基于细胞糖链工程的尼妥珠单抗-NK细胞偶联物的构建及其活性研究

Construction of Nimotuzumab-NK cell conjugates based on cellular glycan engineering and evaluation of its activity

目的 通过基于唾液酸酶-糖基转移酶的细胞糖链工程构建偶联高密度的尼妥珠单抗的NK-92MI细胞,并评价其对肿瘤细胞的靶向和杀伤效果。方法 首先通过化学酶法和点击化学制备含有四嗪标签的GDP-岩藻糖,并探索制备含有反式环辛烯标签的尼妥珠单抗,再利用反式环辛烯-四嗪点击化学构建GDP-岩藻糖-尼妥珠单抗偶联物(GF-Nimo)。然后利用α1,3岩藻糖基转移酶将GF-Nimo转移至经唾液酸酶处理过的NK-92MI细胞表面,构建尼妥珠单抗-NK细胞偶联物,并通过流式细胞术和共聚焦成像实验验证偶联效果。最后以人结肠腺癌SW480为靶细胞,通过乳酸脱氢酶细胞毒性检测评价抗体-细胞偶联物的肿瘤细胞杀伤效果。结果首次成功将基于唾液酸酶-糖基转移酶的偶联方法应用于NK-92MI细胞,成功制备尼妥珠单抗-NK-92MI细胞偶联物。相比之前的基于岩藻基糖转移酶的方法,构建的抗体-细胞偶联物抗体偶联密度更高,具备更好的EGFR+细胞靶向性和更强的细胞杀伤作用。结论 构建抗体药物偶联NK细胞为尼妥珠单抗的拓展应用提供了新的途径,基于唾液酸酶-糖基转移酶的偶联方法学为后续开发阶梯式抗体与海洋糖类药物双分子细胞偶联方法奠定了基础。

Objective To construct NK-92MI cells conjugated with high density of nimotuzumab by sialidaseglycosyltransferase-based cellular glycan engineering and evaluate its targeting and cytotoxicity on tumor cells.Methods GDP-fucose containing tetrazine group was firstly prepared by chemoenzymatic method and click chemistry,and GDP-fucose nimotuzumab conjugate(GF-Nimo) was constructed using transcyclooctenetetrazine click chemistry.Then,GF-Nimo was transferred to the surface of sialidase-treated NK-92MI cells using α1,3 fucosyltransferase to construct nimotuzumab-NK cell conjugate,which was verified by flow cytometry and confocal imaging experiments.Finally,the cytotoxicity of antibody-cell conjugates was evaluated by lactate dehydrogenase cytotoxicity assay using human colon cancer SW480 as the target cells.Results For the first time,the sialidase-glycosyltransferase-based coupling method was successfully applied to NK-92MI cells,and nimotuzumab-NK-92MI cell conjugate were successfully prepared.The antibody-cell conjugate constructed in this paper have a higher antibody coupling density,better EGFR+ cell targeting and stronger cell killing effect than the previous fucosyltransferase-based method.Conclusion The construction of antibody conjugated NK cells provides a new avenue for the expanded application of nimotuzumab,and the sialidase-glycosyltransferase-based coupling methodology provides a foundational framework for the implementation of a stepwise antibody-marine polysaccharide bimolecular cell coupling method.