A Membrane-Activated, Universal T-Cell Receptor Agonist

T cells confer adaptive immunity by sensitively and specifically recognizing antigenic peptides presented on Major Histocompatibility Complex (pMHCs) molecules with their T-cell receptors (TCRs). Each developing T cell expresses a unique TCR that binds productively to only one or a few pMHCs. TCR diversity within an individual facilitates robust, specific immune responses, but complicates efforts to study T-cell activation because the pMHCs that activate T cells are generally unknown. Antibodies are able to universally activate T cells; however they activate T cells by crosslinking TCR and are active from solution, neither of which is true for natural antigen pMHC. When tethered to a membrane surface, but not from solution, pMHC monomers can activate T cells at the single molecule level without crosslinking. Antibodies are likely to effectively short-circuit the TCR signaling system, and thus may not represent natural antigen pMHC activation. We have designed a monovalent, membrane-tethered universal TCR agonist that can trigger early T-cell activation, introducing a synthetic TCR agonist that most closely mimics pMHC. This agonist, an anti-TCRβ Fab’ fragment, is coupled to a supported lipid bilayer via DNA complementation to mimic the physiological cell-cell interface. In our experimental platform, every Fab’-DNA:TCR complex is visualized and mapped to cellular readouts for signal propagation and activation. We demonstrate that Fab’-DNA triggers TCRs and stimulates translocation of the transcription factor Nuclear Factor for the Activation of T Cells (NFAT) with a similar potency to strongly activating pMHCs. Other dimensions of Fab’-DNA triggered T cells will also be discussed, including differences between distinct binding epitopes. Ultimately, a comprehensive characterization of multiple distinct Fab’-DNA constructs may help inform design of cancer immunotheraputics, such as bispecific antibodies, that are structurally similar to this synthetic ligand.