T cell receptor modification by alpha and beta chain domain cross-over for adoptive T cell therapy

Adoptive T cell transfer is a novel approach for cancer treatment in which patient’s T lymphocytes are extracted and genetically modified to redirect these against the cancer cell. However, there are still a number of risks and limitations to this approach. One is the formation of autoreactive T cell: The antigen-recognizing part of the T cell receptor is a dimer of an α- and a β-chain. Accordingly, transduced T cells can express ‘mispaired’ T cell receptors that comprise a native and an exogenous chain. These heterodimers can cause autoimmunity if they target self-antigens. The aim of this study was to investigate a novel approach against mispairing by transferring either the variable or the constant domain of one chain across to the other chain. This concept is already successfully employed in the design of bispecific antibodies to avoid mispairing and may be transferable due to a high degree of homology between T cell receptors and antibodies. The transduction of crossed receptors into murine T cells did not result in any surface expression. We observed an intracellular accumulation of α-chains. After a redesign eliminating an interference with the linker sequence, we found a temporary surface expression of α-chains in one of our constructs. However, when transducing only the single chain, we could not detect surface expression. We concluded that, while undetectable, the β-chain must be expressed when transducing with the bicistronic vector and that the crossed T cell receptor assembles correctly with the CD3 subunits. Finally, we aimed at stabilizing surface expression of the crossed receptor. We added an additional disulfide bond between both chains at different locations and changed the exact location of the domain cross-over in the so-called elbow region, which links the variable and the constant domain of each chain. Yet, a sustained surface expression of crossed T cell receptors could not be achieved. Alternative strategies are thus needed to prevent TCR mispairing.