Compositions for Treating Ectopic Calcification Disorders, and Methods Using Same

Abstract

The present invention includes compositions and methods for treating disease and disorders associated with pathological calcification or pathological ossification.

What is claimed is: 
     
         1 . A method of reducing or preventing progression of pathological calcification of soft tissue in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide comprising a soluble ecto-nucleotide pyrophosphate/phosphodiesterase-3 (ENPP3) polypeptide, whereby pathological calcification in the subject is reduced or progression of pathological calcification in the subject is prevented. 
     
     
         2 . The method of  claim 1 , wherein the polypeptide is administered locally, regionally, parenterally, or systemically to the subject. 
     
     
         3 . The method of  claim 1 , wherein the polypeptide is administered to the subject in a dosage ranging from about 1 ng/kg to about 500 mg/kg of body weight. 
     
     
         4 . The method of  claim 1 , wherein the polypeptide is administered daily, or more than once per day, or every other day, or weekly, or biweekly, or monthly, to the subject. 
     
     
         5 . The method of  claim 1 , wherein the polypeptide comprises soluble human ENPP3 comprising a C-terminal domain selected from the group consisting of a human IgG Fc domain and human serum albumin. 
     
     
         6 . The method of  claim 5 , wherein the human IgG is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. 
     
     
         7 . The method of  claim 5 , wherein the polypeptide is selected from the group consisting of SEQ ID NOs: 19, 21, and 22. 
     
     
         8 . The method of  claim 5 , wherein the extracellular domain of ENPP3 comprises amino acid residues of SEQ ID NO: 1. 
     
     
         9 . The method of  claim 5 , wherein the polypeptide is selected from the group consisting of SEQ ID NOs: 24, 25, and 26. 
     
     
         10 . The method of  claim 1 , wherein the pathological calcification of soft tissue is present in a disease selected from the group consisting of general arterial calcification of infancy (GACI), idiopathic infantile arterial calcification (IIAC), aging related hardening of arteries, progeria, pseudoxanthoma elasticum (PXE), medial wall vascular calcification (MWVC), end state renal disease (ESRD), chronic kidney disease-bone/mineral disorder (CKD-MBD), and calcific uremic arteriolopathy (CUA). 
     
     
         11 . A method of reducing or preventing progression of pathological ossification in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide comprising a soluble ecto-nucleotide pyrophosphate/phosphodiesterase-3 (ENPP3) polypeptide, whereby pathological ossification in the subject is reduced or progression of pathological ossification in the subject is prevented. 
     
     
         12 . The method of  claim 11 , wherein the pathological ossification is present in a disease selected from the group consisting of ossification of posterior longitudinal ligament (OPLL), autosomal recessive hypophosphatemia rickets type-2 (ARHR2), X-linked hypophosphatemia (XLH), age related osteopenia, and hypophosphatemic rickets. 
     
     
         13 . The method of  claim 11 , wherein the soluble ENPP3 polypeptide comprises a C-terminal domain selected from the group consisting of a human IgG Fc domain and human serum albumin. 
     
     
         14 . A method of treating a subject having ecto-nucleotide pyrophosphate/phosphodiesterase-1 (ENPP1) deficiency or ENPP1-associated disease, the method comprising administering to the subject a therapeutically effective amount of an isolated recombinant ecto-nucleotide pyrophosphate/phosphodiesterase-3 (ENPP3) polypeptide. 
     
     
         15 . The method of  claim 14 , wherein the ENPP1-associated disease is selected from the group consisting of general arterial calcification of infancy (GACI), idiopathic infantile arterial calcification (IIAC), aging related hardening of arteries, progeria, pseudoxanthoma elasticum (PXE), medial wall vascular calcification (MWVC), end state renal disease (ESRD), chronic kidney disease-bone/mineral disorder (CKD-MBD), calcific uremic arteriolopathy (CUA), ossification of posterior longitudinal ligament (OPLL), autosomal recessive hypophosphatemia rickets type-2 (ARHR2), X-linked hypophosphatemia (XLH), age related osteopenia, and hypophosphatemic rickets. 
     
     
         16 . A method of treating a subject having low plasma pyrophosphate (PPi) in the range of about 500 nM to 1 μM, the method comprising administering to the subject a therapeutically effective amount of an isolated recombinant ecto-nucleotide pyrophosphate/phosphodiesterase-3 (ENPP3) polypeptide, whereby the administering results in a rise in the level of plasma PPi in the subject to about 3 μM. 
     
     
         17 . The method of  claim 16 , whereby the level of plasma PPi in the subject rises to a level of about 4 μM. 
     
     
         18 . A method of producing soluble ENPP3 polypeptide fusion from cells comprising an ecto-nucleotide pyrophosphate/phosphodiesterase-3 (ENPP3) precursor polypeptide fusion, wherein the ENPP3 precursor polypeptide fusion comprises a signal peptide and a C terminal domain selected from the group consisting of a human IgG Fc domain and human serum albumin, the method comprising culturing the cells in medium to permit secretion from the cells into medium of a soluble ENPP3 polypeptide fusion comprising the human IgG Fc domain or human serum albumin domain, wherein the ENPP3 precursor polypeptide fusion is proteolytically processed upon secretion from the cells to yield the soluble ENPP3 polypeptide fusion. 
     
     
         19 . The method of  claim 18 , wherein the signal peptide is selected from the group consisting of: ecto-nucleotide pyrophosphate/phosphodiesterase-5 (ENPP5) signal sequence, ecto-nucleotide pyrophosphate/phosphodiesterase-7 (ENPP7) signal sequence, ENPP3 signal sequence, and signal sequence of the immunoglobulin kappa or lambda light chain protein. 
     
     
         20 . The method of  claim 18 , wherein the method further comprises the step of purifying the soluble ENPP3 polypeptide by chromatography to greater than 99% purity. 
     
     
         21 . The method of  claim 20 , wherein the soluble ENPP3 polypeptide has a K cat  value of about 2.59 (±0.04) s −1  enzyme −1 . 
     
     
         22 . An isolated cell comprising ecto-nucleotide pyrophosphate/phosphodiesterase-3 (ENPP3) precursor polypeptide fusion, wherein the ENPP3 precursor polypeptide fusion comprises a signal peptide and a C terminal domain selected from the group consisting of a human IgG Fc domain and albumin, wherein the ENPP3 precursor polypeptide fusion protein is proteolytically processed upon secretion from the cell to yield a soluble ENPP3 polypeptide fusion comprising the human IgG Fc domain or albumin domain. 
     
     
         23 . The isolated cell of  claim 22 , wherein the signal peptide is selected from the group consisting of: ecto-nucleotide pyrophosphate/phosphodiesterase-5 (ENPP5) signal sequence, ecto-nucleotide pyrophosphate/phosphodiesterase-7 (ENPP7) signal sequence, ENPP3 signal sequence, and signal sequence of the immunoglobulin kappa or lambda light chain protein. 
     
     
         24 . An isolated polypeptide comprising ectonucleotide pyrophosphate/phosphodiesterase-3 (ENPP3) extracellular domain and a C terminal domain selected from the group consisting of a human IgG Fc domain and human serum albumin. 
     
     
         25 . The isolated polypeptide of  claim 24 , wherein the human IgG is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. 
     
     
         26 . The isolated polypeptide of  claim 24 , wherein the polypeptide is selected from the group consisting of SEQ ID NOs: 19, 21, and 22. 
     
     
         27 . The isolated polypeptide of  claim 24 , wherein the extracellular domain of ENPP3 comprises amino acid residues of SEQ ID NO: 1. 
     
     
         28 . The isolated polypeptide of  claim 24 , wherein the polypeptide is selected from the group consisting of SEQ ID NOs: 24, 25, and 26.