Lee Koon H, Gowrishankar Kavitha, Street Janine, McGuire Helen M, Luciani Fabio, Hughes Brendan, Singh Mandeep, Clancy Leighton E, Gottlieb David J, Micklethwaite Kenneth P, Blyth Emily
Westmead Institute for Medical Research Westmead NSW Australia.
Faculty of Medicine and Health Sydney Medical School Sydney NSW Australia.
Clin Transl Immunology. 2020 Oct 21;9(10):e1200. doi: 10.1002/cti2.1200. eCollection 2020.
Adoptive immunotherapy with expanded tumor-specific T cells has potential as anticancer therapy. Preferentially expressed antigen in melanoma (PRAME) is an attractive target overexpressed in several cancers including melanoma and acute myeloid leukaemia (AML), with low expression in normal tissue outside the gonads. We developed a GMP-compliant manufacturing method for PRAME-specific T cells from healthy donors for adoptive immunotherapy.
Mononuclear cells were pulsed with PRAME 15-mer overlapping peptide mix. After 16 h, activated cells expressing CD137 were isolated with immunomagnetic beads and cocultured with irradiated CD137 fraction in medium supplemented with interleukin (IL)-2, IL-7 and IL-15. Cultured T cells were restimulated with antigen-pulsed autologous cells after 10 days. Cellular phenotype and cytokine response following antigen re-exposure were assessed with flow cytometry, enzyme-linked immunospot (ELISPOT) and supernatant cytokine detection. Detailed phenotypic and functional analysis with mass cytometry and T-cell receptor (TCR) beta clonality studies were performed on selected cultures.
PRAME-stimulated cultures ( = 10) had mean expansion of 2500-fold at day 18. Mean CD3 percentage was 96% with CD4:CD8 ratio of 4:1. Re-exposure to PRAME peptide mixture showed enrichment of CD4 cells expressing interferon (IFN)-γ (mean: 12.2%) and TNF-α (mean: 19.7%). Central and effector memory cells were 23% and 72%, respectively, with 24% T cells expressing PD1. Mass cytometry showed predominance of Th1 phenotype (CXCR3/CCR4/CCR6/Tbet, mean: 73%) and cytokine production including IL-2, IL-4, IL-8, IL-13 and GM-CSF (2%, 6%, 8%, 4% and 11%, respectively).
PRAME-specific T cells for adoptive immunotherapy were enriched from healthy donor mononuclear cells. The products were oligoclonal, exhibited Th1 phenotype and produced multiple cytokines.
用扩增的肿瘤特异性T细胞进行过继性免疫治疗具有作为抗癌疗法的潜力。黑色素瘤优先表达抗原（PRAME）是一个有吸引力的靶点，在包括黑色素瘤和急性髓系白血病（AML）在内的几种癌症中过表达，而在性腺外的正常组织中低表达。我们开发了一种符合药品生产质量管理规范（GMP）的方法，用于从健康供体中制备PRAME特异性T细胞以进行过继性免疫治疗。
用PRAME 15聚体重叠肽混合物刺激单核细胞。16小时后，用免疫磁珠分离表达CD137的活化细胞，并在补充有白细胞介素（IL）-2、IL-7和IL-15的培养基中与经辐照的CD137组分共培养。培养10天后，用抗原刺激的自体细胞对培养的T细胞进行再刺激。用流式细胞术、酶联免疫斑点法（ELISPOT）和上清细胞因子检测评估再次暴露于抗原后的细胞表型和细胞因子反应。对选定的培养物进行了用质谱流式细胞术的详细表型和功能分析以及T细胞受体（TCR）β克隆性研究。
PRAME刺激培养物（n = 10）在第18天平均扩增2500倍。平均CD3百分比为96%，CD4:CD8比例为4:1。再次暴露于PRAME肽混合物显示表达干扰素（IFN）-γ（平均：12.2%）和肿瘤坏死因子-α（平均：19.7%）的CD4细胞富集。中枢记忆细胞和效应记忆细胞分别为23%和72%，24%的T细胞表达程序性死亡蛋白1（PD1）。质谱流式细胞术显示Th1表型（CXCR3/CCR4/CCR6/T盒转录因子，平均：73%）占优势，并产生包括IL-2、IL-4、IL-8、IL-13和粒细胞-巨噬细胞集落刺激因子（GM-CSF）在内（分别为2%、6%、8%、4%和11%）的细胞因子。
用于过继性免疫治疗的PRAME特异性T细胞从健康供体单核细胞中富集得到。这些产物是寡克隆的，表现出Th1表型并产生多种细胞因子。
