Promiscuous Behavior of HPV16E6 Specific T Cell Receptor Beta Chains Hampers Functional Expression in TCR Transgenic T Cells, Which Can Be Restored in Part by Genetic Modification

BACKGROUND T cell receptor gene transfer is a promising strategy to treat patients suffering from HPV induced malignancies. Therefore we isolated the TCRalphabeta open reading frames of an HPV16E6 specific CTL clone and generated TCR transgenic T cells. In general low level expression of the transgenic TCR in recipient human T cells is observed as well as the formation of mixed TCRs dimers. Here we addressed both issues employing three different expression platforms. METHODS We isolated the HVP16E6 specific TCRalpha and TCRbeta open reading frames and retrovirally transduced human T cells with either wild-type (wt), or codon-modified (cm) chains to achieve enhanced TCR expression levels, or used codon-modification in combination with cysteinization (cmCys) of TCRs to facilitate preferential pairing of the introduced TCRalpha and TCRbeta chains. RESULTS Careful analysis of recipient T cells carrying the HPV16E6 TCRbeta and endogenous TCR chains revealed the transgenic TCRbeta chain to behave very promiscuously. Further analysis showed that the percentage of tetramer positive T cells in codon-modified/cysteinized TCR transgenic T cells was four-fold higher compared to wild-type and two-fold higher compared to codon-modification only. Functional activity, as determined by IFN-gamma production, was high in cmCysTCR transgenic T cells, where it was low in cm and wt TCR transgenic T cells. Recognition of endogenously processed HPV16E6 antigen by cmCysTCR transgenic T cells was confirmed in a cytotoxicity assay. CONCLUSION Promiscuous behavior of the HPV16E6 specific TCRbeta chain can in part be forced back into specific action in TCR transgenic T cells by codon modification in combination with the inclusion of an extra cysteine in the TCR chains.