Thermostability and binding properties of single-chained Fv fragments derived from therapeutic antibodies

Abstract

Small antibody fragments have recently been used as alternatives to full-length monoclonal antibodies in therapeutic applications. One of the most popular fragment antibodies is single-chain fragment variables (scFvs), consisting of variable heavy (VH) and variable light (VL) domains linked by a flexible peptide linker. scFvs have small molecular sizes, which enables good tissue penetration and low immunogenicity. Despite these advantages, the use of scFvs, especially for therapeutic purpose, is still limited because of the difficulty to regulate the binding activity and conformational stability. In this study, we constructed and analyzed 10 scFv fragments derived from 10 representatives of FDA-approved mAbs to evaluate their physicochemical properties. Differential scanning calorimetry analysis showed that scFvs exhibited relatively high but varied thermostability, from 50 to 70 degrees of melting temperatures, and different unfolding cooperativity. Surface plasmon resonance analysis revealed that scFvs fragments that exhibit high stability and cooperative unfolding likely tend to maintain antigen binding. This study demonstrated the comprehensive physicochemical properties of scFvs derived from FDA-approved antibodies, providing insights into antibody design and development.

Development, High-Throughput Profiling, and Biopanning of a Large Phage Display Single-Domain Antibody Library

“…To address these challenges, researchers have attempted to develop therapeutic antibodies with smaller single-chain variable fragment (scFv) forms [65]. However, these efforts have faced limitations, such as reduced thermal stability [66,67]. Alternatively, VHHs are known to have improved thermal stability despite their smaller size compared with scFvs, making them a promising candidate for therapeutic antibodies [68,69].…”

“…Alternatively, VHHs are known to have improved thermal stability despite their smaller size compared with scFvs, making them a promising candidate for therapeutic antibodies [68,69]. Tadokoro et al measured the thermal stability of 10 approved antibodies in scFv form, with a T m ranging from 52.3 • C to 71.2 • C [67]. In comparison, selected VHHs obtained through biopanning from the constructed library exhibited a T m range of 60.5 • C to 77.5 • C, with an average of 68.7 • C. These results indicate that the selected VHHs have higher thermal stability compared with scFvs.…”