Strong Immunogenic Potential of a B7 Retroviral Expression Vector: Generation of HLA-B7-Restricted CTL Response Against Selectable Marker Genes

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Abstract
The stimulation of a specific immune response is an attractive goal in cancer therapy. Gene transfer of co-stimulatory molecules and/or cytokine genes into tumor cells and the injection of these genetically modified cells leads to tumor rejection by syngeneic hosts and the induction of tumor immunity. However, the development of host immune response could be either due to the introduced immunomodulatory genes or due to vector components. In this study, human renal cell carcinoma cell lines were modified by a retrovirus to express the co-stimulatory molecule B7-1 together with the hygromycin/thymidine kinase fusion protein (HygTk) as positive and negative selection markers. These B7-1-transduced renal cell carcinoma cell lines were able significantly to activate allogeneic T cell proliferation. The cytolytic activity of these T cells was determined by employing several transduced and nontransduced renal cell carcinoma cell lines as targets. Evidence for a strong vector-specific T cell reactivity induced by the Hyg/Tk protein was obtained in autologous renal cell carcinoma systems. Antibody blocking experiments as well as peptide binding assays demonstrated an HLA-B7-restricted T cell response directed against both the Hyg and the Tk genes. Thus, the vector itself may mask the generation of immune reactivity against tumor antigens and may even detract from it. Vectors with immunogenic potential may be useful for tumor vaccination via cross priming in vivo, whereas antivector reactivities would be detrimental in situations where gene defects are being corrected and where long term expression of a therapeutic protein is required. The in vitro T cell response following stimulation with renal cell carcinoma (RCC) cell lines that have been retrovirally transfected with the co-stimulatory molecule B7-1 and the hygromycin thymidine kinase fusion gene (HygTk) as positive and negative selection markers was analyzed. B7-1-transduced RCC lines are able to induce proliferation of autologous and allogeneic T cells. Interestingly, the CTL response was not directed against specific tumor antigens but against the Hyg and the Tk genes. The specific epitopes of Hyg and Tk, identified by peptide loading assays and the MHC class I blocking experiments, are both presented by HLA-B7. Thus, the B7-1 retroviral expression vector employed to transduce RCC cell lines had the capacity to induce a strong immune response against different vector components, suggesting that vectors expressing the highly immunogenic Hyg and Tk gene should not be employed in gene therapeutical approaches correcting gene defects or requiring long-term expression of proteins.