P08.07.a Comprehensive Evaluation of Car-Engineered Immune Cell Subsets Reveals Therapeutic Synergy in an Immune-Competent Glioblastoma Model

Abstract BACKGROUND Chimeric antigen receptor (CAR)-based therapies have yet to achieve meaningful success in solid tumors such as glioblastoma (GBM). Recent efforts have focused on leveraging alternative immune cell types for CAR engineering, but a systematic side-by-side comparison in an immune-competent glioma model has been lacking. MATERIAL AND METHODS To address this, we developed a transgenic mouse strain on a C57BL/6 background harboring an anti-EGFRvIII CAR downstream of a Lox-Stop-Lox cassette inserted into the Rosa26 locus. Crossing with Vav-Cre mice enabled pan-hematopoietic CAR expression. This platform allowed us to directly compare CAR-T, CAR-NKT, CAR-NK, and CAR-macrophages using the syngeneic, EGFRvIII-expressing SB28 glioma model. RESULTS CAR-NK cells demonstrated robust antigen-specific cytotoxicity in vitro. However, neither intravenous nor intratumoral delivery of memory-like CAR-NK cells suppressed tumor growth in vivo. Co-administration with CD4⁺ CAR-T cells also failed to enhance CAR-NK efficacy, and long-term tumor control was not achieved. These findings highlight the limitations of CAR-NK cells in immunocompetent glioma environments, despite their potent in vitro activity, and were considered to be associated with poor persistence of CAR-NK cells in brain tumors. In contrast, CAR-NKT cells exhibited strong EGFRvIII-specific cytotoxicity in vitro and secreted high levels of perforin and GM-CSF. Intratumoral CAR-NKT injection led to survival benefits in SB28-bearing mouse models comparable to CAR-T monotherapy. In vitro co-cultures showed increased IFN-γ production with combined CAR-T and CAR-NKT cells, suggesting functional synergy. Importantly, co-delivery of CAR-T and CAR-NKT cells resulted in significantly improved survival compared to CAR-T cells alone. To improve CAR signaling in myeloid cells, we engineered CAR-macrophages expressing a CD3ζ-only signaling domain. This modification enhanced antigen-specific TNF production and phagocytic activity in vitro compared to wild-type macrophages, but did not improve antigen presentation capacity. In vivo, CAR-macrophages failed to persist in the tumor or suppress tumor growth and showed no therapeutic advantage over control macrophages. CONCLUSION Our data provide the first comprehensive comparison of CAR-expressing immune cell subsets in an immune-competent glioma model. This platform enables precise functional assessment and supports the development of rational combination strategies—most notably, the synergy between CAR-T and CAR-NKT cells—to improve GBM immunotherapy.