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: 2098582
• 负责人: Malcolm Stuart Mitchell
• 金额: $ 13.41万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 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 1996-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2098582
• 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）的进一步完善将需要 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 这类研究中的重要试剂。 23个新的黑色素瘤基因 已经通过在黑色素瘤和黑色素瘤之间进行分子减法来确定。 肺鳞癌 其中一个基因的mRNA相对 仅限于黑色素瘤的基因：“基因50”。 A17氨基酸 合成基因50的肽。 它引起4 CD 4+细胞增殖 从免疫患者的TIL克隆并被Th细胞识别 5例中4例为术后复发。 基因50的完整DNA序列 被确定。 它和它的各种DNA结构域将被插入到 具有适当病毒载体的自体淋巴母细胞系， 引起T辅助性CD 4细胞（Th）增殖的表位试验 表位）和对T细胞毒性CD 8细胞的细胞毒性敏感的那些 (Tc表位）。 一系列九聚体的合成 免疫原性结构域可以允许黑素瘤表位精确区分。 将尝试用逆转录病毒载体转染完整基因50。 载体，以获得Tc的永久靶细胞和特异性刺激物 和体外Th。 然后将对表位具有特异性的Tc克隆进行克隆。 从TIL或PBL获得。 他们的T细胞受体将被测序 用特异性引物进行PCR分析，看是否有何种程度的 它们与表位之间存在对应关系，特别是对于Tc。 由基因50编码的免疫原的相关性将通过以下来判断： 确定给予ASI的患者如何常见地发展Tc， 基因50个表位。 ASI前后Tc的频率为 在HLA-A2和非A2基因型患者中通过有限稀释测量。 这些研究也可能表明某些Tc表位是否可以被 只有在特定的MHC背景下才能被识别。 Tc的“永生化” 通过转染蛋白识别特异性表位的克隆 激酶C，可能有助于探索其特性。 一旦有效率 筛选和测试已经完善，我们将使用重组 牛痘病毒和后来的逆转录病毒载体到LCL中，以研究 2至3个其他新的黑色素瘤基因的免疫原性， 仅限于黑色素瘤 这种方法可以阐明人类T 细胞-黑色素瘤表位相互作用，并在此过程中可能导致 开发预定的合成治疗性黑素瘤疫苗。