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