IMMU-67. INTRACRANIAL IL12 SECRETING CAR-T CELLS OBVIATE THE NEED FOR LYMPHODEPLETION AND COMBAT HETEROGENEITY VIA MICROGLIAL INTERACTIONS

Abstract INTRODUCTION Epidermal growth factor receptor variant III (EGFRvIII) is a tumor-specific antigen distinctively expressed in glioblastoma multiforme (GBM). However, its heterogeneous expression in GBM complicates its application as a target for Chimeric Antigen Receptor (CAR) T cells. Additionally, Interleukin-12 (IL12) has recently been identified as a potent cytokine in the treatment of solid tumors. In this study, we investigate the potential synergy between CAR-T cells and IL12 in targeting heterogeneous tumor antigens. METHODS To model tumor heterogeneity, mice were injected with a 50/50 mix of CT2A and CT2A EGFRvIII+ cells. EGFRvIII-specific third-generation CAR T cells, engineered with or without single-chain IL12, were cloned using an MSGV retrovirus backbone and transduced into murine T cells. The antitumor efficacy of CAR-T cells was evaluated through survival analysis. Additionally, the capability of CAR-T cells with or without IL12 to prolong survival and alter the tumor microenvironment was analyzed using single-cell sequencing. RESULTS EGFRvIII CARs alone were insufficient to cure mice with heterogeneous tumors, whereas EGFRvIII CARs co-expressing IL12 achieved a 60% cure rate. Interestingly, EGFRvIII CARs secreting IL12 demonstrated longer persistence in vivo compared to EGFRvIII CARs alone and did not require lymphodepletion before administration. We found that EGFRvIII CARs expressing IL12 promoted the elimination of EGFRvIII-negative tumor cells by recruiting endogenous CD8 T cells. Furthermore, among all Antigen Presenting Cell (APC) populations in the tumor microenvironment, we found that IL12 primarily acted on microglia to enhance antigen presentation pathways and support epitope spreading. CONCLUSION EGFRvIII CARs engineered to secrete IL12 effectively treat antigen-heterogeneous tumors by modulating local APCs and recruiting endogenous T cells. IL12-secreting CARs demonstrate longer persistence without the need for lymphodepletion compared to traditional EGFRvIII CARs. Employing CAR-T cells that secrete IL12 is a promising strategy to address tumor heterogeneity, offering therapeutic potential against GBM in clinical settings.