We report a systematic combinatorial exploration of affinity enhancement of antibodies by insertions and deletions (InDels). Transposon-based introduction of InDels via the method TRIAD (transposition-based random insertion and deletion mutagenesis) was used to generate large libraries with random in-frame InDels across the entire single-chain variable fragment gene that were further recombined and screened by ribosome display. Knowledge of potential insertion points from TRIAD libraries formed the basis of exploration of length and sequence diversity of novel insertions by insertional-scanning mutagenesis (InScaM). An overall 256-fold affinity improvement of an anti–IL-13 antibody BAK1 as a result of InDel mutagenesis and combination with known point mutations validates this approach, and suggests that the results of this InDel mutagenesis and conventional exploration of point mutations can synergize to generate antibodies with higher affinity.

我们报告了通过插入和删除（InDels）抗体的亲和力增强的系统组合探索。通过基于TRIAD方法的基于转座子的InDel导入（基于换位的随机插入和缺失诱变）被用于生成大型文库，该文库在整个单链可变片段基因中具有随机符合读框的InDel，并通过核糖体展示进一步重组和筛选。TRIAD库中潜在插入点的知识构成了通过插入扫描诱变（InScaM）探索新颖插入物的长度和序列多样性的基础。通过InDel诱变以及与已知点突变的结合，抗IL-13抗体BAK1的整体亲和力提高256倍，验证了该方法的有效性，