Chen M L, Peng N, Liu X Y, Zhang T, Xu Y X, Tian Z, Xing H Y, Tang K J, Rao Q, Wang J X, Wang M
State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Institute of Hematology & Blood Diseases Hospital, Peking Union Medical University, CAMS & PUMC, Tianjin 300020, China.
Zhonghua Xue Ye Xue Za Zhi. 2021 Mar 14;42(3):217-223. doi: 10.3760/cma.j.issn.0253-2727.2021.03.007.
To prepare a novel tri-specific T cell engager (19TriTE) targeting CD19 antigen, and to investigate its immunotherapeutic effect on CD19-positive hematological malignancies. 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. 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. 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.
制备一种靶向CD19抗原的新型三特异性T细胞衔接子（19TriTE），并研究其对CD19阳性血液系统恶性肿瘤的免疫治疗效果。通过分子克隆技术构建19TriTE，并通过真核表达系统成功表达。验证了19TriTE对T细胞增殖和活化的影响，以及对CD19阳性肿瘤细胞系的特异性细胞毒性。①构建了19TriTE表达质粒，并通过真核表达系统成功表达。②19TriTE能特异性结合T细胞和Nalm6细胞，平衡解离常数分别为19.21 nmol/L和11.67 nmol/L。③在共培养体系中加入2 nmol/L 19TriTE时，CD69阳性T细胞和CD25阳性T细胞的表达率分别为35.4%和49.8%，显著高于对照组。④19TriTE能显著促进T细胞增殖。在第12天，当浓度为1 nmol/L时，T细胞绝对计数从最初的100万扩增至7400万，增加了74倍。⑤19TriTE能以剂量依赖的方式显著介导T细胞杀伤CD19阳性靶细胞。在浓度为10 nmol/L时，靶细胞裂解率达到50%。⑥脱颗粒实验证实，19TriTE在CD19阳性靶细胞存在的情况下能激活T细胞，且T细胞的活化与19TriTE的剂量呈正相关。⑦当19TriTE融合蛋白分别与过表达RFP和荧光素酶基因的T细胞和靶细胞共培养时，19TriTE可通过荧光显微镜或生物发光成像技术显著介导T细胞杀伤CD19阳性靶细胞。在本研究中，我们成功构建并表达了19TriTE融合蛋白，验证了其能在体外有效激活T细胞并促进其增殖。同时，它能结合CD19阳性靶细胞和T细胞，并增强T细胞在体外的抗白血病作用，为进一步的临床研究奠定了基础。
