Structure-based improvement of the biophysical properties of immunoglobulin VH domains with a generalizable approach
Structure-based improvement of the biophysical properties of immunoglobulin VH domains with a generalizable approach
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DOI：
10.1021/bi026448p
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发表时间：
2003-02-18
期刊：
影响因子：
2.9
通讯作者：
Plückthun, A
中科院分区：
文献类型：
--
作者：
Ewert, S;Honegger, A;Plückthun, A
在用共有V-H和V-L结构域单独和以scFv形式组合进行的V基因家族的系统研究中，我们发现在含有人种系家族2、4和6的VH结构域的抗体片段中，相对较低的表达产率和较低的平衡解折叠协同性。从疏水核心的包装分析，电荷簇的完整性，不满意的氢键的发生，以及具有低β-折叠倾向的残基，正（D）角和暴露的疏水侧链，我们查明了可能导致这些种系编码序列的不令人满意的性质的残基。其中有几个是共同的领域之间的偶数分组，但不发生在奇数的。在这项研究中，我们系统地交换了这些残基单独和组合在两个不同的scFv使用V（H）6框架，我们描述了它们对平衡稳定性和折叠产量的影响。我们将稳定性提高了20.9 kJ/mol，表达产率提高了4倍，现在可以使用这些数据来合理地工程化衍生自该和类似种系家族的抗体，以获得更好的生物物理特性。此外，我们提供了一个改进的设计库利用这些框架提供的显着的额外的多样性。本文研究的两种抗体完全保留其结合亲和力，表明CDR构象不受影响。
In a systematic study of V gene families carried out with consensus V-H and V-L domains alone and in combinations in the scFv format, we found comparatively low expression yields and lower cooperativity in equilibrium unfolding in antibody fragments containing VH domains of human germline families 2, 4, and 6. From an analysis of the packing of the hydrophobic core, the completeness of charge clusters, the occurrence of unsatisfied hydrogen bonds, and residues with low beta-sheet propensities, positive (D angles, and exposed hydrophobic side chains, we pinpointed residues potentially responsible for the unsatisfactory properties of these germline-encoded sequences. Several of those are in common between the domains of the even-numbered subgroups, but do not occur in the odd-numbered ones. In this study, we have systematically exchanged those residues alone and in combination in two different scFvs using the V(H)6 framework, and we describe their effect on equilibrium stability and folding yield. We improved the stability by 20.9 kJ/mol and the expression yield by a factor of 4 and can now use these data to rationally engineer antibodies derived from this and similar germline families for better biophysical properties. Furthermore, we provide an improved design for libraries exploiting the significant additional diversity provided by these frameworks. Both antibodies studied here completely retain their binding affinity, demonstrating that the CDR conformations were not affected.