IL-2-inducible T cell kinase deficiency sustains chimeric antigen receptor T cell therapy against tumor cells.

Despite the revolutionary achievements of chimeric antigen receptor (CAR) T cell therapy in treating cancers, especially leukemia, several key challenges still limit its therapeutic efficacy. Of particular relevance is the relapse of cancer in large part as a result of exhaustion and short persistence of CAR-T cells in vivo. IL-2-inducible T cell kinase (ITK) is a critical modulator of the strength of T cell receptor signaling, while its role in CAR signaling is unknown. By electroporation of CRISPR-associated protein 9 (Cas9) ribonucleoprotein (RNP) complex into CAR-T cells, we successfully deleted ITK in CD19-CAR-T cells with high efficiency. Bulk and single-cell RNA sequencing analyses revealed downregulation of exhaustion and upregulation of memory gene signatures in ITK-deficient CD19-CAR-T cells. Our results further demonstrated a significant reduction of T cell exhaustion and enhancement of T cell memory, with significant improvement of CAR-T cell expansion and persistence both in vitro and in vivo. Moreover, ITK-deficient CD19-CAR-T cells showed better control of tumor relapse. Our work provides a promising strategy of targeting ITK to develop sustainable CAR-T cell products for clinical use.