65. CD4+ T Cell Immunity Following Gene Transfer into Hematopoietic Stem Cells

Background: Immunotherapeutic approaches to treat melanoma have become increasingly sophisticated with attempts to manipulate the immune response via high dose IL-2, adoptive transfer, and gene therapy. Although, many of these approaches show promise, clinical response appears modest, with adoptive transfer of melanoma antigen specific CD8+ T-cells showing the most impressive results. CD8+ cytotoxic lymphocyte therapies are limited by lack of support from the remaining components of the immune system such as dendritic cells, memory cells, B cells, and CD4+ T cells. Increasing evidence indicates that CD4+ T helper cells play a crucial function as a facilitator in the anti-tumor response, particularly through the induction and maintenance of CD8+ T cell immunity. Yet despite, their important role there have been limited efforts to exploit CD4+ T cells to enhance the immune response to cancer. Results: in this study, we identified an MHC Class II HLA-DR4 restricted CD4+ T cell receptor (TCR) capable of reacting against a naturally occurring melanocyte differentiation antigen (MDA), Tyrosinase Related Protein-1 (TRP-1). Human lymphocytes specific and reactive to a 21-residue epitope of TRP-1 (positions 277-297) were isolated from the peripheral blood of a metastatic melanoma patient by limiting dilution. We then identified the gene sequence of the individual α and β subunits of the TCR from these lymphocytes via a 5’ race strategy. Next, we constructed a bicistronic lentivirus containing the genes for the α and β subunits, and successfully transduced both transformed human T-cells (Jurkat cells) and peripheral blood mononuclear cells (PBMC). At an MOI of 10:1 we were able to achieve 94% transduction efficiency of our TCR into Jurkat cells, and at a MOI of 100:1 we achieved 56% transduction efficiency into PBMCs. Furthermore, transduced PBMCs were found to recognize TRP-1 peptide, melanoma tumor lysates, and TRP-1 positive tumor lines as measured by specific interferon γ (IFN γ) production on ELISA. Following these in vitro studies, a series of bone marrow transplant experiments were conducted using a TRP-1 TCR transgenic mouse developed by insertion of the TRP-1 TCR gene into fertilized murine oocytes. Initial characterization of the naïve TRP-1 Tg mice by flow cytometry showed no presence of TRP-1 TCR specific lymphocytes in the peripheral blood using a peptide-antibody complex (TRP-1 Pentamer). However, 2.6% of lymphocytes showed pentamer specificity in the thymus. To test the role of the thymus to positively select the transgenic TCR, whole body irradiated DR4 transgenic and C57BL/6 recipient mice received a mix of DR4 Tg and TRP-1 Tg bone marrow. Initial results at the four week time point show a slight increase in TRP-1 pentamer specific CD4+ cells in the DR4 Tg mice (2.3% vs. 0.2%). By 12 weeks, however, expression had dramatically increased to 37%. Conclusions: Herein we demonstrate the development of a CD4+ T cell model for studying gene transfer into hematopoietic stem cells. We demonstrate the efficient transduction of our TCR using lentiviral constructs, the functional expression of our TCR in transduced PBMCs, and the successful transplantation of our TCR Tg bone marrow into MHC specific transplant recipients (DR4 Tg mice). These results establish the basis for a lentiviral delivery system which could be used to deliver MDA specific CD4+ TCRs into hematopoietic stem cells for use in the treatment of patients with metastatic melanoma.