The effects of domain deletion, glycosylation, and long IgG(3) hinge on the biodistribution and serum stability properties of a humanized IgG(1) immunoglobulin, hLL2, and its fragments

Antibody (Ab) fragments are preferred agents for imaging applications becau se of their rapid clearance from the blood, thereby providing high tumor:bl ood ratios within a few hours. Several preclinical studies have also sugges ted that Ab fragments might be preferred for therapeutic applications over an intact IgG. The purpose of this project was to develop engineered Ab fra gments using a humanized anti-carcinoembryonic antigen and anti-CD22 Ab as the parent. Three types of variants were prepared: a Delta CH2 (deletion mu tant missing the CH2), a gamma 3 F(ab')(2) containing the human IgG3 hinge, and three glycosylated variants. The gamma 3 F(ab')(2) and glycosylated va riants were developed because of the potential for site-specific linkage to the Ab in its divalent or monovalent fragment. The gamma 3 F(ab')(2) varia nt contains 10 cysteine residues that could be used for direct coupling usi ng thiol chemistry, whereas the glycosylated variants have N-linked glycosy lation sites engineered in the CH1 domain (two variants) as well as the VK domain (one variant), All of these variants were successfully prepared and shown to react with the target antigen. All Abs could be purified to a sing le peak by size-exclusion HPLC, but the Delta CH2 variant showed two distin ct peaks, which were believed to be both the divalent and monovalent forms of this fragment. The two CH1 glycosylated variants showed differences in t he extent of glycosylation. Modeling studies suggest that one variant would be better suited for site-specific coupling than the other because the car bohydrate chain is extended further away from the antigen-binding site. The Abs were radioiodinated to determine their pharmacokinetic behavior in mic e. All of the humanized Ab divalent fragments cleared nearly 20 times faste r from the blood than the murine parent F(ab')(2) over a 24-h period. The g lycosylated fragments showed some added stability compared to the other fra gments over 4 h, but by 24 h, they had cleared to the same extent. Size-exc lusion high-performance liquid chromatography of blood samples indicated th at the humanized Ab fragments were quickly degraded in the blood. Thus, the re is an inherent instability of the divalent fragments from these humanize d IgG1 constructs that may affect their utility in imaging or therapy appli cations.