ENPP1 polypeptides and methods of using same
Inventors
Stabach, Paul • Braddock, Demetrios
Assignees
Publication Number
US-12344868-B2
Publication Date
2025-07-01
Expiration Date
2039-08-31
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Abstract
The present invention includes ENPP1 polypeptides with improved in vivo half-lives. The invention further provides an ENPP1 polypeptide fusion comprising an ENPP1 polypeptide fused to a Fc region of an immunoglobulin, wherein the polypeptide fusion comprises at least one point mutation.
Core Innovation
The invention provides ENPP1 polypeptides fused to a Fc region of an immunoglobulin, wherein the polypeptide fusion comprises at least one point mutation that improves in vivo half-lives. These mutations include additional N-linked glycosylation sites engineered onto the exterior surface of the ENPP1 tertiary structure to promote glycosylation and shield the polypeptide from degradation, as well as mutations in the Fc domain that enhance affinity for the neonatal Fc receptor (FcRn) to improve recycling and extend half-life. Moreover, expression of the polypeptide fusion in CHO cell lines stably transfected with human ST6 beta-galactosamide alpha-2,6-sialyltransferase (ST6GAL1) and/or growing cells in culture media supplemented with sialic acid and/or its high-flux precursor 1,3,4-O-Bu3ManNAc further enhances sialylation, leading to improved pharmacokinetic profiles.
The problem addressed by the invention arises from the need for ENPP1 polypeptides that can effectively treat diseases involving pathological calcification or ossification with in vivo half-lives suitable for convenient and effective dosing. Existing ENPP1-Fc fusion proteins have limitations in half-life and bioavailability, which restrict their therapeutic utility and dosing frequency.
The invention further provides methods of treatment using the engineered ENPP1-Fc polypeptide fusions and mutant polypeptides to reduce or prevent progression of pathological calcification, ossification, and related disorders by elevating and maintaining plasma pyrophosphate (PPi) levels at or near normal physiological concentrations. This therapeutic approach addresses diseases associated with ENPP1 deficiency and low plasma PPi levels, such as generalized arterial calcification of infancy (GACI), hypophosphatemic rickets, ossification of the posterior longitudinal ligament (OPLL), and others.
Claims Coverage
The patent contains one independent claim defining a fusion polypeptide encompassing at least one mutation in the ENPP1 portion and optionally mutations in the Fc region, covering multiple mutations that confer improved properties including enhanced glycosylation, FcRn binding, and extended half-life.
ENPP1 polypeptide fusion with specific ENPP1 mutations
An ENPP1 polypeptide fusion comprising at least one mutation selected from C25N, K27T, V29N, K369N, I371T, P534N, V536T, R545T, P554L, E592N, R741D, and S766N as relating to SEQ ID NO:7.
Fc region mutations enhancing half-life and recycling
The Fc region comprises at least one mutation selected from S885N, M883Y, M883Y/S885T/T887E, H1064K/N1065F, M883Y, S885T, T887E, H1064K, N1065F, E864N and L866T as relating to SEQ ID NO:7.
Combination mutations in ENPP1 polypeptide and Fc region
Polypeptides comprising combinations of ENPP1 mutations and Fc domain mutations, such as M883Y, S885T, T887E; P534N, V536T, M883Y, S885T, T887E; and E592N, H1064K, N1065F.
Expression and production conditions improving sialylation
ENPP1 polypeptide fusions expressed from CHO cell lines stably transfected with ST6GAL1 and/or grown in culture supplemented with sialic acid or N-acetylmannosamine (1,3,4-O-Bu3ManNAc) to enhance sialylation and improve pharmacokinetics.
Methods of treating diseases involving pathological calcification or ossification
Methods of treating or preventing progression of diseases such as ossification of the posterior longitudinal ligament (OPLL), hypophosphatemic rickets, chronic kidney disease, osteoarthritis, GACI, and calcification of atherosclerotic plaques by administering the polypeptide fusion.
The claims cover engineered ENPP1-Fc fusion polypeptides with defined mutations in the ENPP1 and Fc regions that improve enzymatic activity, glycosylation, FcRn binding, and in vivo half-life, expressed under modified cellular conditions, and methods of using these polypeptides to treat diseases characterized by pathological calcification and ossification.
Stated Advantages
Improved in vivo half-life of ENPP1-Fc polypeptides as compared to existing constructs.
Enhanced bioavailability and pharmacokinetic properties due to increased glycosylation and sialylation.
Increased affinity for the neonatal Fc receptor (FcRn) leading to improved pH-dependent recycling.
Ability to administer at lower doses and/or reduced frequency while maintaining efficacy.
Improved patient compliance and increased therapeutic efficacy due to less frequent dosing.
Documented Applications
Treatment or prevention of pathological calcification disorders, including generalized arterial calcification of infancy (GACI), idiopathic infantile arterial calcification (IIAC), calcification of atherosclerotic plaques, and calcific uremic arteriolopathy.
Treatment or prevention of pathological ossification disorders, such as ossification of the posterior longitudinal ligament (OPLL), hypophosphatemic rickets, osteoarthritis, and aging-related hardening of arteries.
Therapy for diseases caused by ENPP1 deficiency manifested by reduced extracellular pyrophosphate (PPi) concentration.
Therapeutic use for raising and maintaining normal plasma pyrophosphate (PPi) levels in subjects with lower than normal PPi.
Treatment of chronic kidney disease (CKD), end stage renal disease (ESRD), calciphylaxis, osteoarthritis, x-linked hypophosphatemic rickets (XLH), hereditary hypophosphatemic rickets (HHRH), hypophosphatemic bone disease (HBD), autosomal dominant hypophosphatemic rickets (ADHR), and autosomal recessive hypophosphatemic rickets.
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