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An engineered human IgG1 CH2 domain with decreased aggregation and nonspecific binding.
mAbs ( IF 5.6 ) Pub Date : 2019-12-03 , DOI: 10.1080/19420862.2019.1689027
Guangcan Cao 1, 2 , Xinyu Gao 1, 2 , Yancheng Zhan 1, 2 , Qingguang Wang 1, 2 , Zhe Zhang 1 , Dimiter S Dimitrov 3 , Rui Gong 1
Affiliation  

The immunoglobulin (Ig) CH2 domain is a promising scaffold for the development of candidate therapeutics. We have previously shown that the stability of isolated CH2 could be increased by the introduction of an additional disulfide bond and removal of seven N-terminal residues (m01s). However, both isolated CH2 and m01s aggregate, likely due to the existence of aggregation-prone regions (APRs) that we identified by using computational methods. This knowledge was used to generate a phage display library of mutants. The library was incubated at high temperature to remove aggregating CH2 domains, and then panned against a mouse anti-human CH2 monoclonal antibody targeting a conformational epitope to remove misfolded CH2s. After two rounds of panning, one clone, m01s5, with smaller APRs, was identified. After additional mutagenesis one clone, m01s5.4, which aggregated much less than m01s as measured by a turbidity assay and dynamic light scattering, was identified. m01s5.4 also exhibited much lower nonspecific binding than m01s. Engineering of a previously identified m01s-based tumor antigen-specific binder led to a dramatic reduction of its aggregation without affecting its binding. In summary, we describe a new approach for reducing aggregation based on a combination of computational and phage display methodologies, and show that aggregation of CH2-based scaffolds can be significantly reduced by the newly identified mutants, which can improve the developability of potential CH2-based therapeutics.

中文翻译:

经过工程改造的人IgG1 CH2域,具有减少的聚集和非特异性结合。

免疫球蛋白(Ig)CH2结构域是开发候选疗法的有前途的支架。先前我们已经表明,通过引入额外的二硫键并去除七个N末端残基(m01),可以提高分离的CH2的稳定性。但是,孤立的CH2和m01s都会聚集,这很可能是由于我们使用计算方法确定的容易聚集的区域(APR)的存在。该知识被用于产生突变体的噬菌体展示文库。该文库在高温下孵育以去除聚集的CH2域,然后针对靶向构象表位的小鼠抗人CH2单克隆抗体进行淘选,以去除错误折叠的CH2。经过两轮淘选,鉴定出一个具有较小APR的克隆m01s5。经过进一步诱变后,一个克隆m01s5.4 经浊度测定和动态光散射测定,其聚集量远小于m01s。m01s5.4还表现出比m01s低得多的非特异性结合。先前鉴定的基于m01s的肿瘤抗原特异性结合剂的工程设计导致其聚集作用显着降低,而不会影响其结合。总而言之,我们描述了一种基于计算和噬菌体展示方法相结合的减少聚集的新方法,并表明通过新鉴定的突变体可以显着减少基于CH2的支架的聚集,这可以提高潜在的CH2的可开发性。基础疗法。先前鉴定的基于m01s的肿瘤抗原特异性结合剂的工程设计导致其聚集作用显着降低,而不会影响其结合。总而言之,我们描述了一种基于计算和噬菌体展示方法相结合的减少聚集的新方法,并表明通过新鉴定的突变体可以显着减少基于CH2的支架的聚集,这可以提高潜在的CH2的可开发性。基础疗法。先前鉴定的基于m01s的肿瘤抗原特异性结合剂的工程设计导致其聚集作用显着降低,而不会影响其结合。总而言之,我们描述了一种基于计算和噬菌体展示方法相结合的减少聚集的新方法,并表明通过新鉴定的突变体可以显着减少基于CH2的支架的聚集,这可以提高潜在的CH2的可开发性。基础疗法。
更新日期:2020-04-20
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