Building an affinity panel of synthetic polyclonal T-cell receptor ligands that mimic native peptide-MHC

T cells accurately and sensitively discriminate between foreign- and self-peptide antigens presented in Major Histocompatibility Complexes (pMHC). T cells can achieve near single molecule sensitivity to agonist foreign peptides, even in a background of self-peptides that may outnumber agonists more than 1000:1. Each individual T cell receptor (TCR) discriminates its particular agonist ligands from all others based on the binding dwell time of the pMHC:TCR complex, with agonists binding for a few seconds or longer. Overall, even successful pMHC:TCR binding pairs tend to be rather weak binders. A native population of T cells from an organism consists of a polyclonal repertoire of millions of distinct TCRs, each with its own ligand specificity for foreign antigen. Thus from an investigative, or therapeutic perspective, it is not possible to activate TCR, in a polyclonal manner, through the same binding epitope as native pMHC. Additionally, native pMHC ligands do not activate T cells from solution unless cross linked, in which case they yield a different activation profile then native membrane-associated, monovalent pMHC. Our interest here is to construct a monovalent polyclonal TCR activator that exhibits similar activation behavior to pMHC (monomeric, inactive from solution, and single molecule active on membranes). We synthetically couple a monovalent anti-TCR OKT3 Fab’, which strongly binds human T cells, to a supported lipid bilayer (SLB) using short DNA handels. By purifying different OKT3 Fab’ ligands with varying binding affinities we develop a panel of synthetic polyclonal TCR ligands spanning a potency range from null/self to foreign/agonist peptides. This modular OKT3 Fab’-DNA ligand can bind and universally activate human T cells in polyclonal populations.