hongbo Chi, Stephen Gottschalk. B7-H3-CAR T-cell therapy in immune-competent osteosarcoma models: Regnase-1 KO overcomes limited CAR T-cell expansion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1778. |
