Serafini M, Manganini M, Borleri G, Bonamino M, Imberti L, Biondi A, Golay J, Rambaldi A, Introna M
Istituto di Ricerche Farmacologiche Mario Negri, 20157 Milan, Italy.
Hum Gene Ther. 2004 Jan;15(1):63-76. doi: 10.1089/10430340460732463.
We have previously proposed the CD20 molecule as a novel suicide gene for T lymphocytes in the context of allogeneic bone marrow transplantation, because CD20 can be used both as a selection marker and as a killer gene after exposure to the anti-CD20 therapeutic antibody rituximab. We now report on preclinical studies using this novel system, in which the best transduction protocol, reproducibility, yield, feasibility, and functionality of the transduced T lymphocytes have been investigated with a large donor series. Wild-type human CD20 cDNA was transduced into human T lymphocytes, using a Moloney-derived retroviral vector. Alternative protocols were tested by employing either one or four spinoculations (in which cells are centrifuged in the presence of retroviral vector supernatant) and stimulating T cells with phytohemagglutinin (PHA) or anti-CD3/CD28. One spinoculation alone was sufficient to obtain approximately 30% CD20-positive cells within four experimental days. Four spinoculations significantly increased transduction to 60%. A small difference in transduction efficiency was observed between the two stimulation methods, with PHA being superior to anti-CD3/CD28. Transduced cells could be purified on immunoaffinity columns, with purity reaching 98% and yield being on average 50%. Finally, 86-97% of immunoselected T lymphocytes could be killed in vitro with rituximab and complement. More importantly, the CD20 transgene did not alter the functionality of T lymphocytes with respect to allogeneic recognition and cytotoxic response, anti-Epstein-Barr virus cytotoxic response, antigenic response to tetanus toxoid antigen, interleukin 2 (IL-2), IL-4, and interferon gamma production; chemotaxis in the presence of stromal cell-derived factor 1, phenotype for several activation markers including HLA-DR, CD25, CD69, and CD95, and T cell repertoire.
我们之前提出，在异基因骨髓移植背景下，CD20分子可作为T淋巴细胞的一种新型自杀基因，因为CD20在暴露于抗CD20治疗性抗体利妥昔单抗后，既可用作选择标记，也可用作杀伤基因。我们现在报告使用这个新系统的临床前研究，其中用大量供体样本研究了转导T淋巴细胞的最佳转导方案、可重复性、产量、可行性和功能。使用莫洛尼衍生的逆转录病毒载体将野生型人CD20 cDNA转导到人T淋巴细胞中。通过采用一次或四次离心接种（细胞在逆转录病毒载体上清液存在下离心）以及用植物血凝素（PHA）或抗CD3/CD28刺激T细胞来测试替代方案。仅一次离心接种就足以在四个实验日内获得约30%的CD20阳性细胞。四次离心接种显著提高转导率至60%。两种刺激方法之间观察到转导效率存在小差异，PHA优于抗CD3/CD28。转导细胞可在免疫亲和柱上纯化，纯度达到98%，平均产量为50%。最后，86% - 97%经免疫选择的T淋巴细胞可在体外被利妥昔单抗和补体杀死。更重要的是，CD20转基因在同种异体识别和细胞毒性反应、抗爱泼斯坦 - 巴尔病毒细胞毒性反应、对破伤风类毒素抗原的抗原反应、白细胞介素2（IL - 2）、IL - 4和干扰素γ产生方面，并未改变T淋巴细胞的功能；在基质细胞衍生因子1存在下的趋化性、包括HLA - DR、CD25、CD69和CD95在内的几种激活标志物的表型以及T细胞库。
