RNA pulsed Dendritic Cells as Immunotherapy for Melanoma

恶性黑色素瘤对患者、医生和科学家来说仍然是一个巨大的挑战。虽然手术切除是主要的治疗方法，而且通常是可以治愈的，但黑色素瘤在局部和远处都有复发。不幸的是，目前的辅助治疗并没有改善与区域性和转移性疾病相关的惨淡预后。本研究的重点是利用免疫疗法治疗微转移性和复发性黑色素瘤。通过这项临床前研究获得的信息将用于开发有效的临床黑色素瘤疫苗。肿瘤细胞的免疫排斥反应是由识别特异性肿瘤相关抗原（TAA）的细胞毒性t淋巴细胞（CTL）介导的。抗原呈递细胞，如树突状细胞（DC），已被证明在体外和体内用TAA启动后都能产生抗原免疫反应。目前的研究和临床试验正在进行中，在该机构加载DC与肿瘤裂解物和TAA肽。本研究项目将尝试通过方法学研究使用肿瘤RNA诱导DC的方法来优化针对黑色素瘤的CTL反应，从而诱导意想不到的肿瘤特异性CTL反应。具体来说，DC将通过白细胞分离从黑色素瘤患者中分离出来，这些患者可以建立肿瘤细胞系。来自这些患者的DC将用患者自身肿瘤的总肿瘤RNA引物，或用先前分离的黑色素瘤特异性TAA RNA（如MART-1、MAGE-3、肿瘤）组合引物，或用先前分离的黑色素瘤特异性TAA RNA（如MART-1、MAGE-3、肿瘤）组合引物，或用先前分离的黑色素瘤特异性TAA RNA（如MART-1、MAGE-3、酪氨酸酶和gp100）组合引物。将引物的树突状细胞与CTL孵育。这些CTL随后将与黑色素瘤细胞系混合，并使用铬释放试验定量裂解细胞的百分比。产生最大细胞毒性的抗原将直接与目前的脉冲树突状细胞与蛋白质产生的方法进行测试，然后将直接与目前的脉冲树突状细胞与蛋白质裂解物和肽的方法进行测试。从这项临床前工作中获得的数据将指导未来在临床中使用疫苗开发。

Malignant melanoma remains a formidable challenge to patients, physicians, and scientists. Although surgical resection serves as the primary therapy and is often curative, melanoma recurs both locally and at distant sites. Unfortunately, current adjuvant therapies have not improved the dismal prognosis associated with regional and metastatic disease. This research focuses on the use of immunotherapy to treat micrometastatic and recurrent melanoma. Information gained through this preclinical research will be applied to developing an effective clinical melanoma vaccine. Immunologic rejection of tumor cells is mediated by cytotoxic T-lymphocytes (CTL) which recognize specific tumor- associated antigens (TAA). Antigen presenting cells, such as dendritic cells (DC), have been shown to generate an antigen-immunologic response both in vitro and in vivo after priming with TAA. Current research and clinical trials are underway at this institution loading of DC with tumor lysates and TAA peptides. This research project will attempt to optimize the CTL response against melanoma by methodologically studying the methods used to prime DC with tumor RNA induce unexpectedly potent tumor-specific CTL responses. Specifically, DC will be isolated through leukopheresis from patients with melanoma in whom tumor cell lines can be established. The DC from these patients will be primed with total tumor RNA from the patients' own tumor, or primed with combinations of previously isolated RNA for melanoma-specific TAA such as MART-1, MAGE-3, tumor, or primed with combinations of previously isolated RNA for melanoma-specific TAA such as MART-1, MAGE-3, tumor, or primed with combinations of previously isolated RNA for melanoma-specific TAA such as MART-1, MAGE-3, tyrosinase, and gp100. The primed dendritic cells will be incubated with CTL. These CTL will subsequently be mixed with melanoma cell lines and the percentage of lysed cells will be quantified using a chromium release assay. The antigen that yields the greatest cytoxicity will then by directly tested against the current method of pulsing dendritic cells with protein yields the greater cytotoxicity will then be directly tested against the current method of pulsing dendritic cells with protein lysates and peptides. Data gained from this preclinical work will guide future uses of vaccine development in the clinic.