A microbial expression system for high-level production of scFv HIV-neutralizing antibody fragments in Escherichia coli

Marloes L C Petrus,Evert Heemskerk,Dan H Barouch,Gilles P Van Wezel,Pranav Puri,Lukas A Kiefer,Sagrario Arias,Menzo Havenga,Michael S Seaman

doi:10.1007/s00253-019-10145-1

Abstract

Monoclonal antibodies (mABs) are of great biopharmaceutical importance for the diagnosis and treatment of diseases. However, their production in mammalian expression hosts usually requires extensive production times and is expensive. Escherichia coli has become a new platform for production of functional small antibody fragment variants. In this study, we have used a rhamnose-inducible expression system that allows precise control of protein expression levels. The system was first evaluated for the cytoplasmic production of super folder green fluorescence protein (sfGFP) in various production platforms and then for the periplasmic production of the anti-HIV single-chain variable antibody fragment (scFv) of PGT135. Anti-HIV broadly neutralizing antibodies, like PGT135, have potential for clinical use to prevent HIV transmission, to promote immune responses and to eradicate infected cells. Different concentrations of L-rhamnose resulted in the controlled production of both sfGFP and scFv PGT135 antibody. In addition, by optimizing the culture conditions, the amount of scFv PGT135 antibody that was expressed soluble or as inclusions bodies could be modulated. The proteins were produced in batch bioreactors, with yields of 4.9 g/L for sfGFP and 0.8 g/L for scFv. The functionality of the purified antibodies was demonstrated by their ability to neutralize a panel of different HIV variants in vitro. We expect that this expression system will prove very useful for the development of a more cost-effective production process for proteins and antibody fragments in microbial cells.

Highlights

Over the past years, the development of the antibody therapeutic field has made significant progress, driving a sustained increase in the number of antibodies that are granted their first marketingElectronic supplementary material The online version of this article contains supplementary material, which is available to authorized users.approvals each year, with a new record of 10 monoclonal antibody therapeutics approved in 2017 (Kaplon and Reichert 2018)
Cells were grown in low salt Luria-Bertani broth (LB) or on low salt LB agar plates at 37 °C and selection pressure was applied by the addition of 25 μM of Zeocin
The gene coding for super-folded green fluorescent protein (Pédelacq et al 2006) and for PGT135 scFv antibody fragment was codon optimized and synthesized by Invitrogen GeneArt Gene Synthesis (Regensburg, Germany) and cloned into the vector

Summary

Introduction

The development of the antibody therapeutic field has made significant progress, driving a sustained increase in the number of antibodies that are granted their first marketing. Approvals each year, with a new record of 10 monoclonal antibody (mAb) therapeutics approved in 2017 (Kaplon and Reichert 2018). The potential impact of monoclonal antibodies on the entire pharmaceutical industry is illustrated by their global sales in 2017, which grossed $108 billion, and it is expected to continue growing during the upcoming years (Grilo and Mantalaris 2019). Most of the mAbs are produced using mammalian cells, which can perform human-like N-glycosylation as posttranslational modifications. A disadvantage of using mammalian cell culture for heterologous protein production is an inconvenient and time-consuming production process, which is sometimes difficult to scale up. Other issues relate to typically low product yields and growth rates, the risk of viral contamination, and the need for complex growth

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Conclusion