Carbohydrates engineered at antibody constant domains can be used for site-specific conjugation of drugs and chelates

Abstract

To improve the efficiency of site-specific conjugation of chelates and drugs to antibodies, and to minimize the incidence of immunoreactivity perturbation to the resultant immunoconjugates, Asn-linked oligosaccharide moieties were designed and engineered into the constant domains of a humanized anti-CD22 monoclonal antibody, hLL2. From 10 potential glycosylation mutants, two CH 1 domain glycosylation sites, HCN1 and HCN5, were identified that were positioned favorably for glycosylation. The carbohydrate (CHO) chains attached at these sites were differentially processed so that HCN5-CHOs were physically larger than HCN1–CHOs. Although both the CH 1-appended CHOs, and the LL2 V κ-appended CHOs conjugated efficiently with small chelates, the HCN5–CHOs, due to the structural and positional superiority, appear to be a better conjugation site for large drug complexes, such as 18 kDa doxorubicin (DOX)–dextran.