Human TCR that incorporate CD3zeta induce highly preferred pairing between TCRalpha and beta chains following gene transfer

TCR gene therapy is adversely affected by newly formed TCRalphabeta heterodimers comprising exogenous and endogenous TCR chains that dilute expression of transgenic TCRalphabeta dimers and are potentially self-reactive. We have addressed TCR mispairing by using a modified two-chain TCR that encompasses total human CD3zeta with specificities for three different Ags. Transfer of either TCRalpha:CD3zeta or beta:CD3zeta 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 TCRalphabeta:zeta does not compromise surface expression and functions of an endogenous TCRalphabeta. Flow cytometry fluorescence resonance energy transfer and biochemical analyses demonstrate that TCRalphabeta:CD3zeta is the first strategy that results in highly preferred pairing between CD3zeta-modified TCRalpha and beta chains as well as absence of TCR mispairing between TCR:CD3zeta and nonmodified TCR chains. Intracellular assembly and surface expression of TCR:CD3zeta chains is independent of endogenous CD3gamma, delta, and epsilon. Taken together, our data support the use of TCRalphabeta:CD3zeta to prevent TCR mispairing, which may provide an adequate strategy to enhance efficacy and safety of TCR gene transfer.