Chemical Macrocyclization of Peptides Fused to Antibody Fc Fragments
ABSTRACT To extend the plasma half-life of a bicyclic peptide antagonist, we chose to link it to the Fc fragment of the long-lived serum protein IgG1. Instead of chemically conjugating the entire bicyclic peptide, we recombinantly expressed its peptide moiety as a fusion protein to an Fc fragment and subsequently cyclized the peptide by chemically reacting its three cysteine residues with tris-(bromomethyl)benzene. This reaction was efficient and selective, yielding completely modified peptide fusion protein and no side products. After optimization of the linker and the Fc fragment format, the bicyclic peptide was fully functional as an inhibitor (K(i) = 76 nM) and showed an extended terminal half-life of 1.5 days in mice. The unexpectedly clean reaction makes chemical macrocyclization of peptide-Fc fusion proteins an attractive synthetic approach. Its good compatibility with the Fc fragment may lend the bromomethylbenzene-based chemistry also for the generation of antibody-drug conjugates.
- SourceAvailable from: Joycelyn Wüstehube-Lausch[Show abstract] [Hide abstract]
ABSTRACT: Cystine-knot peptides sharing a common fold but displaying a notably large diversity within the primary structure of flanking loops have shown great potential as scaffolds for the development of therapeutic and diagnostic agents. In this study, we demonstrated that the cystine-knot peptide MCoTI-II, a trypsin inhibitor from Momordica cochinchinensis, can be engineered to bind to cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), an inhibitory receptor expressed by T lymphocytes, that has emerged as a target for the treatment of metastatic melanoma. Directed evolution was used to convert a cystine-knot trypsin inhibitor into a CTLA-4 binder by screening a library of variants using yeast surface display. A set of cystine-knot peptides possessing dissociation constants in the micromolar range was obtained; the most potent variant was synthesized chemically. Successive conjugation with neutravidin, fusion to antibody Fc domain or the oligomerization domain of C4b binding protein resulted in oligovalent variants that possessed enhanced (up to 400-fold) dissociation constants in the nanomolar range. Our data indicate that display of multiple knottin peptides on an oligomeric scaffold protein is a valid strategy to improve their functional affinity with ramifications for applications in diagnostics and therapy. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.Journal of Peptide Science 05/2015; DOI:10.1002/psc.2782 · 1.86 Impact Factor
- Chemical Reviews 02/2015; DOI:10.1021/cr500399p · 45.66 Impact Factor
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ABSTRACT: Many naturally occurring cyclic peptides or derivatives thereof are used as therapeutics such as the human hormones vasopressin and oxytocin or the antibiotics vancomycin and daptomycin. The success of cyclic peptide therapeutics is based on their ability to bind with high affinity, their good target selectivity and their low toxicity. As nature provides cyclic peptides to only a small number of disease targets, strategies have been developed to generate cyclic peptide ligands with tailored specificity de novo. Our laboratory is specialized on the directed evolution of bicyclic peptide ligands by phage display. In this article, we review our recent work to in vitro evolve bicyclic peptide antagonists, the binding and pharmacokinetic properties of bicyclic peptides, as well as efforts to generate bicyclic peptides for therapeutic application.CHIMIA International Journal for Chemistry 12/2013; 67(12):910-5. DOI:10.2533/chimia.2013.910 · 1.09 Impact Factor