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

Soichiro Yamauchi,Hiroshi Morioka,Hiroshi Kimura,Yuko Sato,Yuya Toyota,Teru Ogura,Sumio Ohtsuki,Chenjiang Liu,Natsuki Fukuda,Kentaro Noi,Takeshi Masuda,Yuka Ikeguchi,Manaka Teramoto,Yoshihiro Kobashigawa,Takashi Sato

doi:10.3390/molecules24142620

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.

Highlights

IntroductionMonoclonal antibodiesareare widely in medical and industrial applications—e.g., as Monoclonal antibodies widely usedused in medical and industrial applications—e.g., as therapeutic therapeutic and diagnostic agents, biosensor materials, and biochemical tools [1,2,3].In recent years, and diagnostic agents, biosensor materials, and biochemical tools [1,2,3]
The Single-chain Fv (scFv) was mixed with sortase A at a molar ratio of 1:1
The 13C7-scFv was expressed in E. coli strain BL21 (DE3) and obtained as an inclusion body, which was solubilized by 6 M guanidine HCl (GdnHCl), purified by affinity chromatography using Ni-NTA, and refolded by gradually reducing the concentration of GdnHCl by step-wise dialysis

Summary

Introduction

Monoclonal antibodiesareare widely in medical and industrial applications—e.g., as Monoclonal antibodies widely usedused in medical and industrial applications—e.g., as therapeutic therapeutic and diagnostic agents, biosensor materials, and biochemical tools [1,2,3]. Diagnostic agents, biosensor materials, and biochemical tools [1,2,3]. The antibodies market grownmonoclonal rapidly, monoclonal have continued to be the antibodies market has grownhas rapidly, antibodiesantibodies have continued to be produced produced using mammalian cells and technologies, advanced technologies, in high production costs. Using mammalian cells and advanced resulting inresulting high production costs. Depletion of target molecules has shifted development of antibody therapeutics to that depletion of target molecules has shifted development of antibody therapeutics to that which require which require moretechnology, sophisticated technology, for example, bispecific antibodies.

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