表位嵌合抗原受体工程化T细胞治疗CD20阳性的复发难治性血液系统肿瘤的基础及临床研究

The tumor killing activity possessed by CD8 and CD56 double positive T cells has been proved to be significantly higher than that of CD3 positive but CD56 negative T cells. CD3/CD56 dobule positive T cells are enriched in cytokine-induced killer cells (CIK and Nature Killer T-like Cells). Given the lack of specific tumor killing role for these cells, adoptive immunotherapy by transfusion of either CIK or CD3+/CD56- T cells can hardly reach to an ideal clinical output for tumor control, espicially for refractory and chemotherapy-resistant tumors. Recently, it was reported that the proliferative property of T cells upon they interacting with specific antigen and their specifically targeting feature on tumor cells can be endued by the special modification at molecular level. CD20 is an accepted molecular therapeutic target for most of B-cell lymphomas, chronic lymphocyte leukemia, and other several types of malignant hematological disorders. Anti-CD20 antibody has been widely used in the clinic for several years and get better benefit for more than 80% cases of CD20 positive B-cell lymphoma patients, but not for the rest and failed for relapsed cases. In the previous, the applicant has integrated the variable regions of anti-human CD20 antibody molecular heavy and light chains, costimulator CD137, and zata region of CD3 molecule, into one chimeric molecule. By letivirus-mediated transduction, the chimeric antigen CD20 receptor engineered T cells (CART20) were successfully constructed by the applicant. Theoretically, CART20 cells can specifically bind to CD20 positive cells, peoliferate following CD137-CD3 zata activation, and killing the target tumor cells. In this project, the applicant will perform the following studies: 1. develop the highly-efficient culture system for CD3 and CD56 double positive CART20 T cells, and subsequently evaluate and compare their proliferative characteristics, secretory function, and tumor killing roles by using a series of in vitro and in vivo study strategies. Based on these, 10 cases with refractory or other therapy regimen-resistant malignant hematological disease with CD20 positive phenotype of tumor cells will be enrolled for CD8 and CD56 double positive CART20 cell therapy after the ethical approval and international clinical trial registration. The clinical safety and efficiency will be exactly depicted and reported. The completion of this project will undoubtedly provide highlight perspective for CD20 positive refractory tumor patients and produce extensive push power for the wild application of adoptive immunotherapy using engineered T cells.

CD20是B细胞淋巴瘤等多种血液系统恶性疾病的公认的分子治疗靶。申请人在前期已将抗人CD20的重链与轻链可变区、共刺激分子CD137以及CD3zata片段串联，通过转染构建了嵌合抗原CD20受体阳性T细胞（CART-20）。体外实验观察到CART-20对CD20阳性淋巴瘤细胞特异的杀伤能力。应用CAR-T20对7例复发难治CD20阳性的DLBCL进行治疗，最长1例患者治疗后获得了长达11个月以上的完全缓解。本项目将：①优化建立培养稳定的CART-20工程化T细胞（富含CD3+/CD56+T细胞）的技术体系，进而通过体内外等手段研究其增殖效应、分泌效应与杀瘤活性等；②完成15-20例难治复发性CD20+血液肿瘤的临床研究。本项目将为肿瘤特异工程化T细胞的临床应用奠定基础。

CD20是B淋巴细胞表面特有的标识，同时在B细胞淋巴瘤、非霍奇金淋巴瘤等恶性血液肿瘤中表达量异常增高。随着CD20单克隆抗体的临床应用，大多数淋巴瘤患者使用抗体联合化疗治疗能获得较好的临床治疗效果。但仍有10%-20%的患者应用标准化疗无法获得缓解；另外对于化疗获得缓解的患者，又存在5年复发率高的风险。面对复发难治性血液系统恶性肿瘤，临床上缺少长期有效的治疗方案。本课题针对CD20靶点，设计嵌合抗原受体修饰的T淋巴细胞，在体外证实了CART-20对多种CD20阳性的肿瘤细胞的靶向杀伤作用。在临床前实验完成基础上，开展了I期针对复发难治性非霍奇金淋巴瘤的临床试验。7例入组患者，1例获得了完全缓解；2例部分缓解。同时观察到了消化道占位患者消化道出血、溶瘤综合症、CRS、毛细血管渗漏综合症等毒性。在I期临床试验基础上，更改了适应症条件，排除了消化道占位患者，针对肿瘤大负荷的患者固定了预处理方案，进一步开展了11例难治复发NHL的IIa临床试验，CR率54.5%，没有严重不可控的毒性。临床前及2期临床试验证实了CART-20对难治复发性NHL的安全性及有效性。在此研究成果之上，进一步设计了针对霍奇金淋巴瘤的CD30靶点以及实体肿瘤EGFR靶点的CART。针对实体肿瘤靶点存在对正常组织的损伤特点，设计了截短启动子的CAR设计载体，体外证实对高表达EGFR的肿瘤组织杀伤，但对低表达EGFR的正常细胞杀伤低的特点。在基础研究基础上，分别开展了CART-30治疗18例难治复发霍奇金淋巴瘤的I期临床试验，7例PR，6例疾病稳定，观察到不同病理位置的不同反应，没有严重毒副作用，该临床试验结果初步证实了CART-30临床的安全性及有效性；开展了11例CART-EGFR治疗难治复发性非小细胞肺癌的I期临床试验，2例PR，5例疾病稳定，没有出现3级以上的毒副作用，证实了CART临床的安全性及部分有效性。