Lasso-grafting of macrocyclic peptide pharmacophores yields multi-functional proteins

Abstract

Engineering of multifunctional recombinant proteins are a promising approach for devising next-generation proteinous drugs that engage specific receptors on cell(s), but it often requires drastic modifications of the parental protein scaffolds, e.g., additional domains at the N/C-terminus (or termini) or replacement of a domain to another. A discovery platform system, called RaPID (Random non-standard Peptides Integrated Discovery) system, has enabled for a rapid discovery of small de novo macrocyclic peptides that bind a target protein with high binding specificity and affinity. Taking the advantage of such exquisite properties of the RaPID-derived peptides, here we show that their pharmacophore sequences can be implanted to a surface-exposed loop or loops of recombinant proteins and maintain not only the parental peptide binding function(s) but also the host protein function. By applying this method, referred to as lasso-grafting, many different proteins including IgG and serum albumin could be endowed with binding capability toward various receptors, allowing us to quickly formulate bi-, tri-, and even tetra-specific binder molecules. Moreover, lasso-grafting of a receptor-targeting peptide to capsid proteins of adeno-associated virus (AAV) has generated engineered AAV vectors that can infect cells solely dependent on the targeted receptor.