This result suggests that a diverse native pairing library of ~20,000 unique antibody sequences can be assembled in just one 96-well plate PCR reaction, followed by concurrent specificity characterization against 10-20 antigens of choice within days. Such a scale would be truly unprecedented. (11/)

In the update, we tested the limit of our assembly strategy as we increased the # of scFv to assemble in a single PCR. Our one-pot PCR strategy performed amazingly well, as it achieved extremely high reproducibility and coverage even with the assembly of different 200 scFvs in a single tube. (9/)

Through computational design, we overcame this challenge by splitting a defined scFv sequence into fragments and assembling them back through PCR. To ensure precise assembly, we utilized the highly diverse CDR regions to computationally design overlaps that are unique at the nucleotide level. (9/)

The key innovation of oPool+ display lies within the assembly of native paring antibodies, as current technologies does not permit massively parallel oligo synthesis >350nt, which is only 1/3 of the length of a single-chain variable fragment (scFv), the shortest human antibody format. (8/)

We then selected 25 antibodies and thoroughly tested their binding against all 9 HAs by both BLI (using Fab) and ELISA (using IgG). Both validations indicated >70% of true positive rate and >95% of true negative rate, demonstrating the robust performance of oPool+ display. (7/)

Our screening showed that 114 of the 325 antibodies bound to at least one of the nine HA screened, 45 of which were further identified to target the more conserved stem domain through either HA stem screens or binding competition with CR9114, a known HA stem antibody. (6/n)

As a proof-of-concept, we applied oPool+ display to rapidly synthesize >300 uncharacterized and uncommon influenza hemagglutinin (HA) antibodies and measure their binding to 9 HA variants through 16 different screens. Over 5,000 binding tests were performed in < 5 days. (5/)

To address this, we combined oligo pool synthesis with mRNA display to synthesize and screen antibodies with defined sequences at scale. Compared to the conventional approaches, our platform is significantly faster (~3-5 days), costs ~80-90% less, and only requires one person to perform it. (4/)

Considering that hundreds to thousands of natively paired antibody sequences can usually be isolated in a clinical study, this process would often take dedicated research teams weeks to months to complete and cost up to hundreds of thousands of dollars. A bottleneck clearly exists. (3/)

Antibody discovery is crucial to developing therapeutics and understanding adaptive immunity. However, despite years of research, the characterization of monoclonal antibodies today remains low throughput, as it involves cloning, expressing, and characterizing each antibody one by one (2/)