Context-dependent mutations predominate in an engineered high-affinity single chain antibody fragment
Context-dependent mutations predominate in an engineered high-affinity single chain antibody fragment
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DOI：
10.1110/ps.051842406
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发表时间：
2006-02-01
期刊：
影响因子：
8
通讯作者：
Wittrup, KD
中科院分区：
文献类型：
--
作者：
Midelfort, KS;Wittrup, KD
与其野生型祖细胞4-4-20相比，飞摩尔亲和力抗荧光素抗体4M5.3的突变分析表明，背景依赖性和非依赖性突变都是亲和力提高1800倍的原因。4M5.3是通过定向进化从4-4-20改造而来的，含有14个突变。在此研究了鉴定为存在于10个最后一轮亲和力成熟克隆中的每一个中的七个突变。比较了4-4-20单突变体添加和4M5.3单位点回复突变体的亲和力。这些实验确定了这七个突变中的四个突变，它们在对更高亲和力的贡献方面是依赖于上下文的。创建仅包含这七个突变的简化突变体以分析所选突变组的完整双突变体循环。研究的特定突变集包括配体接触突变、重链CDR 3突变、重链CDR 3突变加上位点H108处的相邻残基以及定向进化途径上的早期和晚期获得性突变。显示重链CDR 3突变组和配体接触突变分别提供七位点共有突变体的结合自由能的总约3.5kcal/mol变化的约1.4和约2.0kcal/mol。在定向进化轮中后期获得的突变提供了自由能的大部分变化，而没有较早获得的突变（总-3.5kcal/mol的-3.1kcal/mol）。先前的结构数据和静电计算提出了几种更高亲和力贡献的假设，其中一些得到了这些突变数据的支持。
A mutational analysis of the femtomolar-affinity anti-fluorescein antibody 4M5.3, compared to its wild-type progenitor, 4-4-20, indicates both context-dependent and -independent mutations are responsible for the 1800-fold affinity improvement. 4M5.3 was engineered from 4-4-20 by directed evolution and contains 14 mutations. The seven mutations identified as present in each of 10 final round affinity maturation clones were studied here. Affinities of the 4-4-20 single mutant addition and 4M5.3 single site reversion mutants were compared. These experiments identified four mutations, of these seven, that were context-dependent in their contribution to higher affinity. A simplified mutant containing only these seven mutations was created to analyze complete double mutant cycles of selected sets of mutations. Specific mutational sets studied included the ligand contact mutations, the heavy chain CDR3 mutations, the heavy chain CDR3 mutations plus the neighboring residue at site H108, and the early and late acquired mutations on the directed evolution pathway. The heavy chain CDR3 mutational set and the ligand-contacting mutations were shown to provide -1.4 and -2.0 kcal/mol, respectively, of the total -3.5 kcal/mol change in free energy of binding of the seven-site consensus mutant. The mutations acquired late in the directed evolution rounds provided much of the change in free energy without the earlier acquired mutations (-3.1 kcal/mol of the total -3.5 kcal/mol). Prior structural data and electrostatic calculations presented several hypotheses for the higher affinity contributions, some of which are supported by these mutational data.