IgG78-美登素1(IgG78-DM1)通过靶向杀伤CD248阳性肌纤维母细胞抑制小鼠肺纤维化

IgG78-DM1 inhibits pulmonary fibrosis by targeting and killing CD248-positive myofibroblasts in mice

目的 探究通过靶向杀伤CD248阳性肌纤维母细胞对博来霉素致小鼠肺纤维化的治疗效果。方法 制备靶向CD248的抗体偶联药物IgG78-美登素1(IgG78-DM1),通过紫外分光光度计检测并计算药物偶联效率,通过SDS-PAGE及Western blot法对IgG78-DM1进行鉴定。利用流式细胞术检测IgG78-DM1与CD248阳性肌纤维母细胞的结合情况,CCK-8法检测IgG78-DM1对CD248阳性肌纤维母细胞的杀伤活性。将C57BL/6雄鼠随机分为正常对照组、模型组、人IgG-DM1(hIgG-DM1)阴性对照组和IgG78-DM1治疗组,利用博来霉素构建小鼠肺纤维化模型,建模2周后尾静脉注射IgG78-DM1,治疗2周后取小鼠肺组织进行马松和天狼猩红染色评价纤维化程度,实时荧光定量PCR检测CD248和纤维化标志物α平滑肌肌动蛋白(α-SMA)、Ⅰ型胶原蛋白α1(COL1A1)表达水平。通过肝肾功检测初步评估IgG78-DM1的安全性。结果 成功制备抗体偶联药物IgG78-DM1,药物偶联比为3.2;偶联药物后抗体结构稳定,能够有效结合并杀伤CD248阳性肌纤维母细胞;IgG78-DM1治疗后,小鼠肺纤维化程度显著减轻,CD248和α-SMA、 COL1A1表达降低;与正常对照相比,小鼠肝肾功能正常。结论 抗体偶联药物IgG78-DM1通过靶向杀伤CD248阳性肌纤维母细胞能够有效抑制小鼠肺纤维化,初步评估该策略安全性较好。

Objective To investigate the therapeutic effect of targeting and killing CD248-positive myofibroblasts on bleomycin-induced pulmonary fibrosis in mice.Methods IgG78-DM1,an antibody-maytansine 1(DM1)conjugate targeting CD248,was prepared.The drug conjugation efficiency was measured and calculated by UV spectrophotometer,and the identification of IgG78-DM1 was performed through SDS-PAGE and Western blot analysis.In vitro,the binding activity of IgG78-DM1 on CD248-positive myofibroblasts was detected by flow cytometry and the cytotoxicity of IgG78-DM1 to CD248-positive myofibroblasts was evaluated by CCK-8 assay.In vivo,C57BL/6 male mice were randomly divided into control group,idiopathic pulmonary fibrosis group,human IgG-DM1(hlgG-DM1)control group,and IgG78-DM1 treatment group.Then,the mouse models with pulmonary fibrosis induced by bleomycin were constructed.Two weeks later,the animal models were intravenously injected with IgG78-DM1.After the treatment of two weeks,lung tissues were collected for Masson staining and Sirius Red staining to evaluate the degree of pulmonary fibrosis.Real-time fluorescence quantitative PCR was used to measure the expression levels of CD248,as well as markers of fibroblastic activation including alpha-smooth muscle actin(α-SMA)and type I collagen alpha 1(COL1A1).The safety of IgG78-DM1 was preliminarily assessed by conducting liver and kidney function tests.Results IgG78-DM1 was successfully prepared,and its drug conjugation ratio was 3.2.The antibody structure remained stable after conjugation,allowing effective binding and cytotoxicity against CD248-positive myofibroblasts.After treatment with IgG78-DM1,the degree of pulmonary fibrosis in mice significantly reduced,accompanied by the decrease of the expression of CD248,α-SMA,and COL1A1.The liver and kidney function of the mice remained at normal levels compared to the normal control group.Conclusion IgG78-DM1effectivelyinhibits pulmonary fibrosis in mice by targeting and klling CD248-positive myofibroblasts.The safety of this strategy is preliminarily assessed.