Affinity improvement of the fully human anti‑TSLP recombinant antibody

Chen,Qi; Xian,Dequn; Xu,Wenfeng; Nian,Siji; Yu,Hong; Wu,Yuchuan; Yuan,Qing

doi:10.3892/mmr.2019.10880

Affinity improvement of the fully human anti‑TSLP recombinant antibody

Authors:
- Qi Chen
- Dequn Xian
- Wenfeng Xu
- Siji Nian
- Hong Yu
- Yuchuan Wu
- Qing Yuan
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Affiliations: State Key Laboratory of Quality Research in Chinese Medicines, Macau Institute for Applied Research in Medicine and Health, Macau University of Science and Technology, Taipa, Macau SAR 000853, P.R. China, Immunology Department, The School of Basic Medical Sciences, Southwest Medical University, Luzhou, Sichuan 646000, P.R. China
Published online on: December 12, 2019 https://doi.org/10.3892/mmr.2019.10880
Pages: 759-767
Copyright: © Chen et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Thymic stromal lymphopoietin (TSLP) is a potentially important target for the treatment of asthma and malignancies. However, a fully human antibody reactive with TSLP is currently unavailable for clinical use. In a previous study, a human anti‑TSLP‑single‑chain antibody variable fragment (anti‑TSLP‑scFv) 84 was selected by phage display from a constructed human scFv library. In the present study, a computer simulation method was developed using Discovery Studio 4.5 software, to increase the affinity of anti‑TSLP‑scFv‑84. Specific primers were designed and mutated DNA sequences of anti‑TSLP‑scFvs were obtained by overlap extension PCR. The mutant scFvs were expressed in pLZ16 and affinity‑enhanced anti‑TSLP‑scFv‑M4 was screened using ELISA. However, in general the scFvs have low stability and short half‑lives in vivo. Therefore, scFv‑84 and scFv‑M4 were inserted into eukaryotic expression vectors (pcDNA3.1‑sp‑Fc and PMH3EN‑sp‑Fc) and then transfected into 293F cells to express anti‑TSLP‑scFv‑Fc. ELISA and western blotting results indicated the size of the anti‑TSLP‑scFv‑Fc to be ~50 kDa. Binding of anti‑TSLP‑scFv‑Fc‑M4 to TSLP was enhanced compared with the pre‑mutated scFv‑Fc‑84. The affinity of the mutated recombinant antibody was determined using the BIAcore technique and found to be ~10‑fold greater than the pre‑mutated antibody.

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