Immunogene therapy of cancer and virus infections using immortalized T-cell clones

1.WT1基因编码一个锌指转录因子，在急变期的急性白血病细胞和慢性粒细胞白血病细胞中优先表达，而在大多数正常细胞中不表达。这些发现有力地表明WT1是人类白血病免疫治疗的潜在靶点。我们已经建立了一个CD8细胞毒性T淋巴细胞(CTL)克隆，命名为TAK-1，它是WT1来源的9-肽的特异性克隆，该9-肽由HLA-A24结合锚定基序组成。Tak-1可以裂解携带WT1衍生多肽的同种异体细胞和自体细胞。Tak-1对HLAA24阳性的白血病细胞有细胞毒作用，但对不表达WT1的HLAA24阳性的淋巴瘤细胞、HLAA24阴性的白血病细胞或HLAA24阳性的正常细胞无杀伤作用。用与WT1互补的反义寡核苷酸治疗白血病细胞可降低TAK-1介导的细胞毒性。结论：1.Tak-1不抑制人类白细胞抗原A24阳性的正常骨髓细胞集落形成。由于hTERT在肿瘤细胞中优先表达，而在正常细胞中不表达，因此它被认为是癌症免疫治疗的潜在靶点。我们检测了携带HLA-A24基序的hTERT衍生多肽(HLA-A^*2402)诱导抗白血病细胞毒性T淋巴细胞(CTL)的能力。VYAETKHFL和VYGFVRACL这5个被测试的多肽中，有两个似乎能够产生hTERT多肽特异性和人类白细胞抗原A24限制性CTL。针对这些hTERT多肽的CD8CTL克隆对白血病细胞的杀伤作用是以HLA-A24限制性的方式进行的。加入hTERT多肽的自体LCLS可抑制hTERT的细胞毒作用，提示hTERT多肽是在白血病细胞上自然加工和表达的，并能被这些CTL裂解。结合目前发现的来源于hTERT的人类白细胞抗原A2限制性CTL表位，人类白细胞抗原A24提出的CTL表位的鉴定扩大了利用hTERT衍生肽进行白血病免疫治疗的可行性。

1. The WT1 gene encodes a zinc finger transcription factor, which is preferentially expressed in acute leukemia cells and chronic myelogenous leukemia cells in blast crisis, but not in most normal cells. These findings strongly suggest that WT1 is a potential target of immunotherapy for human leukemia. We have established a CD8+ cytotoxic Tlymphocyte (CTL) clone, designated TAK-1, which is specific for a WT1-derived 9-mer peptide consisting of HLA-A24-binding anchor motifs. TAK-1 lysed both HLA-A24-positive allogeneic cells and autologous cells that were loaded with a WT1-derived peptide. TAK-1 was cytotoxic to HLA-A24-positive leukemia cells, but not to HLA-A24-positive lymphoma cells that did not express WT1, to HLA-A24-negative leukemia cells, or to HLA-A24-positive normal cells. Treating leukemia cells with an antisense oligonucleotide complementary to WT1 reduced TAK-1-mediated cytotoxicity. TAK-1 did not inhibit colony formation of HLA-A24-positive normal bone marrow cells.2. Because hTERT is preferentially expressed in malignant cells but not in normal cells, it is considered to be a potential target for cancer immunotherapy. We examined hTERT-derived peptides carrying motifs for HLA-A24 (HLA-A^*2402) for their capacity to elicit anti-leukemia cytotoxic Tlymphocytes (CTLs). Two of the 5 peptides tested, VYAETKHFL and VYGFVRACL, appeared capable of generating hTERT peptide-specific and HLA-A24-restricted CTLs. The CD8+ CTL clones specific for these hTERT peptides exerted cytotoxicity against leukemia cells in an HLA-A24-restricted manner. The cytotoxicity was inhibited by adding hTERT peptide-loaded autologous-LCLs, suggesting that hTERT peptide is naturally processed and expressed on leukamia cells, and can be lysed by these CTLs. Taken together with the currently identified HLA-A2-restricted CTL epitopes derived from hTERT, identification of CTL epitopes presented by HLA-A24 extends the feasibility of immunotherapy for leukemia using hTERT-derived peptides.