Affinity maturation is a powerful technique in antibody engineering for the in vitro evolution of antigen binding interactions. Key to the success of this process is the expansion of sequence and combinatorial diversity to increase the structural repertoire from which superior binding variants may be selected. However, conventional strategies are often restrictive and only focus on small regions of the antibody at a time. In this study, we used a method that combined antibody chain shuffling and a staggered-extension process to produce unbiased libraries, which recombined beneficial mutations from all six complementarity-determining regions (CDRs) in the affinity maturation of an inhibitory antibody to Arginase 2 (ARG2). We made use of the vast display capacity of ribosome display to accommodate the sequence space required for the diverse library builds. Further diversity was introduced through pool maturation to optimize seven leads of interest simultaneously. This resulted in antibodies with substantial improvements in binding properties and inhibition potency. The extensive sequence changes resulting from this approach were translated into striking structural changes for parent and affinity-matured antibodies bound to ARG2, with a large reorientation of the binding paratope facilitating increases in contact surface and shape complementarity to the antigen. The considerable gains in therapeutic properties seen from extensive sequence and structural evolution of the parent ARG2 inhibitory antibody clearly illustrate the advantages of the unbiased approach developed, which was key to the identification of high-affinity antibodies with the desired inhibitory potency and specificity.

亲和力成熟是抗体工程中用于抗原结合相互作用的体外进化的强大技术。该过程成功的关键是扩展序列和组合多样性，以增加结构库，从中可以选择优良的结合变体。然而，常规策略通常是限制性的，并且一次只关注抗体的小区域。在这项研究中，我们使用了一种将抗体链改组和交错延伸过程相结合的方法来产生无偏文库，该文库在抑制性抗体对精氨酸酶2的亲和力成熟中重组了来自所有六个互补决定区（CDR）的有益突变。 ARG2）。我们利用核糖体展示的巨大展示能力来适应各种文库构建所需的序列空间。通过库成熟引入了进一步的多样性，以同时优化七个感兴趣的潜在客户。这导致抗体在结合特性和抑制能力上有了实质性的改善。这种方法产生的广泛序列变化被转化为与ARG2结合的亲本抗体和亲和力成熟抗体的显着结构变化，结合互补位的大方向改变促进了与抗原的接触表面和形状互补性的增加。