MELANOMA-ASSOCIATED EPITOPES RECOGNIZED BY HUMAN T-CELLS

人类 T 细胞识别的黑色素瘤相关表位

• 批准号: 3202180
• 负责人: Malcolm Stuart Mitchell
• 金额: $ 15.78万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-08-01 至 1994-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3202180
• 关键词: T cell receptor; clone cells; cytotoxic T lymphocyte; genetic transduction; helper T lymphocyte; human tissue; lymphocyte proliferation; melanoma; neoplasm /cancer genetics; nucleic acid sequence; polymerase chain reaction; tissue /cell culture; tumor antigens

Further refinement of active specific immunotherapy (ASI) will require the precise identification of melanoma-associated antigens and epitopes recognized human T cells. T cells from immunized patients can serve as important reagents in such studies. Twenty-three novel melanoma genes have been identified by molecular subtraction between a melanoma and a squamous lung carcinoma. One of the genes whose mRNA was relatively restricted to melanomas has been studied: "gene 50". A 17 amino acid peptide of gene 50 was synthesized. It caused the proliferation of 4 CD4 clones from TIL of an immunized patient and was recognized by Th cells in 4 of 5 patients after ASI. The complete DNA sequence of gene 50 will be determined. It, and its various DNA domains, will be inserted into autologous lymphoblastoid cell lines with appropriate viral vectors, to test for epitopes causing proliferation of T-helper CD4 cells (Th epitopes) and those sensitizing for cytotoxicity by T-cytotoxic CD8 cells (Tc epitopes). Synthesis of a series of nonamers within a limited immunogenic domain may permit exact discrimination of melanoma epitopes. Transfection of complete gene 50 will be attempted with a retroviral vector, to obtain permanent target cells and specific stimulators of Tc and Th in vitro. Tc clones specific for the epitope will then be obtained from TIL or PBL. Their T cell receptors will be sequenced by PCR analysis with specific primers, to see whether what degree of correspondence exists between them and the epitope, particularly for Tc. The relevance of the immunogen encoded by gene 50 will be judged by determining how commonly patients given ASI develop Tc recognizing the gene 50 epitopes. The frequency of Tc before and after ASI will be measured by limited dilutions in patients of HLA-A2 and -non-A2 genotype. These studies may also indicate whether certain Tc epitopes can be recognized only in a particular MHC context. "Immortalization" of Tc clones recognizing specific epitopes, by means of transfected protein kinase C, may be helpful in exploring their properties. Once efficient screening and testing have been perfected, we will use recombinant vaccinia virus and later retroviral vectors into LCL to study the immunogenicity of 2 to 3 other novel melanoma genes that are relatively restricted to melanomas. This approach may elucidate human T cell-melanoma epitope interactions and in the process may lead to the development of a predetermined synthetic therapeutic melanoma vaccine.

主动特异性免疫疗法（ASI）的进一步完善需要 黑色素瘤相关抗原和表位的精确鉴定 识别人类 T 细胞。 来自免疫患者的 T 细胞可以作为 此类研究中的重要试剂。 二十三个新的黑色素瘤基因 已通过黑色素瘤和黑色素瘤之间的分子减法鉴定出 鳞状肺癌。 mRNA 相对较高的基因之一 仅限于黑色素瘤的研究：“基因 50”。 17个氨基酸 合成了基因50的肽。 它引起了4个CD4的增殖 来自免疫患者 TIL 的克隆并被 Th 细胞识别 ASI 后 5 名患者中有 4 名出现这种情况。 基因 50 的完整 DNA 序列将 被确定。 它和它的各种 DNA 结构域将被插入 具有适当病毒载体的自体淋巴母细胞系， 测试导致 T 辅助 CD4 细胞（Th 表位）和那些对 T 细胞毒性 CD8 细胞的细胞毒性敏感的表位 （Tc 表位）。 在有限的范围内合成一系列九聚物 免疫原性结构域可以精确区分黑色素瘤表位。 将尝试用逆转录病毒转染完整的基因 50 载体，获得永久靶细胞和Tc特异性刺激物 和 Th 体外。 然后将针对该表位特异的 Tc 克隆 从 TIL 或 PBL 获得。 他们的 T 细胞受体将被测序 用特异引物进行PCR分析，看看是否达到什么程度 它们和表位之间存在对应关系，特别是 Tc。 基因50编码的免疫原的相关性将通过以下方式判断 确定接受 ASI 治疗的患者发生 Tc 的频率 基因50个表位。 ASI前后的Tc频率为 通过有限稀释对 HLA-A2 和非 A2 基因型患者进行测量。 这些研究还可能表明某些 Tc 表位是否可以 仅在特定的 MHC 环境中被识别。 Tc的“永生化” 通过转染蛋白识别特定表位的克隆 激酶 C，可能有助于探索它们的特性。 一旦高效 筛选和测试已经完善，我们将使用重组 将痘苗病毒和后来的逆转录病毒载体导入 LCL 中以研究 2 至 3 个其他新型黑色素瘤基因的免疫原性相对较高 仅限于黑色素瘤。 这种方法可能会阐明人类 T 细胞-黑色素瘤表位相互作用，在此过程中可能会导致 开发预定的合成治疗性黑色素瘤疫苗。