Enzymatically mediated engineering of multivalent MHC class II-peptide chimeras

We previously reported the genetic engineering of the first soluble, bivale nt major histocompatibility complex (MHC) class II-peptide ligand for T-cel l receptor (TCR). This ligand binds stably and specifically to cognate T-ce lls and exhibits immunomodulatory effects in vitro and in vivo. The increas e in valence of MHC class II-peptide ligands was shown to parallel their av idity for cognate TCRs and potency in stimulating cognate T-cells, We descr ibe a new enzymatic method to increase the valence of MHC-peptide ligands b y cross-linking the N-glycan moieties of dimeric MHC II-peptide units throu gh a flexible, bifunctional polyethylene glycol linker. Using this method, we generated covalently stabilized tetravalent and octavalent MHC II-peptid e ligands which bound stably and specifically to cognate TCR and preserved their structural integrity in blood and lymphoid organs for 72 h. Depending on the TCR/CD4 occupancy and degree of TCR/CD4 co-clustering, the multival ent MHC II-peptide ligands polarized efficiently the antigen-specific CD4() T-cells toward type 2 cell differentiation or induced T-cell anergy and a poptosis. The enzymatically mediated engineering of multivalent MHC-peptide ligands for cognate TCRs may provide rational grounds for the development of new therapeutic agents endowed with strong modulatory effects on antigen -specific T-cells.