ENGINEERING A UNIQUE GLYCOSYLATION SITE FOR SITE-SPECIFIC CONJUGATIONOF HAPTENS TO ANTIBODY FRAGMENTS

A natural N-linked glycosylation site (Asn-Val-Thr) at amino acid posi tions 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 ant i-B cell lymphoma Ab, LL-2. Our earlier studies demonstrated that no c ontact between the V kappa-appended oligosaccharide and the Ag binding site was evident, because glycosylation at this site did not affect t he Ag binding property of the Ab. By using the murine LL-2 F(ab')(2) f ragment (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-ap pended carbohydrate moiety with no reduction in immunoreactivity. The resulting conjugates labeled efficiently with both Y-90 and In-111, wi th no significant effect on Ab affinity. In contrast, conjugation of l ess than five chelates/Ab fragment randomly at lysine residues resulte d in a three- to fivefold reduction in affinity. By a single Arg to As n mutation, an N-linked glycosylation site similar to that of LL-2 was introduced in the FR-1 segment of a nonglycosylated, humanized anti-c arcinoembryonic Ag (CEA) Ab, MN-14 (hMN-14). Glycosylation at the engi neered carbohydrate-addition site was demonstrated by SDS-PAGE analysi s. Neither glycosylation nor site-specific conjugation of chelate at t he V kappa-appended carbohydrate moiety resulted in the loss of immuno reactivity. The glycosylated hMN-14 conjugate labeled efficiently with Y-90.