NMR mapping of the highly flexible regions of 13 C/ 15 N-labeled antibody TTAC-0001-Fab

Monoclonal antibody (mAb) drugs are clinically important for the treatment of various diseases. TTAC-0001 is under development as a new anti-cancer antibody drug targeting VEGFR-2. As the less severe toxicity of TTAC-0001 compared to Bevacizumab, likely due to the decreased in vivo half-life, seems to be related to its structural flexibility, it is important to map the exact flexible regions. Although the 13 C/ 15 N-labeled protein is required for NMR analyses, it is difficult to obtain antibody fragments (Fab and scFv) containing disulfide bonds through general cytosolic expression in Escherichia coli ( E. coli ). Here, we notably increased the periplasmic expression of the 13 C/ 15 N-labeled TTAC-0001-Fab ( 13 C/ 15 N-TTAC-Fab) through simple isopropyl β-D-1-thiogalactopyranoside (IPTG)-induction at an increased optical density (1.5 OD 600nm ). Through NMR triple resonance experiments, two loop insertions (LI-1 between the V H and C H 1; LI-2 between the V L and C L ) were confirmed to be highly flexible. The additional LIs could be another way to engineer the antibody by changing the pharmacokinetic properties.