EXTH-114. Dual CD123 and B7-H3 CAR T Cells Potentiate Immune Responses and Remodel the Tumor Microenvironment in Pediatric Gliomas

Abstract BACKGROUND Pediatric high-grade gliomas (pHGG) are characterized by an immunologically cold and highly immunosuppressive tumor microenvironment (TME). It is dominated by M2-like tumor-associated macrophages (TAMs), which impair CAR T-cell function and persistence. However, broad myeloid targeting can be detrimental, as recent evidence shows that inflammatory macrophages are essential to support CAR T-cell activity. We therefore identified CD123 as selectively expressed on immunosuppressive TAMs, making it a promising target to remodel the TME. Meanwhile, B7-H3 is a tumor-associated antigen that is broadly expressed in pHGGs, making it an ideal target for CAR T-cell therapy. We now hypothesize that dual targeting of CD123+ TAMs and B7-H3+ tumor cells would synergistically deplete suppressive myeloid populations, restore T-cell function, and enhance anti-tumor efficacy. OBJECTIVE To evaluate the therapeutic potential of CD123 and B7-H3 dual CAR T-cell therapy in modulating the TME, preserving supportive myeloid subsets, and enhancing anti-tumor efficacy in pHGG. METHODS Using immunocompetent pHGG models, we tested CD123 and B7-H3 CAR T-cells through in vitro cytotoxicity, antigen stimulation, and co-cultures with bone marrow–derived M1 or M2 macrophages. In vivo, CD123 CAR T-cells were administered intratumorally as monotherapy or in combination with B7-H3 CAR T-cells. Endpoints included TAM depletion, phenotypic and transcriptomic TME remodeling, tumor progression, and survival. RESULTS CD123 CAR T-cells exhibited minimal cytotoxicity against B7-H3+ glioma cells but selectively depleted M2-like TAMs in vitro and in vivo. M2 macrophages suppressed B7-H3 CAR T-cell persistence in vitro; this suppression was reversed by co-treatment with CD123 CAR T-cells. Dual CAR T-cell therapy significantly enhanced anti-tumor activity, reduced myeloid and microglial infiltration, and extended survival in vivo. CONCLUSION Targeted depletion of CD123+ TAMs enhances B7-H3 CAR T-cell efficacy by mitigating immunosuppressive signaling while preserving beneficial myeloid support. This dual-targeting strategy offers a mechanistically guided approach to overcoming TME-driven resistance in DIPG and other pHGG.