IMMUNE RESPONSES INDUCED BY GENE TRANSFER

根据我们在上一个资助期的研究， 能够开发两种基因方法来上调免疫 对肿瘤的反应。 第一种方法涉及体内基因 将同种异体MHC基因转移到肿瘤中， 人类研究发现，可靠地导致转基因表达。 更重要的是，我们能够引发增强的抗肿瘤反应， 在肿瘤和引流淋巴结（LN）内;以及观察到的肿瘤 选择患者的回归。 第二种方法涉及使用 自体肿瘤疫苗经遗传改变以分泌GM-CSF作为 方法，以增强局部免疫反应，引流LN。 我们 目前正在进行第一阶段的研究，以评估使用转基因- CSF转导的肿瘤作为疫苗产生免疫LN细胞用于 过继免疫疗法 该项目的主要重点是开发肿瘤反应性T细胞 其可以离体扩增用于临床治疗。 我们建议 评价来源于以下的TIL的体外和体内肿瘤反应性： 黑色素瘤和结直肠肿瘤的HLA-B7基因 转移 此外，我们计划评估抗原的特异性 诱导的细胞应答的识别和MHC限制 基因转移 该项目的另一个方面将是 树突状细胞（DC）的应用，其将被用作 肿瘤疫苗的组分使LN细胞对随后的 过继免疫疗法 DC有独特的能力来启动天真 用于体外和体内免疫应答的T细胞。 DC研究 从皮肤肿瘤中分离出来的病毒已经证明， 免疫抑制细胞因子可抑制该能力。 这 通过GM-CSF的存在逆转， B7-1和B7-2共刺激分子。 我们计划评估 肿瘤致敏DC致敏LN细胞的抗肿瘤活性 与DM-CSF成纤维细胞混合， 结肠直肠癌 该项目的具体目标是：1）评估 直接修饰肿瘤来源的TIL的免疫反应性 利用同种异体I类MHC基因的体内基因转移，2） 评估体内用肿瘤致敏的T细胞的免疫反应性， 有或没有GM-CSF分泌的混合物的脉冲DC 3）比较TIL与成纤维细胞的免疫反应性， 目的1和2中治疗的患者中的致敏LN。

Based upon our studies from the previous grant period, we have been able to develop two genetic approaches to upregulate immune responsiveness to tumor. The first approach involved the in vivo gene transfer of an allogeneic MHC gene into tumors which in animal and human studies, was found to reliably result in transgene expression. More importantly, we were able to elicit enhanced antitumor reactivity within the tumor and draining lymph nodes (LN); and observed tumor regression in select patients. The second approach involved the use of autologous tumor vaccines genetically altered to secrete GM-CSF as a method to augment regional immune responses in draining LN. We are currently conducting a phase I study to evaluate the use of GM- CSF transduced tumor as a vaccine to generate immune LN cells for adoptive immunotherapy. The primary focus of this project is to develop tumor reactive T cells that can be expanded ex vivo for clinical therapy. We propose to evaluate the in vitro and in vivo tumor reactivity of TIL derived from melanoma and colorectal tumors after intralesional HLA-B7 gene transfer. In addition we plan to evaluate the specificity of antigen recognition and MHC restriction of the cellular responses induced by the gene transfer. Another aspect of this project will be the application of dendritic cells (DC) which will be employed as components of tumor vaccines to sensitize LN cells for subsequent adoptive immunotherapy. DC have the unique capacity to prime naive T cells for immune responses in vitro and in vivo. Studies of DC isolated from skin tumors have demonstrated that their functional capacity can be inhibited by immunosuppressive cytokines. This has been reversed by the presence of GM-CSF with upregulation of both B7-1 and B7-2 co-stimulatory molecules. We plan to evaluate the antitumor reactivity of LN cells primed in vivo with tumor-pulsed DC admixed with DM-CSF fibroblasts in patients with melanoma and colorectal cancer. The specific aims of this project are: 1) To evaluate the immune reactivity of TIL derived from tumors modified by direct gene transfer in vivo utilizing an allogeneic class I MHC gene, 2) to evaluate the immune reactivity of T cells primed in vivo with tumor- pulsed DC with or without the admixture of GM-CSF secreting fibroblasts, and 3) To compare the immune reactivity of TIL versus primed LN in patients being treated in Aims 1 and 2.