TY - JOUR
T1 - Improving the Pharmacodynamics and In Vivo Activity of ENPP1-Fc Through Protein and Glycosylation Engineering
AU - Stabach, Paul R.
AU - Zimmerman, Kristin
AU - Adame, Aaron
AU - Kavanagh, Dillon
AU - Saeui, Christopher T.
AU - Agatemor, Christian
AU - Gray, Shawn
AU - Cao, Wenxiang
AU - De La Cruz, Enrique M.
AU - Yarema, Kevin J.
AU - Braddock, Demetrios T.
N1 - Funding Information:
Funding from Inozyme Pharma and the National Institutes of Health through R01 DK121326 to D.T.B., and from the National Institutes of Health through R01 CA112314 and R21 CA249381 (awarded to K.J.Y.), and R35GM136656 (awarded to E.M.D.L.C.). Glycan analysis was supported in part by NIH grants 4P41GM103490-14 and P41GM10349010 to Parastoo Azadi. D.T.B. acknowledges helpful discussions with Steven Jungles (Inozyme), Denis Schrier (Inozyme), and Christopher Crean (xyzagen).
Funding Information:
Funding from Inozyme Pharma and the National Institutes of Health through R01 DK121326 to D.T.B., and from the National Institutes of Health through R01 CA112314 and R21 CA249381 (awarded to K.J.Y.), and R35GM136656 (awarded to E.M.D.L.C.). Glycan analysis was supported in part by NIH grants 4P41GM103490‐14 and P41GM10349010 to Parastoo Azadi.
Publisher Copyright:
© 2020 The Authors. Clinical and Translational Science published by Wiley Periodicals LLC on behalf of the American Society for Clinical Pharmacology and Therapeutics.
PY - 2021/1
Y1 - 2021/1
N2 - Enzyme replacement with ectonucleotide pyrophosphatase phospodiesterase-1 (ENPP1) eliminates mortality in a murine model of the lethal calcification disorder generalized arterial calcification of infancy. We used protein engineering, glycan optimization, and a novel biomanufacturing platform to enhance potency by using a three-prong strategy. First, we added new N-glycans to ENPP1; second, we optimized pH-dependent cellular recycling by protein engineering of the Fc neonatal receptor; finally, we used a two-step process to improve sialylation by first producing ENPP1-Fc in cells stably transfected with human α-2,6-sialyltransferase (ST6) and further enhanced terminal sialylation by supplementing production with 1,3,4-O-Bu3ManNAc. These steps sequentially increased the half-life of the parent compound in rodents from 37 hours to ~ 67 hours with an added N-glycan, to ~ 96 hours with optimized pH-dependent Fc recycling, to ~ 204 hours when the therapeutic was produced in ST6-overexpressing cells with 1,3,4-O-Bu3ManNAc supplementation. The alterations were demonstrated to increase drug potency by maintaining efficacious levels of plasma phosphoanhydride pyrophosphate in ENPP1-deficient mice when the optimized biologic was administered at a 10-fold lower mass dose less frequently than the parent compound—once every 10 days vs. 3 times a week. We believe these improvements represent a general strategy to rationally optimize protein therapeutics.
AB - Enzyme replacement with ectonucleotide pyrophosphatase phospodiesterase-1 (ENPP1) eliminates mortality in a murine model of the lethal calcification disorder generalized arterial calcification of infancy. We used protein engineering, glycan optimization, and a novel biomanufacturing platform to enhance potency by using a three-prong strategy. First, we added new N-glycans to ENPP1; second, we optimized pH-dependent cellular recycling by protein engineering of the Fc neonatal receptor; finally, we used a two-step process to improve sialylation by first producing ENPP1-Fc in cells stably transfected with human α-2,6-sialyltransferase (ST6) and further enhanced terminal sialylation by supplementing production with 1,3,4-O-Bu3ManNAc. These steps sequentially increased the half-life of the parent compound in rodents from 37 hours to ~ 67 hours with an added N-glycan, to ~ 96 hours with optimized pH-dependent Fc recycling, to ~ 204 hours when the therapeutic was produced in ST6-overexpressing cells with 1,3,4-O-Bu3ManNAc supplementation. The alterations were demonstrated to increase drug potency by maintaining efficacious levels of plasma phosphoanhydride pyrophosphate in ENPP1-deficient mice when the optimized biologic was administered at a 10-fold lower mass dose less frequently than the parent compound—once every 10 days vs. 3 times a week. We believe these improvements represent a general strategy to rationally optimize protein therapeutics.
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U2 - 10.1111/cts.12887
DO - 10.1111/cts.12887
M3 - Article
C2 - 33064927
AN - SCOPUS:85092748857
SN - 1752-8054
VL - 14
SP - 362
EP - 372
JO - Clinical and Translational Science
JF - Clinical and Translational Science
IS - 1
ER -