ABSTRACT

In vitro

affinity maturation of therapeutic monoclonal antibodies is commonly applied to achieve desired properties, such as improved binding kinetics and affinity. Currently there are no universally accepted protocols for generation of variegated antibody libraries or selection thereof. Here, we performed affinity maturation using a yeast-based single-chain variable fragment (scFv) expression system to compare two mutagenesis methods: random mutagenesis across the entire V(D)J region by error-prone PCR, and a novel combinatorial mutagenesis process limited to the complementarity-determining regions (CDRs). We applied both methods of mutagenesis to four human antibodies against well-known immuno-oncology target proteins. Detailed sequence analysis showed an even mutational distribution across the entire length of the scFv for the error-prone PCR method and an almost exclusive targeting of the CDRs for the combinatorial method. Though there were distinct mutagenesis profiles for each target antibody and mutagenesis method, we found that both methods improved scFv affinity with similar efficiency. When a subset of the affinity-matured antibodies was expressed as full-length immunoglobulin, the measured affinity constants were mostly comparable to those of the respective scFv, but the full-length antibodies were inferior to their scFv counterparts for one of the targets. Furthermore, we found that improved affinity for the full-length antibody did not always translate into enhanced binding to cell-surface expressed antigen or improved immune checkpoint blocking ability, suggesting that screening with full-length antibody or antigen-binding fragment formats might be advantageous and the subject of a future study.

中文翻译：

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通过组合密码子诱变与易错PCR进行抗​​体的亲和力成熟。

摘要

体外

治疗性单克隆抗体的亲和力成熟通常用于获得所需的特性，例如改善的结合动力学和亲和力。当前，尚无普遍接受的用于产生杂色抗体文库或其选择的方案。在这里，我们使用基于酵母的单链可变片段（scFv）表达系统进行亲和力成熟，以比较两种诱变方法：通过易错PCR在整个V（D）J区域进行随机诱变，以及新颖的组合诱变过程限于互补决定区（CDR）。我们将这两种诱变方法应用于针对著名免疫肿瘤靶蛋白的四种人类抗体。详细的序列分析显示，对于容易出错的PCR方法，scFv整个长度上的突变分布均匀，而对于组合方法，CDR几乎是靶向的。尽管每种靶抗体和诱变方法都有不同的诱变概况，但我们发现这两种方法均以相似的效率提高了scFv亲和力。当亲和力成熟的抗体的一个子集表达为全长免疫球蛋白时，测得的亲和常数几乎与相应的scFv相当，但对于其中一个靶标，全长抗体却不如其scFv对应物。此外，