Identification of a cluster of tumor antigens recognized by CTh of colon cancer patients

背景：结肠癌是一种主要的恶性肿瘤，需要开发新的治疗方法。为了为结肠癌的特异性免疫治疗提供科学依据，本研究从结肠癌患者的肿瘤浸润性淋巴细胞(TIL)中筛选出一组由人类白细胞抗原A2(HLA-A2)限制性和肿瘤反应性细胞毒性T淋巴细胞(CTL)识别的肿瘤抗原及其表位。采用基因表达克隆的方法鉴定OK-CTLd亚系识别的肿瘤抗原编码基因。为了确定CTL导向的表位，将CTL与T2细胞孵育18小时，每种多肽预先冲击T2细胞2小时，然后收集上清液，用ELISA检测干扰素-γ。结果：共鉴定出27个基因，其中包括9个独特的基因。在已知或推测功能的23个基因中，有13个(56.5%)编码与增殖相关的蛋白。其他主要由编码转运蛋白的基因组成。在27个基因编码的393个具有HLA-A2结合基序的多肽中，68个多肽被亲本CTL识别，其中26个多肽也被结肠癌患者循环中的CTL识别。此外，这26个多肽中有16个具有诱导结肠癌患者外周血单个核细胞中的人类白细胞抗原A2限制性和多肽特异性CTL杀伤结肠癌细胞的能力。另一只手则分别鉴定出5个和7个由2个和4个基因编码的多肽被人类白细胞抗原B46和A31限制性CTL识别。结论：这些抗原和多肽可用于人类白细胞抗原A2+结肠癌患者的特异性免疫治疗。

Background : Colon cancer is one of major malignant tumors to which development of new treatment modality is needed. To provide scientific basis of specific immunotherapy of colon cancer, this study focused on identification of a cluster of tumor antigens and their epitopes recognized by HLA-A2-restricted and tumor-reactive cytotoxic T lymphocytes (CTLs) generated from tumor-infiltrating lymphocytes (TIL) of a colon cancer patient.Methods : The HLA-A2-restricted and tumor-reactive OK-Cm line was established from TILs of a patient with colon cancer. A gene-expression cloning method was used to identify genes coding for tumor antigens recognized by the OK-CTLd subline. To identify CTL-directed epitopes, the CTLs were incubated for 18 hr with T2 cells pre-pulsed with each peptide at different doses for 2 hr followed by harvesting of supernatant to measure IFN-γ by ELISA.Results : We identified 27 genes, including 9 unique genes. Thirteen (56.5%) of the 23 genes with known or presumed functions encoded proliferation-related proteins. The others mainly consisted of genes encoding transporters. Among 393 peptides with HLA-A2 binding motifs encoded by the 27 genes, 68 peptides were recognized by the parental CTLs, and 26 peptides among them were also recognized by CTLs in the circulation of colon cancer patients. Further, 16 of these 26 peptides possessed the ability to induce HLA-A2 restricted and peptide-specific CTLs cytotoxic to colon tumor cells in peripheral blood mononuclear cells of colon cancer patients. In the other hands, 5 and 7 peptides encoded by 2 and 4 genes, respectively, were identified with recognized by HLA-B46 and -A31 restricted CTL.Conclusion : These antigens and peptides could be appropriate in use for specific immunotherapy of HLA-A2^+ colon cancer patients.