ENPP1 polypeptides and methods of use thereof

Abstract

The present invention encompasses ENPP1 polypeptides having an improved half-life in vivo. The present invention further provides an ENPP1 polypeptide fusion comprising an ENPP1 polypeptide fused to the Fc region of an immunoglobulin wherein the polypeptide fusion comprises at least one point mutation.

Description

[0001] This application is a divisional application. The filing date of the original application is August 31, 2019, the application number is (201980070064.X), and the title is "ENPP1 Polypeptides and Methods of Use Thereof".

[0002] Cross - reference to related applications

[0003] According to 35 U.S.C.§119(e), this application claims the priority of U.S. Provisional Patent Application No. 62 / 725,607 filed on August 31, 2018 and U.S. Provisional Patent Application No. 62 / 830,247 filed on April 5, 2019. The entire contents of all these applications are incorporated herein by reference. Background of the invention

[0004] The human ectonucleotide pyrophosphatase (ENPP) protein family consists of seven extracellular glycosylated proteins (i.e., ENPP1 - ENPP7) that hydrolyze phosphodiester bonds. ENPPs are cell - surface enzymes, except for ENPP2, which is exported to the plasma membrane but is cleaved by furin protease and released into the extracellular fluid. ENPP enzymes have a high degree of sequence and structural homology but display diverse substrate specificities ranging from nucleotides to lipids.

[0005] ENPP1 (also known as PC - 1) is a type II extracellular membrane - bound glycoprotein that is located on the mineral - deposition matrix vesicles of osteoblasts and chondrocytes and hydrolyzes extracellular nucleotides (mainly ATP) to adenosine monophosphate (AMP) and inorganic pyrophosphate (PPi). PPi acts as an effective inhibitor of ectopic tissue mineralization: it binds to nascent hydroxyapatite (HA) crystals, thereby preventing the future growth of these crystals. ENPP1 generates PPi through the hydrolysis of nucleoside triphosphate (NTP), progressive ankylosis protein (ANK) transports intracellular PPi to the extracellular space, and tissue - non - specific alkaline phosphatase (TNAP) removes PPi by directly hydrolyzing it to Pi.

[0006] Ectopic tissue mineralization is associated with a variety of human diseases, including chronic joint diseases and acute fatal neonatal syndromes. To prevent harmful tissue calcification, a tight balance must be maintained between factors that promote and inhibit tissue mineralization. The balance of extracellular inorganic pyrophosphate (PPi) and phosphate (Pi) is an important regulator of ectopic tissue mineralization. The activities of three extracellular enzymes, TNAP, ANK, and ENPP1, tightly control the concentrations of Pi and PPi in mammals at 1 - 3 mM and 2 - 3 μM, respectively. PPi is a regulator of biomineralization that inhibits the formation of basic calcium phosphate from amorphous calcium phosphate.

[0007] ENPP1 polypeptides have been shown to be effective in treating certain ectopic tissue calcification diseases. ENPP1-Fc has been shown to reduce systemic arterial calcification in a mouse model of GACI (generalized arterial calcification of infancy), a severe disease that occurs in infants and involves extensive arterial calcification (Albright et al., 2015, Nature Comm. 10006). Fusion proteins of ENPP1 for treating severe tissue calcification diseases have also been described (PCT application publication numbers WO2014 / 126965 and WO2016 / 187408), and fusion proteins of ENPP1 comprising a bone-targeting domain for treating GACI have been described (PCT application publication number WO / 2012 / 125182).

[0008] There is a need in the art for polypeptides that can be used for in vivo treatment of certain calcification or ossification diseases. Such polypeptides should have an in vivo half-life that allows for convenient and effective administration of the polypeptide to a subject in need. The present invention meets this need. SUMMARY OF THE INVENTION

[0009] The present invention provides an ENPP1 polypeptide fusion comprising an ENPP1 polypeptide fused to the Fc region of an immunoglobulin, wherein the polypeptide fusion comprises at least one point mutation as described herein. The present invention further provides an ENPP1 mutant polypeptide comprising at least one point mutation as described elsewhere herein. The present invention further provides a polypeptide fusion and / or a mutant polypeptide, either of which is expressed by a CHO cell line stably transfected with human ST6β-galactoside α-2,6-sialyltransferase (ST6GAL1). The present invention further provides a polypeptide fusion and / or a mutant polypeptide, either of which is grown in a cell culture supplemented with sialic acid and / or N-acetylmannosamine (1,3,4-O-Bu3ManNAc).

[0010] The present invention further provides a method for reducing and / or preventing the progression of pathological calcification in a subject in need, the method comprising administering to the subject a therapeutically effective amount of the polypeptide fusion and / or mutant polypeptide of the present invention.

[0011] The present invention further provides a method for reducing and / or preventing the progression of pathological ossification in a subject in need, the method comprising administering to the subject a therapeutically effective amount of the polypeptide fusion and / or mutant polypeptide of the present invention.

[0012] The present invention further provides a method for reducing and / or preventing the progression of ectopic calcification of soft tissues in a subject in need, the method comprising administering to the subject a therapeutically effective amount of the polypeptide fusion and / or mutant polypeptide of the present invention.

[0013] The present invention further provides a method for treating, reversing, and / or preventing the progression of ossification of the posterior longitudinal ligament (OPLL) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the polypeptide fusion and / or mutant polypeptide of the present invention.

[0014] The present invention further provides a method for treating, reversing, and / or preventing the progression of hypophosphatemic rickets in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the polypeptide fusion and / or mutant polypeptide of the present invention.

[0015] The present invention further provides a method for reducing and / or preventing the progression of at least one disease in a subject diagnosed with at least one disease selected from the following: chronic kidney disease (CKD), end-stage renal disease (ESRD), calcific uremic arteriolopathy (CUA), calciphylaxis, ossification of the posterior longitudinal ligament (OPLL), hypophosphatemic rickets, osteoarthritis, aging-related arteriosclerosis, idiopathic infantile arterial calcification (IIAC), generalized arterial calcification of infancy (GACI), and atherosclerotic plaque calcification, the method comprising administering to the subject a therapeutically effective amount of the polypeptide fusion and / or mutant polypeptide of the present invention.

[0016] The present invention further provides a method for reducing and / or preventing the progression of aging-related arteriosclerosis in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the polypeptide fusion and / or mutant polypeptide of the present invention.

[0017] The present invention further provides a method for increasing the level of pyrophosphate (PPi) in a subject in which the PPi level is lower than the normal PPi level, the method comprising administering to the subject a therapeutically effective amount of the polypeptide fusion and / or mutant polypeptide of the present invention, whereby after administration, the level of PPi in the subject increases to at least the normal level of 2 μM and is maintained at approximately the same level.

[0018] The present invention further provides a method for reducing and / or preventing the progression of pathological calcification and / or ossification in a subject in which the pyrophosphate (PPi) level is lower than the normal PPi level, the method comprising administering to the subject a therapeutically effective amount of the polypeptide fusion and / or mutant polypeptide of the present invention, whereby pathological calcification and / or ossification in the subject is reduced and / or whereby the progression of pathological calcification and / or ossification in the subject is prevented.

[0019] The present invention further provides a method for treating ENPP1 deficiency manifested by a decrease in the concentration of extracellular pyrophosphate (PPi) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the polypeptide fusion and / or mutant polypeptide of the present invention, thereby increasing the level of PPi in the subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The following detailed description of illustrative embodiments of the invention will be better understood when read in conjunction with the accompanying drawings. For purposes of illustration of the invention, exemplary embodiments are shown in the drawings. However, it is to be understood that the invention is not limited to the precise arrangements and instrumentalities shown in the drawings.

[0021] Figure 1 The ENPP1 polypeptide (SEQ ID NO:7) contemplated in the present invention is shown. Point mutations are identified with reference to SEQ ID NO:7, which may also be referred to as the "parent compound" or "construct #770". The labeling scheme identifies amino acid numbers and residues with reference to the numbering scheme shown in SEQ ID NO:7, followed by the amino acid that has replaced the residue in SEQ ID NO:7. For example, the mutation C25N refers to the substitution of asparagine (Asn or N) for cysteine (Cys or C) at position 25 in SEQ ID NO:7. Legend: (A) = N-terminal signal sequence from hENPP7; all regions in black (B) represent sequences from hENPP1 with no formal domain definition; (C) = somatomedin B domain of hENPP1; (D) = catalytic domain of hENPP1; (E) = endonuclease domain of hENPP1; (F) = Fc domain from the Invivogen plasmid pFUSE-hIgG1-Fc; (G) = four-amino acid linker between hENPP1 and the Fc domain; (H) = known glycosylation residues.

[0022] Figure 2 A bar graph is shown summarizing plasma phosphodiesterase activity (measured using the thymidine 5'-monophosphate p-nitrophenyl ester assay or pNP-TMP assay) in mice (n = 3 - 5) after a single injection of certain ENPP1 polypeptides. Phosphodiesterase activity remained elevated in all polypeptides after 25 hours, and higher activity was observed at 75 hours with construct #981 (constructs of interest are listed in a table included elsewhere herein).

[0023] Figure 3 In vivo pharmacokinetic data for construct #981 are shown, measured using the pNP-TMP assay to record enzyme activity in mouse plasma samples after subcutaneous injection of the construct. Based on a single subcutaneous bolus in 5 mice, the half-life was estimated to be about 122 hours. Separate experiments to reach the half-life are described elsewhere herein.

[0024] Figure 4Shows the in vivo pharmacokinetic data of construct #1014, construct #1014 prepared in CHO cells grown in medium supplemented with 1,3,4-O-Bu3ManNAc (designated as "1014A" in the figure), and construct #981. As described elsewhere herein, the half-life of the construct can be derived from Equation 1.

[0025] Figure 5 Shows three known glycosylation sites in ENPP1, all of which are located in random helical regions: (A) = Asn; (B) = N-acetylglucosamine. An additional glycosylation site (identified by surface glycoprotein kinetics measurements) is located in the α-helix and is marked in red. There is a common NLT (Asn Leu Thr) in the PDB unstable region, and its glycosylation status is unknown. Four additional consensus sequences were found in hENPP1, and their glycosylation status is unknown. Calcium atoms (C); 2 zinc atoms (D); ATP molecule (E).

[0026] Figure 6A Shows certain domains of human ENPP1 that have loss-of-function mutations known to cause the human disease "generalized arterial calcification of infancy" (GACI). In certain embodiments, glycosylation sites are not introduced adjacent to regions with known loss-of-function mutations that cause GACI (as Figure 6A shown).

[0027] Figure 6B Shows the crystal structure of ENPP1, with residues known to cause loss-of-function mutations leading to GACI prominently marked (and labeled with *). The residue in (B) is located in the catalytic domain and corresponds to T238A. As Figure 5 in: calcium atoms (C); 2 zinc atoms (D); ATP molecule (E).

[0028] Figures 7A - 7D Shows the selected results for phosphodiesterase activity from a high-throughput TMP-pNP (thymidine monophosphate p-nitrophenyl ester) assay of the ENPP1 polypeptide. This is a high-throughput assay designed by the inventors to rapidly screen glycosylation isoforms introduced into construct #770. The figure illustrates the design and execution of a high-throughput screen capable of rapidly evaluating the biological efficacy of the parent compound - construct #770 - in mutant forms. The construct numbers in (#) represent the original WT clones before the introduction of mutations. The construct numbers in (*) show clones with possible gain-of-function mutations.

[0029] Figure 8AA ribbon diagram showing the Fc domain of human IgG1. This domain was fused to the C-terminal portion of ENPP1 to improve efficacy. Mutations were introduced into the Fc domain to enhance the pH-dependent recycling of FcRn. (A) = sites that eliminate acidic-dependent binding. (B) = sites that enhance binding. (C) = cysteine disulfide bonds. Magenta = known glycosylation sites. Figure 8B Shows mutations in the Fc domain of human IgG1 that are known to enhance the pH-dependent recycling of FcRn.

[0030] Figure 9 Include figures and tables that show the effect of glycosylation on the PK (in hours, based on half-life) and bioavailability of ENPP1 polypeptides. The PK of all mutants is comparable to that in construct #CC07 (770B). Further, the PK value of construct #951 is similar to that of construct #CC07, but construct #951 grown in a cell line stably transfected with ST6GAL1 (construct #951-ST) shows improved PK and bioavailability. Construct #930 has a similar half-life compared to construct #CC07 but lower bioavailability. In contrast, construct #1020 has higher bioavailability than construct #CC07. PK and bioavailability data are given in the table, as Figure 3 、 4 shown in and calculated using Equation 1.

[0031] Figure 10 Include figures and tables that show the effect of glycosylation and the H1064K / N1065F Fc mutation on the half-life (PK, in hours) and bioavailability (AUC) of ENPP1 polypeptides. All constructs containing H1064 / N1065 show improved half-life and AUC values compared to construct #770B. Notably, constructs #1048 and #1051 correspond to the same cDNA in two different clones, demonstrating the reproducibility of the PK / AUC analysis provided herein. Construct #1064 was also grown in a cell line stably transfected with ST6GAL1 (construct #1064-ST). Construct #1057 was also grown in a cell line stably transfected with ST6GAL1 (“-ST”) (construct #1057-ST) and in a cell line stably transfected with ST6GAL1 and supplemented with 1,3,4-O-Bu3-ManNAc (“-A”) (construct #1057-ST-A). Construct #1089 is the same as construct #1014 but with additional mutations to eliminate potential trypsin cleavage sites. Construct #1014 was also grown in a cell line stably transfected with ST6GAL1, but in this case, there was no improvement in PK and bioavailability. PK and bioavailability data are given in the table, as Figure 3 、Figure 4 and Figure 12 determined as shown and calculated using Equation 1.

[0032] Figure 11 including figures and tables that show the effects of glycosylation and the M883Y / S885T / T887E Fc mutation on the PK (in hours, based on half-life) and bioavailability of the ENPP1 polypeptide. The AUC of construct #1030 was lower than that of the other constructs, which may be due to the S766N mutation. When grown in a cell line stably transfected with ST6GAL1, constructs #981 and #1028 showed an increase in both PK and AUC values. PK and bioavailability data are given in the tables as Figure 3 、 Figure 4 and Figure 12 determined as shown and calculated using Equation 1.

[0033] Figure 12 including a set of figures that show the effect of expressing constructs in CHO cells stably transfected with human α-2,6-ST to produce recombinant biologics with terminal sialic acid residues having both α-2,3 and α-2,6 linkages. These cells are referred to as ST6GAL1 cells or ST cells (designated as “-ST”). The figure also shows the effect of growing constructs in ST6GAL1 cells in the presence of sialic acid or a high-throughput sialic acid precursor called 1,3,4-O-Bu3-ManNAc (designated as “-A”). PK and bioavailability data are given in the tables as Figure 3 、 Figure 4 and Figure 12 determined as shown and calculated using Equation 1.

[0034] Figures 13A - 13B shows the domains of ENPP1 and selected point mutations introduced into the parent compound (SEQ ID NO:7). The figure identifies the specific point mutations introduced into SEQ ID NO:7. Constructs that have been stably transfected into CHO cells stably transfected with human α-2,6-ST are referred to as “ST”. PK and bioavailability data are given in the tables as Figure 3 、 Figure 4 and Figure 12 determined as shown and calculated using Equation 1.

[0035] Figure 14 shows the bioavailability (such as area under the curve, or AUC) of certain constructs of the present invention classified by mutation of the signal sequence (N-terminal region) region.

[0036] Figure 15 shows the bioavailability (such as area under the curve, or AUC) of certain constructs of the present invention classified by mutation of the endonuclease region. Detailed implementation mode

[0037] In one aspect, the present invention relates to the discovery that certain ENPP1-Fc derivatives have improved in vivo half-lives compared to ENPP1-Fc polypeptides known in the art.

[0038] In one aspect, glycosylation is promoted to protect the ENPP1-Fc polypeptide from degradation. This is achieved by introducing additional N-glycan consensus sequences onto the outer surface of the predicted tertiary structure under the guidance of a three-dimensional model of ENPP1.

[0039] In another aspect, pH-dependent FcRn-mediated cellular recycling is increased by mutating the Fc domain to enhance the affinity of the fusion protein for the neonatal receptor (FcRn).

[0040] In another aspect, sialylation of the fusion protein is enhanced by expressing ENPP1-Fc in a CHO cell line stably transfected with human ST6β-galactosamide α-2,6-sialyltransferase (also known as ST6GAL1).

[0041] In another aspect, sialic acid capping is enhanced by supplementing the cell culture medium with N-acetylmannosamine (also known as 1,3,4-O-Bu3ManNAc), which is a "high-throughput" precursor of sialic acid.

[0042] In certain embodiments, protein sialylation is enhanced by expressing the biologic in CHO cells stably transfected with human α-2,6-sialyltransferase, which substantially improves the bioavailability of ENPP1-Fc when administered subcutaneously (C max ). In other embodiments, manipulation of the Fc domain to increase pH-dependent FcRn-mediated cellular recycling results in improved in vivo biological half-life. In still other embodiments, binding to CHO cells stably transfected with human α-2,6-sialyltransferase and growing the cells in N-acetylmannosamine results in a significant increase in half-life and / or biological exposure (AUC). In still other embodiments, combining two or more of the methods described herein into a single construct results in a significant increase in half-life and / or biological exposure (AUC).

[0043] In certain embodiments, the polypeptides of the present invention are more highly glycosylated compared to other ENPP1-Fc polypeptides in the art. In other embodiments, the polypeptides of the present invention have a higher affinity for the neonatal orphan receptor (FcRn) compared to other ENPP1-Fc polypeptides in the art. In still other embodiments, the polypeptides of the present invention have a longer in vivo half-life compared to other ENPP1-Fc polypeptides in the art. In still other embodiments, the kinetic properties of the parent compound (Construct #770) are altered such that the change represents a "gain-of-function" change in the enzyme rate constant. In still other embodiments, the in vivo half-life of the ENPP1-Fc polypeptides of the present invention is at least about 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, or 20-fold higher compared to ENPP-1 polypeptides described in the art. In still other embodiments, the polypeptides of the present invention are administered to a subject at a lower dose and / or lower frequency compared to other ENPP1-Fc polypeptides in the art. In still other embodiments, the polypeptides of the present invention are administered to a subject once a month, twice a month, three times a month, and / or four times a month. In still other embodiments, the lower frequency of administration of the polypeptides of the present invention compared to other ENPP1-Fc polypeptides in the art results in better patient compliance and / or increased efficacy.

[0044] In certain embodiments, the ENPP1-Fc polypeptides of the present invention can be used to increase the PPi level in a subject whose pyrophosphate (PPi) level is below the normal level (about 2 μM). In other embodiments, the ENPP1-Fc polypeptides of the present invention can be used to reduce or prevent the progression of pathological calcification or ossification in a subject whose PPi level is below the normal level. In still other embodiments, the ENPP1-Fc polypeptides of the present invention can be used to treat ENPP1 deficiency manifested by a decrease in extracellular PPi concentration in a subject.

[0045] In certain embodiments, the steady-state level of plasma PPi achieved after administration of the first dose of the construct of the present invention is maintained for a period of at least 2 days, at least 4 days, at least one week, or at least one month.

[0046] In certain embodiments, at an appropriate time interval after two days, four days, one week, or one month, a second dose of the construct of the present invention is administered to the subject such that the steady-state level of plasma PPi is maintained at a constant or steady-state level and does not return to the lower level of PPi in the subject before administration of the first dose of the construct of the present invention.

[0047] Without wishing to be bound by theory, it is believed that maintaining the steady-state concentration of plasma PPi at a normal level reduces and / or prevents the progression of pathological calcification and pathological ossification in a subject.

[0048] Certain ENPP1 polypeptides, mutants thereof, or mutant fragments thereof have been previously disclosed in International PCT Application Publication Nos. WO 2012 / 125182, WO 2014 / 126965, WO 2016 / 187408, and WO 2018 / 027024, the entire contents of which are incorporated herein by reference.

[0049] Reference will now be made in detail to certain embodiments of the disclosed subject matter. Although the disclosed subject matter will be described in connection with the recited claims, it should be understood that the illustrated subject matter is not intended to limit the claims to the disclosed subject matter.

[0050] Throughout the document, values expressed in a range format should be interpreted in a flexible manner to include not only the explicitly recited values as the limits of the range, but also all individual values or sub-ranges within that range as if each value and sub-range were explicitly recited. For example, a range of "about 0.1% to about 5%" or "about 0.1% to 5%" should be interpreted to include not only about 0.1% to about 5%, but also the individual values within the specified range (e.g., 1%, 2%, 3%, and 4%) and sub-ranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%). Unless otherwise indicated, the statement "about X to Y" has the same meaning as "about X to about Y". Similarly, unless otherwise indicated, the statement "about X, Y, or about Z" has the same meaning as "about X, about Y, or about Z".

[0051] Definitions

[0052] As used herein, each of the following terms has the meaning associated with it in this section. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures in animal pharmacology, pharmaceutical sciences, separation sciences, and organic chemistry are well known and commonly used in the art. It should be understood that the order of steps or the order of performing certain actions is not important so long as the present teachings remain operable. Any use of section headings is intended to aid in reading the document and should not be construed as restrictive; the information associated with a section heading may occur within or outside of that specific section. All publications, patents, and patent documents cited herein are incorporated herein by reference in their entirety as if each were incorporated by reference individually.

[0053] In this application, where an element or component is considered to be included in and / or selected from a recited listing of elements or components, it should be understood that the element or component can be any one of the recited elements or components and can be selected from two or more of the recited elements or components.

[0054] In the methods described herein, the acts can be performed in any order, unless a temporal or operational sequence is explicitly recited. Additionally, specified acts can be performed concurrently, unless the claim language explicitly states that they are to be performed separately. For example, the claimed act of doing X and the claimed act of doing Y can be performed concurrently in a single operation, and the resulting method will fall within the literal scope of the claimed method.

[0055] As used herein, the terms "a", "an", or "the" are used to include one or more, unless the context clearly dictates otherwise. The term "or" is used to mean non-exclusive "or", unless stated otherwise. The statements "at least one of A and B" or "at least one of A or B" have the same meaning as "A, B, or A and B".

[0056] For clarity, the following symbol convention is applied to this disclosure. In any event, any teachings herein that do not follow this convention are still part of this disclosure and can be fully understood in view of the context of the disclosed teachings. Protein symbols are disclosed in non-italicized capital letters. As a non-limiting example, "ENPP1" refers to a protein. In certain embodiments, if the protein is a human protein, an "h" is used before the protein symbol. In other embodiments, if the protein is a mouse protein, an "m" is used before the symbol. Thus, human ENPP1 is referred to as "hENPP1", while mouse ENPP1 is referred to as "mENPP1". Human gene symbols are disclosed in italicized capital letters. As a non-limiting example, the human gene corresponding to the protein hENPP1 is ENPP1. Mouse gene symbols are disclosed, where the first letter is capitalized and the remaining letters are lower case; further, mouse gene symbols are italicized. As a non-limiting example, the mouse gene that makes the protein mEnpp1 is Enpp1. Symbols for gene mutations are shown in capital text.

[0057] When referring to measurable values such as amounts, time durations, and the like, "about" as used herein means encompassing a variation of ±20% or ±10% of the specified value, in certain embodiments ±5%, in certain embodiments ±1%, and in certain embodiments ±0.1% because such variations are applicable to performing the disclosed methods.

[0058] A disease or disorder is "alleviated" if the severity of the symptoms of the disease or disorder, the frequency with which the patient experiences such symptoms, or both are reduced.

[0059] As used herein, the terms "alteration", "defect", "variant", or "mutation" refer to a mutation in a gene that affects the function, activity, expression (transcription or translation), or conformation of the encoded polypeptide in a cell, including missense and nonsense mutations, insertions, deletions, frameshifts, and premature terminations.

[0060] As used herein, the term "antibody" refers to an immunoglobulin molecule capable of specifically binding to a specific epitope on an antigen. An antibody can be a complete immunoglobulin derived from a natural or recombinant source, or an immunoreactive portion of a complete immunoglobulin.

[0061] The "ATP hydrolysis activity" of ENPP1 can be determined using an ATP cleavage assay. ENPP1 readily hydrolyzes ATP to AMP and PPi. The steady-state Michaelis - Menten enzyme constants of ENPP1 are determined using ATP as a substrate. The cleavage of ATP by ENPP1 can be demonstrated by HPLC analysis of the enzymatic reaction, and the identity of the substrate and reaction products is confirmed using ATP, AMP, and ADP standards. In the presence of ENPP1, the ATP substrate degrades over time with an accumulation of the enzyme product AMP. Using different concentrations of the ATP substrate, the initial rate velocity of ENPP1 is derived in the presence of ATP, and the data are fit to a curve to obtain the enzymatic rate constant. At physiological pH, the kinetic rate constants of NPP1 are K m = 144 μM and k cat = 7.8 s -1 .

[0062] As used herein, the term "AUC" refers to the area under the plasma drug concentration - time curve (AUC) and is related to the actual bodily exposure to a drug after administration of a given dose. In certain embodiments, the AUC is expressed as mg*h / L. The AUC can be used to measure the bioavailability of a drug, which is the fraction of an unchanged drug that is completely absorbed and reaches the site of action or the systemic circulation after administration by any route.

[0063] The linear trapezoidal method or the logarithmic trapezoidal method can be used to calculate the AUC. The linear trapezoidal method uses linear interpolation between data points to calculate the AUC. OGD and FDA require the use of this method, and it is the standard for bioequivalence trials. For a given time interval (t1 - t2), the AUC can be calculated as follows:

[0064]

[0065] where C1 and C2 are the average concentrations within the time interval (t1 and t2).

[0066] The log trapezoidal method uses logarithmic interpolation between data points to calculate the AUC. This method is more accurate when concentrations are decreasing because drug elimination is exponential (which makes it linear on a logarithmic scale). For a given time interval (t1–t2), the AUC can be calculated as follows (assuming C1 > C2):

[0067]

[0068] As used herein, the term "bioavailability" refers to the extent and rate at which the active moiety (protein, drug, or metabolite) enters the systemic circulation and thus reaches the site of action. The bioavailability of the active moiety is largely determined by the nature of the dosage form, which in turn depends in part on its design and manufacture. Differences in bioavailability between formulations of a given drug or protein can be of clinical significance; thus, it is crucial to know whether drug formulations are equivalent. The most reliable measure of the bioavailability of a drug or protein is the area under the plasma concentration-time curve (AUC). The AUC is proportional to the total amount of unchanged drug or therapeutic protein that reaches the systemic circulation. If the plasma concentration curves of a drug or therapeutic protein are substantially superimposable, their extent and rate of absorption can be considered bioequivalent.

[0069] The term "bioavailability" of a drug or therapeutic product is defined as the fraction of the unchanged drug that is completely absorbed and reaches the site of action or the systemic circulation following administration by any route. For an intravenous dose of a drug, the bioavailability is defined as one (unity). For drugs administered by other routes of administration, the bioavailability is usually less than one. Incomplete bioavailability may be due to many factors that can be subdivided into categories of dosage form effects, membrane effects, and site of administration effects. The half-life and AUC provide information about the bioavailability of a drug or biological agent.

[0070] As used herein, the term "conservative variation" or "conservative substitution" refers to the replacement of an amino acid residue with another biologically similar residue. Conservative variations or substitutions are not likely to alter the shape of the peptide chain. Examples of conservative variations or substitutions include replacing one hydrophobic residue with another (e.g., isoleucine, valine, leucine, or methionine), or replacing one polar residue with another, such as replacing arginine with lysine, replacing glutamate with aspartate, or replacing glutamine with asparagine.

[0071] As used herein, a "construct" of the present invention refers to a fusion polypeptide comprising an ENPP1 polypeptide or a fragment or site-directed mutant thereof.

[0072] A "disease" is a health state of an animal in which the animal is unable to maintain homeostasis and in which the animal's health condition will continue to deteriorate if the disease is not ameliorated.

[0073] A "disorder" in an animal is a health state in which the animal is able to maintain homeostasis, but in which the health state of the animal is less favorable compared to the situation without the disorder. If untreated, the disorder does not necessarily lead to a further decline in the animal's health status.

[0074] As used herein, the terms "effective amount", "pharmaceutically effective amount", and "therapeutically effective amount" refer to a dosage of a test substance that is non-toxic but sufficient to provide the desired biological result. The result can be a reduction and / or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. In any individual case, one of ordinary skill in the art can determine the appropriate therapeutic amount using routine experimentation.

[0075] As used herein, the term "ENPP" or "NPP" refers to ectonucleotide pyrophosphatase / phosphodiesterase.

[0076] As used herein, the terms "ENPP1 protein" or "ENPP1 polypeptide" refer to the ectonucleotide pyrophosphatase / phosphodiesterase-1 protein encoded by the ENPP1 gene. The encoded protein is a type II transmembrane glycoprotein and cleaves a variety of substrates, which include the phosphodiester bonds of nucleotides and nucleotide sugars as well as the pyrophosphate bonds of nucleotides and nucleotide sugars. The ENPP1 protein has a transmembrane domain and a soluble extracellular domain. The extracellular domain is further subdivided into a somatomedin B domain, a catalytic domain, and a nuclease domain. The sequence and structure of wild-type ENPP1 are described in detail in PCT application publication number WO2014 / 126965 by Braddock et al., which is incorporated herein by reference in its entirety.

[0077] In the context of functional derivatives of amino acid sequences, the terms "functional equivalent" or "functional derivative" refer to a molecule that retains a biological activity that is substantially similar to the biological activity (function or structure) of the sequence of the ENPP1-Fc construct shown herein. The functional derivative or equivalent can be a natural derivative or a synthetic preparation. The functional equivalent polypeptides of the present invention can also be polypeptides identified using one or more structure and / or sequence alignment techniques known in the art.

[0078] Exemplary functional derivatives include amino acid sequences having one or more amino acid substitutions, deletions or additions, provided that the biological activity of the protein is conserved. The substituted amino acids desirably have chemical-physical properties similar to the amino acids being substituted. Desirable similar chemical-physical properties include similarities in charge, bulkiness, hydrophobicity, hydrophilicity, etc. Generally, greater than 30% identity between two polypeptides is considered an indication of functional equivalence. Preferably, the functionally equivalent polypeptides of the present invention have a degree of sequence identity with the ENPP1-Fc construct greater than 80%. More preferred polypeptides have degrees of identity greater than 85%, 90%, 95%, 98% or 99% respectively. A method for determining whether a functional equivalent or functional derivative has the same or similar or higher biological activity as the ENPP1-Fc construct can be determined by using the enzymatic assay involving ATP cleavage described in WO2016 / 187408.

[0079] As used herein, the term "human ENPP1" refers to the human ENPP1 sequence as described in NCBI accession number NP_006199. As used herein, the term "soluble human ENPP1" refers to a polypeptide corresponding to residues 96 to 925 of NCBI accession number NP_006199. As used herein, the term "enzymatically active" with respect to ENPP1 is defined as being able to bind and hydrolyze ATP to AMP and PPi and / or bind and hydrolyze AP3a to ATP.

[0080] As used herein, the term "ENPP1 precursor protein" refers to ENPP1 having its signal peptide sequence at the N-terminus of ENPP1. After proteolysis, the signal sequence is cleaved from ENPP1 to provide the ENPP1 protein. Signal peptide sequences useful in the present invention include, but are not limited to, the ENPP1 signal peptide sequence, the ENPP2 signal peptide sequence, the ENPP7 signal peptide sequence, and / or the ENPP5 signal peptide sequence.

[0081] As used herein, the term "ENPP1-Fc" refers to ENPP1 recombinantly fused and / or chemically conjugated (including covalent and non-covalent conjugation) to the FcR-binding domain of an IgG molecule (preferably, human IgG). In certain embodiments, the C-terminus of ENPP1 is fused or conjugated to the N-terminus of the FcR-binding domain.

[0082] As used herein, the term "Fc" refers to the Fc domain of human IgG (immunoglobulin). IgG subtypes such as IgG1, IgG2, IgG3 and IgG4 are contemplated for use as the Fc domain.

[0083] As used herein, "Fc region" is a part of an IgG molecule that is related to the crystallizable fragment obtained by pepsin digestion of an IgG molecule. The Fc region contains the C-terminal halves of the two heavy chains of an IgG molecule that are linked by disulfide bonds. It has no antigen-binding activity but contains a carbohydrate moiety as well as 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 the region of an IgG molecule that consists of the Fc region (residues 231-447) and the hinge region (residues 216-230) that extends from the N-terminus of the Fc region. The term "constant domain" refers to a part of an immunoglobulin molecule that has a more conserved amino acid sequence relative to another part of the immunoglobulin, namely the variable domain that has an antigen-binding site. The constant domain contains the CH1, CH2, and CH3 domains of the heavy chain and the CHL domain of the light chain.

[0084] As used herein, the term "Fc receptor" refers to a protein found on the surface of certain cells (including B lymphocytes, follicular dendritic cells, natural killer cells, macrophages, neutrophils, eosinophils, basophils, human platelets, and mast cells, etc.), which contributes to the protective functions of the immune system. The Fc receptor binds to an antibody attached to an infected cell or an invading pathogen. Immunoglobulin Fc receptors (FcRs) are expressed on all hematopoietic cells and play a key role in antibody-mediated immune responses. The binding of an immune complex to an FcR activates effector cells, which leads to phagocytosis, endocytosis of IgG-opsonized particles, release of inflammatory mediators, and antibody-dependent cell cytotoxicity (ADCC). Fc receptors have been described for all types of immunoglobulins: FcγR and neonatal FcR (FcRn) for IgG, FcεR for IgE, FcαR for IgA, FcδR for IgD, and FcμR for IgM. Except for FcRn and FcεRII, all known Fc receptors belong to the immunoglobulin superfamily in structure, and FcRn and FcεRII are structurally related to class I major histocompatibility antigens and C-type lectins, respectively (Fc Receptors, Neil A. Fangera et al., Encyclopedia of Immunology (Second Edition), 1998).

[0085] As used herein, the term "FcRn receptor" refers to the neonatal Fc receptor (FcRn), also known as the Brambell receptor, which is a protein encoded by the FCGRT gene in the human body. FcRn specifically binds to the Fc domain of an antibody. FcRn prolongs the half-life of IgG and serum albumin by reducing lysosomal degradation in endothelial cells. IgG, serum albumin, and other serum proteins are continuously internalized by pinocytosis. Typically, serum proteins are transported from endosomes to lysosomes, where they are degraded. FcRn-mediated transcytosis of IgG across epithelial cells is possible because FcRn binds IgG at acidic pH (<6.5), but not at neutral or higher pH. IgG and serum albumin are bound by FcRn at weakly acidic pH (<6.5) and recycled to the cell surface, where they are released at the neutral pH (>7.0) of the blood. In this way, IgG and serum albumin avoid lysosomal degradation.

[0086] The Fc portion of the IgG molecule is located in the constant region of the heavy chain, particularly in the CH2 domain. The Fc region binds to the Fc receptor (FcRn), which is a surface receptor of B cells and also a protein of the complement system. Binding of the Fc region of the IgG molecule to FcRn activates the cells bearing the receptor and thus activates the immune system. Fc residues crucial for mouse Fc-mouse FcRn and human Fc-human FcRn interactions have been identified (Dall’Acqua et al., 2002, J. Immunol. 169(9):5171-80). The FcRn-binding domain includes the CH2 domain (or its FcRn-binding portion) of the IgG molecule.

[0087] As used herein, when the term "fragment" is applied to a nucleic acid, it refers to a subsequence of a larger nucleic acid. A "fragment" of a nucleic acid can be at least about 15, 50-100, 100-500, 500-1000, 1000-1500 nucleotides, 1500-2500, or 2500 nucleotides (and any integer value therebetween). As used herein, when the term "fragment" is applied to a protein or peptide, it refers to a subsequence of a larger protein or peptide, which can be at least about 20, 50, 100, 200, 300, or 400 amino acids (and any integer value therebetween).

[0088] As used herein, the term "in vivo half-life" for a protein and / or polypeptide contemplated in the present invention (e.g., an ENPP1 polypeptide comprising an FcRn binding site) refers to the time required to clear half of the administered amount from the circulatory system and / or other tissues of an animal. When constructing a clearance curve for an ENPP1-Fc fusion protein as a function of time, the curve is typically biphasic, having a rapid α-phase (which represents the equilibration of the administered molecule between the intravascular and extravascular spaces and depends in part on the size of the molecule) and a longer β-phase (which represents the catabolism of the molecule in the intravascular space). In certain embodiments, the term "in vivo half-life" actually corresponds to the half-life of the molecule in the β-phase.

[0089] The term "directing material" as used herein includes publications, records, charts, or any other expression medium that can be used to convey the usefulness of the nucleic acids, peptides, and / or compounds of the present invention in a kit for identifying or alleviating or treating the various diseases or disorders described herein.

[0090] "Isolated" means altered or removed from its natural state. For example, a nucleic acid or polypeptide that occurs naturally in a living animal is not "isolated," but the same nucleic acid or polypeptide that is partially or completely separated from the coexisting materials in its natural state is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form or can exist in a non-natural environment, such as a host cell.

[0091] "Oligonucleotide" or "polynucleotide" refers to a nucleic acid or a compound that specifically hybridizes to a polynucleotide, having a length ranging from at least 2, and in some embodiments, at least 8, 15, or 25 nucleotides, but can be up to 50, 100, 1000, or 5000 nucleotides in length.

[0092] As used herein, the terms "patient," "individual," or "subject" refer to a human being.

[0093] As used herein, the term "pharmaceutical composition" or "composition" refers to a mixture of at least one compound useful in the present invention and a pharmaceutically acceptable carrier. The pharmaceutical composition facilitates the administration of the compound to a patient. There are various techniques for administering compounds in the art, including but not limited to subcutaneous, intravenous, oral, aerosol, inhalation, rectal, vaginal, transdermal, intranasal, buccal, sublingual, parenteral, intrathecal, intragastric, ocular, pulmonary, and topical administration.

[0094] As used herein, the term "pharmaceutically acceptable" refers to a material, such as a carrier or diluent, that does not eliminate the biological activity or properties of a compound and is relatively non-toxic, i.e., the material can be administered to an individual without causing adverse biological effects or interacting in a harmful manner with any of the components contained in the composition.

[0095] As used herein, the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, stabilizer, dispersant, suspending agent, diluent, excipient, thickening agent, solvent, or encapsulating material, which participates in carrying or transporting a compound useful in the present invention in or to a patient's body such that the compound can perform its intended function. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation, including the compound useful in the present invention, and not injurious to the patient. Some examples of materials that can be used as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives. As used herein, "pharmaceutically acceptable carrier" also includes any and all coatings, antibacterial and antifungal agents, absorption delaying agents, etc. that are compatible with the activity of the compound useful in the present invention and are physiologically acceptable to the patient. "Pharmaceutically acceptable carrier" can further include pharmaceutically acceptable salts of the compound useful in the present invention. Other additional ingredients that may be included in the pharmaceutical compositions used in the practice of the present invention are known in the art and are described, for example, in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference.

[0096] As used herein, the phrase "pharmaceutically acceptable salt" refers to salts of a compound administered that are prepared from pharmaceutically acceptable non-toxic acids and bases (including inorganic acids, inorganic bases, organic acids, inorganic bases, solvates, hydrates, and their inclusion compounds).

[0097] As used herein, the term "plasma pyrophosphate (PPi) level" refers to the amount of pyrophosphate present in the plasma of an animal. In certain embodiments, the animal includes rats, mice, cats, dogs, humans, cows, and horses. Due to release from platelets, it is necessary to measure PPi in plasma rather than in serum. There are multiple ways to measure PPi, one of which is by an enzymatic assay using modified uridine diphosphate glucose (UDPG) pyrophosphorylase (Lust and Seegmiller, 1976, Clin. Chim. Acta 66:241-249; Cheung and Suhadolnik, 1977, Anal. Biochem 83:61-63). The normal PPi level range in healthy subjects is typically from about 1 μM to about 3 μM, and in some cases between 1-2 μM. Subjects with defective ENPP1 expression tend to exhibit lower PPi levels that are at least 10% lower than normal levels, at least 20% lower than normal levels, at least 30% lower than normal levels, at least 40% lower than normal levels, at least 50% lower than normal levels, at least 60% lower than normal levels, at least 70% lower than normal levels, at least 80% lower than normal levels, and any combination thereof. In patients with pathological calcification or ossification diseases, the PPi level in plasma is found to be below 1 μM, and in some cases below the detection level. In some cases, the plasma PPi level in subjects with pathological calcification or ossification diseases is below 0.5 μM (Arterioscler Thromb, Vasc Biol. 2014, 34(9):1985-9; Braddock et al., 2015, Nat Commun. 6:10006).

[0098] As used herein, the term "polypeptide" refers to a polymer composed of amino acid residues linked by peptide bonds, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof.

[0099] As used herein, the term "PPi" refers to pyrophosphate.

[0100] As used herein, the term "prevent" or "prevention" means that, if a disorder or disease does not occur, then no disorder or disease develops, or if a disorder or disease has already developed, then no further disorder or disease develops. Also contemplated is the ability of a human to prevent some or all of the symptoms associated with a disorder or disease.

[0101] As used herein, "sample" or "biological sample" refers to a biological material isolated from a subject. A biological sample can comprise any biological material suitable for detecting mRNA, polypeptide, or other markers of a physiological or pathological process in a subject, and can comprise fluids, tissues, cells, and / or acellular materials obtained from an individual.

[0102] As used herein, the term "signal peptide" refers to a sequence of amino acid residues (e.g., ranging in length from 10 to 30 residues) that binds to 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 signal peptidase following transport across the endoplasmic reticulum (Lodish et al., 2000, Molecular Cell Biology, 4th ed.).

[0103] As used herein, "substantially purified" means substantially free of other components. For example, a substantially purified polypeptide is one that has been separated from other components with which it is normally associated in its native state. Non-limiting embodiments include 95% purity, 99% purity, 99.5% purity, 99.9% purity, and 100% purity.

[0104] As used herein, the term "treatment" is defined as the application or administration of a therapeutic agent, i.e., a compound useful in the present invention (alone or in combination with another agent), to a patient, or the application or administration of a therapeutic agent to an isolated tissue or cell line from a patient (e.g., for diagnostic or ex vivo applications), where the patient has a disease or disorder, symptoms of a disease or disorder, or a predisposition to develop a disease or disorder, with the aim of treating, curing, alleviating, relieving, altering, remedying, ameliorating, improving, or affecting the disease or disorder, symptoms of the disease or disorder, or the predisposition to develop a disease or disorder. Such treatment can be specifically tailored or modified based on knowledge obtained from the field of pharmacogenomics.

[0105] A "vector" is a composition of matter that includes an isolated nucleic acid and can be used to deliver the isolated nucleic acid into a cell interior. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be construed to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, etc. Examples of viral vectors include but are not limited to adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and the like.

[0106] As used herein, the term "wild-type" refers to a gene or gene product isolated from a naturally occurring source. Wild-type genes are the most common in the population and are thus arbitrarily designated as the "normal" or "wild-type" form of the gene. In contrast, the terms "modified" or "mutant" refer to a gene or gene product that shows an alteration in sequence and / or functional properties (i.e., altered characteristics) compared to the wild-type gene or gene product. Naturally occurring mutants can be isolated; they are identified by the fact that they have altered characteristics (including an altered nucleic acid sequence) compared to the wild-type gene or gene product.

[0107] Scope: Throughout this disclosure, aspects of the present invention may 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 present invention. Thus, the description of a range should be considered to have explicitly disclosed all possible sub-ranges and individual numerical values within that range. For example, a range description such as from 1 to 6 should be considered to have explicitly disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and individual numbers within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0108] Polypeptide

[0109] In one aspect, the present invention provides an ENPP1-Fc polypeptide. The present invention contemplates that the polypeptides of the present invention may have one or more mutations as described herein.

[0110] In certain embodiments, the ENPP1 polypeptide comprises at least one mutation in the signal sequence as set forth in Figure 13A and / or Figure 13B . In certain embodiments, the mutations are selected from C25N, K27T, and V29N related to SEQ ID NO:7. In certain embodiments, the mutation is C25N related to SEQ ID NO:7. In certain embodiments, the mutation is K27T related to SEQ ID NO:7. In certain embodiments, the mutation is V29N related to SEQ ID NO:7. In certain embodiments, the ENPP1 polypeptide comprises at least one mutation selected from C25N / K27T and V29N related to SEQ ID NO:7.

[0111] In certain embodiments, the ENPP1 polypeptide comprises at least one mutation in the catalytic region as set forth in Figure 13A and / or Figure 13B . In certain embodiments, the mutations are selected from K369N and I371T related to SEQ ID NO:7. In certain embodiments, the mutation is K369N related to SEQ ID NO:7. In certain embodiments, the mutation is I371T related to SEQ ID NO:7. In certain embodiments, the ENPP1 polypeptide comprises the mutation K369N / I371T related to SEQ ID NO:7.

[0112] In certain embodiments, the ENPP1 polypeptide comprises those shown in Table 1, Table 2, Table 3, Table 4, Table 5, Figure 6A , Figure 13A , Figure 13B , Figure 14 and / or Figure 15At least one mutation in the stated endonuclease domain. In certain embodiments, the mutation is selected from P534N, V536T, R545T, P554L, E592N, R741D, and S766N related to SEQ ID NO:7. In certain embodiments, the mutation is P534N related to SEQ ID NO:7. In certain embodiments, the mutation is V536T related to SEQ ID NO:7. In certain embodiments, the mutation is R545T related to SEQ ID NO:7. In certain embodiments, the mutation is P554L related to SEQ ID NO:7. In certain embodiments, the mutation is E592N related to SEQ ID NO:7. In certain embodiments, the mutation is R741D related to SEQ ID NO:7. In certain embodiments, the mutation is S766N related to SEQ ID NO:7. In certain embodiments, the ENPP1 polypeptide comprises at least one mutation selected from P534N / V536T, P554L / R545T, E592N, E592N / R741D, and S766N related to SEQ ID NO:7.

[0113] In certain embodiments, the ENPP1 polypeptide comprises as Figure 13A and / or Figure 13B at least one mutation in the linker region stated in. In certain embodiments, the mutation is selected from E864N and L866T related to SEQ ID NO:7. In certain embodiments, the mutation is E864N related to SEQ ID NO:7. In certain embodiments, the mutation is L866T related to SEQ ID NO:7.

[0114] In certain embodiments, the ENPP1 polypeptide comprises at least the mutation E864N / L866T related to SEQ ID NO:7.

[0115] In certain embodiments, the polypeptide comprises an ENPP1 polypeptide and an FcRn binding domain, wherein the FcRn binding domain comprises Table 1, Table 2, Figure 6A , Figure 13A , Figure 13B , Figure 14 and / or Figure 15Any mutation recited in [[SEQ ID NO:7]]. In certain embodiments, the mutation is selected from M883Y, S885N, S885T, T887E, H1064K, and N1065F related to SEQ ID NO:7. In certain embodiments, the mutation is M883Y related to SEQ ID NO:7. In certain embodiments, the mutation is S885N related to SEQ ID NO:7. In certain embodiments, the mutation is S885T related to SEQ ID NO:7. In certain embodiments, the mutation is T887E related to SEQ ID NO:7. In certain embodiments, the mutation is H1064K related to SEQ ID NO:7. In certain embodiments, the mutation is N1065F related to SEQ ID NO:7. In certain embodiments, the FcRn binding domain comprises at least one mutation selected from S885N, M883Y, M883Y / S885T / T887E, and H1064K / N1065F related to SEQ ID NO:7.

[0116] In certain embodiments, the ENPP1 polypeptide comprises at least one mutation selected from C25N, K27T, V29N, C25N / K27T, K369N, I371T, K369N / I371T, P534N, V536T, R545T, P554L, E592N, R741D, S766N, P534N / V536T, P554L / R545T, E592N / R741D, E864N, L866T, E864N / L866T, M883Y, S885N, S885T, T887E, H1064K, N1065F, M883Y / S885T / T887E, H1064K / N1065F related to SEQ ID NO:7.

[0117] In certain embodiments, the polypeptide comprises at least one mutation selected from S885N, S766N, M883Y / S885T / T887E, E864N / L866T, P534N / V536T / H1064K / N1065F, P554L / R545T, S766N / H1064K / N1065F, E592N / H1064K / N1065F, and P534N / V536T / M883Y / S885T / T887E related to SEQ ID NO:7.

[0118] In certain embodiments, the polypeptide comprises an ENPP1 polypeptide and an FcRn binding domain, and the polypeptide comprises the mutations M883Y, S885T, and T887E related to SEQ ID NO:7.

[0119] In certain embodiments, the polypeptide comprises an ENPP1 polypeptide and an FcRn binding domain, and the polypeptide comprises mutations P534N, V536T, M883Y, S885T, and T887E related to SEQ ID NO:7.

[0120] In certain embodiments, the polypeptide comprises an ENPP1 polypeptide and an FcRn binding domain, and the polypeptide comprises mutations E592N, H1064K, and N1065F related to SEQ ID NO:7.

[0121] In certain embodiments, the polypeptide comprises an ENPP1 mutant polypeptide, wherein the mutant polypeptide comprises an ENPP1 mutation selected from S766N, P534N, V536T, P554L, R545T, and E592N related to SEQ ID NO:7.

[0122] In certain embodiments, the ENPP1 mutant polypeptide comprises at least one mutation selected from S766N, P534N / V536T, P554L / R545T, and E592N related to SEQ ID NO:7.

[0123] In certain embodiments, the polypeptide further comprises an FcRn binding domain of IgG.

[0124] In certain embodiments, the polypeptide comprises mutations selected from S885N, S766N, M883Y / S885T / T887E, P534N / V536T / H1064K / N1065F, P554L / R545T, S766N / H1064K / N1065F, E592N / H1064K / N1065F, and P534N / V536T / M883Y / S885T / T887E related to SEQ ID NO:7.

[0125] In certain embodiments, the polypeptide comprises an S885N mutation in the FcRn binding domain related to SEQ ID NO:7.

[0126] In certain embodiments, the polypeptide comprises an S766N mutation in the ENPP1 mutant polypeptide related to SEQ ID NO:7.

[0127] In certain embodiments, the polypeptide comprises mutations M883Y, S885T, and T887E in the FcRn binding domain related to SEQ ID NO:7.

[0128] In certain embodiments, the polypeptide comprises the mutations P534N and V536T in the ENPP1 mutant polypeptide related to SEQ ID NO:7, and the mutations H1064K and N1065F in the FcRn binding domain.

[0129] In certain embodiments, the polypeptide comprises the mutations P554L and R545T in the ENPP1 mutant polypeptide related to SEQ ID NO:7.

[0130] In certain embodiments, the polypeptide comprises the mutation S766N in the ENPP1 mutant polypeptide related to SEQ ID NO:7, and the mutations H1064K and N1065F in the FcRn binding domain.

[0131] In certain embodiments, the polypeptide comprises the mutation E592N in the ENPP1 mutant polypeptide related to SEQ ID NO:7, and the mutations H1064K and N1065F in the FcRn binding domain.

[0132] In certain embodiments, the polypeptide comprises the mutations P534N and V536T in the ENPP1 mutant polypeptide related to SEQ ID NO:7, and the mutations M883Y, S885T and T887E in the FcRn binding domain.

[0133] In certain embodiments, the ENPP1 polypeptide lacks the nuclease domain. In other embodiments, the ENPP1 polypeptide is truncated to remove the nuclease domain. In still other embodiments, the ENPP1 polypeptide is truncated to remove the nuclease domain from about residue 524 to about residue 885 relative to SEQ ID NO:1, leaving only the catalytic domain from about residue 186 to about residue 586 relative to SEQ ID NO:1, which is used to maintain the catalytic activity of the protein.

[0134] In certain embodiments, compared to SEQ ID NO:1, the ENPP1 polypeptide is modified by a segment of the extracellular region of ENPP1 that contains a peptidase cleavage site after the signal peptide and between the transmembrane and extracellular domains.

[0135] In certain embodiments, compared to SEQ ID NO:1, the ENPP1 polypeptide is modified by a segment of the extracellular region of ENPP1 that contains a furin cleavage site between the transmembrane and extracellular domains. In other embodiments, compared to SEQ ID NO:1, the ENPP1 polypeptide is not modified by a segment of the extracellular region of ENPP1 that has a furin cleavage site between the transmembrane and extracellular domains.

[0136] In certain embodiments, the ENPP1 polypeptide is modified by a segment of the extracellular region of ENPP2 that contains a signal peptidase cleavage site as compared to SEQ ID NO:1. In other embodiments, the ENPP1 polypeptide is not modified by a segment of the extracellular region of ENPP2 that contains a signal peptidase cleavage site as compared to SEQ ID NO:1.

[0137] In certain embodiments, the polypeptide is soluble. In other embodiments, the polypeptide is a recombinant polypeptide. In still other embodiments, the polypeptide comprises an ENPP1 polypeptide lacking the ENPP1 transmembrane domain. In still other embodiments, the polypeptide comprises an ENPP1 polypeptide in which the ENPP1 transmembrane domain has been removed (and / or truncated) and replaced with the transmembrane domain of another polypeptide, such as (by way of non-limiting example) ENPP2, ENPP5, or ENPP7.

[0138] In certain embodiments, the polypeptide comprises a signal peptide that results in the secretion of a precursor of the ENPP1 polypeptide, which precursor is proteolytically processed to produce a polypeptide comprising the ENPP1 polypeptide. In other embodiments, the signal peptide is selected from the signal peptides of ENPP2, ENPP5, and ENPP7. In still other embodiments, the polypeptide comprises an ENPP1 polypeptide that comprises the transmembrane domain of ENPP1; and another polypeptide, such as (by way of non-limiting example) ENPP2. In still other embodiments, the ENPP1 polypeptide comprises a cleavage product of a precursor ENPP1 polypeptide that comprises the ENPP2 transmembrane domain. In still other embodiments, the ENPP2 transmembrane domain comprises residues 12-30 of SEQ ID NO:7, which corresponds to IISLFTFAVGVNICLGFTA.

[0139] In certain embodiments, the ENPP1 polypeptide is fused at the C-terminus to the Fc domain of human immunoglobulin 1 (IgG1), human immunoglobulin 2 (IgG2), human immunoglobulin 3 (IgG3), and / or human immunoglobulin 4 (IgG4). In other embodiments, the ENPP1 polypeptide is fused at the N-terminus to the Fc domain of human immunoglobulin 1 (IgG1), human immunoglobulin 2 (IgG2), human immunoglobulin 3 (IgG3), and / or human immunoglobulin 4 (IgG4). In still other embodiments, the presence of the Ig Fc domain improves the half-life, solubility, reduces immunogenicity, and increases the activity of the ENPP1 polypeptide.

[0140] In certain embodiments, the ENPP1 polypeptide is fused at the C-terminus to human serum albumin. Human serum albumin can be conjugated to the ENPP1 protein by a chemical linker that includes, but is not limited to, naturally occurring or engineered disulfide bonds, or by genetic fusion to ENPP1 or a fragment and / or variant thereof.

[0141] In certain embodiments, the polypeptide is further PEGylated (fused with a poly(ethylene glycol) chain).

[0142] In certain embodiments, the k cat value of the polypeptide for the substrate ATP is greater than or equal to about 3.4 (±0.4) s -1 enzyme -1 wherein the k cat is determined by measuring the ATP hydrolysis rate of the polypeptide.

[0143] In certain embodiments, the K M value of the polypeptide for the substrate ATP is less than or equal to about 2 μM, wherein the K M is determined by measuring the ATP hydrolysis rate of the polypeptide.

[0144] In certain embodiments, the polypeptide is formulated as a liquid preparation. In other embodiments, the present invention provides a dry product form of a pharmaceutical composition, which comprises a therapeutically effective amount of the polypeptide of the present invention, whereby the dry product can be reconstituted into a solution of the compound in liquid form.

[0145] The present invention provides a kit, which comprises at least one polypeptide of the present invention, or a salt or solvate thereof, and instructions for using the polypeptide in the methods of the present invention.

[0146] In certain embodiments, the polypeptide lacks a negatively charged bone targeting sequence. In still other embodiments, a polyaspartic acid domain (about 2 to about 20 or more contiguous aspartic acid residues) is a non-limiting example of a negatively charged bone targeting sequence. In other embodiments, the polypeptide has a negatively charged bone targeting sequence.

[0147] It should be understood that the ENPP1 polypeptide according to the present invention includes not only the native human protein, but also any fragment, derivative, fusion, conjugate or mutant thereof having the ATP hydrolysis activity of the native protein. As used herein, the phrase "ENPP1 polypeptide, its mutant or mutant fragment" also includes any compound or polypeptide (such as, but not limited to, a fusion protein) comprising an ENPP1 polypeptide, its mutant or mutant fragment. The fusion protein according to the present invention is considered to be a bioequivalent of ENPP1, but is intended to provide a longer half-life or higher potency due to increased in vivo bioexposure (as judged by "area under the curve" (AUC) or increased half-life in pharmacokinetic experiments).

[0148] Vectors and cells

[0149] The present invention further provides an autonomously replicating or integrating mammalian cell vector comprising a recombinant nucleic acid encoding a polypeptide of the present invention. In certain embodiments, the vector comprises a plasmid or a virus. In other embodiments, the vector comprises a mammalian cell expression vector. In still other embodiments, the vector further comprises at least one nucleic acid sequence that directs and / or controls the expression of the polypeptide. In still other embodiments, the recombinant nucleic acid encodes a polypeptide comprising the ENPP1 polypeptide of the present invention and a signal peptide, wherein the polypeptide is proteolytically processed after secretion from the cell to produce the ENPP1 polypeptide of the present invention.

[0150] In still another aspect, the present invention provides an isolated host cell comprising the vector of the present invention. In certain embodiments, the cell is a non-human cell. In other embodiments, the cell is mammalian. In still other embodiments, the vector of the present invention comprises a recombinant nucleic acid encoding a polypeptide comprising the ENPP1 polypeptide of the present invention and a signal peptide. In still other embodiments, the polypeptide is proteolytically processed after secretion from the cell to produce the ENPP1 polypeptide of the present invention.

[0151] Cloning and Expression of ENPP1

[0152] The preparation of ENPP1 or an ENPP1 polypeptide as described in US2015 / 0359858 A1, which is incorporated herein by reference in its entirety. ENPP1 is a transmembrane protein that is located on the cell surface with different intracellular domains. To express ENPP1 as a soluble extracellular protein, the transmembrane domain of ENPP1 can be exchanged with the transmembrane domain of ENPP2, which results in the accumulation of soluble recombinant ENPP1 in the extracellular fluid of baculovirus cultures.

[0153] The signal sequence of any other known protein can also be used to target the extracellular domain of ENPP1 for secretion, such as but not limited to the signal sequences of immunoglobulin κ and λ light chain proteins. Further, the present invention should not be construed as limited to the polypeptides described herein, but also includes any enzymatically active truncated polypeptides comprising the extracellular domain of ENPP1.

[0154] Solubilize ENPP1 by omitting the transmembrane domain. Modify human ENPP1 (SEQ ID NO:1) to express a soluble recombinant protein by replacing the transmembrane region of human ENPP1 (e.g., residues 77 - 98) with the corresponding subdomain of human ENPP2 (NCBI accession number NP_001124335, e.g., residues 12 - 30). The modified ENPP1 sequence is cloned into a modified pFastbac FIT vector with a TEV protease cleavage site followed by a C-terminal 9-F1IS tag and cloned and expressed in insect cells, and both proteins are 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), which results in the accumulation of the soluble recombinant protein in the extracellular fluid.

[0155] Production and purification of ENPP1 and ENPP1 fusion proteins

[0156] In certain embodiments, soluble ENPP1 polypeptides - including an IgG Fc domain or an enzymatic / biologically active fragment thereof - can be effective in treating, reducing, and / or preventing the progression of diseases or disorders contemplated herein. In other embodiments, the soluble ENPP1 polypeptide does not include an osteotropic domain, such as 2 - 20 contiguous polyaspartic acid residues or 2 - 20 contiguous polyglutamic acid residues.

[0157] To produce soluble recombinant ENPP1 for in vitro use, ENPP1 is fused to the Fc domain of IgG (referred to as "NPP1-Fc"), and the fusion protein is expressed in a stable CHO cell line. Using a suitable vector, the protein can also be expressed from HEK293 cells, a baculovirus insect cell system, or a CHO cell or Pichia pastoris expression system. The protein can be produced in adherent cells or suspension cells. Preferably, the fusion protein is expressed in CHO cells. To establish a stable cell line, the nucleic acid sequence encoding the ENPP1 construct is cloned into a suitable vector for large-scale protein production.

[0158] Many expression systems are known to be useful for producing ENPP1 fusion proteins, including bacteria (e.g., Escherichia coli and Bacillus subtilis), yeast (e.g., Saccharomyces cerevisiae, Kluyveromyces lactis, and Pichia pastoris), filamentous fungi (e.g., Aspergillus), plant cells, animal cells, and insect cells. The desired protein can be produced in a conventional manner, such as from a coding sequence inserted into the host chromosome or on a free plasmid.

[0159] Yeast can be transformed with the coding sequence of the desired protein in any conventional manner, such as electroporation. The method of transforming yeast by electroporation is disclosed in Becker and Guarente, 1990, Methods Enzymol. 194:182. Successfully transformed cells, i.e., cells containing the DNA construct of the present invention, can be identified by well-known techniques. For example, cells resulting from the introduction of the expression construct can be grown to produce the desired polypeptide. The cells can be harvested and lysed, and the DNA content of the cells can be examined using methods such as those described in Southern, 1975, J. Mol. Biol. 98:503 and / or Berent et al., 1985, Biotech 3:208 to detect the presence of the DNA. Optionally, the presence of the protein in the supernatant can be detected using an antibody.

[0160] Useful yeast plasmid vectors include pRS403-406 and pRS413-416, and are generally available from Strat:1.geneCloning Systems, La Jolla, CA, USA. Plasmids pRS403, pRS404, pRS405, and pRS406 are yeast integrating plasmids (Y1p) and incorporate the yeast selectable markers I-11S3, TRP1, LEU2, and IJRA3. Plasmids pRS413-416 are yeast centromere plasmids (YCp).

[0161] A variety of methods have been developed to efficiently ligate DNA to a vector via complementary sticky ends. For example, complementary homopolymer tracts can be added to DNA segments for insertion into vector DNA. The vector and the DNA segment are then joined by hydrogen bonding between the complementary homopolymer tails to form a recombinant DNA molecule.

[0162] Synthetic linkers containing one or more restriction sites provide an alternative method for ligating DNA segments to a vector. DNA segments generated by endonuclease restriction digestion are treated with bacteriophage T4 DNA polymerase or Escherichia coli DNA polymerase I, which are enzymes that remove protruding 3'-single-stranded ends with 3'-5'-exonuclease activity and fill in recessed 3'-ends using their polymerase activity.

[0163] The combination of these activities thus results in blunt-ended DNA segments. The blunt-ended segments are then incubated with a large molar excess of linker molecules in the presence of an enzyme capable of catalyzing the ligation of blunt-ended DNA molecules (e.g., bacteriophage T4 DNA ligase). Thus, the product of the reaction is a DNA segment with a polymeric linker sequence at its ends. These DNA segments are subsequently cleaved with an appropriate restriction endonuclease and ligated to an expression vector that has been cleaved with an enzyme that generates ends compatible with the ends of the DNA segments.

[0164] Clones of individual stably transfected cells are then established and high-expressing clones of the desired fusion protein are screened. Screening for single-cell clones expressing the ENPP3 protein can be done in a high-throughput manner in 96-well plates using the synthetic enzyme substrate pNP-TMP as described previously (Albright et al., 2015, Nat. Commun. 6:10006). After identifying high-expressing clones by screening, protein production can be done in shake flasks or in a bioreactor as described in Albright et al., 2015, Nat. Commun. 6:10006.

[0165] Purification of ENPP1 can be accomplished using a combination of standard purification techniques known in the art. Examples thereof are described above in the production of the ENPP3 protein. After purification, ENPP1-Fc is dialyzed into PBS supplemented with Zn 2+ and Mg 2+ (PBSplus) — concentrated to between 5 and 7 mg / ml and frozen in 200 - 500 μl aliquots at -80 °C. Just before use, the aliquots are thawed and the specific activity of the solution is adjusted to 31.25 au / ml (or approximately 0.7 mg / ml, depending on the preparation) by dilution in PBSplus.

[0166] Sequence

[0167] SEQ ID NO:1: hENPP1 amino acid sequence

[0168] MERDGCAGGGSRGGEGGRAPREGPAGNGRDRGRSHAAEAPGDPQAAASLLAPMDVGEEPLEKAARARTAKDPNTYKVLSLVLSVCVLTTILGCIFGLKPSCAKEVKSCKGRCFERTFGNCRCDAACVELGNCCLDYQETCIEPEHIWTCNKFRCGEKRLTRSLCACSDDCKDKGDCCINYSSVCQGEKSWVEEPCESINEPQCPAGFETPPTLLFSLDGFRAEYLHTWGGLLPVISKLKKCGTYTKNMRPVYPTKTFPNHYSIVTGLYPESHGIIDNKMYDPKMNASFSLKSKEKFNPEWYKGEPIWVTAKYQGLKSGTFFWPGSDVEINGIFPDIYKMYNGSVPFEERILAVLQWLQLPKDERPHFYTLYLEEPDSSGHSYGPVSSEVIKALQRVDGMVGMLMDGLKELNLHRCLNLILISDHGMEQGSCKKYIYLNKYLGDVKNIKVIYGPAARLRPSDVPDKYYSFNYEGIARNLSCREPNQHFKPYLKHFLPKRLHFAKSDRIEPLTFYLDPQWQLALNPSERKYCGSGFHGSDNVFSNMQALFVGYGPGFKHGIEADTFENIEVYNLMCDLLNLTPAPNNGTHGSLNHLLKNPVYTPKHPKEVHPLVQCPFTRNPRDNLGCSCNPSILPIEDFQTQFNLTVAEEKIIKHETLPYGRPRVLQKENTICLLSQHQFMSGYSQDILMPLWTSYTVDRNDSFSTEDFSNCLYQDFRIPLSPVHKCSFYKNNTKVSYGFLSPPQLNKNSSGIYSEALLTTNIVPMYQSFQVIWRYFHDTLLRKYAEERNGVNVVSGPVFDFDYDGRCDSLENLRQKRRVIRNQEILIPTHFFIVLTSCKDTSQTPLHCENLDTLAFILPHRTDNSESCVHGKHDSSWVEELLMLHRARITDVEHITGLSFYQQRKEPVSDILKLKTHLPTFSQED

[0169] SEQ ID NO:2: ENPP2 amino acid sequence

[0170] MARRSSFQSCQIISLFTFAVGVNICLGFTAHRIKRAEGWEEGPPTVLSDSPWTNISGSCKGRCFELQEAGPPDCRCDNLCKSYTSCCHDFDELCLKTARGWECTKDRCGEVRNEENACHCSEDCLARGDCCTNYQVVCKGESHWVDDDCEEIKAAECPAGFVRPPLIIFSVDGFRASYMKKGSKVMPNIEKLRSCGTHSPYMRPVYPTKTFPNLYTLATGLYPESHGIVGNSMYDPVFDATFHLRGREKFNHRWWGGQPLWITATKQGVKAGTFFWSVVIPHERRILTILQWLTLPDHERPSVYAFYSEQPDFSGHKYGPFGPEMTNPLREIDKIVGQLMDGLKQLKLHRCVNVIFVGDHGMEDVTCDRTEFLSNYLTNVDDITLVPGTLGRIRSKFSNNAKYDPKAIIANLTCKKPDQHFKPYLKQHLPKRLHYANNRRIEDIHLLVERRWHVARKPLDVYKKPSGKCFFQGDHGFDNKVNSMQTVFVGYGSTFKYKTKVPPFENIELYNVMCDLLGLKPAPNNGTHGSLNHLLRTNTFRPTMPEEVTRPNYPGIMYLQSDFDLGCTCDDKVEPKNKLDELNKRLHTKGSTEAETRKFRGSRNENKENINGNFEPRKERHLLYGRPAVLYRTRYDILYHTDFESGYSEIFLMPLWTSYTVSKQAEVSSVPDHLTSCVRPDVRVSPSFSQNCLAYKNDKQMSYGFLFPPYLSSSPEAKYDAFLVTNMVPMYPAFKRVWNYFQRVLVKKYASERNGVNVISGPIFDYDYDGLHDTEDKIKQYVEGSSIPVPTHYYSIITSCLDFTQPADKCDGPLSVSSFILPHRPDNEESCNSSEDESKWVEELMKMHTARVRDIEHLTSLDFFRKTSRSYPEILTLKTYLHTYESEI

[0171] SEQ ID NO:3: hIgG Fc domain, Fc

[0172] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0173] SEQ ID NO:4: hENPP5 protein export signal sequence

[0174] MTSKFLLVSFILAALSLSTTFS-Xaa 23 Xaa 24 ,

[0175] where Xaa 23 is absent or is L, and

[0176] where if Xaa 23 is absent then Xaa 24 is absent, and if Xaa 23 is L, then Xaa 24 is absent or is Q

[0177] SEQ ID NO:5: hENPP7 protein export signal sequence

[0178] MRGPAVLLTV ALATLLAPGA GA

[0179] SEQ ID NO:6: hENPP7 protein export signal sequence

[0180] MRGPAVLLTV ALATLLAPGA

[0181] SEQ ID NO:7: ENPP1-Fc

[0182]

[0183]

[0184] Bold: Signal sequence

[0185] Regular: ENPP1 extracellular domain

[0186] Underlined: Linker sequence

[0187] Italic: Fc domain

[0188] Method

[0189] The present invention includes a method for reducing or preventing the progression of pathological calcification in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide of the present invention.

[0190] The present invention further includes a method for reducing or preventing the progression of pathological ossification in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide of the present invention.

[0191] The present invention further includes a method for reducing or preventing the progression of ectopic calcification of soft tissues in a subject in need thereof, which includes reducing, improving or preventing vascular calcification, the method comprising administering to the subject a therapeutically effective amount of a polypeptide of the present invention.

[0192] The present invention further includes a method for reducing or preventing the progression of a disease caused by ENPP1 deficiency. ENPP1 deficiency is characterized by a reduced ENPP1 activity level or a defective ENPP1 expression level in a subject in need thereof (compared to the ENPP1 activity level or ENPP1 expression level in a normal healthy subject, respectively), the method comprising administering to the subject a therapeutically effective amount of a polypeptide of the present invention.

[0193] The present invention further includes a method for reducing or preventing the progression of a disease caused by a lower level of plasma PPi in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide of the present invention to increase the subject's plasma PPi to a normal level (1 - 3 μM) or above the normal level (30 - 50% higher than the normal level), and then maintaining the plasma PPi at a constant normal level or above the normal level thereafter. The method further includes administering additional therapeutically effective amounts at intervals of two days, three days, one week or one month to maintain the subject's plasma PPi at a constant normal level or above the normal level to reduce or prevent the progression of pathological calcification or ossification.

[0194] The present invention further includes a method for treating, reversing or preventing the progression of ossification of the posterior longitudinal ligament (OPLL) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide of the present invention.

[0195] The present invention further includes a method for treating, reversing or preventing the progression of hypophosphatemic rickets in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide of the present invention.

[0196] The present invention further includes a method for reducing or preventing the progression of at least one disease in a subject diagnosed with at least one disease selected from the group consisting of: chronic kidney disease (CKD), end-stage renal disease (ESRD), calcific uremic arteriolopathy (CUA), calciphylaxis, ossification of the posterior longitudinal ligament (OPLL), hypophosphatemic rickets, osteoarthritis, age-related arteriosclerosis, idiopathic infantile arterial calcification (IIAC), generalized arterial calcification of infancy (GACI), and atherosclerotic plaque calcification, the method comprising administering to the subject a therapeutically effective amount of the polypeptide of the present invention.

[0197] The present invention further includes a method for reducing and / or preventing the progression of age-related arteriosclerosis in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the polypeptide of the present invention.

[0198] The present invention further includes a method for reducing or preventing the progression of a disease caused by ENPP1 deficiency (e.g., a reduced ENPP1 activity level and / or a defective ENPP1 expression level as compared to the ENPP1 activity level or ENPP1 expression level in a normal healthy subject, respectively) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the polypeptide of the present invention.

[0199] The present invention further includes a method for reducing or preventing the progression of a disease caused by a plasma PPi level lower than the normal level in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the polypeptide of the present invention to increase and / or maintain the plasma PPi of the subject at a level of about 90%, 95%, 100%, 105%, 110%, 120%, 130%, 140%, or 150% of the normal PPi level (about 1 - 3 μM). In certain embodiments, the method further comprises further administering the polypeptide of the present invention every two days, three days, one week, or one month to maintain the plasma PPi level at about 90%, 95%, 100%, 105%, 110%, 120%, 130%, 140%, or 150% of the normal PPi level, thereby preventing the progression of pathological calcification or ossification.

[0200] The present invention further includes a method for treating, reversing, or preventing the progression of pseudoxanthoma elasticum (PXE) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the polypeptide of the present invention.

[0201] The present invention further includes a method for treating, reversing, or preventing the progression of atherosclerotic plaque calcification in arterial vessels in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the polypeptide of the present invention.

[0202] The present invention further includes a method for treating, reversing or preventing the progression of osteoarthritis in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the polypeptide of the present invention.

[0203] The present invention further includes a method for treating, reversing or preventing the progression of arteriosclerosis caused by premature aging in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the polypeptide of the present invention.

[0204] The present invention further includes a method for treating, reversing or preventing the progression of X-linked hypophosphatemic rickets (XLH), hereditary hypophosphatemic rickets (HHRH), hypophosphatemic bone disease (HBD), autosomal dominant hypophosphatemic rickets (ADHR) and / or autosomal recessive hypophosphatemic rickets in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the polypeptide of the present invention.

[0205] The present invention further includes a method for treating, reversing or preventing the progression of age-related osteopenia in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the polypeptide of the present invention.

[0206] The present invention further includes a method for treating, reversing or preventing the progression of ankylosing spondylitis in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the polypeptide of the present invention.

[0207] The present invention further includes a method for treating, reversing or preventing the progression of pediatric sickle cell anemia stroke in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the polypeptide of the present invention.

[0208] In certain embodiments, the pathological calcification is selected from idiopathic infantile arterial calcification (IIAC) and atherosclerotic plaque calcification.

[0209] In certain embodiments, the pathological ossification is selected from ossification of the posterior longitudinal ligament (OPLL), hypophosphatemic rickets and osteoarthritis.

[0210] In certain embodiments, the soft tissue calcification is selected from IIAC and osteoarthritis. In other embodiments, the soft tissue includes atherosclerotic plaques. In still other embodiments, the soft tissue includes muscular arteries. In still other embodiments, the soft tissue is selected from joints and the spine. In still other embodiments, the joints are selected from hand joints and foot joints. In still other embodiments, the soft tissue is selected from articular cartilage and intervertebral disc cartilage. In still other embodiments, the soft tissue includes blood vessels. In still other embodiments, the soft tissue includes connective tissue.

[0211] In certain embodiments, the subject is diagnosed with premature aging.

[0212] In certain embodiments, the polypeptide of the present invention is a secreted product of the ENPP1 precursor protein expressed in mammalian cells. In other embodiments, the ENPP1 precursor protein comprises a signal peptide sequence and an ENPP1 polypeptide, wherein the ENPP1 precursor protein is proteolytically processed to become the polypeptide of the present invention. In still other embodiments, in the ENPP1 precursor protein, the signal peptide sequence is conjugated to the N-terminus of the ENPP1 polypeptide. After proteolysis, the signal sequence is cleaved from the ENPP1 precursor protein to provide the ENPP1 polypeptide. In certain embodiments, the signal peptide sequence is selected from the ENPP1 signal peptide sequence, the ENPP2 signal peptide sequence, the ENPP7 signal peptide sequence, and the ENPP5 signal peptide sequence.

[0213] In certain embodiments, the polypeptide is administered to a subject acutely or chronically. In other embodiments, the polypeptide is administered to the subject locally, regionally, parenterally, or systemically.

[0214] In certain embodiments, the subject is a mammal. In other embodiments, the mammal is a human.

[0215] In certain embodiments, the polypeptide or its precursor protein is administered by at least one route selected from: subcutaneous, oral, aerosol, inhalation, rectal, vaginal, transdermal, intradermal, intranasal, buccal, sublingual, parenteral, intrathecal, intragastric, ocular, pulmonary, and topical. In other embodiments, the polypeptide or its precursor protein is administered to the subject as a pharmaceutical composition, which further comprises at least one pharmaceutically acceptable carrier.

[0216] In certain embodiments, the polypeptide or its precursor protein is administered to a subject acutely or chronically. In other embodiments, the polypeptide or its precursor protein is administered to the subject locally, regionally, or systemically. In still another embodiment, the polypeptide or its precursor protein is delivered on a coding vector, wherein the vector encodes the protein and, after the vector is administered to the subject, it is transcribed and translated from the vector.

[0217] Those skilled in the art will understand that when equipped with the present disclosure including the methods detailed herein, the present invention is not limited to the treatment of a disease or disorder after its determination. Specifically, the symptoms of the disease or disorder need not have manifested to the point of harm to the subject; indeed, it is not necessary to detect the disease or disorder in the subject prior to administration of the treatment. That is, significant pathology of the disease or disorder need not have occurred before the present invention can provide a benefit.

[0218] Accordingly, as more fully described herein, the present invention includes methods for preventing diseases and disorders in a subject, wherein, as discussed elsewhere herein, the polypeptides of the present invention can be administered to a subject prior to the occurrence of the disease or disorder, thereby preventing the development of the disease or disorder. Specifically, when the symptoms of the disease or disorder have not yet manifested to a point of harm to the subject; in fact, it is not necessary to detect the disease or disorder in the subject prior to administration of the treatment. That is, significant pathology of the disease or disorder does not have to have occurred before the present invention can provide a benefit. Accordingly, the present invention includes methods for the prevention or delayed onset, or reduction of the progression or growth, of a disease or disorder in a subject, as the polypeptides of the present invention can be administered to the subject prior to detection of the disease or disorder. In certain embodiments, the polypeptides of the present invention are administered to a subject having a strong family history of the disease or disorder, thereby preventing or delaying the onset or progression of the disease or disorder.

[0219] With the disclosure herein, those skilled in the art will thus understand that preventing a disease or disorder in a subject includes administering the polypeptides of the present invention to the subject as a preventive measure against the disease or disorder.

[0220] Pharmaceutical Compositions and Formulations

[0221] The present invention provides within the methods described herein pharmaceutical compositions comprising the polypeptides of the present invention.

[0222] Such pharmaceutical compositions are in a form suitable for administration to a subject, or the pharmaceutical composition can further comprise one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination thereof. As is well known in the art, the various components of the pharmaceutical composition can be present in the form of physiologically acceptable salts, for example in combination with physiologically acceptable cations or anions.

[0223] In embodiments, the pharmaceutical compositions for practicing the methods of the present invention can be administered to deliver a dose between 1 ng / kg / day and 100 mg / kg / day. In other embodiments, the pharmaceutical compositions for practicing the present invention can be administered to deliver a dose between 1 ng / kg / day and 500 mg / kg / day.

[0224] The relative amounts of the active ingredient, pharmaceutically acceptable carrier, and any additional ingredients in the pharmaceutical compositions of the present invention will vary depending on the identity, size, and condition of the subject being treated, and further depending on the route of administration of the pharmaceutical composition. For example, the composition can comprise between about 0.1% and about 100% (w / w) of the active ingredient.

[0225] The pharmaceutical compositions useful in the methods of the present invention can be appropriately developed for inhalation, oral, rectal, vaginal, parenteral, topical, transdermal, pulmonary, intranasal, buccal, ocular, intrathecal, intravenous, or another route of administration. Other contemplated formulations include projected nanoparticles, liposomal preparations, resealed red blood cells containing the active ingredient, and immunologically-based formulations. One or more routes of administration will be apparent to the person of ordinary skill in the art and will depend on many factors, including the type and severity of the disease being treated, the type and age of the mammalian or human patient being treated, and the like.

[0226] The formulations of the pharmaceutical compositions described herein can be prepared by any method known in the art of pharmacology or developed hereinafter. Generally, such a preparation method includes the step of associating the active ingredient with a carrier or one or more other excipient ingredients, and then, if necessary or desired, shaping or packaging the product into the desired single-dose or multi-dose unit.

[0227] As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition that contains a predetermined amount of the active ingredient. The amount of the active ingredient generally equals the dose of the active ingredient to be administered to a subject or a convenient fraction of that dose, such as one-half or one-third of that dose. The unit dosage form can be 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 for each dose can be the same or different.

[0228] Administration

[0229] The administration regimen may affect what constitutes an effective amount. For example, several divided doses can be administered daily or sequentially and staggered, or continuous infusion doses can be administered, or bolus injections can also be used. Further, the dose of the therapeutic formulation can be increased or decreased proportionally as indicated in an emergency situation in a therapeutic or prophylactic context. In certain embodiments, administration of a compound of the present invention to a subject raises the plasma PPi of the subject to near normal, where the normal level of PPi in a mammal is 1 - 3 μM. "Near normal" means 0 to 1.2 μM or 0 - 40% lower or higher than normal, 30 nM to 0.9 μM or 1 - 30% lower or higher than normal, 0 to 0.6 μM or 0 - 20% lower or higher than normal, 0 to 0.3 μM or 0 - 10% lower or higher than normal.

[0230] The compositions of the present invention can be administered to a patient, such as a mammal, using known procedures, in a dose and for a period of time effective to treat a disease or disorder of the patient. The effective amount of the therapeutic compound necessary to achieve a therapeutic effect can vary depending on a variety of factors, such as the activity of the specific compound used; 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, the age, sex, weight, condition, general health and previous medical history of the patient being treated, and similar factors well known in the medical arts. The dosage regimen can be adjusted to provide the optimum therapeutic response. The dose is determined according to the biological activity of the therapeutic compound, which in turn depends on the half-life and the area under the plasma time curve of the therapeutic compound. The polypeptides according to the present invention can be administered at appropriate time intervals of every 2 days, or every 4 days, or weekly or monthly to achieve a continuous level of plasma PPi that is close to the normal level of PPi (1-3 μM) or higher than the normal level of PPi (higher by 30-50%). The therapeutic dose of the polypeptides according to the present invention can also be determined based on the half-life or the rate of clearance of the therapeutic polypeptide from the body. The polypeptides according to the present invention are administered at appropriate time intervals of every 2 days, or every 4 days, weekly or monthly to achieve a constant level of the enzymatic activity of ENPP1.

[0231] For example, several divided doses can be administered daily or the dose can be proportionally reduced as indicated by the exigencies of the therapeutic situation. Non-limiting examples of the effective dose range of the therapeutic compounds of the present invention are from about 0.01 to 50 mg / kg body weight / day. In some embodiments, the effective dose range of the therapeutic compounds of the present invention is from about 50 ng to 500 ng / kg body weight, preferably from 100 ng to 300 ng / kg body weight. A person of ordinary skill in the art will be able to study the relevant factors and determine the effective amount of the therapeutic compound without undue experimentation.

[0232] The compound can be administered to the patient frequently several times a day, or it can 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 few months or even once a year or less. It should be understood that, in non-limiting examples, the amount of the compound administered daily can be administered every day, every other day, every 2 days, every 3 days, every 4 days or every 5 days. For example, using every other day administration, a dose of 5 mg per day can be started on Monday, the first subsequent dose of 5 mg per day can be administered on Wednesday, the second subsequent dose of 5 mg per day can be administered on Friday, and so on. The frequency of the dose will be apparent to the skilled person and depends on many factors, such as but not limited to the type and severity of the disease being treated and the type and age of the patient.

[0233] The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention can be varied to obtain an amount of the active ingredient that is effective in achieving the desired therapeutic response for a particular patient, composition, and mode of administration and that is non-toxic to the patient.

[0234] A physician having ordinary skill in the art, such as a medical doctor, can readily determine and prescribe the effective amount of the required pharmaceutical composition. For example, a physician or veterinarian can start with a dose of the compound of the present invention used in the pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved.

[0235] In certain embodiments, the compositions of the present invention are administered to a patient in a dosage range of once to five or more times per day. In other embodiments, the compositions of the present invention are administered to a patient in a dosage range including but not limited to once a day, once every two days, once every three days to once a week, and once every two weeks. The frequency of administration of the various compositions of the present invention depends on many factors and varies from subject to subject, which include but are not limited to age, the disease or disorder to be treated, gender, overall health, and other factors. Accordingly, the present invention should not be construed as being limited to any particular dosage regimen and precise dosage, and it is up to the attending physician to determine the composition to be administered to any patient, taking into account all other factors relevant to the patient.

[0236] In certain embodiments, the present invention relates to a packaged pharmaceutical composition comprising a container containing a therapeutically effective amount of the compound of the present invention alone or in combination with a second agent; and instructions for using the compound to treat, prevent, or alleviate one or more symptoms of a disease or disorder in a patient.

[0237] Route of administration

[0238] The route of administration of any composition of the present invention includes inhalation, oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginal), nasal (intranasal), and rectal), intravesical, intralung, intraduodenal, intragastric, intrathecal, subcutaneous, intramuscular, intradermal, intraarterial, intravenous, intratracheal, inhalational, and topical administration.

[0239] Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, lozenges, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, emulsions, 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 useful in the present invention are not limited to the specific formulations and compositions described herein.

[0240] Parenteral administration

[0241] As used herein, "parenteral administration" of a pharmaceutical composition includes any route of administration characterized by physical disruption of the tissue of a subject and administration of the pharmaceutical composition through that disruption in the tissue. Thus, parenteral administration includes, but is not limited to, administering the pharmaceutical composition by injection of the composition, by surgical incision, by non-surgical wound that penetrates the tissue, and the like. Specifically, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intravenous, intraperitoneal, intramuscular, intracardiac injection, and renal dialysis infusion techniques.

[0242] Additional dosage forms

[0243] Additional dosage forms of the present invention include those described in U.S. Patent Nos. 6,340,475, 6,488,962, 6,451,808, 5,972,389, 5,582,837, and 5,007,790. Additional dosage forms of the present invention also include those described in U.S. Patent Application Nos. 20030147952, 20030104062, 20030104053, 20030044466, 20030039688, and 20020051820. Additional dosage forms of the present invention also include those described in PCT Application Nos. WO 03 / 35041, WO 03 / 35040, WO03 / 35029, WO 03 / 35177, WO 03 / 35039, WO 02 / 96404, WO 02 / 32416, WO 01 / 97783, WO 01 / 56544, WO 01 / 32217, WO 98 / 55107, WO 98 / 11879, WO 97 / 47285, WO 93 / 18755, and WO 90 / 11757.

[0244] Controlled release formulations and drug delivery systems

[0245] Controlled release or sustained release formulations of the pharmaceutical compositions of the present invention can be prepared using conventional techniques. In some cases, the dosage forms used can provide slow or controlled release of one or more of the active ingredients, using, for example, hydroxypropyl methylcellulose, other polymeric matrices, gels, osmotic membranes, osmotic systems, multilayer coatings, microparticles, liposomes, or microspheres, or combinations thereof, to provide the desired release characteristics in different proportions. The present invention contemplates single unit dosage forms (e.g., tablets, capsules, softgels, and cachets) suitable for oral administration that are adapted for controlled release.

[0246] In certain embodiments, the formulations of the present invention can be, but are not limited to, short-term, rapid offset, and controlled, e.g., sustained release, delayed release, and pulsatile release, formulations

[0247] The term sustained release, in its conventional sense, refers to a pharmaceutical formulation that provides a gradual release of a drug over an extended period of time, which may, although not necessarily, result in a substantially constant drug blood level over an extended period of time. This period of time may be up to a month or longer and is a longer release than the same amount of the medicament administered in a bolus form. For sustained release, the compound can be prepared with a suitable polymer or a hydrophobic material that provides the compound with sustained release properties. Thus, the compounds using the method of the present invention can be administered in particulate form (e.g., by injection) or in the form of wafers or discs (by implantation). In some embodiments of the present invention, the compounds of the present invention are administered to a patient alone or in combination with another medicament using a sustained release formulation.

[0248] The term delayed release is used herein in its conventional meaning and refers to a pharmaceutical formulation that provides an initial release of a drug after some delay following drug administration, which may, although not necessarily, include a delay of from about 10 minutes up to about 12 hours. The term pulsatile release is used herein in its conventional meaning and refers to a pharmaceutical formulation that provides drug release in a manner that results in a plasma profile of drug pulses following drug administration. The term immediate release is used herein in its conventional meaning and refers to a pharmaceutical formulation that provides drug release immediately following drug administration.

[0249] As used herein, short term refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes or about 10 minutes following drug administration and any or all of its increments.

[0250] As used herein, rapid offset refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes or about 10 minutes following drug administration and any or all of its increments.

[0251] Using only routine experimentation, one of ordinary skill in the art will recognize or be able to ascertain many equivalents to the specific procedures, embodiments, claims and examples described herein. Such equivalents are considered to be within the scope of the present invention and are covered by the appended claims. For example, it should be understood that modifications to reaction and preparation conditions - using recognized alternatives in the art and only routine experimentation - are within the scope of this application.

[0252] It should be understood that wherever numerical values and ranges are provided herein, all numerical values and ranges encompassed by these values and ranges are intended to be covered within the scope of the present invention. In addition, all numerical values falling within these ranges, as well as the upper or lower limits of the value ranges, are also contemplated in this application.

[0253] The following examples further illustrate various aspects of the present invention. However, they are in no way a limitation of the teachings or disclosure of the present invention as described herein.

[0254] Example

[0255] The present invention is now described with reference to the following examples. These examples are provided for illustrative purposes only, and the present invention is not limited to these examples, but encompasses all variations that are obvious from the teachings provided herein.

[0256] Methods and Materials

[0257] Unless otherwise specified, the constructs were expressed in CHO cells or modified CHO cells with or without supplementation using the protocols described elsewhere herein, V max assay, K m / K cat assay, AUC assay, half-life assay.

[0258] Generation of ENPP1-Fc Mutant Constructs

[0259] Human NPP1 (human: NCBI accession number NP 006199) was modified to express a soluble recombinant protein, and the recombinant protein was fused with IgG1 by subcloning into the pFUSE-hlgG1-Fcl or pFUSE-mlgG1-Fcl plasmid (InvivoGen, San Diego CA), respectively. A commercially available kit ( Site-Directed Mutagenesis Kit / New England Biolabs) was used to generate constructs from SEQ ID NO:7 using site-directed mutagenesis. The constructs thus generated were sequenced to verify the nucleic acid sequence and then used for protein expression.

[0260] Expression of Mutant Constructs

[0261] Under bleomycin (Zeocin) / gentamicin selection, stable transfection of the ENPP1-Fc construct was established in CHO K1 cells (Sigma Aldrich, 85051005) and adapted for suspension growth. The adapted cells were used to inoculate liquid cultures and grown in CD FortiCHO TM medium (A1148301, Thermo Fischer) in shake flasks at 37 °C and 5% CO2 with stirring at 120 rpm under high humidity. The cultures were gradually expanded to the desired target volume and then maintained for an additional 2 days to accumulate extracellular protein.

[0262] Expression of ENPP1-Fc Mutant Constructs in Modified CHO Cells

[0263] CHO-K1 cells were modified to generate CHO-K1-MOD cells that stably express human α-2,6-sialyltransferase (α-2,6-ST) enzyme. Stable transfection of the ENPP1-Fc construct was established in CHO K1-MOD cells and the protein was expressed according to the same protocol as above. Optionally, in some constructs, the cell culture medium of CHO-K1-MOD cells expressing the corresponding construct was supplemented with sialic acid or a sialic acid "high-throughput" precursor called 1,3,4-O-Bu3ManNAc to promote higher levels of glycosylation during protein production.

[0264] Purification of ENPP1-Fc mutant constructs

[0265] The liquid culture was centrifuged at 4300×g for 5 min, and the supernatant was filtered through a 0.2 μm membrane and used 30.0.11m 2 A 30 kDa cassette (Millipore, Billerica MA) was concentrated by tangential flow. The concentrated supernatant was then purified by a combination of chromatographic techniques in a multi-step process. These techniques were performed sequentially and could include any of the following: affinity chromatography with protein A or protein G, cation exchange chromatography, anion exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, high performance liquid chromatography (HPLC), precipitation steps, extraction steps, lyophilization steps, and / or crystallization steps. By continuously using any of these steps, one of ordinary skill in the art of protein chemistry could purify the described substance composition to homogeneity such that there were no contaminating protein bands on a silver-stained gel. The resulting protein samples were then tested using a Pierce LAL Chromogenic Endotoxin Quantitation Kit (catalog number 88282) to ensure that all were endotoxin-free.

[0266] To quantify the biological impact of clone optimization, the pharmacodynamic effects of selected ENPP1-Fc isoforms were quantified by measuring plasma PPi concentrations at multiple time points after single subcutaneous administration of each isoform.

[0267] K m / K cat Determination

[0268] The steady-state hydrolysis of ATP of the ENPP1 construct was determined by HPLC. Briefly, the enzyme reaction was initiated by adding 10 nM PPi to different concentrations of ATP in a reaction buffer containing 20 mM Tris (pH 7.4), 150 mM NaCl, 4.5 nM KCl, 14 mM ZnCl2, 1 mM MgCl2, and 1 mM CaCl2. At different time points, 50 μl of the reaction solution was removed and quenched with an equal volume of 3 M formic acid. The quenched reaction solution was loaded onto a C-18 (5 μm, 250×4.6 mm) column (Higgins Analytical) equilibrated in a 5 mM ammonium acetate (pH 6.0) solution and eluted with a 0% to 20% methanol gradient. The substrates and products were monitored by UV absorbance at 259 nm and quantified based on the integration of their corresponding peaks and the standard curve.

[0269] V max Determination

[0270] For each mutant prepared, phosphodiesterase activity was assayed using thymidine 5'-monophosphate p-nitrophenyl ester (pNP-TMP) (Saunders et al., 2008, Mol. Cancer Ther. 7(10):3352-62; Albright et al., 2015, Nat Commun. 6:10006).

[0271] Area under the curve determination

[0272] The area under the plasma concentration-versus-time curve (also known as the area under the curve (AUC)) can be used as a method to evaluate the volume of distribution (V), total elimination clearance (CL), and bioavailability (F) for extravascular drug delivery. The area under the plasma time curve for each expressed and purified ENPP1-Fc construct was performed using standard equations to determine the half-life and bioavailability after a single subcutaneous injection of the biologic, as described in Equation 1.

[0273] Half-life determination

[0274] The drug half-life (t 1 / 2 ) is the time it takes for the plasma concentration or the amount of drug or biologic in the body to decrease by 50%. The half-life values for each expressed and purified ENPP1-Fc construct were performed following the prior art and / or the protocols described herein, such as Equation 1, which allows for the determination of the half-life and bioavailability after a single subcutaneous injection of the biologic.

[0275] The drug half-life can be calculated using Equation 1, which correlates the relationship between the systemic fractional concentration of a drug administered as a single injection to a subcutaneous depot and time. Plotting the data as the fraction of drug absorbed (F) versus time (t) allows determination of the elimination constant (k e ) and the absorption constant (k a ) by fitting the data to the total systemic absorption equation for a drug administered at the subcutaneous depot at time t = 0.

[0276]

[0277] Example 1: Selection and Optimization of Glycosylation Mutations

[0278] The AENPP1-Fc construct was mutated to introduce putative additional glycosylation sites and / or increase the affinity of the Fc for the neonatal Fc receptor (FcRn). The mutations tested are described elsewhere herein, and the specific constructs discussed are described below.

[0279] Improving the pharmacokinetic properties of ENPP1-Fc was sought by introducing additional N-linked glycosylation sites and enhancing the pH-dependent recycling of the fusion protein. As a method to guide the selection of additional N-linked glycosylation sites, an electron density map derived from X-ray diffraction of the mouse ENPP1 crystal was used, which revealed four glycosylation sites in ENPP1. These sites were hypothesized to be present in the highly homologous human ENPP1, and in addition, human ENPP1 also contains four additional N-linked glycosylation consensus sequences, the glycosylation status of which is unknown ( Figure 6B ).

[0280] To identify regions of ENPP1 suitable for hyperglycosylation that would not have an adverse effect on catalytic activity, a combination of the structural model of ENPP1 in GACI patients, clinical data, and genetic data was used. First, N-linked glycosylation consensus sequences were identified in ENPP2, and sequences that were amenable to allowing the introduction of glycosylation sites by altering a single adjacent residue were evaluated. Then ENPP2-7 was modeled structurally using standard software to thread the sequences through the mouse ENPP1 structure (PDB ID code 4GTW). The positions of the proposed glycosylation sites were compared to the positions of known inactivating ENPP1 mutations in GACI ( Figures 6A - 6B ) and the positions of disulfide bonds in the enzyme. If the predicted spatial position of the proposed glycosylation site interfered with either, that site was discarded. These modeling studies led to the identification of 53 potential sites for additional N-linked glycosylation programs that could be readily introduced into ENPP1 without disruption of protein folding or enzymatic activity being expected (Figures 7, 13, and 14).

[0281] Then, additional N-glycosylation consensus sequences were introduced into human ENPP1-Fc (hENPP1-Fc, construct #770) by site-directed mutagenesis. The protein was transiently expressed in CHO cells in 96-well plates and the enzymatic activity of the extracellular supernatant of each clone was screened in triplicate in a high-throughput assay using pNP-TMP as a chromogenic substrate as described in the method ( Figures 7A - 7D ). The rate of pNP-TMP hydrolysis was equal to or better than that of construct #770 in 10 out of 53 possible ENPP1-Fc isoforms ( Figures 7A - 7D ), and these 10 glycoforms were selected for combinatorial optimization with each other and the IgG1 Fc domain as described below.

[0282] FcRn is the major homeostatic regulator of the serum half-life of human IgG1 Fc, and mutations in the Fc domain that enhance the pH-dependent interaction of Fc with FcRn prolong the circulating half-life of biologics. The effects of two Fc mutations reported to enhance pH-dependent recycling, H433K / N434F (hereinafter referred to as the HN mutation) and M242Y / S254T / T246E (hereinafter referred to as the MST mutation), were examined herein ( Figures 8A - 8B ). Either of the two variants of the Fc domain was randomly combined with one or more of the 10 ENPP1-Fc glycoforms demonstrating acceptable hydrolysis rates, thereby creating 12 additional ENPP1-Fc clones (Table 3). Some of these clones were selected to test the effect of multiple glycoforms on the pharmacokinetics of ENPP1-Fc, where two spatially distinct putative glycosylation sites on different protein domains were selected to enhance the potential glycan shielding of the protein surface area (Table 3; constructs #1057, #1064, #1014, #1040). Other clones were tested only for the effect of the Fc mutation on the pK properties alone or in the presence of only a single additional putative glycosylation (Table 3; constructs #981 and #1051, respectively).

[0283] Example 2: Expression Using a CHO Cell Line and Growth Conditions

[0284] In recombinantly produced proteins, non-human Chinese hamster ovary (CHO) cells are widely used for the production of biologics due to the similarity of the CHO and human glycosylation patterns. However, there are glycosylation differences between the two, most notably that the terminal sialic acid residues of human N-glycans have both α-2,3 and α-2,6 linkages, while CHO cells contain only α-2,3 linkages.

[0285] To test whether the terminal sialylation differences between CHO and human cells affect PK and bioavailability in this system, a CHO cell line stably expressing human α-2,6-sialyltransferase (α-2,6-ST) was established as the host, and this clone was used to produce 7 ENPP1 isoforms to compare the effects of α-2,6 linkages on PK and bioavailability in multiple constructs (Table 5; construct numbers ending with '-ST'). To explore the effects of growth conditions on PK and bioavailability, cells stably transfected with the selected ENPP1-Fc isoform (both CHO K1 cells and CHO K1 cells stably transfected with human α-2,6-ST) were supplemented with the "high-throughput" precursor of sialic acid, which is called 1,3,4-O-Bu3ManNAc (Table 5) during protein production.

[0286] The ENPP1-Fc isoforms were purified to homogeneity using the same purification protocol, and Michaelis-Menton enzyme rate constants and pharmacokinetic properties were determined as described elsewhere in this article. Finally, the pharmacodynamic effects of the selected ENPP1-Fc isoforms were quantified by measuring plasma PPi concentrations at multiple time points after single subcutaneous administration of each isoform.

[0287] Example 3: Pharmacokinetic effects of additional N-glycosylation sites

[0288] Adding N-glycosylation sites to the glycoform using the above-described in silico prediction and HTS methods significantly increased in vivo exposure of mice to ENPP1-Fc - 4-fold increase in construct #1020. The sizes of the ENPP1-Fc isoforms in Table 2 were compared by SDS-PAGE gels to determine which sequence variations led to increased glycosylation, and an increase in molecular weight was shown to be consistent with the addition of glycosylation. To determine whether the sequence changes in construct #1020 successfully introduced glycosylation, MALDI-TOF was used, which also confirmed the presence of glycosylation at these sites.

[0289] Example 4: Pharmacokinetic effects of Fc IgG1 mutations ( Figures 10 - 11 )

[0290] Antibodies containing mutations in the Fc domain that enhance its affinity for FcRn and increase pH-dependent antibody recycling have never been used in therapeutic enzymes fused to the Fc domain. Some Fc mutations have successfully increased the affinity of the Fc domain for the FcRn receptor but have led to adverse PK properties in in vivo antibody PK, while others have shown enhanced in vivo PK properties.

[0291] To determine whether similar Fc modifications enhance the PK properties of the enzyme fusion proteins, two specific IgG1 mutations in the Fc of the previously used biologic antibody, H433K / N434F and M242Y / S254T / T246E, were investigated. Typically, the M242Y / S254T / T246E mutation was found to be superior to H433K / N434F in improving the properties of ENPP1-Fc. For example, construct #981, which had only the M242Y / S254T / T246E mutation compared to construct #770, increased the half-life by 3.3-fold and the AUC by 5.8-fold. In contrast, in the case of multiple ENPP1 mutations, the H433K / N434F mutant construct achieved a more modest increase in half-life between 1.2 - 1.7-fold.

[0292] Example 5: Effects of Host Cells and Growth Conditions

[0293] The expression of proteins in CHO cells stably transfected with human α-2,6-ST has been successfully used to produce recombinant biologics with terminal sialic acid residues that possess both α-2,3 and α-2,6 linkages, which have been reported to increase and decrease PK properties depending on the biologic.

[0294] To determine whether the α-2,6 linkage affects the PK properties of ENPP1-Fc, the in vivo exposure (AUC) and half-life (Table 4) of 7 ENPP1-Fc isoforms produced in either CHOK1 cells or CHOK1 cells stably transfected with human α-2,6-ST were directly compared. The overall trend of producing biologics in CHOK1 cells stably transfected with human α-2,6-ST was beneficial. The strongest effect was noted in the organism's exposure to the drug (AUC), indicating a 1.7 - 4.6-fold increase in AUC in the responding isoforms (constructs #1057, #1028, #951, #930, and #981). Another trend was that the magnitude of the AUC effect was greater in isoforms with a lower initial AUC (constructs #951 and #1057). However, the effect was quite substantial in the longer-lasting isoforms (constructs #1028 and #981), which produced AUC values 8 - 10 times greater than the parent compound produced in CHOK1 cells.

[0295] The effect of the α-2,6 linkage on the half-life was more modest, increasing by 20 - 30% in the responding constructs. To understand the different effects of the α-2,6 linkage on AUC and half-life, the change in protein activity over time of the isoforms produced in CHO k1 cells and 1078 cells was compared.

[0296] Example 6: Pharmacokinetic Effects of Growing with High-Throughput Sialic Acid Precursors

[0297] To determine the effect of growth conditions on PK properties, the "high-throughput" sialic acid precursor 1,3,4-O-Bu3ManNAc or sialic acid itself was supplemented to the culture medium of selected clones. Supplementation of CHOK1 cells with 1,3,4-O-Bu3ManNAc had little improvement on the PK properties of ENPP1-Fc, but when the biologic was produced in CHOK1 cells stably transfected with human α-2,6-ST, the effects on half-life and AUC were notable ( Figure 12 and Table 4). For example, supplementation of the cell culture medium of CHOK1 cells producing construct #1014 with 1,3,4-O-Bu3ManNAc had little effect on enhancing the AUC and seemed to decrease the half-life of the isoform. In contrast, when 1,3,4-O-Bu3ManNAc was added to the cell culture medium of construct #1057 produced in CHOK1 cells stably transfected with α-2,6-ST, the effects were more pronounced. Compared to the same isoform produced in CHOK1 grown in medium not supplemented with 1,3,4-O-Bu3ManNAc, these effects produced a net increase in AUC and half-life of 4-fold and 2-fold, respectively ( Figure 12 and Table 4).

[0298] Table 1.

[0299]

[0300] Table 2: Effect of additional N-glycosylation on pharmacokinetics (PK).

[0301]

[0302] Table 3: Effect of Fc mutations on pharmacokinetics (PK).

[0303]

[0304]

[0305] Table 4: Effect of cell line and mutations on pharmacokinetics (PK). Constructs labeled "-ST" were prepared using a modified CHO cell line stably transfected with human α-2,6-sialyltransferase (α-2,6-ST); this increased the amount of sialylation of the fusion protein when compared to the fusion protein expressed in a normal CHO cell line. Enhanced construct sialylation led to improved AUC and half-life values.

[0306]

[0307]

[0308] *: Construct transfected into CHO-K1 cells

[0309] **: Construct transfected into CHO-K1-MOD cells

[0310] Table 5: Effect of sialic acid supplementation on pharmacokinetics (PK). Those constructs labeled "-ST" were prepared using a modified CHO cell line stably transfected with human α-2,6-sialyltransferase (α-2,6-ST); this increased the amount of sialylation of the fusion protein when compared to the fusion protein expressed in the normal CHO cell line. Those constructs labeled "-A" were prepared from cells grown in media supplemented with 1,3,4-O-Bu3ManNAc (a "high-throughput" precursor of sialic acid) during protein production.

[0311]

[0312]

[0313] Table 6: List of polypeptides and corresponding mutants

[0314]

[0315] Table 7: List of mutants in the ENPP1 polypeptide

[0316] Mutated residue Mutated residue C25N P558N K27T E560T V29N E591N E115N E592K P117T E592N P125T P643T A276N S645T L278T S765N D285N S766N R287T S885N Y364T R741A K369N V793N I371T H794S H409T G795T P448L G795N S449T H797T P521L E864N V522T L866T V522N H1064K K526N N1065K P528T M883Y P534N S885T V536T T887E P543L M1059L R544T N1065S R545T I884A G548T H941A P554H H1066A P554L

[0317] Enumerated embodiments:

[0318] The following exemplary embodiments are provided, and their numbers should not be construed as designating an order of importance.

[0319] Embodiment 1 provides an ENPP1 polypeptide fusion comprising an ENPP1 polypeptide fused to the Fc region of an immunoglobulin, wherein the Fc region comprises at least one mutation selected from M883Y, S885N, S885T, T887E, H1064K, and N1065F related to SEQ ID NO:7.

[0320] Embodiment 2 provides the polypeptide fusion according to Embodiment 1, wherein the Fc region comprises at least one mutation selected from S885N, M883Y, M883Y / S885T / T887E, and H1064K / N1065F related to SEQ ID NO:7.

[0321] Embodiment 3 provides an ENPP1 polypeptide fusion comprising an ENPP1 polypeptide fused to the Fc region of an immunoglobulin, wherein the ENPP1 polypeptide comprises at least one mutation selected from C25N, K27T, and V29N related to SEQ ID NO:7.

[0322] Embodiment 4 provides a polypeptide fusion according to Embodiment 3, wherein the ENPP1 polypeptide comprises at least one mutation selected from C25N / K27T and V29N related to SEQ ID NO:7.

[0323] Embodiment 5 provides an ENPP1 polypeptide fusion comprising an ENPP1 polypeptide fused to the Fc region of an immunoglobulin, wherein the ENPP1 polypeptide comprises at least one mutation selected from K369N and I371T related to SEQ ID NO:7.

[0324] Embodiment 6 provides a polypeptide fusion according to Embodiment 5, wherein the ENPP1 polypeptide comprises at least the mutations K369N / I371T related to SEQ ID NO:7.

[0325] Embodiment 7 provides an ENPP1 polypeptide fusion comprising an ENPP1 polypeptide fused to the Fc region of an immunoglobulin, wherein the ENPP1 polypeptide comprises at least one mutation selected from P534N, V536T, R545T, P554L, E592N, R741D, and S766N related to SEQ ID NO:7.

[0326] Embodiment 8 provides a polypeptide fusion according to Embodiment 7, wherein the ENPP1 polypeptide comprises at least one mutation selected from P534N / V536T, P554L / R545T, E592N, E592N / R741D, and S766N related to SEQ ID NO:7.

[0327] Embodiment 9 provides an ENPP1 polypeptide fusion comprising an ENPP1 polypeptide fused to the Fc region of an immunoglobulin, wherein the ENPP1 polypeptide comprises at least one mutation selected from E864N and L866T related to SEQ ID NO:7.

[0328] Embodiment 10 provides a polypeptide fusion according to Embodiment 9, wherein the ENPP1 polypeptide comprises at least the mutations E864N / L866T related to SEQ ID NO:7.

[0329] Embodiment 11 provides a polypeptide fusion according to any one of Embodiments 1-10, comprising at least one mutation selected from C25N, K27T, V29N, C25N / K27T, K369N, I371T, K369N / I371T, P534N, V536T, R545T, P554L, E592N, R741D, S766N, P534N / V536T, P554L / R545T, E592N / R741D, E864N, L866T, E864N / L866T, M883Y, S885N, S885T, T887E, H1064K, N1065F, M883Y / S885T / T887E, H1064K / N1065F related to SEQ ID NO:7.

[0330] Embodiment 12 provides a polypeptide fusion according to any one of Embodiments 1-11, wherein the Fc region is that of IgG.

[0331] Embodiment 13 provides a polypeptide fusion according to any one of Embodiments 1-12, comprising at least one mutation selected from P534N, V536T, R545T, P554L, S766N and E592N related to SEQ ID NO:7.

[0332] Embodiment 14 provides a polypeptide fusion according to any one of Embodiments 1-12, comprising at least one mutation selected from S766N, P534N / Y536T, P554L / R545T and E592N related to SEQ ID NO:7.

[0333] Embodiment 15 provides a polypeptide fusion according to any one of Embodiments 1-12, comprising at least one mutation selected from S885N, S766N, M883Y / S885T / T887E, E864N / L866T, P534N / V536T / H1064K / N1065F, P554L / R545T, S766N / H1064K / N1065F, E592N / H1064K / N1065F and P534N / V536T / M883Y / S885T / T887E related to SEQ ID NO:7.

[0334] Embodiment 16 provides an ENPP1 polypeptide fusion, which comprises an ENPP1 polypeptide and an Fc region of an immunoglobulin, and the polypeptide fusion comprises mutations M883Y, S885T and T887E related to SEQ ID NO:7.

[0335] Embodiment 17 provides an ENPP1 polypeptide fusion comprising an ENPP1 polypeptide and the Fc region of an immunoglobulin, the polypeptide fusion comprising the mutations P534N, V536T, M883Y, S885T, and T887E related to SEQ ID NO:7.

[0336] Embodiment 18 provides an ENPP1 polypeptide fusion comprising an ENPP1 polypeptide and the Fc region of an immunoglobulin, the polypeptide fusion comprising the mutations E592N, H1064K, and N1065F related to SEQ ID NO:7.

[0337] Embodiment 19 provides an ENPP1 mutant polypeptide comprising SEQ ID NO:7, wherein the mutant polypeptide comprises mutations selected from S766N, P534N, V536T, P554L, R545T, and E592N related to SEQ ID NO:7.

[0338] Embodiment 20 provides the mutant polypeptide according to Embodiment 19, wherein the mutant polypeptide comprises at least one mutation selected from S766N, P534N / V536T, P554L / R545T, and E592N related to SEQ ID NO:7.

[0339] Embodiment 21 provides the mutant polypeptide according to Embodiment 19, which comprises mutations selected from S885N, S766N, M883Y / S885T / T887E, P534N / V536T / H1064K / N1065F, P554L / R545T, S766N / H1064K / N1065F, E592N / H1064K / N1065F, and P534N / V536T / M883Y / S885T / T887E related to SEQ ID NO:7.

[0340] Embodiment 22 provides the mutant polypeptide according to Embodiment 19, which comprises the S885N mutation related to SEQ ID NO:7.

[0341] Embodiment 23 provides the mutant polypeptide according to Embodiment 19, which comprises the S766N mutation related to SEQ ID NO:7.

[0342] Embodiment 24 provides the mutant polypeptide according to Embodiment 19, which comprises the mutations M883Y, S885T, and T887E related to SEQ ID NO:7.

[0343] Embodiment 25 provides a mutant polypeptide according to Embodiment 19, which comprises mutations P534N, V536T, H1064K and N1065F related to SEQ ID NO:7.

[0344] Embodiment 26 provides a mutant polypeptide according to Embodiment 19, which comprises mutations P554L and R545T related to SEQ ID NO:7.

[0345] Embodiment 27 provides a mutant polypeptide according to Embodiment 19, which comprises mutations S766N, H1064K and N1065F related to SEQ ID NO:7.

[0346] Embodiment 28 provides a mutant polypeptide according to Embodiment 19, which comprises mutations E592N, H1064K and N1065F related to SEQ ID NO:7.

[0347] Embodiment 29 provides a mutant polypeptide according to Embodiment 19, which comprises mutations P534N, V536T, M883Y, S885T and T887E related to SEQ ID NO:7.

[0348] Embodiment 30 provides a polypeptide fusion according to any one of Embodiments 1-18 or a mutant polypeptide according to any one of Embodiments 19-29, which is expressed by a CHO cell line stably transfected with human ST6β-galactoside α-2,6-sialyltransferase (also known as ST6GAL1).

[0349] Embodiment 31 provides a polypeptide fusion according to any one of Embodiments 1-18 or a mutant polypeptide according to any one of Embodiments 19-29, which is grown in a cell culture supplemented with sialic acid and / or N-acetylmannosamine (also known as 1,3,4-O-Bu3ManNAc).

[0350] Embodiment 32 provides a method for reducing or preventing the progression of pathological calcification in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide fusion according to any one of Embodiments 1-18 and 30-31 or a mutant polypeptide according to any one of Embodiments 19-31.

[0351] Embodiment 33 provides a method for reducing or preventing the progression of pathological ossification in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide fusion according to any one of Embodiments 1-18 and 30-31 or a mutant polypeptide according to any one of Embodiments 19-31.

[0352] Embodiment 34 provides a method for reducing or preventing the progression of soft tissue ectopic calcification in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide fusion according to any one of Embodiments 1-18 and 30-31 or a mutant polypeptide according to any one of Embodiments 19-31.

[0353] Embodiment 35 provides a method for treating, reversing or preventing the progression of ossification of the posterior longitudinal ligament (OPLL) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide fusion according to any one of Embodiments 1-18 and 30-31 or a mutant polypeptide according to any one of Embodiments 19-31.

[0354] Embodiment 36 provides a method for treating, reversing or preventing the progression of hypophosphatemic rickets in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide fusion according to any one of Embodiments 1-18 and 30-31 or a mutant polypeptide according to any one of Embodiments 19-31.

[0355] Embodiment 37 provides a method for reducing or preventing the progression of at least one disease in a subject diagnosed with at least one disease selected from the group consisting of chronic kidney disease (CKD), end-stage renal disease (ESRD), calcific uremic arteriolopathy (CUA), calciphylaxis, ossification of the posterior longitudinal ligament (OPLL), hypophosphatemic rickets, osteoarthritis, age-related arteriosclerosis, idiopathic infantile arterial calcification (IIAC), generalized arterial calcification of infancy (GACI), and atherosclerotic plaque calcification, the method comprising administering to the subject a therapeutically effective amount of a polypeptide fusion according to any one of Embodiments 1-18 and 30-31 or a mutant polypeptide according to any one of Embodiments 19-31.

[0356] Embodiment 38 provides a method for reducing or preventing the progression of age-related arteriosclerosis in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide fusion according to any one of Embodiments 1-18 and 30-31 or a mutant polypeptide according to any one of Embodiments 19-31.

[0357] Embodiment 39 provides the method according to Embodiment 32, wherein the pathological calcification is selected from idiopathic infantile arterial calcification (IIAC) and atherosclerotic plaque calcification.

[0358] Embodiment 40 provides the method according to Embodiment 33, wherein the pathological ossification is selected from ossification of the posterior longitudinal ligament (OPLL), hypophosphatemic rickets, and osteoarthritis.

[0359] Embodiment 41 provides the method according to Embodiment 34, wherein the soft tissue calcification is selected from IIAC and osteoarthritis.

[0360] Embodiment 42 provides the method according to Embodiment 34, wherein the soft tissue is selected from atherosclerotic plaque, muscular artery, joint, spine, articular cartilage, intervertebral disc cartilage, blood vessel, and connective tissue.

[0361] Embodiment 43 provides a method for increasing the level of pyrophosphate (PPi) in a subject whose PPi level is lower than the normal PPi level, the method comprising administering to the subject a therapeutically effective amount of the polypeptide of the polypeptide fusion according to any one of Embodiments 1-18 and 30-31 or the mutant polypeptide according to any one of Embodiments 19-31, whereby after the administration, the level of PPi in the subject is elevated to at least the normal level of 2 μM and maintained at approximately the same level.

[0362] Embodiment 44 provides a method for reducing or preventing the progression of pathological calcification or ossification in a subject whose pyrophosphate (PPi) level is lower than the normal PPi level, the method comprising administering to the subject a therapeutically effective amount of the polypeptide fusion according to any one of Embodiments 1-18 and 30-31 or the mutant polypeptide according to any one of Embodiments 19-31, thereby reducing pathological calcification or ossification in the subject or preventing the progression of pathological calcification or ossification in the subject.

[0363] Embodiment 45 provides a method for treating ENPP1 deficiency manifested by a reduction in the concentration of extracellular pyrophosphate (PPi) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the polypeptide fusion according to any one of Embodiments 1-18 and 30-31 or the mutant polypeptide according to any one of Embodiments 19-31, thereby increasing the level of PPi in the subject.

[0364] Embodiment 46 provides the method according to any one of Embodiments 32-45, wherein the polypeptide fusion or mutant polypeptide is a secreted product of the ENPP1 precursor protein expressed in mammalian cells, wherein the ENPP1 precursor protein comprises a signal peptide sequence and an ENPP1 polypeptide, and wherein the ENPP1 precursor protein is proteolytically processed to produce the ENPP1 polypeptide.

[0365] Embodiment 47 provides the method according to Embodiment 46, wherein in the ENPP1 precursor protein, the signal peptide sequence is conjugated to the N-terminus of the ENPP1 polypeptide.

[0366] Embodiment 48 provides a method according to any one of Embodiments 46-47, wherein the signal peptide sequence is selected from an ENPP1 signal peptide sequence, an ENPP2 signal peptide sequence, an ENPP7 signal peptide sequence, and an ENPP5 signal peptide sequence.

[0367] Embodiment 49 provides a method according to any one of Embodiments 32-48, wherein the polypeptide fusion or mutant polypeptide is administered to the subject acutely or chronically.

[0368] Embodiment 50 provides a method according to any one of Embodiments 32-49, wherein the polypeptide fusion or mutant polypeptide is administered to the subject locally, regionally, parenterally, or systemically.

[0369] Embodiment 51 provides a method according to any one of Embodiments 32-50, wherein the polypeptide fusion or mutant polypeptide is administered to the subject by at least one route selected from: subcutaneous, oral, aerosol, inhalation, rectal, vaginal, transdermal, intradermal, intranasal, buccal, sublingual, parenteral, intrathecal, intragastric, ocular, pulmonary, and topical.

[0370] Embodiment 52 provides a method according to any one of Embodiments 32-51, wherein the polypeptide fusion or mutant polypeptide is administered to the subject as a pharmaceutical composition, the pharmaceutical composition further comprising at least one pharmaceutically acceptable carrier.

[0371] Embodiment 53 provides a method according to any one of Embodiments 32-52, wherein the subject is a mammal.

[0372] Embodiment 54 provides a method according to Embodiment 53, wherein the mammal is a human.

[0373] The respective disclosures of each patent, patent application, and publication cited herein are incorporated herein by reference in their entirety. Although the invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of the invention can be devised by other persons skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

1. An ENPP1 polypeptide fusion comprising an ENPP1 polypeptide fused to the Fc region of an immunoglobulin, wherein the Fc region comprises at least one mutation selected from M883Y, S885N, S885T, T887E, H1064K, and N1065F related to SEQ ID NO:

7.

2. The polypeptide fusion according to claim 1, wherein the Fc region comprises at least one mutation selected from S885N, M883Y, M883Y / S885T / T887E, and H1064K / N1065F related to SEQ ID NO:

7.

3. An ENPP1 polypeptide fusion comprising an ENPP1 polypeptide fused to the Fc region of an immunoglobulin, wherein the ENPP1 polypeptide comprises at least one mutation selected from C25N, K27T, and V29N related to SEQ ID NO:

7.

4. The polypeptide fusion according to claim 3, wherein the ENPP1 polypeptide comprises at least one mutation selected from C25N / K27T and V29N related to SEQ ID NO:

7.

5. An ENPP1 polypeptide fusion comprising an ENPP1 polypeptide fused to the Fc region of an immunoglobulin, wherein the ENPP1 polypeptide comprises at least one mutation selected from K369N and I371T related to SEQ ID NO:

7.

6. The polypeptide fusion according to claim 5, wherein the ENPP1 polypeptide comprises at least the mutation K369N / I371T related to SEQ ID NO:

7.

7. An ENPP1 polypeptide fusion comprising an ENPP1 polypeptide fused to the Fc region of an immunoglobulin, wherein the ENPP1 polypeptide comprises at least one mutation selected from P534N, V536T, R545T, P554L, E592N, R741D, and S766N related to SEQ ID NO:

7.

8. The polypeptide fusion according to claim 7, wherein the ENPP1 polypeptide comprises at least one mutation selected from P534N / V536T, P554L / R545T, E592N, E592N / R741D, and S766N related to SEQ ID NO:

7.

9. An ENPP1 polypeptide fusion comprising an ENPP1 polypeptide fused to the Fc region of an immunoglobulin, wherein the ENPP1 polypeptide comprises at least one mutation selected from E864N and L866T related to SEQ ID NO:

7.

10. The polypeptide fusion according to claim 9, wherein the ENPP1 polypeptide comprises at least the mutation E864N / L866T related to SEQ ID NO:7.

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