ABSTRACT
Plant suspension cells are interesting hosts for the heterologous production of pharmacological proteins such as antibodies. They have the advantage to facilitate the containment and the application of good manufacturing practices. Furthermore, antibodies can be secreted to the extracellular medium, which makes the purification steps much simpler. However, improvements are still to be made regarding the quality and the production yield. For instance, the inactivation of proteases and the humanization of glycosylation are both important targets which require either gene silencing or gene inactivation. To this purpose, CRISPR-Cas9 is a very promising technique which has been used recently in a series of plant species, but not yet in plant suspension cells. Here, we sought to use the CRISPR-Cas9 system for gene inactivation in Nicotiana tabacum BY-2 suspension cells. We transformed a transgenic line expressing a red fluorescent protein (mCherry) with a binary vector containing genes coding for Cas9 and three guide RNAs targeting mCherry restriction sites, as well as a bialaphos-resistant (bar) gene for selection. To demonstrate gene inactivation in the transgenic lines, the mCherry gene was PCR-amplified and analyzed by electrophoresis. Seven out of 20 transformants displayed a shortened fragment, indicating that a deletion occurred between two target sites. We also analyzed the transformants by restriction fragment length polymorphism and observed that the three targeted restriction sites were hit. DNA sequencing of the PCR fragments confirmed either deletion between two target sites or single nucleotide deletion. We therefore conclude that CRISPR-Cas9 can be used in N. tabacum BY2 cells.
ABSTRACT
Nicotiana tabacum suspension cells have been widely used to produce monoclonal antibodies, but the yield of secreted antibodies is usually low probably because of proteolytic degradation. Most IgGs that have been expressed in suspension cells have been of the human IgG1 isotype. In this study, we examined whether other isotypes displayed the same sensitivity to proteolytic degradation and whether the choice of plant host species mattered. Human serum IgG displayed different degradation profiles when incubated in spent culture medium from N. tabacum, Nicotiana benthamiana or Arabidopsis thaliana suspension cells. Zymography showed that the protease profile was host species dependent. Three human isotypes, IgG1, IgG2 and IgG4, and a mouse IgG2a were provided with the same heavy- and light-chain variable regions from an anti-human IgM antibody and expressed in N. tabacum cv. BY-2 and A. thaliana cv. Col-0 cells. Although all tested isotypes were detected in the extracellular medium using SDS-PAGE and a functional ELISA, up to 10-fold differences in the level of intact antibody were found according to the isotype expressed, to the host species and to the culture conditions. In the best combination (BY-2 cells secreting human IgG1), we reported accumulation of more than 30 mg/L of intact antibody in culture medium. The possibility of using IgG constant regions as a scaffold to allow stable accumulation of antibodies with different variable regions was demonstrated for human IgG2 and mouse IgG2a.
