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

共123个肿瘤浸润T淋巴细胞（TIL）建立转移性黑色素瘤患者进行了筛选的T细胞，可能会识别新的黑色素瘤抗原或表位在以前确定的抗原。使用5个可能识别HLA-A1、-A2或-A3背景下的新抗原的TIL，通过cDNA表达克隆分离抗原。鉴定了先前鉴定的抗原的新表位（1个HLA-A1结合酪氨酸酶肽，2个HLA-A2结合gp 100肽，1个HLA-A3结合gp 100肽）。将gp 100肽RLPRIFCSC中的半胱氨酸替换为β-氨基丁酸，增强了T细胞识别，表明未氧化的半胱氨酸存在于肿瘤细胞表面。由于半胱氨酸的氧化在体外培养的合成肽中很容易发生，因此半胱氨酸的修饰可能对基于肽的疫苗的开发具有重要意义。用另一种HLA-A1限制性T细胞分离8B 6抗原。广泛表达的8B 6的cDNA编码具有磷酸结合环（P-环）基序的未表征蛋白。从1362 mel黑色素瘤细胞系中获得的8B 6 cDNA的P环中发现突变，突变的8B 6失去GTP结合能力。鉴定了具有由突变序列编码的谷氨酸的T细胞表位，并且野生型肽不被TIL 1362识别，表明该T细胞应答是自体肿瘤特异性的。由于先前用CD 8 + T细胞分离的许多突变抗原，包括β-连环蛋白、CDK 4和半胱天冬酶8，似乎参与肿瘤发生，因此突变的8B 6也可能通过不能结合GTP而参与黑色素瘤的产生。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了解黑色素瘤的生物学。

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.