SnoopLigase Enables Highly Efficient Generation of C–C-Linked Bispecific Nanobodies Targeting TNF‑α and IL-17A

• Published in: Bioconjugate chemistry 2022-08, Vol.33 (8), p.1446-1455
• Main Authors: Chen, Jiewen, Huang, Chundong, Zhao, Wei, Ren, Jun, Ji, Fangling, Jia, Lingyun
• Format: Article
• Language: English
• Subjects: Antibodies; Arthritis; Bispecific antibodies; C-Terminus; Interleukins; Monomers; Nanobodies; Rheumatoid arthritis; Synoviocytes; Tumor necrosis factor-TNF; Tumor necrosis factor-α
• ISSN: 1043-1802; 1520-4812
• DOI: 10.1021/acs.bioconjchem.2c00143

Bispecific antibodies (bis-Nbs) have been extensively developed since the concept was devised over the decades. Taking advantage of the superior characteristics of nanobodies, bis-Nbs exhibit an emerging tendency to become the new generation of research and diagnostic tools. Traditional strategies to connect the homo- or heterogeneous monomers are commonly applied, but there are still technical issues to generate the bispecific molecules as efficiently as designed. Here, we utilize SnoopLigase to directly tether the C terminus (C–C) of the tagged nanobodies against tumor necrosis factor-α (TNF-α) and interleukin-17A (IL-17A). Under optimal conditions, the yield of C–C-linked bis-Nbs can reach as high as 70% due to the existence of SnoopLigase. The prepared bis-Nbs possessed similar or even higher affinity as the monomers and significantly inhibited the proliferation and migration of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) induced by TNF-α and IL-17A. This study provides an innovative route for using SnoopLigase to realize a highly efficient generation of C–C-linked bis-Nbs. The approach can be applied to different and multicomponent systems for their potential applications in disease diagnosis and treatment.