IMMU-41. Investigating Multi-Targeting CAR T Cells to Overcome Heterogeneity in Adult Glioblastoma

Abstract High-grade gliomas (HGGs) exhibit significant intra- and inter-tumoral heterogeneity, posing a major challenge for chimeric antigen receptor (CAR) T cell therapies that target a single antigen. Multi-targeting CAR T cells offer a promising solution by simultaneously engaging multiple tumour-associated antigens. However, current clinical trials in glioblastoma (GBM) focus on a limited set of antigen targets, restricting therapeutic efficacy. To expand the range of targetable antigens, we performed a comprehensive surface proteomic analysis of primary GBM tumours. This led to the development of a library of 70 CAR constructs targeting a refined pool of novel and clinically validated surface proteins. These CARs were screened in a pooled manner to identify optimal candidates, with top hits validated as mono-CARs in two GBM models, both in vitro and in vivo. The top 10 CAR candidates were then tested in multi-target pools of up to five CARs. Additionally, we incorporated unique protein tags, called Procodes, into the transgene of each CAR. This enabled the tracking and analysis of up to 32 unique sub-populations within a single pooled CAR T cell product via spectral flow cytometry, both in vitro and ex vivo. This facilitated a deeper understanding of multi-CAR dynamics, including the impact of antigen abundance, CAR T cell phenotype, and CAR design on therapeutic efficacy. Through this work, we have identified novel antigen combinations that elicit potent anti-tumour activity in vivo. In parallel, we are uncovering key principles that govern multi-CAR functionality. Notably, our findings suggest that there may be an upper limit to the number of CARs that can be co-expressed before T cell function is compromised. Additionally, certain CAR combinations may be incompatible within the same product, further influencing efficacy. These insights provide a foundation for the rational design of next-generation CAR T cell therapies for brain tumours.