TfR CAR-iCasp9 T细胞的靶向抗瘤效应及安全性研究

Recent years have witnessed much progress in both basic research and clinical trials regarding cancer immunotherapy with chimeric antigen receptor (CAR)-engineered T cells. The unique structure of CAR endows T cell tumor specific cytotoxicity and resistance to immunosuppressive microenvironment in cancers, helps patients to better tackle the issue of immunological tolerance. Adoptive immunotherapy (AIT) using this supernatural T cell have gained momentum after decades of intense debates because of the promising results obtained from preclinical models and clinical trials. Targeting solid tumors is more challenging compared to the treatment of hematological malignancies because of rare target antigens. CAR-based therapy has only been conducted in several tumors, such as neuroblastoma, colorectal, ovarian and prostate cancer. The transferrin receptor (TfR, CD71) is a membrane-bound protein involved in transferrin (Tf) -mediated iron uptake. The high levels of expression of TfRs in cancer cells and their central role in the pathology of human cancer make TfR an attractive target that can be exploited for the delivery of cytotoxic agents into tumor cells and the target of tumor imaging. Hence, TfR specific CAR redirected T cells could be widely used to treat TfR expressing tumors. In this research, gene encoding TfR scFv will be fused sequentially with genes encoding the cytoplasmic domains of CD28, 4-1BB and CD3ζ to constuct TfRscFv28BBζ(TfR CAR). Furthermore, in order to improve the safety of T cell administration, an inducible T-cell safety switch (iCasp9) is constructed to the downstream of bicistronic vector containing TfR CAR. This iCasp9 system use FKBP12-F36V to replace the recruitment domain of caspase9. When exposed to bioinert small-molecule dimerizing agent AP1903, FKBP12-F36V dimerized to ensure the dimerization of caspase9 and subsequent apoptosis of cells. The transposon-based TfR CAR-iCasp9 vector tranfected central memory T cells are called TfR CAR–iCasp9 T cells. In vitro and in vivo studies will be performed to evaluate the specific lysis and the safety of these TfR CAR-iCasp9 T cells. This research will shed new light on tumor adoptive immunotherapy and expand clinical applications of CAR-redirected T cells.

如何选择特异性靶标是CAR T细胞过继免疫治疗需要解决的问题。文献报道及我们前期研究证明，转铁蛋白受体(TfR)在多种恶性肿瘤细胞高表达，是肿瘤免疫显像及治疗的理想靶点。本课题设想将TfR scFv与CD28、4-1BB、CD3ζ链的胞浆段序列融合，构建TfR特异性CAR；为提高体内应用安全性，在表达CAR的载体中引入iCasp9安全开关系统，构建CAR-iCasp9共表达的转座子载体，转染中枢记忆T细胞，有可能建立在体内具有较强抗肿瘤效应和免疫记忆潜能、凋亡可人工调控的TfR CAR-iCasp9 T细胞，同时该T细胞可通过阻止肿瘤细胞铁摄取发挥更强的抗瘤效应。基于此，本项目拟采用分子生物学技术，建立 TfR CAR-iCasp9 T细胞，通过体内外实验，探讨该T细胞的靶向抗瘤效应及iCasp9系统主动调控T细胞凋亡的双重作用，为以TfR为靶点的CAR T细胞过继免疫治疗研究奠定基础。

背景：对抗体单链可变区片段（scFv）的改造和利用在肿瘤免疫治疗中取得了显著的疗效，其策略主要包括构建双特异性T细胞衔接器(BiTE)和嵌合抗原受体(CAR)。这两种策略都使用scFv来引导CTL与肿瘤细胞表面的特异性抗原结合，引发多克隆T细胞活化与增殖，分泌细胞因子。其中，CAR修饰的T细胞（CAR-T细胞）技术将抗体对肿瘤抗原的特异性与T 淋巴细胞的杀伤机制相结合，赋予不同克隆T细胞全新的靶向杀伤活性，能大大提高肿瘤特异性T细胞的数量。而BiTE，可以介导T细胞与肿瘤细胞间形成经典的免疫突触，且无需额外提供T细胞活化所需的第二信号，即可激活自身体内T细胞，进而杀灭肿瘤细胞。转铁蛋白受体(TfR)在多种恶性肿瘤细胞表面高表达，是肿瘤免疫显像及治疗的理想靶点。.主要研究内容：构建靶向TfR的CAR-T细胞及BiTE双特异性抗体，体内外研究TfR CAR-T细胞及TfR-BiTE的靶向抗瘤效应。.重要结果：TfR CAR-T细胞及TfR-BiTE体内外均能靶向结合并介导杀伤多种TfR+肿瘤细胞。.关键数据： .成功制备了靶向TfR的CAR-T细胞及BiTE双特异性抗体；.TfR CAR-T细胞体外能靶向结合并杀伤多种TfR+血液肿瘤细胞，如U266、Molt-4、K562及KG-1a等；在NSG小鼠接种Molt-4细胞制备的白血病动物模型中，单次TfR CAR-T细胞输注能明显抑制Molt-4细胞的增殖浸润，降低模型小鼠的白血病负荷；.TfR-BiTE抗体外能靶向结合并杀伤多种TfR+实体肿瘤细胞，如HepG2、HepG2.215、HT1080及血液肿瘤细胞Molt-4；在NSG小鼠接种HepG2细胞制备的实体肿瘤动物模型中，TfR-BiTE抗体连续注射7天，能显著抑制实体肿瘤的生长..科学意义：在靶向TfR对T细胞重定向进行肿瘤治疗的2种策略中，TfR CAR-T细胞及TfR-BiTE抗体分别显示了对于多种血液肿瘤和实体肿瘤的有效杀伤，证实了以TfR为靶标进行肿瘤免疫治疗的可行性，为临床肿瘤免疫治疗提供了新策略和新靶点。