Locally anchoring enzymes to tissues via extracellular glycan recognition

Shaheen A Farhadi,Dillon T Seroski,Antonietta Restuccia,Evelyn Bracho-Sanchez,Margaret M Fettis,Benjamin G Keselowsky,Gregory A Hudalla,Sabrina L Freeman

doi:10.1038/s41467-018-07129-6

Abstract

Success of enzymes as drugs requires that they persist within target tissues over therapeutically effective time frames. Here we report a general strategy to anchor enzymes at injection sites via fusion to galectin-3 (G3), a carbohydrate-binding protein. Fusing G3 to luciferase extended bioluminescence in subcutaneous tissue to ~7 days, whereas unmodified luciferase was undetectable within hours. Engineering G3-luciferase fusions to self-assemble into a trimeric architecture extended bioluminescence in subcutaneous tissue to 14 days, and intramuscularly to 3 days. The longer local half-life of the trimeric assembly was likely due to its higher carbohydrate-binding affinity compared to the monomeric fusion. G3 fusions and trimeric assemblies lacked extracellular signaling activity of wild-type G3 and did not accumulate in blood after subcutaneous injection, suggesting low potential for deleterious off-site effects. G3-mediated anchoring to common tissue glycans is expected to be broadly applicable for improving local pharmacokinetics of various existing and emerging enzyme drugs.

Highlights

Success of enzymes as drugs requires that they persist within target tissues over therapeutically effective time frames
Fusion to the N-terminus was chosen because the carbohydraterecognition domain (CRD) of G3 is encoded by the C-terminal portion of the protein, while the N-terminal domain (NTD) is an a Enzyme-G3 fusion protein Enzyme Linker Galectin-3
Hydrodynamic diameters, as determined via dynamic light scattering (DLS) number-weighted size distribution, were larger for each nanoassembly when compared to its respective monomeric fusion protein and increased as the molecular weights (MWs) of the enzyme increased (i.e., NanoLucTM luciferase (NL)-G3 < green fluorescent protein (GFP)-G3 < ChABCG3) (Fig. 2c)

Summary

Introduction

Success of enzymes as drugs requires that they persist within target tissues over therapeutically effective time frames. We report a general strategy to anchor enzymes at injection sites via fusion to galectin-3 (G3), a carbohydrate-binding protein. ~15% of all Food and Drug Administration (FDA)-approved proteins are enzymes used to treat various diseases, including lysosomal storage disorders[1], immunodeficiency[2], leukemia[3], hemophilia B4, and thrombosis[5] Despite these notable successes, many attractive enzyme drug candidates fail in clinical trials due to unfavorable pharmacokinetics, pharmacodynamics, and safety profiles. Strategies to improve enzyme pharmacokinetics, pharmacodynamics, and safety profiles by extending half-life, increasing target site accumulation, and minimizing immunogenicity hold promise for increasing the number of FDA-approved enzyme drugs. Fusion to the N-terminus was chosen because the carbohydraterecognition domain (CRD) of G3 is encoded by the C-terminal portion of the protein, while the N-terminal domain (NTD) is an a Enzyme-G3 fusion protein Enzyme Linker Galectin-3

Methods

Results

Conclusion