ENPP1 polypeptides and methods of using the same

Abstract

To provide polypeptides that can be used to treat certain calcification or ossification diseases in vivo, wherein the polypeptides have in vivo half-lives that allow for convenient and effective dosing of the polypeptides to a subject in need thereof.SOLUTION: The present invention provides an ENPP1 polypeptide fusion comprising an ENPP1 polypeptide fused to an Fc region of an immunoglobulin, wherein the polypeptide fusion comprises at least one point mutation.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 62 / 725,607, filed August 31, 2018, and U.S. Provisional Patent Application No. 62 / 830,247, filed April 5, 2019, all of which are incorporated by reference in their entireties. [Background technology]

[0002] 2. Background of the Invention The human ectonucleotide pyrophosphatase (ENPP) protein family comprises seven extracellular glycosylated proteins (i.e., ENPP1-ENPP7) that hydrolyze phosphodiester bonds. ENPPs are cell surface enzymes, except for ENPP2, which is transported to the plasma membrane but is cleaved by furin and released into the extracellular fluid. ENPP enzymes have a high degree of sequence and structural homology but display diverse substrate specificities, including from nucleotides to lipids.

[0003] ENPP1 (also known as PC-1) is a type 2 extracellular membrane-bound glycoprotein located in mineral-accumulating matrix vesicles of osteoblasts and chondrocytes that hydrolyzes extracellular nucleotides (mainly ATP) to adenosine monophosphate (AMP) and inorganic pyrophosphate (PPi). PPi functions as a potent inhibitor of ectopic tissue mineralization by binding to newly developing hydroxyapatite (HA) crystals, thereby preventing the future growth of these crystals. ENPP1 generates PPi through hydrolysis of nucleotide triphosphate (NTP). Progressive ankylosis protein (ANK) transports intracellular PPi to the extracellular space, and tissue nonspecific alkaline phosphatase activity (TNAP) removes PPi through direct hydrolysis of PPi to Pi.

[0004] Ectopic tissue mineralization is associated with many human diseases, including chronic joint disease and acute lethal infantile syndrome. To prevent unwanted tissue calcification, a strict balance must be maintained between factors that promote and inhibit tissue mineralization. The balance of extracellular inorganic pyrophosphate (PPi) and phosphate (Pi) is a key regulator of ectopic tissue mineralization. The activity of three extracellular enzymes, TNAP, ANK, and ENPP1, tightly controls the concentrations of Pi and PPi to 1–3 mM and 2–3 μM, respectively, in mammals. PPi is a regulator of biomineralization and inhibits the formation of basic calcium phosphate from amorphous calcium phosphate.

[0005] ENPP1 polypeptide has 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 in infancy), a serious disease that occurs in infants and involves widespread arterial calcification (Albright, et al., 2015, Nature Comm. 10006). Fusion proteins of ENPP1 have also been reported to treat severe tissue calcification diseases (PCT Application Publication Nos. WO2014 / 126965 (Patent Document 1) and WO2016 / 187408 (Patent Document 2)), and fusion proteins of ENPP1 containing a bone targeting domain have been reported to treat GACI (PCT Application Publication No. WO / 2012 / 125182 (Patent Document 3)).

[0006] There is a need in the art for polypeptides that can be used to treat certain mineralization or ossification diseases in vivo. Such polypeptides should have an in vivo half-life that allows for convenient and effective administration of the polypeptide to subjects in need thereof. The present invention fulfills this need. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] WO2014 / 126965 [Patent Document 2] WO2016 / 187408 [Patent Document 3] WO / 2012 / 125182 [Non-patent literature]

[0008] [Non-Patent Document 1] Albright, et al., 2015, Nature Comm. 10006 Summary of the Invention

[0009] The present invention provides ENPP1 polypeptide fusions comprising an ENPP1 polypeptide fused to an Fc region of an immunoglobulin and comprising at least one point mutation as described herein. The present invention further provides ENPP1 mutant polypeptides comprising at least one point mutation as described elsewhere herein. The present invention further provides polypeptide fusions and / or mutant polypeptides, both expressed from a CHO cell line stably transfected with human ST6β-galactosamide α-2,6-sialyltransferase (ST6GAL1). The present invention further provides polypeptide fusions and / or mutant polypeptides, either of which are grown in 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 thereof, comprising administering to the subject a therapeutically effective amount of a polypeptide fusion and / or variant polypeptide of the invention.

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

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

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

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

[0017] The present invention further provides a method for increasing pyrophosphate (PPi) levels in a subject having lower than normal PPi levels, comprising administering to the subject a therapeutically effective amount of a polypeptide fusion and / or mutant polypeptide of the present invention, whereby, upon administration, the level of PPi in the subject is increased to a normal level of at least 2 μM and 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 having lower than normal pyrophosphate (PPi) levels, comprising administering to the subject a therapeutically effective amount of a polypeptide fusion and / or variant polypeptide of the invention, whereby pathological calcification and / or ossification in the subject is reduced and / or 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 decreased extracellular pyrophosphate (PPi) concentrations in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a 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 exemplary embodiments of the invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, exemplary embodiments are shown in the drawings. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings. [Figure 1]1 shows an ENPP1 polypeptide (SEQ ID NO:7) contemplated in the present invention. 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 indicates the amino acid number and residue with reference to the numbering scheme shown in SEQ ID NO:7, followed by the amino acid that is substituted for the residue in SEQ ID NO:7. For example, the mutation C25N represents a substitution of asparagine (Asn or N) for cysteine ​​(Cys or C) at position 25 of SEQ ID NO:7. Notes: (A) = N-terminal signal sequence from hENPP7; (B) all black regions 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 Invivogen plasmid pFUSE-hIgG1-Fc; (G) = 4 amino acid linker between hENPP1 and the Fc domain; (H) = known glycosylation residues. [Diagram 2] 1 shows a bar graph summarizing plasma phosphodiesterase activity (measured using the thymidine 5'-monophosphate p-nitrophenyl ester assay or pNP-TMP assay) following a single injection of specific ENPP1 polypeptides in mice (n=3-5). For all polypeptides, phosphodiesterase activity remained elevated after 25 hours, with higher activity observed at 75 hours for construct #981 (constructs of interest are outlined in tables contained elsewhere herein). [Diagram 3] Shown is the in vivo pharmacokinetic data of construct #981, measured using pNP-TNP assay to record enzyme activity in plasma samples of mice after subcutaneous injection of the construct. Its half-life was estimated to be approximately 122 hours based on a single subcutaneous bolus injection into five mice. Separate experiments to arrive at the half-life are described elsewhere herein. [Figure 4]1 shows selected in vivo pharmacokinetic data for construct #1014, construct #1014 (indicated as "1014A" in the graph) prepared in CHO cells grown in culture medium supplemented with 1,3,4-O-Bu3ManNAc, and construct #981. The half-lives of the constructs can be derived from Equation 1 described elsewhere herein. [Diagram 5] Shown are three known glycosylation sites in ENPP1, all located in random coil regions: (A) = Asn; (B) = N-acetylglucosamine. One additional glycosylation site (identified by surface glycoprotein dynamics measurements) is located within an α-helix and is labeled in red. One consensus NLT (Asn Leu Thr) is located in a PDB unstable region not yet known to be glycosylated. Four additional consensus sequences are found in hENPP1 whose glycosylation status is unknown: a calcium atom (C); two zinc atoms (D); an ATP molecule (E). [Figure 6A] 6A shows certain domains of human ENPP1 that have known loss-of-function mutations that cause the human disease "Generalized Arterial Calcification of Infancy" (GACI). In certain embodiments, glycosylation sites are not introduced near the regions that have known loss-of-function mutations that cause GACI (shown in FIG. 6A). [Figure 6B] The crystal structure of ENPP1 is shown with residues with known loss-of-function mutations that cause GACI highlighted (marked with *). The residue in (B) is located in the catalytic domain and corresponds to T238A. As in Figure 5, a calcium atom (C); two zinc atoms; (D) an ATP molecule (E). [Figure 7A]Figures 7A-7D show selected results from a high throughput TMP-pNP (thymidine monophosphate-p-nitrophenyl) assay of ENPP1 polypeptide for phosphodiesterase activity. This is a high throughput assay designed by the inventors to rapidly screen for glycosylation isoforms introduced into construct #770. The figure shows the design and implementation of a high throughput screen that allows rapid evaluation of the biological effects of mutant forms of the parent compound, construct #770. Construct numbers with (#) represent the original WT clones before mutations were introduced. Construct numbers with (*) indicate clones with putative gain-of-function mutations. [Figure 7B] See legend to Figure 7A. [Figure 7C] See legend to Figure 7A. [Figure 7D] See legend to Figure 7A. [Figure 8A] Figure 8A is a ribbon diagram showing the Fc domain of human IgG1. This domain is fused to the C-terminal portion of ENPP1 to enhance efficacy. Mutations were introduced into the Fc domain to enhance pH-dependent recycling by FcRn. (A) = sites that eliminate acid-dependent binding. (B) = sites that enhance binding. (C) = cysteine ​​disulfide bonds. Magenta = known glycosylation sites. [Figure 8B] FIG. 8B shows mutations in the Fc domain of human IgG1 that are known to enhance pH-dependent recycling by FcRn. [Figure 9]Included are graphs and tables showing the effect of glycosylation on the PK (half-life, time) and bioavailability of ENPP1 polypeptide. For all mutations, the PK was comparable to that of construct #CC07 (770B). Furthermore, construct #951 showed similar PK values ​​to that of construct #CC07, but construct #951 expanded in a cell line stably transfected with ST6GAL1 (construct #951-ST) showed improved PK and bioavailability. Construct #930 had a similar half-life to construct #CC07, but a lower bioavailability. In contrast, construct #1020 had a higher bioavailability than construct #CC07. PK and bioavailability data measured as shown in Figures 3, 4, and 12 and calculated using Equation 1 are shown in the table. [Figure 10] Contains graphs and tables showing the effect of glycosylation and H1064K / N1065F Fc mutations on the half-life (PK, hours) and bioavailability (AUC) of ENPP1 polypeptide. All H1064 / N1065-containing constructs showed improved half-life and AUC values ​​compared to construct #770B. Note that constructs #1048 and #1051 correspond to the same cDNA in two separate 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 also increased ST6GAL1 in stably transfected cell lines ("-ST") (construct #1057-ST) and in cell lines stably transfected with ST6GAL1 and supplemented with 1,3,4-O-Bu3-ManNAc ("-A") (construct #1064-ST-A). Construct #1089 is identical to construct #1014 except that a mutation was added to remove the putative trypsin cleavage site. Construct #1014 also increased ST6GAL1 in stably transfected cell lines, but in this example PK and bioavailability were not improved. PK and bioavailability data measured as shown in Figures 3, 4, and 12 and calculated using Equation 1 are shown in the table. [Figure 11] Included are graphs and tables showing the effect of glycosylation and M883Y / S885T / T887E Fc mutations on the PK (half-life, hours) and bioavailability of ENPP1 polypeptide. Construct #1030 has a lower AUC than the other constructs, likely due to the S766N mutation. Constructs #981 and #1028 showed increased values ​​of both PK and AUC when grown in cells stably transfected with ST6GAL1. PK and bioavailability data measured as shown in Figures 3, 4, and 12 and calculated using Equation 1 are shown in the tables. [Figure 12] Included is a collection of graphs showing the effect of expressing the constructs in CHO cells stably transfected with human α-2,6-ST to generate recombinant biomaterials with terminal sialic acid residues with both α-2,3 and α-2,6 linkages. These cells are referred to as ST6GAL1 cells or ST cells (represented by "-ST"). The figure also shows the effect of growing the constructs in ST6GAL1 cells in the presence of sialic acid or a high flux precursor of sialic acid known as 1,3,4-O-Bu3-ManNAc (represented by "-A"). PK and bioavailability data measured as shown in Figures 3, 4, and 12 and calculated using Equation 1 are shown in the tables. [Figure 13A] Figures 13A-13B show selected point mutations introduced into the domains of ENPP1 and the parent compound (SEQ ID NO:7). The figures show the specific point mutations introduced into SEQ ID NO:7. Constructs stably transfected into CHO cells stably transfected with human α-2,6-ST are designated "ST". PK and bioavailability data measured as shown in Figures 3, 4, and 12 and calculated using Equation 1 are shown in the tables. [Figure 13B] See legend to Figure 13A. [Figure 14] 1 shows the bioavailability (as area under the curve or AUC) for certain constructs of the invention categorized by mutations in the signal sequence (N-terminal region) region. [Figure 15] 1 shows the bioavailability (as area under the curve or AUC) for certain constructs of the invention categorized by mutations in the endonuclease domain. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Detailed Description of the Invention The present invention relates in one aspect to the discovery of certain ENPP1-Fc polypeptides that have improved in vivo half-lives compared to ENPP1-Fc polypeptides known in the art.

[0022] In one aspect, glycosylation was enhanced to protect the ENPP1-Fc polypeptide from degradation by introducing additional N-glycan consensus sequences on the outer surface of the predicted tertiary structure under the guidance of a three-dimensional model of ENPP1.

[0023] In another aspect, pH-dependent FcRn-mediated cell recycling was increased by mutating the Fc domain to enhance the affinity of the fusion protein for the neonatal receptor (FcRn).

[0024] In yet another aspect, sialylation of the fusion protein was enhanced by expressing ENPP1-Fc in a CHO cell line stably transfected with human ST6 β-galactosamide α-2,6-sialyltransferase (also known as ST6GAL1).

[0025] In yet another aspect, sialic acid capping was enhanced by supplementing cell culture medium with N-acetylmannosamine (also known as 1,3,4-O-Bu3ManNAc), a "high flux" precursor of sialic acid.

[0026] In certain embodiments, enhancing protein sialylation by expressing the bioagent in CHO cells stably transfected with human α-2,6-sialyltransferase increases the bioavailability (C) of ENPP1-Fc when administered subcutaneously. max ) significantly improved. In other embodiments, increasing pH-dependent FcRn-mediated cellular recycling by engineering the Fc domain improved the half-life of a biological entity in vivo. In yet other embodiments, combining CHO cells stably transfected with human α-2,6-sialyltransferase and growing the cells in N-acetylmannosamine dramatically increased the half-life and / or exposure (AUC) of a biological entity. In yet other embodiments, combining two or more methods described herein into a single construct dramatically increased the half-life and / or exposure (AUC) of a biological entity.

[0027] In certain embodiments, the polypeptides of the present invention are more highly glycosylated than 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) than other ENPP1-Fc polypeptides in the art. In yet other embodiments, the polypeptides of the present invention have a longer in vivo half-life than other ENPP1-Fc polypeptides in the art. In yet other embodiments, the kinetic properties of the parent compound (construct #770) are altered such that the change is a "gain of function" modification in the enzymatic rate constant. In yet 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 times longer than ENPP-1 polypeptides reported in the art. In yet other embodiments, the polypeptides of the present invention are administered to a subject at a lower dose and / or less frequently than other ENPP1-Fc polypeptides in the art. In yet other embodiments, the polypeptide of the present invention is administered to a subject once a month, twice a month, three times a month, and / or four times a month. In yet other embodiments, the less frequent administration of the polypeptide of the present invention results in better patient compliance and / or increased efficacy compared to other ENPP1-Fc polypeptides in the art.

[0028] In certain embodiments, the ENPP1-Fc polypeptide of the present invention can be used to increase pyrophosphate (PPi) levels in subjects with lower than normal levels (approximately 2 μM). In other embodiments, the ENPP1-Fc polypeptide of the present invention can be used to reduce or prevent the progression of pathological calcification or ossification in subjects with lower than normal levels of PPi. In yet other embodiments, the ENPP1-Fc polypeptide of the present invention can be used to treat ENPP1 deficiency manifested by a decrease in extracellular PPi concentration in a subject.

[0029] In certain embodiments, the steady state level of plasma PPi achieved following administration of the first dose of the construct of the invention is maintained for a period of at least 2 days, at least 4 days, at least 1 week, or at least 1 month.

[0030] In certain embodiments, a second dose of the construct of the present invention is administered to the subject after an appropriate time interval, such as 2 days, 4 days, 1 week or 1 month, such that the steady state level of plasma PPi is maintained constant or at a steady state level and does not return to the lower PPi level that the subject had prior to administration of the first dose of the construct of the present invention.

[0031] Without wishing to be bound by theory, it is believed that maintaining the steady state concentration of plasma PPi at normal levels reduces and / or prevents the progression of pathological calcification and pathological ossification in a subject.

[0032] Certain ENPP1 polypeptides, variants, or mutated 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, all of which are incorporated by reference in their entireties.

[0033] Reference will now be made in detail to certain aspects of the disclosed subject matter. While the disclosed subject matter may be described in conjunction with the enumerated claims, it will be understood that the illustrated subject matter is not intended to limit the scope of the claims to the disclosed subject matter.

[0034] Throughout this specification, values ​​expressed in range format should be interpreted in a flexible manner to include not only the numerical values ​​explicitly stated as the limits of the range, but also all individual numerical values ​​or subranges contained within the range as if each numerical value and subrange were explicitly stated. For example, the range "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 ​​(e.g., 1%, 2%, 3% and 4%) and subranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the range stated. The phrase "about X to Y" has the same meaning as "about X to about Y" unless otherwise indicated. Similarly, the phrase "about X, Y, or about Z" has the same meaning as "about X, about Y, or about Z" unless otherwise indicated.

[0035] definition 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 and experimental procedures used herein in veterinary medicine, pharmaceutical science, separation science and organic chemistry are well known and widely used in the art. It should be understood that the order of steps or order in which certain actions are taken is not critical so long as the present teachings remain operable. The use of subsection headings is intended to aid in the reading of the document and should not be construed as limiting, and information related to a novel heading may be found within or outside that particular subsection. All publications, patents, and patent documents referenced herein are incorporated by reference in their entirety as if each was individually incorporated by reference.

[0036] In this application, when an element or component is said to be included in and / or selected from a list of referenced elements or components, it is to be understood that the element or component can be any one of the referenced elements or components, and can be selected from a group consisting of two or more of the referenced elements or components.

[0037] In the methods described herein, actions may be performed in any order unless a chronological or action sequence is explicitly indicated. Moreover, actions shown may be performed simultaneously unless the claim language explicitly indicates that they are performed separately. For example, a claimed action of performing X and a claimed action of performing Y may be performed simultaneously in one operation, and the process thereby formed is encompassed within the literal scope of the claimed process.

[0038] In this document, the terms "a," "an," or "the" are used to include one or more, unless the context clearly indicates otherwise. The term "or" is used to indicate a non-exclusive "or," unless otherwise indicated. References to "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."

[0039] The following notation conventions are applied in this disclosure for clarity. In any case, teachings herein that do not follow this convention are also part of this disclosure and can be fully understood from the context in which the teachings are disclosed. Protein symbols are disclosed in non-italic capital letters. As a non-limiting example, "ENPP1" represents the 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 protein symbol. Thus, human ENPP1 is represented as "hENPP1" and mouse ENPP1 is represented as "mENPP1". Human gene symbols are disclosed in italic capital letters. As a non-limiting example, the human gene corresponding to the protein hENPP1 is italic ENPP1. Mouse gene symbols are disclosed with the first letter capitalized and the remaining letters lowercase, and further, the mouse gene symbols are italicized. As a non-limiting example, the mouse gene that produces the protein mEnpp1 is italic Enpp1. Genetic variants are indicated in capital letters.

[0040] "About," when used herein with reference to a measurable value, e.g., an amount, a length of time, etc., is meant to encompass variations of ±20% or ±10%, in certain embodiments ±5%, in certain embodiments ±1%, and in certain embodiments ±0.1% from the indicated value, so long as such variations are appropriate to perform the disclosed methods.

[0041] A disease or disorder is "alleviated" if the severity of a symptom of the disease or disorder is reduced, the frequency with which the patient experiences such symptoms is reduced, or both.

[0042] As used herein, the terms "alteration," "deletion," "variation," or "mutation" refer to a mutation in a gene in a cell that affects the function, activity, expression (transcription or translation), or conformation of the polypeptide it encodes, including missense and nonsense mutations, insertions, deletions, frameshifts, and premature terminations.

[0043] The term "antibody" as used herein refers to an immunoglobulin molecule capable of specifically binding to a particular epitope or antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources, and can be immunoreactive portions of intact immunoglobulins.

[0044] The "ATP hydrolysis activity" of ENPP1 can be measured using an ATP cleavage assay. ENPP1 readily hydrolyzes ATP to AMP and PPi. The steady-state Michaelis-Menten enzyme constant of ENPP1 is measured using ATP as a substrate. ENPP1 can be shown to cleave ATP by HPLC analysis of the enzymatic reaction, and the identity of the substrates and products of the reaction is confirmed by using ATP, AMP, and ADP standards. The ATP substrate degrades over time in the presence of ENPP1, and the enzyme product AMP accumulates. Using various concentrations of ATP substrate, the initial velocity of ENPP1 in the presence of ATP is derived, and the data is curve-fitted to derive the enzyme rate constant. At physiological pH, the kinetic rate constant of NPP1 is K m = 144 μM and k cat =7.8 s -1 It is.

[0045] As used herein, the term "AUC" refers to the area under the plasma drug concentration versus time curve (AUC), which correlates to the actual body exposure to the drug after administration of that dose of drug. In certain embodiments, AUC is expressed in mg*h / L. AUC can be used to measure the bioavailability of a drug, which is the fraction of unchanged drug that is absorbed intact and reaches the site of action, or the fraction that is in the systemic circulation after administration by any route.

[0046] The AUC can be calculated using the linear trapezoidal method or the logarithmic trapezoidal method. The linear trapezoidal method uses linear interpolation between data points to calculate the AUC. This method is required by the OGD and the FDA and is the standard in bioequivalence studies. For a given time interval (t1 to t2), the AUC is calculated as follows: TIFF2025000736000002.tif7128, where C1 and C2 are the average concentrations in the time intervals (t1 and t2).

[0047] The logarithmic trapezoidal method uses logarithmic interpolation between data points to calculate the AUC. This method is more accurate when the concentration decreases because drug elimination is exponential (making it linear on a logarithmic scale). For a given time interval (t1 to t2), the AUC is given by: This can be calculated as follows: TIFF2025000736000003.tif10128 (assuming C1>C2).

[0048] The term "bioavailability", as used herein, describes the extent and rate at which an active moiety (protein or drug or metabolite) enters the systemic circulation and thereby accesses the site of action. The bioavailability of an active moiety is determined, in large part, by the properties 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 have clinical significance, and therefore it is essential to know if drug formulations are equivalent. The most reliable measure of drug or protein bioavailability is the area under the plasma concentration-time curve (AUC). The AUC is directly proportional to the total amount of unchanged drug or therapeutic protein that reaches the systemic circulation. Drugs or therapeutic proteins may be considered bioequivalent with respect to the extent and rate of absorption if their plasma concentration curves are essentially superimposable.

[0049] The term "bioavailability" of a drug or therapeutic product is defined as the fraction of unchanged drug that is absorbed intact and reaches the site of action, or the systemic circulation after administration by any route. For intravenous administration of a drug, the bioavailability is defined as 1. For drugs administered by other routes of administration, the bioavailability is often less than 1. Incomplete bioavailability can be due to many factors that can be subdivided into the categories of dosage form effect, membrane effect, and administration site effect. Half-life and AUC provide information about the bioavailability of drugs and biological substances.

[0050] As used herein, the term "conservative variation" or "conservative substitution" refers to the replacement of an amino acid residue with another biologically similar residue. A conservative variation or substitution is likely not to change the shape of the peptide chain. Examples of conservative variations or substitutions include the replacement of one hydrophobic residue, such as isoleucine, valine, leucine or methionine, with another hydrophobic residue, or the replacement of one polar residue with another, such as arginine with lysine, glutamic acid with aspartic acid, or glutamine with asparagine.

[0051] 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.

[0052] A "disease" is a condition in the health of an animal in which the animal is unable to maintain homeostasis and where, if the disease is not ameliorated, the animal's health continues to deteriorate.

[0053] A "disorder" in an animal is a health condition in which the animal is able to maintain homeostasis, but in which the health condition of the animal is less favorable than it would be in the absence of the disorder. If left untreated, the disorder does not necessarily cause a further deterioration in the health condition of the animal.

[0054] As used herein, the terms "effective amount," "pharmacologically effective amount," and "therapeutically effective amount" refer to a non-toxic but sufficient amount of an agent to provide a desired biological result. The result may be a reduction and / or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. The appropriate therapeutic amount in any individual case may be determined by one of skill in the art using routine experimentation.

[0055] As used herein, the term "ENPP" or "NPP" refers to ectonucleotide pyrophosphatase / phosphodiesterase.

[0056] As used herein, the term "ENPP1 protein" or "ENPP1 polypeptide" refers to the ectonucleotide pyrophosphatase / phosphodiesterase-1 protein encoded by the ENPP1 gene. The encoded protein is a type II transmembrane glycoprotein that cleaves a variety of substrates, including phosphodiester bonds of nucleotides and nucleotide sugars and 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 No. WO 2014 / 126965 to Braddock et al., which is incorporated herein by reference in its entirety.

[0057] The term "functional equivalent" or "functional derivative" refers, in the context of a functional derivative of an amino acid sequence, to a molecule that retains a biological activity (either functional or structural) substantially similar to that of the sequence of the ENPP1-Fc construct presented herein. A functional derivative or equivalent may be a natural derivative or may be prepared synthetically. A functionally equivalent polypeptide of the present invention may also be a polypeptide identified using one or more structural and / or sequence alignment techniques known in the art.

[0058] Exemplary functional derivatives include amino acid sequences with one or more amino acid substitutions, deletions or additions, which preserve the biological activity of the protein. The substituting amino acid desirably has physicochemical properties similar to those of the amino acid to be substituted. Desirable similar physicochemical properties include similarity in charge, bulkiness, hydrophobicity, hydrophilicity, etc. Typically, greater than 30% identity between two polypeptides is considered to indicate functional equivalence. Preferably, the functionally equivalent polypeptides of the present invention have a degree of sequence identity to the ENPP1-Fc construct of greater than 80%. More preferred polypeptides have a degree of identity of 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 an enzymatic assay for ATP cleavage as described in WO2016 / 187408.

[0059] As used herein, the term "human ENPP1" refers to the human ENPP1 sequence set forth in NCBI accession NP_006199. As used herein, the term "soluble human ENPP1" refers to a polypeptide corresponding to residues 96-925 of NCBI accession NP_006199. As used herein, the term "enzymatically active" with respect to ENPP1 is defined as being capable of binding ATP and hydrolyzing it to AMP and PPi and / or being capable of binding AP3a and hydrolyzing it to ATP.

[0060] As used herein, the term "ENPP1 precursor protein" refers to ENPP1 having its signal peptide sequence at the ENPP1 N-terminus. Upon proteolysis, the signal sequence is cleaved from ENPP1 to provide the ENPP1 protein. Signal peptide sequences useful 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.

[0061] As used herein, the term "ENPP1-Fc" refers to ENPP1 recombinantly fused and / or chemically conjugated (including both 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.

[0062] As used herein, the term "Fc" refers to the human IgG (immunoglobulin) Fc domain. It is contemplated that subtypes of IgG, such as IgG1, IgG2, IgG3, and IgG4, may be used as the Fc domain.

[0063] As used herein, an "Fc region" is a portion of an IgG molecule that is associated with a crystallizable fragment obtained by papain digestion of the IgG molecule. The Fc region comprises the C-terminal halves of the two heavy chains of an IgG molecule that are linked by disulfide bonds. It does not have antigen-binding activity, but contains carbohydrate moieties and binding sites for complement and Fc receptors, including the FcRn receptor. The Fc fragment comprises 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 the portion of an immunoglobulin molecule that has a more conserved amino acid sequence than the variable domain, the other part of the immunoglobulin that contains the antigen-binding site. The constant domain includes the CH1, CH2 and CH3 domains of the heavy chain and the CHL domain of the light chain.

[0064] As used herein, the term "Fc receptor" refers to a protein found on the surface of certain cells (including, inter alia, B lymphocytes, follicular dendritic cells, natural killer cells, macrophages, neutrophils, eosinophils, basophils, human platelets and mast cells) that contributes to the protective functions of the immune system. Fc receptors bind antibodies bound to infected cells or invading pathogens. Immunoglobulin Fc receptors (FcRs) are expressed on all hematopoietic cells and play a key role in the immune response through antibodies. Binding of immune complexes to FcRs activates effector cells, resulting in phagocytosis of IgG-opsonized particles, endocytosis, release of inflammatory mediators and antibody-dependent cellular cytotoxicity (ADCC). Fc receptors have been reported for all classes of immunoglobulins: FcγR and neonatal FcR (FcRn) for IgG, FcεR for IgE, FcαR for IgA, FcδR for IgD and FcμR for IgM. All known Fc receptors belong to the immunoglobulin superfamily, except for FcRn and FcεRII, which are structurally related to class I major histocompatibility complexes and C-type lectins, respectively (Fc Receptors, Neil A. Fangera, et al., in Encyclopedia of Immunology (2 nd Edition), 1998).

[0065] 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 humans. FcRn specifically binds to the Fc domain of antibodies. FcRn extends 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 through pinocytosis. In general, serum proteins are transferred from endosomes to lysosomes where they are degraded. Since FcRn binds IgG at acidic pH (<6.5) but not at neutral or higher pH, FcRn-mediated transcytosis of IgG across endothelial cells can occur. IgG and serum albumin are bound by FcRn at slightly acidic pH (<6.5) and recycled to the cell surface, where they are released at the neutral pH (>7.0) of blood. This allows IgG and serum albumin to avoid lysosomal degradation.

[0066] The Fc portion of an IgG molecule is located in the constant region of the heavy chain, especially in the CH2 domain. This Fc region binds to the Fc receptor (FcRn), a surface receptor for B cells and a protein of the complement system. Binding of the Fc region of an IgG molecule to FcRn activates cells that bear this receptor, thus activating the immune system. Fc residues important 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 comprises the CH2 domain (or the FcRn binding portion thereof) of an IgG molecule.

[0067] As used herein, the term "fragment" when applied to a nucleic acid 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 in between). As used herein, the term "fragment" when applied to a protein or peptide refers to a subsequence of a larger protein or peptide and can be at least about 20, 50, 100, 200, 300, or 400 amino acids in length (and any integer value in between).

[0068] As used herein, the term "in vivo half-life" of a protein and / or polypeptide (e.g., an ENPP1 polypeptide comprising an FcRn binding site) envisaged in the present invention refers to the time required for half of the amount administered to an animal to be cleared from the circulation and / or other tissues of the animal. When the clearance curve of an ENPP1-Fc fusion protein is considered as a function of time, the curve is usually biphasic, with a fast α-phase (representing the equilibrium of the administered molecule between the intravascular and extravascular spaces, determined in part by the size of the molecule) and a longer β-phase (representing 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.

[0069] "Instruction material," as that term is used herein, includes publications, records, drawings, or any other medium of expression that can be used to communicate the usefulness of the nucleic acids, peptides, and / or compounds of the invention in the kit in identifying or alleviating or treating the various diseases or disorders referred to herein.

[0070] "Isolated" means altered or removed from its natural state. For example, a nucleic acid or polypeptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or polypeptide that is partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein may be present in a substantially pure form, or may exist in a non-native environment, such as a host cell.

[0071] An "oligonucleotide" or "polynucleotide" is a nucleic acid that ranges from at least 2 nucleotides in length, and in certain embodiments at least 8, 15, or 25 nucleotides in length, but can be up to 50, 100, 1000, or 5000 nucleotides in length, or a compound that specifically hybridizes to a polynucleotide.

[0072] As used herein, the terms "patient," "individual," or "subject" refer to a human.

[0073] 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 pharma- ceutical acceptable carrier. The pharmaceutical composition facilitates administration of the compound to a patient. There are various methods of administering the compound in the art, including, but not limited to, subcutaneous, intravenous, oral, aerosol, inhalation, rectal, vaginal, transdermal, intranasal, buccal, sublingual, parenteral, intrathecal, intragastric, ophthalmic, pulmonary, and topical administration.

[0074] As used herein, the term "pharmacologically acceptable" refers to a substance, such as a carrier or diluent, that does not negate the biological activity or properties of the compound and is relatively non-toxic, i.e., the substance may be administered to an individual without producing undesired biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0075] As used herein, the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable substance, composition or carrier, such as a liquid or solid filler, stabilizer, dispersant, suspending agent, diluent, excipient, thickener, solvent, or encapsulating material, involved in carrying or transporting a compound useful in the present invention into or to a patient so that it 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 harmful to the patient. Some examples of substances that can function as pharmaceutically acceptable carriers include sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and derivatives thereof. As used herein, "pharmaceutically acceptable carrier" also includes any and all coatings, antibacterial and antifungal agents, and 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" may further include pharmaceutically acceptable salts of the compound useful in the present invention. Other additional ingredients that may be included in pharmaceutical compositions used in practicing 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.

[0076] As used herein, the phrase "pharmacologically acceptable salts" refers to salts of the administered compounds prepared from pharma- ceutically acceptable non-toxic acids and bases, including inorganic acids, inorganic bases, organic acids, inorganic bases, solvates, hydrates, and clathrates thereof.

[0077] 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 animals include rats, mice, cats, dogs, humans, cows, and horses. PPi is released from platelets and therefore must be measured in plasma rather than serum. There are various methods for measuring PPi, one of which is by a modified enzyme assay using uridine-diphosphoglucose (UDPG) pyrophosphorylase (Lust & Seegmiller, 1976, Clin. Chim. Acta 66:241-249; Cheung & Suhadolnik, 1977, Anal. Biochem. 83:61-63). Typically, normal PPi levels in healthy subjects range from about 1 μM to about 3 μM, and in some instances, 1-2 μM. The subject with defective ENPP1 expression tends to show low PPi levels, ranging from at least 10% lower than normal, at least 20% lower than normal, at least 30% lower than normal, at least 40% lower than normal, at least 50% lower than normal, at least 60% lower than normal, at least 70% lower than normal, at least 80% lower than normal, and any combination thereof. In patients with pathological calcification or ossification disease, PPi levels in plasma are found to be less than 1 μM, and in some cases below detection level. In some cases, the plasma PPi levels of patients with pathological calcification or ossification disease are less than 0.5 μM (Arterioscler Thromb Vasc Biol. 2014, 34(9):1985-9; Braddock et al., 2015, Nat Commun. 6:10006.).

[0078] As used herein, the term "polypeptide" refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic, non-naturally occurring analogues thereof, linked through peptide bonds.

[0079] As used herein, the term "PPi" refers to pyrophosphate.

[0080] As used herein, the term "prevent" or "prevention" means not allowing a disorder or disease to develop if it has not already developed, or not allowing the disorder or disease to develop further if it has already developed. It is also considered to be within the ability of one of skill in the art to prevent some or all of the symptoms associated with a disorder or disease.

[0081] "Sample" or "biological sample" as used herein means biological material isolated from a subject. A biological sample may include any biological material suitable for detecting mRNA, polypeptides, or other markers of a physiological or pathological process in a subject, and may include bodily fluids, tissues, cells, and / or non-cellular material obtained from an individual.

[0082] As used herein, the term "signal peptide" refers to a sequence of amino acid residues (e.g., ranging from 10 to 30 residues in length) attached to the amino terminus of a newly generated protein of interest during protein translation. Signal peptides are recognized by the signal recognition particle (SRP) and cleaved by signal peptidases after transport to the endoplasmic reticulum (Lodish, et al., 2000, Molecular Cell Biology, 4 th edition).

[0083] As used herein, "substantially purified" refers to being essentially free of other components. For example, a substantially purified polypeptide is one that is separated from other components that normally accompany it in its naturally occurring state. Non-limiting embodiments include 95% purity, 99% purity, 99.5% purity, 99.9% purity, and 100% purity.

[0084] As used herein, the term "treatment" or "treating" 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 having a disease or disorder, a symptom of a disease or disorder, or a likelihood of developing a disease or disorder, or the application or administration of a therapeutic agent to isolated tissues or cell lines derived from a patient (e.g., for diagnostic or ex vivo applications), for the purpose of curing, relieving, alleviating, palliating, altering, treating, ameliorating, improving, or affecting the disease or disorder, the symptom of a disease or disorder, or the likelihood of developing a disease or disorder. Such treatments may be personalized or modified based on knowledge obtained from the field of pharmacogenomics.

[0085] A "vector" is a composition that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid inside a cell. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. This term should also be construed to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acid 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, etc.

[0086] As used herein, the term "wild type" refers to a gene or gene product isolated from a naturally occurring source. A wild type gene is the most commonly observed in a population and is therefore the arbitrarily designated "normal" or "wild type" form of that gene. In contrast, the term "modified" or "mutant" refers to a gene or gene product that exhibits modifications 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 and are identified by the fact that they have altered characteristics (including altered nucleic acid sequences) compared to the wild type gene or gene product.

[0087] Ranges: Throughout this disclosure, various aspects of the 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 considered as an inflexible limitation on the scope of the invention. Thus, the description of a range should be considered to specifically disclose all possible subranges and individual numerical values ​​within that range. For example, the description of a range such as 1-6 should be considered to specifically disclose subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as individual numerical values ​​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.

[0088] Polypeptides In one aspect, the present invention provides an ENPP1-Fc polypeptide. The present invention contemplates that the polypeptide of the present invention may have one or more of the mutations described herein.

[0089] In certain embodiments, the ENPP1 polypeptide comprises at least one mutation in the signal sequence region shown in Figure 13A and / or Figure 13B. In certain embodiments, the mutation is selected from the group consisting of C25N, K27T, and V29N with respect to SEQ ID NO:7. In certain embodiments, the mutation is C25N with respect to SEQ ID NO:7. In certain embodiments, the mutation is K27T with respect to SEQ ID NO:7. In certain embodiments, the mutation is V29N with respect to SEQ ID NO:7. In certain embodiments, the ENPP1 polypeptide comprises at least one mutation selected from the group consisting of C25N / K27T and V29N with respect to SEQ ID NO:7.

[0090] In certain embodiments, the ENPP1 polypeptide comprises at least one mutation in the catalytic region depicted in Figure 13A and / or Figure 13B. In certain embodiments, the mutation is selected from the group consisting of K369N and I371T with respect to SEQ ID NO:7. In certain embodiments, the mutation is K369N with respect to SEQ ID NO:7. In certain embodiments, the mutation is I371T with respect to SEQ ID NO:7. In certain embodiments, the ENPP1 polypeptide comprises the mutations K369N / I371T with respect to SEQ ID NO:7.

[0091] In certain embodiments, the ENPP1 polypeptide comprises at least one mutation in the endonuclease domain depicted in Table 1, Table 2, Table 3, Table 4, Table 5, Figure 6A, Figure 13A, Figure 13B, Figure 14, and / or Figure 15. In certain embodiments, the mutation is selected from the group consisting of P534N, V536T, R545T, P554L, E592N, R741D, and S766N with respect to SEQ ID NO:7. In certain embodiments, the mutation is P534N with respect to SEQ ID NO:7. In certain embodiments, the mutation is V536T with respect to SEQ ID NO:7. In certain embodiments, the mutation is R545T with respect to SEQ ID NO:7. In certain embodiments, the mutation is P554L with respect to SEQ ID NO:7. In certain embodiments, the mutation is E592N with respect to SEQ ID NO:7. In certain embodiments, the mutation is R741D relative to SEQ ID NO:7. In certain embodiments, the mutation is S766N relative to SEQ ID NO:7. In certain embodiments, the ENPP1 polypeptide comprises at least one mutation selected from the group consisting of P534N / V536T, P554L / R545T, E592N, E592N / R741D, and S766N relative to SEQ ID NO:7.

[0092] In certain embodiments, the ENPP1 polypeptide comprises at least one mutation in the linker region shown in Figure 13A and / or Figure 13B. In certain embodiments, the mutation is selected from the group consisting of E864N and L866T with respect to SEQ ID NO:7. In certain embodiments, the mutation is E864N with respect to SEQ ID NO:7. In certain embodiments, the mutation is L866T with respect to SEQ ID NO:7.

[0093] In a particular embodiment, the ENPP1 polypeptide comprises at least the mutations E864N / L866T relative to SEQ ID NO:7.

[0094] In certain embodiments, the polypeptide comprises an ENPP1 polypeptide and an FcRn-binding domain, wherein the FcRn-binding domain comprises any of the mutations depicted in Table 1, Table 2, FIG. 6A, FIG. 13A, FIG. 13B, FIG. 14, and / or FIG. 15. In certain embodiments, the mutation is selected from the group consisting of M883Y, S885N, S885T, T887E, H1064K, and N1065F with respect to SEQ ID NO:7. In certain embodiments, the mutation is M883Y with respect to SEQ ID NO:7. In certain embodiments, the mutation is S885N with respect to SEQ ID NO:7. In certain embodiments, the mutation is S885T with respect to SEQ ID NO:7. In certain embodiments, the mutation is T887E with respect to SEQ ID NO:7. In certain embodiments, the mutation is H1064K with respect to SEQ ID NO:7. In certain embodiments, the mutation is N1065F relative to SEQ ID NO:7. In certain embodiments, the FcRn-binding domain comprises at least one mutation selected from the group consisting of S885N, M883Y, M883Y / S885T / T887E, and H1064K / N1065F relative to SEQ ID NO:7.

[0095] In certain embodiments, the ENPP1 polypeptide is selected from the group consisting of C25N, K27T, V29N, C25N / K27T, K369N, I371T, K369N / I371T, P534N, V536T, R545T, P554L, E592N, R741D, S766N, P534N / V536T, P554L / R545T, E592N / R741D, E864N, Contains at least one mutation selected from the group consisting of L866T, E864N / L866T, M883Y, S885N, S885T, T887E, H1064K, N1065F, M883Y / S885T / T887E, H1064K / N1065F.

[0096] In certain embodiments, the polypeptide comprises at least one mutation selected from the group consisting of 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 with respect to SEQ ID NO:7.

[0097] In certain embodiments, a polypeptide comprises an ENPP1 polypeptide and an FcRn binding domain, wherein the polypeptide comprises the mutations M883Y, S885T, and T887E relative to SEQ ID NO:7.

[0098] In certain embodiments, the polypeptide comprises an ENPP1 polypeptide and an FcRn binding domain, wherein the polypeptide comprises the mutations P534N, V536T, M883Y, S885T, and T887E relative to SEQ ID NO:7.

[0099] In a particular embodiment, the polypeptide comprises an ENPP1 polypeptide and an FcRn binding domain, wherein the polypeptide comprises the mutations E592N, H1064K, and N1065F relative to SEQ ID NO:7.

[0100] In certain embodiments, the polypeptide comprises an ENPP1 mutant polypeptide, wherein the mutant polypeptide comprises an ENPP1 mutation selected from the group consisting of S766N, P534N, V536T, P554L, R545T, and E592N with respect to SEQ ID NO:7.

[0101] In certain embodiments, the ENPP1 mutant polypeptide comprises at least one mutation selected from the group consisting of S766N, P534N / V536T, P554L / R545T, and E592N relative to SEQ ID NO:7.

[0102] In certain embodiments, the polypeptide further comprises an FcRn-binding domain of IgG.

[0103] In certain embodiments, the polypeptide comprises a mutation selected from the group consisting of S885N, S766N, M883Y / S885T / T887E, P534N / V536T / H1064K / N1065F, P554L / R545T, S766N / H1064K / N1065F, E592N / H1064K / N1065F, and P534N / V536T / M883Y / S885T / T887E with respect to SEQ ID NO:7.

[0104] In a particular embodiment, the polypeptide comprises a S885N mutation in the FcRn binding domain relative to SEQ ID NO:7.

[0105] In certain embodiments, the polypeptide comprises an S766N mutation in the ENPP1 mutant polypeptide relative to SEQ ID NO:7.

[0106] In certain embodiments, the polypeptide comprises the mutations M883Y, S885T, and T887E in the FcRn binding domain relative to SEQ ID NO:7.

[0107] In certain embodiments, the polypeptide comprises the mutations P534N and V536T in the ENPP1 mutant polypeptide relative to SEQ ID NO:7 and the mutations H1064K and N1065F in the FcRn binding domain.

[0108] In certain embodiments, the polypeptide comprises the mutations P554L and R545T within the ENPP1 mutant polypeptide relative to SEQ ID NO:7.

[0109] In a particular embodiment, the polypeptide comprises the mutation S766N in the ENPP1 mutant polypeptide relative to SEQ ID NO:7 and the mutations H1064K and N1065F in the FcRn binding domain.

[0110] In a particular embodiment, the polypeptide comprises the mutation E592N in the ENPP1 mutant polypeptide relative to SEQ ID NO:7 and the mutations H1064K and N1065F in the FcRn binding domain.

[0111] In certain embodiments, the polypeptide comprises the mutations P534N and V536T in the ENPP1 mutant polypeptide relative to SEQ ID NO:7 and the mutations M883Y, S885T, and T887E in the FcRn binding domain.

[0112] In certain embodiments, the ENPP1 polypeptide lacks a nuclease domain. In other embodiments, the ENPP1 polypeptide is truncated to remove the nuclease domain. In yet other embodiments, the ENPP1 polypeptide is truncated to remove the nuclease domain from about residue 524 to about residue 885 of SEQ ID NO:1, leaving only the catalytic domain from about residue 186 to about residue 586 of SEQ ID NO:1, such that the catalytic activity of the protein is preserved.

[0113] In certain embodiments, the ENPP1 polypeptide is modified, relative to SEQ ID NO:1, in a segment of the extracellular region of ENPP1 that includes a peptidase cleavage site after the signal peptide and between the transmembrane and extracellular domains.

[0114] In certain embodiments, the ENPP1 polypeptide is modified at a segment of the extracellular region of ENPP1 that includes a furin cleavage site between the transmembrane and extracellular domains, as compared to SEQ ID NO:1. In other embodiments, the ENPP1 polypeptide is not modified at a segment of the extracellular region of ENPP1 that includes a furin cleavage site between the transmembrane and extracellular domains, as compared to SEQ ID NO:1.

[0115] In certain embodiments, the ENPP1 polypeptide is modified at the segment of the extracellular domain of ENPP2 that contains the signal peptidase cleavage site, as compared to SEQ ID NO: 1. In other embodiments, the ENPP1 polypeptide is not modified at the segment of the extracellular domain of ENPP2 that contains the signal peptidase cleavage site, as compared to SEQ ID NO: 1.

[0116] In certain embodiments, the polypeptide is soluble. In other embodiments, the polypeptide is a recombinant polypeptide. In yet other embodiments, the polypeptide comprises an ENPP1 polypeptide lacking the ENPP1 transmembrane domain. In yet other embodiments, the polypeptide comprises an ENPP1 polypeptide in which the ENPP1 transmembrane domain has been removed (and / or shortened) and replaced with the transmembrane domain of another polypeptide, for example, as non-limiting examples, ENPP2, ENPP5, or ENPP7.

[0117] In certain embodiments, the polypeptide comprises a signal peptide that results in the secretion of a precursor of the ENPP1 polypeptide, which is subjected to proteolytic processing to generate a polypeptide comprising the ENPP1 polypeptide. In other embodiments, the signal peptide is selected from the group consisting of the signal peptides of ENPP2, ENPP5, and ENPP7. In yet other embodiments, the polypeptide comprises an ENPP1 polypeptide comprising ENPP1 and another polypeptide, such as, as a non-limiting example, a transmembrane domain of ENPP2. In yet other embodiments, the ENPP1 polypeptide comprises a cleavage product of a precursor ENPP1 polypeptide comprising the ENPP2 transmembrane domain. In yet other embodiments, the ENPP2 transmembrane domain comprises, Contains residues 12 to 30 of SEQ ID NO:7, which corresponds to TIFF2025000736000004.tif3128.

[0118] In certain embodiments, the ENPP1 polypeptide is fused C-terminally 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 N-terminally to the Fc domain of human immunoglobulin 1 (IgG1), human immunoglobulin 2 (IgG2), human immunoglobulin 3 (IgG3), and / or human immunoglobulin 4 (IgG4). In yet other embodiments, the presence of the IgFc domain improves the half-life, solubility, reduces immunogenicity, and increases activity of the ENPP1 polypeptide.

[0119] In certain embodiments, the ENPP1 polypeptide is fused to human serum albumin at the C-terminus. Human serum albumin can be conjugated to the ENPP1 protein through a chemical linker, including but not limited to a naturally occurring or modified disulfide bond, or by genetic fusion to ENPP1 or a fragment and / or variant thereof.

[0120] In certain embodiments, the polypeptide is further pegylated (fused to a poly(ethylene glycol) chain).

[0121] In certain embodiments, the polypeptide has a length of about 3.4 (±0.4) s -1 enzyme -1 k for the substrate ATP cat has a value k cat is determined by measuring the rate of ATP hydrolysis for the polypeptide.

[0122] In certain embodiments, the polypeptide has a K for the substrate ATP of less than or equal to about 2 μM. M has a value of K M is determined by measuring the rate of ATP hydrolysis for the polypeptide.

[0123] In certain embodiments, the polypeptide is formulated as a liquid formulation. In other embodiments, the present invention provides a pharmaceutical composition in the form of a dry product comprising a therapeutic amount of a polypeptide of the present invention, the dry product being reconstituted into a solution of the compound in liquid form.

[0124] The invention provides kits comprising at least one polypeptide of the invention, or a salt or solvate thereof, and instructions for using the polypeptide in the methods of the invention.

[0125] In certain embodiments, the polypeptide lacks a negatively charged bone-targeting sequence. In yet other embodiments, a polyaspartic acid domain (about 2 to about 20 or more consecutive 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.

[0126] It will 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 that has the ATP hydrolysis activity of the native protein.As used herein in this disclosure, the phrase "ENPP1 polypeptide, mutant, or mutant fragment thereof" also includes any compound or polypeptide (such as, but not limited to, fusion protein) that includes ENPP1 polypeptide, mutant, or mutant fragment thereof.The fusion protein according to the present invention is considered a biological equivalent of ENPP1, but is intended to provide a longer half-life or a stronger potency due to increased in vivo biological exposure, as judged by "area under the curve" (AUC) or prolonged half-life in pharmacokinetic experiments.

[0127] Vectors and cells 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 yet other embodiments, the vector further comprises at least one nucleic acid sequence that directs and / or controls 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, which is proteolytically processed to produce the ENPP1 polypeptide of the present invention when secreted from the cell.

[0128] In yet 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 yet other embodiments, the vector of the present invention comprises a recombinant nucleic acid encoding an ENPP1 polypeptide of the present invention and a polypeptide comprising a signal peptide. In yet other embodiments, the polypeptide is proteolytically processed upon secretion from the cell to produce the ENPP1 polypeptide of the present invention.

[0129] Cloning and expression of ENPP1 ENPP1 or ENPP1 polypeptide is prepared as described in US 2015 / 0359858 A1, the entirety of which is incorporated herein by reference. ENPP1 is a transmembrane protein that is localized on the cell surface and has a distinct intramembrane domain. To express ENPP1 as a soluble extracellular protein, the transmembrane domain of ENPP1 can be replaced with the transmembrane domain of ENPP2, thereby soluble recombinant ENPP1 accumulates in the extracellular fluid of baculovirus culture.

[0130] Any other known protein signal sequence, including but not limited to the signal sequences of immunoglobulin kappa and lambda light chain proteins, may also be used to secrete the extracellular domain of ENPP1. Furthermore, the present invention should not be construed as being limited to the polypeptides described herein, but also includes polypeptides comprising any enzymatically active truncations of the ENPP1 extracellular domain.

[0131] ENPP1 is made soluble by removing the transmembrane domain. Human ENPP1 (SEQ ID NO:1) was modified to express a soluble recombinant protein by replacing its transmembrane region (e.g., residues 77-98) with the corresponding subdomain of human ENPP2 (NCBI accession NP_00112433 5, e.g., residues 12-30). The modified ENPP1 sequence was cloned into a modified pFastbac FIT vector with a TEV protease cleavage site followed by a C-terminal 9-F1IS tag, cloned and expressed in insect cells, and both proteins were expressed in the baculovirus system as previously described (Albright, et al., 2012, Blood 120:4432-4440; Saunders, et al., 2011, J. Biol. Chem. 18:994-1004; Saunders, et al., 2008, Mol. Cancer Ther. 7:3352-3362), allowing soluble recombinant proteins to accumulate in the extracellular fluid.

[0132] Production and purification of ENPP1 and ENPP1 fusion proteins In certain embodiments, a soluble ENPP1 polypeptide comprising an IgG Fc domain or an enzymatically / biologically active fragment thereof is effective in treating, alleviating, and / or preventing the progression of a disease or disorder contemplated herein. In other embodiments, the soluble ENPP1 polypeptide does not comprise a bone-targeting domain, e.g., 2-20 consecutive polyaspartic acid residues or 2-20 consecutive polyglutamic acid residues.

[0133] To produce soluble recombinant ENPP1 for in vitro use, ENPP1 was fused to the Fc domain of IgG (referred to as "NPP1-Fc") and the fusion protein was expressed in a stable CHO cell line. The protein can also be expressed from HEK293 cells, baculovirus-insect cell system, or CHO cells or Pichia yeast expression system using a suitable vector. The protein can be produced in either adherent 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.

[0134] Many expression systems are known and can be used to produce ENPP1 fusion proteins, including bacteria (e.g., E. 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, for example, from a coding sequence inserted into the host chromosome or on a free plasmid.

[0135] Yeast can be transformed with the coding sequence of the desired protein by any conventional method, for example, electroporation. Methods for transforming yeast by electroporation are disclosed in Becker & 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 obtained by the introduction of an expression construct can be grown to produce the desired polypeptide. Cells can be harvested and lysed, and their DNA content can be tested for the presence of the DNA using methods such as those described in Southern, 1975, J. Mol. Biol, 98:503 and / or Berent, et al., 1985, Biotech 3:208. Alternatively, the presence of the protein in the supernatant can be detected using antibodies.

[0136] Useful yeast plasmid vectors include pRS403-406 and pRS413-416, and are generally available from Strat:1.gene Cloning Systems, La Jolla, Calif., USA. Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integrating plasmids (Y1ps) and contain yeast selectable markers I-llS3, TRP1, LEU2 and lJRA3. Plasmids pRS413-416 are Yeast Centromeric Plasmids (YCps).

[0137] A variety of methods have been developed to operably link DNA to a vector through complementary cohesive termini. For example, complementary homopolymer tracts can be added to the DNA segment to be inserted into the vector DNA. The vector and DNA segment are then joined by hydrogen bonding between the complementary homopolymer tails to form a recombinant DNA molecule.

[0138] Synthetic linkers containing one or more restriction sites provide an alternative method of joining DNA segments to vectors. DNA segments generated by endonuclease restriction digestion are treated with bacteriophage T4 DNA polymerase or E. coli DNA polymerase I, enzymes that remove protruding 3' single-stranded ends by their 3'-5' exonuclease activity and fill in recessed 3' ends by their polymerization activity.

[0139] The combination of these activities thus produces 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, such as bacteriophage T4 DNA ligase. Thus, the reaction products are DNA segments with polymeric linker sequences at their ends. These DNA segments are then cut with an appropriate restriction enzyme and ligated into an expression vector that has been cut with an enzyme that produces ends compatible with those of the DNA segments.

[0140] Then, single stably transfected cell clones are established and screened for high expression clones of desired fusion protein. Screening of single cell clones for ENPP3 protein expression can be achieved in a high throughput manner in 96-well plates using synthetic enzyme substrate pNP-TMP as previously described (Albright, et al., 2015, Nat. Commun. 6:10006). After identifying high expression clones through screening, protein production can be achieved in shake flasks or bioreactors as previously described in Albright, et al., 2015, Nat. Commun. 6:10006.

[0141] Purification of ENPP1 can be achieved using a combination of standard purification techniques known in the art, examples of which are described above for the production of ENPP3 protein. After purification, ENPP1-Fc is purified by Zn2+ and Mg 2+ Concentrate to 5-7 mg / ml by dialysis into PBS supplemented with 0.1% NaCl (PBSplus) and freeze in 200-500 μl aliquots at -80°C. Immediately before use, thaw an aliquot and adjust the specific activity of the solution to 31.25 au / ml (or approximately 0.7 mg / ml depending on the preparation) by dilution in PBSplus.

[0142] array TIFF2025000736000005.tif136149TIFF2025000736000006.tif183149Bold: signal sequence Standard: ENPP1 extracellular domain Underline: linker sequence Italics: Fc domain

[0143] method The present invention includes a method of reducing or preventing the progression of pathological calcification in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polypeptide of the invention.

[0144] The present invention further includes a method of reducing or preventing the progression of pathological ossification in a subject in need thereof, comprising the step of administering to the subject a therapeutically effective amount of a polypeptide of the present invention.

[0145] The present invention further includes a method of reducing or preventing the progression of ectopic soft tissue calcification, including reducing, ameliorating, or preventing vascular calcification, in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polypeptide of the invention.

[0146] The present invention further includes a method for alleviating or preventing the progression of a disease caused by an ENPP1 deficiency, characterized by a reduced level of ENPP1 activity and / or defective ENPP1 expression in a subject in need thereof (compared to that of the ENPP1 activity level or ENPP1 expression level, respectively, in a normal healthy subject), comprising administering to the subject a therapeutically effective amount of a polypeptide of the present invention.

[0147] The invention further includes a method of alleviating or preventing the progression of a disease caused by low plasma PPi levels in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polypeptide of the invention that increases the subject's plasma PPi to normal (1-3 μM) or supranormal (30%-50% higher) levels and thereafter maintains the plasma PPi at the constant normal or supranormal level. The invention further includes administering additional therapeutically effective amounts at 2-day, 3-day, 1-week, or 1-month intervals to maintain the subject's plasma PPi at a constant normal or supranormal level to reduce or prevent the progression of pathological calcification or ossification.

[0148] The present invention further includes a method of treating, reversing, or preventing the progression of ossification of the posterior longitudinal ligament (OPLL) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polypeptide of the present invention.

[0149] The present invention further includes a method of treating, reversing, or preventing the progression of hypophosphatemic rickets in a subject in need thereof, comprising the step of administering to the subject a therapeutically effective amount of a polypeptide of the present invention.

[0150] The present invention further includes a method for alleviating or preventing the progression of at least one disease selected from the group consisting of chronic kidney disease (CKD), end stage renal disease (ESRD), uremic arteriolar calcification (CUA), calciphylaxis, ossification of the posterior longitudinal ligament (OPLL), hypophosphatemic rickets, osteoarthritis, age-related arteriosclerosis, idiopathic infantile arterial calcification (IIAC), generalized infantile arterial calcification (GACI), and atherosclerotic plaque calcification in a subject diagnosed with the at least one disease, comprising administering to the subject a therapeutically effective amount of a polypeptide of the present invention.

[0151] The present invention further includes a method for reducing or preventing the progression of age-related arteriosclerosis in a subject in need thereof, comprising the step of administering to the subject a therapeutically effective amount of a polypeptide of the present invention.

[0152] The present invention further includes a method for alleviating or preventing the progression of a disease caused by an ENPP1 deficiency (e.g., a reduced level of ENPP1 activity and / or defective ENPP1 expression compared to that of the ENPP1 activity level or ENPP1 expression level, respectively, in a normal healthy subject) in a subject in need thereof, comprising the step of administering to the subject a therapeutically effective amount of a polypeptide of the present invention.

[0153] The present invention further includes a method for alleviating or preventing the progression of a disease caused by lower than normal plasma PPi levels in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polypeptide of the present invention that increases and / or maintains the subject's plasma PPi at a level that is 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 includes further administration of a polypeptide of the present invention every 2 days, 3 days, 1 week, or 1 month to maintain the plasma PPi level at a level that is 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.

[0154] The present invention further includes a method of treating, reversing, or preventing the progression of pseudoxanthoma elasticum (PXE) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polypeptide of the present invention.

[0155] The present invention further includes a method for treating, reversing, or preventing the progression of atherosclerotic plaque calcification in an arterial vessel in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polypeptide of the present invention.

[0156] The present invention further includes a method of treating, reversing, or preventing the progression of osteoarthritis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polypeptide of the present invention.

[0157] The present invention further includes a method for treating, reversing, or preventing the progression of arteriosclerosis due to progeria in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polypeptide of the present invention.

[0158] The present invention further includes a method of 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, comprising the step of administering to the subject a therapeutically effective amount of a polypeptide of the invention.

[0159] The present invention further includes a method of treating, reversing, or preventing the progression of age-related osteopenia in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polypeptide of the present invention.

[0160] The present invention further includes a method of treating, reversing, or preventing the progression of ankylosing spondylitis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polypeptide of the present invention.

[0161] The present invention further includes a method of treating, reversing, or preventing the progression of pediatric sickle cell anemia episodes in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polypeptide of the present invention.

[0162] In certain embodiments, the pathological calcification is selected from the group consisting of idiopathic infantile arterial calcification (IIAC) and calcification of atherosclerotic plaque.

[0163] In certain embodiments, the pathological ossification is selected from the group consisting of ossification of the posterior longitudinal ligament (OPLL), hypophosphatemic rickets, and osteoarthritis.

[0164] In certain embodiments, the soft tissue calcification is selected from the group consisting of IIAC and osteoarthritis. In other embodiments, the soft tissue comprises arteriosclerotic plaque, and in still other embodiments, the soft tissue comprises muscular arteries. In still other embodiments, the soft tissue is selected from the group consisting of joints and the spine. In still other embodiments, the joints are selected from the group consisting of hand joints and foot joints. In still other embodiments, the soft tissue is selected from the group consisting of articular cartilage and vertebral disk cartilage. In still other embodiments, the soft tissue comprises blood vessels. In still other embodiments, the soft tissue comprises connective tissue.

[0165] In certain embodiments, the subject has been diagnosed with progeria.

[0166] In certain embodiments, the polypeptide of the present invention is a secreted product of ENPP1 precursor protein expressed in mammalian cells. In other embodiments, the ENPP1 precursor protein comprises a signal peptide sequence and an ENPP1 polypeptide, and the ENPP1 precursor protein undergoes a proteolytic process to become the polypeptide of the present invention. In yet other embodiments, in the ENPP1 precursor protein, the signal peptide sequence is conjugated to the N-terminus of the ENPP1 polypeptide. Proteolysis cleaves the signal peptide sequence from the ENPP1 precursor protein to provide the ENPP1 polypeptide. In certain embodiments, the signal peptide sequence is selected from the group consisting of an ENPP1 signal peptide sequence, an ENPP2 signal peptide sequence, an ENPP7 signal peptide sequence, and an ENPP5 signal peptide sequence.

[0167] In certain embodiments, the polypeptide is administered to the subject acutely or chronically, hi other embodiments, the polypeptide is administered to the subject topically, locally, parenterally, or systemically.

[0168] In certain embodiments, the subject is a mammal, hi other embodiments, the mammal is a human.

[0169] In certain embodiments, the polypeptide or precursor protein thereof is administered by at least one route selected from the group consisting of subcutaneous, oral, aerosol, inhalation, rectal, vaginal, transdermal, subcutaneous, intranasal, buccal, sublingual, parenteral, intrathecal, intragastric, ophthalmic, pulmonary, and topical. In other embodiments, the polypeptide or precursor protein thereof is administered to the subject as a pharmaceutical composition further comprising at least one pharma- ceutically acceptable carrier.

[0170] 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 a subject locally, regionally, or systemically. In yet another embodiment, the polypeptide or its precursor protein is delivered on an encoded vector, where the vector encodes a protein that is transcribed and translated from the vector upon administration of the vector to a subject.

[0171] Armed with the present disclosure, including the methods detailed herein, it will be understood by those skilled in the art that the present invention is not limited to treating a disease or disorder once established. In particular, symptoms of the disease or disorder need not be manifested to the point of being harmful to the subject, and in fact the disease or disorder need not be detected in the subject before treatment is administered. That is, substantial symptoms from the disease or disorder do not have to occur before the present invention can provide benefits.

[0172] Thus, the present invention includes a method of preventing disease and disorder in a subject, as fully described herein, in that the polypeptide of the present invention can be administered to a subject before the onset of a disease or disorder, as discussed elsewhere herein, thereby preventing the progression of the disease or disorder. In fact, a disease or disorder does not need to be detected in a subject before treatment is performed, particularly if the symptoms of the disease or disorder have not yet manifested to the point of being harmful to the subject. That is, substantial symptoms from the disease or disorder do not need to occur before the present invention can provide benefits. Thus, the present invention includes a method of preventing or delaying the onset of a disease or disorder in a subject, or reducing the progression or growth of a disease or disorder, in that the polypeptide of the present invention can be administered to a subject before the detection of the disease or disorder. In a particular embodiment, the polypeptide of the present invention is administered to a subject with a strong family history of the disease or disorder, thereby preventing or delaying the onset or progression of the disease or disorder.

[0173] Thus, one of skill in the art armed with the disclosure herein will understand that preventing a disease or disorder in a subject includes administration to the subject of a polypeptide of the invention as a prophylactic measure against that disease or disorder.

[0174] Pharmaceutical Compositions and Formulations The invention provides pharmaceutical compositions comprising the polypeptides of the invention, which are included in the methods described herein.

[0175] Such pharmaceutical compositions are in a form suitable for administration to a subject, or the pharmaceutical compositions may further comprise one or more pharma- ceutical acceptable carriers, one or more additional components, or some combination thereof.Various components of the pharmaceutical compositions may be prepared in physiologically acceptable salt form, e.g., in combination with physiologically acceptable cations or anions, as is well known in the art.

[0176] In one embodiment, pharmaceutical compositions useful for practicing the methods of the invention may be administered to deliver a dose of 1 ng / kg / day to 100 mg / kg / day. In other embodiments, pharmaceutical compositions useful for practicing the methods of the invention may be administered to deliver a dose of 1 ng / kg / day to 500 mg / kg / day.

[0177] The relative amounts of active ingredient, pharma- ceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will depend on the identity, size, and condition of the subject being treated, and may also vary depending on the route by which the composition is administered. By way of example, a composition may contain from about 0.1% to about 100% (w / w) active ingredient.

[0178] Pharmaceutical compositions useful in the methods of the present invention may be suitably developed for inhalation, oral, rectal, vaginal, parenteral, topical, transdermal, pulmonary, intranasal, buccal, ophthalmic, intrathecal, intravenous, or another route of administration. Other contemplated formulations include projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immune-based formulations. The route of administration will be readily apparent to one of skill in the art and will depend on any number of factors, including the type and severity of the disease being treated, the type and age of the animal or human patient being treated, and the like.

[0179] The formulation of pharmaceutical composition described herein can be prepared by any method known or subsequently developed in the pharmaceutical field.In general, such preparation method comprises the step of combining active ingredient with carrier or one or more other auxiliary ingredients, and then, if necessary or desired, shaping or packaging the product into desired single or multiple dose unit.

[0180] As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition containing a predetermined amount of active ingredient. The amount of active ingredient is usually equal to the dose of active ingredient administered to a subject or a convenient fraction of such a dose, such as one-half or one-third of such a dose. The unit dosage form can be for one of a single daily dose or multiple daily doses (e.g., about 1-4 or more times per day). When multiple daily doses are used, the unit dosage form can be the same or different for each dose.

[0181] Administration / Dosage The dosing regimen may affect what the effective amount will be. For example, multiple divided doses and staggered doses may be administered daily or continuously, or doses may be continuously infused or bolus injected. Furthermore, the dosage of the therapeutic formulation may be increased or decreased in proportion to the exigencies of the treatment or prophylactic situation. In certain embodiments, administration of the compound of the present invention to a subject increases the subject's plasma PPi to near-normal levels, where the normal level of PPi in a mammal is 1-3 μM. "Near-normal" refers to 0-1.2 μM or 0-40% below or above normal, 30 nM-0.9 μM or 1-30% below or above normal, 0-0.6 μM or 0-20% below or above normal, or 0-0.3 μM or 0-10% below or above normal.

[0182] Administration of the compounds of the invention to a patient, e.g., a mammal, e.g., a human, can be performed using known methods at a dosage and for a period of time effective to treat a disease or disorder in the patient. The effective amount of the therapeutic compound required to achieve a therapeutic effect can vary depending on factors such as the activity of the particular compound used; the time of administration; the excretion rate of the compound; the duration of treatment; other drugs, compounds or substances 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 art. Dosage regimens can be adjusted to provide an optimal therapeutic response. The dosage is determined based on the biological activity of the therapeutic compound, which depends on the half-life and area under the plasma time curve of the therapeutic compound. The polypeptide according to the invention is administered at appropriate time intervals, such as every 2 days, or every 4 days, or every week, or every month, to achieve sustained levels of plasma PPi that are either close to or above normal (1-3 μM) levels of PPi (30-50% higher). The therapeutic dose of the polypeptide of the invention can also be determined based on the half-life or the rate at which the therapeutic polypeptide is cleared from the body. The polypeptide according to the invention is administered at appropriate time intervals, such as every 2 days, or every 4 days, or every week, or every month, to achieve a constant level of enzymatic activity of ENPP1.

[0183] For example, various divided doses may be administered daily or the dose may be reduced in proportion to the exigencies of the therapeutic situation. A non-limiting example of an effective dose range of the therapeutic compound of the present invention is about 0.01-50 mg / kg body weight / day. In some embodiments, the effective dose range of the therapeutic compound of the present invention is about 50 ng-500 ng / kg, preferably 100 ng-300 ng / kg body weight. It is believed that one of ordinary skill in the art can study the relevant factors and make a determination regarding the effective amount of the therapeutic compound without undue experimentation.

[0184] The compound may be administered to a patient several times a day, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every few months or once a year or less frequently. It is understood that the amount of compound administered per day may be administered, as a non-limiting example, every day, every other day, every 2nd day, every 3rd day, every 4th day, or every 5th day. For example, in the case of administration every other day, a dose of 5 mg per day is administered starting on Monday, a first subsequent dose of 5 mg per day is administered on Wednesday, a second subsequent dose of 5 mg per day is administered on Friday, etc. The frequency of administration will be readily apparent to those skilled in the art, and it depends on any number of factors, such as the type and severity of the disease being treated and the type and age of the patient.

[0185] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied to obtain an amount of the active ingredient effective to achieve the desired therapeutic response for a particular patient, composition and mode of administration without causing toxicity to the patient.

[0186] A physician, e.g., a doctor, having ordinary skill in the art, can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the doctor or veterinarian can start the dosage of the compound of the present invention used in the pharmaceutical composition at a level lower than required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0187] In certain embodiments, the compositions of the present invention are administered to patients in dosages ranging from 1 to 5 or more times per day. In other embodiments, the compositions of the present invention are administered to patients in dosage ranges including, but not limited to, once a day, every 2 days, every 3 days to once a week, and once every 2 weeks. The frequency of administration of the various combination compositions of the present invention will vary from patient to patient depending on many factors including, but not limited to, age, disease or disorder being treated, sex, general health, and other factors. Thus, the present invention should not be construed as being limited to any particular dosing regimen, and the exact dose and composition administered to any patient will be determined by the attending physician in consideration of all other factors for that patient.

[0188] In certain embodiments, the invention relates to a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound of the 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.

[0189] Route of administration Routes of administration of any of the compounds of the invention include inhalation, oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, ​​vaginal (e.g., vaginal and perivaginal), (intravaginal), and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastric, intrathecal, subcutaneous, intramuscular, intradermal, intraarterial, intravenous, intrabronchial, inhalation, and topical administration.

[0190] Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gelcaps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration, etc. The formulations and compositions that may be useful in the present invention are not limited to the specific formulations and compositions described herein.

[0191] Parenteral Administration As used herein, "parenteral administration" of a pharmaceutical composition includes any administration route characterized by the physical rupture of the subject's tissue and administration of the pharmaceutical composition through a break in the tissue. Parenteral administration thus includes, but is not limited to, administration of the pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a non-surgical wound that penetrates the tissue, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intravenous, intraperitoneal, intramuscular, intrasternal injection, and kidney dialysis infusion techniques.

[0192] Further dosage forms 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, WO 03 / 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.

[0193] Controlled Release Formulations and Drug Delivery Systems The controlled or sustained release formulation of the pharmaceutical composition of the present invention can be made using conventional technology.In some instances, the dosage form used can be provided as a sustained or controlled release of one or more active ingredients therein, for example, using hydropropylmethylcellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multi-layer coatings, microparticles, liposomes or microspheres or combinations thereof in various ratios to provide desired release profile.The unit dosage form suitable for oral administration, such as tablets, capsules, gel caps and caplets, that is adapted for controlled release, is included in the present invention.

[0194] In certain embodiments, the formulations of the present invention can be, but are not limited to, short-term, rapid-offset, and controlled, including sustained release, delayed release, and pulsatile release formulations.

[0195] The term sustained release is used in its conventional sense to refer to a drug formulation that provides a gradual release of drug over an extended period of time, resulting in, but not necessarily, a substantially constant blood level of drug over an extended period of time. The period can be as long as one month or longer, and should be longer release than the same amount of agent administered in bolus form. For sustained release, the compound can be formulated with a suitable polymer or hydrophobic material that provides the compound with sustained release properties. In that case, the compound used in the method of the present invention can be administered, for example, by injection, in the form of microparticles, or by implantation, in the form of wafers or disks. In certain embodiments of the present invention, the compound of the present invention is administered to a patient using a sustained release formulation, alone or in combination with another agent.

[0196] The term delayed release is used herein in its conventional sense to refer to a drug formulation that provides an initial release of drug after a certain delay after drug administration, which may include, but is not required to include, a delay of about 10 minutes up to about 12 hours.The term pulsatile release is used herein in its conventional sense to refer to a drug formulation that provides a release of drug after drug administration in a manner that produces a pulsatile plasma profile of drug.The term immediate release is used herein in its conventional sense to refer to a drug formulation that provides a release of drug immediately after drug administration.

[0197] 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 after drug administration, and any or all full or partial increments thereof.

[0198] As used herein, rapid clearance 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 after drug administration, and any and all whole or partial increments therein.

[0199] It is believed that those skilled in the art can recognize and ascertain, using only routine experimentation, many equivalents to the individual procedures, aspects, claims, and examples described herein.Such equivalents are considered to be within the scope of the present invention and to be encompassed by the appended claims.For example, modifications of reaction and preparation conditions using only routine experimentation with substitutes known in the art should be understood to be within the scope of this application.

[0200] It is to be understood that whatever values ​​and ranges are provided herein, all values ​​and ranges subsumed within those values ​​and ranges are meant to be encompassed within the scope of the present invention. Moreover, all values ​​subsumed within those ranges, as well as the upper or lower limits of value ranges, are also contemplated by this application.

[0201] The following examples further illustrate aspects of the present invention, but do not in any way serve to limit the present teachings or disclosure herein. EXAMPLES

[0202] The invention will now be described with reference to the following examples, which are provided for illustrative purposes only, and the invention is not limited to these examples, but rather encompasses all variations that become evident as a result of the teachings provided herein.

[0203] Methods and Materials Unless specifically mentioned, expression of constructs in CHO cells or modified CHO cells with and without supplementation, V max Assay, K m / K cat The assays, AUC assays, and half-life assays were performed using protocols described elsewhere herein.

[0204] Generation of ENPP1-Fc mutant constructs Human NPP1 (human: NCBI accession NP 006199) was modified to express a soluble recombinant protein fused to IgG1 by subcloning into pFUSE-hlgGl-Fcl or pFUSE-mlgGl-Fcl plasmids (InvivoGen, San Diego CA), respectively. The constructs were generated from SEQ ID NO:7 using site-directed mutagenesis with a commercially available kit (Q5® Site-Directed Mutagenesis Kit / New England Biolabs). The constructs thus generated were sequenced to verify the nucleic acid sequence and then used for protein expression.

[0205] Expression of ENPP1-Fc mutant constructs Stable transfection of the ENPPl-Fc construct was established in CHO K1 cells (Sigma Aldrich, 85051005) under Zeocin / Gentamicin selection and adapted to suspension growth. The adapted cells were used to inoculate liquid cultures grown in CD FortiCHO™ medium (A1148301, Thermo Fischer) in shake flasks at 37° C. and 5% CO2, 120 rpm agitation, and high humidity. The cultures were expanded stepwise to the desired target volume and then maintained for an additional 2 days to allow extracellular protein accumulation.

[0206] Expression of ENPP1-Fc mutant constructs in engineered CHO cells CHO-K1 cells were modified to generate CHO-K1-MOD cells stably expressing the 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 following the same protocol as above. For some constructs, the culture medium of CHO-K1-MOD cells expressing the corresponding construct was supplemented with sialic acid or a "high flux" precursor of sialic acid called 1,3,4-O-Bu3ManNAc, as required, to promote high levels of glycosylation during protein production.

[0207] Purification of ENPP1-Fc mutant constructs The liquid culture was centrifuged at 4300×g for 5 min and the supernatant was filtered through a 0.2 μm membrane and centrifuged using a Pellicon® 3 0.011 μm filter. 2 Concentration was performed through tangential flow using an Ultracell® 30 D cassette (Millipore, Billerica MA). The concentrated supernatant was then purified by a combination of chromatographic techniques in a multi-step process. These techniques were performed sequentially and may include any of the following: affinity chromatography using Protein A or Protein G, cation exchange chromatography, anion exchange chromatography, size exclusion chromatography, hydrophobic exchange chromatography, high pressure liquid chromatography (HPLC), precipitation, extraction, lyophilization and / or crystallization steps. Using any one of these steps in sequence, one skilled in the art of protein chemistry can purify the described composition of interest to homogeneity showing no contaminating protein bands in silver stained gels. The resulting protein samples were then tested using the Pierce LAL Chromogenic Endotoxin Quantitation Kit (cat. no. 88282) to confirm that all were endotoxin-free.

[0208] To quantify the biological impact of clonal optimization, the pharmacokinetic effects of selected ENPP1-Fc isoforms were quantified by measuring plasma PPi concentrations at multiple time points following a single subcutaneous dose of each isoform.

[0209] K m / K cat decision Steady-state hydrolysis of ATP by ENPP1 constructs was measured by HPLC. Briefly, the enzymatic reaction was initiated by adding 10 nM PPi to various 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 various 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 mt 250 × 4.6 mm) column (Higgins Analytical) equilibrated with 5 mM ammonium acetate (pH 6.0) solution and eluted with a 0% to 20% methanol gradient. Substrates and products were monitored by UV absorption at 259 nm and quantified according to the integration of their corresponding peaks and standard curves.

[0210] V max Assay Each of the prepared mutants was analyzed for phosphodiesterase activity 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).

[0211] Area under the curve assay The area under the curve of plasma concentration versus time, also called area under the curve (AUC), can be used as a measure 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 a standard equation, as described in Equation 1, to measure the half-life and bioavailability after a single subcutaneous injection of the biological material.

[0212] Half-life determination Drug half-life (t 1 / 2) is the time required for the plasma concentration or amount of a drug or biological substance in the body to decrease by 50%. Half-life values ​​for each expressed and purified ENPP1-Fc construct were performed according to protocols in the art and / or described herein that allow for the determination of half-life and bioavailability after a single subcutaneous injection of a biological substance, e.g., Equation 1.

[0213] Drug half-life can be calculated using Equation 1, which corresponds to the relationship between time and fractional systemic concentration of a drug administered in a single injection at a subcutaneous injection site. A plot of this data as fraction of drug absorbed (F) versus time (t) gives the elimination (k) by fitting this data to the equation for total systemic absorption of a drug administered at a subcutaneous injection site at time t = 0. e ) and absorption (k a ) constant determination. TIFF2025000736000007.tif11128

[0214] Example 1: Selection and optimization of glycosylation mutations The ENPP1-Fc construct was subjected to mutations that introduced putative additional glycosylation sites and / or increased the affinity of the Fc for the neonatal orphan receptor (FcRn). The mutations tested are presented elsewhere herein, and the specific constructs of that discussion are described below.

[0215] We attempted to improve the pharmacokinetic properties of ENPP1-Fc by introducing additional N-linked glycosylation sites and enhancing the pH-dependent recycling of the fusion protein. As a guide to the selection of additional N-linked glycosylation sites, we used electron density maps obtained from X-ray diffraction of mouse Enpp1 crystals, which revealed four glycosylation sites in Enpp1. We presumed that these sites are present in the highly homologous human ENPP1, which in addition contains four additional N-linked glycosylation consensus sequences whose glycosylation status is unknown (Figure 6B).

[0216] To identify regions of ENPP1 amenable to hyperglycosylation that would not adversely affect catalytic activity, we used a combination of structural modeling, clinical data, and genetic data on ENPP1 from GACI patients. First, N-linked glycosylation consensus sequences were identified in ENPP2-7 to evaluate sequences that could facilitate the introduction of a glycosylation site through the modification of one adjacent residue. We then structurally modeled ENPP2-7 using standard software and threaded the sequence through the mouse Enpp1 structure (PDB ID code 4GTW). The location of the proposed glycosylation sites was compared to the sites of known inactivating ENPP1 mutations in GACI (Figures 6A-6B) and the location of disulfide bonds within this enzyme. If the spatial location of a proposed glycosylation site was predicted to interfere with either, the site was rejected. These modeling studies identified 53 putative sites for additional N-linked glycosylation steps that could be readily introduced into ENPP1 that would not be expected to interfere with its protein folding or enzymatic activity (Figures 7, 13, and 14).

[0217] Additional N-linked glycosylation consensus sequences were then introduced into human ENPP1-Fc (hENPP1-Fc, construct #770) through site-directed mutagenesis. The protein was transiently expressed in CHO cells in 96-well plates, and the enzymatic activity of extracellular supernatants from each clone was screened in triplicate in a high-throughput assay using pNP-TMP as a chromogenic substrate as described in the methods (Figures 7A-7D). The rates of pNP-TMP hydrolysis in 10 of the 53 putative ENPP1-Fc isoforms were comparable or greater than that of construct #770 (Figures 7A-7D), and these 10 glycoforms were selected for combinatorial optimization with each other and with the IgG1 Fc domain, as described below.

[0218] FcRn is the main homeostatic regulator of human IgG1 Fc serum half-life, and mutations in the Fc domain that enhance the pH-dependent interaction of Fc to FcRn extended the circulating half-life of biological antibodies. The effects of two Fc mutations reported to enhance pH-dependent recycling, H433K / N434F, hereafter referred to as the HN mutant, and M242Y / S254T / T246E, hereafter referred to as the MST mutant, were tested here (Figures 8A-8B). Either of the two variants of the Fc domain were randomly combined with one or more of the 10 ENPP1-Fc glycoforms that showed acceptable hydrolysis rates, generating 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, with two spatially distinct putative glycosylation sites on different protein domains chosen to enhance potential glycan shielding effects on the protein surface region (Table 3; constructs #1057, #1064, #1014, #1040). Other clones only tested the effect of Fc mutations alone or in the presence of one additional putative glycosylation on pK properties (Table 3; constructs #981 and #1051, respectively).

[0219] Example 2: Expression using CHO cell lines and growth conditions Non-human Chinese Hamster Ovary (CHO) cells are widely used for the production of biomaterials due to the similarity of CHO and human glycosylation patterns in recombinantly produced proteins. However, differences in glycosylation exist between the two, most notably that human N-linked glycans contain terminal sialic acid residues with both α-2,3 and α-2,6 linkages, whereas CHO cells contain only α-2,3 linkages.

[0220] To test whether the difference in terminal sialylation between CHO and human cells affects PK and bioavailability in the present system, a CHO cell line stably expressing human α-2,6-sialyltransferase (α-2,6-ST) was established as a host, and this clone was used to produce seven ENPP1 isoforms to compare the effect of α-2,6 linkage on PK and bioavailability in various constructs (Table 5; construct numbers ending with "-ST"). To investigate the effect of growth conditions on PK and bioavailability, cells stably transfected with selected ENPP1-Fc isoforms (both CHO K1 cells and CHO K1 cells stably transfected with human α-2,6-ST) were supplemented with a "high flux" precursor of sialic acid called 1,3,4-O-Bu3ManNAc during protein production (Table 5).

[0221] ENPP1-Fc isoforms were purified to homogeneity using an identical purification scheme, and Michaelis-Menten enzyme kinetic constants and pharmacokinetic properties were determined as described elsewhere herein. Finally, to quantify the biological impact of clonal optimization, the pharmacokinetic effects of selected ENPP1-Fc isoforms were quantified by measuring plasma PPi concentrations at multiple time points following a single subcutaneous dose of each isoform.

[0222] Example 3: Pharmacokinetic effects of additional N-linked glycosylation sites Addition of N-linked glycosylation sites using the in silico prediction and HTS methods described above significantly increased in vivo exposure of mice to ENPP1-Fc in glycoforms, 4-fold in construct #1020. To determine which sequence variations increased glycosylation and showed an increase in molecular weight corresponding to the addition of glycosylation, the sizes of the ENPP1-Fc isoforms in Table 2 were compared by SDS-PAGE gel. 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.

[0223] Example 4: Pharmacokinetic effects of Fc IgG1 mutations (Figures 10-11) Antibodies containing mutations in the Fc domain that enhance their affinity for FcRn and increase pH-dependent antibody recycling have not been used to date with therapeutic enzymes fused to the Fc domain. Some Fc mutations have been shown to successfully increase the affinity of the Fc domain for the FcRn receptor but result in unfavorable PK properties in antibody PK in vivo, while others have been shown to enhance PK properties in vivo.

[0224] To determine whether similar Fc changes would enhance the PK properties of enzyme fusion proteins, two specific IgG1 mutations in Fc previously used in biological antibodies were investigated: H433K / N434F and M242Y / S254T / T246E. In general, 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 has only the M242Y / S254T / T246E mutations, increased half-life by 3.3-fold and AUC by 5.8-fold when compared to construct #770. In contrast, constructs with the H433K / N434F mutations in the context of various ENPP1 mutations achieved smaller half-life increases of 1.2-1.7-fold.

[0225] Example 5: Effect of host cells and growth conditions Expression of proteins in CHO cells stably transfected with human α-2,6-ST producing recombinant biomaterials with terminal sialic acid residues with both α-2,3 and α-2,6 linkages has been met with varying degrees of success, with increased and decreased PK properties reported depending on the biomaterial.

[0226] To determine whether α-2,6 binding affects the PK properties of ENPP1-Fc, the in vivo exposure (AUC) and half-life of seven ENPP1-Fc isoforms produced in either CHOK1 cells or CHOK1 cells stably transfected with human α-2,6-ST were directly compared (Table 4). An overall trend was confirmed that production of biomaterials in CHOK1 cells stably transfected with human α-2,6-ST is beneficial. The strongest effect was seen in the exposure (AUC) of the organism to the drug, showing a 1.7- to 4.6-fold increase in AUC in the responsive isoforms (constructs #1057, #1028, #951, #930, and #981). Another trend was that the impact in AUC was greater in isoforms with lower initial AUC (constructs #951 and #1057). However, the efficacy of the longer acting isoforms (constructs #1028 and #981) was also greater, showing AUC values ​​8-10 fold higher than their parent compounds produced in CHOK1 cells.

[0227] The effect of α-2,6 linkage on half-life was smaller, with a 20-30% increase in the responsive constructs. To understand the difference in the effect of α-2,6 linkage on AUC over time and protein activity half-life, the isoforms produced in CHOk1 and 1078 cells were compared.

[0228] Example 6: Pharmacokinetic effects of proliferation with high flux sialic acid precursors To determine the effect of growth conditions on PK properties, the culture medium of selected clones was supplemented with 1,3,4-O-Bu3ManNAc, a "high flux" precursor of sialic acid, or with sialic acid itself. Supplementation of CHOK1 cells with 1,3,4-O-Bu3ManNAc did not significantly improve the PK properties of ENPP1-Fc, but the effect on half-life and AUC was significant when this biomaterial was produced in CHOK1 cells stably transfected with human α-2,6-ST (FIG. 12 and Table 4). For example, supplementation of cell culture medium of CHOK1 cells producing construct #1014 with 1,3,4-O-Bu3ManNAc did not significantly enhance AUC and appeared to decrease the half-life of the isoform. In contrast, the effects were more dramatic 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, resulting in a net increase in AUC and half-life of 4-fold and 2-fold compared to the same isoforms produced in CHOK1 grown in medium not supplemented with 1,3,4-O-Bu3ManNAc (FIG. 12 and Table 4).

[0229] (Table 1) TIFF2025000736000008.tif154128

[0230] Table 2. Effect of additional N-linked glycosylation on pharmacokinetics (PK) TIFF2025000736000009.tif100146

[0231] Table 3. Effect of Fc mutations on pharmacokinetics (PK) TIFF2025000736000010.tif149154

[0232] Table 4. Effect of cell line and mutations on pharmacokinetics (PK). Constructs marked with "-ST" were prepared using a modified CHO cell line stably transfected with human α-2,6-sialyltransferase (α-2,6-ST); this enhances the amount of sialylation of the fusion protein when compared to fusion proteins expressed in normal CHO cell lines. The enhanced sialylation of the constructs resulted in improved AUC and half-life values. TIFF2025000736000011.tif117143*: Construct transfected into CHO-K1 cells **: constructs transfected into CHO-K1-MOD cells

[0233] Table 5: Effect of sialic acid supplementation on pharmacokinetics (PK). Constructs marked "-ST" were prepared using a modified CHO cell line stably transfected with human α-2,6-sialyltransferase (α-2,6-ST); this enhances the amount of sialylation of the fusion protein when compared to fusion proteins expressed in normal CHO cell lines. Constructs marked "-A" were prepared in cells grown in culture medium supplemented with 1,3,4-O-Bu3ManNAc, a "high flux" precursor of sialic acid during protein production. TIFF2025000736000012.tif86137

[0234] Table 6: List of polypeptides and corresponding mutations TIFF2025000736000013.tif211149

[0235] Table 7. List of mutations in ENPP1 polypeptide TIFF2025000736000014.tif143128

[0236] List of Aspects The following exemplary aspects are provided, but the numbering should not be construed as designating a level of importance. Embodiment 1 provides an ENPP1 polypeptide fusion comprising an ENPP1 polypeptide fused to an Fc region of an immunoglobulin, wherein the Fc region comprises at least one mutation selected from the group consisting of M883Y, S885N, S885T, T887E, H1064K, and N1065F relative to SEQ ID NO:7. Embodiment 2 provides the polypeptide fusion of embodiment 1, wherein said Fc region comprises at least one mutation selected from the group consisting of S885N, M883Y, M883Y / S885T / T887E, and H1064K / N1065F with respect to SEQ ID NO:7. Embodiment 3 provides an ENPP1 polypeptide fusion comprising an ENPP1 polypeptide fused to an Fc region of an immunoglobulin, wherein the ENPP1 polypeptide comprises at least one mutation selected from the group consisting of C25N, K27T, and V29N relative to SEQ ID NO:7. Embodiment 4 provides the polypeptide fusion of embodiment 3, wherein said ENPP1 polypeptide comprises at least one mutation selected from the group consisting of C25N / K27T and V29N relative to SEQ ID NO:7. Embodiment 5 provides an ENPP1 polypeptide fusion comprising an ENPP1 polypeptide fused to an Fc region of an immunoglobulin, wherein the ENPP1 polypeptide comprises at least one mutation selected from the group consisting of K369N and I371T relative to SEQ ID NO:7. Embodiment 6 provides the polypeptide fusion of embodiment 5, wherein said ENPP1 polypeptide comprises at least the mutations K369N / I371T relative to SEQ ID NO:7. Embodiment 7 provides an ENPP1 polypeptide fusion comprising an ENPP1 polypeptide fused to an Fc region of an immunoglobulin, wherein the ENPP1 polypeptide comprises at least one mutation selected from the group consisting of P534N, V536T, R545T, P554L, E592N, R741D, and S766N relative to SEQ ID NO:7. Example 8 provides the polypeptide fusion of example 7, wherein said ENPP1 polypeptide comprises at least one mutation selected from the group consisting of P534N / V536T, P554L / R545T, E592N, E592N / R741D, and S766N relative to SEQ ID NO:7. Embodiment 9 provides an ENPP1 polypeptide fusion comprising an ENPP1 polypeptide fused to an Fc region of an immunoglobulin, wherein the ENPP1 polypeptide comprises at least one mutation selected from the group consisting of E864N and L866T relative to SEQ ID NO:7. Embodiment 10 provides the polypeptide fusion of embodiment 9, wherein said ENPP1 polypeptide comprises at least the mutations E864N / L866T relative to SEQ ID NO:7. Aspect 11 is a sequence ID 11. The polypeptide fusion of any of aspects 1-10 is provided, comprising at least one mutation selected from the group consisting of 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 for NO:7. Aspect 12 provides the polypeptide fusion of any of aspects 1 to 11, wherein the Fc region is an IgG Fc region. Embodiment 13 provides a polypeptide fusion of any of embodiments 1 to 12, comprising at least one mutation selected from the group consisting of P534N, V536T, R545T, P554L, S766N, and E592N with respect to SEQ ID NO:7. Embodiment 14 provides a polypeptide fusion of any of embodiments 1 to 12, comprising at least one mutation selected from the group consisting of S766N, P534N / Y536T, P554L / R545T, and E592N relative to SEQ ID NO:7. Embodiment 15 provides a polypeptide fusion of any of embodiments 1 to 12, comprising at least one mutation selected from the group consisting of 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 with respect to SEQ ID NO:7. Embodiment 16 provides an ENPP1 polypeptide fusion comprising an ENPP1 polypeptide and an Fc region of an immunoglobulin, the ENPP1 polypeptide fusion comprising the mutations M883Y, S885T, and T887E relative to SEQ ID NO:7. Embodiment 17 provides an ENPP1 polypeptide fusion comprising an ENPP1 polypeptide and an Fc region of an immunoglobulin, the ENPP1 polypeptide fusion comprising the mutations P534N, V536T, M883Y, S885T, and T887E relative to SEQ ID NO:7. Embodiment 18 provides an ENPP1 polypeptide fusion comprising an ENPP1 polypeptide and an Fc region of an immunoglobulin, the ENPP1 polypeptide fusion comprising the mutations E592N, H1064K, and N1065F relative to SEQ ID NO:7. Embodiment 19 provides an ENPP1 mutant polypeptide comprising SEQ ID NO:7 and comprising a mutation selected from the group consisting of S766N, P534N, V536T, P554L, R545T, and E592N with respect to SEQ ID NO:7. Embodiment 20 provides a mutant polypeptide of embodiment 19, comprising at least one mutation selected from the group consisting of S766N, P534N / V536T, P554L / R545T, and E592N relative to SEQ ID NO:7. Embodiment 21 provides a mutant polypeptide of embodiment 19, comprising a mutation selected from the group consisting of S885N, S766N, M883Y / S885T / T887E, P534N / V536T / H1064K / N1065F, P554L / R545T, S766N / H1064K / N1065F, E592N / H1064K / N1065F, and P534N / V536T / M883Y / S885T / T887E with respect to SEQ ID NO:7. Embodiment 22 provides a mutant polypeptide of embodiment 19, which comprises a S885N mutation relative to SEQ ID NO:7. Embodiment 23 provides a mutant polypeptide of embodiment 19, which comprises a S766N mutation relative to SEQ ID NO:7. Embodiment 24 provides a mutant polypeptide of embodiment 19, comprising the mutations M883Y, S885T, and T887E relative to SEQ ID NO:7. Embodiment 25 provides a mutant polypeptide of embodiment 19, comprising the mutations P534N, V536T, H1064K, and N1065F relative to SEQ ID NO:7. Embodiment 26 provides a mutant polypeptide of embodiment 19, which comprises the mutations P554L and R545T relative to SEQ ID NO:7. Embodiment 27 provides a mutant polypeptide of embodiment 19, comprising the mutations S766N, H1064K, and N1065F relative to SEQ ID NO:7. Embodiment 28 provides a mutant polypeptide of embodiment 19, comprising the mutations E592N, H1064K, and N1065F relative to SEQ ID NO:7. Embodiment 29 provides a mutant polypeptide of embodiment 19, comprising the mutations P534N, V536T, M883Y, S885T, and T887E relative to SEQ ID NO:7. Embodiment 30 provides a polypeptide fusion of any of embodiments 1 to 18 or a mutant polypeptide of any of embodiments 19 to 29, expressed from a CHO cell line stably transfected with human ST6 β-galactosamide α-2,6-sialyltransferase (also known as ST6GAL1). Embodiment 31 provides a polypeptide fusion of any of embodiments 1 to 18 or a variant polypeptide of any of embodiments 19 to 29 grown in a cell culture supplemented with sialic acid and / or N-acetylmannosamine (also known as 1,3,4-O-Bu3ManNAc). 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 said subject a therapeutically effective amount of a polypeptide fusion of any of embodiments 1 to 18 and 30 to 31 or a mutant polypeptide of any of embodiments 19 to 31. 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 said subject a therapeutically effective amount of a polypeptide fusion of any of embodiments 1 to 18 and 30 to 31 or a mutant polypeptide of any of embodiments 19 to 31. Embodiment 34 provides a method of reducing or preventing the progression of ectopic calcification of soft tissue in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a polypeptide fusion of any of embodiments 1-18 and 30-31 or a mutant polypeptide of any of embodiments 19-31. Embodiment 35 provides a method of treating, reversing or preventing the progression of ossification of the posterior longitudinal ligament (OPLL) in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a polypeptide fusion of any of embodiments 1-18 and 30-31 or a mutant polypeptide of any of embodiments 19-31. Embodiment 36 provides a method of treating, reversing or preventing the progression of hypophosphatemic rickets in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a polypeptide fusion of any of embodiments 1-18 and 30-31 or a mutant polypeptide of any of embodiments 19-31. Embodiment 37 provides a method for alleviating or preventing the progression of at least one disease selected from the group consisting of chronic kidney disease (CKD), end stage renal disease (ESRD), uremic arteriolar calcification (CUA), calciphylaxis, ossification of the posterior longitudinal ligament (OPLL), hypophosphatemic rickets, osteoarthritis, age-related arteriosclerosis, idiopathic infantile arterial calcification (IIAC), generalized infantile arterial calcification (GACI), and atherosclerotic plaque calcification in a subject diagnosed with the at least one disease, the method comprising administering to the subject a therapeutically effective amount of a polypeptide fusion of any of embodiments 1-18 and 30-31 or a mutant polypeptide of any of embodiments 19-31. Embodiment 38 provides a method for alleviating or preventing the progression of age-related arteriosclerosis in a subject in need thereof, the method comprising the step of administering to the subject a therapeutically effective amount of a polypeptide fusion of any of Embodiments 1 to 18 and 30 to 31, or a mutant polypeptide of any of Embodiments 19 to 31. Example 39 provides the method of example 32, wherein said pathological calcification is selected from the group consisting of idiopathic infantile arterial calcification (IIAC) and atherosclerotic plaque calcification. Example 40 provides the method of example 33, wherein said pathological ossification is selected from the group consisting of ossification of the posterior longitudinal ligament (OPLL), hypophosphatemic rickets, and osteoarthritis. Example 41 provides the method of example 34, wherein said soft tissue calcification is selected from the group consisting of IIAC and osteoarthritis. Example 42 provides the method of Example 34, wherein the soft tissue is selected from the group consisting of atherosclerotic plaque, muscular arteries, joints, vertebrae, articular cartilage, intervertebral disc cartilage, blood vessels, and connective tissue. Embodiment 43 provides a method of increasing pyrophosphate (PPi) levels in a subject having a lower than normal PPi level, comprising administering to the subject a therapeutically effective amount of a polypeptide fusion of any of embodiments 1-18 and 30-31 or a mutant polypeptide of any of embodiments 19-31, whereby, upon administration, the level of PPi in the subject is increased to a normal level of at least 2 μM and maintained at approximately the same level. Embodiment 44 provides a method for reducing or preventing the progression of pathological calcification or ossification in a subject having a lower than normal pyrophosphate (PPi) level, the method comprising administering to the subject a therapeutically effective amount of a polypeptide fusion of any of embodiments 1-18 and 30-31 or a mutant polypeptide of any of embodiments 19-31, whereby pathological calcification or ossification in the subject is reduced or the progression of pathological calcification or ossification in the subject is prevented. Embodiment 45 provides a method of treating ENPP1 deficiency manifested by reduced extracellular pyrophosphate (PPi) concentrations in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a polypeptide fusion of any of embodiments 1-18 and 30-31 or a mutant polypeptide of any of embodiments 19-31, thereby increasing the level of PPi in said subject. Example 46 provides the method of any of Examples 32 to 45, wherein the polypeptide fusion or mutant polypeptide is a secreted product of an ENPP1 precursor protein expressed in a mammalian cell, the ENPP1 precursor protein comprising a signal peptide sequence and an ENPP1 polypeptide, and the ENPP1 precursor protein is proteolytically processed to produce the ENPP1 polypeptide. Example 47 provides the method of example 46, wherein in said ENPP1 precursor protein, said signal peptide sequence is conjugated to the N-terminus of said ENPP1 polypeptide. Example 48 provides the method of any of Examples 46 to 47, wherein the signal peptide sequence is selected from the group consisting of an ENPP1 signal peptide sequence, an ENPP2 signal peptide sequence, an ENPP7 signal peptide sequence, and an ENPP5 signal peptide sequence. Example 49 provides the method of any of Examples 32 to 48, wherein the polypeptide fusion or mutant polypeptide is administered to the subject acutely or chronically. Embodiment 50 provides the method of any of embodiments 32 to 49, wherein the polypeptide fusion or variant polypeptide is administered to the subject locally, topically, parenterally, or systemically. Example 51 provides the method of any of Examples 32 to 50, wherein the polypeptide fusion or variant polypeptide is administered to the subject by at least one route selected from the group consisting of subcutaneous, oral, aerosol, inhalation, rectal, vaginal, transdermal, subcutaneous, intranasal, buccal, sublingual, parenteral, intrathecal, intragastric, ophthalmic, pulmonary, and topical. Example 52 provides the method of any of Examples 32 to 51, wherein the polypeptide fusion or mutant polypeptide is administered to the subject as a pharmaceutical composition further comprising at least one pharma- ceutically acceptable carrier. Example 53 provides the method of any one of Examples 32 to 52, wherein the subject is a mammal. Example 54 provides the method of Example 53, wherein the mammal is a human.

[0237] The disclosures of each and every patent, patent application, and publication cited herein are incorporated herein by reference in their entirety. Although the present invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of the present invention may be devised by those skilled in the art without departing from the true spirit and scope of the present invention. It is intended that the appended claims be deemed to cover all such embodiments and equivalent variations.

[0238] Sequence information SEQUENCE LISTING <110> Yale University <120> ENPP1 Polypeptides and Methods of Using Same <150> US 62 / 725,607 <151> 2018-08-31 <150> US 62 / 830,247 <151> 2019-04-05 <160> 7 <170> PatentIn version 3.5 <210> 1 <211> 925 <212> PRT <213> Homo sapiens <400> 1 Met Glu Arg Asp Gly Cys Ala Gly Gly Gly Ser Arg Gly Gly Glu Gly 1 5 10 15 Gly Arg Ala Pro Arg Glu Gly Pro Ala Gly Asn Gly Arg Asp Arg Gly 20 25 30 Arg Ser His Ala Ala Glu Ala Pro Gly Asp Pro Gln Ala Ala Ala Ser 35 40 45 Leu Leu Ala Pro Met Asp Val Gly Glu Glu Pro Leu Glu Lys Ala Ala 50 55 60 Arg Ala Arg Thr Ala Lys Asp Pro Asn Thr Tyr Lys Val Leu Ser Leu 65 70 75 80 Val Leu Ser Val Cys Val Leu Thr Thr Ile Leu Gly Cys Ile Phe Gly 85 90 95 Leu Lys Pro Ser Cys Ala Lys Glu Val Lys Ser Cys Lys Gly Arg Cys 100 105 110 Phe Glu Arg Thr Phe Gly Asn Cys Arg Cys Asp Ala Ala Cys Val Glu 115 120 125 Leu Gly Asn Cys Cys Leu Asp Tyr Gln Glu Thr Cys Ile Glu Pro Glu 130 135 140 His Ile Trp Thr Cys Asn Lys Phe Arg Cys Gly Glu Lys Arg Leu Thr 145 150 155 160 Arg Ser Leu Cys Ala Cys Ser Asp Asp Cys Lys Asp Lys Gly Asp Cys 165 170 175 Cys Ile Asn Tyr Ser Ser Val Cys Gln Gly Glu Lys Ser Trp Val Glu 180 185 190 Glu Pro Cys Glu Ser Ile Asn Glu Pro Gln Cys Pro Ala Gly Phe Glu 195 200 205 Thr Pro Pro Thr Leu Leu Phe Ser Leu Asp Gly Phe Arg Ala Glu Tyr 210 215 220 Leu His Thr Trp Gly Gly Leu Leu Pro Val Ile Ser Lys Leu Lys Lys 225 230 235 240 Cys Gly Thr Tyr Thr Lys Asn Met Arg Pro Val Tyr Pro Thr Lys Thr 245 250 255 Phe Pro Asn His Tyr Ser Ile Val Thr Gly Leu Tyr Pro Glu Ser His 260 265 270 Gly Ile Ile Asp Asn Lys Met Tyr Asp Pro Lys Met Asn Ala Ser Phe 275 280 285 Ser Leu Lys Ser Lys Glu Lys Phe Asn Pro Glu Trp Tyr Lys Gly Glu 290 295 300 Pro Ile Trp Val Thr Ala Lys Tyr Gln Gly Leu Lys Ser Gly Thr Phe 305 310 315 320 Phe Trp Pro Gly Ser Asp Val Glu Ile Asn Gly Ile Phe Pro Asp Ile 325 330 335 Tyr Lys Met Tyr Asn Gly Ser Val Pro Phe Glu Glu Arg Ile Leu Ala 340 345 350 Val Leu Gln Trp Leu Gln Leu Pro Lys Asp Glu Arg Pro His Phe Tyr 355 360 365 Thr Leu Tyr Leu Glu Glu Pro Asp Ser Ser Gly His Ser Tyr Gly Pro 370 375 380 Val Ser Ser Glu Val Ile Lys Ala Leu Gln Arg Val Asp Gly Met Val 385 390 395 400 Gly Met Leu Met Asp Gly Leu Lys Glu Leu Asn Leu His Arg Cys Leu 405 410 415 Asn Leu Ile Leu Ile Ser Asp His Gly Met Glu Gln Gly Ser Cys Lys 420 425 430 Lys Tyr Ile Tyr Leu Asn Lys Tyr Leu Gly Asp Val Lys Asn Ile Lys 435 440 445 Val Ile Tyr Gly Pro Ala Ala Arg Leu Arg Pro Ser Asp Val Pro Asp 450 455 460 Lys Tyr Tyr Ser Phe Asn Tyr Glu Gly Ile Ala Arg Asn Leu Ser Cys 465 470 475 480 Arg Glu Pro Asn Gln His Phe Lys Pro Tyr Leu Lys His Phe Leu Pro 485 490 495 Lys Arg Leu His Phe Ala Lys Ser Asp Arg Ile Glu Pro Leu Thr Phe 500 505 510 Tyr Leu Asp Pro Gln Trp Gln Leu Ala Leu Asn Pro Ser Glu Arg Lys 515 520 525 Tyr Cys Gly Ser Gly Phe His Gly Ser Asp Asn Val Phe Ser Asn Met 530 535 540 Gln Ala Leu Phe Val Gly Tyr Gly Pro Gly Phe Lys His Gly Ile Glu 545 550 555 560 Ala Asp Thr Phe Glu Asn Ile Glu Val Tyr Asn Leu Met Cys Asp Leu 565 570 575 Leu Asn Leu Thr Pro Ala Pro Asn Asn Gly Thr His Gly Ser Leu Asn 580 585 590 His Leu Leu Lys Asn Pro Val Tyr Thr Pro Lys His Pro Lys Glu Val 595 600 605 His Pro Leu Val Gln Cys Pro Phe Thr Arg Asn Pro Arg Asp Asn Leu 610 615 620 Gly Cys Ser Cys Asn Pro Ser Ile Leu Pro Ile Glu Asp Phe Gln Thr 625 630 635 640 Gln Phe Asn Leu Thr Val Ala Glu Glu Lys Ile Ile Lys His Glu Thr 645 650 655 Leu Pro Tyr Gly Arg Pro Arg Val Leu Gln Lys Glu Asn Thr Ile Cys 660 665 670 Leu Leu Ser Gln His Gln Phe Met Ser Gly Tyr Ser Gln Asp Ile Leu 675 680 685 Met Pro Leu Trp Thr Ser Tyr Thr Val Asp Arg Asn Asp Ser Phe Ser 690 695 700 Thr Glu Asp Phe Ser Asn Cys Leu Tyr Gln Asp Phe Arg Ile Pro Leu 705 710 715 720 Ser Pro Val His Lys Cys Ser Phe Tyr Lys Asn Asn Thr Lys Val Ser 725 730 735 Tyr Gly Phe Leu Ser Pro Pro Gln Leu Asn Lys Asn Ser Ser Gly Ile 740 745 750 Tyr Ser Glu Ala Leu Leu Thr Thr Asn Ile Val Pro Met Tyr Gln Ser 755 760 765 Phe Gln Val Ile Trp Arg Tyr Phe His Asp Thr Leu Leu Arg Lys Tyr 770 775 780 Ala Glu Glu Arg Asn Gly Val Asn Val Val Ser Gly Pro Val Phe Asp 785 790 795 800 Phe Asp Tyr Asp Gly Arg Cys Asp Ser Leu Glu Asn Leu Arg Gln Lys 805 810 815 Arg Arg Val Ile Arg Asn Gln Glu Ile Leu Ile Pro Thr His Phe Phe 820 825 830 Ile Val Leu Thr Ser Cys Lys Asp Thr Ser Gln Thr Pro Leu His Cys 835 840 845 Glu Asn Leu Asp Thr Leu Ala Phe Ile Leu Pro His Arg Thr Asp Asn 850 855 860 Ser Glu Ser Cys Val His Gly Lys His Asp Ser Ser Trp Val Glu Glu 865 870 875 880 Leu Leu Met Leu His Arg Ala Arg Ile Thr Asp Val Glu His Ile Thr 885 890 895 Gly Leu Ser Phe Tyr Gln Gln Arg Lys Glu Pro Val Ser Asp Ile Leu 900 905 910 Lys Leu Lys Thr His Leu Pro Thr Phe Ser Gln Glu Asp 915 920 925 <210> 2 <211> 888 <212> PRT <213> Homo sapiens <400> 2 Met Ala Arg Arg Ser Ser Phe Gln Ser Cys Gln Ile Ile Ser Leu Phe 1 5 10 15 Thr Phe Ala Val Gly Val Asn Ile Cys Leu Gly Phe Thr Ala His Arg 20 25 30 Ile Lys Arg Ala Glu Gly Trp Glu Glu Gly Pro Pro Thr Val Leu Ser 35 40 45 Asp Ser Pro Trp Thr Asn Ile Ser Gly Ser Cys Lys Gly Arg Cys Phe 50 55 60 Glu Leu Gln Glu Ala Gly Pro Pro Asp Cys Arg Cys Asp Asn Leu Cys 65 70 75 80 Lys Ser Tyr Thr Ser Cys Cys His Asp Phe Asp Glu Leu Cys Leu Lys 85 90 95 Thr Ala Arg Gly Trp Glu Cys Thr Lys Asp Arg Cys Gly Glu Val Arg 100 105 110 Asn Glu Glu Asn Ala Cys His Cys Ser Glu Asp Cys Leu Ala Arg Gly 115 120 125 Asp Cys Cys Thr Asn Tyr Gln Val Val Cys Lys Gly Glu Ser His Trp 130 135 140 Val Asp Asp Asp Cys Glu Glu Ile Lys Ala Ala Glu Cys Pro Ala Gly 145 150 155 160 Phe Val Arg Pro Pro Leu Ile Ile Phe Ser Val Asp Gly Phe Arg Ala 165 170 175 Ser Tyr Met Lys Lys Gly Ser Lys Val Met Pro Asn Ile Glu Lys Leu 180 185 190 Arg Ser Cys Gly Thr His Ser Pro Tyr Met Arg Pro Val Tyr Pro Thr 195 200 205 Lys Thr Phe Pro Asn Leu Tyr Thr Leu Ala Thr Gly Leu Tyr Pro Glu 210 215 220 Ser His Gly Ile Val Gly Asn Ser Met Tyr Asp Pro Val Phe Asp Ala 225 230 235 240 Thr Phe His Leu Arg Gly Arg Glu Lys Phe Asn His Arg Trp Trp Gly 245 250 255 Gly Gln Pro Leu Trp Ile Thr Ala Thr Lys Gln Gly Val Lys Ala Gly 260 265 270 Thr Phe Phe Trp Ser Val Val Ile Pro His Glu Arg Arg Ile Leu Thr 275 280 285 Ile Leu Gln Trp Leu Thr Leu Pro Asp His Glu Arg Pro Ser Val Tyr 290 295 300 Ala Phe Tyr Ser Glu Gln Pro Asp Phe Ser Gly His Lys Tyr Gly Pro 305 310 315 320 Phe Gly Pro Glu Met Thr Asn Pro Leu Arg Glu Ile Asp Lys Ile Val 325 330 335 Gly Gln Leu Met Asp Gly Leu Lys Gln Leu Lys Leu His Arg Cys Val 340 345 350 Asn Val Ile Phe Val Gly Asp His Gly Met Glu Asp Val Thr Cys Asp 355 360 365 Arg Thr Glu Phe Leu Ser Asn Tyr Leu Thr Asn Val Asp Asp Ile Thr 370 375 380 Leu Val Pro Gly Thr Leu Gly Arg Ile Arg Ser Lys Phe Ser Asn Asn 385 390 395 400 Ala Lys Tyr Asp Pro Lys Ala Ile Ile Ala Asn Leu Thr Cys Lys Lys 405 410 415 Pro Asp Gln His Phe Lys Pro Tyr Leu Lys Gln His Leu Pro Lys Arg 420 425 430 Leu His Tyr Ala Asn Asn Arg Arg Ile Glu Asp Ile His Leu Leu Val 435 440 445 Glu Arg Arg Trp His Val Ala Arg Lys Pro Leu Asp Val Tyr Lys Lys 450 455 460 Pro Ser Gly Lys Cys Phe Phe Gln Gly Asp His Gly Phe Asp Asn Lys 465 470 475 480 Val Asn Ser Met Gln Thr Val Phe Val Gly Tyr Gly Ser Thr Phe Lys 485 490 495 Tyr Lys Thr Lys Val Pro Pro Phe Glu Asn Ile Glu Leu Tyr Asn Val 500 505 510 Met Cys Asp Leu Leu Gly Leu Lys Pro Ala Pro Asn Asn Gly Thr His 515 520 525 Gly Ser Leu Asn His Leu Leu Arg Thr Asn Thr Phe Arg Pro Thr Met 530 535 540 Pro Glu Glu Val Thr Arg Pro Asn Tyr Pro Gly Ile Met Tyr Leu Gln 545 550 555 560 Ser Asp Phe Asp Leu Gly Cys Thr Cys Asp Asp Lys Val Glu Pro Lys 565 570 575 Asn Lys Leu Asp Glu Leu Asn Lys Arg Leu His Thr Lys Gly Ser Thr 580 585 590 Glu Ala Glu Thr Arg Lys Phe Arg Gly Ser Arg Asn Glu Asn Lys Glu 595 600 605 Asn Ile Asn Gly Asn Phe Glu Pro Arg Lys Glu Arg His Leu Leu Tyr 610 615 620 Gly Arg Pro Ala Val Leu Tyr Arg Thr Arg Tyr Asp Ile Leu Tyr His 625 630 635 640 Thr Asp Phe Glu Ser Gly Tyr Ser Glu Ile Phe Leu Met Pro Leu Trp 645 650 655 Thr Ser Tyr Thr Val Ser Lys Gln Ala Glu Val Ser Ser Val Pro Asp 660 665 670 His Leu Thr Ser Cys Val Arg Pro Asp Val Arg Val Ser Pro Ser Phe 675 680 685 Ser Gln Asn Cys Leu Ala Tyr Lys Asn Asp Lys Gln Met Ser Tyr Gly 690 695 700 Phe Leu Phe Pro Pro Tyr Leu Ser Ser Ser Pro Glu Ala Lys Tyr Asp 705 710 715 720 Ala Phe Leu Val Thr Asn Met Val Pro Met Tyr Pro Ala Phe Lys Arg 725 730 735 Val Trp Asn Tyr Phe Gln Arg Val Leu Val Lys Lys Tyr Ala Ser Glu 740 745 750 Arg Asn Gly Val Asn Val Ile Ser Gly Pro Ile Phe Asp Tyr Asp Tyr 755 760 765 Asp Gly Leu His Asp Thr Glu Asp Lys Ile Lys Gln Tyr Val Glu Gly 770 775 780 Ser Ser Ile Pro Val Pro Thr His Tyr Tyr Ser Ile Ile Thr Ser Cys 785 790 795 800 Leu Asp Phe Thr Gln Pro Ala Asp Lys Cys Asp Gly Pro Leu Ser Val 805 810 815 Ser Ser Phe Ile Leu Pro His Arg Pro Asp Asn Glu Glu Ser Cys Asn 820 825 830 Ser Ser Glu Asp Glu Ser Lys Trp Val Glu Glu Leu Met Lys Met His 835 840 845 Thr Ala Arg Val Arg Asp Ile Glu His Leu Thr Ser Leu Asp Phe Phe 850 855 860 Arg Lys Thr Ser Arg Ser Tyr Pro Glu Ile Leu Thr Leu Lys Thr Tyr 865 870 875 880 Leu His Thr Tyr Glu Ser Glu Ile 885 <210> 3 <211> 227 <212> PRT <213> Homo sapiens <400> 3 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 4 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> Chemically synthesized <220> <221> MOD_RES <222> (23)..(23) <223> Xaa23 is absent or L <220> <221> MOD_RES <222> (24)..(24) <223> Xaa24 is absent if Xaa23 is absent, and Xaa24 is absent or Q if Xaa23 is L <400> 4 Met Thr Ser Lys Phe Leu Leu Val Ser Phe Ile Leu Ala Ala Leu Ser 1 5 10 15 Leu Ser Thr Thr Phe Ser Xaa Xaa 20 <210> 5 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Chemically synthesized <400> 5 Met Arg Gly Pro Ala Val Leu Leu Thr Val Ala Leu Ala Thr Leu Leu 1 5 10 15 Ala Pro Gly Ala Gly Ala 20 <210> 6 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Chemically synthesized <400> 6 Met Arg Gly Pro Ala Val Leu Leu Thr Val Ala Leu Ala Thr Leu Leu 1 5 10 15 Ala Pro Gly Ala 20 <210> 7 <211> 1078 <212> PRT <213> Artificial Sequence <220> <223> ENPP1-Fc <400> 7 Met Arg Gly Pro Ala Val Leu Leu Thr Val Ala Leu Ala Thr Leu Leu 1 5 10 15 Ala Pro Gly Ala Gly Ala Pro Ser Cys Ala Lys Glu Val Lys Ser Cys 20 25 30 Lys Gly Arg Cys Phe Glu Arg Thr Phe Gly Asn Cys Arg Cys Asp Ala 35 40 45 Ala Cys Val Glu Leu Gly Asn Cys Cys Leu Asp Tyr Gln Glu Thr Cys 50 55 60 Ile Glu Pro Glu His Ile Trp Thr Cys Asn Lys Phe Arg Cys Gly Glu 65 70 75 80 Lys Arg Leu Thr Arg Ser Leu Cys Ala Cys Ser Asp Asp Cys Lys Asp 85 90 95 Lys Gly Asp Cys Cys Ile Asn Tyr Ser Ser Val Cys Gln Gly Glu Lys 100 105 110 Ser Trp Val Glu Glu Pro Cys Glu Ser Ile Asn Glu Pro Gln Cys Pro 115 120 125 Ala Gly Phe Glu Thr Pro Pro Thr Leu Leu Phe Ser Leu Asp Gly Phe 130 135 140 Arg Ala Glu Tyr Leu His Thr Trp Gly Gly Leu Leu Pro Val Ile Ser 145 150 155 160 Lys Leu Lys Lys Cys Gly Thr Tyr Thr Lys Asn Met Arg Pro Val Tyr 165 170 175 Pro Thr Lys Thr Phe Pro Asn His Tyr Ser Ile Val Thr Gly Leu Tyr 180 185 190 Pro Glu Ser His Gly Ile Ile Asp Asn Lys Met Tyr Asp Pro Lys Met 195 200 205 Asn Ala Ser Phe Ser Leu Lys Ser Lys Glu Lys Phe Asn Pro Glu Trp 210 215 220 Tyr Lys Gly Glu Pro Ile Trp Val Thr Ala Lys Tyr Gln Gly Leu Lys 225 230 235 240 Ser Gly Thr Phe Phe Trp Pro Gly Ser Asp Val Glu Ile Asn Gly Ile 245 250 255 Phe Pro Asp Ile Tyr Lys Met Tyr Asn Gly Ser Val Pro Phe Glu Glu 260 265 270 Arg Ile Leu Ala Val Leu Gln Trp Leu Gln Leu Pro Lys Asp Glu Arg 275 280 285 Pro His Phe Tyr Thr Leu Tyr Leu Glu Glu Pro Asp Ser Ser Gly His 290 295 300 Ser Tyr Gly Pro Val Ser Ser Glu Val Ile Lys Ala Leu Gln Arg Val 305 310 315 320 Asp Gly Met Val Gly Met Leu Met Asp Gly Leu Lys Glu Leu Asn Leu 325 330 335 His Arg Cys Leu Asn Leu Ile Leu Ile Ser Asp His Gly Met Glu Gln 340 345 350 Gly Ser Cys Lys Lys Tyr Ile Tyr Leu Asn Lys Tyr Leu Gly Asp Val 355 360 365 Lys Asn Ile Lys Val Ile Tyr Gly Pro Ala Ala Arg Leu Arg Pro Ser 370 375 380 Asp Val Pro Asp Lys Tyr Tyr Ser Phe Asn Tyr Glu Gly Ile Ala Arg 385 390 395 400 Asn Leu Ser Cys Arg Glu Pro Asn Gln His Phe Lys Pro Tyr Leu Lys 405 410 415 His Phe Leu Pro Lys Arg Leu His Phe Ala Lys Ser Asp Arg Ile Glu 420 425 430 Pro Leu Thr Phe Tyr Leu Asp Pro Gln Trp Gln Leu Ala Leu Asn Pro 435 440 445 Ser Glu Arg Lys Tyr Cys Gly Ser Gly Phe His Gly Ser Asp Asn Val 450 455 460 Phe Ser Asn Met Gln Ala Leu Phe Val Gly Tyr Gly Pro Gly Phe Lys 465 470 475 480 His Gly Ile Glu Ala Asp Thr Phe Glu Asn Ile Glu Val Tyr Asn Leu 485 490 495 Met Cys Asp Leu Leu Asn Leu Thr Pro Ala Pro Asn Asn Gly Thr His 500 505 510 Gly Ser Leu Asn His Leu Leu Lys Asn Pro Val Tyr Thr Pro Lys His 515 520 525 Pro Lys Glu Val His Pro Leu Val Gln Cys Pro Phe Thr Arg Asn Pro 530 535 540 Arg Asp Asn Leu Gly Cys Ser Cys Asn Pro Ser Ile Leu Pro Ile Glu 545 550 555 560 Asp Phe Gln Thr Gln Phe Asn Leu Thr Val Ala Glu Glu Lys Ile Ile 565 570 575 Lys His Glu Thr Leu Pro Tyr Gly Arg Pro Arg Val Leu Gln Lys Glu 580 585 590 Asn Thr Ile Cys Leu Leu Ser Gln His Gln Phe Met Ser Gly Tyr Ser 595 600 605 Gln Asp Ile Leu Met Pro Leu Trp Thr Ser Tyr Thr Val Asp Arg Asn 610 615 620 Asp Ser Phe Ser Thr Glu Asp Phe Ser Asn Cys Leu Tyr Gln Asp Phe 625 630 635 640 Arg Ile Pro Leu Ser Pro Val His Lys Cys Ser Phe Tyr Lys Asn Asn 645 650 655 Thr Lys Val Ser Tyr Gly Phe Leu Ser Pro Pro Gln Leu Asn Lys Asn 660 665 670 Ser Ser Gly Ile Tyr Ser Glu Ala Leu Leu Thr Thr Asn Ile Val Pro 675 680 685 Met Tyr Gln Ser Phe Gln Val Ile Trp Arg Tyr Phe His Asp Thr Leu 690 695 700 Leu Arg Lys Tyr Ala Glu Glu Arg Asn Gly Val Asn Val Val Ser Gly 705 710 715 720 Pro Val Phe Asp Phe Asp Tyr Asp Gly Arg Cys Asp Ser Leu Glu Asn 725 730 735 Leu Arg Gln Lys Arg Arg Val Ile Arg Asn Gln Glu Ile Leu Ile Pro 740 745 750 Thr His Phe Phe Ile Val Leu Thr Ser Cys Lys Asp Thr Ser Gln Thr 755 760 765 Pro Leu His Cys Glu Asn Leu Asp Thr Leu Ala Phe Ile Leu Pro His 770 775 780 Arg Thr Asp Asn Ser Glu Ser Cys Val His Gly Lys His Asp Ser Ser 785 790 795 800 Trp Val Glu Glu Leu Leu Met Leu His Arg Ala Arg Ile Thr Asp Val 805 810 815 Glu His Ile Thr Gly Leu Ser Phe Tyr Gln Gln Arg Lys Glu Pro Val 820 825 830 Ser Asp Ile Leu Lys Leu Lys Thr His Leu Pro Thr Phe Ser Gln Glu 835 840 845 Asp Arg Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 850 855 860 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 865 870 875 880 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 885 890 895 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 900 905 910 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 915 920 925 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 930 935 940 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 945 950 955 960 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 965 970 975 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 980 985 990 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 995 1000 1005 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 1010 1015 1020 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 1025 1030 1035 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 1040 1045 1050 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 1055 1060 1065 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 1070 1075

Claims

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

7.

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

7.

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

7.

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

7.

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

7.

6. The polypeptide fusion of claim 5, wherein the ENPP1 polypeptide comprises at least the mutations K369N / I371T relative to SEQ ID NO:

7.

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

7.

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

7.

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

7.

10. The polypeptide fusion of claim 9, wherein the ENPP1 polypeptide comprises at least the mutations E864N / L866T relative to SEQ ID NO:

7.

11. C25N, K27T, V29N, C25N / K27T, K369N, I371T, K369N / I371T, P534N, V536T, R545T, P554L, E592N, R741D, S766N, P534N / V536T, P554L / R545T, E592N / R741D, E864N, L86 10. The polypeptide fusion of any one of claims 1, 3, 5, 7, and 9, comprising at least one mutation selected from the group consisting of 6T, E864N / L866T, M883Y, S885N, S885T, T887E, H1064K, N1065F, M883Y / S885T / T887E, H1064K / N1065F.

12. The polypeptide fusion of any one of claims 1, 3, 5, 7, and 9, wherein the Fc region is an IgG Fc region.

13. The polypeptide fusion of any one of claims 1, 3, 5, 7 and 9, comprising at least one mutation selected from the group consisting of P534N, V536T, R545T, P554L, S766N and E592N with respect to SEQ ID NO:

7.

14. The polypeptide fusion of any one of claims 1, 3, 5, 7, and 9, comprising at least one mutation selected from the group consisting of S766N, P534N / Y536T, P554L / R545T, and E592N relative to SEQ ID NO:

7.

15. The polypeptide fusion of any one of claims 1, 3, 5, 7, and 9, comprising at least one mutation selected from the group consisting of 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 with respect to SEQ ID NO:

7.

16. An ENPP1 polypeptide fusion comprising an ENPP1 polypeptide and an Fc region of an immunoglobulin, The ENPP1 polypeptide fusion comprising the mutations M883Y, S885T, and T887E relative to SEQ ID NO:

7.

17. An ENPP1 polypeptide fusion comprising an ENPP1 polypeptide and an Fc region of an immunoglobulin, The ENPP1 polypeptide fusion comprising the mutations P534N, V536T, M883Y, S885T, and T887E relative to SEQ ID NO:

7.

18. An ENPP1 polypeptide fusion comprising an ENPP1 polypeptide and an Fc region of an immunoglobulin, The ENPP1 polypeptide fusion comprising the mutations E592N, H1064K, and N1065F relative to SEQ ID NO:

7.

19. An ENPP1 mutant polypeptide comprising SEQ ID NO:7 and comprising a mutation selected from the group consisting of S766N, P534N, V536T, P554L, R545T, and E592N relative to SEQ ID NO:

7.

20. 20. The mutant polypeptide of claim 19, comprising at least one mutation selected from the group consisting of S766N, P534N / V536T, P554L / R545T, and E592N relative to SEQ ID NO:

7.

21. 20. The mutant polypeptide of claim 19, comprising a mutation selected from the group consisting of S885N, S766N, M883Y / S885T / T887E, P534N / V536T / H1064K / N1065F, P554L / R545T, S766N / H1064K / N1065F, E592N / H1064K / N1065F, and P534N / V536T / M883Y / S885T / T887E with respect to SEQ ID NO:

7.

22. 20. The mutant polypeptide of claim 19, comprising an S885N mutation relative to SEQ ID NO:

7.

23. 20. The mutant polypeptide of claim 19, comprising an S766N mutation relative to SEQ ID NO:

7.

24. 20. The mutant polypeptide of claim 19, comprising the mutations M883Y, S885T, and T887E relative to SEQ ID NO:

7.

25. 20. The mutant polypeptide of claim 19, comprising the mutations P534N, V536T, H1064K, and N1065F relative to SEQ ID NO:

7.

26. 20. The mutant polypeptide of claim 19, comprising the mutations P554L and R545T relative to SEQ ID NO:

7.

27. 20. The mutant polypeptide of claim 19, comprising the mutations S766N, H1064K, and N1065F relative to SEQ ID NO:

7.

28. 20. The mutant polypeptide of claim 19, comprising the mutations E592N, H1064K, and N1065F relative to SEQ ID NO:

7.

29. 20. The mutant polypeptide of claim 19, comprising the mutations P534N, V536T, M883Y, S885T, and T887E relative to SEQ ID NO:

7.

30. A polypeptide fusion according to any one of claims 1, 3, 5, 7, 9, 16, 17 and 18 or a mutant polypeptide according to claim 19, which is expressed from a CHO cell line stably transfected with human ST6β-galactosamide α-2,6-sialyltransferase (also known as ST6GAL1).

31. Sialic acid and / or N-acetylmannosamine (1,3,4-O-Bu 3 The polypeptide fusion of any one of claims 1, 3, 5, 7, 9, 16, 17, and 18 or the mutant polypeptide of claim 19, grown in a cell culture supplemented with ManNAc (also known as ManNAc).

32. 1. A method of reducing or preventing the progression of pathological calcification in a subject in need thereof, comprising: The method comprises administering to the subject a therapeutically effective amount of a polypeptide fusion described in any one of claims 1, 3, 5, 7, 9, 16, 17, and 18 or a mutant polypeptide described in claim 19.

33. 1. A method of reducing or preventing the progression of pathological ossification in a subject in need thereof, comprising: The method comprises administering to the subject a therapeutically effective amount of a polypeptide fusion described in any one of claims 1, 3, 5, 7, 9, 16, 17, and 18 or a mutant polypeptide described in claim 19.

34. 1. A method of reducing or preventing the progression of ectopic soft tissue calcification in a subject in need thereof, comprising: The method comprises administering to the subject a therapeutically effective amount of a polypeptide fusion described in any one of claims 1, 3, 5, 7, 9, 16, 17, and 18 or a mutant polypeptide described in claim 19.

35. 1. A method of treating, reversing, or preventing the progression of ossification of the posterior longitudinal ligament (OPLL) in a subject in need thereof, comprising: The method comprises administering to the subject a therapeutically effective amount of a polypeptide fusion described in any one of claims 1, 3, 5, 7, 9, 16, 17, and 18 or a mutant polypeptide described in claim 19.

36. 1. A method of treating, reversing, or preventing the progression of hypophosphatemic rickets in a subject in need thereof, comprising: The method comprises administering to the subject a therapeutically effective amount of a polypeptide fusion described in any one of claims 1, 3, 5, 7, 9, 16, 17, and 18 or a mutant polypeptide described in claim 19.

37. 1. A method for alleviating or preventing the progression of at least one disease selected from the group consisting of chronic kidney disease (CKD), end stage renal disease (ESRD), uremic arteriolar calcification (CUA), calciphylaxis, ossification of the posterior longitudinal ligament (OPLL), hypophosphatemic rickets, osteoarthritis, age-related arteriosclerosis, idiopathic infantile arterial calcification (IIAC), generalized infantile arterial calcification (GACI), and atherosclerotic plaque calcification in a subject diagnosed with the at least one disease, comprising: The method comprises administering to the subject a therapeutically effective amount of a polypeptide fusion described in any one of claims 1, 3, 5, 7, 9, 16, 17, and 18 or a mutant polypeptide described in claim 19.

38. 1. A method for reducing or preventing the progression of age-related arteriosclerosis in a subject in need thereof, comprising: The method comprises administering to the subject a therapeutically effective amount of a polypeptide fusion described in any one of claims 1, 3, 5, 7, 9, 16, 17, and 18 or a mutant polypeptide described in claim 19.

39. 33. The method of claim 32, wherein the pathological calcification is selected from the group consisting of idiopathic infantile arterial calcification (IIAC) and atherosclerotic plaque calcification.

40. 34. The method of claim 33, wherein the pathological ossification is selected from the group consisting of ossification of the posterior longitudinal ligament (OPLL), hypophosphatemic rickets, and osteoarthritis.

41. 35. The method of claim 34, wherein the soft tissue calcification is selected from the group consisting of IIAC and osteoarthritis.

42. 35. The method of claim 34, wherein the soft tissue is selected from the group consisting of atherosclerotic plaque, muscular arteries, joints, vertebrae, articular cartilage, vertebral disk cartilage, blood vessels, and connective tissue.

43. 1. A method for increasing pyrophosphate (PPi) levels in a subject having a lower than normal PPi level, comprising: The method comprises administering to the subject a therapeutically effective amount of a polypeptide fusion described in any one of claims 1, 3, 5, 7, 9, 16, 17, and 18 or a mutant polypeptide described in claim 19, whereby upon administration, the level of PPi in the subject is increased to a normal level of at least 2 μM and maintained at approximately the same level.

44. 1. A method for reducing or preventing the progression of pathological calcification or ossification in a subject having a lower than normal level of pyrophosphate (PPi), comprising: The method comprises administering to a subject a therapeutically effective amount of a polypeptide fusion described in any one of claims 1, 3, 5, 7, 9, 16, 17, and 18 or a mutant polypeptide described in claim 19, thereby reducing pathological calcification or ossification in the subject or preventing the progression of pathological calcification or ossification in the subject.

45. 1. A method of treating ENPP1 deficiency manifested by a decrease in extracellular pyrophosphate (PPi) concentration in a subject in need thereof, comprising: The method comprises administering to the subject a therapeutically effective amount of a polypeptide fusion described in any one of claims 1, 3, 5, 7, 9, 16, 17, and 18 or a mutant polypeptide described in claim 19, thereby increasing the level of PPi in the subject.

46. The method of any one of claims 32-38 and 43-45, wherein the polypeptide fusion or mutant polypeptide is a secreted product of an ENPP1 precursor protein expressed in a mammalian cell, the ENPP1 precursor protein comprising a signal peptide sequence and an ENPP1 polypeptide, and the ENPP1 precursor protein is proteolytically processed to produce the ENPP1 polypeptide.

47. The method of claim 46, wherein in the ENPP1 precursor protein, the signal peptide sequence is conjugated to the N-terminus of the ENPP1 polypeptide.

48. 47. The method of claim 46, wherein the signal peptide sequence is selected from the group consisting of an ENPP1 signal peptide sequence, an ENPP2 signal peptide sequence, an ENPP7 signal peptide sequence, and an ENPP5 signal peptide sequence.

49. The method of any one of claims 32-38 and 43-45, wherein said polypeptide fusion or mutant polypeptide is administered to said subject acutely or chronically.

50. The method of any one of claims 32-38 and 43-45, wherein the polypeptide fusion or variant polypeptide is administered to the subject locally, topically, parenterally, or systemically.

51. The method of any one of claims 32-38 and 43-45, wherein the polypeptide fusion or variant polypeptide is administered to the subject by at least one route selected from the group consisting of subcutaneous, oral, aerosol, inhalation, rectal, vaginal, transdermal, subcutaneous, intranasal, buccal, sublingual, parenteral, intrathecal, intragastric, ophthalmic, pulmonary, and topical.

52. The method of any one of claims 32-38 and 43-45, wherein the polypeptide fusion or mutant polypeptide is administered to the subject as a pharmaceutical composition further comprising at least one pharma- ceutically acceptable carrier.

53. The method of any one of claims 32-38 and 43-45, wherein the subject is a mammal.

54. 54. The method of claim 53, wherein the mammal is a human.

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