Identification of novel cancer-specific antigens and application for cellular imunotherapy of hematological malignancies

为了鉴定新的癌症相关抗原和开发新的免疫治疗血液系统恶性肿瘤，本研究进行。从一系列实验中得到的数据如下。1) wt1特异性，HLA- a24限制性CTL克隆（指定TAK-1）对携带HLA- a24的肺癌细胞系显示细胞毒性，但不裂解缺乏该HLA的细胞。将TAK-1过继转移到移植了hla - a24阳性肺癌细胞系的裸鼠体内，可抑制癌细胞生长，延长存活时间。这些发现有力地表明，WT1是一种普遍的肿瘤相关抗原，WT1靶向免疫疗法为肺癌和白血病提供了一种潜在的有效治疗选择。2)建立了一种新的wt1衍生肽特异性CD8^+ CTL，命名为nim1。nim1能溶解HLA-A24阳性白血病细胞，但不能溶解HLA-A24阴性白血病细胞或正常细胞。3)未成熟树突状细胞（D…More Cs）装载t（6; 9）或t（9; 22）的白血病细胞，然后与dek-can融合肽特异性或bcr-abl融合肽特异性CD4^+ t淋巴细胞克隆共培养。dek-can肽特异性和bcr-abl肽特异性CD4^+ t淋巴细胞克隆在与hla - dr匹配的dc共培养时产生干扰素-γ(IFN-γ)，而与t（6; 9）和t（9; 22）分别装载凋亡和坏死白血病细胞的错配dc共培养时则不产生干扰素-γ。这些数据表明，急性髓性白血病相关融合蛋白dek-can和慢性髓性白血病相关融合蛋白bcr-abl都被dc加工并提呈给融合肽特异性CD4^+ T淋巴细胞。4)为了明确穿孔素在人CD4^+ ctl介导的抗原特异性细胞毒性中的作用，我们建立了遗传性穿孔素缺乏症患者的抗原特异性CD4^+ t淋巴细胞克隆，并对其细胞毒活性进行了研究。数据表明，穿孔素阴性CD4^+ ctl可对fas敏感靶细胞发挥细胞毒性；然而，穿孔素在人类CD4^+和CD8^+ ctl介导的抗原特异性细胞毒性中起着重要作用。少

To identify the novel cancer-associated antigens and to develop the novel immunotherapy for hematological malignancies, this study was performed. The data obtained from the series of experiments are as follows.1) A WT1-specific, HLA-A24-restricted CTL clone (designated TAK-1) exhibited cytotoxicity against lung cancer cell lines bearing HLA-A24 but did not lyse cells lacking this HLA. Adoptive transfer of TAK-1 into nude mice that had been engrafted with an HLA-A24-positive lung cancer cell line resulted in inhibition of the cancer cell growth and prolonged survival. These findings strongly suggest that WT1 is a universal tumor-associated antigen and that WT1 -targeting immunotherapy offers a potentially effective treatment option for lung cancer as well as leukemia.2) A novel WT1-derived peptide-specific CD8^+ CTL line, designated NIM-1 was established. NIM-1 lysed HLA-A24-positive leukaemia cells, but not HLA-A24 negative leukaemia cells or normal cells.3) Immature dendritic cells (D … More Cs) were loaded with leukemia cells with t(6 ; 9) or t(9 ; 22) and then cocultured with the dek-can fusion peptide-specific or the bcr-abl fusion peptide-specific CD4^+ T-lymphocyte clone. The dek-can peptide-specific and bcr-abl peptide-specific CD4^+ T-lymphocyte clones produced interferon-γ(IFN-γ) when they were cocultured with HLA-DR-matched but not with mismatched DCs which had been loaded with apoptotic as well as necrotic leukemia cells with t(6 ; 9) and t(9 ; 22), respectively. These data indicate that the acute myelogenous leukemia-associated fusion protein, dek-can, and chronic myelogenous leukemia-associated fusion protein, bcr-abl, are both processed and presented by DCs to the fusion peptide-specific CD4^+ T lymphocytes.4) In order to clarify the roles of perforin in antigen-specific cytotoxicity mediated by human CD4^+ CTLs, antigen-specific human CD4^+ T-lymphocyte clones were established from a patient with hereditary perforin deficiency and their cytotoxic activities were investigated. The data demonstrated that perforin-negative CD4^+ CTLs can exert cytotoxicity against Fas-sensitive target cells ; however, perforin plays essential roles in antigen-specific cytotoxicity mediated by human CD4^+ as well as CD8^+ CTLs. Less