溶瘤新城疫病毒增强靶向CD147分子CAR-T细胞治疗的策略及分子机制研究

Chimeric antigen receptor T (CAR-T) cell therapy has shown a promising future for tumor immunotherapy in recent years. While there still has some challenges to use CAR-T cells for the treatment of solid tumor. To promote CAR-T cell infiltration into tumor tissue and to avoid "on-target/off-tumor" toxicity are two main issues to improve the therapeutic effects of CAR-T cell therapy..Oncolytic virotherapy, which uses oncolytic virus as agents for tumor therapy, was concerned as the best strategy to improve the CAR-T cell therapy effect. Oncolytic virus could infect tumor cells specifically and induce the immunogenic tumor cell death. DAMPs and PAMAs will be released with the infection and tumor death, which could enhance the antitumor immune response, such as enhancing the presentation of tumor-associated antigen, releasing cytokines and chemokines and promote lymphocytic infiltration. These will facilitate the infiltration to of CAR-T cell tumor tissue and avoid the "on-target/off-tumor" toxicity..Based on the previous work, we will use oncolytic Newcastle disease virus (NDV) to enhance the therapeutic effects of CAR-T cell targeting CD147 for liver cancer. We will construct humanized immune system mice with subcutaneous human liver tumor to evaluate the combination therapy effects and its mechanism. We will use quantitative proteomics, PCR array and fluorescence-activated cell sorting to identify the cell signal pathway which involved in tumor cell death and CAR-T cell therapy. These results will expound the mechanism of combination therapy of oncolytic virus with CAR-T cells, and will also facilitate the further clinical study of CAR-T cell therapy for solid tumor.

嵌合抗原受体T细胞（CAR-T）治疗被认为是最有前景的肿瘤免疫治疗策略，然而在对实体瘤治疗上仍面临较大困难。如何促进CAR-T细胞浸润肿瘤组织，并避免CAR-T治疗中的脱靶效应以提高治疗的安全性和有效性，都是CAR-T治疗实体瘤需要解决的重大问题。.溶瘤病毒治疗被认为是解决上述CAR-T治疗面临问题的理想策略。溶瘤病毒感染肿瘤细胞后引起肿瘤细胞免疫源性死亡、增强肿瘤抗原呈递、激活局部免疫反应并释放多种细胞因子及趋化因子，这都非常有利于CAR-T细胞的活化及向肿瘤组织中的趋化，有望增强CAR-T细胞治疗实体瘤的效果。.在前期工作基础上，我们拟利用溶瘤新城疫病毒来增强靶向CD147的CAR-T细胞在肝癌中的治疗效果，并在肿瘤/免疫双人源化小鼠模型的基础上，利用定量蛋白质质谱技术、PCR芯片技术、流式细胞分选技术对其治疗的的分子机制予以深入研究，为今后开展相关临床试验提供理论依据和候选治疗方案。

CAR-T细胞治疗在血液系统肿瘤治疗中取得了成功，是目前最有前景的肿瘤免疫治疗策略。但其在实体肿瘤的疗效尚不佳，主要是缺乏肿瘤特异性靶点、CAR-T细胞静脉回输后难以有效浸润肿瘤，及肿瘤局部的免疫抑制微环境对CAR-T细胞功能的抑制。溶瘤病毒被认为是提高CAR-T治疗实体肿瘤效果的理想策略。溶瘤病毒能够特异性的杀伤肿瘤细胞，通过引起肿瘤细胞免疫源性死亡、增强肿瘤抗原呈递等多种机制提高抗肿瘤免疫反应；重组溶瘤病毒还可表达外源基因进一步增强疗效。.本课题拟通过联合溶瘤病毒和CAR-T细胞，探索CAR-T治疗实体肿瘤的新策略。通过构建转录调控型的靶向肿瘤相关抗原CD147分子的CAR-T细胞，联合表达人趋化因子CCL19的重组溶瘤新城疫病毒，感染杀伤肿瘤细胞的同时促进CART浸润肿瘤组织，提高治疗效果。同时对溶瘤病毒感染肿瘤细胞后的细胞分子特征进行研究，确定下一步优化方向。.本课题成功构建基于受Dox调控的靶向CD147的CART细胞（Teton-CD147-CART），实现特定时间段CAR片段表达，降低CAR-T细胞与正常细胞的非特异性结合，提高安全性。以瘤内注射的方式治疗荷瘤小鼠后，能够显著抑制肿瘤体积增加，延长生存时间。成功构建了表达人CCL19分子的重组溶瘤新城疫病毒rNDV-hCCL19，在保留特异性杀伤肿瘤细胞特性的同时，趋化以CD8+T细胞为主的淋巴细胞。联合上述CART细胞与溶瘤病毒，以静脉给药治疗在原位肺癌模型小鼠，可以显著延长小鼠生存期。通过病毒感染细胞转录组分析，表明细胞免疫和溶酶体通路相关信号通路变化显著。.本课题证实了联合CAR-T细胞和溶瘤病毒用于实体肿瘤治疗的可行性，获得了针对在肝癌、肺癌等多种肿瘤中高表达的CD147分子的新型CAR-T细胞制剂，以及表达人CCL19的重组溶瘤新城疫病毒，为临床实体肿瘤的生物治疗提供了新的候选策略和制剂。