Paediatric high-grade gliomas (pHGGs) are aggressive brain tumours with a median survival of only 11–18 months. Chimeric antigen receptor (CAR) T cell therapy has shown early potential in targeting pHGGs; however, its effectiveness is limited by antigen heterogeneity, highlighting the need for novel therapeutic targets. EphB1 was identified as a promising candidate due to its high expression in fresh primary pHGGs samples and cell lines, based on cell surface proteomic analysis. This study aims to develop EphB1-targeting CAR T cells as a potential immunotherapy for pHGGs. The goal is to lay the groundwork for a viable, precision-based treatment for pHGGs. The research holds strong potential for translational impact and may lead to multiple commercialisation opportunities.
EphB1-reactive antibody clones were generated using a human phage display screen. These clones, in various orientations, along with different CAR modules (spacers, transmembrane domains (TMDs), and signalling tail variants), were used to construct a library of CAR constructs, which were subsequently transduced into primary human T cells. To identify the most functional constructs, the transduced T cells were co-cultured with EphB1-expressing pHGGs cells, and CAR T cell activation was assessed by CD137 expression using flow cytometry. Oxford Nanopore sequencing (ONS) was then employed to profile and track activated CAR constructs within the library.
Twelve antibody clones were reactive to recombinant EphB1; ten were converted into single-chain variable fragments (scFvs) with an 8-fold variation in binding affinity. The resulting 432-second-generation EphB1 CAR library showed robust activation. ONS revealed that CAR T cell activation was primarily influenced by scFv clones and signalling tail variants, rather than by spacers, TMDs, or scFv orientations.
We identified critical design parameters influencing EphB1-CAR T cell activation, providing a rational strategy for screening diverse CAR libraries. Further in vitro evaluations are ongoing to identify lead CAR constructs for therapeutic development.