Morgan Richard A, Dudley Mark E, Yu Yik Y L, Zheng Zhili, Robbins Paul F, Theoret Marc R, Wunderlich John R, Hughes Marybeth S, Restifo Nicholas P, Rosenberg Steven A
Surgery Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
J Immunol. 2003 Sep 15;171(6):3287-95. doi: 10.4049/jimmunol.171.6.3287.
The alpha- and beta-chains of the TCR from a highly avid anti-gp100 CTL clone were isolated and used to construct retroviral vectors that can mediate high efficiency gene transfer into primary human lymphocytes. Expression of this TCR gene was confirmed by Western blot analysis, immunocytometric analysis, and HLA Ag tetramer staining. Gene transfer efficiencies of >50% into primary lymphocytes were obtained without selection for transduced cells using a method of prebinding retroviral vectors to cell culture vessels before the addition of lymphocytes. The biological activity of transduced cells was confirmed by cytokine production following coculture with stimulator cells pulsed with gp100 peptides, but not with unrelated peptides. The ability of this anti-gp100 TCR gene to transfer high avidity Ag recognition to engineered lymphocytes was confirmed in comparison with highly avid antimelanoma lymphocytes by the high levels of cytokine production (>200,000 pg/ml IFN-gamma), by recognition of low levels of peptide (<200 pM), and by HLA class I-restricted recognition and lysis of melanoma tumor cell lines. CD4(+) T cells engineered with this anti-gp100 TCR gene were Ag reactive, suggesting CD8-independent activity of the expressed TCR. Finally, nonmelanoma-reactive tumor-infiltrating lymphocyte cultures developed antimelanoma activity following anti-gp100 TCR gene transfer. In addition, tumor-infiltrating lymphocytes with reactivity against non-gp100 melanoma Ags acquired gp100 reactivity and did not lose the recognition of autologous melanoma Ags following gp100 TCR gene transfer. These results suggest that lymphocytes genetically engineered to express anti-gp100 TCR may be of value in the adoptive immunotherapy of patients with melanoma.
从一个高亲和力的抗gp100细胞毒性T淋巴细胞（CTL）克隆中分离出T细胞受体（TCR）的α链和β链，并用于构建逆转录病毒载体，该载体可介导高效基因转移至原代人淋巴细胞。通过蛋白质免疫印迹分析、免疫细胞分析和HLA抗原四聚体染色证实了该TCR基因的表达。在不使用选择转导细胞的方法的情况下，通过在添加淋巴细胞之前将逆转录病毒载体预结合到细胞培养容器上，获得了超过50%的原代淋巴细胞基因转移效率。在用gp100肽脉冲刺激的细胞与转导细胞共培养后，通过细胞因子产生证实了转导细胞的生物学活性，但与无关肽共培养时则无此现象。与高亲和力的抗黑色素瘤淋巴细胞相比，通过高水平的细胞因子产生（>200,000 pg/ml干扰素-γ）、对低水平肽（<200 pM）的识别以及HLA I类限制性识别和对黑色素瘤肿瘤细胞系的裂解，证实了该抗gp100 TCR基因将高亲和力抗原识别转移至工程化淋巴细胞的能力。用该抗gp100 TCR基因工程改造的CD4(+) T细胞具有抗原反应性，表明所表达的TCR具有不依赖CD8的活性。最后，非黑色素瘤反应性肿瘤浸润淋巴细胞培养物在抗gp100 TCR基因转移后产生了抗黑色素瘤活性。此外，对非gp100黑色素瘤抗原具有反应性的肿瘤浸润淋巴细胞在gp100 TCR基因转移后获得了gp100反应性，并且没有丧失对自体黑色素瘤抗原的识别。这些结果表明，经基因工程改造以表达抗gp100 TCR的淋巴细胞在黑色素瘤患者的过继性免疫治疗中可能具有价值。
