glycan optimization of human monoclonal antibody in the aquatic plant lemna minor elleke bosma...
Post on 21-Dec-2015
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Glycan optimization of human monoclonal antibody in the aquatic plant lemna minor
Elleke BosmaHarmen KloosterboerRutger Mantingh
Prof.Dr. Dirk Bosch
Introduction
Xylose 1,3-Fucose
Plant glycosylationHuman Ab
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
Research goals
Designing an optimized plant model for the efficient production of therapeutic mAbs
-Eliminating immunogenic glycans Xylose and Fucose
Results (1a)Lex system (Lemna expression system)
- MDX-060 LEX
-MDX-060 LEXOPT
RNAi against Fucose and Xylosetransferases
Results (1b)
Purifying the Abs : Protein A binds IgG
- Flow Trough: no IgG- Eluate: IgG
SDS-PAGE
Results (2a)MALDI TOF MS analysis
Results (2b)NP-HPLC-QTOF MS analysis
Results (3a)Flow cytometry
FITC sec Ab
Results (3b)Equilibrium binding of Ab to FcR
FcR
Results (3c)ADCC Activity (cell lysis)
(experimental release – spontaneous release) X 100
(maximal release – spontaneous release)
Homozygote Heterozygote
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
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.