Abstract
Antibodies developed for research and clinical applications may exhibit suboptimal stability, expressibility, or affinity. Existing optimization strategies focus on surface mutations, whereas natural affinity maturation also introduces mutations in the antibody core, simultaneously improving stability and affinity. To systematically map the mutational tolerance of an antibody variable fragment (Fv), we performed yeast display and applied deep mutational scanning to an anti-lysozyme antibody and found that many of the affinity-enhancing mutations clustered at the variable light-heavy chain interface, within the antibody core. Rosetta design combined enhancing mutations, yielding a variant with tenfold higher affinity and substantially improved stability. To make this approach broadly accessible, we developed AbLIFT, an automated web server that designs multipoint core mutations to improve contacts between specific Fv light and heavy chains (http://AbLIFT.weizmann.ac.il). We applied AbLIFT to two unrelated antibodies targeting the human antigens VEGF and QSOX1. Strikingly, the designs improved stability, affinity, and expression yields. The results provide proof-of-principle for bypassing laborious cycles of antibody engineering through automated computational affinity and stability design.
Highlights
High-affinity natural antibodies are generated through an iterative process of mutation and selection for antigen binding known as affinity maturation
Many of the affinity-enhancing mutations clustered at the variable light-heavy chain interface
The positions encompassed most of the complementarity-determining regions (CDRs), the vL-vH interface and additional peripheral positions (Fig 1A)
Summary
High-affinity natural antibodies are generated through an iterative process of mutation and selection for antigen binding known as affinity maturation. Synthetic antibody repertoires have been widely adopted in antibody discovery and optimization, providing greater control over the selection process than animal immunization In this approach, a library of antibody variable fragments (Fv) is displayed, for instance on yeast cells, and screened to select high-affinity binders or to improve the affinity of existing antibodies [3]. A library of antibody variable fragments (Fv) is displayed, for instance on yeast cells, and screened to select high-affinity binders or to improve the affinity of existing antibodies [3] These methods are powerful [4,5], but a large fraction of high-affinity antibodies isolated from synthetic repertoires exhibits impaired stability [6]. General methods to improve antibody stability while maintaining or even increasing affinity are urgently needed to reduce the attrition rate in antibody development pipelines [11]
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