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, and a method of reducing or preventing progression of pathological calcification in a subject.SOLUTION: The present invention provides an ENPP1 polypeptide fusion comprising an ENPP1 polypeptide fused to a Fc region of an immunoglobulin, wherein the ENPP1 polypeptide comprises the mutation I256T relating to a specific amino acid sequence.SELECTED DRAWING: None

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 / 830,230, filed April 5, 2019, No. 62 / 983,142, filed February 28, 2020, and No. 62 / 984,650, filed March 3, 2020, 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] WO2012 / 125182 [Non-patent literature]

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

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

[0010] The present disclosure further provides a method of 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 mutant polypeptide of the present disclosure.

[0011] The present disclosure 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 present disclosure.

[0012] The present disclosure 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 mutant polypeptide of the present disclosure.

[0013] The present disclosure 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 disclosure.

[0014] The present disclosure further provides a method of 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 present disclosure.

[0015] The present disclosure further provides a method of 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 the at least one disease, comprising administering to the subject a therapeutically effective amount of a polypeptide fusion and / or mutant polypeptide of the present disclosure.

[0016] The present disclosure further provides a method for reducing and / or preventing the progression of age-related arteriosclerosis 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 disclosure.

[0017] The present disclosure 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 disclosure, 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 disclosure further provides a method of reducing and / or preventing the progression of pathological calcification 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 mutant polypeptide of the present disclosure, whereby pathological calcification or ossification in the subject is reduced and / or the progression of pathological calcification or ossification in the subject is prevented.

[0019] The present disclosure further provides a method of 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 disclosure, thereby increasing the level of PPi in the subject. [The present invention 1001] An ENPP1 polypeptide fusion comprising an ENPP1 polypeptide fused to an Fc region of an immunoglobulin, wherein the ENPP1 polypeptide comprises the mutation I256T relative to SEQ ID NO:7. [The present invention 1002] The polypeptide fusion of the present invention 1001, 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. [The present invention 1003] The polypeptide fusion of the present invention 1001, 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. [The present invention 1004] The polypeptide fusion of the present invention 1001, wherein the ENPP1 polypeptide further comprises at least one mutation selected from the group consisting of C25N, K27T, and V29N relative to SEQ ID NO:7. [The present invention 1005] The polypeptide fusion of the present invention 1004, 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. [The present invention 1006] The polypeptide fusion of the present invention 1001, wherein the ENPP1 polypeptide further comprises at least one mutation selected from the group consisting of K369N and I371T relative to SEQ ID NO:7. [The present invention 1007] The polypeptide fusion of the present invention 1006, wherein the ENPP1 polypeptide comprises the mutations K369N / I371T relative to SEQ ID NO:7. [The present invention 1008] The polypeptide fusion of the present invention 1001, wherein the ENPP1 polypeptide further 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. [The present invention 1009] The polypeptide fusion of the present invention 1008, 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. [The present invention 1010] The polypeptide fusion of the present invention 1001, wherein the ENPP1 polypeptide further comprises at least one mutation selected from the group consisting of E864N and L866T relative to SEQ ID NO:7. [The present invention 1011] The polypeptide fusion of the present invention 1010, wherein the ENPP1 polypeptide comprises at least the mutations E864N / L866T relative to SEQ ID NO:7. [The present invention 1012] SEQ ID The polypeptide fusion of the present invention 1001, 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. [The present invention 1013] The polypeptide fusion of the present invention, wherein the Fc region is an IgG Fc region. [The present invention 1014] The polypeptide fusion of the present invention 1001, 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. [The present invention 1015] The polypeptide fusion of the present invention 1001, comprising at least one mutation selected from the group consisting of S766N, P534N / Y536T, P554L / R545T, and E592N relative to SEQ ID NO:7. [The present invention 1016] The polypeptide fusion of the present invention 1001, 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. [The present invention 1017] An ENPP1 polypeptide fusion comprising an ENPP1 polypeptide and an Fc region of an immunoglobulin, the ENPP1 polypeptide fusion comprising the mutations I256T, M883Y, S885T, and T887E relative to SEQ ID NO:7. [The present invention 1018] An ENPP1 polypeptide fusion comprising an ENPP1 polypeptide and an Fc region of an immunoglobulin, the ENPP1 polypeptide fusion comprising the mutations I256T, P534N, V536T, M883Y, S885T, and T887E relative to SEQ ID NO:7. [The present invention 1019] An ENPP1 polypeptide fusion comprising an ENPP1 polypeptide and an Fc region of an immunoglobulin, the ENPP1 polypeptide fusion comprising the mutations I256T, E592N, H1064K, and N1065F relative to SEQ ID NO:7. [The present invention 1020] ENPP1 mutant polypeptide comprising amino acids 23 to 849 of SEQ ID NO:7, comprising the mutation I256T relative to SEQ ID NO:7, and further comprising a mutation selected from the group consisting of S766N, P534N, V536T, P554L, R545T, and E592N; ENPP1 mutant polypeptide. [The present invention 1021] A variant polypeptide of the present invention comprising the amino acid sequence of SEQ ID NO:7. [The present invention 1022] The mutant polypeptide of any of 1020 to 1021 of the present invention, which lacks a signal peptide sequence. [The present invention 1023] 1020. A mutant polypeptide of the present invention comprising at least one mutation selected from the group consisting of S766N, P534N / V536T, P554L / R545T, and E592N relative to SEQ ID NO:7. [The present invention 1024] A mutant polypeptide of the present invention 1021 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. [The present invention 1025] A mutant polypeptide of the present invention 1021 comprising an S885N mutation relative to SEQ ID NO:7. [The present invention 1026] A mutant polypeptide of the present invention 1020 comprising a S766N mutation relative to SEQ ID NO:7. [The present invention 1027] A mutant polypeptide of the present invention 1021 comprising the mutations M883Y, S885T, and T887E relative to SEQ ID NO:7. [The present invention 1028] A mutant polypeptide of the present invention 1021 comprising the mutations P534N, V536T, H1064K, and N1065F relative to SEQ ID NO:7. [The present invention 1029] A mutant polypeptide of the present invention 1020 comprising the mutations P554L and R545T relative to SEQ ID NO:7. [The present invention 1030] A mutant polypeptide of the present invention 1021 comprising the mutations S766N, H1064K, and N1065F relative to SEQ ID NO:7. [The present invention 1031] A mutant polypeptide of the present invention 1021 comprising the mutations E592N, H1064K, and N1065F relative to SEQ ID NO:7. [The present invention 1032] A mutant polypeptide of the present invention 1021 comprising the mutations P534N, V536T, M883Y, S885T, and T887E relative to SEQ ID NO:7. [The present invention 1033] A polypeptide fusion of any of 1001, 1017, 1018, and 1019 or a mutant polypeptide of 1020 of the invention expressed from a CHO cell line stably transfected with human ST6 beta-galactoside alpha-2,6-sialyltransferase (also known as ST6GAL1). [The present invention 1034] Sialic acid and / or N-acetylmannosamine (1,3,4-O-Bu 3 A polypeptide fusion of any of 1001, 1017, 1018, and 1019 or a mutant polypeptide of 1020 of the invention grown in a cell culture supplemented with 10NAc (also known as ManNAc). [The present invention 1035] A method for 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 fusion of any of 1001, 1017, 1018, and 1019 of the present invention or a mutant polypeptide of 1020 of the present invention. [The present invention 1036] A method for reducing 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 of any of 1001, 1017, 1018, and 1019 of the present invention or a mutant polypeptide of 1020 of the present invention. [The present invention 1037] A method for reducing or preventing the progression of ectopic calcification of soft tissue in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polypeptide fusion of any of 1001, 1017, 1018, and 1019 of the present invention or a mutant polypeptide of 1020 of the present invention. [The present invention 1038] A method for 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 fusion of any of 1001, 1017, 1018, and 1019 of the present invention or a mutant polypeptide of 1020 of the present invention. [The present invention 1039] A method for treating, reversing, 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 of any of 1001, 1017, 1018, and 1019 of the present invention or a mutant polypeptide of 1020 of the present invention. [The present invention 1040] A method for reducing 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: A method comprising the step of administering to a subject a therapeutically effective amount of any of the polypeptide fusions of 1001, 1017, 1018, and 1019 of the present invention or the mutant polypeptide of 1020 of the present invention. [The present invention 1041] A method for reducing or preventing the progression of age-related atherosclerosis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any of the polypeptide fusions of 1001, 1017, 1018, and 1019 of the present invention or the mutant polypeptide of 1020 of the present invention. [The present invention 1042] The method of claim 1035, wherein the pathological calcification is selected from the group consisting of idiopathic infantile arterial calcification (IIAC) and atherosclerotic plaque calcification. [The present invention 1043] The method of claim 1036, wherein the pathological ossification is selected from the group consisting of ossification of the posterior longitudinal ligament (OPLL), hypophosphatemic rickets, and osteoarthritis. [The present invention 1044] The method of claim 1037, wherein the soft tissue calcification is selected from the group consisting of IIAC and osteoarthritis. [The present invention 1045] The method of claim 1037, 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. [The present invention 1046] 1. A method for increasing pyrophosphate (PPi) levels in a subject having a lower than normal PPi level, comprising: Administering to a subject a therapeutically effective amount of a polypeptide of any of the polypeptide fusions of the invention 1001, 1017, 1018, and 1019 or a mutant polypeptide of the invention 1020, wherein said administration increases the level of PPi in the subject to a normal level of at least 2 μM and maintains it at about the same level. A method comprising: [The present invention 1047] 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: Administering to a subject a therapeutically effective amount of any of the polypeptide fusions of 1001, 1017, 1018, and 1019 of the invention or the mutant polypeptide of 1020 of the invention, thereby reducing pathological calcification or ossification in the subject or preventing the progression of pathological calcification or ossification in the subject. A method comprising: [The present invention 1048] 1. A method of treating an ENPP1 deficiency manifested by a decrease in extracellular pyrophosphate (PPi) concentration in a subject in need thereof, comprising: administering to a subject a therapeutically effective amount of a polypeptide fusion of any of 1001, 1017, 1018, and 1019 of the present invention or a mutant polypeptide of 1020 of the present invention, thereby increasing the level of PPi in the subject; A method comprising: [The present invention 1049] Any of the methods of claims 1035 to 1041 and 1046 to 1048, 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 undergoes proteolytic processing to produce the ENPP1 polypeptide. [The present invention 1050] The method of the present invention 1049, wherein in the ENPP1 precursor protein, the signal peptide sequence is linked to the N-terminus of the ENPP1 polypeptide. [The present invention 1051] The method of claim 1049, 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. [The present invention 1052] The method of any of claims 1035 to 1041 and 1046 to 1048, wherein the polypeptide fusion or mutant polypeptide is administered to the subject on a short-term or long-term basis. [The present invention 1053] The method of any of claims 1035 to 1041 and 1046 to 1048, wherein the polypeptide fusion or mutant polypeptide is administered locally, regionally, parenterally, or systemically to the subject. [The present invention 1054] Any of the methods of claims 1035 to 1041 and 1046 to 1048, wherein the polypeptide fusion or mutant 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, ocular, pulmonary, and topical. [The present invention 1055] The method of any of claims 1035 to 1041 and 1046 to 1048, 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. [The present invention 1056] The method according to any one of claims 1035 to 1041 and 1046 to 1048, wherein the subject is a mammal. [The present invention 1057] The method of claim 1056, wherein the mammal is a human. [The present invention 1058] An ENPP1 mutant polypeptide comprising one or more amino acid substitutions relative to SEQ ID NO:7, comprising an amino acid substitution at position 256 relative to SEQ ID NO:7. [The present invention 1059] 1058. An ENPP1 mutant polypeptide of the present invention, wherein the amino acid sequence of the ENPP1 mutant polypeptide is at least 90% identical to amino acids 23 to 849 of SEQ ID NO:7. [The present invention 1060] ENPP1 mutant polypeptide comprising amino acids 23 to 849 of SEQ ID NO:7, There are 10 or fewer amino acid substitutions relative to amino acids 23-849 of SEQ ID NO:7, containing an amino acid substitution at position 256 relative to SEQ ID NO:7, ENPP1 mutant polypeptide. [The present invention 1061] 1058 to 1060. The ENPP1 mutant polypeptide of any of claims 1058 to 1060, wherein the amino acid substitution is a substitution of isoleucine (I) with threonine (T) at position 256 relative to SEQ ID NO:7. [The present invention 1062] 1058 to 1060. The ENPP1 mutant polypeptide of any of claims 1058 to 1060, wherein the amino acid substitution is a substitution of isoleucine (I) with serine (S) at position 256 relative to SEQ ID NO:7. [The present invention 1063] 1. An ENPP1 variant polypeptide comprising an amino acid sequence that is at least 90% identical to amino acids 23 to 849 of SEQ ID NO:7, containing the mutation I256T with respect to SEQ ID NO:7, Further comprising a mutation selected from the group consisting of S766N, P534N, V536T, P554L, R545T, and E592N with respect to SEQ ID NO:7; ENPP1 mutant polypeptide. [The present invention 1064] 1063. An ENPP1 mutant polypeptide of the present invention, comprising at least one amino acid substitution selected from the group consisting of S766N, P534N / V536T, P554L / R545T, and E592N relative to SEQ ID NO:7. [The present invention 1065] 1063. An ENPP1 mutant polypeptide of the invention comprising the amino acid substitution V29N. [The present invention 1066] Any of ENPP1 mutant polypeptides of the present invention, 1058 to 1061, comprising the amino acid sequence of SEQ ID NO:11. [The present invention 1067] An ENPP1 mutant polypeptide fusion comprising any one of the ENPP1 mutant polypeptides of the present invention 1058 to 1066 and a heterologous protein. [The present invention 1068] The ENPP1 mutant polypeptide fusion of the present invention 1067, wherein the heterologous protein is an FcRn binding domain. [The present invention 1069] The ENPP1 mutant polypeptide fusion of any one of 1067 to 1068, wherein the heterologous protein is located on the carboxy-terminal side of the ENPP1 mutant polypeptide of the fusion. [The present invention 1070] An ENPP1 mutant polypeptide fusion of any of claims 1067 to 1068, wherein the heterologous protein is located on the amino terminal side of the ENPP1 mutant polypeptide of the fusion. [The present invention 1071] The ENPP1 mutant polypeptide fusion of any one of 1068 to 1070 of the present invention, wherein the FcRn-binding domain is an albumin polypeptide. [The present invention 1072] The ENPP1 mutant polypeptide fusion of any one of 1068 to 1070 of the present invention, wherein the FcRn binding domain is the Fc portion of an immunoglobulin molecule. [The present invention 1073] The ENPP1 mutant polypeptide fusion of the present invention 1072, wherein the immunoglobulin molecule is IgG1. [The present invention 1074] An ENPP1 mutant polypeptide fusion of any of claims 1068 to 1073, wherein the FcRn-binding domain comprises one or more amino acid substitutions relative to the wild-type FcRn-binding domain. [The present invention 1075] An ENPP1 mutant polypeptide fusion of any of 1068 to 1070 and 1072 to 1074 of the present invention, wherein the FcRn binding domain is the Fc portion of a human IgG1 molecule and contains the following amino acid substitutions relative to SEQ ID NO:7: M883Y, S885T, and T887E, respectively. [The present invention 1076] ENPP1 mutant polypeptide fusions of any of 1068-1070 and 1072-1075 of the present invention, each comprising one or more of the following substitutions with respect to SEQ ID NO:7: S885N, S766N, M883Y / S885T / T887E, P534N / V536T / H1064K / N1065F, P554L / R545T, S766N / H1064K / N1065F, E592N / H1064K / N1065F, or P534N / V536T / M883Y / S885T / T887E. [The present invention 1077] An ENPP1 mutant polypeptide fusion of any of 1068-1070 and 1072-1076 of the present invention, comprising an S885N mutation relative to SEQ ID NO:7. [The present invention 1078] An ENPP1 mutant polypeptide fusion of any of 1068-1070 and 1072-1077 of the present invention, comprising an S766N mutation relative to SEQ ID NO:7. [The present invention 1079] An ENPP1 mutant polypeptide fusion of any of 1068-1070 and 1072-1078 of the present invention, comprising the mutations M883Y, S885T, and T887E relative to SEQ ID NO:7. [The present invention 1080] An ENPP1 mutant polypeptide fusion of any of 1068-1070 and 1072-1079 of the present invention, comprising the mutations P534N, V536T, H1064K, and N1065F relative to SEQ ID NO:7. [The present invention 1081] An ENPP1 mutant polypeptide fusion of any of 1068-1070 and 1072-1080 of the present invention, comprising the mutations P554L and R545T with respect to SEQ ID NO:7. [The present invention 1082] An ENPP1 mutant polypeptide fusion of any of 1068-1070 and 1072-1081 of the present invention, comprising the mutations S766N, H1064K, and N1065F relative to SEQ ID NO:7. [The present invention 1083] An ENPP1 mutant polypeptide fusion of any of 1068-1070 and 1072-1082 of the present invention, comprising the mutations E592N, H1064K, and N1065F relative to SEQ ID NO:7. [The present invention 1084] An ENPP1 mutant polypeptide fusion of any of 1068-1070 and 1072-1083 of the present invention, comprising the mutations P534N, V536T, M883Y, S885T, and T887E relative to SEQ ID NO:7. [The present invention 1085] An ENPP1 mutant polypeptide fusion of any of claims 1068 to 1070 and 1072 to 1084, 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. [The present invention 1086] An ENPP1 mutant polypeptide fusion of any of 1068 to 1070 and 1072 to 1085 of the present invention, wherein the Fc region includes at least one mutation selected from the group consisting of S885N, M883Y, M883Y / S885T / T887E, and H1064K / N1065F relative to SEQ ID NO:7. [The present invention 1087] An ENPP1 mutant polypeptide fusion of any of 1068 to 1070 and 1072 to 1086 of the present invention or an ENPP1 mutant polypeptide of any of 1058 to 1065 of the present invention, comprising at least one mutation selected from the group consisting of C25N, K27T, and V29N relative to SEQ ID NO:7. [The present invention 1088] An ENPP1 mutant polypeptide fusion of any of 1068 to 1070 and 1072 to 1087 of the present invention or an ENPP1 mutant polypeptide of any of 1058 to 1065 and 1085 of the present invention, comprising at least one mutation selected from the group consisting of C25N / K27T and V29N relative to SEQ ID NO:7. [The present invention 1089] An ENPP1 mutant polypeptide fusion of any one of 1068 to 1070 and 1072 to 1088 of the present invention or an ENPP1 mutant polypeptide of any one of 1058 to 1065 and 1087 to 1088 of the present invention, comprising one mutation selected from the group consisting of K369N and I371T with respect to SEQ ID NO:7. [The present invention 1090] An ENPP1 mutant polypeptide fusion of any one of 1068 to 1070 and 1072 to 1089 of the present invention or an ENPP1 mutant polypeptide of any one of 1058 to 1065 and 1087 to 1089 of the present invention, comprising the mutation K369N / I371T relative to SEQ ID NO:7. [The present invention 1091] An ENPP1 mutant polypeptide fusion of any of 1068 to 1070 and 1072 to 1090 of the present invention or an ENPP1 mutant polypeptide of any of 1058 to 1065 and 1087 to 1090 of the present invention, comprising at least one mutation selected from the group consisting of P534N, V536T, R545T, P554L, E592N, R741D, and S766N with respect to SEQ ID NO:7. [The present invention 1092] An ENPP1 mutant polypeptide fusion of any of claims 1068 to 1070 and 1072 to 1091 of the present invention or an ENPP1 mutant polypeptide of any of claims 1058 to 1065 and 1087 to 1091 of the present invention, comprising at least one mutation selected from the group consisting of P534N / V536T, P554L / R545T, E592N, E592N / R741D, and S766N with respect to SEQ ID NO:7. [The present invention 1093] SEQ ID C25N, K27T, V29N, C25N / K27T, K369N, I371T, K369N / I371T, P534N, V536T, R545T, P554L, E592N regarding NO:7 , R741D, S766N, P534N / V536T, P554L / R545T, E592N / R741D, E864N, L866T, E864N / L866T, M883Y, S885N, S8 An ENPP1 mutant polypeptide fusion of any of 1068 to 1070 and 1072 to 1092 of the present invention or an ENPP1 mutant polypeptide of any of 1058 to 1065 and 1087 to 1092 of the present invention, comprising at least one mutation selected from the group consisting of 85T, T887E, H1064K, N1065F, M883Y / S885T / T887E, and H1064K / N1065F. [The present invention 1094] An ENPP1 mutant polypeptide fusion of any of claims 1068 to 1070 and 1072 to 1093 of the present invention or an ENPP1 mutant polypeptide of any of claims 1058 to 1065 and 1087 to 1093 of the present invention, 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. [The present invention 1095] An ENPP1 mutant polypeptide fusion of any one of 1068 to 1070 and 1072 to 1094 of the present invention or an ENPP1 mutant polypeptide of any one of 1058 to 1065 and 1087 to 1094 of the present invention, comprising at least one mutation selected from the group consisting of S766N, P534N / Y536T, P554L / R545T, and E592N relative to SEQ ID NO:7. [The present invention 1096] An ENPP1 mutant polypeptide fusion of any one of claims 1068 to 1070 and 1072 to 1095 of the present invention or an ENPP1 mutant polypeptide of any one of claims 1058 to 1065 and 1087 to 1095 of the present invention, 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. [The present invention 1097] An ENPP1 mutant polypeptide fusion of any of 1068-1070 and 1072-1096 of the present invention, comprising the mutations I256T, M883Y, S885T, and T887E relative to SEQ ID NO:7. [The present invention 1098] An ENPP1 mutant polypeptide fusion of any of 1068-1070 and 1072-1096 of the present invention, comprising the mutations I256T, P534N, V536T, M883Y, S885T, and T887E relative to SEQ ID NO:7. [This invention 1099] An ENPP1 mutant polypeptide fusion of any of 1068-1070 and 1072-1096 of the present invention, comprising the mutations I256T, E592N, H1064K, and N1065F relative to SEQ ID NO:7. [The present invention 1100] A fusion of any of 1067 to 1099 of the present invention, comprising a linker amino acid sequence. [The present invention 1101] The fusion of the present invention 1100, wherein a linker amino acid sequence connects the ENPP1 mutant polypeptide portion of the fusion and the heterologous protein. [The present invention 1102] The fusion of any one of claims 1100 to 1101, wherein the linker amino acid sequence comprises SEQ ID NO:8 or SEQ ID NO:9. [The present invention 1103] A nucleic acid encoding any one of the ENPP1 mutant polypeptides of the present invention as set forth in 1058 to 1066 or a fusion thereof as set forth in any one of the ENPP1 mutant polypeptides of the present invention as set forth in 1067 to 1102. [The present invention 1104] A vector comprising the nucleic acid of the present invention. [The present invention 1105] An expression vector comprising the nucleic acid of the present invention. [The present invention 1106] A cell or a plurality of cells, each comprising a nucleic acid of the invention 1103, a vector of the invention 1104, and / or an expression vector of the invention 1105. [The present invention 1107] The cell or cells of the invention 1106, which are CHO cells and / or NS0 cells. [The present invention 1108] The cell or cells of the invention 1107, wherein the CHO cells are stably transfected with human ST6 beta-galactoside alpha-2,6-sialyltransferase. [The present invention 1109] A method for producing an ENPP1 mutant polypeptide or fusion, comprising the step of culturing a cell or a plurality of cells according to any one of claims 1106 to 1108 of the present invention under conditions suitable for expression of the ENPP1 mutant polypeptide or fusion by the cell or the plurality of cells. [The present invention 1110] The method of claim 1109, wherein said cells are cultured in a medium supplemented with sialic acid and / or N-acetylmannosamine. [The present invention 1111] The method of any of claims 1109 to 1110, further comprising the step of purifying the ENPP1 mutant polypeptide or the fusion from the cell, the plurality of cells, or the medium in which the cell or the plurality of cells has been cultured. [The present invention 1112] 1111. An ENPP1 mutant polypeptide or fusion purified by the method of the present invention. [The present invention 1113] (i) any one of ENPP1 mutant polypeptides 1058 to 1066, 1087 to 1096 and 1112 of the present invention, and / or any one of ENPP1 mutant polypeptides 1067 to 1102 and 1112 of the present invention fused thereto; and (ii) dissimilar parts; A conjugate comprising: [The present invention 1114] The conjugate of the present invention 1113, wherein the heterologous moiety is polyethylene glycol. [The present invention 1115] A pharmaceutical composition comprising any one of the ENPP1 mutant polypeptides of the present invention 1058-1066, 1087-1096, and 1112, any one of the fusions of the present invention 1067-1102 and 1112, any one of the nucleic acid of the present invention 1103, any one of the vectors of the present invention 1104, any one of the expression vectors of the present invention 1105, and / or any one of the conjugates of the present invention 1113-1114, and a pharma- ceutically acceptable carrier. [The present invention 1116] 1. A method for reducing or preventing the progression of pathological calcification in a subject in need thereof, comprising: (a) any one of the ENPP1 mutant polypeptides of the present invention, 1058 to 1066, 1087 to 1096, and 1112; (b) a fusion of any of 1067 to 1102 and 1112 of the present invention; (c) any one of the conjugates 1113 to 1114 of the present invention; and / or (d) Pharmaceutical Compositions of the Invention to the subject in a therapeutically effective amount, thereby reducing or preventing the progression of pathological calcification in the subject. A method comprising: [The present invention 1117] 1. A method for reducing or preventing the progression of pathological ossification in a subject in need thereof, comprising: (a) any one of the ENPP1 mutant polypeptides of the present invention, 1058 to 1066, 1087 to 1096, and 1112; (b) a fusion of any of 1067 to 1102 and 1112 of the present invention; (c) any one of the conjugates 1113 to 1114 of the present invention; and / or (d) Pharmaceutical Compositions of the Invention to the subject in a therapeutically effective amount, thereby reducing or preventing the progression of pathological ossification in the subject. A method comprising: [The present invention 1118] 1. A method for reducing or preventing the progression of ectopic calcification of soft tissue in a subject in need thereof, comprising: (a) any one of the ENPP1 mutant polypeptides of the present invention, 1058 to 1066, 1087 to 1096, and 1112; (b) a fusion of any of 1067 to 1102 and 1112 of the present invention; (c) any one of the conjugates 1113 to 1114 of the present invention; and / or (d) Pharmaceutical Compositions of the Invention to the subject in a therapeutically effective amount, thereby reducing or preventing the progression of ectopic soft tissue calcification in the subject. A method comprising: [The present invention 1119] 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: (a) any one of the ENPP1 mutant polypeptides of the present invention, 1058 to 1066, 1087 to 1096, and 1112; (b) a fusion of any of 1067 to 1102 and 1112 of the present invention; (c) any one of the conjugates 1113 to 1114 of the present invention; and / or (d) Pharmaceutical Compositions of the Invention to the subject in a therapeutically effective amount, thereby reducing, reversing, or preventing ossification of the posterior longitudinal ligament (OPLL) in the subject. A method comprising: [The present invention 1120] 1. A method of treating, reversing, or preventing the progression of hypophosphatemic rickets in a subject in need thereof, comprising: (a) any one of the ENPP1 mutant polypeptides of the present invention, 1058 to 1066, 1087 to 1096, and 1112; (b) a fusion of any of 1067 to 1102 and 1112 of the present invention; (c) any one of the conjugates 1113 to 1114 of the present invention; and / or (d) Pharmaceutical Compositions of the Invention to the subject in a therapeutically effective amount, thereby reducing, reversing, or preventing the progression of hypophosphatemic rickets in the subject. A method comprising: [The present invention 1121] A method for reducing 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: (a) any one of the ENPP1 mutant polypeptides of the present invention, 1058 to 1066, 1087 to 1096, and 1112; (b) a fusion of any of 1067 to 1102 and 1112 of the present invention; (c) any one of the conjugates 1113 to 1114 of the present invention; and / or (d) Pharmaceutical Compositions of the Invention to the subject in a therapeutically effective amount, thereby reducing or preventing the progression of the disease. A method comprising: [The present invention 1122] 1. A method for reducing or preventing the progression of age-related arteriosclerosis in a subject in need thereof, comprising: (a) any one of the ENPP1 mutant polypeptides of the present invention, 1058 to 1066, 1087 to 1096, and 1112; (b) a fusion of any of 1067 to 1102 and 1112 of the present invention; (c) any one of the conjugates 1113 to 1114 of the present invention; and / or (d) Pharmaceutical Compositions of the Invention administering to a subject a therapeutically effective amount of A method comprising: [The present invention 1123] 1. A method for increasing pyrophosphate (PPi) levels in a subject having a lower than normal PPi level, comprising: (a) any one of the ENPP1 mutant polypeptides of the present invention, 1058 to 1066, 1087 to 1096, and 1112; (b) a fusion of any of 1067 to 1102 and 1112 of the present invention; (c) any one of the conjugates 1113 to 1114 of the present invention; and / or (d) Pharmaceutical Compositions of the Invention to a subject in a therapeutically effective amount, wherein said administration increases the level of PPi in the subject to a normal level of at least 2 μM and maintains it at about the same level. A method comprising: [The present invention 1124] 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: (a) any one of the ENPP1 mutant polypeptides of the present invention, 1058 to 1066, 1087 to 1096, and 1112; (b) a fusion of any of 1067 to 1102 and 1112 of the present invention; (c) any one of the conjugates 1113 to 1114 of the present invention; and / or (d) Pharmaceutical Compositions of the Invention to a subject in a therapeutically effective amount, thereby reducing pathological calcification or ossification in the subject or preventing the progression of pathological calcification or ossification in the subject. A method comprising: [The present invention 1125] 1. A method of treating an ENPP1 deficiency manifested by a decrease in extracellular pyrophosphate (PPi) concentration in a subject in need thereof, comprising: (a) any one of the ENPP1 mutant polypeptides of the present invention, 1058 to 1066, 1087 to 1096, and 1112; (b) a fusion of any of 1067 to 1102 and 1112 of the present invention; (c) any one of the conjugates 1113 to 1114 of the present invention; and / or (d) Pharmaceutical Compositions of the Invention to a subject in a therapeutically effective amount, thereby increasing PPi levels in the subject. A method comprising: [The present invention 1126] The method of any of claims 1116 to 1125, wherein the mutant polypeptide, fusion, conjugate, or pharmaceutical composition is administered to the subject acutely or chronically. [The present invention 1127] The method of any of claims 1116 to 1126, wherein the mutant polypeptide, fusion, conjugate, or pharmaceutical composition is administered locally, regionally, parenterally, or systemically to the subject. [The present invention 1128] The method according to any one of claims 1116 to 1127, wherein the subject is a human. [Brief description of the drawings]

[0020] The following detailed description of exemplary embodiments of the present disclosure will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the present disclosure, exemplary embodiments are shown in the drawings. It should be understood, however, that the present disclosure is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings. (FIG. 1) shows an ENPP1 polypeptide (SEQ ID NO:7) contemplated in the present disclosure. Point mutations are identified with reference to SEQ ID NO:7, which may also be referred to as the "parent compound," "parent polypeptide," 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. (Figure 2A) Figure 2A shows the domain structure of parent construct #770. The two somatomedin B domains, catalytic domain and endonuclease domain of human ENPP1 were fused to the signal sequence of human ENPP7 at the N-terminus and to the Fc domain of human IgG1 at the C-terminus. Figure 2B shows the pharmacokinetic analysis of parent construct #770. After an initial rise in plasma activity 17 hours after subcutaneous injection, the plasma activity drops sharply with a calculated half-life of 34 hours. Figure 2C shows the non-limiting effect of the additional N-glycosylation consensus sequence incorporated into parent construct #770. Pharmacokinetic plots of AUC (bars, left y-axis) and half-life in hours (line, right y-axis) show that clone 7 with the I256T mutation has a significant increase in both AUC and half-life compared to parent construct #770. FIG. 2D shows digested peptide fragments showing abundant sialoglycopeptide peaks (bottom) associated with ENPP1-Fc clone 19 but not with parental construct #770. Mass spectrum of TIFF2024178422000001.tif5128 is shown. Figure 2E shows the finding that Michaelis-Menten kinetics assays show that the rates of the enzyme at various substrate concentrations are nearly identical at the two different enzyme concentrations in the two I256T-containing clones (clone 17 in yellow and clone 19 in red) compared to the parental construct #770 in black. (Figure 2B) See legend to Figure 2A. (Figure 2C) See legend to Figure 2A. (Figure 2D-1) See the legend to Figure 2A. (Figure 2D-2) See the legend to Figure 2A. (Figure 2E) See legend to Figure 2A. (FIG. 3) 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). (Figure 4) shows the in vivo pharmacokinetic data of construct #981, measured using a pNP-TNP assay to record the 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 5) Construct #1014, 1,3,4-O-Bu 3 Selected in vivo pharmacokinetic data for construct #1014 (indicated in the graph as "1014A") and construct #981 prepared in CHO cells grown in culture medium supplemented with ManNAc are shown. The half-life of the constructs can be derived from Equation 1 described elsewhere herein. (Figure 6) Shows 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). (FIG. 7A) Specific domains of human ENPP1 with known loss-of-function mutations that cause the human disease "Generalized Arterial Calcification of Infancy" (GACI) are shown. In certain embodiments, glycosylation sites are not introduced near the regions with known loss-of-function mutations that cause GACI (as shown in FIG. 7A). (Figure 7B) Crystal structure of ENPP1 with residues with known loss-of-function mutations causing GACI highlighted (marked with *). The residue in (B) is located in the catalytic domain and corresponds to T238A. As in Figure 6, a calcium atom (C); two zinc atoms; (D) an ATP molecule (E). (FIG. 8A) FIGS. 8A-8D show selected results from a high-throughput TMP-pNP (thymidine monophosphate-p-nitrophenyl) assay of the 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 polypeptide, i.e., 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 8B) See legend to Figure 8A. (Figure 8C) See legend to Figure 8A. (Figure 8D) See legend to Figure 8A. (FIG. 9A) FIG. 9A is a ribbon diagram showing the Fc domain of human IgG1. This domain is fused to the C-terminal portion of ENPP1 to enhance efficiency. Mutations were introduced in the Fc domain to enhance pH-dependent recycling by FcRn. (A) = sites that eliminate acidity-dependent binding. (B) = sites that enhance binding. (C) = cysteine ​​disulfide bonds. Magenta = known glycosylation sites. FIG. 9B shows mutations in the Fc domain of human IgG1 that are known to enhance pH-dependent recycling by FcRn. (Figure 9B) See legend to Figure 9A. (FIG. 10) includes graphs and tables showing the effect of glycosylation on the PK (half-life, hours) and bioavailability of the ENPP1 polypeptide. For all mutations, PK was comparable to that of construct #CC07 (770B), except for the I256T mutation in construct #922. This mutation (located in the insertion loop of the catalytic domain) was modeled after the equivalent glycosylation site in ENPP3. Furthermore, construct #951 showed similar PK values ​​to that of construct #CC07, but construct #951 grown in a cell line stably transfected with ST6GAL1 (construct #951-ST) showed improved PK and bioavailability. Bioavailability was improved for construct #922 containing the I256T mutation. Construct #930 had a half-life similar 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 4, 5 and 13 and calculated using Equation 1 are shown in the Table. (FIG. 11) includes 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 4, 5, and 13 and calculated using Equation 1 are shown in the table. (FIG. 12) includes graphs and tables showing the effect of glycosylation and M883Y / S885T / T887E Fc mutations on 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, #1028, and #1101 showed increased values ​​of both PK and AUC when grown in cells stably transfected with ST6GAL1. Construct #1101 had improved PK and AUC values. PK and bioavailability data measured as shown in FIGS. 4, 5, and 13 and calculated using Equation 1 are shown in the table. (FIG. 13) includes 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"). This 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 FIGS. 4, 5, and 13 and calculated using Equation 1 are shown in the table. (FIG. 14A) FIG. 14A shows the pharmacokinetic analysis of clones containing Fc-HR mutations (clones 9, 10, 11, 12 and 15) and Fc-MST mutations (clones 8, 13, 14, 16 and 17) compared to parental clone 770 and I256T-containing clone 7. The area under the curve (left y-axis) is represented by the bars and the half-life in hours (right y-axis) is represented by the lines. Clone 7 has only a slight increase compared to clones 16 and 17 (lines), but it has an almost equivocal AUC as a result of its high initial activity after injection (bars). FIG. 14B shows the bioavailability represented by the slope of the area under the curve. Clone 7 containing only the I256T mutation is initially very active in plasma but decays quickly, AUC in red. Clone 14-ST, which contains only the Fc MST mutation, is initially less active than clone 7, but has a longer half-life as indicated by the shallower slope (grey AUC). Clone 19-ST, which combines the two mutations into one clone, indicated by the yellow AUC, resulted in an enzyme with both higher initial activity and a longer half-life. Figure 14C shows the activity of the enzyme in unmodified CHO cells or in CHO cells overexpressing α-2,6-sialyltransferase (α-2,6-ST), or 1,3,4-O-Bu 3AUC (bars, left axis) and half-life in hours (lines, right axis) are shown for clones grown in CHO cells overexpressing α-2,6-ST combined with ManNAc supplementation. In all cases, the addition of α-2,6-ST (clones 1, 2, 9, 10, 14, 15, 17 and 18) increased the half-life and AUC of that clone. Clone 9 (left three arrows) was grown in CHO cells overexpressing α-2,6-ST and 1,3,4-O-Bu 3 Both CHO cells overexpressing α-2,6-ST combined with ManNAc supplementation showed increased AUC and half-life. Compared to clone 7, clone 19 (right three arrows) also showed increased AUC and half-life in 1,3,4-O-Bu, as well as clone 9. 3 The half-life and AUC were increased by ManNAc supplementation. Figure 14D shows that the sialic acid precursor 1,3,4-O-Bu was activated in CHO K1 cells alone or in human α-2,6-ST or α-2,6-ST in combination with the sialic acid precursor 1,3,4-O-Bu 3 Figure 14E shows the finding that anion exchange chromatography with pulsed amperometric detection (HPAE-PAD) of clone 9 grown in CHO K1 cells stably transfected with a combination of ManNAc shows a progressive increase in the percentage of N-acetylneuraminic acid content with each treatment. 3 Shown is the AUC pharmacokinetic analysis of parental construct #770 (designated 770) grown in CHO cells alone compared to clone 9 grown in CHO cells overexpressing α-2,6-ST in combination with ManNAc supplementation (designated 9(ST)A). Clone 9 contains the Fc-HN mutation that provides a small increase in half-life and AUC over parental clone 770. However, there is no significant difference in the AUC between clone 9 and CHO cells overexpressing α-2,6-ST or α-2,6-ST and 1,3,4-O-Bu. 3 When grown in combination with ManNAc supplementation, it shows a cumulatively large increase in AUC and half-life. (Figure 14B) See legend to Figure 14A. (Figure 14C) See legend to Figure 14A. (Figure 14D) See legend to Figure 14A. (Figure 14E) See legend to Figure 14A. (FIG. 15A) FIG. 15A shows the AUC pharmacokinetic analysis of Fc-MST-containing clone 14-ST compared to Fc-MST / I256T-containing clones 17-ST, 19-ST and 19-ST-A. The AUC of the Fc-MST-containing clones could be further enhanced by the I256T mutation, and 1,3,4-O-Bu 3 This can be further increased when grown in the presence of ManNAc precursors. Figure 15B shows the AUC pharmacokinetics of parental clone 770 compared to clone 19-ST-A. Clone 19, which contains both the Fc-MST and I256T mutations, was treated with 1,3,4-O-Bu in CHO cells overexpressing α-2,6-ST. 3 When grown with supplementation of ManNAc, bioavailability increases by approximately 18-fold. Figure 15C shows the finding that MALDI-TOF / TOF analysis for N-glycan profiling revealed a higher % sialic acid-containing glycans in clone 19-ST-A (99.2%) compared to parental clone 770 (78.4%), calculated based on structures containing at least one galactose for sialic acid transfer. Figure 15D shows the results of Enpp1. asj / asj Figure 1 shows the pharmacodynamic effects after a single dose of 0.3 mg / kg of either parental clone 770 (red squares) or optimized ENPP1-Fc clone 19-ST (red circles) as measured by the generation of PPi (left y-axis) in mice. Physiological levels of PPi in normal mice (shaded grey) are 1.5-2.5 um PPi, whereas Enpp1 asj / asjMice have nearly undetectable amounts. A single dose of clone 770 restores physiological levels of PPi, which return to baseline before 89 hours, while clone 19-ST is able to maintain near-physiological levels up to 263 hours. The error bars for PPi production appear to be much larger than those for the % activity of this enzyme (right y-axis) (compare red and black circles). (Figure 15B) See legend to Figure 15A. (Figure 15C) See legend to Figure 15A. (Figure 15D) See legend to Figure 15A. (FIG. 16A) FIGS. 16A-16B show domains of ENPP1 and selected mutations introduced into the parent polypeptide (SEQ ID NO:7). The figures identify the individual point mutations introduced into SEQ ID NO:7. Constructs stably transfected into CHO cells stably transfected with human α-2,6-ST are referenced with "ST". PK and bioavailability data determined as shown in FIGS. 4, 5 and 13 and calculated using Equation 1 are shown in the tables. (Figure 16B) See legend to Figure 16A. (FIG. 17) Shows the bioavailability (as area under the curve or AUC) of certain constructs of the disclosure, categorized by mutations in the signal sequence (N-terminal region) region. (FIG. 18) Shows the bioavailability (as area under the curve or AUC) of certain constructs of the disclosure, categorized by mutations in the endonuclease region. (FIG. 19) FIG. 19A includes images showing stable CHO cell subclones co-transfected with plasmid cDNA for both ENPP1-Fc and hST6GAL1, which were screened for expression of alpha-2,6-sialyltransferase by immunofluorescence of paraformaldehyde-fixed cells using rabbit anti-hST6GAl1 antibody (R&D Systems catalog number AF5924) followed by donkey polyclonal goat IgG Alexa Fluor594 (Abcam Ab150140). Characteristic Golgi localization was observed in these cell clones that were also positive by Western blot using the same antibody. FIG. 19B shows a representative Western blot using the same primary antibody as FIG. 19A, showing a single band of approximately 48 kD indicating expression of alpha-2,6-sialyltransferase of various intensities in some of the CHO cell subclones used in this study. The first lane is a non-transfected CHO cell lysate as a negative control. The next three lanes are three individual subclones of the unmodified parent plasmid 770, designated A, B, and C. The other lanes are a selection of subclones used in the literature for clones 1, 2, 10, 14, and 18. "X" represents a subclone of the construct that was not used further herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Detailed Description of the Invention The present disclosure 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 non-limiting 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 non-limiting 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 non-limiting aspect, sialylation of the fusion protein was enhanced by expressing ENPP1-Fc in a CHO cell line stably transfected with human ST6 beta-galactoside alpha-2,6-sialyltransferase (also known as ST6GAL1).

[0025] In yet another non-limiting aspect, the cell culture medium is provided with N-acetylglucosamine (1,3,4-O-mannosamine), a "high flux" precursor of sialic acid. 3 Sialic acid capping was enhanced by supplementation with sialic acid (also known as ManNAc).

[0026] In a particular embodiment, enhancing protein sialylation by expressing this bioagent in CHO cells stably transfected with human alpha-2,6-sialyltransferase improved 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 the biological material 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 biological exposure (AUC). In yet other embodiments, combining two or more methods described herein into a single construct dramatically increased the half-life and / or biological exposure (AUC).

[0027] In certain embodiments, the polypeptides of the present disclosure are more highly glycosylated than other ENPP1-Fc polypeptides in the art. In other embodiments, the polypeptides of the present disclosure 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 disclosure 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 polypeptide (construct #770) are altered such that the change is a "gain of function" change in the enzyme rate constant. In yet other embodiments, the kinetic properties of the parent polypeptide (construct #770) are not significantly altered by specific site-directed mutagenesis, and thus the resulting mutant enzyme has substantially the same enzyme rate constant as the parent polypeptide. In yet other embodiments, specific point mutations in the parent polypeptide introduce glycans at the mutated residues, increasing the biological exposure of the mutant polypeptide. In yet other non-limiting embodiments, the increased biological exposure of the mutant polypeptide is due to increased biological absorption and / or circulation of the mutant polypeptide.

[0028] In certain embodiments, any ENPP1 mutant polypeptide described herein retains ENPP1 catalytic activity compared to a soluble ENPP1 polypeptide comprising or consisting of amino acids 23-849 of SEQ ID NO:7. In certain embodiments, any ENPP1 mutant polypeptide described herein retains at least about 30% (e.g., at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8% or 100%) of the catalytic activity of a soluble ENPP1 polypeptide comprising or consisting of amino acids 23-849 of SEQ ID NO:7. In certain embodiments, any one ENPP1 mutant polypeptide described herein has a higher catalytic activity than a soluble ENPP1 polypeptide comprising or consisting of amino acids 23-849 of SEQ ID NO:7.

[0029] In certain aspects, any one of the ENPP1 mutant polypeptides described herein has improved pharmacokinetic and / or bioavailability properties in a mammal compared to a soluble ENPP1 polypeptide comprising or consisting of amino acids 23-849 of SEQ ID NO:7. In certain embodiments, any ENPP1 mutant polypeptide has a circulating half-life in a mammal that is at least 30% (e.g., at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 9%7, 98%, 99%, 99.5%, 99.8%, 100%, 120%, 140%, 160%, 180%, 200%, 250%, 300%, 350%, 400%, 450%, 500% or more) of the circulating half-life of a soluble ENPP1 polypeptide comprising or consisting of amino acids 23 to 849 of SEQ ID NO:7. In certain aspects, any one of the ENPP1 mutant polypeptides described herein has an AUC greater than that of a soluble ENPP1 polypeptide comprising or consisting of amino acids 23 to 849 of SEQ ID NO:7.

[0030] In certain embodiments, the in vivo half-life of the ENPP1-Fc polypeptide of the present disclosure 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 the ENPP-1 polypeptides reported in the art. In other embodiments, the polypeptide of the present disclosure is administered to the 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 disclosure is administered to the 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 disclosure results in better patient compliance and / or higher efficacy compared to other ENPP1-Fc polypeptides in the art.

[0031] In certain embodiments, the ENPP1-Fc polypeptide of the present disclosure 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 disclosure 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 disclosure can be used to treat ENPP1 deficiency manifested by a decrease in extracellular PPi concentration in a subject.

[0032] In certain embodiments, the steady state level of plasma PPi achieved following administration of the first dose of a construct of the present disclosure is maintained for a period of at least 2 days, at least 4 days, at least 1 week, or at least 1 month.

[0033] In certain embodiments, a second dose of the construct of the present disclosure is administered to the subject after an appropriate time interval of 2 days, 4 days, 1 week or 1 month so 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 disclosure.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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 disclosure belongs. Generally, the nomenclature and experimental procedures in veterinary medicine, pharmaceutical science, separation science and organic chemistry used herein are those well known and widely used in the art. It should be understood that the order of steps or order of taking certain actions is not important so long as the present teachings are 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 individually incorporated by reference.

[0039] 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.

[0040] 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.

[0041] 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."

[0042] 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.

[0043] "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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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 (t 1 ~t 2 ), the AUC is as follows: TIFF2024178422000002.tif9128, where C 1 and C 2 is the time interval (t 1 and t 2 ) is the average concentration at

[0050] The log-trapezoidal method uses logarithmic interpolation between data points to calculate the AUC. This method is more accurate when the concentration decreases because the drug elimination is exponential (making it linear on a log scale). 1 ~t 2 ), the AUC is as follows: TIFF2024178422000003.tif12128(C 1 >C 2 It can be calculated as follows (assuming

[0051] The term "bioavailability", as used herein, refers to 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 or enters the systemic circulation after administration by any route. 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 the bioavailability of a drug or protein 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 when their plasma concentration curves are essentially superimposable. 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, which can be subdivided into the categories of dosage form effects, membrane effects, and administration site effects. Half-life and AUC provide information about the bioavailability of drugs and biological substances.

[0052] 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.

[0053] As used herein, a "construct" of the present disclosure refers to a fusion polypeptide comprising an ENPP1 polypeptide or a fragment or site-directed mutant thereof.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] As used herein, the term "ENPP" or "NPP" refers to ectonucleotide pyrophosphatase / phosphodiesterase.

[0058] 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.

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

[0069] Exemplary functional derivatives include amino acid sequences with one or more amino acid substitutions, deletions, or additions, so long as the biological activity of the protein is preserved. The substituting amino acid desirably has physicochemical properties similar to those of the amino acid being 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, functionally equivalent polypeptides of the present disclosure 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.

[0070] "Gene transfer" and "gene delivery" refer to a method or system for the reliable insertion of specific nucleic acid sequences into target cells.

[0071] An "inducible" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes that gene product to be produced in a cell only when an inducer corresponding to that promoter is present in the cell.

[0072] 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) contemplated in the present disclosure 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 a clearance curve of an ENPP1-Fc fusion protein is generated 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.

[0073] "Instruction materials," as that term is used herein, include publications, records, diagrams, or any other medium of expression that can be used to communicate the usefulness of the disclosed nucleic acids, peptides and / or compounds in the kit in identifying or alleviating or treating the various diseases or disorders referred to herein.

[0074] "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.

[0075] "Isolated nucleic acid" refers to a nucleic acid segment or fragment that is separated from sequences that naturally flank it, i.e., a DNA fragment that has been removed from the sequences that normally flank it, i.e., the sequences that flank it in the genome in which it naturally occurs. The term also applies to nucleic acids that have been substantially purified from other components that naturally accompany it, i.e., RNA or DNA or proteins that naturally accompany it in the cell. Thus, the term includes recombinant DNA that exists, for example, in a vector, an autonomously replicating plasmid or virus, or in the genomic DNA of a prokaryote or eukaryote, or as a separate molecule independent of other sequences (i.e., as a cDNA or as a genomic or cDNA fragment generated by PCR or restriction enzyme digestion). It also includes recombinant DNA that is part of a hybrid gene that encodes an additional polypeptide sequence.

[0076] An "oligonucleotide" or "polynucleotide" is a nucleic acid that ranges from at least 2, 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.

[0077] The term "operably linked" refers to a functional link between a regulatory sequence and a heterologous nucleic acid sequence that results in the expression of the latter. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous, and where necessary, two protein coding regions are connected in the same reading frame.

[0078] As used herein, the terms "patient," "individual," or "subject" refer to a human.

[0079] As used herein, the term "pharmaceutical composition" or "composition" refers to a mixture of at least one compound useful in the present disclosure 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.

[0080] 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.

[0081] 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 disclosure 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 disclosure, 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 disclosure and are physiologically acceptable to the patient. "Pharmaceutically acceptable carrier" may further include pharmaceutically acceptable salts of the compound useful in the present disclosure. Other additional ingredients that may be included in pharmaceutical compositions used in practicing the present disclosure are known in the art and described, for example, in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, Pa.), which is incorporated herein by reference.

[0082] 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.

[0083] 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.).

[0084] 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.

[0085] As used herein, the term "PPi" refers to pyrophosphate.

[0086] 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.

[0087] The term "promoter" as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell or introduced synthetic machinery required to initiate the individual transcription of a polynucleotide sequence.

[0088] As used herein, the term "promoter / regulatory sequence" refers to a nucleic acid sequence required for expression of a gene product operably linked to the promoter / regulatory sequence. In some instances, this sequence may be a core promoter sequence, and in other instances, this sequence may also include an enhancer sequence and other regulatory elements required for expression of the gene product. The promoter / regulatory sequence may, for example, be one that expresses the gene product in a tissue-specific manner.

[0089] The term "recombinant polypeptide," as used herein, is defined as a polypeptide produced through the use of recombinant DNA methods.

[0090] The term "recombinant DNA," as used herein, is defined as DNA that is produced by joining pieces of DNA from different sources.

[0091] "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.

[0092] 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) that is 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).

[0093] 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 native state. Non-limiting embodiments include 95% purity, 99% purity, 99.5% purity, 99.9% purity, and 100% purity.

[0094] A "tissue-specific" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specified by a gene, causes the product of that gene to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to that promoter.

[0095] The phrases "under transcriptional control" or "operably linked" as used herein mean that a promoter is positioned and oriented correctly relative to a polynucleotide to control the initiation of transcription and expression of that polynucleotide by RNA polymerase.

[0096] The terms "transfect" or "transformation" or "transduction" as used herein refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the original subject cell and its progeny.

[0097] 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 disclosure (alone or in combination with another agent), to a patient having a disease or disorder, a symptom of a disease or disorder, or the likelihood of developing a disease or disorder, or the application or administration of a therapeutic agent to a tissue or cell line isolated from the patient (e.g., for diagnostic or ex vivo applications), for the purpose of curing, relieving, alleviating, mitigating, altering, treating, ameliorating, improving, or affecting the disease or disorder, the symptom of the disease or disorder, or the likelihood of developing the disease or disorder. Such treatments may be personalized or modified based on knowledge obtained from the field of pharmacogenomics.

[0098] A "variant", as the term is used herein, is a nucleic acid sequence or peptide sequence that differs in sequence from a reference nucleic acid sequence or peptide sequence, respectively, but retains essential properties of the reference molecule. Changes in the sequence of a nucleic acid variant may not change the amino acid sequence of the peptide encoded by the reference nucleic acid, or may result in amino acid substitutions, additions, deletions, fusions, and truncations. Changes in the sequence of a peptide variant are typically limited or conservative, such that the sequences of the reference peptide and variant are overall very similar, and in many regions identical. The variant and reference peptide may differ in amino acid sequence by one or more substitutions, additions, or deletions in any combination. A variant of a nucleic acid or peptide may be naturally occurring, e.g., an allelic variant, or a variant that is not known to occur naturally. Non-naturally occurring variants of nucleic acids and peptides may be made by mutagenesis techniques or by direct synthesis.

[0099] 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. The 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.

[0100] As used herein, the term "virus" is defined as a particle consisting of nucleic acid (RNA or DNA) surrounded by a protein coat, with or without an outer lipid envelope, that is capable of transfecting its nucleic acid into a cell.

[0101] As used herein, the term "wild type" refers to a gene or gene product isolated from a naturally occurring source. A wild type gene is the most frequently observed in a population and is therefore the arbitrarily designed "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 may 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.

[0102] The following abbreviations are used herein: 1,3,4-O-Bu 3 ManNAc, N-acetylmannosamine; ST6GAL1, ST6 beta-galactoside alpha-2,6-sialyltransferase.

[0103] Ranges: Throughout this disclosure, various aspects of the disclosure 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 disclosure. 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.

[0104] Polypeptides In one aspect, the present disclosure provides an ENPP1-Fc polypeptide. The present disclosure contemplates that the polypeptide of the present disclosure can have one or more of the mutations described herein.

[0105] In another aspect, the disclosure provides an ENPP1 mutant polypeptide comprising at least one amino acid substitution at position 256 relative to SEQ ID NO:7. In certain embodiments, the amino acid substitution is a substitution of isoleucine (I) with threonine (T) at position 256 relative to SEQ ID NO:7. In certain embodiments, the amino acid substitution is a substitution of isoleucine (I) with serine (S) at position 256 relative to SEQ ID NO:7.

[0106] In certain embodiments, the ENPP1 mutant polypeptide comprises the catalytic domain of ENPP1. In certain embodiments, the ENPP1 mutant polypeptide comprises the endonuclease domain of ENPP1. In certain embodiments, the ENPP1 mutant polypeptide lacks the nuclease domain of ENPP1. In certain embodiments, the ENPP1 mutant polypeptide lacks the transmembrane domain of ENPP1. In certain embodiments, the ENPP1 mutant polypeptide lacks the intracellular domain of ENPP1. In certain embodiments, the ENPP1 mutant polypeptide lacks both the intracellular domain and the transmembrane domain of ENPP1. In certain embodiments, the ENPP1 mutant polypeptide lacks a signal sequence. In certain embodiments, the ENPP1 mutant polypeptide comprises an amino acid sequence that is at least about 90% (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to amino acids 23-849 of SEQ ID NO:7.

[0107] In another aspect, the disclosure provides an ENPP1 mutant polypeptide comprising an amino acid sequence at least about 90% (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to amino acids 23-849 of SEQ ID NO:7, the ENPP1 mutant polypeptide comprising an amino acid substitution at position 256 relative to SEQ ID NO:7. In certain embodiments, the amino acid substitution is I256T. In certain embodiments, the amino acid substitution is I256S.

[0108] In yet another aspect, the disclosure provides an ENPP1 mutant polypeptide comprising amino acids 23-849 of SEQ ID NO:7, wherein there are 10 or fewer (e.g., 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 or fewer) amino acid substitutions relative to amino acids 23-849 of SEQ ID NO:7. In certain embodiments, the ENPP1 mutant polypeptide comprises an amino acid substitution at position 256 relative to SEQ ID NO:7. In certain embodiments, the amino acid substitution is I256T. In certain embodiments, the amino acid substitution is I256S.

[0109] In certain embodiments, the ENPP1 polypeptide comprises at least one mutation within the signal sequence region shown in Figure 16A and / or Figure 16B.

[0110] In a particular embodiment, the polypeptide comprises the mutation I256T relative to SEQ ID NO:7.

[0111] In certain embodiments, the mutation is selected from the group consisting of C25N, K27T, and V29N relative to SEQ ID NO:7. In certain embodiments, the mutation is C25N relative to SEQ ID NO:7. In certain embodiments, the mutation is K27T relative to SEQ ID NO:7. In certain embodiments, the mutation is V29N relative 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 relative to SEQ ID NO:7.

[0112] In certain embodiments, the ENPP1 polypeptide comprises at least one mutation in the catalytic region depicted in Figure 16A and / or Figure 16B. In certain embodiments, the mutation is selected from the group consisting of I256T, K369N, and I371T with respect to SEQ ID NO:7. In certain embodiments, the mutation is I256Y 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 at least one mutation selected from the group consisting of the mutations I256T and K369N / I371T with respect to SEQ ID NO:7.

[0113] 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 7A, Figure 16A, Figure 16B, Figure 17, and / or Figure 18. 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.

[0114] In certain embodiments, the ENPP1 polypeptide comprises at least one mutation in the linker region shown in Figure 16A and / or Figure 16B. 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 ENPP1 polypeptide comprises at least the mutations E864N / 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.

[0115] 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. 7A, FIG. 16A, FIG. 16B, FIG. 17, and / or FIG. 18. 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.

[0116] In certain embodiments, the ENPP1 polypeptide is selected from the group consisting of C25N, K27T, V29N, C25N / K27T, I256T, 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.

[0117] 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.

[0118] 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.

[0119] 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.

[0120] 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.

[0121] 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.

[0122] 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.

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

[0124] 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.

[0125] In a particular embodiment, the polypeptide comprises a S885N mutation in the FcRn binding domain relative to SEQ ID NO:7.

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

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

[0128] 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.

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

[0130] 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.

[0131] 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.

[0132] 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.

[0133] In a particular embodiment, the polypeptide comprises the mutation I256T relative to SEQ ID NO:7.

[0134] In certain embodiments, the polypeptide comprises the mutations I256T, M883Y, S885T, and T887E relative to SEQ ID NO:7.

[0135] In certain embodiments, the polypeptide comprises the mutations V29N, I256T, P534N, V536T, M883Y, S885T, and T887E relative to SEQ ID NO:7.

[0136] In another aspect, the disclosure features an ENPP1 mutant polypeptide that includes an amino acid sequence that is at least about 90% (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to amino acid sequence 23-849 of SEQ ID NO:7, the ENPP1 mutant polypeptide including a mutation I256T relative to SEQ ID NO:7, and further including a mutation selected from the group consisting of S766N, P534N, V536T, P554L, R545T, and E592N relative to SEQ ID NO:7.

[0137] In certain embodiments, any mutant polypeptide described herein comprises at least one amino acid substitution selected from the group consisting of S766N, P534N / V536T, P554L / R545T, and E592N relative to SEQ ID NO:7.

[0138] In certain embodiments, any of the mutant polypeptides described herein comprises the amino acid substitution V29N.

[0139] In certain embodiments, the mutant polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO:11.

[0140] Also featured is an ENPP1 mutant polypeptide fusion comprising any of the ENPP1 mutant polypeptides described herein and a heterologous protein, such as an FcRn binding domain. In certain embodiments, the heterologous protein is located at the carboxy-terminal end of the ENPP1 mutant polypeptide portion of the fusion. In certain embodiments, the heterologous protein is located at the amino-terminal end of the ENPP1 mutant polypeptide portion of the fusion.

[0141] In certain embodiments of any of the fusions described herein, the FcRn-binding domain is an albumin polypeptide. In certain embodiments, the FcRn-binding domain is the Fc portion of an immunoglobulin molecule, such as an IgG1 immunoglobulin molecule.

[0142] In certain embodiments of any of the fusions described herein, the FcRn-binding domain comprises one or more amino acid substitutions relative to the wild-type FcRn-binding domain. In certain embodiments, the FcRn-binding domain is the Fc portion of a human IgG1 molecule, each of which comprises the following amino acid substitutions relative to SEQ ID NO:7: M883Y, S885T, and T887E (MST / YTE substitutions).

[0143] In certain embodiments, the fusions described herein each include one or more of the following substitutions relative to SEQ ID NO:7: S885N, S766N, M883Y / S885T / T887E, P534N / V536T / H1064K / N1065F, P554L / R545T, S766N / H1064K / N1065F, E592N / H1064K / N1065F, or P534N / V536T / M883Y / S885T / T887E.

[0144] In certain embodiments of any of the ENPP1 mutant polypeptides or fusions described herein, the ENPP1 mutant polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO:11.

[0145] In certain embodiments of any of the ENPP1 mutant polypeptides or fusions described herein, the ENPP1 mutant polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO:12.

[0146] In certain embodiments, any fusion described herein comprises (a) an ENPP1 mutant polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO:11 or SEQ ID NO:12, (b) a variant human IgG1 Fc region carboxy-terminal to the ENPP1 mutant polypeptide, e.g., the amino acid sequence set forth in SEQ ID NO:14, and (c) a linker amino acid sequence separating (a) and (b), wherein the linker sequence is LIN (SEQ ID NO:8) or GGGGS (SEQ ID NO:9).

[0147] Conjugates of any one of the ENPP1 mutant polypeptides or ENPP1 mutant polypeptide fusions described herein and a heterologous moiety, such as, but not limited to, a small molecule, are also features of the present invention. In certain embodiments, the heterologous moiety increases or further increases the pharmacokinetics and / or bioavailability of the mutant polypeptide in a mammal. In certain embodiments, the heterologous moiety is an oligomer of ethylene glycol and / or propylene glycol, such as, but not limited to, polyethylene glycol (PEG) and / or polypropylene glycol (PPG).

[0148] In certain embodiments, any ENPP1 mutant polypeptide fusion or conjugate described herein comprises an S885N mutation relative to SEQ ID NO:7.

[0149] In certain embodiments, any ENPP1 mutant polypeptide, fusion or conjugate described herein comprises an S766N mutation relative to SEQ ID NO:7.

[0150] In certain embodiments, any ENPP1 mutant polypeptide fusion or conjugate described herein comprises the mutations M883Y, S885T, and T887E relative to SEQ ID NO:7.

[0151] In certain embodiments, any ENPP1 mutant polypeptide, fusion or conjugate described herein comprises the mutations P534N, V536T, H1064K, and N1065F relative to SEQ ID NO:7.

[0152] In certain embodiments, any ENPP1 mutant polypeptide, fusion or conjugate described herein comprises the mutations P554L and R545T relative to SEQ ID NO:7.

[0153] In certain embodiments, any ENPP1 mutant polypeptide, fusion or conjugate described herein comprises the mutations S766N, H1064K, and N1065F relative to SEQ ID NO:7.

[0154] In certain embodiments, any ENPP1 mutant polypeptide, fusion or conjugate described herein comprises the mutations E592N, H1064K, and N1065F relative to SEQ ID NO:7.

[0155] In certain embodiments, any ENPP1 mutant polypeptide, fusion or conjugate described herein comprises the mutations P534N, V536T, M883Y, S885T, and T887E with respect to SEQ ID NO:7.

[0156] In another aspect, the disclosure provides an ENPP1 mutant polypeptide fusion comprising an ENPP1 mutant polypeptide fused to an Fc region of an immunoglobulin, wherein the ENPP1 mutant polypeptide comprises a substitution at position 256 relative to SEQ ID NO:7.

[0157] In certain embodiments of any of the fusions described herein, 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.

[0158] In certain embodiments of any of the fusions described herein, 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.

[0159] In certain embodiments, the ENPP1 mutant polypeptide further comprises at least one mutation selected from the group consisting of C25N, K27T, and V29N relative to SEQ ID NO:7.

[0160] In certain embodiments, the ENPP1 mutant polypeptide or fusion described herein comprises at least one mutation selected from the group consisting of C25N / K27T and V29N relative to SEQ ID NO:7.

[0161] In certain embodiments, the ENPP1 mutant polypeptide described herein further comprises at least one mutation selected from the group consisting of K369N and I371T relative to SEQ ID NO:7.

[0162] In certain embodiments, the ENPP1 mutant polypeptide described herein or a fusion comprising such a mutant polypeptide comprises the mutations K369N / I371T relative to SEQ ID NO:7.

[0163] In certain embodiments, the ENPP1 mutant polypeptide described herein or a fusion comprising such a mutant polypeptide further comprises at least one mutation selected from the group consisting of P534N, V536T, R545T, P554L, E592N, R741D, and S766N with respect to SEQ ID NO:7.

[0164] In certain embodiments, the ENPP1 mutant polypeptide described herein or a fusion comprising such a mutant polypeptide comprises at least one mutation selected from the group consisting of P534N / V536T, P554L / R545T, E592N, E592N / R741D, and S766N with respect to SEQ ID NO:7.

[0165] In certain embodiments, any ENPP1 mutant polypeptide or fusion described herein further comprises at least one mutation selected from the group consisting of E864N and L866T relative to SEQ ID NO:7.

[0166] In certain embodiments, any ENPP1 mutant polypeptide or fusion described herein comprises at least the mutations E864N / L866T relative to SEQ ID NO:7.

[0167] In certain embodiments, any ENPP1 mutant polypeptide or fusion described herein has the structure represented by SEQ ID NO: The gene includes 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, and H1064K / N1065F with respect to NO:7.

[0168] In certain embodiments, any fusion described herein comprises the Fc region of an IgG, such as an IgG1.

[0169] In certain embodiments, any ENPP1 mutant polypeptide described herein or a fusion protein comprising such an ENPP1 mutant polypeptide comprises at least one mutation selected from the group consisting of P534N, V536T, R545T, P554L, S766N, and E592N with respect to SEQ ID NO:7.

[0170] In certain embodiments, any ENPP1 mutant polypeptide described herein or a fusion protein comprising such an ENPP1 mutant polypeptide comprises at least one mutation selected from the group consisting of S766N, P534N / Y536T, P554L / R545T, and E592N relative to SEQ ID NO:7.

[0171] In certain embodiments, any ENPP1 mutant polypeptide described herein or a fusion protein comprising such an ENPP1 mutant 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.

[0172] In certain embodiments, any of the fusions described herein contain the mutations I256T, M883Y, S885T, and T887E relative to SEQ ID NO:7.

[0173] In certain embodiments, any fusion described herein comprises an ENPP1 polypeptide and an Fc region of an immunoglobulin, and includes the mutations I256T, P534N, V536T, M883Y, S885T, and T887E relative to SEQ ID NO:7.

[0174] In certain embodiments, any of the fusions described herein includes an ENPP1 polypeptide fusion comprising an ENPP1 polypeptide and an Fc region of an immunoglobulin, and including the mutations I256T, E592N, H1064K, and N1065F relative to SEQ ID NO:7.

[0175] In certain embodiments, the ENPP1 mutant polypeptide fusions described herein comprise a linker amino acid sequence, e.g., between the ENPP1 mutant polypeptide portion and the heterologous protein portion of the fusion. In certain embodiments, the linker amino acid sequence comprises or consists of SEQ ID NO:8. In certain embodiments, the linker amino acid sequence comprises or consists of SEQ ID NO:9, where n=1, n=2, n=3, n=4, n=5, n=6, n=7, n=8, n=9, or n=10.

[0176] In yet another aspect, the disclosure features an ENPP1-containing polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO:15 or SEQ ID NO:16, and conjugates thereof.

[0177] 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.

[0178] 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.

[0179] 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.

[0180] 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.

[0181] In yet another aspect, the disclosure provides an ENPP1 mutant polypeptide, an ENPP1-containing polypeptide, or a fusion expressed from a CHO cell line stably transfected with human ST6 beta-galactoside alpha-2,6-sialyltransferase (also known as ST6GAL1).

[0182] In yet another aspect, the disclosure provides an ENPP1 mutant polypeptide, an ENPP1-containing polypeptide, or a fusion grown in a cell culture supplemented with sialic acid and / or N-acetylmannosamine (also known as 1,3,4-O-Bu3ManNAc).

[0183] Also provided herein are pharmaceutical compositions comprising any one of the ENPP1 mutant polypeptides, ENPP1 mutant polypeptide fusions, conjugates or other polypeptides and proteins described herein and a pharma- ceutically acceptable carrier.

[0184] 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.

[0185] 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 still other embodiments, the polypeptide comprises an ENPP1 polypeptide comprising ENPP1 and another polypeptide, such as, by way of non-limiting example, a transmembrane domain of ENPP2. In still other embodiments, the ENPP1 polypeptide comprises a cleavage product of a precursor ENPP1 polypeptide comprising the ENPP2 transmembrane domain. In still other embodiments, the ENPP2 transmembrane domain comprises residues 12-30 of SEQ ID NO:7, which corresponds to IISLFTFAVGVNICLGFTA.

[0186] 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.

[0187] 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.

[0188] In certain embodiments, the polypeptide is further pegylated (fused to a poly(ethylene glycol) chain).

[0189] 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.

[0190] 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.

[0191] In certain embodiments, the polypeptide is formulated as a liquid formulation. In other embodiments, the present disclosure provides a pharmaceutical composition in the form of a dry product comprising a therapeutic amount of a polypeptide of the present disclosure, the dry product being reconstituted into a solution of the compound in liquid form.

[0192] The present disclosure provides kits comprising at least one polypeptide of the present disclosure, or a salt or solvate thereof, and instructions for using the polypeptide in the methods of the present disclosure.

[0193] 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.

[0194] It will be understood that the ENPP1 polypeptide according to the present disclosure 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 (e.g., but not limited to, fusion protein) that includes the ENPP1 polypeptide, mutant, or mutant fragment thereof.The fusion protein according to the present disclosure 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 extended half-life in pharmacokinetic experiments.

[0195] Vectors and cells Also provided herein is a nucleic acid encoding any one of the ENPP1 mutant polypeptides, ENPP1-containing polypeptides or fusions described herein. The disclosure further provides a vector, e.g., an expression vector, comprising such a nucleic acid. Also provided is a cell, cells or a plurality of cells (e.g., mammalian cells) comprising any one of the nucleic acids, vectors or expression vectors described herein. Also provided is a method of producing a protein (e.g., any one of the ENPP1 mutant polypeptides, ENPP1-containing polypeptides or fusions described herein), which in certain embodiments comprises culturing the cell, cells or a plurality of cells under conditions suitable for expression of a protein by the cell or cells from the nucleic acid, vector or expression vector. The method may also comprise purifying the protein from the cell, cells or a plurality of cells or from the medium in which the cell, cells or a plurality of cells was cultured. In addition, the disclosure provides a protein purified by any such method.

[0196] The present disclosure further provides an autonomously replicating or integrating mammalian cell vector comprising a recombinant nucleic acid encoding a polypeptide of the present disclosure. 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 the expression of the polypeptide. In still other embodiments, the recombinant nucleic acid encodes a polypeptide comprising an ENPP1 polypeptide of the present disclosure and a signal peptide, which is proteolytically processed to produce an ENPP1 polypeptide of the present disclosure when secreted from the cell.

[0197] In yet another aspect, the present disclosure provides an isolated host cell comprising the vector of the present disclosure. In certain embodiments, the cell is a non-human cell. In other embodiments, the cell is a mammalian cell. In yet other embodiments, the vector of the present disclosure comprises a recombinant nucleic acid encoding an ENPP1 polypeptide of the present disclosure and a polypeptide comprising a signal peptide. In yet other embodiments, the polypeptide is proteolytically processed when secreted from the cell to produce the ENPP1 polypeptide of the present disclosure.

[0198] 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.

[0199] 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 disclosure 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.

[0200] 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.

[0201] 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.

[0202] 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.

[0203] 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.

[0204] 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 disclosure, 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.

[0205] 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).

[0206] 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.

[0207] 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.

[0208] 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.

[0209] Then, single stably transfected cell clones are established and screened for high expression clones of desired fusion protein. Screening of single cell clones for ENPP1 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.

[0210] Purification of ENPP1 can be achieved using a combination of standard purification techniques known in the art, examples of which are described above with respect to the production of ENPP1 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.

[0211] Gene therapy Nucleic acids encoding the polypeptides useful in this disclosure can be used in gene therapy protocols for the treatment of diseases or disorders contemplated herein. Improved constructs encoding the polypeptides can be inserted into appropriate gene therapy vectors and administered to patients to treat or prevent the disease or disorder of interest.

[0212] Vectors, such as viral vectors, have been used in the art to introduce genes into a wide variety of different target cells.Typically, the vector is exposed to the target cells so that a sufficient proportion of cells are transformed to provide useful therapeutic or prophylactic effects from the expression of the desired polypeptide (e.g., receptor).The transfected nucleic acid may be permanently integrated into the genome of each of the targeted cells, providing a long-lasting effect, or the treatment may need to be repeated periodically.In certain embodiments, a (viral) vector is transfected in vivo into liver cells with genetic material that codes for the polypeptide of the present disclosure.

[0213] A variety of vectors, both viral and plasmid vectors, are known in the art (see, for example, U.S. Pat. No. 5,252,479 and WO 93 / 07282). In particular, many viruses, including papovaviruses, such as SV40, vaccinia virus, herpes viruses, including HSV and EBV, and retroviruses, have been used as gene transfer vectors. Many gene therapy protocols in the prior art use disabled murine retroviruses. Several recently issued patents relate to methods and compositions for carrying out gene therapy (see, for example, U.S. Pat. Nos. 6,168,916; 6,135,976; 5,965,541 and 6,129,705). Each of the above patents is incorporated herein by reference in its entirety.

[0214] AAV-mediated gene therapy AAV, a parvovirus belonging to the Dependovirus genus, has various characteristics that make it particularly suitable for gene therapy applications. For example, AAV can infect a wide range of host cells, including non-dividing cells. In addition, AAV can infect cells from various species. Importantly, AAV is not associated with any human or animal disease, and does not appear to change the physiological properties of host cells upon integration. Finally, AAV is stable in a wide range of physical and chemical conditions, which makes it suitable for manufacturing, storage, and transportation requirements.

[0215] The AAV genome is a linear, single-stranded DNA molecule containing approximately 4,700 nucleotides (the AAV-2 genome is composed of 4,681 nucleotides, and the AAV-4 genome is composed of 4,767 nucleotides), and typically contains an internal non-repeated segment flanked at each end by inverted terminal repeats (ITRs). The ITRs are approximately 145 nucleotides in length (AAV-1 has an ITR of 143 nucleotides) and have multiple functions, including serving as origins of replication and as packaging signals for the viral genome.

[0216] This internal non-repetitive portion of the genome contains two large open reading frames (ORFs) known as the AAV replication (rep) and capsid (cap) regions. These ORFs code for the replication and capsid gene products that allow replication, assembly and packaging of complete AAV virions. More specifically, a family of at least four viral proteins, Rep 78, Rep 68, Rep 52 and Rep 40, all so named for their apparent molecular weight, are expressed from the AAV rep region. The AAV cap region codes for at least three proteins, VP1, VP2 and VP3.

[0217] AAV is a helper-dependent virus, i.e., it requires co-infection with a helper virus (e.g., adenovirus, herpesvirus, or vaccinia virus) to form a functionally complete AAV virion. In the absence of helper virus co-infection, AAV enters a latent state in which the viral genome is inserted into the host cell chromosome or exists in an extrachromosomal form, but no infectious virus is produced. Subsequent helper virus infection "rescues" the integrated genome, allowing it to replicate and be packaged into viral capsids, thereby reconstituting infectious virions. Although AAV can infect cells from different species, the helper virus must be of the same species as its host cell. Thus, for example, human AAV replicates in canine cells co-infected with canine adenovirus.

[0218] To generate an infectious recombinant AAV (rAAV) containing a heterologous nucleic acid sequence, an AAV vector containing the heterologous nucleic acid sequence but lacking the AAV helper function genes, rep and cap, can be transfected into a suitable host cell line. The AAV helper function genes can then be provided on a separate vector. Also, rather than providing a replication-competent helper virus (e.g., adenovirus, herpesvirus, or vaccinia), only the helper virus genes (i.e., accessory function genes) required for AAV production can be provided on the vector.

[0219] In summary, AAV helper function genes (i.e., rep and cap) and accessory function genes can be provided in one or more vectors. The helper and accessory function gene products can then be expressed in the host cell, where they can act in trans on the rAAV vector containing the heterologous nucleic acid sequence. The rAAV vector containing the heterologous nucleic acid sequence can then be replicated and packaged as if it were a wild-type (wt) AAV genome to form a recombinant virion. When the resulting rAAV virion is infected into the patient's cells, the heterologous nucleic acid sequence enters and is expressed in the patient's cells. Because the patient's cells lack the rep and cap genes and accessory function genes, the rAAV cannot further replicate and package their genomes. Furthermore, without a source of rep and cap genes, wtAAV cannot be formed in the patient's cells.

[0220] There are 11 known AAV serotypes, AAV-1 through AAV-11 (Mori, et al., 2004, Virology 330(2):375-83). AAV-2 is the most prevalent serotype in the human population, with one study estimating that at least 80% of the general population is infected with wt AAV-2 (Berns and Linden, 1995, Bioessays 17:237-245). AAV-3 and AAV-5 are also prevalent in the human population, with infection rates of up to 60% (Georg-Fries, et al., 1984, Virology 134:64-71). AAV-1 and AAV-4 are simian isolates, but both serotypes can transduce human cells (Chiorini, et al., 1997, J Virol 71:6823-6833; Chou, et al., 2000, Mol Ther 2:619-623). Of the six known serotypes, AAV-2 is the best characterized. For example, AAV-2 has been used in a wide range of in vitro transduction experiments, including in mice (U.S. Pat. No. 5,858,351; U.S. Pat. No. 6,093,392); dog muscle; mouse liver (Couto, et al., 1999, Proc. Natl. Acad. Sci. USA 96:12725-12730; Couto, et al., 1997, J. Virol. 73:5438-5447; Nakai, et al., 1999, J. Virol. 73:5438-5447; and Snyder, et al., 1997, Nat. Genet. 16:270-276); mouse heart (Su, et al., 2000, Proc. Natl. Acad. Sci. USA 97:13801-13806); rabbit lung (Flotte, et al., 1993, Proc. Natl. Acad. Sci. USA 90:10613-10617); and rodent photoreceptors (Flannery et al., 1997, Proc. Natl. Acad. Sci. USA 94:6916-6921).

[0221] The broad tissue tropism of AAV-2 can be exploited to deliver tissue-specific transgenes. For example, AAV-2 vectors have been used to deliver the following genes: cystic fibrosis transmembrane conductance regulator to rabbit lungs (Flotte, et al., 1993, Proc. Natl. Acad. Sci. USA 90:10613-10617); factor NIII gene (Burton, et al., 1999, Proc. Natl. Acad. Sci. USA 96:12725-12730) and factor IX gene (Nakai, et al., 1999, J. Virol. 73:5438-5447; Snyder, et al., 1997, Nat. Genet. 16:270-276; U.S. Patent No. 6,093,392) in mouse liver, dog and mouse muscle (U.S. Patent No. 6,093,392); the erythropoietin gene in mouse muscle (U.S. Patent No. 5,858,351); the vascular endothelial growth factor (VEGF) gene in mouse heart (Su, et al., 2000, Proc. Natl. Acad. Sci. USA 97:13801-13806); and the aromatic 1-amino acid decarboxylase gene in monkey neural cells. Expression of certain rAAV-delivered transgenes has shown therapeutic efficacy in experimental animals, for example, expression of factor IX was reported to restore phenotypic normality in a dog model of hemophilia B (U.S. Patent No. 6,093,392). Furthermore, expression of NEGF delivered by rAAV to mouse myocardium resulted in cardiovascular angiogenesis (Su, et al., 2000, Proc. Natl. Acad. Sci. USA 97:13801-13806), and expression of AADC delivered by rAAV to the brains of Parkinson's monkeys resulted in restoration of dopaminergic function.

[0222] The delivery of a protein of interest to a mammalian cell is achieved by first preparing an AAV vector containing DNA encoding the protein of interest, and then administering the vector to a mammal.Thus, the present disclosure should be considered to include an AAV vector containing DNA encoding a polypeptide of interest.In view of the present disclosure, the preparation of an AAV vector containing DNA encoding this / these polypeptides will be obvious to one skilled in the art.

[0223] In certain embodiments, the rAAV vector of the present disclosure comprises various essential DNA elements.In certain embodiments, these DNA elements comprise at least two copies of the AAV ITR sequence, promoter / enhancer element, transcription termination signal, any necessary 5' or 3' untranslated region adjacent to the DNA encoding the protein of interest or its biologically active fragment.The rAAV vector of the present disclosure can also comprise a part of the intron of the protein of interest.Optionally, the rAAV vector of the present disclosure also comprises the DNA encoding the mutant polypeptide of interest.

[0224] In certain embodiments, the vector comprises a promoter / regulatory sequence, including a promiscuous promoter that can induce high levels of expression of heterologous genes in many different cell types. Such promoters include, but are not limited to, cytomegalovirus (CMV) immediate early promoter / enhancer sequence, Rous sarcoma virus promoter / enhancer sequence, and the like. In certain embodiments, the promoter / regulatory sequence in the rAAV vector of the present disclosure is a CMV immediate early promoter / enhancer. However, the promoter sequence used to induce expression of heterologous genes can also be an inducible promoter, such as, but not limited to, a steroid-inducible promoter, or a tissue-specific promoter, such as, but not limited to, the skeletal alpha-actin promoter and the muscle creatine kinase promoter / enhancer, which are specific for muscle tissue.

[0225] In certain embodiments, the rAAV vectors of the present disclosure include a transcription termination signal. Although any transcription termination signal can be included in the vectors of the present disclosure, in certain embodiments, the transcription termination signal is an SV40 transcription termination signal.

[0226] In certain embodiments, the rAAV vector of the present disclosure comprises an isolated DNA encoding a polypeptide of interest or a biologically active fragment of a polypeptide of interest.The present disclosure should be interpreted as encompassing any mammalian sequence of a polypeptide of interest, whether known or unknown.Thus, the present disclosure should be interpreted as encompassing a gene from a mammal other than human, whose polypeptide functions in a manner substantially similar to that of a human polypeptide.Preferably, the nucleotide sequence comprising the gene encoding the polypeptide of interest is about 50% homologous to the gene encoding the polypeptide of interest, more preferably about 70% homologous, even more preferably about 80% homologous, and most preferably about 90% homologous.

[0227] Furthermore, the present disclosure should be construed to encompass naturally occurring or recombinantly derived variants of the wild-type protein sequences that are as therapeutically effective as, or even more therapeutically effective than, the full-length polypeptides in gene therapy methods of the present disclosure.

[0228] The present disclosure should also be construed to encompass DNA encoding variants that retain the biological activity of the polypeptide. Such variants include proteins or polypeptides that have been modified or can be modified using recombinant DNA technology to have additional properties that enhance their suitability for use in the methods described herein, including, but not limited to, variants that confer enhanced stability or enhanced specific activity to the protein in plasma. Analogs can differ from naturally occurring proteins or peptides by conservative amino acid sequence differences or by modifications that do not affect sequence, or both. For example, conservative amino acid changes can be made that change the primary sequence of the protein or peptide but do not usually change its function.

[0229] This disclosure is not limited to the particular rAAV vectors exemplified in the experimental examples, and this disclosure should be construed to encompass any suitable AAV vector, including, but not limited to, vectors based on AAV-1, AAV-3, AAV-4, and AAV-6.

[0230] Also included in the present disclosure is a method for treating a mammal with a disease or disorder in an amount effective to provide a therapeutic effect.The method comprises administering to the mammal an rAAV vector encoding a polypeptide of interest.Preferably, the mammal is a human.

[0231] Typically, the number of viral vector genomes administered per injection per mammal is approximately 1 x 10 8 ~Approx. 5×10 16 Preferably, the number of viral vector genomes administered per injection per mammal is in the range of about 1×10 10 ~Approx. 1×10 15 and more preferably, the number of viral vector genomes administered per injection per mammal is about 5×10 10 ~Approx. 5×10 15 and most preferably, the number of viral vector genomes administered to a mammal in a single injection is about 5×10 11~Approx. 5×10 14 There are 10 pieces.

[0232] When the methods of the present disclosure involve multiple site simultaneous injections or several multiple site injections, including injections at different sites over the course of several hours (e.g., less than about 1 hour to about 2 or 3 hours), the total number of viral vector genomes administered can be the same as that referred to in the single site injection method, or a fraction or multiple thereof.

[0233] For administration of the rAAV vectors of the present disclosure in a single site injection, in certain embodiments, a composition containing the virus is injected directly into an organ of the subject (for example, but not limited to, the liver of the subject).

[0234] For administration to mammals, the rAAV vector can be suspended in a pharma- ceutically acceptable carrier, such as HEPES-buffered saline at a pH of about 7.8. Other useful pharma- ceutically acceptable carriers include, but are not limited to, glycerol, water, saline, ethanol, and other pharma- ceutically acceptable salt solutions, such as phosphates and salts of organic acids. Examples of these and other pharma- ceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey).

[0235] The rAAV vectors of the disclosure can also be provided in the form of a kit comprising, for example, a lyophilized preparation of the vector in a dry salt formulation, sterile water for suspension of the vector / salt composition, and instructions for suspension of the vector and its administration to a mammal.

[0236] array SEQ ID NO:1: hENPP1 amino acid sequence TIFF2024178422000004.tif98149

[0237] SEQ ID NO:2: ENPP2 amino acid sequence TIFF2024178422000005.tif98149

[0238] SEQ ID NO:3: hIgG Fc domain, Fc TIFF2024178422000006.tif24148

[0239] SEQ ID NO:4: hENPP5 protein export signal sequence TIFF2024178422000007.tif3128, where Xaa 23 is absent or is L, where Xaa 23 If Xaa does not exist, 24 does not exist, and Xaa 23 If L, then Xaa 24 is absent or Q.

[0240] SEQ ID NO:5: hENPP7 protein export signal sequence TIFF2024178422000008.tif3128

[0241] SEQ ID NO:6: hENPP7 protein export signal sequence TIFF2024178422000009.tif3128

[0242] SEQ ID NO:7:ENPP1-Fc TIFF2024178422000010.tif118149Bold: Signal sequence Standard: ENPP1 extracellular domain Underline: linker sequence Italics: Fc domain

[0243] SEQ ID NO:8: Exemplary amino acid linker sequences LIN

[0244] SEQ ID NO: 9: Exemplary amino acid linker sequences (GGGGS) n n is an integer between 1 and 10, inclusive, for example, n=1, n=2, n=3, n=4, n=5, n=6, n=7, n=8, n=9 or n=10.

[0245] SEQ ID NO: 10: Exemplary extracellular domain of human ENPP1 TIFF2024178422000011.tif91148

[0246] SEQ ID NO:11: Exemplary ENPP1 mutant polypeptides (substitutions relative to wild-type human ENPP1 are shown in bold / underlined) TIFF2024178422000012.tif91148

[0247] SEQ ID NO:12: Exemplary ENPP1 mutant polypeptides (substitutions relative to wild-type human ENPP1 are shown in bold / underlined) TIFF2024178422000013.tif91148

[0248] SEQ ID NO: 13: Exemplary human IgG1 Fc region TIFF2024178422000014.tif24148

[0249] SEQ ID NO: 14: Exemplary variant human IgG1 Fc region (including MST / YTE substitutions (bold / underlined)) TIFF2024178422000015.tif24148

[0250] SEQ ID NO: 15: Exemplary ENPP1-containing fusion: (GGGGS) fused at its C-terminus to a variant human IgG Fc region (SEQ ID NO: 14; unmodified letters). 1 (SEQ ID NO:9 (n=1); double underlined) fused at its C-terminus to the ENPP1 extracellular domain (SEQ ID NO:10; italics) TIFF2024178422000016.tif118149 Italics: ENPP1 extracellular domain Double underline: linker sequence Standard: IgG Fc region

[0251] SEQ ID NO: 16: Exemplary ENPP1-containing fusion: ENPP1 extracellular domain (SEQ ID NO: 10; italics) fused at its C-terminus to the amino acid sequence LIN (SEQ ID NO: 8; double underlined) fused at its C-terminus to a variant human IgG Fc region (SEQ ID NO: 14; unmodified letters). TIFF2024178422000017.tif118149 Italics: ENPP1 extracellular domain Double underline: linker sequence Standard: IgG Fc region

[0252] SEQ ID NO: 17: Exemplary ENPP1 mutant polypeptide fusion: ENPP1 mutant polypeptide (SEQ ID NO: 11; italics) fused at its C-terminus to LIN (SEQ ID NO: 8; double underlined) fused at its C-terminus to a variant human IgG Fc region (SEQ ID NO: 14; unmodified letters) TIFF2024178422000018.tif118149

[0253] SEQ ID NO: 18: Exemplary ENPP1 mutant polypeptide fusion: (GGGGS) fused at its C-terminus to a variant human IgG Fc region (SEQ ID NO: 14; unmodified letters). 1 (SEQ ID NO:9, n=1; double underlined) fused at its C-terminus to the ENPP1 mutant polypeptide (SEQ ID NO:11; italics) TIFF2024178422000019.tif121150

[0254] method The present disclosure 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 present disclosure.

[0255] The present disclosure further includes a method of reducing or preventing the progression of pathologic ossification in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polypeptide of the present disclosure.

[0256] The present disclosure 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 present disclosure.

[0257] The present disclosure further includes a method for alleviating or preventing the progression of a disease caused by ENPP1 deficiency, characterized by a reduced level of ENPP1 activity and / or defective ENPP1 expression in a subject in need thereof (compared to 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 disclosure.

[0258] The disclosure further includes a method of reducing 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 disclosure 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 method 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.

[0259] The present disclosure 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 disclosure.

[0260] The present disclosure further includes a method of treating, reversing, 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 of the present disclosure.

[0261] The present disclosure further includes a method of 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 disclosure.

[0262] The present disclosure further includes a method of reducing or preventing the progression of age-related arteriosclerosis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polypeptide of the present disclosure.

[0263] The present disclosure further includes a method of 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 administering to the subject a therapeutically effective amount of a polypeptide of the present disclosure.

[0264] The present disclosure further includes a method of 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 disclosure 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 disclosure 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.

[0265] The present disclosure 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 disclosure.

[0266] The present disclosure further includes a method of treating, reversing, or preventing the progression of atherosclerotic plaque calcification in an arterial blood vessel in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polypeptide of the present disclosure.

[0267] The present disclosure 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 disclosure.

[0268] The present disclosure further includes a method of 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 disclosure.

[0269] The present disclosure further includes methods 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 administering to the subject a therapeutically effective amount of a polypeptide of the present disclosure.

[0270] The present disclosure 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 disclosure.

[0271] The present disclosure 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 disclosure.

[0272] The present disclosure 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 disclosure.

[0273] The disclosure further 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 any one of the ENPP1 mutant polypeptides, fusions, ENPP-1-containing polypeptides or conjugates described herein, thereby reducing or preventing the progression of pathological calcification in the subject.

[0274] The disclosure further includes a method of reducing or preventing the progression of pathological ossification in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any one of the ENPP1 mutant polypeptides, fusions, ENPP-1-containing polypeptides or conjugates described herein, thereby reducing or preventing the progression of pathological ossification in the subject.

[0275] The disclosure further includes a method of reducing or preventing the progression of ectopic calcification of soft tissue in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any one of the ENPP1 mutant polypeptides, fusions, ENPP-1-containing polypeptides or conjugates described herein, thereby reducing or preventing the progression of ectopic calcification of soft tissue in the subject.

[0276] The present disclosure further includes a method of treating, ameliorating, 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 any one of the ENPP1 mutant polypeptides, fusions, ENPP-1-containing polypeptides or conjugates described herein, thereby reducing, ameliorating, or preventing ossification of the posterior longitudinal ligament (OPLL) in the subject.

[0277] The present disclosure further includes a method of treating, reversing, or preventing the progression of hypophosphatemic rickets in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any one of the ENPP1 mutant polypeptides, fusions, ENPP-1-containing polypeptides or conjugates described herein, thereby reducing, reversing, or preventing the progression of hypophosphatemic rickets in the subject.

[0278] The present disclosure further includes a method of reducing 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 any one of the ENPP1 mutant polypeptides, fusions, ENPP-1-containing polypeptides or conjugates described herein, thereby reducing or preventing the progression of the disease.

[0279] The present disclosure further includes a method of reducing or preventing the progression of age-related atherosclerosis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any one of the ENPP1 mutant polypeptides, fusions, ENPP-1-containing polypeptides or conjugates described herein, thereby reducing or preventing the progression of age-related atherosclerosis in the subject.

[0280] The present disclosure further includes a method of increasing pyrophosphate (PPi) levels in a subject having lower than normal PPi levels, comprising administering to the subject a therapeutically effective amount of any one of the ENPP1 mutant polypeptides, fusions, ENPP-1-containing polypeptides or conjugates described herein, whereby 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.

[0281] The disclosure further includes a method of reducing or preventing the progression of pathological calcification or ossification in a subject having a lower than normal pyrophosphate (PPi) level, comprising administering to the subject a therapeutically effective amount of any one of the ENPP1 mutant polypeptides, fusions, ENPP-1-containing polypeptides or conjugates described herein, thereby reducing pathological calcification or ossification in the subject or preventing the progression of pathological calcification or ossification in the subject.

[0282] The disclosure further includes a method of treating an ENPP1 deficiency manifested by a decrease in extracellular pyrophosphate (PPi) concentration in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any one of the ENPP1 mutant polypeptides, fusions, ENPP-1-containing polypeptides or conjugates described herein, thereby increasing PPi levels in the subject.

[0283] In certain embodiments, the pathological calcification is selected from the group consisting of idiopathic infantile arterial calcification (IIAC) and atherosclerotic plaque calcification.

[0284] In certain embodiments, the pathological ossification is selected from the group consisting of ossification of the posterior longitudinal ligament (OPLL), hypophosphatemic rickets, and osteoarthritis.

[0285] In certain embodiments, the soft tissue calcification is selected from the group consisting of IIAC and osteoarthritis.

[0286] In certain embodiments of any of the methods described herein, 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. In other embodiments, the soft tissue comprises atherosclerotic plaque. In yet other embodiments, the soft tissue comprises muscular arteries. In yet other embodiments, the soft tissue is selected from the group consisting of joints and vertebrae. In yet other embodiments, the joints are selected from the group consisting of hand joints and foot joints. In yet other embodiments, the soft tissue is selected from the group consisting of articular cartilage and intervertebral disc cartilage. In yet other embodiments, the soft tissue comprises blood vessels. In yet other embodiments, the soft tissue comprises connective tissue.

[0287] In certain embodiments, the subject has been diagnosed with progeria.

[0288] In certain embodiments of any of the methods described herein, the ENPP1 mutant polypeptide, fusion or ENPP1-containing 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 undergoes proteolytic processing to produce the ENPP1 polypeptide. In certain embodiments, the polypeptide of the present disclosure is a secreted product of an ENPP1 precursor protein expressed in a mammalian cell. In other embodiments, the ENPP1 precursor protein comprises a signal peptide sequence and an ENPP1 polypeptide, and the ENPP1 precursor protein undergoes proteolytic processing to become the polypeptide of the present disclosure. In yet other embodiments, in the ENPP1 precursor protein, the signal peptide sequence is linked 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.

[0289] In certain embodiments, the polypeptide is administered to the subject acutely or chronically, hi other embodiments, the polypeptide is administered to the subject locally, regionally, parenterally or systemically.

[0290] In certain embodiments, the subject is a mammal, hi other embodiments, the mammal is a human.

[0291] In certain embodiments, the ENPP1 mutant polypeptide, ENPP1-containing polypeptide or fusion, 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, ocular, pulmonary, and topical. In other embodiments, the ENPP1 mutant polypeptide, ENPP1-containing polypeptide or fusion, or precursor protein thereof is administered to the subject as a pharmaceutical composition further comprising at least one pharma- ceutically acceptable carrier.

[0292] In certain embodiments, the ENPP1 mutant polypeptide, ENPP1-containing polypeptide or fusion, or precursor protein thereof is administered to a subject short-term or long-term. In other embodiments, the ENPP1 mutant polypeptide, ENPP1-containing polypeptide or fusion, or precursor protein thereof is administered to a subject locally, regionally, or systemically. In yet another embodiment, the polypeptide or precursor protein thereof is delivered on an encoded vector, which encodes a protein that is transcribed and translated from the vector after the vector is administered to a subject.

[0293] Armed with the present disclosure, including the methods detailed herein, it will be understood by those skilled in the art that the present disclosure is not limited to the treatment of disease or disorder once established.In particular, the symptoms of the disease or disorder do not have to be manifested to the point of being harmful to the subject, and in fact the disease or disorder does not have to 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 disclosure can provide benefits.

[0294] Thus, the present disclosure includes a method of preventing disease and disorder in a subject, as fully described herein, in that the polypeptide of the present disclosure can be administered to a subject before the onset of disease or disorder, as discussed elsewhere herein, thereby preventing the progression of disease or disorder.In fact, disease or disorder does not need to be detected in a subject before treatment is performed, particularly if the symptoms of disease or disorder have not yet manifested to the point of being harmful to the subject.That is, substantial symptoms from disease or disorder do not need to occur before the present disclosure can provide benefits.Thus, the present disclosure includes a method of preventing or delaying the onset of disease or disorder in a subject, or reducing the progression or growth of disease or disorder, in that the polypeptide of the present disclosure can be administered to a subject before the detection of disease or disorder.In certain embodiments, the polypeptide of the present disclosure is administered to a subject with a strong family history of disease or disorder, thereby preventing or delaying the onset or progression of disease or disorder.

[0295] 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 present disclosure as a prophylactic measure against that disease or disorder.

[0296] Pharmaceutical Compositions and Formulations The present disclosure provides pharmaceutical compositions comprising the polypeptides of the present disclosure for inclusion in the methods described herein.

[0297] 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.

[0298] In one embodiment, pharmaceutical compositions useful for practicing the methods of the present disclosure 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 present disclosure may be administered to deliver a dose of 1 ng / kg / day to 500 mg / kg / day.

[0299] The relative amounts of active ingredient, pharma- ceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the present disclosure may vary depending on the identity, size, and condition of the subject being treated, and also 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.

[0300] Pharmaceutical compositions useful in the methods of the present disclosure 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.

[0301] 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.

[0302] 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.

[0303] 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 disclosure 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.

[0304] Administration of the compounds of the present disclosure 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 polypeptides according to the present disclosure are 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 polypeptides of the present disclosure can also be determined based on the half-life or the rate at which the therapeutic polypeptide is cleared from the body. A polypeptide according to the present disclosure 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.

[0305] 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 disclosure is about 0.01-50 mg / kg body weight / day. In certain embodiments, the effective dose range of the therapeutic compound of the present disclosure 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.

[0306] 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.

[0307] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present disclosure may be varied to provide 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.

[0308] A physician, such as 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 disclosure 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.

[0309] In certain embodiments, the compositions of the present disclosure are administered to patients in dosages ranging from 1 to 5 times per day or more. In other embodiments, the compositions of the present disclosure are administered to patients in dosage ranges including, but not limited to, once per day, every 2 days, every 3 days to once per week, and once per 2 weeks. The frequency of administration of the various combination compositions of the present disclosure 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 disclosure 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.

[0310] In certain embodiments, the disclosure relates to packaged pharmaceutical compositions comprising a container holding a therapeutically effective amount of a compound of the disclosure, 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.

[0311] Route of administration Routes of administration of any of the compounds of the present disclosure 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.

[0312] 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 disclosure are not limited to the specific formulations and compositions described herein.

[0313] 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.

[0314] Further dosage forms Additional dosage forms of the present disclosure 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 disclosure also include those described in U.S. Patent Application Nos. 20030147952, 20030104062, 20030104053, 20030044466, 20030039688 and 20020051820. Additional dosage forms of the present disclosure 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.

[0315] Controlled Release Formulations and Drug Delivery Systems The controlled or sustained release formulation of the pharmaceutical composition of the present disclosure 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 encompassed by the present disclosure.

[0316] In certain embodiments, the formulations of the present disclosure can be, but are not limited to, short-term, rapid-offset, and controlled, including sustained-release, delayed-release, and pulsed-release formulations.

[0317] 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 disclosure 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 disclosure, the compound of the present disclosure is administered to a patient using a sustained release formulation, alone or in combination with another agent.

[0318] 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.

[0319] 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.

[0320] 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.

[0321] It is believed that those skilled in the art can recognize and ascertain many equivalents to the individual procedures, aspects, claims, and examples described herein using only routine experimentation.Such equivalents are considered to be within the scope of this disclosure and encompassed by the appended claims.For example, it should be understood that modifications of reaction and preparation conditions using only routine experimentation with substitutes known in the art are within the scope of this application.

[0322] 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 disclosure. Moreover, all values ​​subsumed within those ranges, as well as the upper or lower limits of value ranges, are also contemplated by this application.

[0323] The following examples further illustrate aspects of the present disclosure, but do not in any way constitute a limitation of the teachings or disclosure presented herein. EXAMPLES

[0324] The present disclosure will now be illustrated with reference to the following examples, which are provided for illustrative purposes only, and the present disclosure is not limited to these examples, but rather encompasses all variations that become evident as a result of the teachings provided herein.

[0325] 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.

[0326] 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.

[0327] Expression of ENPP1-Fc mutant constructs Stable transfection of the ENPP1-Fc construct was established in CHO K1 cells (Sigma Aldrich, 85051005) under zeocin / gentamicin selection and adapted for suspension growth. The adapted cells were incubated at 37°C and 5% CO 2 Inoculation of liquid cultures grown in CD FortiCHO™ medium (A1148301, Thermo Fischer) or PEPROGROW™ AF-CHO (PeproTech AF-CHO) in shake flasks was performed at 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 for accumulation of extracellular protein.

[0328] 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.

[0329] 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.

[0330] 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.

[0331] K m / K cat decision Steady-state hydrolysis of ATP by ENPP1 constructs was measured by HPLC. Briefly, 20 mM Tris, pH 7.4, 150 mM NaCl, 4.5 nM KCl, 14 mM ZnCl 2 , 1 mM MgCl 2 , and 1 mM CaCl 2 The enzymatic reaction was initiated by adding 10 nM PPi to various concentrations of ATP in a reaction buffer containing 100 mM ATP. 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.

[0332] 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).

[0333] 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.

[0334] 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.

[0335] 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. TIFF2024178422000020.tif13128

[0336] 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.

[0337] 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 7B).

[0338] 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 7A-7B) 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 several 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 8A-8D, 16A-16B, and 17).

[0339] 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 the ten ENPP1-Fc isoforms were comparable or superior to construct #770 (Figures 7A-7D), and these ten glycoforms were selected for combinatorial optimization with each other and with the IgG1 Fc domain as described elsewhere herein.

[0340] 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 9A-9B). 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, #1101). 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).

[0341] 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.

[0342] 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, 1,3,4-O-Bu was added to cells stably transfected with selected ENPP1-Fc isoforms (both CHO K1 cells and CHO K1 cells stably transfected with human α-2,6-ST) during protein production. 3 Supplemented with a "high flux" precursor of sialic acid called ManNAc (Table 5).

[0343] The ENPP1-Fc isoforms were purified to homogeneity using an identical purification scheme, and the Michaelis-Menten enzyme kinetic constants and pharmacokinetic properties were determined as described elsewhere herein. Finally, to quantify the biological impact of the clonal optimization, the pharmacodynamic effects of selected ENPP1-Fc isoforms were quantified by determining plasma PPi concentrations at multiple time points following a single subcutaneous administration of each isoform. Half-lives and areas under the curve were determined by plotting the fraction of drug absorbed (F) per time (t) and its elimination (k e ) and absorption (k a) constants were obtained by fitting this data to Equation 1 for the total systemic absorption of a drug administered at a subcutaneous injection site at time t=0.

[0344] Example 3: Pharmacokinetic effects of additional N-linked glycosylation sites A representative plot of the parent isoform, which showed a half-life of 34 hours and an area under the curve (AUC) of 3,027 (construct #770, Table), is shown in Figure 2B. 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 two glycoforms - 4-fold in construct #1020 and 7.7-fold in construct #922 (Figure 10 and Table 2), and the I256T mutation introduced in construct #922 further increased the half-life by 160%. Residue 256 is close to the catalytic threonine of human ENPP1, which is responsible for the nucleophilic addition to the phosphate anhydride substrate. Without being bound by any theory, this sequence variation exists at an analogous site in human ENPP3. This may be a modulator of substrate preference: ENPP2 lacks this loop and may accommodate larger lipid substrates in its catalytic pocket; both ENPP1 and ENPP3 have this loop, but only ENPP3 has an N-glycan consensus sequence (N-GCS).

[0345] 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.

[0346] In one aspect, not all N-GCSs are actually glycosylated: there may be steric hindrances related to the position of the N-GCS such that Asn residues cannot accept glycans due to certain adjacent amino acids. Therefore, unless the purified protein is analyzed for glycan content, it is not possible to ascertain whether the Pk effect of a particular N-GCS is due to a new glycan shielding phenomenon or whether the amino acid change alters the enzyme kinetics, or both. For this reason, the effect on PK associated with the N-GCS should be verified by glycan analysis. Mass spectrometry can be used to analyze the digested peptide fragments. We confirmed that ENPP1-Fc clone 19, which harbors the I256T mutation located within TIFF2024178422000021.tif5128, is indeed glycosylated at Asn254, as indicated by the abundance of sialoglycopeptide peaks compared to the parental ENPP1-Fc clone lacking the I256T mutation (Figure 2D).

[0347] To determine whether the 10-fold increased bioavailability was due to enhanced absorption and retention of this biomaterial or due to a gain of function in the enzyme resulting in higher activity in plasma, the Michaelis-Menten rate constants of the parent construct (construct #770) as well as those of the two I256T-containing constructs (clone 17 and clone 19) were determined at two different concentrations, and the K m or K cat No significant difference was observed between the glycan at position 256 and the glycan at position 260 (FIG. 2E). In certain non-limiting embodiments, the increased biological exposure induced by the addition of a glycan at position 256 is associated with increased biological absorption and / or circulation of the biological material. In certain non-limiting embodiments, the increased biological exposure induced by the addition of a glycan at position 256 is not due to a gain of function of the enzyme.

[0348] Example 4: Pharmacokinetic effects of Fc IgG1 mutations (Figures 11-12) 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.

[0349] FcRn is the major homeostatic regulator of human IgG1 Fc serum half-life. 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 only has the M242Y / S254T / T246E mutation, 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 mutation for various ENPP1 mutations achieved smaller half-life increases of 1.2-1.7-fold.

[0350] Overall, Fc MST mutations increased biological exposure to a greater extent than Fc HN mutations (Table 3, Figures 14A-14E). For example, the addition of MST mutations to the parent isoform increased the AUC by 6-fold and the half-life by approximately 2.5-fold compared to the HN mutation, which increased the AUC by 4.5-fold in the presence of additional glycans (compare clone 14 with clones 9-12 in Table 3 and Figures 14A-14E). However, in some instances, specific N-GCS mutations actually decreased bioavailability for certain Fc mutations, i.e., the N-GCS mutation at residue 766 decreased the AUC for MST-Fc containing constructs (compare clone 8 with clone 14, Table 3, Figure 14A) and HN-Fc containing constructs (compare clone 9 with clone 11, Table 3, Figure 14A). As proof of experimental design and reproducibility, two independent CHO cell clones with the same mutations were constructed and showed little variation in their pharmacodynamic properties (compare clones 11 and 12 in Table 3). It is noteworthy that the PK improvement induced by the Fc mutation did not reach the level of that achieved by the addition of the N-glycan at residue 256, thereby highlighting the importance of the selected glycosylation on pharmacokinetics. To compare the PK effects of the MST Fc mutations with the effect of an additional glycan at position 256 (through the I256T mutation), the plasma activity of the ENPP1-Fc isoform was plotted against time (Figure 14B). This figure shows that the Fc mutation enhances PK by increasing the half-life of this bioagent in plasma, as indicated by the reduced slope of the activity vs. time curve in clone 14 vs. clone 7. In contrast, the addition of the I256T glycosylation increases drug absorption into plasma, i.e., C, as indicated by the greater maximum activity present in clone 7. max(Figure 14B). Although combining the MST Fc mutation with I256T hyperglycosylation only increases the overall biological exposure (AUC) by 16% compared to the effect of the I256T mutation alone, the net effect on the parent isoform is substantially 11.5-fold, supporting the use of both approaches together to maximize bioavailability (compare clones 7 and 17, Table 3 and Figure 14A).

[0351] 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. Differences in glycosylation exist between hamsters and humans, specifically, 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.

[0352] To determine whether alpha-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 alpha-2,6-ST were directly compared (Table 4). An overall trend was confirmed that production of biomaterials in CHOK1 cells stably transfected with human alpha-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 polypeptides produced in CHOK1 cells.

[0353] The effect of α-2,6 linkage on half-life was smaller, with a 20-30% increase in responsive constructs. To understand the difference in the effect of α-2,6 linkage on AUC and protein activity half-life vs. time, isoforms produced in CHOk1 and 1078 cells were compared.

[0354] 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-Bu, a “high flux” precursor of sialic acid. 3 ManNAc or sialic acid itself was supplemented. 3Supplementation with ManNAc 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 (Figure 13 and Table 4). The improvement in PK was primarily due to increased systemic absorption of the subcutaneously administered biomaterial and not due to an increase in half-life (C for clones 7 and 14). max Note the difference, Figure 14B).

[0355] For example, 1,3,4-O-Bu was added to the cell culture medium of CHOK1 cells producing construct #1014 (clone 15). 3 Supplementation with ManNAc did not significantly enhance the AUC and appeared to decrease the half-life of the isoform.

[0356] In contrast, 1,3,4-O-Bu 3 The effect was more dramatic when ManNAc was added to the cell culture medium of construct #1057 (clone 9 with two additional glycosylations in the signal sequence and nuclease domain as well as HN Fc mutations) produced in CHOK1 cells stably transfected with α-2,6-ST. The biological exposure in this clone was increased 2.6-fold by expressing it in CHO cells containing human α-2,6-ST and could be increased a further 1.4-fold by supplementing the growth medium of these cells with sialic acid precursors. These effects were enhanced by the addition of 1,3,4-O-Bu 3 This resulted 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 ManNAc (Figure 13 and Table 4). The percentage of N-acetylneuraminic acid content of ENPP1-Fc was significantly increased in CHOK1 cells stably transfected with human α-2,6-ST and sialic acid precursor 1,3,4-O-Bu 3When expressed in CHO K1 cells grown in the presence of ManNAc, it was progressively increased (Figure 14E), consistent with the notion that these methods can increase the sialic acid content in the enzyme biomaterial and thereby favorably affect pharmacokinetics.

[0357] The effect of expressing the biomaterials in CHO cells containing only human α-2,6-ST ranges from 4.5-fold in the least potent isoform (clone 1 vs. clone 1-ST, Table 5 and Figure 14C) to a more mild effect in the most potent construct. However, these mild effects resulted in a substantial overall increase, as shown by clone 17. Expression of clone 17 in CHO cells containing human α-2,6-ST resulted in a mild 28% increase in AUC, yet this is an absolute 3-fold effect over the AUC of the starting clone due to the enhanced nature of these optimized biomaterials. The final enhancement over clone 770 is 11.5-fold in clone 17 and 14.5-fold in clone 17-ST (Table 5 and Figure 15A). Removal of glycosylation in the signal sequence and nuclease domain of clone 17 did not result in loss of bioavailability, indicating that these glycosylations are disposable for the performance of the clones (Figure 15A, clones 17-ST and 19-ST). 3 Expression of clone 19-ST in culture medium containing ManNAc resulted in a highly active polypeptide. 3The increase resulting from expression of this protein in medium containing ManNAc is represented by the region shaded in dark grey in FIG. 15A, resulting in a net increase in bioavailability of nearly 18-fold compared to the parental construct (FIG. 15B). Mass spectrometry analysis of the sialylation content in the parental clone and the final product reveals that only 78.4% of the available sites (glycans with at least one galactose for the transfer of sialic acid) in the parental clone are sialylated, compared to 99.2% of the sites in clone 19-ST-A (FIG. 15C). The combined findings demonstrate the importance of the sialic acid cap of glycosylation in enzyme biomaterials and the ability of the described method to enhance the bioavailability of glycan-optimized enzyme biomaterials.

[0358] Example 6: Pharmacokinetic Effects ENPP1-Fc is the only enzyme capable of generating plasma PPi in mammals, and plasma PPi is therefore a biomarker that predicts the efficacy of ENPP1 enzyme replacement therapy in the setting of ENPP1 deficiency. To determine the pharmacodynamic effects of the optimized ENPP1-Fc isoform, we performed a pharmacokinetic study of Enpp1. asj / asj Mice were administered a subcutaneous dose of 0.3 mg / Kg of either construct #770 or clone 19-ST, and plasma PPi and enzyme presence were measured in plasma for 263 hours (Figure 15D). Plasma PPi in mice administered construct #770 rose to the normal range 24 hours after administration but returned to baseline by 48 hours, whereas clone 19-ST elevated plasma PPi to approximately 2-fold above the normal range and maintained elevated above or within the normal range for approximately 250 hours. These experiments demonstrate that the pharmacokinetic effects observed in the optimized ENPP1-Fc isoform directly translate to enhanced pharmacodynamic activity.

[0359] (Table 1) TIFF2024178422000022.tif218156TIFF2024178422000023.tif104156

[0360] Table 2. Effect of additional N-linked glycosylation on pharmacokinetics (PK) TIFF2024178422000024.tif100143

[0361] Table 3. Effect of Fc mutations on pharmacokinetics (PK) TIFF2024178422000025.tif163149

[0362] 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 compared to the fusion protein expressed in a normal CHO cell line. The enhanced sialylation of the constructs improved the AUC and half-life values. TIFF2024178422000026.tif228154

[0363] 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 compared to fusion proteins expressed in regular CHO cell lines. Constructs marked "-A" were prepared using 1,3,4-O-Bu, a "high flux" precursor of sialic acid during protein production. 3 It was prepared in cells grown in culture medium supplemented with ManNAc. TIFF2024178422000027.tif132154

[0364] Table 6: List of polypeptides and corresponding mutations TIFF2024178422000028.tif234151

[0365] Table 7. List of mutations in the ENPP1 polypeptide TIFF2024178422000029.tif143128

[0366] List of Aspects The following exemplary aspects are provided, but the numbering should not be construed as designating a level of importance.

[0367] Embodiment 1 provides an ENPP1 polypeptide fusion comprising an ENPP1 polypeptide fused to an Fc region of an immunoglobulin, wherein the ENPP1 polypeptide comprises the mutation I256T relative to SEQ ID NO:7.

[0368] Embodiment 2 provides the polypeptide fusion of embodiment 1, wherein the Fc region comprises at least one mutation selected from the group consisting of M883Y, S885N, S885T, T887E, H1064K, and N1065F with respect to SEQ ID NO:7.

[0369] Embodiment 3 provides the polypeptide fusion of embodiment 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.

[0370] Embodiment 4 provides the polypeptide fusion of any of embodiments 1 to 3, wherein the ENPP1 polypeptide further comprises at least one mutation selected from the group consisting of C25N, K27T, and V29N relative to SEQ ID NO:7.

[0371] Embodiment 5 provides the polypeptide fusion of any of embodiments 1 to 4, 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.

[0372] Embodiment 6 provides the polypeptide fusion of any of embodiments 1 to 5, wherein the ENPP1 polypeptide further comprises at least one mutation selected from the group consisting of K369N and I371T relative to SEQ ID NO:7.

[0373] Embodiment 7 provides the polypeptide fusion of any of embodiments 1 to 6, wherein the ENPP1 polypeptide comprises the mutations K369N / I371T relative to SEQ ID NO:7.

[0374] Embodiment 8 provides the polypeptide fusion of any of embodiments 1 to 7, wherein the ENPP1 polypeptide further 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.

[0375] Embodiment 9 provides a polypeptide fusion of any of embodiments 1 to 8, 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.

[0376] Embodiment 10 provides the polypeptide fusion of any of embodiments 1 to 9, wherein the ENPP1 polypeptide further comprises at least one mutation selected from the group consisting of E864N and L866T relative to SEQ ID NO:7.

[0377] Embodiment 11 provides the polypeptide fusion of any of embodiments 1 to 10, wherein the ENPP1 polypeptide comprises at least the mutations E864N / L866T relative to SEQ ID NO:7.

[0378] Aspect 12 is a sequence ID 12. The polypeptide fusion of any of embodiments 1-11, 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.

[0379] Embodiment 13 provides the polypeptide fusion of any of embodiments 1 to 12, wherein the Fc region is an IgG Fc region.

[0380] Embodiment 14 provides a polypeptide fusion of any of embodiments 1 to 13, 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.

[0381] Embodiment 15 provides a polypeptide fusion of any of embodiments 1 to 13, comprising at least one mutation selected from the group consisting of S766N, P534N / Y536T, P554L / R545T, and E592N relative to SEQ ID NO:7.

[0382] Embodiment 16 provides a polypeptide fusion of any of embodiments 1 to 13, 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.

[0383] 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 I256T, M883Y, S885T, and T887E relative to SEQ ID NO:7.

[0384] 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 I256T, P534N, V536T, M883Y, S885T, and T887E relative to SEQ ID NO:7.

[0385] Embodiment 19 provides an ENPP1 polypeptide fusion comprising an ENPP1 polypeptide and an Fc region of an immunoglobulin, the ENPP1 polypeptide fusion comprising the mutations I256T, E592N, H1064K, and N1065F relative to SEQ ID NO:7.

[0386] A twenty-first aspect is an ENPP1 mutant polypeptide comprising amino acids 23 to 849 of SEQ ID NO:7, comprising the mutation I256T relative to SEQ ID NO:7, and further comprising a mutation selected from the group consisting of S766N, P534N, V536T, P554L, R545T, and E592N; ENPP1 mutant polypeptides are provided.

[0387] Embodiment 21 provides the variant polypeptide of embodiment 20, comprising the amino acid sequence of SEQ ID NO:7.

[0388] Example 22 provides a variant polypeptide of any of Examples 20 to 21, comprising the amino acid sequence of SEQ ID NO:7.

[0389] Embodiment 23 provides a mutant polypeptide of any of embodiments 20 to 23, comprising at least one mutation selected from the group consisting of S766N, P534N / V536T, P554L / R545T and E592N relative to SEQ ID NO:7.

[0390] Embodiment 24 provides a mutant polypeptide of any of embodiments 21 to 23, 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.

[0391] Embodiment 25 provides a mutant polypeptide of any of embodiments 21 to 24, which comprises a S885N mutation relative to SEQ ID NO:7.

[0392] Embodiment 26 provides a mutant polypeptide of any of embodiments 20 to 25, which comprises a S766N mutation relative to SEQ ID NO:7.

[0393] Embodiment 27 provides a mutant polypeptide of any of embodiments 21 to 26, comprising the mutations M883Y, S885T and T887E relative to SEQ ID NO:7.

[0394] Embodiment 28 provides a mutant polypeptide of any of embodiments 21 to 27, comprising the mutations P534N, V536T, H1064K and N1065F relative to SEQ ID NO:7.

[0395] Embodiment 29 provides a mutant polypeptide of any of embodiments 20 to 28, which comprises the mutations P554L and R545T relative to SEQ ID NO:7.

[0396] Embodiment 30 provides a mutant polypeptide of any of embodiments 21 to 29, comprising the mutations S766N, H1064K and N1065F relative to SEQ ID NO:7.

[0397] Embodiment 31 provides a mutant polypeptide of any of embodiments 21 to 30, which comprises the mutations E592N, H1064K and N1065F relative to SEQ ID NO:7.

[0398] Example 32 provides a mutant polypeptide of any of Examples 21 to 31, comprising the mutations P534N, V536T, M883Y, S885T and T887E relative to SEQ ID NO:7.

[0399] Embodiment 33 provides a polypeptide fusion of any of embodiments 1 to 19 or a mutant polypeptide of any of embodiments 20 to 32, expressed from a CHO cell line stably transfected with human ST6 beta-galactoside alpha-2,6-sialyltransferase (also known as ST6GAL1).

[0400] Embodiment 34 is directed to sialic acid and / or N-acetylmannosamine (1,3,4-O-Bu 3 The present invention provides a polypeptide fusion of any of embodiments 1-19 or a mutant polypeptide of any of embodiments 20-32, grown in a cell culture supplemented with NADH (also known as ManNAc).

[0401] Embodiment 35 provides a method for reducing or preventing the progression of pathological calcification in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide fusion of any of embodiments 1 to 19 and 33 to 34 or a mutant polypeptide of any of embodiments 20 to 34.

[0402] Embodiment 36 provides a method for reducing or preventing the progression of pathological ossification in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide fusion of any of embodiments 1 to 19 and 33 to 34 or a mutant polypeptide of any of embodiments 20 to 34.

[0403] Embodiment 37 provides a method for reducing or preventing the progression of ectopic calcification of soft tissue in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polypeptide fusion of any of embodiments 1-19 and 33-34 or a mutant polypeptide of any of embodiments 20-34.

[0404] Embodiment 38 provides a method of treating, ameliorating 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 of any of embodiments 1-19 and 33-34 or a mutant polypeptide of any of embodiments 20-34.

[0405] Embodiment 39 provides a method of treating, reversing 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 of any of embodiments 1-19 and 33-34 or a mutant polypeptide of any of embodiments 20-34.

[0406] Aspect 40 is a method of reducing 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 includes the step of administering to a subject a therapeutically effective amount of a polypeptide fusion according to any of embodiments 1 to 19 and 33 to 34 or a mutant polypeptide according to any of embodiments 20 to 34.

[0407] Embodiment 41 provides a method for reducing 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 19 and 33 to 34, or a mutant polypeptide of any of embodiments 20 to 34.

[0408] Example 42 provides the method of example 35, wherein the pathological calcification is selected from the group consisting of idiopathic infantile arterial calcification (IIAC) and atherosclerotic plaque calcification.

[0409] Example 43 provides the method of example 36, wherein the pathological ossification is selected from the group consisting of ossification of the posterior longitudinal ligament (OPLL), hypophosphatemic rickets, and osteoarthritis.

[0410] Example 44 provides the method of example 37, wherein the soft tissue calcification is selected from the group consisting of IIAC and osteoarthritis.

[0411] Example 45 provides the method of Example 37, 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.

[0412]

[0036] Embodiment 46 is a method of increasing pyrophosphate (PPi) levels in a subject having a lower than normal PPi level, comprising: Administering to a subject a therapeutically effective amount of a polypeptide of a polypeptide fusion of any of embodiments 1-19 and 33-34 or a mutant polypeptide of any of embodiments 20-34, wherein said administration increases the level of PPi in the subject to a normal level of at least 2 μM and maintains it at about the same level. The present invention provides a method comprising:

[0413]

[0023] Aspect 47 is a method for reducing or preventing the progression of pathological calcification or ossification in a subject having a lower than normal pyrophosphate (PPi) level, comprising: Administering to a subject a therapeutically effective amount of a polypeptide fusion of any of embodiments 1-19 and 33-34 or a mutant polypeptide of any of embodiments 20-34, thereby reducing pathological calcification or ossification in the subject or preventing the progression of pathological calcification or ossification in the subject. The present invention provides a method comprising:

[0414]

[0023] Embodiment 48 is a method of treating an ENPP1 deficiency manifested by a decrease in extracellular pyrophosphate (PPi) concentration in a subject in need thereof, comprising: Administering to a subject a therapeutically effective amount of a polypeptide fusion of any of embodiments 1-19 and 33-34 or a mutant polypeptide of any of embodiments 20-34, thereby increasing the level of PPi in the subject. The present invention provides a method comprising:

[0415] Example 49 provides the method of any of Examples 35 to 48, 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 undergoes proteolytic processing to produce the ENPP1 polypeptide.

[0416] Embodiment 50 provides the method of embodiment 49, wherein in the ENPP1 precursor protein, a signal peptide sequence is linked to the N-terminus of the ENPP1 polypeptide.

[0417] Example 51 provides the method of any of Examples 49 to 50, 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.

[0418] Embodiment 52 provides the method of any of embodiments 35 to 51, wherein the polypeptide fusion or mutant polypeptide is administered to the subject acutely or chronically.

[0419] Embodiment 53 provides the method of any of embodiments 35 to 52, wherein the polypeptide fusion or variant polypeptide is administered locally, regionally, parenterally or systemically to the subject.

[0420] Embodiment 54 provides the method of any of embodiments 35 to 53, 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, ocular, pulmonary, and topical.

[0421] Embodiment 55 provides the method of any of embodiments 35 to 54, 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.

[0422] Embodiment 56 provides the method of any of embodiments 35 to 55, wherein the subject is a mammal.

[0423] Embodiment 57 provides the method of embodiment 56, wherein the mammal is a human.

[0424] Embodiment 58 provides an ENPP1 mutant polypeptide comprising one or more amino acid substitutions relative to SEQ ID NO:7, comprising an amino acid substitution at position 256 relative to SEQ ID NO:7.

[0425] Example 59 provides the ENPP1 mutant polypeptide of example 58, wherein the amino acid sequence of the ENPP1 mutant polypeptide is at least 90% identical to amino acids 23 to 849 of SEQ ID NO:7.

[0426] Aspect 60 is an ENPP1 mutant polypeptide comprising amino acids 23 to 849 of SEQ ID NO:7, There are 10 or fewer amino acid substitutions relative to amino acids 23-849 of SEQ ID NO:7, containing an amino acid substitution at position 256 relative to SEQ ID NO:7, ENPP1 mutant polypeptides are provided.

[0427] Embodiment 61 provides the ENPP1 mutant polypeptide of any one of embodiments 58 to 60, wherein the amino acid substitution is a substitution of isoleucine (I) with threonine (T) at position 256 relative to SEQ ID NO:7.

[0428] Embodiment 62 provides an ENPP1 mutant polypeptide of any one of embodiments 58 to 60, wherein the amino acid substitution is a substitution of isoleucine (I) with serine (S) at position 256 relative to SEQ ID NO:7.

[0429] Embodiment 63 is an ENPP1 variant polypeptide comprising an amino acid sequence that is at least 90% identical to amino acids 23 to 849 of SEQ ID NO:7, containing the mutation I256T with respect to SEQ ID NO:7, Further comprising a mutation selected from the group consisting of S766N, P534N, V536T, P554L, R545T, and E592N with respect to SEQ ID NO:7; ENPP1 mutant polypeptides are provided.

[0430] Embodiment 64 provides an ENPP1 mutant polypeptide of embodiment 63, comprising at least one amino acid substitution selected from the group consisting of S766N, P534N / V536T, P554L / R545T, and E592N relative to SEQ ID NO:7.

[0431] Embodiment 65 provides the ENPP1 mutant polypeptide of embodiment 63, which comprises the amino acid substitution V29N.

[0432] Example 66 provides the ENPP1 mutant polypeptide of any one of Examples 58 to 61, comprising the amino acid sequence of SEQ ID NO:11.

[0433] Embodiment 67 provides an ENPP1 mutant polypeptide fusion comprising the ENPP1 mutant polypeptide of any one of embodiments 58 to 66 and a heterologous protein.

[0434] Embodiment 68 provides the ENPP1 mutant polypeptide fusion of embodiment 67, wherein the heterologous protein is an FcRn binding domain.

[0435] Embodiment 69 provides an ENPP1 mutant polypeptide fusion of any one of embodiments 67 to 68, wherein the heterologous protein is carboxy-terminal to the ENPP1 mutant polypeptide of the fusion.

[0436] Embodiment 70 provides the ENPP1 mutant polypeptide fusion of any one of embodiments 67 to 68, wherein the heterologous protein is amino-terminal to the ENPP1 mutant polypeptide of the fusion.

[0437] Embodiment 71 provides the ENPP1 mutant polypeptide fusion of any one of embodiments 68 to 70, wherein the FcRn binding domain is an albumin polypeptide.

[0438] Embodiment 72 provides the ENPP1 mutant polypeptide fusion of any one of embodiments 68 to 70, wherein the FcRn binding domain is the Fc portion of an immunoglobulin molecule.

[0439] Embodiment 73 provides the ENPP1 mutant polypeptide fusion of embodiment 72, wherein the immunoglobulin molecule is IgG1.

[0440] Embodiment 74 provides the ENPP1 mutant polypeptide fusion of any one of embodiments 68 to 73, wherein the FcRn-binding domain comprises one or more amino acid substitutions relative to the wild-type FcRn-binding domain.

[0441] Embodiment 75 provides an ENPP1 mutant polypeptide fusion of any one of embodiments 68 to 70 and 72 to 74, wherein the FcRn binding domain is the Fc portion of a human IgG1 molecule and comprises the following amino acid substitutions relative to SEQ ID NO:7: M883Y, S885T, and T887E, respectively.

[0442] Embodiment 76 provides an ENPP1 mutant polypeptide fusion of any one of embodiments 68 to 70 and 72 to 75, each comprising one or more of the following substitutions relative to SEQ ID NO:7: S885N, S766N, M883Y / S885T / T887E, P534N / V536T / H1064K / N1065F, P554L / R545T, S766N / H1064K / N1065F, E592N / H1064K / N1065F, or P534N / V536T / M883Y / S885T / T887E.

[0443] Embodiment 77 provides an ENPP1 mutant polypeptide fusion of any one of embodiments 68 to 70 and 72 to 76, comprising a S885N mutation relative to SEQ ID NO:7.

[0444] Embodiment 78 provides an ENPP1 mutant polypeptide fusion of any one of embodiments 68 to 70 and 72 to 77, comprising a S766N mutation relative to SEQ ID NO:7.

[0445] Embodiment 79 provides an ENPP1 mutant polypeptide fusion of any one of embodiments 68 to 70 and 72 to 78, comprising the mutations M883Y, S885T, and T887E relative to SEQ ID NO:7.

[0446] Embodiment 80 provides an ENPP1 mutant polypeptide fusion of any one of embodiments 68 to 70 and 72 to 79, comprising the mutations P534N, V536T, H1064K, and N1065F relative to SEQ ID NO:7.

[0447] Embodiment 81 provides an ENPP1 mutant polypeptide fusion of any one of embodiments 68 to 70 and 72 to 80, comprising the mutations P554L and R545T relative to SEQ ID NO:7.

[0448] Embodiment 82 provides an ENPP1 mutant polypeptide fusion of any one of embodiments 68 to 70 and 72 to 81, comprising the mutations S766N, H1064K, and N1065F relative to SEQ ID NO:7.

[0449] Embodiment 83 provides an ENPP1 mutant polypeptide fusion of any one of embodiments 68 to 70 and 72 to 82, comprising the mutations E592N, H1064K, and N1065F relative to SEQ ID NO:7.

[0450] Embodiment 84 provides an ENPP1 mutant polypeptide fusion of any one of embodiments 68 to 70 and 72 to 83, comprising the mutations P534N, V536T, M883Y, S885T, and T887E relative to SEQ ID NO:7.

[0451] Embodiment 85 provides an ENPP1 mutant polypeptide fusion of any one of embodiments 68 to 70 and 72 to 84, 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.

[0452] Embodiment 86 provides an ENPP1 mutant polypeptide fusion of any one of embodiments 68 to 70 and 72 to 85, 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.

[0453] Embodiment 87 provides an ENPP1 mutant polypeptide fusion of any one of embodiments 68 to 70 and 72 to 86 or an ENPP1 mutant polypeptide of any one of embodiments 58 to 65, which comprises at least one mutation selected from the group consisting of C25N, K27T, and V29N relative to SEQ ID NO:7.

[0454] Embodiment 88 provides an ENPP1 mutant polypeptide fusion of any one of embodiments 68 to 70 and 72 to 87 or an ENPP1 mutant polypeptide of any one of embodiments 58 to 65 and 85, which comprises at least one mutation selected from the group consisting of C25N / K27T and V29N relative to SEQ ID NO:7.

[0455] Embodiment 89 provides an ENPP1 mutant polypeptide fusion of any one of embodiments 68 to 70 and 72 to 88 or an ENPP1 mutant polypeptide of any one of embodiments 58 to 65 and 87 to 88, which comprises one mutation selected from the group consisting of K369N and I371T relative to SEQ ID NO:7.

[0456] Embodiment 90 provides an ENPP1 mutant polypeptide fusion of any one of embodiments 68 to 70 and 72 to 89 or an ENPP1 mutant polypeptide of any one of embodiments 58 to 65 and 87 to 89, comprising the mutations K369N / I371T relative to SEQ ID NO:7.

[0457] Embodiment 91 provides an ENPP1 mutant polypeptide fusion of any one of embodiments 68 to 70 and 72 to 90 or an ENPP1 mutant polypeptide of any one of embodiments 58 to 65 and 87 to 90, comprising at least one mutation selected from the group consisting of P534N, V536T, R545T, P554L, E592N, R741D and S766N with respect to SEQ ID NO:7.

[0458] Embodiment 92 provides an ENPP1 mutant polypeptide fusion of any one of embodiments 68 to 70 and 72 to 91 or an ENPP1 mutant polypeptide of any one of embodiments 58 to 65 and 87 to 91, comprising 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.

[0459] Embodiment 93 is directed to 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, S885Y, and S885Y, respectively, with respect to SEQ ID NO:7. The present invention provides an ENPP1 mutant polypeptide fusion of any one of aspects 68 to 70 and 72 to 92 or an ENPP1 mutant polypeptide of any one of aspects 58 to 65 and 87 to 92, comprising at least one mutation selected from the group consisting of N, S885T, T887E, H1064K, N1065F, M883Y / S885T / T887E, H1064K / N1065F.

[0460] Embodiment 94 provides an ENPP1 mutant polypeptide fusion of any one of embodiments 68 to 70 and 72 to 93 or an ENPP1 mutant polypeptide of any one of embodiments 58 to 65 and 87 to 93, comprising at least one mutation selected from the group consisting of P534N, V536T, R545T, P554L, S766N and E592N relative to SEQ ID NO:7.

[0461] Embodiment 95 provides an ENPP1 mutant polypeptide fusion of any one of embodiments 68 to 70 and 72 to 94 or an ENPP1 mutant polypeptide of any one of embodiments 58 to 65 and 87 to 94, comprising at least one mutation selected from the group consisting of S766N, P534N / Y536T, P554L / R545T, and E592N relative to SEQ ID NO:7.

[0462] Embodiment 96 provides an ENPP1 mutant polypeptide fusion of any one of embodiments 68 to 70 and 72 to 95 or an ENPP1 mutant polypeptide of any one of embodiments 58 to 65 and 87 to 95, 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.

[0463] Embodiment 97 provides an ENPP1 mutant polypeptide fusion of any one of embodiments 68 to 70 and 72 to 96, comprising the mutations I256T, M883Y, S885T, and T887E relative to SEQ ID NO:7.

[0464] Embodiment 98 provides an ENPP1 mutant polypeptide fusion of any one of embodiments 68 to 70 and 72 to 96, comprising the mutations I256T, P534N, V536T, M883Y, S885T, and T887E relative to SEQ ID NO:7.

[0465] Embodiment 99 provides an ENPP1 mutant polypeptide fusion of any one of embodiments 68 to 70 and 72 to 96, comprising the mutations I256T, E592N, H1064K, and N1065F relative to SEQ ID NO:7.

[0466] Embodiment 100 provides a fusion of any one of embodiments 67 to 99, comprising a linker amino acid sequence.

[0467] Embodiment 101 provides the fusion of embodiment 100, wherein a linker amino acid sequence connects the ENPP1 mutant polypeptide portion of the fusion and the heterologous protein.

[0468] Embodiment 102 provides the fusion of any one of embodiments 100 to 101, wherein the linker amino acid sequence comprises SEQ ID NO:8 or SEQ ID NO:9.

[0469] Embodiment 103 provides a nucleic acid encoding the ENPP1 mutant polypeptide of any one of embodiments 58 to 66 or the fusion of any one of embodiments 67 to 102.

[0470] Embodiment 104 provides a vector comprising the nucleic acid of embodiment 103.

[0471] Embodiment 105 provides an expression vector comprising the nucleic acid of embodiment 103.

[0472] Embodiment 106 provides a cell or a plurality of cells, each comprising the nucleic acid of embodiment 103, the vector of embodiment 104, and / or the expression vector of embodiment 105.

[0473] Embodiment 107 provides a cell or a plurality of cells of embodiment 106, which is a CHO cell and / or an NS0 cell.

[0474] Embodiment 108 provides a cell or a plurality of cells according to embodiment 107, wherein the CHO cells are stably transfected with human ST6 beta-galactoside alpha-2,6-sialyltransferase.

[0475] Aspect 109 provides a method for producing an ENPP1 mutant polypeptide or fusion comprising culturing a cell or a plurality of cells of any one of aspects 106 to 108 under conditions suitable for expression of the ENPP1 mutant polypeptide or fusion by the cell or a plurality of cells.

[0476] Embodiment 110 provides the method of embodiment 109, wherein said cells are cultured in a medium supplemented with sialic acid and / or N-acetylmannosamine.

[0477] Example 111 provides the method of any one of Examples 109 to 110, further comprising the step of purifying the ENPP1 mutant polypeptide or fusion from the cell, the plurality of cells, or the medium in which the cell or the plurality of cells has been cultured.

[0478] Embodiment 112 provides an ENPP1 mutant polypeptide or fusion purified by the method of embodiment 111.

[0479] Aspect 113 is (i) an ENPP1 mutant polypeptide according to any one of embodiments 58 to 66, 87 to 96 and 112, and / or an ENPP1 mutant polypeptide fusion according to any one of embodiments 67 to 102 and 112, and (ii) dissimilar parts; The present invention provides a conjugate comprising:

[0480] Embodiment 114 provides the conjugate of embodiment 113, wherein the heterologous moiety is polyethylene glycol.

[0481] Embodiment 115 provides a pharmaceutical composition comprising an ENPP1 mutant polypeptide of any one of embodiments 58 to 66, 87 to 96 and 112, a fusion of any one of embodiments 67 to 102 and 112, a nucleic acid of embodiment 103, a vector of embodiment 104, an expression vector of embodiment 105 and / or a conjugate of any one of embodiments 113 to 114, and a pharma- ceutically acceptable carrier.

[0482] Embodiment 116 provides a method for 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) an ENPP1 mutant polypeptide of any one of embodiments 58-66, 87-96 and 112; (b) a fusion of any one of embodiments 67-102 and 112; (c) a conjugate of any one of embodiments 113-114; and / or (d) a pharmaceutical composition of embodiment 115, thereby reducing or preventing the progression of pathological calcification in the subject.

[0483] Embodiment 117 provides a method for reducing or preventing the progression of pathological ossification in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of (a) an ENPP1 mutant polypeptide of any one of embodiments 58-66, 87-96 and 112; (b) a fusion of any one of embodiments 67-102 and 112; (c) a conjugate of any one of embodiments 113-114; and / or (d) a pharmaceutical composition of embodiment 115, thereby reducing or preventing the progression of pathological ossification in the subject.

[0484] Embodiment 118 provides a method for reducing or preventing the progression of ectopic calcification of soft tissue in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of (a) an ENPP1 mutant polypeptide of any one of embodiments 58-66, 87-96 and 112; (b) a fusion of any one of embodiments 67-102 and 112; (c) a conjugate of any one of embodiments 113-114; and / or (d) a pharmaceutical composition of embodiment 115, thereby reducing or preventing the progression of ectopic calcification of soft tissue in the subject.

[0485] Embodiment 119 provides a method of treating, ameliorating 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) an ENPP1 mutant polypeptide of any one of embodiments 58-66, 87-96 and 112; (b) a fusion of any one of embodiments 67-102 and 112; (c) a conjugate of any one of embodiments 113-114; and / or (d) a pharmaceutical composition of embodiment 115, thereby reducing, ameliorating or preventing ossification of the posterior longitudinal ligament (OPLL) in the subject.

[0486] Embodiment 120 provides a method of treating, reversing or preventing the progression of hypophosphatemic rickets in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of (a) an ENPP1 mutant polypeptide of any one of embodiments 58-66, 87-96 and 112; (b) a fusion of any one of embodiments 67-102 and 112; (c) a conjugate of any one of embodiments 113-114; and / or (d) a pharmaceutical composition of embodiment 115, thereby reducing, reversing or preventing the progression of hypophosphatemic rickets in the subject.

[0487] Aspect 121 is a method for reducing 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 present invention provides a method for reducing or preventing the progression of the disease, the method comprising the step of administering to a subject a therapeutically effective amount of (a) an ENPP1 mutant polypeptide of any one of embodiments 58 to 66, 87 to 96 and 112; (b) a fusion of any one of embodiments 67 to 102 and 112; (c) a conjugate of any one of embodiments 113 to 114; and / or (d) a pharmaceutical composition of embodiment 115, thereby reducing or preventing the progression of the disease.

[0488] Embodiment 122 provides 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) an ENPP1 mutant polypeptide of any one of embodiments 58 to 66, 87 to 96 and 112; (b) a fusion of any one of embodiments 67 to 102 and 112; (c) a conjugate of any one of embodiments 113 to 114; and / or (d) a pharmaceutical composition of embodiment 115, thereby reducing or preventing the progression of age-related arteriosclerosis in the subject.

[0489] Embodiment 123 provides a method for increasing pyrophosphate (PPi) levels in a subject having a PPi level lower than the normal level, comprising administering to the subject a therapeutically effective amount of (a) an ENPP1 mutant polypeptide of any one of embodiments 58 to 66, 87 to 96 and 112; (b) a fusion of any one of embodiments 67 to 102 and 112; (c) a conjugate of any one of embodiments 113 to 114; and / or (d) a pharmaceutical composition of embodiment 115, wherein said administration increases the level of PPi in the subject to a normal level of at least 2 μM and maintains it at approximately the same level.

[0490] Embodiment 124 provides a method for reducing or preventing the progression of pathological calcification or ossification in a subject having a pyrophosphate (PPi) level lower than normal, comprising administering to the subject a therapeutically effective amount of (a) an ENPP1 mutant polypeptide of any one of embodiments 58-66, 87-96 and 112; (b) a fusion of any one of embodiments 67-102 and 112; (c) a conjugate of any one of embodiments 113-114; and / or (d) a pharmaceutical composition of embodiment 115, whereby pathological calcification or ossification in the subject is reduced or the progression of pathological calcification or ossification in the subject is prevented.

[0491] Embodiment 125 provides a method for treating an ENPP1 defect manifested by a decrease in extracellular pyrophosphate (PPi) concentration in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of (a) an ENPP1 mutant polypeptide of any one of embodiments 58-66, 87-96 and 112; (b) a fusion of any one of embodiments 67-102 and 112; (c) a conjugate of any one of embodiments 113-114; and / or (d) a pharmaceutical composition of embodiment 115, thereby increasing PPi levels in the subject.

[0492] Example 126 provides the method of any one of Examples 116 to 125, wherein the mutant polypeptide, fusion, conjugate or pharmaceutical composition is administered to the subject acutely or chronically.

[0493] Embodiment 127 provides the method of any one of embodiments 116 to 126, wherein the mutant polypeptide, fusion, conjugate or pharmaceutical composition is administered locally, regionally, parenterally or systemically to the subject.

[0494] Example 128 provides the method of any one of Examples 116 to 127, wherein the subject is a human.

[0495] The disclosures of each and every patent, patent application and publication cited herein are incorporated herein by reference in their entirety. Although the present disclosure has been disclosed with reference to specific embodiments, it is clear that other embodiments and variations of the present disclosure may be devised by those skilled in the art without departing from the true spirit and scope of the present disclosure. It is intended that the appended claims be deemed to encompass all such embodiments and equivalent variations.

[0496] Sequence information SEQUENCE LISTING <110> Yale University <120> ENPP1 Polypeptides and Methods of Using Same <150> US 62 / 830,230 <151> 2019-04-05 <150> US 62 / 983,142 <151> 2020-02-28 <150> US 62 / 984,650 <151> 2020-03-03 <160> 18 <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> Homo sapiens <220> <221> MOD_RES <222> (23)..(23) <223> Xaa23 is absent or Leu <220> <221> MOD_RES <222> (24)..(24) <223> Xaa24 is absent if Xaa23 is absent, and Xaa24 is absent or Gln if Xaa23 is Leu <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> Homo sapiens <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> Homo sapiens <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> Recombinantly synthesized <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 <210> 8 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Linker <400> 8 Leu Ile Asn 1 <210> 9 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Linker <400> 9 Gly Gly Gly Gly Ser 1 5 <210> 10 <211> 827 <212> PRT <213> Artificial Sequence <220> <223> Recombinantly synthesized <400> 10 Pro Ser Cys Ala Lys Glu Val Lys Ser Cys Lys Gly Arg Cys Phe Glu 1 5 10 15 Arg Thr Phe Gly Asn Cys Arg Cys Asp Ala Ala Cys Val Glu Leu Gly 20 25 30 Asn Cys Cys Leu Asp Tyr Gln Glu Thr Cys Ile Glu Pro Glu His Ile 35 40 45 Trp Thr Cys Asn Lys Phe Arg Cys Gly Glu Lys Arg Leu Thr Arg Ser 50 55 60 Leu Cys Ala Cys Ser Asp Asp Cys Lys Asp Lys Gly Asp Cys Cys Ile 65 70 75 80 Asn Tyr Ser Ser Val Cys Gln Gly Glu Lys Ser Trp Val Glu Glu Pro 85 90 95 Cys Glu Ser Ile Asn Glu Pro Gln Cys Pro Ala Gly Phe Glu Thr Pro 100 105 110 Pro Thr Leu Leu Phe Ser Leu Asp Gly Phe Arg Ala Glu Tyr Leu His 115 120 125 Thr Trp Gly Gly Leu Leu Pro Val Ile Ser Lys Leu Lys Lys Cys Gly 130 135 140 Thr Tyr Thr Lys Asn Met Arg Pro Val Tyr Pro Thr Lys Thr Phe Pro 145 150 155 160 Asn His Tyr Ser Ile Val Thr Gly Leu Tyr Pro Glu Ser His Gly Ile 165 170 175 Ile Asp Asn Lys Met Tyr Asp Pro Lys Met Asn Ala Ser Phe Ser Leu 180 185 190 Lys Ser Lys Glu Lys Phe Asn Pro Glu Trp Tyr Lys Gly Glu Pro Ile 195 200 205 Trp Val Thr Ala Lys Tyr Gln Gly Leu Lys Ser Gly Thr Phe Phe Trp 210 215 220 Pro Gly Ser Asp Val Glu Ile Asn Gly Ile Phe Pro Asp Ile Tyr Lys 225 230 235 240 Met Tyr Asn Gly Ser Val Pro Phe Glu Glu Arg Ile Leu Ala Val Leu 245 250 255 Gln Trp Leu Gln Leu Pro Lys Asp Glu Arg Pro His Phe Tyr Thr Leu 260 265 270 Tyr Leu Glu Glu Pro Asp Ser Ser Gly His Ser Tyr Gly Pro Val Ser 275 280 285 Ser Glu Val Ile Lys Ala Leu Gln Arg Val Asp Gly Met Val Gly Met 290 295 300 Leu Met Asp Gly Leu Lys Glu Leu Asn Leu His Arg Cys Leu Asn Leu 305 310 315 320 Ile Leu Ile Ser Asp His Gly Met Glu Gln Gly Ser Cys Lys Lys Tyr 325 330 335 Ile Tyr Leu Asn Lys Tyr Leu Gly Asp Val Lys Asn Ile Lys Val Ile 340 345 350 Tyr Gly Pro Ala Ala Arg Leu Arg Pro Ser Asp Val Pro Asp Lys Tyr 355 360 365 Tyr Ser Phe Asn Tyr Glu Gly Ile Ala Arg Asn Leu Ser Cys Arg Glu 370 375 380 Pro Asn Gln His Phe Lys Pro Tyr Leu Lys His Phe Leu Pro Lys Arg 385 390 395 400 Leu His Phe Ala Lys Ser Asp Arg Ile Glu Pro Leu Thr Phe Tyr Leu 405 410 415 Asp Pro Gln Trp Gln Leu Ala Leu Asn Pro Ser Glu Arg Lys Tyr Cys 420 425 430 Gly Ser Gly Phe His Gly Ser Asp Asn Val Phe Ser Asn Met Gln Ala 435 440 445 Leu Phe Val Gly Tyr Gly Pro Gly Phe Lys His Gly Ile Glu Ala Asp 450 455 460 Thr Phe Glu Asn Ile Glu Val Tyr Asn Leu Met Cys Asp Leu Leu Asn 465 470 475 480 Leu Thr Pro Ala Pro Asn Asn Gly Thr His Gly Ser Leu Asn His Leu 485 490 495 Leu Lys Asn Pro Val Tyr Thr Pro Lys His Pro Lys Glu Val His Pro 500 505 510 Leu Val Gln Cys Pro Phe Thr Arg Asn Pro Arg Asp Asn Leu Gly Cys 515 520 525 Ser Cys Asn Pro Ser Ile Leu Pro Ile Glu Asp Phe Gln Thr Gln Phe 530 535 540 Asn Leu Thr Val Ala Glu Glu Lys Ile Ile Lys His Glu Thr Leu Pro 545 550 555 560 Tyr Gly Arg Pro Arg Val Leu Gln Lys Glu Asn Thr Ile Cys Leu Leu 565 570 575 Ser Gln His Gln Phe Met Ser Gly Tyr Ser Gln Asp Ile Leu Met Pro 580 585 590 Leu Trp Thr Ser Tyr Thr Val Asp Arg Asn Asp Ser Phe Ser Thr Glu 595 600 605 Asp Phe Ser Asn Cys Leu Tyr Gln Asp Phe Arg Ile Pro Leu Ser Pro 610 615 620 Val His Lys Cys Ser Phe Tyr Lys Asn Asn Thr Lys Val Ser Tyr Gly 625 630 635 640 Phe Leu Ser Pro Pro Gln Leu Asn Lys Asn Ser Ser Gly Ile Tyr Ser 645 650 655 Glu Ala Leu Leu Thr Thr Asn Ile Val Pro Met Tyr Gln Ser Phe Gln 660 665 670 Val Ile Trp Arg Tyr Phe His Asp Thr Leu Leu Arg Lys Tyr Ala Glu 675 680 685 Glu Arg Asn Gly Val Asn Val Val Ser Gly Pro Val Phe Asp Phe Asp 690 695 700 Tyr Asp Gly Arg Cys Asp Ser Leu Glu Asn Leu Arg Gln Lys Arg Arg 705 710 715 720 Val Ile Arg Asn Gln Glu Ile Leu Ile Pro Thr His Phe Phe Ile Val 725 730 735 Leu Thr Ser Cys Lys Asp Thr Ser Gln Thr Pro Leu His Cys Glu Asn 740 745 750 Leu Asp Thr Leu Ala Phe Ile Leu Pro His Arg Thr Asp Asn Ser Glu 755 760 765 Ser Cys Val His Gly Lys His Asp Ser Ser Trp Val Glu Glu Leu Leu 770 775 780 Met Leu His Arg Ala Arg Ile Thr Asp Val Glu His Ile Thr Gly Leu 785 790 795 800 Ser Phe Tyr Gln Gln Arg Lys Glu Pro Val Ser Asp Ile Leu Lys Leu 805 810 815 Lys Thr His Leu Pro Thr Phe Ser Gln Glu Asp 820 825 <210> 11 <211> 827 <212> PRT <213> Artificial Sequence <220> <223> Recombinantly synthesized <400> 11 Pro Ser Cys Ala Lys Glu Val Lys Ser Cys Lys Gly Arg Cys Phe Glu 1 5 10 15 Arg Thr Phe Gly Asn Cys Arg Cys Asp Ala Ala Cys Val Glu Leu Gly 20 25 30 Asn Cys Cys Leu Asp Tyr Gln Glu Thr Cys Ile Glu Pro Glu His Ile 35 40 45 Trp Thr Cys Asn Lys Phe Arg Cys Gly Glu Lys Arg Leu Thr Arg Ser 50 55 60 Leu Cys Ala Cys Ser Asp Asp Cys Lys Asp Lys Gly Asp Cys Cys Ile 65 70 75 80 Asn Tyr Ser Ser Val Cys Gln Gly Glu Lys Ser Trp Val Glu Glu Pro 85 90 95 Cys Glu Ser Ile Asn Glu Pro Gln Cys Pro Ala Gly Phe Glu Thr Pro 100 105 110 Pro Thr Leu Leu Phe Ser Leu Asp Gly Phe Arg Ala Glu Tyr Leu His 115 120 125 Thr Trp Gly Gly Leu Leu Pro Val Ile Ser Lys Leu Lys Lys Cys Gly 130 135 140 Thr Tyr Thr Lys Asn Met Arg Pro Val Tyr Pro Thr Lys Thr Phe Pro 145 150 155 160 Asn His Tyr Ser Ile Val Thr Gly Leu Tyr Pro Glu Ser His Gly Ile 165 170 175 Ile Asp Asn Lys Met Tyr Asp Pro Lys Met Asn Ala Ser Phe Ser Leu 180 185 190 Lys Ser Lys Glu Lys Phe Asn Pro Glu Trp Tyr Lys Gly Glu Pro Ile 195 200 205 Trp Val Thr Ala Lys Tyr Gln Gly Leu Lys Ser Gly Thr Phe Phe Trp 210 215 220 Pro Gly Ser Asp Val Glu Ile Asn Gly Thr Phe Pro Asp Ile Tyr Lys 225 230 235 240 Met Tyr Asn Gly Ser Val Pro Phe Glu Glu Arg Ile Leu Ala Val Leu 245 250 255 Gln Trp Leu Gln Leu Pro Lys Asp Glu Arg Pro His Phe Tyr Thr Leu 260 265 270 Tyr Leu Glu Glu Pro Asp Ser Ser Gly His Ser Tyr Gly Pro Val Ser 275 280 285 Ser Glu Val Ile Lys Ala Leu Gln Arg Val Asp Gly Met Val Gly Met 290 295 300 Leu Met Asp Gly Leu Lys Glu Leu Asn Leu His Arg Cys Leu Asn Leu 305 310 315 320 Ile Leu Ile Ser Asp His Gly Met Glu Gln Gly Ser Cys Lys Lys Tyr 325 330 335 Ile Tyr Leu Asn Lys Tyr Leu Gly Asp Val Lys Asn Ile Lys Val Ile 340 345 350 Tyr Gly Pro Ala Ala Arg Leu Arg Pro Ser Asp Val Pro Asp Lys Tyr 355 360 365 Tyr Ser Phe Asn Tyr Glu Gly Ile Ala Arg Asn Leu Ser Cys Arg Glu 370 375 380 Pro Asn Gln His Phe Lys Pro Tyr Leu Lys His Phe Leu Pro Lys Arg 385 390 395 400 Leu His Phe Ala Lys Ser Asp Arg Ile Glu Pro Leu Thr Phe Tyr Leu 405 410 415 Asp Pro Gln Trp Gln Leu Ala Leu Asn Pro Ser Glu Arg Lys Tyr Cys 420 425 430 Gly Ser Gly Phe His Gly Ser Asp Asn Val Phe Ser Asn Met Gln Ala 435 440 445 Leu Phe Val Gly Tyr Gly Pro Gly Phe Lys His Gly Ile Glu Ala Asp 450 455 460 Thr Phe Glu Asn Ile Glu Val Tyr Asn Leu Met Cys Asp Leu Leu Asn 465 470 475 480 Leu Thr Pro Ala Pro Asn Asn Gly Thr His Gly Ser Leu Asn His Leu 485 490 495 Leu Lys Asn Pro Val Tyr Thr Pro Lys His Pro Lys Glu Val His Pro 500 505 510 Leu Val Gln Cys Pro Phe Thr Arg Asn Pro Arg Asp Asn Leu Gly Cys 515 520 525 Ser Cys Asn Pro Ser Ile Leu Pro Ile Glu Asp Phe Gln Thr Gln Phe 530 535 540 Asn Leu Thr Val Ala Glu Glu Lys Ile Ile Lys His Glu Thr Leu Pro 545 550 555 560 Tyr Gly Arg Pro Arg Val Leu Gln Lys Glu Asn Thr Ile Cys Leu Leu 565 570 575 Ser Gln His Gln Phe Met Ser Gly Tyr Ser Gln Asp Ile Leu Met Pro 580 585 590 Leu Trp Thr Ser Tyr Thr Val Asp Arg Asn Asp Ser Phe Ser Thr Glu 595 600 605 Asp Phe Ser Asn Cys Leu Tyr Gln Asp Phe Arg Ile Pro Leu Ser Pro 610 615 620 Val His Lys Cys Ser Phe Tyr Lys Asn Asn Thr Lys Val Ser Tyr Gly 625 630 635 640 Phe Leu Ser Pro Pro Gln Leu Asn Lys Asn Ser Ser Gly Ile Tyr Ser 645 650 655 Glu Ala Leu Leu Thr Thr Asn Ile Val Pro Met Tyr Gln Ser Phe Gln 660 665 670 Val Ile Trp Arg Tyr Phe His Asp Thr Leu Leu Arg Lys Tyr Ala Glu 675 680 685 Glu Arg Asn Gly Val Asn Val Val Ser Gly Pro Val Phe Asp Phe Asp 690 695 700 Tyr Asp Gly Arg Cys Asp Ser Leu Glu Asn Leu Arg Gln Lys Arg Arg 705 710 715 720 Val Ile Arg Asn Gln Glu Ile Leu Ile Pro Thr His Phe Phe Ile Val 725 730 735 Leu Thr Ser Cys Lys Asp Thr Ser Gln Thr Pro Leu His Cys Glu Asn 740 745 750 Leu Asp Thr Leu Ala Phe Ile Leu Pro His Arg Thr Asp Asn Ser Glu 755 760 765 Ser Cys Val His Gly Lys His Asp Ser Ser Trp Val Glu Glu Leu Leu 770 775 780 Met Leu His Arg Ala Arg Ile Thr Asp Val Glu His Ile Thr Gly Leu 785 790 795 800 Ser Phe Tyr Gln Gln Arg Lys Glu Pro Val Ser Asp Ile Leu Lys Leu 805 810 815 Lys Thr His Leu Pro Thr Phe Ser Gln Glu Asp 820 825 <210> 12 <211> 827 <212> PRT <213> Artificial Sequence <220> <223> Recombinantly synthesized <400> 12 Pro Ser Cys Ala Lys Glu Asn Lys Ser Cys Lys Gly Arg Cys Phe Glu 1 5 10 15 Arg Thr Phe Gly Asn Cys Arg Cys Asp Ala Ala Cys Val Glu Leu Gly 20 25 30 Asn Cys Cys Leu Asp Tyr Gln Glu Thr Cys Ile Glu Pro Glu His Ile 35 40 45 Trp Thr Cys Asn Lys Phe Arg Cys Gly Glu Lys Arg Leu Thr Arg Ser 50 55 60 Leu Cys Ala Cys Ser Asp Asp Cys Lys Asp Lys Gly Asp Cys Cys Ile 65 70 75 80 Asn Tyr Ser Ser Val Cys Gln Gly Glu Lys Ser Trp Val Glu Glu Pro 85 90 95 Cys Glu Ser Ile Asn Glu Pro Gln Cys Pro Ala Gly Phe Glu Thr Pro 100 105 110 Pro Thr Leu Leu Phe Ser Leu Asp Gly Phe Arg Ala Glu Tyr Leu His 115 120 125 Thr Trp Gly Gly Leu Leu Pro Val Ile Ser Lys Leu Lys Lys Cys Gly 130 135 140 Thr Tyr Thr Lys Asn Met Arg Pro Val Tyr Pro Thr Lys Thr Phe Pro 145 150 155 160 Asn His Tyr Ser Ile Val Thr Gly Leu Tyr Pro Glu Ser His Gly Ile 165 170 175 Ile Asp Asn Lys Met Tyr Asp Pro Lys Met Asn Ala Ser Phe Ser Leu 180 185 190 Lys Ser Lys Glu Lys Phe Asn Pro Glu Trp Tyr Lys Gly Glu Pro Ile 195 200 205 Trp Val Thr Ala Lys Tyr Gln Gly Leu Lys Ser Gly Thr Phe Phe Trp 210 215 220 Pro Gly Ser Asp Val Glu Ile Asn Gly Thr Phe Pro Asp Ile Tyr Lys 225 230 235 240 Met Tyr Asn Gly Ser Val Pro Phe Glu Glu Arg Ile Leu Ala Val Leu 245 250 255 Gln Trp Leu Gln Leu Pro Lys Asp Glu Arg Pro His Phe Tyr Thr Leu 260 265 270 Tyr Leu Glu Glu Pro Asp Ser Ser Gly His Ser Tyr Gly Pro Val Ser 275 280 285 Ser Glu Val Ile Lys Ala Leu Gln Arg Val Asp Gly Met Val Gly Met 290 295 300 Leu Met Asp Gly Leu Lys Glu Leu Asn Leu His Arg Cys Leu Asn Leu 305 310 315 320 Ile Leu Ile Ser Asp His Gly Met Glu Gln Gly Ser Cys Lys Lys Tyr 325 330 335 Ile Tyr Leu Asn Lys Tyr Leu Gly Asp Val Lys Asn Ile Lys Val Ile 340 345 350 Tyr Gly Pro Ala Ala Arg Leu Arg Pro Ser Asp Val Pro Asp Lys Tyr 355 360 365 Tyr Ser Phe Asn Tyr Glu Gly Ile Ala Arg Asn Leu Ser Cys Arg Glu 370 375 380 Pro Asn Gln His Phe Lys Pro Tyr Leu Lys His Phe Leu Pro Lys Arg 385 390 395 400 Leu His Phe Ala Lys Ser Asp Arg Ile Glu Pro Leu Thr Phe Tyr Leu 405 410 415 Asp Pro Gln Trp Gln Leu Ala Leu Asn Pro Ser Glu Arg Lys Tyr Cys 420 425 430 Gly Ser Gly Phe His Gly Ser Asp Asn Val Phe Ser Asn Met Gln Ala 435 440 445 Leu Phe Val Gly Tyr Gly Pro Gly Phe Lys His Gly Ile Glu Ala Asp 450 455 460 Thr Phe Glu Asn Ile Glu Val Tyr Asn Leu Met Cys Asp Leu Leu Asn 465 470 475 480 Leu Thr Pro Ala Pro Asn Asn Gly Thr His Gly Ser Leu Asn His Leu 485 490 495 Leu Lys Asn Pro Val Tyr Thr Pro Lys His Pro Lys Glu Val His Asn 500 505 510 Leu Thr Gln Cys Pro Phe Thr Arg Asn Pro Arg Asp Asn Leu Gly Cys 515 520 525 Ser Cys Asn Pro Ser Ile Leu Pro Ile Glu Asp Phe Gln Thr Gln Phe 530 535 540 Asn Leu Thr Val Ala Glu Glu Lys Ile Ile Lys His Glu Thr Leu Pro 545 550 555 560 Tyr Gly Arg Pro Arg Val Leu Gln Lys Glu Asn Thr Ile Cys Leu Leu 565 570 575 Ser Gln His Gln Phe Met Ser Gly Tyr Ser Gln Asp Ile Leu Met Pro 580 585 590 Leu Trp Thr Ser Tyr Thr Val Asp Arg Asn Asp Ser Phe Ser Thr Glu 595 600 605 Asp Phe Ser Asn Cys Leu Tyr Gln Asp Phe Arg Ile Pro Leu Ser Pro 610 615 620 Val His Lys Cys Ser Phe Tyr Lys Asn Asn Thr Lys Val Ser Tyr Gly 625 630 635 640 Phe Leu Ser Pro Pro Gln Leu Asn Lys Asn Ser Ser Gly Ile Tyr Ser 645 650 655 Glu Ala Leu Leu Thr Thr Asn Ile Val Pro Met Tyr Gln Ser Phe Gln 660 665 670 Val Ile Trp Arg Tyr Phe His Asp Thr Leu Leu Arg Lys Tyr Ala Glu 675 680 685 Glu Arg Asn Gly Val Asn Val Val Ser Gly Pro Val Phe Asp Phe Asp 690 695 700 Tyr Asp Gly Arg Cys Asp Ser Leu Glu Asn Leu Arg Gln Lys Arg Arg 705 710 715 720 Val Ile Arg Asn Gln Glu Ile Leu Ile Pro Thr His Phe Phe Ile Val 725 730 735 Leu Thr Ser Cys Lys Asp Thr Ser Gln Thr Pro Leu His Cys Glu Asn 740 745 750 Leu Asp Thr Leu Ala Phe Ile Leu Pro His Arg Thr Asp Asn Ser Glu 755 760 765 Ser Cys Val His Gly Lys His Asp Ser Ser Trp Val Glu Glu Leu Leu 770 775 780 Met Leu His Arg Ala Arg Ile Thr Asp Val Glu His Ile Thr Gly Leu 785 790 795 800 Ser Phe Tyr Gln Gln Arg Lys Glu Pro Val Ser Asp Ile Leu Lys Leu 805 810 815 Lys Thr His Leu Pro Thr Phe Ser Gln Glu Asp 820 825 <210> 13 <211> 227 <212> PRT <213> Homo sapiens <400> 13 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 G...

Claims

1. An ENPP1 polypeptide fusion comprising an ENPP1 polypeptide fused to an Fc region of an immunoglobulin, wherein the ENPP1 polypeptide comprises the mutation I256T 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 M883Y, S885N, S885T, T887E, H1064K, and N1065F relative to SEQ ID NO:

7.

3. 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.

4. The polypeptide fusion of claim 1, wherein the ENPP1 polypeptide further comprises at least one mutation selected from the group consisting of C25N, K27T, and V29N relative to SEQ ID NO:

7.

5. The polypeptide fusion of claim 4, 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.

6. The polypeptide fusion of claim 1, wherein the ENPP1 polypeptide further comprises at least one mutation selected from the group consisting of K369N and I371T relative to SEQ ID NO:

7.

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

7.

8. 2. The polypeptide fusion of claim 1, wherein the ENPP1 polypeptide further 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.

9. The polypeptide fusion of claim 8, 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.

10. The polypeptide fusion of claim 1, wherein the ENPP1 polypeptide further comprises at least one mutation selected from the group consisting of E864N and L866T relative to SEQ ID NO:

7.

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

7.

12. 2. The polypeptide fusion of claim 1, 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 with respect to SEQ ID NO:

7.

13. The polypeptide fusion of claim 1, wherein the Fc region is an IgG Fc region.

14. 2. The polypeptide fusion of claim 1, 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.

15. 2. The polypeptide fusion of claim 1, comprising at least one mutation selected from the group consisting of S766N, P534N / Y536T, P554L / R545T, and E592N relative to SEQ ID NO:

7.

16. 2. The polypeptide fusion of claim 1, 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.

17. An ENPP1 polypeptide fusion comprising an ENPP1 polypeptide and an Fc region of an immunoglobulin, the ENPP1 polypeptide fusion comprising the mutations I256T, 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 I256T, P534N, V536T, M883Y, S885T, and T887E relative to SEQ ID NO:

7.

19. An ENPP1 polypeptide fusion comprising an ENPP1 polypeptide and an Fc region of an immunoglobulin, the ENPP1 polypeptide fusion comprising the mutations I256T, E592N, H1064K, and N1065F relative to SEQ ID NO:

7.

20. 1. An ENPP1 mutant polypeptide comprising amino acids 23 to 849 of SEQ ID NO:7, comprising the mutation I256T relative to SEQ ID NO:7, and further comprising a mutation selected from the group consisting of S766N, P534N, V536T, P554L, R545T, and E592N; ENPP1 mutant polypeptide.

21. 21. The mutant polypeptide of claim 20, comprising the amino acid sequence of SEQ ID NO:

7.

22. 22. The mutant polypeptide of any one of claims 20 to 21, which lacks a signal peptide sequence.

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

7.

24. 22. The mutant polypeptide of claim 21, 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.

25. 22. The mutant polypeptide of claim 21, comprising an S885N mutation relative to SEQ ID NO:

7.

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

7.

27. 22. The mutant polypeptide of claim 21, comprising the mutations M883Y, S885T, and T887E relative to SEQ ID NO:

7.

28. 22. The mutant polypeptide of claim 21, comprising the mutations P534N, V536T, H1064K, and N1065F relative to SEQ ID NO:

7.

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

7.

30. 22. The mutant polypeptide of claim 21, comprising the mutations S766N, H1064K, and N1065F relative to SEQ ID NO:

7.

31. 22. The mutant polypeptide of claim 21, comprising the mutations E592N, H1064K, and N1065F relative to SEQ ID NO:

7.

32. 22. The mutant polypeptide of claim 21, comprising the mutations P534N, V536T, M883Y, S885T, and T887E relative to SEQ ID NO:

7.

33. A polypeptide fusion described in any one of claims 1, 17, 18 and 19 or a mutant polypeptide described in claim 20, which is expressed from a CHO cell line stably transfected with human ST6 beta-galactoside alpha-2,6-sialyltransferase (also known as ST6GAL1).

34. Sialic acid and / or N-acetylmannosamine (1,3,4-O-Bu 3 The polypeptide fusion of any one of claims 1, 17, 18 and 19 or the mutant polypeptide of claim 20, which is grown in a cell culture supplemented with ManNAc (also known as ManNAc).

35. A method for 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 fusion described in any one of claims 1, 17, 18, and 19 or a mutant polypeptide described in claim 20.

36. A method for reducing 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 described in any one of claims 1, 17, 18, and 19 or a mutant polypeptide described in claim 20.

37. A method for reducing or preventing the progression of ectopic calcification of soft tissue in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polypeptide fusion described in any one of claims 1, 17, 18, and 19 or a mutant polypeptide described in claim 20.

38. A method for 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 fusion described in any one of claims 1, 17, 18, and 19 or a mutant polypeptide described in claim 20.

39. A method for treating, reversing, 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 described in any one of claims 1, 17, 18, and 19 or a mutant polypeptide described in claim 20.

40. 1. A method for reducing 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: A method comprising administering to a subject a therapeutically effective amount of a polypeptide fusion described in any one of claims 1, 17, 18, and 19 or a mutant polypeptide described in claim 20.

41. A method for reducing 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 described in any one of claims 1, 17, 18, and 19 or a mutant polypeptide described in claim 20.

42. 36. The method of claim 35, wherein the pathological calcification is selected from the group consisting of idiopathic infantile arterial calcification (IIAC) and atherosclerotic plaque calcification.

43. 37. The method of claim 36, wherein the pathological ossification is selected from the group consisting of ossification of the posterior longitudinal ligament (OPLL), hypophosphatemic rickets, and osteoarthritis.

44. 38. The method of claim 37, wherein the soft tissue calcification is selected from the group consisting of IIAC and osteoarthritis.

45. 38. The method of claim 37, 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.

46. 1. A method for increasing pyrophosphate (PPi) levels in a subject having a lower than normal PPi level, comprising: Administering to a subject a therapeutically effective amount of a polypeptide of the polypeptide fusion of any one of claims 1, 17, 18, and 19 or a mutant polypeptide of claim 20, wherein said administration increases the level of PPi in the subject to a normal level of at least 2 μM and maintains it at about the same level. A method comprising:

47. 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: Administering to a subject a therapeutically effective amount of a polypeptide fusion of any one of claims 1, 17, 18, and 19 or a mutant polypeptide of claim 20, thereby reducing pathological calcification or ossification in the subject or preventing the progression of pathological calcification or ossification in the subject. A method comprising:

48. 1. A method of treating an ENPP1 deficiency manifested by a decrease in extracellular pyrophosphate (PPi) concentration in a subject in need thereof, comprising: Administering to a subject a therapeutically effective amount of a polypeptide fusion of any one of claims 1, 17, 18, and 19 or a mutant polypeptide of claim 20, thereby increasing the level of PPi in the subject. A method comprising:

49. The method of any one of claims 35-41 and 46-48, 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 undergoes proteolytic processing to produce the ENPP1 polypeptide.

50. The method of claim 49, wherein in the ENPP1 precursor protein, the signal peptide sequence is linked to the N-terminus of the ENPP1 polypeptide.

51. 50. The method of claim 49, 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.

52. The method of any one of claims 35-41 and 46-48, wherein the polypeptide fusion or mutant polypeptide is administered to the subject acutely or chronically.

53. The method of any one of claims 35-41 and 46-48, wherein the polypeptide fusion or variant polypeptide is administered locally, regionally, parenterally, or systemically to the subject.

54. The method of any one of claims 35-41 and 46-48, 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, ocular, pulmonary, and topical.

55. The method of any one of claims 35-41 and 46-48, 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.

56. The method of any one of claims 35-41 and 46-48, wherein the subject is a mammal.

57. 57. The method of claim 56, wherein the mammal is a human.

58. An ENPP1 mutant polypeptide comprising one or more amino acid substitutions relative to SEQ ID NO:7, comprising an amino acid substitution at position 256 relative to SEQ ID NO:

7.

59. 59. The ENPP1 mutant polypeptide of claim 58, wherein the amino acid sequence of the ENPP1 mutant polypeptide is at least 90% identical to amino acids 23 to 849 of SEQ ID NO:

7.

60. 1. An ENPP1 mutant polypeptide comprising amino acids 23 to 849 of SEQ ID NO:7, There are 10 or fewer amino acid substitutions relative to amino acids 23-849 of SEQ ID NO:7; containing an amino acid substitution at position 256 relative to SEQ ID NO:7, ENPP1 mutant polypeptide.

61. 61. The ENPP1 mutant polypeptide of any one of claims 58 to 60, wherein the amino acid substitution is a substitution of isoleucine (I) with threonine (T) at position 256 relative to SEQ ID NO:

7.

62. 61. The ENPP1 mutant polypeptide of any one of claims 58 to 60, wherein the amino acid substitution is a substitution of isoleucine (I) with serine (S) at position 256 relative to SEQ ID NO:

7.

63. 1. An ENPP1 variant polypeptide comprising an amino acid sequence that is at least 90% identical to amino acids 23 to 849 of SEQ ID NO:7, containing the mutation I256T with respect to SEQ ID NO:7, Further comprising a mutation selected from the group consisting of S766N, P534N, V536T, P554L, R545T, and E592N with respect to SEQ ID NO:7; ENPP1 mutant polypeptide.

64. 64. The ENPP1 mutant polypeptide of claim 63, comprising at least one amino acid substitution selected from the group consisting of S766N, P534N / V536T, P554L / R545T, and E592N relative to SEQ ID NO:

7.

65. 64. The ENPP1 mutant polypeptide of claim 63, comprising the amino acid substitution V29N.

66. 62. The ENPP1 mutant polypeptide of any one of claims 58 to 61, comprising the amino acid sequence of SEQ ID NO:

11.

67. 67. An ENPP1 mutant polypeptide fusion comprising an ENPP1 mutant polypeptide according to any one of claims 58 to 66 and a heterologous protein.

68. The ENPP1 mutant polypeptide fusion of claim 67, wherein the heterologous protein is an FcRn binding domain.

69. The ENPP1 mutant polypeptide fusion of any one of claims 67 to 68, wherein the heterologous protein is located carboxy-terminal to the ENPP1 mutant polypeptide of the fusion.

70. The ENPP1 mutant polypeptide fusion of any one of claims 67 to 68, wherein the heterologous protein is amino-terminal to the ENPP1 mutant polypeptide of the fusion.

71. The ENPP1 mutant polypeptide fusion of any one of claims 68 to 70, wherein the FcRn binding domain is an albumin polypeptide.

72. The ENPP1 mutant polypeptide fusion of any one of claims 68 to 70, wherein the FcRn binding domain is the Fc portion of an immunoglobulin molecule.

73. The ENPP1 mutant polypeptide fusion of claim 72, wherein the immunoglobulin molecule is IgG1.

74. The ENPP1 mutant polypeptide fusion of any one of claims 68 to 73, wherein the FcRn-binding domain comprises one or more amino acid substitutions relative to the wild-type FcRn-binding domain.

75. The ENPP1 mutant polypeptide fusion of any one of claims 68 to 70 and 72 to 74, wherein the FcRn binding domain is the Fc portion of a human IgG1 molecule and comprises the following amino acid substitutions relative to SEQ ID NO:7: M883Y, S885T, and T887E, respectively.

76. 76. The ENPP1 mutant polypeptide fusion of any one of claims 68 to 70 and 72 to 75, comprising one or more of the following substitutions with respect to SEQ ID NO:7: S885N, S766N, M883Y / S885T / T887E, P534N / V536T / H1064K / N1065F, P554L / R545T, S766N / H1064K / N1065F, E592N / H1064K / N1065F, or P534N / V536T / M883Y / S885T / T887E.

77. The ENPP1 mutant polypeptide fusion of any one of claims 68 to 70 and 72 to 76, comprising an S885N mutation relative to SEQ ID NO:

7.

78. The ENPP1 mutant polypeptide fusion of any one of claims 68 to 70 and 72 to 77, comprising an S766N mutation relative to SEQ ID NO:

7.

79. The ENPP1 mutant polypeptide fusion of any one of claims 68 to 70 and 72 to 78, comprising the mutations M883Y, S885T, and T887E relative to SEQ ID NO:

7.

80. 80. The ENPP1 mutant polypeptide fusion of any one of claims 68 to 70 and 72 to 79, comprising the mutations P534N, V536T, H1064K, and N1065F with respect to SEQ ID NO:

7.

81. The ENPP1 mutant polypeptide fusion of any one of claims 68 to 70 and 72 to 80, comprising the mutations P554L and R545T with respect to SEQ ID NO:

7.

82. The ENPP1 mutant polypeptide fusion of any one of claims 68 to 70 and 72 to 81, comprising the mutations S766N, H1064K, and N1065F relative to SEQ ID NO:

7.

83. The ENPP1 mutant polypeptide fusion of any one of claims 68 to 70 and 72 to 82, comprising the mutations E592N, H1064K, and N1065F relative to SEQ ID NO:

7.

84. The ENPP1 mutant polypeptide fusion of any one of claims 68 to 70 and 72 to 83, comprising the mutations P534N, V536T, M883Y, S885T, and T887E with respect to SEQ ID NO:

7.

85. The ENPP1 mutant polypeptide fusion of any one of claims 68 to 70 and 72 to 84, 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.

86. The ENPP1 mutant polypeptide fusion of any one of claims 68 to 70 and 72 to 85, 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.

87. An ENPP1 mutant polypeptide fusion according to any one of claims 68 to 70 and 72 to 86 or an ENPP1 mutant polypeptide according to any one of claims 58 to 65, comprising at least one mutation selected from the group consisting of C25N, K27T, and V29N relative to SEQ ID NO:

7.

88. An ENPP1 mutant polypeptide fusion according to any one of claims 68 to 70 and 72 to 87 or an ENPP1 mutant polypeptide according to any one of claims 58 to 65 and 85, comprising at least one mutation selected from the group consisting of C25N / K27T and V29N relative to SEQ ID NO:

7.

89. An ENPP1 mutant polypeptide fusion according to any one of claims 68 to 70 and 72 to 88 or an ENPP1 mutant polypeptide according to any one of claims 58 to 65 and 87 to 88, comprising one mutation selected from the group consisting of K369N and I371T with respect to SEQ ID NO:

7.

90. An ENPP1 mutant polypeptide fusion according to any one of claims 68 to 70 and 72 to 89 or an ENPP1 mutant polypeptide according to any one of claims 58 to 65 and 87 to 89, comprising the mutations K369N / I371T with respect to SEQ ID NO:

7.

91. An ENPP1 mutant polypeptide fusion according to any one of claims 68 to 70 and 72 to 90 or an ENPP1 mutant polypeptide according to any one of claims 58 to 65 and 87 to 90, comprising at least one mutation selected from the group consisting of P534N, V536T, R545T, P554L, E592N, R741D and S766N with respect to SEQ ID NO:

7.

92. An ENPP1 mutant polypeptide fusion according to any one of claims 68 to 70 and 72 to 91 or an ENPP1 mutant polypeptide according to any one of claims 58 to 65 and 87 to 91, comprising at least one mutation selected from the group consisting of P534N / V536T, P554L / R545T, E592N, E592N / R741D, and S766N with respect to SEQ ID NO:

7.

93. SEQ ID C25N, K27T, V29N, C25N / K27T, K369N, I371T, K369N / I371T, P534N, V536T, R545T, P554L, E59 regarding NO:7 2N, R741D, S766N, P534N / V536T, P554L / R545T, E592N / R741D, E864N, L866T, E864N / L866T, M883Y, S885 An ENPP1 mutant polypeptide fusion according to any one of claims 68 to 70 and 72 to 92 or an ENPP1 mutant polypeptide according to any one of claims 58 to 65 and 87 to 92, comprising at least one mutation selected from the group consisting of N, S885T, T887E, H1064K, N1065F, M883Y / S885T / T887E, H1064K / N1065F.

94. An ENPP1 mutant polypeptide fusion according to any one of claims 68 to 70 and 72 to 93 or an ENPP1 mutant polypeptide according to any one of claims 58 to 65 and 87 to 93, 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.

95. An ENPP1 mutant polypeptide fusion according to any one of claims 68 to 70 and 72 to 94 or an ENPP1 mutant polypeptide according to any one of claims 58 to 65 and 87 to 94, comprising at least one mutation selected from the group consisting of S766N, P534N / Y536T, P554L / R545T, and E592N with respect to SEQ ID NO:

7.

96. ENPP1 mutant polypeptide fusion according to any one of claims 68 to 70 and 72 to 95 or ENPP1 mutant polypeptide according to any one of claims 58 to 65 and 87 to 95, 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.

97. The ENPP1 mutant polypeptide fusion of any one of claims 68 to 70 and 72 to 96, comprising the mutations I256T, M883Y, S885T, and T887E relative to SEQ ID NO:

7.

98. The ENPP1 mutant polypeptide fusion of any one of claims 68 to 70 and 72 to 96, comprising the mutations I256T, P534N, V536T, M883Y, S885T, and T887E with respect to SEQ ID NO:

7.

99. The ENPP1 mutant polypeptide fusion of any one of claims 68 to 70 and 72 to 96, comprising the mutations I256T, E592N, H1064K, and N1065F relative to SEQ ID NO:

7.

100. The fusion of any one of claims 67 to 99, comprising a linker amino acid sequence.

101. The fusion of claim 100, wherein a linker amino acid sequence connects the ENPP1 mutant polypeptide portion of the fusion and the heterologous protein.

102. The fusion of any one of claims 100 to 101, wherein the linker amino acid sequence comprises SEQ ID NO:8 or SEQ ID NO:

9.

103. A nucleic acid encoding an ENPP1 mutant polypeptide according to any one of claims 58 to 66 or a fusion according to any one of claims 67 to 102.

104. A vector comprising the nucleic acid of claim 103.

105. An expression vector comprising the nucleic acid of claim 103.

106. 107. A cell or a plurality of cells, each comprising the nucleic acid of claim 103, the vector of claim 104, and / or the expression vector of claim 105.

107. 107. The cell or cells of claim 106, which are CHO cells and / or NS0 cells.

108. The cell or cells of claim 107, wherein the CHO cell is stably transfected with human ST6 beta-galactoside alpha-2,6-sialyltransferase.

109. A method for producing an ENPP1 mutant polypeptide or fusion, comprising culturing a cell or a plurality of cells according to any one of claims 106 to 108 under conditions suitable for expression of the ENPP1 mutant polypeptide or fusion by the cell or a plurality of cells.

110. The method of claim 109, wherein the cells are cultured in a medium supplemented with sialic acid and / or N-acetylmannosamine.

111. The method of any one of claims 109 to 110, further comprising purifying the ENPP1 mutant polypeptide or fusion from said cell, said plurality of cells, or the medium in which said cell or said plurality of cells was cultured.

112. 112. An ENPP1 mutant polypeptide or fusion purified by the method of claim 111.

113. (i) an ENPP1 mutant polypeptide according to any one of claims 58 to 66, 87 to 96 and 112, and / or an ENPP1 mutant polypeptide fusion according to any one of claims 67 to 102 and 112, and (ii) heterogeneous parts; A conjugate comprising:

114. 114. The conjugate of claim 113, wherein the heterologous moiety is polyethylene glycol.

115. A pharmaceutical composition comprising an ENPP1 mutant polypeptide according to any one of claims 58 to 66, 87 to 96 and 112, a fusion according to any one of claims 67 to 102 and 112, a nucleic acid according to claim 103, a vector according to claim 104, an expression vector according to claim 105 and / or a conjugate according to any one of claims 113 to 114, and a pharma- ceutically acceptable carrier.

116. 1. A method for reducing or preventing the progression of pathological calcification in a subject in need thereof, comprising: (a) an ENPP1 mutant polypeptide according to any one of claims 58 to 66, 87 to 96, and 112; (b) a fusion according to any one of claims 67 to 102 and 112; (c) a conjugate according to any one of claims 113 to 114; and / or (d) The pharmaceutical composition of claim 115. to the subject in a therapeutically effective amount, thereby reducing or preventing the progression of pathological calcification in the subject. A method comprising:

117. 1. A method for reducing or preventing the progression of pathological ossification in a subject in need thereof, comprising: (a) an ENPP1 mutant polypeptide according to any one of claims 58 to 66, 87 to 96, and 112; (b) a fusion according to any one of claims 67 to 102 and 112; (c) a conjugate according to any one of claims 113 to 114; and / or (d) The pharmaceutical composition of claim 115. to the subject in a therapeutically effective amount, thereby reducing or preventing the progression of pathological ossification in the subject. A method comprising:

118. 1. A method for reducing or preventing the progression of ectopic calcification of soft tissue in a subject in need thereof, comprising: (a) an ENPP1 mutant polypeptide according to any one of claims 58 to 66, 87 to 96, and 112; (b) a fusion according to any one of claims 67 to 102 and 112; (c) a conjugate according to any one of claims 113 to 114; and / or (d) The pharmaceutical composition of claim 115. to the subject in a therapeutically effective amount, thereby reducing or preventing the progression of ectopic soft tissue calcification in the subject. A method comprising:

119. 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: (a) an ENPP1 mutant polypeptide according to any one of claims 58 to 66, 87 to 96, and 112; (b) a fusion according to any one of claims 67 to 102 and 112; (c) a conjugate according to any one of claims 113 to 114; and / or (d) The pharmaceutical composition of claim 115. to the subject in a therapeutically effective amount, thereby reducing, reversing, or preventing ossification of the posterior longitudinal ligament (OPLL) in the subject. A method comprising:

120. 1. A method of treating, reversing, or preventing the progression of hypophosphatemic rickets in a subject in need thereof, comprising: (a) an ENPP1 mutant polypeptide according to any one of claims 58 to 66, 87 to 96, and 112; (b) a fusion according to any one of claims 67 to 102 and 112; (c) a conjugate according to any one of claims 113 to 114; and / or (d) The pharmaceutical composition of claim 115. to the subject in a therapeutically effective amount, thereby reducing, reversing, or preventing the progression of hypophosphatemic rickets in the subject. A method comprising:

121. 1. A method for reducing 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: (a) an ENPP1 mutant polypeptide according to any one of claims 58 to 66, 87 to 96, and 112; (b) a fusion according to any one of claims 67 to 102 and 112; (c) a conjugate according to any one of claims 113 to 114; and / or (d) The pharmaceutical composition of claim 115. to the subject in a therapeutically effective amount, thereby reducing or preventing the progression of the disease. A method comprising:

122. 1. A method for reducing or preventing the progression of age-related arteriosclerosis in a subject in need thereof, comprising: (a) an ENPP1 mutant polypeptide according to any one of claims 58 to 66, 87 to 96, and 112; (b) a fusion according to any one of claims 67 to 102 and 112; (c) a conjugate according to any one of claims 113 to 114; and / or (d) The pharmaceutical composition of claim 115. to a subject in a therapeutically effective amount, thereby reducing or preventing the progression of age-related arteriosclerosis in the subject. A method comprising:

123. 1. A method for increasing pyrophosphate (PPi) levels in a subject having a lower than normal PPi level, comprising: (a) an ENPP1 mutant polypeptide according to any one of claims 58 to 66, 87 to 96, and 112; (b) a fusion according to any one of claims 67 to 102 and 112; (c) a conjugate according to any one of claims 113 to 114; and / or (d) The pharmaceutical composition of claim 115. to a subject in a therapeutically effective amount, wherein said administration increases the level of PPi in the subject to a normal level of at least 2 μM and maintains it at about the same level. A method comprising:

124. 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: (a) an ENPP1 mutant polypeptide according to any one of claims 58 to 66, 87 to 96, and 112; (b) a fusion according to any one of claims 67 to 102 and 112; (c) a conjugate according to any one of claims 113 to 114; and / or (d) The pharmaceutical composition of claim 115. to a subject in a therapeutically effective amount, thereby reducing pathological calcification or ossification in the subject or preventing the progression of pathological calcification or ossification in the subject. A method comprising:

125. 1. A method of treating an ENPP1 deficiency manifested by a decrease in extracellular pyrophosphate (PPi) concentration in a subject in need thereof, comprising: (a) an ENPP1 mutant polypeptide according to any one of claims 58 to 66, 87 to 96, and 112; (b) a fusion according to any one of claims 67 to 102 and 112; (c) a conjugate according to any one of claims 113 to 114; and / or (d) The pharmaceutical composition of claim 115. to a subject in a therapeutically effective amount, thereby increasing PPi levels in the subject. A method comprising:

126. The method of any one of claims 116-125, wherein the mutant polypeptide, fusion, conjugate, or pharmaceutical composition is administered to the subject acutely or chronically.

127. The method of any one of claims 116-126, wherein the mutant polypeptide, fusion, conjugate, or pharmaceutical composition is administered locally, regionally, parenterally, or systemically to the subject.

128. The method of any one of claims 116 to 127, wherein the subject is a human.

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