GENE THERAPY OF MELANOMA BY PARTICLE BOMBARDMENT

虽然在理解有效执行《公约》的要求方面取得了进展， T细胞识别人类恶性黑色素瘤，这种疾病仍然存在 一旦转移就无法治愈临床上的当务之急是翻译， 将T细胞识别应用于黑素瘤的有效治疗 患者该项目旨在提供一个关键的联系， T细胞识别和癌基因启动的基础科学 刺激黑色素瘤中抗黑色素瘤T细胞免疫的治疗试验 患者 T细胞有效识别人类免疫缺陷病毒的必要成分 黑素瘤包括在黑素瘤中优先表达肽， 由适当的MHC分子呈递。人类共有的编码基因 已经克隆了刺激溶细胞性T细胞的黑素瘤抗原， 都是可用的。这些包括酪氨酸酶和MART-1基因，两者都是 其在HLA-A2的背景下呈现。大约50%的 HLA-A2+的人，这在临床上是非常相关的。此外，本发明还提供了一种方法， 需要存在足够的MHC表面分子表达， 有效地将抗原呈递给T细胞。干扰素γ（IFN-γ）的应用 γ）以增加MHC分子的表达，以及其他重要的 抗原加工/呈递组件，mad扩增呈递 这些免疫原性肽。这种肽的呈递仍然可以 降低刺激T细胞溶解自体黑色素瘤，除非给予 必要的协同刺激。人类B7-1和B7-2的基因共同 刺激分子已被克隆并可获得。 我们假设HLA-A2+细胞表达高水平的MHC分子， 共刺激B7-1和B7-2分子，以及酪氨酸酶或MART-1 分子，将更好地诱导抗黑色素瘤T细胞免疫比黑色素瘤 这些分子表达水平低或检测不到的细胞。我们将 最初基因修饰HLA-A2+黑素瘤细胞、上皮细胞和B细胞 使其成为抗黑色素瘤T细胞免疫的有效刺激物。我们 然后将使用健康志愿者的外周血淋巴细胞（PBL） 以确定这些基因修饰的细胞的体外免疫原性。 外周血淋巴细胞、淋巴结和远处转移 将评估黑色素瘤患者的基因， 修饰的细胞可以刺激T细胞对自体未修饰的 黑素瘤细胞。然后，我们将在体内基因修饰人类HLA-A2+黑色素瘤 在SCID小鼠中生长为皮下结节的细胞， 评价它们刺激体外T细胞增殖的能力， 对自体未修饰的黑素瘤细胞的细胞毒性反应。基因转移 这些体外和体内研究将利用新技术 粒子介导的基因转移， 基因靶向细胞刺激抗黑色素瘤T细胞免疫。这 项目应提供一个基础，以启动临床基因治疗 转移性黑色素瘤患者的临床试验。

While progress has been made understanding the requirements for effective T-cell recognition of human malignant melanoma, this disease remains incurable once metastatic. The clinical imperative is to translate and apply T-cell recognition into an effective treatment for melanoma patients. This project is intended to provide a critical link between the basic science ofT-cell recognition and the initiation of a cancer gene therapy trial to stimulate anti-melanoma T-cell immunity in melanoma patients. The necessary components for effective T-cell recognition of human melanoma include peptide that is preferentially expressed in melanoma and presented by appropriate MHC molecules. Genes encoding shared human melanoma antigens that stimulate cytolytic T cells have been cloned and are available. These include the genes for tyrosinase and MART-1, both of which are presented in the context of HLA-A2. As approximately 50% of individuals are HLA-A2+, this is clinically quite relevant. In addition, sufficient MHC surface molecule expression needs to be present to effectively present antigen to T cells. The use of interferon gamma (IFN- gamma) to increase expression of MHC molecules, and other important components for antigen processing/ presentation, mad augment presentation of these immunogenic peptides. This presentation of peptides may still fall to stimulate T cells to lyse autologous melanoma unless given with necessary co-stimulation. The genes for the human B7-1 and B7-2 co- stimulatory molecules have been cloned and are available. We hypothesize that HLA-A2+ cells expressing high levels of MHC molecules, the co-stimulatory B7-1 and B7-2 molecules, and tyrosinase or MART-1 molecules, will better induce anti-melanoma T-cell immunity than melanoma cells expressing low or undetectable levels of these molecules. We will initially gene-modify HLA-A2+ melanoma cells, epithelial cells, and B-cell lines to make them potent stimulators of anti-melanoma T-cell immunity. We will then use peripheral blood lymphocytes (PBL) from healthy volunteers to determine the in vitro immunogenicity of these gene-modified cells. Lymphocytes from the peripheral blood, lymph bodes and distant metastases from melanoma patients will be evaluated to determine if these same gene- modified cells can stimulate T-cell reactivity to autologous unmodified melanoma cells. We will then gene-modify in vivo human HLA-A2+ melanoma cells that are growing as subcutaneous nodules in SCID mice, and will evaluate their ability to stimulate in vitro T-cell proliferative and cytotoxic responses to autologous unmodified melanoma cells. Gene transfer for these in vitro and in vivo studies will utilize the novel technology of particle-mediated gene transfer to deliver combinations of up to four genes to target cells to stimulate anti-melanoma T-cell immunity. This project should provide a foundation to initiate a clinical gene therapy trial for patients with metastatic melanoma.