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Isolation of human melanoma antigens recognized bt T cells for development of immunotherapy and gene therapy

分离人类黑色素瘤抗原识别的 BT T 细胞，用于开发免疫疗法和基因疗法

基本信息

批准号：
10557083
负责人：
KAWAKAMI Yutaka
金额：
$ 8.7万
依托单位：
Keio University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10557083/
关键词：
Melanoma Tumor antigens T cells cDNA cloning Melanosomal proteins P-loop Mutation Immunotherapy 突然変異チロシナーゼ gp100 HLA

项目摘要

A total of 123 tumor infiltrating T lymphocytes (TIL) established from patients with metastatic melanoma was screened for identification of T cells that might recognize novel melanoma antigens or epitopes in previously identified antigens. Using 5 TIL that possibly recognized new antigens in the context of HLA-A 1, -A2 or -A3, antigens were isolated by cDNA expression cloning. New epitopes of the previously identified antigens (1 HLA-A1 binding tyrosinase peptide, 2 HLA-A2 binding gp100 peptides, 1 HLA-A3 binding gp100 peptide) were identified. Replacement of either cysteine to β-amino butyric acid in the gp100 peptide, RLPRIFCSC, enhanced the T cell recognition, suggesting that unoxidized cysteines were presented on the tumor cell surface. Since oxidation of the cysteines may easily occur in the synthetic peptides in in vitro culture, the modification of cysteines may have important implications for the development of peptide based vaccines. Using another HLA-A 1 restricted T cells, 8B6 antigen was isolated. The cDNA for the ubiquitously expressed 8B6 encoded an uncharacterized protein with a phosphate binding loop (P-loop) motif. A mutation was found in the P-loop of the 8B6 cDNA obtained from the 1362mel melanoma cell line, and the mutated 8B6 lost GTP binding ability. A T cell epitope with a glutamic acid encoded by the mutated sequence was identified, and the wild type peptide was not recognized by TIL 1362, suggesting that this T cell response was autologous tumor specific. Since many of the mutated antigens previously isolated with CD8+ T cells, including β-catenin, CDK4 and caspase 8, appeared to be involved in tumorigenesis, the mutated 8B6 may also be involved in the generation of melanoma possibly through inability to bind GTP. These newly identified melanoma antigens may be useful for development of immunotherapy as well as for understanding the biology of melanoma.

对123例转移性黑色素瘤患者建立的肿瘤浸润性T淋巴细胞(TIL)进行筛选，以确定可能识别新的黑色素瘤抗原或已知抗原表位的T细胞。以5个可能识别人类白细胞抗原A1、A2或A3中新抗原的TIL为材料，通过基因表达克隆的方法分离抗原。确定了新的抗原表位(1个与HLAA1结合的酪氨酸酶多肽，2个结合HLAA2的gp100多肽，1个结合HLAA3的gp100多肽)。用β-氨基丁酸取代半胱氨酸可增强T细胞的识别能力，提示肿瘤细胞表面存在未氧化的半胱氨酸。由于人工合成的多肽在体外培养过程中容易发生半胱氨酸的氧化，半胱氨酸的修饰对多肽疫苗的研制具有重要意义。用另一种人类白细胞抗原A1限制性T细胞，分离出8B6抗原。无处不在表达的8B6基因编码一个带有磷酸结合环(P-loop)基序的未知蛋白。从1362mel黑色素瘤细胞系中获得的8B6基因的P-环发生突变，突变的8B6失去了GTP结合能力。该突变序列编码谷氨酸的T细胞表位被鉴定，TIL 1362不能识别野生型多肽，表明这种T细胞反应是自体肿瘤特异性的。由于以前从CD8+T细胞中分离到的许多突变抗原，包括β-连环蛋白、CDK4和Caspase8，似乎与肿瘤的发生有关，突变的8B6也可能通过不能与GTP结合而参与黑色素瘤的发生。这些新发现的黑色素瘤抗原可能有助于免疫治疗的发展以及对黑色素瘤生物学的了解。