Tadokoro, T. | Search bioRxiv

bioRxiv2024

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

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}C 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.

bioengineering↗

bioRxiv2019

Structure of HIV-2 Nef reveals unique features distinct from HIV-1 involved in immune regulation

The HIV accessory protein Nef plays a major role in establishing and maintaining infection, particularly through immune evasion. Many HIV-2 infected people experience long-term viral control and survival, resembling HIV-1 elite control. HIV-2 Nef has overlapping but also distinct functions from HIV-1 Nef. Here we report the crystal structure of HIV-2 Nef core. The dileucine sorting motif forms a helix bound to neighboring molecules, and moreover, isothermal titration calorimetry demonstrated that the CD3 endocytosis motif can directly bind to HIV-2 Nef, ensuring AP-2 mediated endocytosis for CD3. The highly-conserved C-terminal region forms a -helix, absent from HIV-1. We further determined the structure of SIV Nef harboring this region, demonstrating similar C-terminal -helix, which may contribute to AP-1 binding for MHC-I downregulation. These results provide new insights into the distinct pathogenesis of HIV-2 infection.

biophysics↗