STRUCTURE DETERMINATION OF N-LINKED OLIGOSACCHARIDES ENGINEERED AT THE CH1 DOMAIN OF HUMANIZED LL2

Two humanized antibody mutants, hLL2HCN1 and hLL2HCN5, engineered with CH1 domain-appended carbohydrates (CHOs) were generated to facilitate site-specific conjugation of radionuclides and anti-cancer drugs to a ntibodies, Such site-specific conjugation may minimize the incidence o f immunoreactivity perturbation as is often observed with random conju gation, Since the compositions and structures of CHOs are important in determining the chemistry, efficiency, and extent of conjugation, the sequences of the CH1-appended CHOs were determined by exoglycosidase digestions and fluorophore-assisted CHO electrophoresis (FACE), The CH O species attached at HCN1 and HCN5 sites in hLL2HCN1 and hLL2HCN5, re spectively, were distinct from each other, heterogeneous, and extensiv ely processed, All of these CHOs were core-fucosylated complex-type ol igosaccharides and contained Gal (galactose) and GlcNAc (N-acetylgluco samine) residues in the outer branches, Some of the outer branches wer e composed of Gal alpha 1-3Gal beta 1-4GlcNAc structure, also known as alpha-galactosyl epitope. Most of the CHOs were sialylated, While all HCN1-CHOs were biantennary, the majority of HCN5-CHOs (>60%) were tri antennary, The CH1-appended CHOs have favorable structural characteris tics suitable for site-specific conjugation, For efficient conjugation of large drug complexes, hLL2HCN5 is preferable to hLL2HCN1 because t he attached CHO is larger in size and more remotely positioned from th e V region, The effects of the a-galactosyl epitope found in these CHO s on the immunological properties of the immunoconjugates as efficient cancer diagnostics and therapeutics are being studied.