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.
De novo macrocyclic peptides for inhibiting, stabilizing, and probing the function of the retromer endosomal trafficking complex