GENE THERAPY OF MELANOMA BY PARTICLE BOMBARDMENT

虽然已经取得了进展，但理解有效的 T细胞识别人类恶性黑色素瘤，本病残留 一旦转移就无法治愈。临床上的当务之急是翻译和 将T细胞识别应用于黑色素瘤的有效治疗 病人。该项目旨在提供一条关键的纽带， 细胞识别与癌症基因启动的基础科学 激发黑色素瘤患者抗黑色素瘤T细胞免疫的治疗试验 病人。 人类T细胞有效识别的必要成分 黑色素瘤包括在黑色素瘤中优先表达的多肽和 由合适的MHC分子提供。编码共享人类的基因 刺激细胞溶解T细胞的黑色素瘤抗原已经被克隆并 都是可用的。这些基因包括酪氨酸酶和MART-1基因，两者都是 这些都是在人类白细胞抗原A2的背景下提出的。作为大约50%的 个体是人类白细胞抗原A2+，这在临床上是非常相关的。此外, 需要有足够的MHC表面分子表达才能 有效地将抗原呈递给T细胞。干扰素-伽马干扰素的使用 Gamma)来增加MHC分子的表达，以及其他重要的 抗原处理/呈递组件、MAD增强呈递组件 这些免疫原性多肽。这种多肽的呈现形式可能仍然 Fall刺激T细胞溶解自体黑色素瘤，除非给予 必要的共同刺激。人类B7-1和B7-2联合感染的基因 刺激分子已经被克隆并可用。 我们假设人类白细胞抗原A2+细胞表达高水平的MHC分子， 共刺激的B7-1和B7-2分子，以及酪氨酸酶或MART-1 分子，比黑色素瘤更能诱导抗黑色素瘤T细胞免疫 表达低水平或检测不到这些分子的细胞。我们会 最初基因修饰的人类白细胞抗原-A2+黑色素瘤细胞、上皮细胞和B细胞 使它们成为抗黑色素瘤T细胞免疫的有效刺激物。我们 然后使用健康志愿者的外周血淋巴细胞(PBL) 检测这些基因修饰细胞的体外免疫原性。 外周血、淋巴组织和远处转移的淋巴细胞 将对黑色素瘤患者的基因进行评估，以确定这些相同的基因- 修饰细胞可刺激T细胞对自体未修饰细胞的反应性 黑色素瘤细胞。然后我们将在体内对人类HLA-A2+黑色素瘤进行基因修饰 在SCID小鼠体内以皮下结节的形式生长的细胞，并将 评估他们在体外刺激T细胞增殖和 对自体未修饰黑色素瘤细胞的细胞毒反应。基因转移 对于这些在体外和体内的研究将利用新技术 通过粒子介导的基因转移来传递最多四种组合 基因靶向细胞，激发抗黑色素瘤T细胞免疫。这 项目应为启动临床基因治疗提供基础 转移性黑色素瘤患者的试验。

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.