Enhancing the Secretion of a Glyco-Engineered Anti-CD20 scFv-Fc Antibody in Hairy Root Cultures

Abstract

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 is to enhance the accumulation of a tumor-targeting antibody with a human-compatible glycosylation profile in HR culture medium. To this aim, the authors produce 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) is found to induce high accumulation levels (334 mg L ) of RFP in the culture medium. The same protocol is used to improve the secretion of the tumor-targeting, CD20-specific 2B8-FcΔXF recombinant antibody from glyco-engineered ΔXTFT N. benthamiana HR cultures. The addition of 2,4-D determine a 28-fold increase of the accumulation of fully functional 2B8-FcΔXF in the culture medium, at levels of ≈16 mg L . Antibody N-glycosylation profiling reveal 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.

Optimised production of an anti-fungal antibody in Solanaceae hairy roots to develop new formulations against Candida albicans

“…Almost certainly both bands derived from a proteolytic cleavage in the hinge region connecting constant domains (CH2-CH3) to scFv. This is not surprising since it was demonstrated that antibodies expressed in plants are particularly prone to proteolysis in the hinge region [20] and in a previous work, in which a scFvFc was expressed in HR of N. benthamiana, the purified antibody showed a similar degradation pattern [21].…”

Expression of Single Chain Variable Fragment (scFv) Molecules in Plants: A Comprehensive Update

“…The biological activity studies with purified scFv-Fc exhibited an improved binding affinity compared to the non-engineered form of scFv and rituximab. Cytotoxicity induced through the antibody-dependent cell-mediated pathway and the complementary dependent pathway was strongly enhanced [18,126]. The data indicate that the major factor which prevents the use of plant system is the cost associated with the downstream purification of the recombinant protein rather than the post-translational changes associated with plant expression.…”

“…Addition of the plant growth regulator 2,4-D was found to be enhancing the secretion of recombinant antibodies about 28-fold to the culture vessels. The purified CD20 2B8-Fc effectively bound to the CD20 antigen on the surface of human Daudi cells [18]. Transient expression is favored when the protein is needed within a short span of time in a comparatively large quantity [53,93,94] and for the expression of proteins which have an adverse effect on the host cells when overexpressed [95].…”

“…To synthesize mammalian biosimilar proteins glycosylation machinery of the host plant need to be manipulated. Glycosylation mutants developed in A. thaliana and other model plants for protein expression had similar glycosylation as that of mammalian cells [16,18,75]. Even though there were minor differences in the glycosylation, scFv expressed in A. thaliana showed biological effects similar to the parental antibody [56,112].…”

“…The differences in the posttranslational modifications between mammalian cells and the plant cells were a serious concern in using plants for the recombinant antibody expression. Genetic manipulations leading to the mammalian-like post-translational modifications in the model plant systems resulted in the biosynthesis of antibodies equivalent to mammalian products [16][17][18]. Even though biologically active mAbs can be expressed successfully in plants, a more feasible approach would be the expression of antibody fragments.…”