Project 1 focuses on developing reporter gene imaging methods that can be translated into clinical applications. Specifically, we wish to test whether sequential imaging of adoptively transferred antigenspecific T cells can be used to predict responses of targeted tumors, early after transfer, and to identify T cell interactions affecting anti-tumor activity. We propose to initially develop and test a series of vectors encoding both a constitutive and an inducible reporter gene, and to assess their capacity to distinguish subpopulations of EBV-specific T cells during their specific antigen-induced activation-proliferation or apoptosis (Aim 1). We will then evaluate whether and to what degree different subpopulations of transduced antigen-specific T-cells can be distinguished in vivo by sequential imaging of co-administered T-cells, adoptively transferred into NOD/SCID mice bearing human EBV lymphoma xenografts. We will also examine the contributions of different functional subsets of T-cells, transduced to express distinguishable reporter genes, to target and accumulate in EBV lymphoma xenografts, to proliferate and survive in these tumors, and to assess their tumoricidal activity against targeted tumor cells at sequential intervals after adoptive transfer (Aim 2).
In Aim 3, we propose to conduct a phase I clinical trial of EBV-specific T-cells transduced with a new vector encoding two human genes, a mutant LNGFR and the human iodide symporter (hNIS), in the treatment of EBV lymphomas complicating allogeneic hematopoietic progenitor cell transplants or organ allografts. Thereafter, we will incorporate this vector into a phase II trial testing and imaging EBV-specific T-cells differing in repertoire and duration of selection in vitro, as well as in their content of CD4 and CDS T-cells that will be identifiable following their transduction with distinguishable reporter constructs. One of these vectors, termed NIT, is a retroviral vector encoding a biologically inactive mutant of the human nerve growth factor receptor and an IRES-linked HSV-thymidine kinase gene. This vector being used in a current clinical trial. The new dicistronic vector being introduced, is derived from the NIT vector, but substitutes the hNIS gene for HSVItk because HSVItk (and fusion genes incorporating HSVItk) currently in use as reporters are limited by their potential immunogenicity in man. This new dicistronic vector, which exclusively encodes human genes for in vitro selection and in vivo imaging, is likely to be far less immunogenic, and should therefore not compromise the life span of transduced T-cells following adoptive transfer. The planned phase II trial should also provide a direct comparison of the activity and persistence of antigen-specific T-cells selected early or late in the course of in vitro culture.
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