Engineering a unique glycosylation site for site-specific conjugation of haptens to antibody fragments.
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.
Leung
S
S
Immunomedics, Inc., Morris Plains, NJ 07950, USA.
Losman
M J
MJ
Govindan
S V
SV
Griffiths
G L
GL
Goldenberg
D M
DM
Hansen
H J
HJ
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology; D000915:Antibody Affinity / Q000276:immunology; D001483:Base Sequence; D002614:Chelating Agents; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006241:Haptens / Q000737:chemistry* / Q000276:immunology; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000276:immunology; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry* / Q000276:immunology; D015202:Protein Engineering; D014162:Transfection
D000911:Antibodies, Monoclonal; D002614:Chelating Agents; D006241:Haptens; D007140:Immunoglobulin Fab Fragments; D009844:Oligosaccharides