基于唾液酸酶和岩藻糖转移酶的抗体—细胞偶联物构建

A sialyldase and fucosyltransferase based method for the construction of antibody-cell conjugates

目的 通过基于唾液酸酶和岩藻糖转移酶的糖链工程将曲妥珠单抗高密度偶联到人外周血白血病T细胞Jurkat细胞,并评价偶联细胞对HER2+靶细胞的识别活性。方法 首先通过凝集素染色和流式细胞术探究去唾液酸化对细胞上α-1,3FucT催化的岩藻糖编辑效率的影响。然后制备含有叠氮标签的曲妥珠单抗,与GDP-炔基岩藻糖通过生物正交反应构建GDP-岩藻糖-抗体偶联物。再利用α-1,3FucT将GDP-岩藻糖-抗体偶联物转移至唾液酸酶处理过的细胞表面,构建抗体-细胞偶联物,并通过流式细胞术和共聚焦成像验证。最后通过荧光成像观察抗体-细胞偶联物对靶细胞SK-BR-3细胞的识别。结果 细胞表面糖链的末端α-2,6唾液酸切除后能够显著提升α-1,3FucT催化的岩藻糖编辑的效率,成功制备GDP-岩藻糖-抗体偶联物并转移至细胞表面得到曲妥珠单抗-Jurkat偶联物,去唾液酸化显著增加了Jurkat细胞偶联曲妥珠单抗的容量,构建的曲妥珠单抗-Jurkat偶联物具备HER2+细胞靶向性。结论 细胞糖链工程可实现细胞偶联分子容量的调节,应用于抗体对免疫细胞的功能化,也为构建海洋糖类活性化合物偶联细胞提供了新的思路。

Objective To construct engineered Jurkat cells conjugated with high density of Trastuzumab by sialidase and fucosyltransferase based glycoengineering, and evaluate its ability to recognize target cells.Methods The effect of pre-desialylation on α-1,3FucT catalyzed fucose editing on cell surface was first explored by lectin blot and flow cytometry. Then alkynyl GDP-fucose and azide modified Trastuzumab were prepared, which were linked together to form GDP-fucose-Trastuzumab conjugates by bioorthogonal reactions.Then GDP-fucose-Trastuzumab were transferred to sialidase pre-treated Jurkat cells by α-1,3FucT to generate antibody-cell conjugates. The conjugation efficiency was verified by flow cytometry and confocal microscopy.Finally, the targeting and binding of antibody-cell conjugates to the target cells, SK-BR-3 cells, were investigated by fluorescence microscope. Results The removal of terminal α 2,6 sialic acids from cell surface N-glycans could significantly increase fucose editing efficiency by α-1,3Fuc T. GDP fucose-antibody conjugates were successfully prepared and transferred to the cell surface to construct trastuzumab-Jurkat conjugates, and desialylation significantly increased the capacity of Jurkat cells to conjugate trastuzumab. Trastuzumab-Jurkat conjugates could target and bind HER2+ cancer cells compared with regular Jurkat cells. Conclusion Cellular glycoengineering was a promising strategy which could tune the capacity of cells to conjugate molecules and be applied to functionalization of immune cells with antibodies. This would provide a new idea for the construction of marine carbohydrates-cells conjugates.