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.

Functional interrogation and mining of natively paired human V_H:V_L antibody repertoires.

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