钙网蛋白突变体作为骨髓增殖性肿瘤检测标志物和CAR-T细胞治疗靶向抗原的应用及机制研究

经典的骨髓增殖性肿瘤（MPN）是一组起源于造血干细胞的恶性克隆性疾病，目前无法治愈。钙网蛋白（CALR）突变见于大多数无JAK2或MPL基因突变的MPN患者，被纳入WHO诊断指南。检测CALR基因突变的方法无法兼顾突变类型多、灵敏高、检测快和成本效益好等需求。所有形式的CALR突变都会因+1bp移码导致共有的异常C末端肽段，我们对此肽段制备了特异性单抗，拟利用此抗体通过流式细胞术检测患者外周血细胞表面上的CALR突变蛋白，为MPN诊断提供一种简便、快速、可检测所有CALR突变类型的新方法；同时获取单抗单链可变区序列，以CD137为共刺激分子，T细胞受体(TCR)/CD3的-δ链为胞内信号结构域来构建第二代嵌合抗原受体。从而探索靶向突变CALR抗原的CAR-T细胞在体内、体外对CALR突变阳性血液肿瘤细胞的靶向杀伤作用及细胞因子分泌引起的炎性反应，为晚期或高危MPN患者提供新的治疗策略。

The myeloproliferative neoplasms (MPNs) are chronic myeloid cancers characterized by the overproduction of mature blood cells. There is no known cure for the disease. CALR mutations are the founder gene alteration in the JAK2- and MPL-negative MPNs according to the WHO diagnostic guidelines. Methods that detect CALR mutations at the DNA levels cannot accommodate the performance needs of mutation diversity, sensitivity, rapidness, and cost-effectiveness. All CALR mutations lead to +1 base-pair frame-shifts in the open reading frame of the gene and result in a novel C-terminal mutant peptide consisting of 36 amino acids. With an in-house made specific monoclonal antibody targeting this peptide, we propose to detect the CALR mutation protein on peripheral blood cells surface by the flow cytometry. We intend to provide a simple, fast and quantitative method for the MPNs detection with any types of CALR mutation. We will also validate the tumor-killing effect of an anti-CALR chimeric antigen receptor T cell targeting the CALR mutant peptide both in vitro and in vivo, and the inflammatory response induced by cytokine secretion, thus provide a new therapeutic strategy for the patients with late stage MPNs.