摘要目的:研究靶向CD33抗原双特异性及三特异性T细胞衔接器对T细胞增殖及其抗白血病作用。方法:构建抗CD33scFv-抗CD3scFv的双特异性T细胞衔接器（CD33-BiTE）及在BiTE基础上加入CD80胞外段的三特异性T细胞衔接器（CD33-TriTE）表达载体，使用真核细胞表达系统表达蛋白并进行亲和层析纯化。检测CD33-BiTE及CD33-TriTE对T细胞活化增殖功能及对白血病细胞杀伤功能活性的影响。结果:①成功构建了CD33-BiTE及CD33-TriTE表达载体，在真核细胞中表达，纯化得到的融合蛋白能与相同靶抗原流式抗体竞争结合于靶细胞表面。②CD33-BiTE及CD33-TriTE分别与人T细胞共培养12 d后，T细胞数扩增至基线值的（33.89±19.46）倍和（81.54±23.62）倍，CD33-TriTE促T细胞增殖能力明显优于CD33-BiTE（ P<0.05）。③体外实验证实CD33-BiTE和CD33-TriTE均可增强T细胞对表达CD33白血病细胞的特异性杀伤作用，且在一定浓度范围内，浓度越高，抗体的杀伤作用越强。④与CD33-TriTE相比，CD33-BiTE杀伤白血病细胞的同时增加其PD-L1表达，而TriTE对过表达PD-L1的Molm13细胞具有更强的杀伤作用。 结论:该研究构建了CD33-BiTE及CD33-TriTE表达载体，并在真核细胞表达了融合蛋白，体外实验证实其促T细胞增殖活化的特性，并具有促T细胞抗白血病作用。其中CD33-TriTE较CD33-BiTE促增殖效果更强，且对PD-L1高表达的白血病细胞杀伤效果更强。

abstractsObjective: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.