Influence of Escherichia coli chaperone DnaK on protein immunogenicity.
Influence of Escherichia coli chaperone DnaK on protein immunogenicity.
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DOI：
10.1111/imm.12689
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发表时间：
2017-03
期刊：
影响因子：
6.4
通讯作者：
Dearman RJ
中科院分区：
文献类型：
--
作者：
Ratanji KD;Derrick JP;Kimber I;Thorpe R;Wadhwa M;Dearman RJ
抗药抗体的产生会对生物治疗药物的安全性和有效性产生重大影响。已知各种因素（包括聚集和工艺相关杂质的存在）可改变和增强蛋白质的免疫原性潜力。本文报告的研究旨在在小鼠中表征聚集和宿主细胞蛋白杂质对人源化单链抗体可变片段（scFv）和小鼠白蛋白免疫原性的影响。从大肠杆菌纯化的scFv制剂中的宿主细胞蛋白质杂质在BALB/c品系小鼠中显示出对scFv应答的佐剂样活性。70 000 MW E.大肠杆菌伴侣蛋白DnaK通过质谱分析被鉴定为scFv的关键污染物。缺乏可检测DnaK的scFv的制备物用重组E.大肠杆菌DnaK以模拟工艺相关杂质。用单体和聚集的制剂免疫小鼠，含和不含按质量计0.1%的DnaK。单独的聚集增强IgM和IgG 2a抗体反应，但对总IgG或IgG 1反应没有显著影响。DnaK的加入进一步增强了IgG和IgG 2a抗体应答，但仅在聚集蛋白存在的情况下。通过Western印迹分析显示DnaK与聚集的scFv相关。用小鼠白蛋白进行的实验显示，单独使用蛋白质聚集的免疫原性总体增加，并且DnaK的存在进一步增加了IgG 2a抗体应答的活力。总的来说，这些数据表明，DnaK有可能修改和增强免疫原性时，与聚集的蛋白质。
The production of anti‐drug antibodies can impact significantly upon the safety and efficacy of biotherapeutics. It is known that various factors, including aggregation and the presence of process‐related impurities, can modify and augment the immunogenic potential of proteins. The purpose of the investigations reported here was to characterize in mice the influence of aggregation and host cell protein impurities on the immunogenicity of a humanized single‐chain antibody variable fragment (scFv), and mouse albumin. Host cell protein impurities within an scFv preparation purified from Escherichia coli displayed adjuvant‐like activity for responses to the scFv in BALB/c strain mice. The 70 000 MW E. coli chaperone protein DnaK was identified as a key contaminant of scFv by mass spectrometric analysis. Preparations of scFv lacking detectable DnaK were spiked with recombinant E. coli DnaK to mimic the process‐related impurity. Mice were immunized with monomeric and aggregated preparations, with and without 0·1% DnaK by mass. Aggregation alone enhanced IgM and IgG2a antibody responses, but had no significant effect on total IgG or IgG1 responses. The addition of DnaK further enhanced IgG and IgG2a antibody responses, but only in the presence of aggregated protein. DnaK was shown to be associated with the aggregated scFv by Western blot analysis. Experiments with mouse albumin showed an overall increase in immunogenicity with protein aggregation alone, and the presence of DnaK increased the vigour of the IgG2a antibody response further. Collectively these data reveal that DnaK has the potential to modify and enhance immunogenicity when associated with aggregated protein.