IMMUNE RESPONSES INDUCED BY GENE TRANSFER

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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.