抗PD-L1抗体的心脏毒性及甲状腺素的救治作用实验研究

Cardiotoxicity of Anti-PD-L1 Antibody and the Effect of Levothyroxine in Attenuating the Related Mortality in Mice

背景与目的免疫检查点抑制剂在肿瘤治疗中表现出了显著的疗效,但也可能会引起免疫相关的不良反应(immune-related adverse events,ir AEs)。近来国际上报道了数百例免疫检查点抑制剂相关心肌炎,患者死亡率达50%。本研究目的是在动物水平重现临床情况,通过多次静脉注射抗程序性细胞死亡受体1(programmed cell death 1,PD-1)抗体、抗程序性细胞死亡受体配体1(programmed cell death ligand 1,PD-L1)抗体,观察药物对正常小鼠所产生的心脏毒性,并探讨L-甲状腺素的保护作用。方法通过尾静脉向6周龄-8周龄的C57BL/6小鼠分别注射抗PD-1抗体(剂量为12.5μg/g,每5天注射1次,共6次)、抗PD-L1抗体(10μg/g,每周注射1次,共6次)、抗PD-L1抗体(用法用量同上)联合L-甲状腺素(注射抗体前30 min腹腔注射,剂量为0.25μg/g)或同型对照免疫球蛋白IgG(10μg/g,每周1次,共6次)。通过超声心动图检测小鼠心脏射血功能;通过无创鼠尾动脉测压仪、小动物生命体征监护仪检测小鼠血压、心电特征与体温;通过酶联免疫标记法检测小鼠血清中游离甲状腺素浓度。结果小鼠心脏表达不同水平的PD-L1。12只接受同型对照免疫球蛋白、12只接受抗PD-1抗体的小鼠无一发生死亡。12只接受3次-6次抗PD-L1抗体注射的小鼠的心重胫骨比显著升高,心肌细胞出现变性、透明化及血管外炎症细胞浸润等改变。心脏超声及心电图检测结果显示,小鼠的心脏射血功能严重受损、心肌缺血。此外,小鼠注射抗PD-L1抗体后血压下降、体温异常降低,血清甲状腺素(tetraiodothyronine,T4)水平降至对照组的1/3。这些小鼠中有8只死亡,死亡率达66.7%。在注射抗PD-L1抗体前30 min经腹腔注射L-甲状腺素可明显降低小鼠死亡率。结论抗PD-1抗体不会引起小鼠心脏毒性及小鼠死亡。抗PD-L1抗体可引起心脏毒性导致小鼠死亡,而L-甲状腺素有明显的保护作用。

Background and objective Immune checkpoint inhibitors(ICIs)such as antibodies against programmed cell death 1(PD-1)and programmed cell death ligand 1(PD-L1),have shown remarkable efficacies in many subtypes of cancers.However,ICIs may also cause severe immune-related adverse events in the recipient patients.Recently,ICIassociated myocarditis have been reported in hundreds of patients worldwide,with a mortality rate of approximately 50%in these cases.This study aims to recapitulate the cardiotoxicity and explore the detoxicifying approaches to attenuate mortality caused by PD-1/PD-L1 inhibitors in healthy mice.Methods Six to eight-week-old C57 BL/6 mice were inoculated with antiPD-1 antibody(12.5μg/g every 5 days for 6 injections),anti-PD-L1 antibody(10μg/g once a week for 6 weeks),anti-PD-L1 antibody(with the same dosage described above)in combination with levothyroxine(0.25μg/g,intraperitoneally injected half an hour before anti-PD-L1 antibody injection),or isotype control immunoglobulin IgG(10μg/g once a week for 6 weeks).The ejection function of the hearts was detected by echocardiography,body temperature and blood pressure were detected by Mouse MonitorTM and non-invassive blood pressure minotor,and serum free thyroxine concentration was detected by The enzyme linked immunosorbent assay(ELISA).Results PD-L1 was expressed at different levels by the cardiomyocytes of the mice.The isotype control immunoglobulin and anti-PD-1 antibody did not cause death of the mice.The 12 mice receiving 3-6 injections of anti-PD-L1 antibody showed a significant increase in the heart-to-tibial ratio and cardiomyoctye degeneration,hyalinization and extravascular inflammatory cell infiltration.In addition,the serum thyroxine was mardedly decreased to 1/3 of that in the control group mice,and the blood pressure and body temperature were abnormally decreased in mice upon treatment with PD-L1 blockade.Eight of the 12(66.7%)mice died from multiple intravenous injection of anti-PD-L1 antibody.Intraperitoneal injection of levothyroxine 30 min before the injection of anti-PD-L1 antibody significantly attenuated the mortality rate of the anti-PD-L1 antibody-treated mice.Conclusion The anti-PD-L1 antibody is cardiotoxic and lethal,and levothyroxine is able to rescue the mice from this immune checkpoint inhibitor-caused mortality.