A Single C226S Mutation Rescues Fragmentation and Enhances Antigen-Binding Activity of a Giant Panda Chimeric Antibody Against Canine Distemper Virus

Background/Objectives: Canine distemper virus (CDV) poses a major threat to the conservation of the endangered giant panda (Ailuropoda melanoleuca). Although monoclonal antibodies hold great therapeutic promise, the development of species-specific antibody therapeutics for wildlife remains challenging. In this study, we aimed to construct and optimize a giant panda chimeric antibody targeting the CDV H protein through Fc engineering. Methods: A chimeric antibody (6G4-gpIgG1) was constructed by fusing the variable regions of a murine anti-CDV H antibody with giant panda IgG1b Fc. Structural analysis using AlphaFold identified excessive hinge exposure as a potential cause of instability. A C226S mutation in the hinge region was designed and introduced. The wild-type and mutant antibodies were expressed in CHO-S cells, purified, and characterized by SDS-PAGE, ELISA, biolayer interferometry (BLI), and SEC-HPLC. Results: The wild-type murine–giant panda chimeric antibody exhibited degradation fragmentation and a nanomolar-range binding affinity (EC50 = 0.62 nM; BLI KD = 4.27 × 10−9 M). The C226S mutation (6G4-gpIgG1-Opt2) reduced fragmentation, increased monomer purity from 88.38% to 96.99%, and increased antigen-binding activity by over 30-fold (EC50 = 0.019 nM), reaching levels comparable to the murine–canine chimeric control, which exhibited a canonical Y-shaped conformation. BLI also revealed stronger binding affinity for Opt2 (KD = 1.33 × 10−10 M), associated with a slower dissociation rate (kdis = 7.80 × 10−5 s−1 versus 1.15 × 10−3 s−1 for the wild type). Conclusions: A single C226S mutation in the giant panda IgG1 hinge region substantially improves structural stability, reducing degradation fragments, increasing monomer purity, and unexpectedly enhancing antigen-binding affinity by over 30-fold. This study provides a structure-guided Fc engineering strategy for developing chimeric antibodies in giant pandas, offering a promising candidate for CDV protection and a generalizable framework for conservation medicine.