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Abstract
Biopharmaceuticals are a large and fast-growing sector of the total pharmaceutical market with antibody-based therapeutics accounting for over 100 billion USD in sales yearly. Mammalian cells are traditionally used for monoclonal antibody production, however plant-based expression systems have significant advantages. In this work, we showcase recent advances made in plant transient expression systems using optimized geminiviral vectors that can efficiently produce heteromultimeric proteins. Two, three, or four fluorescent proteins were coexpressed simultaneously, reaching high yields of 3–5 g/kg leaf fresh weight or ~50% total soluble protein. As a proof-of-concept for this system, various antibodies were produced using the optimized vectors with special focus given to the creation and production of a chimeric broadly neutralizing anti-flavivirus antibody. The variable regions of this murine antibody, 2A10G6, were codon optimized and fused to a human IgG1. Analysis of the chimeric antibody showed that it was efficiently expressed in plants at 1.5 g of antibody/kilogram of leaf tissue, can be purified to near homogeneity by a simple one-step purification process, retains its ability to recognize the Zika virus envelope protein, and potently neutralizes Zika virus. Two other monoclonal antibodies were produced at similar levels (1.2–1.4 g/kg). This technology will be a versatile tool for the production of a wide spectrum of pharmaceutical multi-protein complexes in a fast, powerful, and cost-effective way.
Highlights
Antibody-based therapeutics are the largest sector of the global biopharmaceutical market, with sales exceeding 100 billion USD worldwide and sales predicted to reach 137–200 billion USD by 2022 (Grilo and Mantalaris, 2019)
In order to study the co-expression of multiple genes, we created an optimized vector by the following method: the coding sequences for green fluorescent protein (GFP) and DsRed were incorporated into vectors containing a single bean yellow dwarf virus (BeYDV) replicon, the NbPsaK 5′ UTR, a double terminator consisting of the intronless tobacco extensin terminator fused to the NbACT3 3′ UTR, and the Rb7 matrix attachment region (Figure 1) (Diamos et al, 2016; Diamos and Mason, 2018a,c)
As plants already contained the required glycoform, plantbased production of glucocerebrosidase reduced costs as well as potentially improved consistency and efficacy (Zimran et al, 2011). These advances led to the first FDA approval of a plantbased therapeutic (Fox, 2012) while further research has led to the development of a lyophilized carrot cell juice which restores healthy enzyme levels in patients when consumed orally (Shaaltiel et al, 2015)
Summary
Antibody-based therapeutics are the largest sector of the global biopharmaceutical market, with sales exceeding 100 billion USD worldwide and sales predicted to reach 137–200 billion USD by 2022 (Grilo and Mantalaris, 2019). While most biopharmaceuticals have traditionally been produced in mammalian cell culture systems, plant-based recombinant expression systems have demonstrated significant advantages. Plant systems do not need to be grown in sterile conditions and, as plants lack animal pathogens, plant-based biotechnology has improved intrinsic safety over mammalian expression systems (Sack et al, 2015b). These factors allow highly scalable production of biopharmaceutical proteins with substantially reduced costs (Tusé et al, 2014; Walwyn et al, 2015; Nandi et al, 2016; Alam et al, 2018; Mir-Artigues et al, 2019). The cost-effectiveness of plant-based systems may especially benefit developing countries (Ma et al, 2013)
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