title: Coiled coil peptides as universal linkers for the attachment of recombinant proteins to polymer therapeutics.
abstract: We have designed, synthesized, and characterized peptides containing four repeats of the sequences VAALEKE (peptide E) or VAALKEK (peptide K). While the peptides alone adopt in aqueous solutions a random coil conformation, their equimolar mixture forms heterodimeric coiled coils as confirmed by CD spectroscopy. 5-Azidopentanoic acid was connected to the N-terminus of peptide E via a short poly(ethylene glycol) spacer. The terminal azide group enabled conjugation of the peptide with a synthetic drug carrier based on the N-(2-hydroxypropyl)methacrylamide copolymer containing propargyl groups using "click" chemistry. When incorporated into the polymer drug carrier, peptide E formed a stable noncovalent complex with peptide K belonging to a recombinant single-chain fragment (scFv) of the M75 antibody. The complex thereby mediates a noncovalent linkage between the polymer drug carrier and the protein. The recombinant scFv antibody fragment was selected as a targeting ligand against carbonic anhydrase IX-a marker overexpressed by tumor cells of various human carcinomas. The antigen binding affinity of the polymer-scFv complex was confirmed by ELISA. This approach offers a well-defined, specific, and nondestructive universal method for the preparation of protein (antibody)-targeted polymer drug and gene carriers designed for cell-specific delivery.
authors.affiliation: Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovskeho nam 2, 162 06 Prague 6, Czech Republic.
authors.forename: Michal
authors.initials: M
authors.lastname: Pechar
authors.forename: Robert
authors.initials: R
authors.lastname: Pola
authors.forename: Richard
authors.initials: R
authors.lastname: Laga
authors.forename: Karel
authors.initials: K
authors.lastname: Ulbrich
authors.forename: Lucie
authors.initials: L
authors.lastname: Bednárová
authors.forename: Petr
authors.initials: P
authors.lastname: Maloň
authors.forename: Irena
authors.initials: I
authors.lastname: Sieglová
authors.forename: Vlastimil
authors.initials: V
authors.lastname: Král
authors.forename: Milan
authors.initials: M
authors.lastname: Fábry
authors.forename: Ondřej
authors.initials: O
authors.lastname: Vaněk
mesh_terms: D000178:Acrylamides / Q000737:chemistry*; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D000951:Antigens, Neoplasm / Q000276:immunology / Q000378:metabolism; D014408:Biomarkers, Tumor / Q000276:immunology / Q000378:metabolism; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases / Q000276:immunology / Q000378:metabolism; D002277:Carcinoma / Q000188:drug therapy / Q000201:enzymology / Q000276:immunology / Q000473:pathology; D002942:Circular Dichroism; D057930:Click Chemistry / Q000379:methods*; D003001:Cloning, Molecular; D019281:Dimerization; D004337:Drug Carriers / Q000138:chemical synthesis* / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli; D006801:Humans; D018796:Immunoconjugates / Q000737:chemistry* / Q000276:immunology / Q000494:pharmacology; D008968:Molecular Conformation; D009842:Oligopeptides / Q000138:chemical synthesis* / Q000276:immunology / Q000494:pharmacology; D010957:Plasmids; D011092:Polyethylene Glycols / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D014169:Transformation, Bacterial
chemical_list: D000178:Acrylamides; D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; D004337:Drug Carriers; D018796:Immunoconjugates; C475102:M75 monoclonal antibody; D009842:Oligopeptides; D011994:Recombinant Proteins; D011092:Polyethylene Glycols; C089226:CA9 protein, human; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases; C032976:N-(2-hydroxypropyl)methacrylamide