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《山东大学学报(理学版)》 ›› 2024, Vol. 59 ›› Issue (11): 51-63.doi: 10.6040/j.issn.1671-9352.0.2023.504

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氧化应激对一款半胱氨酸偶联型抗体偶联药物的影响

曲耀成1,2(),姚雪静2,孙允东1,*()   

  1. 1. 山东大学基础医学院，山东 济南 250012
    2. 荣昌生物制药(烟台)股份有限公司，山东 烟台 264006
  • 收稿日期:2023-11-29 出版日期:2024-11-20 发布日期:2024-11-29
  • 通讯作者: 孙允东 E-mail:quyaocheng@remegen.com;syd@sdu.edu.cn
  • 作者简介:曲耀成(1989—)，男，硕士研究生，研究方向为生物制药质量. E-mail: quyaocheng@remegen.com

Effect of oxidative stress on a cysteine-conjugated antibody-drug conjugate

Yaocheng QU1,2(),Xuejing YAO2,Yundong SUN1,*()   

  1. 1. School of Basic Medical Sciences, Shandong University, Jinan 250012, Shandong, China
    2. RemeGen Co., Ltd., Yantai 264006, Shandong, China
  • Received:2023-11-29 Online:2024-11-20 Published:2024-11-29
  • Contact: Yundong SUN E-mail:quyaocheng@remegen.com;syd@sdu.edu.cn

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摘要：

探索氧化应激对一款半胱氨酸(cysteine，Cys)偶联型抗体偶联药物(antibody-drug conjugate，ADC)结构和功能的影响, 研究对象为一款基于Cys偶联的靶向c-Met的ADC(简称c-Met ADC)。首先，将研究样本置于不同比例H2O2溶液中孵育进行强制氧化。然后，采用多种分析技术对氧化样本的结构和功能变化进行深度表征。H2O2诱导的氧化应激造成c-Met ADC抗体骨架上的Met氧化和连接头中的硫醚键裂解。Met氧化进一步导致了c-Met ADC的热稳定性和新生儿受体(neonatal Fc receptor, FcRn)亲和力降低，且均主要与抗体可结晶片段(fragment crystallizable，Fc)上的Met257氧化有关；而硫醚键裂解则导致c-Met ADC的生物学活性降低。氧化应激是该ADC稳定性的关键影响因素，由其导致的Met257氧化以及硫醚键裂解均应作为其关键质量属性(critical quality attributes，CQAs)在日常放行和稳定性研究中严格监控。同时，还应避免将其暴露于氧化环境中或通过优化环境因素来减轻氧化应激对其结构和功能的影响。

关键词: 抗体偶联药物, 氧化应激, 强制降解, 甲硫氨酸氧化, 脱靶毒性

Abstract:

We investigated the effects of oxidative stress on the structure and function of a cysteine (Cys)-conjugated antibody-drug conjugate (ADC). The research object was a Cys-conjugated, c-Met ADC (referred to as c-Met ADC). Firstly, the samples were incubated in different proportions of H2O2 solution for forced oxidation. Then, multiple analytical techniques were used to deeply characterize the structural and functional changes of the oxidized samples. H2O2 induced oxidative stress resulted in Met oxidation on the c-Met ADC antibody backbone and thioether bond cleavage in the linker. Met oxidation further led to a decrease in the thermal stability and neonatal Fc receptor (FcRn) affinity of the c-Met ADC, which were mainly related to Met257 oxidation on the antibody Fc. The cleavage of thioether bonds led to a decrease in the biological activity of the c-Met ADC. Oxidative stress was a key influencing factor on the stability of c-Met ADC, and the Met257 oxidation and thioether bond cleavage caused by it should be strictly monitored as its critical quality attributes in daily release and stability studies. At the same time, it should also be avoided to be exposed to oxidative environments or to mitigate the impact of oxidative stress on its structure and function by optimizing environmental factors.

Key words: antibody-drug conjugate, oxidative stress, forced degradation, methionine oxidation, off-target toxicity

中图分类号: 

  • R917

图1

c-Met ADC结构简图"

图2

分子筛纯度随H2O2含量增加的变化"

图3

各位点Met氧化随H2O2含量增加的变化"

图4

抗c-Met ADC和mAb对应位点Met氧化变化的比对"

图5

H2O2处理的c-Met ADC样本与未处理对照的镜像质谱图"

图6

DAR值随H2O2增加的变化"

图7

热稳定性随H2O2含量增加的变化"

图8

Tm, 1和Met257的相关性分析"

图9

结合活性随H2O2含量增加的变化"

图10

内吞活性随H2O2含量增加的变化"

图11

生物学活性随H2O2含量增加的变化( ***P＜0.001, ****P＜0.000 1)"

图12

ADCC活性随H2O2含量增加的变化(nsP＞0.05)"

图13

FcRn亲和力性随H2O2含量增加的变化( nsP＞0.05, **P＜0.01, ****P＜0.000 1)"

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