17 Conversely, Dabrafenib the 2A peptide linker results in a single mRNA molecule, but during translation ribosomal skipping generates two separate proteins from the single mRNA.18 The majority of constructs currently in clinical and preclinical development use the 2A sequence to link the TCR-α and TCR-β chains as a result of the improved equimolar expression of both genes, compared to vectors with an IRES element separating the TCR genes. Importantly, it has been shown by ourselves and others that T cells transduced with constructs containing the TCR genes linked by a 2A sequence express higher levels of cell-surface TCR and demonstrate improved antigen-specific function, as measured by IFN-γ secretion,

compared with constructs containing identical TCR sequences

separated by an IRES element.19 Efficient cell-surface TCR expression requires the formation of a stable TCR–CD3 complex.11 In mTOR inhibitor the absence of CD3, TCRs do not assemble properly and are degraded. Therefore, the availability of CD3 molecules for TCR–CD3 complex assembly is a major rate-limiting effect when introducing additional exogenous TCRs into T cells. Competition may reduce cell-surface expression of the introduced TCR and impair the avidity of antigen recognition of the transduced cells. We have recently demonstrated that the double transduction of CD8+ T cells with a vector encoding the desired TCR-α and TCR-β chain genes, together with a second vector encoding the CD3 gamma, delta, epsilon and zeta genes (linked by 2A sequences), can enhance the avidity of CD8+ T cells (King J, Ahmadi M, personal communication). This may be a mechanism to enhance the functional avidity of transduced T cells expressing low-affinity TCRs. It is common for the introduced TCRs to be expressed at lower levels than the endogenous TCRs, which may impair the ability of the transduced T cell to respond to low concentrations of the TCR-recognized antigen, as

discussed above. This observation is consistent with the introduced TCR competing with the endogenous TCR for limited CD3 molecules. Heemskerk et al.20 Carbohydrate have recently shown that the expression levels of the introduced TCR can be influenced by the ‘strength’ of the endogenous TCR by introducing the same TCR into different antigen-specific T-cell clones. It is currently unclear whether TCR-specific molecular motifs exist to determine the ‘competitiveness’ of a given TCR-αβ chain. Primary T cells transduced with exogenous TCRs have the potential to express four different TCR-αβ heterodimers on the recipient T-cell surface: (i) the endogenous αβ heterodimer; (ii) the introduced αβ heterodimer; (iii) the endogenous α chain paired with the introduced β chain; and, finally, (iv) the introduced β chain paired with the endogenous α chain. These possibilities are indicated in the schematic diagram shown in Fig. 2.