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Glycan optimization of human monoclonal antibody in the aquatic plant lemna minor

Glycan optimization of human monoclonal antibody in the aquatic plant lemna minor. Elleke Bosma Harmen Kloosterboer Rutger Mantingh Prof.Dr. Dirk Bosch. Introduction. Plant glycosylation. Human Ab. Xylose. 1,3-Fucose. Introduction. - ADCC: Antibody Dependent

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Glycan optimization of human monoclonal antibody in the aquatic plant lemna minor

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  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 Plant glycosylation Human Ab Xylose 1,3-Fucose

  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 LEXOPT RNAi against Fucose and Xylose transferases

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

  7. Results (2a) MALDI TOF MS analysis

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

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

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

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

  12. 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

  13. 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.

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