NMR mapping of the highly flexible regions of ¹³C/¹⁵N-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 ¹³C/¹⁵N-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 ¹³C/¹⁵N-labeled TTAC-0001-Fab (¹³C/¹⁵N-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_H1; 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.