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.