Abstract
Immunization-based antibody discovery platforms require robust and effective protocols for the amplification, cloning, expression, and screening of antibodies from large numbers of B-cells in order to effectively capture the diversity of an experienced Ig-repertoire. Multiplex PCR using a series of forward and reverse primers designed to recover antibodies from a range of different germline sequences is challenging because primer design requires the recovery of full length antibody sequences, low starting template concentrations, and the need for all the primers to function under the same PCR conditions. Here we demonstrate several advantages to incorporating RNase H2-dependent PCR (rh-PCR) into a high-throughput, antibody-discovery platform. Firstly, rh-PCR eliminated primer dimer synthesis to below detectable levels, thereby eliminating clones with a false positive antibody titer. Secondly, by increasing the specificity of PCR, the rh-PCR primers increased the recovery of cognate antibody variable regions from single B-cells, as well as downstream recombinant antibody titers. Finally, we demonstrate that rh-PCR primers provide a more homogeneous sample pool and greater sequence quality in a Next Generation Sequencing-based approach to obtaining DNA sequence information from large numbers of cloned antibody cognate pairs. Furthermore, the higher specificity of the rh-PCR primers allowed for a better match between native antibody germline sequences and the VL/VH fragments amplified from single B-cells.
Highlights
In 2018, there were 64 FDA-approved therapeutic antibodies on the market with total sales exceeding $100 billion
Following cDNA synthesis, a separate amplification reaction (PCR #1) is done using primers that anneal to the signal peptide sequences upstream of the antibodies variable light (VL) or heavy variable heavy chain (VH) (VH) sequences and the constant kappa/lambda or IgG domains
A nested PCR (PCR #2) is performed using primers that anneal to the framework 1 (FR1) and J-region sequences of the antibody variable regions
Summary
In 2018, there were 64 FDA-approved therapeutic antibodies on the market with total sales exceeding $100 billion. As additional antibody-based therapeutics come to market, global sales are projected to reach $300 billion by 2025 [1,2,3]. Productive antibody discovery platforms will continue to play a critical role towards delivering on this important class of therapeutics. RNase H-dependent PCR and high-throughput antibody discovery no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Eli Lilly & Company approved the publication of this work
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