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

• Published in: Journal of biomolecular NMR 2020-07, Vol.74 (6-7), p.311-319
• Main Authors: Cha, Soyoung, Lee, Weon Sup, Choi, Joonhyeok, Jeong, Jong Geun, Nam, Ju Ryoung, Kim, Jihong, Kim, Hak-Nam, Lee, Joon-Hwa, Yoo, Jin-San, Ryu, Kyoung-Seok
• Format: Article
• Language: English
• Subjects: Antibodies; Bevacizumab; Biochemistry; Biocompatibility; Biological and Medical Physics; Biophysics; Disulfide bonds; E coli; Fab; Mapping; Monoclonal antibodies; Nitrogen isotopes; NMR; Nuclear magnetic resonance; Optical density; Pharmacokinetics; Physics; Physics and Astronomy; Spectroscopy/Spectrometry; Targeted cancer therapy; Therapeutic targets; Toxicity; Vascular endothelial growth factor receptors
• ISSN: 0925-2738; 1573-5001
• DOI: 10.1007/s10858-020-00313-1

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.