In vitro modelling of immune effector cell-associated neurotoxicity syndrome (ICANS) resulting from CAR T-cell therapy treating haematological cancer

Introduction of chimeric antigen receptor (CAR) T-cell therapy has transformed haematological cancer treatment by directing T cells to target tumour antigens e.g. CD19, inducing tumour cell death. However, its use is limited by neurotoxicity, most notably immune effector cell-associated neurotoxicity syndrome (ICANS). ICANS presents with wide-ranging neurological symptoms including seizures and altered consciousness, which can progress to coma and death. ICANS is linked to excessive cytokine release and blood-brain barrier dysfunction, yet its precise mechanisms remain unclear. We aim to elucidate how CAR-T cell activity induces ICANS. We are establishing an in vitro model comprising activated CAR-T cells co-cultured with CNS cells to assess phenotypic and functional changes. We generated a clinically-relevant CD19-targeting CAR construct (UCART19) and a cognate non-signalling CAR construct serving as control. Co-culturing healthy-donor-derived UCART19 CAR-T cells with CD19-expressing B-cell lymphoma cell line, NALM6, showed CAR-T activation, effector cytokine production and tumour cell killing. In contrast, the non-signalling UCART19 cells showed no response. Conditioned media from UCART19:NALM6 co-culture induced activation of human umbilical vein endothelial cells (HUVECs) and upregulated pro-inflammatory genes in induced pluripotent stem cell (iPSC) derived microglia. Analysis of activated UCART19 conditioned media identified nine cytokines that were absent in non-signalling UCART19 conditioned media. One prime candidate is tumour necrosis factor alpha (TNF-α), which is known to activate microglia. In response, iPSC-derived microglia released T-cell chemoattractants, CXCL9 and CXCL10, potentially facilitating CAR-T cell trafficking into the brain. Understanding these mechanisms could inform strategies for preventing ICANS, thereby enhancing the safety of CAR-T cell therapy.