IMMUNE RESPONSES INDUCED BY GENE TRANSFER

基于我们在上一次资助期间的研究，我们一直在 能够开发出两种提高免疫力的遗传方法 对肿瘤的反应性。第一种方法涉及体内基因。 将同种异体MHC基因转移到动物和小鼠肿瘤中 人类研究，被发现可靠地导致转基因表达。 更重要的是，我们能够激发增强的抗肿瘤反应性 肿瘤内和引流淋巴结(LN)内；并观察到肿瘤 在选定的患者中进行回归。第二种方法涉及使用 基因改造的自体肿瘤疫苗分泌GM-CSF作为一种 在引流LN时增强局部免疫反应的方法。我们 目前正在进行一项第一阶段的研究，以评估转基因技术的使用 脑脊液转导的肿瘤作为疫苗产生免疫LN细胞 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 它可以在体外扩大，用于临床治疗。我们建议 来源的TIL的体内外肿瘤活性评价 肿瘤内植入人类白细胞抗原B7基因后的黑色素瘤和结直肠肿瘤 调职。此外，我们计划评估抗原的特异性 MHC诱导的细胞反应的识别和限制 基因转移。该项目的另一个方面将是 树突状细胞(DC)的应用 用于致敏LN细胞的肿瘤疫苗的成分 过继免疫疗法。DC有独一无二的能力来激发天真 T细胞在体外和体内的免疫反应。关于DC的研究 从皮肤肿瘤中分离出来的研究表明，它们的功能 能力可被免疫抑制细胞因子抑制。这有 可通过GM-CSF的存在逆转，同时上调两者 B7-1和B7-2共刺激分子。我们计划评估 肿瘤致敏DC体内致敏的LN细胞的抗肿瘤活性 与DM-CSF成纤维细胞混合的黑色素瘤患者和 结直肠癌。本项目的具体目标是：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基因进行体内基因转移，2) 评价体内T细胞对肿瘤的免疫反应性 有或无GM-CSF混合分泌物的脉冲DC 3)比较TIL和TIL的免疫反应性 在AIMS 1和AIMS 2中接受治疗的患者的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.