Compositions and methods for treating pathological calcification and ossification

Abstract

To provide methods for treating disease and disorders associated with pathological calcification or pathological ossification.SOLUTION: The method comprises administering a composition comprising at least one agent selected from the group consisting of Ecto-Nucleotide pyrophosphate / Phosphodiesterase-1 (NPP1) polypeptide and a fragment, derivative, mutant or mutant fragment thereof, and an activator of NPP1 polypeptide and a fragment, mutant or mutant fragment thereof. Preferably, the NPP1 polypeptide or a fragment, mutant or mutant fragment thereof is a soluble recombinant NPP1 polypeptide or a fragment, mutant or mutant fragment thereof.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 61 / 904,786, filed on 15 November 2013, and U.S. Provisional Patent Application No. 61 / 764,297, filed on 13 February 2013, under Section 119(e) of the U.S. Patent Act, all of which are incorporated herein by reference as a whole. [Background technology]

[0002] Background of the Invention Ectopic tissue mineralization is associated with many human diseases, including chronic joint diseases and acute fatal neonatal syndromes. To prevent unwanted tissue calcification, a tight balance of factors that promote and inhibit tissue mineralization must be maintained. Genetic analysis of human and animal models of diseases associated with ectopic calcification has identified the balance of extracellular inorganic pyrophosphates (PPi) and inorganic phosphates (Pi) as important regulators of ectopic tissue mineralization (Terkeltaub, 2001, Am. J. Phys. Cell Phys., 281:C1-C11 (Non-patent Literature 1)). The concentrations of Pi and PPi in mammals are tightly regulated to 1–3 mM and 2–3 μM, respectively, by the action of three extracellular enzymes: tissue-nonspecific alkaline phosphatase (TNAP), progressive rigidus protein (ANK), and ectonucleotide pyrophosphatase / phosphodiesterase-1 (NPP1). PPi is a regulator of biomineralization and inhibits the formation of basic calcium phosphate from amorphous calcium phosphate.

[0003] Ectopic calcification disorders range from extremely rare and fatal diseases in infants to common age-related illnesses in a large proportion of the human population. Infant systemic arterial calcification (GACI), also known as IIAC, is a rare and fatal form of ectopic calcification present in a very small cohort of unrelated individuals (approximately 200 reported cases), characterized by calcification of the internal elastic membrane of muscular arteries and stenosis due to myointimal proliferation. While the clinical presentation of these patients varies, the disease typically leads to death in the neonatal period, usually by 6 months of age. OPLL is a common form of human myelopathy caused by compression of the spinal cord due to ectopic ossification of the spinal ligaments (Stapleton et al., 2011. Neurosurgical Focus 30:E6 (Non-patent Literature 2)). This disorder most frequently occurs in the cervical spine and was first described in the Japanese population, with a prevalence of 1.9–4.3% of the overall population. While disease presentations fluctuate, several genes and proteins have emerged as promising targets for etiological investigations over the past few years.

[0004] The human NPP family consists of seven extracellular glycosylated proteins (i.e., NPP1, NPP2, NPP3, NPP4, NPP5, NPP6, and NPP7) that hydrolyze phosphodiester bonds (Bollen et al., 2000, Crit. Rev. Biochem. Mol. Biol. 35:393-432 (Non-Patent Literature 3); Stefan et al., 2005, Trends Biochem. Sci. 30:542-550 (Non-Patent Literature 4); Goding et al., 2003, Biochim. Biophys. Acta 1638:1-19 (Non-Patent Literature 5)). These enzymes are numbered in the order in which they were discovered. NPPs are cell surface enzymes, with the exception of NPP2, which is exported to the cell membrane but is cleaved by furin and released into the extracellular fluid (Jansen et al., 2005, J. Cell Sci. 118:3081-3089 (Non-Patent Literature 6)). These enzymes exhibit a high degree of homology in terms of sequence and structure, but show diverse substrate specificity, ranging from nucleotides to lipids.

[0005] NPP1 (also known as PC-1) is a type 2 extracellular membrane-bound glycoprotein located in mineral deposition matrix vesicles of osteoblasts and chondrocytes, and hydrolyzes extracellular nucleotides (mainly ATP) into AMP and PPi (Bollen et al., 2000, Crit. Rev. Biochem. Mol. Biol. 35:393-432 (Non-Patent Literature 3); Terkeltaub, 2006, Purinergic signaling 2:371-377 (Non-Patent Literature 7)). PPi functions as a potent inhibitor of ectopic tissue mineralization by binding to newly formed hydroxyapatite (HA) crystals, thereby preventing the future growth of these crystals (Terkeltaub, 2006, Purinergic signaling 2:371-377 (Non-Patent Literature 7); Addison et al., 2007, J. Biol. Chem. 282:15872-15873 (Non-Patent Literature 8)). NPP1 generates PPi by hydrolysis of nucleotide triphosphates (NTPs), ANK transports intracellular PPi to the extracellular space, and TNAP removes PPi by direct hydrolysis of PPi to Pi (Figure 1).

[0006] NPP2 is a lysophospholipase-D enzyme that produces lysophosphatidic acid (LPA) from lysophosphocholine (Umezu-Goto et al., 2002, J. Cell Biol. 158:227-233 (Non-patent Literature 9)). NPP4 has recently been shown to be a diadenosine triphosphate (Ap3A) hydrolase and a potent procoagulant on the surface of blood vessels (Albright et al., 2012, Blood 120:4432-4440 (Non-patent Literature 10)). NPP5 remains uncharacterized, while both NPP6 and NPP7 hydrolyze lipid substrates; NPP6 is a lysophospholipase-C enzyme, and NPP7 is an alkaline sphingomyelinase (Duan et al., 2003, J. Biol. Chem. 278:38528-36 (Non-patent Literature 11); Sakagami et al., 2005, J. Biol. Chem. 23084-93 (Non-patent Literature 12)).

[0007] The lack of substantial production and purification of biologically active NPP proteins in this membrane-bound protein family has hindered their study and characterization. Expression systems for soluble NPP4 and NPP1 have not been demonstrated for large-scale protein production and purification. No mutations altering the enzyme's Ap3A-to-ATP activity in NPP4 have been reported.

[0008] Extracellular nucleotides, by binding to purine receptors on the cell surface, are involved in paracrine and autocrine cell signaling, resulting in a wide range of physiological responses, including platelet aggregation (Offermanns, 2006, Circ. Res. 99:1293-1304 (Non-Patent Literature 13)), bone development and reformation (Terkeltaub, 2006, Purinergic Signalling 2:371-377 (Non-Patent Literature 6)), and endocrine disorders such as diabetes and obesity (Omatsu-Kanbe et al., 2002, Exper. Physiol. 87:643-652 (Non-Patent Literature 14); Schodel et al., 2004, Biochem. Biophys. Res. Comm. 321:767-773 (Non-Patent Literature 15)). P2X purine receptors on the cell surface are ion channels that primarily bind to ATP, while P2Y receptors are cell surface G protein-coupled receptors that interact with a wider range of nucleotides. The concentration of extracellular purine substrates that drive purinergic signaling is determined by the release of ectonucleotides by degranulation or cell lysis, the rate of ectonucleotide synthesis, and the metabolism of ectonucleotides by exocenes.

[0009] Platelet aggregation involves extracellular ADP and platelet P2Y1 and P2Y1. 12Induced by interaction with purinergic receptors, it leads to rapid calcium influx, followed by further platelet activation, degranulation, and irreversible shape change, expanding the growing thrombus. Through the metabolism of extracellular ADP by membrane-bound CD39 on vascular endothelial cells and soluble phosphohydrolases in the platelet microenvironment, ADP is rapidly degraded to AMP and Pi, and the expansion of the ADP aggregation burst is restricted to platelets in the immediate vicinity of activated degranulating platelets. AMP is further metabolized by membrane-bound CD73 to adenosine, a potent antithrombotic signaling molecule that modulates vasoconstriction, reduces leukocyte adhesion, and limits thrombus formation. The release of platelet dense core granules ejects high concentrations of ADP into the thrombus microenvironment, further stimulating platelet aggregation.

[0010] Platelet dense core granules also contain high concentrations of the dinucleotide Ap3A, which can reach local concentrations exceeding 100 μM in the presence of platelet degranulation. Although the role of Ap3A in hemostasis has not been fully defined, Ap3A has long been considered to correspond to a more stable "chemically masked" ADP that can be released into the thrombus microenvironment to sustain platelet aggregation. Ap3A hydrolytic activity has been identified on the vascular surface of endothelial cells from both bovine and porcine sources.

[0011] There is a need in the art for novel compositions and methods for treating diseases and disorders associated with pathologic calcification and / or pathologic ossification. Such compositions and methods should not unduly interfere with other physiological processes. The present invention meets this need.

Prior Art Documents

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[0013] Brief summary of the invention In one aspect, the present invention includes a method for treating or preventing a disease or disorder associated with pathological calcification or pathological ossification in a subject requiring such treatment, the method comprising administering to the subject a therapeutically effective amount of a composition comprising at least one active substance selected from the group consisting of ectonucleotide pyrophosphate / phosphodiesterase-1 (NPP1) polypeptide and its fragments, derivatives, variants, or variant fragments, and activators of NPP1 polypeptide and its fragments, variants, or variant fragments, thereby treating or preventing the disease or disorder in the subject.

[0014] In another aspect, the present invention includes a method for treating a disease or disorder associated with pathological calcification or pathological ossification in a subject requiring such treatment, the method comprising administering to the subject a therapeutically effective amount of a composition comprising at least one active substance selected from the group consisting of ectonucleotide pyrophosphate / phosphodiesterase-4 (NPP4) polypeptide and its fragments, derivatives, variants, or variant fragments, and activators of NPP4 polypeptide or its fragments or variants.

[0015] In yet another aspect, the present invention includes a composition comprising at least one active substance selected from the group consisting of ectonucleotide pyrophosphate / phosphodiesterase-1 (NPP1) polypeptide, NPP1 polypeptide fragment, NPP1 polypeptide derivative, mutant NPP1 polypeptide, and mutant NPP1 polypeptide fragment.

[0016] In yet another aspect, the present invention includes a composition comprising at least one active substance selected from the group consisting of ectonucleotide pyrophosphate / phosphodiesterase-4 (NPP4) polypeptide, NPP4 polypeptide fragment, NPP4 polypeptide derivative, mutant NPP4 polypeptide, and mutant NPP4 polypeptide fragment.

[0017] In various aspects of any of the above aspects or any other aspects of the present invention described herein, the NPP1 polypeptide or its fragments, variants, or variant fragments include soluble recombinant NPP1 polypeptide or its fragments, variants, or variant fragments. In certain aspects of the aspects listed herein, the NPP1 polypeptide or its fragments, variants, or variant fragments lack the NPP1 transmembrane domain. In other aspects of the aspects listed herein, the NPP1 polypeptide or its fragments, variants, or variant fragments include the IgG Fc domain. In yet another aspect of the aspects listed herein, the mutant NPP1 polypeptide or its fragments has lower Ap3A hydrolysis activity compared to the corresponding wild-type NPP1 polypeptide or its fragments. In yet another aspect of the aspects listed herein, the mutant NPP1 polypeptide or its fragments has substantially the same ATP hydrolysis activity compared to the corresponding wild-type NPP1 polypeptide or its fragments. In yet another aspect of the aspects listed herein, the mutant NPP1 polypeptide or its fragments has lower Ap3A hydrolysis activity and substantially the same ATP hydrolysis activity compared to the corresponding wild-type NPP1 polypeptide or its fragments. In yet another embodiment of the aspects described herein, the mutant NPP1 polypeptide or fragment thereof has a mutation at at least one position selected from the group consisting of Ser 532, Tyr 529, Tyr 451, Ile 450, Ser 381, Tyr 382, ​​Ser 377, Phe 346, Gly 531, Ser 289, Ser 287, Ala 454, Gly 452, Gln 519, Glu 526, Lys 448, Glu 508, Arg 456, Asp 276, Tyr 434, Gln 519, Ser 525, Gly 342, Ser 343, and Gly 536, compared to SEQ ID NO: 1.

[0018] In various embodiments of any of the above aspects or any other aspects of the present invention described herein, the NPP1 polypeptide or its fragment, variant, or variant fragment comprises a polyaspartate domain. In certain embodiments of the aspects cited herein, the polyaspartate domain comprises about 2 to about 20 or more consecutive aspartate residues. In other embodiments of the aspects cited herein, the NPP1 polypeptide or its fragment, variant, or variant fragment comprises an NPP2 transmembrane domain. In yet another embodiment of the aspects cited herein, the activator of NPP1 activates at least one selected from the group consisting of the expression of the NPP1 polypeptide or its fragment, variant, or variant fragment, and the activity of the NPP1 polypeptide or its fragment, variant, or variant fragment. In yet another embodiment of the aspects cited herein, the activator of NPP1 is at least one selected from the group consisting of chemical compounds, proteins, peptides, peptide mimes, and small molecule chemical compounds.

[0019] In certain embodiments of the aspects described herein, at least one active agent is administered acutely or chronically. In other embodiments of the aspects described herein, at least one active agent is administered locally, locally, or systemically. In yet another embodiment of the aspects described herein, the subject is human. In yet another embodiment of the aspects described herein, the disease or disorder is at least one selected from the group consisting of idiopathic infantile arterial calcification (IIAC), ossification of the posterior longitudinal ligament (OPLL), hypophosphatemic rickets, osteoarthritis, and calcification of atherosclerotic plaques.

[0020] In any of the above aspects or in various aspects of the invention described herein, the NPP4 polypeptide or its fragment, variant, or variant fragment is a soluble recombinant NPP4 polypeptide or its fragment, variant, or variant fragment. In a particular aspect of the aspects cited herein, the mutant NPP4 polypeptide or its fragment comprises at least one mutation selected from the group consisting of D335, S92, D264, L265, S330, Q331, K332, and T323, compared to SEQ ID NO: 3. In another aspect of the aspects cited herein, the mutant NPP4 polypeptide or its fragment comprises at least one mutation that increases the ATP hydrolysis activity of the mutant NPP4 polypeptide or its fragment compared to the corresponding wild-type NPP4 or its fragment. In yet another aspect of the aspects cited herein, the mutant NPP4 polypeptide or its fragment comprises at least one mutation that increases the NPP1-like hydrolysis activity of the mutant NPP4 polypeptide or its fragment compared to the corresponding wild-type NPP4 or its fragment. In yet another embodiment of the aspects cited herein, a mutant NPP4 polypeptide or fragment thereof comprises at least one mutation that increases the substrate selectivity of the mutant NPP4 polypeptide for ATP compared to the corresponding wild-type NPP4 or fragment thereof. In yet another embodiment of the aspects cited herein, a mutant NPP4 polypeptide or fragment thereof comprises at least one mutation that decreases the Ap3A hydrolysis activity of the enzyme compared to the corresponding wild-type NPP4 or fragment thereof. In yet another embodiment of the aspects cited herein, a mutant NPP4 polypeptide or fragment thereof comprises at least one mutation that substantially increases the ATP hydrolysis activity of the enzyme and decreases the Ap3A hydrolysis activity of the enzyme compared to the corresponding wild-type NPP4 or fragment thereof. In yet another embodiment of the aspects cited herein, the NPP4 polypeptide or fragment thereof, a variant, or a variant fragment lacks the NPP4 transmembrane domain.In further embodiments of the aspects described herein, the NPP4 polypeptide or a fragment, variant, or variant fragment thereof comprises an IgG Fc domain.

[0021] In various embodiments of any of the above aspects or any other aspects of the present invention described herein, the NPP4 polypeptide or its fragment, variant, or variant fragment comprises a polyaspartate domain. In certain embodiments of the aspects cited herein, the polyaspartate domain comprises about 2 to about 20 or more consecutive aspartate residues. In other embodiments of the aspects cited herein, the activator of NPP4 activates at least one selected from the group consisting of the expression of the NPP4 polypeptide or its fragment, variant, or variant fragment, and the activity of the NPP4 polypeptide or its fragment, variant, or variant fragment. In yet another embodiment of the aspects cited herein, the activator of NPP4 is at least one selected from the group consisting of chemical compounds, proteins, peptides, peptide mimes, and small molecule chemical compounds. [Invention 1001] A method for treating or preventing a disease or disorder associated with pathological calcification or pathological ossification in a subject requiring such treatment, comprising the step of administering to the subject a therapeutically effective amount of a composition comprising at least one active substance selected from the group consisting of ectonucleotide pyrophosphate / phosphodiesterase-1 (NPP1) polypeptide and its fragments, derivatives, variants, or variant fragments, and activators of NPP1 polypeptide and its fragments, variants, or variant fragments, thereby treating or preventing the disease or disorder in the subject. [Invention 1002] The method of the present invention 1001, wherein the NPP1 polypeptide or a fragment, variant, or variant fragment comprises a soluble recombinant NPP1 polypeptide or a fragment, variant, or variant fragment. [Invention 1003] The method of the present invention 1001, wherein the NPP1 polypeptide or a fragment, variant, or variant fragment lacks the NPP1 transmembrane domain. [Invention 1004] The method of the present invention 1003, wherein the NPP1 polypeptide or a fragment, variant, or variant fragment thereof contains an IgG Fc domain. [Invention 1005] The method of the present invention 1001, wherein the mutant NPP1 polypeptide or a fragment thereof has lower Ap3A hydrolysis activity compared to the corresponding wild-type NPP1 polypeptide or a fragment thereof. [Invention 1006] The method of the present invention 1001, wherein the mutant NPP1 polypeptide or a fragment thereof has substantially the same ATP hydrolysis activity as the corresponding wild-type NPP1 polypeptide or a fragment thereof. [Invention 1007] The method of the present invention 1001, wherein the mutant NPP1 polypeptide or fragment thereof has lower Ap3A hydrolysis activity and substantially the same ATP hydrolysis activity compared to the corresponding wild-type NPP1 polypeptide or fragment thereof. [Invention 1008] The method of the present invention 1001, wherein the mutated NPP1 polypeptide or fragment thereof has a mutation at at least one position selected from the group consisting of Ser 532, Tyr 529, Tyr 451, Ile 450, Ser 381, Tyr 382, ​​Ser 377, Phe 346, Gly 531, Ser 289, Ser 287, Ala 454, Gly 452, Gln 519, Glu 526, Lys 448, Glu 508, Arg 456, Asp 276, Tyr 434, Gln 519, Ser 525, Gly 342, Ser 343, and Gly 536, compared to SEQ ID NO: 1. [Invention 1009] The method of the present invention 1001, wherein the NPP1 polypeptide or a fragment, variant, or variant fragment thereof contains a polyaspartate domain. [Invention 1010] The method of the present invention 1009, wherein the polyaspartate domain contains approximately 2 to approximately 20 or more consecutive aspartate residues. [Invention 1011] The method of the present invention 1001, wherein the NPP1 polypeptide or a fragment, variant, or variant fragment thereof contains the NPP2 transmembrane domain. [Invention 1012] The method of the present invention 1001, wherein at least one of the active substances is administered acutely or chronically. [Invention 1013] The method of the present invention 1001, wherein at least one of the active substances is administered locally, locally, or systemically. [Invention 1014] The method of the present invention 1001, wherein the activator of NPP1 activates at least one selected from the group consisting of the expression of the NPP1 polypeptide or a fragment, variant, or variant fragment thereof, and the activity of the NPP1 polypeptide or a fragment, variant, or variant fragment thereof. [Invention 1015] The method of the present invention 1014, wherein the activator of NPP1 is at least one selected from the group consisting of chemical compounds, proteins, peptides, peptide mimes, and small molecule chemical compounds. [Invention 1016] The method of the present invention 1001, wherein the subject is a human. [Invention 1017] The method of the present invention 1001, wherein the disease or disorder is at least one selected from the group consisting of idiopathic infantile arterial calcification (IIAC), ossification of the posterior longitudinal ligament (OPLL), hypophosphatemic rickets, osteoarthritis, and calcification of atherosclerotic plaques. [Invention 1018] A method for treating a disease or disorder associated with pathological calcification or pathological ossification in a subject requiring such treatment, comprising the step of administering to the subject a therapeutically effective amount of a composition containing at least one active substance selected from the group consisting of ectonucleotide pyrophosphate / phosphodiesterase-4 (NPP4) polypeptide and its fragments, derivatives, variants, or variant fragments, and activators of NPP4 polypeptide or its fragments or variants. [Invention 1019] The method of the present invention 1018, wherein the NPP4 polypeptide or its fragment, variant, or variant fragment is a soluble recombinant NPP4 polypeptide or its fragment, variant, or variant fragment. [Invention 1020] The method of the present invention 1018, wherein the mutated NPP4 polypeptide or a fragment thereof contains at least one mutation selected from the group consisting of D335, S92, D264, L265, S330, Q331, K332, and T323, compared to SEQ ID NO: 3. [Invention 1021] The method of the present invention 1018, wherein the mutant NPP4 polypeptide or fragment thereof contains at least one mutation that increases the ATP hydrolysis activity of the mutant NPP4 polypeptide or fragment thereof compared to the corresponding wild-type NPP4 polypeptide or fragment thereof. [Invention 1022] The method of the present invention 1018, wherein the mutant NPP4 polypeptide or fragment thereof contains at least one mutation that increases the NPP1-like hydrolytic activity of the mutant NPP4 polypeptide or fragment thereof compared to the corresponding wild-type NPP4 polypeptide or fragment thereof. [Invention 1023] The method of the present invention 1018, wherein the mutant NPP4 polypeptide or fragment thereof comprises at least one mutation that increases the substrate selectivity or hydrolytic activity of the mutant NPP4 polypeptide or fragment thereof toward ATP compared with the corresponding wild-type NPP4 polypeptide or fragment thereof. [Invention 1024] The method of the present invention 1018, wherein the mutant NPP4 polypeptide or fragment thereof comprises at least one mutation that reduces the Ap3A hydrolysis activity of the mutant NPP4 polypeptide or fragment thereof compared to the corresponding wild-type NPP4 polypeptide or fragment thereof. [Invention 1025] The method of the present invention 1018, wherein the mutant NPP4 polypeptide or fragment comprises at least one mutation that reduces the Ap3A hydrolysis activity of the mutant NPP4 polypeptide or fragment and substantially increases the ATP hydrolysis activity of the mutant NPP4 polypeptide or fragment compared to the corresponding wild-type NPP4 polypeptide or fragment. [Invention 1026] The method of the present invention 1018, wherein the NPP4 polypeptide or a fragment, variant, or variant fragment lacks the NPP4 transmembrane domain. [Invention 1027] The method of the present invention 1018, wherein the NPP4 polypeptide or a fragment, variant, or variant fragment thereof contains an IgG Fc domain. [Invention 1028] The method of the present invention 1018, wherein the NPP4 polypeptide or a fragment, variant, or variant fragment thereof contains a polyaspartate domain. [Invention 1029] The method of the present invention 1028, wherein the polyaspartate domain contains approximately 2 to approximately 20 or more consecutive aspartate residues. [Invention 1030] The method of the present invention 1018, wherein at least one of the active substances is administered acutely or chronically. [Invention 1031] The method of the present invention 1018, wherein at least one of the active substances is administered locally, locally, or systemically. [Invention 1032] The method of the present invention 1018, wherein the activator of NPP4 activates at least one selected from the group consisting of the expression of the NPP4 polypeptide or a fragment, variant, or variant fragment thereof, and the activity of the NPP4 polypeptide or a fragment, variant, or variant fragment thereof. [Invention 1033] The method of the present invention 1032, wherein the activator of NPP4 is at least one selected from the group consisting of chemical compounds, proteins, peptides, peptide mimes, and small molecule chemical compounds. [Invention 1034] The method of the present invention 1018, wherein the subject is a human. [Invention 1035] The method of the present invention 1018, wherein the disease or disorder is at least one selected from the group consisting of idiopathic infantile arterial calcification (IIAC), ossification of the posterior longitudinal ligament (OPLL), hypophosphatemic rickets, osteoarthritis, and calcification of atherosclerotic plaques. [Invention 1036] A composition comprising at least one active substance selected from the group consisting of ectonucleotide pyrophosphate / phosphodiesterase-1 (NPP1) polypeptide, NPP1 polypeptide fragment, NPP1 polypeptide derivative, mutant NPP1 polypeptide, and mutant NPP1 polypeptide fragment. [Invention 1037] The composition of the present invention 1036, wherein the NPP1 polypeptide or a fragment, variant, or variant fragment comprises a soluble recombinant NPP1 polypeptide or a fragment, variant, or variant fragment. [Invention 1038] The composition of the present invention 1036, wherein the NPP1 polypeptide or a fragment, variant, or variant fragment lacks the NPP1 transmembrane domain. [Invention 1039] The composition of the present invention 1036, wherein the NPP1 polypeptide or a fragment, variant, or variant fragment thereof contains an IgG Fc domain. [Invention 1040] The composition of the present invention 1036, wherein the mutant NPP1 polypeptide or a fragment thereof has lower Ap3A hydrolysis activity compared to the corresponding wild-type NPP1 polypeptide or a fragment thereof. [Invention 1041] The composition of the present invention 1036, wherein the mutant NPP1 polypeptide or a fragment thereof has substantially the same ATP hydrolysis activity as the corresponding wild-type NPP1 polypeptide or a fragment thereof. [Invention 1042] The composition of the present invention 1036, wherein the mutant NPP1 polypeptide or fragment thereof has lower Ap3A hydrolysis activity and substantially the same ATP hydrolysis activity compared to the corresponding wild-type NPP1 polypeptide or fragment thereof. [Invention 1043] The composition of the present invention 1036, wherein the mutant NPP1 polypeptide or fragment thereof has a mutation at at least one position selected from the group consisting of Ser 532, Tyr 529, Tyr 451, Ile 450, Ser 381, Tyr 382, ​​Ser 377, Phe 346, Gly 531, Ser 289, Ser 287, Ala 454, Gly 452, Gln 519, Glu 526, Lys 448, Glu 508, Arg 456, Asp 276, Tyr 434, Gln 519, Ser 525, Gly 342, Ser 343, and Gly 536, compared to SEQ ID NO: 1. [Invention 1044] The composition of the present invention 1036, wherein the NPP1 polypeptide or a fragment, variant, or variant fragment thereof contains a polyaspartate domain. [Invention 1045] The composition of the present invention 1044, wherein the polyaspartate domain contains about 2 to about 20 or more consecutive aspartate residues. [Invention 1046] The composition of the present invention 1036, wherein the NPP1 polypeptide or a fragment, variant, or variant fragment thereof contains the NPP2 transmembrane domain. [Invention 1047] A composition comprising at least one active substance selected from the group consisting of ectonucleotide pyrophosphate / phosphodiesterase-4 (NPP4) polypeptide, NPP4 polypeptide fragment, NPP4 polypeptide derivative, mutant NPP4 polypeptide, and mutant NPP4 polypeptide fragment. [Invention 1048] The composition of the present invention 1047, wherein the NPP4 polypeptide or its fragment, variant, or variant fragment is a soluble recombinant NPP4 polypeptide or its fragment, variant, or variant fragment. [Invention 1049] The composition of the present invention 1047, wherein the mutated NPP4 polypeptide or a fragment thereof contains at least one mutation selected from the group consisting of D335, S92, D264, L265, S330, Q331, K332, and T323, compared to SEQ ID NO: 3. [Invention 1050] The composition of the present invention 1047, wherein the mutant NPP4 polypeptide or fragment thereof contains at least one mutation that increases the ATP hydrolysis activity of the mutant NPP4 polypeptide or fragment thereof compared to the corresponding wild-type NPP4 polypeptide or fragment thereof. [Invention 1051] The composition of the present invention 1047, wherein the mutant NPP4 polypeptide or fragment thereof contains at least one mutation that increases the NPP1-like hydrolytic activity of the mutant NPP4 polypeptide or fragment thereof compared to the corresponding wild-type NPP4 polypeptide or fragment thereof. [Invention 1052] The composition of the present invention 1047, wherein the mutant NPP4 polypeptide or fragment thereof contains at least one mutation that increases the substrate selectivity of the mutant NPP4 polypeptide or fragment thereof for ATP compared with the corresponding wild-type NPP4 polypeptide or fragment thereof. [Invention 1053] The composition of the present invention 1047, wherein the NPP4 polypeptide or a fragment, variant, or variant fragment lacks the NPP4 transmembrane domain. [Invention 1054] The composition of the present invention 1047, wherein the NPP4 polypeptide or a fragment, variant, or variant fragment thereof contains an IgG Fc domain. [Invention 1055] The composition of the present invention 1047, wherein the NPP4 polypeptide or a fragment, variant, or variant fragment thereof contains a polyaspartate domain. [Invention 1056] The composition of the present invention 1055, wherein the polyaspartate domain contains about 2 to about 20 or more consecutive aspartic acid residues. [Brief explanation of the drawing]

[0022] The following detailed description of preferred embodiments of the present invention will be better understood when read in conjunction with the accompanying drawings. For illustrative purposes of the present invention, currently preferred embodiments are shown in the drawings. However, it should be understood that the present invention is not limited to the exact configuration and means of the embodiments shown in the drawings. [Figure 1] This is an explanatory diagram illustrating proteins that regulate the extracellular balance of inorganic pyrophosphates (PPi) and inorganic phosphates (Pi). Inorganic pyrophosphates (PPi) are produced by the cleavage of extracellular nucleotide triphosphates (NTPs) by NPP1, or by the transport of PPi from the intracellular to the extracellular space by Ank. TNAP degrades PPi to produce Pi. [Figure 2]Figure 2, including Figures 2A-2B, is a series of explanatory diagrams illustrating the cloning strategy for NPP1. Figure 2A: NPP1 is replaced with a signal peptide derived from NPP2, and in this way, it can be expressed as a secreted protein. NPP1 fragments on either side of the transmembrane domain (indicated by 1 and 2) are separately PCR amplified and replaced with the NPP2 signal peptide sequence (indicated by 3, indicated by the arrow). Figure 2B: Schematic diagram of the domain structure and expressed protein used in this study. NPP1 is a type II transmembrane protein, while NPP4 is a type I transmembrane protein. The protein is represented by a colored schematic diagram illustrating the domain structure, with the transmembrane domain shown as 1, the somatomedin B domain as 2, the catalytic domain as 3, the nuclease domain as 4, and the NPP4 signal peptide as 5. The expressed NPP1 protein consists of human sequence residues 96-925, including the entire extracellular sequence of the protein, which contains both somatomedin B domains, the catalytic domain, and the nuclease domain. The secreted NPP4 protein also consists of the entire extracellular portion of the protein, which contains human sequence amino acids 16-407. [Figure 3] This is a photograph of a gel illustrating the expression and purification of NPP1 in baculovirus. Baculovirus cells were infected with the NPP1 virus, and the extracellular medium was collected, concentrated, and flowed onto a nickel column two days after incubation. After washing with buffer, the protein was eluted with imidazole, and 5 ml fractions were collected and flowed onto an SDS-PAGE gel (E1-E9). [Figure 4] This is a photograph of a gel illustrating the expression and purification of NPP1 in mammalian cells. To express mammalian glycosylated NPP1, NPP1 was produced in HEK293 kidney cells using a similar expression and purification procedure described for baculoviruses. The SDS-PAGE gel of the purified protein is shown. The concentration of the NPP1 stock solution, determined by amino acid analysis, is 2.15 mg / ml. [Figure 5-1] Figure 5, including Figures 5A-5E, illustrates the results of experiments assessing ATP cleavage and hydrolysis. Figure 5A is a graph illustrating the HPLC analysis of ATP cleavage by NPP1 (nM NPP1 and 500 μM ATP were added, and suppression occurred at 3, 6, and 30 minutes (from bottom to top)). The enzyme product of ATP hydrolysis by NPP1 was confirmed by HPLC. Figure 5B is a graph illustrating a comparison of ATP hydrolysis between NPP1 and NPP4. Comparison of the ATP hydrolysis activity of NPP1 and NPP4 (which share 38% sequence identity) revealed that only NPP1 hydrolyzes ATP, demonstrating the controlled substrate specificity of the NPP family. Figure 5C is a graph illustrating the results of experiments assessing steady-state ATP cleavage by NPP1. The initial rate velocity at each ATP concentration was derived using the time course of ATP cleavage monitored at absorbance of 259 nm after mixing with 200 nM NPP1, and the data was fitted to a rectangular hyperbola. The smooth line through the data is the best fit for the hyperbola, yielding KM = 144.5 (±36.0) μM and kcat = 468 (±48) min⁻¹ = 7.8 (±0.8) s⁻¹. Figure 5D: Ap3A concentration dependence of initial steady-state Ap3A substrate cleavage rates for NPP1 and NPP4 obtained from the best linear fit for absorbance change over time. The smooth line through the data is the best fit for the hyperbola, with KM = 20 (±3) μM and kcat = 7.2 (±0.3) s⁻¹ NPP⁻¹ for NPP1, while with KM = 685 (±108) μM and kcat = 8.0 (±0.1) s⁻¹ NPP⁻¹ for NPP4. [Figure 5-2]Figure 5, including Figures 5A-5E, illustrates the results of experiments assessing ATP cleavage and hydrolysis. Figure 5E: Inhibition of NPP4 cleavage activity by nucleotide monophosphates. Solid lines passing through data points represent the best fit to the rectangular hyperbola. Symbols: AMP: magenta diamond, CMP: black square, GMP: blue circle. The resulting IC50s from strongest to weakest inhibition are AMP 129±73 μM, CMP 322±39 μM, UMP 2.11±0.37 mM (not shown for clarity), and GMP 2.98±0.38 mM. [Figure 6A] Figure 6, including Figures 6A-6C, is a series of explanatory diagrams illustrating the NPP4-AMP structure and its comparison with other NPP catalytic domains. The three-dimensional structure of NPP4 with the bound enzyme product (AMP) was determined by X-ray crystallography at a resolution of 1.5 Å. Figure 6A: The protein is presented as a ribbon with AMP in stick form, color-coded by atomic type. The active site zinc ion is illustrated as a sphere. [Figure 6B]Figure 6, including Figures 6A-6C, is a series of explanatory diagrams regarding the NPP4-AMP structure and its comparison with other NPP catalytic domains. The three-dimensional structure of NPP4 with the bound enzyme product (AMP) was determined by X-ray crystallography for a resolution of 1.5 Å. Figure 6B: Superposition of Cα-traces of human NPP4 and other NPP catalytic domains reveals the highest degree of structural conservation (red, 0.68 Å rmsd for all four molecules as shown) across the central β-sheet core and the active site near the zinc ion, including the α-helix where the catalytic threonine is located and the main chain near the hydrophobic groove. Regions of moderate similarity are yellow (1.33 Å rmsd), and those of lowest similarity are blue-green (2.51 Å rmsd). Conserved subdomains were superimposed using the ccp4 program Superpose (red-yellow, 1.17 Å rmsd). Despite the high degree of structural conservation near the active site, various NPPs can exhibit diverse substrate specificities. The four superimposed catalytic domains are derived from human NPP4 (presented here), mouse NPP1 (4B56), mouse NPP2 (3NKN), and bacterial NPP (2GSU). To accommodate lipid substrates, NPP2 lacks the region found in all other NPP catalytic domains; therefore, the 8-residue linker (residues 272-279) in mouse NPP2 has been removed in this comparison diagram. The superposition of the entire catalytic domain of NPP4 with those of NPP1, NPP2, and bacterial NPP yields rmsd values ​​of 1.54 Å, 1.43 Å, and 1.43 Å, respectively. [Figure 6C]Figure 6, including Figures 6A-6C, is a series of explanatory diagrams illustrating the NPP4-AMP structure and its comparison with other NPP catalytic domains. The three-dimensional structure of NPP4 with the bound enzyme product (AMP) was determined by X-ray crystallography at a resolution of 1.5 Å. Figure 6C: NPP4-product complex with AMP. The mFo-DFc difference densities are shown for each, with contour lines drawn at 3σ. Ligands and water molecules within the binding pocket were not included in the electron density map calculation. All molecular images were generated using PyMOL (Molecular Graphics System, version 1.2r3pre, Schrodinger, LLC). [Figure 7A] Figure 7, including Figures 7A-7D, illustrates the base recognition of the substrate at the NPP4 catalytic site. Figure 7A: The active site of NPP4 contains a pre-formed hydrophobic groove, which gives it its specificity for 5' nucleotide-containing substrates. In the NPP4-AMP complex, the adenine ring stacks with Tyr154 on one wall of the pocket and, on the other hand, receives favorable VDW interactions from the tip of Phe71 along the opposite wall. For compatibility, the molecular surface of NPP4 (network) and the VDW surface of AMP (translucent shape) are shown. [Figure 7B] Figure 7, including Figures 7A-7D, illustrates the base recognition of the substrate in the NPP4 catalytic site. Figure 7B: Positioning of AMP in the active site with highlighted key residues and bound metal ions. The two bound zinc ions (spheres) play different roles: Zn2 activates Thr70 (green) for nucleophilic attack on the substrate, while Zn1 electrostatically attracts the substrate's phosphate group to a very close position. The nucleotides Tyr154 and Phe71 are cyan. [Figure 7C] Figure 7, including Figures 7A-7D, illustrates the base recognition of the substrate at the NPP4 catalytic site. Figure 7C: Superposition of the NPP4-AMP complex and apoNPP4, illustrating the very slight change when the product is bound. The citrate anion bound at Zn1 in the apo structure has been removed for visual clarity. [Figure 7D] Figure 7, including Figures 7A-7D, illustrates substrate base recognition at the NPP4 catalytic site. Figure 7D: Superposition of the NPP4-AMP and NPP1-AMP complexes illustrating similar shapes within this half of their active sites, both possessing grooves for nucleotide binding. [Figure 8] This is a diagram illustrating a non-restrictive enzymatic mechanism of Ap3A hydrolysis by NPP4. It is a proposed reaction mechanism for NPP4 hydrolysis of Ap3A, originally proposed by Gijsbers et al. based on the topology isomorphism of the active site to AP. The steps in this mechanism, for which a crystal structure has been obtained, are enclosed in a box. [Figure 9A] Figure 9, including Figures 9A-9F, illustrates a non-restrictive model of the molecular basis for substrate discrimination in NPP1 and NPP4. Based on the AMP cocrystal structure for each enzyme, models of ATP bound in an AMP-like orientation are shown for NPP1 (left column) and NPP4 (right column). Figure 9A: In NPP1, the γ-phosphate of ATP is simultaneously stabilized by three lysine residues, two of which line the upper edge of the pocket and become regular only when a substrate is present due to electrostatics. As a result of this three-part lysine claw, the bound γ-phosphate of ATP is preferably charge-stabilized and greatly shielded from the solvent by an inductively fitting lid formed by the long hydrophobic side chains of these two lysine residues together with the adjacent tyrosine ring. In contrast, NPP4 provides a less favorable γ-phosphate environment for similarly bound ATP, with lower charge stabilization, a more open structural mode lacking a lid mechanism, and two neighboring aspartate residues for charge repulsion. Consequently, ATP is unlikely to bind to NPP4 very frequently in this orientation. Figures 9A and 9B: Stick diagrams of bound ATP modeled from AMP cocrystal structures. [Figure 9B]Figure 9, including Figures 9A-9F, illustrates a non-restrictive model of the molecular basis for substrate discrimination in NPP1 and NPP4. Based on the AMP co-crystal structure for each enzyme, models of ATP bound in an AMP-like orientation are shown for NPP1 (left column) and NPP4 (right column). Figures 9A and 9B: Stick diagrams of bound ATP modeled from the AMP co-crystal structure. [Figure 9C] Figure 9, including Figures 9A–9F, illustrates a non-restrictive model of the molecular basis for substrate discrimination in NPP1 and NPP4. Based on the AMP cocrystal structure for each enzyme, models of ATP bound in an AMP-like orientation are shown for NPP1 (left column) and NPP4 (right column). Figures 9C and 9D: The same, but as molecular surfaces with neighboring charged side chains (positive or negative, as shown) at the tips. [Figure 9D] Figure 9, including Figures 9A–9F, illustrates a non-restrictive model of the molecular basis for substrate discrimination in NPP1 and NPP4. Based on the AMP cocrystal structure for each enzyme, models of ATP bound in an AMP-like orientation are shown for NPP1 (left column) and NPP4 (right column). Figures 9C and 9D: The same, but as molecular surfaces with neighboring charged side chains (positive or negative, as shown) at the tips. [Figure 9E] Figure 9, including Figures 9A-9F, illustrates a non-restrictive model of the molecular basis for substrate discrimination of NPP1 and NPP4. Based on the AMP co-crystal structure for each enzyme, models of ATP bound in an AMP-like orientation are shown for NPP1 (left column) and NPP4 (right column). Figures 9E and 9F: rotated approximately 90°. Sequence alignment shows that human NPP1 retains all the characteristics derived from the mouse NPP1 structure. [Figure 9F]Figure 9, including Figures 9A-9F, illustrates a non-restrictive model of the molecular basis for substrate discrimination of NPP1 and NPP4. Based on the AMP co-crystal structure for each enzyme, models of ATP bound in an AMP-like orientation are shown for NPP1 (left column) and NPP4 (right column). Figures 9E and 9F: rotated approximately 90°. Sequence alignment shows that human NPP1 retains all the characteristics derived from the mouse NPP1 structure. [Figure 10A] Figure 10, including Figures 10A-10B, is a series of graphs illustrating the effect of NPP1 on platelet aggregation. Platelet aggregation in platelet-rich plasma in response to increasing concentrations of NPP1 and NPP4, as well as 80 μM Ap3A, was assessed using light-transmittance aggregation ability measurements. Data are presented graphically as percentages of light transmittance (y-axis) over time (x-axis). Figure 10A: In the absence of NPP1, 80 μM Ap3A elicited only a primary wave of aggregation, followed by rapid dissociation; this pattern was similarly observed with the addition of NPP1 at concentrations of 300 pM and 500 pM. In contrast, 1 nM NPP1 in the presence of 80 μM Ap3A stimulated a measurable secondary wave of aggregation. [Figure 10B] Figure 10, including Figures 10A-10B, is a series of graphs illustrating the effect of NPP1 on platelet aggregation. Platelet aggregation in platelet-rich plasma was assessed using light-transmittance aggregation ability measurements in response to increasing concentrations of NPP1 and NPP4, as well as 80 μM Ap3A. Data are presented graphically as percentages of light transmittance (y-axis) over time (x-axis). Figure 10B: NPP4 exhibited primary aggregation in the presence of either Ap3A alone or Ap3A containing 1 nM NPP4. Significant secondary platelet aggregation occurred in the presence of 20 nM NPP4 and higher concentrations. This finding suggests that, in the presence of physiological concentrations of Ap3A, either protein can directly stimulate platelet aggregation at low nM concentrations. [Figure 11] This is an illustrative model of Ap3A docked within human NPP4. [Figure 12]This is a series of photographs illustrating protein crystals containing the inactive form of NPP4 bound to Ap3A. [Modes for carrying out the invention]

[0023] Detailed description of the invention This invention relates to the discovery that NPP1 polypeptide, its fragments, derivatives, variants, or variant fragments, and mutant NPP4 polypeptide or its fragments are useful in treating diseases and disorders involving pathological calcification and / or ossification.

[0024] Accordingly, in certain embodiments, the present invention relates to compositions and methods for increasing the level or activity of NPP1 polypeptide, its fragments, derivatives, variants, or variant fragments; on the other hand, in other embodiments, the present invention relates to compositions and methods for increasing the level or activity of mutant NPP4 polypeptide, its fragments, or derivatives. In yet another embodiment, the present invention relates to compositions and methods for increasing the level or activity of NPP1 polypeptide, its fragments, derivatives, variants, or variant fragments, and mutant NPP4 polypeptide or its fragments.

[0025] In one particular embodiment, the present invention relates to a method for eliminating and / or reducing the thrombogenic activity of at least one active substance selected from the group consisting of ectonucleotide pyrophosphate / phosphodiesterase-1 (NPP1) polypeptide, and its fragments, derivatives, variants, or variant fragments, while maintaining ATP hydrolysis activity. In another embodiment, the present invention relates to a method for eliminating and / or reducing the thrombogenic activity of at least one active substance selected from the group consisting of ectonucleotide pyrophosphate / phosphodiesterase-1 (NPP4) polypeptide, and its fragments, derivatives, variants, or variant fragments, while also increasing ATP hydrolysis activity. In yet another embodiment, the method of the present invention enables the safe treatment of ectopic mineralization without inducing unintended risks associated with thrombogenic conditions.

[0026] In various embodiments, mutant NPP1 polypeptides, fragments thereof, or derivatives useful in the methods of the present invention have lower Ap3A hydrolysis activity compared to the corresponding wild-type NPP1 polypeptides, fragments thereof, or derivatives. In various embodiments, mutant NPP1 polypeptides, fragments thereof, or derivatives useful in the methods of the present invention have substantially the same ATP hydrolysis activity compared to the corresponding wild-type NPP1 polypeptides, fragments thereof, or derivatives. In various embodiments, mutant NPP1 polypeptides, fragments thereof, or derivatives useful in the methods of the present invention have lower Ap3A hydrolysis activity and substantially the same ATP hydrolysis activity compared to the corresponding wild-type NPP1 polypeptides, fragments thereof, or derivatives.

[0027] In various embodiments, the present invention relates to compositions and methods for increasing the level or activity of NPP1 polypeptide, its fragments, derivatives, variants, or variant fragments. The compositions and methods of the present invention include compositions and methods for treating or preventing disorders and diseases in which an increase in the activity or level of NPP1 polypeptide, its fragments, derivatives, variants, or variant fragments is desirable. In various embodiments, disorders and diseases include diseases and disorders involving pathological calcification and / or pathological ossification. Diseases and disorders involving pathological calcification and / or pathological ossification that can be treated by the compositions and methods of the present invention include, but are not limited to, idiopathic infantile arterial calcification (IIAC), ossification of the posterior longitudinal ligament (OPLL), hypophosphatemic rickets, osteoarthritis, and calcification of atherosclerotic plaques.

[0028] In other embodiments, the present invention relates to compositions and methods for increasing the level or activity of mutant NPP4 polypeptides or fragments thereof. The compositions and methods of the present invention include compositions and methods for treating or preventing disorders and diseases in which an increase in the activity or level of mutant NPP4 polypeptides or fragments thereof is desirable. In various embodiments, disorders and diseases include diseases and disorders involving pathological calcification and / or pathological ossification. Diseases and disorders involving pathological calcification and / or pathological ossification that can be treated by the compositions and methods of the present invention include, but are not limited to, idiopathic infantile arterial calcification (IIAC), ossification of the posterior longitudinal ligament (OPLL), hypophosphatemic rickets, osteoarthritis, and calcification of atherosclerotic plaques.

[0029] Both hemostasis and bone development are precisely balanced, essential physiological processes regulated by the extracellular metabolism of purine-mediated signals. To better understand the roles and atomic details of purine-mediated signal metabolism by NPP1 and NPP4, as described herein, the high-resolution structure of NPP4, the first human NPP to be resolved, was determined as a means of structurally and enzymatically characterizing NPP4 versus NPP1. NPP1 has been shown to hydrolyze Ap3A at low nM concentrations, and either NPP1 or NPP4 at low nM concentrations promoted irreversible platelet aggregation in human PRP in vitro.

[0030] In addition, the effect of NPP1 on platelet aggregation in the presence of physiological levels of Ap3A was directly measured. Despite high sequence identity and homology, as well as shared structural characteristics, which enable targeting of nearly identical sets of nucleotide-containing substrates, these two enzymes also possess important structural differences that explain the distinct substrate specificity central to their biological functions. NPP1 was found not to induce platelet aggregation at physiological concentrations reported in human blood, but it could stimulate platelet aggregation when localized at low nM concentrations on vascular endothelium. This study describes the molecular basis of substrate discrimination by NPP4 and NPP1, provides insights into their physiological roles governing bone mineralization and platelet aggregation, and offers clear mechanisms by which NPP1 polymorphisms may act in connection with stroke protection.

[0031] Without wishing to be limited by any theory, this study suggests an alternative mechanism by which polymorphisms in NPP1 are protective against thrombotic stroke compared to the mechanism proposed in the prior art. The data described herein support the idea that loss-of-function mutations, rather than gain-of-function mutations proposed in the prior art, which reduce Ap3A hydrolysis in the thrombotic microenvironment, contribute to the mechanism by which these polymorphisms provide stroke protection. This difference plays a crucial role in designing modifications of NPP1 and NPP4 proteins that are useful as therapeutic agents for ectopic bone mineralization without inducing unintended side effects of pro-thrombus formation in treated individuals. In certain embodiments, a decrease in NPP1 activity in cerebral capillaries leads to a decrease in ADP concentration in the cerebral capillary bed, reducing platelet aggregation and thrombus formation.

[0032] Furthermore, the recognition of NPP1 as a thrombus-promoting enzyme suggests that recombinant enzyme replacement therapy using NPP1 or NPP4 poses a significant thrombosis risk to patients treated with recombinant NPP1 and / or NPP4 enzymes. Patients in a pro-thrombus state are at risk of sudden death from stroke due to coronary and / or pulmonary artery thrombosis, as well as cerebral artery thrombosis. As demonstrated herein, the inventors have established biochemical and physiological arguments supporting the idea that patients treated with recombinant unmodified NPP1 enzymes are at increased risk of these unintended effects. The inventors' recognition of the role of NPP1 in thrombosis establishes arguments for inducing specific point mutations in NPP1 and / or NPP4 that eliminate and / or improve the enzyme's thrombus-promoting activity, while retaining the enzyme's ability to produce PPi. In certain embodiments, the specific modifications to NPP1 and NPP4 proposed herein achieve the goal of establishing safe therapeutic methods for the treatment of GACI and other diseases of ectopic mineralization using recombinant NPP4 and NPP1 enzymes.

[0033] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in which the invention pertains. Any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of the invention, but preferred methods and materials are described.

[0034] As used herein, each of the following terms shall have the meanings relating to those in this chapter.

[0035] The articles “a” and “an” are used herein to refer to one or more (i.e., at least one) grammatical objects of the articles. For example, “an element” means one or more elements.

[0036] As used herein when referring to measurable values ​​such as quantity or duration, “about” is intended to include variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the specified value, for such variations are appropriate for carrying out the disclosed method.

[0037] As used herein, the term "Ap3P" refers to adenosine-(5')-triphospho-(5')-adenosine or a salt thereof.

[0038] As used herein, the term "NPP" refers to ectonucleotide pyrophosphatase / phosphodiesterase.

[0039] A "disease" is a state of animal health in which the animal is unable to maintain homeostasis and whose health continues to deteriorate if the disease is not improved.

[0040] In animals, a “disorder” is defined as a state of health in which the animal can maintain homeostasis, but the state of health is less desirable than it would be in the absence of the disorder. If left untreated, a disorder does not necessarily lead to a further deterioration of the animal’s health.

[0041] A disease or disorder is "reduced" if the severity of its symptoms, the frequency with which such symptoms are experienced by the patient, or both are reduced.

[0042] When used in the context of organisms, tissues, cells, or their components, the term “abnormal” means that such organism, tissue, cell, or their component differs from such organism, tissue, cell, or their component in that it exhibits at least one observable or detectable feature (e.g., age, treatment, date and time) that is “normal” (expected). A feature that is normal or expected for one type of cell or tissue may be abnormal for a different type of cell or tissue.

[0043] "Isolated" means that it has been modified or removed from its natural state. For example, nucleic acids or polypeptides that are naturally present in living animals are not "isolated," but the same nucleic acids or polypeptides that are partially or completely separated from the coexisting material in their natural state are "isolated." Isolated nucleic acids or proteins may exist in a substantially purified form or in a non-natural environment, such as a host cell.

[0044] As used herein, “substantially purified” means essentially free from other components. For example, a substantially purified polypeptide is a polypeptide that has been separated from the other components that would normally be associated with it in its naturally occurring state.

[0045] As used herein, “sample” or “biological sample” means biological material isolated from a subject. A biological sample may contain any biological material suitable for detecting mRNA, polypeptides, or other markers of physiological or pathological processes in a subject, and may include bodily fluids, tissues, cellular and / or non-cellular materials obtained from an individual.

[0046] As used herein, the term “wild-type” refers to a gene or gene product isolated from a naturally occurring source. A wild-type gene is the most frequently observed form in a population and is therefore optionally intended to be the “normal” or “wild-type” form of that gene. In contrast, the terms “modified” or “mutant” refer to a gene or gene product that exhibits alterations in sequence and / or functional properties (i.e., altered features) compared to a wild-type gene or gene product. It should be noted that naturally occurring mutants can be isolated and are identified by the fact that they have altered features (including altered nucleic acid sequences) compared to a wild-type gene or gene product.

[0047] As used herein, the term "polypeptide" refers to a polymer composed of amino acid residues, associated naturally occurring structural variants, and non-naturally occurring synthetic analogs linked by peptide bonds. Synthetic polypeptides can be synthesized, for example, using an automated polypeptide synthesizer. As used herein, the term "protein" typically refers to a large polypeptide. As used herein, the term "peptide" typically refers to a short polypeptide. Conventional notation is used herein to represent polypeptide sequences, where the left end of a polypeptide sequence is the amino terminus and the right end is the carboxyl terminus.

[0048] In this specification, amino acids are represented by their formal names, their corresponding three-letter codes, or their corresponding one-letter codes, as shown below. TIFF0007876894000001.tif96128

[0049] The term "amino acid sequence variant" refers to a polypeptide that has an amino acid sequence that differs to some extent from the native sequence polypeptide. Typically, an amino acid sequence variant has at least approximately 70%, at least approximately 80%, at least approximately 90%, or at least approximately 95% homology to the native polypeptide. An amino acid sequence variant has substitutions, deletions, and / or insertions at specific positions within the amino acid sequence of the native amino acid sequence.

[0050] As used herein, the terms “conservative variation” or “conservative substitution” refer to the substitution of an amino acid residue with another biologically similar residue. Conservative variations or substitutions are unlikely to alter the shape of the peptide chain. Examples of conservative variations or substitutions include the substitution of a single hydrophobic residue, such as isoleucine, valine, leucine, or methionine, for another, or the substitution of a single polar residue, such as arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine.

[0051] As used herein, the term “domain” refers to a portion of a molecule or structure that shares common physicochemical properties, including but not limited to hydrophobic, polar, spherical, and helical domains or properties. Specific examples of binding domains include, but are not limited to, DNA-binding domains and ATP-binding domains.

[0052] "Nucleic acids" refers to polynucleotides, including polyribonucleotides and polydeoxyribonucleotides. Nucleic acids according to the present invention may include any polymers or oligomers of pyrimidines and purine bases, preferably cytosine, thymine, and uracil, respectively, as well as adenine and guanine. (See Albert L. Lehninger, Principles of Biochemistry, 793-800 (Worth Pub. 1982), which is incorporated herein by reference as a whole for all purposes.) In fact, the present invention aims to provide any deoxyribonucleotide, ribonucleotide, or peptide nucleic acid components, and any chemical variations thereof, such as methylated, hydroxymethylated, or glucosylated forms of these bases. Polymers or oligomers may be heterogeneous or homogeneous in composition and may be isolated from naturally occurring sources or produced artificially or synthetically. In addition, nucleic acids may be DNA or RNA, or mixtures thereof, and may exist permanently or transiently in single-stranded or double-stranded forms, including homoduplex, heteroduplex, and hybrid states.

[0053] An “oligonucleotide” or “polynucleotide” is a nucleic acid having a length of at least two nucleotides, preferably at least eight, fifteen, or twenty-five nucleotides, but may be up to 50, 100, 1,000, or 5,000 nucleotides, or may be a compound that specifically hybridizes with a polynucleotide. Polynucleotides include sequences of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), or their mimics, which may be isolated from natural sources, produced by recombinant processes, or synthesized artificially. A further example of a polynucleotide in the present invention may be a peptide nucleic acid (PNA). (See U.S. Patent No. 6,156,501, which is incorporated herein by reference as a whole.) The present invention also encompasses situations in which there are non-traditional base pairs, such as Hoogsteen base pairs, which are identified in certain tRNA molecules and assumed to exist in a triple-helix state. “Polynucleotide” and “oligonucleotide” are used interchangeably herein. Where a nucleotide sequence is represented herein by a DNA sequence (e.g., A, T, G, and C), this is understood to also include the corresponding RNA sequence (e.g., A, U, G, C) where "U" replaces "T".

[0054] As used herein, “polynucleotide” includes both sense and antisense strands of cDNA, RNA, DNA / RNA hybrids, antisense RNA, ribozymes, genomic DNA, synthetic forms, and mixed polymers, and may be chemically or biochemically modified to contain unnatural or derivatized, synthetic, or semi-synthetic nucleotide bases. Furthermore, modifications of wild-type or synthetic genes are intended, including but not limited to the deletion, insertion, substitution, or fusion of one or more nucleotides into other polynucleotide sequences.

[0055] "Isolated nucleic acid" refers to a nucleic acid segment or fragment that has been separated from adjacent sequences in its naturally occurring state, for example, a DNA fragment that has been separated from sequences normally adjacent to the fragment in a naturally occurring genome. The term also applies to nucleic acids that have been substantially purified from other components naturally associated with nucleic acids, for example, RNA or DNA or proteins naturally associated with them in cells. Therefore, the term includes recombinant DNA that is incorporated into, for example, vectors, self-replicating plasmids or viruses, or into the genomic DNA of prokaryotes or eukaryotes, or that exists as a distinct molecule independently of other sequences (for example, cDNA produced by PCR or restriction enzyme digestion, or as a genomic fragment or cDNA fragment). It also includes recombinant DNA that is part of a hybrid gene encoding further polypeptide sequences.

[0056] An "allele" refers to a specific form of a gene sequence (such as a gene) within a cell, individual, or population, which differs from other forms of the same gene in at least one, and more frequently two or more, variant sites within the gene sequence. These variant sites that differ between different alleles are called "variants," "polymorphisms," or "mutations."

[0057] As used herein, the terms “modification,” “deletion,” “variation,” or “mutation” refer to changes in a gene within a cell that affect the function, activity, expression (transcription or translation), or conformation of the polypeptide it encodes. Mutations encompassed by the present invention may be any changes in a gene within a cell that result in enhancement or interference of the function, activity, expression, or conformation of the encoded polypeptide, including the complete absence of expression of the encoded protein, and may include, for example, missense and nonsense mutations, insertions, deletions, frameshifts, and immature terminations. Without limiting so, mutations encompassed by the present invention may alter the splicing of mRNA (splicing site mutations) or cause a shift in the reading frame (frameshift).

[0058] As used herein, the term “antibody” refers to an immunoglobulin molecule capable of specifically binding to a specific epitope on an antigen. Antibodies may be intact immunoglobulins derived from natural or recombinant sources, or they may be the immunoreactive portion of intact immunoglobulins. Antibodies as used herein can exist in a variety of forms, including, for example, polyclonal antibodies, monoclonal antibodies, intracellular antibodies ("intrabodies"), Fv, Fab, and F(ab)2, as well as single-chain antibodies (scFv), heavy-chain antibodies such as camel antibodies, synthetic antibodies, chimeric antibodies, and humanized antibodies (Harlow et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0059] As used herein, "immunoassay" refers to any binding assay method that uses an antibody capable of specifically binding to a target molecule for the detection and quantification of that target molecule.

[0060] As used herein, the terms “immunoglobulin” or “Ig” are defined as a class of proteins that function as antibodies. Antibodies expressed by B cells are sometimes referred to as BCRs (B cell receptors) or antigen receptors. The five members of this class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is the primary antibody found in bodily secretions such as saliva, tears, breast milk, gastrointestinal secretions, and mucus secretions of the respiratory and urogenital tracts. IgG is the most commonly found circulating antibody. IgM is the primary immunoglobulin produced in major immune responses in most targets. It is the most efficient immunoglobulin in agglutination, complement fixation, and other antibody responses, and is important in defense against bacteria and viruses. IgD is an immunoglobulin that does not have known antibody function but can function as an antigen receptor. IgE is an immunoglobulin that mediates immediate hypersensitivity by triggering the release of mediating factors from mast cells and basophils upon exposure to allergens.

[0061] "Code" refers to the inherent properties of a specific sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, that act as a template for the synthesis of other polymers and macromolecules in a biological process having either a predetermined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a predetermined sequence of amino acids, and the biological properties derived therefrom. Thus, a gene codes for a protein when the transcription and translation of the mRNA corresponding to that gene produces a protein in a cell or other biological system. Both the coding strand, which is the nucleotide sequence identical to the mRNA sequence and typically provided in a sequence listing, and the non-coding strand, which is used as a template for the transcription of a gene or cDNA, may be said to code for a protein or other product of that gene or cDNA.

[0062] As used herein, the term “coding sequence” means a sequence of nucleic acid, its complement, or a part thereof that can be transcribed and / or translated to produce mRNA and / or polypeptides, or fragments thereof. A coding sequence includes exons in genomic DNA or immature primary RNA transcripts, which are joined together by the cell’s biochemical mechanisms to provide mature mRNA. The antisense strand is the complement of such nucleic acid, and the coding sequence can be inferred from it. In contrast, as used herein, the term “non-coding sequence” means a sequence of nucleic acid, its complement, or a part thereof that is not translated into amino acids in vivo, or does not interact with or attempt to interact with tRNA to place amino acids. Non-coding sequences include both intron sequences in genomic DNA or immature primary RNA transcripts, and gene-associated sequences such as promoters, enhancers, and silencers.

[0063] As used herein with respect to antibodies, the term “specifically binding” means an antibody that recognizes a specific antigen in a sample but substantially does not recognize or bind to other molecules. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. However, such interspecies reactivity itself does not change the classification of the antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allele forms of that antigen. However, such cross-reactivity itself does not change the classification of the antibody as specific. In some cases, the terms “specific binding” or “specifically binding” may be used in relation to the interaction between an antibody, protein, or peptide and a second chemical species, meaning that the interaction depends on the presence of a specific structure (e.g., an antigenic determinant or epitope) on the chemical species, for example, an antibody recognizes and binds to a specific protein structure rather than to proteins in general. If the antibody is specific to epitope "A", then in a reaction involving labeled "A" and the antibody, the presence of molecules containing epitope A (or free, unlabeled A) is thought to reduce the amount of labeled A bound to the antibody.

[0064] As used herein, the terms “complementary” or “complementarity” are used in relation to polynucleotides (i.e., sequences of nucleotides) linked by base pairing rules. For example, the sequence “AGT” is complementary to the sequence “TCA”. Complementarity can be “partial,” where only some of the bases of the nucleic acid match according to base pairing rules. Alternatively, there can be “complete” or “all” complementarity between nucleic acids. The degree of complementarity between nucleic acid strands has a significant impact on the efficiency and strength of hybridization between nucleic acid strands. This is particularly important in amplification reactions and in detection methods that rely on binding between nucleic acids.

[0065] As used herein, the term “fragment” applied to nucleic acids refers to a subsequence of a larger nucleic acid. A “fragment” of nucleic acid may be a length of at least about 15 nucleotides, e.g., at least about 50 to about 100 nucleotides, at least about 100 to about 500 nucleotides, at least about 500 to about 1000 nucleotides, at least about 1000 to about 1500 nucleotides, at about 1500 to about 2500 nucleotides, or about 2500 nucleotides (and any integer value in between). As used herein, the term “fragment” applied to proteins or peptides refers to a subsequence of a larger protein or peptide. A “fragment” of protein or peptide may be a length of at least about 20 amino acids, e.g., at least about 50 amino acids, at least about 100 amino acids, at least about 200 amino acids, at least about 300 amino acids, or at least about 400 amino acids (and any integer value in between).

[0066] "Homologous" refers to sequence similarity or identity between two polypeptides or two nucleic acid molecules. If the positions in both compared sequences are occupied by the same base or amino acid monomer subunit—for example, if the positions in each of two DNA molecules are occupied by adenine—then the molecules are homologous at that position. The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences (divided by the number of positions compared) × 100. For example, if 6 out of 10 positions in two sequences match or are homologous, then the two sequences are 60% homologous. As an example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, comparisons are made when the two sequences are aligned to give the greatest possible homology.

[0067] Where the term “Educational Materials” is used herein, “Educational Materials” include publications, records, diagrams, or any other expressive medium that can be used to convey the usefulness of the nucleic acids, peptides, and / or compounds of the present invention in the kit for identifying, mitigating, or treating the various diseases or disorders listed herein. Optionally or alternatively, the educational materials may describe one or more methods for identifying or mitigating a disease or disorder in the target cells or tissues. The educational materials of the kit may, for example, be affixed to the container containing the nucleic acids, polypeptides, and / or compounds of the present invention, or shipped together with the container containing the nucleic acids, polypeptides, and / or compounds. Alternatively, the educational materials may be shipped separately from the container, with the intention that the recipient use the educational materials and compounds in conjunction.

[0068] As used herein, the term “modulate” means mediating a detectable increase or decrease in the activity and / or level of mRNA, polypeptide, or response in a subject compared to the activity and / or level of mRNA, polypeptide, or response in the subject in the absence of the treatment or compound, and compared to the activity and / or level of mRNA, polypeptide, or response in a subject that is otherwise identical but not treated. The term encompasses activating, inhibiting, and / or otherwise affecting a native signal or response, thereby mediating a beneficial therapeutic response in a subject, preferably a human.

[0069] As used herein, the terms “treatment” or “to treat” are defined as the application or administration (alone or in combination with another pharmaceutically active agent) of a therapeutic agent to a patient, i.e., a compound useful in the present invention, for the purpose of curing, healing, reducing, mitigating, altering, correcting, improving, enhancing, or influencing a disease or disorder, symptoms of a disease or disorder, or the likelihood of developing a disease or disorder, or the application or administration of a therapeutic agent to isolated tissue or cell lines derived from a patient having a disease or disorder, symptoms of a disease or disorder, or the likelihood of developing a disease or disorder (for example, for diagnostic or ex vivo application). Such treatments may be specifically adapted or modified based on knowledge derived from the field of genomic pharmacology.

[0070] As used herein, the terms “prevent” or “prevention” mean, in the absence of any occurrence, the absence of the onset of a disability or disease, or, in the absence of an existing disability or disease, the absence of further onset of a disability or disease. The ability of something to prevent some or all of the symptoms associated with a disability or disease is also taken into consideration.

[0071] As used herein, the terms “patient,” “individual,” or “subject” refer to human or non-human mammals. Non-human mammals include livestock and pets such as sheep, cattle, pigs, dogs, cats, and rodents. Preferably, the patient, individual, or subject is human.

[0072] As used herein, the terms “effective dose,” “pharmaceutical effective dose,” and “therapeutic effective dose” refer to a non-toxic but sufficient amount of an active substance that provides the desired biological outcome. This outcome may be a reduction and / or mitigation of the signs, symptoms, or causes of a disease, or any other desired modification of a biological system. The appropriate therapeutic dose in any individual case can be determined by those skilled in the art using routine experiments.

[0073] As used herein, the term “pharmaceutically acceptable” means a material, such as a carrier or diluent, that does not negate the biological activity or properties of a compound and is relatively non-toxic, i.e., the material can be administered to an individual without causing undesirable biological effects or interacting in a harmful manner with any of the components of a composition containing it.

[0074] As used herein, the term “pharmaceutically acceptable salt” refers to a salt of the compound to be administered, prepared from pharmaceutically acceptable, non-toxic acids and bases, including inorganic acids, inorganic bases, organic acids, inorganic bases, their solvates, hydrates, and inclusion compounds. A suitable pharmaceutically acceptable acid addition salt may be prepared from an inorganic or organic acid. Examples of inorganic acids include sulfates, hydrogen sulfates, hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, carbonic acid, sulfuric acid, and phosphoric acid (including hydrogen phosphates and dihydrogen phosphates). Suitable organic acids can be selected from fatty acids, cycloaliphatic acids, aromatic acids, araliphatic acids, heterocyclic acids, carboxylic acids, and sulfonic acids, examples of which include formic acid, acetic acid, propionic acid, succinic acid, glycolic acid, gluconic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, glucuronic acid, maleic acid, fumaric acid, pyruvic acid, aspartic acid, glutamic acid, and benzoic acid. Acids include anthranilic acid, 4-hydroxybenzoic acid, phenylacetic acid, mandelic acid, embonic acid (pamoic acid), methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, pantothenic acid, trifluoromethanesulfonic acid, 2-hydroxyethanesulfonic acid, p-toluenesulfonic acid, sulfanilic acid, cyclohexylaminosulfonic acid, stearic acid, alginic acid, β-hydroxybutyric acid, salicylic acid, galactaric acid, and galacturonic acid. Suitable pharmaceutically acceptable base addition salts of the compounds of the present invention include metal salts, such as alkali metal salts, alkaline earth metal salts, and transition metal salts, such as calcium salts, magnesium salts, potassium salts, sodium salts, and zinc salts. Pharmaceutically acceptable base addition salts also include organic salts made from basic amines such as N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine), and procaine. All of these salts can be prepared from the corresponding compounds, for example, by reacting the compound with a suitable acid or base.

[0075] As used herein, the terms “composition” or “pharmaceutical composition” refer to a mixture of at least one compound useful in the present invention and a pharmaceutically acceptable carrier. Pharmaceutical compositions facilitate the administration of compounds to patients. Numerous techniques exist in the art for administering compounds, including but not limited to intravenous, oral, aerosol, inhalation, rectal, vaginal, transdermal, intranasal, oral, sublingual, parenteral, intrathecal, gastric, ophthalmic, intrapulmonary, and local administration.

[0076] As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition, or carrier, such as a liquid or solid filler, stabilizer, dispersant, suspending agent, diluent, excipient, thickener, solvent, or encapsulating material, that accompanies the transport or delivery of a compound useful in the present invention into or to a patient, so that it may perform its intended function. Typically, such a construct is transported or delivered from one organ or part of the body to another. Each carrier must be “acceptable” in the sense that it is compatible with the other components of the formulation, including the compound useful in the present invention, and is not harmful to the patient. Some examples of materials that can act as pharmaceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose, and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; surfactants; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer; and other non-toxic, suitable substances used in pharmaceutical formulations. As used herein, “pharmaceutically acceptable carrier” includes any and all coatings, antimicrobial and antifungal agents, as well as absorption retarders, etc., that are active and compatible with the compounds useful in the present invention and are physiologically acceptable to the patient. Auxiliary active compounds may also be incorporated into the composition. “pharmaceutically acceptable carrier” may further include pharmaceutically acceptable salts of the compounds useful in the present invention.Further components that may be included in pharmaceutical compositions used in practice of the present invention are known in the art and are described, for example, in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference.

[0077] Range: Throughout this disclosure, various aspects of the invention may be presented in range form. It should be understood that the range form is merely for convenience and brevity and should not be considered a firm limitation on the scope of the invention. Therefore, a range description should be considered to specifically disclose all possible subranges, as well as the individual numbers within those ranges. For example, a range description such as 1-6 should be considered to specifically disclose subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, as well as the individual numbers within those ranges, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the range width.

[0078] explanation The current high-resolution structural determination of human NPP4 has enabled detailed structure-function comparative studies with NPP1. These membrane-bound cell surface enzymes are involved in the metabolism of extracellular purinergic signals and nucleotide reuptake. Both exogenous enzymes possess a narrow, hydrophobic groove adjacent to two bound zinc ions, which explains the targeting of nucleotide-containing substrates. There, nucleotide bases bind within the groove, and hydrolysis yields a nucleotide monophosphate as one of the products. Adenine is the preferred base type for both enzymes, and their co-crystal structures with the bound AMP molecular product highlight their functional similarity in that region of the binding site. Both enzymes can hydrolyze Ap3A, but were found to exhibit surprisingly different responses to ATP.

[0079] NPP4 and NPP1 differ significantly in their responses to ATP. NPP1 rapidly hydrolyzes ATP to AMP and PPi, the latter being a potent inhibitor of extraosseous mineralization, and variants of NPP1 may be involved in diseases involving bone or soft tissue calcification, as outlined previously. To produce the observed products, ATP must bind to NPP1 in the same orientation as seen in the NPP1-AMP cocrystal structure. In stark contrast, NPP4 cleaves ATP very slowly, even though it binds to AMP in a manner very similar to NPP1. The superposition of these two enzymes reveals a significant structural difference in the area of ​​the terminal phosphate of ATP when bound like AMP. Energy minimization simulations of the ATP complex reveal that NPP1 provides a favorable environment for the γ-phosphate of ATP due to the presence of triplicate lysine claws that provide charge stabilization by inducible fit. In the absence of the substrate, the two lysine residues (Lys260 and Lys510, numbered by mouse) lining the upper ridge of the binding pocket are mobile, as demonstrated in the NPP1-AMP product complex (4GTW) where they are irregular, or in the NPP1-vanadate complex (4B56) where they exhibit high factor B and extend into the solvent. In the presence of ATP substrate binding, the highly negatively charged γ-phosphate should electrostatically attract these lysine residues, which, along with the fixed Lys237 on the floor of the binding pocket, should effectively encapsulate the terminal phosphate with a positive charge. Since PPi is even more negatively charged, this may also promote hydrolysis through product stabilization. The role of these lysine residues in NPP1 hydrolysis of ATP has not been previously understood and has been revealed through detailed structural comparisons with NPP4.

[0080] The superposition of corresponding regions of NPP4 has shown to be significantly less favorable to the similarly bound ATP, possessing a local structural mode that is more open, contains fewer positively charged residues, and incorporates negatively charged residues such as Asp335 protruding into the active site very close to the γ-phosphate of ATP. Consistent with the observation that NPP4 provides an unfavorable environment in the γ-phosphate region, attempts at co-crystallization with an uncleavable ATP analog revealed no recognizable binding. Similarly, attempts at co-crystallization with ATP or a cleavable ATP analog have shown that the AMP complex is produced over several days, taking several days for the crystal to grow, and although the ATP binding is weak, it occasionally comes very close enough to be hydrolyzed. The AMP product molecule can bind under identical conditions, reflecting a good, strong affinity. Product inhibition may be an inherent characteristic of the NPP reaction, as the phosphate group adjacent to Zn1 is, by definition, converted from a phosphodiester to a terminal phosphate with a greater negative charge. These data suggest that NPP4 is unlikely to effectively hydrolyze ATP in vivo, supporting the view that NPP1 is the major extracellular enzyme that metabolizes purinergic signals regulating bone regeneration and extracellular calcification.

[0081] In contrast, both NPP4 and NPP1 hydrolyze Ap3A to AMP and ADP, and NPP1 has a Michaelis constant for Ap3A that is approximately 30 times tighter than that of NPP4. This higher affinity is reflected in the lower concentrations of NPP1 required to induce platelet aggregation at the same concentration of Ap3A (Figure 10). Upon platelet activation, high concentrations of Ap3A stored within the dense granules of circulating platelets are released. Without wishing to be limited by any theory, by identifying NPP4 on cerebral vascular endothelium that can induce platelet aggregation via hydrolysis of physiologically concentrated Ap3A, vascularized NPPs may contribute to cerebral platelet aggregation. Ap3A has long been hypothesized to support stable thrombus formation by having a significantly longer lifetime in whole blood than ADP and by acting as a "chemically masked" source of ADP.

[0082] The ability of NPP1 to rapidly hydrolyze Ap3A to ADP raises the question of whether NPP1 can play a role in hemostasis. In this study, NPP1 was shown to be able to hydrolyze Ap3A at low nM concentrations, and either NPP1 or NPP4 at low nM concentrations promoted irreversible platelet aggregation in human PRP in vitro. This work suggests that either enzyme, when present at low nM concentrations in a thrombus-promoting environment, may significantly contribute to in vivo platelet aggregation.

[0083] Recently, polymorphisms in NPP1 have been identified as providing stroke protection in pediatric patients with sickle cell anemia. These results suggest that the stroke-protective NPP1 polymorphisms correspond to loss-of-function mutations that reduce Ap3A hydrolysis in the thrombotic microenvironment. Reduced NPP1 activity in cerebral capillaries is thought to lead to decreased ADP concentration in the cerebral capillary bed, thus providing a direct mechanism to explain the reduction in platelet aggregation and thrombus formation.

[0084] The K173Q mutation in human NPP1 is not found in the catalytic domain, but rather in the somatomedin B-2 domain near the transmembrane region (Figure 2B). Loss-of-function mutations within the NPP1 catalytic domain are consistent with their absence in populations screened for stroke protection rather than compatibility with human survival beyond the neonatal period. Without wishing to be limited by any theory, the NPP1 K173Q mutation may increase NPP1 serum concentrations similarly to the K121Q mutation observed in IDDM-2, but the hypothesis of gain-of-function NPP1 is not supported by these serum increases, particularly in light of kinetic and aggregation measurement data. The NPP1 serum concentration in the K121Q polymorphism (28 pM) is far below what is required to activate NPP1-induced platelet aggregation in vitro, and the Michaelis constants of NPP1 for both ATP and Ap3A are approximately 10 times lower than the 4 pM increase induced by the K121Q polymorphism. 6 The high levels suggest that this increase is unlikely to affect either PPi or ADP systemic concentration. Without wishing to be constrained by any particular theory, a possible mechanism for the changes attributable to the NPP1 K173Q polymorphism is that it may increase ectodomain shedding of vascular endothelial NPP1, which could explain both the increased serum levels of this protein and the loss of NPP1 activity on the vascular endothelium exposed at this point. In addition, although the means by which the K173Q mutation impairs NPP1 catalytic activity remains unclear, these findings support the idea that NPP1 polymorphisms protective against stroke are more likely to be loss-of-function mutations that reduce Ap3A hydrolysis on the endothelial surface of the cerebral capillary bed than gain-of-function mutations that increase PPi concentration.

[0085] In some embodiments, the present invention relates to compositions and methods for increasing the ATP hydrolysis level or activity of NPP1 polypeptides, fragments thereof, derivatives, variants, or variant fragments. In other embodiments, the present invention relates to compositions and methods for inducing and increasing the ATP hydrolysis level or activity of mutant NPP4 polypeptides or fragments thereof.

[0086] In some embodiments, the present invention relates to compositions and methods for reducing the Ap3A hydrolysis level or activity of NPP1 polypeptides, fragments thereof, derivatives, variants, or variant fragments. In other embodiments, the present invention relates to compositions and methods for reducing the Ap3A hydrolysis level or activity of mutant NPP4 polypeptides or fragments thereof.

[0087] The methods of the present invention include methods for treating or preventing disorders and diseases in which an increase in the activity or level of the NPP1 polypeptide, its fragments, derivatives, variants, or variant fragments is desirable. Accordingly, in some embodiments, the compositions of the present invention relate to activators of the NPP1 polypeptide, its fragments, derivatives, variants, or variant fragments. In various embodiments, disorders and diseases include, but are not limited to, IIAC, OPLL, hypophosphatemic rickets, osteoarthritis, and calcification of atherosclerotic plaques.

[0088] In various embodiments, mutant NPP1 polypeptides or fragments useful in the methods of the present invention have lower Ap3A hydrolysis activity compared to the corresponding wild-type NPP1 polypeptide or fragment. In various embodiments, mutant NPP1 polypeptides or fragments useful in the methods of the present invention have substantially the same ATP hydrolysis activity compared to the corresponding wild-type NPP1 polypeptide or fragment. In various embodiments, mutant NPP1 polypeptides or fragments useful in the methods of the present invention have lower Ap3A hydrolysis activity and substantially the same ATP hydrolysis activity compared to the corresponding wild-type NPP1 polypeptide or fragment.

[0089] In other embodiments, the methods of the present invention include methods for treating or preventing disorders and diseases in which an increase in the activity or level of a mutant NPP4 polypeptide or its fragments is desirable. Accordingly, in some embodiments, the compositions of the present invention relate to activators of mutant NPP4 polypeptides or their fragments. In various embodiments, disorders and diseases include, but are not limited to, IIAC, OPLL, hypophosphatemic rickets, osteoarthritis, and calcification of atherosclerotic plaques. In some embodiments, the present invention relates to compositions and methods for changing the enzymatic activity of a mutant NPP4 polypeptide or its fragments to a hydrolase, i.e., an enzyme having the same enzymatic activity as the NPP1 polypeptide, its fragments, derivatives, variants, or variant fragments.

[0090] In various embodiments, the mutant NPP4 polypeptide or fragment useful in the methods of the present invention has lower Ap3A hydrolysis activity compared to the corresponding wild-type NPP4 polypeptide or fragment. In other embodiments, the mutant NPP4 polypeptide or fragment useful in the methods of the present invention has substantially increased ATP hydrolysis activity compared to the corresponding wild-type NPP4 polypeptide or fragment. In yet another embodiment, the mutant NPP4 polypeptide or fragment useful in the methods of the present invention has lower Ap3A hydrolysis activity and substantially increased ATP hydrolysis activity compared to the corresponding wild-type NPP4 polypeptide or fragment.

[0091] In a further embodiment, the present invention relates to compositions and methods for increasing the level or activity of NPP1 polypeptides, fragments thereof, derivatives, variants, or variant fragments, and mutant NPP4 polypeptides or fragments thereof.

[0092] NPP1 therapeutic activator composition and method The present invention comprises NPP1 activator compositions and methods for increasing the level or activity of NPP1 or its variants. In various embodiments, the NPP1 activator compositions and therapeutic methods of the present invention increase the amount of NPP1 polypeptide, NPP1 mRNA, NPP1 enzyme activity, NPP1 substrate binding activity, its variants, or combinations thereof. In various embodiments, diseases and disorders in which a reduction in pathological calcification or ossification may improve therapeutic outcomes include, but are not limited to, IIAC, OPLL, hypophosphatemic rickets, osteoarthritis, and calcification of atherosclerotic plaques.

[0093] In various embodiments, mutant NPP1 useful in the methods of the present invention has lower Ap3A hydrolysis activity compared to the corresponding wild-type NPP1. In various embodiments, mutant NPP1 useful in the methods of the present invention has substantially the same ATP hydrolysis activity compared to the corresponding wild-type NPP1. In various embodiments, mutant NPP1 useful in the methods of the present invention has lower Ap3A hydrolysis activity and substantially the same ATP hydrolysis activity compared to the corresponding wild-type NPP1. In various embodiments, mutant NPP1 has a mutation at at least one position selected from the group consisting of Ser 532, Tyr 529, Tyr 451, Ile 450, Ser 381, Tyr 382, ​​Ser 377, Phe 346, Gly 531, Ser 289, Ser 287, Ala 454, Gly 452, Gln 519, Glu 526, Lys 448, Glu 508, Arg 456, Asp 276, Tyr 434, Gln 519, Ser 525, Gly 342, Ser 343, and Gly 536. Without wishing to be limited by any theory, the mutations envisioned within this invention are informed by the inventors' high-resolution structural determination of NPP4 and by the correct interpretation of the lysine graft in NPP1 that facilitates ATP hydrolysis by NPP1 (Figures 6-9).

[0094] Based on the disclosures provided herein, it will be understood by those skilled in the art that increasing the level of NPP1 or its variants includes increasing the expression, including transcription, translation, or both, of NPP1 or its variants. Those skilled in the art will also understand, once they have acquired the teachings of the present invention, that increasing the level of NPP1 or its variants also includes increasing the activity of NPP1 or its variants (e.g., enzyme activity, substrate binding activity, etc.). Therefore, increasing the level or activity of NPP1 or its variants includes, but is not limited to, increasing the amount of the NPP1 polypeptide or its variants, and increasing the transcription, translation, or both, of the nucleic acid encoding NPP1 or its variants; and similarly includes increasing any activity of the NPP1 polypeptide or its variants. The compositions and methods of the present invention may selectively activate NPP1 or its variants, or may activate both NPP1 or its variants and, in non-limiting examples, another molecule such as mutant NPP4.

[0095] Based on the disclosures provided herein, including the three-dimensional structure reported herein, an increase in the level of NPP1 activity or its variants is due to the affinity of this enzyme to ATP (K m ) or increase ATP PP by NPP1 i Metabolic turnover rate (k cat Those skilled in the art will understand that this involves manipulating specific residues in NPP1 to increase the Michaelis-Menten constant. Those skilled in the art will also understand that, once the teachings of the present invention and the three-dimensional structure reported herein are provided, this can be achieved by substitution of residues within the active site of NPP1, which is made possible by the disclosures, findings, and knowledge contained herein and described herein. Thus, the mutation of this effect is claimed to be a method of the art of this application.

[0096] Those skilled in the art will know that, once the three-dimensional structure described in the teachings of the present invention and the three-dimensional structure reported herein is present, reducing the thrombogenic activity of NPP1 or its variants depends on the affinity of NPP1 to Ap3A (K m ) reduces or reduces the turnover rate of Ap3A to ADP by NPP1 (k cat It will also be understood that this involves manipulating specific residues in NPP1 to result in a change in the Michaelis-Menten constant of this enzyme, thereby reducing the adenine-binding pocket of Ap3A. Therefore, mutations to achieve this effect are claimed as methods of the art of this application. Those skilled in the art will also understand that, once equipped with the teachings of the present invention and the three-dimensional structures reported herein, including the molecule of Ap3A docked within NPP1 as detailed in Figures 9 and 11, this can be achieved by substitution or modification of amino acids lining the adenine-binding pocket of NPP4 to amino acids that increase the occupied space of amino acids that reduce or eliminate the adenine-binding pocket of Ap3A, as made possible by the disclosures, knowledge, molecular structure of NPP1, and the molecular structure of the model of NPP1 bound to Ap3A as illustrated in Figures 6-7, 9, and 11, as well as other knowledge. Therefore, mutations to achieve this effect are claimed as methods of the art of this application.

[0097] Accordingly, the present invention relates to the prevention and treatment of diseases or disorders by administration of NPP1 polypeptides, recombinant NPP1 polypeptides, mutant NPP1 polypeptides, active NPP1 polypeptide fragments, or activators of NPP1 expression or activity. In one embodiment, the NPP1 polypeptide or its variants are soluble. In another embodiment, the NPP1 polypeptide or its variants are recombinant NPP1 polypeptides. In one embodiment, the NPP1 polypeptide or its variants include NPP1 polypeptides lacking the NPP1 transmembrane domain. In another embodiment, the NPP1 polypeptide or its variants include NPP1 polypeptides in which the NPP1 transmembrane domain has been removed and replaced, in non-limiting examples, with the transmembrane domain of another polypeptide, such as NPP2.

[0098] In some embodiments, the NPP1 polypeptide or its variants include an IgG Fc domain. In other embodiments, the NPP1 polypeptide or its variants include a polyaspartate domain containing approximately 2 to 20 or more consecutive aspartate residues to enable the NPP1 polypeptide to target bone. In some embodiments, the NPP1 polypeptide or its variants include an IgG Fc domain and a polyaspartate domain containing approximately 2 to 20 or more consecutive aspartate residues. In other embodiments, the NPP1 protein or its variants are truncated to remove a nuclease domain. In certain embodiments, the NPP1 protein or its variants are truncated to remove a nuclease domain of approximately 524 to 885 residues relative to SEQ ID NO: 1, leaving only a catalytic domain of approximately 186 to 586 residues relative to SEQ ID NO: 1, which helps to prevent the loss of catalytic activity of the protein.

[0099] Those skilled in the art will understand that an increase in the level of NPP1 or its variants includes an increase in the amount of NPP1 or its variants (e.g., by increasing NPP1 protein expression through administration of NPP1, its fragments, or its variants). In addition, those skilled in the art will understand that an increase in the level of NPP1 or its variants includes an increase in NPP1 activity. Thus, increasing the level or activity of NPP1 or its variants includes, but is not limited to, administration of NPP1, its fragments, or its variants, and increasing the transcription, translation, or both of the nucleic acids encoding NPP1 or its variants; and it also includes increasing any activity of NPP1 or its variants.

[0100] Increases in the level or activity of NPP1 or its variants can be assessed using a wide variety of methods, including those disclosed herein, as well as methods known in the art or to be developed in the future. In other words, a routine worker will understand, based on the disclosures provided herein, that increases in the level or activity of NPP1 or its variants can be easily assessed using methods that assess the level of nucleic acid (e.g., mRNA) encoding NPP1 or its variants, the level of NPP1 polypeptide or its variants, and / or the level of activity in biological samples obtained from the subject, thereby altering the Michaelis-Menten reaction kinetics for the turnover of substrates and products described herein.

[0101] Those skilled in the art will understand, based on the disclosures provided herein, that the present invention is useful in subjects that are being treated or can be treated for pathological calcification or ossification holistically (e.g., systemically) or partially (e.g., locally, in tissues, organs). In one embodiment, the present invention is useful in treating or preventing pathological calcification or ossification. Those skilled in the art will understand, based on the teachings provided herein, that the diseases and disorders treatable by the compositions and methods described herein include any diseases or disorders in which a reduction in calcification or ossification is considered to promote a positive therapeutic outcome.

[0102] Those skilled in the art will realize that, in addition to directly activating NPP1 or its variants, reducing the amount or activity of molecules that themselves reduce the amount or activity of NPP1 or its variants can also increase the amount or activity of NPP1 or its variants. Therefore, activators may include, but should not be considered limited to, chemical compounds, proteins, peptide mimetic molecules, antibodies, ribozymes, and antisense nucleic acid molecules. Those skilled in the art will readily understand, based on the disclosures provided herein, that activators include chemical compounds that increase the level, enzyme activity, or substrate-binding activity of NPP1 or its variants. In addition, as is well known to those skilled in the art of chemistry, activators also include chemically modified compounds and derivatives.

[0103] Based on the disclosures provided herein, it will be understood by those skilled in the art that increasing the level of NPP1 or its variants includes increasing the expression of NPP1 or its variants, including transcription, translation, or both. Those skilled in the art will also understand, once they have acquired the teachings of the present invention, that increasing the level of NPP1 or its variants also includes increasing the activity of NPP1 or its variants (e.g., enzyme activity, substrate binding activity, etc.). Therefore, increasing the level or activity of NPP1 or its variants includes, but is not limited to, increasing the amount of the NPP1 polypeptide or its variants, increasing the transcription, translation, or both of the nucleic acids encoding NPP1 or its variants; and similarly includes increasing any activity of the NPP1 polypeptide or its variants. The activator compositions and methods of the present invention may selectively activate NPP1 or its variants, or may activate both NPP1 or its variants and, in non-limited examples, other molecules such as NPP4.

[0104] Accordingly, the present invention relates to the administration of NPP1 polypeptides, recombinant NPP1 polypeptides, mutant NPP1 polypeptides, active NPP1 polypeptide fragments, or activators of NPP1 expression or activity. In one embodiment, the NPP1 polypeptide or its variants are soluble. In another embodiment, the NPP1 polypeptide or its variants are recombinant polypeptides. In one embodiment, the NPP1 polypeptide or its variants include NPP1 polypeptides or their variants that lack the NPP1 transmembrane domain. In another embodiment, the NPP1 polypeptide or its variants include NPP1 polypeptides or their variants in which the NPP1 transmembrane domain or its variants have been removed and replaced, in non-limiting examples, with the transmembrane domain of another polypeptide, such as NPP2.

[0105] Furthermore, those skilled in the art, given the methods illustrated herein and herein, will understand that NPP1 activators include activators that may be identified by well-known standards in the art of pharmacology, such as physiological results of NPP1 activation described herein and / or known in the art, as described herein in detail. Accordingly, the present invention is by no means limited to any specific NPP1 activator or mutant NPP1 activator illustrated or disclosed herein, but rather encompasses such activators, both known in the art and those that may be discovered in the art, that are understood to be useful to those who perform their work routinely.

[0106] Further methods for identifying and producing NPP1 activators are well known to those skilled in the art, including but not limited to obtaining activators from naturally occurring sources (e.g., Streptomyces, Pseudomonas, Stylotella aurantium, etc.). Alternatively, NPP1 activators can be chemically synthesized. Furthermore, anyone working routinely will understand, based on the teachings provided herein, that NPP1 activators can be obtained from recombinant protein expression systems, including but not limited to mammalian protein expression systems, insect cell protein expression systems, and yeast protein expression systems. Compositions and methods for chemically synthesizing NPP1 activators or mutant NPP1 activators, and for obtaining them from natural sources, are well known and described in the art.

[0107] Those skilled in the art will understand that activators can be administered as small molecule chemicals, proteins, protein-coding nucleic acid constructs, or combinations thereof. Many vectors and other compositions and methods are well known for administering proteins or protein-coding nucleic acid constructs to cells or tissues. Therefore, the present invention includes methods for administering a protein or protein-coding nucleic acid that is an activator of NPP1 or its variants (Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York; Ausubel et al., 1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York).

[0108] Those skilled in the art will notice that reducing the amount or activity of a molecule that itself reduces the amount or activity of NPP1 or its variants can have the effect of increasing the amount or activity of NPP1 or its variants. An antisense oligonucleotide is a DNA or RNA molecule that is complementary to a portion of an mRNA molecule. When present in a cell, an antisense oligonucleotide hybridizes to an existing mRNA molecule and inhibits its translation into a gene product. Inhibiting gene expression using antisense oligonucleotides is well known in the art, as is the method of expressing antisense oligonucleotides in cells (Inoue, U.S. Patent No. 5,190,931) (Marcus-Sekura, 1988, Anal. Biochem. 172:289). The method of the present invention involves the use of an antisense oligonucleotide to reduce the amount of a molecule that causes a decrease in the amount or activity of NPP1 or its variants, thereby increasing the amount or activity of NPP1 or its variants. An antisense oligonucleotide synthesized and supplied to cells by a method well known to those skilled in the art is contemplated in the present invention. As an example, antisense oligonucleotides can be synthesized to have a nucleotide length of about 10 to about 100, more preferably about 15 to about 50. The synthesis of nucleic acid molecules is well known in the art, as is the synthesis of modified antisense oligonucleotides that have improved biological activity compared to unmodified antisense oligonucleotides (U.S. Patent No. 5,023,243).

[0109] Similarly, gene expression can be inhibited by the hybridization of antisense molecules to the gene's promoter or other regulatory elements, thereby affecting the transcription of the gene. Methods for identifying promoters or other regulatory elements that interact with genes of interest are well known in this field, including methods such as the yeast two-hybrid system (Bartel and Fields, eds., The Yeast Two Hybrid System, Oxford University Press, Cary, NC).

[0110] Alternatively, inhibition of genes expressing proteins that reduce the level or activity of NPP1 or its variants can be achieved by the use of ribozymes. The use of ribozymes to inhibit gene expression is well known to those skilled in the art (see, for example, Cech et al., 1992, J. Biol. Chem. 267:17479; Hampel et al., 1989, Biochemistry 28:4929; Altman et al., U.S. Patent No. 5,168,053). Ribozymes are catalytic RNA molecules that have the ability to cleave other single-stranded RNA molecules. Ribozymes are known to be sequence-specific and can therefore be modified to recognize specific nucleotide sequences (Cech, 1988, J. Amer. Med. Assn. 260:3030), enabling selective cleavage of specific mRNA molecules. With the information provided in this disclosure and the references incorporated herein, a person skilled in the art could synthesize antisense oligonucleotides or ribozymes without excessive experimentation, taking into account the nucleotide sequence of the molecule.

[0111] Those skilled in the art will understand that NPP1 activators, NPP1 polypeptides, recombinant NPP1 polypeptides, mutant NPP1 polypeptides, or active NPP1 polypeptide fragments may be administered alone or in any combination thereof. Those skilled in the art will also understand that appropriate administration may be acute (e.g., over a short period such as a day, a week, or a month) or chronic (e.g., over a long period such as several weeks, several months, a year, or longer). Furthermore, NPP1 polypeptides, recombinant NPP1 polypeptides, mutant NPP1 polypeptides, or active NPP1 polypeptide fragments may be administered alone or in any combination thereof in a temporal sense, in that they may be administered simultaneously, before and / or after each other. Those skilled in the art will understand, based on the disclosures provided herein, that pathological calcification or ossification can be treated or prevented using NPP1 polypeptides, recombinant NPP1 polypeptides, mutant NPP1 polypeptides, or active NPP1 polypeptide fragments, and that therapeutic results can be obtained using activators alone or in any combination with other NPP1 polypeptides, recombinant NPP1 polypeptides, active NPP1 polypeptide fragments, or NPP1 activators.

[0112] With the present disclosure, including the methods detailed herein, it will be understood by those skilled in the art that the present invention is not limited to the treatment of a disease or disorder once established. In particular, the symptoms of the disease or disorder do not need to be present to the point in time when they are harmful to the subject, and in fact, the disease or disorder does not need to be detected in the subject before treatment is administered. That is, a serious pathological condition from the disease or disorder does not need to occur before the present invention can provide any benefit. Accordingly, as more fully described herein, the present invention includes methods for preventing disease and disorder in a subject, in that an NPP1 polypeptide, its fragments, derivatives, or variants, or an NPP1 activator or mutant NPP1 activator, as discussed elsewhere herein, can be administered to the subject before the onset of the disease or disorder, thereby preventing the onset of the disease or disorder.

[0113] Those skilled in the art will understand, with the disclosures herein, that prevention of disease or impairment in a subject includes administering to the subject an NPP1 polypeptide, recombinant NPP1 polypeptide, mutant NPP1 polypeptide, active NPP1 polypeptide fragment, or NPP1 activator as a preventive measure against disease or impairment. In one embodiment, the NPP1 polypeptide is soluble. In another embodiment, the NPP1 polypeptide is recombinant NPP1 polypeptide. In one embodiment, the NPP1 polypeptide includes an NPP1 polypeptide lacking the NPP1 transmembrane domain. In another embodiment, the NPP1 polypeptide includes an NPP1 polypeptide in which the NPP1 transmembrane domain has been removed and replaced, in non-limiting examples, with the transmembrane domain of another polypeptide, such as NPP2.

[0114] In some embodiments, the NPP1 polypeptide includes an IgG Fc domain. In other embodiments, the NPP1 polypeptide includes a polyaspartate domain containing approximately 2 to 20 or more consecutive aspartate residues to enable the NPP1 polypeptide to target bone. In some embodiments, the NPP1 polypeptide includes an IgG Fc domain and a polyaspartate domain containing approximately 2 to 20 or more consecutive aspartate residues. In other embodiments, the NPP1 protein is truncated to remove the nuclease domain. In certain embodiments, the NPP1 protein is truncated to remove the nuclease domain of approximately 524 to 885 residues relative to SEQ ID NO: 1, leaving only the catalytic domain of approximately 186 to 586 residues relative to SEQ ID NO: 1, which helps to prevent the loss of catalytic activity of the protein.

[0115] As will be discussed more thoroughly elsewhere in this specification, methods for increasing the level or activity of NPP1 encompass a wide range of techniques for increasing not only NPP1 activity but also the expression of the nucleic acid encoding NPP1. In addition, as will be disclosed elsewhere in this specification, those skilled in the art will understand, once they have acquired the teachings provided herein, that the present invention encompasses methods for preventing a wide variety of diseases or disorders in which the disease or disorder is modulated, treated, or prevented by increasing the expression and / or activity of NPP1. Furthermore, the present invention encompasses the treatment or prevention of such diseases or disorders that may be discovered in the future.

[0116] The present invention encompasses the administration of NPP1 polypeptide, recombinant NPP1 polypeptide, mutant NPP1 polypeptide, active NPP1 polypeptide fragment, NPP1 activator, or mutant NPP4 polypeptide modified to exhibit NPP1-like ATP hydrolase activity for practicing the methods of the present invention, and those skilled in the art will understand, based on the disclosures provided herein, how to formulate and administer an appropriate NPP1 polypeptide, recombinant NPP1 polypeptide, active NPP1 polypeptide fragment, or NPP1 activator to a subject. However, the present invention is not limited to any particular method of administration or therapeutic regimen. This is especially true when it will be understood by those skilled in the art, having received the disclosures provided herein, that the method of administering NPP1 polypeptide, recombinant NPP1 polypeptide, mutant NPP1 polypeptide, active NPP1 polypeptide fragment, or NPP1 activator can be determined by those skilled in the art in the pharmacological art, including practice using models recognized in the art for pathological calcification or ossification.

[0117] NPP4 therapeutic activator composition and method In various embodiments, the present invention comprises NPP4 activator compositions and methods for increasing the level or activity of NPP4. In various embodiments, the NPP4 activator compositions and therapeutic methods of the present invention increase the amount of NPP4 polypeptide, the amount of NPP4 mRNA, the amount of NPP4 enzyme activity, the amount of NPP4 substrate binding activity, or a combination thereof. In various embodiments, diseases and disorders in which a reduction in pathological calcification or ossification may improve therapeutic outcomes include, but are not limited to, IIAC, OPLL, hypophosphatemic rickets, osteoarthritis, and calcification of atherosclerotic plaques.

[0118] Based on the disclosures provided herein, including the three-dimensional structure reported herein, increasing the ATP hydrolysis activity of NPP4 or its variants involves increasing the affinity of this enzyme to ATP (K m(increasing), or increasing the metabolic turnover rate of ATP by NPP4 (k i increasing)), the manipulation of specific amino acids in NPP4 to affect the change of the Michaelis-Menten constant will be understood by those skilled in the art. Once those skilled in the art have the teachings of the present invention, including the three-dimensional structure reported herein, they will be able to perform a detailed analysis of the active sites of the two enzymes illustrated in FIG. 9, and reproduce the lysine claws in NPP1 within NPP4. It will also be understood that this can be achieved by substituting residues within the active site of NPP1 into the active site of NPP4, which is enabled by the disclosure, findings, and knowledge contained and described herein, including but not limited to those. Therefore, the mutations of this effect are claimed as a method of the technology according to this application.

[0119] Once those skilled in the art have the teachings of the present invention and the three-dimensional structure reported herein, to reduce the thrombus-forming promoting activity of NPP4 or its variants, it is necessary to reduce the affinity of NPP4 for Ap3A (K m decreasing), or decreasing the metabolic turnover rate of Ap3A to ADP by NPP1 (k cat decreasing)), it will also be understood that the change of the Michaelis-Menten constant of this enzyme is included. Once those skilled in the art have the teachings of the present invention and the three-dimensional structure reported herein, including the molecule of Ap3A docked within NPP4, they will be able to substitute or change the amino acids lining the adenine-binding pocket of NPP4 with amino acids that increase the occupancy space of the amino acids that reduce or eliminate the adenine-binding pocket of Ap3A within NPP4, which is enabled by the disclosure, findings, the molecular structure of NPP4, and the molecular structure of the model of NPP4 binding to Ap3A detailed in FIGS. 6-7, 9, and 11, and other knowledge described herein. It will also be understood that this can be achieved. Therefore, the mutations of this effect are claimed as a method of the technology according to this application.

[0120] ​​Based on the disclosures provided herein, it will be understood by those skilled in the art that an increase in NPP4 levels includes an increase in NPP4 expression, including transcription, translation, or both. Those skilled in the art will also understand, once they have acquired the teachings of the present invention, that an increase in NPP4 levels also includes an increase in NPP4 activity (e.g., enzyme activity, substrate binding activity, etc.). Therefore, increasing the level or activity of NPP4 includes, but is not limited to, increasing the amount of NPP4 polypeptide and increasing the transcription, translation, or both of the nucleic acid encoding NPP4; and similarly includes increasing any activity of the NPP4 polypeptide. The NPP4 activator compositions and methods of the present invention may selectively activate NPP4, or may activate both NPP4 and, in non-limiting examples, another molecule such as NPP1.

[0121] Accordingly, the present invention relates to the prevention and treatment of diseases or disorders by administration of NPP4, including NPP4 polypeptide, recombinant NPP4 polypeptide, mutant NPP4 polypeptide, active NPP4 polypeptide fragment, or activators of NPP4 expression or activity. In one embodiment, NPP4 is soluble. In another embodiment, NPP4 is a recombinant NPP4 polypeptide. In one embodiment, NPP4 includes an NPP4 polypeptide lacking the NPP4 transmembrane domain. In another embodiment, NPP4 includes an NPP4 polypeptide in which the NPP4 transmembrane domain has been removed and replaced with the transmembrane domain of another polypeptide. In one embodiment, NPP4 includes an NPP4 polypeptide lacking the NPP4 cytoplasmic domain. In another embodiment, NPP4 includes an NPP4 polypeptide in which the NPP4 cytoplasmic domain has been removed and replaced with the cytoplasmic domain of another polypeptide. In yet another embodiment, NPP4 is modified to exhibit NPP1-like ATP hydrolysis activity. In one embodiment, the mutant NPP4 polypeptide is modified to exhibit NPP1-like ATP hydrolysis activity and is fused to an IgG Fc and / or polyaspartate domain, as described elsewhere in this specification. In one embodiment, the mutant NPP4 polypeptide includes at least one mutation that alters the substrate selectivity of the NPP4 polypeptide from Ap3A to ATP. In various embodiments, the mutant NPP4 polypeptide includes, as a non-limiting example, at least one mutation that alters the substrate selectivity of the NPP4 polypeptide from Ap3A to ATP, such as D335, S92, D264, L265, S330, Q331, K332, or T323 for SEQ NO ID: 3.

[0122] Those skilled in the art will understand that an increase in NPP4 levels includes an increase in the amount of NPP4 (for example, by increasing NPP4 protein expression through administration of NPP4 or its variants or fragments). In addition, those skilled in the art will understand that an increase in NPP4 levels includes an increase in NPP4 activity. Therefore, increasing the level or activity of NPP4 includes, but is not limited to, administration of NPP4 or its variants or fragments, and increasing the transcription, translation, or both of the nucleic acid encoding NPP4, and similarly includes increasing any activity of NPP4.

[0123] An increase in NPP4 levels or activity can be assessed using a wide variety of methods, including those disclosed herein and those known or to be developed in the art. In other words, a routine worker would understand, based on the disclosures provided herein, that an increase in NPP4 levels or activity can be easily assessed using methods for assessing the levels of the nucleic acid encoding NPP4 (e.g., mRNA), the NPP4 polypeptide, and / or the level of NPP4 activity in a biological sample obtained from a subject.

[0124] Those skilled in the art will understand, based on the disclosures provided herein, that the present invention is useful in subjects being treated or potentially treated for pathological calcification or ossification, either holistically (e.g., systemically) or partially (e.g., locally, in tissues, organs). In one embodiment, the present invention is useful in treating or preventing pathological calcification or ossification. Those skilled in the art will understand, based on the teachings provided herein, that the diseases and disorders treatable by the compositions and methods described herein include any diseases or disorders in which a reduction in calcification or ossification is considered to promote a positive therapeutic outcome.

[0125] Those skilled in the art will notice that, in addition to directly activating NPP4, reducing the amount or activity of molecules that themselves reduce the amount or activity of NPP4 can also increase the amount or activity of NPP4. Therefore, NPP4 activators may include, but should not be considered limited to, chemical compounds, proteins, peptide mimetic molecules, antibodies, ribozymes, and antisense nucleic acid molecules. Those skilled in the art will readily understand, based on the disclosures provided herein, that NPP4 activators encompass chemical compounds that increase the level, enzyme activity, or substrate-binding activity of NPP4. Furthermore, as is well known to those skilled in the art of chemistry, NPP4 activators also encompass chemically modified compounds and derivatives.

[0126] Based on the disclosures provided herein, it will be understood by those skilled in the art that an increase in NPP4 levels includes an increase in NPP4 expression, including transcription, translation, or both. Those skilled in the art will also understand, once they have acquired the teachings of the present invention, that an increase in NPP4 levels also includes an increase in NPP4 activity (e.g., enzyme activity, substrate binding activity, etc.). Therefore, increasing the level or activity of NPP4 includes, but is not limited to, increasing the amount of NPP4 polypeptide, increasing the transcription, translation, or both of the nucleic acid encoding NPP4; and similarly includes increasing any activity of the NPP4 polypeptide. The NPP4 activator compositions and methods of the present invention may selectively activate NPP4, or activate both NPP4 and, in non-limiting examples, another molecule such as NPP1. Accordingly, the present invention relates to the administration of NPP4 polypeptides, recombinant NPP4 polypeptides, mutant NPP4 polypeptides, active NPP4 polypeptide fragments, or activators of NPP4 expression or activity. In one embodiment, NPP4 is soluble. In another embodiment, NPP4 is a recombinant NPP4 polypeptide. In one embodiment, NPP4 includes an NPP4 polypeptide lacking the NPP4 transmembrane domain. In another embodiment, NPP4 includes an NPP4 polypeptide in which the NPP4 transmembrane domain has been removed and replaced with the transmembrane domain of another polypeptide. In one embodiment, NPP4 includes an NPP4 polypeptide lacking the NPP4 cytoplasmic domain. In another embodiment, NPP4 includes an NPP4 polypeptide in which the NPP4 cytoplasmic domain has been removed and replaced with the cytoplasmic domain of another polypeptide. In yet another embodiment, NPP4 is modified to exhibit NPP1-like ATP hydrolysis activity. In one embodiment, NPP4 modified to exhibit NPP1-like ATP hydrolysis activity, as described elsewhere in this specification, is fused to an IgG Fc and / or polyaspartate domain.In one embodiment, the mutant NPP4 polypeptide comprises at least one mutation that alters the substrate selectivity of the NPP4 polypeptide from Ap3A to ATP. In various embodiments, the NPP4 polypeptide comprises, as a non-limiting example, at least one mutation that alters the substrate selectivity of the NPP4 polypeptide from Ap3A to ATP, such as D335, S92, D264, L265, S330, Q331, K332, or T323 for SEQ ID NO: 3.

[0127] Furthermore, those skilled in the art, given the methods illustrated herein and herein, will understand that NPP4 activators include activators that may be identified by well-known standards in the art of pharmacology, such as physiological results of NPP4 activation described herein and / or known in the art, as described herein in detail. Accordingly, the present invention is by no means limited to any specific NPP4 activator illustrated or disclosed herein, but rather encompasses such activators, both known in the art and those that may be discovered in the art, that are understood to be useful to those who perform the job routinely.

[0128] Further methods for identifying and producing NPP4 activators are well known to those skilled in the art, including but not limited to obtaining activators from naturally occurring sources (e.g., Streptomyces, Pseudomonas, Stylotera aurantium, etc.). Alternatively, NPP4 activators can be chemically synthesized. Furthermore, anyone working routinely will understand, based on the teachings provided herein, that NPP4 activators can be obtained from recombinant organisms. Compositions and methods for chemically synthesizing NPP4 activators and obtaining them from natural sources are well known and described in the art.

[0129] Those skilled in the art will understand that activators can be administered as small molecule chemicals, proteins, protein-coding nucleic acid constructs, or combinations thereof. Many vectors and other compositions and methods are well known for administering proteins or protein-coding nucleic acid constructs to cells or tissues. Therefore, the present invention includes a method for administering a protein or protein-coding nucleic acid that is an activator of NPP4 (Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York; Ausubel et al., 1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York).

[0130] Those skilled in the art will notice that reducing the amount or activity of a molecule that itself reduces the amount or activity of NPP4 can have the effect of increasing the amount or activity of NPP4. An antisense oligonucleotide is a DNA or RNA molecule that is complementary to a portion of an mRNA molecule. When present in a cell, an antisense oligonucleotide hybridizes to an existing mRNA molecule and inhibits its translation into a gene product. Inhibiting gene expression using antisense oligonucleotides is well known in the art, as is the method of expressing antisense oligonucleotides in cells (Inoue, U.S. Patent No. 5,190,931) (Marcus-Sekura, 1988, Anal. Biochem. 172:289). The method of the present invention involves the use of an antisense oligonucleotide to reduce the amount of a molecule that causes a decrease in the amount or activity of NPP4, thereby increasing the amount or activity of NPP4. An antisense oligonucleotide synthesized and supplied to cells by a method well known to those skilled in the art is intended in the present invention. As an example, an antisense oligonucleotide may be synthesized to have a nucleotide length of about 10 to about 100, more preferably about 15 to about 50. The synthesis of nucleic acid molecules is well known in the art, as is the synthesis of modified antisense oligonucleotides that have improved biological activity compared to unmodified antisense oligonucleotides (Tullis, 1991; U.S. Patent No. 5,023,243).

[0131] Similarly, gene expression can be inhibited by the hybridization of antisense molecules to the gene's promoter or other regulatory elements, thereby affecting the transcription of the gene. Methods for identifying promoters or other regulatory elements that interact with genes of interest are well known in this field, including methods such as the yeast two-hybrid system (Bartel and Fields, eds., The Yeast Two Hybrid System, Oxford University Press, Cary, NC).

[0132] Alternatively, inhibition of genes expressing proteins that reduce NPP4 levels or activity can be achieved by the use of ribozymes. The use of ribozymes to inhibit gene expression is well known to those skilled in the art (see, for example, Cech et al., 1992, J. Biol. Chem. 267:17479; Hampel et al., 1989, Biochemistry 28:4929; Altman et al., U.S. Patent No. 5,168,053). Ribozymes are catalytic RNA molecules that have the ability to cleave other single-stranded RNA molecules. Ribozymes are known to be sequence-specific and can therefore be modified to recognize specific nucleotide sequences (Cech, 1988, J. Amer. Med. Assn. 260:3030), enabling selective cleavage of specific mRNA molecules. With the information provided in this disclosure and the references incorporated herein, a person skilled in the art could synthesize antisense oligonucleotides or ribozymes without excessive experimentation, taking into account the nucleotide sequence of the molecule.

[0133] Those skilled in the art will understand that NPP4 activators, NPP4 polypeptides, recombinant NPP4 polypeptides, mutant NPP4 polypeptides, or active NPP4 polypeptide fragments may be administered alone or in any combination thereof. Those skilled in the art will also understand that appropriate administration may be acute (e.g., over a short period such as a day, a week, or a month) or chronic (e.g., over a long period such as several weeks, several months, a year, or longer). Furthermore, NPP4 polypeptides, recombinant NPP4 polypeptides, mutant NPP4 polypeptides, or active NPP4 polypeptide fragments may be administered alone or in any combination thereof in a temporal sense, in that they may be administered simultaneously, before and / or after each other. Those skilled in the art will understand, based on the disclosures provided herein, that pathological calcification or ossification can be treated or prevented using NPP4 polypeptides, recombinant NPP4 polypeptides, mutant NPP4 polypeptides, or active NPP4 polypeptide fragments, and that therapeutic results can be obtained using activators alone or in any combination with other NPP4 polypeptides, recombinant NPP4 polypeptides, active NPP4 polypeptide fragments, or NPP4 activators.

[0134] With the present disclosure, including the methods detailed herein, it will be understood by those skilled in the art that the present invention is not limited to the treatment of a disease or disorder once established. In particular, the symptoms of the disease or disorder do not need to be present to the point in time when they are harmful to the subject, and in fact, the disease or disorder does not need to be detected in the subject before treatment is administered. That is, a serious pathological condition from the disease or disorder does not need to occur before the present invention can provide any benefit. Accordingly, as more fully described herein, the present invention includes methods for preventing diseases and disorders in a subject, in that an NPP4 polypeptide, or a fragment, derivative, or variant thereof, or an NPP4 activator, as discussed elsewhere herein, can be administered to the subject before the onset of the disease or disorder, thereby preventing the onset of the disease or disorder.

[0135] Those skilled in the art will understand, with the disclosures herein, that prevention of disease or impairment in a subject includes administering NPP4 to the subject as a preventive measure against disease or impairment, including NPP4 polypeptide, recombinant NPP4 polypeptide, mutant NPP4 polypeptide, active NPP4 polypeptide fragment, or NPP4 activator. In one embodiment, NPP4 is soluble. In another embodiment, NPP4 is recombinant NPP4 polypeptide. In one embodiment, NPP4 includes an NPP4 polypeptide lacking the NPP4 transmembrane domain. In another embodiment, NPP4 includes an NPP4 polypeptide in which the NPP4 transmembrane domain has been removed and replaced with the transmembrane domain of another polypeptide. In one embodiment, NPP4 includes an NPP4 polypeptide lacking the NPP4 cytoplasmic domain. In another embodiment, NPP4 includes an NPP4 polypeptide in which the NPP4 cytoplasmic domain has been removed and replaced with the cytoplasmic domain of another polypeptide. In yet another embodiment, NPP4 is modified to exhibit NPP1-like ATP hydrolysis activity. In one embodiment, NPP4 modified to exhibit NPP1-like ATP hydrolysis activity, as described elsewhere in this specification, is fused to an IgG Fc and / or polyaspartate domain. In one embodiment, the mutant NPP4 polypeptide includes at least one mutation that alters the substrate selectivity of the NPP4 polypeptide from Ap3A to ATP. In various embodiments, the mutant NPP4 polypeptide includes, as a non-limiting example, at least one mutation that alters the substrate selectivity of the NPP4 polypeptide from Ap3A to ATP, such as D335, S92, D264, L265, S330, Q331, K332, or T323 for SEQ ID NO: 3.

[0136] As will be discussed more thoroughly elsewhere in this specification, methods for increasing the level or activity of NPP4 encompass a wide range of techniques for increasing not only NPP4 activity but also the expression of the nucleic acid encoding NPP4. In addition, as will be disclosed elsewhere in this specification, those skilled in the art will understand, once they have acquired the teachings provided herein, that the present invention encompasses methods for preventing a wide variety of diseases or disorders in which the disease or disorder is modulated, treated, or prevented by increasing the expression and / or activity of NPP4. Furthermore, the present invention encompasses the treatment or prevention of such diseases or disorders that may be discovered in the future.

[0137] The present invention encompasses the administration of NPP4, including NPP4 polypeptides, recombinant NPP4 polypeptides, mutant NPP4 polypeptides, active NPP4 polypeptide fragments, or NPP4 activators, for practicing the methods of the present invention, and those skilled in the art will understand, based on the disclosures provided herein, how to formulate and administer appropriate NPP4 polypeptides, recombinant NPP4 polypeptides, active NPP4 polypeptide fragments, or NPP4 activators to a subject. However, the present invention is not limited to any particular method of administration or therapeutic regimen. This is especially true when it will be understood by those skilled in the art, having received the disclosures provided herein, including the practice using models recognized in the art for pathological calcification or ossification, that the method of administering NPP4 polypeptides, recombinant NPP4 polypeptides, mutant NPP4 polypeptides, active NPP4 polypeptide fragments, or NPP4 activators can be determined by those skilled in the art in the pharmacological art.

[0138] Those skilled in the art will recognize or be able to find that, at most, routine experiments will be used, and many equivalents of the specific procedures, embodiments, claims, and examples described herein will be used. Such equivalents are considered to be within the scope of the present invention and covered by the accompanying claims. For example, modifications of reaction time, reaction size / volume, and experimental reagents such as solvents and catalysts, pressure, atmospheric conditions such as nitrogen atmosphere, and reducing / oxidizing agents, using substitutes recognized in the art and, at most, routine experiments, should be understood to be within the scope of this application.

[0139] Whenever values ​​and ranges are provided herein, it should be understood that all values ​​and ranges encompassed by these values ​​and ranges are included within the scope of the present invention. Furthermore, all values ​​that fall within these ranges, as well as any upper or lower limits on the ranges of values, are also contemplated in this application.

[0140] The following embodiments further illustrate aspects of the present invention. However, they are by no means limitations of the teachings or disclosures of the present invention expressed herein. [Examples]

[0141] The present invention is described herein by reference to the following embodiments. These embodiments are provided for illustrative purposes only, and the present invention is not limited to these embodiments, but rather encompasses all variations that become apparent as a result of the teachings provided herein.

[0142] Cloning and expression NPP1 and NPP4 are transmembrane proteins that are localized to the cell surface and possess separate intramembrane domains. For example, NPP1 is type II oriented, while NPP4 is type I oriented. In contrast, NPP2 is synthesized as a preproprotein and, after proteolytic processing, is secreted as a soluble protein after cleavage of the extracellular domain by furin (Jansen et al., 2005, J. Cell Sci. 118:3081-3089). To express NPP4 as a soluble recombinant protein in baculovirus, the cytoplasmic and transmembrane domains were removed from the protein construct. In contrast, to express NPP1 as a soluble extracellular protein, the transmembrane domain of NPP1 was replaced with the transmembrane domain of NPP2, resulting in the accumulation of soluble recombinant NPP1 in the extracellular fluid of baculovirus cultures.

[0143] NPP1 can be made soluble by removing the transmembrane domain. Human NPP1 (NCBI accession NP_006199) was modified to express a soluble recombinant protein by replacing its transmembrane region (e.g., residues 77-98) with the corresponding subdomain of human NPP2 (NCBI accession NP_001124335, e.g., residues 12-30). The modified NPP1 sequence was cloned into a modified pFastbac HT vector containing a TEV protease cleavage site followed by a C-terminal 9-HIS tag, and then cloned and expressed in insect cells. Both proteins were also expressed in a baculovirus system as previously described (Albright et al., 2012, Blood 120:4432-4440; Saunders et al., 2011, J. Biol. Chem. 18:994-1004; Saunders et al., 2008, Mol. Cancer Ther. 7:3352-3362), resulting in the accumulation of soluble recombinant proteins in the extracellular fluid (Figures 2-3).

[0144] array NPP1 amino acid sequence (NCBI accession NP_006199) (SEQ ID NO: 1) TIFF0007876894000002.tif114150NPP2 amino acid sequence (NCBI accession NP_001124335) (SEQ ID NO: 2) TIFF0007876894000003.tif106150NPP4 amino acid sequence (NCBI accession AAH18054.1) (SEQ ID NO: 3) TIFF0007876894000004.tif52150

[0145] purification The protein was purified using a nickel affinity column, and after elution with imidazole, the C-terminal histidine tag was cleaved from the protein using tobacco ecchi disease virus (TEV) protease. After cleavage of the histidine tag, a second round of purification was performed using a nickel column to remove the C-terminal histidine tag and any contaminating proteins that had nonspecifically bound to the nickel column during the first round of purification. Soluble NPP1 eluted during pass-through, was recovered, and concentrated by spin concentration. The entire purification process yielded approximately 2 mg of pure protein per 1 L of cell culture (Figures 3-4). Similar general purification schemes have been described for biochemical, biophysical, and physiological studies of several members of the NPP family (Albright et al., 2012, Blood 120:4432-4440; Saunders et al., 2011, J. Biol. Chem. 18:994-1004; Saunders et al., 2008, Mol. Cancer Ther. 7:3352-3362). As is well known to those experienced in the art and the science of protein purification, the purification of proteins containing the Fc domain of IgG can also be achieved by binding to a protein A or protein G column.

[0146] Mammalian expression systems for NPP1 NPP1 is a glycosylated protein, and the sugar portion of insect cells is expected to induce a strong immunogenic response from mammalian hosts. To reduce the likelihood of inducing an immune response by treating animals with recombinant NPP1, the insect cell glycosylation pattern was replaced with a mammalian glycosylation pattern using a mammalian expression system. The same NPP1 construct was cloned into a mammalian expression vector, followed by stable transfection into HEK293 cells, thereby expressing the protein in the HEK293 mammalian kidney cell line. Stable clones were identified by immunoblotting against His antibody. Strongly expressing clones were expanded, and the culture medium was collected and processed as described in the baculovirus purification scheme described elsewhere in this specification. The total protein yield was approximately 1.5 mg per L of culture medium, and the sample purity was over 95% (Figure 4).

[0147] ATP hydrolysis activity of NPP1 and NPP4 To demonstrate the enzymatic activity of the extracellular soluble domain of NPP1, the steady-state Michaelis-Menten enzyme constant for NPP1 was determined using ATP as a substrate. In addition, to illustrate substrate specificity among NPP family members, the ATP hydrolysis of NPP1 was directly compared with that of NPP4, a protein with 38% sequence identity to NPP1. To prove that NPP1 cleaved ATP, HPLC analysis of the enzymatic reaction was used, and the characteristics of the substrate and product of the reaction were confirmed using ATP, AMP, and ADP standards (Figure 5). The ATP substrate degrades over time in the presence of NPP1, resulting in the accumulation of the enzyme product AMP (Figure 5). The initial velocity for NPP1 was derived in the presence of ATP using various concentrations of ATP substrate, and the enzyme rate constant was derived by fitting the data to a curve (Figure 5C). Despite considerable sequence identity between NPP4 and NPP1, NPP4 lacked ATP hydrolysis activity, while NPP1 readily hydrolyzed ATP to AMP and PPi (Figure 5C). At physiological pH, the reaction rate constants for NPP1 were Km = 144 μM and k cat =7.8s -1That is the case.

[0148] HPLC protocol The HPLC protocol used to measure ATP cleavage by NPP1 and NPP4, and for product identification, was modified from the literature (Stocchi et al., 1985, Anal. Biochem. 146:118-124). Reactions containing various concentrations of ATP in 50 mM Tris pH 8.0, 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, and 1 mM CaCl2 buffer were initiated by adding 0.2–1 μM NPP1, and then suppressed at various time points with equal volumes of 3 M formic acid or 0.5 N KOH, and re-acidified to pH 6 with glacial acetic acid. The suppressed reaction solutions were systematically diluted and loaded into an HPLC system (Waters, Milford MA), and the substrates and products were monitored by UV absorbance at 254 or 259 nm. The substrate and product were separated using a 15 mM ammonium acetate pH 6.0 solution with a methanol gradient of 0% to 10% (or 20%) on a 5 μm 250 × 4.6 mm HPLC column C18 (Higgins Analytical, Mountain View, CA). The product and substrate were quantified by integrating their corresponding peaks and according to the formula. In formula TIFF0007876894000005.tif12128, [substrate]0 is the initial substrate concentration. The extinction coefficients of AMP, ADP, and ATP used in the formula are 15.4 mM. -1 cm -1 When monitored at 254 nm, the integrated product / substrate peak area was converted to concentration using standard materials of the substrate and product that were run on the same day as the reaction.

[0149] Mouse model TTW mice were discovered in sibling mating of Institute of Cancer Research (ICR, Japan) mice and develop multiple progressive abnormal calcifications, ultimately dying with severe malformations and joint ankylosis. These mice serve as an established animal model for both OPLL and osteoarthritis due to specific spinal and joint abnormalities. The gene deletion explaining the ectopic tissue mineralization phenotype has been identified as an immature stop codon (glycine to stop) at position 568 in NPP1, which truncates NPP1 by approximately 350 amino acids. C57BL / 6J-Enpp1 asj / Grsr, i.e., NPP1 asj The TTW mouse variant has a point mutation in the NPP1 protein-coding region at exon 7, position 737, resulting in a point mutation of valine to alanine, and exhibits spinal arthritis at 12 weeks and osteoarthritis of many joints that become stiff and unable to bend by 7 months of age. These mouse models are valuable reagents for mimicking human diseases of ectopic calcification, including osteoarthritis, and are suitable animal models for testing the hypotheses of this study.

[0150] This study investigates the therapeutic efficacy of recombinant NPP1 in two mouse models of ectopic calcification, both of which are variants of toe-walking (ttw) mice. The first model is the original ttw mouse model described by Japanese researchers and is available through the Central Institute for Experimental Animals in Japan. This mouse carries a spontaneously occurring recessive mutation that predisposes the animal to systemic articular ankylosis of the axial and appendage skeleton, which begins at 3 weeks of age in the small distal joints of the hands and feet. In addition to cartilage in the articular and intervertebral discs, ttw mice also exhibit calcification of blood vessels and connective tissue. After gene mapping, the mutation responsible for the phenotype was identified as truncation at position 568 of NPP1. The second mouse model, C57BL / 6J-Enpp1, is available directly from Jackson Laboratories. asjThese mice are named / GrsrJ mice. They have a point mutation in the NPP1 protein-coding region at exon 7, position 737, which results in a point mutation of valine to alanine, and they exhibit spinal arthritis at 12 weeks of age and osteoarthritis of many joints that become stiff and unable to bend by 7 months of age.

[0151] Establishment of NPP1 dosage levels To establish the NPP1 intraperitoneal (IP) administration level that normalizes serum PPi concentration in ttw mice. To conduct initial pharmacokinetic experiments to establish the appropriate NPP1 pathway and concentration required to affect PPi levels in vivo. A single dose of NPP1 will be administered to wild-type (C57bl / 6) animals at a starting concentration of 0.03 mg / kg (e.g., ip, iv). This initial concentration is chosen because the physiological concentration of NPP1 in human serum is considerably lower (100-300 ρM). A mammalian NPP1 concentration of 2.15 mg / ml in aqueous buffer is very close to physiological conditions (50 mM Tris pH 8.0, 150 mM NaCl, 0.8 mM ZnCl2, 0.4 mM CaCl2, 0.4 mM MgCl2). For example, approximately 0.5 μl of concentrated stock will be injected into a 40 g mouse. Adjust the NPP1 dose as needed to reach a dosage level that directly affects serum PPi concentration.

[0152] Approximately three animals are administered per experimental group. Initial administration is performed at concentrations of approximately 0, 0.5 mg / kg, 2 mg / kg, and 8.0 mg / kg. Animals are sacrificed approximately 1, 4, and 8 hours after treatment, and blood is collected by cardiac puncture after terminal anesthesia. Serum is isolated, and serum PPi concentrations are directly measured using a commercially available fluorescence-generating pyrophosphate sensor whose fluorescence intensity is proportionally dependent on the pyrophosphate concentration (see Abcam product number ab112155). Serum pyrophosphate concentrations are compared between treated and untreated animals to determine the initial NPP1 dose level that physiologically alters serum PPi levels in mice.

[0153] Once the NPP1 dosage level is established in wild-type mice, pharmacokinetic experiments using ttw mice are initiated. To begin, the NPP1 concentration that modulates PPi levels in wild-type mice is used. Approximately three animals are used per experimental group, and the animals are administered at levels of approximately 0, 1×, and 10×, where × is the minimum NPP1 concentration observed to modulate serum PPi levels in wild-type (c57bl / 6) mice. As described elsewhere in this specification, the animals are sacrificed approximately 1, 4, and 8 hours after treatment, blood is collected, and PPi concentrations are analyzed. To establish the final starting concentration of NPP1 to be used in efficacy experiments, serum PPi levels of treated and untreated animals are compared to those of wild-type animals in experiments described elsewhere in this specification.

[0154] Efficacy of recombinant NPP1 in a mouse model of ectopic calcification As described elsewhere in this specification, TTW and C57BL / 6J-Enpp1 asj The effect of recombinant NPP1 on a mouse model of ectopic calcification will be evaluated using / GrsrJ mice. Four breeding pairs will be established to create separate genetic colonies. Heterozygous females will be bred with homozygous males, as the development of progressive physical impairment impairs the ability of homozygous females to maintain a litter. The animals will be genotyped by the Yale Animal Resource Center at 3 weeks of age and, once weaned, will be separated into cages according to their genotype and age. Strict record keeping will be maintained to ensure that the animals are correctly identified before the experiment. At 6 weeks of age, the mice will be separated into cohorts of approximately 6 mice and treated with increasing concentrations of NPP1, starting at the lowest concentration observed to normalize serum PPi levels, as described elsewhere in this specification.

[0155] We will compare the onset of symptoms between treated and untreated animals, focusing on the following phenotypes.

[0156] Walking: C57BL / 6J-Enpp1asj Slow, dragging gait in / GrsrJ mice begins around 2 months of age. TTW mice also develop abnormal gait, spinal rigidity, and limb joint stiffness around 2 months of age.

[0157] Abnormal resting posture: C57BL / 6J-Enpp1 asj / GrsrJ mice develop a stiff posture with forelimbs that remain fixed in a body-facing position by approximately two months of age. TTW mice also develop limb joint stiffness at approximately two months of age.

[0158] Skeletal phenotype: C57BL / 6J-Enpp1 asj / GrsrJ mice develop hyperplastic joint spaces in the knees and elbows at approximately 11 weeks, spinal arthritis at approximately 12 weeks, and osteoarthritis in many joints at approximately 12 weeks. By approximately 7 months, C57BL / 6J-Enpp1 asj The joints of / GrsrJ mice become stiff and unable to bend. TTW mice develop spinal rigidity, as well as generalized arthroknic arthrosis of the axial and appendage skeletons, which begins at approximately 3 weeks of age.

[0159] Osteoarthritis: Osteoarthritis is diagnosed around 12 weeks of age with ttw and C57BL / 6J-Enpp1 asj Found in both / GrsrJ mice.

[0160] Hearing loss: Auditory brainstem response is observed by approximately 3 months of age, C57BL / 6J-Enpp1 asj This shows severe hearing loss in / GrsrJ mice.

[0161] Imaging Investigation: Sites of active mineralization in mice and humans can be observed by radioactive nucleotide scanning using various active agents, including but not limited to Tc99m-pyrophosphate. Increased Tc99m-pyrophosphate levels may be observed in animals with increased mineralization. Radionuclide imaging scans are performed on animals on days 0, 7, and 14 of treatment with subclavian injection of radiolabeled tracers. Increased PPi deposition correlates with NPP1 administration levels. In certain embodiments, as observed in radioactive nucleotide scanning, PPi deposition increases in NPP1 mutant animals at the lowest concentration levels of recombinant NPP1 or NPP4 enzymes, but decreases or eliminates PPi deposition at therapeutic levels of recombinant NPP1 or NPP4.

[0162] At least one of the above phenotypes is monitored and / or quantified in treated and untreated animals by recording the animals' gait on a treadmill to track walking and posture, imaging studies to track in vivo skeletal changes during the experiment, auditory assessment of mice to demonstrate hearing loss, and histological examination of the skeleton and soft tissues of mice at the end of the experiment to demonstrate ectopic calcification and skeletal abnormalities. The degree of soft tissue mineralization and skeletal abnormalities in animals during the experiment is tracked using MRI imaging. Prior to imaging, mice are anesthetized with isoflurane in an anesthesia chamber and then transferred to an IVIS imaging box during the imaging process. Mice are kept anesthetized through a nose cone present in the MRI imaging box during the imaging process. After the procedure, animals are observed for approximately 30 minutes for signs of pain and distress after reversal of anesthesia. Mice require 1-2 minutes to recover from anesthesia, and mice can be safely imaged 2-3 times per week for up to 1 month without health sequelae (sequella). Mice will be periodically recorded on a mechanized treadmill to observe and analyze changes in their gait and posture. Auditory brain response and lick suppression tests will be used to monitor hearing loss in animals, and histology of tissues and bones will be used to demonstrate the presence of soft tissue calcification and bone abnormalities.

[0163] As described elsewhere in this specification, the procedure is selected to minimize the immune response to recombinant human NPP1 in immune-responsive ttw mice. In the event of an unexpected decrease in serum PPi concentration observed, consistent with a decrease in NPP1 half-life suggesting immune-mediated disruption of the protein, standard immunotolerance techniques may be used, such as those used in mice to generate monoclonal antibodies specific to alternative protein isoforms (Matthew et al., 2987, J. Immuno. Methods 100:73-82; Salata et al., 1992, Anal. Biochem. 207:142-149). These procedures involve immunizing the animals with cyclophosphamide concurrently with an initial dose of NPP1 protein, which may induce tolerance to the human isoform. If tolerance fails, the mouse isoform of NPP1 is cloned and expressed in the same manner as described for humans, and the described experiments are performed using the mouse isoform of the protein. One of these alternative procedures provides a method for treating mice with NPP1 in a manner that does not induce an immune response, thereby achieving a stable and effective NPP1 concentration sufficient to delay or reverse the ectopic mineralization that characterizes their genetic disorders.

[0164] Crystallization, data collection and processing, structure determination NPP4 was replaced with 50 mM Tris pH 8.0, 150 mM NaCl, 0.8 mM ZnCl2, 0.4 mM CaCl2, and 0.4 mM MgCl2, and A 280The protein concentration was calculated using []. NPP4 at 6 mg / ml (0.14 mM) and the well solution (200 mM diammonium citrate, 17.5% - 19.5% (w / v) PEG 3350) were mixed in a 1:1 ratio, and the best diffraction crystals were obtained by the hanging drop method, in which 2 μl droplets were suspended in a well solution of more than 600 μl in a sealed chamber. Protein crystals typically appeared within 4 - 6 days and continued to grow slowly during the following week, reaching final dimensions of up to 500 μm × 150 μm × 50 μm. Cryoprotection was achieved by rapidly passing the crystals through a series of mixtures consisting of the above well solution, 0.6 mM ZnCl2, 5 mM ligand (if present), and 5% (v / v) increments of glycerol up to a final concentration of up to 20% - 25%, and then immediately flash-freezing in liquid nitrogen. Apo crystals of NPP4 were difficult to obtain and hampered efforts to obtain the structure of NPP4 with ligand by soaking. Co-crystallization with ATP or a cleavable ATP analogue (Sigma M7510) resulted in an AMP product complex, reflecting the slow hydrolysis during crystallization. Attempts at co-crystallization with a non-cleavable ATP analogue (Sigma M6517) showed no recognizable binding.

[0165] The synchrotron diffraction data reported herein were collected at APS (Argonne National Laboratory, Advanced Photon Source, NE-CAT beamlines ID-24-C and ID-24-E) and CHESS (Cornell High Energy Synchrotron Source, beamline A1). HKL2000 was used for indexing and reduction of NPP4-AMP diffraction data. Initial phases were obtained by molecular substitution using PHENIX AutoMR and AutoBuild with a search model consisting only of the protein portion of Xanthomonas axonopodis NPP(2GSU) (Zalatan et al., 2006, Biochem. 45:9788-9803). COOT was employed for model construction and high-quality initial electron density maps, allowing for clear correction of areas with originally problematic or out-of-order structures. PHENIX was used for maximum likelihood refinement of iterative rounds, during which ligands, water, and several glycosylations were incorporated. As described above, however, the protein-only portion of the NPP4-AMP complex was used as a starting point to elucidate the structures of apoNPP4 and all subsequent structures. Each entire structure was systematically checked using an unbiased electron density map (omit map) in which local atoms are excluded from the map calculation. The atomic arrangement for all residues 24-402 was determined. The adjacent residues at the terminals remained irregular. Statistical values ​​for the diffraction data of the final structures are illustrated in Table 1. The structures of human NPP4-AMP at 1.54 Å resolution (4LQY) and apoNPP4 at 1.50 angstrom resolution (4LR2) are deposited in the Protein Data Bank.

[0166] Molecular modeling of the NPP1:ATP complex Mouse NPP1 (RCSB code 4B56) was loaded into MOE (Molecular Operating Environment, Chemical Computing Group Inc., Montreal, Canada), and the A chain was deleted while residues K169 - E905 were retained in the B chain. The asparagine - linked glycosylation sites were excised and capped with methyl groups. Water was deleted, the atom types were fixed and protonated with Protonate 3D. Zinc ions were made immobile with respect to the distances and shapes of their ligands. The metal charge was modeled as +1. ATP was manually positioned by placing an adenine nucleus between the cleft formed by phenylalanine 239 and tyrosine 322 along with the phosphate moiety in the extended conformation of the protein along a large channel to make the protein better. The protein was constrained at a distance of 4.5 Å from the ligand, and minimization was performed on the entire system using AMBER12 (AMBER12:EHT) with the extended Huckel treatment of the ligand.

[0167] Molecular Modeling of the NPP4:ATP Complex NPP4 was modeled in a similar fashion to NPP1, including glycosyl stripping and capping, and the placement of ATP. For the placement of adenine, the corresponding residues in NPP4 are phenylalanine 71 and tyrosine 154. Protein protonation, restraint, and minimization were carried out in a similar format using the AMBER12:EHT force field treatment.

[0168] Enzymology The steady-state enzyme activity of human NPP1 and NPP4 was determined by either absorbance (Ap3A substrate) or HPLC (ATP substrate). The affinity of nucleotide monophosphates (NMPs) for NPP4 was estimated from the [NMP] dependence of the steady-state pNP-TMP (p-nitrophenyl 5'-thymidine monophosphate) cleavage rate, monitored by the absorbance change at 405 nm (Saunders et al., 2008, Mol. Cancer Ther. 7:3352-3362). [NPP4] was 5 nM and [pNP-TMP] was 20 mM. From the best fit of the nucleotide concentration-dependent NPP4 cleavage activity to the rectangular hyperbola, IC was calculated. 50 The value (i.e., the nucleotide concentration that exhibits half of the maximum activity) was determined. 50 This reflects the weighted average affinity for mixed inhibitors (Saunders et al., 2008, Mol. Cancer Ther. 7:3352-3362).

[0169] Preparation of human platelets and measurement of platelet aggregation ability Platelet preparation and platelet aggregation ability measurements were performed as described in Albright et al., 2012, Blood 120:4432-4440.

[0170] Example 1: Enzyme replacement therapy for idiopathic infant arterial calcification (IIAC) and ossification of the posterior longitudinal ligament (OPLL) As described herein, we have developed soluble forms of nucleotide pyrophosphatase / phosphodiesterase-1 (NPP-1) and mutant NPP-4, which may be useful therapeutic agents for diseases and disorders involving pathological calcification and / or ossification. Direct demonstration of the physiological activity of this enzyme in disease states of NPP1 dysfunction is also described herein, thereby establishing the practical applicability of NPP1 enzyme replacement therapy in the selective treatment of disorders of ectopic calcification.

[0171] To develop a soluble form of NPP1, the sequences of NPP2 and NPP1 were combined to obtain a soluble, secreted NPP1 protein. It was discovered that soluble and active recombinant NPP1 can be produced by replacing the transmembrane domain of NPP2 with a homologous region of NPP1. Using such constructs, several variant forms of NPP1 occurring in OPLL and IIAC were cloned and expressed. In some embodiments, NPP1 can be made soluble by removing the entire transmembrane domain of the protein.

[0172] A soluble and fully active form of NPP1, and a mutant NPP4 engineered to change the specificity of NPP4 from Ap3A to ATP, are useful protein therapies for OPLL, IIAC, and other diseases resulting from an imbalance of PPi. Specifically, this protein construct consists of the soluble domain of NPP1, the constant region (Fc) of the human IgG Fc domain, and approximately 10 or more consecutive aspartic acid residues designed to cause the protein to target bone.

[0173] These constructs yield soluble forms of NPP1 and mutant NPP4 in high yield, resulting in large quantities of soluble, high-purity, recombinant, enzymatically active NPP1 and mutant NPP4 capable of hydrolyzing ATP to AMP and PPi. These constructs may be useful in treating human diseases and disorders involving improper PPi balance, which are also associated with SNPs in NPP1.

[0174] The structure of NPP1 was modeled using the previously determined high-resolution structure of NPP4, and it was found that many of the disease-associated SNPs described elsewhere in this specification are located at the active site of NPP1.

[0175] While we do not wish to be bound by any particular theory, the data described herein are consistent with the explanation that reduced NPP1 activity is involved in the disruption of PPi / Pi homeostasis in many of the disorders described elsewhere herein, and with the explanation that restoring PPi balance through the use of soluble recombinant NPP1 protein improves the skeletal and joint abnormalities and ectopic tissue mineralization seen in these disorders.

[0176] We investigate OPLL using a standard animal model of human disease, so-called "tweezers" (ttw) mice. These mice develop progressive abnormal calcification of the spine and joints, exhibiting striking similarities to OPLL disease in humans, and ultimately dying from severe spinal malformations and joint ankylosis. This spontaneously occurring gene deletion introduces a missense mutation within the coding region of NPP1 at position 568, resulting in the immature truncation of this protein. These mice are an accepted animal model for OPLL.

[0177] We will establish administration routes and concentrations of recombinant NPP1 in these mice to normalize their serum PPi concentrations and test whether these doses can delay, improve, or reverse the signs and / or symptoms of the severe phenotype experienced by these animals. We will investigate NPP1 as a protein therapy for animal models of ectopic tissue mineralization, including OPLL and osteoarthritis. We will establish the efficacy of recombinant NPP1 in diseases of ectopic tissue calcification by measuring the rate of joint and spinal calcification in toe-walking (ttw) mice treated with recombinant NPP1 protein.

[0178] To determine the appropriate NPP1 dose required to normalize PPi levels, administer increased NPP1 concentrations to ttw and wild-type (c57bl / 6, genetic background) mice and measure serum PPi concentrations in treated and untreated animals using established methods for PPi determination. To determine the effectiveness of recombinant NPP1 in the disease of ectopic calcification, treat ttw mice with recombinant NPP1 and compare the incidence of symptoms associated with ectopic tissue calcification between treated and untreated animals.

[0179] Example 2: Substrate discrimination between NPP4 and NPP1 To determine the degree of substrate discrimination exhibited by highly homologous NPP family members, the steady-state enzyme rates of human NPP4 and NPP1 against their putative in vivo substrates, Ap3A and ATP, were measured, respectively (Figure 5). Human NPP4 shares 40% sequence identity with NPP1 across its catalytic domain, and the structure of mouse NPP1 has been determined (Jansen et al., 2012, Structure 20:1948-1959), providing an opportunity to identify the structural origin of substrate discrimination at the atomic level. Human and mouse NPP1 are 79% identical, and sequence mapping of the human sequence onto the mouse NPP1 structure has shown that all sequence differences are outside the substrate binding and active sites. Although the Michaelis constant of NPP1 is >30 times tighter than that of NPP4, NPP4 and NPP1 have comparable maximum turnover rates (k cat Approximately 7-8 seconds -1 Ap3A is hydrolyzed by NPP4. In contrast, the rate at which NPP4 hydrolyzes ATP to AMP and PPi is negligible compared to the hydrolysis by NPP1.

[0180] Example 3: Structural Overview To understand the molecular basis of NPP4 substrate specificity and to gain detailed insights into its similarities and differences with NPP1, high-resolution three-dimensional structures of both the apo and AMP-bound forms of human NPP4 were determined using X-ray crystallography at resolutions of 1.50 Å and 1.54 Å, respectively (Table 1 and Figure 6A). These were then compared to the recently determined structures of mouse NPP1 complexes, including mNPP1-AMP at 2.70 Å resolution, to define the structural characteristics involved in the observed substrate specificity for each. The bimetallic catalytic domains of NPP4 and NPP1 contain two bound zinc ions, share a similar overall folding pattern, and employ a conserved catalytic mechanism to hydrolyze the substrate at the same site, yielding a nucleotide monophosphate product. Hydrolysis of Ap3A or ATP by either of these enzymes produces the AMP product molecule. Therefore, the hNPP4-AMP and mNPP1-AMP structures are product complexes. The superposition of the entire catalytic domain of NPP4 with those of NPP1, NPP2, and bacterial NPP yields rmsd values ​​of 1.54 Å, 1.43 Å, and 1.43 Å, respectively. The structural characteristics observed in NPP4, which favor nucleotide binding, are largely conserved in bacterial enzymes.

[0181] NPP4 is a monomeric enzyme with a binding pocket that remains essentially unchanged in the presence or absence of the bound product. Disulfide bonds link residues 254–287 and 394–401, and three N-linked glycosylations are observed at asparagine residues 155, 166, and 386. Due to their location, these glycosylations are unlikely to significantly affect enzymatic activity. This is the first human NPP structure to be determined and shares a common overall folding pattern with the catalytic domain of all other structurally determined NPPs (Figure 6B).

[0182] Example 4: Substrate recognition and active site shape NPP4 targets a phosphodiester substrate with a 5'-nucleotide group at the end and exhibits the greatest preference for the adenine ring. This specificity for nucleotide-containing substrates is the result of a preformed hydrophobic groove on the protein surface, approximately 6.8 Å wide, consisting of the toroidal surface of Tyr154 on one side and the tip of Phe71 on the other (Figures 7A - 7B). The nucleotide base bound therein experiences favorable π-π stacking interactions with the tyrosine ring and van der Waals (VDW) interactions with phenylalanine, each approximately 3.4 Å away. The back wall of the groove is just out of reach of the nucleotide base, making any direct hydrogen bond interaction with the protein impossible, and only a few water-mediated hydrogen bonds are observed between the edge of the AMP ring and NPP4.

[0183] Such a groove is expected to prefer purines over pyrimidines due to their larger size, but this pattern is not strictly maintained. The relative affinity of nucleotide monophosphates for NPP4, determined by TMP-pNP substrate inhibition, is AMP > CMP > UMP > GMP (Figure 5E). The corresponding measurements reported for another family member, NPP1, which has a similar nucleotide groove, revealed AMP > CMP > GMP > UMP (Kato et al., 2012, Proc. Natl. Acad. Sci. U S A 109:16876 - 16881). This model building has revealed that the N2 atom of the guanine ring of similarly bound GMP experiences some steric clashes with NPP4 at residues 104 and 105, which protrude slightly into the nucleotide groove. Inspection of the NPP1 - GMP co-crystal structure revealed similar steric clashes that force the guanine ring to rotate slightly.

[0184] From this point onward, two bound zinc ions, referred to as Zn1 and Zn2, are in close proximity to a hydrophobic groove and are maintained at approximately 4.5 Å apart by interactions with six invariant residues found in all NPPs and alkaline phosphatases (APs). Both zinc ions exhibit a tetrahedral coordination configuration. Zn1 is ligated by Asp189, His193, and His336 and has four-coordinate to the phosphate oxygen atom in the NPP4-AMP complex. Alternatively, in the empty pocket, this coordination can be provided by a water molecule. Zn2 is maintained by Asp34, Asp237, and His238 and has four-coordinate to the Oγ atom of Thr70, the "catalyst residue" of NPP4. As described for APs, this close association likely activates Thr70 for nucleophilic attack on the substrate molecule, presumably by disordering its pKa. Thr70 is located at the N-terminus of an α-helix directly facing the phosphate binding site near the partially exposed Zn1, thereby the helix-dipole force complements the positively charged zinc ion when electrostatically drawing the negatively charged phosphodiester group into the active site. The narrow nucleotide groove and short spacing between the groove and the zinc ion are primarily involved in substrate selection, sterically favoring the terminal 5'-nucleotide monophosphate group.

[0185] In comparison, the other end of the NPP4 binding pocket appears relatively unremarkable and significantly more exposed to the solvent, consistent with NPP4's ability to hydrolyze substrates of varying lengths and chemical characteristics. Although the crystal structure of any NPP with a bound, intact substrate has not been reported to date, our substrate docking simulations indicate that the NPP4 binding pocket runs along a shallow groove on the surface of this protein.

[0186] Example 5: NPP4-AMP vs. ApoNPP4 The apo structure of NPP4 is essentially identical to that of the AMP-bound structure (Figure 7C). Since no changes are observed in the empty hydrophobic groove or at the catalytic residue, the binding site is pre-formed and is not subject to inducible adaptation adjustment even when substrate binding is present. The apo structure has a citrate anion derived from the crystallization conditions, bound at Zn1 via a chelate-like interaction. Asn91 is the only significantly mobile active site residue, swirling to provide room for the citrate molecule. In the NPP4-AMP complex, Asn91 confers a hydrogen bond to the phosphate group bound near Zn1. The swirling ability of Asn91 may allow for the maintenance of hydrogen bonding interactions with the phosphate oxygen via the catalytic intermediate of the reaction, or provide flexibility to facilitate ligand entry or exit.

[0187] Example 6: NPP4-AMP vs. NPP1-AMP The overall similarity in how AMP binds within the groove region of NPP4 and NPP1 is illustrated in Figure 7D. NPP1 contains conserved residues corresponding to Tyr154 and Phe71 of NPP4, and therefore also targets substrates with a 5'-nucleotide terminus. The phosphate group of AMP binds near Zn1 in both enzymes. It is unknown whether the small differences observed at the AMP position are real or simply reflect a significantly lower resolution for the NPP1-AMP structure (2.70 Å compared to 1.54 Å for NPP4-AMP).

[0188] Example 7: Catalytic Mechanism NPP is a member of the AP superfamily and shares many important structural characteristics, residues, and structural modes at the center of the catalyst-generating active site. Figure 8 illustrates the general reaction mechanism for NPP, as it is applied to the NPP4 hydrolysis of Ap3A, along with the corresponding crystal structure or binding simulation model for each step.

[0189] In NPP4, the relative distance between the pre-formed hydrophobic groove and the two bound zinc ions determines that the α-phosphate group of the substrate is located close to Zn1 and Thr70, and its Oγ atom is constantly activated for nucleophilic attack due to its extremely close proximity to Zn2. When Ap3A binds, the α-phosphate is attacked by Thr70, causing the breakdown of the ester bond on the opposite side and releasing ADP. A water molecule immediately enters the empty space next to Zn1, becomes activated, and attacks the α-phosphate from the opposite direction, causing the breakdown of the transient covalent NPP4-AMP bond, releasing AMP, and NPP4 recovers to its original state. The release of the two product molecules is sequential, with the nucleotide monophosphate remaining last. As such, NPP4-AMP and NPP1-AMP are product complexes.

[0190] Example 8: Molecular determinants of substrate specificity of NPP4 and NPP1 NPP1 rapidly cleaves ATP into AMP and PPi, while NPP4 does so only very slowly. Since both enzymes target nucleotide-containing substrates with a preference for the adenine ring and bind to AMP in a similar manner (Figure 7D), the clue to this misalignment must lie elsewhere within the binding pocket. Because NPP1 efficiently hydrolyzes ATP into AMP and PPi, ATP is likely to bind to NPP1 in the same orientation as observed with respect to AMP. Therefore, by adding two phosphate groups to the NPP1-AMP cocrystal coordination, we modeled ATP within the NPP1 active site (4B56) (Figures 9A, 9C, 9E), and then subjected the complex to energy minimization as described. The superposition of the NPP1 binding site and NPP4 reveals good overlap between most of the side chains within 4.5 Å of this ATP substrate, although a significant difference occurs near the terminal γ-phosphate. In NPP1, the addition of Phe516 (numbered by mouse) to the protein core creates more space for the γ-phosphate of ATP, which is thought to be charge-stabilized by three lysine residues (Lys237, Lys260, and Lys510) referred to as the lysine claws. Two of these lysine residues (Lys260 and Lys510) line the upper edge of the binding pocket and remain irregular in the absence of γ-phosphate, as observed in the NPP1-AMP structure. Simulations illustrate that when a new substrate enters the NPP1 binding site, these lysine residues are electrostatically attracted to the γ-phosphate and become regular in this process. The bound γ-phosphate of ATP appears to be under an inductively compatible lid composed of Tyr433, Lys260, and Lys510, where it should be charge-stabilized by the three lysine residues (Figure 9A). Once the PPi product is released, Lys260 and Lys510 should become irregular again until a new substrate is drawn into this site.

[0191] In a similar manner, ATP was modeled within the NPP4 active site by adding two phosphates to the NPP4-AMP structure, and the complex was then energy-minimized. In contrast to NPP1, the region of NPP4 near the γ-phosphate is much more open and exposed to the solvent, contains additional negatively charged residues and fewer positively charged residues, and lacks the ability to form a lysine claw or lid (Figures 9B, 9D, and 9F). The γ-phosphate of the bound ATP in this orientation is thought to be adjacent to the negatively charged Asp335, which corresponds to Phe516 of NPP1, but at this point is oriented directly into the binding pocket. Asp264 (corresponding to mouse NPP1, Val432) is also nearby. In summary, the local electrostatic environment of this region of the NPP4 binding pocket is significantly less favorable than that of NPP1.

[0192] The ability to stabilize the charge at this position may be more pronounced with ATP than with other substrates such as Ap3A, because terminal phosphate groups (phosphomonoesters) inherently carry more negative charge than non-terminal phosphate groups (phosphodiesters). For example, the terminal γ-phosphate of ATP carries more negative charge than the corresponding linearly aligned γ-phosphates of Ap3A. The highly effective charge stabilization of NPP1 at this position may be a clue to its ability to readily hydrolyze ATP, while the less favorable local environment found at this same position in NPP4 is detrimental, and hydrolysis occurs only very slowly.

[0193] Example 9: NPP1 and platelet aggregation The hydrolysis of Ap3A by NPP1 raises the question of whether NPP1 can play a role in platelet aggregation under physiological conditions. Given that this enzyme is active against the Ap3A substrate, the relative abundance of NPP1 in the vascular lumen may determine its role in coagulation. NPP1 has been reported to be present on the cerebral capillary endothelium but not on capillaries elsewhere. In addition, NPP1 is present on the membrane surface of plasma cells, osteoblasts, chondrocytes, and matrix vesicles (MVs) expelled from osteoblasts and chondrocytes. NPP1 is also present as a soluble protein in the vascular system at very low concentrations of 10-30 ng / mL (i.e., 100-300 pM).

[0194] To determine the concentration of NPP1 that can induce platelet aggregation, increasing concentrations of NPP1 were titrated in platelet-rich plasma (PRP) with physiological levels of Ap3A. Addition of NPP1 to 80 μM Ap3A did not induce platelet aggregation until the NPP1 concentration reached 1 nM (Figure 10). Although this concentration is approximately three times higher than the highest reported plasma concentration for NPP1, it is below the threshold required for NPP4 to induce platelet aggregation (Figure 10) and well within the concentration range for membrane-bound endothelial proteins. Therefore, while plasma concentrations of NPP1 are unlikely to induce platelet-mediated primary hemostasis in vivo, endothelial-bound NPP1 at anatomical locations with local concentrations exceeding 1 nM may induce significant platelet aggregation and thrombus formation.

[0195] Example 10: Ap3A bound to NPP4 Human NPP4 (rcsb code 4LR2) was loaded into a Molecular Operating Environment (MOE, Chemical Computing Group Inc., Montreal, Canada), and the asparagine-binding glycosylation site was excised and capped with a methyl group. Water was removed, and the atomic type was fixed and protonation was performed with Protonate 3D. Zinc ions were immobilized with respect to the distance and shape of their ligands. Metal charges were modeled at +1. Ap3A was manually positioned by placing an adenine nucleus between phenylalanine 239 and tyrosine 322, along with the phosphate moiety in the expanded three-dimensional structure along a large channel that makes this protein better. The remainder of the Ap3A molecule was left free in solution. The protein was constrained to a distance of 4.5 Å from the ligand, and minimization of the entire system was performed using AMBER12 (AMBER12:EHT) along with the ligand's expanded Huckel treatment. The docked Ap3A molecule (Figure 11) revealed the location of a second adenine-binding site. This binding site is formed by a narrow groove on the protein surface, which, in certain embodiments, can be eliminated by altering the residues lining the surface of the pocket.

[0196] Example 11: Crystal of Ap3A bound to NPP4 To directly determine the position of Ap3A in the NPP4 protein, an inactive mutant of NPP4 was crystallized together with Ap3A (Figure 12). This structure can be used to refine mutations in NPP4 and NPP1 designed to reduce the thrombus-promoting effects of these enzymes.

[0197] (Table 1) Data collection and refined statistics TIFF0007876894000006.tif163147 a The values ​​for the highest resolution bin are shown in parentheses. b R sym =Σ hkl Σ i |Ii (hkl)-<I(hkl)> | / Σ hkl Σ i I i (hkl) c I / σI is the average intensity of reflections within this resolution bin, divided by the mean standard deviation (sigma) of reflections in the same group. d AMP is 5'-adenosine monophosphate. FLC is a citrate anion derived from the crystallization conditions. e R work =Σ||F (obs) |-|F (calc) || / Σ|F (obs) | f R free =As a value for Rwork, it is calculated for 5.0% of total internal reflections that are randomly selected and excluded from refinement.

[0198] Any patents, patent applications, and publications disclosed herein by reference are incorporated herein by reference in their entirety. While the present invention is disclosed with reference to specific embodiments, it will be apparent that other embodiments and variations of the present invention can be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be considered to include all such embodiments and equivalent variations.

[0199] Sequence information SEQUENCE LISTING <110> Yale Plaza <120> Compositions and Methods for Treating Pathological Calcification and Ossification <150> US 61 / 904,786 <151> 2013-11-15 <150> US 61 / 764,297 <151> 2013-02-13 <160> 3 <170> PatentIn version 3.5 <210> 1 <211> 925 <212> PRT <213> Homo sapiens <220> <221> misc_feature <223> NPP1 Amino Acid Sequence <400> 1 Met Glu Arg Asp Gly Cys Ala Gly Gly Gly Ser Arg Gly Gly Glu Gly 1 5 10 15 Gly Arg Ala Pro Arg Glu Gly Pro Ala Gly Asn Gly Arg Asp Arg Gly 20 25 30 Arg Ser His Ala Ala Glu Ala Pro Gly Asp Pro Gln Ala Ala Ala Ser 35 40 45 Leu Leu Ala Pro Met Asp Val Gly Glu Glu Pro Leu Glu Lys Ala Ala 50 55 60 Arg Ala Arg Thr Ala Lys Asp Pro Asn Thr Tyr Lys Val Leu Ser Leu 65 70 75 80 Val Leu Ser Val Cys Val Leu Thr Thr Ile Leu Gly Cys Ile Phe Gly 85 90 95 Leu Lys Pro Ser Cys Ala Lys Glu Val Lys Ser Cys Lys Gly Arg Cys 100 105 110 Phe Glu Arg Thr Phe Gly Asn Cys Arg Cys Asp Ala Ala Cys Val Glu 115 120 125 Leu Gly Asn Cys Cys Leu Asp Tyr Gln Glu Thr Cys Ile Glu Pro Glu 130 135 140 His Ile Trp Thr Cys Asn Lys Phe Arg Cys Gly Glu Lys Arg Leu Thr 145 150 155 160 Arg Ser Leu Cys Ala Cys Ser Asp Asp Cys Lys Asp Lys Gly Asp Cys 165 170 175 Cys Ile Asn Tyr Ser Ser Val Cys Gln Gly Glu Lys Ser Trp Val Glu 180 185 190 Glu Pro Cys Glu Ser Ile Asn Glu Pro Gln Cys Pro Ala Gly Phe Glu 195 200 205 Thr Pro Pro Thr Leu Leu Phe Ser Leu Asp Gly Phe Arg Ala Glu Tyr 210 215 220 Leu His Thr Trp Gly Gly Leu Leu Pro Val Ile Ser Lys Leu Lys Lys 225 230 235 240 Cys Gly Thr Tyr Thr Lys Asn Met Arg Pro Val Tyr Pro Thr Lys Thr 245 250 255 Phe Pro Asn His Tyr Ser Ile Val Thr Gly Leu Tyr Pro Glu Ser His 260 265 270 Gly Ile Ile Asp Asn Lys Met Tyr Asp Pro Lys Met Asn Ala Ser Phe 275 280 285 Ser Leu Lys Ser Lys Glu Lys Phe Asn Pro Glu Trp Tyr Lys Gly Glu 290 295 300 Pro Ile Trp Val Thr Ala Lys Tyr Gln Gly Leu Lys Ser Gly Thr Phe 305 310 315 320 Phe Trp Pro Gly Ser Asp Val Glu Ile Asn Gly Ile Phe Pro Asp Ile 325 330 335 Tyr Lys Met Tyr Asn Gly Ser Val Pro Phe Glu Glu Arg Ile Leu Ala 340 345 350 Val Leu Gln Trp Leu Gln Leu Pro Lys Asp Glu Arg Pro His Phe Tyr 355 360 365 Thr Leu Tyr Leu Glu Glu Pro Asp Ser Ser Gly His Ser Tyr Gly Pro 370 375 380 Val Ser Ser Glu Val Ile Lys Ala Leu Gln Arg Val Asp Gly Met Val 385 390 395 400 Gly Met Leu Met Asp Gly Leu Lys Glu Leu Asn Leu His Arg Cys Leu 405 410 415 Asn Leu Ile Leu Ile Ser Asp His Gly Met Glu Gln Gly Ser Cys Lys 420 425 430 Lys Tyr Ile Tyr Leu Asn Lys Tyr Leu Gly Asp Val Lys Asn Ile Lys 435 440 445 Val Ile Tyr Gly Pro Ala Ala Arg Leu Arg Pro Ser Asp Val Pro Asp 450 455 460 Lys Tyr Tyr Ser Phe Asn Tyr Glu Gly Ile Ala Arg Asn Leu Ser Cys 465 470 475 480 Arg Glu Pro Asn Gln His Phe Lys Pro Tyr Leu Lys His Phe Leu Pro 485 490 495 Lys Arg Leu His Phe Ala Lys Ser Asp Arg Ile Glu Pro Leu Thr Phe 500 505 510 Tyr Leu Asp Pro Gln Trp Gln Leu Ala Leu Asn Pro Ser Glu Arg Lys 515 520 525 Tyr Cys Gly Ser Gly Phe His Gly Ser Asp Asn Val Phe Ser Asn Met 530 535 540 Gln Ala Leu Phe Val Gly Tyr Gly Pro Gly Phe Lys His Gly Ile Glu 545 550 555 560 Ala Asp Thr Phe Glu Asn Ile Glu Val Tyr Asn Leu Met Cys Asp Leu 565 570 575 Leu Asn Leu Thr Pro Ala Pro Asn Asn Gly Thr His Gly Ser Leu Asn 580 585 590 His Leu Leu Lys Asn Pro Val Tyr Thr Pro Lys His Pro Lys Glu Val 595 600 605 His Pro Leu Val Gln Cys Pro Phe Thr Arg Asn Pro Arg Asp Asn Leu 610 615 620 Gly Cys Ser Cys Asn Pro Ser Ile Leu Pro Ile Glu Asp Phe Gln Thr 625 630 635 640 Gln Phe Asn Leu Thr Val Ala Glu Glu Lys Ile Ile Lys His Glu Thr 645 650 655 Leu Pro Tyr Gly Arg Pro Arg Val Leu Gln Lys Glu Asn Thr Ile Cys 660 665 670 Leu Leu Ser Gln His Gln Phe Met Ser Gly Tyr Ser Gln Asp Ile Leu 675 680 685 Met Pro Leu Trp Thr Ser Tyr Thr Val Asp Arg Asn Asp Ser Phe Ser 690 695 700 Thr Glu Asp Phe Ser Asn Cys Leu Tyr Gln Asp Phe Arg Ile Pro Leu 705 710 715 720 Ser Pro Val His Lys Cys Ser Phe Tyr Lys Asn Asn Thr Lys Val Ser 725 730 735 Tyr Gly Phe Leu Ser Pro Pro Gln Leu Asn Lys Asn Ser Ser Gly Ile 740 745 750 Tyr Ser Glu Ala Leu Leu Thr Thr Asn Ile Val Pro Met Tyr Gln Ser 755 760 765 Phe Gln Val Ile Trp Arg Tyr Phe His Asp Thr Leu Leu Arg Lys Tyr 770 775 780 Ala Glu Glu Arg Asn Gly Val Asn Val Val Ser Gly Pro Val Phe Asp 785 790 795 800 Phe Asp Tyr Asp Gly Arg Cys Asp Ser Leu Glu Asn Leu Arg Gln Lys 805 810 815 Arg Arg Val Ile Arg Asn Gln Glu Ile Leu Ile Pro Thr His Phe Phe 820 825 830 Ile Val Leu Thr Ser Cys Lys Asp Thr Ser Gln Thr Pro Leu His Cys 835 840 845 Glu Asn Leu Asp Thr Leu Ala Phe Ile Leu Pro His Arg Thr Asp Asn 850 855 860 Ser Glu Ser Cys Val His Gly Lys His Asp Ser Ser Trp Val Glu Glu 865 870 875 880 Leu Leu Met Leu His Arg Ala Arg Ile Thr Asp Val Glu His Ile Thr 885 890 895 Gly Leu Ser Phe Tyr Gln Gln Arg Lys Glu Pro Val Ser Asp Ile Leu 900 905 910 Lys Leu Lys Thr His Leu Pro Thr Phe Ser Gln Glu Asp 915 920 925 <210> 2 <211> 888 <212> PRT <213> Homo sapiens <220> <221> misc_feature <223> NPP2 Amino Acid Sequence <400> 2 Met Ala Arg Arg Ser Ser Phe Gln Ser Cys Gln Ile Ile Ser Leu Phe 1 5 10 15 Thr Phe Ala Val Gly Val Asn Ile Cys Leu Gly Phe Thr Ala His Arg 20 25 30 Ile Lys Arg Ala Glu Gly Trp Glu Glu Gly Pro Pro Thr Val Leu Ser 35 40 45 Asp Ser Pro Trp Thr Asn Ile Ser Gly Ser Cys Lys Gly Arg Cys Phe 50 55 60 Glu Leu Gln Glu Ala Gly Pro Pro Asp Cys Arg Cys Asp Asn Leu Cys 65 70 75 80 Lys Ser Tyr Thr Ser Cys Cys His Asp Phe Asp Glu Leu Cys Leu Lys 85 90 95 Thr Ala Arg Gly Trp Glu Cys Thr Lys Asp Arg Cys Gly Glu Val Arg 100 105 110 Asn Glu Glu Asn Ala Cys His Cys Ser Glu Asp Cys Leu Ala Arg Gly 115 120 125 Asp Cys Cys Thr Asn Tyr Gln Val Val Cys Lys Gly Glu Ser His Trp 130 135 140 Val Asp Asp Asp Cys Glu Glu Ile Lys Ala Ala Glu Cys Pro Ala Gly 145 150 155 160 Phe Val Arg Pro Pro Leu Ile Ile Phe Ser Val Asp Gly Phe Arg Ala 165 170 175 Ser Tyr Met Lys Lys Gly Ser Lys Val Met Pro Asn Ile Glu Lys Leu 180 185 190 Arg Ser Cys Gly Thr His Ser Pro Tyr Met Arg Pro Val Tyr Pro Thr 195 200 205 Lys Thr Phe Pro Asn Leu Tyr Thr Leu Ala Thr Gly Leu Tyr Pro Glu 210 215 220 Ser His Gly Ile Val Gly Asn Ser Met Tyr Asp Pro Val Phe Asp Ala 225 230 235 240 Thr Phe His Leu Arg Gly Arg Glu Lys Phe Asn His Arg Trp Trp Gly 245 250 255 Gly Gln Pro Leu Trp Ile Thr Ala Thr Lys Gln Gly Val Lys Ala Gly 260 265 270 Thr Phe Phe Trp Ser Val Val Ile Pro His Glu Arg Arg Ile Leu Thr 275 280 285 Ile Leu Gln Trp Leu Thr Leu Pro Asp His Glu Arg Pro Ser Val Tyr 290 295 300 Ala Phe Tyr Ser Glu Gln Pro Asp Phe Ser Gly His Lys Tyr Gly Pro 305 310 315 320 Phe Gly Pro Glu Met Thr Asn Pro Leu Arg Glu Ile Asp Lys Ile Val 325 330 335 Gly Gln Leu Met Asp Gly Leu Lys Gln Leu Lys Leu His Arg Cys Val 340 345 350 Asn Val Ile Phe Val Gly Asp His Gly Met Glu Asp Val Thr Cys Asp 355 360 365 Arg Thr Glu Phe Leu Ser Asn Tyr Leu Thr Asn Val Asp Asp Ile Thr 370 375 380 Leu Val Pro Gly Thr Leu Gly Arg Ile Arg Ser Lys Phe Ser Asn Asn 385 390 395 400 Ala Lys Tyr Asp Pro Lys Ala Ile Ile Ala Asn Leu Thr Cys Lys Lys 405 410 415 Pro Asp Gln His Phe Lys Pro Tyr Leu Lys Gln His Leu Pro Lys Arg 420 425 430 Leu His Tyr Ala Asn Asn Arg Arg Ile Glu Asp Ile His Leu Leu Val 435 440 445 Glu Arg Arg Trp His Val Ala Arg Lys Pro Leu Asp Val Tyr Lys Lys 450 455 460 Pro Ser Gly Lys Cys Phe Phe Gln Gly Asp His Gly Phe Asp Asn Lys 465 470 475 480 Val Asn Ser Met Gln Thr Val Phe Val Gly Tyr Gly Ser Thr Phe Lys 485 490 495 Tyr Lys Thr Lys Val Pro Pro Phe Glu Asn Ile Glu Leu Tyr Asn Val 500 505 510 Met Cys Asp Leu Leu Gly Leu Lys Pro Ala Pro Asn Asn Gly Thr His 515 520 525 Gly Ser Leu Asn His Leu Leu Arg Thr Asn Thr Phe Arg Pro Thr Met 530 535 540 Pro Glu Glu Val Thr Arg Pro Asn Tyr Pro Gly Ile Met Tyr Leu Gln 545 550 555 560 Ser Asp Phe Asp Leu Gly Cys Thr Cys Asp Asp Lys Val Glu Pro Lys 565 570 575 Asn Lys Leu Asp Glu Leu Asn Lys Arg Leu His Thr Lys Gly Ser Thr 580 585 590 Glu Ala Glu Thr Arg Lys Phe Arg Gly Ser Arg Asn Glu Asn Lys Glu 595 600 605 Asn Ile Asn Gly Asn Phe Glu Pro Arg Lys Glu Arg His Leu Leu Tyr 610 615 620 Gly Arg Pro Ala Val Leu Tyr Arg Thr Arg Tyr Asp Ile Leu Tyr His 625 630 635 640 Thr Asp Phe Glu Ser Gly Tyr Ser Glu Ile Phe Leu Met Pro Leu Trp 645 650 655 Thr Ser Tyr Thr Val Ser Lys Gln Ala Glu Val Ser Ser Val Pro Asp 660 665 670 His Leu Thr Ser Cys Val Arg Pro Asp Val Arg Val Ser Pro Ser Phe 675 680 685 Ser Gln Asn Cys Leu Ala Tyr Lys Asn Asp Lys Gln Met Ser Tyr Gly 690 695 700 Phe Leu Phe Pro Pro Tyr Leu Ser Ser Ser Pro Glu Ala Lys Tyr Asp 705 710 715 720 Ala Phe Leu Val Thr Asn Met Val Pro Met Tyr Pro Ala Phe Lys Arg 725 730 735 Val Trp Asn Tyr Phe Gln Arg Val Leu Val Lys Lys Tyr Ala Ser Glu 740 745 750 Arg Asn Gly Val Asn Val Ile Ser Gly Pro Ile Phe Asp Tyr Asp Tyr 755 760 765 Asp Gly Leu His Asp Thr Glu Asp Lys Ile Lys Gln Tyr Val Glu Gly 770 775 780 Ser Ser Ile Pro Val Pro Thr His Tyr Tyr Ser Ile Ile Thr Ser Cys 785 790 795 800 Leu Asp Phe Thr Gln Pro Ala Asp Lys Cys Asp Gly Pro Leu Ser Val 805 810 815 Ser Ser Phe Ile Leu Pro His Arg Pro Asp Asn Glu Glu Ser Cys Asn 820 825 830 Ser Ser Glu Asp Glu Ser Lys Trp Val Glu Glu Leu Met Lys Met His 835 840 845 Thr Ala Arg Val Arg Asp Ile Glu His Leu Thr Ser Leu Asp Phe Phe 850 855 860 Arg Lys Thr Ser Arg Ser Tyr Pro Glu Ile Leu Thr Leu Lys Thr Tyr 865 870 875 880 Leu His Thr Tyr Glu Ser Glu Ile 885 <210> 3 <211> 453 <212> PRT <213> Homo sapiens <220> <221> misc_feature <223> NPP4 Amino Acid Sequence <400> 3 Met Lys Leu Leu Val Ile Leu Leu Phe Ser Gly Leu Ile Thr Gly Phe 1 5 10 15 Arg Ser Asp Ser Ser Ser Ser Leu Pro Pro Lys Leu Leu Leu Val Ser 20 25 30 Phe Asp Gly Phe Arg Ala Asp Tyr Leu Lys Asn Tyr Glu Phe Pro His 35 40 45 Leu Gln Asn Phe Ile Lys Glu Gly Val Leu Val Glu His Val Lys Asn 50 55 60 Val Phe Ile Thr Lys Thr Phe Pro Asn His Tyr Ser Ile Val Thr Gly 65 70 75 80 Leu Tyr Glu Glu Ser His Gly Ile Val Ala Asn Ser Met Tyr Asp Ala 85 90 95 Val Thr Lys Lys His Phe Ser Asp Ser Asn Asp Lys Asp Pro Phe Trp 100 105 110 Trp Asn Glu Ala Val Pro Ile Trp Val Thr Asn Gln Leu Gln Glu Asn 115 120 125 Arg Ser Ser Ala Ala Ala Met Trp Pro Gly Thr Asp Val Pro Ile His 130 135 140 Asp Thr Ile Ser Ser Tyr Phe Met Asn Tyr Asn Ser Ser Val Ser Phe 145 150 155 160 Glu Glu Arg Leu Asn Asn Ile Thr Met Trp Leu Asn Asn Ser Asn Pro 165 170 175 Pro Val Thr Phe Ala Thr Leu Tyr Trp Glu Glu Pro Asp Ala Ser Gly 180 185 190 His Lys Tyr Gly Pro Glu Asp Lys Glu Asn Met Ser Arg Val Leu Lys 195 200 205 Lys Ile Asp Asp Leu Ile Gly Asp Leu Val Gln Arg Leu Lys Met Leu 210 215 220 Gly Leu Trp Glu Asn Leu Asn Val Ile Ile Thr Ser Asp His Gly Met 225 230 235 240 Thr Gln Cys Ser Gln Asp Arg Leu Ile Asn Leu Asp Ser Cys Ile Asp 245 250 255 His Ser Tyr Tyr Thr Leu Ile Asp Leu Ser Pro Val Ala Ala Ile Leu 260 265 270 Pro Lys Ile Asn Arg Thr Glu Val Tyr Asn Lys Leu Lys Asn Cys Ser 275 280 285 Pro His Met Asn Val Tyr Leu Lys Glu Asp Ile Pro Asn Arg Phe Tyr 290 295 300 Tyr Gln His Asn Asp Arg Ile Gln Pro Ile Ile Leu Val Ala Asp Glu 305 310 315 320 Gly Trp Thr Ile Val Leu Asn Glu Ser Ser Gln Lys Leu Gly Asp His 325 330 335 Gly Tyr Asp Asn Ser Leu Pro Ser Met His Pro Phe Leu Ala Ala His 340 345 350 Gly Pro Ala Phe His Lys Gly Tyr Lys His Ser Thr Ile Asn Ile Val 355 360 365 Asp Ile Tyr Pro Met Met Cys His Ile Leu Gly Leu Lys Pro His Pro 370 375 380 Asn Asn Gly Thr Phe Gly His Thr Lys Cys Leu Leu Val Asp Gln Trp 385 390 395 400 Cys Ile Asn Leu Pro Glu Ala Ile Ala Ile Val Ile Gly Ser Leu Leu 405 410 415 Val Leu Thr Met Leu Thr Cys Leu Ile Ile Ile Met Gln Asn Arg Leu 420 425 430 Ser Val Pro Arg Pro Phe Ser Arg Leu Gln Leu Gln Glu Asp Asp Asp 435 440 445 Asp Pro Leu Ile Gly 450

Claims

1. A composition for use in the treatment, improvement, and / or prevention of diseases associated with pathological calcification and / or ossification in a subject, wherein the composition is (1) Recombinant ectonucleotide pyrophosphatase / phosphodiesterase-1 (ENPP1) polypeptide lacking the ENPP1 transmembrane domain, (2) IgG Fc domain and It contains a polypeptide that includes, The polypeptide lacks 4 to 20 consecutive aspartic acid residues, The disease associated with the aforementioned pathological calcification and / or ossification is at least one of the following: idiopathic infantile arterial calcification (IIAC), generalized infantile arterial calcification (GACI), ossification of the posterior longitudinal ligament (OPLL), hypophosphatemic rickets, osteoarthritis, and calcification of atherosclerotic plaques. composition.

2. The composition for use according to claim 1, wherein the recombinant ENPP1 polypeptide comprises residues 96 to 925 of human ENPP1 (NCBI accession NP_006199, SEQ ID NO: 1).

3. The composition for use according to claim 1 or 2, wherein the subject is a human.

4. The composition for use according to any one of claims 1 to 3, wherein the disease associated with the pathological calcification and / or ossification is generalized arterial calcification of infants (GACI).

5. The composition for use according to any one of claims 1 to 4, wherein the serum pyrophosphate (PPi) concentration is normalized in a subject to which the composition is administered.

6. The use of polypeptides in the manufacture of drugs for the treatment, improvement, and / or prevention of diseases associated with pathological calcification and / or ossification in subjects, wherein the polypeptide is (1) Recombinant ectonucleotide pyrophosphatase / phosphodiesterase-1 (ENPP1) polypeptide lacking the ENPP1 transmembrane domain, (2) IgG Fc domain and including, and, The polypeptide lacks 4 to 20 consecutive aspartic acid residues, The disease associated with the aforementioned pathological calcification and / or ossification is at least one of the following: idiopathic infantile arterial calcification (IIAC), generalized infantile arterial calcification (GACI), ossification of the posterior longitudinal ligament (OPLL), hypophosphatemic rickets, osteoarthritis, and calcification of atherosclerotic plaques. use.

7. The use according to claim 6, wherein the recombinant ENPP1 polypeptide consists of residues 96 to 925 of human ENPP1 (NCBI accession NP_006199, SEQ ID NO: 1).

8. The use according to claim 6 or 7, wherein the subject is a human.

9. The use according to any one of claims 6 to 8, wherein the disease is generalized arterial calcification in infants (GACI).

10. The use according to any one of claims 6 to 9, wherein serum pyrophosphate (PPi) concentration is normalized in the subject to whom the drug is administered.

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