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Glycobiology Advance Access published online on September 26, 2003
Glycobiology, doi:10.1093/glycob/cwh001
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Submitted on June 18, 2003
Revised on August 31, 2003
Accepted on August 31, 2003

© 2003 Oxford University Press

ORIGINAL ARTICLES

Glycoforms obtained by expression in Pichia pastoris improve cancer targeting potential of a recombinant antibody-enzyme fusion protein

Katalin F. Medzihradszky 1, Daniel I. R. Spencer 2, Surinder K. Sharma 2, Jeetendra Bhatia 2, R. Barbara Pedley 2, David A. Read 2, Richard H. J. Begent 2, and Kerry A. Chester 2*

1 Mass Spectrometry Facility, Department of Pharmaceutical Chemistry, School of Pharmacy, University of California San Francisco, San Francisco, CA-94143-0446, USA
2 Cancer Research UK Targeting and Imaging Group, Department of Oncology, Royal Free and University College Medical School, University of London, Royal Free Campus, Rowland Hill Street, London, NW3 2PF, UK

* To whom correspondence should be addressed. E-mail: k.chester{at}ucl.ac.uk.

Abstract

MFE-CP is a recombinant antibody-enzyme fusion protein used for antibody-mediated delivery of an enzyme to cancer deposits. After clearance from normal tissues, the tumour-targeted enzyme is used to activate a subsequently administered pro-drug to give a potent cytotoxic in the tumour. MFE-CP localizes to cancer deposits in vivo but we proposed that its therapeutic potential could be improved by N-glycosylation, obtained by expression in Pichia pastoris (P. pastoris). Glycosylation could enhance clearance from healthy tissue and result in better tumour:normal tissue ratios. To test this, glycosylated MFE-CP was expressed and purified from P. pastoris. The resultant MFE-CP fusion protein was enzymatically active and showed enhanced clearance from normal tissues in vivo. Furthermore, it showed effective tumour localisation. This favourable glycosylation pattern was analysed by tandem mass spectrometry. High resolution, high detection sensitivity collision-induced dissociation experiments proved essential for this task. Results showed that of the three potential N-glycosylation sites only two were consistently occupied with oligomannose structures. Asn-442 appeared the most heterogeneously populated with oligomannose carbohydrates extending from 5 to 13 units in length. Asn-484 was found only in its non-glycosylated form. There was less heterogeneity at Asn-492 which was glycosylated with oligosaccharide structures ranging from 8 to 10 mannose units. Non-glycosylated forms of Asn-442 and Asn 492 were not observed.


ADEPT, CID, mass spectrometry, N-glycosylation, Pichia pastoris
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