Abstract A039-PR007: Reprogramming the Myeloid Tumor Microenvironment with CD123 CAR T Cells Enhances B7-H3 CAR T Cell Therapy in DIPG

Abstract Background: Despite promising safety profiles, B7-H3 CAR T cell therapies have yet to demonstrate meaningful clinical efficacy in diffuse intrinsic pontine glioma (DIPG), a devastating pediatric brain cancer characterized by a profoundly immunosuppressive tumor microenvironment (TME). This TME is dominated by tumor-associated macrophages (TAMs) and suppressive resident microglia that restrict CAR T-cell activation and persistence. Importantly, broad myeloid targeting risks eliminating macrophages essential for supporting T cell activation and anti-tumor immunity. We identified CD123 as a selective marker of suppressive TAMs in DIPG and hypothesized that dual targeting of CD123+ TAMs alongside B7-H3+ tumor cells would effectively remodel the TME, restore T cell function, and enhance therapeutic efficacy. Methods: Using immunocompetent DIPG models, we evaluated a sequential combination of CD123 and B7-H3 CAR T cells to remodel the TME and improve tumor control. In vitro assays included cytotoxicity, repeated antigen stimulation, and co-cultures with M1- or M2-polarized macrophages. Endpoints included TAM depletion, TME remodeling, tumor burden, and survival. Results: In vitro, M2 macrophages suppressed B7-H3 CAR T cell proliferation, persistence, and cytokine secretion; these effects were reversed by co-treatment with CD123 CAR T cells. CD123 CAR T cells selectively depleted M2-like TAMs and microglia without cytotoxicity against B7-H3+ DIPG tumor cells. In vivo, CD123 CAR T cell treatment reduced immunosuppressive TAMs and shifted the tumor microenvironment toward a pro-inflammatory phenotype, as demonstrated by intracellular cytokine staining and comprehensive flow cytometry analyses. Notably, administration of CD123 CAR T cells after B7-H3 CAR T cell infusion failed to rescue antitumor activity. However, administering CD123 CAR T cells within a four-day window prior to B7-H3 CAR T cell infusion significantly enhanced tumor control. Dual CAR therapy substantially decreased tumor burden, limited suppressive myeloid and microglial infiltration, and improved T cell activation and persistence, resulting in extended survival compared to monotherapies, with some mice achieving durable tumor remission. Ongoing transcriptomic studies are underway to elucidate the mechanisms driving this robust, lasting antitumor immunity. Conclusions: Priming the TME with CD123 CAR T cells significantly improves B7-H3 CAR T cell efficacy in aggressive DIPG models. Dual targeting of CD123+ TAMs and B7-H3+ tumor cells reprograms the DIPG TME and enhances CAR T-cell function by overcoming immunosuppressive barriers while preserving supportive immune populations. This strategy offers a promising mechanistic approach to improving CAR T therapy in DIPG. Citation Format: Khatereh Khorsandi, Kaleem Coleman, Jaquelyn T. Zoine, M. Paulina Velasquez, Dalia Haydar. Reprogramming the Myeloid Tumor Microenvironment with CD123 CAR T Cells Enhances B7-H3 CAR T Cell Therapy in DIPG abstract. In: Proceedings of the AACR Special Conference in Cancer Research: Discovery and Innovation in Pediatric Cancer— From Biology to Breakthrough Therapies; 2025 Sep 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85 (18Suppl₂): Abstract nr A039-PR007.