Antibody (Ab) fragments are preferred agents for imaging applications because of their rapid clearance from the blood, thereby providing high tumor:blood ratios within a few hours. Several preclinical studies have also suggested that Ab fragments might be preferred for therapeutic applications over an intact IgG. The purpose of this project was to develop engineered Ab fragments using a humanized anti-carcinoembryonic antigen and anti-CD22 Ab as the parent. Three types of variants were prepared: a deltaCH2 (deletion mutant missing the CH2), a gamma3 F(ab')2 containing the human IgG3 hinge, and three glycosylated variants. The gamma3 F(ab')2 and glycosylated variants were developed because of the potential for site-specific linkage to the Ab in its divalent or monovalent fragment. The gamma3 F(ab')2 variant contains 10 cysteine residues that could be used for direct coupling using thiol chemistry, whereas the glycosylated variants have N-linked glycosylation 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 single peak by size-exclusion HPLC, but the deltaCH2 variant showed two distinct peaks, which were believed to be both the divalent and monovalent forms of this fragment. The two CH1 glycosylated variants showed differences in the extent of glycosylation. Modeling studies suggest that one variant would be better suited for site-specific coupling than the other because the carbohydrate chain is extended further away from the antigen-binding site. The Abs were radioiodinated to determine their pharmacokinetic behavior in mice. All of the humanized Ab divalent fragments cleared nearly 20 times faster from the blood than the murine parent F(ab')2 over a 24-h period. The glycosylated fragments showed some added stability compared to the other fragments over 4 h, but by 24 h, they had cleared to the same extent. Size-exclusion high-performance liquid chromatography of blood samples indicated that the humanized Ab fragments were quickly degraded in the blood. Thus, there is an inherent instability of the divalent fragments from these humanized IgG1 constructs that may affect their utility in imaging or therapy applications.