Human TCR that incorporate CD3ζ induce highly preferred pairing between TCRα and β chains following gene transfer

TCR gene therapy is adversely affected by newly formed TCR alpha beta heterodimers comprising exogenous and endogenous TCR chains that dilute expression of transgenic TCR alpha beta dimers and are potentially self-reactive. We have addressed TCR mispairing by using a modified two-chain TCR that encompasses total human CD3 with specificities for three different Ags. Transfer of either TCR alpha:CD3 zeta or beta:CD3 zeta genes alone does not result in surface expression, whereas transfer of both modified TCR chains results in high surface expression, binding of peptide-MHC complexes and Ag-specific T cell functions. Genetic introduction of TCR alpha beta:CD3 zeta does not compromise surface expression and functions of an endogenous TCR alpha beta. Flow cytometry fluorescence resonance energy transfer and biochemical analyses demonstrate that TCR alpha beta:CD3 zeta is the first strategy that results in highly preferred pairing between CD3 zeta-modified TCR alpha and beta chains as well as absence of TCR mispairing between TCR:CD3 zeta and nonmodified TCR chains. Intracellular assembly and surface expression of TCR:CD3 zeta chains is independent of endogenous CD3 gamma, 8 delta, and epsilon. Taken together, our data support the use of TCR alpha beta:CD3 zeta to prevent TCR mispairing, which may provide an adequate strategy to enhance efficacy and safety of TCR gene transfer.