An integral approach towards a practical application for a plant-made monoclonal antibody in vaccine purification.

The use of transgenic plants for the production of pharmaceutical compounds has received increasing attention in the last few years. However, many technological and regulatory issues regarding the practical exploitation of this alternative system of production remain to be solved; a situation that explains the lack of commercial products derived from such a system. This paper reports the expression in transgenic plants and cells of a single-chain antibody variable-region fragment (scFv) and a mouse monoclonal antibody to the hepatitis B virus surface antigen (HBsAg). The large-scale purification of the scFv from plants and its use for immunopurification of HBsAg are also described, together with elements concerning regulatory issues and technologies for compliance with good manufacturing and agricultural practices.

Plant-derived mouse IgG monoclonal antibody fused to KDEL endoplasmic reticulum-retention signal is N-glycosylated homogeneously throughout the plant with mostly high-mannose-type N-glycans.

Plants are potential hosts for the expression of recombinant glycoproteins intended for therapeutic purposes. However, N-glycans of mammalian glycoproteins produced in transgenic plants differ from their natural counterparts. The use of the endoplasmic reticulum (ER)-retention signal has been proposed to restrict glycosylation of plantibodies to only high-mannose-type N-glycans. Furthermore, little is known about the influence of plant development and growth conditions on N-linked glycosylation. Here, we report a detailed N-glycosylation profiling study of CB.Hep1, a mouse IgG2b monoclonal antibody (mAb) against hepatitis B surface antigen (HBsAg) currently expressed in tobacco plants (Nicotiana tabacum L.). The KDEL ER-retention signal was fused to the C-terminal of both light and heavy chains. The structures of the N-linked glycans of this mAb produced in transgenic tobacco plants at various growth stages were analysed by high-performance liquid chromatography (HPLC) profiling techniques and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and compared with those of murine origin. The high-mannose-type oligosaccharides accounted for more than 80% of the total N-glycans, with Man7GlcNAc2 being the most abundant species. Some complex N-glycans bearing xylose and small amounts of oligosaccharides with both xylose and fucose were identified. No appreciable differences were detected when comparing glycosylation at different leaf ages, e.g. from seedling leaves up to 8 weeks old and top or basal leaves of mature plants, or between leaves, stems and whole plants. A strict retention of glycoproteins to ER by the use of the tetrapeptide KDEL was not sufficient, even though the majority of the resulting N-glycosylation was of the high-mannose type. It is highly likely to be dependent on other factors, which are most probably protein specific.

Transient expression in tobacco leaves of an aglycosylated recombinant antibody against the epidermal growth factor receptor.

When generating stably transformed transgenic plants, transient gene expression experiments are especially useful to rapidly confirm that the foreign molecule of interest is correctly assembled and retains its biological activity. TheraCIM(R) (CIMAB S.A., Havana) is a recombinant humanized antibody against the Epidermal Growth Factor receptor (EGF-R), now in clinical trials for cancer therapy in Cuba and other countries. An aglycosylated version (Asn 297 was mutated for Gln 297) of this antibody was transiently expressed in tobacco leaves after vacuum-mediated infiltration of recombinant Agrobacterium tumefaciens that carried a binary plasmid bearing the antibody heavy and light chain genes and plant regulation signals. Protein extracts from "agroinfiltrated" leaves were tested by ELISA and Western blot, showing that the fully assembled antibody was accumulated in plant tissues. The absence of plant specific glycans did not interfere in the assembling or in the activity of the plantibody, as demonstrated in this work. Indirect immunofluorescence demonstrated that the aglycosylated antibody expressed in plants recognizes the EGF-R expressed on the surface of A431 human tumor culture cells.