抗前蛋白转化酶枯草溶菌素9单克隆抗体的体外活性表征  

Characterization of in Vitro Activity of PCSK9 Monoclonal Antibodies

作　　者：朱馨婷 韩冷 郭澄[1] 杨全军[1] ZHU Xinting;HAN Leng;GUO Cheng;YANG Quanjun(Shanghai Sixth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine,Shanghai,China 200233)

机构地区：[1]上海交通大学医学院附属第六人民医院,上海200233

出　　处：《中国药业》2024年第23期45-51,共7页China Pharmaceuticals

基　　金：国家自然科学基金[81872494]。

摘　　要：目的探讨托莱西单抗、依洛尤单抗和阿利西尤单抗与前蛋白转化酶枯草溶菌素9(PCSK9)的亲和力。方法采用表面等离子共振法和生物膜层干涉法检测托莱西单抗、依洛尤单抗、阿利西尤单抗3种PCSK9抑制剂对PCSK9的亲和力;采用免疫印迹法(Western blot)法检测HepG2细胞中低密度脂蛋白受体(LDLR)的表达水平,考察不同质量浓度(0,6,60,180μg/mL)托莱西单抗阻断重组人PCSK9(D374Y)与LDLR的结合能力。结果表面等离子共振法和生物膜层干涉法测得托莱西单抗与PCSK9的平衡解离常数分别为2.90×10^(-10)mmol/L和5.05×10^(-10)mmol/L;托莱西单抗与重组人PCSK9(D374Y)的亲和力是依洛尤单抗的1.85倍、2.57倍,是阿利西单抗的5.38倍、4.71倍。托莱西单抗阻断重组人PCSK9(D374Y)与LDLR结合的半数抑制浓度(IC50)为64.42 nmol/L,与重组人PCSK9(D374Y)结合恢复低密度脂蛋白胆固醇(LDL-C)内吞作用的半数最大效应浓度(EC50)分别为(15.03±0.63)μg/mL和(7.22±0.16)μg/mL;Western blot法试验结果显示,不同质量浓度(0,6,60,180μg/mL)托莱西单抗中HepG2细胞灰度值分别为0.76,0.83,3.21,3.28。结论托莱西单抗与PCSK9的亲和力强于阿利西尤单抗和依洛尤单抗,能阻止LDLR的降解,从而浓度依赖性地增加肝细胞表面LDLR的表达。Objective To investigate the affinity of tafolecimab,evolocumab and alirocumab to proprotein convertase subtilisin/kexin type 9(PCSK9).Methods The affinity of three PCSK9 monoclonal antibodies(tafolecimab,evolocumab and alirocumab)for PCSK9 was examined by the surface plasmon resonance and biolayer interferometry methods.The expression level of low-density lipoprotein receptor(LDLR)in HepG2 cells was detected by the Western blot,and the ability of different mass concentrations(0,6,60,180μg/mL)of tafolecimab to block recombinant human PCSK9(D374Y)binding to LDLR was investigated.Results The equilibrium dissociation constants of tafolecimab with PCSK9 determined by the surface plasmon resonance and biolayer interferometry methods were 2.90×10^(-10)mmol/L and 5.05×10^(-10)mmol/L,respectively.The affinity of tafolecimab to recombinant human PCSK9(D374Y)was 1.85 times and 2.57 times higher than that of evolocumab,and 5.38 times and 4.71 times higher than that of alirocumab.The half maximal inhibitory concentration(IC50)of tafolecimab for blocking the binding of recombinant human PCSK9(D374Y)to LDLR was 64.42 nmol/L.The concentration for 50%of maximal effect(EC50)of tafolecimab for restoring low-density lipoprotein cholesterin(LDL-C)endocytosis by binding human PCSK9 and recombinant human PCSK9(D374Y)was(15.03±0.63)μg/mL and(7.22±0.63)μg/mL,respectively.Western blot test results showed that the grayscale values of HepG2 cells in different concentrations(0,6,60,180μg/mL)of tafolecimab were 0.76,0.83,3.21,and 3.28,respectively.Conclusion Tafolecimab,which has a stronger affinity for PCSK9 than alirocumab and evolocumab,can prevent the degradation of LDLR and increase the expression of LDLR on the surface of hepatocytes in a concentration-dependent manner.

关 键 词：前蛋白转化酶枯草溶菌素9 托莱西单抗 依洛尤单抗 阿利西尤单抗 抗体亲和力 体外活性 

分 类 号：R965[医药卫生—药理学] R979.1[医药卫生—药学]