嵌合抗原受体
电穿孔
细胞毒性T细胞
生物
CD19
T细胞
细胞培养
淋巴母细胞
免疫学
抗原
癌症研究
分子生物学
免疫系统
体外
基因
生物化学
遗传学
作者
Leonardo Chicaybam,Luiza Abdo,Mayra Carneiro,Barbara Peixoto,Mariana Saldanha Viegas,Priscila de Sousa,Marcia C. Fornazin,Maria C. Spago,Angelo Brunelli Albertoni Laranjeira,Pedro O. de Campos-Lima,Alexandre E. Nowill,Luciana Rodrigues Carvalho Barros,Martín Hernán Bonamino
出处
期刊:Human Gene Therapy
[Mary Ann Liebert]
日期:2019-04-04
卷期号:30 (4): 511-522
被引量:20
摘要
Chimeric antigen receptor (CAR) T cell immunotherapy for the treatment of cancer is now an approved treatment for B cell malignancies. However, the use of viral vectors to provide long-term CAR expression is associated with high production costs and cumbersome quality controls, impacting the final cost of CAR T cell therapies. Nonviral integrative vectors, such as Sleeping Beauty (SB) transposons, provide an alternative to modify primary T cells. Therefore, we developed a protocol to expand SB-transfected 19BBζ CAR T cells using a lymphoblastoid cell line, and evaluated T cell phenotype as well as function along the T cell expansion. Electroporation of PBMCs with transposon plasmid decreased cell viability on day 1 but had a minor impact on the frequency of memory subpopulations when compared to mock condition. CAR+ lymphocytes showed increased proliferation compared to mock control and high cytotoxic activity towards CD19+ cells without significant differences in exhaustion markers expression. Moreover, CAR+ lymphocytes showed an increased frequency by the end of the stimulation cycle compared with day 1, suggesting that CAR expression confers a selective proliferation advantage. Immunodeficient NOD scid gamma chain knockout (NSG) mice engrafted with the human pre-B leukemic cell line RS4;11 and treated with 19BBζ CAR T cells showed improved overall survival when compared to mock T cells treated animals. The results showed that electroporation using the SB system is a simple and affordable method for inducing long-term CAR expression in T lymphocytes. Expansion of gene-modified T cells with the lymphoblastoid cell line provided up to 2 cycles of stimulations, generating effective T cells against leukemia in vitro and in vivo.
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