基于枯否细胞吞噬作用研究抗体-毒素偶联药物T-DM1肝脏毒性的机制

As an antibody-drug conjugate (ADC), T-DM1 showes significant clinical effects, while severe hepatotoxicity limits its clinical applications, and related research on the mechanisms of the toxicity has yet not been reported without rational intervention strategies. We have found in previous studies that T-DM1 caused significant hepatotoxicity in SD rats, characterized by liver parenchymal cells injuried posterior to the Kupffer cells which are one kind of macrophages residing in livers responsible for clearing exogenous substances, suggesting that Kupffer cells may be involved in hepatoxicity and regulating its phagocytosis maybe one of valid intervention strategies. It is also known that antibody drugs may be endocytosed into cells mediated by its receptor FcRn which is also expressed in Kupffer cells. Based on above discussions, this project intends firstly to confirm the importent roles of Kupffer cells in liver parenchymal cells injury, and then investigate mechanisms of hepatotoxicity caused by T-DM1 from the perspective of phagocytosis of Kupffer cells, what’s more, to explore the potential to reduce toxicity by regulation the interaction of T-DM1 and its receptor FcRn. It is expected through this study that the mechanisms of hepatotoxicity involved by T-DM1 will be clarified and a valid intervention strategy will be found to mitigate hepatotoxicity by regulating hagocytosis of Kuffer cells, hence providing theoretical ideas for research of hepatotoxicity of other ADC and scientific basises for rationally and safely medication of T-DM1.

抗体-毒素偶联药物T-DM1具有显著的临床治疗效果，但严重的肝脏毒性限制了其临床应用，关于其毒性机制的研究尚未见报道且缺乏有效的干预手段。前期研究发现，在T-DM1引起的肝脏毒性中枯否细胞先于肝实质细胞产生病变，而后者作为肝脏特有的巨噬细胞在外源物质清除中起重要作用，这提示枯否细胞可能参与了肝脏毒性的发生，降低其吞噬作用可能减轻肝脏毒性。此外已知抗体类药物可通过受体FcRn的介导进入细胞，且枯否细胞表达该受体。基于此，本课题拟从枯否细胞对T-DM1的吞噬作用入手研究肝脏毒性的机制，并确认枯否细胞在肝脏毒性中的关键作用，进一步探索调控T-DM1与其受体FcRn的结合对减轻肝脏毒性的作用。预期通过本研究，深入阐明T-DM1肝脏毒性的分子机制，明确降低枯否细胞吞噬作用作为T-DM1肝脏毒性干预手段的可行性，为其他ADC药物肝脏毒性研究提供思路并为T-DM1的合理安全用药提供科学依据。

抗体-毒素偶联药物T-DM1具有显著的临床治疗效果，但严重的肝脏毒性限制了其临床应用，关于其毒性机制尚不明确且缺乏有效的干预手段。本研究对T-DM1引起的肝脏毒性的机制进行了考察。.通过PET-CT考察了食蟹猴静脉注射给予89Zr-T-DM1（905.4μCi）后在主要脏器的分布情况。肝脏中放射性暴露量AUC0-456h最高，放射性摄取持续时间最长。该结果提示，肝脏可能通过某种机制参与了对T-DM1的摄取。.大鼠静脉注射给予3.6~36mg/kg T-DM1后24h，ALT、AST、TBIL和ALP可见升高趋势，96h升至最高；168h可见下降（ALP除外），肝脏湿重高于对照组，高剂量组枯否细胞病变和部分肝实质细胞坏死。该结果初步提示了T-DM1引起肝脏毒性的时间规律。进一步研究发现，大鼠以36mg/kg给药，血清miR-122的变化早于传统肝损伤标志物；2h可见肝脏枯否细胞坏死和单个核细胞浸润，持续至24h。.采用anti-DM1抗体通过免疫组化考察了DM1（或含有DM1的片段）在大鼠肝脏中的分布情况。给药后1h可在枯否细胞中检测到DM1；8h可观察到DM1在枯否细胞中大量分布，小部分肝实质细胞可检测到DM1；48h枯否细胞形态发生改变且较多肝实质细胞可检测到DM1，伴肝损伤标志物的明显升高。提示枯否细胞的吞噬作用导致了DM1（或含有DM1的片段）的释放并对肝实质细胞造成损伤。.T-DM1与枯否细胞孵育后的上清液对肝实质细胞的损伤作用大于相同浓度的T-DM1；在枯否细胞-肝实质细胞共培养体系中，T-DM1对肝实质细胞的损伤作用更为明显。.本研究确认了枯否细胞在肝脏毒性中发挥关键作用，减少枯否细胞吞噬作用有助于缓解T-DM1肝脏毒性，为ADC药物肝脏毒性研究和安全用药提供了科学依据。