An Effective Way of Producing Fully Assembled Antibody in Transgenic Tobacco Plants by Linking Heavy and Light Chains via a Self-Cleaving 2A Peptide

Abstract

Therapeutic monoclonal antibodies (mAbs) have evolved into an important class of effective medicine in treatment of various diseases. Since the antibody molecule consists of two identical heavy chains (HC) and two light chains (LC), each chain encoded by two different genes, their expressions at similar levels are critical for efficient assembly of functional recombinant mAbs. Although the plant-based expression system has been tested to produce fully assembled recombinant mAbs, coordinately expressing HC and LC at similar levels in a transgenic plant remains a challenge. A sequence coding for a foot-and-mouth disease virus (FMDV) 2A peptide has been successfully used to link two or more genes, which enable the translated polyprotein to be “self-cleaved” into individual protein in various genetically modified organisms. In the present study, we exploited the usage of F2A in Ebola virus monoclonal antibody (EBOV mAb) production. We found that transgenic tobacco plants carrying a transcription unit containing HC and LC linked by 2A not only produced similar levels of HC and LC but also rendered a higher yield of fully assembled EBOV mAb compared to those expressing HC and LC in two independent transcription units. Purified EBOV mAb bound to an Ebola epitope peptide with apparent Kd-values of 90.13–149.2 nM, indicating its proper assembly and high affinity binding to Ebola epitope peptide. To our knowledge, this is the first report showing mAb production by overexpressing a single transcription unit consisting of HC, LC and 2A in stable transformed tobacco plants.

Nicotiana spp. for the Expression and Purification of Functional IgG3 Antibodies Directed Against the Staphylococcus aureus Alpha Toxin

“…IgG3 heavy chains are more susceptible to degradation than other subtypes, particularly the hinge region (Saito et al, 2019), and unassembled heavy and light chain monomers are in general more exposed to proteolysis than assembled mAbs (Gardner et al, 1993;Chen et al, 2016;Zischewski et al, 2016). To minimize the latter issue, we attempted to support mAb assembly with a theoretically optimal 1:1 ratio (Lin et al, 2018) of heavy and light chains by creating single-cassette expression vectors using an F2A linker peptide (Supplementary Figure S1) (Ryan et al, 1991). However, given that similar mRNA levels for each chain do not necessarily translate to similar protein levels (e.g., due to differences in translation, folding or degradation), the effect of excess IgG3 heavy and light chain monomers should be investigated more carefully in the future (Schlatter et al, 2005).…”

Contributions of the international plant science community to the fight against human infectious diseases – part 1: epidemic and pandemic diseases

“…However, plants have been used to produce monoclonal antibodies against the virus which can help with the treatment of patients who have already contracted the disease (Olinger et al, 2012;Zeitlin et al, 2011). Single monoclonal antibodies and cocktails of two or three different types have been produced in N. benthamiana and transplastomic lettuce leaves, achieving the neutralization of Ebola virus in pre-clinical tests (Fulton et al, 2015;Lai et al, 2012;Lin et al, 2018;Phoolcharoen et al, 2011). The light and heavy chains of monoclonal antibody 6D8 were produced with yields of up to 0.5 g/kg by transient expression (Phoolcharoen et al, 2011).…”

“…Single monoclonal antibodies and cocktails of two or three different types have been produced in N . benthamiana and transplastomic lettuce leaves, achieving the neutralization of Ebola virus in pre‐clinical tests (Fulton et al ., 2015 ; Lai et al ., 2012 ; Lin et al ., 2018 ; Phoolcharoen et al ., 2011 ). The light and heavy chains of monoclonal antibody 6D8 were produced with yields of up to 0.5 g/kg by transient expression (Phoolcharoen et al ., 2011 ).…”

2A and 2A-like Sequences: Distribution in Different Virus Species and Applications in Biotechnology

“…To construct the co-expression vectors, the 2A/2A-like sequences are usually incorporated into an adenovirus [75], adeno-associated virus (AAV) [12], retrovirus [76], lentivirus [77,78], or plasmid vector [79,80]. Many other biotechnological applications that depend on the co-expression of multiple genes use 2A/2A-like sequences, e.g., the production of antibodies and antigens that can be used in vaccine production [80][81][82][83][84][85], observation of chromatin dynamics and genome (DNA and RNA) editing in the application of cell/gene therapies [78,79,[86][87][88][89][90], and development of optogenetic tools [91][92][93]. More examples of viral 2As applications can be found in [94].…”