Abstract Glypican-2 (GPC2) is highly expressed across paediatric malignancies, including high-risk neuroblastomas and medulloblastomas, and GPC2-directed CART-cells have now entered phase 1 trials (NCT05650749, NCT07087002). Yet durable responses remain limited due to heterogeneous antigen expression, treatment-driven antigen loss, and suboptimal CART persistence. Because B7-H3 is broadly expressed in GPC2+medulloblastoma, it represents a compelling partner target for a dual GPC2.B7H3 CAR approach. To enable dual targeting, we generated seven bicistronic GPC2/B7-H3 CAR constructs incorporating CD28, 4-1BB, or OX40 costimulatory domains and either two (dual, d1–3) or one (parallel, p1–4) CD3-ζ signalling domain. Among these, constructs d1 and p2 were prioritised for further study. Both induced strong activation of Jurkat NFAT-GFP CART-cells when co-cultured with isogenic NALM-6 cells expressing either or both antigens and produced higher cytokine secretion than singleantigen B7-H3 (B) or GPC2 (G) CARs. In medulloblastoma in vitro models, repeated tumourstimulation assays consistently showed superior activity for d1 and p2. These findings translated in vivo. In a GPC2-low medulloblastoma PDX, dual CARs matched the efficacy of B7H3-2G-CARs and outperformed GPC2-2G-CARs. In an aggressive group3 medulloblastoma PDX where all GPC2 CAR–treated mice relapsed, the p2-CAR achieved the strongest therapeutic effect, yielding 16week cure rates of 100% versus 71.4% for d1 and 60% for the single B7H3-CAR, demonstrating the advantage of dual GPC2.B7H3 targeting and suggesting a benefit of parallel CD3ζ–sharing designs. Dual-targeting CART-cells also drove potent regression of COG-N-453x neuroblastoma PDXs, with 16-week cure rates of 83.3%, 83.3%, and 100%, exceeding single-antigen CARs. Long-term persistence of human CART-cells in bone marrow and spleen correlated with durable remissions. Upon tumour rechallenge, only d1 and p2 prevented recurrence, with sustained CART-cell persistence through 36-weeks. Overall, GPC2.B7-H3 bicistronic CARs generated durable and often curative responses across neuroblastoma and medulloblastoma models, supporting this strategy as a next-generation GPC2-directed therapy for clinical evaluation.
Draper et al. (Tue,) studied this question.