基于RTCA技术研究抗体偶联药物的旁观者效应的检测方法  

Detection of bystander effect in antibody-drug conjugates based on real-time label-free cell analysis technique

作　　者：曹天助 牛庆田 刘兵 昌根琼 林军 罗顺 CAO Tian-zhu;NIU Qing-tian;LIU Bing;CHANG Gen-qiong;LIN Jun;LUO Shun(School of Pharmacy,Jiangxi University of Chinese Medicine,Nanchang 330004;Nantong Thousand Oaks Biopharma,Nantong Jiangsu 226133;Yangtze Delta Institute for Advanced Pharmaceutical Research,Nantong Jiangsu 226133)

机构地区：[1]江西中医药大学药学院,南昌330004 [2]澳斯康生物(南通)股份有限公司,江苏南通226133 [3]长三角药物高等研究院,江苏南通226133

出　　处：《中南药学》2024年第5期1227-1231,共5页Central South Pharmacy

摘　　要：目的 利用实时无标记细胞分析(RTCA)系统评估抗体偶联药物(ADC)的旁观者效应。方法 采用RTCA系统监测抗原阳性细胞(Ag^(+))和抗原阴性细胞(Ag^(-))的共培养状态,观察Ag^(-)的细胞存活情况;采用重复实验验证结果的可靠性和一致性,并采用单因素方差分析比较不同细胞比例培养组在单个时间点的Ag^(-)存活率。结果 加入抗体偶联药物后,随着共培养时间的延长,Ag^(-)存活率下降;此外,在单个时间点(96 h)上,与0%Ag^(+)细胞的Ag^(-)细胞存活率相比,10%Ag^(+)细胞的Ag^(-)细胞存活率为89.07%(P=0.156),25%Ag^(+)细胞的Ag^(-)存活率为68.93%(P=0.0026),50%Ag^(+)、75%Ag^(+)和90%Ag^(+)细胞的Ag^(-)存活率分别为35.28%、13.99%和12.02%(P<0.0001)。在 Ag^(+)细胞比例为25%~90%时,旁杀伤效果显著;重复性结果显示不同Ag^(+)细胞的旁杀伤率的相对标准偏差(RSD)均小于30%。结论 结合RTCA技术和共培养方式的方法,可以更准确地模拟旁观者效应的生物学过程,为后续的抗体偶联药物开发提供有价值的参考。Objective To evaluate the bystander effect of antibody-drug conjugates with a real-time label-free cell analysis(RTCA).Methods RTCA was used to monitor the co-cultivation of Ag^(+)(antigen-positive cells)and Ag^(-)(antigen-negative cells)to observe the survival of Ag^(-)cells.The experiment was repeated to validate the reliability and consistency of the results.Additionally,one-way ANOVA was used to compare the Ag^(-)survival rates at a single time point among cultivation groups with different cell ratios.Results After adding antibody-drug conjugates drugs,the survival rate of Ag^(-)decreased with the prolongation of co-culture time.In addition,at 96 h,compared to the Ag^(-)cell survival rate with 0%Ag^(+)cells,the Ag^(-)cell survival rate of 10%Ag^(+)cells was 89.07%(P=0.156),the Ag^(-)cell survival rate with 25%Ag^(+)cells was 68.93%(P=0.0026),and the Ag^(-)cell survival rates with 50%Ag^(+),75% Ag^(+),and 90%Ag^(+)cells were 35.28%,13.99%,and 12.02%,respectively(P<0.0001).The bystander killing effect was significant within 25%~90%the proportion of Ag^(+)cells.The repeatability test showed that the relative standard deviation of the bystander killing rates of different Ag^(+)cells was less than 30%.Conclusion This method,which combines RTCA technology and co-cultivation,can more accurately simulate the bystander effect in biological processes,providing valuable reference for subsequent antibody-drug conjugate development.

关 键 词：抗体偶联药物 实时无标记细胞分析 旁观者效应 细胞存活 旁杀伤率 

分 类 号：R917[医药卫生—药物分析学] R927[医药卫生—药学]