Structure determination of N-linked oligosaccharides engineered at the CH1 domain of humanized LL2

Two humanized antibody mutants, hLL2HCN1 and hLL2HCN5, engineeredwith CH₁ domain-appended carbohydrates (CHOs) were generatedto facilitate site-specific conjugation of radionudides andanti-cancer drugs to antibodies. Such site-specific conjugationmay minimize the incidence of immunoreactlvity perturbationas is often observed with random conjugation. Since the compositionsand structures of CHOs are important in determining the chemistry,efficiency, and extent of conjugation, the sequences of theCH₁-appended CHOs were determined by exoglycosidase digestionsand fluorophore-assisted CHO electrophoresis (FACE). The CHOspecies attached at HCN1 and HCN5 sites in hLL2HCN1 and IJLL2HCN5,respectively, were distinct from each other, heterogeneous,and extensively processed. All of these CHOs were corefucosylatedcomplex-type oligosaccharides and contained Gal (galactose)and GlcNAc (N-acetylglucosamine) residues in the outer branches.Some of the outer branches were composed of Gal     

Surrogate target cells expressing surface anti-idiotype antibody for the clinical evaluation of an internalizing CD22-specific antibody

SM03, a chimeric antibody that targets the B-cell restricted antigen CD22, is currently being clinically evaluated for the treatment of lymphomas and other autoimmune diseases in China. SM03 binding to surface CD22 leads to rapid internalization, making the development of an appropriate cell-based bioassay for monitoring changes in SM03 bioactivities during production, purification, storage, and clinical trials difficult. We report herein the development of an anti-idiotype antibody against SM03. Apart from its being used as a surrogate antigen for monitoring SM03 binding affinities, the anti-idiotype antibody was engineered to express as fusion proteins on cell surfaces in a non-internalizing manner, and the engineered cells were used as novel “surrogate target cells” for SM03. SM03-induced complement-mediated cytotoxicity (CMC) against these “surrogate target cells” proved to be an effective bioassay for monitoring changes in Fc functions, including those resulting from minor structural modifications borne within the Fc-appended carbohydrates. The approach can be generally applied for antibodies that target rapidly internalizing or non-surface bound antigens. The combined use of the anti-idiotype antibody and the surrogate target cells could help evaluate clinical parameters associated with safety and efficacies, and possibly the mechanisms of action of SM03.