Target-independent Immune-cell Activation by Aggregates of T Cell-redirecting Bispecific Antibodies

T cell-redirecting bispecific antibodies (bsAbs) have been under development as a new class of biotherapeutics for cancer immunotherapy. T cell-redirecting bsAbs simultaneously bind tumor-associated antigens on tumor cells and CD3 on T cells, resulting in T cell-mediated cytotoxicity against tumor cells. In this study, we prepared a tandem scFv-typed bsAb targeting HER2 and CD3 (HER2-CD3), and evaluated the impact of aggregation of HER2-CD3 on the in vitro immunotoxicity. A cell-based assay using CD3-expressing reporter cells revealed that the aggregates of HER2-CD3 directly activated CD3-expressing immune cells in the absence of target antigen (HER2)-expressing cells. Comparison of the aggregates generated under various stress conditions indicated the possibility that insoluble protein particles, which were detected by qLD analysis and contained non-denatured functional domains, contributed to the activation of CD3-expressing immune cells. In addition, HER2-CD3 aggregates stimulated hPBMCs and strongly induced the secretion of inflammatory cytokines and chemokines. The cytokine/chemokine-release profiles suggested that the aggregates could induce inflammatory responses not only by CD3-mediated T cell activation but also by other immune cell activations. These results indicated the potential risk of aggregation of T cell-redirecting bsAbs, which could induce unwanted immune cell activation and inflammation and thereby immune-mediated adverse reactions.