基于双靶分子识别的Dox调控的增强型IL13 CAR-T在恶性脑胶质瘤动物模型中的实验治疗研究

Although chimeric antigen receptor T (CAR-T) therapy plays an important role in tumor immunotherapy, it has some shortcomings. The project will establish a novel anti-HER2 scFv-tTA-synNotch receptor –based, Dox regulated and activity enhanced CAR-T by solving some key issues during CAR-T mediated tumor therapy: When the killing activity of CAR-T for tumors is enhanced, its side effects are also reduced. The novel CAR-T could be activated only the simultaneous existence of two molecules, Her2 and IL13Rα2, on the surface of target cell, and regulated by Dox, which hence could reduce off-target and on-target side effects caused by CAR-T activation. The novel CAR-T expresses Dox-controllable PD-1 dominant negative receptor that could block the inhibitory signal of CAR-T activation produced by the interaction between PD-1 and PD-L1, which therefore could further enhance CAR-T activity for killing tumor. The project will further investigate the therapeutic effects of the novel CAR-T in vivo using glioblastoma animal modelon the basis of validating the biological activity of CAR-T in vitro, which would lay a foundation for the clinical application of CAR-T in glioblastoma or other tumors. The project explores the role of CAR-T immunotherapy in glioblastoma on the basis of the previously finished project, which would open a new way for the therapy of glioblastoma.

本课题将重点针对CAR-T免疫治疗中存在的关键问题，构建一种新型的基于双分子识别的Dox调控的增强型IL13 CAR-T。该新型CAR-T只有在靶细胞表面同时存在HER2及IL13Rα2两种分子时才能被激活，因此可以有效降低CAR-T 脱靶所导致的副作用；该CAR-T还同时表达PD-1 DNR （dominant negative receptor, DNR）分子，降低肿瘤细胞表面分子PD-L1对CAR-T的抑制作用，从而进一步增强CAR-T杀伤肿瘤的活性。此外，双分子识别所控制的CAR-T激活及PD-1 DNR 所致的CAR-T活性增强均受Dox的负调控，因此可以降低CAR-T打靶所致的副作用。本课题将在体外测试该新型CAR-T的基础上，采用恶性脑胶质瘤的动物模型在体内进一步评估该新型CAR-T的肿瘤免疫治疗效果，从而为CAR-T在恶性脑胶质瘤及其它肿瘤的临床免疫治疗中的应用奠定基础。

恶性脑胶质母细胞瘤血管生长丰富，侵蚀性强，与脑组织无明显分界，手术难以彻底切除、易复发、生存期短、死亡率高，是严重危害人类健康的肿瘤疾病之一。近年来，CAR-T 疗法在非实体瘤的血液系统肿瘤治疗中具有非常好的治疗效果，但是CAR-T 细胞在实体瘤治疗中，特别是在恶性脑胶质母细胞瘤治疗中，效果还不是十分理想。本研究围绕“在提高CAR-T 杀伤活性的前提下，最大程度降低CAR-T在肿瘤免疫治疗中所导致的毒副作用这一关键科学问题”，成功构建了一种新型双特异性增强型低毒性IL13(E13K.R66D.S69D.R109K)-EphA2-scFv-TetR-VP64-TansynNotch-NFAT-luci-HER2-scFv CAR-T。该新型CAR-T只有在靶细胞表面HER2分子与IL13R胞表和EphA2两者其中的一种或两种分子同时存在时才能被激活。体外实验中，这种新型双特异性增强型低毒性TansynNotchR的CAR除了具有潜在可以通过降低抗原逃逸来提高免疫治疗效果外，同时还具有潜在降低由CAR活化所引起的脱靶效应和靶向毒性的作用。恶性脑胶质瘤动物模型的体内实验研究表明，新型双特异性增强型低毒性TansynNotchR的CAR具有明显的抗肿瘤活性，同时也具有明显降低off-target所致细胞毒性作用及降低肿瘤抗原免疫逃逸的作用。该研究的主要结果发表SCI文章15篇，其中SCI 1区文章1 篇，SCI 2区文章1篇，SCI 3区文章7篇。同时获得国家授权发明专利7项。培养毕业博士生3人，毕业硕士研究生6人。本课题的研究提供的重要的数据可以作为指导新型CAR-T开发的重要依据，可提供给相关企业，市场巨大，有十分可观的后期经济效益和重要的社会效益。本课题的研究将会对恶性脑胶质瘤及其它肿瘤的免疫治疗产生重要作用。