[Preparation of CD33 targeted bispecific- and trispecific-T cell engagers and their cytotoxicity on leukemia cells]

Objective: To investigate the effect of CD33-targeted bi-specific and tri-specific T-cell engagers on T-cell proliferation and explore their cytotoxicity on leukemia cells. Methods: The CD33-targeted bi-specific T-cell engager (CD33-BiTE) and tri-specific T-cell engager (CD33-TriTE) expression vectors were successfully constructed and expressed through a eukaryotic cell expression system. CD33-BiTE and CD33-TriTE were purified by affinity chromatography. The effects of CD33-BiTE and CD33-TriTE on T cells were analyzed through in vitro experiments. Results: ① CD33-BiTE and CD33-TriTE were successfully constructed and purified and could compete with flow cytometry antibodies for binding to the target cells. ② After 12 days of co-culture with CD33-BiTE and CD33-TriTE, the number of human T cells were expanded to 33.89±19.46 and 81.56±23.62 folds, respectively. CD33-TriTE induced a stronger proliferation of T cells than CD33-BiTE (P<0.05) . ③ Both CD33-BiTE and CD33-TriTE induced specific dose-dependent cytotoxicity on CD33(+) leukemia cells. ④ Compared to CD33-TriTE, leukemia cells were prone to express PD-L1 when co-cultured with T cells and CD33-BiTE. CD33-TriTE induced powerful cytotoxicity on leukemia cells with high PD-L1 expression. Conclusion: CD33-BiTE and CD33-TriTE expression vectors were constructed, and fusion proteins were expressed in eukaryotic cells. Our results support the proliferative and activating effects of BiTE and TriTE on T cells. Compared to that of CD33-BiTE, CD33-TriTE induced a stronger proliferative effect on T cells and a more powerful cytotoxicity on leukemia cells with high PD-L1 expression.