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

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Abstract

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 OD600nm). Through NMR triple resonance experiments, two loop insertions (LI-1 between the VH and CH1; LI-2 between the VL and CL) were confirmed to be highly flexible. The additional LIs could be another way to engineer the antibody by changing the pharmacokinetic properties.

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Acknowledgements

This research was supported by a Grant from the Korea Basic Science Institute (T39632 to K.S.R.) and by a Grant from the National Research Foundation of Korea (2016R1A2B4015436 to K.S.R.). Additionally, this work was supported, in part, by the Samsung Science & Technology Foundation (SSTF-BA1701-10 to J.W.L.).

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Author notes

  1. Soyoung Cha, Weon Sup Lee and Kyoung-Seok Ryu have contributed equally to this paper.

Authors and Affiliations

  1. Protein Structure Research Team, Korea Basic Science Institute, 162 Yeongudanji-Ro, Ochang-Eup, Cheongju-Si, Chungcheongbuk-Do, 28119, South Korea

    Soyoung Cha, Joonhyeok Choi, Hak-Nam Kim & Kyoung-Seok Ryu

  2. PharmAbcine, 2F, Research Building 2, 70, Yuseong-daero 1689 Beon-gil, Yuseong-gu, Daejeon, 34047, South Korea

    Weon Sup Lee, Jong Geun Jeong, Ju Ryoung Nam & Jin-San Yoo

  3. New Drug Development Center, Osong Medical Innovation Foundation, 123 Osongsaengmyeong-Ro, Osong-Eup, Cheongju-Si, Chungcheongbuk-Do, 28160, South Korea

    Jihong Kim

  4. Department of Chemistry and RINS, Gyeongsang National University, Jinju-si, Gyeongsangnam-Do, 52828, South Korea

    Joon-Hwa Lee

  5. Department of Bio-Analytical Science, University of Science and Technology, 217 Gajeong-ro, Yuseong-gu, Daejeon, 34113, South Korea

    Soyoung Cha, Weon Sup Lee & Kyoung-Seok Ryu

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  1. Soyoung Cha
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  2. Weon Sup Lee
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Correspondence to Jin-San Yoo or Kyoung-Seok Ryu.

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Cha, S., Lee, W.S., Choi, J. et al. NMR mapping of the highly flexible regions of 13C/15N-labeled antibody TTAC-0001-Fab. J Biomol NMR 74, 311–319 (2020). https://doi.org/10.1007/s10858-020-00313-1

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  • DOI: https://doi.org/10.1007/s10858-020-00313-1

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