MELANOMA-ASSOCIATED EPITOPES RECOGNIZED BY HUMAN T-CELLS

进一步完善主动特异性免疫疗法(ASI)将需要 黑色素瘤相关抗原和表位的精确鉴定 可识别的人类T细胞。来自免疫病人的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 是通过黑色素瘤和黑色素瘤之间的分子减法来鉴定的 肺鳞癌。基因中的一种，其mRNA相对 仅限于黑色素瘤已被研究过：“基因50”。A 17个氨基酸 合成了基因50的多肽。它引起了4个CD4细胞的增殖 从免疫患者的TIL中克隆并被Th细胞识别 在5例ASI患者中有4例。基因50的完整DNA序列将 要下定决心。它和它的各种DNA结构域将被插入到 携带适当病毒载体的自体淋巴母细胞系 检测导致T辅助细胞(Th)增殖的表位 表位)和T细胞毒性CD8细胞的细胞毒作用 (TC表位)。在有限的条件下合成一系列九聚体 免疫原域可以准确区分黑色素瘤表位。 将尝试用逆转录病毒感染完整的基因50 载体构建，获得永久性靶细胞和特异性的TC刺激物 和体外培养的Th。然后，针对该表位的TC克隆将 从TIL或PBL获得。他们的T细胞受体将通过 用特定的聚合酶链式反应进行分析，看看是否有多大程度的 它们与表位之间存在对应关系，特别是对于T_c。 由基因50编码的免疫原的相关性将通过 确定接受ASI的患者在认识到 基因50表位。超声心动图检查前后心肌梗死的频率为 以人类白细胞抗原A2和非A2基因携带者的有限稀释法测定。 这些研究还可能表明，某些TC表位是否可以 仅在特定的MHC上下文中识别。TC的“永垂不朽” 通过转基因蛋白识别特定表位的克隆 激酶C可能有助于探索它们的性质。一度高效 筛选和检测已经完善，我们将使用重组 痘苗病毒和后来的逆转录病毒载体导入LCL进行研究 其他2~3个黑色素瘤新基因的免疫原性 仅限于黑色素瘤。这一方法可能会阐明人类T细胞 细胞-黑色素瘤表位相互作用并在此过程中可能导致 一种预先确定的合成治疗性黑色素瘤疫苗的开发。

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.