Evidence suggests that the level of TCR mispairing is also affected by the variable region of the endogenous TCR chains (Fig. 3).12 An additional approach to prevent TCR mispairing, as demonstrated by Voss et al.,26 was the identification and inversion of a pair of specifically interacting amino acids in the TCR-α and TCR-β constant-domain interface. Mutational inversion of these two amino acids changed a ‘knob-into-hole’ configuration into a charged ‘hole-into-knob’ configuration and by so doing increased the preferential pairing of the transduced mutated TCRs. This approach was effective in both human and murine TCR gene-transfer

systems. An alternative method to completely abolish TCR mispairing is the development of chimeric antigen receptors (CARs), which consist of a single chain Fv fused to CD3 signalling elements. However, the functional activity of CARs is dependent on TAM Receptor inhibitor the sensitivity of the signalling elements, which in some constructs contain additional costimulatory molecules and/or cytokines. Early Everolimus research with CAR-expressing T cells suggested that they were less sensitive to peptide than T cells expressing αβ TCR heterodimers.27,28 It is possible that the described modified TCRs will be immunogenic in an immunocompetent

host, resulting in reduced persistence or elimination of the transduced T cells. Whilst the lymphodepleting regimens currently used before adoptive T-cell transfer are likely to permit T-cell engraftment, it is still necessary to consider strategies to minimize the possible immunogenicity of the modified TCRs. An alternative and novel method of eliminating TCR mispairing is to transduce TCR-αβ genes into γδ T cells. Using this system, T cells must either be transduced with CD8 or CD4 co-receptor independent TCRs, or TCRs and co-receptors must be co-transferred together. These TCR-transduced γδ T cells demonstrate peptide-specific lysis and cytokine release in vitro and also peptide-specific proliferation, persistence and recall responses in vivo.29–31 Achieving

T cells with a high functional avidity is one of the major Reverse transcriptase challenges in current TCR gene-therapy protocols. One means of attaining T cells capable of recognizing and effectively killing tumour cells is to confer the manipulated T cells with TCRs with a high affinity. As the majority of currently available tumour-associated antigens (TAAs) are self-antigens that are expressed at elevated levels in tumours, T cells expressing high-affinity TCRs to tumour antigens may be deleted in the thymus or rendered unresponsive by central or peripheral tolerance. As a result, TAA-specific T cells identifiable within the autologous repertoire are often of low frequency and low-to-moderate functional avidity.