SPECIFIC ACTIVE IMMUNOTHERAPY OF HUMAN MELANOMA

人类黑色素瘤的特异性主动免疫治疗

• 批准号: 3173748
• 负责人: Malcolm Stuart Mitchell
• 金额: $ 23.89万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-03-01 至 1993-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3173748
• 关键词: CD4 molecule; CD8 molecule; T cell receptor; cell adhesion molecules; cell mediated lymphocytolysis test; chimeric proteins; chromium; clinical trials; cyclophosphamide; cytotoxic T lymphocyte; human subject; human therapy evaluation; immunocytochemistry; interleukin 2; leukocyte adhesion molecules; lung neoplasms; lymphocyte proliferation; major histocompatibility complex; melanoma; molecular cloning; monoclonal antibody; neoplasm /cancer immunotherapy; neoplasm /cancer vaccine; nucleic acid hybridization; polymerase chain reaction; psoralens; radionuclides; tissue /cell culture; ultraviolet radiation; western blottings

The goal of this proposal is to optimize specific active immunotherapy for melanoma. Towards that end, several aspects of the immunology of human melanoma will be studied, to determine the mechanisms by which some patients achieve excellent clinical remissions with a therapeutic vaccine ("theraccine"). Theraccine has caused remissions of metastatic melanoma in 20% of patients, with a median response of 17 mo, associated with a rise in precursors of cytotoxic T lymphocytes (pCTL). Modifications of theraccine, such as lysates of 8-methoxypsoralen-sensitized UV light-irradiated cells, lyophilized lysates, and membrane vesicles, will be assessed by clinical trials, monitored closely by in vitro immunological assays. Low-dose cyclophosphamide and interleukin-2 will be combined with theraccine to try to increase CTL generation and thus, clinical response rates. A randomized Phase III trial in early stage melanoma (lb-lll) will be conducted in an attempt to eradicate microscopic residual diseases. To improve and simplify immunological correlations, additional assays will be compared with our estimate of the frequency of pCTL, including a proliferation assay and an assay for "lytic units" of cytotoxicity. Leukocytes and cytokines present in regressing lesions will be detected by immunohistochemistry. The antigens recognized by CTL and the interaction of CTL with melanomas will be studied in several ways. Specificity of tumor-infiltrating lymphocytes (TIL) and CTL from peripheral blood will be analyzed by cold target competition assays with melanoma lines and histologically different tumors. CTL clones will be established, phenotyped for surface markers and T cell receptor chains, and their specificity examined by direct cytotoxicity and cold target competition. CD8+ "classical" CTL and the broadly reactive (CD4+ only?) CTL found after theraccine treatment will be compared. The melanoma peptides recognized by CTL clones will be identified after Western immunoblotting by proliferation and competition assays. Immunogenic peptides will then be sequenced. The importance of accessory molecules and major histocompatibility complex alleles, especially HLA-A2, in the interaction of CTL and melanomas will be studied by statistical correlations with response, and by sorting or blocking of cytotoxicity by monoclonal antibodies in vitro. T cell receptor genes of cloned CTL from the blood or tumor and of uncloned TIL from regressing lesions will be examined with antibodies to gene products and by the polymerase chain reaction. Limited use of V region genes unique to melanoma will be identified by subtractive hybridization: of normal melanocytes and melanoma cells, and of squamous lung carcinoma and melanoma. Immunogenicity of fusion proteins will be tested by proliferation, competition assays, and cytotoxicity assays with protein- treated non-melanoma cells. A preselected combination of highly immunogenic MAA from cDNA clones will constitute the ultimate version of the melanoma theraccine.

该提案的目标是优化特异性主动免疫疗法 黑色素瘤。 为此，人类免疫学的几个方面 将研究黑色素瘤，以确定某些黑色素瘤的机制 患者通过治疗性疫苗获得良好的临床缓解 （“theracine”）。 Theraccine 已使转移性黑色素瘤得到缓解 20% 的患者（中位缓解时间为 17 个月）与 细胞毒性 T 淋巴细胞 (pCTL) 的前体细胞。 Theraccine的修饰， 例如8-甲氧基补骨脂素致敏的紫外线照射细胞的裂解物， 冻干裂解物和膜囊泡将通过临床评估 试验，通过体外免疫学测定密切监测。 低剂量 环磷酰胺和白细胞介素2将与theraccine联合尝试 增加 CTL 的产生，从而提高临床反应率。 随机 早期黑色素瘤 (lb-lll) 的 III 期试验将在 尝试根除微小残留疾病。 为了改善和 简化免疫学相关性，将比较其他测定 我们对 pCTL 频率的估计，包括增殖测定 以及细胞毒性“裂解单位”的测定。 白细胞和细胞因子 存在于消退病变中的细胞将通过免疫组织化学检测。 CTL识别的抗原及其与黑色素瘤的相互作用 将通过多种方式进行研究。 肿瘤浸润的特异性 外周血中的淋巴细胞 (TIL) 和 CTL 将通过冷分析 黑色素瘤系和组织学上不同的靶标竞争测定 肿瘤。 将建立 CTL 克隆，对表面标记进行表型分析，并 T 细胞受体链及其通过直接检查的特异性 细胞毒性和冷靶竞争。 CD8+“经典”CTL 和 Theraccine 治疗后发现的广泛反应性（仅 CD4+？）CTL 将 比较的。 CTL克隆识别的黑色素瘤肽将是 通过增殖和竞争进行蛋白质免疫印迹后鉴定 化验。 然后对免疫原性肽进行测序。 的重要性 辅助分子和主要组织相容性复合体等位基因， 特别是HLA-A2，将研究CTL和黑色素瘤的相互作用 通过与响应的统计相关性，以及通过对 单克隆抗体的体外细胞毒性。 T细胞受体基因 来自血液或肿瘤的克隆 CTL 以及来自回归的非克隆 TIL 病变将用基因产物的抗体进行检查，并通过 聚合酶链式反应。 限制使用独特的V区基因 黑色素瘤将通过消减杂交来鉴定： 黑色素细胞和黑色素瘤细胞，以及鳞状肺癌和 黑色素瘤。 融合蛋白的免疫原性将通过 增殖、竞争分析和细胞毒性分析 处理过的非黑色素瘤细胞。 高度预选的组合 来自 cDNA 克隆的免疫原性 MAA 将构成最终版本 黑色素瘤Theraccine。