Preparation,characterization,and in vivo muscle-promoting function of an anti-GDF8 monoclonal antibody(P249337)

YU Duo; XIE Fei; ZHAO Bochen; HE Lei; LI Meng; LEI Xiaoying

doi:10.13276/j.issn.2097-1656.2026.07.008

Abstract：Objective To prepare monoclonal antibodies against growth differentiation factor 8(GDF8)with excellent properties,and systematically evaluate their physicochemical properties,in vitro neutralizing activity and ability to promote skeletal muscle growth in animal models.Methods BALB/c mice were immunized with recombinant human GDF8,and hybridoma cell bank were constructed to screen for cell lines secreting positive monoclonal antibodies.The antibodies were purified using Protein A affinity chromatography.The purity,aggregation state,and endotoxin level of the antibodies were detected by SDS-PAGE,size exclusion chromatography-high performance liquid chromatography,and limulus amebocyte lysate test,respectively.The binding activity of the antibodies was detected by ELISA,and the half maximal effective concentration(EC50)was calculated.The expression level of p-SMAD3 in myoblast C2C12 cells was detected by Western blotting to evaluate the in vitro neutralizing ability of the antibodies.Eight-week-old male Kunming mice were intraperitoneally injected with the antibody at a dose of 10 mg/kg twice a week for 4 weeks.The body mass,forelimb grip strength,wet weight of bilateral gastrocnemius muscle,and cross-sectional area of muscle fibers of the mice were dynamically monitored.The related indicators of cardiac function were simultaneously detected,and the content of GDF8 in serum was detected by ELISA.Results A hybridoma cell line GYM329 that stably secretes anti-GDF8 antibodies was obtained.The purified antibody P249337 had a complete molecular composition,with a monomer content of ≥99％and an endotoxin content of＜0.1 EU/μg,meeting the standards for injectable biological products.The EC50 of P249337 was 0.069 40 mg/L.In vitro,it could dose-dependently block the phosphorylation of SMAD3 in C2C12 cells.Compared with the control group,the body mass,absolute and relative forelimb grip strength,wet weight of bilateral gastrocnemius muscle,and cross-sectional area of muscle fibers of the antibody treatment group were significantly increased(P＜0.05).The content of GDF8 in serum was significantly decreased(P＜0.01).There were no significant differences in heart rate,cardiac output,left ventricular ejection fraction,and ventricular systolic function(P＞0.05).Conclusion The anti-GDF8 monoclonal antibody P249337 has good molecular integrity and homogeneity,strong antigen binding ability,and in vitro neutralizing activity,and can significantly promote skeletal muscle growth.It has no effect on cardiac function.This study lays a foundation for the development of new biological preparations for the treatment of muscle atrophy diseases.