Application of blood brain barrier models in pre-clinical assessment of glioblastoma-targeting CAR-T based immunotherapies

Abstract

Abstract Human blood brain barrier (BBB) models derived from induced pluripotent stem cells (iPSCs) have become an important tool for the discovery and preclinical evaluation of central nervous system (CNS) targeting cell and gene-based therapies. Chimeric antigen receptor (CAR)-T cell therapy is a revolutionary form of gene-modified cell-based immunotherapy with potential for targeting solid tumors, such as glioblastomas. Crossing the BBB is an important step in the systemic application of CAR-T therapy for the treatment of glioblastomas and other CNS malignancies. In addition, even CAR-T therapies targeting non-CNS antigens, such as the well-known CD19-CAR-T therapies, are known to trigger CNS side-effects including brain swelling due to BBB disruption. In this study, we used iPSC-derived brain endothelial-like cell (iBEC) transwell co-culture model to assess BBB extravasation of CAR-T based immunotherapies targeting U87MG human glioblastoma (GBM) cells overexpressing the tumor-specific mutated protein EGFRvIII (U87vIII). Two types of anti-EGFRvIII targeting CAR-T cells, with varying tonic signaling profiles (CAR-F263 and CAR-F269), and control Mock T cells were applied on the luminal side of BBB model in vitro. CAR-F263 and CAR-F269 T cells triggered a decrease in transendothelial electrical resistance (TEER) and an increase in BBB permeability. CAR-T cell extravasation and U87vIII cytotoxicity were assessed from the abluminal compartment using flow cytometry and Incucyte real-time viability imaging, respectively. A significant decrease in U87vIII cell viability was observed over 48 h, with the most robust cytotoxicity response observed for the constitutively activated CAR-F263. CAR-F269 T cells showed a similar cytotoxic profile but were approximately four fold less efficient at killing the U87vIII cells compared to CAR-F263, despite similar transmigration rates. Visualization of CAR-T cell extravasation across the BBB was further confirmed using BBTB-on-CHIP models. The described BBB assay was able to discriminate the cytotoxic efficacies of different EGFRvIII-CARs and provide a measure of potential alterations to BBB integrity. Collectively, we illustrate how BBB models in vitro can be a valuable tool in deciphering the mechanisms of CAR-T–induced BBB disruption, accompanying toxicity and effector function on post-barrier target cells.