靶向CD93的嵌合抗原受体修饰T细胞疗法治疗携带MLL重排的急性髓系白血病的临床前研究

Acute myeloid leukemia with MLL rearrangement represents a unique subgroup of leukemia, which is associated with a dismal response to chemotherapy, poor prognosis, and a higher rate of relapse. Targeted therapy is tremendously investigated in this field, but the actionable target and modalities of treatments are still elusive. The pioneer studies have shown that CD93 is a phenotypic and functional biomarker of leukemia stem cells of MLLr AML, Thus targeting CD93 would lead to eliminate the malicious clones. Chimeric antigen receptor-modified T-cell therapy is a perfect therapeutic tool for the cancers in which the membrane functional markers have been identified. In this scenario, we initiate the pre-clinical experiments of CD93 CAR-T therapy for MLLr AML. We plan to apply our research on human MLLr AML cell lines, primary cells from MLLr AML patients, NOD/SCID xenograft mouse model and conditional MLL-AF6 knockin mouse model. Combining these in vitro and in vivo data, we try to thoroughly evaluate the efficacy and safety of applying CD93 CAR-T cell therapy for MLLr AML, at last, we are going to investigate whether the suicide gene ”HSV-TK” will improve our therapeutic results which will be important for clinical application in future. Our study will provide a whole new adoptive immunotherapy for MLLr AML, develop novel targeting and safer strategies and bring hope to the dismal disease.

MLL基因/11q23易位聚集于白血病患者中，是强烈提示治疗失败和复发的高危因素，是一种亟待攻克的白血病独立亚型，其病理生理机制和靶向干预一直该领域研究的热点。最新研究表明，CD93是MLLr AML特异性的LSCs表面功能性标志和清除的潜在靶点。我们启动了CD93 CAR-T疗法的临床前研究，试图利用人MLLr AML细胞株和原代细胞、免疫缺陷小鼠白血病异种移植模型和条件性MLL-AF6基因敲入小鼠模型等诸多手段，在体内、外全面评价CD93 CAR-T疗法治疗MLLr AML的有效性和安全性，并尝试引入自杀基因”HSV-TK”以优化治疗的安全性，探讨未来临床应用的可行性。该研究有望提出针对AML独特亚型的过继性细胞免疫治疗技术，发展和充实MLLr AML的个体化治疗策略，以期翦灭LSCs和清除微小残留病灶，克服复发并根治疾病，为其转化医学研究带来新的希望。

MLL基因/11q23易位聚集于白血病患者中，是强烈提示治疗失败和复发的高危因素，是一种亟待攻克的白血病独立亚型。本研究以携带MLL重排的急性髓系白血病（MLL-rearranged acute myeloid leukemia，MLLr AML）为研究对象，探索针对AML独特亚型或遗传学畸变的过继性细胞免疫治疗技术，拟利用人MLLr AML细胞株和原代细胞、免疫缺陷小鼠白血病异种移植模型等多种手段，在体内、外全面评价靶向CD93的嵌合抗原受体（chimeric antigen receptor，CAR）T细胞疗法治疗MLLr AML的有效性和安全性，探讨其未来临床应用的可行性，发展和充实AML的靶向治疗策略。在项目资助下，我们克隆并表达了人CD93真核重组蛋白，通过杂交瘤技术免疫Balb/c小鼠获得两株单克隆抗体。进而克隆高亲和力抗体克隆株的scFv序列，构建相应慢病毒载体并制备病毒，制备了靶向CD93的CAR T细胞。我们发现CAR T细胞疗法能特异性地杀伤AML细胞株、原代细胞和CD34+的白血病干细胞，并在NSG小鼠体内显著降低治疗组小鼠在不同时间点的肿瘤负荷和死亡率，明显延长小鼠生存。不仅如此，CAR T细胞对CD34+ 正常造血干、祖细胞的杀伤与未转染的对照T细胞相比没有差别，提示CAR T细胞无明显血液学毒性，初步完成了该疗法安全性的评价。从而利用人AML细胞株、原代细胞、免疫缺陷小鼠白血病异种移植模型等多种手段，在体内、外评价了CAR T疗法治疗MLLr AML的有效性、安全性和可行性。我们还评价了IL1RAP在包括MLLr AML在内多种AML细胞株、原代细胞和CD34+ 细胞上的表达，丰富了AML的靶向治疗选择。我们还发表了多篇论著，比较、总结了CAR T细胞制备和保存的不同实验方法，总结了CAR T细胞疗法治疗B细胞肿瘤的疗效和CAR T细胞治疗中乙肝病毒再激活的防治，进一步拓展了课题的研究广度，具有一定的科学意义和应用前景。

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