Wang Jingyu, Wei Ming, Li Zhengxuan, Zhou Yike, Han Donghui
4th Cadet Regiment, Basic Medical Science Academy, Air Force Medical University, Xi'an 710032, China.
Department of Urology, 989th Hospital of Joint Logistic Support Force of Chinese People's Liberation Army, Luoyang 471032, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2023 Sep;39(9):769-776.
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 (hIgG-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-DM1 effectively inhibits pulmonary fibrosis in mice by targeting and killing CD248-positive myofibroblasts. The safety of this strategy is preliminarily assessed.
目的 探讨靶向杀伤CD248阳性肌成纤维细胞对博来霉素诱导的小鼠肺纤维化的治疗作用。方法 制备靶向CD248的抗体-美登素1（DM1）偶联物IgG78-DM1。用紫外分光光度计测定并计算药物偶联效率，通过SDS-PAGE和蛋白质免疫印迹分析对IgG78-DM1进行鉴定。体外实验，采用流式细胞术检测IgG78-DM1对CD248阳性肌成纤维细胞的结合活性，采用CCK-8法评估IgG78-DM1对CD248阳性肌成纤维细胞的细胞毒性。体内实验，将C57BL/6雄性小鼠随机分为对照组、特发性肺纤维化组、人IgG-DM1（hIgG-DM1）对照组和IgG78-DM1治疗组。然后，构建博来霉素诱导的小鼠肺纤维化模型。两周后，对动物模型静脉注射IgG78-DM1。治疗两周后，收集肺组织进行Masson染色和天狼星红染色，以评估肺纤维化程度。采用实时荧光定量PCR检测CD248以及成纤维细胞活化标志物α-平滑肌肌动蛋白（α-SMA）和I型胶原α1（COL1A1）的表达水平。通过进行肝肾功能检测初步评估IgG78-DM1的安全性。结果 成功制备IgG78-DM1，其药物偶联率为3.2。偶联后抗体结构保持稳定，对CD248阳性肌成纤维细胞具有有效的结合和细胞毒性。用IgG78-DM1治疗后，小鼠肺纤维化程度显著降低，同时CD248、α-SMA和COL1A1的表达下降。与正常对照组相比，小鼠的肝肾功能保持在正常水平。结论 IgG78-DM1通过靶向杀伤CD248阳性肌成纤维细胞有效抑制小鼠肺纤维化。对该策略的安全性进行了初步评估。
