【作者】 李丹；

【导师】 刘晗；

【作者基本信息】 上海交通大学 ， 生物学， 2020， 博士

【摘要】 近年来,应用靶向CD19的嵌合抗原受体重组T细胞(CAR-T)治疗B细胞急性淋巴细胞白血病(B-ALL)获得了重大进展,在临床上取得了颠覆性治疗效果。但是众多报道显示针对CD19的CAR-T治疗之后,易发生CD19抗原逃逸,尤其高发于MLL白血病中。在对MLL白血病的研究过程中,我们发现B-ALL型的MLL白血病细胞中表面抗原CD133高表达,而其它B-ALL细胞或正常B细胞不表达CD133。CD133是常见的干细胞标记物,而正常的干细胞是不表达CD19的。基于此,我们设计出针对CD19/CD133双靶点的TanCAR,只有B-ALL型的MLL白血病细胞同时表达CD19和CD133两种表面抗原,才能有效激活TanCAR-T细胞,因此靶向CD19/CD133的双特异CAR-T将特异地杀死这一类白血病细胞,而在造血干细胞中CD133表达量较低,所以TanCAR-T细胞并不会攻击造血干细胞。并且当MLL白血病细胞发生CD19抗原逃逸时,TanCAR-T仍然能够有效识别并杀伤这群细胞。因此针对CD19和CD133的双特异性CAR-T能够有效治疗MLL白血病并降低发生抗原逃逸的几率。目前针对CD19抗原逃逸的机制主要有四种假说,包括CD19 mRNA的可变剪接;CAR-T细胞对肿瘤细胞的选择或诱导;CAR分子对CD19抗原的封闭;CAR-T细胞的胞啃。我们研究发现CD19抗原逃逸还存在着其他机制,即肿瘤细胞对CD19抗原的内吞,当CD19抗原接触到CD19scFv之后会通过胞膜窖的方式快速被内吞,然后通过溶酶体途径被降解。当CD19缺失会导致依赖CD19存活的肿瘤细胞发生凋亡,因此可以认为CD19 CAR-T通过两种机制“杀伤”肿瘤细胞,分别是CAR-T细胞的直接杀伤作用和肿瘤细胞由于丢失CD19,导致磷酸化的Akt下调,进一步导致c-Myc降低,从而引起的自发凋亡。根据CD19丢失导致凋亡的原理,我们设计出通用型的CD19scFv-MSC细胞治疗白血病,该治疗方案不仅可以降低CRS对病人的损伤,降低CRS引起肿瘤细胞转分化的几率,而且可以大规模制备,有效降低成本,减轻病人负担。更多还原

【Abstract】 CD19-directed chimeric antigen receptor(CAR)T-cell therapies have achieved great clinical success but they still suffer from some challenges such as CD19-negative relapses.CD19-negative relapses are frequently observed in MLL-rearranged B-ALL,which is characteristically associated with a lymphoid-to-myeloid lineage switch.We demonstrated that CD133 can serve as an ideal MLL leukemia associated marker.Based on this,we developed a CD19/CD133 TanCAR,which triggered robust cytotoxicity against CD19+CD133+MLL leukemic cells,but exhibited diminished activity against normal cells with single antigen such as HSPCs or B cells.Importantly,the CD19/CD133 TanCAR retained cytotoxic activity towards CD19-negtive MLL leukemic cells due to their high CD133 expression levels.These results suggested that the TanCAR may be an effective solution for treating MLL-rearranged B-ALL,with the additional benefit of preventing antigen escape relapse.Furthermore,our proof-of-principle design can be utilized to construct novel TanCARs targeting different antigen combinations,increasing the efficacy of T cell therapy for other types of cancer.There are four main mechanisms for CD19 antigen escape at the moment,including variable splicing of CD19 mRNA,selection of CAR-T cells on tumor cells or transdifferentiation of tumor cells,blocking of CD19 antigen by CARs and trogocytosis of CAR-T cells.In our research,we found that there was other mechanism for escaping of CD19.As CD19 antigen contacts CD19scFv,it would be internalized through the way of caveolae,and then degraded by lysosome rapidly.When CD19 lost,tumor cells that depend on CD19 will apoptosis.That means there were two mechanisms involved in the process of CD19 CAR-T cells killing tumor cells.On the one hand,CAR-T cells killed tumor cells directly.And on the other,decreased level of c-MYC was followed by reduced expression of phosphor-Akt in CD19 lost tumor cells,which resultantly induced T cells-independent apoptosis in tumor cells.According to the mechanism of CD19 lost tumor cells,we described a universal strategy in which antigen recognition domain-expressing MSCs could induce tumor cells apoptosis.We conclude that proper use of universal CD19scFv-cells could not only potentially avoid CRS,reduce the possibility of transdifferentiation induced by CRS,which can furtherly serve as the“off-the-shelf”ready,ubiquitous therapeutic agents for large-scale clinical application.更多还原