Antibody production in plant cell suspension cultures : searching for new host species and identification of active genomic loci

Plant suspension cells are considered as an interesting platform to produce monoclonal antibodies (mAb) and other pharmacological proteins. However, to date, the expression yields obtained are still relatively low in comparison with other expression platforms. So far suspension cells from very few plant species have been used as hosts. In this study, we screened cell suspension lines derived from the 13 following dicot species for their capacity to express an antibody: Ocimum basilicum, Lavandula angustifolia, Linum usitatissimum, Mentha x piperita, Brassica rapa, Dianthus caryophyllus, Citrus x sinensis, Phytolacca acinosa, Solanum tuberosum, Malus domestica, Phytolacca americana, Helianthus annuus, and Vitis vinifera. We succeeded in setting up Agrobacterium-mediated transformation and expressing intact mAbs in suspension cells derived from two of them, L. usitatissimum (flax) and L. angustifolia (lavender). We showed that, unlike the cauliflower mosaic virus 35S promoter, the constitutive promoter of Nicotiana plumaginifolia PMA4 was not able to efficiently drive gene transcription in L. usitatissimum cells. Mass spectrometry analysis of mAbs secreted from L. angustifolia cells revealed a high proportion of N-glycosylation with plant-specific glycans. In the second part, we sought to localize highly active genomic loci for heterologous expression. We screened 1595 Nicotiana tabacum and 430 Arabidopsis thaliana transformants expressing a fluorescent protein (mCherry) to select the highest expressing cell lines. The high mCherry expression observed in the selected lines seems to result from several T-DNA insertions among which active ones will have to be identified. The genomic environment of 20 insertion events was analyzed. In the last part, we showed that culture conditions (e.g. medium composition, culture vessel) influence plant cell growth as well as mAb accumulation in the extracellular medium of a transgenic cell line.(AGRO - Sciences agronomiques et ingénierie biologique) -- UCL, 201

Gene Inactivation by CRISPR-Cas9 in Nicotiana tabacum BY-2 Suspension Cells

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 (GMPs). 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

Accumulation of secreted antibodies in plant cell cultures varies according to the isotype, host species and culture conditions.

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