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New Technology Paired with NGS, Paves the Way for Novel Therapeutic Antibodies

Jun 08, 2018

Novel Therapeutic Antibodies Picture Next generation sequencing is paving the way for scientists to reach new heights in antibody engineering. In a study published in Nature, a lab used NGS and their own technology to screen millions of B-cells for natively paired human antibody repertories. Further analysis identified a novel therapeutic antibody against the Ebola virus.

The human B-cell receptor repertoire has become an invaluable resource for discovering new therapeutic antibodies. These B-cells have been collected using different techniques such as cloning individual B cells by immortalization and expansion or by a technique called single cell sorting. Single cell sorting of individual B-cells obtained by fluorescence-activated cell sorting (FACS) or by limiting dilution has been used to discover anti-infective antibodies including broadly neutralizing antibodies (bNAbs) for viral disease such as influenza or HIV-1. Using this technique, combinatorial libraries generating random pairings of amplified V_H and V genes from human B-cells have yielded thousands of antibodies. There are some drawbacks to single cell cloning, 1) the process is time consuming, 2) the process is resource intensive and 3) because of 1 and 2 only a small fraction of the human antibody repertoire can be studied at a time. In this study, scientists have designed a new technology for large scale functional interrogation of the natively paired V_H: V_L antibody repertoire. First since V_H and V_L genes are encoded by separate mRNA transcripts, they can be physically linked to a single amplicon and cloned into an expression vector. V_H and V_L linkage is accomplished using two step single-cell emulsion lysis and oligo-dT capture of the V_H and V_L transcripts from a single B-cell. The process is followed by reverse transcriptase and overlap-extension PCR to create amplicons.

In these amplicons, V_H and V_L genes are joined through a linker that is designed to enable one-step cloning into a yeast Fab surface expression vector. Using a yeast display platform originally optimized for human Fab, scientists were able to discover an HIV-1 bNAbs from an HIV-1 slow progressor and a high-affinity neutralizing antibodies against Ebola virus glycoprotein and influenza hemagglutinin. This new technology used a combinantion of next generation sequencing, recombinant antibody production, and novel technology to discover new therapeutic antibodies for a variety of diseases.