Identifying CD19-targeted CAR-T cell immune pathways in an in vitro human immune mimetic cytokine release assay.

Abstract

CD19-targeted chimeric antigen receptor-modified T (CAR-T) cells have shown success in clinical studies, with several CD19 CAR-T cell products now having been approved for market use. However, this cell therapy can be associated with side effects such as cytokine release syndrome (CRS). Therefore, pre-clinical test systems are highly desired to permit the evaluation of these unwanted effects before clinical trials begin. In this study, we evaluated cytokine secretion and cell phenotype changes induced by human CD4⁺ and CD8⁺ CD19-targeted CAR-T cells in the cytokine release assay (CRA) module of a pre-clinical human in vitro 3D co-culture platform. The in vitro CRA data showed that CD19-targeted CAR-T cells induced a diverse and concentration-dependent cytokine response led by a T_H1-profile (IFNγ, IL-2) and pro-inflammatory cytokines (IL-6, TNFα, MCP-1, IL-8, MIP-1b). It was also shown that different cellular components in this 3D co-culture system contributed to the CAR-T cell cytokine response. In particular, whole blood-derived cell populations were necessary to drive the production of T cell cytokines, and endothelial cells were required to generate pro-inflammatory cytokines. CD19-targeted CAR-T cells also triggered cell phenotype changes, including the activation of whole blood-derived CD4⁺ and CD8⁺ T-cells and activation/maturation of antigen-presenting cells, during treatment of the in vitro CRA platform. Additionally, the observation of a CD19-targeted CAR-T cell concentration-dependent reduction in the B-cell compartment in this study is aligned with the expected pharmacology and clinical profile of this compound. Overall, this dataset shows the utility of an in vitro CRA model as a pre-clinical platform for evaluating cytokine release potential and analysis of mechanisms of action of CD19-targeted CAR-T cells.