Coiled Coil Peptides as\nUniversal Linkers for the\nAttachment of Recombinant Proteins to Polymer Therapeutics

Abstract

We have designed, synthesized, and characterized peptides\ncontaining\nfour repeats of the sequences VAALEKE (peptide E) or VAALKEK (peptide\nK). While the peptides alone adopt in aqueous solutions a random coil\nconformation, their equimolar mixture forms heterodimeric coiled coils\nas confirmed by CD spectroscopy. 5-Azidopentanoic acid was connected\nto the N-terminus of peptide E via a short poly­(ethylene glycol) spacer.\nThe terminal azide group enabled conjugation of the peptide with a\nsynthetic drug carrier based on the <i>N</i>-(2-hydroxypropyl)­methacrylamide\ncopolymer containing propargyl groups using “click”\nchemistry. When incorporated into the polymer drug carrier, peptide\nE formed a stable noncovalent complex with peptide K belonging to\na recombinant single-chain fragment (scFv) of the M75 antibody. The\ncomplex thereby mediates a noncovalent linkage between the polymer\ndrug carrier and the protein. The recombinant scFv antibody fragment\nwas selected as a targeting ligand against carbonic anhydrase IXa\nmarker overexpressed by tumor cells of various human carcinomas. The\nantigen binding affinity of the polymer–scFv complex was confirmed\nby ELISA. This approach offers a well-defined, specific, and nondestructive\nuniversal method for the preparation of protein (antibody)-targeted\npolymer drug and gene carriers designed for cell-specific delivery.