COMPOSITIONS AND METHODS FOR TREATING ALLOGRAFT VASCULOPATHY, MOYAMOYA DISEASE, MOYAMOYA SYNDROME AND INTIMAL PROLIFERATION

Abstract

The present disclosure provides compositions and methods for treating allograft vasculopathy, for treating Moyamoya Diseases (MMD) and Moyamoya Syndrome (MMS), for treating inhibiting or preventing unwanted intimal proliferation in a subject by administering an ectonucleotide pyrophosphatase phosphodiesterase-1 (ENPP1) agent or an ectonucleotide pyrophosphatase phosphodiesterase-3 (ENPP3).

Claims

1-155. (canceled)

156. A method for treating a subject at risk for developing Moyamoya disease or a subject afflicted with Moyamoya disease, the method comprising: administering to the subject an ENPP1 agent in an amount sufficient to thereby treat the subject.

157. The method of claim 156, wherein the ENPP1 agent comprises an ENPP1 polypeptide or a nucleic acid encoding an ENPP1 polypeptide or a viral vector comprising a nucleic acid encoding an ENPP1 polypeptide.

158. The method of claim 157, wherein the ENPP1 polypeptide comprises the extracellular domain of ENPP1.

159. The method of claim 157, wherein the ENPP1 polypeptide comprises amino acids 99 to 925 of SEQ ID NO: 1.

160. The method of claim 157, wherein the ENPP1 polypeptide comprises a heterologous protein and said heterologous protein increases the circulating half-life of the ENPP1 polypeptide in mammal.

161. The method of claim 160, wherein the heterologous protein is an Fc region of an immunoglobulin molecule or an albumin molecule.

162. The method of claim 160, wherein the heterologous protein is carboxy-terminal to the ENPP1 polypeptide.

163. The method of claim 160, wherein the ENPP1 agent comprises a linker and the linker separates the ENPP1 polypeptide and the heterologous protein.

164. The method of claim 160, wherein the ENPP1 agent is administered to the subject subcutaneously or intravenously.

165. The method of claim 160, wherein the ENPP1 agent comprises ENPP1 variants that retain enzymatic activity.

166. A method for inhibiting or preventing cerebral vascular occlusion or unwanted vascular smooth muscle cell proliferation in a subject at risk for developing Moyamoya disease or is afflicted with Moyamoya disease, the method comprising: administering to the subject an ENPP1 agent in an amount sufficient to thereby inhibit or prevent cerebral vascular occlusion or unwanted vascular smooth muscle cell proliferation in the subject.

167. The method of claim 166, wherein said subject is one who is expected to receive or who has received a surgical intervention as a treatment for Moyamoya disease.

168. The method of claim 166, wherein the subject bears the RNF213 R4810K mutation.

169. The method of claim 166, wherein said subject experiences stenosis, thrombosis, embolism and/or hemorrhage in the brain.

170. The method of claim 166, wherein the surgical intervention is a vascular bypass graft or cerebral revascularization.

171. The method of claim 170, wherein the ENPP1 agent or the ENPP3 agent is administered to the subject prior to the surgical intervention or concurrently with the surgical intervention or following the surgical intervention.

172. The method of claim 166, wherein the ENPP1 agent comprises an ENPP1 polypeptide or a nucleic acid encoding an ENPP1 polypeptide or a viral vector comprising a nucleic acid encoding an ENPP1 polypeptide.

173. The method of claim 172, wherein the ENPP1 polypeptide comprises the extracellular domain of ENPP1.

174. The method of claim 172, wherein the ENPP1 polypeptide comprises a heterologous protein and said heterologous protein increases the circulating half-life of the ENPP1 polypeptide in mammal.

175. The method of claim 174, wherein the heterologous protein is an Fc region of an immunoglobulin molecule or an albumin molecule.

176. The method of claim 174, wherein the ENPP1 agent comprises a linker and the linker separates the ENPP1 polypeptide and the heterologous protein.

177. The method of claim 174, wherein the ENPP1 agent is administered to the subject subcutaneously or intravenously.

178. The method of claim 174, wherein the ENPP1 agent comprises ENPP1 variants that retain enzymatic activity.

179. A method for reducing and/or preventing allograft vasculopathy in a subject having an allograft, the method comprising administering to the subject an effective amount of an ENPP1 agent to thereby reduce and/or prevent allograft vasculopathy in said subject.

180. The method of claim 179, wherein the ENPP1 agent is administered to the subject prior to the surgical intervention or concurrently with the surgical intervention or following the surgical intervention.

181. The method of claim 179, wherein the ENPP1 agent comprises an ENPP1 polypeptide or a nucleic acid encoding an ENPP1 polypeptide or viral vector comprising a nucleic acid encoding an ENPP1 polypeptide.

182. The method of claim 181, wherein the ENPP1 polypeptide comprises the extracellular domain of ENPP1.

183. The method of claim 181, wherein the ENPP1 polypeptide comprises a heterologous protein and said heterologous protein increases the circulating half-life of the ENPP1 polypeptide in mammal.

184. The method of claim 183, wherein the heterologous protein is an Fc region of an immunoglobulin molecule or an albumin molecule.

185. The method of claim 184, wherein the ENPP1 agent is administered to the subject subcutaneously or intravenously.

186. The method of claim 184, wherein the ENPP1 agent comprises ENPP1 variants that retain enzymatic activity.

187. A method for reducing and/or preventing progression of vascular smooth muscle cell proliferation in a subject's peripheral vessel at or around the site at which an arterio-venous dialysis shunt has been placed, the method comprising: administering to the subject an effective amount of an ENPP1 agent to thereby reduce and/or prevent progression of vascular smooth muscle cell proliferation in said peripheral vessel at or around the site the arterio-venous dialysis shunt has been placed.

188. The method of claim 187, wherein the agent is administered prior to, during and/or after said surgery or said shunt placement.

189. The method of claim 187, wherein the ENPP1 agent comprises an ENPP1 polypeptide or a nucleic acid encoding an ENPP1 polypeptide or a viral vector comprising a nucleic acid encoding an ENPP1 polypeptide.

190. The method of claim 187, wherein the ENPP1 polypeptide comprises a heterologous protein and said heterologous protein increases the circulating half-life of the ENPP1 polypeptide in mammal.

191. The method of claim 190, wherein the heterologous protein is an Fc region of an immunoglobulin molecule or an albumin molecule.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0148] FIG. 1 shows the schematic diagram of prophylactic treatment regimen of control and experimental mice prior and after transplant. The experimental mice are treated 7 days prior to aortic transplantation with ENPP1-Fc at an exemplary dosage of 10 mg/kg weight by subcutaneous injection every day. The control cohorts are injected with vehicle containing tris buffered saline, at pH 7.4. All mice are then dissected at 28 days after transplantation and the mice are approximately 10 weeks of age.

[0149] FIG. 2 shows a schematic diagram of heart transplant in mouse. It also shows morphometrical measurements of 5 m sections of the transplanted aorta. The medial area, the intimal area and the intima/media ratio (I/M ratio) of each section are calculated.

[0150] FIG. 3 shows a schematic version of Porcine model of heterotopic heart transplantation. 3 (A) shows the donor heart is harvested after cardiac standstill achieved by using cold cardioplegic solution (Plegisol). 3(B) shows that the graft is maintained in the ice-saline slurry and prepared for implantation by creating an atrial septal defect and defunction the mitral valve to minimize left ventricular atrophy and intracavity thrombus formation. 3(C) shows the recipient's inferior vena cava (IVC) and the infrarenal aorta were isolated. 3(D) shows the graft heart is implanted by anastomosing the donor pulmonary artery to the recipient's IVC and the donor ascending aorta to the abdominal aorta of the recipient. Graft function was monitored by using (E) electrocardiography (ECG) and (F) echocardiography (UCG). Arrows indicate electrical spikes attributed to heterotopic cardiac allograft. (Hsu et al., Transplantation. 2018 December; 102(12): 2002-2011.)

[0151] FIG. 4 is a series of photographs of representative profunda artery images captured by angiography at day 14 and day 42 post stent implantation. The two control images illustrate a narrowing of the profunda due to intimal proliferation at day 42 relative to the morphology of the vessel at day 14. By contrast, in animals treated with ENPP1-Fc little visible change in profunda morphology was observed between day 14 and day 42. The upper and lower boundary of the stent within the vessel is identified in each photograph by rectangles.

[0152] FIG. 5 is a series of photographs of representative profunda artery images captured by Optical Coherence Tomography (OCT) at day 14 and day 42 post stent implantation. The two control images illustrate a pronounced intimal thickening within the profunda at day 42 relative to the morphology of the vessel at day 14. By contrast, in animals treated with ENPP1-Fc little visible intimal thickening was observed between day 14 and day 42. The extent of stenosis is highlighted in the day 42 photographs.

[0153] FIG. 6 is a bar graph depicting the percent area of stenosis at day 14 and day 42 in the profunda of pigs treated with ENPP1-Fc (Treatment) or given vehicle control (Control), as measured by OCT.

[0154] FIG. 7 shows the schematic diagram of prophylactic treatment regimen of control and experimental mice prior and after brain surgery to induce MMD. The experimental mice are treated 7 days prior to surgery with ENPP1-Fc at an exemplary dosage of 10 mg/kg weight by subcutaneous injection every day. The control cohorts are injected with vehicle containing tris buffered saline, at pH 7.4. All mice are then dissected at 28 days after transplantation and the mice are approximately 10 weeks of age.

[0155] FIG. 8 shows the process of creating MMD model by Internal Carotid Artery Stenosis. 8A) shows orientation of the mouse during the surgical procedure. Head (teeth), forepaws and tail are restrained, and incision is made in the midline of the neck (red dashed line). White box indicates region of images that follow. 8B) shows opening of the cervical region exposing the trachea, sternocleidomastoid (SCM) muscle and posterior belly of the digastric (PBD) muscle. 8C) shows suture (S1-2) placement retracting the SCM and PBD to expose the common, internal and external carotid (CCA, ICA, ECA) arteries. 8D) shows Identification of the occipital artery (OA), vagus nerve (VN) and ICA. 8E) shows suture ligation of the OA and dashed line showing cut to better expose the ICA. 8F) shows cut OA with ICA exposed and isolated using 60 suture. 8G) shows micro-coil placement on ICA deep to ECA (as seen in H). (Roberts et al., Internal carotid artery stenosis: A novel surgical model for moyamoya syndrome, PLoS One. 2018; 13(1): e0191312.)

[0156] FIG. 9 is a diagram of hemodialysis blood flow from a subject's arm, which contains a dialysis shunt, into a tube, past a pressure monitor, a blood pump, and a heparin pump, which prevents clotting. Blood flows past another pressure monitor before entering the dialyzer, or filter. Filtered blood continues past a venous pressure monitor, an air trap and air detector, and an air detector clamp, and returns to the subject's arm.

[0157] FIG. 10 is a view of an implantable shunt 2 positioned in the upper right chest area 100 of a subject. The implantable dialysis shunt 2 may also be implanted into other areas of the body, so long as it is implanted in reasonable proximity to a medium sized artery, typically between 6 and 8 mm, for use with the implantable dialysis shunt 2. The implantable dialysis shunt preferably comprises an arterial port 4 and a venous port 6 connected to each other in a single structure. In other embodiments, the ports 4, 6 may be separate structures which may include features to permit their attachment to each other. An arterial graft 12 generally extends through the arterial port 4 while a venous graft 18 extends from the venous port 6. During the implantation process, the arterial graft 12 is preferably connected at each of its ends to the sidewall of an artery 26 while the end of the venous graft 18 is connected to a vein 34. In other embodiments, the arterial graft 12 may be connected to the artery 26 by a pair of end-to-end anasomoses. Additionally, the venous graft 18 may take the form of a venous catheter which is inserted into the vein 34 such that it may enter the central venous system. Dialysis may be conducted by tapping the arterial port 4 with an arterial catheter 102 and the venous port with a venous catheter 104. Each of the arterial and venous catheters 102, 104 are connected to a dialysis machine.

DETAILED DESCRIPTION

Definitions

[0158] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure, the preferred methods and materials are described.

[0159] For clarity, NPP1 and ENPP1 refer to the same protein and are used interchangeably herein. As used herein, the term ENPP1 protein or ENPP1 polypeptide refers to ectonucleotide pyrophosphatase/phosphodiesterase-1 protein encoded by the ENPP1 gene that is capable of cleaving ATP to generate PPi and also reduces ectopic calcification in soft tissue.

[0160] ENPP1 protein is a type II transmembrane glycoprotein and cleaves a variety of substrates, including phosphodiester bonds of nucleotides and nucleotide sugars and pyrophosphate bonds of nucleotides and nucleotide sugars. ENPP1 protein has a transmembrane domain and soluble extracellular domain. The extracellular domain is further subdivided into somatomedin B domain, catalytic domain and the nuclease domain. The sequence and structure of wild-type ENPP1 is described in detail in PCT Application Publication No. WO 2014/126965 to Braddock, et al., which is incorporated herein in its entirety by reference.

[0161] ENPP1 polypeptides as used herein encompasses polypeptides that exhibit ENPP1 enzymatic activity, mutants of ENPP1 that retain ENPP1 enzymatic activity, fragments of ENPP1 or variants of ENPP1 including deletion variants that exhibit ENPP1 enzymatic activity. ENPP1 enzymatic activity refers to the ability of the ENPP1 polypeptide to cleave Adenosine Triphosphate (ATP) into plasma pyrophosphate (PPi), as noted below.

[0162] ENPP3 polypeptides as used herein encompasses polypeptides that exhibit ATP cleavage enzymatic activity, mutants of ENPP3 that retain ATP cleavage enzymatic activity, fragments of ENPP3 or variants of ENPP3 including deletion variants that exhibit ATP cleavage enzymatic activity. ATP cleavage enzymatic activity refers to the ability of the ENPP3 polypeptide to cleave Adenosine Triphosphate (ATP) into plasma pyrophosphate (PPi), as noted below.

[0163] Some examples of ENPP1 and ENPP3 polypeptides, mutants, or mutant fragments thereof, have been previously disclosed in International PCT Application Publications No. WO/2014/126965Braddock et al., WO/2016/187408-Braddock et al., WO/2017/087936-Braddock et al., and WO2018/027024-Braddock et al., all of which are incorporated by reference in their entireties herein.

[0164] Enzymatically active with respect to an ENPP1 polypeptide or an ENPP3 polypeptide is defined as possessing ATP hydrolytic activity into AMP and PPi and/or AP3a hydrolysis to ADP and AMP. NPP1 and NPP3 readily hydrolyze ATP into AMP and PPi. The steady-state Michaelis-Menten enzymatic constants of NPP1 are determined using ATP as a substrate. NPP1 can be demonstrated to cleave ATP by HPLC analysis of the enzymatic reaction, and the identity of the substrates and products of the reaction are confirmed by using ATP, AMP, and ADP standards. The ATP substrate degrades over time in the presence of NPP1, with the accumulation of the enzymatic product AMP. Using varying concentrations of ATP substrate, the initial rate velocities for NPP1 are derived in the presence of ATP, and the data is fit to a curve to derive the enzymatic rate constants. At physiologic pH, the kinetic rate constants of NPP1 are Km=144 M and kcat.sub.t=7.8 s.sup.1.

[0165] As used herein, the term ENPP1 precursor protein refers to ENPP1 with its signal peptide sequence at the ENPP1 N-terminus. Upon proteolysis, the signal sequence is cleaved from ENPP1 to provide the ENPP1 protein. Signal peptide sequences useful within the disclosure include, but are not limited to, Albumin signal sequence, Azurocidin signal sequence, ENPP1 signal peptide sequence, ENPP2 signal peptide sequence, ENPP7 signal peptide sequence, and/or ENPP5 signal peptide sequence.

[0166] As used herein, the term ENPP3 precursor protein refers to ENPP3 with its signal peptide sequence at the ENPP3 N-terminus. Upon proteolysis, the signal sequence is cleaved from ENPP3 to provide the ENPP3 protein. Signal peptide sequences useful within the disclosure include, but are not limited to, Albumin signal peptide sequence, Azurocidin signal peptide sequence, ENPP1 signal peptide sequence, ENPP2 signal peptide sequence, ENPP7 signal peptide sequence, and/or ENPP5 signal peptide sequence.

[0167] As used herein, the term Azurocidin signal peptide sequence refers to the signal peptide derived from human azurocidin. Azurocidin, also known as cationic antimicrobial protein CAP37 or heparin-binding protein (HBP), is a protein that in humans is encoded by the AZU1 gene. The nucleotide sequence encoding Azurocin signal peptide MTRLTVLALLAGLLASSRA (SEQ ID NO: 42) is fused with the nucleotide sequence of NPP1 or NPP3 gene which when encoded generates ENPP1 precursor protein or ENPP3 precursor protein. (Optimized signal peptides for the development of high expressing CHO cell lines, Kober et al., Biotechnol Bioeng. 2013 April; 110(4):1164-73)

[0168] As used herein, the term ENPP1-Fc construct refers to ENPP1 (e.g., the extracellular domain of ENPP1) recombinantly fused and/or chemically conjugated (including both covalent and non-covalent conjugations) to an FcR binding domain of an IgG molecule (preferably, a human IgG). In certain embodiments, the C-terminus of ENPP1 is fused or conjugated to the N-terminus of the FcR binding domain.

[0169] As used herein, the term ENPP3-Fc construct refers to ENPP3 recombinantly fused and/or chemically conjugated (including both covalent and non-covalent conjugations) to an FcR binding domain of an IgG molecule (preferably, a human IgG). In certain embodiments, the C-terminus of ENPP1 is fused or conjugated to the N-terminus of the FcR binding domain.

[0170] As used herein, the term Fc refers to a human IgG (immunoglobulin) Fc domain. Subtypes of IgG such as IgG1, IgG2, IgG3, and IgG4 are contemplated for use as Fc domains. The Fc region or Fe polypeptide is the portion of an IgG molecule that correlates to a crystallizable fragment obtained by papain digestion of an IgG molecule. The Fc region comprises the C-terminal half of the two heavy chains of an IgG molecule that are linked by disulfide bonds. It has no antigen binding activity but contains the carbohydrate moiety and the binding sites for complement and Fc receptors, including the FcRn receptor. The Fc fragment contains the entire second constant domain CH2 (residues 231-340 of human IgG1, according to the Kabat numbering system) and the third constant domain CH3 (residues 341-447). The term IgG hinge-Fc region or hinge-Fc fragment refers to a region of an IgG molecule consisting of the Fc region (residues 231-447) and a hinge region (residues 216-230) extending from the N-terminus of the Fc region. The term constant domain refers to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable domain, which contains the antigen binding site. The constant domain contains the CHL CH2 and CH3 domains of the heavy chain and the CHL domain of the light chain.

[0171] As used herein the term functional equivalent variant, as used herein, relates to a polypeptide substantially homologous to the sequences of ENPP1 or ENPP3 (defined above) and that preserves the enzymatic and biological activities of ENPP1 or ENPP3, respectively. Methods for determining whether a variant preserves the biological activity of the native ENPP1 or ENPP3 are widely known to the skilled person and include any of the assays used in the experimental part of said application. Particularly, functionally equivalent variants of ENPP1 or ENPP3 delivered by viral vectors is encompassed by the present disclosure.

[0172] The functionally equivalent variants of ENPP1 or ENPP3 are polypeptides substantially homologous to the native ENPP1 or ENPP3 respectively. The expression substantially homologous, relates to a protein sequence when said protein sequence has a degree of identity with respect to the ENPP1 or ENPP3 sequences described above of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% respectively and still retaining at least 50%, 55%, 60%, 70%, 80% or 90% activity of wild type ENPP1 or ENPP3 protein with respect to ATP cleavage.

[0173] The degree of identity between two polypeptides is determined using computer algorithms and methods that are widely known for the persons skilled in the art. The identity between two amino acid sequences is preferably determined by using the BLASTP algorithm (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990)), though other similar algorithms can also be used. BLAST and BLAST 2.0 are used, with the parameters described herein, to determine percent sequence identity. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.

[0174] Functionally equivalent variants of ENPP1 or ENPP3 may be obtained by replacing nucleotides within the polynucleotide accounting for codon preference in the host cell that is to be used to produce the ENPP1 or ENPP3 respectively. Such codon optimization can be determined via computer algorithms which incorporate codon frequency tables such as Human high.cod for codon preference as provided by the University of Wisconsin Package Version 9.0, Genetics Computer Group, Madison, Wis. The variants of ENPP1 or ENPP3 polypeptides are expected to retain at least 50%, 55%, 60%, 70%, 80% or 90% activity of wild type ENPP1 or ENPP3 protein with respect to ATP cleavage.

[0175] As used herein the term ENPP1 fragment refers to a fragment or a portion of ENPP1 protein or an active subsequence of the full-length NPP1 having at least an ENPP1 catalytic domain administered in protein form or in the form of a nucleic acid encoding the same.

[0176] As used herein, the term ENPP1 agent refers to ENPP1 polypeptide or fusion protein or ENPP1 fragment comprising at least catalytic domain capable of producing plasma pyrophosphate (Ppi) by cleavage of adenosine triphosphate (ATP) or a polynucleotide such as cDNA or RNA encoding ENPP1 fusion protein or ENPP1 fragment comprising at least catalytic domain capable of producing PPi by enzymatic cleavage of ATP or a vector such as a viral vector containing a polynucleotide encoding the same.

[0177] 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 most frequently observed in a population and is thus arbitrarily designed the normal or wild-type form of the human NPP1 or NPP3 genes. In contrast, the term functionally equivalent refers to a NPP1 or NPP3 gene or gene product that displays modifications in sequence and/or functional properties (i.e., altered characteristics) when compared to the wild-type gene or gene product. Naturally occurring mutants can be isolated; these are identified by the fact that they have altered characteristics (including altered nucleic acid sequences) when compared to the wild-type gene or gene product.

[0178] About as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of +20% or +10%, more preferably +5%, even more preferably +1%, and still more preferably +0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0179] As defined herein, the term moiety refers to a chemical component or biological molecule that can be covalently or non-covalently linked to ENPP1 or ENPP3 polypeptide and has the ability to confer a desired property to the protein to which it is attached. For example, the term moiety can refer to a bone targeting peptide such as polyaspartic acid or polyglutamic acid (of 4-20 consecutive asp or glu residues) or a molecule that extends the half-life of ENPP1 or ENPP3 polypeptide. Some other examples of moieties include Fc, albumin, transferrin, polyethylene glycol (PEG), homo-amino acid polymer (HAP), proline-alanine-serine polymer (PAS), elastin-like peptide (ELP), and gelatin-like protein (GLK).

[0180] As defined herein, the term subject, individual or patient refers to mammal preferably a human who does not possess a loss of function mutation in the NPP1 gene, such as those loss of function mutations that result in pathological calcification and pathological ossification diseases such as Generalized Arterial Calcification of Infancy (GACI), Autosomal Recessive Hypophosphatemic Rickets Type 2 (ARHR2), Infantile idiopathic arterial calcification (IIAC), Ossification of the Posterior Longitudinal Ligament (OPLL), hypophosphatemic rickets, osteoarthritis, calcification of atherosclerotic plaques, hereditary and non-hereditary forms of osteoarthritis, ankylosing spondylitis, hardening of the arteries occurring with aging, calciphylaxis resulting from end stage renal disease and progeria. Such a patient will have a normal level of NPP1 in serum which refers to the amount of NPP1 required to maintain a normal level of plasma pyrophosphate (PPi) in a healthy subject. A normal level of PPi corresponds to 2-3 M.

[0181] As used herein the term plasma pyrophosphate (PPi) levels refers to the amount of pyrophosphate present in plasma of animals. In certain embodiments, animals include rat, mouse, cat, dog, human, cow and horse. There are several ways to measure PPi, one of which is by enzymatic assay using uridine-diphosphoglucose (UDPG) pyrophosphorylase (Lust & Seegmiller, 1976, Clin. Chim. Acta 66:241-249; Cheung & Suhadolnik, 1977, Anal. Biochem. 83:61-63) with modifications.

[0182] Typically, plasma PPi levels in healthy human subjects range from about 1 m to about 3 M, in some cases between 1-2 m. Subjects who have defective ENPP1 expression tend to exhibit low ppi levels which range from at least 10% below normal levels, at least 20% below normal levels, at least 30% below normal levels, at least 40% below normal levels, at least 50% below normal levels, at least 60% below normal levels, at least 70% below normal levels, at least 80% below normal levels and combinations thereof. In patients afflicted with Generalized Arterial Calcification of Infancy (GACI), the ppi levels are found to be less than 1 m and in some cases are below the level of detection. In patients afflicted with Pseudoxanthoma Elasticum (PXE), the ppi levels are below 0.5 m. (Arterioscler Thromb Vasc Biol. 2014 September; 34(9):1985-9; Braddock et al., Nat Commun. 2015; 6: 10006.)

[0183] As used herein, the term PPi refers to inorganic pyrophosphate.

[0184] A low level of PPi refers to a condition in which the subject has at least 0.1%-0.99% less than 2%-5% of normal levels of plasma pyrophosphate (PPi). Normal levels of Plasma PPi in healthy human subjects are in the range of 1.8 to 2.6 M.+/0.1 M (Arthritis and Rheumatism, Vol. 22, No. 8 (August 1979))

[0185] As used herein the term non-surgical tissue injury refers to injuries sustained to a tissue or blood vessel during a traumatic event including but not limited to physical altercations involving use of blunt force or sharp objects such as knife, mechanical injury such fall from elevation, workplace injury due to heavy machinery or vehicular injury such as car accidents.

[0186] As used herein, the term myocardial infarction refers to a permanent damage to the heart muscle that occurs due to the formation of plaques in the interior walls of the arteries resulting in reduced blood flow to the heart and injuring heart muscles because of lack of oxygen supply. The symptoms of MI include chest pain, which travels from left arm to neck, shortness of breath, sweating, nausea, vomiting, abnormal heart beating, anxiety, fatigue, weakness, stress, depression, and other factors.

[0187] As used herein, the term moyamoya disease or moyamoya syndrome refers to a steno-occlusive disease of the cerebral arteries, involving smooth muscle cell proliferation with intima hyperplasia causing arterial stenosis and occlusion around the circle of Willis. It involves development of new blood vessels resembling a puff of smoke (moyamoya) in the subcortical region. MMD occurs in children and adults with two peaksat around age 5-10 and a second peak between the third and fifth decade of life. Common symptoms include headache or dizziness, weakness or paralysis in a limb or on one side of the body, problems with speech inability to speak or recall words, sensory or cognitive impairment, involuntary movements, seizures or loss of consciousness, vision problems, stroke, and cerebral hemorrhage. 80% of MMD cases are carriers of RNF213 and or R4810K mutations. Treatment options for both MMD and MMS involve daily aspirin use, lifestyle modifications to maximize cerebral perfusion, and surgical direct or indirect bypass to restore blood flow.

[0188] Diagnostic criteria for definitive MMD were revised to include patients with both bilateral and unilateral presentation of terminal carotid artery stenosis (ICA) with an abnormal vascular network at the base of the brain. Suzuki system of grading the patient population has been used for MMD. Definitive diagnosis of MMD requires catheter angiography in unilateral cases, whereas bilateral cases can be promptly diagnosed by either catheter angiography or magnetic resonance imaging/angiography (MRI/MRA).

[0189] As used herein, the phrase cerebral vascular occlusion refers to the temporary or permanent blockage of blood vessels in the brain. Restrictions in blood flow may occur from vessel narrowing (stenosis), clot formation (thrombosis), blockage (embolism) or blood vessel rupture (hemorrhage). Lack of sufficient blood flow (ischemia) affects brain tissue and may cause a stroke.

[0190] As used herein the term Suzuki classification System refers to classification system developed by Suzuki et al. (Suzuki J, Takaku A. Cerebrovascular moyamoya disease. Disease showing abnormal net-like vessels in base of brain. Arch Neurol. 1969; 20(3):28899.). This classification system grades the clinical presentation of patients to four stages. The vast majority of patients will progress through some or all of the Suzuki stages, although progression may occur at different rates, and appears to occur more rapidly in children than in adolescents or adults. The system is solely based on conventional angiography and is as shown in table below.

TABLE-US-00001 GRADE SYMPTOMS Stage I Narrowing of the carotid fork; narrowed Internal carotid artery (ICA) bifurcation Stage II Initiation of the Moyamoya; dilated Anterior cerebral artery (ACA), Middle Cerebral artery (MCA) and narrowed ICA bifurcation with Moyamoya change Stage III Intensification of the Moyamoya, further increase in Moyamoya change of the ICA bifurcation and narrowed ACA and MCA Stage IV Minimization of the Moyamoya, Moyamoya change reducing with occlusive changes in ICA and tenuous ACA and MCA Stage V Reduction of the Moyamoya, further decrease in Moyamoya change with occlusion of ICA, ACA and MCA Stage VI Disappearance of the Moyamoya, ICA essentially disappeared with supply of brain from External Carotid artery (ECA)

[0191] As used herein, the term internal carotid artery (ICA) refers to the artery that is located in the inner side of the neck and supplies blood to the brain and eyes.

[0192] As used herein, the term external carotid artery (ECA) refers to a major artery of the head and neck. It arises from the common carotid artery when it splits into the external and internal carotid artery. ECA supplies blood to face, scalp, skull, and meninges. As used herein, the term anterior Cerebral Artery (ACA) refers is an artery on the brain that supplies oxygenated blood to most midline portions of the frontal lobes and superior medial parietal lobes. A pair of anterior cerebral arteries arise from the internal carotid artery and are part of the circle of Willis.

[0193] As used herein, the term medial cerebral artery (MCA) refers to is one of the three major paired arteries that supply blood to the cerebrum. The MCA arises from the internal carotid and continues into the lateral sulcus where it then branches and projects to many parts of the lateral cerebral cortex. It also supplies blood to the anterior temporal lobes and the insular cortices.

[0194] As used herein, the term conventional angiography refers to Angiography or arteriography is a medical imaging technique used to visualize the inside, or lumen, of blood vessels and organs of the body, with particular interest in the arteries, veins, and the heart chambers. This is traditionally done by injecting a radio-opaque contrast agent into the blood vessel and imaging using X-ray based techniques such as fluoroscopy.

[0195] As used herein, the term catheter angiography refers to a medical procedure wherein a catheter, x-ray imaging guidance and an injection of contrast material to examine blood vessels in key areas of the body such as brain or heart for abnormalities such as aneurysms and disease such as atherosclerosis (plaque).

[0196] As used herein, the phrase magnetic resonance angiography (MRA) refers to a medical process wherein magnetic resonance imaging scanner is used to visualize the blockages in the blood vessels of critical regions such as brain, lungs and heart with aid of a contrast agent administered with an intravenous needle. It is non-invasive method to diagnose blockages or occlusions in the blood vessels.

[0197] As used herein, the term subject who requires surgery refers to a patient who is not ENPP1 deficient and has arterial occlusion in the peripheral arteries such as femoral, femoropopliteal or tibial-peroneal arteries.

[0198] As used herein, the term site of surgery refers to the region of the artery upon which a tissue injury has occurred either due to vascular trauma or accidental trauma.

[0199] As used herein, the term brain calcification (BC) refers to a nonspecific neuropathology wherein deposition of calcium and other mineral in blood vessel walls and tissue parenchyma occurs leading to neuronal death and gliosis. Brain calcification is often associated with various chronic and acute brain disorders including Down's syndrome, Lewy body disease, Alzheimer's disease, Parkinson's disease, vascular dementia, brain tumors, and various endocrinologic conditions Calcification of heart tissue refers to accumulation of deposits of calcium (possibly including other minerals) in tissues of the heart, such as aorta tissue and coronary tissue.

[0200] As used herein with respect to use of a dialysis shunt, stenosis slows and reduces blood flow through an AV fistula, causing problems with the quality of dialysis treatment, prolonged bleeding after puncture, or pain in the fistula. Stenosis can also lead to a blocked or clotted access.

[0201] As used herein the term scapel incision refers to incision made in a tissue using a sharp object such as a scapel during surgical procedure. An incision is a cut made into the tissues of the body to expose the underlying tissue, bone, or organ so that a surgical procedure can be performed.

[0202] As used herein, the term site of surgery refers to the region of the artery upon which a tissue injury has occurred either due to vascular trauma or accidental trauma.

[0203] The term arterio-venous shunt or AV shunt or simply shunt refers to an implanted device which includes a tube to which an artery and vein is attached. A shunt connects the arterial and venous cannulas and provides a larger than normal volume of blood flow for effective hemodialysis. A shunt can be located in any part of the body, and is most often located in an arm, a leg or the chest area below the right collarbone.

[0204] As used herein, the term coated shunt refers to shunts that are capable of slowly eluting therapeutic compounds or polypeptides such as ENPP1 or ENPP3 to reduce the amount of vascular smooth muscle cell proliferation at the site of surgery, typically performed to remove blockage of the arteries.

[0205] As used herein, the term hemodialysis refers to a treatment that is required to compensate for abnormal kidney function, in which wastes and water are filtered out of blood and the filtered cleaner blood is returned to the body. Hemodialysis helps control blood pressure and balance important minerals, such as potassium, sodium, and calcium, in a subject's blood.

[0206] As used herein, the term fistula refers to an abnormal or surgically made passage between a hollow or tubular organ and the body surface, or between two hollow or tubular organs.

[0207] The term stent refers to a tubular support placed inside a blood vessel, canal, or duct to aid healing or relieve an obstruction.

[0208] The term vessel refers to a tubular structure carrying blood through the tissues and organs; a vein, artery, or capillary.

[0209] As used herein, the term complement inhibitor refer to a molecule (e.g., a protein (such as an antibody), a small molecule, or a peptide) that prevents or reduces activation and/or propagation of the complement cascade that results in the formation of C3a or signaling through the C3a receptor, C5a or signaling through the C5a receptor, or formation of terminal complement. Complement inhibitors are well known in the art and described in, e.g., Zipfel et al. (2019) Front Immunol 10:2166. See also, e.g., U.S. Pat. No. 5,679,345, the disclosure of which is incorporated by reference in its entirety.

[0210] As used herein the terms alteration, defect, variation or mutation refer to a mutation in a gene in a cell that affects the function, activity, expression (transcription or translation) or conformation of the polypeptide it encodes, including missense and nonsense mutations, insertions, deletions, frameshifts and premature terminations.

[0211] As defined herein, the phrase medial area is the area between lamina elastica externa and lamina elastica interna of an artery.

[0212] As defined herein, the phrase intimal area and said intimal area is the area between said lamina elastica interna and lumen of an artery.

[0213] As defined herein, the phrase lamina elastica externa refers to a layer of elastic connective tissue lying immediately outside the smooth muscle of the tunica media of an artery.

[0214] As defined herein, the phrase lamina elastica interna refers to a layer of elastic tissue that forms the outermost part of the tunica intima of blood vessels.

[0215] As defined herein, the phrase lumen refers to the interior of a vessel, such as the central space in an artery, vein or capillary through which blood flow occurs.

[0216] As defined herein, the phrase vasculopathy refers to disease of the vasculature. Vasculature refers to the arrangement of blood vessels in the body or in an organ, such as a solid organ transplant, or in a body part. A blood vessel refers to one or more of an artery, arteriole, capillary and vein in the body of a subject or of a solid organ allograft of a subject. Vasculitis refers to inflammation of veins, arteries, capillaries, or lymph vessels. A vascularized graft refers to a graft after the recipient vasculature has been connected with the vessels in the graft.

[0217] As defined herein, the phrase cardiac allograft vasculopathy (CAV) refers to a vascular complication of allograft or solid organ transplantation such as heart wherein the blood vessels supplying the transplanted heart gradually narrow and restrict its blood flow, subsequently leading to impairment of the heart muscle or sudden death. Diagnosis of CAV is by regular follow-up and monitoring of the transplanted organ such as heart for early signs of disease. This involves invasive diagnostics including coronary angiography and intravascular ultrasound, and non-invasive investigations including dobutamine stress echocardiography, positron emission tomography, computed tomographic angiography (CT angiography) and the levels of a variety of biomarkers such as C-reactive protein, serum brain natriuretic peptide, troponin and serum microRNA 628-5p.

[0218] As defined herein, allograft refers to the transplant of an organ or tissue from a donor to a recipient of the same species. Allografts account for many human organ and tissue transplants, including those from cadaveric, living related, and living unrelated donors.

[0219] As defined herein, a solid organ allograft refers to an allograft of a solid organ. A solid organ is an internal organ that has a firm tissue consistency and is neither hollow (such as the organs of the gastrointestinal tract) nor liquid (such as blood). A solid organ includes but is not limited to kidney, liver, cornea, intestines, heart, lung and pancreas.

[0220] As defined herein, the phrase graft rejection or transplant rejection refers to a condition wherein the transplanted organ or tissue is rejected by the recipient's immune system, which destroys the allograft and results in long-term loss of function in transplanted organs via fibrosis of the transplanted tissue blood vessels.

[0221] As defined herein, the phrase prolonging the survival of an allograft refers to the prevention of rejection of a transplanted donor organ or tissue by the recipient immune system and to improve the lifespan of the transplanted organ. Survival of an allograft may be prolonged by at least 12 months, 18 months, 2 years, 3 years, 4 years, 5 years, 8 years, 10 years or longer relative to allograft survival absent treatment.

[0222] As defined herein, the phrase heart allograft refers to a solid organ transplant involving a donor heart transplanted into a recipient or grafting of one or more donor arteries or veins into a recipient's heart. Graft rejection in heart allografts is commonly diagnosed by performing Endomyocardial biopsy.

[0223] As defined herein, the phrase kidney allograft refers to a solid organ transplant involving a donor kidney transplanted into a recipient or grafting of one or more donor arteries or veins into a recipient's kidney. Graft rejection in kidney allografts is commonly diagnosed by monitoring Urine protein levels such total protein-to-creatinine ratio, albumin-to-creatinine ratio, serum creatinine level and glomerular filtration rate.

[0224] As defined herein, the phrase liver allograft refers to a solid organ transplant involving a donor liver transplanted into a recipient or grafting of one or more donor arteries or veins into a recipient's liver. Graft rejection in liver allografts is diagnosed by monitoring Transaminase, bilirubin, and alkaline phosphatase levels.

[0225] As defined herein, the phrase lung allograft refers to refers to a solid organ transplant involving a donor lung transplanted into a recipient or grafting of one or more donor arteries or veins into a recipient's lung. Graft rejection in lung allografts is diagnosed by bronchoscopy with transbronchial biopsies and pulmonary function testing.

[0226] As defined herein, the phrase allografted vessel or Allografted vasculature refers to the grafting of one or more donor blood vessels such as artery, vein, capillary and/or arteriole into the recipient.

[0227] As defined herein, the phrase allografted artery refers to the grafting of one or more donor arteries into the recipient.

[0228] As defined herein, the phrase allografted vein refers to the grafting of one or more donor veins into the recipient.

[0229] As defined herein, the phrase endomyocardial biopsy refers to a procedure that percutaneously obtains small amounts of myocardial tissue for diagnostic, therapeutic, and research purposes. It is primarily used to (1) follow the transplanted heart for myocardial rejection; (2) diagnose specific inflammatory, infiltrative, or familial myocardial disorders; and (3) sample unknown myocardial masses.

[0230] As defined herein, the phrase transbronchial lung biopsy refers to a biopsy from the lung obtained by endoscopically-guided forceps, which is useful in evaluating lesions in the transplant distributed along bronchovascular bundles and in the central lung zones.

[0231] As defined herein, the phrase surgery refers to an invasive medical procedure that involves vascular interventions which result in tissue injury by scapel incision or radiofrequency ablation or cryoablation or laser ablation.

[0232] As defined herein, the phrase tissue injury refers to proliferation or onset of proliferation and migration of vascular smooth muscle eventually resulting in the thickening of arterial walls and decreased arterial lumen space resulting restenosis after percutaneous vascular interventions such as stenting or angioplasty.

[0233] As defined herein, the phrase deficient for NPP1 or ENPP 1 deficiency refers to a reduction in an amount of NPP1 protein or in NPP1 activity relative to a normal serum level of NPP1 protein or normal activity of NPP1, wherein such a reduction results in a disease or disorder of pathological calcification and/or pathological ossification. Such pathological diseases include but are not limited to GACI and ARHR2. ENPP1 deficiency, as used herein, does not refer to small reductions in an amount of NPP1 protein and/or NPP1 activity that do not result in a disease or disorder of pathological calcification and/or pathological ossification.

[0234] As defined herein the phrase restenosis refers to recurrence of stenosis. Stenosis refers to the narrowing of a blood vessel, leading to restricted blood flow. Restenosis usually pertains to an artery or other large blood vessel that has become narrowed, received treatment to clear the blockage and subsequently become re-narrowed. Restenosis is commonly detected by using one or more of ultrasound, X-ray computed tomography (CT), nuclear imaging, optical imaging or contrast enhanced image or immunohistochemical detection. As defined herein the phrase myointimal proliferation refers to the proliferation of vascular smooth muscle cells that occurs at the tunica intima of an arterial wall of an individual.

[0235] As used herein, the phrase reduce or prevent myointimal proliferation refers to the ability of soluble NPP1 upon administration to reduce the level of proliferation vascular smooth muscle cells at the site of tissue injury thereby reducing the thickening of arterial walls and prevent the occurrence of or reduce the level of restenosis of the artery.

[0236] As used herein, the term treatment or treating is defined as the application or administration of soluble NPP1 (alone or in combination with another pharmaceutical agent), to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient (e.g., for diagnosis or ex vivo applications), who has a disease or disorder, a symptom of a disease or disorder or the potential to develop a disease or disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease or disorder, the symptoms of the disease or disorder, or the potential to develop the disease or disorder. Such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics.

[0237] As used herein, the term prevent or prevention or reduce means no disorder or disease development if none had occurred, or no further disorder or disease development if there had already been development of the disorder or disease. Also considered is the ability of one to prevent some or all of the symptoms associated with the disorder or disease.

[0238] As used herein, the term effective amount refers to an amount of an agent (e.g., NPP1 fusion or NPP3 fusion polypeptides) which, as compared to a corresponding subject who has not received such an amount, sufficient to provide improvement of a condition, disorder, disease, or to provide a decrease in progression or advancement of a condition, disorder, or disease. An effective amount also may result in treating, healing, preventing or ameliorating a condition, disease, or disorder. The term also includes within its scope amounts effective to enhance normal physiological function. As used herein, the term polypeptide refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof linked via peptide bonds.

[0239] As used here the term Isolated means altered or removed from the natural state. For example, a nucleic acid or a polypeptide naturally present in a living animal is not isolated, but the same nucleic acid or polypeptide partially or completely separated from the coexisting materials of its natural state is isolated. An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0240] As used herein, substantially purified refers to being essentially free of other components. For example, a substantially purified polypeptide is a polypeptide that has been separated from other components with which it is normally associated in its naturally occurring state. Non-limiting embodiments include 95% purity, 99% purity, 99.5% purity, 99.9% purity and 100% purity.

[0241] As used herein the term oligonucleotide or polynucleotide is a nucleic acid ranging from at least 2, in certain embodiments at least 8, 15 or 25 nucleotides in length, but may be up to 50, 100, 1000, or 5000 nucleotides long or a compound that specifically hybridizes to a polynucleotide.

[0242] As used herein, the term pharmaceutical composition or composition refers to a mixture of at least one compound useful within the disclosure with a pharmaceutically acceptable carrier. The pharmaceutical composition facilitates administration of the compound to a patient. Multiple techniques of administering a compound exist in the art including, but not limited to, subcutaneous, intravenous, oral, aerosol, inhalational, rectal, vaginal, transdermal, intranasal, buccal, sublingual, parenteral, intrathecal, intragastrical, ophthalmic, pulmonary, and topical administration.

[0243] As used herein, the term pharmaceutically acceptable refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively non-toxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained; for example, phosphate-buffered saline (PBS)

[0244] As used herein, the term pathological calcification refers to the abnormal deposition of calcium salts in blood vessels, soft tissues, secretory and excretory passages of the body causing it to harden. There are two types, dystrophic calcification which occurs in dying and dead tissue and metastatic calcification which elevated extracellular levels of calcium (hypercalcemia), exceeding the homeostatic capacity of cells and tissues. Calcification can involve cells as well as extracellular matrix components such as collagen in basement membranes and elastic fibers in arterial walls. Some examples of tissues prone to calcification include: Gastric mucosathe inner epithelial lining of the stomach, Kidneys and lungs, Cornea, heart valves, Systemic arteries and Pulmonary veins.

[0245] As used herein, the term pathological ossification refers to a pathological condition in which bone arises in tissues not in the osseous system and in connective tissues usually not manifesting osteogenic properties. Ossification is classified into three types depending on the nature of the tissue or organ being affected, endochondral ossification is ossification that occurs in and replaces cartilage. Intramembranous ossification is ossification of bone that occurs in and replaces connective tissue. Metaplastic ossification the development of bony substance in normally soft body structures; called also heterotrophic ossification.

[0246] As used herein, reduction of calcification is observed by using non-invasive methods like X-rays, micro CT and Mill. Reduction of calcification is also inferred by using radio imaging with 99mTc-pyrophosphate (99mPYP) uptake. The presence of calcifications in mice are evaluated via post-mortem by micro-computed tomography (CT) scans and histologic sections taken from the heart, aorta and kidneys with the use of dyes such as Hematoxylin and Eosin (H&E) and Alizarin red by following protocols established by Braddock et al. (Nature Communications volume 6, Article number: 10006 (2015))

[0247] As used herein the term ectopic calcification refers to a condition characterized by a pathologic deposition of calcium salts in tissues or bone growth in soft tissues.

[0248] As used herein the term ectopic calcification of soft tissue refers to inappropriate biomineralization, typically composed of calcium phosphate, hydroxyapatite, calcium oxalates and ocatacalcium phosphates occurring in soft tissues leading to loss of hardening of soft tissues. Arterial calcification refers to ectopic calcification that occurs in arteries and heart valves leading to hardening and or narrowing of arteries. Calcification in arteries is correlated with atherosclerotic plaque burden and increased risk of myocardial infarction, increased ischemic episodes in peripheral vascular disease, and increased risk of dissection following angioplasty.

[0249] As used herein, the term venous calcification refers to ectopic calcification that occurs in veins that reduces the elasticity of the veins and restricts blood flow which can then lead to increase in blood pressure and coronary defects

[0250] As used herein, the term vascular calcification refers to the pathological deposition of mineral in the vascular system. It has a variety of forms, including intimal calcification and medial calcification, but can also be found in the valves of the heart. Vascular calcification is associated with atherosclerosis, diabetes, certain heredity conditions, and kidney disease, especially CKD. Patients with vascular calcification are at higher risk for adverse cardiovascular events. Vascular calcification affects a wide variety of patients. Idiopathic infantile arterial calcification is a rare form of vascular calcification where the arteries of neonates calcify.

[0251] The terms adeno-associated viral vector, AAV vector, adeno-associated virus, AAV virus, AAV virion, AAV viral particle and AA V particle, as used interchangeably herein, refer to a viral particle composed of at least one AAV capsid protein (preferably by all of the capsid proteins of a particular AAV serotype) and an encapsidated recombinant viral genome. The particle comprises a recombinant viral genome having a heterologous polynucleotide comprising a sequence encoding human ENPP1 or human ENPP3 or a functionally equivalent variant thereof,) and a transcriptional regulatory region that at least comprises a promoter flanked by the AAV inverted terminal repeats. The particle is typically referred to as an AAV vector particle or AAV vector.

[0252] As used herein, the term vector means a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In some embodiments, the vector is a plasmid, i.e., a circular double stranded DNA loop into which additional DNA segments may be ligated. In some embodiments, the vector is a viral vector, wherein additional nucleotide sequences may be ligated into the viral genome. In some embodiments, the vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). In other embodiments, the vectors (e.g., non-episomal mammalian vectors) is integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors (expression vectors) are capable of directing the expression of genes to which they are operatively linked.

[0253] As used herein, the term recombinant host cell (or simply host cell), as used herein, means a cell into which an exogenous nucleic acid and/or recombinant vector has been introduced. It should be understood that recombinant host cell and host cell mean not only the particular subject cell but also the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term host cell as used herein.

[0254] The term recombinant viral genome, as used herein, refers to an AAV genome in which at least one extraneous expression cassette polynucleotide is inserted into the naturally occurring AAV genome. The genome of the AAV according to the disclosure typically comprises the cis-acting 5 and 3 inverted terminal repeat sequences (ITRs) and an expression cassette.

[0255] The term expression cassette, as used herein, refers to a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements, which permit transcription of a particular nucleic acid in a target cell. The expression cassette of the recombinant viral genome of the AAV vector according to the disclosure comprises a transcriptional regulatory region operatively linked to a nucleotide sequence encoding ENPP1 or ENPP3 or a functionally equivalent variant thereof.

[0256] The term transcriptional regulatory region, as used herein, refers to a nucleic acid fragment capable of regulating the expression of one or more genes. The transcriptional regulatory region according to the disclosure includes a promoter and, optionally, an enhancer.

[0257] The term promoter, as used herein, refers to a nucleic acid fragment that functions to control the transcription of one or more polynucleotides, located upstream the polynucleotide sequence(s), and which is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites, and any other DNA sequences including, but not limited to, transcription factor binding sites, repressor, and activator protein binding sites, and any other sequences of nucleotides known in the art to act directly or indirectly to regulate the amount of transcription from the promoter. Any kind of promoters may be used in the disclosure including inducible promoters, constitutive promoters and tissue-specific promoters.

[0258] The term enhancer, as used herein, refers to a DNA sequence element to which transcription factors bind to increase gene transcription. Examples of enhancers may be, without limitation, RSV enhancer, CMV enhancer, HCR enhancer, etc. In another embodiment, the enhancer is a liver-specific enhancer, more preferably a hepatic control region enhancer (HCR).

[0259] The term operatively linked, as used herein, refers to the functional relation and location of a promoter sequence with respect to a polynucleotide of interest (e.g. a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence). Generally, a promoter operatively linked is contiguous to the sequence of interest. However, an enhancer does not have to be contiguous to the sequence of interest to control its expression. In another embodiment, the promoter and the nucleotide sequence encoding ENPP1 or ENPP3 or a functionally equivalent variant thereof.

[0260] The term effective amount refers to a nontoxic but sufficient amount of a viral vector encoding ENPP1 or ENPP3 to provide the desired biological result. That result may be reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system.

[0261] The term Cap protein, as used herein, refers to a polypeptide having at least one functional activity of a native AAV Cap protein (e.g. VP1, VP2, VP3). Examples of functional activities of Cap proteins include the ability to induce formation of a capsid, facilitate accumulation of single-stranded DNA, facilitate AAV DNA packaging into capsids (i.e. encapsidation), bind to cellular receptors, and facilitate entry of the virion into host cells. In principle, any Cap protein can be used in the context of the present disclosure.

[0262] The term capsid, as used herein, refers to the structure in which the viral genome is packaged. A capsid consists of several oligomeric structural subunits made of proteins. For instance, AAV have an icosahedral capsid formed by the interaction of three capsid proteins: VP1, VP2 and VP3.

[0263] The term Rep protein, as used herein, refers to a polypeptide having at least one functional activity of a native AAV Rep protein (e.g. Rep 40, 52, 68, 78). A functional activity of a Rep protein is any activity associated with the physiological function of the protein, including facilitating replication of DNA through recognition, binding and nicking of the AAV origin of DNA replication as well as DNA helicase activity.

[0264] The term adeno-associated virus ITRs or AAV ITRs, as used herein, refers to the inverted terminal repeats present at both ends of the DNA strand of the genome of an adeno-associated virus. The ITR sequences are required for efficient multiplication of the AAV genome. Another property of these sequences is their ability to form a hairpin. This characteristic contributes to its self-priming which allows the primase-independent synthesis of the second DNA strand. Procedures for modifying these ITR sequences are known in the art (Brown T, Gene Cloning, Chapman & Hall, London, G B, 1995; Watson R, et al., Recombinant DNA, 2.sup.nd Ed. Scientific American Books, New York, N.Y., US, 1992; Alberts B, et al., Molecular Biology of the Cell, Garland Publishing Inc., New York, N.Y., US, 2008; Innis M, et al., Eds., PCR Protocols. A Guide to Methods and Applications, Academic Press Inc., San Diego, Calif., US, 1990; and Schleef M, Ed., Plasmid for Therapy and Vaccination, Wiley-VCH Verlag GmbH, Weinheim, Del., 2001).

[0265] The term tissue-specific promoter is only active in specific types of differentiated cells or tissues. Typically, the downstream gene in a tissue-specific promoter is one which is active to a much higher degree in the tissue(s) for which it is specific than in any other. In this case there may be little or substantially no activity of the promoter in any tissue other than the one(s) for which it is specific.

[0266] The term inducible promoter, as used herein, refers to a promoter that is physiologically or developmentally regulated, e.g. by the application of a chemical inducer. For example, it can be a tetracycline-inducible promoter, a mifepristone (RU-486)-inducible promoter and the like.

[0267] The term constitutive promoter, as used herein, refers to a promoter whose activity is maintained at a relatively constant level in all cells of an organism, or during most developmental stages, with little or no regard to cell environmental conditions. In another embodiment, the transcriptional regulatory region allows constitutive expression of ENPP1. Examples of constitutive promoters include, without limitation, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer), the SV40 promoter, the dihydrofolate reductase promoter, the -actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1a promoter (Boshart M, et al., Cell 1985; 41:521-530).

[0268] The term polyadenylation signal, as used herein, relates to a nucleic acid sequence that mediates the attachment of a polyadenine stretch to the 3 terminus of the mRNA. Suitable polyadenylation signals include, without limitation, the SV40 early polyadenylation signal, the SV40 late polyadenylation signal, the HSV thymidine kinase polyadenylation signal, the protamine gene polyadenylation signal, the adenovirus 5 EIb polyadenylation signal, the bovine growth hormone polyadenylation signal, the human variant growth hormone polyadenylation signal and the like.

[0269] The term signal peptide, as used herein, refers to a sequence of amino acid residues (ranging in length from 10-30 residues) bound at the amino terminus of a nascent protein of interest during protein translation. The signal peptide is recognized by the signal recognition particle (SRP) and cleaved by the signal peptidase following transport at the endoplasmic reticulum. (Lodish et al., 2000, Molecular Cell Biology, 4th edition).

[0270] As used herein, the term immune response or immune reaction refers to the host's immune system to antigen in an invading (infecting) pathogenic organism, or to introduction or expression of foreign protein. The immune response is generally humoral and local; antibodies produced by B cells combine with antigen in an antigen-antibody complex to inactivate or neutralize antigen. Immune response is often observed when human proteins are injected into mouse model systems. Generally, the mouse model system is made immune tolerant by injecting immune suppressors prior to the introduction of a foreign antigen to ensure better viability.

[0271] As used herein, the term immunesuppression is a deliberate reduction of the activation or efficacy of the host immune system using immunesuppresant drugs to facilitate immune tolerance towards foreign antigens such as foreign proteins, organ transplants, bone marrow and tissue transplantation. Non limiting examples of immunosuppressant drugs include anti-CD4(GK1.5) antibody, Cyclophosphamide, Azathioprine (Imuran), Mycophenolate mofetil (Cellcept), Cyclosporine (Neoral, Sandimmune, Gengraf), Methotrexate (Rheumatrex), Leflunomide (Arava), Cyclophosphamide (Cytoxan) and Chlorambucil (Leukeran).

[0272] Ranges: throughout this disclosure, various aspects of the disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from lto 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from lto 4, from lto 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

Methods of Treatment

[0273] The present disclosure relates to administration of an ENPP1 or ENPP3 agent to treat PAD, which includes administering sNPP1 and sNPP3 polypeptides and fusion proteins thereof to a subject, and to administration of nucleic acids encoding such polypeptides. Sequences of such polypeptides include the following, without limitation.

TABLE-US-00002 Sequences SEQIDNO:1-ENPP1AminoAcidSequence-WildType MetGluArgAspGlyCysAlaGlyGlyGlySerArgGlyGlyGluGly 151015 GlyArgAlaProArgGluGlyProAlaGlyAsnGlyArgAspArgGly 202530 ArgSerHisAlaAlaGluAlaProGlyAspProGlnAlaAlaAlaSer 354045 LeuLeuAlaProMetAspValGlyGluGluProLeuGluLysAlaAla 505560 ArgAlaArgThrAlaLysAspProAsnThrTyrLysValLeuSerLeu 65707580 ValLeuSerValCysValLeuThrThrIleLeuGlyCysIlePheGly 859095 LeuLysProSerCysAlaLysGluValLysSerCysLysGlyArgCys 100105110 PheGluArgThrPheGlyAsnCysArgCysAspAlaAlaCysValGlu 115120125 LeuGlyAsnCysCysLeuAspTyrGlnGluThrCysIleGluProGlu 130135140 HisIleTrpThrCysAsnLysPheArgCysGlyGluLysArgLeuThr 145150155160 ArgSerLeuCysAlaCysSerAspAspCysLysAspLysGlyAspCys 165170175 CysIleAsnTyrSerSerValCysGlnGlyGluLysSerTrpValGlu 180185190 GluProCysGluSerIleAsnGluProGlnCysProAlaGlyPheGlu 195200205 ThrProProThrLeuLeuPheSerLeuAspGlyPheArgAlaGluTyr 210215220 LeuHisThrTrpGlyGlyLeuLeuProValIleSerLysLeuLysLys 225230235240 CysGlyThrTyrThrLysAsnMetArgProValTyrProThrLysThr 245250255 PheProAsnHisTyrSerIleValThrGlyLeuTyrProGluSerHis 260265270 GlyIleIleAspAsnLysMetTyrAspProLysMetAsnAlaSerPhe 275280285 SerLeuLysSerLysGluLysPheAsnProGluTrpTyrLysGlyGlu 290295300 ProIleTrpValThrAlaLysTyrGlnGlyLeuLysSerGlyThrPhe 305310315320 PheTrpProGlySerAspValGluIleAsnGlyIlePheProAspIle 325330335 TyrLysMetTyrAsnGlySerValProPheGluGluArgIleLeuAla 340345350 ValLeuGlnTrpLeuGlnLeuProLysAspGluArgProHisPheTyr 355360365 ThrLeuTyrLeuGluGluProAspSerSerGlyHisSerTyrGlyPro 370375380 ValSerSerGluValIleLysAlaLeuGlnArgValAspGlyMetVal 385390395400 GlyMetLeuMetAspGlyLeuLysGluLeuAsnLeuHisArgCysLeu 405410415 AsnLeuIleLeuIleSerAspHisGlyMetGluGlnGlySerCysLys 420425430 LysTyrIleTyrLeuAsnLysTyrLeuGlyAspValLysAsnIleLys 435440445 ValIleTyrGlyProAlaAlaArgLeuArgProSerAspValProAsp 450455460 LysTyrTyrSerPheAsnTyrGluGlyIleAlaArgAsnLeuSerCys 465470475480 ArgGluProAsnGlnHisPheLysProTyrLeuLysHisPheLeuPro 485490495 LysArgLeuHisPheAlaLysSerAspArgIleGluProLeuThrPhe 500505510 TyrLeuAspProGlnTrpGlnLeuAlaLeuAsnProSerGluArgLys 515520525 TyrCysGlySerGlyPheHisGlySerAspAsnValPheSerAsnMet 530535540 GlnAlaLeuPheValGlyTyrGlyProGlyPheLysHisGlyIleGlu 545550555560 AlaAspThrPheGluAsnIleGluValTyrAsnLeuMetCysAspLeu 565570575 LeuAsnLeuThrProAlaProAsnAsnGlyThrHisGlySerLeuAsn 580585590 HisLeuLeuLysAsnProValTyrThrProLysHisProLysGluVal 595600605 HisProLeuValGlnCysProPheThrArgAsnProArgAspAsnLeu 610615620 GlyCysSerCysAsnProSerIleLeuProIleGluAspPheGlnThr 625630635640 GlnPheAsnLeuThrValAlaGluGluLysIleIleLysHisGluThr 645650655 LeuProTyrGlyArgProArgValLeuGlnLysGluAsnThrIleCys 660665670 LeuLeuSerGlnHisGlnPheMetSerGlyTyrSerGlnAspIleLeu 675680685 MetProLeuTrpThrSerTyrThrValAspArgAsnAspSerPheSer 690695700 ThrGluAspPheSerAsnCysLeuTyrGlnAspPheArgIleProLeu 705710715720 SerProValHisLysCysSerPheTyrLysAsnAsnThrLysValSer 725730735 TyrGlyPheLeuSerProProGlnLeuAsnLysAsnSerSerGlyIle 740745750 TyrSerGluAlaLeuLeuThrThrAsnIleValProMetTyrGlnSer 755760765 PheGlnValIleTrpArgTyrPheHisAspThrLeuLeuArgLysTyr 770775780 AlaGluGluArgAsnGlyValAsnValValSerGlyProValPheAsp 785790795800 PheAspTyrAspGlyArgCysAspSerLeuGluAsnLeuArgGlnLys 805810815 ArgArgValIleArgAsnGlnGluIleLeuIleProThrHisPhePhe 820825830 IleValLeuThrSerCysLysAspThrSerGlnThrProLeuHisCys 835840845 GluAsnLeuAspThrLeuAlaPheIleLeuProHisArgThrAspAsn 850855860 SerGluSerCysValHisGlyLysHisAspSerSerTrpValGluGlu 865870875880 LeuLeuMetLeuHisArgAlaArgIleThrAspValGluHisIleThr 885890895 GlyLeuSerPheTyrGlnGlnArgLysGluProValSerAspIleLeu 900905910 LysLeuLysThrHisLeuProThrPheSerGlnGluAsp 915920925 SEQIDNo:2-Azurocidin-ENPP1-FC MTRLTVLALLAGLLASSRA**APSCAKEVKSCKGRCFERTFGNCRCDAACVELGNCCLDYQETCIEPEHI WTCNKFRCGEKRLTRSLCACSDDCKDKGDCCINYSSVCQGEKSWVEEPCESINEPQCPAGFETPPTLLES LDGFRAEYLHTWGGLLPVISKLKKCGTYTKNMRPVYPTKTFPNHYSIVTGLYPESHGIIDNKMYDPKMNA SFSLKSKEKENPEWYKGEPIWVTAKYQGLKSGTFFWPGSDVEINGIFPDIYKMYNGSVPFEERILAVLQW LQLPKDERPHFYTLYLEEPDSSGHSYGPVSSEVIKALQRVDGMVGMLMDGLKELNLHRCLNLILISDHGM EQGSCKKYIYLNKYLGDVKNIKVIYGPAARLRPSDVPDKYYSENYEGIARNLSCREPNQHFKPYLKHFLP KRLHFAKSDRIEPLTFYLDPQWQLALNPSERKYCGSGFHGSDNVFSNMQALFVGYGPGFKHGIEADTFEN IEVYNLMCDLLNLTPAPNNGTHGSLNHLLKNPVYTPKHPKEVHPLVQCPFTRNPRDNLGCSCNPSILPIE DFQTQFNLTVAEEKIIKHETLPYGRPRVLQKENTICLLSQHQFMSGYSQDILMPLWTSYTVDRNDSESTE DFSNCLYQDFRIPLSPVHKCSFYKNNTKVSYGELSPPQLNKNSSGIYSEALLTTNIVPMYQSFQVIWRYF HDTLLRKYAEERNGVNVVSGPVFDEDYDGRCDSLENLRQKRRVIRNQEILIPTHFFIVLTSCKDTSQTPL HCENLDTLAFILPHRTDNSESCVHGKHDSSWVEELLMLHRARITDVEHITGLSFYQQRKEPVSDILKLKT HLPTFSQEDLINDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK Singleunderline-Azurocidinsignalsequence,Doubleunderline- BeginningandendofENPP1sequence,Boldresidues-Fcsequence, **indicatesthecleavagepointofthesignalsequence. SEQIDNo:3-Azurocidin-ENPP1-Alb MTRLTVLALLAGLLASSRA**APSCAKEVKSCKGRCFERTFGNCRCDAACVELGNCCLDYQETCIEPEHI WTCNKFRCGEKRLTRSLCACSDDCKDKGDCCINYSSVCQGEKSWVEEPCESINEPQCPAGFETPPTLLES LDGFRAEYLHTWGGLLPVISKLKKCGTYTKNMRPVYPTKTFPNHYSIVTGLYPESHGIIDNKMYDPKMNA SFSLKSKEKENPEWYKGEPIWVTAKYQGLKSGTFFWPGSDVEINGIFPDIYKMYNGSVPFEERILAVLQW LQLPKDERPHFYTLYLEEPDSSGHSYGPVSSEVIKALQRVDGMVGMLMDGLKELNLHRCLNLILISDHGM EQGSCKKYIYLNKYLGDVKNIKVIYGPAARLRPSDVPDKYYSENYEGIARNLSCREPNQHFKPYLKHELP KRLHFAKSDRIEPLTFYLDPQWQLALNPSERKYCGSGFHGSDNVFSNMQALFVGYGPGFKHGIEADTFEN IEVYNLMCDLLNLTPAPNNGTHGSLNHLLKNPVYTPKHPKEVHPLVQCPFTRNPRDNLGCSCNPSILPIE DFQTQFNLTVAEEKIIKHETLPYGRPRVLQKENTICLLSQHQFMSGYSQDILMPLWTSYTVDRNDSESTE DESNCLYQDFRIPLSPVHKCSFYKNNTKVSYGELSPPQLNKNSSGIYSEALLTTNIVPMYQSFQVIWRYF HDTLLRKYAEERNGVNVVSGPVEDEDYDGRCDSLENLRQKRRVIRNQEILIPTHFFIVLTSCKDTSQTPL HCENLDTLAFILPHRTDNSESCVHGKHDSSWVEELLMLHRARITDVEHITGLSFYQQRKEPVSDILKLKT HLPTFSQEDLINMKWVTFLLLLFVSGSAFSRGVFRREAHKSEIAHRYNDLGEQHFKGLVLIAFSQYLQKC SYDEHAKLVQEVTDFAKTCVADESAANCDKSLHTLFGDKLCAIPNLRENYGELADCCTKQEPERNECFLQ HKDDNPSLPPFERPEAEAMCTSFKENPTTFMGHYLHEVARRHPYFYAPELLYYAEQYNEILTQCCAEADK ESCLTPKLDGVKEKALVSSVRQRMKCSSMQKFGERAFKAWAVARLSQTFPNADFAEITKLATDLTKVNKE CCHGDLLECADDRAELAKYMCENQATISSKLQTCCDKPLLKKAHCLSEVEHDTMPADLPAIAADFVEDQE VCKNYAEAKDVFLGTFLYEYSRRHPDYSVSLLLRLAKKYEATLEKCCAEANPPACYGTVLAEFQPLVEEP KNLVKTNCDLYEKLGEYGFQNAILVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPEDQRLPCVEDYLS AILNRVCLLHEKTPVSEHVTKCCSGSLVERRPCFSALTVDETYVPKEFKAETFTFHSDICTLPEKEKQIK KQTALAELVKHKPKATAEQLKTVMDDFAQFLDTCCKAADKDTCFSTEGPNLVTRCKDALARSWSHPQFEK Singleunderline-Azurocidinsignalsequence,Doubleunderline- BeginningandendofENPP1sequence,Boldresidues-Albuminsequence, **indicatesthecleavagepointofthesignalsequence. SEQIDNo:4-Azurocidin-ENPP1 MTRLTVLALLAGLLASSRA**APSCAKEVKSCKGRCFERTFGNCRCDAACVELGNCCLDYQETCIEPEHI WTCNKFRCGEKRLTRSLCACSDDCKDKGDCCINYSSVCQGEKSWVEEPCESINEPQCPAGFETPPTLLES LDGFRAEYLHTWGGLLPVISKLKKCGTYTKNMRPVYPTKTFPNHYSIVTGLYPESHGIIDNKMYDPKMNA SFSLKSKEKENPEWYKGEPIWVTAKYQGLKSGTFFWPGSDVEINGIFPDIYKMYNGSVPFEERILAVLQW LQLPKDERPHFYTLYLEEPDSSGHSYGPVSSEVIKALQRVDGMVGMLMDGLKELNLHRCLNLILISDHGM EQGSCKKYIYLNKYLGDVKNIKVIYGPAARLRPSDVPDKYYSFNYEGIARNLSCREPNQHFKPYLKHELP KRLHFAKSDRIEPLTFYLDPQWQLALNPSERKYCGSGFHGSDNVFSNMQALFVGYGPGFKHGIEADTFEN IEVYNLMCDLLNLTPAPNNGTHGSLNHLLKNPVYTPKHPKEVHPLVQCPFTRNPRDNLGCSCNPSILPIE DFQTQFNLTVAEEKIIKHETLPYGRPRVLQKENTICLLSQHQFMSGYSQDILMPLWTSYTVDRNDSESTE DFSNCLYQDFRIPLSPVHKCSFYKNNTKVSYGELSPPQLNKNSSGIYSEALLTTNIVPMYQSFQVIWRYF HDTLLRKYAEERNGVNVVSGPVFDFDYDGRCDSLENLRQKRRVIRNQEILIPTHFFIVLTSCKDTSQTAP SCAKEVKSCKGRCFERTEGNCRCDAACVELGNCCLDYQETCIEPEHIWTCNKERCGEKRLTRSLCACSDD CKDKGDCCINYSSVCQGEKSWVEEPCESINEPQCPAGFETPPTLLESLDGFRAEYLHTWGGLLPVISKLK KCGTYTKNMRPVYPTKTFPNHYSIVTGLYPESHGIIDNKMYDPKMNASESLKSKEKENPEWYKGEPIWVT AKYQGLKSGTFFWPGSDVEINGIFPDIYKMYNGSVPFEERILAVLQWLQLPKDERPHFYTLYLEEPDSSG HSYGPVSSEVIKALQRVDGMVGMLMDGLKELNLHRCLNLILISDHGMEQGSCKKYIYLNKYLGDVKNIKV IYGPAARLRPSDVPDKYYSENYEGIARNLSCREPNQHFKPYLKHELPKRLHFAKSDRIEPLTFYLDPQWQ LALNPSERKYCGSGFHGSDNVFSNMQALFVGYGPGFKHGIEADTFENIEVYNLMCDLLNLTPAPNNGTHG SLNHLLKNPVYTPKHPKEVHPLVQCPFTRNPRDNLGCSCNPSILPIEDEQTQENLTVAEEKIIKHETLPY GRPRVLQKENTICLLSQHQFMSGYSQDILMPLWTSYTVDRNDSESTEDESNCLYQDFRIPLSPVHKCSFY KNNTKVSYGFLSPPQLNKNSSGIYSEALLTTNIVPMYQSFQVIWRYFHDTLLRKYAEERNGVNVVSGPVF DEDYDGRCDSLENLRQKRRVIRNQEILIPTHFFIVLTSCKDTSQTPLHCENLDTLAFILPHRTDNSESCV HGKHDSSWVEELLMLHRARITDVEHITGLSFYQQRKEPVSDILKLKTHLPTFSQED Singleunderline-Azurocidinsignalsequence,Doubleunderline- BeginningandendofENPP1sequence, **indicatesthecleavagepointofthesignalsequence. SEQIDNO:5-ENPP2AminoAcidSequence-WildType MetAlaArgArgSerSerPheGlnSerCysGlnIleIleSerLeuPhe 151015 ThrPheAlaValGlyValAsnIleCysLeuGlyPheThrAlaHisArg 202530 IleLysArgAlaGluGlyTrpGluGluGlyProProThrValLeuSer 354045 AspSerProTrpThrAsnIleSerGlySerCysLysGlyArgCysPhe 505560 GluLeuGlnGluAlaGlyProProAspCysArgCysAspAsnLeuCys 65707580 LysSerTyrThrSerCysCysHisAspPheAspGluLeuCysLeuLys 859095 ThrAlaArgGlyTrpGluCysThrLysAspArgCysGlyGluValArg 100105110 AsnGluGluAsnAlaCysHisCysSerGluAspCysLeuAlaArgGly 115120125 AspCysCysThrAsnTyrGlnValValCysLysGlyGluSerHisTrp 130135140 ValAspAspAspCysGluGluIleLysAlaAlaGluCysProAlaGly 145150155160 PheValArgProProLeuIleIlePheSerValAspGlyPheArgAla 165170175 SerTyrMetLysLysGlySerLysValMetProAsnIleGluLysLeu 180185190 ArgSerCysGlyThrHisSerProTyrMetArgProValTyrProThr 195200205 LysThrPheProAsnLeuTyrThrLeuAlaThrGlyLeuTyrProGlu 210215220 SerHisGlyIleValGlyAsnSerMetTyrAspProValPheAspAla 225230235240 ThrPheHisLeuArgGlyArgGluLysPheAsnHisArgTrpTrpGly 245250255 GlyGlnProLeuTrpIleThrAlaThrLysGlnGlyValLysAlaGly 260265270 ThrPhePheTrpSerValValIleProHisGluArgArgIleLeuThr 275280285 IleLeuGlnTrpLeuThrLeuProAspHisGluArgProSerValTyr 290295300 AlaPheTyrSerGluGlnProAspPheSerGlyHisLysTyrGlyPro 305310315320 PheGlyProGluMetThrAsnProLeuArgGluIleAspLysIleVal 325330335 GlyGlnLeuMetAspGlyLeuLysGlnLeuLysLeuHisArgCysVal 340345350 AsnValIlePheValGlyAspHisGlyMetGluAspValThrCysAsp 355360365 ArgThrGluPheLeuSerAsnTyrLeuThrAsnValAspAspIleThr 370375380 LeuValProGlyThrLeuGlyArgIleArgSerLysPheSerAsnAsn 385390395400 AlaLysTyrAspProLysAlaIleIleAlaAsnLeuThrCysLysLys 405410415 ProAspGlnHisPheLysProTyrLeuLysGlnHisLeuProLysArg 420425430 LeuHisTyrAlaAsnAsnArgArgIleGluAspIleHisLeuLeuVal 435440445 GluArgArgTrpHisValAlaArgLysProLeuAspValTyrLysLys 450455460 ProSerGlyLysCysPhePheGlnGlyAspHisGlyPheAspAsnLys 465470475480 ValAsnSerMetGlnThrValPheValGlyTyrGlySerThrPheLys 485490495 TyrLysThrLysValProProPheGluAsnIleGluLeuTyrAsnVal 500505510 MetCysAspLeuLeuGlyLeuLysProAlaProAsnAsnGlyThrHis 515520525 GlySerLeuAsnHisLeuLeuArgThrAsnThrPheArgProThrMet 530535540 ProGluGluValThrArgProAsnTyrProGlyIleMetTyrLeuGln 545550555560 SerAspPheAspLeuGlyCysThrCysAspAspLysValGluProLys 565570575 AsnLysLeuAspGluLeuAsnLysArgLeuHisThrLysGlySerThr 580585590 GluAlaGluThrArgLysPheArgGlySerArgAsnGluAsnLysGlu 595600605 AsnIleAsnGlyAsnPheGluProArgLysGluArgHisLeuLeuTyr 610615620 GlyArgProAlaValLeuTyrArgThrArgTyrAspIleLeuTyrHis 625630635640 ThrAspPheGluSerGlyTyrSerGluIlePheLeuMetProLeuTrp 645650655 ThrSerTyrThrValSerLysGlnAlaGluValSerSerValProAsp 660665670 HisLeuThrSerCysValArgProAspValArgValSerProSerPhe 675680685 SerGlnAsnCysLeuAlaTyrLysAsnAspLysGlnMetSerTyrGly 690695700 PheLeuPheProProTyrLeuSerSerSerProGluAlaLysTyrAsp 705710715720 AlaPheLeuValThrAsnMetValProMetTyrProAlaPheLysArg 725730735 ValTrpAsnTyrPheGlnArgValLeuValLysLysTyrAlaSerGlu 740745750 ArgAsnGlyValAsnValIleSerGlyProIlePheAspTyrAspTyr 755760765 AspGlyLeuHisAspThrGluAspLysIleLysGlnTyrValGluGly 770775780 SerSerIleProValProThrHisTyrTyrSerIleIleThrSerCys 785790795800 LeuAspPheThrGlnProAlaAspLysCysAspGlyProLeuSerVal 805810815 SerSerPheIleLeuProHisArgProAspAsnGluGluSerCysAsn 820825830 SerSerGluAspGluSerLysTrpValGluGluLeuMetLysMetHis 835840845 ThrAlaArgValArgAspIleGluHisLeuThrSerLeuAspPhePhe 850855860 ArgLysThrSerArgSerTyrProGluIleLeuThrLeuLysThrTyr 865870875880 LeuHisThrTyrGluSerGluIle 885 SEQ.IDNO:6-ExtracellularDomainofENPP3: GluLysGlnGlySerCysArgLysLysCysPheAspAlaSerPheArg 151015 GlyLeuGluAsnCysArgCysAspValAlaCysLysAspArgGlyAsp 202530 CysCysTrpAspPheGluAspThrCysValGluSerThrArgIleTrp 354045 MetCysAsnLysPheArgCysGlyGluThrArgLeuGluAlaSerLeu 505560 CysSerCysSerAspAspCysLeuGlnArgLysAspCysCysAlaAsp 65707580 TyrLysSerValCysGlnGlyGluThrSerTrpLeuGluGluAsnCys 859095 AspThrAlaGlnGlnSerGlnCysProGluGlyPheAspLeuProPro 100105110 ValIleLeuPheSerMetAspGlyPheArgAlaGluTyrLeuTyrThr 115120125 TrpAspThrLeuMetProAsnIleAsnLysLeuLysThrCysGlyIle 130135140 HisSerLysTyrMetArgAlaMetTyrProThrLysThrPheProAsn 145150155160 HisTyrThrIleValThrGlyLeuTyrProGluSerHisGlyIleIle 165170175 AspAsnAsnMetTyrAspValAsnLeuAsnLysAsnPheSerLeuSer 180185190 SerLysGluGlnAsnAsnProAlaTrpTrpHisGlyGlnProMetTrp 195200205 LeuThrAlaMetTyrGlnGlyLeuLysAlaAlaThrTyrPheTrpPro 210215220 GlySerGluValAlaIleAsnGlySerPheProSerIleTyrMetPro 225230235240 TyrAsnGlySerValProPheGluGluArgIleSerThrLeuLeuLys 245250255 TrpLeuAspLeuProLysAlaGluArgProArgPheTyrThrMetTyr 260265270 PheGluGluProAspSerSerGlyHisAlaGlyGlyProValSerAla 275280285 ArgValIleLysAlaLeuGlnValValAspHisAlaPheGlyMetLeu 290295300 MetGluGlyLeuLysGlnArgAsnLeuHisAsnCysValAsnIleIle 305310315320 LeuLeuAlaAspHisGlyMetAspGlnThrTyrCysAsnLysMetGlu 325330335 TyrMetThrAspTyrPheProArgIleAsnPhePheTyrMetTyrGlu 340340345350 GlyProAlaProArgIleArgAlaHisAsnIleProHisAspPhePhe 355360365 SerPheAsnSerGluGluIleValArgAsnLeuSerCysArgLysPro 370375380 AspGlnHisPheLysProTyrLeuThrProAspLeuProLysArgLeu 385390395400 HisTyrAlaLysAsnValArgIleAspLysValHisLeuPheValAsp 405410415 GlnGlnTrpLeuAlaValArgSerLysSerAsnThrAsnCysGlyGly 420425430 GlyAsnHisGlyTyrAsnAsnGluPheArgSerMetGluAlaIlePhe 435440445 LeuAlaHisGlyProSerPheLysGluLysThrGluValGluProPhe 450455460 GluAsnIleGluValTyrAsnLeuMetCysAspLeuLeuArgIleGln 465470475480 ProAlaProAsnAsnGlyThrHisGlySerLeuAsnHisLeuLeuLys 485490495 ValProPheTyrGluProSerHisAlaGluGluValSerLysPheSer 500505510 ValCysGlyPheAlaAsnProLeuProThrGluSerLeuAspCysPhe 515520525 CysProHisLeuGlnAsnSerThrGlnLeuGluGlnValAsnGlnMet 530535540 LeuAsnLeuThrGlnGluGluIleThrAlaThrValLysValAsnLeu 545550555560 ProPheGlyArgProArgValLeuGlnLysAsnValAspHisCysLeu 565570575 LeuTyrHisArgGluTyrValSerGlyPheGlyLysAlaMetArgMet 580585590 ProMetTrpSerSerTyrThrValProGlnLeuGlyAspThrSerPro 595600605 LeuProProThrValProAspCysLeuArgAlaAspValArgValPro 610615620 ProSerGluSerGlnLysCysSerPheTyrLeuAlaAspLysAsnIle 625630635640 ThrHisGlyPheLeuTyrProProAlaSerAsnArgThrSerAspSer 645650655 GlnTyrAspAlaLeuIleThrSerAsnLeuValProMetTyrGluGlu 660665670 PheArgLysMetTrpAspTyrPheHisSerValLeuLeuIleLysHis 675680685 AlaThrGluArgAsnGlyValAsnValValSerGlyProIlePheAsp 690695700 TyrAsnTyrAspGlyHisPheAspAlaProAspGluIleThrLysHis 705710715720 LeuAlaAsnThrAspValProIleProThrHisTyrPheValValLeu 725730735 ThrSerCysLysAsnLysSerHisThrProGluAsnCysProGlyTrp 740745750 LeuAspValLeuProPheIleIleProHisArgProThrAsnValGlu 755760765 SerCysProGluGlyLysProGluAlaLeuTrpValGluGluArgPhe 770775780 ThrAlaHisIleAlaArgValArgAspValGluLeuLeuThrGlyLeu 785790795800 AspPheTyrGlnAspLysValGlnProValSerGluIleLeuGlnLeu 805810815 LysThrTyrLeuProThrPheGluThrThrIle 820825 SEQ.IDNO:7-NPP3AminoAcidSequence: MetGluSerThrLeuThrLeuAlaThrGluGlnProValLysLysAsn 151015 ThrLeuLysLysTyrLysIleAlaCysIleValLeuLeuAlaLeuLeu 202530 ValIleMetSerLeuGlyLeuGlyLeuGlyLeuGlyLeuArgLysLeu 354045 GluLysGlnGlySerCysArgLysLysCysPheAspAlaSerPheArg 505560 GlyLeuGluAsnCysArgCysAspValAlaCysLysAspArgGlyAsp 65707580 CysCysTrpAspPheGluAspThrCysValGluSerThrArgIleTrp 859095 MetCysAsnLysPheArgCysGlyGluThrArgLeuGluAlaSerLeu 100105110 CysSerCysSerAspAspCysLeuGlnArgLysAspCysCysAlaAsp 115120125 TyrLysSerValCysGlnGlyGluThrSerTrpLeuGluGluAsnCys 130135140 AspThrAlaGlnGlnSerGlnCysProGluGlyPheAspLeuProPro 145150155160 ValIleLeuPheSerMetAspGlyPheArgAlaGluTyrLeuTyrThr 165170175 TrpAspThrLeuMetProAsnIleAsnLysLeuLysThrCysGlyIle 180185190 HisSerLysTyrMetArgAlaMetTyrProThrLysThrPheProAsn 195200205 HisTyrThrIleValThrGlyLeuTyrProGluSerHisGlyIleIle 210215220 AspAsnAsnMetTyrAspValAsnLeuAsnLysAsnPheSerLeuSer 225230235240 SerLysGluGlnAsnAsnProAlaTrpTrpHisGlyGlnProMetTrp 245250255 LeuThrAlaMetTyrGlnGlyLeuLysAlaAlaThrTyrPheTrpPro 260265270 GlySerGluValAlaIleAsnGlySerPheProSerIleTyrMetPro 275280285 TyrAsnGlySerValProPheGluGluArgIleSerThrLeuLeuLys 290295300 TrpLeuAspLeuProLysAlaGluArgProArgPheTyrThrMetTyr 305310315320 PheGluGluProAspSerSerGlyHisAlaGlyGlyProValSerAla 325330335 ArgValIleLysAlaLeuGlnValValAspHisAlaPheGlyMetLeu 340345350 MetGluGlyLeuLysGlnArgAsnLeuHisAsnCysValAsnIleIle 355360365 LeuLeuAlaAspHisGlyMetAspGlnThrTyrCysAsnLysMetGlu 370375380 TyrMetThrAspTyrPheProArgIleAsnPhePheTyrMetTyrGlu 385390395400 GlyProAlaProArgIleArgAlaHisAsnIleProHisAspPhePhe 405410415 SerPheAsnSerGluGluIleValArgAsnLeuSerCysArgLysPro 420425430 AspGlnHisPheLysProTyrLeuThrProAspLeuProLysArgLeu 435440445 HisTyrAlaLysAsnValArgIleAspLysValHisLeuPheValAsp 450455460 GlnGlnTrpLeuAlaValArgSerLysSerAsnThrAsnCysGlyGly 465470475480 GlyAsnHisGlyTyrAsnAsnGluPheArgSerMetGluAlaIlePhe 485490495 LeuAlaHisGlyProSerPheLysGluLysThrGluValGluProPhe 500505510 GluAsnIleGluValTyrAsnLeuMetCysAspLeuLeuArgIleGln 515520525 ProAlaProAsnAsnGlyThrHisGlySerLeuAsnHisLeuLeuLys 530535540 ValProPheTyrGluProSerHisAlaGluGluValSerLysPheSer 545550555560 ValCysGlyPheAlaAsnProLeuProThrGluSerLeuAspCysPhe 565570575 CysProHisLeuGlnAsnSerThrGlnLeuGluGlnValAsnGlnMet 580585590 LeuAsnLeuThrGlnGluGluIleThrAlaThrValLysValAsnLeu 595600605 ProPheGlyArgProArgValLeuGlnLysAsnValAspHisCysLeu 610615620 LeuTyrHisArgGluTyrValSerGlyPheGlyLysAlaMetArgMet 625630635640 ProMetTrpSerSerTyrThrValProGlnLeuGlyAspThrSerPro 645650655 LeuProProThrValProAspCysLeuArgAlaAspValArgValPro 660665670 ProSerGluSerGlnLysCysSerPheTyrLeuAlaAspLysAsnIle 675680685 ThrHisGlyPheLeuTyrProProAlaSerAsnArgThrSerAspSer 690695700 GlnTyrAspAlaLeuIleThrSerAsnLeuValProMetTyrGluGlu 705710715720 PheArgLysMetTrpAspTyrPheHisSerValLeuLeuIleLysHis 725730735 AlaThrGluArgAsnGlyValAsnValValSerGlyProIlePheAsp 740745750 TyrAsnTyrAspGlyHisPheAspAlaProAspGluIleThrLysHis 755760765 LeuAlaAsnThrAspValProIleProThrHisTyrPheValValLeu 770775780 ThrSerCysLysAsnLysSerHisThrProGluAsnCysProGlyTrp 785790795800 LeuAspValLeuProPheIleIleProHisArgProThrAsnValGlu 805810815 SerCysProGluGlyLysProGluAlaLeuTrpValGluGluArgPhe 820825830 ThrAlaHisIleAlaArgValArgAspValGluLeuLeuThrGlyLeu 835840845 AspPheTyrGlnAspLysValGlnProValSerGluIleLeuGlnLeu 850855860 LysThrTyrLeuProThrPheGluThrThrIle 865870875 SEQIDNo:8-Azurocidin-ENPP3-FC MTRLTVLALLAGLLASSRA**AKQGSCRKKCFDASFRGLENCRCDVACKDRGDCCWDFEDTCVES TRIWMCNKFRCGETRLEASLCSCSDDCLQRKDCCADYKSVCQGETSWLEENCDTAQQSQCPEGFDLPPVI LFSMDGFRAEYLYTWDTLMPNINKLKTCGIHSKYMRAMYPTKTFPNHYTIVTGLYPESHGIIDNNMYDVN LNKNFSLSSKEQNNPAWWHGQPMNLTAMYQGLKAATYFWPGSEVAINGSEPSIYMPYNGSVPFEERISTL LKWLDLPKAERPRFYTMYFEEPDSSGHAGGPVSARVIKALQVVDHAFGMLMEGLKQRNLHNCVNIILLAD HGMDQTYCNKMEYMTDYFPRINFFYMYEGPAPRIRAHNIPHDFFSENSEEIVRNLSCRKPDQHFKPYLTP DLPKRLHYAKNVRIDKVHLFVDQQWLAVRSKSNTNCGGGNHGYNNEFRSMEAIFLAHGPSFKEKTEVEPF ENIEVYNLMCDLLRIQPAPNNGTHGSLNHLLKVPFYEPSHAEEVSKESVCGFANPLPTESLDCFCPHLQN STQLEQVNQMLNLTQEEITATVKVNLPFGRPRVLQKNVDHCLLYHREYVSGFGKAMRMPMWSSYTVPQLG DTSPLPPTVPDCLRADVRVPPSESQKCSFYLADKNITHGFLYPPASNRTSDSQYDALITSNLVPMYEEER KMWDYFHSVLLIKHATERNGVNVVSGPIFDYNYDGHEDAPDEITKHLANTDVPIPTHYFVVLTSCKNKSH TPENCPGWLDVLPFIIPHRPTNVESCPEGKPEALWVEERFTAHIARVRDVELLTGLDFYQDKVQPVSEIL QLKTYLPTFETTIDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK Singleunderline-Azurocidinsignalsequence,Doubleunderline- BeginningandendofENPP3sequence,Boldresidues-Fcsequence, **indicatesthecleavagepointofthesignalsequence. SEQIDNo:9-Azurocidin-ENPP3-Albumin MTRLTVLALLAGLLASSRA**AKQGSCRKKCFDASFRGLENCRCDVACKDRGDCCWDFEDTCVES TRIWMCNKFRCGETRLEASLCSCSDDCLQRKDCCADYKSVCQGETSWLEENCDTAQQSQCPEGEDLPPVI LFSMDGFRAEYLYTWDTLMPNINKLKTCGIHSKYMRAMYPTKTFPNHYTIVTGLYPESHGIIDNNMYDVN LNKNFSLSSKEQNNPAWWHGQPMNLTAMYQGLKAATYFWPGSEVAINGSFPSIYMPYNGSVPFEERISTL LKWLDLPKAERPRFYTMYFEEPDSSGHAGGPVSARVIKALQVVDHAFGMLMEGLKQRNLHNCVNIILLAD HGMDQTYCNKMEYMTDYFPRINFFYMYEGPAPRIRAHNIPHDFFSENSEEIVRNLSCRKPDQHFKPYLTP DLPKRLHYAKNVRIDKVHLFVDQQWLAVRSKSNTNCGGGNHGYNNEFRSMEAIFLAHGPSFKEKTEVEPF ENIEVYNLMCDLLRIQPAPNNGTHGSLNHLLKVPFYEPSHAEEVSKESVCGFANPLPTESLDCFCPHLQN STQLEQVNQMLNLTQEEITATVKVNLPFGRPRVLQKNVDHCLLYHREYVSGFGKAMRMPMWSSYTVPQLG DTSPLPPTVPDCLRADVRVPPSESQKCSFYLADKNITHGFLYPPASNRTSDSQYDALITSNLVPMYEEFR KMWDYFHSVLLIKHATERNGVNVVSGPIFDYNYDGHEDAPDEITKHLANTDVPIPTHYFVVLTSCKNKSH TPENCPGWLDVLPFIIPHRPTNVESCPEGKPEALWVEERFTAHIARVRDVELLTGLDFYQDKVQPVSEIL QLKTYLPTFETTIMKWVTFLLLLFVSGSAFSRGVFRREAHKSEIAHRYNDLGEQHFKGLVLIAFSQYLQK CSYDEHAKLVQEVTDFAKTCVADESAANCDKSLHTLFGDKLCAIPNLRENYGELADCCTKQEPERNECFL QHKDDNPSLPPFERPEAEAMCTSFKENPTTFMGHYLHEVARRHPYFYAPELLYYAEQYNEILTQCCAEAD KESCLTPKLDGVKEKALVSSVRQRMKCSSMQKFGERAFKAWAVARLSQTFPNADFAEITKLATDLTKVNK ECCHGDLLECADDRAELAKYMCENQATISSKLQTCCDKPLLKKAHCLSEVEHDTMPADLPAIAADFVEDQ EVCKNYAEAKDVFLGTFLYEYSRRHPDYSVSLLLRLAKKYEATLEKCCAEANPPACYGTVLAEFQPLVEE PKNLVKTNCDLYEKLGEYGFQNAILVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPEDQRLPCVEDYL SAILNRVCLLHEKTPVSEHVTKCCSGSLVERRPCFSALTVDETYVPKEFKAETFTFHSDICTLPEKEKQI KKQTALAELVKHKPKATAEQLKTVMDDFAQFLDTCCKAADKDTCFSTEGPNLVTRCKDALARSWSHPQFE K Singleunderline-Azurocidinsignalsequence,Doubleunderline- BeginningandendofENPP3sequence,Boldresidues-Albuminsequence, **indicatesthecleavagepointofthesignalsequence. SEQIDNo:10-Azurocidin-ENPP3 MTRLTVLALLAGLLASSRA**AKQGSCRKKCFDASFRGLENCRCDVACKDRGDCCWDFEDTCVES TRIWMCNKFRCGETRLEASLCSCSDDCLQRKDCCADYKSVCQGETSWLEENCDTAQQSQCPEGFDLPPVI LFSMDGFRAEYLYTWDTLMPNINKLKTCGIHSKYMRAMYPTKTFPNHYTIVTGLYPESHGIIDNNMYDVN LNKNFSLSSKEQNNPAWWHGQPMNLTAMYQGLKAATYFWPGSEVAINGSFPSIYMPYNGSVPFEERISTL LKWLDLPKAERPRFYTMYFEEPDSSGHAGGPVSARVIKALQVVDHAFGMLMEGLKQRNLHNCVNIILLAD HGMDQTYCNKMEYMTDYFPRINFFYMYEGPAPRIRAHNIPHDFFSENSEEIVRNLSCRKPDQHFKPYLTP DLPKRLHYAKNVRIDKVHLFVDQQWLAVRSKSNTNCGGGNHGYNNEFRSMEAIFLAHGPSFKEKTEVEPF ENIEVYNLMCDLLRIQPAPNNGTHGSLNHLLKVPFYEPSHAEEVSKESVCGFANPLPTESLDCFCPHLQN STQLEQVNQMLNLTQEEITATVKVNLPFGRPRVLQKNVDHCLLYHREYVSGFGKAMRMPMWSSYTVPQLG DTSPLPPTVPDCLRADVRVPPSESQKCSFYLADKNITHGFLYPPASNRTSDSQYDALITSNLVPMYEEER KMWDYFHSVLLIKHATERNGVNVVSGPIFDYNYDGHFDAPDEITKHLANTDVPIPTHYFVVLTSCKNKSH TPENCPGWLDVLPFIIPHRPTNVESCPEGKPEALWVEERFTAHIARVRDVELLTGLDFYQDKVQPVSEIL QLKTYLPTFETTI Singleunderline-Azurocidinsignalsequence,Doubleunderline- BeginningandendofENPP3sequence,**indicatesthecleavagepoint ofthesignalsequence. SEQ.IDNO:11-ENPP4AminoAcidSequence-WildType MetLysLeuLeuValIleLeuLeuPheSerGlyLeuIleThrGlyPhe 151015 ArgSerAspSerSerSerSerLeuProProLysLeuLeuLeuValSer 202530 PheAspGlyPheArgAlaAspTyrLeuLysAsnTyrGluPheProHis 354045 LeuGlnAsnPheIleLysGluGlyValLeuValGluHisValLysAsn 505560 ValPheIleThrLysThrPheProAsnHisTyrSerIleValThrGly 65707580 LeuTyrGluGluSerHisGlyIleValAlaAsnSerMetTyrAspAla 859095 ValThrLysLysHisPheSerAspSerAsnAspLysAspProPheTrp 100100105110 TrpAsnGluAlaValProIleTrpValThrAsnGlnLeuGlnGluAsn 115120125 ArgSerSerAlaAlaAlaMetTrpProGlyThrAspValProIleHis 130135140 AspThrIleSerSerTyrPheMetAsnTyrAsnSerSerValSerPhe 145150155160 GluGluArgLeuAsnAsnIleThrMetTrpLeuAsnAsnSerAsnPro 165170175 ProValThrPheAlaThrLeuTyrTrpGluGluProAspAlaSerGly 180185190 HisLysTyrGlyProGluAspLysGluAsnMetSerArgValLeuLys 195200205 LysIleAspAspLeuIleGlyAspLeuValGlnArgLeuLysMetLeu 210215220 GlyLeuTrpGluAsnLeuAsnValIleIleThrSerAspHisGlyMet 225230235240 ThrGlnCysSerGlnAspArgLeuIleAsnLeuAspSerCysIleAsp 245250255 HisSerTyrTyrThrLeuIleAspLeuSerProValAlaAlaIleLeu 260265270 ProLysIleAsnArgThrGluValTyrAsnLysLeuLysAsnCysSer 275280285 ProHisMetAsnValTyrLeuLysGluAspIleProAsnArgPheTyr 290295300 TyrGlnHisAsnAspArgIleGlnProIleIleLeuValAlaAspGlu 305310315320 GlyTrpThrIleValLeuAsnGluSerSerGlnLysLeuGlyAspHis 325330335 GlyTyrAspAsnSerLeuProSerMetHisProPheLeuAlaAlaHis 340345350 GlyProAlaPheHisLysGlyTyrLysHisSerThrIleAsnIleVal 355360365 AspIleTyrProMetMetCysHisIleLeuGlyLeuLysProHisPro 370375380 AsnAsnGlyThrPheGlyHisThrLysCysLeuLeuValAspGlnTrp 385390395400 CysIleAsnLeuProGluAlaIleAlaIleValIleGlySerLeuLeu 405410415 ValLeuThrMetLeuThrCysLeuIleIleIleMetGlnAsnArgLeu 420425430 SerValProArgProPheSerArgLeuGlnLeuGlnGluAspAspAsp 435440445 AspProLeuIleGly 450 SEQ.IDNO:12-ENPP51AminoAcidSequence MetThrSerLysPheLeuLeuValSerPheIleLeuAlaAlaLeuSer 151015 LeuSerThrThrPheSerLeuGln**ProSerCysAlaLysGluValLys 202530 SerCysLysGlyArgCysPheGluArgThrPheSerAsnCysArgCys 354045 AspAlaAlaCysValSerLeuGlyAsnCysCysLeuAspPheGlnGlu 505560 ThrCysValGluProThrHisIleTrpThrCysAsnLysPheArgCys 65707580 GlyGluLysArgLeuSerArgPheValCysSerCysAlaAspAspCys 859095 LysThrHisAsnAspCysCysIleAsnTyrSerSerValCysGlnAsp 100105110 LysLysSerTrpValGluGluThrCysGluSerIleAspThrProGlu 115120125 CysProAlaGluPheGluSerProProThrLeuLeuPheSerLeuAsp 130135140 GlyPheArgAlaGluTyrLeuHisThrTrpGlyGlyLeuLeuProVal 145150155160 IleSerLysLeuLysAsnCysGlyThrTyrThrLysAsnMetArgPro 165170175 MetTyrProThrLysThrPheProAsnHisTyrSerIleValThrGly 180185190 LeuTyrProGluSerHisGlyIleIleAspAsnLysMetTyrAspPro 195200205 LysMetAsnAlaSerPheSerLeuLysSerLysGluLysPheAsnPro 210215220 LeuTrpTyrLysGlyGlnProIleTrpValThrAlaAsnHisGlnGlu 225230235240 ValLysSerGlyThrTyrPheTrpProGlySerAspValGluIleAsp 245250255 GlyIleLeuProAspIleTyrLysValTyrAsnGlySerValProPhe 260265270 GluGluArgIleLeuAlaValLeuGluTrpLeuGlnLeuProSerHis 275280285 GluArgProHisPheTyrThrLeuTyrLeuGluGluProAspSerSer 290295300 GlyHisSerHisGlyProValSerSerGluValIleLysAlaLeuGln 305310315320 LysValAspArgLeuValGlyMetLeuMetAspGlyLeuLysAspLeu 325330335 GlyLeuAspLysCysLeuAsnLeuIleLeuIleSerAspHisGlyMet 340345350 GluGlnGlySerCysLysLysTyrValTyrLeuAsnLysTyrLeuGly 355360365 AspValAsnAsnValLysValValTyrGlyProAlaAlaArgLeuArg 370375380 ProThrAspValProGluThrTyrTyrSerPheAsnTyrGluAlaLeu 385390395400 AlaLysAsnLeuSerCysArgGluProAsnGlnHisPheArgProTyr 405410415 LeuLysProPheLeuProLysArgLeuHisPheAlaLysSerAspArg 420425430 IleGluProLeuThrPheTyrLeuAspProGlnTrpGlnLeuAlaLeu 435440445 AsnProSerGluArgLysTyrCysGlySerGlyPheHisGlySerAsp 450455460 AsnLeuPheSerAsnMetGlnAlaLeuPheIleGlyTyrGlyProAla 465470475480 PheLysHisGlyAlaGluValAspSerPheGluAsnIleGluValTyr 485490495 AsnLeuMetCysAspLeuLeuGlyLeuIleProAlaProAsnAsnGly 500505510 SerHisGlySerLeuAsnHisLeuLeuLysLysProIleTyrAsnPro 515520525 SerHisProLysGluGluGlyPheLeuSerGlnCysProIleLysSer 530535540 ThrSerAsnAspLeuGlyCysThrCysAspProTrpIleValProIle 545550555560 LysAspPheGluLysGlnLeuAsnLeuThrThrGluAspValAspAsp 565570575 IleTyrHisMetThrValProTyrGlyArgProArgIleLeuLeuLys 580585590 GlnHisArgValCysLeuLeuGlnGlnGlnGlnPheLeuThrGlyTyr 595600605 SerLeuAspLeuLeuMetProLeuTrpAlaSerTyrThrPheLeuSer 610615620 AsnAspGlnPheSerArgAspAspPheSerAsnCysLeuTyrGlnAsp 625630635640 LeuArgIleProLeuSerProValHisLysCysSerTyrTyrLysSer 645650655 AsnSerLysLeuSerTyrGlyPheLeuThrProProArgLeuAsnArg 660665670 ValSerAsnHisIleTyrSerGluAlaLeuLeuThrSerAsnIleVal 675680685 ProMetTyrGlnSerPheGlnValIleTrpHisTyrLeuHisAspThr 690695700 LeuLeuGlnArgTyrAlaHisGluArgAsnGlyIleAsnValValSer 705710715720 GlyProValPheAspPheAspTyrAspGlyArgTyrAspSerLeuGlu 725730735 IleLeuLysGlnAsnSerArgValIleArgSerGlnGluIleLeuIle 740745750 ProThrHisPhePheIleValLeuThrSerCysLysGlnLeuSerGlu 755760765 ThrProLeuGluCysSerAlaLeuGluSerSerAlaTyrIleLeuPro 770775780 HisArgProAspAsnIleGluSerCysThrHisGlyLysArgGluSer 785790795800 SerTrpValGluGluLeuLeuThrLeuHisArgAlaArgValThrAsp 805810815 ValGluLeuIleThrGlyLeuSerPheTyrGlnAspArgGlnGluSer 820825830 ValSerGluLeuLeuArgLeuLysThrHisLeuProIlePheSerGln 835840845 GlnAsp 850 Singlyunderlined:signalpeptidesequence;double-underlined: beginningandendofNPP1;**=cleavagepositionatthesignal peptidesequence SEQ.IDNO:13-ENPP51-ALBAminoAcidSequence: MetThrSerLysPheLeuLeuValSerPheIleLeuAlaAlaLeuSer 151015 LeuSerThrThrPheSerLeuGln**ProSerCysAlaLysGluValLys 202530 SerCysLysGlyArgCysPheGluArgThrPheSerAsnCysArgCys 354045 AspAlaAlaCysValSerLeuGlyAsnCysCysLeuAspPheGlnGlu 505560 ThrCysValGluProThrHisIleTrpThrCysAsnLysPheArgCys 65707580 GlyGluLysArgLeuSerArgPheValCysSerCysAlaAspAspCys 859095 LysThrHisAsnAspCysCysIleAsnTyrSerSerValCysGlnAsp 100105110 LysLysSerTrpValGluGluThrCysGluSerIleAspThrProGlu 115120125 CysProAlaGluPheGluSerProProThrLeuLeuPheSerLeuAsp 130135140 GlyPheArgAlaGluTyrLeuHisThrTrpGlyGlyLeuLeuProVal 145150155160 IleSerLysLeuLysAsnCysGlyThrTyrThrLysAsnMetArgPro 165170175 MetTyrProThrLysThrPheProAsnHisTyrSerIleValThrGly 180185190 LeuTyrProGluSerHisGlyIleIleAspAsnLysMetTyrAspPro 195200205 LysMetAsnAlaSerPheSerLeuLysSerLysGluLysPheAsnPro 210215220 LeuTrpTyrLysGlyGlnProIleTrpValThrAlaAsnHisGlnGlu 225230235240 ValLysSerGlyThrTyrPheTrpProGlySerAspValGluIleAsp 245250255 GlyIleLeuProAspIleTyrLysValTyrAsnGlySerValProPhe 260265270 GluGluArgIleLeuAlaValLeuGluTrpLeuGlnLeuProSerHis 275280285 GluArgProHisPheTyrThrLeuTyrLeuGluGluProAspSerSer 290295300 GlyHisSerHisGlyProValSerSerGluValIleLysAlaLeuGln 305310315320 LysValAspArgLeuValGlyMetLeuMetAspGlyLeuLysAspLeu 325330335 GlyLeuAspLysCysLeuAsnLeuIleLeuIleSerAspHisGlyMet 340345350 GluGlnGlySerCysLysLysTyrValTyrLeuAsnLysTyrLeuGly 355360365 AspValAsnAsnValLysValValTyrGlyProAlaAlaArgLeuArg 370375380 ProThrAspValProGluThrTyrTyrSerPheAsnTyrGluAlaLeu 385390395400 AlaLysAsnLeuSerCysArgGluProAsnGlnHisPheArgProTyr 405410415 LeuLysProPheLeuProLysArgLeuHisPheAlaLysSerAspArg 420425430 IleGluProLeuThrPheTyrLeuAspProGlnTrpGlnLeuAlaLeu 435440445 AsnProSerGluArgLysTyrCysGlySerGlyPheHisGlySerAsp 450455460 AsnLeuPheSerAsnMetGlnAlaLeuPheIleGlyTyrGlyProAla 465470475480 PheLysHisGlyAlaGluValAspSerPheGluAsnIleGluValTyr 485490495 AsnLeuMetCysAspLeuLeuGlyLeuIleProAlaProAsnAsnGly 500505510 SerHisGlySerLeuAsnHisLeuLeuLysLysProIleTyrAsnPro 515520525 SerHisProLysGluGluGlyPheLeuSerGlnCysProIleLysSer 530535540 ThrSerAsnAspLeuGlyCysThrCysAspProTrpIleValProIle 545550555560 LysAspPheGluLysGlnLeuAsnLeuThrThrGluAspValAspAsp 565570575 IleTyrHisMetThrValProTyrGlyArgProArgIleLeuLeuLys 580585590 GlnHisArgValCysLeuLeuGlnGlnGlnGlnPheLeuThrGlyTyr 595600605 SerLeuAspLeuLeuMetProLeuTrpAlaSerTyrThrPheLeuSer 610615620 AsnAspGlnPheSerArgAspAspPheSerAsnCysLeuTyrGlnAsp 625630635640 LeuArgIleProLeuSerProValHisLysCysSerTyrTyrLysSer 645650655 AsnSerLysLeuSerTyrGlyPheLeuThrProProArgLeuAsnArg 660665670 ValSerAsnHisIleTyrSerGluAlaLeuLeuThrSerAsnIleVal 675680685 ProMetTyrGlnSerPheGlnValIleTrpHisTyrLeuHisAspThr 690695700 LeuLeuGlnArgTyrAlaHisGluArgAsnGlyIleAsnValValSer 705710715720 GlyProValPheAspPheAspTyrAspGlyArgTyrAspSerLeuGlu 725730735 IleLeuLysGlnAsnSerArgValIleArgSerGlnGluIleLeuIle 740745750 ProThrHisPhePheIleValLeuThrSerCysLysGlnLeuSerGlu 755760765 ThrProLeuGluCysSerAlaLeuGluSerSerAlaTyrIleLeuPro 770775780 HisArgProAspAsnIleGluSerCysThrHisGlyLysArgGluSer 785790795800 SerTrpValGluGluLeuLeuThrLeuHisArgAlaArgValThrAsp 805810815 ValGluLeuIleThrGlyLeuSerPheTyrGlnAspArgGlnGluSer 820825830 ValSerGluLeuLeuArgLeuLysThrHisLeuProIlePheSerGln 835840845 GluAspGlyGlySerGlyGlySerMetLysTrpValThrPheLeuLeu 850855860 LeuLeuPheValSerGlySerAlaPheSerArgGlyValPheArgArg 865870875880 GluAlaHisLysSerGluIleAlaHisArgTyrAsnAspLeuGlyGlu 885890895 GlnHisPheLysGlyLeuValLeuIleAlaPheSerGlnTyrLeuGln 900905910 LysCysSerTyrAspGluHisAlaLysLeuValGlnGluValThrAsp 915920925 PheAlaLysThrCysValAlaAspGluSerAlaAlaAsnCysAspLys 930935940 SerLeuHisThrLeuPheGlyAspLysLeuCysAlaIleProAsnLeu 945950955960 ArgGluAsnTyrGlyGluLeuAlaAspCysCysThrLysGlnGluPro 965970975 GluArgAsnGluCysPheLeuGlnHisLysAspAspAsnProSerLeu 980985990 ProProPheGluArgProGluAlaGluAlaMetCysThrSerPheLys 99510001005 GluAsnProThrThrPheMetGlyHisTyrLeuHisGluValAla 101010151020 ArgArgHisProTyrPheTyrAlaProGluLeuLeuTyrTyrAla 102510301035 GluGlnTyrAsnGluIleLeuThrGlnCysCysAlaGluAlaAsp 104010451050 LysGluSerCysLeuThrProLysLeuAspGlyValLysGluLys 105510601065 AlaLeuValSerSerValArgGlnArgMetLysCysSerSerMet 107010751080 GlnLysPheGlyGluArgAlaPheLysAlaTrpAlaValAlaArg 108510901095 LeuSerGlnThrPheProAsnAlaAspPheAlaGluIleThrLys 110011051110 LeuAlaThrAspLeuThrLysValAsnLysGluCysCysHisGly 111511201125 AspLeuLeuGluCysAlaAspAspArgAlaGluLeuAlaLysTyr 113011351140 MetCysGluAsnGlnAlaThrIleSerSerLysLeuGlnThrCys 114511501155 CysAspLysProLeuLeuLysLysAlaHisCysLeuSerGluVal 116011651170 GluHisAspThrMetProAlaAspLeuProAlaIleAlaAlaAsp 117511801185 PheValGluAspGlnGluValCysLysAsnTyrAlaGluAlaLys 119011951200 AspValPheLeuGlyThrPheLeuTyrGluTyrSerArgArgHis 120512101215 ProAspTyrSerValSerLeuLeuLeuArgLeuAlaLysLysTyr 122012251230 GluAlaThrLeuGluLysCysCysAlaGluAlaAsnProProAla 123512401245 CysTyrGlyThrValLeuAlaGluPheGlnProLeuValGluGlu 125012551260 ProLysAsnLeuValLysThrAsnCysAspLeuTyrGluLysLeu 126512701275 GlyGluTyrGlyPheGlnAsnAlaIleLeuValArgTyrThrGln 128012851290 LysAlaProGlnValSerThrProThrLeuValGluAlaAlaArg 129513001305 AsnLeuGlyArgValGlyThrLysCysCysThrLeuProGluAsp 131013151320 GlnArgLeuProCysValGluAspTyrLeuSerAlaIleLeuAsn 132513301335 ArgValCysLeuLeuHisGluLysThrProValSerGluHisVal 134013451350 ThrLysCysCysSerGlySerLeuValGluArgArgProCysPhe 1355 SerAlaLeuThrValAspGluThrTyrValProLysGluPheLys 137013751380 AlaGluThrPheThrPheHisSerAspIleCysThrLeuProGlu 138513901395 LysGluLysGlnIleLysLysGlnThrAlaLeuAlaGluLeuVal 140014051410 LysHisLysProLysAlaThrAlaGluGlnLeuLysThrValMet 141514201425 AspAspPheAlaGlnPheLeuAspThrCysCysLysAlaAlaAsp 143014351440 LysAspThrCysPheSerThrGluGlyProAsnLeuValThrArg 144514501455 CysLysAspAlaLeuAlaArgSerTrpSerHisProGlnPheGlu 146014651470 Lys Singlyunderlined:signalpeptidesequence;double-underlined: beginningandendofNPP1;**=cleavagepositionatthesignal peptidesequence;boldresiduesindicatealbuminsequence SEQ.IDNO:14-ENPP5-NPP3-Fcsequence MetThrSerLysPheLeuLeuValSerPheIleLeuAlaAlaLeuSer 151015 LeuSerThrThrPheSer**LysGlnGlySerCysArgLysLysCysPhe 202530 AspAlaSerPheArgGlyLeuGluAsnCysArgCysAspValAlaCys 354045 LysAspArgGlyAspCysCysTrpAspPheGluAspThrCysValGlu 505560 SerThrArgIleTrpMetCysAsnLysPheArgCysGlyGluArgLeu 65707580 GluAlaSerLeuCysSerCysSerAspAspCysLeuGlnArgLysAsp 859095 CysCysAlaAspTyrLysSerValCysGlnGlyGluThrSerTrpLeu 100105110 GluGluAsnCysAspThrAlaGlnGlnSerGlnCysProGluGlyPhe 115120125 AspLeuProProValIleLeuPheSerMetAspGlyPheArgAlaGlu 130135140 TyrLeuTyrThrTrpAspThrLeuMetProAsnIleAsnLysLeuLys 145150155160 ThrCysGlyIleHisSerLysTyrMetArgAlaMetTyrProThrLys 165170175 ThrPheProAsnHisTyrThrIleValThrGlyLeuTyrProGluSer 180185190 HisGlyIleIleAspAsnAsnMetTyrAspValAsnLeuAsnLysAsn 195200205 PheSerLeuSerSerLysGluGlnAsnAsnProAlaTrpTrpHisGly 210215220 GlnProMetTrpLeuThrAlaMetTyrGlnGlyLeuLysAlaAlaThr 225230235240 TyrPheTrpProGlySerGluValAlaIleAsnGlySerPheProSer 245250255 IleTyrMetProTyrAsnGlySerValProPheGluGluArgIleSer 260265270 ThrLeuLeuLysTrpLeuAspLeuProLysAlaGluArgProArgPhe 275280285 TyrThrMetTyrPheGluGluProAspSerSerGlyHisAlaGlyGly 290295300 ProValSerAlaArgValIleLysAlaLeuGlnValValAspHisAla 305310315320 PheGlyMetLeuMetGluGlyLeuLysGlnArgAsnLeuHisAsnCys 325330335 ValAsnIleIleLeuLeuAlaAspHisGlyMetAspGlnThrTyrCys 340345350 AsnLysMetGluTyrMetThrAspTyrPheProArgIleAsnPhePhe 355360365 TyrMetTyrGluGlyProAlaProArgIleArgAlaHisAsnIlePro 370375380 HisAspPhePheSerPheAsnSerGluGluIleValArgAsnLeuSer 385390395400 CysArgLysProAspGlnHisPheLysProTyrLeuThrProAspLeu 405410415 ProLysArgLeuHisTyrAlaLysAsnValArgIleAspLysValHis 420425430 LeuPheValAspGlnGlnTrpLeuAlaValArgSerLysSerAsnThr 435440445 AsnCysGlyGlyGlyAsnHisGlyTyrAsnAsnGluPheArgSerMet 450455460 GluAlaIlePheLeuAlaHisGlyProSerPheLysGluLysThrGlu 465470475480 ValGluProPheGluAsnIleGluValTyrAsnLeuMetCysAspLeu 485490495 LeuArgIleGlnProAlaProAsnAsnGlyThrHisGlySerLeuAsn 500505510 HisLeuLeuLysValProPheTyrGluProSerHisAlaGluGluVal 515520525 SerLysPheSerValCysGlyPheAlaAsnProLeuProThrGluSer 530535540 LeuAspCysPheCysProHisLeuGlnAsnSerThrGlnLeuGluGln 545550555560 ValAsnGlnMetLeuAsnLeuThrGlnGluGluIleThrAlaThrVal 565570575 LysValAsnLeuProPheGlyArgProArgValLeuGlnLysAsnVal 580585590 AspHisCysLeuLeuTyrHisArgGluTyrValSerGlyPheGlyLys 595600605 AlaMetArgMetProMetTrpSerSerTyrThrValProGlnLeuGly 610615620 AspThrSerProLeuProProThrValProAspCysLeuArgAlaAsp 625630635640 ValArgValProProSerGluSerGlnLysCysSerPheTyrLeuAla 645650655 AspLysAsnIleThrHisGlyPheLeuTyrProProAlaSerAsnArg 660665670 ThrSerAspSerGlnTyrAspAlaLeuIleThrSerAsnLeuValPro 675680685 MetTyrGluGluPheArgLysMetTrpAspTyrPheHisSerValLeu 690695700 LeuIleLysHisAlaThrGluArgAsnGlyValAsnValValSerGly 705710715720 ProIlePheAspTyrAsnTyrAspGlyHisPheAspAlaProAspGlu 725730735 IleThrLysHisLeuAlaAsnThrAspValProIleProThrHisTyr 740745750 PheValValLeuThrSerCysLysAsnLysSerHisThrProGluAsn 755760765 CysProGlyTrpLeuAspValLeuProPheIleIleProHisArgPro 770775780 ThrAsnValGluSerCysProGluGlyLysProGluAlaLeuTrpVal 785790795800 GluGluArgPheThrAlaHisIleAlaArgValArgAspValGluLeu 805810815 LeuThrGlyLeuAspPheTyrGlnAspLysValGlnProValSerGlu 820825830 IleLeuGlnLeuLysThrTyrLeuProThrPheGluThrThrIleAsp 835840845 LysThrHisThrCysProProCysProAlaProGluLeuLeuGlyGly 850855860 ProSerValPheLeuPheProProLysProLysAspThrLeuMetIle 865870875880 SerArgThrProGluValThrCysValValValAspValSerHisGlu 885890895 AspProGluValLysPheAsnTrpTyrValAspGlyValGluValHis 900905910 AsnAlaLysThrLysProArgGluGluGlnTyrAsnSerThrTyrArg 915920925 ValValSerValLeuThrValLeuHisGlnAspTrpLeuAsnGlyLys 930935940 GluTyrLysCysLysValSerAsnLysAlaLeuProAlaProIleGlu 945950955960 LysThrIleSerLysAlaLysGlyGlnProArgGluProGlnValTyr 965970975 ThrLeuProProSerArgGluGluMetThrLysAsnGlnValSerLeu 980985990 ThrCysLeuValLysGlyPheTyrProSerAspIleAlaValGluTrp 99510001005 GluSerAsnGlyGlnProGluAsnAsnTyrLysThrThrProPro 101010151020 ValLeuAspSerAspGlySerPhePheLeuTyrSerLysLeuThr 102510301035 ValAspLysSerArgTrpGlnGlnGlyAsnValPheSerCysSer 104010451050 ValMetHisGluAlaLeuHisAsnHisTyrThrGlnLysSerLeu 105510601065 SerLeuSerProGlyLys 1070 Singlyunderlined:signalpeptidesequence;double-underlined: beginningandendofNPP3;**=cleavagepositionatthesignal peptidesequence;boldresiduesindicatealbuminsequence SEQ.IDNO:15-ENPP5-NPP3-Albuminsequence MetThrSerLysPheLeuLeuValSerPheIleLeuAlaAlaLeuSer 151015 LeuSerThrThrPheSer**LysGlnGlySerCysArgLysLysCysPhe 202530 AspAlaSerPheArgGlyLeuGluAsnCysArgCysAspValAlaCys 354045 LysAspArgGlyAspCysCysTrpAspPheGluAspThrCysValGlu 505560 SerThrArgIleTrpMetCysAsnLysPheArgCysGlyGluArgLeu 65707580 GluAlaSerLeuCysSerCysSerAspAspCysLeuGlnArgLysAsp 859095 CysCysAlaAspTyrLysSerValCysGlnGlyGluThrSerTrpLeu 100105110 GluGluAsnCysAspThrAlaGlnGlnSerGlnCysProGluGlyPhe 115120125 AspLeuProProValIleLeuPheSerMetAspGlyPheArgAlaGlu 130135140 TyrLeuTyrThrTrpAspThrLeuMetProAsnIleAsnLysLeuLys 145150155160 ThrCysGlyIleHisSerLysTyrMetArgAlaMetTyrProThrLys 165170175 ThrPheProAsnHisTyrThrIleValThrGlyLeuTyrProGluSer 180185190 HisGlyIleIleAspAsnAsnMetTyrAspValAsnLeuAsnLysAsn 195200205 PheSerLeuSerSerLysGluGlnAsnAsnProAlaTrpTrpHisGly 210215220 GlnProMetTrpLeuThrAlaMetTyrGlnGlyLeuLysAlaAlaThr 225230235240 TyrPheTrpProGlySerGluValAlaIleAsnGlySerPheProSer 245250255 IleTyrMetProTyrAsnGlySerValProPheGluGluArgIleSer 260265270 ThrLeuLeuLysTrpLeuAspLeuProLysAlaGluArgProArgPhe 275280285 TyrThrMetTyrPheGluGluProAspSerSerGlyHisAlaGlyGly 290295300 ProValSerAlaArgValIleLysAlaLeuGlnValValAspHisAla 305310315320 PheGlyMetLeuMetGluGlyLeuLysGlnArgAsnLeuHisAsnCys 325330335 ValAsnIleIleLeuLeuAlaAspHisGlyMetAspGlnThrTyrCys 340345350 AsnLysMetGluTyrMetThrAspTyrPheProArgIleAsnPhePhe 355360365 TyrMetTyrGluGlyProAlaProArgIleArgAlaHisAsnIlePro 370375380 HisAspPhePheSerPheAsnSerGluGluIleValArgAsnLeuSer 385390395400 CysArgLysProAspGlnHisPheLysProTyrLeuThrProAspLeu 405410415 ProLysArgLeuHisTyrAlaLysAsnValArgIleAspLysValHis 420425430 LeuPheValAspGlnGlnTrpLeuAlaValArgSerLysSerAsnThr 435440445 AsnCysGlyGlyGlyAsnHisGlyTyrAsnAsnGluPheArgSerMet 450455460 GluAlaIlePheLeuAlaHisGlyProSerPheLysGluLysThrGlu 465470475480 ValGluProPheGluAsnIleGluValTyrAsnLeuMetCysAspLeu 485490495 LeuArgIleGlnProAlaProAsnAsnGlyThrHisGlySerLeuAsn 500505510 HisLeuLeuLysValProPheTyrGluProSerHisAlaGluGluVal 515520525 SerLysPheSerValCysGlyPheAlaAsnProLeuProThrGluSer 530535540 LeuAspCysPheCysProHisLeuGlnAsnSerThrGlnLeuGluGln 545550555560 ValAsnGlnMetLeuAsnLeuThrGlnGluGluIleThrAlaThrVal 565570575 LysValAsnLeuProPheGlyArgProArgValLeuGlnLysAsnVal 580585590 AspHisCysLeuLeuTyrHisArgGluTyrValSerGlyPheGlyLys 595600605 AlaMetArgMetProMetTrpSerSerTyrThrValProGlnLeuGly 610615620 AspThrSerProLeuProProThrValProAspCysLeuArgAlaAsp 625630635640 ValArgValProProSerGluSerGlnLysCysSerPheTyrLeuAla 645650655 AspLysAsnIleThrHisGlyPheLeuTyrProProAlaSerAsnArg 660665670 ThrSerAspSerGlnTyrAspAlaLeuIleThrSerAsnLeuValPro 675680685 MetTyrGluGluPheArgLysMetTrpAspTyrPheHisSerValLeu 690695700 LeuIleLysHisAlaThrGluArgAsnGlyValAsnValValSerGly 705710715720 ProIlePheAspTyrAsnTyrAspGlyHisPheAspAlaProAspGlu 725730735 IleThrLysHisLeuAlaAsnThrAspValProIleProThrHisTyr 740745750 PheValValLeuThrSerCysLysAsnLysSerHisThrProGluAsn 755760765 CysProGlyTrpLeuAspValLeuProPheIleIleProHisArgPro 770775780 ThrAsnValGluSerCysProGluGlyLysProGluAlaLeuTrpVal 785790795800 GluGluArgPheThrAlaHisIleAlaArgValArgAspValGluLeu 805810815 LeuThrGlyLeuAspPheTyrGlnAspLysValGlnProValSerGlu 820825830 IleLeuGlnLeuLysThrTyrLeuProThrPheGluThrThrIleGly 835840845 GlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySerMetLysTrp 850855860 ValThrPheLeuLeuLeuLeuPheValSerGlySerAlaPheSerArg 865870875880 GlyValPheArgArgGluAlaHisLysSerGluIleAlaHisArgTyr 885890895 AsnAspLeuGlyGluGlnHisPheLysGlyLeuValLeuIleAlaPhe 900905910 SerGlnTyrLeuGlnLysCysSerTyrAspGluHisAlaLysLeuVal 915920925 GlnGluValThrAspPheAlaLysThrCysValAlaAspGluSerAla 930935940 AlaAsnCysAspLysSerLeuHisThrLeuPheGlyAspLysLeuCys 945950955960 AlaIleProAsnLeuArgGluAsnTyrGlyGluLeuAlaAspCysCys 965970975 ThrLysGlnGluProGluArgAsnGluCysPheLeuGlnHisLysAsp 980985990 AspAsnProSerLeuProProPheGluArgProGluAlaGluAlaMet 99510001005 CysThrSerPheLysGluAsnProThrThrPheMetGlyHisTyr 101010151020 LeuHisGluValAlaArgArgHisProTyrPheTyrAlaProGlu 102510301035 LeuLeuTyrTyrAlaGluGlnTyrAsnGluIleLeuThrGlnCys 104010451050 CysAlaGluAlaAspLysGluSerCysLeuThrProLysLeuAsp 105510601065 GlyValLysGluLysAlaLeuValSerSerValArgGlnArgMet 107010751080 LysCysSerSerMetGlnLysPheGlyGluArgAlaPheLysAla 108510901095 TrpAlaValAlaArgLeuSerGlnThrPheProAsnAlaAspPhe 110011051110 AlaGluIleThrLysLeuAlaThrAspLeuThrLysValAsnLys 111511201125 GluCysCysHisGlyAspLeuLeuGluCysAlaAspAspArgAla 113011351140 GluLeuAlaLysTyrMetCysGluAsnGlnAlaThrIleSerSer 114511501155 LysLeuGlnThrCysCysAspLysProLeuLeuLysLysAlaHis 116011651170 CysLeuSerGluValGluHisAspThrMetProAlaAspLeuPro 117511801185 AlaIleAlaAlaAspPheValGluAspGlnGluValCysLysAsn 119011951200 TyrAlaGluAlaLysAspValPheLeuGlyThrPheLeuTyrGlu 120512101215 TyrSerArgArgHisProAspTyrSerValSerLeuLeuLeuArg 122012251230 LeuAlaLysLysTyrGluAlaThrLeuGluLysCysCysAlaGlu 123513401245 AlaAsnProProAlaCysTyrGlyThrValLeuAlaGluPheGln 125012551260 ProLeuValGluGluProLysAsnLeuValLysThrAsnCysAsp 126512701275 LeuTyrGluLysLeuGlyGluTyrGlyPheGlnAsnAlaIleLeu 128012851290 ValArgTyrThrGlnLysAlaProGlnValSerThrProThrLeu 129513001305 ValGluAlaAlaArgAsnLeuGlyArgValGlyThrLysCysCys 131013151320 ThrLeuProGluAspGlnArgLeuProCysValGluAspTyrLeu 132513301335 SerAlaIleLeuAsnArgValCysLeuLeuHisGluLysThrPro 134013451350 ValSerGluHisValThrLysCysCysSerGlySerLeuValGlu 135513601365 ArgArgProCysPheSerAlaLeuThrValAspGluThrTyrVal 137013751380 ProLysGluPheLysAlaGluThrPheThrPhe1395SerAspIle 138513901395 CysThrLeuProGluLysGluLysGlnIleLysLysGlnThrAla 140014051410 LeuAlaGluLeuValLysHisLysProLysAlaThrAlaGluGln 141514201425 LeuLysThrValMetAspAspPheAlaGlnPheLeuAspThrCys 143014351440 CysLysAlaAlaAspLysAspThrCysPheSerThrGluGlyPro 144514501455 AsnLeuValThrArgCysLysAspAlaLeuAla 14601465 Singlyunderlined:signalpeptidesequence;double-underlined: beginningandendofNPP3;**=cleavagepositionatthesignal peptidesequence;boldresiduesindicatealbuminsequence SEQ.IDNO:16-ENPP5ProteinExportSignalSequence MetThrSerLysPheLeuLeuValSerPheIleLeuAlaAlaLeuSer 151015 LeuSerThrThrPheSerXaa 20 SEQ.IDNO:17-ENPP51-Fc MetThrSerLysPheLeuLeuValSerPheIleLeuAlaAlaLeuSer 151015 LeuSerThrThrPheSer**GlyLeuLysProSerCysAlaLysGluVal 202530 LysSerCysLysGlyArgCysPheGluArgThrPheGlyAsnCysArg 354045 CysAspAlaAlaCysValGluLeuGlyAsnCysCysLeuAspTyrGln 505560 GluThrCysIleGluProGluHisIleTrpThrCysAsnLysPheArg 65707580 CysGlyGluLysArgLeuThrArgSerLeuCysAlaCysSerAspAsp 859095 CysLysAspLysGlyAspCysCysIleAsnTyrSerSerValCysGln 100105110 GlyGluLysSerTrpValGluGluProCysGluSerIleAsnGluPro 115120125 GlnCysProAlaGlyPheGluThrProProThrLeuLeuPheSerLeu 130135140 AspGlyPheArgAlaGluTyrLeuHisThrTrpGlyGlyLeuLeuPro 145150155160 ValIleSerLysLeuLysLysCysGlyThrTyrThrLysAsnMetArg 165170175 ProValTyrProThrLysThrPheProAsnHisTyrSerIleValThr 180185190 GlyLeuTyrProGluSerHisGlyIleIleAspAsnLysMetTyrAsp 195200205 ProLysMetAsnAlaSerPheSerLeuLysSerLysGluLysPheAsn 210215220 ProGluTrpTyrLysGlyGluProIleTrpValThrAlaLysTyrGln 225230235240 GlyLeuLysSerGlyThrPhePheTrpProGlySerAspValGluIle 245250255 AsnGlyIlePheProAspIleTyrLysMetTyrAsnGlySerValPro 260265270 PheGluGluArgIleLeuAlaValLeuGlnTrpLeuGlnLeuProLys 275280285 AspGluArgProHisPheTyrThrLeuTyrLeuGluGluProAspSer 290295300 SerGlyHisSerTyrGlyProValSerSerGluValIleLysAlaLeu 305310315320 GlnArgValAspGlyMetValGlyMetLeuMetAspGlyLeuLysGlu 325330335 LeuAsnLeuHisArgCysLeuAsnLeuIleLeuIleSerAspHisGly 340345350 MetGluGlnGlySerCysLysLysTyrIleTyrLeuAsnLysTyrLeu 355360365 GlyAspValLysAsnIleLysValIleTyrGlyProAlaAlaArgLeu 370375380 ArgProSerAspValProAspLysTyrTyrSerPheAsnTyrGluGly 385390395400 IleAlaArgAsnLeuSerCysArgGluProAsnGlnHisPheLysPro 405410415 TyrLeuLysHisPheLeuProLysArgLeuHisPheAlaLysSerAsp 420425430 ArgIleGluProLeuThrPheTyrLeuAspProGlnTrpGlnLeuAla 435440445 LeuAsnProSerGluArgLysTyrCysGlySerGlyPheHisGlySer 450455460 AspAsnValPheSerAsnMetGlnAlaLeuPheValGlyTyrGlyPro 465470475480 GlyPheLysHisGlyIleGluAlaAspThrPheGluAsnIleGluVal 485490495 TyrAsnLeuMetCysAspLeuLeuAsnLeuThrProAlaProAsnAsn 500505510 GlyThrHisGlySerLeuAsnHisLeuLeuLysAsnProValTyrThr 515520525 ProLysHisProLysGluValHisProLeuValGlnCysProPheThr 530535540 ArgAsnProArgAspAsnLeuGlyCysSerCysAsnProSerIleLeu 545550555560 ProIleGluAspPheGlnThrGlnPheAsnLeuThrValAlaGluGlu 565570575 LysIleIleLysHisGluThrLeuProTyrGlyArgProArgValLeu 580585590 GlnLysGluAsnThrIleCysLeuLeuSerGlnHisGlnPheMetSer 595600605 GlyTyrSerGlnAspIleLeuMetProLeuTrpThrSerTyrThrVal 610615620 AspArgAsnAspSerPheSerThrGluAspPheSerAsnCysLeuTyr 625630635640 GlnAspPheArgIleProLeuSerProValHisLysCysSerPheTyr 645650655 LysAsnAsnThrLysValSerTyrGlyPheLeuSerProProGlnLeu 660665670 AsnLysAsnSerSerGlyIleTyrSerGluAlaLeuLeuThrThrAsn 675680685 IleValProMetTyrGlnSerPheGlnValIleTrpArgTyrPheHis 690695700 AspThrLeuLeuArgLysTyrAlaGluGluArgAsnGlyValAsnVal 705710715720 ValSerGlyProValPheAspPheAspTyrAspGlyArgCysAspSer 725730735 LeuGluAsnLeuArgGlnLysArgArgValIleArgAsnGlnGluIle 740745750 LeuIleProThrHisPhePheIleValLeuThrSerCysLysAspThr 755760765 SerGlnThrProLeuHisCysGluAsnLeuAspThrLeuAlaPheIle 770775780 LeuProHisArgThrAspAsnSerGluSerCysValHisGlyLysHis 785790795800 AspSerSerTrpValGluGluLeuLeuMetLeuHisArgAlaArgIle 805810815 ThrAspValGluHisIleThrGlyLeuSerPheTyrGlnGlnArgLys 820825830 GluProValSerAspIleLeuLysLeuLysThrHisLeuProThrPhe 835840845 SerGlnGluAspAspLysThrHisThrCysProProCysProAlaPro 850855860 GluLeuLeuGlyGlyProSerValPheLeuPheProProLysProLys 865870875880 AspThrLeuMetIleSerArgThrProGluValThrCysValValVal 885890895 AspValSerHisGluAspProGluValLysPheAsnTrpTyrValAsp 900905910 GlyValGluValHisAsnAlaLysThrLysProArgGluGluGlnTyr 915920925 AsnSerThrTyrArgValValSerValLeuThrValLeuHisGlnAsp 930935940 TrpLeuAsnGlyLysGluTyrLysCysLysValSerAsnLysAlaLeu 945950955960 ProAlaProIleGluLysThrIleSerLysAlaLysGlyGlnProArg 965970975 GluProGlnValTyrThrLeuProProSerArgGluGluMetThrLys 980985990 AsnGlnValSerLeuThrCysLeuValLysGlyPheTyrProSerAsp 99510001005 IleAlaValGluTrpGluSerAsnGlyGlnProGluAsnAsnTyr 101010151020 LysThrThrProProValLeuAspSerAspGlySerPhePheLeu 102510301035 TyrSerLysLeuThrValAspLysSerArgTrpGlnGlnGlyAsn 104010451050 ValPheSerCysSerValMetHisGluAlaLeuHisAsnHisTyr 105510601065 ThrGlnLysSerLeuSerLeuSerProGlyLys 10701075 Singlyunderlined:signalpeptidesequence;double-underlined: beginningandendofNPP1;**=cleavagepositionatthesignal peptidesequence;boldresiduesindicateFcsequence SEQ.IDNO:18-ENPP71-FcAminoAcidSequence MetArgGlyProAlaValLeuLeuThrValAlaLeuAlaThrLeuLeu 151015 AlaProGlyAlaGlyAla**GlyLeuLysProSerCysAlaLysGluVal 202530 LysSerCysLysGlyArgCysPheGluArgThrPheGlyAsnCysArg 354045 CysAspAlaAlaCysValGluLeuGlyAsnCysCysLeuAspTyrGln 505560 GluThrCysIleGluProGluHisIleTrpThrCysAsnLysPheArg 65707580 CysGlyGluLysArgLeuThrArgSerLeuCysAlaCysSerAspAsp 859095 CysLysAspLysGlyAspCysCysIleAsnTyrSerSerValCysGln 100105110 GlyGluLysSerTrpValGluGluProCysGluSerIleAsnGluPro 115120125 GlnCysProAlaGlyPheGluThrProProThrLeuLeuPheSerLeu 130135140 AspGlyPheArgAlaGluTyrLeuHisThrTrpGlyGlyLeuLeuPro 145150155160 ValIleSerLysLeuLysLysCysGlyThrTyrThrLysAsnMetArg 165170175 ProValTyrProThrLysThrPheProAsnHisTyrSerIleValThr 180185190 GlyLeuTyrProGluSerHisGlyIleIleAspAsnLysMetTyrAsp 195200205 ProLysMetAsnAlaSerPheSerLeuLysSerLysGluLysPheAsn 210215220 ProGluTrpTyrLysGlyGluProIleTrpValThrAlaLysTyrGln 225230235240 GlyLeuLysSerGlyThrPhePheTrpProGlySerAspValGluIle 245250255 AsnGlyIlePheProAspIleTyrLysMetTyrAsnGlySerValPro 260265270 PheGluGluArgIleLeuAlaValLeuGlnTrpLeuGlnLeuProLys 275280285 AspGluArgProHisPheTyrThrLeuTyrLeuGluGluProAspSer 290295300 SerGlyHisSerTyrGlyProValSerSerGluValIleLysAlaLeu 305310315320 GlnArgValAspGlyMetValGlyMetLeuMetAspGlyLeuLysGlu 325330335 LeuAsnLeuHisArgCysLeuAsnLeuIleLeuIleSerAspHisGly 340345350 MetGluGlnGlySerCysLysLysTyrIleTyrLeuAsnLysTyrLeu 355360365 GlyAspValLysAsnIleLysValIleTyrGlyProAlaAlaArgLeu 370375380 ArgProSerAspValProAspLysTyrTyrSerPheAsnTyrGluGly 385390395400 IleAlaArgAsnLeuSerCysArgGluProAsnGlnHisPheLysPro 405410415 TyrLeuLysHisPheLeuProLysArgLeuHisPheAlaLysSerAsp 420425430 ArgIleGluProLeuThrPheTyrLeuAspProGlnTrpGlnLeuAla 435440445 LeuAsnProSerGluArgLysTyrCysGlySerGlyPheHisGlySer 450455460 AspAsnValPheSerAsnMetGlnAlaLeuPheValGlyTyrGlyPro 465470475480 GlyPheLysHisGlyIleGluAlaAspThrPheGluAsnIleGluVal 485490495 TyrAsnLeuMetCysAspLeuLeuAsnLeuThrProAlaProAsnAsn 500505510 GlyThrHisGlySerLeuAsnHisLeuLeuLysAsnProValTyrThr 515520525 ProLysHisProLysGluValHisProLeuValGlnCysProPheThr 530535540 ArgAsnProArgAspAsnLeuGlyCysSerCysAsnProSerIleLeu 545550555560 ProIleGluAspPheGlnThrGlnPheAsnLeuThrValAlaGluGlu 565570575 LysIleIleLysHisGluThrLeuProTyrGlyArgProArgValLeu 580585590 GlnLysGluAsnThrIleCysLeuLeuSerGlnHisGlnPheMetSer 595600605 GlyTyrSerGlnAspIleLeuMetProLeuTrpThrSerTyrThrVal 610615620 AspArgAsnAspSerPheSerThrGluAspPheSerAsnCysLeuTyr 625630635640 GlnAspPheArgIleProLeuSerProValHisLysCysSerPheTyr 645650655 LysAsnAsnThrLysValSerTyrGlyPheLeuSerProProGlnLeu 660665670 AsnLysAsnSerSerGlyIleTyrSerGluAlaLeuLeuThrThrAsn 675680685 IleValProMetTyrGlnSerPheGlnValIleTrpArgTyrPheHis 690695700 AspThrLeuLeuArgLysTyrAlaGluGluArgAsnGlyValAsnVal 705710715720 ValSerGlyProValPheAspPheAspTyrAspGlyArgCysAspSer 725730735 LeuGluAsnLeuArgGlnLysArgArgValIleArgAsnGlnGluIle 740745750 LeuIleProThrHisPhePheIleValLeuThrSerCysLysAspThr 755760765 SerGlnThrProLeuHisCysGluAsnLeuAspThrLeuAlaPheIle 770775780 LeuProHisArgThrAspAsnSerGluSerCysValHisGlyLysHis 785790795800 AspSerSerTrpValGluGluLeuLeuMetLeuHisArgAlaArgIle 805810815 ThrAspValGluHisIleThrGlyLeuSerPheTyrGlnGlnArgLys 820825830 GluProValSerAspIleLeuLysLeuLysThrHisLeuProThrPhe 835840845 SerGlnGluAspLeuIleAsnAspLysThrHisThrCysProProCys 850855860 ProAlaProGluLeuLeuGlyGlyProSerValPheLeuPheProPro 865870875880 LysProLysAspThrLeuMetIleSerArgThrProGluValThrCys 885890895 ValValValAspValSerHisGluAspProGluValLysPheAsnTrp 900905910 TyrValAspGlyValGluValHisAsnAlaLysThrLysProArgGlu 915920925 GluGlnTyrAsnSerThrTyrArgValValSerValLeuThrValLeu 930935940 HisGlnAspTrpLeuAsnGlyLysGluTyrLysCysLysValSerAsn 945950955960 LysAlaLeuProAlaProIleGluLysThrIleSerLysAlaLysGly 965970975 GlnProArgGluProGlnValTyrThrLeuProProSerArgGluGlu 980985990 MetThrLysAsnGlnValSerLeuThrCysLeuValLysGlyPheTyr 99510001005 ProSerAspIleAlaValGluTrpGluSerAsnGlyGlnProGlu 101010151020 AsnAsnTyrLysThrThrProProValLeuAspSerAspGlySer 102510301035 PhePheLeuTyrSerLysLeuThrValAspLysSerArgTrpGln 104010451050 GlnGlyAsnValPheSerCysSerValMetHisGluAlaLeuHis 105510601065 AsnHisTyrThrGlnLysSerLeuSerLeuSerProGlyLys 107010751080 Singlyunderlined:signalpeptidesequence;double-underlined: beginningandendofNPP1;**=cleavagepositionatthesignal peptidesequence;boldresiduesindicateFcsequence SEQ.IDNO:19-ENPP71(lackingNPP1N-TerminusGLK)AminoAcid Sequence: MetArgGlyProAlaValLeuLeuThrValAlaLeuAlaThrLeuLeu 151015 AlaProGlyAlaGlyAla**ProSerCysAlaLysGluValLysSerCys 202530 LysGlyArgCysPheGluArgThrPheGlyAsnCysArgCysAspAla 354045 AlaCysValGluLeuGlyAsnCysCysLeuAspTyrGlnGluThrCys 505560 IleGluProGluHisIleTrpThrCysAsnLysPheArgCysGlyGlu 65707580 LysArgLeuThrArgSerLeuCysAlaCysSerAspAspCysLysAsp 859095 LysGlyAspCysCysIleAsnTyrSerSerValCysGlnGlyGluLys 100105110 SerTrpValGluGluProCysGluSerIleAsnGluProGlnCysPro 115120125 AlaGlyPheGluThrProProThrLeuLeuPheSerLeuAspGlyPhe 130135140 ArgAlaGluTyrLeuHisThrTrpGlyGlyLeuLeuProValIleSer 145150155160 LysLeuLysLysCysGlyThrTyrThrLysAsnMetArgProValTyr 165170175 ProThrLysThrPheProAsnHisTyrSerIleValThrGlyLeuTyr 180185190 ProGluSerHisGlyIleIleAspAsnLysMetTyrAspProLysMet 195200205 AsnAlaSerPheSerLeuLysSerLysGluLysPheAsnProGluTrp 210215220 TyrLysGlyGluProIleTrpValThrAlaLysTyrGlnGlyLeuLys 225230235240 SerGlyThrPhePheTrpProGlySerAspValGluIleAsnGlyIle 245250255 PheProAspIleTyrLysMetTyrAsnGlySerValProPheGluGlu 260265270 ArgIleLeuAlaValLeuGlnTrpLeuGlnLeuProLysAspGluArg 275280285 ProHisPheTyrThrLeuTyrLeuGluGluProAspSerSerGlyHis 290295300 SerTyrGlyProValSerSerGluValIleLysAlaLeuGlnArgVal 305310315320 AspGlyMetValGlyMetLeuMetAspGlyLeuLysGluLeuAsnLeu 325330335 HisArgCysLeuAsnLeuIleLeuIleSerAspHisGlyMetGluGln 340345350 GlySerCysLysLysTyrIleTyrLeuAsnLysTyrLeuGlyAspVal 355360365 LysAsnIleLysValIleTyrGlyProAlaAlaArgLeuArgProSer 370375380 AspValProAspLysTyrTyrSerPheAsnTyrGluGlyIleAlaArg 385390395400 AsnLeuSerCysArgGluProAsnGlnHisPheLysProTyrLeuLys 405410415 HisPheLeuProLysArgLeuHisPheAlaLysSerAspArgIleGlu 420425430 ProLeuThrPheTyrLeuAspProGlnTrpGlnLeuAlaLeuAsnPro 435440445 SerGluArgLysTyrCysGlySerGlyPheHisGlySerAspAsnVal 450455460 PheSerAsnMetGlnAlaLeuPheValGlyTyrGlyProGlyPheLys 465470475480 HisGlyIleGluAlaAspThrPheGluAsnIleGluValTyrAsnLeu 485490495 MetCysAspLeuLeuAsnLeuThrProAlaProAsnAsnGlyThrHis 500505510 GlySerLeuAsnHisLeuLeuLysAsnProValTyrThrProLysHis 515520525 ProLysGluValHisProLeuValGlnCysProPheThrArgAsnPro 530535540 ArgAspAsnLeuGlyCysSerCysAsnProSerIleLeuProIleGlu 545550555560 AspPheGlnThrGlnPheAsnLeuThrValAlaGluGluLysIleIle 565570575 LysHisGluThrLeuProTyrGlyArgProArgValLeuGlnLysGlu 580585590 AsnThrIleCysLeuLeuSerGlnHisGlnPheMetSerGlyTyrSer 595600605 GlnAspIleLeuMetProLeuTrpThrSerTyrThrValAspArgAsn 610615620 AspSerPheSerThrGluAspPheSerAsnCysLeuTyrGlnAspPhe 625630635640 ArgIleProLeuSerProValHisLysCysSerPheTyrLysAsnAsn 645650655 ThrLysValSerTyrGlyPheLeuSerProProGlnLeuAsnLysAsn 660665670 SerSerGlyIleTyrSerGluAlaLeuLeuThrThrAsnIleValPro 675680685 MetTyrGlnSerPheGlnValIleTrpArgTyrPheHisAspThrLeu 690695700 LeuArgLysTyrAlaGluGluArgAsnGlyValAsnValValSerGly 705710715720 ProValPheAspPheAspTyrAspGlyArgCysAspSerLeuGluAsn 725730735 LeuArgGlnLysArgArgValIleArgAsnGlnGluIleLeuIlePro 740745750 ThrHisPhePheIleValLeuThrSerCysLysAspThrSerGlnThr 755760765 ProLeuHisCysGluAsnLeuAspThrLeuAlaPheIleLeuProHis 770775780 ArgThrAspAsnSerGluSerCysValHisGlyLysHisAspSerSer 785790795800 TrpValGluGluLeuLeuMetLeuHisArgAlaArgIleThrAspVal 805810815 GluHisIleThrGlyLeuSerPheTyrGlnGlnArgLysGluProVal 820825830 SerAspIleLeuLysLeuLysThrHisLeuProThrPheSerGlnGlu 835840845 Asp Singlyunderlined:signalpeptidesequence;double-underlined: beginningandendofNPP1;**=cleavagepositionatthesignal peptidesequence SEQ.IDNO:20-ENPP71(lackingNPP1N-TerminusGLK)-FcAmino AcidSequence: MetArgGlyProAlaValLeuLeuThrValAlaLeuAlaThrLeuLeu 151015 AlaProGlyAlaGlyAla**ProSerCysAlaLysGluValLysSerCys 202530 LysGlyArgCysPheGluArgThrPheGlyAsnCysArgCysAspAla 354045 AlaCysValGluLeuGlyAsnCysCysLeuAspTyrGlnGluThrCys 505560 IleGluProGluHisIleTrpThrCysAsnLysPheArgCysGlyGlu 65707580 LysArgLeuThrArgSerLeuCysAlaCysSerAspAspCysLysAsp 859095 LysGlyAspCysCysIleAsnTyrSerSerValCysGlnGlyGluLys 100105110 SerTrpValGluGluProCysGluSerIleAsnGluProGlnCysPro 115120125 AlaGlyPheGluThrProProThrLeuLeuPheSerLeuAspGlyPhe 130135140 ArgAlaGluTyrLeuHisThrTrpGlyGlyLeuLeuProValIleSer 145150155160 LysLeuLysLysCysGlyThrTyrThrLysAsnMetArgProValTyr 165170175 ProThrLysThrPheProAsnHisTyrSerIleValThrGlyLeuTyr 180185190 ProGluSerHisGlyIleIleAspAsnLysMetTyrAspProLysMet 195200205 AsnAlaSerPheSerLeuLysSerLysGluLysPheAsnProGluTrp 210215220 TyrLysGlyGluProIleTrpValThrAlaLysTyrGlnGlyLeuLys 225230235240 SerGlyThrPhePheTrpProGlySerAspValGluIleAsnGlyIle 245250255 PheProAspIleTyrLysMetTyrAsnGlySerValProPheGluGlu 260265270 ArgIleLeuAlaValLeuGlnTrpLeuGlnLeuProLysAspGluArg 275280285 ProHisPheTyrThrLeuTyrLeuGluGluProAspSerSerGlyHis 290295300 SerTyrGlyProValSerSerGluValIleLysAlaLeuGlnArgVal 305310315320 AspGlyMetValGlyMetLeuMetAspGlyLeuLysGluLeuAsnLeu 325330335 HisArgCysLeuAsnLeuIleLeuIleSerAspHisGlyMetGluGln 340345350 GlySerCysLysLysTyrIleTyrLeuAsnLysTyrLeuGlyAspVal 355360365 LysAsnIleLysValIleTyrGlyProAlaAlaArgLeuArgProSer 370375380 AspValProAspLysTyrTyrSerPheAsnTyrGluGlyIleAlaArg 385390395400 AsnLeuSerCysArgGluProAsnGlnHisPheLysProTyrLeuLys 405410415 HisPheLeuProLysArgLeuHisPheAlaLysSerAspArgIleGlu 420425430 ProLeuThrPheTyrLeuAspProGlnTrpGlnLeuAlaLeuAsnPro 435440445 SerGluArgLysTyrCysGlySerGlyPheHisGlySerAspAsnVal 450455460 PheSerAsnMetGlnAlaLeuPheValGlyTyrGlyProGlyPheLys 465470475480 HisGlyIleGluAlaAspThrPheGluAsnIleGluValTyrAsnLeu 485490495 MetCysAspLeuLeuAsnLeuThrProAlaProAsnAsnGlyThrHis 500505510 GlySerLeuAsnHisLeuLeuLysAsnProValTyrThrProLysHis 515520525 ProLysGluValHisProLeuValGlnCysProPheThrArgAsnPro 530535540 ArgAspAsnLeuGlyCysSerCysAsnProSerIleLeuProIleGlu 545550555560 AspPheGlnThrGlnPheAsnLeuThrValAlaGluGluLysIleIle 565570575 LysHisGluThrLeuProTyrGlyArgProArgValLeuGlnLysGlu 580585590 AsnThrIleCysLeuLeuSerGlnHisGlnPheMetSerGlyTyrSer 595600605 GlnAspIleLeuMetProLeuTrpThrSerTyrThrValAspArgAsn 610615620 AspSerPheSerThrGluAspPheSerAsnCysLeuTyrGlnAspPhe 625630635640 ArgIleProLeuSerProValHisLysCysSerPheTyrLysAsnAsn 645650655 ThrLysValSerTyrGlyPheLeuSerProProGlnLeuAsnLysAsn 660665670 SerSerGlyIleTyrSerGluAlaLeuLeuThrThrAsnIleValPro 675680685 MetTyrGlnSerPheGlnValIleTrpArgTyrPheHisAspThrLeu 690695700 LeuArgLysTyrAlaGluGluArgAsnGlyValAsnValValSerGly 705710715720 ProValPheAspPheAspTyrAspGlyArgCysAspSerLeuGluAsn 725730735 LeuArgGlnLysArgArgValIleArgAsnGlnGluIleLeuIlePro 740745750 ThrHisPhePheIleValLeuThrSerCysLysAspThrSerGlnThr 755760765 ProLeuHisCysGluAsnLeuAspThrLeuAlaPheIleLeuProHis 770775780 ArgThrAspAsnSerGluSerCysValHisGlyLysHisAspSerSer 785790795800 TrpValGluGluLeuLeuMetLeuHisArgAlaArgIleThrAspVal 805810815 GluHisIleThrGlyLeuSerPheTyrGlnGlnArgLysGluProVal 820825830 SerAspIleLeuLysLeuLysThrHisLeuProThrPheSerGlnGlu 835840845 AspLeuIleAsnAspLysThrHisThrCysProProCysProAlaPro 850855860 GluLeuLeuGlyGlyProSerValPheLeuPheProProLysProLys 865870875880 AspThrLeuMetIleSerArgThrProGluValThrCysValValVal 885890895 AspValSerHisGluAspProGluValLysPheAsnTrpTyrValAsp 900905910 GlyValGluValHisAsnAlaLysThrLysProArgGluGluGlnTyr 915920925 AsnSerThrTyrArgValValSerValLeuThrValLeuHisGlnAsp 930935940 TrpLeuAsnGlyLysGluTyrLysCysLysValSerAsnLysAlaLeu 945950955960 ProAlaProIleGluLysThrIleSerLysAlaLysGlyGlnProArg 965970975 GluProGlnValTyrThrLeuProProSerArgGluGluMetThrLys 980985990 AsnGlnValSerLeuThrCysLeuValLysGlyPheTyrProSerAsp 99510001005 IleAlaValGluTrpGluSerAsnGlyGlnProGluAsnAsnTyr 101010151020 LysThrThrProProValLeuAspSerAspGlySerPhePheLeu 102510301035 TyrSerLysLeuThrValAspLysSerArgTrpGlnGlnGlyAsn 104010451050 ValPheSerCysSerValMetHisGluAlaLeuHisAsnHisTyr 105510601065 ThrGlnLysSerLeuSerLeuSerProGlyLys 10701075 Singlyunderlined:signalpeptidesequence;double-underlined: beginningandendofNPP1;**=cleavagepositionatthesignal peptidesequence;boldresiduesindicateFcsequence SEQ.IDNO:21-ENPP71(lackingNPP1N-TerminusGLK)-ALBAmino AcidSequence MetArgGlyProAlaValLeuLeuThrValAlaLeuAlaThrLeuLeu 151015 AlaProGlyAlaGlyAla**ProSerCysAlaLysGluValLysSerCys 202530 LysGlyArgCysPheGluArgThrPheGlyAsnCysArgCysAspAla 354045 AlaCysValGluLeuGlyAsnCysCysLeuAspTyrGlnGluThrCys 505560 IleGluProGluHisIleTrpThrCysAsnLysPheArgCysGlyGlu 65707580 LysArgLeuThrArgSerLeuCysAlaCysSerAspAspCysLysAsp 859095 LysGlyAspCysCysIleAsnTyrSerSerValCysGlnGlyGluLys 100105110 SerTrpValGluGluProCysGluSerIleAsnGluProGlnCysPro 115120125 AlaGlyPheGluThrProProThrLeuLeuPheSerLeuAspGlyPhe 130135140 ArgAlaGluTyrLeuHisThrTrpGlyGlyLeuLeuProValIleSer 145150155160 LysLeuLysLysCysGlyThrTyrThrLysAsnMetArgProValTyr 165170175 ProThrLysThrPheProAsnHisTyrSerIleValThrGlyLeuTyr 180185190 ProGluSerHisGlyIleIleAspAsnLysMetTyrAspProLysMet 195200205 AsnAlaSerPheSerLeuLysSerLysGluLysPheAsnProGluTrp 210215220 TyrLysGlyGluProIleTrpValThrAlaLysTyrGlnGlyLeuLys 225230235240 SerGlyThrPhePheTrpProGlySerAspValGluIleAsnGlyIle 245250255 PheProAspIleTyrLysMetTyrAsnGlySerValProPheGluGlu 260265270 ArgIleLeuAlaValLeuGlnTrpLeuGlnLeuProLysAspGluArg 275280285 ProHisPheTyrThrLeuTyrLeuGluGluProAspSerSerGlyHis 290295300 SerTyrGlyProValSerSerGluValIleLysAlaLeuGlnArgVal 305310315320 AspGlyMetValGlyMetLeuMetAspGlyLeuLysGluLeuAsnLeu 325330335 HisArgCysLeuAsnLeuIleLeuIleSerAspHisGlyMetGluGln 340345350 GlySerCysLysLysTyrIleTyrLeuAsnLysTyrLeuGlyAspVal 355360365 LysAsnIleLysValIleTyrGlyProAlaAlaArgLeuArgProSer 370375380 AspValProAspLysTyrTyrSerPheAsnTyrGluGlyIleAlaArg 385390395400 AsnLeuSerCysArgGluProAsnGlnHisPheLysProTyrLeuLys 405410415 HisPheLeuProLysArgLeuHisPheAlaLysSerAspArgIleGlu 420425430 ProLeuThrPheTyrLeuAspProGlnTrpGlnLeuAlaLeuAsnPro 435440445 SerGluArgLysTyrCysGlySerGlyPheHisGlySerAspAsnVal 450455460 PheSerAsnMetGlnAlaLeuPheValGlyTyrGlyProGlyPheLys 465470475480 HisGlyIleGluAlaAspThrPheGluAsnIleGluValTyrAsnLeu 485490495 MetCysAspLeuLeuAsnLeuThrProAlaProAsnAsnGlyThrHis 500505510 GlySerLeuAsnHisLeuLeuLysAsnProValTyrThrProLysHis 515520525 ProLysGluValHisProLeuValGlnCysProPheThrArgAsnPro 530535540 ArgAspAsnLeuGlyCysSerCysAsnProSerIleLeuProIleGlu 545550555560 AspPheGlnThrGlnPheAsnLeuThrValAlaGluGluLysIleIle 565570575 LysHisGluThrLeuProTyrGlyArgProArgValLeuGlnLysGlu 580585590 AsnThrIleCysLeuLeuSerGlnHisGlnPheMetSerGlyTyrSer 595600605 GlnAspIleLeuMetProLeuTrpThrSerTyrThrValAspArgAsn 610615620 AspSerPheSerThrGluAspPheSerAsnCysLeuTyrGlnAspPhe 625630635640 ArgIleProLeuSerProValHisLysCysSerPheTyrLysAsnAsn 645650655 ThrLysValSerTyrGlyPheLeuSerProProGlnLeuAsnLysAsn 660665670 SerSerGlyIleTyrSerGluAlaLeuLeuThrThrAsnIleValPro 675680685 MetTyrGlnSerPheGlnValIleTrpArgTyrPheHisAspThrLeu 690695700 LeuArgLysTyrAlaGluGluArgAsnGlyValAsnValValSerGly 705710715720 ProValPheAspPheAspTyrAspGlyArgCysAspSerLeuGluAsn 725730735 LeuArgGlnLysArgArgValIleArgAsnGlnGluIleLeuIlePro 740745750 ThrHisPhePheIleValLeuThrSerCysLysAspThrSerGlnThr 755760765 ProLeuHisCysGluAsnLeuAspThrLeuAlaPheIleLeuProHis 770775780 ArgThrAspAsnSerGluSerCysValHisGlyLysHisAspSerSer 785790795800 TrpValGluGluLeuLeuMetLeuHisArgAlaArgIleThrAspVal 805810815 GluHisIleThrGlyLeuSerPheTyrGlnGlnArgLysGluProVal 820825830 SerAspIleLeuLysLeuLysThrHisLeuProThrPheSerGlnGlu 835840845 AspArgSerGlySerGlyGlySerMetLysTrpValThrPheLeuLeu 850855860 LeuLeuPheValSerGlySerAlaPheSerArgGlyValPheArgArg 865870875880 GluAlaHisLysSerGluIleAlaHisArgTyrAsnAspLeuGlyGlu 885890895 GlnHisPheLysGlyLeuValLeuIleAlaPheSerGlnTyrLeuGln 900905910 LysCysSerTyrAspGluHisAlaLysLeuValGlnGluValThrAsp 915920925 PheAlaLysThrCysValAlaAspGluSerAlaAlaAsnCysAspLys 930935940 SerLeuHisThrLeuPheGlyAspLysLeuCysAlaIleProAsnLeu 945950955960 ArgGluAsnTyrGlyGluLeuAlaAspCysCysThrLysGlnGluPro 965970975 GluArgAsnGluCysPheLeuGlnHisLysAspAspAsnProSerLeu 980985990 ProProPheGluArgProGluAlaGluAlaMetCysThrSerPheLys 99510001005 GluAsnProThrThrPheMetGlyHisTyrLeuHisGluValAla 101010151020 ArgArgHisProTyrPheTyrAlaProGluLeuLeuTyrTyrAla 102510301035 GluGlnTyrAsnGluIleLeuThrGlnCysCysAlaGluAlaAsp 104010451050 LysGluSerCysLeuThrProLysLeuAspGlyValLysGluLys 105510601065 AlaLeuValSerSerValArgGlnArgMetLysCysSerSerMet 107010751080 GlnLysPheGlyGluArgAlaPheLysAlaTrpAlaValAlaArg 108510901095 LeuSerGlnThrPheProAsnAlaAspPheAlaGluIleThrLys 110011051110 LeuAlaThrAspLeuThrLysValAsnLysGluCysCysHisGly 111511201125 AspLeuLeuGluCysAlaAspAspArgAlaGluLeuAlaLysTyr 113011351140 MetCysGluAsnGlnAlaThrIleSerSerLysLeuGlnThrCys 114511501155 CysAspLysProLeuLeuLysLysAlaHisCysLeuSerGluVal 116011651170 GluHisAspThrMetProAlaAspLeuProAlaIleAlaAlaAsp 117511801185 PheValGluAspGlnGluValCysLysAsnTyrAlaGluAlaLys 119011951200 AspValPheLeuGlyThrPheLeuTyrGluTyrSerArgArgHis 120512101215 ProAspTyrSerValSerLeuLeuLeuArgLeuAlaLysLysTyr 122012251230 GluAlaThrLeuGluLysCysCysAlaGluAlaAsnProProAla 123513401245 CysTyrGlyThrValLeuAlaGluPheGlnProLeuValGluGlu 125012551260 ProLysAsnLeuValLysThrAsnCysAspLeuTyrGluLysLeu 126512701275 GlyGluTyrGlyPheGlnAsnAlaIleLeuValArgTyrThrGln 128012851290 LysAlaProGlnValSerThrProThrLeuValGluAlaAlaArg 129513001305 AsnLeuGlyArgValGlyThrLysCysCysThrLeuProGluAsp 131013151320 GlnArgLeuProCysValGluAspTyrLeuSerAlaIleLeuAsn 132513301335 ArgValCysLeuLeuHisGluLysThrProValSerGluHisVal 134013451350 ThrLysCysCysSerGlySerLeuValGluArgArgProCysPhe 135513601365 SerAlaLeuThrValAspGluThrTyrValProLysGluPheLys 137013751380 AlaGluThrPheThrPheHisSerAspIleCysThrLeuProGlu 138513901395 LysGluLysGlnIleLysLysGlnThrAlaLeuAlaGluLeuVal 140014051410 LysHisLysProLysAlaThrAlaGluGlnLeuLysThrValMet 141514201425 AspAspPheAlaGlnPheLeuAspThrCysCysLysAlaAlaAsp 143014351440 LysAspThrCysPheSerThrGluGlyProAsnLeuValThrArg 144514501455 CysLysAspAlaLeuAlaArgSerTrpSerHisProGlnPheGlu 146014651470 Singlyunderlined:signalpeptidesequence;double-underlined: beginningandendofNPP1;**=cleavagepositionatthesignal peptidesequence;boldresiduesindicatealbuminsequence SEQ.IDNO:22-ENPP7-NPP3-Fcsequence: MetArgGlyProAlaValLeuLeuThrValAlaLeuAlaThrLeuLeu 151015 AlaProGlyAla**LysGlnGlySerCysArgLysLysCysPheAspAla 202530 SerPheArgGlyLeuGluAsnCysArgCysAspValAlaCysLysAsp 354045 ArgGlyAspCysCysTrpAspPheGluAspThrCysValGluSerThr 505560 ArgIleTrpMetCysAsnLysPheArgCysGlyGluArgLeuGluAla 65707580 SerLeuCysSerCysSerAspAspCysLeuGlnArgLysAspCysCys 859095 AlaAspTyrLysSerValCysGlnGlyGluThrSerTrpLeuGluGlu 100105110 AsnCysAspThrAlaGlnGlnSerGlnCysProGluGlyPheAspLeu 115120125 ProProValIleLeuPheSerMetAspGlyPheArgAlaGluTyrLeu 130135140 TyrThrTrpAspThrLeuMetProAsnIleAsnLysLeuLysThrCys 145150155160 GlyIleHisSerLysTyrMetArgAlaMetTyrProThrLysThrPhe 165170175 ProAsnHisTyrThrIleValThrGlyLeuTyrProGluSerHisGly 180185190 IleIleAspAsnAsnMetTyrAspValAsnLeuAsnLysAsnPheSer 195200205 LeuSerSerLysGluGlnAsnAsnProAlaTrpTrpHisGlyGlnPro 210215220 MetTrpLeuThrAlaMetTyrGlnGlyLeuLysAlaAlaThrTyrPhe 225230235240 TrpProGlySerGluValAlaIleAsnGlySerPheProSerIleTyr 245250255 MetProTyrAsnGlySerValProPheGluGluArgIleSerThrLeu 260265270 LeuLysTrpLeuAspLeuProLysAlaGluArgProArgPheTyrThr 275280285 MetTyrPheGluGluProAspSerSerGlyHisAlaGlyGlyProVal 290295300 SerAlaArgValIleLysAlaLeuGlnValValAspHisAlaPheGly 305310315320 MetLeuMetGluGlyLeuLysGlnArgAsnLeuHisAsnCysValAsn 325330335 IleIleLeuLeuAlaAspHisGlyMetAspGlnThrTyrCysAsnLys 340345350 MetGluTyrMetThrAspTyrPheProArgIleAsnPhePheTyrMet 355360365 TyrGluGlyProAlaProArgIleArgAlaHisAsnIleProHisAsp 370375380 PhePheSerPheAsnSerGluGluIleValArgAsnLeuSerCysArg 385390395400 LysProAspGlnHisPheLysProTyrLeuThrProAspLeuProLys 405410415 ArgLeuHisTyrAlaLysAsnValArgIleAspLysValHisLeuPhe 420425430 ValAspGlnGlnTrpLeuAlaValArgSerLysSerAsnThrAsnCys 435440445 GlyGlyGlyAsnHisGlyTyrAsnAsnGluPheArgSerMetGluAla 450455460 IlePheLeuAlaHisGlyProSerPheLysGluLysThrGluValGlu 465470475480 ProPheGluAsnIleGluValTyrAsnLeuMetCysAspLeuLeuArg 485490495 IleGlnProAlaProAsnAsnGlyThrHisGlySerLeuAsnHisLeu 500505510 LeuLysValProPheTyrGluProSerHisAlaGluGluValSerLys 515520525 PheSerValCysGlyPheAlaAsnProLeuProThrGluSerLeuAsp 530535540 CysPheCysProHisLeuGlnAsnSerThrGlnLeuGluGlnValAsn 545550555560 GlnMetLeuAsnLeuThrGlnGluGluIleThrAlaThrValLysVal 565570575 AsnLeuProPheGlyArgProArgValLeuGlnLysAsnValAspHis 580585590 CysLeuLeuTyrHisArgGluTyrValSerGlyPheGlyLysAlaMet 595600605 ArgMetProMetTrpSerSerTyrThrValProGlnLeuGlyAspThr 610615620 SerProLeuProProThrValProAspCysLeuArgAlaAspValArg 625630635640 ValProProSerGluSerGlnLysCysSerPheTyrLeuAlaAspLys 645650655 AsnIleThrHisGlyPheLeuTyrProProAlaSerAsnArgThrSer 660665670 AspSerGlnTyrAspAlaLeuIleThrSerAsnLeuValProMetTyr 675680685 GluGluPheArgLysMetTrpAspTyrPheHisSerValLeuLeuIle 690695700 LysHisAlaThrGluArgAsnGlyValAsnValValSerGlyProIle 705710715720 PheAspTyrAsnTyrAspGlyHisPheAspAlaProAspGluIleThr 725730735 LysHisLeuAlaAsnThrAspValProIleProThrHisTyrPheVal 740745750 ValLeuThrSerCysLysAsnLysSerHisThrProGluAsnCysPro 755760765 GlyTrpLeuAspValLeuProPheIleIleProHisArgProThrAsn 770775780 ValGluSerCysProGluGlyLysProGluAlaLeuTrpValGluGlu 785790795800 ArgPheThrAlaHisIleAlaArgValArgAspValGluLeuLeuThr 805810815 GlyLeuAspPheTyrGlnAspLysValGlnProValSerGluIleLeu 820825830 GlnLeuLysThrTyrLeuProThrPheGluThrThrIleAspLysThr 835840845 HisThrCysProProCysProAlaProGluLeuLeuGlyGlyProSer 850855860 ValPheLeuPheProProLysProLysAspThrLeuMetIleSerArg 865870875880 ThrProGluValThrCysValValValAspValSerHisGluAspPro 885890895 GluValLysPheAsnTrpTyrValAspGlyValGluValHisAsnAla 900905910 LysThrLysProArgGluGluGlnTyrAsnSerThrTyrArgValVal 915920925 SerValLeuThrValLeuHisGlnAspTrpLeuAsnGlyLysGluTyr 930935940 LysCysLysValSerAsnLysAlaLeuProAlaProIleGluLysThr 945950955960 IleSerLysAlaLysGlyGlnProArgGluProGlnValTyrThrLeu 965970975 ProProSerArgGluGluMetThrLysAsnGlnValSerLeuThrCys 980985990 LeuValLysGlyPheTyrProSerAspIleAlaValGluTrpGluSer 99510001005 AsnGlyGlnProGluAsnAsnTyrLysThrThrProProValLeu 101010151020 AspSerAspGlySerPhePheLeuTyrSerLysLeuThrValAsp 102510301035 LysSerArgTrpGlnGlnGlyAsnValPheSerCysSerValMet 104010451050 HisGluAlaLeuHisAsnHisTyrThrGlnLysSerLeuSerLeu 105510601065 SerProGlyLys 1070 Singlyunderlined:signalpeptidesequence;double-underlined: beginningandendofNPP3;**=cleavagepositionatthesignal peptidesequence;boldresiduesindicateFcsequence SEQ.IDNO:23-ENPP71-Albumin MetArgGlyProAlaValLeuLeuThrValAlaLeuAlaThrLeuLeu 151015 AlaProGlyAlaGlyLeuLys**ProSerCysAlaLysGluValLysSer 202530 CysLysGlyArgCysPheGluArgThrPheGlyAsnCysArgCysAsp 354045 AlaAlaCysValGluLeuGlyAsnCysCysLeuAspTyrGlnGluThr 505560 CysIleGluProGluHisIleTrpThrCysAsnLysPheArgCysGly 65707580 GluLysArgLeuThrArgSerLeuCysAlaCysSerAspAspCysLys 859095 AspLysGlyAspCysCysIleAsnTyrSerSerValCysGlnGlyGlu 100105110 LysSerTrpValGluGluProCysGluSerIleAsnGluProGlnCys 115120125 ProAlaGlyPheGluThrProProThrLeuLeuPheSerLeuAspGly 130135140 PheArgAlaGluTyrLeuHisThrTrpGlyGlyLeuLeuProValIle 145150155160 SerLysLeuLysLysCysGlyThrTyrThrLysAsnMetArgProVal 165170175 TyrProThrLysThrPheProAsnHisTyrSerIleValThrGlyLeu 180185190 TyrProGluSerHisGlyIleIleAspAsnLysMetTyrAspProLys 195200205 MetAsnAlaSerPheSerLeuLysSerLysGluLysPheAsnProGlu 210215220 TrpTyrLysGlyGluProIleTrpValThrAlaLysTyrGlnGlyLeu 225230235240 LysSerGlyThrPhePheTrpProGlySerAspValGluIleAsnGly 245250255 IlePheProAspIleTyrLysMetTyrAsnGlySerValProPheGlu 260265270 GluArgIleLeuAlaValLeuGlnTrpLeuGlnLeuProLysAspGlu 275280285 ArgProHisPheTyrThrLeuTyrLeuGluGluProAspSerSerGly 290295300 HisSerTyrGlyProValSerSerGluValIleLysAlaLeuGlnArg 305310315320 ValAspGlyMetValGlyMetLeuMetAspGlyLeuLysGluLeuAsn 325330335 LeuHisArgCysLeuAsnLeuIleLeuIleSerAspHisGlyMetGlu 340345350 GlnGlySerCysLysLysTyrIleTyrLeuAsnLysTyrLeuGlyAsp 355360365 ValLysAsnIleLysValIleTyrGlyProAlaAlaArgLeuArgPro 370375380 SerAspValProAspLysTyrTyrSerPheAsnTyrGluGlyIleAla 385390395400 ArgAsnLeuSerCysArgGluProAsnGlnHisPheLysProTyrLeu 405410415 LysHisPheLeuProLysArgLeuHisPheAlaLysSerAspArgIle 420425430 GluProLeuThrPheTyrLeuAspProGlnTrpGlnLeuAlaLeuAsn 435440445 ProSerGluArgLysTyrCysGlySerGlyPheHisGlySerAspAsn 450455460 ValPheSerAsnMetGlnAlaLeuPheValGlyTyrGlyProGlyPhe 465470475480 LysHisGlyIleGluAlaAspThrPheGluAsnIleGluValTyrAsn 485490495 LeuMetCysAspLeuLeuAsnLeuThrProAlaProAsnAsnGlyThr 500505510 HisGlySerLeuAsnHisLeuLeuLysAsnProValTyrThrProLys 515520525 HisProLysGluValHisProLeuValGlnCysProPheThrArgAsn 530535540 ProArgAspAsnLeuGlyCysSerCysAsnProSerIleLeuProIle 545550555560 GluAspPheGlnThrGlnPheAsnLeuThrValAlaGluGluLysIle 565570575 IleLysHisGluThrLeuProTyrGlyArgProArgValLeuGlnLys 580585590 GluAsnThrIleCysLeuLeuSerGlnHisGlnPheMetSerGlyTyr 595600605 SerGlnAspIleLeuMetProLeuTrpThrSerTyrThrValAspArg 610615620 AsnAspSerPheSerThrGluAspPheSerAsnCysLeuTyrGlnAsp 625630635640 PheArgIleProLeuSerProValHisLysCysSerPheTyrLysAsn 645650655 AsnThrLysValSerTyrGlyPheLeuSerProProGlnLeuAsnLys 660665670 AsnSerSerGlyIleTyrSerGluAlaLeuLeuThrThrAsnIleVal 675680685 ProMetTyrGlnSerPheGlnValIleTrpArgTyrPheHisAspThr 690695700 LeuLeuArgLysTyrAlaGluGluArgAsnGlyValAsnValValSer 705710715720 GlyProValPheAspPheAspTyrAspGlyArgCysAspSerLeuGlu 725730735 AsnLeuArgGlnLysArgArgValIleArgAsnGlnGluIleLeuIle 740745750 ProThrHisPhePheIleValLeuThrSerCysLysAspThrSerGln 755760765 ThrProLeuHisCysGluAsnLeuAspThrLeuAlaPheIleLeuPro 770775780 HisArgThrAspAsnSerGluSerCysValHisGlyLysHisAspSer 785790795800 SerTrpValGluGluLeuLeuMetLeuHisArgAlaArgIleThrAsp 805810815 ValGluHisIleThrGlyLeuSerPheTyrGlnGlnArgLysGluPro 820825830 ValSerAspIleLeuLysLeuLysThrHisLeuProThrPheSerGln 835840845 GluAspGlyGlySerGlyGlySerMetLysTrpValThrPheLeuLeu 850855860 LeuLeuPheValSerGlySerAlaPheSerArgGlyValPheArgArg 865870875880 GluAlaHisLysSerGluIleAlaHisArgTyrAsnAspLeuGlyGlu 885890895 GlnHisPheLysGlyLeuValLeuIleAlaPheSerGlnTyrLeuGln 900905910 LysCysSerTyrAspGluHisAlaLysLeuValGlnGluValThrAsp 915920925 PheAlaLysThrCysValAlaAspGluSerAlaAlaAsnCysAspLys 930935940 SerLeuHisThrLeuPheGlyAspLysLeuCysAlaIleProAsnLeu 945950955960 ArgGluAsnTyrGlyGluLeuAlaAspCysCysThrLysGlnGluPro 965970975 GluArgAsnGluCysPheLeuGlnHisLysAspAspAsnProSerLeu 980985990 ProProPheGluArgProGluAlaGluAlaMetCysThrSerPheLys 99510001005 GluAsnProThrThrPheMetGlyHisTyrLeuHisGluValAla 101010151020 ArgArgHisProTyrPheTyrAlaProGluLeuLeuTyrTyrAla 102510301035 GluGlnTyrAsnGluIleLeuThrGlnCysCysAlaGluAlaAsp 104010451050 LysGluSerCysLeuThrProLysLeuAspGlyValLysGluLys 105510601065 AlaLeuValSerSerValArgGlnArgMetLysCysSerSerMet 107010751080 GlnLysPheGlyGluArgAlaPheLysAlaTrpAlaValAlaArg 108510901095 LeuSerGlnThrPheProAsnAlaAspPheAlaGluIleThrLys 110011051110 LeuAlaThrAspLeuThrLysValAsnLysGluCysCysHisGly 111511201125 AspLeuLeuGluCysAlaAspAspArgAlaGluLeuAlaLysTyr 113011351140 MetCysGluAsnGlnAlaThrIleSerSerLysLeuGlnThrCys 114511501155 CysAspLysProLeuLeuLysLysAlaHisCysLeuSerGluVal 116011651170 GluHisAspThrMetProAlaAspLeuProAlaIleAlaAlaAsp 117511801185 PheValGluAspGlnGluValCysLysAsnTyrAlaGluAlaLys 119011951200 AspValPheLeuGlyThrPheLeuTyrGluTyrSerArgArgHis 120512101215 ProAspTyrSerValSerLeuLeuLeuArgLeuAlaLysLysTyr 122012251230 GluAlaThrLeuGluLysCysCysAlaGluAlaAsnProProAla 123513401245 CysTyrGlyThrValLeuAlaGluPheGlnProLeuValGluGlu 125012551260 ProLysAsnLeuValLysThrAsnCysAspLeuTyrGluLysLeu 126512701275 GlyGluTyrGlyPheGlnAsnAlaIleLeuValArgTyrThrGln 128012851290 LysAlaProGlnValSerThrProThrLeuValGluAlaAlaArg 129513001305 AsnLeuGlyArgValGlyThrLysCysCysThrLeuProGluAsp 131013151320 GlnArgLeuProCysValGluAspTyrLeuSerAlaIleLeuAsn 132513301335 ArgValCysLeuLeuHisGluLysThrProValSerGluHisVal 134013451350 ThrLysCysCysSerGlySerLeuValGluArgArgProCysPhe 135513601365 SerAlaLeuThrValAspGluThrTyrValProLysGluPheLys 137013751380 AlaGluThrPheThrPheHisSerAspIleCysThrLeu 138513901395 Singlyunderlined:signalpeptidesequence;double-underlined: beginningandendofNPP3;**=cleavagepositionatthesignal peptidesequence;boldresiduesindicateFcsequence SEQ.IDNO:24-ENPP7-NPP3-Albumin MetArgGlyProAlaValLeuLeuThrValAlaLeuAlaThrLeuLeu 151015 AlaProGlyAla**LysGlnGlySerCysArgLysLysCysPheAspAla 202530 SerPheArgGlyLeuGluAsnCysArgCysAspValAlaCysLysAsp 354045 ArgGlyAspCysCysTrpAspPheGluAspThrCysValGluSerThr 505560 ArgIleTrpMetCysAsnLysPheArgCysGlyGluArgLeuGluAla 65707580 SerLeuCysSerCysSerAspAspCysLeuGlnArgLysAspCysCys 859095 AlaAspTyrLysSerValCysGlnGlyGluThrSerTrpLeuGluGlu 100105110 AsnCysAspThrAlaGlnGlnSerGlnCysProGluGlyPheAspLeu 115120125 ProProValIleLeuPheSerMetAspGlyPheArgAlaGluTyrLeu 130135140 TyrThrTrpAspThrLeuMetProAsnIleAsnLysLeuLysThrCys 145150155160 GlyIleHisSerLysTyrMetArgAlaMetTyrProThrLysThrPhe 165170175 ProAsnHisTyrThrIleValThrGlyLeuTyrProGluSerHisGly 180185190 IleIleAspAsnAsnMetTyrAspValAsnLeuAsnLysAsnPheSer 195200205 LeuSerSerLysGluGlnAsnAsnProAlaTrpTrpHisGlyGlnPro 210215220 MetTrpLeuThrAlaMetTyrGlnGlyLeuLysAlaAlaThrTyrPhe 225230235240 TrpProGlySerGluValAlaIleAsnGlySerPheProSerIleTyr 245250255 MetProTyrAsnGlySerValProPheGluGluArgIleSerThrLeu 260265270 LeuLysTrpLeuAspLeuProLysAlaGluArgProArgPheTyrThr 275280285 MetTyrPheGluGluProAspSerSerGlyHisAlaGlyGlyProVal 290295300 SerAlaArgValIleLysAlaLeuGlnValValAspHisAlaPheGly 305310315320 MetLeuMetGluGlyLeuLysGlnArgAsnLeuHisAsnCysValAsn 325330335 IleIleLeuLeuAlaAspHisGlyMetAspGlnThrTyrCysAsnLys 340345350 MetGluTyrMetThrAspTyrPheProArgIleAsnPhePheTyrMet 355360365 TyrGluGlyProAlaProArgIleArgAlaHisAsnIleProHisAsp 370375380 PhePheSerPheAsnSerGluGluIleValArgAsnLeuSerCysArg 385390395400 LysProAspGlnHisPheLysProTyrLeuThrProAspLeuProLys 405410415 ArgLeuHisTyrAlaLysAsnValArgIleAspLysValHisLeuPhe 420425430 ValAspGlnGlnTrpLeuAlaValArgSerLysSerAsnThrAsnCys 435440445 GlyGlyGlyAsnHisGlyTyrAsnAsnGluPheArgSerMetGluAla 450455460 IlePheLeuAlaHisGlyProSerPheLysGluLysThrGluValGlu 465470475480 ProPheGluAsnIleGluValTyrAsnLeuMetCysAspLeuLeuArg 485490495 IleGlnProAlaProAsnAsnGlyThrHisGlySerLeuAsnHisLeu 500505510 LeuLysValProPheTyrGluProSerHisAlaGluGluValSerLys 515520525 PheSerValCysGlyPheAlaAsnProLeuProThrGluSerLeuAsp 530535540 CysPheCysProHisLeuGlnAsnSerThrGlnLeuGluGlnValAsn 545550555560 GlnMetLeuAsnLeuThrGlnGluGluIleThrAlaThrValLysVal 565570575 AsnLeuProPheGlyArgProArgValLeuGlnLysAsnValAspHis 580585590 CysLeuLeuTyrHisArgGluTyrValSerGlyPheGlyLysAlaMet 595600605 ArgMetProMetTrpSerSerTyrThrValProGlnLeuGlyAspThr 610615620 SerProLeuProProThrValProAspCysLeuArgAlaAspValArg 625630635640 ValProProSerGluSerGlnLysCysSerPheTyrLeuAlaAspLys 645650655 AsnIleThrHisGlyPheLeuTyrProProAlaSerAsnArgThrSer 660665670 AspSerGlnTyrAspAlaLeuIleThrSerAsnLeuValProMetTyr 675680685 GluGluPheArgLysMetTrpAspTyrPheHisSerValLeuLeuIle 690695700 LysHisAlaThrGluArgAsnGlyValAsnValValSerGlyProIle 705710715720 PheAspTyrAsnTyrAspGlyHisPheAspAlaProAspGluIleThr 725730735 LysHisLeuAlaAsnThrAspValProIleProThrHisTyrPheVal 740745750 ValLeuThrSerCysLysAsnLysSerHisThrProGluAsnCysPro 755760765 GlyTrpLeuAspValLeuProPheIleIleProHisArgProThrAsn 770775780 ValGluSerCysProGluGlyLysProGluAlaLeuTrpValGluGlu 785790795800 ArgPheThrAlaHisIleAlaArgValArgAspValGluLeuLeuThr 805810815 GlyLeuAspPheTyrGlnAspLysValGlnProValSerGluIleLeu 820825830 GlnLeuLysThrTyrLeuProThrPheGluThrThrIleGlyGlyGly 835840845 SerGlyGlyGlyGlySerGlyGlyGlyGlySerMetLysTrpValThr 850855860 PheLeuLeuLeuLeuPheValSerGlySerAlaPheSerArgGlyVal 865870875880 PheArgArgGluAlaHisLysSerGluIleAlaHisArgTyrAsnAsp 885890895 LeuGlyGluGlnHisPheLysGlyLeuValLeuIleAlaPheSerGln 900905910 TyrLeuGlnLysCysSerTyrAspGluHisAlaLysLeuValGlnGlu 915920925 ValThrAspPheAlaLysThrCysValAlaAspGluSerAlaAlaAsn 930935940 CysAspLysSerLeuHisThrLeuPheGlyAspLysLeuCysAlaIle 945950955960 ProAsnLeuArgGluAsnTyrGlyGluLeuAlaAspCysCysThrLys 965970975 GlnGluProGluArgAsnGluCysPheLeuGlnHisLysAspAspAsn 980985990 ProSerLeuProProPheGluArgProGluAlaGluAlaMetCysThr 995100010055 SerPheLysGluAsnProThrThrPheMetGlyHisTyrLeuHis 101010151020 GluValAlaArgArgHisProTyrPheTyrAlaProGluLeuLeu 102510301035 TyrTyrAlaGluGlnTyrAsnGluIleLeuThrGlnCysCysAla 104010451050 GluAlaAspLysGluSerCysLeuThrProLysLeuAspGlyVal 105510601065 LysGluLysAlaLeuValSerSerValArgGlnArgMetLysCys 107010751080 SerSerMetGlnLysPheGlyGluArgAlaPheLysAlaTrpAla 108510901095 ValAlaArgLeuSerGlnThrPheProAsnAlaAspPheAlaGlu 110011051110 IleThrLysLeuAlaThrAspLeuThrLysValAsnLysGluCys 111511201125 CysHisGlyAspLeuLeuGluCysAlaAspAspArgAlaGluLeu 113011351140 AlaLysTyrMetCysGluAsnGlnAlaThrIleSerSerLysLeu 114511501155 GlnThrCysCysAspLysProLeuLeuLysLysAlaHisCysLeu 116011651170 SerGluValGluHisAspThrMetProAlaAspLeuProAlaIle 117511801185 AlaAlaAspPheValGluAspGlnGluValCysLysAsnTyrAla 119011951200 GluAlaLysAspValPheLeuGlyThrPheLeuTyrGluTyrSer 120512101215 ArgArgHisProAspTyrSerValSerLeuLeuLeuArgLeuAla 122012251230 LysLysTyrGluAlaThrLeuGluLysCysCysAlaGluAlaAsn 123513401245 ProProAlaCysTyrGlyThrValLeuAlaGluPheGlnProLeu 125012551260 ValGluGluProLysAsnLeuValLysThrAsnCysAspLeuTyr 126512701275 GluLysLeuGlyGluTyrGlyPheGlnAsnAlaIleLeuValArg 128012851290 TyrThrGlnLysAlaProGlnValSerThrProThrLeuValGlu 129513001305 AlaAlaArgAsnLeuGlyArgValGlyThrLysCysCysThrLeu 131013151320 ProGluAspGlnArgLeuProCysValGluAspTyrLeuSerAla 132513301335 IleLeuAsnArgValCysLeuLeuHisGluLysThrProValSer 134013451350 GluHisValThrLysCysCysSerGlySerLeuValGluArgArg 135513601365 ProCysPheSerAlaLeuThrValAspGluThrTyrValProLys 137013751380 GluPheLysAlaGluThrPheThrPheHisSerAspIleCysThr 138513901395 LeuProGluLysGluLysGlnIleLysLysGlnThrAlaLeuAla 140014051410 GluLeuValLysHisLysProLysAlaThrAlaGluGlnLeuLys 141514201425 ThrValMetAspAspPheAlaGlnPheLeuAspThrCysCysLys 143014351440 AlaAlaAspLysAspThrCysPheSerThrGluGlyProAsnLeu 144514501455 ValThrArgCysLysAspAlaLeuAla 14601465 Singlyunderlined:signalpeptidesequence;double-underlined: beginningandendofNPP3;**=cleavagepositionatthesignal peptidesequence;boldresiduesindicatealbuminsequence SEQ.IDNO:25-ENPP7-ENPP3-Albumin MetArgGlyProAlaValLeuLeuThrValAlaLeuAlaThrLeuLeu 151015 AlaProGlyAla**LysGlnGlySerCysArgLysLysCysPheAspAla 202530 SerPheArgGlyLeuGluAsnCysArgCysAspValAlaCysLysAsp 354045 ArgGlyAspCysCysTrpAspPheGluAspThrCysValGluSerThr 505560 ArgIleTrpMetCysAsnLysPheArgCysGlyGluArgLeuGluAla 65707580 SerLeuCysSerCysSerAspAspCysLeuGlnArgLysAspCysCys 859095 AlaAspTyrLysSerValCysGlnGlyGluThrSerTrpLeuGluGlu 100105110 AsnCysAspThrAlaGlnGlnSerGlnCysProGluGlyPheAspLeu 115120125 ProProValIleLeuPheSerMetAspGlyPheArgAlaGluTyrLeu 130135140 TyrThrTrpAspThrLeuMetProAsnIleAsnLysLeuLysThrCys 145150155160 GlyIleHisSerLysTyrMetArgAlaMetTyrProThrLysThrPhe 165170175 ProAsnHisTyrThrIleValThrGlyLeuTyrProGluSerHisGly 180185190 IleIleAspAsnAsnMetTyrAspValAsnLeuAsnLysAsnPheSer 195200205 LeuSerSerLysGluGlnAsnAsnProAlaTrpTrpHisGlyGlnPro 210215220 MetTrpLeuThrAlaMetTyrGlnGlyLeuLysAlaAlaThrTyrPhe 225230235240 TrpProGlySerGluValAlaIleAsnGlySerPheProSerIleTyr 245250255 MetProTyrAsnGlySerValProPheGluGluArgIleSerThrLeu 260265270 LeuLysTrpLeuAspLeuProLysAlaGluArgProArgPheTyrThr 275280285 MetTyrPheGluGluProAspSerSerGlyHisAlaGlyGlyProVal 290295300 SerAlaArgValIleLysAlaLeuGlnValValAspHisAlaPheGly 305310315320 MetLeuMetGluGlyLeuLysGlnArgAsnLeuHisAsnCysValAsn 325330335 IleIleLeuLeuAlaAspHisGlyMetAspGlnThrTyrCysAsnLys 340345350 MetGluTyrMetThrAspTyrPheProArgIleAsnPhePheTyrMet 355360365 TyrGluGlyProAlaProArgIleArgAlaHisAsnIleProHisAsp 370375380 PhePheSerPheAsnSerGluGluIleValArgAsnLeuSerCysArg 385390395400 LysProAspGlnHisPheLysProTyrLeuThrProAspLeuProLys 405410415 ArgLeuHisTyrAlaLysAsnValArgIleAspLysValHisLeuPhe 420425430 ValAspGlnGlnTrpLeuAlaValArgSerLysSerAsnThrAsnCys 435440445 GlyGlyGlyAsnHisGlyTyrAsnAsnGluPheArgSerMetGluAla 450455460 IlePheLeuAlaHisGlyProSerPheLysGluLysThrGluValGlu 465470475480 ProPheGluAsnIleGluValTyrAsnLeuMetCysAspLeuLeuArg 485490495 IleGlnProAlaProAsnAsnGlyThrHisGlySerLeuAsnHisLeu 500505510 LeuLysValProPheTyrGluProSerHisAlaGluGluValSerLys 515520525 PheSerValCysGlyPheAlaAsnProLeuProThrGluSerLeuAsp 530535540 CysPheCysProHisLeuGlnAsnSerThrGlnLeuGluGlnValAsn 545550555560 GlnMetLeuAsnLeuThrGlnGluGluIleThrAlaThrValLysVal 565570575 AsnLeuProPheGlyArgProArgValLeuGlnLysAsnValAspHis 580585590 CysLeuLeuTyrHisArgGluTyrValSerGlyPheGlyLysAlaMet 595600605 ArgMetProMetTrpSerSerTyrThrValProGlnLeuGlyAspThr 610615620 SerProLeuProProThrValProAspCysLeuArgAlaAspValArg 625630635640 ValProProSerGluSerGlnLysCysSerPheTyrLeuAlaAspLys 645650655 AsnIleThrHisGlyPheLeuTyrProProAlaSerAsnArgThrSer 660665670 AspSerGlnTyrAspAlaLeuIleThrSerAsnLeuValProMetTyr 675680685 GluGluPheArgLysMetTrpAspTyrPheHisSerValLeuLeuIle 690695700 LysHisAlaThrGluArgAsnGlyValAsnValValSerGlyProIle 705710715720 PheAspTyrAsnTyrAspGlyHisPheAspAlaProAspGluIleThr 725730735 LysHisLeuAlaAsnThrAspValProIleProThrHisTyrPheVal 740745750 ValLeuThrSerCysLysAsnLysSerHisThrProGluAsnCysPro 755760765 GlyTrpLeuAspValLeuProPheIleIleProHisArgProThrAsn 770775780 ValGluSerCysProGluGlyLysProGluAlaLeuTrpValGluGlu 785790795800 ArgPheThrAlaHisIleAlaArgValArgAspValGluLeuLeuThr 805810815 GlyLeuAspPheTyrGlnAspLysValGlnProValSerGluIleLeu 820825830 GlnLeuLysThrTyrLeuProThrPheGluThrThrIleAspLysThr 835840845 HisThrCysProProCysProAlaProGluLeuLeuGlyGlyProSer 850855860 ValPheLeuPheProProLysProLysAspThrLeuMetIleSerArg 865870875880 ThrProGluValThrGlyGlyGlySerGlyGlyGlyGlySerGlyGly 885890895 GlyGlySerMetLysTrpValThrPheLeuLeuLeuLeuPheValSer 900905910 GlySerAlaPheSerArgGlyValPheArgArgGluAlaHisLysSer 915920925 GluIleAlaHisArgTyrAsnAspLeuGlyGluGlnHisPheLysGly 930935940 LeuValLeuIleAlaPheSerGlnTyrLeuGlnLysCysSerTyrAsp 945950955960 GluHisAlaLysLeuValGlnGluValThrAspPheAlaLysThrCys 965970975 ValAlaAspGluSerAlaAlaAsnCysAspLysSerLeuHisThrLeu 980985990 PheGlyAspLysLeuCysAlaIleProAsnLeuArgGluAsnTyrGly 99510001005 GluLeuAlaAspCysCysThrLysGlnGluProGluArgAsnGlu 101010151020 CysPheLeuGlnHisLysAspAspAsnProSerLeuProProPhe 102510301035 GluArgProGluAlaGluAlaMetCysThrSerPheLysGluAsn 104010451050 ProThrThrPheMetGlyHisTyrLeuHisGluValAlaArgArg 105510601065 HisProTyrPheTyrAlaProGluLeuLeuTyrTyrAlaGluGln 107010751080 TyrAsnGluIleLeuThrGlnCysCysAlaGluAlaAspLysGlu 108510901095 SerCysLeuThrProLysLeuAspGlyValLysGluLysAlaLeu 110011051110 ValSerSerValArgGlnArgMetLysCysSerSerMetGlnLys 111511201125 PheGlyGluArgAlaPheLysAlaTrpAlaValAlaArgLeuSer 113011351140 GlnThrPheProAsnAlaAspPheAlaGluIleThrLysLeuAla 114511501155 ThrAspLeuThrLysValAsnLysGluCysCysHisGlyAspLeu 116011651170 LeuGluCysAlaAspAspArgAlaGluLeuAlaLysTyrMetCys 117511801185 GluAsnGlnAlaThrIleSerSerLysLeuGlnThrCysCysAsp 119011951200 LysProLeuLeuLysLysAlaHisCysLeuSerGluValGluHis 120512101215 AspThrMetProAlaAspLeuProAlaIleAlaAlaAspPheVal 122012251230 GluAspGlnGluValCysLysAsnTyrAlaGluAlaLysAspVal 123513401245 PheLeuGlyThrPheLeuTyrGluTyrSerArgArgHisProAsp 125012551260 TyrSerValSerLeuLeuLeuArgLeuAlaLysLysTyrGluAla 126512701275 ThrLeuGluLysCysCysAlaGluAlaAsnProProAlaCysTyr 128012851290 GlyThrValLeuAlaGluPheGlnProLeuValGluGluProLys 129513001305 AsnLeuValLysThrAsnCysAspLeuTyrGluLysLeuGlyGlu 131013151320 TyrGlyPheGlnAsnAlaIleLeuValArgTyrThrGlnLysAla 132513301335 ProGlnValSerThrProThrLeuValGluAlaAlaArgAsnLeu 134013451350 GlyArgValGlyThrLysCysCysThrLeuProGluAspGlnArg 135513601365 LeuProCysValGluAspTyrLeuSerAlaIleLeuAsnArgVal 137013751380 CysLeuLeuHisGluLysThrProValSerGluHisValThrLys 138513901395 CysCysSerGlySerLeuValGluArgArgProCysPheSerAla 140014051410 LeuThrValAspGluThrTyrValProLysGluPheLysAlaGlu 141514201425 ThrPheThrPheHisSerAspIleCysThrLeuProGluLysGlu 143014351440 LysGlnIleLysLysGlnThrAlaLeuAlaGluLeuValLysHis 144514501455 LysProLysAlaThrAlaGluGlnLeuLysThrValMetAspAsp 146014651470 PheAlaGlnPheLeuAspThrCysCysLysAlaAlaAspLysAsp 147514801485 ThrCysPheSerThrGluGlyProAsnLeuValThrArgCysLys 149014951500 AspAlaLeuAla 1505 Singlyunderlined:signalpeptidesequence;double-underlined: beginningandendofNPP3;**=cleavagepositionatthesignal peptidesequence;boldresiduesindicatealbuminsequence SEQ.IDNO:26-ENPP71AminoAcidSequence MetArgGlyProAlaValLeuLeuThrValAlaLeuAlaThrLeuLeu 151015 AlaProGlyAlaGlyAla**GlyLeuLysProSerCysAlaLysGluVal 202530 LysSerCysLysGlyArgCysPheGluArgThrPheGlyAsnCysArg 354045 CysAspAlaAlaCysValGluLeuGlyAsnCysCysLeuAspTyrGln 505560 GluThrCysIleGluProGluHisIleTrpThrCysAsnLysPheArg 65707580 CysGlyGluLysArgLeuThrArgSerLeuCysAlaCysSerAspAsp 859095 CysLysAspLysGlyAspCysCysIleAsnTyrSerSerValCysGln 100105110 GlyGluLysSerTrpValGluGluProCysGluSerIleAsnGluPro 115120125 GlnCysProAlaGlyPheGluThrProProThrLeuLeuPheSerLeu 130135140 AspGlyPheArgAlaGluTyrLeuHisThrTrpGlyGlyLeuLeuPro 145150155160 ValIleSerLysLeuLysLysCysGlyThrTyrThrLysAsnMetArg 165170175 ProValTyrProThrLysThrPheProAsnHisTyrSerIleValThr 180185190 GlyLeuTyrProGluSerHisGlyIleIleAspAsnLysMetTyrAsp 195200205 ProLysMetAsnAlaSerPheSerLeuLysSerLysGluLysPheAsn 210215220 ProGluTrpTyrLysGlyGluProIleTrpValThrAlaLysTyrGln 225230235240 GlyLeuLysSerGlyThrPhePheTrpProGlySerAspValGluIle 245250255 AsnGlyIlePheProAspIleTyrLysMetTyrAsnGlySerValPro 260265270 PheGluGluArgIleLeuAlaValLeuGlnTrpLeuGlnLeuProLys 275280285 AspGluArgProHisPheTyrThrLeuTyrLeuGluGluProAspSer 290295300 SerGlyHisSerTyrGlyProValSerSerGluValIleLysAlaLeu 305310315320 GlnArgValAspGlyMetValGlyMetLeuMetAspGlyLeuLysGlu 325330335 LeuAsnLeuHisArgCysLeuAsnLeuIleLeuIleSerAspHisGly 340345350 MetGluGlnGlySerCysLysLysTyrIleTyrLeuAsnLysTyrLeu 355360365 GlyAspValLysAsnIleLysValIleTyrGlyProAlaAlaArgLeu 370375380 ArgProSerAspValProAspLysTyrTyrSerPheAsnTyrGluGly 385390395400 IleAlaArgAsnLeuSerCysArgGluProAsnGlnHisPheLysPro 405410415 TyrLeuLysHisPheLeuProLysArgLeuHisPheAlaLysSerAsp 420425430 ArgIleGluProLeuThrPheTyrLeuAspProGlnTrpGlnLeuAla 435440445 LeuAsnProSerGluArgLysTyrCysGlySerGlyPheHisGlySer 450455460 AspAsnValPheSerAsnMetGlnAlaLeuPheValGlyTyrGlyPro 465470475480 GlyPheLysHisGlyIleGluAlaAspThrPheGluAsnIleGluVal 485490495 TyrAsnLeuMetCysAspLeuLeuAsnLeuThrProAlaProAsnAsn 500505510 GlyThrHisGlySerLeuAsnHisLeuLeuLysAsnProValTyrThr 515520525 ProLysHisProLysGluValHisProLeuValGlnCysProPheThr 530535540 ArgAsnProArgAspAsnLeuGlyCysSerCysAsnProSerIleLeu 545550555560 ProIleGluAspPheGlnThrGlnPheAsnLeuThrValAlaGluGlu 565570575 LysIleIleLysHisGluThrLeuProTyrGlyArgProArgValLeu 580585590 GlnLysGluAsnThrIleCysLeuLeuSerGlnHisGlnPheMetSer 595600605 GlyTyrSerGlnAspIleLeuMetProLeuTrpThrSerTyrThrVal 610615620 AspArgAsnAspSerPheSerThrGluAspPheSerAsnCysLeuTyr 625630635640 GlnAspPheArgIleProLeuSerProValHisLysCysSerPheTyr 645650655 LysAsnAsnThrLysValSerTyrGlyPheLeuSerProProGlnLeu 660665670 AsnLysAsnSerSerGlyIleTyrSerGluAlaLeuLeuThrThrAsn 675680685 IleValProMetTyrGlnSerPheGlnValIleTrpArgTyrPheHis 690695700 AspThrLeuLeuArgLysTyrAlaGluGluArgAsnGlyValAsnVal 705710715720 ValSerGlyProValPheAspPheAspTyrAspGlyArgCysAspSer 725730735 LeuGluAsnLeuArgGlnLysArgArgValIleArgAsnGlnGluIle 740745750 LeuIleProThrHisPhePheIleValLeuThrSerCysLysAspThr 755760765 SerGlnThrProLeuHisCysGluAsnLeuAspThrLeuAlaPheIle 770775780 LeuProHisArgThrAspAsnSerGluSerCysValHisGlyLysHis 785790795800 AspSerSerTrpValGluGluLeuLeuMetLeuHisArgAlaArgIle 805810815 ThrAspValGluHisIleThrGlyLeuSerPheTyrGlnGlnArgLys 820825830 GluProValSerAspIleLeuLysLeuLysThrHisLeuProThrPhe 835840845 SerGlnGluAsp 850 Singlyunderlined:signalpeptidesequence;double-underlined: beginningandendofNPP1;**=cleavagepositionatthesignal peptidesequence SEQ.IDNO:27-ENPP121AminoAcidSequence MetGluArgAspGlyCysAlaGlyGlyGlySerArgGlyGlyGluGly 151015 GlyArgAlaProArgGluGlyProAlaGlyAsnGlyArgAspArgGly 202530 ArgSerHisAlaAlaGluAlaProGlyAspProGlnAlaAlaAlaSer 354045 LeuLeuAlaProMetAspValGlyGluGluProLeuGluLysAlaAla 505560 ArgAlaArgThrAlaLysAspProAsnThrTyrLysIleIleSerLeu 65707580 PheThrPheAlaValGlyValAsnIleCysLeuGly**PheThrAlaGly 859095 LeuLysProSerCysAlaLysGluValLysSerCysLysGlyArgCys 100105110 PheGluArgThrPheGlyAsnCysArgCysAspAlaAlaCysValGlu 115120125 LeuGlyAsnCysCysLeuAspTyrGlnGluThrCysIleGluProGlu 130135140 HisIleTrpThrCysAsnLysPheArgCysGlyGluLysArgLeuThr 145150155160 ArgSerLeuCysAlaCysSerAspAspCysLysAspLysGlyAspCys 165170175 CysIleAsnTyrSerSerValCysGlnGlyGluLysSerTrpValGlu 180185190 GluProCysGluSerIleAsnGluProGlnCysProAlaGlyPheGlu 195200205 ThrProProThrLeuLeuPheSerLeuAspGlyPheArgAlaGluTyr 210215220 LeuHisThrTrpGlyGlyLeuLeuProValIleSerLysLeuLysLys 225230235240 CysGlyThrTyrThrLysAsnMetArgProValTyrProThrLysThr 245250255 PheProAsnHisTyrSerIleValThrGlyLeuTyrProGluSerHis 260265270 GlyIleIleAspAsnLysMetTyrAspProLysMetAsnAlaSerPhe 275280285 SerLeuLysSerLysGluLysPheAsnProGluTrpTyrLysGlyGlu 290295300 ProIleTrpValThrAlaLysTyrGlnGlyLeuLysSerGlyThrPhe 305310315320 PheTrpProGlySerAspValGluIleAsnGlyIlePheProAspIle 325330335 TyrLysMetTyrAsnGlySerValProPheGluGluArgIleLeuAla 340345350 ValLeuGlnTrpLeuGlnLeuProLysAspGluArgProHisPheTyr 355360365 ThrLeuTyrLeuGluGluProAspSerSerGlyHisSerTyrGlyPro 370375380 ValSerSerGluValIleLysAlaLeuGlnArgValAspGlyMetVal 385390395400 GlyMetLeuMetAspGlyLeuLysGluLeuAsnLeuHisArgCysLeu 405410415 AsnLeuIleLeuIleSerAspHisGlyMetGluGlnGlySerCysLys 420425430 LysTyrIleTyrLeuAsnLysTyrLeuGlyAspValLysAsnIleLys 435440445 ValIleTyrGlyProAlaAlaArgLeuArgProSerAspValProAsp 450455460 LysTyrTyrSerPheAsnTyrGluGlyIleAlaArgAsnLeuSerCys 465470475480 ArgGluProAsnGlnHisPheLysProTyrLeuLysHisPheLeuPro 485490495 LysArgLeuHisPheAlaLysSerAspArgIleGluProLeuThrPhe 500505510 TyrLeuAspProGlnTrpGlnLeuAlaLeuAsnProSerGluArgLys 515520525 TyrCysGlySerGlyPheHisGlySerAspAsnValPheSerAsnMet 530535540 GlnAlaLeuPheValGlyTyrGlyProGlyPheLysHisGlyIleGlu 545550555560 AlaAspThrPheGluAsnIleGluValTyrAsnLeuMetCysAspLeu 565570575 LeuAsnLeuThrProAlaProAsnAsnGlyThrHisGlySerLeuAsn 580585590 HisLeuLeuLysAsnProValTyrThrProLysHisProLysGluVal 595600605 HisProLeuValGlnCysProPheThrArgAsnProArgAspAsnLeu 610615620 GlyCysSerCysAsnProSerIleLeuProIleGluAspPheGlnThr 625630635640 GlnPheAsnLeuThrValAlaGluGluLysIleIleLysHisGluThr 645650655 LeuProTyrGlyArgProArgValLeuGlnLysGluAsnThrIleCys 660665670 LeuLeuSerGlnHisGlnPheMetSerGlyTyrSerGlnAspIleLeu 675680685 MetProLeuTrpThrSerTyrThrValAspArgAsnAspSerPheSer 690695700 ThrGluAspPheSerAsnCysLeuTyrGlnAspPheArgIleProLeu 705710715720 SerProValHisLysCysSerPheTyrLysAsnAsnThrLysValSer 725730735 TyrGlyPheLeuSerProProGlnLeuAsnLysAsnSerSerGlyIle 740745750 TyrSerGluAlaLeuLeuThrThrAsnIleValProMetTyrGlnSer 755760765 PheGlnValIleTrpArgTyrPheHisAspThrLeuLeuArgLysTyr 770775780 AlaGluGluArgAsnGlyValAsnValValSerGlyProValPheAsp 785790795800 PheAspTyrAspGlyArgCysAspSerLeuGluAsnLeuArgGlnLys 805810815 ArgArgValIleArgAsnGlnGluIleLeuIleProThrHisPhePhe 820825830 IleValLeuThrSerCysLysAspThrSerGlnThrProLeuHisCys 835840845 GluAsnLeuAspThrLeuAlaPheIleLeuProHisArgThrAspAsn 850855860 SerGluSerCysValHisGlyLysHisAspSerSerTrpValGluGlu 865870875880 LeuLeuMetLeuHisArgAlaArgIleThrAspValGluHisIleThr 885890895 GlyLeuSerPheTyrGlnGlnArgLysGluProValSerAspIleLeu 900905910 LysLeuLysThrHisLeuProThrPheSerGlnGluAsp 915920925 Singlyunderlined:signalpeptidesequence;double-underlined: beginningandendofNPP1;**=cleavagepositionatthesignal peptidesequence SEQ.ID.NO:28-ENPP121-FcAminoAcidSequence MetGluArgAspGlyCysAlaGlyGlyGlySerArgGlyGlyGluGly 151015 GlyArgAlaProArgGluGlyProAlaGlyAsnGlyArgAspArgGly 202530 ArgSerHisAlaAlaGluAlaProGlyAspProGlnAlaAlaAlaSer 354045 LeuLeuAlaProMetAspValGlyGluGluProLeuGluLysAlaAla 505560 ArgAlaArgThrAlaLysAspProAsnThrTyrLysIleIleSerLeu 65707580 PheThrPheAlaValGlyValAsnIleCysLeuGly**PheThrAlaGly 859095 LeuLysProSerCysAlaLysGluValLysSerCysLysGlyArgCys 100105110 PheGluArgThrPheGlyAsnCysArgCysAspAlaAlaCysValGlu 115120125 LeuGlyAsnCysCysLeuAspTyrGlnGluThrCysIleGluProGlu 130135140 HisIleTrpThrCysAsnLysPheArgCysGlyGluLysArgLeuThr 145150155160 ArgSerLeuCysAlaCysSerAspAspCysLysAspLysGlyAspCys 165170175 CysIleAsnTyrSerSerValCysGlnGlyGluLysSerTrpValGlu 180185190 GluProCysGluSerIleAsnGluProGlnCysProAlaGlyPheGlu 195200205 ThrProProThrLeuLeuPheSerLeuAspGlyPheArgAlaGluTyr 210215220 LeuHisThrTrpGlyGlyLeuLeuProValIleSerLysLeuLysLys 225230235240 CysGlyThrTyrThrLysAsnMetArgProValTyrProThrLysThr 245250255 PheProAsnHisTyrSerIleValThrGlyLeuTyrProGluSerHis 260265270 GlyIleIleAspAsnLysMetTyrAspProLysMetAsnAlaSerPhe 275280285 SerLeuLysSerLysGluLysPheAsnProGluTrpTyrLysGlyGlu 290295300 ProIleTrpValThrAlaLysTyrGlnGlyLeuLysSerGlyThrPhe 305310315320 PheTrpProGlySerAspValGluIleAsnGlyIlePheProAspIle 325330335 TyrLysMetTyrAsnGlySerValProPheGluGluArgIleLeuAla 340345350 ValLeuGlnTrpLeuGlnLeuProLysAspGluArgProHisPheTyr 355360365 ThrLeuTyrLeuGluGluProAspSerSerGlyHisSerTyrGlyPro 370375380 ValSerSerGluValIleLysAlaLeuGlnArgValAspGlyMetVal 385390395400 GlyMetLeuMetAspGlyLeuLysGluLeuAsnLeuHisArgCysLeu 405410415 AsnLeuIleLeuIleSerAspHisGlyMetGluGlnGlySerCysLys 420425430 LysTyrIleTyrLeuAsnLysTyrLeuGlyAspValLysAsnIleLys 435440445 ValIleTyrGlyProAlaAlaArgLeuArgProSerAspValProAsp 450455460 LysTyrTyrSerPheAsnTyrGluGlyIleAlaArgAsnLeuSerCys 465470475480 ArgGluProAsnGlnHisPheLysProTyrLeuLysHisPheLeuPro 485490495 LysArgLeuHisPheAlaLysSerAspArgIleGluProLeuThrPhe 500505510 TyrLeuAspProGlnTrpGlnLeuAlaLeuAsnProSerGluArgLys 515520525 TyrCysGlySerGlyPheHisGlySerAspAsnValPheSerAsnMet 530535540 GlnAlaLeuPheValGlyTyrGlyProGlyPheLysHisGlyIleGlu 545550555560 AlaAspThrPheGluAsnIleGluValTyrAsnLeuMetCysAspLeu 565570575 LeuAsnLeuThrProAlaProAsnAsnGlyThrHisGlySerLeuAsn 580585590 HisLeuLeuLysAsnProValTyrThrProLysHisProLysGluVal 595600605 HisProLeuValGlnCysProPheThrArgAsnProArgAspAsnLeu 610615620 GlyCysSerCysAsnProSerIleLeuProIleGluAspPheGlnThr 625630635640 GlnPheAsnLeuThrValAlaGluGluLysIleIleLysHisGluThr 645650655 LeuProTyrGlyArgProArgValLeuGlnLysGluAsnThrIleCys 660665670 LeuLeuSerGlnHisGlnPheMetSerGlyTyrSerGlnAspIleLeu 675680685 MetProLeuTrpThrSerTyrThrValAspArgAsnAspSerPheSer 690695700 ThrGluAspPheSerAsnCysLeuTyrGlnAspPheArgIleProLeu 705710715720 SerProValHisLysCysSerPheTyrLysAsnAsnThrLysValSer 725730735 TyrGlyPheLeuSerProProGlnLeuAsnLysAsnSerSerGlyIle 740745750 TyrSerGluAlaLeuLeuThrThrAsnIleValProMetTyrGlnSer 755760765 PheGlnValIleTrpArgTyrPheHisAspThrLeuLeuArgLysTyr 770775780 AlaGluGluArgAsnGlyValAsnValValSerGlyProValPheAsp 785790795800 PheAspTyrAspGlyArgCysAspSerLeuGluAsnLeuArgGlnLys 805810815 ArgArgValIleArgAsnGlnGluIleLeuIleProThrHisPhePhe 820825830 IleValLeuThrSerCysLysAspThrSerGlnThrProLeuHisCys 835840845 GluAsnLeuAspThrLeuAlaPheIleLeuProHisArgThrAspAsn 850855860 SerGluSerCysValHisGlyLysHisAspSerSerTrpValGluGlu 865870875880 LeuLeuMetLeuHisArgAlaArgIleThrAspValGluHisIleThr 885890895 GlyLeuSerPheTyrGlnGlnArgLysGluProValSerAspIleLeu 900905910 LysLeuLysThrHisLeuProThrPheSerGlnGluAspLeuIleAsn 915920925 AspLysThrHisThrCysProProCysProAlaProGluLeuLeuGly 930935940 GlyProSerValPheLeuPheProProLysProLysAspThrLeuMet 945950955960 IleSerArgThrProGluValThrCysValValValAspValSerHis 965970975 GluAspProGluValLysPheAsnTrpTyrValAspGlyValGluVal 980985990 HisAsnAlaLysThrLysProArgGluGluGlnTyrAsnSerThrTyr 99510001005 ArgValValSerValLeuThrValLeuHisGlnAspTrpLeuAsn 101010151020 GlyLysGluTyrLysCysLysValSerAsnLysAlaLeuProAla 102510301035 ProIleGluLysThrIleSerLysAlaLysGlyGlnProArgGlu 104010451050 ProGlnValTyrThrLeuProProSerArgGluGluMetThrLys 105510601065 AsnGlnValSerLeuThrCysLeuValLysGlyPheTyrProSer 107010751080 AspIleAlaValGluTrpGluSerAsnGlyGlnProGluAsnAsn 108510901095 TyrLysThrThrProProValLeuAspSerAspGlySerPhePhe 110011051110 LeuTyrSerLysLeuThrValAspLysSerArgTrpGlnGlnGly 111511201125 AsnValPheSerCysSerValMetHisGluAlaLeuHisAsnHis 113011351140 TyrThrGlnLysSerLeuSerLeuSerProGlyLys 114511501155 Singlyunderlined:signalpeptidesequence;double-underlined: beginningandendofNPP1;**=cleavagepositionatthesignal peptidesequence;boldresiduesindicateFcsequence SEQ.IDNO:29-ENPP121-ALBAminoAcidSequence: MetGluArgAspGlyCysAlaGlyGlyGlySerArgGlyGlyGluGly 151015 GlyArgAlaProArgGluGlyProAlaGlyAsnGlyArgAspArgGly 202530 ArgSerHisAlaAlaGluAlaProGlyAspProGlnAlaAlaAlaSer 354045 LeuLeuAlaProMetAspValGlyGluGluProLeuGluLysAlaAla 505560 ArgAlaArgThrAlaLysAspProAsnThrTyrLysIleIleSerLeu 65707580 PheThrPheAlaValGlyValAsnIleCysLeuGly**PheThrAlaGly 859095 LeuLysProSerCysAlaLysGluValLysSerCysLysGlyArgCys 100105110 PheGluArgThrPheGlyAsnCysArgCysAspAlaAlaCysValGlu 115120125 LeuGlyAsnCysCysLeuAspTyrGlnGluThrCysIleGluProGlu 130135140 HisIleTrpThrCysAsnLysPheArgCysGlyGluLysArgLeuThr 145150155160 ArgSerLeuCysAlaCysSerAspAspCysLysAspLysGlyAspCys 165170175 CysIleAsnTyrSerSerValCysGlnGlyGluLysSerTrpValGlu 180185190 GluProCysGluSerIleAsnGluProGlnCysProAlaGlyPheGlu 195200205 ThrProProThrLeuLeuPheSerLeuAspGlyPheArgAlaGluTyr 210215220 LeuHisThrTrpGlyGlyLeuLeuProValIleSerLysLeuLysLys 225230235240 CysGlyThrTyrThrLysAsnMetArgProValTyrProThrLysThr 245250255 PheProAsnHisTyrSerIleValThrGlyLeuTyrProGluSerHis 260265270 GlyIleIleAspAsnLysMetTyrAspProLysMetAsnAlaSerPhe 275280285 SerLeuLysSerLysGluLysPheAsnProGluTrpTyrLysGlyGlu 290295300 ProIleTrpValThrAlaLysTyrGlnGlyLeuLysSerGlyThrPhe 305310315320 PheTrpProGlySerAspValGluIleAsnGlyIlePheProAspIle 325330335 TyrLysMetTyrAsnGlySerValProPheGluGluArgIleLeuAla 340345350 ValLeuGlnTrpLeuGlnLeuProLysAspGluArgProHisPheTyr 355360365 ThrLeuTyrLeuGluGluProAspSerSerGlyHisSerTyrGlyPro 370375380 ValSerSerGluValIleLysAlaLeuGlnArgValAspGlyMetVal 385390395400 GlyMetLeuMetAspGlyLeuLysGluLeuAsnLeuHisArgCysLeu 405410415 AsnLeuIleLeuIleSerAspHisGlyMetGluGlnGlySerCysLys 420425430 LysTyrIleTyrLeuAsnLysTyrLeuGlyAspValLysAsnIleLys 435440445 ValIleTyrGlyProAlaAlaArgLeuArgProSerAspValProAsp 450455460 LysTyrTyrSerPheAsnTyrGluGlyIleAlaArgAsnLeuSerCys 465470475480 ArgGluProAsnGlnHisPheLysProTyrLeuLysHisPheLeuPro 485490495 LysArgLeuHisPheAlaLysSerAspArgIleGluProLeuThrPhe 500505510 TyrLeuAspProGlnTrpGlnLeuAlaLeuAsnProSerGluArgLys 515520525 TyrCysGlySerGlyPheHisGlySerAspAsnValPheSerAsnMet 530535540 GlnAlaLeuPheValGlyTyrGlyProGlyPheLysHisGlyIleGlu 545550555560 AlaAspThrPheGluAsnIleGluValTyrAsnLeuMetCysAspLeu 565570575 LeuAsnLeuThrProAlaProAsnAsnGlyThrHisGlySerLeuAsn 580585590 HisLeuLeuLysAsnProValTyrThrProLysHisProLysGluVal 595600605 HisProLeuValGlnCysProPheThrArgAsnProArgAspAsnLeu 610615620 GlyCysSerCysAsnProSerIleLeuProIleGluAspPheGlnThr 625630635640 GlnPheAsnLeuThrValAlaGluGluLysIleIleLysHisGluThr 645650655 LeuProTyrGlyArgProArgValLeuGlnLysGluAsnThrIleCys 660665670 LeuLeuSerGlnHisGlnPheMetSerGlyTyrSerGlnAspIleLeu 675680685 MetProLeuTrpThrSerTyrThrValAspArgAsnAspSerPheSer 690695700 ThrGluAspPheSerAsnCysLeuTyrGlnAspPheArgIleProLeu 705710715720 SerProValHisLysCysSerPheTyrLysAsnAsnThrLysValSer 725730735 TyrGlyPheLeuSerProProGlnLeuAsnLysAsnSerSerGlyIle 740745750 TyrSerGluAlaLeuLeuThrThrAsnIleValProMetTyrGlnSer 755760765 PheGlnValIleTrpArgTyrPheHisAspThrLeuLeuArgLysTyr 770775780 AlaGluGluArgAsnGlyValAsnValValSerGlyProValPheAsp 785790795800 PheAspTyrAspGlyArgCysAspSerLeuGluAsnLeuArgGlnLys 805810815 ArgArgValIleArgAsnGlnGluIleLeuIleProThrHisPhePhe 820825830 IleValLeuThrSerCysLysAspThrSerGlnThrProLeuHisCys 835840845 GluAsnLeuAspThrLeuAlaPheIleLeuProHisArgThrAspAsn 850855860 SerGluSerCysValHisGlyLysHisAspSerSerTrpValGluGlu 865870875880 LeuLeuMetLeuHisArgAlaArgIleThrAspValGluHisIleThr 885890895 GlyLeuSerPheTyrGlnGlnArgLysGluProValSerAspIleLeu 900905910 LysLeuLysThrHisLeuProThrPheSerGlnGluAspArgSerGly 915920925 SerGlyGlySerMetLysTrpValThrPheLeuLeuLeuLeuPheVal 930935940 SerGlySerAlaPheSerArgGlyValPheArgArgGluAlaHisLys 945950955960 SerGluIleAlaHisArgTyrAsnAspLeuGlyGluGlnHisPheLys 965970975 GlyLeuValLeuIleAlaPheSerGlnTyrLeuGlnLysCysSerTyr 980985990 AspGluHisAlaLysLeuValGlnGluValThrAspPheAlaLysThr 99510001005 CysValAlaAspGluSerAlaAlaAsnCysAspLysSerLeuHis 101010151020 ThrLeuPheGlyAspLysLeuCysAlaIleProAsnLeuArgGlu 102510301035 AsnTyrGlyGluLeuAlaAspCysCysThrLysGlnGluProGlu 104010451050 ArgAsnGluCysPheLeuGlnHisLysAspAspAsnProSerLeu 105510601065 ProProPheGluArgProGluAlaGluAlaMetCysThrSerPhe 107010751080 LysGluAsnProThrThrPheMetGlyHisTyrLeuHisGluVal 108510901095 AlaArgArgHisProTyrPheTyrAlaProGluLeuLeuTyrTyr 110011051110 AlaGluGlnTyrAsnGluIleLeuThrGlnCysCysAlaGluAla 111511201125 AspLysGluSerCysLeuThrProLysLeuAspGlyValLysGlu 113011351140 LysAlaLeuValSerSerValArgGlnArgMetLysCysSerSer 114511501155 MetGlnLysPheGlyGluArgAlaPheLysAlaTrpAlaValAla 116011651170 ArgLeuSerGlnThrPheProAsnAlaAspPheAlaGluIleThr 117511801185 LysLeuAlaThrAspLeuThrLysValAsnLysGluCysCysHis 119011951200 GlyAspLeuLeuGluCysAlaAspAspArgAlaGluLeuAlaLys 120512101215 TyrMetCysGluAsnGlnAlaThrIleSerSerLysLeuGlnThr 122012251230 CysCysAspLysProLeuLeuLysLysAlaHisCysLeuSerGlu 123513401245 ValGluHisAspThrMetProAlaAspLeuProAlaIleAlaAla 125012551260 AspPheValGluAspGlnGluValCysLysAsnTyrAlaGluAla 126512701275 LysAspValPheLeuGlyThrPheLeuTyrGluTyrSerArgArg 128012851290 HisProAspTyrSerValSerLeuLeuLeuArgLeuAlaLysLys 129513001305 TyrGluAlaThrLeuGluLysCysCysAlaGluAlaAsnProPro 131013151320 AlaCysTyrGlyThrValLeuAlaGluPheGlnProLeuValGlu 132513301335 GluProLysAsnLeuValLysThrAsnCysAspLeuTyrGluLys 134013451350 LeuGlyGluTyrGlyPheGlnAsnAlaIleLeuValArgTyrThr 135513601365 GlnLysAlaProGlnValSerThrProThrLeuValGluAlaAla 137013751380 ArgAsnLeuGlyArgValGlyThrLysCysCysThrLeuProGlu 138513901395 AspGlnArgLeuProCysValGluAspTyrLeuSerAlaIleLeu 140014051410 AsnArgValCysLeuLeuHisGluLysThrProValSerGluHis 141514201425 ValThrLysCysCysSerGlySerLeuValGluArgArgProCys 143014351440 PheSerAlaLeuThrValAspGluThrTyrValProLysGluPhe 144514501455 LysAlaGluThrPheThrPheHisSerAspIleCysThrLeuPro 146014651470 GluLysGluLysGlnIleLysLysGlnThrAlaLeuAlaGluLeu 147514801485 ValLysHisLysProLysAlaThrAlaGluGlnLeuLysThrVal 149014951500 MetAspAspPheAlaGlnPheLeuAspThrCysCysLysAlaAla 150515101515 AspLysAspThrCysPheSerThrGluGlyProAsnLeuValThr 152015251530 ArgCysLysAspAlaLeuAlaArgSerTrpSerHisProGlnPhe 153515401545 GluLys 1550 Singlyunderlined:signalpeptidesequence;double-underlined: beginningandendofNPP1;**=cleavagepositionatthesignal peptidesequence;boldresiduesindicatealbuminsequence SEQ.IDNO:30-ENPP121-NPP3-Fcsequence MetGluArgAspGlyCysAlaGlyGlyGlySerArgGlyGlyGluGly 151015 GlyArgAlaProArgGluGlyProAlaGlyAsnGlyArgAspArgGly 202530 ArgSerHisAlaAlaGluAlaProGlyAspProGlnAlaAlaAlaSer 354045 LeuLeuAlaProMetAspValGlyGluGluProLeuGluLysAlaAla 505560 ArgAlaArgThrAlaLysAspProAsnThrTyrLysIleIleSerLeu 65707580 PheThrPheAlaValGlyValAsnIleCysLeuGlyPheThrAla**Lys 859095 GlnGlySerCysArgLysLysCysPheAspAlaSerPheArgGlyLeu 100105110 GluAsnCysArgCysAspValAlaCysLysAspArgGlyAspCysCys 115120125 TrpAspPheGluAspThrCysValGluSerThrArgIleTrpMetCys 130135140 AsnLysPheArgCysGlyGluArgLeuGluAlaSerLeuCysSerCys 145150155160 SerAspAspCysLeuGlnArgLysAspCysCysAlaAspTyrLysSer 165170175 ValCysGlnGlyGluThrSerTrpLeuGluGluAsnCysAspThrAla 180185190 GlnGlnSerGlnCysProGluGlyPheAspLeuProProValIleLeu 195200205 PheSerMetAspGlyPheArgAlaGluTyrLeuTyrThrTrpAspThr 210215220 LeuMetProAsnIleAsnLysLeuLysThrCysGlyIleHisSerLys 225230235240 TyrMetArgAlaMetTyrProThrLysThrPheProAsnHisTyrThr 245250255 IleValThrGlyLeuTyrProGluSerHisGlyIleIleAspAsnAsn 260265270 MetTyrAspValAsnLeuAsnLysAsnPheSerLeuSerSerLysGlu 275280285 GlnAsnAsnProAlaTrpTrpHisGlyGlnProMetTrpLeuThrAla 290295300 MetTyrGlnGlyLeuLysAlaAlaThrTyrPheTrpProGlySerGlu 305310315320 ValAlaIleAsnGlySerPheProSerIleTyrMetProTyrAsnGly 325330335 SerValProPheGluGluArgIleSerThrLeuLeuLysTrpLeuAsp 340345350 LeuProLysAlaGluArgProArgPheTyrThrMetTyrPheGluGlu 355360365 ProAspSerSerGlyHisAlaGlyGlyProValSerAlaArgValIle 370375380 LysAlaLeuGlnValValAspHisAlaPheGlyMetLeuMetGluGly 385390395400 LeuLysGlnArgAsnLeuHisAsnCysValAsnIleIleLeuLeuAla 405410415 AspHisGlyMetAspGlnThrTyrCysAsnLysMetGluTyrMetThr 420425430 AspTyrPheProArgIleAsnPhePheTyrMetTyrGluGlyProAla 435440445 ProArgIleArgAlaHisAsnIleProHisAspPhePheSerPheAsn 450455460 SerGluGluIleValArgAsnLeuSerCysArgLysProAspGlnHis 465470475480 PheLysProTyrLeuThrProAspLeuProLysArgLeuHisTyrAla 485490495 LysAsnValArgIleAspLysValHisLeuPheValAspGlnGlnTrp 500505510 LeuAlaValArgSerLysSerAsnThrAsnCysGlyGlyGlyAsnHis 515520525 GlyTyrAsnAsnGluPheArgSerMetGluAlaIlePheLeuAlaHis 530535540 GlyProSerPheLysGluLysThrGluValGluProPheGluAsnIle 545550555560 GluValTyrAsnLeuMetCysAspLeuLeuArgIleGlnProAlaPro 565570575 AsnAsnGlyThrHisGlySerLeuAsnHisLeuLeuLysValProPhe 580585590 TyrGluProSerHisAlaGluGluValSerLysPheSerValCysGly 595600605 PheAlaAsnProLeuProThrGluSerLeuAspCysPheCysProHis 610615620 LeuGlnAsnSerThrGlnLeuGluGlnValAsnGlnMetLeuAsnLeu 625630635640 ThrGlnGluGluIleThrAlaThrValLysValAsnLeuProPheGly 645650655 ArgProArgValLeuGlnLysAsnValAspHisCysLeuLeuTyrHis 660665670 ArgGluTyrValSerGlyPheGlyLysAlaMetArgMetProMetTrp 675680685 SerSerTyrThrValProGlnLeuGlyAspThrSerProLeuProPro 690695700 ThrValProAspCysLeuArgAlaAspValArgValProProSerGlu 705710715720 SerGlnLysCysSerPheTyrLeuAlaAspLysAsnIleThrHisGly 725730735 PheLeuTyrProProAlaSerAsnArgThrSerAspSerGlnTyrAsp 740745750 AlaLeuIleThrSerAsnLeuValProMetTyrGluGluPheArgLys 755760765 MetTrpAspTyrPheHisSerValLeuLeuIleLysHisAlaThrGlu 770775780 ArgAsnGlyValAsnValValSerGlyProIlePheAspTyrAsnTyr 785790795800 AspGlyHisPheAspAlaProAspGluIleThrLysHisLeuAlaAsn 805810815 ThrAspValProIleProThrHisTyrPheValValLeuThrSerCys 820825830 LysAsnLysSerHisThrProGluAsnCysProGlyTrpLeuAspVal 835840845 LeuProPheIleIleProHisArgProThrAsnValGluSerCysPro 850855860 GluGlyLysProGluAlaLeuTrpValGluGluArgPheThrAlaHis 865870875880 IleAlaArgValArgAspValGluLeuLeuThrGlyLeuAspPheTyr 885890895 GlnAspLysValGlnProValSerGluIleLeuGlnLeuLysThrTyr 900905910 LeuProThrPheGluThrThrIleAspLysThrHisThrCysProPro 915920925 CysProAlaProGluLeuLeuGlyGlyProSerValPheLeuPhePro 930935940 ProLysProLysAspThrLeuMetIleSerArgThrProGluValThr 945950955960 CysValValValAspValSerHisGluAspProGluValLysPheAsn 965970975 TrpTyrValAspGlyValGluValHisAsnAlaLysThrLysProArg 980985990 GluGluGlnTyrAsnSerThrTyrArgValValSerValLeuThrVal 99510001005 LeuHisGlnAspTrpLeuAsnGlyLysGluTyrLysCysLysVal 101010151020 SerAsnLysAlaLeuProAlaProIleGluLysThrIleSerLys 102510301035 AlaLysGlyGlnProArgGluProGlnValTyrThrLeuProPro 104010451050 SerArgGluGluMetThrLysAsnGlnValSerLeuThrCysLeu 105510601065 ValLysGlyPheTyrProSerAspIleAlaValGluTrpGluSer 107010751080 AsnGlyGlnProGluAsnAsnTyrLysThrThrProProValLeu 108510901095 AspSerAspGlySerPhePheLeuTyrSerLysLeuThrValAsp 110011051110 LysSerArgTrpGlnGlnGlyAsnValPheSerCysSerValMet 111511201125 HisGluAlaLeuHisAsnHisTyrThrGlnLysSerLeuSerLeu 113011351140 SerProGlyLys 1145 Singlyunderlined:signalpeptidesequence;double-underlined: beginningandendofNPP1;**=cleavagepositionatthesignal peptidesequence;boldresiduesindicateFcsequence SEQ.IDNO:31-ENPP121-NPP3-Albuminsequence MetGluArgAspGlyCysAlaGlyGlyGlySerArgGlyGlyGluGly 151015 GlyArgAlaProArgGluGlyProAlaGlyAsnGlyArgAspArgGly 202530 ArgSerHisAlaAlaGluAlaProGlyAspProGlnAlaAlaAlaSer 354045 LeuLeuAlaProMetAspValGlyGluGluProLeuGluLysAlaAla 505560 ArgAlaArgThrAlaLysAspProAsnThrTyrLysIleIleSerLeu 65707580 PheThrPheAlaValGlyValAsnIleCysLeuGlyPheThrAla**Lys 859095 GlnGlySerCysArgLysLysCysPheAspAlaSerPheArgGlyLeu 100105110 GluAsnCysArgCysAspValAlaCysLysAspArgGlyAspCysCys 115120125 TrpAspPheGluAspThrCysValGluSerThrArgIleTrpMetCys 130135140 AsnLysPheArgCysGlyGluArgLeuGluAlaSerLeuCysSerCys 145150155160 SerAspAspCysLeuGlnArgLysAspCysCysAlaAspTyrLysSer 165170175 ValCysGlnGlyGluThrSerTrpLeuGluGluAsnCysAspThrAla 180185190 GlnGlnSerGlnCysProGluGlyPheAspLeuProProValIleLeu 195200205 PheSerMetAspGlyPheArgAlaGluTyrLeuTyrThrTrpAspThr 210215220 LeuMetProAsnIleAsnLysLeuLysThrCysGlyIleHisSerLys 225230235240 TyrMetArgAlaMetTyrProThrLysThrPheProAsnHisTyrThr 245250255 IleValThrGlyLeuTyrProGluSerHisGlyIleIleAspAsnAsn 260265270 MetTyrAspValAsnLeuAsnLysAsnPheSerLeuSerSerLysGlu 275280285 GlnAsnAsnProAlaTrpTrpHisGlyGlnProMetTrpLeuThrAla 290295300 MetTyrGlnGlyLeuLysAlaAlaThrTyrPheTrpProGlySerGlu 305310315320 ValAlaIleAsnGlySerPheProSerIleTyrMetProTyrAsnGly 325330335 SerValProPheGluGluArgIleSerThrLeuLeuLysTrpLeuAsp 340345350 LeuProLysAlaGluArgProArgPheTyrThrMetTyrPheGluGlu 355360365 ProAspSerSerGlyHisAlaGlyGlyProValSerAlaArgValIle 370375380 LysAlaLeuGlnValValAspHisAlaPheGlyMetLeuMetGluGly 385390395400 LeuLysGlnArgAsnLeuHisAsnCysValAsnIleIleLeuLeuAla 405410415 AspHisGlyMetAspGlnThrTyrCysAsnLysMetGluTyrMetThr 420425430 AspTyrPheProArgIleAsnPhePheTyrMetTyrGluGlyProAla 435440445 ProArgIleArgAlaHisAsnIleProHisAspPhePheSerPheAsn 450455460 SerGluGluIleValArgAsnLeuSerCysArgLysProAspGlnHis 465470475480 PheLysProTyrLeuThrProAspLeuProLysArgLeuHisTyrAla 485490495 LysAsnValArgIleAspLysValHisLeuPheValAspGlnGlnTrp 500505510 LeuAlaValArgSerLysSerAsnThrAsnCysGlyGlyGlyAsnHis 515520525 GlyTyrAsnAsnGluPheArgSerMetGluAlaIlePheLeuAlaHis 530535540 GlyProSerPheLysGluLysThrGluValGluProPheGluAsnIle 545550555560 GluValTyrAsnLeuMetCysAspLeuLeuArgIleGlnProAlaPro 565570575 AsnAsnGlyThrHisGlySerLeuAsnHisLeuLeuLysValProPhe 580585590 TyrGluProSerHisAlaGluGluValSerLysPheSerValCysGly 595600605 PheAlaAsnProLeuProThrGluSerLeuAspCysPheCysProHis 610615620 LeuGlnAsnSerThrGlnLeuGluGlnValAsnGlnMetLeuAsnLeu 625630635640 ThrGlnGluGluIleThrAlaThrValLysValAsnLeuProPheGly 645650655 ArgProArgValLeuGlnLysAsnValAspHisCysLeuLeuTyrHis 660665670 ArgGluTyrValSerGlyPheGlyLysAlaMetArgMetProMetTrp 675680685 SerSerTyrThrValProGlnLeuGlyAspThrSerProLeuProPro 690695700 ThrValProAspCysLeuArgAlaAspValArgValProProSerGlu 705710715720 SerGlnLysCysSerPheTyrLeuAlaAspLysAsnIleThrHisGly 725730735 PheLeuTyrProProAlaSerAsnArgThrSerAspSerGlnTyrAsp 740745750 AlaLeuIleThrSerAsnLeuValProMetTyrGluGluPheArgLys 755760765 MetTrpAspTyrPheHisSerValLeuLeuIleLysHisAlaThrGlu 770775780 ArgAsnGlyValAsnValValSerGlyProIlePheAspTyrAsnTyr 785790795800 AspGlyHisPheAspAlaProAspGluIleThrLysHisLeuAlaAsn 805810815 ThrAspValProIleProThrHisTyrPheValValLeuThrSerCys 820825830 LysAsnLysSerHisThrProGluAsnCysProGlyTrpLeuAspVal 835840845 LeuProPheIleIleProHisArgProThrAsnValGluSerCysPro 850855860 GluGlyLysProGluAlaLeuTrpValGluGluArgPheThrAlaHis 865870875880 IleAlaArgValArgAspValGluLeuLeuThrGlyLeuAspPheTyr 885890895 GlnAspLysValGlnProValSerGluIleLeuGlnLeuLysThrTyr 900905910 LeuProThrPheGluThrThrIleGlyGlyGlySerGlyGlyGlyGly 915920925 SerGlyGlyGlyGlySerMetLysTrpValThrPheLeuLeuLeuLeu 930935940 PheValSerGlySerAlaPheSerArgGlyValPheArgArgGluAla 945950955960 HisLysSerGluIleAlaHisArgTyrAsnAspLeuGlyGluGlnHis 965970975 PheLysGlyLeuValLeuIleAlaPheSerGlnTyrLeuGlnLysCys 980985990 SerTyrAspGluHisAlaLysLeuValGlnGluValThrAspPheAla 99510001005 LysThrCysValAlaAspGluSerAlaAlaAsnCysAspLysSer 101010151020 LeuHisThrLeuPheGlyAspLysLeuCysAlaIleProAsnLeu 102510301035 ArgGluAsnTyrGlyGluLeuAlaAspCysCysThrLysGlnGlu 104010451050 ProGluArgAsnGluCysPheLeuGlnHisLysAspAspAsnPro 105510601065 SerLeuProProPheGluArgProGluAlaGluAlaMetCysThr 107010751080 SerPheLysGluAsnProThrThrPheMetGlyHisTyrLeuHis 108510901095 GluValAlaArgArgHisProTyrPheTyrAlaProGluLeuLeu 110011051110 TyrTyrAlaGluGlnTyrAsnGluIleLeuThrGlnCysCysAla 111511201125 GluAlaAspLysGluSerCysLeuThrProLysLeuAspGlyVal 113011351140 LysGluLysAlaLeuValSerSerValArgGlnArgMetLysCys 114511501155 SerSerMetGlnLysPheGlyGluArgAlaPheLysAlaTrpAla 116011651170 ValAlaArgLeuSerGlnThrPheProAsnAlaAspPheAlaGlu 117511801185 IleThrLysLeuAlaThrAspLeuThrLysValAsnLysGluCys 119011951200 CysHisGlyAspLeuLeuGluCysAlaAspAspArgAlaGluLeu 120512101215 AlaLysTyrMetCysGluAsnGlnAlaThrIleSerSerLysLeu 122012251230 GlnThrCysCysAspLysProLeuLeuLysLysAlaHisCysLeu 123513401245 SerGluValGluHisAspThrMetProAlaAspLeuProAlaIle 125012551260 AlaAlaAspPheValGluAspGlnGluValCysLysAsnTyrAla 126512701275 GluAlaLysAspValPheLeuGlyThrPheLeuTyrGluTyrSer 128012851290 ArgArgHisProAspTyrSerValSerLeuLeuLeuArgLeuAla 129513001305 LysLysTyrGluAlaThrLeuGluLysCysCysAlaGluAlaAsn 131013151320 ProProAlaCysTyrGlyThrValLeuAlaGluPheGlnProLeu 132513301335 ValGluGluProLysAsnLeuValLysThrAsnCysAspLeuTyr 134013451350 GluLysLeuGlyGluTyrGlyPheGlnAsnAlaIleLeuValArg 135513601365 TyrThrGlnLysAlaProGlnValSerThrProThrLeuValGlu 137013751380 AlaAlaArgAsnLeuGlyArgValGlyThrLysCysCysThrLeu 138513901395 ProGluAspGlnArgLeuProCysValGluAspTyrLeuSerAla 140014051410 IleLeuAsnArgValCysLeuLeuHisGluLysThrProValSer 141514201425 GluHisValThrLysCysCysSerGlySerLeuValGluArgArg 143014351440 ProCysPheSerAlaLeuThrValAspGluThrTyrValProLys 144514501455 GluPheLysAlaGluThrPheThrPheHisSerAspIleCysThr 146014651470 LeuProGluLysGluLysGlnIleLysLysGlnThrAlaLeuAla 147514801485 GluLeuValLysHisLysProLysAlaThrAlaGluGlnLeuLys 149014951500 ThrValMetAspAspPheAlaGlnPheLeuAspThrCysCysLys 150515101515 AlaAlaAspLysAspThrCysPheSerThrGluGlyProAsnLeu 152015251530 ValThrArgCysLysAspAlaLeuAla 15351540 Singlyunderlined:signalpeptidesequence;double-underlined: beginningandendofNPP3;**=cleavagepositionatthesignal peptidesequence;boldresiduesindicatealbuminsequence SEQ.IDNO:32-ENPP121GLKProteinExportSignalSequence MetGluArgAspGlyCysAlaGlyGlyGlySerArgGlyGlyGluGly 151015 GlyArgAlaProArgGluGlyProAlaGlyAsnGlyArgAspArgGly 202530 ArgSerHisAlaAlaGluAlaProGlyAspProGlnAlaAlaAlaSer 354045 LeuLeuAlaProMetAspValGlyGluGluProLeuGluLysAlaAla 505560 ArgAlaArgThrAlaLysAspProAsnThrTyrLysIleIleSerLeu 65707580 PheThrPheAlaValGlyValAsnIleCysLeuGlyPheThrAlaGly 859095 LeuLys SEQ.IDNO:33-AlbuminSequence GlyGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySerMet 151015 LysTrpValThrPheLeuLeuLeuLeuPheValSerGlySerAlaPhe 202530 SerArgGlyValPheArgArgGluAlaHisLysSerGluIleAlaHis 354045 ArgTyrAsnAspLeuGlyGluGlnHisPheLysGlyLeuValLeuIle 505560 AlaPheSerGlnTyrLeuGlnLysCysSerTyrAspGluHisAlaLys 65707580 LeuValGlnGluValThrAspPheAlaLysThrCysValAlaAspGlu 859095 SerAlaAlaAsnCysAspLysSerLeuHisThrLeuPheGlyAspLys 100105110 LeuCysAlaIleProAsnLeuArgGluAsnTyrGlyGluLeuAlaAsp 115120125 CysCysThrLysGlnGluProGluArgAsnGluCysPheLeuGlnHis 130135140 LysAspAspAsnProSerLeuProProPheGluArgProGluAlaGlu 145150155160 AlaMetCysThrSerPheLysGluAsnProThrThrPheMetGlyHis 165170175 TyrLeuHisGluValAlaArgArgHisProTyrPheTyrAlaProGlu 180185190 LeuLeuTyrTyrAlaGluGlnTyrAsnGluIleLeuThrGlnCysCys 195200205 AlaGluAlaAspLysGluSerCysLeuThrProLysLeuAspGlyVal 210215220 LysGluLysAlaLeuValSerSerValArgGlnArgMetLysCysSer 225230235240 SerMetGlnLysPheGlyGluArgAlaPheLysAlaTrpAlaValAla 245250255 ArgLeuSerGlnThrPheProAsnAlaAspPheAlaGluIleThrLys 260265270 LeuAlaThrAspLeuThrLysValAsnLysGluCysCysHisGlyAsp 275280285 LeuLeuGluCysAlaAspAspArgAlaGluLeuAlaLysTyrMetCys 290295300 GluAsnGlnAlaThrIleSerSerLysLeuGlnThrCysCysAspLys 305310315320 ProLeuLeuLysLysAlaHisCysLeuSerGluValGluHisAspThr 325330335 MetProAlaAspLeuProAlaIleAlaAlaAspPheValGluAspGln 340345350 GluValCysLysAsnTyrAlaGluAlaLysAspValPheLeuGlyThr 355360365 PheLeuTyrGluTyrSerArgArgHisProAspTyrSerValSerLeu 370375380 LeuLeuArgLeuAlaLysLysTyrGluAlaThrLeuGluLysCysCys 385390395400 AlaGluAlaAsnProProAlaCysTyrGlyThrValLeuAlaGluPhe 405410415 GlnProLeuValGluGluProLysAsnLeuValLysThrAsnCysAsp 420425430 LeuTyrGluLysLeuGlyGluTyrGlyPheGlnAsnAlaIleLeuVal 435440445 ArgTyrThrGlnLysAlaProGlnValSerThrProThrLeuValGlu 450455460 AlaAlaArgAsnLeuGlyArgValGlyThrLysCysCysThrLeuPro 465470475480 GluAspGlnArgLeuProCysValGluAspTyrLeuSerAlaIleLeu 485490495 AsnArgValCysLeuLeuHisGluLysThrProValSerGluHisVal 500505510 ThrLysCysCysSerGlySerLeuValGluArgArgProCysPheSer 515520525 AlaLeuThrValAspGluThrTyrValProLysGluPheLysAlaGlu 530535540 ThrPheThrPheHisSerAspIleCysThrLeuProGluLysGluLys 545550555560 GlnIleLysLysGlnThrAlaLeuAlaGluLeuValLysHisLysPro 565570575 LysAlaThrAlaGluGlnLeuLysThrValMetAspAspPheAlaGln 580585590 PheLeuAspThrCysCysLysAlaAlaAspLysAspThrCysPheSer 595600605 ThrGluGlyProAsnLeuValThrArgCysLysAspAlaLeuAla 610615620 SEQ.IDNO:34-HumanIgGFcdomain,Fc AspLysThrHisThrCysProProCysProAlaProGluLeuLeuGly 151015 GlyProSerValPheLeuPheProProLysProLysAspThrLeuMet 202530 IleSerArgThrProGluValThrCysValValValAspValSerHis 354045 GluAspProGluValLysPheAsnTrpTyrValAspGlyValGluVal 505560 HisAsnAlaLysThrLysProArgGluGluGlnTyrAsnSerThrTyr 65707580 ArgValValSerValLeuThrValLeuHisGlnAspTrpLeuAsnGly 859095 LysGluTyrLysCysLysValSerAsnLysAlaLeuProAlaProIle 100105110 GluLysThrIleSerLysAlaLysGlyGlnProArgGluProGlnVal 115120125 TyrThrLeuProProSerArgGluGluMetThrLysAsnGlnValSer 130135140 LeuThrCysLeuValLysGlyPheTyrProSerAspIleAlaValGlu 145150155160 TrpGluSerAsnGlyGlnProGluAsnAsnTyrLysThrThrProPro 165170175 ValLeuAspSerAspGlySerPhePheLeuTyrSerLysLeuThrVal 180185190 AspLysSerArgTrpGlnGlnGlyAsnValPheSerCysSerValMet 195200205 HisGluAlaLeuHisAsnHisTyrThrGlnLysSerLeuSerLeuSer 210215220 ProGlyLys 225 SEQ.IDNO:35-AlbuminSequence MetLysTrpValThrPheLeuLeuLeuLeuPheValSerGlySerAla 151015 PheSerArgGlyValPheArgArgGluAlaHisLysSerGluIleAla 202530 HisArgTyrAsnAspLeuGlyGluGlnHisPheLysGlyLeuValLeu 354045 IleAlaPheSerGlnTyrLeuGlnLysCysSerTyrAspGluHisAla 505560 LysLeuValGlnGluValThrAspPheAlaLysThrCysValAlaAsp 65707580 GluSerAlaAlaAsnCysAspLysSerLeuHisThrLeuPheGlyAsp 859095 LysLeuCysAlaIleProAsnLeuArgGluAsnTyrGlyGluLeuAla 100105110 AspCysCysThrLysGlnGluProGluArgAsnGluCysPheLeuGln 115120125 HisLysAspAspAsnProSerLeuProProPheGluArgProGluAla 130135140 GluAlaMetCysThrSerPheLysGluAsnProThrThrPheMetGly 145150155160 HisTyrLeuHisGluValAlaArgArgHisProTyrPheTyrAlaPro 165170175 GluLeuLeuTyrTyrAlaGluGlnTyrAsnGluIleLeuThrGlnCys 180185190 CysAlaGluAlaAspLysGluSerCysLeuThrProLysLeuAspGly 195200205 ValLysGluLysAlaLeuValSerSerValArgGlnArgMetLysCys 210215220 SerSerMetGlnLysPheGlyGluArgAlaPheLysAlaTrpAlaVal 225230235240 AlaArgLeuSerGlnThrPheProAsnAlaAspPheAlaGluIleThr 245250255 LysLeuAlaThrAspLeuThrLysValAsnLysGluCysCysHisGly 260265270 AspLeuLeuGluCysAlaAspAspArgAlaGluLeuAlaLysTyrMet 275280285 CysGluAsnGlnAlaThrIleSerSerLysLeuGlnThrCysCysAsp 290295300 LysProLeuLeuLysLysAlaHisCysLeuSerGluValGluHisAsp 305310315320 ThrMetProAlaAspLeuProAlaIleAlaAlaAspPheValGluAsp 325330335 GlnGluValCysLysAsnTyrAlaGluAlaLysAspValPheLeuGly 340345350 ThrPheLeuTyrGluTyrSerArgArgHisProAspTyrSerValSer 355360365 LeuLeuLeuArgLeuAlaLysLysTyrGluAlaThrLeuGluLysCys 370375380 CysAlaGluAlaAsnProProAlaCysTyrGlyThrValLeuAlaGlu 385390395400 PheGlnProLeuValGluGluProLysAsnLeuValLysThrAsnCys 405410415 AspLeuTyrGluLysLeuGlyGluTyrGlyPheGlnAsnAlaIleLeu 420425430 ValArgTyrThrGlnLysAlaProGlnValSerThrProThrLeuVal 435440445 GluAlaAlaArgAsnLeuGlyArgValGlyThrLysCysCysThrLeu 450455460 ProGluAspGlnArgLeuProCysValGluAspTyrLeuSerAlaIle 465470475480 LeuAsnArgValCysLeuLeuHisGluLysThrProValSerGluHis 485490495 ValThrLysCysCysSerGlySerLeuValGluArgArgProCysPhe 500505510 SerAlaLeuThrValAspGluThrTyrValProLysGluPheLysAla 515520525 GluThrPheThrPheHisSerAspIleCysThrLeuProGluLysGlu 530535540 LysGlnIleLysLysGlnThrAlaLeuAlaGluLeuValLysHisLys 545550555560 ProLysAlaThrAlaGluGlnLeuLysThrValMetAspAspPheAla 565570575 GlnPheLeuAspThrCysCysLysAlaAlaAspLysAspThrCysPhe 580585590 SerThrGluGlyProAsnLeuValThrArgCysLysAspAlaLeuAla 595600605 ArgSerTrpSerHisProGlnPheGluLys 610615 SEQ.IDNO:36-ENPP2SignalPeptide LeuPheThrPheAlaValGlyValAsnIleCysLeuGly 151015 PheThrAla SEQ.IDNO:37-SignalSequenceENPP7 MetArgGlyProAlaValLeuLeuThrValAlaLeuAlaThrLeuLeu 151015 AlaProGlyAla 20 SEQ.IDNO:38-SignalsequenceENPP7 MetArgGlyProAlaValLeuLeuThrValAlaLeuAlaThrLeuLeu 151015 AlaProGlyAlaGlyAla 20 SEQ.IDNO:39-SignalSequenceENPP1-2-1 MetGluArgAspGlyCysAlaGlyGlyGlySerArgGlyGlyGluGly 151015 GlyArgAlaProArgGluGlyProAlaGlyAsnGlyArgAspArgGly 202530 ArgSerHisAlaAlaGluAlaProGlyAspProGlnAlaAlaAlaSer 354045 LeuLeuAlaProMetAspValGlyGluGluProLeuGluLysAlaAla 505560 ArgAlaArgThrAlaLysAspProAsnThrTyrLysIleIleSerLeu 65707580 PheThrPheAlaValGlyValAsnIleCysLeuGlyPheThrAla 859095 SEQ.IDNO:40-exENPP3 LeuLeuValIleMetSerLeuGlyLeuGlyLeuGlyLeuGlyLeuArg 151015 Lys SEQ.IDNO:41-SignalSequenceENPP5: MetThrSerLysPheLeuLeuValSerPheIleLeuAlaAlaLeuSer 151015 LeuSerThrThrPheSer 20 SEQIDNO:42-SignalSequence-Azurocidin MetThrArgLeuThrValLeuAlaLeuLeuAlaGlyLeuLeuAlaSer SerArgAla SEQ.IDNO:43-Linker AspSerSer SEQ.IDNO:44-Linker GluSerSer SEQ.IDNO:45-Linker ArgGlnGln SEQ.IDNO:46-Linker LysArg SEQ.IDNO:47-Linker (Arg).sub.m;m=015 SEQ.IDNO:48-Linker AspSerSerSerGluGluLysPheLeuArgArgIleGlyArgPheGly SEQ.IDNO:49-Linker GluGluGluGluGluGluGluProArgGlyAspThr 1510 SEQ.IDNO:50-Linker AlaProTrpHisLeuSerSerGlnTyrSerArgThr 1510 SEQ.IDNO:51-Linker SerThrLeuProIleProHisGluPheSerArgGlu 1510 SEQ.IDNO:52-Linker ValThrLysHisLeuAsnGlnIleSerGlnSerTyr 1510 SEQ.IDNO:53-Linker (Glu).sub.m;m=115 SEQ.IDNO:54-Linker LeuIleAsn SEQ.IDNO:55-Linker GlyGlySerGlyGlySer 15 SEQ.IDNO:56-Linker ArgSerGlySerGlyGlySer 15 SEQ.IDNO:57-Linker (Asp).sub.m;m=115 1 SEQ.IDNO:58-Linker LeuValIleMetSerLeuGlyLeuGlyLeuGlyLeuGlyLeuArgLys 151015 SEQ.IDNO:59-Linker ValIleMetSerLeuGlyLeuGlyLeuGlyLeuGlyLeuArgLys 151015 SEQ.IDNO:60-Linker IleMetSerLeuGlyLeuGlyLeuGlyLeuGlyLeuArgLys 1510 SEQ.IDNO:61-Linker MetSerLeuGlyLeuGlyLeuGlyLeuGlyLeuArgLys 1510 SEQ.IDNO:62-Linker SerLeuGlyLeuGlyLeuGlyLeuGlyLeuArgLys 1510 SEQ.IDNO:63-Linker LeuGlyLeuGlyLeuGlyLeuGlyLeuArgLys 1510 SEQ.IDNO:64-Linker GlyLeuGlyLeuGlyLeuGlyLeuArgLys 1510 SEQ.IDNO:65-Linker LeuGlyLeuGlyLeuGlyLeuArgLys 15 SEQ.IDNO:66-Linker GlyLeuGlyLeuGlyLeuArgLys 15 SEQ.IDNO:67-Linker LeuGlyLeuGlyLeuArgLys 15 SEQ.IDNO:68-Linker GlyLeuGlyLeuArgLys 15 SEQ.IDNO:69-Linker LeuGlyLeuArgLys 15 SEQ.IDNO:70-Linker GlyLeuArgLys 1 SEQ.IDNO:71-Linker LeuArgLys 1 SEQ.IDNO:72-Linker ArgLys 1 SEQ.IDNO:73-Linker (Lys).sub.m;m=015 1 SEQ.IDNO:74-Linker D.sub.m;m=115 SEQ.IDNO:75-Linker (GGGGS).sub.n;n=110 SEQ.IDNO:76-ENPP3Nucleotidesequence atggaatctacgttgactttagcaacggaacaacctgttaagaagaacactcttaagaaa 60 tataaaatagcttgcattgttcttcttgctttgctggtgatcatgtcacttggattaggc 120 ctggggcttggactcaggaaactggaaaagcaaggcagctgcaggaagaagtgctttgat 180 gcatcatttagaggactggagaactgccggtgtgatgtggcatgtaaagaccgaggtgat 240 tgctgctgggattttgaagacacctgtgtggaatcaactcgaatatggatgtgcaataaa 300 tttcgttgtggagagaccagattagaggccagcctttgctcttgttcagatgactgtttg 360 cagaggaaagattgctgtgctgactataagagtgtttgccaaggagaaacctcatggctg 420 gaagaaaactgtgacacagcccagcagtctcagtgcccagaagggtttgacctgccacca 480 gttatcttgttttctatggatggatttagagctgaatatttatacacatgggatacttta 540 atgccaaatatcaataaactgaaaacatgtggaattcattcaaaatacatgagagctatg 600 tatcctaccaaaaccttcccaaatcattacaccattgtcacgggcttgtatccagagtca 660 catggcatcattgacaataatatgtatgatgtaaatctcaacaagaatttttcactttct 720 tcaaaggaacaaaataatccagcctggtggcatgggcaaccaatgtggctgacagcaatg 780 tatcaaggtttaaaagccgctacctacttttggcccggatcagaagtggctataaatggc 840 tcctttccttccatatacatgccttacaacggaagtgtcccatttgaagagaggatttct 900 acactgttaaaatggctggacctgcccaaagctgaaagacccaggttttataccatgtat 960 tttgaagaacctgattcctctggacatgcaggtggaccagtcagtgccagagtaattaaa 1020 gccttacaggtagtagatcatgcttttgggatgttgatggaaggcctgaagcagcggaat 1080 ttgcacaactgtgtcaatatcatccttctggctgaccatggaatggaccagacttattgt 1140 aacaagatggaatacatgactgattattttcccagaataaacttcttctacatgtacgaa 1200 gggcctgccccccgcatccgagctcataatatacctcatgacttttttagttttaattct 1260 gaggaaattgttagaaacctcagttgccgaaaacctgatcagcatttcaagccctatttg 1320 actcctgatttgccaaagcgactgcactatgccaagaacgtcagaatcgacaaagttcat 1380 ctctttgtggatcaacagtggctggctgttaggagtaaatcaaatacaaattgtggagga 1440 ggcaaccatggttataacaatgagtttaggagcatggaggctatctttctggcacatgga 1500 cccagttttaaagagaagactgaagttgaaccatttgaaaatattgaagtctataaccta 1560 atgtgtgatcttctacgcattcaaccagcaccaaacaatggaacccatggtagtttaaac 1620 catcttctgaaggtgcctttttatgagccatcccatgcagaggaggtgtcaaagttttct 1680 gtttgtggctttgctaatccattgcccacagagtctcttgactgtttctgccctcaccta 1740 caaaatagtactcagctggaacaagtgaatcagatgctaaatctcacccaagaagaaata 1800 acagcaacagtgaaagtaaatttgccatttgggaggcctagggtactgcagaagaacgtg 1860 gaccactgtctcctttaccacagggaatatgtcagtggatttggaaaagctatgaggatg 1920 cccatgtggagttcatacacagtcccccagttgggagacacatcgcctctgcctcccact 1980 gtcccagactgtctgcgggctgatgtcagggttcctccttctgagagccaaaaatgttcc 2040 ttctatttagcagacaagaatatcacccacggcttcctctatcctcctgccagcaataga 2100 acatcagatagccaatatgatgctttaattactagcaatttggtacctatgtatgaagaa 2160 ttcagaaaaatgtgggactacttccacagtgttcttcttataaaacatgccacagaaaga 2220 aatggagtaaatgtggttagtggaccaatatttgattataattatgatggccattttgat 2280 gctccagatgaaattaccaaacatttagccaacactgatgttcccatcccaacacactac 2340 tttgtggtgctgaccagttgtaaaaacaagagccacacaccggaaaactgccctgggtgg 2400 ctggatgtcctaccctttatcatccctcaccgacctaccaacgtggagagctgtcctgaa 2460 ggtaaaccagaagctctttgggttgaagaaagatttacagctcacattgcccgggtccgt 2520 gatgtagaacttctcactgggcttgacttctatcaggataaagtgcagcctgtctctgaa 2580 attttgcaactaaagacatatttaccaacatttgaaaccactatt 2625 SEQ.IDNO:77-ENPP1Nucleotidesequence: atggaacgggacggctgtgccggcggaggatcaagaggcggagaaggcggcagagcccct 60 agagaaggacctgccggcaacggcagagacagaggcagatctcatgccgccgaagcccct 120 ggcgatcctcaggctgctgcttctctgctggcccccatggatgtgggcgaggaacctctg 180 gaaaaggccgccagagccagaaccgccaaggaccccaacacctacaaggtgctgagcctg 240 gtgctgtccgtgtgcgtgctgaccaccatcctgggctgcatcttcggcctgaagcccagc 300 tgcgccaaagaagtgaagtcctgcaagggccggtgcttcgagcggaccttcggcaactgc 360 agatgcgacgccgcctgtgtggaactgggcaactgctgcctggactaccaggaaacctgc 420 atcgagcccgagcacatctggacctgcaacaagttcagatgcggcgagaagcggctgacc 480 agatccctgtgtgcctgcagcgacgactgcaaggacaagggcgactgctgcatcaactac 540 agcagcgtgtgccagggcgagaagtcctgggtggaagaaccctgcgagagcatcaacgag 600 ccccagtgccctgccggcttcgagacacctcctaccctgctgttcagcctggacggcttt 660 cgggccgagtacctgcacacatggggaggcctgctgcccgtgatcagcaagctgaagaag 720 tgcggcacctacaccaagaacatgcggcccgtgtaccccaccaagaccttccccaaccac 780 tactccatcgtgaccggcctgtaccccgagagccacggcatcatcgacaacaagatgtac 840 gaccccaagatgaacgccagcttcagcctgaagtccaaagagaagttcaaccccgagtgg 900 tataagggcgagcccatctgggtcaccgccaagtaccagggcctgaaaagcggcacattc 960 ttttggcccggcagcgacgtggaaatcaacggcatcttccccgacatctataagatgtac 1020 aacggcagcgtgcccttcgaggaacggatcctggctgtgctgcagtggctgcagctgccc 1080 aaggatgagcggccccacttctacaccctgtacctggaagaacctgacagcagcggccac 1140 agctacggccctgtgtccagcgaagtgatcaaggccctgcagcgggtggacggcatggtg 1200 ggaatgctgatggacggcctgaaagagctgaacctgcacagatgcctgaacctgatcctg 1260 atcagcgaccacggcatggaacagggatcctgcaagaagtacatctacctgaacaagtac 1320 ctgggcgacgtgaagaacatcaaagtgatctacggcccagccgccagactgaggcctagc 1380 gacgtgcccgacaagtactacagcttcaactacgagggaatcgcccggaacctgagctgc 1440 agagagcccaaccagcacttcaagccctacctgaagcacttcctgcccaagcggctgcac 1500 ttcgccaagagcgacagaatcgagcccctgaccttctacctggacccccagtggcagctg 1560 gccctgaatcccagcgagagaaagtactgcggcagcggcttccacggctccgacaacgtg 1620 ttcagcaacatgcaggccctgttcgtgggctacggacccggctttaagcacggcatcgag 1680 gccgacaccttcgagaacatcgaggtgtacaatctgatgtgcgacctgctgaatctgacc 1740 cctgcccccaacaatggcacccacggcagcctgaaccatctgctgaagaaccccgtgtac 1800 acccctaagcaccccaaagaggtgcaccccctggtgcagtgccccttcaccagaaacccc 1860 agagacaacctgggctgtagctgcaaccccagcatcctgcccatcgaggacttccagacc 1920 cagttcaacctgaccgtggccgaggaaaagatcatcaagcacgagacactgccctacggc 1980 agaccccgggtgctgcagaaagagaacaccatctgcctgctgagccagcaccagttcatg 2040 agcggctactcccaggacatcctgatgcccctgtggaccagctacaccgtggaccggaac 2100 gacagcttctccaccgaggatttcagcaactgcctgtaccaggatttccggatccccctg 2160 agccccgtgcacaagtgcagcttctacaagaacaacaccaaggtgtcctacggcttcctg 2220 agccctccccagctgaacaagaacagctccggcatctacagcgaggccctgctgactacc 2280 aacatcgtgcccatgtaccagagcttccaagtgatctggcggtacttccacgacaccctg 2340 ctgcggaagtacgccgaagaacggaacggcgtgaacgtggtgtccggcccagtgttcgac 2400 ttcgactacgacggcagatgtgacagcctggaaaatctgcggcagaaaagaagagtgatc 2460 cggaaccaggaaattctgatccctacccacttctttatcgtgctgacaagctgcaaggat 2520 accagccagacccccctgcactgcgagaacctggataccctggccttcatcctgcctcac 2580 cggaccgacaacagcgagagctgtgtgcacggcaagcacgacagctcttgggtggaagaa 2640 ctgctgatgctgcaccgggccagaatcaccgatgtggaacacatcaccggcctgagcttt 2700 taccagcagcggaaagaacccgtgtccgatatcctgaagctgaaaacccatctgcccacc 2760 ttcagccaggaagat 2775 SEQIDNO:78-Azurocidin-ENPP1-FCNucleotidesequence ggtaccgccaccatgacaagactgacagtgctggctctgctggccggactgttggcctcttctagagctg ctccttcctgcgccaaagaagtgaagtcctgcaagggcagatgcttcgagcggaccttcggcaactgtag atgtgacgccgcttgcgtggaactgggcaactgctgcctggactaccaagagacatgcatcgagcccgag cacatctggacctgcaacaagttcagatgcggcgagaagcggctgaccagatctctgtgcgcctgctctg acgactgcaaggacaagggcgactgctgcatcaactactcctctgtgtgccagggcgagaagtcctgggt tgaagaaccctgcgagtccatcaacgagcctcagtgtcctgccggcttcgagacacctcctactctgctg ttctccctggatggcttcagagccgagtacctgcatacttggggaggcctgctgccagtgatctccaagc tgaagaagtgcggcacctacaccaagaacatgaggcctgtgtaccctaccaagacattccccaaccacta ctccatcgtgaccggcctgtatcctgagagccacggcatcatcgacaacaagatgtacgaccccaagatg aacgcctccttcagcctgaagtccaaagagaagttcaaccccgagtggtataagggcgagcctatctggg tcaccgctaagtaccagggactgaagtctggcaccttcttttggcctggctccgacgtggaaatcaacgg catcttccccgacatctataagatgtacaacggctccgtgcctttcgaggaacgcattctggctgttctg cagtggctgcagctgcctaaggatgagaggcctcacttctacaccctgtacctggaagaacctgactcct ccggccactcttatggccctgtgtcctctgaagtgatcaaggccctgcagcgagtggacggaatggtcgg aatgctgatggacggcctgaaagagctgaacctgcacagatgcctgaacctgatcctgatctccgaccac ggcatggaacaggggagctgcaagaagtacatctacctgaacaagtacctgggcgacgtgaagaacatca aagtgatctacggcccagccgccagactgaggccttctgatgtgcctgacaagtactactccttcaacta cgagggaatcgcccggaacctgtcctgcagagagcctaaccagcacttcaagccctacctgaagcacttt ctgcctaagcggctgcacttcgccaagtctgacagaatcgagcccctgaccttctatctggaccctcagt ggcagctggccctgaatcctagcgagagaaagtactgtggctccggcttccacggctccgacaacgtgtt ctctaatatgcaggccctgttcgtcggctacggccctggctttaaacacggcatcgaggccgacaccttc gagaacatcgaggtgtacaatctgatgtgtgacctgctgaatctgacccctgctcctaacaacggcaccc acggatctctgaaccatctgctgaagaatcccgtgtacacccctaagcaccccaaagaggttcaccctct ggtccagtgtcctttcaccagaaatcctcgggacaacctgggctgctcttgcaacccttctatcctgcct atcgaggactttcagacccagttcaacctgaccgtggccgaggaaaagatcatcaagcacgagacactgc cctacggcagacctagagtgctgcagaaagagaacaccatctgcctgctgtcccagcaccagttcatgtc cggctactcccaggacatcctgatgcctctgtggacctcctacaccgtggaccggaacgatagcttctcc accgaggacttcagcaactgcctgtaccaggatttcagaatccctctgagccccgtgcacaagtgcagct tctacaagaacaacaccaaggtgtcctacggcttcctgtctcctccacagctgaacaagaactccagcgg catctactctgaggccctgctgaccaccaacatcgtgcccatgtaccagtccttccaagtgatctggcgg tacttccacgacaccctgctgaggaagtacgccgaagaaagaaacggcgtgaacgtggtgtctggccccg tgttcgacttcgactacgacggcagatgcgactctctggaaaacctgcggcagaaaagacgagtgatccg gaatcaagagatcctgattcctacacacttctttatcgtgctgaccagctgcaaggatacctctcagacc cctctgcactgcgagaatctggacaccctggccttcattctgcctcacagaaccgacaactccgagtcct gtgtgcacggcaagcacgactcctcttgggtcgaagaactgctgatgctgcaccgggccagaatcaccga tgtggaacacatcaccggcctgagcttctaccagcagcggaaagaacctgtgtccgatatcctgaagctg aaaacccatctgccaaccttcagccaagaggacctgatcaacgacaagacccacacctgtcctccatgtc ctgctccagaactgctcggaggcccctctgtgttcctgtttccacctaagccaaaggacacactgatgat ctctcggacccctgaagtgacctgcgtggtggtggatgtgtctcacgaagatcccgaagtcaagttcaat tggtacgtggacggcgtggaagtgcacaacgccaagaccaagcctagagaggaacagtacaactccacct acagagtggtgtccgtgctgactgtgctgcaccaggattggctgaacggcaaagagtacaagtgcaaagt gtccaacaaggctctgcccgctcctatcgaaaagaccatctccaaggctaagggccagcctcgggaacct caggtttacaccctgcctccatctcgggaagagatgaccaagaaccaggtgtccctgacctgcctggtca agggcttctacccttccgatatcgccgtggaatgggagtccaatggccagcctgagaacaactacaagac aacccctcctgtgctggacagcgacggctcattcttcctgtactctaagctgacagtggacaagtcccgg tggcagcaaggcaatgtgttttcctgctctgtgatgcacgaggccctccacaatcactacacccagaagt ccctgtctctgtcccctggcaaatgatagctcgag Legend-bold=start/stopcodon;underlined=nucleotide sequenceofsignalpeptide. SEQIDNO:79-Azurocidin-ENPP3-FCNucleotidesequence atgaccagactgaccgtgctggccctgctggccggcctgctggccagcagcagagccgccaagca gggcagctgcagaaagaagtgcttcgacgccagcttcagaggcctggagaactgcagatgcgacgtggcc tgcaaggacagaggcgactgctgctgggacttcgaggacacctgcgtggagagcaccagaatctggatgt gcaacaagttcagatgcggcgagaccagactggaggccagcctgtgcagctgcagcgacgactgcctgca gagaaaggactgctgcgccgactacaagagcgtgtgccagggcgagaccagctggctggaggagaactgc gacaccgcccagcagagccagtgccccgagggcttcgacctgccccccgtgatcctgttcagcatggacg gcttcagagccgagtacctgtacacctgggacaccctgatgcccaacatcaacaagctgaagacctgcgg catccacagcaagtacatgagagccatgtaccccaccaagaccttccccaaccactacaccatcgtgacc ggcctgtaccccgagagccacggcatcatcgacaacaacatgtacgacgtgaacctgaacaagaacttca gcctgagcagcaaggagcagaacaaccccgcctggtggcacggccagcccatgaacctgaccgccatgta ccagggcctgaaggccgccacctacttctggcccggcagcgaggtggccatcaacggcagcttccccagc atctacatgccctacaacggcagcgtgcccttcgaggagagaatcagcaccctgctgaagtggctggacc tgcccaaggccgagagacccagattctacaccatgtacttcgaggagcccgacagcagcggccacgccgg cggccccgtgagcgccagagtgatcaaggccctgcaggtggtggaccacgccttcggcatgctgatggag ggcctgaagcagagaaacctgcacaactgcgtgaacatcatcctgctggccgaccacggcatggaccaga cctactgcaacaagatggagtacatgaccgactacttccccagaatcaacttcttctacatgtacgaggg ccccgcccccagaatcagagcccacaacatcccccacgacttcttcagcttcaacagcgaggagatcgtg agaaacctgagctgcagaaagcccgaccagcacttcaagccctacctgacccccgacctgcccaagagac tgcactacgccaagaacgtgagaatcgacaaggtgcacctgttcgtggaccagcagtggctggccgtgag aagcaagagcaacaccaactgcggcggcggcaaccacggctacaacaacgagttcagaagcatggaggcc atcttcctggcccacggccccagcttcaaggagaagaccgaggtggagcccttcgagaacatcgaggtgt acaacctgatgtgcgacctgctgagaatccagcccgcccccaacaacggcacccacggcagcctgaacca cctgctgaaggtgcccttctacgagcccagccacgccgaggaggtgagcaagttcagcgtgtgcggcttc gccaaccccctgcccaccgagagcctggactgcttctgcccccacctgcagaacagcacccagctggagc aggtgaaccagatgctgaacctgacccaggaggagatcaccgccaccgtgaaggtgaacctgcccttcgg cagacccagagtgctgcagaagaacgtggaccactgcctgctgtaccacagagagtacgtgagcggcttc ggcaaggccatgagaatgcccatgtggagcagctacaccgtgccccagctgggcgacaccagccccctgc cccccaccgtgcccgactgcctgagagccgacgtgagagtgccccccagcgagagccagaagtgcagctt ctacctggccgacaagaacatcacccacggcttcctgtacccccccgccagcaacagaaccagcgacagc cagtacgacgccctgatcaccagcaacctggtgcccatgtacgaggagttcagaaagatgtgggactact tccacagcgtgctgctgatcaagcacgccaccgagagaaacggcgtgaacgtggtgagcggccccatctt cgactacaactacgacggccacttcgacgcccccgacgagatcaccaagcacctggccaacaccgacgtg cccatccccacccactacttcgtggtgctgaccagctgcaagaacaagagccacacccccgagaactgcc ccggctggctggacgtgctgcccttcatcatcccccacagacccaccaacgtggagagctgccccgaggg caagcccgaggccctgtgggtggaggagagattcaccgcccacatcgccagagtgagagacgtggagctg ctgaccggcctggacttctaccaggacaaggtgcagcccgtgagcgagatcctgcagctgaagacctacc tgcccaccttcgagaccaccatcgacaagacccacacctgccccccctgccccgcccccgagctgctggg cggccccagcgtgttcctgttcccccccaagcccaaggacaccctgatgatcagcagaacccccgaggtg acctgcgtggtggtggacgtgagccacgaggaccccgaggtgaagttcaactggtacgtggacggcgtgg aggtgcacaacgccaagaccaagcccagagaggagcagtacaacagcacctacagagtggtgagcgtgct gaccgtgctgcaccaggactggctgaacggcaaggagtacaagtgcaaggtgagcaacaaggccctgccc gcccccatcgagaagaccatcagcaaggccaagggccagcccagagagccccaggtgtacaccctgcccc ccagcagagaggagatgaccaagaaccaggtgagcctgacctgcctggtgaagggcttctaccccagcga catcgccgtggagtgggagagcaacggccagcccgagaacaactacaagaccaccccccccgtgctggac agcgacggcagcttcttcctgtacagcaagctgaccgtggacaagagcagatggcagcagggcaacgtgt tcagctgcagcgtgatgcacgaggccctgcacaaccactacacccagaagagcctgagcctgagccccgg caag

Cloning and Expression of ENPP1 and ENPP3 Fusion Polypeptides

[0274] ENPP1, or an ENPP1 polypeptide, is prepared as described in US 2015/0359858 A1, which is incorporated herein in its entirety by reference. ENPP1 is a transmembrane protein localized to the cell surface with distinct intramembrane domains. In order to express ENPP1 as a soluble extracellular protein, the transmembrane domain of ENPP1 may be swapped for the transmembrane domain of ENPP2 or a signal peptide sequence such as Azurocidin, which results in the accumulation of soluble, recombinant ENPP1 in the extracellular fluid of the baculovirus cultures. Signal sequences of any other known proteins may be used to target the extracellular domain of ENPP1 for secretion as well, such as but not limited to the signal sequence of the immunoglobulin kappa and lambda light chain proteins. Further, the disclosure should not be construed to be limited to the polypeptides described herein, but also includes polypeptides comprising any enzymatically active truncation of the ENPP1 extracellular domain.

[0275] ENPP1 is made soluble by omitting the transmembrane domain. Human ENPP1 (SEQ ID NO:1) was modified to express a soluble, recombinant protein by replacing its transmembrane region (e.g., residues 77-98) with the corresponding subdomain of human ENPP2 (NCBI accession NP 00112433 5, e.g., residues 12-30) or Azurocidin signal sequence (SEQ ID 42).

[0276] The modified ENPP1 sequence was cloned into a modified pFastbac FIT vector possessing a TEV protease cleavage site followed by a C-terminus 9-F HS tag, and cloned and expressed in insect cells, and both proteins were expressed in a baculovirus system as described previously (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 protein in the extracellular fluid.

[0277] ENPP3 is poorly exported to the cell surface. Soluble ENPP3 polypeptide is constructed by replacing the signal sequence of ENPP3 with the native signal sequence of other ENPPs or Azurocidin or suitable signal sequences. Several examples of ENPP3 fusion constructs are disclosed in WO 2017/087936. Soluble ENPP3 constructs are prepared by using the signal export signal sequence of other ENPP enzymes, such as but not limited to ENPP7 and/or ENPP5. Soluble ENPP3 constructs are prepared using a signal sequence comprised of a combination of the signal sequences of ENPP1 and ENPP2 (ENPP1-2-1 or ENPP121 hereinafter). Signal sequences of any other known proteins may be used to target the extracellular domain of ENPP3 for secretion as well, such as but not limited to the signal sequence of the immunoglobulin kappa and lambda light chain proteins. Further, the disclosure should not be construed to be limited to the constructs described herein, but also includes constructs comprising any enzymatically active truncation of the ENPP3 extracellular domain.

[0278] In certain embodiments, the ENPP3 polypeptide is soluble. In some embodiments, the polypeptide of the disclosure includes an ENPP3 polypeptide that lacks the ENPP3 transmembrane domain. In another embodiment, the polypeptide of the disclosure includes an ENPP3 polypeptide wherein the ENPP3 transmembrane domain has been removed and replaced with the transmembrane domain of another polypeptide, such as, by way of non-limiting example, ENPP2, ENPPS or ENPP7 or Azurocidin signal sequence.

[0279] In some embodiments, the polypeptide of the disclosure comprises an IgG Fc domain. In certain embodiments, the polypeptide of the disclosure comprises an albumin domain. In other embodiments, the albumin domain is located at the C terminal region of the ENPP3 polypeptide. In yet other embodiments, the IgG Fc domain is located at the C terminal region of the ENPP3 polypeptide. In yet other embodiments, the presence of IgG Fc domain or albumin domain improves half-life, solubility, reduces immunogenicity and increases the activity of the ENPP3 polypeptide.

[0280] In certain embodiments, the polypeptide of the disclosure comprises a signal peptide resulting in the secretion of a precursor of the ENPP3 polypeptide, which undergoes proteolytic processing to yield the ENPP3 polypeptide. In other embodiments, the signal peptide is selected from the group consisting of signal peptides of ENPP2, ENPP5 and ENPP7. In yet other embodiments, the signal peptide is selected from the group consisting of SEQ ID NOs: 36-42.

[0281] In certain embodiments, the IgG Fc domain or the albumin domain is connected to the C terminal region of the ENPP3 polypeptide by a linker region. In other embodiments, the linker is selected from SEQ ID NOs: 43-75, where n is an integer ranging from 1-20.

Production and Purification of ENPP1 and ENPP3 Fusion Polypeptides

[0282] To produce soluble, recombinant ENPP1 polypeptide for in vitro use, polynucleotide encoding the extracellular domain of ENPP1 (Human NPP1 (NCBI accession NP 006199)) was fused to the Fc domain of IgG (referred to as ENPP1-Fc) and was expressed in stable CHO cell lines. In some embodiments, ENPP1 polynucleotide encoding residues 96 to 925 of NCBI accession NP 006199 were fused to Fc domain to generate ENPP1 polypeptide.

[0283] Alternately the ENPP1 polypeptide can also be expressed from HEK293 cells, Baculovirus insect cell system or CHO cells or Yeast Pichia expression system using suitable vectors. The ENPP1 polypeptide can be produced in either adherent or suspension cells. Preferably the ENPP1 polypeptide is expressed in CHO cells. To establish stable cell lines the nucleic acid sequence encoding ENPP1 constructs are cloned into an appropriate vector for large scale protein production.

[0284] ENPP3 is produced by establishing stable transfections in either CHO or HEK293 mammalian cells. ENPP3 polynucleotide encoding ENPP3 (Human NPP3 (UniProtKB/Swiss-Prot: O14638.2) was fused to the Fc domain of IgG (referred to as ENPP3-Fc) and was expressed in stable CHO cell lines. In some embodiments, ENPP3 polynucleotide encoding residues 49-875 of UniProtKB/Swiss-Prot: O14638.2 was fused to Fc domain to generate ENPP3 polypeptide. The ENPP3 polypeptide can be produced in either adherent or suspension cells. To establish stable cell lines the nucleic acid sequence encoding NPP3 fusion polypeptides of the disclosure into an appropriate vector for large scale protein production. There are a variety of these vectors available from commercial sources and any of those can be used. ENPP3 polypeptides are produced following the protocols established in WO 2017/087936, the contents of which are hereby incorporated by reference in their entirety. ENPP1 polypeptides are produced following the protocols established in Albright, et al, 2015, Nat Commun. 6:10006, the contents of which are hereby incorporated by reference in their entirety.

[0285] A suitable plasmid containing the desired polypeptide constructs of ENPP1 or ENPP3 can be stably transfected into expression plasmid using established techniques such as electroporation or lipofectamine, and the cells can be grown under antibiotic selection to enhance for stably transfected cells. Clones of single, stably transfected cells are then established and screened for high expressing clones of the desired fusion protein. Screening of the single cell clones for ENPP1 or ENPP3 polypeptide expression can be accomplished in a high-throughput manner in 96 well plates using the synthetic enzymatic substrate pNP-TMP as previously described (Saunders, et al, 2008, Mol. Cancer Therap. 7(10):3352-62; Albright, et al, 2015, Nat Commun. 6:10006).

[0286] Upon identification of high expressing clones for ENPP3 or ENPP1 polypeptides through screening, protein production can be accomplished in shaking flasks or bio-reactors previously described for ENPP1 (Albright, et al, 2015, Nat Commun. 6:10006). Purification of ENPP3 or ENPP1 polypeptides can be accomplished using a combination of standard purification techniques known in the art. These techniques are well known in art and are selected from techniques such as column chromatograph, ultracentrifugation, filtration, and precipitation. Column chromatographic purification is accomplished using affinity chromatography such as protein-A and protein-G resins, metal affinity resins such as nickel or copper, hydrophobic exchange chromatography, and reverse-phase high-pressure chromatography (HPLC) using C8-C14 resins. Ion exchange may also be employed, such as anion and cation exchange chromatography using commercially available resins such as Q-sepharose (anion exchange) and SP-sepharose (cation exchange), blue sepharose resin and blue-sephadex resin, and hydroxyapatite resins. Size exclusion chromatography using commercially available S-75 and S200 Superdex resins can also be employed, as known in the art. Buffers used to solubilize the protein and provide the selection media for the above described chromatographic steps, are standard biological buffers known to practitioners of the art and science of protein chemistry.

[0287] Some examples of buffers that are used in preparation include citrate, phosphate, acetate, tris(hydroxymemyl)aminomethane, saline buffers, glycine-HCL buffers, Cacodylate buffers, and sodium barbital buffers, which are well known in art. Using a single techniques, or a series of techniques in combination, and the appropriate buffer systems purified ENPP3 and the crude starting material side by side on a Coomasie stained polyacrylamide gel after a single purification step. The ENPP3 protein can then be additionally purified using additional techniques and/or chromatographic steps as described above, to reach substantially higher purity such as 99% purity adjusted to the appropriate pH, one can purify the ENPP1 or ENPP3 polypeptides described to greater than 99% purity from crude material.

[0288] Following purification, ENPP1-Fc or ENPP3-Fc was dialyzed into PBS supplemented with Zn2+ and Mg2+(PBSplus) concentrated to between 5 and 7 mg/ml, and frozen at 80 C. in aliquots of 200-500 l. Aliquots were thawed immediately prior to use and the specific activity of the solution was adjusted to 31.25 au/ml (or about 0.7 mg/ml depending on the preparation) by dilution in PBSplus.

Dosage & Mode of Administration

[0289] In another embodiment, the hsNPP1 or hsNPP3 is administered in one or more doses containing about 1.0 mg/kg to about 5.0 mg/kg NPP1 or about 1.0 mg/kg to about 5.0 mg/kg NPP3 respectively. In another embodiment, the hsNPP1 or hsNPP3 is administered in one or more doses containing about 1.0 mg/kg to about 10.0 mg/kg NPP1 or about 1.0 mg/kg to about 10.0 mg/kg NPP3.

[0290] The time period between doses of the hsNPP1 or hsNPP3 is at least 2 days and can be longer, for example at least 3 days, at least 1 week, 2 weeks or 1 month. In one embodiment, the administration is weekly, bi-weekly, or monthly.

[0291] The recombinant hsNPP1 or hsNPP3 can be administered in any suitable way, such as intravenously, subcutaneously, or intraperitoneally.

[0292] The recombinant hsNPP1 or hsNPP3 can be administered in combination with one or more additional therapeutic agents. Exemplary therapeutic agents include, but are not limited to Bisphosphonate, Statins, Fibrates, Niacin, Aspirin, Clopidogrel, and warfarin.

[0293] In some embodiments, the recombinant hsNPP1 or hsNPP3 and additional therapeutic agent are administered separately and are administered concurrently or sequentially. In some embodiments, the recombinant hsNPP1 or hsNPP3 is administered prior to administration of the additional therapeutic agent. In some embodiments, the recombinant hsNPP1 or hsNPP3 is administered after administration of the additional therapeutic agent. In other embodiments, the recombinant hsNPP1 or hsNPP3 and additional therapeutic agent are administered together.

Nucleic Acid Administration and Therapy

Viral Vectors for In Vivo Expression of ENPP1 and ENPP3

[0294] The nucleic acids encoding the polypeptide(s) useful within the disclosure may be used in gene therapy protocols for the treatment of the diseases or disorders contemplated herein. The improved construct encoding the polypeptide(s) can be inserted into the appropriate gene therapy vector and administered to a patient to treat or prevent the diseases or disorder of interest.

[0295] Vectors, such as viral vectors, have been used in the prior art to introduce genes into a wide variety of different target cells. Typically, the vectors are exposed to the target cells so that transformation can take place in a sufficient proportion of the cells to provide a useful therapeutic or prophylactic effect from the expression of the desired polypeptide (e.g., a receptor). The transfected nucleic acid may be permanently incorporated into the genome of each of the targeted cells, providing long lasting effect, or alternatively the treatment may have to be repeated periodically. In certain embodiments, the (viral) vector transfects liver cells in vivo with genetic material encoding the polypeptide(s) of the disclosure.

[0296] A variety of vectors, both viral vectors and plasmid vectors are known in the art (see for example U.S. Pat. No. 5,252,479 and WO 93/07282). In particular, a number of viruses have been used as gene transfer vectors, including papovaviruses, such as SV40, vaccinia virus, herpes viruses including HSV and EBV, and retroviruses. Many gene therapy protocols in the prior art have employed disabled murine retroviruses. Several recently issued patents are directed to methods and compositions for performing gene therapy (see for example U.S. Pat. Nos. 6,168,916; 6,135,976; 5,965,541 and 6,129,705). Each of the foregoing patents is incorporated by reference in its entirety herein. Hence, genetic material such as a polynucleotide comprising an NPP1 or an NPP3 sequence can be introduced to a mammal in order to treat VSMC proliferation.

[0297] Certain modified viruses are often used as vectors to carry a coding sequence because after administration to a mammal, a virus infects a cell and expresses the encoded protein. Modified viruses useful according to the disclosure are derived from viruses which include, for example: parvovirus, picornavirus, pseudorabies virus, hepatitis virus A, B or C, papillomavirus, papovavirus (such as polyoma and SV40) or herpes virus (such as Epstein-Barr Virus, Varicella Zoster Virus, Cytomegalovirus, Herpes Zoster and Herpes Simplex Virus types 1 and 2), an RNA virus or a retrovirus, such as the Moloney murine leukemia virus or a lentivirus (i.e. derived from Human Immunodeficiency Virus, Feline Immunodeficiency Virus, equine infectious anemia virus, etc.). Among DNA viruses useful according to the disclosure are: Adeno-associated viruses adenoviruses, Alphaviruses, and Lentiviruses.

[0298] A viral vector is generally administered by injection, most often intravenously (by IV) directly into the body, or directly into a specific tissue, where it is taken up by individual cells. Alternately, a viral vector may be administered by contacting the viral vector ex vivo with a sample of the patient's cells, thereby allowing the viral vector to infect the cells, and cells containing the vector are then returned to the patient. Once the viral vector is delivered, the coding sequence expressed and results in a functioning protein. Generally, the infection and transduction of cells by viral vectors occur by a series of sequential events as follows: interaction of the viral capsid with receptors on the surface of the target cell, internalization by endocytosis, intracellular trafficking through the endocytic/proteasomal compartment, endosomal escape, nuclear import, virion uncoating, and viral DNA double-strand conversion that leads to the transcription and expression of the recombinant coding sequence interest. (Colella et al., Mol Ther Methods Clin Dev. 2017 Dec. 1; 8:87-104.).

[0299] Adeno-Associated Viral Vectors According to the Disclosure

[0300] AAV refers to viruses belonging to the genus Dependovirus of the Parvoviridae family. The AAV genome is approximately 4.7 kilobases long and is composed of linear single-stranded deoxyribonucleic acid (ssDNA) which may be either positive- or negative-sensed. The genome comprises inverted terminal repeats (ITRs) at both ends of the DNA strand, and two open reading frames (ORFs): rep and cap. The rep frame is made of four overlapping genes encoding non-structural replication (Rep) proteins required for the AAV life cycle. The cap frame contains overlapping nucleotide sequences of structural VP capsid proteins: VP1, VP2 and VP3, which interact together to form a capsid of an icosahedral symmetry.

[0301] The terminal 145 nucleotides are self-complementary and are organized so that an energetically stable intramolecular duplex forming a T-shaped hairpin may be formed. These hairpin structures function as an origin for viral DNA replication, serving as primers for the cellular DNA polymerase complex. Following wild type AAV infection in mammalian cells the rep genes (i.e. Rep78 and Rep52) are expressed from the P5 promoter and the P19 promoter, respectively, and both Rep proteins have a function in the replication of the viral genome. A splicing event in the rep ORF results in the expression of actually four Rep proteins (i.e. Rep78, Rep68, Rep52 and Rep40). However, it has been shown that the unspliced mRNA, encoding Rep78 and Rep52 proteins, in mammalian cells are sufficient for AAV vector production. Also in insect cells the Rep78 and Rep52 proteins suffice for AAV vector production.

[0302] AAV is a helper-dependent virus, that is, it requires co-infection with a helper virus (e.g., adenovirus, herpesvirus, or vaccinia virus) in order to form functionally complete AAV virions. In the absence of co-infection with a helper virus, AAV establishes a latent state in which the viral genome inserts into a host cell chromosome or exists in an episomal form, but infectious virions are not produced. Subsequent infection by a helper virus rescues the integrated genome, allowing it to be replicated and packaged into viral capsids, thereby reconstituting the infectious virion. While AAV can infect cells from different species, the helper virus must be of the same species as the host cell. Thus, for example, human AAV replicates in canine cells that have been co-infected with a canine adenovirus.

[0303] To produce infectious recombinant AAV (rAAV) containing a heterologous nucleic acid sequence, a suitable host cell line can be transfected with an AAV vector containing the heterologous nucleic acid sequence, but lacking the AAV helper function genes, rep and cap. The AAV-helper function genes can then be provided on a separate vector. Also, only the helper virus genes necessary for AAV production (i.e., the accessory function genes) can be provided on a vector, rather than providing a replication-competent helper virus (such as adenovirus, herpesvirus, or vaccinia).

[0304] Collectively, the AAV helper function genes (i.e., rep and cap) and accessory function genes can be provided on one or more vectors. Helper and accessory function gene products can then be expressed in the host cell where they will act in trans on rAAV vectors containing the heterologous nucleic acid sequence. The rAAV vector containing the heterologous nucleic acid sequence will then be replicated and packaged as though it were a wild-type (wt) AAV genome, forming a recombinant virion. When a patient's cells are infected with the resulting rAAV virions, the heterologous nucleic acid sequence enters and is expressed in the patient's cells.

[0305] Because the patient's cells lack the rep and cap genes, as well as the accessory function genes, the rAAV cannot further replicate and package their genomes. Moreover, without a source of 5 rep and cap genes, wtAAV cannot be formed in the patient's cells.

[0306] The AAV vector typically lacks rep and cap frames. Such AAV vectors can be replicated and packaged into infectious viral particles when present in a host cell that has been transfected with a vector encoding and expressing rep and cap gene products (i.e. AAV Rep and Cap proteins), and wherein the host cell has been transfected with a vector which encodes and expresses a protein from the adenovirus open reading frame E4orf6.

[0307] Delivery of a protein of interest to the cells of a mammal is accomplished by first generating an AAV vector comprising DNA encoding the protein of interest and then administering the vector to the mammal. Thus, the disclosure should be construed to include AAV vectors comprising DNA encoding the polypeptide(s) of interest. Once armed with the present disclosure, the generation of AAV vectors comprising DNA encoding this/these polypeptide(s)s will be apparent to the skilled artisan.

[0308] In one embodiment, the disclosure relates to an adeno-associated viral (AAV) expression vector comprising a sequence encoding mammal ENPP1 or mammal ENPP3, and upon administration to a mammal the vector expresses an ENPP1 or ENPP3 precursor in a cell, the precursor including an Azurocidin signal peptide fused at its carboxy terminus to the amino terminus of ENPP1 or ENPP3. The ENPP1 or ENPP3 precursor may include a stabilizing domain, such as an IgG Fc region or human albumin. Upon secretion of the precursor from the cell, the signal peptide is cleaved off and enzymatically active soluble mammal ENPP1 or ENPP3 is provided extracellularly.

[0309] An AAV expression vector may include an expression cassette comprising a transcriptional regulatory region operatively linked to a nucleotide sequence comprising a transcriptional regulatory region operatively linked to a recombinant nucleic acid sequence encoding a polypeptide comprising a Azurocidin signal peptide sequence and an ectonucleotide pyrophosphatase/phosphodiesterase (ENPP1) polypeptide sequence.

[0310] In some embodiments, the expression cassette comprises a promoter and enhancer, the Kozak sequence GCCACCATGG, a nucleotide sequence encoding mammal NPP1 protein or a nucleotide sequence encoding mammal NPP3 protein, other suitable regulatory elements and a polyadenylation signal.

[0311] In some embodiments, the AAV recombinant genome of the AAV vector according to the disclosure lacks the rep open reading frame and/or the cap open reading frame.

[0312] The AAV vector according to the disclosure comprises a capsid from any serotype. In general, the AAV serotypes have genomic sequences of significant homology at the amino acid and the nucleic acid levels, provide an identical set of genetic functions, and replicate and assemble through practically identical mechanisms. In particular, the AAV of the present disclosure may belong to the serotype 1 of AAV (AAV1), AAV2, AAV3 (including types 3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh10, AAV11, avian AAV, bovine AAV, canine AAV, equine AAV, or ovine AAV.

[0313] Examples of the sequences of the genome of the different AAV serotypes may be found in the literature or in public databases such as GenBank. For example, GenBank accession numbers NC 001401.2 (AAV2), NC 001829.1 (AAV4), NC 006152.1 (AAV5), AF028704.1 (AAV6), NC 006260.1 (AAV7), NC 006261.1 (AAV8), AX753250.1 (AAV9) and AX753362.1 (AAV10).

[0314] In some embodiments, the adeno-associated viral vector according to the disclosure comprises a capsid derived from a serotype selected from the group consisting of the AAV2, AAV5, AAV7, AAV8, AAV9, AAV10 and AAVrhl 0 serotypes. In another embodiment, the serotype of the AAV is AAV8. If the viral vector comprises sequences encoding the capsid proteins, these may be modified so as to comprise an exogenous sequence to direct the AAV to a particular cell type or types, or to increase the efficiency of delivery of the targeted vector to a cell, or to facilitate purification or detection of the AAV, or to reduce the host response.

[0315] In certain embodiments, the rAAV vector of the disclosure comprises several essential DNA elements. In certain embodiments, these DNA elements include at least two copies of an AAV ITR sequence, a promoter/enhancer element, a transcription termination signal, any necessary 5 or 3 untranslated regions which flank DNA encoding the protein of interest or a biologically active fragment thereof. The rAAV vector of the disclosure may also include a portion of an intron of the protein on interest. Also, optionally, the rAAV vector of the disclosure comprises DNA encoding a mutated polypeptide of interest.

[0316] In certain embodiments, the vector comprises a promoter/regulatory sequence that comprises a promiscuous promoter which is capable of driving expression of a heterologous gene to high levels in many different cell types. Such promoters include but are not limited to the cytomegalovirus (CMV) immediate early promoter/enhancer sequences, the Rous sarcoma virus promoter/enhancer sequences and the like. In certain embodiments, the promoter/regulatory sequence in the rAAV vector of the disclosure is the CMV immediate early promoter/enhancer. However, the promoter sequence used to drive expression of the heterologous gene may also be an inducible promoter, for example, but not limited to, a steroid inducible promoter, or may be a tissue specific promoter, such as, but not limited to, the skeletal a-actin promoter which is muscle tissue specific and the muscle creatine kinase promoter/enhancer, and the like.

[0317] In certain embodiments, the rAAV vector of the disclosure comprises a transcription termination signal. While any transcription termination signal may be included in the vector of the disclosure, in certain embodiments, the transcription termination signal is the SV40 transcription termination signal.

[0318] In certain embodiments, the rAAV vector of the disclosure comprises isolated DNA 5 encoding the polypeptide of interest, or a biologically active fragment of the polypeptide of interest. The disclosure should be construed to include any mammalian sequence of the polypeptide of interest, which is either known or unknown. Thus, the disclosure should be construed to include genes from mammals other than humans, which polypeptide functions in a substantially similar manner to the human polypeptide. Preferably, the nucleotide sequence comprising the gene encoding the polypeptide of interest is about 50% homologous, more preferably about 70% homologous, even more preferably about 80% homologous and most preferably about 90% homologous to the gene encoding the polypeptide of interest.

[0319] Further, the disclosure should be construed to include naturally occurring variants or recombinantly derived mutants of wild type protein sequences, which variants or mutants render the polypeptide encoded thereby either as therapeutically effective as full-length polypeptide, or even more therapeutically effective than full-length polypeptide in the gene therapy methods of the disclosure.

[0320] The disclosure should also be construed to include DNA encoding variants which retain the polypeptide's biological activity. Such variants include proteins or polypeptides which have been or may be modified using recombinant DNA technology, such that the protein or polypeptide possesses additional properties which enhance its suitability for use in the methods described herein, for example, but not limited to, variants conferring enhanced stability on the protein in plasma and enhanced specific activity of the protein. Analogs can differ from naturally occurring proteins or peptides by conservative amino acid sequence differences or by modifications which do not affect sequence, or by both. For example, conservative amino acid changes may be made, which although they alter the primary sequence of the protein or peptide, do not normally alter its function.

[0321] The disclosure is not limited to the specific rAAV vector exemplified in the experimental examples; rather, the disclosure should be construed to include any suitable AAV vector, including, but not limited to, vectors based on AAV-1, AAV-3, AAV-4 and AAV-6, and the like. Also included in the disclosure is a method of treating a mammal having a disease or disorder in an amount effective to provide a therapeutic effect.

[0322] The method comprises administering to the mammal an rAAV vector encoding the polypeptide of interest. Preferably, the mammal is a human. Typically, the number of viral vector genomes/mammal which are administered in a single injection ranges from about 1108 to about 51016. Preferably, the number of viral vector genomes/mammal which are administered in a single injection is from about 110.sup.10 to about 110.sup.15; more preferably, the number of viral vector genomes/mammal which are administered in a single injection is from about 510.sup.10 to about 510.sup.15; and, most preferably, the number of viral vector genomes which are administered to the mammal in a single injection is from about 510.sup.10 to about 510.sup.14.

[0323] When the method of the disclosure comprises multiple site simultaneous injections, or several multiple site injections comprising injections into different sites over a period of several hours (for example, from about less than one hour to about two or three hours) the total number of viral vector genomes administered may be identical, or a fraction thereof or a multiple thereof, to that recited in the single site injection method.

[0324] For administration of the rAAV vector of the disclosure in a single site injection, in certain embodiments a composition comprising the virus is injected directly into an organ of the subject (such as, but not limited to, the liver of the subject).

[0325] For administration to the mammal, the rAAV vector may be suspended in a pharmaceutically acceptable carrier, for example, HEPES buffered saline at a pH of about 7.8. Other useful pharmaceutically acceptable carriers include, but are not limited to, glycerol, water, saline, ethanol and other pharmaceutically acceptable salt solutions such as phosphates and salts of organic acids. Examples of these and other pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey). The rAAV vector of the disclosure may also be provided in the form of a kit, the kit comprising, for example, a freeze-dried preparation of vector in a dried salts formulation, sterile water for suspension of the vector/salts composition and instructions for suspension of the vector and administration of the same to the mammal

[0326] The published application, US 2017/0290926Smith et al., the contents of which are incorporated by reference in their entirety herein, describes in detail the process by which AAV vectors are generated, delivered and administered.

RNA Based In Vivo Expression of ENPP1 and ENPP3 Polypeptides

[0327] The present disclosure provides compositions and methods for the production and delivery of recombinant double-stranded RNA molecules (dsRNA that encode ENPP1 or ENPP3 polypeptides described herein. The double stranded RNA particle (dsRP) can contain a dsRNA molecule enclosed in a capsid or coat protein. The dsRNA molecule can be a viral genome or portion of a genome, which can be derived from a wild-type viral genome. The RNA molecule can encode an RNA-dependent RNA polymerase (RDRP) and a polyprotein that forms at least part of a capsid or coat protein. The RNA molecule can also contain an RNA sub-sequence that encodes an ENPP1 or ENPP3 polypeptides that are translated by the cellular components of a host cell. When the dsRP is transfected into a host cell the sub-sequence can be translated by the cellular machinery of the host cell to produce the ENPP1 or ENPP3 polypeptides.

[0328] In another aspect the disclosure provides a method of producing a protein product in a host cell. The method includes transfecting a host cell with a dsRP having a recombinant double-stranded RNA molecule (dsRNA) and a capsid or coat protein. The RNA molecule can encode an RNA-dependent RNA polymerase and a polyprotein that forms at least part of the capsid or coat protein, and the dsRP can be able to replicate in the host cell. The RNA molecule has at least one RNA sub-sequence that encodes ENPP1 or ENPP3 polypeptides that is translated by cellular components of the host cell.

[0329] In another aspect the disclosure provides an RNA molecule translatable by a host cell. The RNA molecule can be any RNA molecule that encodes the ENPP1 or ENPP3 polypeptides described herein. In one embodiment the RNA molecule encodes an RNA-dependent RNA polymerase and a polyprotein that forms at least part of a capsid or coat protein of a dsRP and, optionally, can have at least one sub-sequence of RNA that encodes an additional protein product.

[0330] Production of dsRP

[0331] A dsRP of the disclosure can also be produced by presenting to a host cell a plasmid or other DNA molecule encoding a dsRP of the disclosure or encoding the genes of the dsRP. The plasmid or DNA molecule containing nucleotide sequences encoding desired protein such as ENPP1 or ENPP3 polypeptide is then transfected into the host cell and the host cell begins producing the dsRP of the disclosure. The dsRP can also be produced in the host cell by presenting to the host cell an RNA molecule encoding the genes of the dsRP. The RNA molecule can be (+)-strand RNA.

[0332] Once the dsRP of the disclosure has been presented to the host cell (or a plasmid encoding the genes of the dsRP of the disclosure, or an RNA molecule encoding the genes of the dsRP), the dsRP will be produced within the host cell using the cellular components of the host cell. The dsRP of the disclosure is therefore self-sustaining within the host cell and is propagated within the host cell. The host cell can be any suitable host cell such as, for example, a eukaryotic cell, a mammalian cell, a fungal cell, a bacterial cell, an insect cell, or a yeast cell. The host cell can propagate a recombinant dsRP after a recombinant dsRNA molecule of the disclosure or a DNA molecule encoding a dsRP of the disclosure is presented to and taken up by the host cell.

[0333] Methods of Producing a dsRNA Virus or dsRP

[0334] The disclosure also provides methods of producing a dsRP of the disclosure. A double-stranded or single-stranded RNA or DNA molecule can be presented to a host cell. The amplification of the dsRNA molecules in the host cell utilizes the natural production and assembly processes already present in many types of host cells (e.g., yeast). The disclosure can thus be applied by presenting to a host cell a single-stranded or double-stranded RNA or DNA molecule of the disclosure, which is taken up by the host cell and is utilized to produce the recombinant dsRP and protein or peptide encoded by the RNA sub-sequence using the host cell's cellular components. The disclosure can also be applied by providing to the host cell a linear or circular DNA molecule (e.g., a plasmid or vector) containing one or more sequences coding for an RNA-dependent RNA polymerase, a polyprotein that forms at least part of the capsid or coat protein of the dsRP, and a sub-sequence encoding the protein of interest such as ENPP1 or ENPP3 polypeptides as disclosed herein.

[0335] The presentation of a dsRNA or ssRNA molecule of the disclosure can be performed in any suitable way such as, for example, by presenting an RNA molecule of the disclosure directly to the host cell as naked or unmodified single-stranded or double-stranded RNA. The RNA molecule can be transfected (or transformed) into a yeast, bacterial, or mammalian host cell by any suitable method, for example by electroporation, exposure of the host cell to calcium phosphate, or by the production of liposomes that fuse with the cell membrane and deposit the viral sequence inside. It can also be performed by a specific mechanism of direct introduction of dsRNA from killer viruses or heterologous dsRNA into the host cell. This step can be optimized using a reporter system, such as red fluorescent protein (RFP), or by targeting a specific constitutive gene transcript within the host cell genome. This can be done by using a target with an obvious phenotype or by monitoring by quantitative reverse transcriptase PCR (RT-PCR).

[0336] In some embodiments a DNA molecule (e.g., a plasmid or other vector) that encodes an RNA molecule of the disclosure is introduced into the host cell. The DNA molecule can contain a sequence coding for the RNA molecule of a dsRP of the disclosure. The DNA molecule can code for an entire genome of the dsRP, or a portion thereof. The DNA molecule can further code for the at least one sub-sequence of RNA that produces the additional (heterologous) protein product. The DNA sequence can also code for gag protein or gag-pol protein, and as well as any necessary or desirable promoters or other sequences supporting the expression and purpose of the molecule. The DNA molecule can be a linear DNA, a circular DNA, a plasmid, a yeast artificial chromosome, or may take another form convenient for the specific application.

[0337] In one embodiment the DNA molecule can further comprise T7 ends for producing concatamers and hairpin structures, thus allowing for propagation of the virus or dsRP sequence in the host cell. The DNA molecule can be transfected or transformed into the host cell and then, using the host cellular machinery, transcribed and thus provide the dsRNA molecule having the at least one sub-sequence of RNA to the host cell. The host cell can then produce the encoded desired ENPP1 or ENPP3 polypeptide. The dsRNA can be packaged in the same manner that a wild-type virus would be, using the host cell's metabolic processes and machinery. The ENPP1 or ENPP3 polypeptide is also produced using the host cell's metabolic processes and cellular components.

[0338] The patent, U.S. Ser. No. 10/266,834 by Brown et al., the contents of which are incorporated by reference in their entirety herein, describes in detail the process by which dsRNA particles that encode polypeptides are generated, delivered and administered

Pharmaceutical Compositions and Formulations

[0339] The disclosure provides pharmaceutical compositions comprising a polypeptide of the disclosure within the methods described herein. Such a pharmaceutical composition is in a form suitable for administration to a subject, or the pharmaceutical composition may further comprise one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these. The various components of the pharmaceutical composition may be present in the form of a physiologically acceptable salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.

[0340] In an embodiment, the pharmaceutical compositions useful for practicing the method of the disclosure may be administered to deliver a dose of between 1 ng/kg/day and 100 mg/kg/day. In other embodiments, the pharmaceutical compositions useful for practicing the disclosure may be administered to deliver a dose of between 1 ng/kg/day and 500 mg/kg/day.

[0341] The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the disclosure will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between about 0.1% and about 100% (w/w) active ingredient.

[0342] Pharmaceutical compositions that are useful in the methods of the disclosure may be suitably developed for inhalational, oral, rectal, vaginal, parenteral, topical, transdermal, pulmonary, intranasal, buccal, ophthalmic, intrathecal, intravenous or another route of administration. Other contemplated formulations include projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immunologically-based formulations. The route(s) of administration is readily apparent to the skilled artisan and depends upon any number of factors including the type and severity of the disease being treated, the type and age of the veterinary or human patient being treated, and the like.

[0343] The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.

[0344] As used herein, a unit dose is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage. The unit dosage form may be for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose.

[0345] The regimen of administration may affect what constitutes an effective amount. For example, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation. In certain embodiments, administration of the compound of the disclosure to a subject elevates the subject's plasma PPi to a level that is close to normal, where a normal level of PPi in mammals is 1-3 M. Close to normal refers to 0 to 1.2 M or 0-40% below or above normal, 30 nM to 0.9 M or 1-30% 15 below or above normal, 0 to 0.6 M or 0-20% below or above normal, or 0 to 0.3 M or 0-10% below or above normal.

[0346] Administration of the compositions of the present disclosure to a patient, such as a mammal, such as a human, may be carried out using known procedures, at dosages and for periods of time effective to treat a disease or disorder in the patient. An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the activity of the particular compound employed; the time of administration; the rate of excretion of the compound; the duration of the treatment; other drugs, compounds or materials used in combination with the compound; the state of the disease or disorder, age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well-known in the medical arts. Dosage regimens may be adjusted to provide the optimum therapeutic response. Dosage is determined based on the biological activity of the therapeutic compound which in turn depends on the half-life and the area under the plasma time of the therapeutic compound curve. The polypeptide according to the disclosure is administered at an appropriate time interval of every 2 days, or every 4 days, or every week or every month so as to achieve a continuous level of plasma PPi that is either close to the normal (1-3 M) level or above (30-50% higher than) normal levels of PPi. Therapeutic dosage of the polypeptides of the disclosure may also be determined based on half-life or the rate at which the therapeutic polypeptide is cleared out of the body. The polypeptide according to the disclosure is administered at appropriate time intervals of either every 2 days, or every 4 days, every week or every month so as to achieve a constant level of enzymatic activity of ENPP1 or ENPP3 polypeptides.

[0347] For example, several divided doses may be administered daily, or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. A non-limiting example of an effective dose range for a therapeutic compound of the disclosure is from about and 50 mg/kg of body weight/per day. In certain embodiments, the effective dose range for a therapeutic compound of the disclosure is from about 50 ng to 500 ng/kg, preferably 100 ng to 300 ng/kg of bodyweight. One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation.

[0348] The compound can be administered to a patient as frequently as several times daily, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every several months or even once a year or less. It is understood that the amount of compound dosed per day may be administered, in non-limiting examples, every day, every other day, every 2 days, every 3 days, every 4 days, or every days. For example, with every other day administration, a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on. The frequency of the dose is readily apparent to the skilled artisan and depends upon any number of factors, such as, but not limited to, the type and severity of the disease being treated, and the type and age of the patient. Actual dosage levels of the active ingredients in the pharmaceutical compositions of this disclosure may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0349] A medical doctor, e.g., physician, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the disclosure employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0350] In certain embodiments, the compositions of the disclosure are administered to the patient in dosages that range from one to five times per day or more. In other embodiments, the compositions of the disclosure are administered to the patient in range of dosages that include, but are not limited to, once every day, every two, days, every three days to once a week, and once every two weeks. The frequency of administration of the various combination compositions of the disclosure varies from subject to subject depending on many factors including, but not limited to, age, disease or disorder to be treated, gender, overall health, and other factors. Thus, the disclosure should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any patient will be determined by the attending physical taking all other factors about the patient into account.

[0351] In certain embodiments, the present disclosure is directed to a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound of the disclosure, alone or in combination with a second pharmaceutical agent; and instructions for using the compound to treat, prevent, or reduce one or more symptoms of a disease or disorder in a patient.

Routes of Administration

[0352] Routes of administration of any of the compositions of the disclosure include inhalational, oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal, and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.

[0353] Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. The formulations and compositions that would be useful in the present disclosure are not limited to the particular formulations and compositions that are described herein.

[0354] Parenteral administration of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intravenous, intraperitoneal, intramuscular, intrasternal injection, and kidney dialytic infusion techniques.

EXAMPLES

[0355] The present disclosure is further exemplified by the following examples. The examples are for illustrative purpose only and are not intended, nor should they be construed as limiting the disclosure in any manner.

Example 1: Efficacy of ENPP1 and ENPP1-Fc Fusion Protein in Mouse Aortic Allografts

[0356] Allograft vasculopathy remains one of the main complications hindering long-term graft survival, thus representing a major risk factor for mortality in patients subjected to solid organ transplantation. The aim of the example is to evaluate the efficacy of an ENPP1-Fc fusion protein or ENPP1 protein in a mouse model for aortic allografts. Therapeutic effects of the ENPP1 or ENPP1-Fc fusion protein are assessed with respect to the ability to inhibit stenosis after solid organ transplant.

[0357] Female DBA/2 (H-2.sup.d) and C57BL/6J (H-2.sup.b) mice ages of 5-6 weeks are used as donor and recipient mice respectively. (Bickerstaff et al, Murine renal allografts: spontaneous acceptance is associated with regulated T cell-mediated immunity, 2001, J. Immunol. 167, 4821-4827). Descending thoracic aortae of DBA/2 mice are transplanted into C57CL/6 mice in the infrarenal position, as already described. (Seppelt et al., Loss of Endothelial Barrier in Marfan Mice (mgR/mgR) Results in Severe Inflammation after Adenoviral Gene Therapy, 2016, PLoS ONE 11, e0148012.)

[0358] Donor mice are euthanized with CO.sub.2. The thoracic cavity is opened, left ventricle is punctured, and the arterial circulatory system is perfused with 5 mL NaCl (4 C., 0.9%). The descending aorta is harvested and transplanted into the recipient mice to create the model for aortic allografts. Alternatively, the entire heart of the donor mice can be harvested and transplanted into the recipient mice as shown in FIG. 2 to create a solid organ transplant mouse model.

[0359] Recipient C57BL/6J mice are anesthetized by inhalation of 5% isoflurane. Novalgin (500 mg/mL; 200 mg/kg body weight) and Carprieve (50 mg/mL carprofen, 5 mg/kg body weight) are injected intraperitoneally. The abdominal cavity of recipient mice is opened and the infrarenal aorta was dissected. Titanium clips are applied, and the aorta was transected. Grafts are connected to recipient aorta with two end-to-end anastomoses (Prolene 11-0, nylon black, S&T, Neuhausen, Switzerland). After removal of the clips the graft was re-perfused. (Remes et al., Molecular Therapy: Methods & Clinical Development Vol. 15 Dec. 2019).

[0360] A control subset of recipient mice containing the transplanted aorta (n=5) is treated with tris buffered saline and the experimental subset of recipient mice with the transplanted aorta (n=5) is treated with ENPP1 or ENPP1-Fc to determine the effect of ENPP1 or ENPP1-Fc on vascular smooth muscle cell proliferation in the allografts. ENPP1 or ENPP1-Fc treatment (ENPP1 or ENPP1-Fc at 10 mg/kg body weight subcutaneously injected every day) is initiated after the aortic transplant in the experimental mice group and continued for 28 days until the transplanted aorta is harvested. Similarly, the control mice group are treated with Tris buffered saline, pH 7.4 after aortic transplant by subcutaneous injection every day continued for 28 days until the transplanted aorta is harvested. The arteries are then fixed with 4% paraformaldehyde in PBS for morphological analyses.

[0361] Serial sections (sections of 5 m each) are collected. 5-m thick frozen aortic sections (Microtom, HM 500 O) are randomly chosen from various intervals throughout the transplanted grafts and are stained by using Elastica van Gieson stain (Roth, Karlsruhe, Germany). ImageJ software is used to measure the circumference of the external elastic lamina, the internal elastic lamina and the luminal border. Afterward, ImageJ (Fiji version 1.51p, NIH, USA) is used to measure neointimal and medial areas with two investigators blinded toward the treatment regimen. The ratio of the two analyzed parameters is used as a measure of lumen obstruction. The medial area, the intimal area and the intima/media ratio (I/M ratio) were calculated.

[0362] Statistical analyses are performed using Student's t test (unpaired two-sample testing for means). Comparisons of multiple groups used one-way ANOVA, followed by the Bonferroni's post hoc test, are performed with GraphPad Prism software version 7. Probability values of p<0.05 are considered significant. Morphometric analysis shows that control non-treated mice developed vascular lesions and intense remodeling, accompanied by high degrees of vessel lumen obstruction.

[0363] In experimental mice treated with ENPP1 or ENPP1-Fc post transplantation, the degree of intimal hyperplasia is compared to control mice which received no ENPP1 or ENPP1-Fc. Quantitative analyses of sequential sections of transplanted aorta from untreated control mice are expected to exhibit significantly increased neointimal proliferation and this also is compared to ENPP1 or ENPP1-Fc treated mice at or after 28 days post-transplant. Control mice are expected to show thickening of arterial intima and this is compared to treated mice. Correspondingly, the I/M ratios of control and treated mice are compared.

Example 2: Prophylactic Effect of ENPP1-Fc

[0364] The same experiment as described in Example 1 is modified to determine the prophylactic effect of ENPP1 or ENPP1-Fc in preventing or reducing allograft vasculopathy by administering ENPP1 or ENPP1-Fc to the experimental group one week prior to aortic transplantation, as shown in FIG. 1. Likewise, the control group is administered Tris buffered saline a week prior to the aortic transplant. The process is then repeated as above with the experimental group after the transplant being treated with 10 mg/kg dosage of ENPP1 or ENPP1-Fc and control group being treated with Tris buffered saline post-transplant. Morphological analysis is expected to show that the intimal area of experimental mice receiving subcutaneous ENPP1 or ENPP1-Fc is expected to be significantly reduced compared to control mice, whereas the medial area, between the external and internal lamina remains constant. The I/M ratio shows a statistically significant decrease in ENPP1 or ENPP1-Fc treated experimental mice compared to vehicle-treated control mice indicating that the prophylactic treatment of ENPP1 or ENPP1-Fc prior to aortic transplant exhibits a protective effect by lowering the level of VSMC proliferation.

Example 3: Rat Model of Aortic Allograft

[0365] The same experiment as described in Example 1 can be performed using a rat model instead of a mouse model. A rat model for transplantation is described in Bogossian et al. (2016) Cardiovasc Ther 34(4):183. ENPP1 or ENPP1-Fc treated rats and control rats (receiving Tris buffer saline) having aortic allograft transplants are compared at 28 days after transplant surgery.

Example 4: Efficacy of ENPP1 or ENPP1-Fc Fusion Protein in Cardiac Allograft Vasculopathy (CAV) in Swine Heart Transplant Model

[0366] The selection of donor-recipient pairs is based upon major histocompatibility complex incompatibility by mixed lymphocyte reaction (MLR). The stimulation index (SI) is calculated through the following formula: (mean cpm of allogeneic MLR)/(mean cpm of autologous MLR). The donor heart is heterotopically transplanted into the recipient swine abdomen by infrarenal allografting. The selected transplant donors and recipients are anesthetized using Zoletil (tiletamine plus zolazepam, 5 mg/kg), succinylcholine (1.1 mg/kg), and atropine (0.6 mg/kg,), and they are maintained under anesthesia using isoflurane (3%/1.5 L/min) administered through a ventilator after intubation. The recipient is placed in the left decubitus position, and vascular access was established for the administration of immunosuppressive drugs.

[0367] A right flank incision is created, and through a retroperitoneal approach, the infrarenal aorta and inferior vena cava are isolated (See FIG. 3). Next, the donor is heparinized (300 IU/kg intravenous injection (i.v.), and the donor heart is harvested after cardiac standstill is achieved using cold (4 C.) cardioplegic solution. An atrial septal defect was created in each donor heart, and the mitral valve is defunctionalized to minimize left ventricular atrophy and intracavitary thrombus formation. The recipient is heparinized (300 IU/kg i.v.), and the donor's pulmonary artery is anastomosed end-to-side to a 1 to 2 cm venotomy in the inferior vena cava with a continuous 5-0 polypropylene suture. Subsequently, the ascending aorta of the donor heart is anastomosed to the recipient's abdominal aorta in a similar manner, followed by the administration of protamine (1.5 mg/kg;) to stop bleeding. (Hsu et al., Transplantation. 2018 December; 102(12): 2002-2011.)

[0368] The beating rate of cardiac allograft was monitored daily through palpation, and electrocardiography is performed twice per week. When the beating rate of the allograft decreased, echocardiography is performed to assess systolic function. Follow-up is continued to the time of allograft arrest or the study end date (150 days).

[0369] A control subset of recipient pigs containing the transplanted heart (n=12) is treated with tris buffered saline and the experimental subset of recipient pigs with the transplanted heart (n=12) is treated with ENPP1 or ENPP1-Fc to determine the effect of ENPP1 on vascular smooth muscle cell proliferation in the solid organ transplants. ENPP1 or ENPP1-Fc treatment (ENPP1-Fc or ENPP1 at 10 mg/kg body weight subcutaneously injected every four days) is initiated after the heart transplant in the experimental pigs group and continued for 150 days until the transplanted heart is harvested. Similarly, the control pig group are treated with Tris buffered saline, pH 7.4 after heart transplant by intraperitoneal injection every 4 days is continued for 150 days until the transplanted heart is harvested.

[0370] Formalin-fixed cardiac specimens are embedded in paraffin, cross-sectioned, deparaffinized, rehydrated, and then subjected to hematoxylin and eosin (HE) or orcein staining. Intimal hyperplasia of the vascular grafts is examined using a Zeiss microscope and determined from computer images of orcein-stained cross sections. The area surrounded by the internal elastic lamina (IELA) and the luminal area (LA) are calculated using an image analysis program (Image J, Version 1.46r, NIH Image). The severity of intimal hyperplasia is calculated using the following formula: [(IELALA)/IELA]100%. After calculation, the severity of intimal hyperplasia for each graft is evaluated in 3 randomly chosen fields per coronary section for 5 cross sections in a blinded manner, and the evaluated severity levels are averaged for statistical analysis.

[0371] Statistical analyses are performed using Student's t test (unpaired two-sample testing for means). Comparisons of multiple groups used one-way ANOVA, followed by the Bonferroni's post hoc test, are performed with GraphPad Prism software version 7. Probability values of p<0.05 are considered significant. Morphometric analysis shows that control non-treated pigs developed vascular lesions and intense remodeling, accompanied by high degrees of vessel lumen obstruction.

[0372] In experimental pigs treated with ENPP1 or ENPP1-Fc post transplantation, the degree of intimal hyperplasia is determined for control and ENPP1 or ENPP1-treated pigs by performing quantitative and qualitative analyses of sequential sections. Control pigs are expected to exhibit significantly increased neointimal proliferation at 150 days post-transplant. Control pigs are expected to show thickening of arterial intima and treated pigs are compared to control. Correspondingly, the I/M ratios of control and treated pigs are compared. Median survival time also is determined for the control and ENPP1 or ENPP1-treated groups. Graft survival time also is determined for control and ENPP1 or ENPP1-treated groups.

Example 5: Efficacy of ENPP3 or ENPP3-Fc Fusion Protein in Mouse Aortic Allografts

[0373] Female DBA/2 (H-2.sup.d) and C57BL/6J (H-2.sup.b) mice ages of 5-6 weeks are used as donor and recipient mice respectively. (Bickerstaff et al, Murine renal allografts: spontaneous acceptance is associated with regulated T cell-mediated immunity, 2001, J. Immunol. 167, 4821-4827). Descending thoracic aortae of DBA/2 mice are transplanted into C57CL/6 mice in the infrarenal position, as already described. (Seppelt et al., Loss of Endothelial Barrier in Marfan Mice (mgR/mgR) Results in Severe Inflammation after Adenoviral Gene Therapy, 2016, PLoS ONE 11, e0148012.)

[0374] Donor mice are euthanized with CO.sub.2. The thoracic cavity is opened, left ventricle is punctured, and the arterial circulatory system is perfused with 5 mL NaCl (4 C., 0.9%). The descending aorta is harvested and transplanted into the recipient mice to create the model for aortic allografts. Alternatively, the entire heart of the donor mice can be harvested and transplanted into the recipient mice as shown in FIG. 2 to create a solid organ transplant mouse model.

[0375] Recipient C57BL/6J mice are anesthetized by inhalation of 5% isoflurane. Novalgin (500 mg/mL; 200 mg/kg body weight) and Carprieve (50 mg/mL carprofen, 5 mg/kg body weight) are injected intraperitoneally. The abdominal cavity of recipient mice is opened and the infrarenal aorta was dissected. Titanium clips are applied, and the aorta was transected. Grafts are connected to recipient aorta with two end-to-end anastomoses (Prolene 11-0, nylon black, S&T, Neuhausen, Switzerland). After removal of the clips the graft was re-perfused. (Remes et al., Molecular Therapy: Methods & Clinical Development Vol. 15 Dec. 2019).

[0376] A control subset of recipient mice containing the transplanted aorta (n=5) is treated with tris buffered saline and the experimental subset of recipient mice with the transplanted aorta (n=5) is treated with ENPP3 or ENPP3-Fc to determine the effect of ENPP3-Fc on vascular smooth muscle cell proliferation in the allografts. ENPP3-Fc treatment (ENPP3-Fc at 10 mg/kg body weight subcutaneously injected every day) is initiated after the aortic transplant in the experimental mice group and continued for 28 days until the transplanted aorta is harvested. Similarly, the control mice group are treated with Tris buffered saline, pH 7.4 after aortic transplant by subcutaneous injection every day continued for 28 days until the transplanted aorta is harvested. The arteries are then fixed with 4% paraformaldehyde in PBS for morphological analyses.

[0377] Serial sections (sections of 5 m each) are collected. 5-m thick frozen aortic sections (Microtom, HM 500 O) are randomly chosen from various intervals throughout the transplanted grafts and are stained by using Elastica van Gieson stain (Roth, Karlsruhe, Germany). ImageJ software is used to measure the circumference of the external elastic lamina, the internal elastic lamina and the luminal border. Afterward, ImageJ (Fiji version 1.51p, NIH, USA) is used to measure neointimal and medial areas with two investigators blinded toward the treatment regimen. The ratio of the two analyzed parameters is used as a measure of lumen obstruction. The medial area, the intimal area and the intima/media ratio (UM ratio) were calculated.

[0378] Statistical analyses are performed using Student's t test (unpaired two-sample testing for means). Comparisons of multiple groups used one-way ANOVA, followed by the Bonferroni's post hoc test, are performed with GraphPad Prism software version 7. Probability values of p<0.05 are considered significant. Morphometric analysis shows that control non-treated mice developed vascular lesions and intense remodeling, accompanied by high degrees of vessel lumen obstruction.

[0379] In experimental mice treated with ENPP3 or ENPP3-Fc post transplantation, the degree of intimal hyperplasia is compared to control mice which received no ENPP3 or ENPP3-Fc. Quantitative analyses of sequential sections of transplanted aorta from untreated control mice are expected to exhibit significantly increased neointimal proliferation and this also is compared to ENPP3 or ENPP3-Fc treated mice at or after 28 days post-transplant. Control mice are expected to show thickening of arterial intima and this is compared to treated mice. Correspondingly, the TIM ratios of control and treated mice are compared.

Example 6: Prophylactic Effect of ENPP3 or ENPP3-Fc

[0380] The same experiment as described in Example 5 is modified to determine the prophylactic effect of ENPP3 or ENPP3-Fc in preventing or reducing allograft vasculopathy by administering ENPP3 or ENPP3-Fc to the experimental group one week prior to aortic transplantation, as shown in FIG. 1. Likewise, the control group is administered Tris buffered saline a week prior to the aortic transplant. The process is then repeated as above with the experimental group after the transplant being treated with 10 mg/kg dosage of ENPP3 or ENPP3-Fc and control group being treated with Tris buffered saline post-transplant. Morphological analysis is expected to show that the intimal area of experimental mice receiving subcutaneous ENPP3 or ENPP3-Fc is expected to be significantly reduced compared to control mice, whereas the medial area, between the external and internal lamina remains constant. The FM ratio shows a statistically significant decrease in ENPP3 or ENPP3-Fc treated experimental mice compared to vehicle-treated control mice indicating that the prophylactic treatment of ENPP3 or ENPP3-Fc prior to aortic transplant exhibits a protective effect by lowering the level of VSMC proliferation.

Example 7: Rat Model of Aortic Allograft

[0381] The same experiment as described in Example 5 can be performed using a rat model instead of a mouse model. A rat model for transplantation is described in Bogossian et al. (2016) Cardiovasc Ther 34(4):183. ENPP3 or ENPP3-Fc treated rats and control rats (receiving Tris buffer saline) having aortic allograft transplants are compared at 28 days after transplant surgery.

Example 8: Efficacy of ENPP3-Fc Fusion Protein in Cardiac Allograft Vasculopathy (CAV) in Swine Heart Transplant Model

[0382] CAV remains the leading cause of allograft failure 1 year after transplantation. Cardiac allograft vasculopathy manifests as accelerated, diffuse coronary arteriosclerosis that has different pathogenesis than conventional native coronary artery disease (CAD). The efficacy of an ENPP3 or ENPP3-Fc fusion protein is evaluated in a large animal model of an organ transplantspecifically, heart transplant of domestic (Yorkshire) swine. Therapeutic effects of the ENPP3 or ENPP3-Fc fusion protein were assessed with respect to the ability to inhibit stenosis after heart transplant in Yorkshire swine.

[0383] The selection of donor-recipient pairs is based upon major histocompatibility complex incompatibility by mixed lymphocyte reaction (MLR). The stimulation index (SI) is calculated through the following formula: (mean cpm of allogeneic MLR)/(mean cpm of autologous MLR). The donor heart is heterotopically transplanted into the recipient swine abdomen by infrarenal allografting. The selected transplant donors and recipients are anesthetized using Zoletil (tiletamine plus zolazepam, 5 mg/kg), succinylcholine (1.1 mg/kg), and atropine (0.6 mg/kg,), and they are maintained under anesthesia using isoflurane (3%/1.5 L/min) administered through a ventilator after intubation. The recipient is placed in the left decubitus position, and vascular access was established for the administration of immunosuppressive drugs.

[0384] A right flank incision is created, and through a retroperitoneal approach, the infrarenal aorta and inferior vena cava are isolated (See FIG. 3). Next, the donor is heparinized (300 IU/kg intravenous injection (i.v.), and the donor heart is harvested after cardiac standstill is achieved using cold (4 C.) cardioplegic solution. An atrial septal defect was created in each donor heart, and the mitral valve is defunctionalized to minimize left ventricular atrophy and intracavitary thrombus formation. The recipient is heparinized (300 IU/kg i.v.), and the donor's pulmonary artery is anastomosed end-to-side to a 1 to 2 cm venotomy in the inferior vena cava with a continuous 5-0 polypropylene suture. Subsequently, the ascending aorta of the donor heart is anastomosed to the recipient's abdominal aorta in a similar manner, followed by the administration of protamine (1.5 mg/kg;) to stop bleeding. (Hsu et al., Transplantation. 2018 December; 102(12): 2002-2011.)

[0385] The beating rate of cardiac allograft was monitored daily through palpation, and electrocardiography is performed twice per week. When the beating rate of the allograft decreased, echocardiography is performed to assess systolic function. Follow-up is continued to the time of allograft arrest or the study end date (150 days).

[0386] A control subset of recipient pigs containing the transplanted heart (n=12) is treated with tris buffered saline and the experimental subset of recipient pigs with the transplanted heart (n=12) is treated with ENPP3 or ENPP3-Fc to determine the effect of ENPP3 polypeptides on vascular smooth muscle cell proliferation in the solid organ transplants. ENPP3 or ENPP3-Fc treatment (ENPP3 or ENPP3-Fc at 10 mg/kg body weight subcutaneously injected every four days) is initiated after the heart transplant in the experimental pigs group and continued for 150 days until the transplanted heart is harvested. Similarly, the control pig group are treated with Tris buffered saline, pH 7.4 after heart transplant by intraperitoneal injection every 4 days is continued for 150 days until the transplanted heart is harvested.

[0387] Formalin-fixed cardiac specimens are embedded in paraffin, cross-sectioned, deparaffinized, rehydrated, and then subjected to hematoxylin and eosin (HE) or orcein staining. Intimal hyperplasia of the vascular grafts is examined using a Zeiss microscope and determined from computer images of orcein-stained cross sections. The area surrounded by the internal elastic lamina (IELA) and the luminal area (LA) are calculated using an image analysis program (Image J, Version 1.46r, NIH Image). The severity of intimal hyperplasia is calculated using the following formula: [(IELALA)/IELA]100%. After calculation, the severity of intimal hyperplasia for each graft is evaluated in 3 randomly chosen fields per coronary section for 5 cross sections in a blinded manner, and the evaluated severity levels are averaged for statistical analysis.

[0388] Statistical analyses are performed using Student's t test (unpaired two-sample testing for means). Comparisons of multiple groups used one-way ANOVA, followed by the Bonferroni's post hoc test, are performed with GraphPad Prism software version 7. Probability values of p<0.05 are considered significant. Morphometric analysis shows that control non-treated pigs developed vascular lesions and intense remodeling, accompanied by high degrees of vessel lumen obstruction.

[0389] In experimental pigs treated with ENPP3 or ENPP3-Fc post transplantation, the degree of intimal hyperplasia is determined for control and ENPP3-treated pigs by performing quantitative and qualitative analyses of sequential sections. Control pigs are expected to exhibit significantly increased neointimal proliferation at 150 days post-transplant. Control pigs are expected to show thickening of arterial intima and treated pigs are compared to control. Correspondingly, the TIM ratios of control and treated pigs are compared. Median survival time also is determined for the control and ENPP3-treated groups. Graft survival time also is determined for control and ENPP3-treated groups.

Example 9: Efficacy of ENPP1-Fc Fusion Protein in In-Stent Restenosis Model

[0390] The efficacy of an ENPP1-Fc fusion protein was evaluated in large animal model of peripheral vascular injuryspecifically, in-stent restenosis lesions in the peripheral vasculature of domestic (Yorkshire) swine. Therapeutic effects of the ENPP1-Fc fusion protein were assessed with respect to the ability to inhibit stenosis after angioplasty in previously injured and stented peripheral arteries of Yorkshire swine.

[0391] Four peripheral arterial sites were created for induction of neointimal response in each animal; one site was selected in each of four arteries (bilateral profunda and superficial femoral arteries).

[0392] All target sites were injured on Day 0 to create the in-stent restenosis model, 10 days prior to the first dose of ENPP1-Fc or a vehicle only control, and 14 days before repeat injury. The four peripheral artery sites were mapped using quantitative vascular angiography (QVA) in order to select the treatment site and correctly sized balloon and stent. The injury was created by overstretch of the artery with a standard angioplasty balloon catheter at a target 130% overstretch; three inflations were performed. Immediately following injury, a bare metal stent was deployed. Peripheral stents were self-expandable, targeting approximately a 120% overstretch.

[0393] ENPP1-Fc treatment occurred systemically starting on Day 10 and was dosed every 4 days subcutaneously until termination. On Day 14, all vessels were assessed by angiography and Optical Coherence Tomography (OCT). Then the previously injured and stented artery sites were subjected to a re-injury event consisting of overstretch of the artery with a single inflation of a standard angioplasty balloon catheter at the same pressure/diameter as the original pre-stent injury was done (130% of the baseline reference diameter). Following re-injury interventions, final post-procedural angiography and OCT were also recorded for select peripheral sites.

[0394] Four weeks following the re-injury event on Day 14, arteries underwent repeat imaging with angiography and endovascular imaging (OCT). The treated peripheral segments were explanted and stored in 10% neutral buffered formalin.

[0395] As shown in FIG. 4, angiography revealed a pronounced narrowing of the profunda at day 42 relative to the morphology of the vessel at day 14 in animals given the vehicle control. By contrast, in animals treated with ENPP1-Fc little visible change in profunda morphology was observed between day 14 and day 42. Similarly, as measured by OCT, pronounced intimal thickening was observed within the profunda at day 42 relative to the morphology of the vessel at day 14 in animals treated with the vehicle control. By contrast, little visible intimal thickening was observed between day 14 and day 42 in the profunda of animals treated with ENPP1-Fc (FIG. 5).

[0396] Tables 1 and 2 (below) summarizes the mean OCT values in all profunda arteries by treatment group.

TABLE-US-00003 TABLE 1 Day 14 Re-Injury, Profunda Lumen Stent Neointimal Neointimal % Area of Area (mm.sup.2) Area (mm.sup.2) Thickness (mm) Area (mm.sup.2) Stenosis ENPP1-Fc 12.10 1.09 14.59 1.24 0.19 0.04 2.49 0.52 17 3 Control 10.82 1.06 13.60 0.82 0.23 0.04 2.78 0.40 21 4

TABLE-US-00004 TABLE 2 Day 42 Termination, Profunda Lumen Stent Neointimal Neointimal % Area of Area (mm.sup.2) Area (mm.sup.2) Thickness (mm) Area (mm.sup.2) Stenosis ENPP1-Fc 12.95 0.70 16.47 0.89 0.27 0.08 3.52 1.07 21 5 Control 9.51 2.24 14.60 1.40 0.45 0.16 5.10 1.34 35 11
As set forth in the Table, the profunda arteries of animals treated with ENPP1-Fc had a higher lumen area at day 42 compared to the vehicle control group. The stent area was similar between both groups. Neointimal thickness and neointimal area were also reduced at day 42 in animals treated with ENPP1-Fc relative to the vehicle control animals. In additional, animals treated with ENPP1-Fc had a markedly lower % area of stenosis as compared to the vehicle control group (see FIG. 6). These data indicate that ENPP1 polypeptides are useful for, among other things, inhibiting the intimal thickening associated with injury of and/or to peripheral vessels:

Example 10: Efficacy of ENPP3-Fc Fusion Protein in In-Stent Restenosis Model

[0397] The efficacy of an ENPP3-Fc fusion protein is evaluated in a large animal model of peripheral vascular injuryspecifically, in-stent restenosis lesions in the peripheral vasculature of domestic (Yorkshire) swine. Therapeutic effects of the ENPP3-Fc fusion protein are assessed with respect to the ability to inhibit stenosis after angioplasty in previously injured and stented peripheral arteries of Yorkshire swine.

[0398] Four peripheral arterial sites are created for induction of neointimal response in each animal; one site is selected in each of four arteries (bilateral profunda and superficial femoral arteries).

[0399] All target sites are injured on Day 0 to create the in-stent restenosis model, 10 days prior to the first dose of ENPP3-Fc or a vehicle only control, and 14 days before repeat injury. The four peripheral artery sites are mapped using quantitative vascular angiography (QVA) in order to select the treatment site and correctly sized balloon and stent. The injury is created by overstretch of the artery with a standard angioplasty balloon catheter at a target 130% overstretch; three inflations are performed. Immediately following injury, a bare metal stent is deployed. Peripheral stents are self-expandable, targeting approximately a 120% overstretch.

[0400] ENPP3-Fc treatment will be systemically starting on Day 10 and dosed every 4 days subcutaneously until termination. On Day 14, all vessels are assessed by angiography and Optical Coherence Tomography (OCT). Then the previously injured and stented artery sites are subjected to a re-injury event consisting of overstretch of the artery with a single inflation of a standard angioplasty balloon catheter at the same pressure/diameter as the original pre-stent injury (130% of the baseline reference diameter). Following re-injury interventions, final post-procedural angiography and OCT are also recorded for select peripheral sites.

[0401] Four weeks following the re-injury event on Day 14, arteries will be subject to repeat imaging with angiography and endovascular imaging (OCT). The treated peripheral segments will be explanted and stored in 10% neutral buffered formalin.

Example 11: Efficacy of ENPP1 or ENPP1-Fc Fusion Protein in MMD Mouse Model

[0402] Moyamoya is a cerebrovascular disorder characterized by progressive stenosis of the intracranial internal carotid arteries leading to both hemorrhagic and ischemic strokes Restriction of blood flow through the ICA leads to eventual development of new blood vessels resembling a puff of smoke (moyamoya in Japanese) in the subcortical region. The aim of the example is to evaluate the efficacy of an ENPP1-Fc fusion protein or ENPP1 for treatment in a mouse model for MMD. Therapeutic effects of the ENPP1-Fc fusion protein or ENPP1 are assessed with respect to the ability to inhibit vascular smooth muscle cell proliferation in MMD and reduce or prevent cerebral occlusions.

[0403] Generation of MMD Phenotype

[0404] C57Bl/6 male mice (5-6 weeks old) obtained from Jackson Laboratories are anesthetized with a cocktail of ketamine and xylazine using a weight based ratio. Once the mice are anesthetized their cervical region are shaved, and the mouse is placed in the supine position with their head, forepaws and tail restrained (FIG. 8). With the mouse in the supine position, the shaved area is cleaned with alcohol and betadine. A midline incision is made from the angle of the mandible to the sternum exposing the trachea, common carotid artery (CCA) and bifurcation of the CCA into the internal carotid and external carotid artery (ICA/ECA). A retractor is used to hold the skin and separated salivary glands from impeding the surgical area. To increase the visual field, the sternocleidomastoid (SCM) muscle and the posterior belly of the digastric (PBD) muscle are exposed inferiorly and superiorly, respectively. The tip of a pair of curved forceps is gently placed under the SCM medial to lateral and one length of 40 suture was transferred underneath. The suture is looped around the SCM and secured using tape. This procedure is repeated with the PBD.

[0405] With the ICA isolated, the 60 suture is used as an anchor for coil placement. Fine tipped forceps are used to grasp the coil at one end and place it at an angle to the ICA so that the vessel inserts into the last rung of the coil. With the vessel in the last rung of the coil, the coil is inverted so that it is parallel to the ICA. Using the 60 suture, the vessel is gently rotated around the coil so that a length of vessel is placed in each rung of the coil. Vessel placement is assessed to ensure that it is not skipping a rung; if so, the vessel is uncoiled and repositioned until the coil completely encompassed the vessel. Thus, the MMD phenotype in both control and experimental subset of mice is induced by following the procedures discussed in Roberts et al. ((Roberts et al., Internal carotid artery stenosis: A novel surgical model for moyamoya syndrome, PLoS One. 2018; 13(1): e0191312.)

[0406] A control subset of MMD model mice (n=5) is treated with tris buffered saline and the experimental subset of MMD mice (n=5) is treated with ENPP1 or ENPP1-Fc post induction of MMD phenotype to determine the effect of ENPP1 or ENPP1-Fc on vascular smooth muscle cell proliferation and cerebral occlusions in the brain of the MMD mice. ENPP1 or ENPP1-Fc treatment (ENPP1 or ENPP1-Fc at 10 mg/kg body weight, subcutaneously injected every day) is initiated after the induction of MMD phenotype by surgery as described above in the experimental mice group and ENPP1 or ENPP1-Fc administration is continued for 28 days until the cerebral artery is harvested.

[0407] Similarly, the control mice group are treated with Tris buffered saline, pH 7.4 after the induction of MMD phenotype by surgery as described and Tris buffered saline is administered via subcutaneous injection every day and continued for 28 days until the cerebral artery is harvested. The arteries of both control and experimental group mice with MMD are then fixed with 4% paraformaldehyde in PBS for morphological analyses.

[0408] Visualization of Cerebrovasculature

[0409] To visualize the cerebrovasculature, all animals from each group are perfused with the fluorescent dye Di I (Li et al., Direct labeling and visualization of blood vessels with lipophilic carbocyanine dye Dd. Nat Protoc. 2008; 3(11):17038). Mice are subjected to a transcardial perfusion using a perfusion pump (set to 1 ml/min) to perfuse (room temperature) 5 ml of PBS, immediately followed by 10 ml of Di I working solution and then 10 ml of 10% buffered formalin. Brains are carefully removed from the skull ensuring that the Circle of Willis (CoW) remained intact.

[0410] The extracted brain is then post-fixed overnight at 4 C. with 10% buffered formalin. The brains are then transferred into PBS for long-term storage at 4 C. and protected from light. Fluorescently labelled brains were imaged using a 1microscope (Nikon Eclipse E800/Nikon DS-Ril). Images of the cortical vasculature are taken to examine anastomoses and images of the CoW were used to measure vessel diameter. Image analysis is performed using Nikon NES Analysis software to measure vessel diameter (m).

[0411] Diameter measurements are taken approximately 20 m from the bifurcation of the supraclinoid internal carotid artery, M1 segment of the middle cerebral artery, and the A1 segment of the anterior cerebral artery. Anastomoses analysis is performed by counting the number of anastomoses (circle placed over each connection point on a magnified image) between the ACA and the MCA of both the ipsilateral and contralateral hemispheres. Diameters of the ICA, ACA and MCA vessels are examined by measuring the width of each vessel near the bifurcation point on both the ipsilateral and contralateral sides to determine if there was any difference in size between the experimental and control groups.

[0412] Morphological Analysis

[0413] Serial sections (sections of 5 m each) of cerebral arteries such as MCA, ACA and ICA are collected for both control and experimental group. 5-m thick frozen aortic sections (Microtome, HM 500 O) are stained by using Elastica van Gieson stain (Roth, Karlsruhe, Germany). ImageJ software is used to measure the circumference of the external elastic lamina, the internal elastic lamina and the luminal border. Afterward, ImageJ (Fiji version 1.51p, NIH, USA) is used to measure neointimal and medial areas with two investigators blinded toward the treatment regimen. The ratio of the two analyzed parameters is used as a measure of lumen obstruction. The medial area, the intimal area and the intima/media ratio (FM ratio) were calculated. (See FIG. 2).

[0414] Statistical analyses are performed using Student's t test (unpaired two-sample testing for means). Comparisons of multiple groups used one-way ANOVA, followed by the Bonferroni's post hoc test, are performed with GraphPad Prism software version 7. Probability values of p<0.05 are considered significant. Morphometric analysis shows that the control non-treated mice with MMD phenotype develop vascular lesions, occlusions and narrowing accompanied by high degrees of vessel lumen obstruction.

[0415] In experimental mice treated with ENPP1 or ENPP1-Fc post induction of MMD phenotype, the degree of intimal hyperplasia is compared to control mice which received no ENPP1 or ENPP1-Fc. Quantitative analyses of cerebral arteries from untreated control mice with MMD phenotype are expected to exhibit significantly increased neointimal proliferation and this also is compared to ENPP1 or ENPP1-Fc treated mice at or after 28 days post-surgery. Control mice are expected to exhibit thickening of arterial intima and this is compared to treated mice. Correspondingly, the I/M ratios of control and treated mice are also compared.

Example 12: Prophylactic Effect of ENPP1 or ENPP1-Fc

[0416] The same experiment as described in Example 11 is modified to determine the prophylactic effect of ENPP1 or ENPP1-Fc in preventing or reducing vascular smooth muscle proliferation and cerebral occlusions by administering ENPP1 or ENPP1-Fc to the experimental group one week prior to induction of MMD phenotype, as shown in FIG. 7. Likewise, the control group is administered Tris buffered saline a week prior to induction of MMD phenotype. The process is then repeated as above with the experimental group after surgery being treated with 10 mg/kg dosage of ENPP1 or ENPP1-Fc and control group being treated with Tris buffered saline post-surgery.

[0417] Morphological analysis is expected to show that the intimal area of experimental mice with MMD phenotype receiving subcutaneous ENPP1 or ENPP1-Fc is expected to be significantly reduced compared to control mice, whereas the medial area, between the external and internal lamina remains constant. The I/M ratio is expected to decrease in ENPP1 or ENPP1-Fc treated experimental mice compared to vehicle-treated control mice. The prophylactic treatment of ENPP1 or ENPP1-Fc prior to induction of MMD phenotype is expected to confer protective effect by lowering the level of VSMC proliferation.

Example 13: Efficacy of ENPP3 or ENPP3-Fc Fusion Protein in a Mouse Model of MMD

[0418] Moyamoya is a cerebrovascular disorder characterized by progressive stenosis of the intracranial internal carotid arteries leading to both hemorrhagic and ischemic strokes Restriction of blood flow through the ICA leads to eventual development of new blood vessels resembling a puff of smoke (moyamoya in Japanese) in the subcortical region. The aim of the example is to evaluate the efficacy of an ENPP3-Fc fusion protein or ENPP3 for treatment in a mouse model for MMD. Therapeutic effects of the ENPP3-Fc fusion protein or ENPP3 are assessed with respect to the ability to inhibit vascular smooth muscle cell proliferation in MMD and reduce or prevent cerebral occlusions.

[0419] Generation of MMD Phenotype

[0420] C57Bl/6 male mice (5-6 weeks old) obtained from Jackson Laboratories are anesthetized with a cocktail of ketamine and xylazine using a weight based ratio. Once the mice are anesthetized their cervical region are shaved, and the mouse is placed in the supine position with their head, forepaws and tail restrained (FIG. 8). With the mouse in the supine position, the shaved area is cleaned with alcohol and betadine. A midline incision is made from the angle of the mandible to the sternum exposing the trachea, common carotid artery (CCA) and bifurcation of the CCA into the internal carotid and external carotid artery (ICA/ECA). A retractor is used to hold the skin and separated salivary glands from impeding the surgical area. To increase the visual field, the sternocleidomastoid (SCM) muscle and the posterior belly of the digastric (PBD) muscle are exposed inferiorly and superiorly, respectively. The tip of a pair of curved forceps is gently placed under the SCM medial to lateral and one length of 40 suture was transferred underneath. The suture is looped around the SCM and secured using tape. This procedure is repeated with the PBD.

[0421] With the ICA isolated, the 60 suture is used as an anchor for coil placement. Fine tipped forceps are used to grasp the coil at one end and place it at an angle to the ICA so that the vessel inserts into the last rung of the coil. With the vessel in the last rung of the coil, the coil is inverted so that it is parallel to the ICA. Using the 60 suture, the vessel is gently rotated around the coil so that a length of vessel is placed in each rung of the coil. Vessel placement is assessed to ensure that it is not skipping a rung; if so, the vessel is uncoiled and repositioned until the coil completely encompassed the vessel. Thus, the MMD phenotype in both control and experimental subset of mice is induced by following the procedures discussed in Roberts et al. ((Roberts et al., Internal carotid artery stenosis: A novel surgical model for moyamoya syndrome, PLoS One. 2018; 13(1): e0191312.)

[0422] A control subset of MMD model mice (n=5) is treated with tris buffered saline and the experimental subset of MMD mice (n=5) is treated with ENPP3-Fc or ENPP3 post induction of MMD phenotype to determine the effect of ENPP3-Fc or ENPP3 on vascular smooth muscle cell proliferation and cerebral occlusions in the brain of the MMD mice. ENPP3-Fc treatment (ENPP3 or ENPP3-Fc at 10 mg/kg body weight, subcutaneously injected every day) is initiated after the induction of MMD phenotype by surgery as described above in the experimental mice group and ENPP3-Fc or ENPP3 administration is continued for 28 days until the cerebral artery is harvested.

[0423] Similarly, the control mice group are treated with Tris buffered saline, pH 7.4 after the induction of MMD phenotype by surgery as described and Tris buffered saline is administered via subcutaneous injection every day and continued for 28 days until the cerebral artery is harvested. The arteries of both control and experimental group mice with MMD are then fixed with 4% paraformaldehyde in PBS for morphological analyses.

[0424] Visualization of Cerebrovasculature

[0425] To visualize the cerebrovasculature, all animals from each group are perfused with the fluorescent dye Di I (Li et al., Direct labeling and visualization of blood vessels with lipophilic carbocyanine dye DiI. Nat Protoc. 2008; 3(11): 17038). Mice are subjected to a transcardial perfusion using a perfusion pump (set to 1 ml/min) to perfuse (room temperature) 5 ml of PBS, immediately followed by 10 ml of Di I working solution and then 10 ml of 10% buffered formalin. Brains are carefully removed from the skull ensuring that the Circle of Willis (CoW) remained intact.

[0426] The extracted brain is then post-fixed overnight at 4 C. with 10% buffered formalin. The brains are then transferred into PBS for long-term storage at 4 C. and protected from light. Fluorescently labelled brains were imaged using a 1microscope (Nikon Eclipse E800/Nikon DS-Ril). Images of the cortical vasculature are taken to examine anastomoses and images of the CoW were used to measure vessel diameter. Image analysis is performed using Nikon NES Analysis software to measure vessel diameter (m).

[0427] Diameter measurements are taken approximately 20 m from the bifurcation of the supraclinoid internal carotid artery, M1 segment of the middle cerebral artery, and the A1 segment of the anterior cerebral artery. Anastomoses analysis is performed by counting the number of anastomoses (circle placed over each connection point on a magnified image) between the ACA and the MCA of both the ipsilateral and contralateral hemispheres. Diameters of the ICA, ACA and MCA vessels are examined by measuring the width of each vessel near the bifurcation point on both the ipsilateral and contralateral sides to determine if there was any difference in size between the experimental and control groups.

[0428] Measurements of the distal ICA and proximal ACA in control mice with MMD phenotype are expected to exhibit severe narrowing of vessel diameter post-surgery, and this is compared with the vessel diameter of ENPP3 or ENPP3-Fc treated mice with MMD phenotype.

[0429] Morphological Analysis

[0430] Serial sections (sections of 5 m each) of cerebral arteries such as MCA, ACA and ICA are collected for both control and experimental group. 5-m thick frozen aortic sections (Microtome, HM 500 O) are stained by using Elastica van Gieson stain (Roth, Karlsruhe, Germany). ImageJ software is used to measure the circumference of the external elastic lamina, the internal elastic lamina and the luminal border. Afterward, ImageJ (Fiji version 1.51p, NIH, USA) is used to measure neointimal and medial areas with two investigators blinded toward the treatment regimen. The ratio of the two analyzed parameters is used as a measure of lumen obstruction. The medial area, the intimal area and the intima/media ratio (FM ratio) were calculated. (See FIG. 2).

[0431] Statistical analyses are performed using Student's t test (unpaired two-sample testing for means). Comparisons of multiple groups used one-way ANOVA, followed by the Bonferroni's post hoc test, are performed with GraphPad Prism software version 7. Probability values of p<0.05 are considered significant. Morphometric analysis shows that the control non-treated mice with MMD phenotype develop vascular lesions, occlusions and narrowing accompanied by high degrees of vessel lumen obstruction.

[0432] In experimental mice treated with ENPP3 or ENPP3-Fc post induction of MMD phenotype, the degree of intimal hyperplasia is compared to control mice which received no ENPP3 or ENPP3-Fc. Quantitative analyses of cerebral arteries from untreated control mice with MMD phenotype are expected to exhibit significantly increased neointimal proliferation and this also is compared to ENPP3 or ENPP3-Fc treated mice at or after 28 days post-surgery. Control mice are expected to exhibit thickening of arterial intima and this is compared to treated mice. Correspondingly, the I/M ratios of control and treated mice are also compared.

Example 14: Prophylactic Effect of ENPP3 or ENPP3-Fc

[0433] The same experiment as described in Example 13 is modified to determine the prophylactic effect of ENPP3 or ENPP3-Fc in preventing or reducing vascular smooth muscle proliferation and cerebral occlusions by administering ENPP3 or ENPP3-Fc to the experimental group one week prior to induction of MMD phenotype, as shown in FIG. 7. Likewise, the control group is administered Tris buffered saline a week prior to induction of MMD phenotype. The process is then repeated as above with the experimental group after surgery being treated with 10 mg/kg dosage of ENPP3 or ENPP3-Fc and control group being treated with Tris buffered saline post-surgery.

[0434] Morphological analysis is expected to show that the intimal area of experimental mice with MMD phenotype receiving subcutaneous ENPP3 or ENPP3-Fc is expected to be significantly reduced compared to control mice, whereas the medial area, between the external and internal lamina remains constant. The I/M ratio is expected to decrease in ENPP3 or ENPP3-Fc treated experimental mice compared to vehicle-treated control mice. The prophylactic treatment of ENPP3 or ENPP3-Fc prior to induction of MMD phenotype is expected to confer protective effect by lowering the level of VSMC proliferation.

Example 15: Efficacy of ENPP1 or ENPP1-Fc Fusion Protein in a Mouse Model of AV Fistula

[0435] Failure

[0436] The efficacy of an ENPP1 or ENPP1-Fc fusion protein is evaluated in a mouse model of arterio-venous fistula failure as described in, e.g., Wong et al. (2014) J Vasc Surg 59:192-201. Unilateral AVFs are created between the external jugular vein and common carotid artery in male C57bl6 mice. The mice are divided into four cohorts: (1) mice that receive chronic subcutaneous treatment with an ENPP1-Fc fusion protein or ENPP1 prior to and after the AVF is created; (2) mice that receive a vehicle control treatment subcutaneously prior to and after the AVF is created; (3) mice that begin chronic subcutaneous treatment with an ENPP1-Fc fusion protein or ENPP1 following AVF creation; and (4) mice that receive a vehicle control treatment subcutaneously after the AVF creation.

[0437] The mice are followed over time and euthanized at various time points (such as one, two, and/or three weeks after AVF creation). Histological analysis is performed on sections of blood vessels at or promixal to the site of AVF.

[0438] It is anticipated that the extent of intimal hyperplasia in the AVF adjacent vessels of mice treated with ENPP1-Fc fusion protein will be markedly reduced as compared to those mice receiving the vehicle control.

Example 16: Efficacy of ENPP3 or ENPP3-Fc Fusion Protein in a Mouse Model of AV Fistula Failure

[0439] The efficacy of an ENPP3-Fc fusion protein or ENPP3 is evaluated in a mouse model of arterio-venous fistula failure as described in, e.g., Wong et al. (2014) J Vasc Surg 59:192-201. Unilateral AVFs are created between the external jugular vein and common carotid artery in male C57bl6 mice. The mice are divided into four cohorts: (1) mice that receive chronic subcutaneous treatment with an ENPP3-Fc fusion protein or ENPP3 prior to and after the AVF is created; (2) mice that receive a vehicle control treatment subcutaneously prior to and after the AVF is created; (3) mice that begin chronic subcutaneous treatment with an ENPP3-Fc fusion protein or ENPP3 following AVF creation; and (4) mice that receive a vehicle control treatment subcutaneously after the AVF creation.

[0440] The mice are followed over time and euthanized at various time points (such as one, two, and/or three weeks after AVF creation). Histological analysis is performed on sections of blood vessels at or proximal to the site of AVF.

[0441] It is anticipated that the extent of intimal hyperplasia in the AVF adjacent vessels of mice treated with ENPP3-Fc fusion protein will be markedly reduced as compared to those mice receiving the vehicle control.

Example 17: Treatment of a Human Cardiac Transplant Patient Suffering from Cardiac Allograft Vasculopathy

[0442] A human adult heart allograft recipient is identified by a medical practitioner as having CAV. The recipient administers or is administered chronically a pharmaceutical composition comprising a fusion protein comprising a soluble form of ENPP1 fused to a Fc region. Medical professionals monitor the recipient over time for cessation of unwanted intimal proliferation in one or more vessels of the allografted heart and/or partial or full resolution over time of vessel occlusion in the allografted heart. Treatment with the fusion protein is expected to halt or substantially reduce unwanted intimal proliferation in one or more vessels of the allografted heart and/or partially or fully resolve over time vessel occlusion in the allografted heart.

[0443] In another example, a pharmaceutical composition comprising a fusion protein comprising a soluble form of ENPP1 fused to a Fc region is chronically administered to the recipient of a cardiac allograft beginning at or around the time of transplantation to prevent, lessen the likelihood of occurrence of, or reduce the extent of unwanted intimal proliferation in one or more vessels of the allografted heart. Medical professionals monitor the recipient over time for the presence and/or level of unwanted intimal proliferation in one or more vessels of the allografted heart. Treatment with the fusion protein is expected to halt or substantially reduce unwanted intimal proliferation in one or more vessels of the allografted heart.

Example 18: Treatment of a Human Suffering from MoyaMoya Disease

[0444] A human adult patient is identified by a medical practitioner as having Moyamoya disease. The recipient administers or is administered chronically a pharmaceutical composition comprising a fusion protein comprising a soluble form of ENPP1 fused to a Fc region. Medical professionals monitor the recipient over time for cessation of unwanted intimal proliferation in one or more vessels feeding the brain and/or partial or full resolution over time of the occlusion of such vessel or vessels. Treatment with the fusion protein is expected to halt or substantially reduce unwanted intimal proliferation in one or more vessels and/or partially or fully resolve over time vessel occlusion.

Example 19: Treatment of a Dialysis Patient Who has Received a Hemodialysis Shunt

[0445] A pharmaceutical composition comprising a fusion protein comprising a soluble form of ENPP1 fused to a Fc region is chronically administered to a hemodialysis patient at or around the time that a hemodialysis shunt is placed in the subject to thereby prevent, lessen the likelihood of occurrence of, or reduce the extent of unwanted intimal proliferation in one or more vessels connected to or involved in the shunt. Medical professionals monitor the recipient over time for the presence and/or level of unwanted intimal proliferation in one or more of the vessels. Treatment with the fusion protein is expected to halt or substantially reduce unwanted intimal proliferation in one or more of the vessels.

INCORPORATION BY REFERENCE

[0446] The disclosure of each and every U.S. and foreign patent and pending patent application and publication referred to herein is specifically incorporated herein by reference in its entirety, as are the contents of Sequence Listing and Figures.

EQUIVALENTS

[0447] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents of the specific embodiments described herein. Such equivalents are intended to be encompassed by the following claims. Any combination of the embodiments disclosed in the any plurality of the dependent claims or Examples is contemplated to be within the scope of the disclosure.

Other Embodiments

[0448] From the foregoing description, it will be apparent that variations and modifications may be made to the disclosure described herein to adopt it to various usages and conditions, including the use of different signal sequences to express functional variants of ENPP1 or ENPP3 or combinations thereof in different viral vectors having different promoters or enhancers or different cell types known in art to treat any diseases characterized by the presence of pathological calcification or ossification are within the scope according to the disclosure. Other embodiments according to the disclosure are within the following claims.

[0449] Recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or sub combination) of listed elements. Recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[0450] All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this disclosure pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0451] Other embodiments are within the following claims.