Identification of tumor-specific antigens recognized by human T cells

本课题的研究目的如下。1)确定肿瘤特异性抗原在肿瘤组织中的强表达，并与各种正常组织进行比较；2)探讨这些肿瘤抗原在癌症诊断和治疗中的可能作用。在这五年的研究期间，我们确定了以下六种对癌症诊断和/或治疗有用的肿瘤特异性抗原。1)Glypcan-3(GPC3)：GPC3是一种癌胎儿抗原，是一种GPI锚定的膜蛋白。GPC3在肝细胞癌和黑色素瘤中强表达，但在各种正常成人组织中不表达。约40%的肝细胞癌和黑色素瘤患者血清中可检测到可溶性GPC3。值得注意的是，这些癌症的早期患者血清GPC3均为阳性。体外培养的人外周血单个核细胞(PBMC)的GPC3多肽或Stim…可产生GPC3多肽特异性和MHC I类限制性CTL更多的小鼠在体内繁殖。用GPC3多肽冲击的小鼠骨髓来源的树突状细胞(DC)预免疫小鼠，以CD8^+CTL依赖的方式保护C26移植的结肠癌细胞株C26生长。2)增殖潜能相关蛋白(PP-RP)：PP-RP是有丝分裂过程中与染色体共定位的核蛋白。PP-RP在食道癌细胞中强表达，在睾丸和胎盘中有中等表达，在许多其他正常组织中不表达。RNAi下调PP-RP基因表达可抑制细胞增殖，且PP-RP强表达与患者预后不良相关。用PP-RP多肽刺激PBMCs可产生人CTL，在裸鼠体内和体内外对人癌细胞均有杀伤作用。3)KM-HN-1、HSP-105、CLP和KM-PA2，用SEREX方法从肿瘤患者血清和从癌细胞或睾丸中构建的表达文库中鉴定这些抗原。这些抗原在几种类型的癌细胞和几种较低水平的正常组织中都有强烈的表达。产生了针对来自这些抗原的多肽的CTL，它们杀死了人类和小鼠的癌细胞。4)胚胎干细胞来源的DC疫苗：我们用同时表达抗原和趋化因子基因的ES细胞来源的DC免疫小鼠，成功地增强了小鼠的抗肿瘤免疫。较少

The aims of this research project are as follows. 1) to identify tumor-specific antigens strongly expressed in cancer tissues as compared with various normal tissues, 2) to investigate possible usefulness of these tumor antigens for diagnosis and treatment of cancers. In these five years of research period, we identified the following six tumor-specific antigens useful for the cancer diagnosis and/or therapy. 1) Glypican-3 (GPC3); GPC3 is an oncofetal antigen and a GPI-anchored membrane protein. GPC3 is strongly expressed in hepatocellular carcinoma (HCC) and melanoma, but not expressed in various normal adult tissues. The serum soluble GPC3 was detected in about 40% of patients with HCC as well as melanoma. It is worth to note that the patients with even early stage of these cancers were positive for serum GPC3. GPC3 peptide-specific and MHC class I-restricted CTLs could be generated by stimulation with GPC3 peptides of human peripheral blood mononuclear cells (PBMCs) in vitro or stim … More ulation of mice in vivo. The mice pre-immunized with bone marrow-derived dendritic cells (DCs) pulsed with the GPC3 peptides were protected from the growth of transplanted colon cancer cell line, C26 transfected with mouse GPC3, in a CD8^+ CTL-dependent manner. 2) Proliferation potential related protein (PP-RP); PP-RP is a nuclear protein co-localized with chromosome during mitosis. PP-RP is strongly expressed in esophageal cancer cells and moderate expression was observed in testis and placenta but not in many other normal tissues. Knock down of PP-RP gene expression by RNAi inhibited the cell proliferation of esophageal cancer cell line, and the strong expression of PP-RP correlated with poor prognosis of the patients. Human CTLs could be generated by stimulation of PBMCs with PP-RP-derived peptides and these CTLs killed esophageal cancer cell line both in vitro and in vivo in nude mice transplanted with human cancer cells. 3) KM-HN-1, HSP-105, CLP and KM-PA2 ; these antigens were identified by SEREX method using cancer patients sera and cDNA expression libraries established from cancer cells or testis. These antigens are strongly expressed in several types of cancer cells and in several normal tissues at a lower level. CTLs specific to peptides derived from these antigens were generated and they killed cancer cells in both human and mice. 4) Embryonic stem (ES) cell-derived DC vaccine; we succeeded in augmentation of anti-tumor immunity by immunization of mice with ES cell-derived DCs expressing simultaneously genes encoding for an antigen and chemokines. Less