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Engineering potent long-acting variants of the Wnt inhibitor DKK2.

ABSTRACT: Wnt signaling pathways are required for a wide variety of biological processes ranging from embryonic development to tissue repair and regeneration. Dickkopf-2 (DKK2) is classically defined as a canonical Wnt inhibitor, though it may play a role in activating non-canonical Wnt pathways in the context of endothelial network formation after acute injury. Here we report the discovery of a fusion partner for a DKK2 polypeptide that significantly improves the expression, biochemical properties and pharmacokinetics (PK) of the DKK2 polypeptide. Specifically, human serum albumin (HSA) was identified as a highly effective fusion partner. Substitution of selected amino acid residues in DKK2 designed to decrease heparan sulfate binding by HSA-DKK2 variants, further improved the PK properties of the molecule in rodents. The HSA-DKK2 variants were monomeric, as thermally stable as wild type, and active as measured by their ability to bind to and prevent phosphorylation of the Wnt coreceptor LRP6. Our engineering efforts resulted in potent long-lived variants of the canonical Wnt inhibitor DKK2, applicable for Wnt pathway manipulation either by systematic delivery or focused administration at sites of tissue injury.

SUBMITTER: Sopko R

PROVIDER: S-EPMC5425732 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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Engineering potent long-acting variants of the Wnt inhibitor DKK2.

Sopko Richelle R Mugford Joshua W JW Lehmann Andreas A Shapiro Renée I RI Rushe Mia M Kulkarni Abhishek A Worrall Joseph J Amatucci Joseph J Wen Dingyi D Pederson Nels E NE Minesinger Brenda K BK Arndt Joseph W JW Pepinsky Blake B

Protein engineering, design & selection : PEDS 20170501 5

Wnt signaling pathways are required for a wide variety of biological processes ranging from embryonic development to tissue repair and regeneration. Dickkopf-2 (DKK2) is classically defined as a canonical Wnt inhibitor, though it may play a role in activating non-canonical Wnt pathways in the context of endothelial network formation after acute injury. Here we report the discovery of a fusion partner for a DKK2 polypeptide that significantly improves the expression, biochemical properties and ph ...[more]

PMID: 28180900

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Project description:Modulating the complement system is a promising strategy in drug discovery for disorders with uncontrolled complement activation. Although some of these disorders can be effectively treated with an antibody that inhibits complement C5, the high plasma concentration of C5 requires a huge dosage and frequent intravenous administration. Moreover, a conventional anti-C5 antibody can cause C5 to accumulate in plasma by reducing C5 clearance when C5 forms an immune complex (IC) with the antibody, which can be salvaged from endosomal vesicles by neonatal Fc receptor (FcRn)-mediated recycling. In order to neutralize the increased C5, an even higher dosage of the antibody would be required. This antigen accumulation can be suppressed by giving the antibody a pH-dependent C5-binding property so that C5 is released from the antibody in the acidic endosome and then trafficked to the lysosome for degradation, while the C5-free antibody returns back to plasma. We recently demonstrated that a pH-dependent C5-binding antibody, SKY59, exhibited long-lasting neutralization of C5 in cynomolgus monkeys, showing potential for subcutaneous delivery or less frequent administration. Here we report the details of the antibody engineering involved in generating SKY59, from humanizing a rabbit antibody to improving the C5-binding property. Moreover, because the pH-dependent C5-binding antibodies that we first generated still accumulated C5, we hypothesized that the surface charges of the ICs partially contributed to a slow uptake rate of the C5-antibody ICs. This idea motivated us to engineer the surface charges of the antibody. Our surface-charge engineered antibody consequently exhibited a high capacity to sweep C5 and suppressed the C5 accumulation in vivo by accelerating the cycle of sweeping: uptake of ICs into cells, release of C5 from the antibody in endosomes, and salvage of the antigen-free antibody. Thus, our engineered anti-C5 antibody, SKY59, is expected to provide significant benefits for patients with complement-mediated disorders.

Dataset Information

Engineering potent long-acting variants of the Wnt inhibitor DKK2.

Publications

Engineering potent long-acting variants of the Wnt inhibitor DKK2.

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