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Site-specific Modifications of Light Chain Glycosylated Antilymphoma (LL2) and Anti-Carcinoembryonic Antigen (hImmu-14-N) Antibody Divalent Fragments¹

Immunomedics, Inc., Morris Plains, New Jersey 07950 [S. V. G., G. L. G., S. O. L., M. J. L., H. J. H.], and Garden State Cancer Center at the Center for Molecular Medicine and Immunology, Newark, New Jersey 07103 [D. M. G.]

Site-specific introduction of metal-chelating groups into F(ab')₂ fragments of an antilymphoma antibody (LL2) possessing a natural Asnlinked light chain carbohydrate and an anti-carcinoembryonic antigen antibody (hImmu-14-N) grafted with a light chain carbohydrate site is described. For this purpose, four yttrium- (and indium)-chelating agents were used, containing a primary amino group for antibody binding and 1-(4-substituted benzyl)diethylenetriaminepentaacetic acid as the metal-chelator, separated by structurally different additional linkers. Conjugates were prepared by reacting excess chelator with oxidized carbohydrate of F(ab')₂ fragments, with or without a subsequent reduction step. The conjugates, with up to an average of 5.5 chelating groups attached to a F(ab')₂ fragment, were readily labeled with ⁹⁰Y and ¹¹¹In and were found to retain antigen-binding ability in in vitro assays. Tumor targeting was demonstrated using a ⁸⁸Y-labeled hImmu-14-N F(ab')₂ carbohydrate-modified conjugate. 2-Pyridyldithiopropionic hydrazide was conjugated to the carbohydrate region, and the disulfide was selectively deprotected to the thiol group, which is reactive with reduced ^99mTc. These initial experiments establish that light chain carbohydrate modification of F(ab')₂ is as facile as with the Fc-region carbohydrate of intact IgG, and thereby offer the possibility of designing site-specifically substituted F(ab')₂ fragments with favorable pharmacokinetic properties.