Engineering potent long-acting variants of the Wnt inhibitor DKK2

Area of Science:

• Molecular Biology
• Biochemistry
• Regenerative Medicine

Background:

• Wnt signaling pathways are crucial for embryonic development, tissue repair, and regeneration.
• Dickkopf-2 (DKK2) is known as a canonical Wnt pathway inhibitor, but its role in non-canonical pathways, like endothelial network formation, is also recognized.

Purpose of the Study:

• To discover a fusion partner for DKK2 to enhance its expression, biochemical properties, and pharmacokinetics (PK).
• To engineer potent and long-lived variants of DKK2 for therapeutic Wnt pathway modulation.

Main Methods:

• Human serum albumin (HSA) was identified as a fusion partner for the DKK2 polypeptide.
• Amino acid substitutions were introduced into DKK2 to reduce heparan sulfate binding.
• Biochemical assays were used to assess the properties of the engineered HSA-DKK2 variants, including binding to LRP6 and inhibition of Wnt signaling.

Main Results:

• HSA proved to be a highly effective fusion partner for DKK2, significantly improving its expression and PK.
• Engineered HSA-DKK2 variants exhibited improved PK properties in rodents, particularly after modifications to decrease heparan sulfate binding.
• The resulting HSA-DKK2 variants were monomeric, thermally stable, and retained biological activity by inhibiting Wnt coreceptor LRP6 phosphorylation.

Conclusions:

• Fusion with HSA creates potent, long-lived variants of the Wnt inhibitor DKK2.
• These engineered DKK2 variants offer potential for Wnt pathway manipulation in tissue injury and regeneration contexts.
• The study highlights a successful protein engineering strategy to enhance the therapeutic potential of Wnt pathway modulators.