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A Trade-off Between Thermostability and Binding Affinity of Anti-(4-hydroxy-3-nitrophenyl)Acetyl Antibodies During the Course of Affinity Maturation

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Abstract

Somatic hypermutation (SHM) is one of the driving forces that increases antibody (Ab) affinity. We studied the effects of SHM on thermostability and affinity using three single-chain Fv fragments (scFvs) of anti-(4-hydroxy-3-nitrophenyl)acetyl Abs, namely 9TG, 9T7, and E11. 9TG has a germline structure that lacks SHM and is an ancestor of 9T7 with 11 mutations. E11, which has 21 mutations, is a mature Ab and has its own ancestor. The thermostabilities and antigen-Ab interactions were analyzed by circular dichroism (CD), differential scanning calorimetry (DSC), and isothermal titration calorimetry (ITC). Far-UV CD spectra showed that all scFvs were folded into a structure referred to as immunoglobulin-fold and were unfolded by heating at different melting temperatures. Comparison of thermodynamic parameters obtained from DSC and ITC revealed that the magnitude of stabilization free energy at 37 °C was in the order, 9TG > 9T7 > E11, while that of the free energy of interaction with antigen was 9TG < 9T7 < E11, suggesting that Abs make a trade-off between stability and affinity during affinity maturation.

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Acknowledgements

The authors thank to Dr. Harumi Fukada for helpful discussion. This work was performed in part under the Collaborative Research Program of Institute for Protein Research, Osaka University, CR-20-02, and was partly supported by Nanotechnology Platform Program <Molecule and Material Synthesis> (JPMXP09S21MS1006) of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. This study was supported by JSPS KAKENHI Grant Number 18K06161.

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Authors and Affiliations

  1. Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, 1-5 Shimogamo Hangi-cho, Sakyo-ku, Kyoto, 606-8522, Japan

    Akihiro Nishiguchi & Masayuki Oda

  2. Graduate School of Biomedical Sciences, Tokushima University, 3-18-15 Kuramoto, Tokushima, 770-8504, Japan

    Akikazu Murakami

  3. Antibody Technology Research Center, Inc, 2361-1Yamazaki, Noda, Chiba, 278-0022, Japan

    Takachika Azuma

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  1. Akihiro Nishiguchi
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  2. Akikazu Murakami
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  3. Takachika Azuma
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  4. Masayuki Oda
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Correspondence to Masayuki Oda.

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Nishiguchi, A., Murakami, A., Azuma, T. et al. A Trade-off Between Thermostability and Binding Affinity of Anti-(4-hydroxy-3-nitrophenyl)Acetyl Antibodies During the Course of Affinity Maturation. Protein J 41, 293–303 (2022). https://doi.org/10.1007/s10930-022-10053-w

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