We propose to develop a novel method for the delivery of gene-engineered proteins to tumors by targeting the proteins to the granules of tumor infiltrating lymphocytes (TIL). The method is designed to increase tumor selectivity by taking advantage of the granule-release mechanisms that are triggered by specific interactions of lymphocytes with their targets. We have shown in preliminary studies that small transgenic human peptide defensin HNP-1. is targeted to granules of murine granulocytes and cytotoxic T-lymphocytes. Deletion mutagenesis of HNP-1 suggests that a limited segment of this small peptide is essential for granule targeting. In this project we will apply and extend these studies to human TIL with the aim of identifying a peptide sequence, the """"""""targeting motif"""""""", that can direct other gene-engineered proteins to granules. We will first study the expression and subcellular distribution of defensin HNP-1 engineered into human TIL. We will then employ deletion mutagenesis and reporter fusion constructs to characterize the defensin targeting motif and its ability to direct reporter proteins (CAT. IL-8) into granules of TIL. Finally, we will examine the effect of the granule environment on the bioactivity of the reporter proteins and compare the selectivity of reporter protein release by granule-targeted and non-targeted TIL in vitro and in vivo, in human tumor-bearing SCID mice. Successful completion of this project will facilitate future clinical trials of tumor-selective delivery by TIL of therapeutic proteins or immunostimulatory cytokines.
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