TCR CAR

TCR CAR (S. Wälchli/C. Forcados):

Effector T cells equipped with engineered antigen receptors specific for cancer targets have proven to be very efficient. Two methods have emerged: the Chimeric Antigen Receptors (CARs) and T-cell Receptor (TCR) redirection. Although very potent, CAR recognition is limited to membrane antigens which represent around 1% of the total proteins expressed, whereas TCRs have the advantage of targeting any peptide resulting from cellular protein degradation. However, TCRs depend on heavy signalling machinery only present in T cells which restricts the type of eligible therapeutic cells. Hence, an introduced therapeutic TCR will compete with the endogenous TCR for the signalling proteins and carries the potential risk of mixed dimer formation giving rise to a new TCR with unpredictable specificity. We have fused a soluble TCR construct to a CAR-signalling tail and named the final product TCR-CAR. In this project we prove that, if expressed, the TCR-CAR conserved the specificity and the functionality of the original TCR. In addition, we demonstrate that TCR-CAR redirection was not restricted to T cells. Indeed, after transduction, the NK cell line NK-92 became TCR positive and reacted against pMHC target. This opens therapeutic avenues combing the killing efficiency of NK cells with the diversified target recognition of TCRs.

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Design of the TCR-CAR constructs. (a) TCR-CAR gene design was based on the strategy previously used to produce soluble TCR (sTCR) in mammalian cells²⁴: TCRα and β chain were truncated at the level of their TM region, cysteines were added on their constant domains and the two chains were linked by a 2A peptide sequence. The artificial STOP codon of the TCRβ chain sTCR was replaced by the transmembrane (TM) domain of CD28 followed by a second generation CAR signalling tail composed of CD28 and CD3ζ signalling domains. The expected product of the TCR-CAR coding sequence should be two separated proteins released in the ER at equimolar amounts. (b) sTCR was produce as a soluble protein which, probably following the vesicular secretion pathway, was released in the cellular medium (left). TCR-CAR is expected to be exported to the cell surface as an TCRα/β heterodimer. Correct folding should ensure specific binding to a peptide-MHC (pMHC) complex and signal transduction through CD28-CD3 signalling tail (right).