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Enhanced Bioactivity of the Anti-LOX-1 scFv Engineered by Multimerization Strategy

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Abstract

Single-chain variable fragment (scFv) antibodies as therapeutic agents have the potential to reduce the production cost and immunogenicity relative to monoclonal antibodies, but their monovalency and lack of a fragment crystallizable region can lead to reduced function. Multimerization is one strategy for recovering the function; however, their application is limited by the production of multimeric proteins. In our previous study, an anti-lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) scFv showed potential use in diagnosis and therapy of atherosclerotic diseases, but is limited by its inherent low antigen-binding activity. In this study, to improve the efficacy of the anti-LOX-1 scFv, we constructed the anti-LOX-1 scFv multimers by modifying the linker length between the variable domains of the scFv or by fusing the scFv with self-merization domains and expressed these scFv multimers in Brevibacillus choshinensis hosts. After optimization, all of the scFv multimers obtained efficient secretion expression. Compared with the scFv monomer, the multimers that are successfully fractionated displayed increased neutralization activity and showed elevated antigen-binding avidity, especially the tetramer, which improved the antigen avidity by two orders of magnitude. Moreover, the scFv dimer and the tetramer both displayed better stability and longer half-life in serum, which can be attractive candidates for the next-generation anti-LOX-1 therapeutic antibody.

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Funding

This work was supported by the National Natural Science Foundation of China [81072564], the Science and Technology Development Planning of Jilin [20140203001YY], and the Jilin Province Development and Reform Commission [2014Y080].

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Authors and Affiliations

  1. School of Life Science, Jilin University, Changchun, 130012, Jilin, People’s Republic of China

    Wei Hu, Qiuhong Xie, Ling Liu & Hongyu Xiang

  2. National Engineering Laboratory for AIDS Vaccine, School of Life Science, Jilin University, Changchun, 130012, Jilin, People’s Republic of China

    Qiuhong Xie & Hongyu Xiang

  3. Key Laboratory for Molecular Enzymology and Engineering, the Ministry of Education, Jilin University, Changchun, 130012, Jilin, People’s Republic of China

    Qiuhong Xie & Hongyu Xiang

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  1. Wei Hu
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  2. Qiuhong Xie
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Correspondence to Hongyu Xiang.

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Electronic supplementary material

ESM 1

(DOC 119 kb)

Supplementary figure 2

Structural characterization of the scFv multimers. (a) Fluorescence spectroscopy excited at the wavelength of 295 nm. (b) Far-UV CD spectrum in the range of 200-240 nm (JPG 65.8 KB)

High Resolution image (TIF 350 KB)

ESM 3

(DOC 78 kb)

Supplementary figure 4

The residual LOX-1 binding activity of the scFv mltimers incubated in mouse serum (JPG 48.5 KB)

High Resolution image (TIF 2.55 MB)

Supplementary figure 5

The mode chart of the engineered scFv proteins. (a) monomer; (b) dimer; (c) trimer; (d) tetramer; (e) heptamer in front vew; (f) heptemer in side view. These structures (N-terminus to C-terminus) were marked with a gradient color (blue to red) (JPG 162 KB)

High Resolution image (TIF 2.19 MB)

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Hu, W., Xie, Q., Liu, L. et al. Enhanced Bioactivity of the Anti-LOX-1 scFv Engineered by Multimerization Strategy. Appl Biochem Biotechnol 185, 233–247 (2018). https://doi.org/10.1007/s12010-017-2649-3

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  • DOI: https://doi.org/10.1007/s12010-017-2649-3

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