Biophysical properties of human antibody variable domains
Biophysical properties of human antibody variable domains
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DOI：
10.1016/s0022-2836(02)01237-8
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发表时间：
2003-01-17
影响因子：
5.6
通讯作者：
Plückthun, A
中科院分区：
文献类型：
--
作者：
Ewert, S;Huber, T;Plückthun, A
对抗体片段的稳定性、表达量和抗聚集性有很高的要求。为了从结构域相互作用中解开内在结构域效应，我们首先提出了对分离的人免疫球蛋白可变重链（V-H）和轻链（V-L）种系家族共有结构域的系统评价，然后提出了scFv形式的V-H-V-L组合的系统系列。根据它们的表达行为、溶液中的寡聚状态和非还原条件下变性剂诱导的解折叠平衡来评价构建体。七个V-H和七个V-L结构域代表主要人种系亚类的共有序列，来源于人组合抗体文库（HuCAL（R））。分离的具有最高热力学稳定性和可溶性蛋白产量的V-H和V-L结构域分别为V（H）3和V（kappa）3。对scFv片段中所有结构域组合的类似测量允许根据热力学稳定性和体内折叠产率对scFv片段进行分类。含有可变结构域组合H3 kappa 3、H1bkappa 3、H5 kappa 3和H3 kappa 1的scFv片段显示出关于产率和稳定性的上级性质。域的相互作用减少了域的内在差异。含有V-λ结构域的ScFv片段显示出高水平的稳定性，即使V-λ结构域本身令人惊讶地不稳定。这是由于与V-H结构域的强相互作用，并且取决于CDR-L3的氨基酸序列。在这些分析和模型结构的基础上，我们提出了进一步改善个人框架的生物物理特性的可能性，并为图书馆设计提出了建议。(C)2003爱思唯尔科技有限公司版权所有。
There are great demands on the stability expression yield and resistance to aggregation of antibody fragments. To untangle intrinsic domain effects from domain interactions, we present first a systematic evaluation of the isolated human immunoglobulin variable heavy (V-H) and light (V-L) germline family consensus domains and then a systematic series Of V-H-V-L combinations in the scFv format. The constructs were evaluated in terms of their expression behavior, oligomeric state in solution and denaturant-induced unfolding equilibria under non-reducing conditions. The seven V-H and seven V-L domains represent the consensus sequences of the major human germline subclasses, derived from the Human Combinatorial Antibody Library (HuCAL(R)). The isolated V-H and V-L domains with the highest thermodynamic stability and yield of soluble protein were V(H)3 and V(kappa)3, respectively. Similar measurements on all domain combinations in scFv fragments allowed the scFv fragments to be classified according to thermodynamic stability and in vivo folding yield. The scFv fragments containing the variable domain combinations H3kappa3, H1bkappa3, H5kappa3 and H3kappa1 show superior properties concerning yield and stability. Domain interactions diminish the intrinsic differences of the domains. ScFv fragments containing V-lambda domains show high levels of stability, even though V-lambda domains are surprisingly unstable by themselves. This is due to a strong interaction with the V-H domain and depends on the amino acid sequence of the CDR-L3. On the basis of these analyses and model structures, we suggest possibilities for further improvement of the biophysical properties of individual frameworks and give recommendations for library design. (C) 2003 Elsevier Science Ltd. All rights reserved.