Enhancing the secretion of a glyco-engineered anti-CD20 scFv-Fc antibody in hairy root cultures.

Hairy root (HR) cultures represent an attractive platform for the production of heterologous proteins, due to the possibility of secreting the molecule of interest in the culture medium. The main limitation is the low accumulation yields of heterologous proteins. The aim of this study was to enhance the accumulation of a tumor-targeting antibody with a human-compatible glycosylation profile in HR culture medium. To this aim, we produced Nicotiana benthamiana HR cultures expressing the red fluorescent protein (RFP) to easily screen for different auxins able to induce heterologous protein secretion in the medium. The hormone 2,4-dichlorophenoxyacetic acid (2,4-D) was found to induce high accumulation levels (334 mg/L) of RFP in the culture medium. The same protocol was used to improve the secretion of the tumor-targeting, CD20-specific 2B8-Fc?XF recombinant antibody from glyco-engineered ?XTFT N. benthamiana HR cultures. The addition of 2,4-D determined a 28-fold increase of the accumulation of fully functional 2B8-Fc?XF in the culture medium, at levels of ~16 mg/L. Antibody N-glycosylation profiling revealed the prominent occurrence of GnGn structures and low levels of xylose- and fucose-containing counterparts. This result is the first example of the expression of an engineered anti-CD20 antibody with a scFv-Fc format at high levels in HR.