转染CAR的T淋巴细胞对靶抗原阴性的白血病起始细胞杀伤作用及机制研究

Infusions of antigen-specific T cells have yielded therapeutic responses in patients with pathogens and tumors. The study results of several groups abroad and also our own have already confirmed that T lymphocytes transduced with tumor antigen specific chimeric antigen receptors（CAR）can efficiently eradicate tumor cells by cytokines releasing and cytotoxic effects without the restriction of MHC/HLA. The results of clinical trials in multiple cancer centers in USA showed that transfusion of CD19 targeted CAR modified T lymphocytes can repeatedly induce complete remissions in relapsed pre-B acute lymphoblastic leukemia, and minimal residual disease done by QT-PCR also became negative in the molecular level. It has already been shown that the immunologic characteristics of leukemia initiating cells in pre-B-ALL is CD34+/CD19-. We intended to study the effects and detailed mechnisms that the CD19 targeted CAR transduced T lymphocytes eradicates the CD34+/CD19- leukemia initiating cells by in vitro cell culture and in mouse models in vivo.Three possible mechnisms were suggested and will be studied in detail, which includes: a) The by-stander effects: how do the cytokines released during the CAR transduced T-cells attack the CD19+ leukemia cells affects the growth and survival of the CD34+/CD19-LICs. b) The effects of the released cytokines and lymphotoxins to the leukemic microevironment that supports and maintains the growth of leukemia initiating cells in the bone marrow, and c) The relationship between the molecular level of CD19 expression on the cell surface of the leukemia initiating cells and the cytotoxic effects indued by CD19-CAR tranduced T lymphocytes. The results of this project will demonstrate the mechanisms involved in the eradication of CD34+/CD19-LIC cells by the CD19-CAR-T lymphocytes and make solid background for CAR mediated immunotheray in clinical trial.

我们前期研究及国外文献均已证实，嵌合抗原受体(CAR)转染的T细胞通过释放细胞因子及CTL效应可高效杀伤靶抗原阳性的肿瘤细胞，且不受MHC的限制。近年欧美多个癌症中心的临床试验表明：CD19靶向的CAR-T细胞输注不仅可使复发状态的B急淋（B-ALL）患者达完全缓解，且分子水平的微小残留病（MRD）也转阴性。而早先研究表明B-ALL白血病起始细胞（LIC）表型特征为CD34+/CD19-。那么，CD19靶向的CAR-T细胞又是通过何种机制清除这种CD34+/CD19-的LIC的呢？本课题拟通过体外细胞培养及小鼠模型探讨CD19-CAR-T细胞杀伤靶细胞过程中的旁观者效应及对骨髓白血病微环境的影响，应用蛋白质谱及流式细胞分析探讨B-ALL中LIC表面CD19的表达量与CD19-CAR-T细胞杀伤效应间关系。阐明CD19-CAR-T细胞清除CD19-LIC的确切机制，为进一步临床应用奠定基础。

CD19-CART在B细胞血液系统恶性肿瘤的治疗中取得了显著的疗效，一部分患者CD19-CART治疗后能够完全缓解并长期生存。研究表明CD34+/CD19-的白血病起始细胞（LIC）是白血病复发的根源。研究首先在体外多角度探究了CD19-CART对CD19阴性细胞的杀伤作用。发现单纯的混合CD19-CART细胞与靶抗原阴性细胞，无论是直接接触或是通过transwell小室隔开培养，或者是同时将CD19-CART与靶抗原阳性或阴性靶细胞混合培养，均不能实现CART细胞对肿瘤细胞的直接杀伤，提示体内观察到的CD19-CART治疗导致靶抗原阴性中肿瘤的消退，需要其他细胞或其他机制的参与。进一步在临床中摸索提高CD19-CART中对CD19-肿瘤的杀伤进而提高CD19-CART治疗临床缓解率，降低复发率方案。并通过临床研究证实通过CD19-CART与造血干细胞移植联合，彻底清除LIC，显著提高了患者的缓解率和生存时间。研究还创造性地提出了CD19-CART联合自体干细胞移植治疗B-淋巴瘤/白血病的治疗策略，利用CART本身识别靶抗原并激活，对肿瘤进行特异性杀伤；另一方面，CART对肿瘤杀伤的过程中，同时激活自体新生T细胞通过特异性TCR识别，进一步分化成针对该肿瘤其他多种抗原的特异性CTL,进而提高对肿瘤细胞的杀伤效果，包括对靶抗原阴性肿瘤细胞的杀伤，最终提高了CART对淋巴瘤患者的疗效，为白血病/淋巴瘤治疗提出了新的治疗思路及其机制说明。后续研究将进一步联合其他固有免疫细胞，比如NK细胞、单核细胞、巨噬细胞、粒细胞等来观察CD19-CART对CD19阳性杀伤同时，对靶抗原阴性细胞的旁杀伤效应及具体机制并进行干预，进一步促进CD19-CART 对LIC的杀伤,提高B细胞血液系统恶性肿瘤治疗疗效。