1. Glycan optimization of human monoclonal antibody in the aquatic plant lemna minor Elleke Bosma Harmen Kloosterboer Rutger Mantingh Prof.Dr. Dirk Bosch

2. Introduction Xylose1,3-Fucose Plant glycosylation Human Ab

3. Introduction - ADCC: Antibody Dependent Cell-mediated Cytotoxicity -mAbs as therapeutics: effectiveness depends on FcR and for example ADCC -ADCC and FcR binding dependent on N-glycan structures

4. Research goals Designing an optimized plant model for the efficient production of therapeutic mAbs -Eliminating immunogenic glycans Xylose and Fucose

5. Results (1a) Lex system (Lemna expression system) - MDX-060 LEX -MDX-060 LEX OPT RNAi against Fucose and Xylose transferases

6. Results (1b) Purifying the Abs : Protein A binds IgG - Flow Trough: no IgG - Eluate: IgG SDS-PAGE

7. Results (2a) MALDI TOF MS analysis

8. Results (2b) NP-HPLC-QTOF MS analysis

10. Results (3a) Flow cytometry FITC sec Ab

11. Results (3b) Equilibrium binding of Ab to FcR FcR

12. Results (3c) ADCC Activity (cell lysis) (experimental release – spontaneous release) X 100 (maximal release – spontaneous release) Homozygote Heterozygote

13. Summary/Conclusion Fucose and xylose can be succesfully removed by RNAi against their transferases The produced Abs effectively bind antigens The produced Abs show increased FcR binding The produced Abs show increased ADCC activation The produced Abs show glycosylation homogeneity

14. Discussion/Future The produced Abs show glycosylation homogeneity ? - Are all Abs glycosylated ? - Is glycan removal 100% effective? Use whole Abs as control in MS analysis Abs purification on a large scale? - No secretion by plant cells.