title: Self-assembled "dock and lock" system for linking payloads to targeting proteins.
abstract: Random conjugation of therapeutic or diagnostic payloads to targeting proteins generates functionally heterogeneous products. Conjugation of payloads to an adapter that binds to a peptide tag engineered into a targeting protein provides an alternative strategy. To progress into clinical development, an adapter/docking tag system should include humanized components and be stable in circulation. We describe here an adapter/docking tag system based on mutated fragments of human RNase I that spontaneously bind to each other and form a conjugate with a disulfide bond between complimentary cysteine residues. This self-assembled "dock and lock" system utilizes the previously described fusion C-tag, a 1-15 aa fragment of human RNase I with the R4C amino acid substitution, and a newly engineered adapter protein (Ad-C), a 21-127-aa fragment of human RNase I with the V118C substitution. Two vastly different C-tagged recombinant proteins, human vascular endothelial growth factor (VEGF) and a 254-aa long N-terminal fragment of anthrax lethal factor (LFn), retain functional activities after spontaneous conjugation of Ad-C to N-terminal or C-terminal C-tag, respectively. Ad-C modified with pegylated phospolipid and inserted into the lipid membrane of drug-loaded liposomes (Doxil) retained the ability to conjugate C-tagged proteins, yielding targeted liposomes decorated with functionally active proteins. To further optimize the system, we engineered an adapter with an additional cysteine residue at position 88 for site-specific modification, conjugated it to C-tagged VEGF, and labeled with a near-infrared fluorescent dye Cy5.5, yielding a unique functionally active probe for in vivo molecular imaging. We expect that this self-assembled "dock and lock" system will provide new opportunities for using functionally active proteins for biomedical purposes.
authors.affiliation: SibTech, Inc., Newington, Connecticut 06111, USA. mbacker@sibtech.com
authors.forename: Marina V
authors.initials: MV
authors.lastname: Backer
authors.forename: Vimal
authors.initials: V
authors.lastname: Patel
authors.forename: Brian T
authors.initials: BT
authors.lastname: Jehning
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Backer
mesh_terms: D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D051379:Mice; D008807:Mice, Inbred BALB C; D016297:Mutagenesis, Site-Directed; D011506:Proteins / Q000737:chemistry*
chemical_list: D017931:DNA Primers; D011506:Proteins