Development of a workflow for in vitro on- and off-target cytotoxicity assessment of CAR T cell therapies to support first-in-human clinical trials: An orthogonal approach using human induced pluripotent stem cell-derived cells as a surrogate for normal vital organ systems

Chimeric antigen receptor (CAR) T cell therapy has revolutionized cancer therapy and is showing promise for other non-cancer disease indications. CAR T cells are directed to a target antigen that is overexpressed by tumor cells. On-target toxicity can arise if the targeted protein is also expressed in normal cell populations. Additionally, off-target toxicity may occur if the CAR T cell recognizes or cross-reacts with a non-target protein thereby activating the T cells leading to subsequent adverse sequela. Given the nature of this biological class as a human cellular product, standard safety assessments in animals are largely not appropriate as the drug product is derived from human cells and the associated CAR often lacks reactivity with the animal homolog of the target protein. Undesired targeting of healthy/normal tissue that express the intended target antigen (on-target/off-tumor), as well as unintended targeting of other antigens expressed on healthy/normal tissue is a safety concern that may be explored using human induced pluripotent stem cell (hiPSC)-derived test systems as surrogates for various normal cell types. The panel of hiPSC-derived cells is intended to broadly represent different human cell types from vital organs that can be targets for severe adverse events. Herein the development of an orthogonal approach to an in vitro co-culture assay used to assess unintended CAR T cell cytotoxicity in normal cells is described. Experimental considerations including assay and cell model qualification are presented, and an orthogonal workflow described. Finally, an illustrative case of experimental CD33 CAR T cells co-cultured with a select panel of hiPSC-derived normal cells serves as a springboard for other CAR T cell developers to consider in their nonclinical safety programs. © 2025 The Authors. Published by Elsevier B.V.

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