MELANOMA ANTIGENS INDENTIFIED FROM GMCSF VACCINATION

操纵编码潜在免疫调节剂的基因的能力， 作为细胞因子和肿瘤抗原， 尝试. 免疫原性差的鼠肿瘤模型系统证明 接种经过辐射的肿瘤细胞， 粒细胞-巨噬细胞集落刺激（GM-CSF）产生有效的， 特异性和持久的免疫力。 为了在病人身上测试这个策略 对于晚期黑色素瘤，申请人的实验室进行了 用致死剂量辐照的自体 表达人GM-CSF的黑素瘤细胞。 接种疫苗会引起罢工 既存转移性病灶浸润，伴有大量 淋巴细胞、浆细胞、巨噬细胞和嗜酸性粒细胞，导致 肿瘤破坏、纤维化和水肿。体液的持续产生 并且细胞免疫应答通过肿瘤特异性免疫应答 细胞毒性和细胞因子产生表征肿瘤浸润 淋巴细胞和含有IgG抗体的接种后血清， 识别细胞内和细胞表面黑色素瘤决定簇。 这些 结果为进行分子分析提供了坚实的基础 刺激特异性T和B细胞应答的黑色素瘤抗原 接种疫苗的患者 许多候选抗原已经被 从用cDNA的自体血清的初始筛选中鉴定 在一个实施方案中，本发明涉及从患者的肿瘤细胞系构建的表达文库。 的 首次揭示了与MUC-1具有结构相似性的新产物， 免疫原性细胞表面分子家族的一员，表达于 各种癌症。 这种基于抗体的策略可能会 用于鉴定许多黑素瘤抗原。 的 申请人设计了几种方法来表征免疫 针对每个候选抗原的应答。 体液反应将是 通过免疫印迹CRIP包装细胞系的裂解物进行评价 用表达每种候选物的基于MFG的逆转录病毒载体转染。 用MFG转染的自体EBV转化的成淋巴细胞 逆转录病毒将用于测定T细胞的细胞毒性， 增殖和细胞因子的产生 抗原的 将通过北方分析确定表达模式 和RT-PCR从各种肿瘤及其正常组织 同行 许多研究人员试图利用以前的 在疫苗策略中鉴定的抗原证明了 对这些抗原产生显著的免疫反应。 的 对申请人的分析应有助于理解 抗肿瘤中常见靶点与独特靶点的相对重要性 免疫力 黑色素瘤免疫原性抗原的进一步鉴定 为未来的治疗提供了希望。

The ability to manipulate genes encoding potential immunomodulators such as cytokines and tumor antigens has opened a new era of research attempts. Poorly immunogenic murine tumor model systems demonstrate that vaccination with irradiated tumor cells engineered to secrete granulocyte-macrophage colony stimulating (GM-CSF) generates potent, specific, and long-lasting immunity. To test this strategy in patients with advanced melanoma, the laboratory of the applicants performed a clinical trial of vaccination with lethally irradiated, autologous melanoma cells expressing human GM-CSF. Vaccination elicits a striking infiltrate of pre-existing metastatic lesions with large numbers of lymphocytes, plasma cells, macrophages, and eosinophils, resulting in tumor destruction, fibrosis, and edema. Consistent production of humoral and cellular immune responses is exemplified by tumor specific cytotoxicity and cytokine production characterizing tumor-infiltrating lymphocytes, and post-vaccination sera containing IgG antibodies that recognize intracellular and cell surface melanoma determinants. These results provide a solid foundation for undertaking a molecular analysis of the melanoma antigens stimulating specific T and B cell responses in vaccinated patients. A number of candidate antigens have been identified from initial screening with autologous sera of a cDNA expression library constructed from a patient's tumor cell line. The first revealed a novel product with structural similarities to MUC-1, a member of a family of immunogenic cell surface molecules expressed in a variety of cancers. This antibody-based strategy will likely be productive for identifying a number of melanoma antigens. The applicants have designed several approaches to characterize immune responses against each candidate antigen. Humoral responses will be evaluated by immunoblotting lysates of CRIP packaging cell lines transfected with MFG based retroviral vectors expressing each candidate. Autologous EBV transformed lymphoblasts transfected with MFG retroviruses will be used to determine T cell cytotoxicity, proliferation, and cytokine production in response to each candidate antigen. Expression patterns will be determined by Northern analyses and RT-PCR from a variety of tumors and their normal tissue counterparts. Attempts by numerous investigators to utilize previously identified antigens in vaccine strategies demonstrate an ability to develop significant immune responses against these antigens. The analysis of the applicants should contribute to the understanding of the relative importance of common versus unique targets in anti-tumor immunity. Further identification of immunogenic antigens in melanoma offers promise for future therapies.