QS319. Enhancing Tumor Immunity Through the Transfer of a Lentiviral Construct into Hematopoietci Stem Cells

Background: Over the past 25 years, substantial progress has been made in understanding the molecular and cellular basis of T-cell-mediated antitumor responses to melanoma. Despite early promise with IL-2, tumor infiltrating lymphocytes (TIL) and vaccine therapy, immune responses have been largely weak and ineffective in the eradication of large multi-visceral melanoma metastases. Most current therapies focus on the CD8+ T-cell arm which possesses the ability to directly destroy tumor. CD4+ T-cells, known for their ability to induce and maintain CD8+ T-cell responses (CTL), have been largely uninvestigated. Our project focuses on how CD4+ T-cells may propagate a therapeutic immune response to melanoma by amplifying the CTL immune response. Results: To accomplish this, a high affinity T-cell receptor derived from CD4+ T-cells, previously shown to be extremely reactive against an HLA-DR4-restricted peptide epitope from the melanoma differentiation antigen gp100, has been identified and cloned. The cloning protocol involved the creation of T-cell hybridoma by fusing a polyclonal CD4+ T-cell population with a murine thymoma cell line. When presented with the specific gp100 peptide epitope, the hybridoma elicited a four-fold increase in IL-2 production when compared with stimulation using a negative control antigen. With the T-cell clone now isolated, the alpha and beta chains of T-cell receptor (TCR) were amplified and cloned into mono-cistronic vector linked between the a Furin-2a peptide sequence allowing for the post-translational cleavage and expression of each TCR subunit. We have verified expression of this lentiviral-TCR construct in transformed cell lines in vitro. To further study the stability and distribution of gene transfer in vivo a model transplantation of gene modified murine hematopoietic stem cells (HSC) was performed. Lineage depleted cells (Lin−) were transduced with a lentiviral-GFP construct and competitively combined with whole bone marrow from C57BL/6 (CD45.2) mice into lethally irradiated C57BL/6 recipients. A negative control transplant was performed using unmanipulated congenic CD 45.1 Lin− cells. The competitive cell distribution of CD 45.2 whole bone marrow and CD 45.1-GFP containing lineage depleted cells was analyzed in peripheral blood and organs of hematopoiesis at set four week intervals by flow cytometry. Peripheral blood from transplanted mice was followed for six months and exhibit high level expression of GFP (greater than 45% in all donor derived cells) and high/stable GFP expression in CD4+ T-cell compartment. The bone marrow, spleen and thymus of sacrificed recipient mice all show equally stable and high levels of GFP expression and demonstrate that CD 45.1-GFP expressing cells out compete whole bone marrow CD 45.2 cells. To continue to evaluate durable gene expression and gene stability of our GFP transduced HSC a secondary bone marrow transplant was performed. At 2 months 36% of all cell types in the peripheral blood express GFP. Conclusion: We have shown that lentivirally transduced HSC exhibit high-level stable long term expression in vivo. This provides the scientific background for further experimentation using our newly cloned CD4+ TCR. Our future plans involve the transfer of this TCR into HSC as a means of reconfiguring the host T-cell repertoire with the theoretical goal of enhancing effector CTL responses to metastatic melanoma.