抗GDF8单克隆抗体P249337的制备、鉴定及体内促肌肉生长功能研究

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

摘要

目的 制备性质优良的生长分化因子8(GDF8)单克隆抗体, 系统评价其理化性质、体外中和活性及在动物模型中对骨骼肌的促肌肉生长功能。方法 以重组人GDF8蛋白免疫BALB/c小鼠构建杂交瘤细胞库, 筛选分泌阳性单克隆抗体的细胞株; 应用Protein A亲和层析纯化抗体; SDS-PAGE、高效尺寸排阻色谱及鲎试剂法分别检测抗体纯度、聚合状态及内毒素水平; 通过ELISA检测抗体结合活性并计算半效结合浓度(EC₅₀); 采用Western blotting检测成肌细胞C2C12中p-SMAD3表达水平评价抗体体外中和能力; 取8周龄雄性昆明小鼠按10 mg/kg剂量腹腔注射抗体, 每周2次, 持续4周, 动态监测小鼠体质量、前肢抓力, 检测腓肠肌湿质量、肌纤维横截面积, 同步检测心功能相关指标; ELISA试剂盒检测血清中GDF8含量。结果 获得可稳定分泌抗GDF8抗体的杂交瘤细胞株GYM329; 纯化后抗体P249337的分子组成完整, 单体含量≥99%, 内毒素含量<0.1 EU/μg, 符合注射用生物制品标准; P249337的EC₅₀为0.069 40 mg/L; 在体外可剂量依赖性地阻断C2C12细胞SMAD3的磷酸化; 与对照组相比, 抗体治疗组小鼠体质量、前肢绝对及相对抓力、双侧腓肠肌湿质量、肌纤维横截面积均显著增加(P<0.05); 血清中活性形式GDF8含量显著降低(P<0.01); 心率、心搏输出量、左心室射血分数及心室收缩功能无显著差异(P>0.05)。结论 抗GDF8单克隆抗体P249337具有良好的分子完整性和均一性, 具有很强的抗原结合能力和体外中和活性, 能显著促进骨骼肌生长, 且对心脏功能无影响, 为研发肌肉萎缩性疾病治疗用新型生物制剂奠定了基础。

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.

关键词

GDF8 / 单克隆抗体 / 肌肉萎缩 / P249337 / 杂交瘤技术 / C2C12细胞 / 腓肠肌 / 前肢抓力

Key words

GDF8 / monoclonal antibody / muscle atrophy / P249337 / hybridoma technology / C2C12 cells / gastrocnemius muscle / forelimb grip strength

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基金资助

国家自然科学基金青年科学基金(82204259)