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

• Published in: Molecules (Basel, Switzerland) Vol. 24; no. 14; p. 2620
• Main Authors: Yamauchi, Soichiro, Kobashigawa, Yoshihiro, Fukuda, Natsuki, Teramoto, Manaka, Toyota, Yuya, Liu, Chenjiang, Ikeguchi, Yuka, Sato, Takashi, Sato, Yuko, Kimura, Hiroshi, Masuda, Takeshi, Ohtsuki, Sumio, Noi, Kentaro, Ogura, Teru, Morioka, Hiroshi
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 18.07.2019; MDPI
• Subjects: aggregation propensity; Antigens; Atomic force microscopy; C-Terminus; Calorimetry, Differential Scanning; Chains; Chromatography; cyclic scFv; Cyclization; Domains; Dynamic Light Scattering; Fluorimetry; Fluorometry; Gram-positive bacteria; high-speed atomic force microscopy; Homogeneity; Humans; Immunoglobulin Heavy Chains - chemistry; Immunoglobulin Heavy Chains - metabolism; Immunoglobulin Light Chains - chemistry; Immunoglobulin Light Chains - metabolism; Immunoglobulin Variable Region - chemistry; Immunoglobulin Variable Region - metabolism; Light scattering; Microscopy; Microscopy, Atomic Force; Monoclonal antibodies; N-Terminus; Photon correlation spectroscopy; Polypeptides; Protein Aggregates; Protein Binding; Proteins; Resonance scattering; Single-Chain Antibodies - chemistry; Single-Chain Antibodies - metabolism; single-chain Fv; Sortase; sortase A; Surface Plasmon Resonance; Therapeutic applications; Thermal stability; high-speed atomic force microscopy; dynamic light scattering; cyclic scFv; single-chain Fv; sortase A; aggregation propensity
• ISSN: 1420-3049; 1420-3049
• DOI: 10.3390/molecules24142620

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 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.