Designing and constructing a phage display synthesized single domain antibodies library based on camel VHHs frame for screening and identifying humanized TNF-α-specific nanobody

Jifan Nie; Xingyuan Ma; Fabiao Hu; Hui Miao; Xin Feng; Peiwen Zhang; Myong Hun Han; Fang You; Yi Yang; Wenlian Zhang; Wenyun Zheng

doi:10.1016/j.biopha.2021.111328

Designing and constructing a phage display synthesized single domain antibodies library based on camel VHHs frame for screening and identifying humanized TNF-α-specific nanobody

Biomed Pharmacother. 2021 May:137:111328. doi: 10.1016/j.biopha.2021.111328. Epub 2021 Feb 8.

Authors

Jifan Nie¹, Xingyuan Ma¹, Fabiao Hu¹, Hui Miao¹, Xin Feng¹, Peiwen Zhang², Myong Hun Han³, Fang You⁴, Yi Yang⁵, Wenlian Zhang⁶, Wenyun Zheng⁷

Affiliations

¹ State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, PR China.
² Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, PR China.
³ State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, PR China; Department of Genetic, Faculty of Life Science, KIM IL SUNG University, Pyongyang 999093, Democratic People's Republic of Korea.
⁴ Department of Chemical and Biomolecular Engineering, National University of Singapore, Singapore 117585, Singapore.
⁵ Department of Chemical and Biomolecular Engineering, National University of Singapore, Singapore 117585, Singapore; SinGENE Biotech Pte Ltd, Singapore Science Park, Singapore 118258, Singapore. Electronic address: yangyi@singenebio.com.
⁶ State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, PR China; Center of Translational Biomedical Research, University of North Carolina at Greensboro, Greensboro, NC 27310, USA.
⁷ Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, PR China. Electronic address: zwy@ecust.edu.edu.cn.

PMID: 33571835
DOI: 10.1016/j.biopha.2021.111328

Abstract

Tumor necrosis factor (TNF-α) is an important clinically tested cytokine that could induce autoimmune diseases and inflammation. Therefore, the anti-TNF-α therapy strategy was developed and used therapeutically in various diseases, especially in the cytokine storm associated chimeric antigen receptor (CAR) T-cell therapy and antiviral therapy. Compare with other anti-TNF-α inhibitors, anti-TNF-α Nb (nanobody) has many unique advantages. Herein, we reported a novel humanized scaffold for library construction, which could be soluble and expressed in Escherichia coli (E.coli), and the efficiency capacity could reach as high as 2.01 × 10⁹. Meanwhile, an anti-TNF-α Nb was selected for further study after 4 rounds of screening, NT-3, as the optimal Nb could effectively inhibit TNF-mediated cytotoxicity. The IC50 of NT-3 was determined as 0.804 μM, and its apoptosis inhibition rate was 62.47 % in L929 cells. Furthermore, the molecular docking results showed that complementarity-determining regions (CDRs) of NT-3 could connect to TNF for blocking function through strong hydrogen bonds and salt bridges. In general, our study not only provided a good Nb screening platform in vitro without animal immunization, but also generated a series of novel humanized anti-TNF-α Nb candidates with potential applications.

Keywords: Designing and constructing; Humanized TNF-α-specific Nb; Phage display library; Screening and identifyin; Single domain antibodies.

MeSH terms

Amino Acid Sequence
Animals
Antibodies / chemistry*
Apoptosis
Camelus / immunology*
Cell Line, Tumor
Cell Survival / drug effects
Computational Biology
Escherichia coli / metabolism
Humans
Models, Molecular
Molecular Docking Simulation
Peptide Library*
Single-Domain Antibodies / chemistry*
Tumor Necrosis Factor-alpha / chemistry*

Substances

Antibodies
Peptide Library
Single-Domain Antibodies
Tumor Necrosis Factor-alpha