The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Leung, S.
Right arrow Articles by Hansen, H. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Leung, S.
Right arrow Articles by Hansen, H. J.

The Journal of Immunology, Vol 154, Issue 11 5919-5926, Copyright © 1995 by American Association of Immunologists

ARTICLES

Engineering a unique glycosylation site for site-specific conjugation of haptens to antibody fragments

S Leung, MJ Losman, SV Govindan, GL Griffiths, DM Goldenberg and HJ Hansen
Immunomedics, Inc., Morris Plains, NJ 07950, USA.

A natural N-linked glycosylation site (Asn-Val-Thr) at amino acid positions 18-20 (Kabat's numbering) was identified in the framework-1 (FR-1) region of the light chain variable (V kappa) domain of a murine anti-B cell lymphoma Ab, LL-2. Our earlier studies demonstrated that no contact between the V kappa-appended oligosaccharide and the Ag binding site was evident, because glycosylation at this site did not affect the Ag binding property of the Ab. By using the murine LL-2 F(ab')2 fragment (which is devoid of constant region-appended oligosaccharide) as substrate, as much as five bifunctional chelator molecules per F(ab')2 fragment could be site specifically conjugated at the V kappa- appended carbohydrate moiety with no reduction in immunoreactivity. The resulting conjugates labeled efficiently with both 90Y and 111In, with no significant effect on Ab affinity. In contrast, conjugation of less than five chelates/Ab fragment randomly at lysine residues resulted in a three- to fivefold reduction in affinity. By a single Arg to Asn mutation, an N-linked glycosylation site similar to that of LL-2 was introduced in the FR-1 segment of a nonglycosylated, humanized anti- carcinoembryonic Ag (CEA) Ab, MN-14 (hMN-14). Glycosylation at the engineered carbohydrate-addition site was demonstrated by SDS-PAGE analysis. Neither glycosylation nor site-specific conjugation of chelate at the V kappa-appended carbohydrate moiety resulted in the loss of immunoreactivity. The glycosylated hMN-14 conjugate labeled efficiently with 90Y.


This article has been cited by other articles:


Home page
 Protein Eng Des SelHome page
T. Olafsen, C.-w. Cheung, P. J. Yazaki, L. Li, G. Sundaresan, S. S. Gambhir, M. A. Sherman, L. E. Williams, J. E. Shively, A. A. Raubitschek, et al.
Covalent disulfide-linked anti-CEA diabody allows site-specific conjugation and radiolabeling for tumor targeting applications
Protein Eng. Des. Sel., January 1, 2004; 17(1): 21 - 27.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
M. J. Coloma, R. K. Trinh, A. R. Martinez, and S. L. Morrison
Position Effects of Variable Region Carbohydrate on the Affinity and In Vivo Behavior of an Anti-(1->6) Dextran Antibody
J. Immunol., February 15, 1999; 162(4): 2162 - 2170.
[Abstract] [Full Text] [PDF]


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Website Copyright © 1995 by The American Association of Immunologists, Inc. All rights reserved.
All Contents Copyright © 1995 by The American Association of Immunologists, Inc. All rights reserved.