A trispecific T cell engager CD19xCD3xCD28 induces potent tumor-directed T cell activation and antitumor activity by simultaneously engagement of TCR and a co-stimulatory receptor CD28

Abstract T-cells, being the major player in adaptive immune system mediating cellular immunity, have become a central focus for engaging the immune system in fighting against cancer. Their activation is initiated by recognition of antigen/major histocompatibility complexes by the T-cell receptor (TCR) and enhanced by engagement of co-stimulatory receptor. In recent years, various immunotherapies deploying T-cell immunity have made immense progress, including chimeric antigen receptor (CAR)-T therapy, checkpoint blockage, and CD3-based bispecific antibodies. Here we report the development of a novel trispecific antibody to CD19, CD3, and CD28, with an emphasis on the co-engagement of both CD3/TCR complex and CD28 co-stimulatory receptor for sustained T cell activation. Compared with the BiTE-CD19xCD3 lacking the co-stimulatory domain, the trispecific antibody augmented T cell proliferation and differentiation into memory T cells. It induced faster tumor cell lysis than the bispecific antibody, preferably through direct tumor lysis by CD8 T cells. RNAseq analysis revealed that the trispecific antibody modulates TCR complex-derived signal at multiple aspects, including amplification of transcriptional factors, promoting production of cytokines, positive regulation of cell cycle, and upregulation of anti-apoptotic factors to influence survival of T-cells. Markedly increased tumor inhibition effects and prolonged survival were observed in vivo in humanized mouse model. By extra engagement of a co-stimulatory receptor to prolong the survival of activated T cells, our study has proposed another possibility to extend the pharmacology effect of tandem single-chain variable fragments (scFv)-based antibody, which is worthy of further exploration in clinical trial settings.