Rapid generation of potent antibodies by autonomous hypermutation in yeast

Abstract

The predominant approach for antibody generation remains animal immunization, which can yield exceptionally selective and potent antibody clones owing to the powerful evolutionary process of somatic hypermutation. However, animal immunization is inherently slow, not always accessible and poorly compatible with many antigens. Here, we describe 'autonomous hypermutation yeast surface display' (AHEAD), a synthetic recombinant antibody generation technology that imitates somatic hypermutation inside engineered yeast. By encoding antibody fragments on an error-prone orthogonal DNA replication system, surface-displayed antibody repertoires continuously mutate through simple cycles of yeast culturing and enrichment for antigen binding to produce high-affinity clones in as little as two weeks. We applied AHEAD to generate potent nanobodies against the SARS-CoV-2 S glycoprotein, a G-protein-coupled receptor and other targets, offering a template for streamlined antibody generation at large.

Evaluation and Selection of Potent Fluorescent Immunosensors by Combining Fluorescent Peptide and Nanobodies Displayed on Yeast Surface

“…This is probably because we used only 30 clones, which were selected by phage display but were not selected by the performance as a Q-body. Therefore, future work will focus on the construction of a synthetic library suitable for potent Q-bodies and directed evolution for various Qbodies that do not achieve su cient sensitivity in practical applications by combining recent novel evolutionary approaches using yeast surface display method 29 .…”

Full speed AHEAD in antibody discovery

“…Foreshadowing the promise of their new system in end-to-end antibody development, Wellner et al 1 adapted a 2 × 10 5 member nanobody library 8 onto the AHEAD platform and performed a successful discovery and affinity maturation campaign. This exciting proof of concept establishes compatibility of their technique with isolating binders from a naïve pool of antibodies and affinity maturation of these clones within the same selection scheme.…”

“…However, these approaches are limited with respect to their efficacy, speed, and scalability. In a new study, Wellner et al 1 addressed these limitations by developing a synthetic recombinant antibody generation technology that integrates an orthogonal DNA replication system (OrthoRep) 2 with yeast surface display 3 to allow continuous gene mutation and accelerated antibody evolution.…”

“…Building on exciting advances in the protein engineering field, Wellner et al 1 sought to develop an accelerated evolutionary framework for antibody discovery that synthesizes OrthoRep with yeast surface display technology.…”

“…The error rate in replicating the p1 plasmid is 100,000-fold higher than that of the yeast genome, leading to targeted diversification of the gene of interest without compromising fitness of the yeast cell host. The platform developed by Wellner et al 1 , known as autonomous hypermutation yeast surface display (AHEAD), entails iterative rounds of evolution with minimal user intervention. The antibody of interest is encoded on the p1 plasmid to allow continuous mutagenesis, and fluorescence-activated cell sorting (FACS) selections are interspersed with generations of yeast cell growth to orchestrate the enrichment of high-affinity antibodies against the desired target protein (Fig.…”