Description:
Background and ObjectivesWe recently demonstrated the efficacy of an enzyme replacement therapy for a lethal neonatal metabolic disorder of vascular calcification called ‘Generalized Arterial Calcification of Infancy’ (GACI). The therapeutic biologic (ENPP1‐Fc) is a recombinant fusion protein consisting of the extracellular domain of Ectonucleotide Pyrophosphatase Phospodiesterase‐1 (ENPP‐1) fused with the Fc domain of IgG1. The fusion protein exhibited exceptional stability and had an in vivo half‐life of 35 hours in rodents. The objective of this study was to investigate protein engineering and bioprocessing methods to further enhance the pharmacokinetic properties of this biotherapeutic enzyme.Approach and MethodsTo optimize the pharmacokinetic properties of ENPP1‐Fc we embarked on a 4‐prong strategy: First, we increased glycosylation to shield the protein from degradation by the introducing additional N‐glycan consensus sequences onto the exterior surface of the predicted tertiary structure, guided by three‐dimensional models of ENPP1, other ENPP enzymes in the superfamily, and known inactivating mutations in ENPP1 resulting in GACI. Second, we increased pH‐dependent FcRn‐mediated cellular recycling by mutating the Fc domain to enhance the affinity of the fusion protein for the neonatal receptor (FcRn). Third, we enhanced sialyation of the fusion protein by expressing ENPP1‐Fc in CHO cell lines stably transfected with human alpha‐2,6‐sialyltransferase. Finally, we enhanced sialic acid capping by supplementing the cell culture media with N‐acetylmannosamine or 1,3,4‐O‐Bu3ManNAc, a “high‐flux” precursor of sialic acid.ConclusionsUsing a combination of the above methods we collectively increased the in vivo half‐life of ENPP1‐Fc by over 4‐fold and the in vivo exposure of animal to ENPP1‐Fc (i.e., Area Under the Curve) by over 10‐fold. We additionally found that expressing ENPP1‐Fc in CHO cells stably transfected with sialytransferase dramatically increased the bioavailability (Cmax) of ENPP1‐Fc when the biologic was administered as a subcutaneous depot. Our results demonstrate the ability of protein engineering and bioprocessing methods to dramatically improve the pharmacokinetics and bioavailability of enzyme replacement biologics for use in humans.Support or Funding InformationInozyme PharmaENPP1‐FcimageENPP1‐FcThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.