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Article

Cyclization of Single-Chain Fv Antibodies Markedly Suppressed Their Characteristic Aggregation Mediated by Inter-Chain VH-VL Interactions

by
Soichiro Yamauchi
1,†,
Yoshihiro Kobashigawa
1,†,
Natsuki Fukuda
1,†,
Manaka Teramoto
1,
Yuya Toyota
1,
Chenjiang Liu
1,
Yuka Ikeguchi
1,
Takashi Sato
1,
Yuko Sato
2,
Hiroshi Kimura
2,
Takeshi Masuda
3,
Sumio Ohtsuki
3,
Kentaro Noi
4,5,
Teru Ogura
6,7 and
Hiroshi Morioka
1,*
1
Department of Analytical and Biophysical Chemistry, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan
2
Cell Biology Center, Institute of Innovative Research, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8503, Japan
3
Department of Pharmaceutical Microbiology, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan
4
Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita 565-0871, Japan
5
Institute for NanoScience Design, Osaka University, 1-3 Machikaneyama, Toyonaka 560-8531, Japan
6
Department of Molecular Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto 860-0811, Japan
7
CREST, JST, 4-1-8, Honcho, Kawaguchi, Saitama 332-0012, Japan
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to the present work.
Molecules 2019, 24(14), 2620; https://doi.org/10.3390/molecules24142620
Submission received: 25 June 2019 / Revised: 16 July 2019 / Accepted: 17 July 2019 / Published: 18 July 2019
(This article belongs to the Special Issue Protein Domains: Structures and Molecular Functions)
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Abstract

Single-chain Fv (scFv) antibodies are recombinant proteins in which the variable regions of the heavy chain (VH) and light chain (VL) are connected by a short flexible polypeptide linker. ScFvs have the advantages of easy genetic manipulation and low-cost production using Escherichia coli compared with monoclonal antibodies, and are thus expected to be utilized as next-generation medical antibodies. However, the practical use of scFvs has been limited due to low homogeneity caused by their aggregation propensity mediated by inter-chain VH-VL interactions. Because the interactions between the VH and VL domains of antibodies are generally weak, individual scFvs are assumed to be in equilibrium between a closed state and an open state, in which the VH and VL domains are assembled and disassembled, respectively. This dynamic feature of scFvs triggers the formation of dimer, trimer, and larger aggregates caused by the inter-chain VH-VL interactions. To overcome this problem, the N-terminus and C-terminus were herein connected by sortase A-mediated ligation to produce a cyclic scFv. Open-closed dynamics and aggregation were markedly suppressed in the cyclic scFv, as judged from dynamic light scattering and high-speed atomic force microscopy analyses. Surface plasmon resonance and differential scanning fluorometry analysis revealed that neither the affinity for antigen nor the thermal stability was disrupted by the scFv cyclization. Generality was confirmed by applying the present method to several scFv proteins. Based on these results, cyclic scFvs are expected to be widely utilized in industrial and therapeutic applications.
Keywords: single-chain Fv; aggregation propensity; sortase A; cyclic scFv; high-speed atomic force microscopy; dynamic light scattering single-chain Fv; aggregation propensity; sortase A; cyclic scFv; high-speed atomic force microscopy; dynamic light scattering
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MDPI and ACS Style

Yamauchi, S.; Kobashigawa, Y.; Fukuda, N.; Teramoto, M.; Toyota, Y.; Liu, C.; Ikeguchi, Y.; Sato, T.; Sato, Y.; Kimura, H.; et al. Cyclization of Single-Chain Fv Antibodies Markedly Suppressed Their Characteristic Aggregation Mediated by Inter-Chain VH-VL Interactions. Molecules 2019, 24, 2620. https://doi.org/10.3390/molecules24142620

AMA Style

Yamauchi S, Kobashigawa Y, Fukuda N, Teramoto M, Toyota Y, Liu C, Ikeguchi Y, Sato T, Sato Y, Kimura H, et al. Cyclization of Single-Chain Fv Antibodies Markedly Suppressed Their Characteristic Aggregation Mediated by Inter-Chain VH-VL Interactions. Molecules. 2019; 24(14):2620. https://doi.org/10.3390/molecules24142620

Chicago/Turabian Style

Yamauchi, Soichiro, Yoshihiro Kobashigawa, Natsuki Fukuda, Manaka Teramoto, Yuya Toyota, Chenjiang Liu, Yuka Ikeguchi, Takashi Sato, Yuko Sato, Hiroshi Kimura, and et al. 2019. "Cyclization of Single-Chain Fv Antibodies Markedly Suppressed Their Characteristic Aggregation Mediated by Inter-Chain VH-VL Interactions" Molecules 24, no. 14: 2620. https://doi.org/10.3390/molecules24142620

APA Style

Yamauchi, S., Kobashigawa, Y., Fukuda, N., Teramoto, M., Toyota, Y., Liu, C., Ikeguchi, Y., Sato, T., Sato, Y., Kimura, H., Masuda, T., Ohtsuki, S., Noi, K., Ogura, T., & Morioka, H. (2019). Cyclization of Single-Chain Fv Antibodies Markedly Suppressed Their Characteristic Aggregation Mediated by Inter-Chain VH-VL Interactions. Molecules, 24(14), 2620. https://doi.org/10.3390/molecules24142620

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