215. Detail Analysis of Non-Specific Activation of CD19 CAR-T Cells Caused by CAR Design

Abstract

Adoptive immunotherapy using the chimeric antigen receptor (CAR) gene-modified T cells is a promising strategy to treat patients with malignancy and autoimmune diseases. The CAR-T cells can target any cell surface antigens in a HLA independent manner, therefore CAR-T therapy is applicable to a broad range of patients irrespective of HLA phenotype. Typically CARs have higher affinities compared to TCRs, and T cell activation and cytotoxic effect on target cells mediated by CARs correlated with the affinity until it reach the thresholds. The latest results of CD19 CAR-T immunotherapy clinical trials have demonstrated impressive potential in a range of B-lymphoid malignancies. In spite of the recent great success, serious adverse events occurred after infusion of CAR-T cells including “on-target off-organ” activation, and cytokine release syndrome has been observed in a number of CAR-T cell therapies as a result of excessive T cell activation. To improve the efficacy and safety of CAR-T cells, selection of the target tumor-associated antigen, that are expressed only on tumor cells, and the suitable CAR constructs showing the optimal T cell activities are essential, thereby minimizing the risk of side effects.Previously, we have found CAR-T cells acquired to express T cell activation marker CD25 without specific antigen stimulation during in vitro culture. To validate of consequence of CD25-expression, we have analyzed the immune phenotype and antigen specific activities of CAR-T cells, and found the excess activation of CAR-T cells caused T cells differentiation with much less Naive T cell populations, resulting reduction of T cell activities against specific tumor cells. All CAR-T cells we have tested were activated without specific antigen stimulation, although the intensity of non-specific activation were correlated with the expression levels of CARs and depended on the scFvs and the extracellular-spacer domains.In this study, aiming to select the optimal design of CD19 CAR for effective and safe immunotherapy, using anti-CD19 antibody, clone FMC63, we performed detail analysis of non-specific activation of CD19 CAR-T cells caused by the design of scFv (e.g.the leader sequences, the order of VH and VL, the spacer sequences between VH and VL, and the extracellular-spacer domains). In vitro culture of CD19-CAR T cells without antigen specific stimulation induced elevation of T cell activation marker expression, such as CD25 and CD69 on CAR T cells, and reduction of Naive phenotype T cell populations (CCR7+/CD45RA+), as the level of non-specific activation of CAR-T cells was depended on the structure of scFvs (the order of VH and VL, and the spacer sequences) and the extracellular-spacer domains. Especially, the CAR-T cells with CD8 hinge domain were highly activated without specific activation, and the excessive non-specific activation lowered the cytokine secretion and the cytotoxic activities against CD19-expressing tumor cells. Although the low level activation of CAR-T cells may contribute the long term survival of T cells in vivo, it is desired to select the optimal antibodies and CAR constructs which shows high antigen-specific activities with low non-specific activities for the safe and effective CAR-T cell therapy.