The influence of the framework core residues on the biophysical properties of immunoglobulin heavy chain variable domains
The influence of the framework core residues on the biophysical properties of immunoglobulin heavy chain variable domains
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DOI：
10.1093/protein/gzn077
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发表时间：
2009-03-01
影响因子：
2.4
通讯作者：
Plueckthun, Andreas
中科院分区：
文献类型：
--
作者：
Honegger, Annemarie;Malebranche, Alain Daniel;Plueckthun, Andreas
抗体可变区的稳定性差异很大。来源于天然谱系的单链抗体(Single Chain Fv，ScFv)片段往往缺乏治疗应用所需的高度稳定性，这就需要对其进行重组，不仅使其序列人性化，而且还改善其生物物理性质。人类V(H)3结构域被认为是人类亚型中生物物理性质最好的。然而，从高度分散的V-H结构域到huv(H)3的互补决定区(CDR)移植到huv(H)3上往往无法达到其优越的稳定性。在以往从稳定性很差的小鼠V(H)9结构域到huv(H)3的CDR接枝实验中，已经获得了将Muv(H)9的低核心残基和huv(H)3的表面残基结合在一起的杂化V-H骨架。这导致了一种生物物理性质远远好于共识huv(H)3骨架的相应移植物的单链抗体。为了更好地了解杂交框架优越属性的起源，我们构建了更多的杂交体，但现在是在原始人类V(H)3结构域的CDR-H1和-H2的共识背景下。新的杂交物包括来自小鼠V(H)9、人V(H)1或人V(H)5结构域的元件。通过氯化胍诱导的平衡变性实验、动力学变性实验、热诱导聚集实验和在大肠杆菌中可溶性表达产量的比较，我们得出结论：最优的V-H骨架是CDR依赖的。本工作查明了导致这种依赖的结构特征，并有助于解释为什么免疫系统使用一个以上的框架，在框架1中具有不同的结构亚型，以最佳地支持广泛不同的CDR。
Antibody variable domains differ considerably in stability. Single-chain Fv (scFv) fragments derived from natural repertoires frequently lack the high stability needed for therapeutic application, necessitating reengineering not only to humanize their sequence, but also to improve their biophysical properties. The human V(H)3 domain has been identified as having the best biophysical properties among human subtypes. However, complementarity determining region (CDR) grafts from highly divergent V-H domains to huV(H)3 frequently fail to reach its superior stability. In previous experiments involving a CDR graft from a murine V(H)9 domain of very poor stability to huV(H)3, a hybrid V-H framework was obtained which combines the lower core residues of muV(H)9 with the surface residues of huV(H)3. It resulted in a scFv with far better biophysical properties than the corresponding grafts to the consensus huV(H)3 framework. To better understand the origin of the superior properties of the hybrid framework, we constructed further hybrids, but now in the context of the consensus CDR-H1 and -H2 of the original human V(H)3 domain. The new hybrids included elements from either murine V(H)9, human V(H)1 or human V(H)5 domains. From guanidinium chloride-induced equilibrium denaturation measurements, kinetic denaturation experiments, measurements of heat-induced aggregation and comparison of soluble expression yield in Escherichia coli, we conclude that the optimal V-H framework is CDR-dependent. The present work pinpoints structural features responsible for this dependency and helps to explain why the immune system uses more than one framework with different structural subtypes in framework 1 to optimally support widely different CDRs.