Objective: To prepare a novel tri-specific T cell engager (19TriTE) targeting CD19 antigen, and to investigate its immunotherapeutic effect on CD19-positive hematological malignancies. Methods: 19TriTE was constructed by molecular cloning technology and successfully expressed through the eukaryotic expressing system. The effects of 19TriTE on the proliferation and activation of T cells, as well as the specific cytotoxicity against CD19 positive tumor cell lines were verified. Results: ①19TriTE expressing plasmid was constructed and successfully expressed through the eukaryotic expressing system. ②19TriTE can specifically bind to T cells and Nalm6 cells, with equilibrium dissociation constants of 19.21 nmol/L and 11.67 nmol/L, respectively. ③The expression rates of CD69 positive T cells and CD25 positive T cells were 35.4% and 49.8% respectively, when 2 nmol/L 19TriTE were added in the co-culture system, which were significantly higher than those in the control group. ④19TriTE can significantly promote the proliferation of T cells. The absolute count of T cells expanded from the initial one million to 74 million with an 74 fold increase at the concentration of 1 nmol/L on day 12. ⑤19TriTE can significantly mediate T cells killing of CD19 positive target cells in a dose-dependent manner. At the concentration of 10 nmol/L, the target cells lysis reached 50%. ⑥Degranulation experiment verified that 19TriTE can activate T cells in the presence of CD19 positive target cells, and the activation of T cells positively correlated with the dose of 19TriTE. ⑦When 19TriTE fusion protein co-cultured with T cells and target cells overexpression RFP and luciferase genes respectively, 19TriTE can notably mediate T cells killing of CD19 positive target cells through fluorescent microscope or bioluminescence imaging technology. Conclusion: In this study, we successfully constructed and expressed 19TriTE fusion protein and verified that it can effectively activate T cells and promote their proliferation in vitro. At the same time, it can bind to CD19 positive target cells and T cells, as well as enhance T cells anti-leukemia effect in vitro, providing the foundation for further clinical research.
目的： 制备一种新的靶向CD19的三特异性T细胞衔接器融合蛋白（19TriTE），研究其对CD19阳性血液肿瘤的免疫治疗作用。 方法： 通过分子克隆技术构建19TriTE表达质粒，并通过真核蛋白表达系统成功表达该融合蛋白。体外实验验证19TriTE对T细胞的活化与增殖作用及介导T细胞发挥对CD19阳性肿瘤细胞的特异性细胞毒作用。 结果： ①成功构建19TriTE融合蛋白表达质粒，并通过真核蛋白表达系统成功表达。②19TriTE可以特异性结合T细胞和Nalm6细胞，与T细胞的平衡解离常数为19.21 nmol/L，与Nalm6细胞的平衡解离常数为11.67 nmol/L。③2 nmol/L浓度的19TriTE与T细胞及Nalm6细胞共培养时，T细胞中CD69阳性表达率为35.4%，CD25阳性表达率为49.8%，较对照组显著升高。④19TriTE在浓度为1 nmol/L时即可显著促进T细胞增殖，第12天时，T细胞绝对计数由初始1×10(6)扩增至7.4×10(7)，增殖74倍。⑤19TriTE融合蛋白能明显介导T细胞杀伤CD19阳性靶细胞且与剂量呈正相关，浓度为10 nmol/L时，靶细胞裂解率达50%。⑥脱颗粒实验验证，CD19阳性靶细胞存在时，19TriTE可以显著激活T细胞且与剂量呈正相关。⑦将19TriTE融合蛋白分别与过表达RFP及Luciferase基因的靶细胞及T细胞共培养，19TriTE能够介导T细胞杀伤CD19阳性的靶细胞。 结论： 本研究成功构建及表达19TriTE融合蛋白，体外实验验证其可以有效活化T细胞，并促进T细胞增殖。同时，能够结合CD19阳性靶细胞和T细胞，促进T细胞发挥体外抗白血病作用，为进一步临床研究奠定基础。.
Keywords: CD19; Fusion protein; Immunotherapy; Leukemia, lymphoblastic, acute; T cell engager, tri-specific.