Abstract A038: Overcoming myeloid-mediated immunosuppression and enhancing blood-brain barrier access to advance B7H3 CAR T cell therapy in high-risk Medulloblastoma

Abstract Background: Group 3 medulloblastoma (G3MB) is the most aggressive medulloblastoma subgroup. Although CAR T-cell therapy is promising, it has shown limited efficacy in G3MB due to poor T-cell persistence, largely driven by the immunosuppressive tumor microenvironment (TME) and an intact blood–brain barrier (BBB). M2-like tumor-associated macrophages (TAMs) are major contributors to this immunosuppression, whereas inflammatory TAMs can support CAR T-cell function. We identified Resiquimod, a Toll-like receptor (TLR) 7/8 agonist, as a potent immunomodulator capable of reprogramming TAMs toward a pro-inflammatory phenotype. In parallel, low-intensity focused ultrasound (LIFU) provides a noninvasive method to transiently disrupt the BBB, potentially enhancing both immunomodulatory effects and CAR T-cell infiltration. The objective of this study is to improve the efficacy of B7-H3-directed CAR T-cell therapy in G3MB by optimizing CAR design, reprogramming the TME using POx-encapsulated Resiquimod (POx-R), and enhancing tumor accessibility with LIFU. Methods: We engineered B7-H3 CARs with CD28, 4-1BB, or dual costimulatory domains and tested their efficacy in vitro and in vivo. POx-R was administered systemically to enable brain penetration and TAM repolarization. LIFU was applied with microbubbles to transiently open the BBB and facilitate delivery of CAR T-cells. Immune profiling included flow cytometry and RNA sequencing. Results: In vitro, CAR T-cells with dual CD28 and 4-1BB costimulatory domains demonstrated superior cytotoxicity and persistence. In vivo, systemically administered POx-R effectively crossed the BBB and induced pro-inflammatory TAM repolarization, as evidenced by upregulation of M1 markers and inflammatory cytokines detected by bulk RNA sequencing and flow cytometry. Preliminary data also indicate enhanced CAR T-cell infiltration and TME remodeling when combined with LIFU-mediated BBB disruption. Ongoing studies aim to elucidate the underlying mechanisms and optimize treatment scheduling to maximize therapeutic synergy. Conclusions: This multimodal strategy integrating CAR engineering, myeloid reprogramming, and BBB modulation offers a promising path to overcome the key barriers limiting CAR T-cell therapy in G3MB. Citation Format: Serge Yaacoub, Stefanyda Maslova, Ying-ting Hsu, Zhongzhen Yi, Kaleem Coleman, Martine F. Roussel, Timothy Gershon, Marina Sokolsky-Papkov, Cheng-Chia Wu, Natasha Sheybani, Dalia Haydar. Overcoming myeloid-mediated immunosuppression and enhancing blood-brain barrier access to advance B7H3 CAR T cell therapy in high-risk Medulloblastoma [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(18_Suppl_2):Abstract nr A038.