Abstract
The human antibody repertoire is increasingly being recognized as a valuable source of therapeutic grade antibodies. However, methods for mining primary antibody-expressing B cells are limited in their ability to rapidly isolate rare and antigen-specific binders. Here we show the encapsulation of two million primary B cells into picoliter-sized droplets, where their cognate V genes are fused in-frame to form a library of scFv cassettes. We used this approach to construct natively paired phage-display libraries from healthy donors and drove selection towards cross-reactive antibodies targeting influenza hemagglutinin. Within 4 weeks we progressed from B cell isolation to a panel of unique monoclonal antibodies, including seven that displayed broad reactivity to different clinically relevant influenza hemagglutinin subtypes. Most isolated antibody sequences were not detected by next-generation sequencing of the paired repertoire, illustrating how this method can isolate extremely rare leads not likely found by existing technologies.
Highlights
The human antibody repertoire is increasingly being recognized as a valuable source of therapeutic grade antibodies
Both of these methods suffer from low screening throughput that overwhelmingly undersamples the ~107 B cells obtained from a typical blood draw
Cognate chain pairing from encapsulated primary B cells
Summary
The human antibody repertoire is increasingly being recognized as a valuable source of therapeutic grade antibodies. The B cells producing these therapeutic antibodies tend to be rare in convalescent patients, making their discovery very challenging Adding to this challenge is the fact that antibodies are heterodimeric proteins whose specificities are encoded by unique pairs of heavy-chain and light-chain transcripts. Validating leads requires gene synthesis, cloning, and expression which can create a severe bottleneck in the number of candidates that can be functionally assessed[14] Both of these methods suffer from low screening throughput that overwhelmingly undersamples the ~107 B cells obtained from a typical blood draw. We report the creation of a microfluidic platform that pairs cognate VH and VL transcripts from millions of single cells into “expression-ready” scFv libraries, while still maintaining the ability to profile the paired repertoire by NGS (Fig. 1a) We coupled these recombinant repertoires with the enormous screening power of phage-display to rapidly enrich for antigenspecific clones. We interrogated the antibody repertoires from healthy individuals by screening for influenza hemagglutinin (HA) binding and selected for a panel of crossreactive leads targeting multiple HA subtypes
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