HLA-A2 ASSOCIATED PEPTIDE EPITOPES FOR CTL ON MELANOMA

拟议项目的长远目标是确定 由人黑素瘤细胞表达的用作表位的抗原 黑色素瘤特异性细胞毒性T淋巴细胞（CTL）。 临床 这些抗原的鉴定和合成的意义 包括（1）创造安全的重组疫苗的可能性， 黑色素瘤，（2）体外刺激人淋巴细胞的可能性 用于黑色素瘤患者的过继转移，使用特定的兴奋剂 黑色素瘤特异性CTL，和（3）识别黑色素瘤特异性CTL的可能性。 蛋白质）和衍生这些抗原的遗传物质， 这本身就能帮助我们理解恶性肿瘤 黑色素瘤的转化。 HLA-A2抗原将从细胞表面和细胞质中纯化， 至少10（9）个HLA-A2（+）人黑素瘤细胞。 筹备工作作为 对照将从A2（+）非黑素瘤细胞中纯化。 的肽 结合在A2分子的裂缝中的蛋白质将通过酸化而洗脱 和过滤离心。 所得肽混合物将是 在电喷雾电离条件下通过质谱分析。 通过质谱法鉴定的黑色素瘤上存在的肽 细胞但非黑素瘤细胞上不存在的细胞将通过质量进行测序 光谱法 测序的肽将被合成， 测试它们使A2（+）非黑色素瘤对以下物质敏感的能力： 通过HLA-A2限制性黑素瘤特异性细胞毒性T淋巴细胞的裂解。

The long-term objective of the proposed project is to identify the antigen(s) expressed by human melanoma cells that serve as epitope(s) for melanoma-specific cytotoxic T-lymphocytes (CTL). The clinical implications of the identification and synthesis of these antigens include (1) the possibility of creating a safe recombinant vaccine for melanoma, (2) the possibility of stimulating human lymphocytes in vitro for adoptive transfer to melanoma patients, using a specific stimulant of melanoma-specific CTL, and (3) the possibility of identifying the proteins) and genetic material from which these antigens are derived, which would in itself have implications for understanding malignant transformation of melanoma. HLA-A2 antigens will be purified from the cell surface and cytoplasm of a least 10(9) HLA-A2(+) human melanoma cells. Preparations serving as controls will be purified from A2(+) non-melanoma cells. The peptides bound in the cleft of the A2 molecules will be eluted by acidification and filtration centrifugation. The resulting peptide mixtures will be analyzed by mass spectrometry under electrospray ionization conditions. Peptides identified by mass spectrometry that are present on melanoma cells but absent on non-melanoma cells will be sequenced by mass spectrometric methods. The sequenced peptides will be synthesized and tested for their ability to render A2(+) non-melanomas sensitive to lysis by HLA-A2-restricted melanoma-specific cytotoxic T lymphocytes.