DMG-07. Enhancing CAR T-Cell Therapy in DIPG Through Dual Targeting of CD123 and B7-H3

Background Tumor-associated macrophages (TAMs) are key contributors to the immunosuppressive tumor microenvironment (TME) in aggressive, cold tumors like Diffuse Intrinsic Pontine Glioma (DIPG). TAMs limit the efficacy and persistence of CAR T cells while driving T-cell exhaustion. This study investigates whether depleting TAMs using anti-CD123 CAR T cells can enhance the anti-tumor efficacy of B7-H3 CAR T cells in glioma models. Objective To evaluate the therapeutic potential of dual CAR T-cell therapy targeting CD123 to deplete immunosuppressive TAMs and B7-H3 to directly eliminate glioma cells, aiming to overcome TME-induced barriers to CAR T-cell efficacy in DIPG. Methods Immunocompetent DIPG models were used to assess murine CD123 CAR and B7-H3 CAR T cells. In vitro assays, including cytotoxicity, repeat stimulation, and co-culture systems with macrophages (M0, M1, M2 phenotypes), were conducted to assess functional outcomes. In vivo studies involved intratumoral injection of CD123 CAR T cells to evaluate TAM depletion and anti-tumor effects. Results CD123 CAR T cells alone exhibited minimal cytotoxicity against B7-H3+ glioma cells. However, combining CD123 and B7-H3 CAR T cells preserved anti-tumor activity, with efficacy dependent on the number of B7-H3 CAR T cells. Specific ratios of dual CAR T cells enhanced tumor cell elimination and modulated the TME to augment the cytotoxicity of B7-H3 CAR T cells. M2-polarized TAMs suppressed B7-H3 CAR T-cell function, but CD123 CAR T cells restored efficacy by depleting TAMs. In vivo, CD123 CAR T cells reduced TAMs but showed limited direct anti-tumor activity. Ongoing studies are examining the extent to which TAM depletion enhances B7-H3 CAR T-cell activity in DIPG models. Conclusion Dual CAR T-cell therapy targeting CD123 and B7-H3 enhances anti-tumor efficacy by modulating the TME. These findings suggest that TAM depletion can overcome TME-mediated immunosuppression and improve outcomes for pediatric brain tumor patients, particularly in DIPG.