Abstract
Immunoglobulin subclass IgG1 is bound and neutralized effectively by Staphylococcus aureus protein A, allowing the bacterium to evade the host’s adaptive immune response. In contrast, the IgG3 subclass is not bound by protein A and can be used to treat S. aureus infections, including drug-resistant strains such as methicillin-resistant Staphylococcus aureus (MRSA). However, the yields of recombinant IgG3 are generally low because this subclass is prone to degradation, and recovery is hindered by the inability to use protein A as an affinity ligand for antibody purification. Here, we investigated plants (Nicotiana spp.) as an alternative to microbes and mammalian cell cultures for the production of an IgG3 antibody specific for the S. aureus alpha toxin. We targeted recombinant IgG3 to different subcellular compartments and tested different chromatography conditions to improve recovery and purification. Finally, we tested the antigen-binding capacity of the purified antibodies. The highest IgG3 levels in planta (>130 mg kg−1 wet biomass) were achieved by targeting the endoplasmic reticulum or apoplast. Although the purity of IgG3 exceeded 95% following protein G chromatography, product recovery requires further improvement. Importantly, the binding affinity of the purified antibodies was in the nanomolar range and thus comparable to previous studies using murine hybridoma cells as the production system.
Highlights
Antibiotic-resistant bacteria are increasingly recognized as a major threat to human health (Conlon et al, 2013)
IgG3 heavy chains are more susceptible to degradation than other subtypes, the hinge region (Saito et al, 2019), and unassembled heavy and light chain monomers are in general more exposed to proteolysis than assembled monoclonal antibody (mAb) (Gardner et al, 1993; Chen et al, 2016; Zischewski et al, 2016)
Because the self-cleaving peptide derived from foot-and-mouth disease virus (F2A) linker can generate a non-native N-terminus on the downstream protein, which would attract regulatory scrutiny, we used a refined self-excising linker composed of an intein and F2A peptide (IntF2A) yielding native heavy and light chain sequences (Zhang et al, 2017)
Summary
Antibiotic-resistant bacteria are increasingly recognized as a major threat to human health (Conlon et al, 2013). Such bacteria are predicted to become more prevalent due to the continual evolution of resistance and the lack of new antibiotics with novel mechanisms of action in the development pipeline (Guo et al, 2020). Penicillin-resistant S. aureus emerged in the mid-1940s (DeLeo and Chambers, 2009), followed by MRSA in the 1960s and vancomycin-resistant S. aureus in the early 2000s (De Lencastre et al, 2007). Strict policies for the use of antibiotics initially reduced the frequency of MRSA in hospitals, but these strains started to emerge in the community in the 1990s (De Lencastre et al, 2007)
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