Engineered Antibody-Like Bivalent Binders for Manipulating Receptor Functions

Intracellular signal transduction mediated by most cell-surface single-pass transmembrane receptors depends on the formation of receptor dimers with defined geometries. Consequently, bivalent binders capable of positioning two receptor molecules in specific spatial configurations can serve as useful molecular tools for manipulating receptor activity. We previously developed Mirabodies, antibody-like bivalent binders generated by grafting peptide-derived receptor-binding sequences into loop regions of the human IgG1 Fc scaffold. This format combines the high efficiency of macrocyclic peptide selection in identifying high-affinity target-binding peptide sequences with the high producibility, structural rigidity, and favorable pharmacokinetic properties of Fc-based molecules. In this chapter, we describe practical protocols for the design, expression, and purification of Mirabodies using mammalian cell expression systems. These methods provide a straightforward workflow for converting receptor-binding peptides into functional Fc-based agonists or antagonists.