海洋细胞毒药物与抗体偶联物的制备及生物活性评价

Preparation and bioactivity evaluation of antibody marine-derived cytotoxic drug conjugate

目的以抗体N-糖链为偶联位点,制备曲妥珠单抗与海洋细胞毒药物单甲基澳瑞他汀E(MMAE)偶联物,并研究其对乳腺癌细胞BT474的杀伤作用。方法合成含叠氮和炔基修饰的唾液酸化寡糖,采用化学酶法制备含叠氮或炔基修饰的曲妥珠单抗,利用生物正交反应分别制备相应的抗体药物偶联物。利用SDS-PAGE对转糖基及偶联过程进行监测,表面等离子共振技术(SPR)对叠氮或炔基修饰前后抗体与FcγRIIIa亲和力进行检测。利用CCK-8法评价2种抗体药物偶联物对乳腺癌细胞株BT474的杀伤作用。结果成功制备了含叠氮或炔基修饰的曲妥珠单抗及N-糖链唾液酸位点抗体药物偶联物,修饰抗体与FcγRIIIa的亲和力较曲妥珠单抗增加,2种抗体MMAE偶联物对BT474有比曲妥珠单抗更明显的生长抑制作用。结论基于抗体糖链末端唾液酸位点偶联的方法可有效应用于海洋细胞毒抗体偶联药物的开发。

Objective To prepare trastuzumab-marine derived monomethyl auristatin E(MMAE)conjugates by N-glycan specific coupling and evaluate their cytotoxicity against breast cancer cells BT474.Methods Azide and alkyne-modified homogeneous sialic acid oligosaccharides were synthesized as the donors.Then azide and alkyne modified trastuzumab were prepared via chemoenzymatic transglycosylation.By different biological orthogonal strategies,MMAE were conjugated to modified trastuzumab to form two kinds of trastuzumabMMAE conjugates.The processes of transglycosylation and coupling reaction were monitored by SDS-PAGE.Surface plasmon resonance(SPR)technology was used to analyze the affinity between antibodies with different modification and FcγRIIIa.The cytotoxicities of two kinds of trastuzumab-MMAE conjugates against the breast cancer cell lines BT474 was evaluated by CCK-8 assay.Results The azide or alkyne modified trastuzumab and trastuzumab-MMAE conjugates were prepared successfully.The affinity with FcγRIIIa of modified trastuzumab was elevated and compared with commercial trastuzumab.The cytotoxicity assays showed that trastuzumabMMAE conjugates had more inhibitory activities against BT474 than trastuzumab.Conclusion The method for N-glycan specific coupling at sialic acid of antibody glycan could be effectively applied for the development of marine-derived cytotoxic drugs based ADCs.