CAR-T治疗所致凝血功能改变及其干预措施研究

Although the treatment of leukemia with chimeric antigen receptor T-cell immunotherapy (CAR-T) has achieved major breakthrough in recent years, cytokine release syndrome (CRS) as an adverse reaction limits the wide application of this novel technique. CRS occurs in 2/3 of patients and can cause multi-organ injury. Currently, using cytokines as molecular markers still cannot accurately predict CRS. In addition, glucocorticoids or cytokine receptor antagonists are used in the treatment of CRS, which can reduce the maximum effect of CAR-T to tumor while inhibiting systemic inflammatory response. Our recent clinical trial has found that all the patients after CAR-T therapy have coagulation disorder in the early stage of CRS. Therefore, it is speculated that abnormal coagulation and microcirculation injury induced by abnormal coagulation are immediate causes for.multiple organ dysfunction, and using anticoagulant protein to block the inflammation-induced excessive activation of coagulation is expected to control CRS while not influencing the anti-tumor effect. This project aims to explore the prediction system for CRS using global coagulation-fibrinolysis tests, CloFAL and STP in combination with changes of cytokines. We have previously found that the anticoagulant activity of protein C (PC) with p.Lys192del mutation is reduced, but its local anti-inflammatory effect is not influenced. On this basis, the project plans to explore the therapeutic effect of rAPC-Lys192del on CRS using a CAR-T mouse model. It is expected to improve the early diagnostic rate of CRS and provide new directions for more effective prevention and treatment of CRS.

嵌合抗原受体T细胞技术（CAR-T）在白血病的治疗中取得重大突破，但细胞因子释放综合症（CRS）这一不良反应限制了该技术的推广。目前以细胞因子水平无法对CRS准确预测。CRS的治疗采用糖皮质激素或细胞因子受体拮抗剂，药物在抑制全身炎症反应的同时也会削弱CAR-T对肿瘤的最大杀伤力。在我们最近开展的CAR-T临床试验中，患者在CRS早期均出现凝血功能异常，推测凝血异常及其引起的微循环损伤是多器官功能障碍的直接原因，利用抗凝蛋白阻断炎症性凝血过度激活有望在不影响抗肿瘤效应的情况下控制CRS。本项目利用凝血-纤溶的总体分析法CloFAL与STP，结合细胞因子变化，探索CRS的预测体系；在既往发现变异蛋白C（PC）p.Lys192del抗凝活性下降但局部抗炎作用不改变的基础上，利用CAR-T大鼠模型探索rAPC-Lys192del治疗CRS的效果。项目有望为CRS的早期诊断和更有效防治指明新方向。

嵌合抗原受体T细胞（CAR-T）治疗显著改善了血液肿瘤患者的预后，其主要不良反应包括细胞因子释放综合征（CRS）和出凝血功能障碍。本项目通过DNA片段合成、慢病毒转染等技术成功构建出安全有效的CD19 CAR-T细胞，并招募患者参与CAR-T治疗临床试验。通过检测细胞因子及免疫指标等，发现血清IL-6的峰值水平和出现时间与CRS等级呈正相关，细胞表面CD57、PD-1、CD27、CD28、CD127等免疫指标异常提示不良预后，完善了CRS预测体系。通过动态监测凝血指标，发现重度CRS患者发生弥散性血管内凝血的几率显著高于轻度CRS患者，且轻重度CRS患者间的D-二聚体、纤维蛋白降解产物及抗凝血酶III水平存在显著差异，提示出凝血功能障碍与CRS之间存在密切联系。通过检测内皮功能指标，发现重度CRS患者的血浆组织因子（TF）及血小板内皮细胞黏附因子（PECAM）-1水平显著高于轻度CRS患者，且TF及PECAM-1水平与IL-6水平的变化趋势具有一致性，提示血管内皮损伤是CRS相关出凝血功能障碍的关键机制，并为CRS早期治疗提供了新靶点。最后，本项目根据临床及基础研究成果，总结出了CRS相关出凝血障碍的防治策略：早期识别，准确评估，去除诱因，分层干预。综上，本项目为CRS及CRS相关出凝血功能障碍的早期诊断和有效防治指明了新方向，提高了CAR-T治疗安全性。