ENPP1 polypeptide and its usage

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 of related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 830,230, filed on 5 April 2019, No. 62 / 983,142, filed on 28 February 2020, and No. 62 / 984,650, filed on 3 March 2020, all of which are incorporated herein by reference in their entirety. [Background technology]

[0002] 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, with the exception of ENPP2, which is transported to the plasma membrane but cleaved by furin and released into the extracellular fluid. ENPP enzymes exhibit a high degree of sequence and structural homology, but show diverse substrate specificity ranging from nucleotides to lipids.

[0003] ENPP1 (also known as PC-1) is a type 2 extracellular membrane-bound glycoprotein located in mineral storage matrix vesicles of osteoblasts and chondrocytes, which hydrolyzes extracellular nucleotides (mainly ATP) to adenosine monophosphate (AMP) and inorganic pyrophosphate (PPi). PPi binds to newly formed hydroxyapatite (HA) crystals, thereby preventing the future growth of these crystals and functioning as a potent inhibitor of ectopic tissue mineralization. ENPP1 produces PPi through the hydrolysis of nucleotide triphosphates (NTPs). Progressive ankylosing protein (ANK) transports intracellular PPi into the extracellular space, and tissue-nonspecific alkaline phosphatase activity (TNAP) removes PPi through direct hydrolysis from PPi to Pi.

[0004] Ectopic tissue mineralization is associated with many human diseases, including chronic joint diseases and acute fatal infant syndromes. 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 crucial regulator of ectopic tissue mineralization. The activity of three extracellular enzymes, TNAP, ANK, and ENPP1, strictly controls the concentrations of Pi and PPi in mammals to 1–3 mM and 2–3 μM, respectively. PPi is a regulator of in vivo mineralization, inhibiting the formation of basic calcium phosphate from amorphous calcium phosphate.

[0005] ENPP1 polypeptides have been shown to be effective in treating certain ectopic tissue calcification diseases. ENPP1-Fc has been shown to reduce systemic arterial calcification in a mouse model of GACI (generalized arterial calcification in infants), a serious disease that occurs in infants and involves widespread arterial calcification (Albright, et al., 2015, Nature Comm. 10006 (Non-Patent Literature 1)). ENPP1 fusion proteins have also been reported to treat severe tissue calcification diseases (PCT application numbers WO2014 / 126965 (Patent Literature 1) and WO2016 / 187408 (Patent Literature 2)), and ENPP1 fusion proteins containing a bone-targeting domain have been reported to treat GACI (PCT application number WO / 2012 / 125182 (Patent Literature 3)).

[0006] There is a need in the art for polypeptides that can be used in vivo to treat certain calcification or ossification disorders. Such polypeptides should have an in vivo half-life that allows for convenient and effective administration of the polypeptide to the target in need. The present invention satisfies 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 Document]

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

[0009] Brief Summary of the Invention The present disclosure provides an ENPP1 polypeptide fusion comprising an ENPP1 polypeptide fused to the Fc region of an immunoglobulin and comprising at least one point mutation described herein. The present disclosure further provides an ENPP1 mutant polypeptide comprising at least one point mutation described elsewhere herein. The present disclosure further provides a polypeptide fusion or mutant polypeptide, each 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, any of which is increased in a cell culture supplemented with sialic acid and / or N-acetylmannosamine (l,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, the method comprising administering to the subject a therapeutically effective amount of the polypeptide fusion and / or mutant polypeptide of the present disclosure.

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

[0012] The Disclosure further provides a method for reducing and / or preventing the progression of ectopic calcification of soft tissue in a subject where such reduction is needed, the method comprising administering a therapeutically effective amount of the polypeptide fusion and / or mutant polypeptide of the Disclosure to the subject.

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

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

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

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

[0017] The Disclosure further provides a method for increasing the PPi level in a subject having a PPi level lower than the normal level, comprising the step of administering a therapeutically effective amount of the polypeptide fusion and / or mutant polypeptide of the Disclosure to the subject, thereby increasing the PPi level in the subject to at least the normal level of 2 μM and maintaining it at substantially the same level.

[0018] The Disclosure further provides a method for reducing and / or preventing the progression of pathological calcification or ossification in a subject having pyrophosphate (PPi) levels lower than normal levels, comprising the step of administering a therapeutically effective amount of the polypeptide fusion and / or mutant polypeptide of the Disclosure to the subject, thereby reducing and / or preventing the progression of pathological calcification or ossification in the subject.

[0019] The Disclosure further provides a method for treating ENPP1 deficiency manifested by a decrease in extracellular pyrophosphate (PPi) concentration in a subject in need thereof, comprising the step of administering a therapeutically effective amount of the polypeptide fusion and / or mutant polypeptide of the Disclosure to the subject, thereby increasing the level of PPi in the subject. [Invention 1001] An ENPP1 polypeptide fusion comprising an ENPP1 polypeptide fused to the Fc region of an immunoglobulin, wherein the ENPP1 polypeptide contains the I256T mutation related to SEQ ID NO:7. [Invention 1002] A polypeptide fusion of the present invention 1001, wherein the Fc region contains at least one mutation selected from the group consisting of M883Y, S885N, S885T, T887E, H1064K, and N1065F relating to SEQ ID NO:7. [Invention 1003] A polypeptide fusion of the present invention 1001, wherein the Fc region contains at least one mutation selected from the group consisting of S885N, M883Y, M883Y / S885T / T887E, and H1064K / N1065F with respect to SEQ ID NO:7. [Invention 1004] A 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 with respect to SEQ ID NO:7. [Invention 1005] A polypeptide fusion of the present invention 1004, wherein the ENPP1 polypeptide contains at least one mutation selected from the group consisting of C25N / K27T and V29N with respect to SEQ ID NO:7. [Invention 1006] A 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 with respect to SEQ ID NO:7. [Invention 1007] A polypeptide fusion of the present invention 1006, comprising the ENPP1 polypeptide containing the mutation K369N / I371T related to SEQ ID NO:7. [Invention 1008] A 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 relating to SEQ ID NO:7. [Invention 1009] A polypeptide fusion of the present invention 1008, wherein the ENPP1 polypeptide contains 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. [Invention 1010] A 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 with respect to SEQ ID NO:7. [Invention 1011] A polypeptide fusion of the present invention 1010, wherein the ENPP1 polypeptide contains at least the E864N / L866T mutation related to SEQ ID NO:7. [Invention 1012] SEQ ID A 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 with respect to NO:7. [Invention 1013] A polypeptide fusion of the present invention 1001, wherein the Fc region is the Fc region of IgG. [Invention 1014] A 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. [Invention 1015] A 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 with respect to SEQ ID NO:7. [Invention 1016] A 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. [Invention 1017] An ENPP1 polypeptide fusion comprising an ENPP1 polypeptide and an immunoglobulin Fc region, comprising mutations I256T, M883Y, S885T, and T887E relating to SEQ ID NO:7. [Invention 1018] An ENPP1 polypeptide fusion comprising an ENPP1 polypeptide and an immunoglobulin Fc region, comprising mutations I256T, P534N, V536T, M883Y, S885T, and T887E relating to SEQ ID NO:7. [Invention 1019] An ENPP1 polypeptide fusion comprising an ENPP1 polypeptide and an immunoglobulin Fc region, comprising mutations I256T, E592N, H1064K, and N1065F relating to SEQ ID NO:7. [Invention 1020] An ENPP1 mutant polypeptide containing amino acids 23-849 of SEQ ID NO:7, The mutation I256T related to SEQ ID NO:7 is included, and further includes mutations selected from the group consisting of S766N, P534N, V536T, P554L, R545T, and E592N. ENPP1 mutant polypeptide. [Invention 1021] A mutant polypeptide of the present invention 1020, containing the amino acid sequence of SEQ ID NO:7. [Invention 1022] A mutant polypeptide according to any of the invention 1020-1021, lacking a signal peptide sequence. [Invention 1023] A mutant polypeptide of the present invention 1020 comprising at least one mutation selected from the group consisting of S766N, P534N / V536T, P554L / R545T, and E592N with respect to SEQ ID NO:7. [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 related to SEQ ID NO:7. [Invention 1025] A mutant polypeptide of the present invention 1021, comprising the S885N mutation related to SEQ ID NO:7. [Invention 1026] A mutant polypeptide of the present invention 1020, comprising the S766N mutation related to SEQ ID NO:7. [Invention 1027] Mutant polypeptides of the present invention 1021, comprising mutations M883Y, S885T, and T887E related to SEQ ID NO:7. [Invention 1028] Mutant polypeptides of the present invention 1021, comprising mutations P534N, V536T, H1064K, and N1065F related to SEQ ID NO:7. [Invention 1029] Mutant polypeptides of the present invention 1020, including mutations P554L and R545T related to SEQ ID NO:7. [Invention 1030] Mutant polypeptides of the present invention 1021, comprising mutations S766N, H1064K, and N1065F related to SEQ ID NO:7. [Invention 1031] Mutant polypeptides of Invention 1021, comprising mutations E592N, H1064K, and N1065F related to SEQ ID NO:7. [Invention 1032] Mutant polypeptides of the present invention 1021, comprising mutations P534N, V536T, M883Y, S885T, and T887E related to SEQ ID NO:7. [Invention 1033] A polypeptide fusion of any of Invention 1001, 1017, 1018, and 1019 or a mutant polypeptide of Invention 1020, expressed from a CHO cell line stably transfected with human ST6 beta-galactoside alpha-2,6-sialyltransferase (also known as ST6GAL1). [Invention 1034] Polypeptide fusions of any of Invention 1001, 1017, 1018, and 1019 or mutant polypeptides of Invention 1020 are grown in cell cultures supplemented with sialic acid and / or N-acetylmannosamine (also known as 1,3,4-O-Bu3ManNAc). [Invention 1035] A method for reducing or preventing the progression of pathological calcification in a subject requiring such reduction, comprising the step of administering to the subject a polypeptide fusion of any of Invention 1001, 1017, 1018, and 1019 or a mutant polypeptide of Invention 1020 in a therapeutically effective dose. [Invention 1036] A method for reducing or preventing the progression of pathological ossification in a subject requiring such reduction, comprising the step of administering to the subject a therapeutically effective dose of a polypeptide fusion of any of Invention 1001, 1017, 1018, and 1019 or a mutant polypeptide of Invention 1020. [Invention 1037] A method for reducing or preventing the progression of ectopic calcification of soft tissue in a subject requiring such reduction, comprising the step of administering to the subject a therapeutically effective dose of a polypeptide fusion of any of the inventions 1001, 1017, 1018, and 1019 or a mutant polypeptide of the invention 1020. [Invention 1038] A method for treating, improving, or preventing the progression of ossification of the posterior longitudinal ligament (OPLL) in a subject requiring such treatment, comprising the step of administering to the subject a therapeutically effective dose of a polypeptide fusion of any of Invention 1001, 1017, 1018, and 1019 or a mutant polypeptide of Invention 1020. [Invention 1039] A method for treating, reversing, or preventing the progression of hypophosphatemic rickets in a subject requiring such treatment, comprising the step of administering to the subject a therapeutically effective dose of a polypeptide fusion of any of Invention 1001, 1017, 1018, and 1019 or a mutant polypeptide of Invention 1020. [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 arteriosclerosis (CUA), calciphylaxis, ossification of the posterior longitudinal ligament (OPLL), hypophosphatemic rickets, osteoarthritis, age-related arteriosclerosis, idiopathic infantile arteriosclerosis (IIAC), generalized infantile arteriosclerosis (GACI), and arteriosclerotic plaque calcification, in a subject diagnosed with at least one of the said diseases, A method comprising the step of administering to a subject a therapeutically effective dose of a polypeptide fusion of any of the inventions 1001, 1017, 1018, and 1019 or a mutant polypeptide of the invention 1020. [Invention 1041] A method for reducing or preventing the progression of age-related arteriosclerosis in a subject requiring such reduction, comprising the step of administering to the subject a polypeptide fusion of any of Invention 1001, 1017, 1018, and 1019 or a mutant polypeptide of Invention 1020 in a therapeutically effective amount. [Invention 1042] The method of the present invention 1035, wherein the pathological calcification is selected from the group consisting of idiopathic infantile arterial calcification (IIAC) and atherosclerotic plaque calcification. [Invention 1043] The method of the present invention 1036, wherein the pathological ossification is selected from the group consisting of ossification of the posterior longitudinal ligament (OPLL), hypophosphatemic rickets, and osteoarthritis. [Invention 1044] The method of the present invention 1037, wherein soft tissue calcification is selected from the group consisting of IIAC and osteoarthritis. [Invention 1045] The method of the present invention 1037, wherein the soft tissue is selected from the group consisting of arteriosclerotic plaque, muscular arteries, joints, vertebrae, articular cartilage, intervertebral disc cartilage, blood vessels, and connective tissue. [Invention 1046] A method for increasing pyrophosphate (PPi) levels in subjects having PPi levels lower than normal levels, A step of administering to a subject a polypeptide of any polypeptide fusion of Invention 1001, 1017, 1018, and 1019 or a mutant polypeptide of Invention 1020 in a therapeutically effective dose, wherein the administration raises the level of PPi in the subject to a normal level of at least 2 μM and maintains it at substantially the same level. Methods that include... [Invention 1047] A method for reducing or preventing the progression of pathological calcification or ossification in subjects with pyrophosphate (PPi) levels lower than normal levels, A step of administering to a subject a therapeutically effective dose of a polypeptide fusion of any of Invention 1001, 1017, 1018, and 1019 or a mutant polypeptide of Invention 1020, wherein pathological calcification or ossification in the subject is reduced or the progression of pathological calcification or ossification in the subject is prevented. Methods that include... [Invention 1048] A method for treating ENPP1 deficiency that manifests due to a decrease in extracellular pyrophosphate (PPi) concentration in subjects requiring it, A step of administering to a subject a polypeptide fusion of any of Invention 1001, 1017, 1018, and 1019 or a mutant polypeptide of Invention 1020 in a therapeutically effective dose, wherein the level of PPi in the subject increases. Methods that include... [Invention 1049] The method according to any one of the present invention 1035-1041 and 1046-1048, wherein the polypeptide fusion or mutant polypeptide is a secretion product of an ENPP1 precursor protein expressed in mammalian cells, the ENPP1 precursor protein comprises a signal peptide sequence and an ENPP1 polypeptide, and the ENPP1 precursor protein undergoes proteolytic processing to produce an ENPP1 polypeptide. [Invention 1050] The method of the present invention 1049, wherein in the ENPP1 precursor protein, the signal peptide sequence is ligated to the N-terminus of the ENPP1 polypeptide. [Invention 1051] The method of the present invention 1049, wherein the signal peptide sequence is selected from the group consisting of the ENPP1 signal peptide sequence, the ENPP2 signal peptide sequence, the ENPP7 signal peptide sequence, and the ENPP5 signal peptide sequence. [Invention 1052] A method according to any one of items 1035-1041 and 1046-1048 of the present invention, wherein a polypeptide fusion or mutant polypeptide is administered to a subject for a short or long period of time. [Invention 1053] A method according to any of items 1035-1041 and 1046-1048 of the present invention, wherein a polypeptide fusion or mutant polypeptide is administered to a target topically, regionally, parenterally, or systemically. [Invention 1054] A method according to any one of items 1035-1041 and 1046-1048 of the present invention, wherein a polypeptide fusion or mutant polypeptide is administered to a subject by at least one route selected from the group consisting of subcutaneous, oral, aerosol, inhalation, rectal, vaginal, transdermal, subcutaneous, intranasal, oral, sublingual, parenteral, intrathecal, intragastric, ocular, pulmonary, and topical. [Invention 1055] A method according to any one of items 1035-1041 and 1046-1048 of the present invention, wherein a polypeptide fusion or mutant polypeptide is administered to a subject as a pharmaceutical composition further comprising at least one pharmaceutically acceptable carrier. [Invention 1056] Any method of the present invention 1035-1041 and 1046-1048, wherein the subject is a mammal. [Invention 1057] The method of the present invention 1056, wherein the mammal is a human. [Invention 1058] An ENPP1 mutant polypeptide comprising one or more amino acid substitutions related to SEQ ID NO:7, wherein the amino acid substitution is located at position 256 with respect to SEQ ID NO:7. [Invention 1059] The ENPP1 mutant polypeptide of the present invention 1058, wherein the amino acid sequence of the ENPP1 mutant polypeptide is at least 90% identical to amino acids 23-849 of SEQ ID NO:7. [Invention 1060] An ENPP1 mutant polypeptide containing amino acids 23-849 of SEQ ID NO:7, There are 10 or fewer amino acid substitutions between amino acids 23-849 of SEQ ID NO:7. SEQ ID NO:7 contains an amino acid substitution at position 256. ENPP1 mutant polypeptide. [Invention 1061] An ENPP1 mutant polypeptide according to any of Invention 1058-1060, wherein the amino acid substitution is a substitution of isoleucine (I) with threonine (T) at position 256 relative to SEQ ID NO:7. [Invention 1062] An ENPP1 mutant polypeptide according to any of Invention 1058-1060, wherein the amino acid substitution is a substitution of serine (S) with isoleucine (I) at position 256 relative to SEQ ID NO:7. [Invention 1063] An ENPP1 mutant polypeptide containing an amino acid sequence that is at least 90% identical to amino acids 23-849 of SEQ ID NO:7, Includes mutation I256T related to SEQ ID NO:7, Further including mutations selected from the group consisting of S766N, P534N, V536T, P554L, R545T, and E592N related to SEQ ID NO:7, ENPP1 mutant polypeptide. [Invention 1064] An ENPP1 mutant polypeptide of the present invention 1063, comprising at least one amino acid substitution selected from the group consisting of S766N, P534N / V536T, P554L / R545T, and E592N for SEQ ID NO:7. [Invention 1065] An ENPP1 mutant polypeptide of the present invention 1063, containing the amino acid substitution V29N. [Invention 1066] An ENPP1 mutant polypeptide according to any of the present invention 1058-1061, containing the amino acid sequence of SEQ ID NO:11. [Invention 1067] An ENPP1 mutant polypeptide fusion comprising any of the ENPP1 mutant polypeptides of Invention 1058 to 1066 and a heterologous protein. [Invention 1068] An ENPP1 mutant polypeptide fusion of the present invention 1067, wherein the heterologous protein is an FcRn-binding domain. [Invention 1069] An ENPP1 mutant polypeptide fusion according to any of the present invention 1067 to 1068, wherein a heterologous protein is located at the carboxyl terminus of the ENPP1 mutant polypeptide of the fusion. [Invention 1070] An ENPP1 mutant polypeptide fusion according to any of invention 1067 to 1068, wherein a heterologous protein is located at the amino-terminal end of the ENPP1 mutant polypeptide of the fusion. [Invention 1071] An ENPP1 mutant polypeptide fusion according to any of the present invention 1068 to 1070, wherein the FcRn binding domain is an albumin polypeptide. [Invention 1072] An ENPP1 mutant polypeptide fusion according to any of the present inventions 1068 to 1070, wherein the FcRn binding domain is the Fc portion of an immunoglobulin molecule. [Invention 1073] An ENPP1 mutant polypeptide fusion of the present invention 1072, wherein the immunoglobulin molecule is IgG1. [Invention 1074] An ENPP1 mutant polypeptide fusion according to any of invention 1068 to 1073, wherein the FcRn binding domain comprises one or more amino acid substitutions relative to the wild-type FcRn binding domain. [Invention 1075] An ENPP1 mutant polypeptide fusion according to any of the inventions 1068-1070 and 1072-1074, wherein the FcRn binding domain is the Fc portion of a human IgG1 molecule, and each contains the following amino acid substitutions relative to SEQ ID NO:7: M883Y, S885T, and T887E. [Invention 1076] An ENPP1 mutant polypeptide fusion of any of the inventions 1068-1070 and 1072-1075, each comprising one or more of the following substitutions for 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. [Invention 1077] An ENPP1 mutant polypeptide fusion of any of the invention 1068-1070 and 1072-1076, containing the S885N mutation related to SEQ ID NO:7. [Invention 1078] An ENPP1 mutant polypeptide fusion of any of the invention 1068-1070 and 1072-1077, containing the S766N mutation related to SEQ ID NO:7. [Invention 1079] An ENPP1 mutant polypeptide fusion of any of the invention 1068-1070 and 1072-1078, comprising mutations M883Y, S885T, and T887E relating to SEQ ID NO:7. [Invention 1080] An ENPP1 mutant polypeptide fusion according to any of invention 1068-1070 and 1072-1079, comprising mutations P534N, V536T, H1064K, and N1065F related to SEQ ID NO:7. [Invention 1081] An ENPP1 mutant polypeptide fusion of any of the invention 1068-1070 and 1072-1080, comprising mutants P554L and R545T related to SEQ ID NO:7. [Invention 1082] An ENPP1 mutant polypeptide fusion of any of the invention 1068-1070 and 1072-1081, comprising mutations S766N, H1064K, and N1065F related to SEQ ID NO:7. [Invention 1083] An ENPP1 mutant polypeptide fusion of any of the invention 1068-1070 and 1072-1082, comprising mutations E592N, H1064K, and N1065F related to SEQ ID NO:7. [Invention 1084] An ENPP1 mutant polypeptide fusion according to any of invention 1068-1070 and 1072-1083, comprising mutations P534N, V536T, M883Y, S885T, and T887E related to SEQ ID NO:7. [Invention 1085] An ENPP1 mutant polypeptide fusion according to any of the inventions 1068-1070 and 1072-1084, wherein the Fc region contains at least one mutation selected from the group consisting of M883Y, S885N, S885T, T887E, H1064K, and N1065F relating to SEQ ID NO:7. [Invention 1086] An ENPP1 mutant polypeptide fusion according to any of the inventions 1068-1070 and 1072-1085, wherein the Fc region contains at least one mutation selected from the group consisting of S885N, M883Y, M883Y / S885T / T887E, and H1064K / N1065F relating to SEQ ID NO:7. [Invention 1087] An ENPP1 mutant polypeptide fusion of any of Invention 1068-1070 and 1072-1086, or an ENPP1 mutant polypeptide of any of Invention 1058-1065, comprising at least one mutation selected from the group consisting of C25N, K27T, and V29N with respect to SEQ ID NO:7. [Invention 1088] An ENPP1 mutant polypeptide fusion of any of Invention 1068-1070 and 1072-1087, or an ENPP1 mutant polypeptide of any of Invention 1058-1065 and 1085, comprising at least one mutation selected from the group consisting of C25N / K27T and V29N with respect to SEQ ID NO:7. [Invention 1089] An ENPP1 mutant polypeptide fusion of any of Invention 1068-1070 and 1072-1088, or an ENPP1 mutant polypeptide of any of Invention 1058-1065 and 1087-1088, comprising one mutation selected from the group consisting of K369N and I371T with respect to SEQ ID NO:7. [Invention 1090] An ENPP1 mutant polypeptide fusion of any of Invention 1068-1070 and 1072-1089, or an ENPP1 mutant polypeptide of any of Invention 1058-1065 and 1087-1089, containing the mutation K369N / I371T related to SEQ ID NO:7. [Invention 1091] An ENPP1 mutant polypeptide fusion of any of Invention 1068-1070 and 1072-1090, or an ENPP1 mutant polypeptide of any of Invention 1058-1065 and 1087-1090, 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. [Invention 1092] An ENPP1 mutant polypeptide fusion of any of Invention 1068-1070 and 1072-1091, or an ENPP1 mutant polypeptide of any of Invention 1058-1065 and 1087-1091, 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. [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 Invention 1068-1070 and 1072-1092, or an ENPP1 mutant polypeptide of any of Invention 1058-1065 and 1087-1092, comprising at least one mutation selected from the group consisting of 85T, T887E, H1064K, N1065F, M883Y / S885T / T887E, and H1064K / N1065F. [Invention 1094] An ENPP1 mutant polypeptide fusion of any of Invention 1068-1070 and 1072-1093, or an ENPP1 mutant polypeptide of any of Invention 1058-1065 and 1087-1093, 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. [Invention 1095] An ENPP1 mutant polypeptide fusion of any of Invention 1068-1070 and 1072-1094, or an ENPP1 mutant polypeptide of any of Invention 1058-1065 and 1087-1094, 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. [Invention 1096] An ENPP1 mutant polypeptide fusion of any of Invention 1068-1070 and 1072-1095, or an ENPP1 mutant polypeptide of any of Invention 1058-1065 and 1087-1095, 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, relating to SEQ ID NO:7. [Invention 1097] An ENPP1 mutant polypeptide fusion of any of the inventions 1068-1070 and 1072-1096, comprising mutations I256T, M883Y, S885T, and T887E related to SEQ ID NO:7. [Invention 1098] An ENPP1 mutant polypeptide fusion of any of the invention 1068-1070 and 1072-1096, comprising mutations I256T, P534N, V536T, M883Y, S885T, and T887E related to SEQ ID NO:7. [Invention 1099] An ENPP1 mutant polypeptide fusion of any of the invention 1068-1070 and 1072-1096, comprising mutations I256T, E592N, H1064K, and N1065F related to SEQ ID NO:7. [Invention 1100] A fusion of any of invention 1067 to 1099, comprising a linker amino acid sequence. [Invention 1101] A fusion of the present invention 1100, wherein the linker amino acid sequence connects the ENPP1 mutant polypeptide portion of the fusion to a heterologous protein. [Invention 1102] A fusion of any of the inventions 1100 to 1101, wherein the linker amino acid sequence includes SEQ ID NO:8 or SEQ ID NO:9. [Invention 1103] A nucleic acid encoding any ENPP1 mutant polypeptide of any of Invention 1058 to 1066 or any fusion of any of Invention 1067 to 1102. [Invention 1104] A vector comprising the nucleic acid of the present invention 1103. [Invention 1105] An expression vector containing the nucleic acid of the present invention 1103. [Invention 1106] A cell or a plurality of cells, each containing the nucleic acid of Invention 1103, the vector of Invention 1104, and / or the expression vector of Invention 1105. [Invention 1107] Cells or a plurality of cells of the present invention 1106, which are CHO cells and / or NS0 cells. [Invention 1108] Cells or a plurality of cells according to Invention 1107, wherein CHO cells are stably transfected with human ST6 beta-galactoside alpha-2,6-sialyltransferase. [Invention 1109] A method for producing an ENPP1 mutant polypeptide or fusion, comprising the step of culturing any of the cells or a plurality of cells described in 1106 to 1108 of the present invention under conditions suitable for the expression of the ENPP1 mutant polypeptide or fusion by said cells or a plurality of cells. [Invention 1110] The method of the present invention 1109, wherein the cells are cultured in a medium supplemented with sialic acid and / or N-acetylmannosamine. [Invention 1111] Any method 1109 to 1110 of the present invention further comprises the step of purifying an ENPP1 mutant polypeptide or fusion from the aforementioned cells, the plurality of cells, or a culture medium in which the aforementioned cells or the plurality of cells are cultured. [Invention 1112] An ENPP1 mutant polypeptide or fusion obtained by the method of Invention 1111. [Invention 1113] (i) any ENPP1 mutant polypeptide of Invention 1058-1066, 1087-1096 and 1112, and / or any ENPP1 mutant polypeptide fusion of Invention 1067-1102 and 1112, and (ii) dissimilar parts; A conjugate that includes this. [Invention 1114] A conjugate according to the present invention 1113, wherein the dissimilar portion is polyethylene glycol. [Invention 1115] A pharmaceutical composition comprising any ENPP1 mutant polypeptide of Invention 1058-1066, 1087-1096, and 1112, a fusion of any of Invention 1067-1102 and 1112, a nucleic acid of Invention 1103, a vector of Invention 1104, an expression vector of Invention 1105, and / or a conjugate of any of Invention 1113-1114, and a pharmaceutically acceptable carrier. [Invention 1116] A method for reducing or preventing the progression of pathological calcification in subjects requiring it, (a) Any ENPP1 mutant polypeptide of the present invention 1058-1066, 1087-1096, and 1112; (b) A fusion of any of the inventions 1067-1102 and 1112; (c) Any conjugate of the present invention 1113 to 1114; and / or (d) Pharmaceutical composition of the present invention 1115 A process of administering a therapeutically effective dose to a subject, thereby reducing or preventing the progression of pathological calcification in the subject. Methods that include... [Invention 1117] A method for reducing or preventing the progression of pathological ossification in subjects requiring it, (a) Any ENPP1 mutant polypeptide of the present invention 1058-1066, 1087-1096, and 1112; (b) A fusion of any of the inventions 1067-1102 and 1112; (c) Any conjugate of the present invention 1113 to 1114; and / or (d) Pharmaceutical composition of the present invention 1115 A process of administering a therapeutically effective dose to a subject, thereby reducing or preventing the progression of pathological ossification in the subject. Methods that include... [Invention 1118] A method for reducing or preventing the progression of ectopic calcification of soft tissue in subjects requiring it, (a) Any ENPP1 mutant polypeptide of the present invention 1058-1066, 1087-1096, and 1112; (b) A fusion of any of the inventions 1067-1102 and 1112; (c) Any conjugate of the present invention 1113 to 1114; and / or (d) Pharmaceutical composition of the present invention 1115 A process of administering a therapeutically effective dose to a subject, thereby reducing or preventing the progression of ectopic calcification of soft tissues in the subject. Methods that include... [Invention 1119] A method for treating, improving, or preventing the progression of ossification of the posterior longitudinal ligament (OPLL) in patients who require it, (a) Any ENPP1 mutant polypeptide of the present invention 1058-1066, 1087-1096, and 1112; (b) A fusion of any of the inventions 1067-1102 and 1112; (c) Any conjugate of the present invention 1113 to 1114; and / or (d) Pharmaceutical composition of the present invention 1115 A process of administering a therapeutically effective dose to a subject, thereby reducing, improving, or preventing ossification of the posterior longitudinal ligament (OPLL) in the subject. Methods that include... [Invention 1120] A method for treating, reversing, or preventing the progression of hypophosphatemic rickets in a person who requires it, (a) Any ENPP1 mutant polypeptide of the present invention 1058-1066, 1087-1096, and 1112; (b) A fusion of any of the inventions 1067-1102 and 1112; (c) Any conjugate of the present invention 1113 to 1114; and / or (d) Pharmaceutical composition of the present invention 1115 A process of administering a therapeutically effective dose to a subject, thereby reducing, improving, or preventing the progression of hypophosphatemic rickets in the subject. Methods that include... [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 arteriosclerosis (CUA), calciphylaxis, ossification of the posterior longitudinal ligament (OPLL), hypophosphatemic rickets, osteoarthritis, age-related arteriosclerosis, idiopathic infantile arteriosclerosis (IIAC), generalized infantile arteriosclerosis (GACI), and arteriosclerotic plaque calcification, in a subject diagnosed with at least one of the said diseases, (a) Any ENPP1 mutant polypeptide of the present invention 1058-1066, 1087-1096, and 1112; (b) A fusion of any of the inventions 1067-1102 and 1112; (c) Any conjugate of the present invention 1113 to 1114; and / or (d) Pharmaceutical composition of the present invention 1115 A process of administering a therapeutically effective dose to a target, thereby reducing the disease or preventing its progression. Methods that include... [Invention 1122] A method for reducing or preventing the progression of age-related arteriosclerosis in those who require it, (a) Any ENPP1 mutant polypeptide of the present invention 1058-1066, 1087-1096, and 1112; (b) A fusion of any of the inventions 1067-1102 and 1112; (c) Any conjugate of the present invention 1113 to 1114; and / or (d) Pharmaceutical composition of the present invention 1115 A process of administering a therapeutically effective dose to a subject, thereby reducing or preventing the progression of age-related arteriosclerosis in the subject. Methods that include... [Invention 1123] A method for increasing pyrophosphate (PPi) levels in subjects having PPi levels lower than normal levels, (a) Any ENPP1 mutant polypeptide of the present invention 1058-1066, 1087-1096, and 1112; (b) A fusion of any of the inventions 1067-1102 and 1112; (c) Any conjugate of the present invention 1113 to 1114; and / or (d) Pharmaceutical composition of the present invention 1115 A step of administering to a subject in a therapeutically effective dose, wherein the administration raises the level of PPi in the subject to a normal level of at least 2 μM and maintains it at approximately the same level. Methods that include... [Invention 1124] A method for reducing or preventing the progression of pathological calcification or ossification in subjects with pyrophosphate (PPi) levels lower than normal levels, (a) Any ENPP1 mutant polypeptide of the present invention 1058-1066, 1087-1096, and 1112; (b) A fusion of any of the inventions 1067-1102 and 1112; (c) Any conjugate of the present invention 1113 to 1114; and / or (d) Pharmaceutical composition of the present invention 1115 A process of administering a therapeutically effective dose to a subject, thereby reducing pathological calcification or ossification in the subject, or preventing the progression of pathological calcification or ossification in the subject. Methods that include... [Invention 1125] A method for treating ENPP1 deficiency that manifests due to a decrease in extracellular pyrophosphate (PPi) concentration in subjects requiring it, (a) Any ENPP1 mutant polypeptide of the present invention 1058-1066, 1087-1096, and 1112; (b) A fusion of any of the inventions 1067-1102 and 1112; (c) Any conjugate of the present invention 1113 to 1114; and / or (d) Pharmaceutical composition of the present invention 1115 A process of administering a therapeutically effective dose to a subject, thereby increasing the PPi level in the subject. Methods that include... [Invention 1126] A method according to any one of the present invention 1116 to 1125, wherein a mutant polypeptide, fusion, conjugate, or pharmaceutical composition is administered to a subject for a short or long period of time. [Invention 1127] A method according to any one of the present invention 1116 to 1126, wherein a mutant polypeptide, fusion, conjugate, or pharmaceutical composition is administered to a subject topically, regionally, parenterally, or systemically. [Invention 1128] Any method 1116 to 1127 of the present invention, wherein the subject is a human. [Brief explanation of the drawing]

[0020] The following detailed description of exemplary embodiments of this disclosure will be better understood when read in conjunction with the accompanying drawings. For illustrative purposes, exemplary embodiments are shown in the drawings. However, it should be understood that this disclosure is not limited to the exact arrangement and means of the embodiments shown in the drawings. (Figure 1) The ENPP1 polypeptide intended in this disclosure (SEQ ID NO:7) is shown. Point mutations are identified by reference to SEQ ID NO:7, which may also be referred to as “Parent Compound,” “Parent Polypeptide,” or “Construction #770.” The labeling scheme shows the amino acid number and residue, referring to the numbering scheme shown in SEQ ID NO:7, followed by the residue in SEQ ID NO:7 and the substituted amino acid. For example, mutation C25N represents the 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 that do not have a 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 glycosylated residues. (Figure 2A) Figure 2A shows the domain structure of parental construct #770. Two somatomedin B domains, a catalytic domain, and an endonuclease domain from human ENPP1 were fused at the N-terminus to the signal sequence of human ENPP7 and at the C-terminus to the Fc domain of human IgG1. Figure 2B shows the pharmacokinetic analysis of parental construct #770. After an initial increase in plasma activity 17 hours after subcutaneous injection, plasma activity plummeted, with a calculated half-life of 34 hours. Figure 2C shows the non-limiting effect of the additional N-glycosylation consensus sequence incorporated into parental construct #770. Pharmacokinetic plots of AUC (bar, left y-axis) and half-life in time units (line, right y-axis) show that clone 7 with the I256T mutation has a significant increase in both AUC and half-life compared to parental construct #770. Figure 2D shows a digested peptide fragment exhibiting abundant sialycopeptide peaks (bottom) associated with ENPP1-Fc clone 19 but not with the parent construct #770. The mass spectrum of TIFF0007874896000001.tif5128 is shown. Figure 2E shows the Michaelis-Menten kinetic assay finding that the enzyme rates at various substrate concentrations are nearly identical at two different enzyme concentrations in two I256T-containing clones (yellow clone 17 and red clone 19) compared to the black parent construct #770. (Figure 2B) See the explanation for Figure 2A. (Figure 2C) See the explanation for Figure 2A. (Figure 2D-1) See the explanation for Figure 2A. (Figure 2D-2) See the explanation for Figure 2A. (Figure 2E) See the explanation for Figure 2A. (Figure 3) A bar graph summarizing plasma phosphodiesterase activity (measured using thymidine 5'-monophosphate p-nitrophenyl ester assay or pNP-TMP assay) after 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 elsewhere herein). (Figure 4) In vivo pharmacokinetic data of construct #981 are shown, measured using a pNP-TNP assay to record enzyme activity in mouse plasma samples after subcutaneous injection of the construct. Its half-life was estimated to be approximately 122 hours based on a single subcutaneous bolus injection into 5 mice. Another experiment reaching half-life is described elsewhere in this specification. (Figure 5) Selected in vivo pharmacokinetic data for construct #1014 (indicated as "1014A" in the graph) and construct #981 prepared in CHO cells grown in culture medium supplemented with construct #1014 and 1,3,4-O-Bu3ManNAc. The half-lives of the constructs can be derived from Equation 1, which is described elsewhere in this specification. (Figure 6) Shows three known glycosylation sites in ENPP1, all located in the random coil region: (A)=Asn; (B)=N-acetylglucosamine. One further glycosylation site (identified by surface glycoprotein dynamics measurement) is located within an α-helix and is labeled in red. One consensus NLT (Asn Leu Thr) is located in a PDB unstable region where glycosylation is not yet known. Four further consensus sequences are found in hENPP1, whose glycosylation state is unknown: calcium atom (C); two zinc atoms (D); ATP molecule (E). (Figure 7A) Shows a specific domain of human ENPP1 with a known loss-of-function mutation that causes the human disease "systemic arterial calcification in infants" (GACI). In certain embodiments, the glycosylation site is not introduced near the region with the known loss-of-function mutation that causes GACI (shown in Figure 7A). (Figure 7B) The crystal structure of ENPP1 is shown, including the residues with known loss-of-function mutations that cause GACI, highlighted (*). The residue in (B) is located in the catalytic domain and corresponds to T238A. As shown in Figure 6, it contains a calcium atom (C); two zinc atoms; (D) an ATP molecule (E). (Figure 8A) Figures 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 glycosylated isoforms introduced into construct #770. The figure illustrates the design and implementation of a high-throughput screen that can rapidly evaluate the biological effects of variant forms of the parent polypeptide, i.e., construct #770. The construct number (#) represents the original WT clone before the introduction of the mutation. The construct number (*) indicates a clone with a putative gain-of-function mutation. (Figure 8B) See the explanation for Figure 8A. (Figure 8C) See the explanation for Figure 8A. (Figure 8D) See the explanation for Figure 8A. (Figure 9A) Figure 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 its effect. Mutations were introduced within the Fc domain to enhance pH-dependent recycling by FcRn. (A) = Site that eliminates acid-dependent binding. (B) = Site that strengthens binding. (C) = Cysteine ​​disulfide bond. Magenta = Known glycosylation site. Figure 9B shows mutations within the Fc domain of human IgG1 that are known to enhance pH-dependent recycling by FcRn. (Figure 9B) See the explanation for Figure 9A. (Figure 10) Includes graphs and tables showing the effect of glycosylation on the PK (half-life, time) and bioavailability of the ENPP1 polypeptide. For all mutations except the I256T mutation in construct #922, the PK was equivalent to that of construct #CC07(770B). This mutation (located in the insertion loop of the catalytic domain) was modeled after an equivalent amount of glycosylation site in ENPP3. Furthermore, construct #951 showed a similar PK value to that of construct #CC07, but construct #951 (construct #951-ST) increased in a cell line stably transfected with ST6GAL1 showed improved PK and bioavailability. Bioavailability was improved for construct #922 containing the I256T mutation. Constructor #930 had a similar half-life to construct #CC07 but lower bioavailability. In contrast, construct #1020 exhibited higher bioavailability than construct #CC07. The PK and bioavailability data, measured as shown in Figures 4, 5, and 13 and calculated using Equation 1, are shown in the table. (Figure 11) Includes graphs and tables showing the effects of glycosylation and H1064K / N1065F Fc mutations on the half-life (PK, time) and bioavailability (AUC) of the ENPP1 polypeptide. All H1064 / N1065-containing constructs showed improved half-life and AUC values ​​compared to construct #770B. It should be noted that constructs #1048 and #1051 correspond to identical cDNAs within two separate clones, demonstrating the reproducibility of the PK / AUC analysis provided herein. Construct #1064 was also augmented in a cell line stably transfected with ST6GAL1 (construct #1064-ST). Architect #1057 also increased PK in cell lines stably transfected with ST6GAL1 ("-ST") (architecture #1057-ST) and in cell lines stably transfected with ST6GAL1 and supplemented with 1,3,4-O-Bu3-ManNAc ("-A") (architecture #1064-ST-A). Architect #1089 is identical to Architect #1014 except that a mutation removing a putative trypsin cleavage site was added. Architect #1014 also increased PK in cell lines stably transfected with ST6GAL1, but in this case, 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. (Figure 12) Includes graphs and tables showing the effects of glycosylation and the M883Y / S885T / T887E Fc mutation on the PK (half-life, time) and bioavailability of the ENPP1 polypeptide. Constructor #1030 has a lower AUC than the other constructs, likely due to the S766N mutation. Constructors #981, #1028, and #1101 showed increases in both PK and AUC values ​​when increased in cells stably transfected with ST6GAL1. Constructor #1101 showed improved PK and AUC values. PK and bioavailability data, measured as shown in Figures 4, 5, and 13 and calculated using Equation 1, are shown in the table. (Figure 13) This figure includes a collection of graphs showing the effect of expressing the construct in CHO cells stably transfected with human α-2,6-ST to produce recombinant biomaterials having terminal sialic acid residues with both α-2,3 and α-2,6 bindings. These cells are referred to as ST6GAL1 cells or ST cells (represented by "-ST"). This figure also shows the effect of increasing the construct in ST6GAL1 cells in the presence of a high-flux precursor of sialic acid known as sialic acid or 1,3,4-O-Bu3-ManNAc (represented by "-A"). PK and bioavailability data, measured as shown in Figures 4, 5, and 13 and calculated using Equation 1, are shown in the table. (Figure 14A) Figure 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 the parent clone 770 and clone 7 containing I256T. The area under the curve (left y-axis) is represented by bars, and the half-life in units of time (right y-axis) is represented by lines. Clones 7 have only a slight increase compared to clones 16 and 17 (lines), but it has an almost ambiguous AUC as a result of its high initial activity after injection (bars). Figure 14B shows bioavailability, represented by the slope of the area under the curve. Clones 7 containing only the I256T mutation are initially very active in plasma but quickly decay, as shown by the red AUC. Clone 14-ST, containing only the Fc MST mutation, is initially less active than clone 7 but has a longer half-life, indicated by the shallower slope (gray AUC). Clone 19-ST, which combines two mutations into one clone and is shown by the yellow AUC, resulted in an enzyme with both higher initial activity and a longer half-life. Figure 14C shows the AUC (bars, left axis) and half-life in units of time (line, right axis) for clones grown in unmodified CHO cells, CHO cells overexpressing α-2,6-sialyltransferase (α-2,6-ST), or CHO cells overexpressing α-2,6-ST combined with 1,3,4-O-Bu3ManNAc supplementation. In all examples, 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 (three arrows on the left) showed increased AUC and half-life in both CHO cells overexpressing α-2,6-ST and CHO cells overexpressing α-2,6-ST combined with 1,3,4-O-Bu3ManNAc supplementation. Compared to clone 7, clone 19 (three arrows on the right) also showed increased half-life and AUC with 1,3,4-O-Bu3ManNAc supplementation, similar to clone 9.Figure 14D shows the finding that anion exchange chromatography (HPAE-PAD) using pulsed amperometric detection of clone 9 grown in CHO K1 cells alone or in CHO K1 cells stably transfected with human α-2,6-ST or a combination of α-2,6-ST and the sialic acid precursor 1,3,4-Bu3ManNAc, shows a gradual increase in the ratio of N-acetylneuraminic acid content with each treatment. Figure 14E shows the AUC pharmacokinetic analysis of parental construct #770 (shown as 770) grown in CHO cells alone (shown as 9), CHO cells overexpressing α-2,6-ST (shown as 9(ST)), or CHO cells overexpressing α-2,6-ST combined with 1,3,4-O-Bu3ManNAc supplementation (shown as 9(ST)A). Clone 9 contains an Fc-HN mutation that provides a small increase in half-life and AUC compared to the parent clone 770. However, when clone 9 is grown in CHO cells overexpressing α-2,6-ST or in a combination of α-2,6-ST and 1,3,4-O-Bu3ManNAc supplementation, it exhibits a cumulatively large increase in AUC and half-life. (Figure 14B) See the explanation for Figure 14A. (Figure 14C) See the explanation for Figure 14A. (Figure 14D) See the explanation for Figure 14A. (Figure 14E) See the explanation for Figure 14A. (Figure 15A) Figure 15A shows the AUC pharmacokinetic analysis of Fc-MST-containing clone 14-ST compared with Fc-MST / I256T-containing clones 17-ST, 19-ST, and 19-ST-A. The AUC of Fc-MST-containing clones can be further enhanced by the I256T mutation and can be further increased when grown in the presence of a 1,3,4-O-Bu3ManNAc precursor. Figure 15B shows the AUC pharmacokinetics of parental clone 770 compared with clone 19-ST-A. When clone 19, containing both the Fc-MST and I256T mutations, was grown in α-2,6-ST overexpressing CHO cells supplemented with 1,3,4-O-Bu3ManNAc, its bioavailability increased by almost 18-fold. Figure 15C shows the findings of MALDI-TOF / TOF analysis for N-glycan profiling, which revealed that clone 19-ST-A (99.2%) had a higher % sialic acid content compared to parent clone 770 (78.4%), based on calculations based on structures containing at least one galactose for sialic acid transfer. Figure 15D shows Enpp1 asj / asj This graph shows the pharmacodynamic effects after a single dose of either parent clone 770 (red square) or optimized ENPP1-Fc clone 19-ST (red circle) at a dose of 0.3 mg / kg, as measured by PPi generation in mice (left y-axis). Physiological levels of PPi in normal mice (shaded gray) are 1.5–2.5 μm PPi, while in Enpp1 mice... asj / asj Mice have nearly undetectable levels. A single dose of clone 770 restores physiological levels of PPi, which return to baseline 89 hours prior, while clone 19-ST can maintain nearly physiological levels up to 263 hours. The error bars for PPi production appear to be much larger than the error bars for the % activity of this enzyme (right y-axis) (comparison of red and black circles). (Figure 15B) See the explanation for Figure 15A. (Figure 15C) See the explanation for Figure 15A. (Figure 15D) See the explanation for Figure 15A. (Figure 16A) Figures 16A-16B show selected mutations introduced into the ENPP1 domain and the parent polypeptide (SEQ ID NO:7). These figures identify the individual point mutations introduced into SEQ ID NO:7. Constructs stably transfected with human α-2,6-ST into stably transfected CHO cells are referred to as "ST". PK and bioavailability data, determined as shown in Figures 4, 5, and 13 and calculated using Equation 1, are shown in the table. (Figure 16B) See the explanation for Figure 16A. (Figure 17) The bioavailability (as area under the curve or AUC) of specific constructs of this disclosure, classified by mutations in the signal sequence (N-terminal region) area, is shown. (Figure 18) The bioavailability (as area under the curve or AUC) of specific constructs of this disclosure, classified by mutations in the endonuclease region, is shown. (Figure 19) Figure 19A includes an image showing stable CHO cell subclones co-transfected with plasmid cDNA for both ENPP1-Fc and hST6GAL1. These were screened for alpha-2,6-sialyltransferase expression by immunofluorescence of paraformaldehyde-fixed cells using rabbit anti-hST6GAl1 antibody (R&D Systems catalog no. AF5924), followed by donkey polyclonal goat IgG Alexa Fluor594 (Abcam Ab150140). Characteristic Golgi localization was observed in these cell clones, which were also positive by Western blotting with the same antibody. Figure 19B shows a representative Western blot using the same primary antibody as in Figure 19A, showing a single band of approximately 48 kD indicating varying intensities of alpha-2,6-sialyltransferase expression in some of the CHO cell subclones used in this study. The first lane is the untransfected CHO cell lysate as a negative control. The following three lanes are three distinct subclones of the unmodified parent plasmid 770, indicated by A, B, and C. The other lane is a selection of subclones used in the literature for clones 1, 2, 10, 14, and 18. "X" represents a subclone of a construct not used further herein. [Modes for carrying out the invention]

[0021] Detailed description of the invention This disclosure relates to the discovery of a specific ENPP1-Fc polypeptide having an improved in vivo half-life compared to ENPP1-Fc polypeptides known in the art, in one aspect.

[0022] In one non-limiting aspect, glycosylation was promoted to protect the ENPP1-Fc polypeptide from degradation. This was achieved by introducing an additional N-glycan consensus sequence to the outer surface of the expected tertiary structure, guided by a three-dimensional model of ENPP1.

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

[0024] Furthermore, in another non-limiting context, we enhanced the sialylation of the fusion protein 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 another, non-limiting context, sialic acid capping was enhanced by supplementing the cell culture medium with N-acetylmannosamine (also known as 1,3,4-O-Bu3ManNAc), a "high-flux" precursor of sialic acid.

[0026] In a particular embodiment, enhancing protein sialylation by expressing this biomaterial in CHO cells stably transfected with human alpha-2,6-sialyltransferase enhances the bioavailability (C) of ENPP1-Fc when administered subcutaneously. max ) was greatly improved. In another embodiment, increased pH-dependent FcRn-mediated cell recycling by manipulating the Fc domain improved the half-life of the biomaterial in vivo. In yet another embodiment, combining CHO cells stably transfected with human α-2,6-sialyltransferase with proliferation of said cells in N-acetylmannosamine dramatically increased the half-life and / or biological exposure (AUC). In yet another embodiment, combining two or more of the 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 Disclosure are more glycosylated than other ENPP1-Fc polypeptides in the Art. In other embodiments, the polypeptides of the Disclosure have a higher affinity for neonatal orphan receptors (FcRn) than other ENPP1-Fc polypeptides in the Art. In yet another embodiment, the polypeptides of the Disclosure have a longer in vivo half-life than other ENPP1-Fc polypeptides in the Art. In yet another embodiment, the kinetic properties of the parent polypeptide (construct #770) are modified such that the change results in a "gain-of-function" change in the enzyme rate constant. In yet another embodiment, the kinetic properties of the parent polypeptide (construct #770) are not significantly altered by specific site-directed mutagenesis, and the resulting mutant enzyme has substantially the same enzyme rate constant as the parent polypeptide. In yet another embodiment, a specific point mutation in the parent polypeptide introduces a glycan at the mutant residue, increasing the biological exposure of the mutant polypeptide. In yet another non-limiting embodiment, 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 containing or comprising 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 containing or comprising amino acids 23-849 of SEQ ID NO:7. In certain embodiments, any one ENPP1 mutant polypeptide described herein has higher catalytic activity than a soluble ENPP1 polypeptide containing or comprising amino acids 23-849 of SEQ ID NO:7.

[0029] In certain embodiments, any one of the ENPP1 mutant polypeptides described herein has improved pharmacokinetic and / or bioavailability properties in mammals compared to a soluble ENPP1 polypeptide containing or comprising amino acids 23-849 of SEQ ID NO:7. In a particular embodiment, any ENPP1 mutant polypeptide has a cyclic half-life in mammals 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%, 98%, 99%, 99.5%, 99.8%, 100%, 120%, 140%, 160%, 180%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, or greater than 500%) of a soluble ENPP1 polypeptide containing or comprising amino acids 23-849 of SEQ ID NO:7. In a particular embodiment, any one ENPP1 mutant polypeptide described herein has a larger AUC than that of a soluble ENPP1 polypeptide containing or consisting of amino acids 23-849 of SEQ ID NO:7.

[0030] In certain embodiments, the in vivo half-life of the ENPP1-Fc polypeptide of the 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 that of ENPP-1 polypeptides reported in the Art. In other embodiments, the polypeptide of the Disclosure is administered to subjects at lower doses and / or less frequently than other ENPP1-Fc polypeptides in the Art. In yet another embodiment, the polypeptide of the Disclosure is administered to subjects once a month, twice a month, three times a month, and / or four times a month. In yet another embodiment, the low-frequency administration of the polypeptide of the 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 Disclosure may be used to increase pyrophosphate (PPi) levels in subjects having pyrophosphate (PPi) levels lower than normal levels (approximately 2 μM). In other embodiments, the ENPP1-Fc polypeptide of the Disclosure may be used to reduce or prevent the progression of pathological calcification or ossification in subjects having PPi levels lower than normal levels. In yet another embodiment, the ENPP1-Fc polypeptide of the Disclosure may be used to treat ENPP1 deficiency manifested by decreased extracellular PPi concentration in subjects.

[0032] In certain embodiments, the steady-state level of plasma PPi achieved after administration of a first dose of the construct of the present disclosure is maintained for a period of at least two days, at least four days, at least one week, or at least one month.

[0033] In certain embodiments, a second dose of the construct of the Disclosure is administered to a subject at appropriate time intervals of 2 days, 4 days, 1 week, or 1 month, such that the steady-state level of plasma PPi remains constant or is maintained at a steady-state level and does not revert to a lower PPi level than that the subject had before administration of the first dose of the construct of the Disclosure.

[0034] While we do not wish to be bound by theory, maintaining steady-state plasma PPi concentrations at normal levels is thought to reduce and / or prevent the progression of pathological calcification and ossification in the subject.

[0035] Certain ENPP1 polypeptides, variants, or variant fragments thereof have been previously disclosed in International PCT Patent Publications WO 2012 / 125182, WO 2014 / 126965, WO 2016 / 187408, and WO 2018 / 027024, all of which are incorporated herein by reference in their entirety.

[0036] Here, specific aspects of the subject matter to be disclosed are to be referenced in detail. While the subject matter to be disclosed may be described in relation to the enumerated claims, it is understood that the subject matter illustrated is not intended to limit the scope of the claims to the subject matter to be disclosed.

[0037] Throughout this book, values ​​expressed in range form should be interpreted flexibly, including not only the numerical limit explicitly stated as the limit of that range, but also all individual numerical values ​​or subranges within that range, as if each numerical value and subrange were explicitly stated. For example, the range "approximately 0.1% to approximately 5%" or "approximately 0.1% to approximately 5%" should be interpreted to include not only approximately 0.1% to approximately 5%, but also the individual values ​​within the indicated range (e.g., 1%, 2%, 3%, and 4%) and subranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, and 3.3% to 4.4%). The expression "approximately X to Y" has the same meaning as "approximately X to approximately Y" unless otherwise indicated. Similarly, the expression "approximately X, Y, or approximately Z" has the same meaning as "approximately X, approximately Y, or approximately Z" unless otherwise indicated.

[0038] definition Where used herein, the following terms have the meanings associated with them in this section. Unless otherwise defined, all technical and scientific terms used herein have the same meanings generally understood by a person of ordinary skill in the art to which this disclosure belongs. Generally, the nomenclature and experimental procedures in veterinary pharmacognosy, pharmaceutical science, separation science, and organic chemistry used herein are well known and widely used in the art. It should be understood that the order of steps or the order in which certain actions are taken is not important as long as this instruction is feasible. The use of subsection titles is intended to aid in the reading of the document and should not be construed as limitation, and information related to the subsection title may be present in or outside of that particular subsection. All publications, patents, and patent documents referenced herein are incorporated by reference in whole, as they are incorporated by reference individually.

[0039] In this application, when it is said that an element or component is included in and / or selected from the list of elements or components mentioned, it should be understood that the element or component may be any one of the elements or components mentioned, and may be selected from a group of two or more of the elements or components mentioned.

[0040] In the methods described herein, actions may be performed in any order unless a time series or sequence of actions is explicitly indicated. Furthermore, the actions described may be performed simultaneously unless the language of the claims explicitly indicates that they are performed separately. For example, the action described in the claim to perform X and the action described in the claim to perform Y may be performed simultaneously in a single operation, and the process formed thereby is encompassed within the literal scope of the process described in the claim.

[0041] In this book, the terms “a,” “an,” or “the” are used to refer to one or more unless the context explicitly indicates otherwise. The term “or” is used to refer to a non-exclusive “or” unless it is indicated otherwise. The phrases “at least one of A and B” or “at least one of A or B” are synonymous with “A, B, or A and B.”

[0042] The following notational conventions apply to this disclosure for clarity. In no event are teachings herein that do not follow these conventions also part of this disclosure and can be fully understood from the context in which they are disclosed. Protein symbols are disclosed in non-italicized uppercase letters. As a non-limiting example, “ENPP1” represents a protein. In certain embodiments, if the protein is a human protein, “h” is used before the protein symbol. In other embodiments, if the protein is a mouse protein, “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 italicized uppercase letters. As a non-limiting example, the human gene corresponding to the protein hENPP1 is italicized ENPP1. Mouse gene symbols are disclosed with the first letter capitalized and the rest lowercase, and furthermore, mouse gene symbols are italicized. As a non-limiting example, the mouse gene that produces the protein mEnpp1 is italicized Enpp1. Genetic mutations are indicated in capital letters.

[0043] When "approximately" is used herein in reference to a measurable value, such as a quantity, length of time, etc., it means to include variations of ±20% or ±10%, in a particular embodiment ±5%, in a particular embodiment ±1%, and in a particular embodiment ±0.1% from the indicated value, insofar as such variations are appropriate for carrying out the disclosed method.

[0044] A disease or disorder is “relieved” if the severity of its symptoms is reduced, the frequency with which the patient experiences such symptoms is reduced, or both are reduced.

[0045] As used herein, the terms “modification,” “deletion,” “variation,” or “mutation” refer to changes in a gene within a cell that affect the function, activity, expression (transcription or translation), or conformation of the polypeptide it encodes, including missense and nonsense mutations, insertions, deletions, frameshifts, and immature terminations.

[0046] As used herein, the term "antibody" refers to an immunoglobulin molecule that can specifically bind to a particular epitope or antigen. Antibodies may be intact immunoglobulins of natural or recombinant sources, or they may be the immunoreactive portion of intact immunoglobulins.

[0047] The "ATP hydrolysis activity" of ENPP1 can be measured using an ATP cleavage assay. ENPP1 immediately hydrolyzes ATP to AMP and PPi. The steady-state Michaelis-Menten enzyme constant of ENPP1 is measured using ATP as the substrate. ENPP1 can be shown to cleave ATP by HPLC analysis of its enzymatic reactants, and the identity of the substrate and products of the reaction can be confirmed using ATP, AMP, and ADP standards. The ATP substrate degrades over time in the presence of ENPP1, and the enzymatic product AMP accumulates. The initial rate of ENPP1 in the presence of ATP is derived using various concentrations of ATP substrate, and this data is fitted to a curve 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 That is the case.

[0048] As used herein, the term "AUC" represents the area under the plasma drug concentration-time curve (AUC), which correlates with the actual physical exposure to the drug after administration of that dose. In certain embodiments, AUC is expressed as mg*h / L. AUC can be used to measure the bioavailability of a drug, which is the proportion of the drug absorbed intact and reaches the site of action, or the proportion circulating systemically after administration by any route.

[0049] AUC can be calculated using the linear trapezoidal or log-trapezoidal method. The linear trapezoidal method uses linear interpolation between data points to calculate AUC. This method is required by the OGD and FDA and is the standard in bioequivalence testing. Given a time interval (t1~t2), the AUC is: It can be calculated as shown in TIFF0007874896000002.tif9128, where C1 and C2 are the average concentrations over the time intervals (t1 and t2).

[0050] The log-trapezoid method uses logarithmic interpolation between data points to calculate the AUC. This method is more accurate when the concentration decreases because drug elimination is exponential (making it linear on a logarithmic scale). Given a time interval (t1~t2), the AUC is: It can be calculated as shown in TIFF0007874896000003.tif12128 (assuming C1 > C2).

[0051] When used herein, the term "bioavailability" refers to the extent and rate to which an active portion (protein or drug or metabolite) enters the systemic circulation, thereby accessing the site of action, or to the extent and rate to which it enters the systemic circulation after administration via any route. The bioavailability of an active portion is largely determined by the characteristics of the dosage form, which depends in part on its design and manufacture. Differences in bioavailability between formulations of a given drug or protein can have clinical importance, and it is therefore essential to know whether drug formulations are equivalent. The most reliable measure of the bioavailability of a drug or protein is the area under the plasma concentration-time curve (AUC). AUC is directly proportional to the total amount of unchanged drug or therapeutic protein that reaches the systemic circulation. Drugs or therapeutic proteins can be considered bioequivalent in terms of the degree and rate of absorption if their plasma concentration curves are essentially superimposed. For intravenous administration of a drug, bioavailability is defined as 1. For drugs administered by other routes of administration, bioavailability is often less than 1. Incomplete bioavailability can be attributed to numerous factors, which can be subdivided into dosage form effects, membrane effects, and administration site effects. Half-life and AUC provide information about the bioavailability of drugs and biomolecules.

[0052] As used herein, the terms “conservative variation” or “conservative substitution” refer to the substitution of one amino acid residue with another biologically similar residue. Conservative variations or substitutions are highly likely to not alter the shape of the peptide chain. Examples of conservative variations or substitutions include the substitution of one hydrophobic residue, such as isoleucine, valine, leucine, or methionine, with another hydrophobic residue, or the substitution of one polar residue with another polar residue, such as arginine and lysine, glutamic acid and aspartic acid, or glutamine and asparagine.

[0053] As used herein, “construction” in this disclosure refers to a fusion polypeptide comprising the ENPP1 polypeptide or a fragment or site-specific variant thereof.

[0054] A "disease" is a state of animal health in which the animal is unable to maintain homeostasis, and if the disease is not improved, the animal's health continues to deteriorate.

[0055] In animals, a "disorder" is a health condition in which the animal can maintain homeostasis, but its health is less favorable than it would be in the absence of the disorder. Leaving the disorder untreated does not necessarily lead to a further decline in the animal's health.

[0056] As used herein, the terms “effective dose,” “pharmaceutical effective dose,” and “therapeutic effective dose” refer to an amount of an active substance that is non-toxic but sufficient to deliver the desired biological effect. The effect may be a reduction and / or alleviation of signs, symptoms, or causes of a disease, or any other desired change in the biological system. The appropriate therapeutic dose in any individual case can be determined by those skilled in the art using conventional experiments.

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

[0058] As used herein, the terms “ENPP1 protein” or “ENPP1 polypeptide” refer to the ectonucleotide pyrophosphatase / phosphodiesterase-1 protein encoded by the ENPP1 gene. The encoded protein is a type II transmembrane glycoprotein that cleaves a variety of substrates, including nucleotides and nucleotide sugars with phosphodiester bonds, as well as nucleotides and nucleotide sugars with pyrophosphate bonds. The ENPP1 protein has a transmembrane domain and a soluble extracellular domain. Its 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 Braddock et al., PCT application number WO 2014 / 126965, which is incorporated herein by reference in its entirety.

[0059] As used herein, the term “human ENPP1” refers to the human ENPP1 sequence represented by NCBI accession NP_006199. As used herein, the term “soluble human ENPP1” refers to the 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 able to bind to ATP and hydrolyze it to AMP and PPi, and / or bind to AP3a and hydrolyze it to ATP.

[0060] As used herein, the term “ENPP1 precursor protein” refers to ENPP1 having its signal peptide sequence at the N-terminus of ENPP1. Proteolytic degradation cleaves the signal sequence from ENPP1, providing the ENPP1 protein. Useful signal peptide sequences in this 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 conjugations) 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. Subtypes of IgG, such as IgG1, IgG2, IgG3, and IgG4, are assumed to be used as Fc domains.

[0063] As used herein, “Fc region” refers to the portion of an IgG molecule associated with a crystalline fragment obtained by papain digestion of an IgG molecule. The Fc region comprises the C-terminal half of the two heavy chains of the IgG molecule linked by disulfide bonds. It does not possess antigen-binding activity but contains a carbohydrate portion as well as a binding site for the Fc receptor, including its complement and 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 terms “IgG hinge-Fc region” or “hinge-Fc fragment” refer to the region of the IgG molecule consisting of the Fc region (residues 231-447) and a hinge region (residues 216-230) extending from the N-terminus of the Fc region. The term “constant domain” refers to the portion of an immunoglobulin molecule that has a more conserved amino acid sequence than the variable domain, which is the other portion of the immunoglobulin that contains the antigen-binding site. The constant domain includes the CH1, CH2, and CH3 domains of the heavy chain, as well as 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 (particularly B lymphocytes, follicular dendritic cells, natural killer cells, macrophages, neutrophils, eosinophils, basophils, human platelets, and mast cells) that contributes to the protective function of the immune system. Fc receptors bind to antibodies conjugated to infected cells or invading pathogens. Immunoglobulin Fc receptors (FcRs) are expressed in all hematopoietic cells and play a crucial role in antibody-mediated immune responses. Binding of immune complexes to FcRs activates effector cells, leading to phagocytosis of IgG opsonized particles, endocytosis, release of inflammatory mediators, and antibody-dependent 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, with the exception of FcRn and FcεRII, which are structurally associated with major class I histocompatibility antigens and type C lectins, respectively, belong to the immunoglobulin superfamily (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 translocated internally through endosomes. Generally, serum proteins are transferred from endosomes to lysosomes, where they are degraded. Since FcRn binds to IgG at acidic pH (<6.5) and 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 weakly acidic pH (<6.5) and recycled to the cell surface, where they are released at the neutral pH (>7.0) of the blood. This allows IgG and serum albumin to avoid lysosomal degradation.

[0066] The Fc region of the IgG molecule is located in the constant region of the heavy chain, particularly the CH2 domain. This Fc region binds to the Fc receptor (FcRn), which is a surface receptor on B cells and a complement system protein. Binding of the IgG molecule's Fc region to FcRn activates cells possessing this receptor, and therefore activates the immune system. Key Fc residues for mouse Fc-mouse FcRn and human Fc-human FcRn interactions have been identified (Dall'Acqua et al., 2002, J. Immunol. 169(9):5171-80). The FcRn-binding domain includes the CH2 domain of the IgG molecule (or its FcRn-binding portion).

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

[0068] The terms “functional equivalent” or “functional derivative” mean, in the context of functional derivatives of amino acid sequences, molecules that possess substantially similar biological activity (either functional or structural) to that of the ENPP1-Fc constructs presented herein. Functional derivatives or equivalents may be naturally occurring derivatives or may be prepared by synthesis. Functionally equivalent polypeptides in this disclosure may also be polypeptides identified using one or more structural and / or sequence alignment techniques known in the art.

[0069] Exemplary functional derivatives include amino acid sequences having one or more amino acid substitutions, deletions, or additions, provided that the biological activity of the protein is conserved. The substituted amino acid preferably has similar physicochemical properties to that of the substituted amino acid. Desired similar physicochemical properties include similarities in charge, bulk, hydrophobicity, hydrophilicity, etc. Typically, more 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 of more than 80% to the ENPP1-Fc construct. More preferred polypeptides have a degree of identity of more than 85%, 90%, 95%, 98%, or 99%, respectively. A method for determining whether a functional equivalent or functional derivative has the same, similar, or higher biological activity as the ENPP1-Fc construct can be determined by using an enzymatic assay for ATP cleavage described in WO2016 / 187408.

[0070] "Genetic transfer" and "gene delivery" refer to methods or systems for reliably inserting a specific nucleic acid sequence into target cells.

[0071] An "inducible" promoter is a nucleotide sequence that, when functionally linked to a polynucleotide encoding or identifying a gene product, ensures that the gene product is produced in the cell only when the inducer corresponding to that promoter is present in the cell.

[0072] Where used herein, the term “in vivo half-life” of a protein and / or polypeptide (e.g., an ENPP1 polypeptide containing an FcRn binding site) as envisioned herein represents the time required for half of the administered amount to be eliminated from the circulation and / or other tissues of an animal. When a clearance curve for an ENPP1-Fc fusion protein is constructed as a function of time, the curve is typically biphasic, comprising a fast α-phase (representing the equilibrium of the administered molecule between intravascular and extravascular spaces, and partly determined 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] When this term is used herein, “Instructional Materials” includes any publications, records, figures, or any other medium of expression that may be used to communicate the usefulness of the nucleic acids, peptides, and / or compounds of this disclosure in the kit in identifying, mitigating, or treating the various diseases or disorders referred to herein.

[0074] "Isolation" means that something has been modified or removed from its natural state. For example, a nucleic acid or polypeptide that is naturally present within a living animal is not "isolated," but the same nucleic acid or polypeptide that has been partially or completely separated from its naturally occurring coexisting substances is "isolated." Isolated nucleic acids or proteins may exist in a substantially pure form or in a non-natural environment, such as within a host cell.

[0075] "Isolated nucleic acid" refers to a nucleic acid segment or fragment that, in its natural state, is separated from its adjacent sequences; that is, a DNA fragment typically isolated from sequences adjacent to that fragment within the genome in which it naturally exists. This term also applies to nucleic acids substantially purified from other components naturally associated with that nucleic acid, i.e., RNA or DNA or proteins naturally associated with it in the cell. Therefore, this term includes recombinant DNA, for example, that is incorporated into vectors, autonomously replicating plasmids or viruses, or into the genomic DNA of prokaryotes or eukaryotes, or that exists as a separate molecule independent of other sequences (i.e., as cDNA produced by PCR or restriction enzyme digestion, or as a genome or cDNA fragment). It also includes recombinant DNA that is part of a hybrid gene encoding further polypeptide sequences.

[0076] An "oligonucleotide" or "polynucleotide" is a nucleic acid or compound that specifically hybridizes to a polynucleotide, having a length of at least 2, and in certain embodiments at least 8, 15, or 25 nucleotides, but which may be up to 50, 100, 1000, or 5000 nucleotides.

[0077] The term "functionally linked" refers to a functional link between a regulatory sequence and a heterogeneous nucleic acid sequence that results in the expression of the latter. For example, when a first nucleic acid sequence is functionally related to a second nucleic acid sequence, the first nucleic acid sequence is functionally linked to the second nucleic acid sequence. For example, when a promoter affects the transcription or expression of a coding sequence, the promoter is functionally linked to the coding sequence. In general, functionally linked DNA sequences are contiguous, and where necessary, two protein-coding regions are connected within the same reading frame.

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

[0079] As used herein, the terms “pharmaceutical composition” or “composition” refer to a mixture of at least one compound useful in this disclosure and a pharmaceutically acceptable carrier. Pharmaceutical compositions facilitate the administration of compounds to patients. Various methods of administering compounds exist 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 “pharmaceutically acceptable” means a substance, such as a carrier or diluent, that does not negate the biological activity or properties of a compound and is relatively non-toxic; that is, the substance can be administered to an individual without producing an undesirable biological effect or interacting in a harmful manner with any component of the composition in which it is contained.

[0081] As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable substance, composition, or carrier, such as a liquid or solid filler, stabilizer, dispersant, suspending agent, diluent, excipient, thickener, solvent, or encapsulating agent, that is involved in transporting or delivering a compound useful in this disclosure into or to a patient so that it can perform its intended function. Each carrier must be “acceptable” in the sense that it is compatible with the other components of its formulation containing the compound useful in this disclosure and is not harmful to the patient. Some examples of substances that can function as a pharmaceutically acceptable carrier include sugars, e.g., lactose, glucose, and sucrose; starches, e.g., corn starch and potato starch; cellulose, and their derivatives. As used herein, “pharmaceutically acceptable carrier” also includes any and all coatings, antibacterial and antifungal agents, and absorption retarders, etc., that are compatible with the activity of the compound useful in this disclosure and are physiologically acceptable to the patient. “pharmaceutically acceptable carrier” may further include pharmaceutically acceptable salts of the compound useful in this disclosure. Other additional components that may be included in the pharmaceutical compositions used in carrying out this disclosure are known in the art and are described, for example, in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference.

[0082] As used herein, the term "pharmaceutically acceptable salt" refers to salts of administered compounds prepared from pharmaceutically acceptable nontoxic acids and bases, including inorganic acids, inorganic bases, organic acids, and inorganic bases, as well as their solvates, hydrates, and clathrates.

[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, animals include rats, mice, cats, dogs, humans, cattle, and horses. Since PPi is released from platelets, it must be measured in plasma, not serum. There are various methods for measuring PPi, one of which is an enzymatic assay using a modified 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 approximately 1 μm to 3 μM, and in some cases from 1 to 2 μM. Subjects with deficient ENPP1 expression tend to exhibit low PPi levels in the range of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, and any combination thereof, below normal levels. In patients with pathological calcification or ossification disorders, plasma PPi levels are found to be less than 1 μM, and in some cases, below the detection level. In some cases, plasma PPi levels in patients with pathological calcification or ossification disorders 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, associated naturally occurring structural variants, and synthetic, non-naturally occurring analogs thereof, linked together by peptide bonds.

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

[0086] As used herein, the terms “prevent” or “prevent” mean to prevent the onset of a disability or disease if it has not yet developed, or to prevent the further progression of a disability or disease if it has already developed. Furthermore, preventing some or all of the symptoms associated with a disability or disease is considered the ability of a person skilled in the art.

[0087] As used herein, the term “promoter” is defined as a DNA sequence recognized by a cellular synthetic mechanism or introduced synthetic mechanism that is required to initiate the individual transcription of a polynucleotide sequence.

[0088] As used herein, the term “promoter / regulatory sequence” means a nucleic acid sequence required for the expression of a gene product functionally linked to that promoter / regulatory sequence. In some examples, this sequence may be a core promoter sequence, and in other examples, this sequence may also include enhancer sequences and other regulatory elements required for the expression of the gene product. A promoter / regulatory sequence may, for example, express its gene product in a tissue-specific manner.

[0089] As used herein, the term "recombinant polypeptide" is defined as a polypeptide produced by using recombinant DNA methods.

[0090] As used herein, the term "recombinant DNA" is defined as DNA produced by joining together fragments of DNA from different sources.

[0091] Where used herein, “sample” or “biological sample” means a biological substance isolated from a subject. A biological sample may include mRNA, polypeptides, or any other biological substance suitable for detecting markers of physiological or pathological processes 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., in the range of 10–30 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 signal recognition particles (SRPs) 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” means essentially free from other components. For example, a substantially purified polypeptide is a polypeptide isolated from other components that would normally be present in its natural 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 functionally linked to a polynucleotide that codes for or is identified by a gene, causes the cell to produce its gene product only when it is substantially the tissue type corresponding to that promoter.

[0095] When used herein, the phrases “transcriptionally controlled” or “functionally linked” mean that the promoter is positioned and oriented correctly to control the initiation of transcription and expression of the polynucleotide by RNA polymerase.

[0096] The terms “transfect,” “transform,” or “transform” as used herein refer to the process by which an exogenous nucleic acid is transferred to or introduced into a host cell. A “transfected,” “transformed,” or “transformed” cell is one that has been transfected, transformed, or transformed with an exogenous nucleic acid. Cells include the original subject cell and its offspring.

[0097] As used herein, the terms “treatment” or “to treat” are defined as the application or administration (alone or in combination with other agents) of a therapeutic agent to a patient having a disease or disorder, symptoms of a disease or disorder, or the possibility of developing a disease or disorder, for the purpose of curing, restoring, reducing, mitigating, altering, treating, improving, or influencing the disease or disorder, symptoms of a disease or disorder, or the possibility of developing a disease or disorder, or the application or administration of a therapeutic agent to tissue or cell lines isolated from a patient (for example, for diagnostic or ex vivo application). Such treatments may be tailored or modified to suit the individual based on knowledge derived from the field of genomic pharmacology.

[0098] A “variant” is a nucleic acid sequence or peptide sequence, respectively, that differs in sequence from a reference nucleic acid sequence or peptide sequence but retains the essential properties of the reference molecule, as the term is used herein. Changes in the sequence of a nucleic acid variant may not alter the amino acid sequence of the peptide encoded by the reference nucleic acid, or they may result in amino acid substitutions, additions, deletions, fusions, and shortenings. Changes in the sequence of a peptide variant are typically limited or conserved, such that the sequences of the reference peptide and the variant are very similar as a whole and identical in many regions. The amino acid sequences of the variant and the reference peptide may differ due to one or more substitutions, additions, or deletions in any combination. Variants of nucleic acids or peptides may be spontaneously occurring, for example, allelic variants, or they may be variants not known to occur spontaneously. Non-spontaneously occurring variants of nucleic acids and peptides may be produced by mutagenesis or direct synthesis.

[0099] A “vector” is a composition containing isolated nucleic acid that can be used to deliver the isolated nucleic acid into 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. Therefore, the term “vector” includes autonomously replicating plasmids or viruses. The term should also be interpreted 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, adenovirus vectors, adeno-associated virus vectors, and retroviral vectors.

[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 external lipid envelope, that can transfect its nucleic acid into cells.

[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 and therefore arbitrarily designed “normal” or “wild-type” form of that gene. In contrast, the terms “modified” or “mutant” refer to a gene or gene product that exhibits modifications (i.e., altered features) in terms of sequence and / or functional characteristics compared to a wild-type gene or gene product. Naturally occurring mutants may be isolated, and these are identified by the fact that they have altered features (including altered nucleic acid sequences) compared to a wild-type gene or gene product.

[0102] The following abbreviations are used herein: 1,3,4-O-Bu3ManNAc, N-acetylmannosamine; ST6GAL1, ST6 beta-galactoside alpha-2,6-sialyltransferase.

[0103] Scope: Throughout this disclosure, various aspects of this disclosure may be indicated in scope form. It should be understood that scope form is for convenience and conciseness only and should not be considered a rigid limitation on the scope of this disclosure. Therefore, scope statements should be considered to specifically disclose all possible subranges and individual numbers within that range. For example, a scope statement 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, and individual numbers within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the range.

[0104] polypeptide In one aspect, the Disclosure provides an ENPP1-Fc polypeptide. The Disclosure assumes that the polypeptide of the Disclosure may have one or more of the mutations described herein.

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

[0106] In certain embodiments, the ENPP1 mutant polypeptide contains the catalytic domain of ENPP1. In certain embodiments, the ENPP1 mutant polypeptide contains 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 and transmembrane domains of ENPP1. In certain embodiments, the ENPP1 mutant polypeptide lacks a signal sequence. In certain embodiments, the ENPP1 mutant polypeptide contains an amino acid sequence that is at least approximately 90% (e.g., at least approximately 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 present disclosure provides an ENPP1 mutant polypeptide comprising 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, wherein the ENPP1 mutant polypeptide comprises an amino acid substitution at position 256 with respect to SEQ ID NO:7. In a particular embodiment, the amino acid substitution is I256T. In a particular embodiment, the amino acid substitution is I256S.

[0108] In 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 amino acid substitutions (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) relative to amino acids 23-849 of SEQ ID NO:7. In a particular embodiment, the ENPP1 mutant polypeptide includes an amino acid substitution at position 256 relative to SEQ ID NO:7. In a particular embodiment, the amino acid substitution is I256T. In a particular embodiment, the amino acid substitution is I256S.

[0109] In a particular embodiment, the ENPP1 polypeptide contains at least one mutation within the signal sequence region shown in Figure 16A and / or Figure 16B.

[0110] In certain embodiments, the polypeptide contains the mutation I256T related to SEQ ID NO:7.

[0111] In certain embodiments, the mutation is selected from the group consisting of C25N, K27T, and V29N with respect to SEQ ID NO:7. In certain embodiments, the mutation is C25N with respect to SEQ ID NO:7. In certain embodiments, the mutation is K27T with respect to SEQ ID NO:7. In certain embodiments, the mutation is V29N with respect to SEQ ID NO:7. In certain embodiments, the ENPP1 polypeptide contains at least one mutation selected from the group consisting of C25N / K27T and V29N with respect to SEQ ID NO:7.

[0112] In certain embodiments, the ENPP1 polypeptide contains at least one mutation in the catalytic region shown 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 contains at least one mutation selected from the group consisting of mutations I256T and K369N / I371T with respect to SEQ ID NO:7.

[0113] In certain embodiments, the ENPP1 polypeptide contains at least one mutation in the endonuclease domain shown in Tables 1, 2, 3, 4, 5, Figure 7A, 16A, 16B, 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 with respect to SEQ ID NO:7. In certain embodiments, the mutation is S766N with respect to SEQ ID NO:7. In certain embodiments, the ENPP1 polypeptide contains 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.

[0114] In certain embodiments, the ENPP1 polypeptide contains 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 contains at least the E864N / L866T mutation 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, the FcRn-binding domain comprising any mutation shown in Table 1, Table 2, Figure 7A, Figure 16A, Figure 16B, Figure 17, and / or Figure 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 a particular embodiment, the mutation is N1065F with respect to SEQ ID NO:7. In a particular embodiment, the FcRn binding domain contains at least one mutation selected from the group consisting of S885N, M883Y, M883Y / S885T / T887E, and H1064K / N1065F with respect to SEQ ID NO:7.

[0116] In a particular embodiment, the ENPP1 polypeptide is C25N, K27T, V29N, C25N / K27T, I256T, K369N, I371T, K369N / I371T, P534N, V536T, R545T, P554L, E592N, R741D, S766N, P534N / V536T, P554L / R545T, E592N / R741D, E864N, It contains at least one mutation selected from the group consisting of L866T, E864N / L866T, M883Y, S885N, S885T, T887E, H1064K, N1065F, M883Y / S885T / T887E, and H1064K / N1065F.

[0117] In a particular embodiment, the polypeptide contains 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, the polypeptide comprises an ENPP1 polypeptide and an FcRn-binding domain, wherein the polypeptide includes mutations M883Y, S885T, and T887E relating to SEQ ID NO:7.

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

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

[0121] In certain embodiments, the polypeptide comprises an ENPP1 mutant polypeptide, the mutant polypeptide comprising 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 contains at least one mutation selected from the group consisting of S766N, P534N / V536T, P554L / R545T, and E592N with respect to SEQ ID NO:7.

[0123] In a particular embodiment, the polypeptide further comprises an IgG FcRn binding domain.

[0124] In certain embodiments, the polypeptide contains 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 certain embodiments, the polypeptide contains the S885N mutation in the FcRn-binding domain related to SEQ ID NO:7.

[0126] In a particular embodiment, the polypeptide contains the S766N mutation within the ENPP1 mutant polypeptide relating to SEQ ID NO:7.

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

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

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

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

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

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

[0133] In certain embodiments, the polypeptide contains the mutation I256T related to SEQ ID NO:7.

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

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

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

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

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

[0140] The specification also features any ENPP1 mutant polypeptide and heterologous protein, such as an FcRn-binding domain, as described herein. In certain embodiments, the heterologous protein is located at the carboxy-terminal end of the ENPP1 mutant polypeptide moiety of the fusion. In certain embodiments, the heterologous protein is located at the amino-terminal end of the ENPP1 mutant polypeptide moiety of the fusion.

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

[0142] In certain embodiments of any fusion 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 comprising the following amino acid substitutions relative to SEQ ID NO:7: M883Y, S885T, and T887E (MST / YTE substitution).

[0143] In certain embodiments, the fusions described herein include 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.

[0144] In any particular embodiment of any ENPP1 mutant polypeptide or fusion described herein, the ENPP1 mutant polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO:11.

[0145] In any particular embodiment of any ENPP1 mutant polypeptide or fusion described herein, the ENPP1 mutant polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO:12.

[0146] In a particular embodiment, any fusion described herein comprises (a) an ENPP1 mutant polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO:11 or SEQ ID NO:12, (b) a variant human IgG1 Fc region at the carboxyl terminus of the ENPP1 mutant polypeptide, for example, the amino acid sequence shown in SEQ ID NO:14, and (c) a linker amino acid sequence that segregates (a) and (b) (where the linker sequence is LIN (SEQ ID NO:8) or GGGGS (SEQ ID NO:9)).

[0147] Any one of the ENPP1 mutant polypeptides or ENPP1 mutant polypeptide fusions described herein and a heterologous moiety, for example, but not limited to, a small molecule conjugate is also a feature 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, for example, 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 the S885N mutation with respect to SEQ ID NO:7.

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

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

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

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

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

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

[0158] In certain embodiments of any fusion described herein, the Fc region comprises at least one mutation selected from the group consisting of S885N, M883Y, M883Y / S885T / T887E, and H1064K / N1065F with respect to SEQ ID NO:7.

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

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

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

[0167] In certain embodiments, any ENPP1 mutant polypeptide or fusion described herein is SEQ ID It contains 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 for NO:7.

[0168] In certain embodiments, any fusion described herein includes an IgG, for example, the Fc region of IgG1.

[0169] In certain embodiments, any ENPP1 mutant polypeptide described herein or a fusion protein comprising such an ENPP1 mutant polypeptide contains 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 contains at least one mutation selected from the group consisting of S766N, P534N / Y536T, P554L / R545T, and E592N with respect to SEQ ID NO:7.

[0171] In certain embodiments, any ENPP1 mutant polypeptide described herein or a fusion protein that includes such an ENPP1 mutant polypeptide includes 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 fusion described herein includes mutations I256T, M883Y, S885T, and T887E with respect to SEQ ID NO:7.

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

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

[0175] In certain embodiments, an ENPP1 mutant polypeptide fusion described herein includes, for example, a linker amino acid sequence between the ENPP1 mutant polypeptide portion and the heterologous protein portion of the fusion. In certain embodiments, the linker amino acid sequence includes or consists of SEQ ID NO:8. In certain embodiments, the linker amino acid sequence includes 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 another aspect, the present disclosure features ENPP1-containing polypeptides and their conjugates comprising or consisting of the amino acid sequences shown in SEQ ID NO:15 or SEQ ID NO:16.

[0177] In certain embodiments, the ENPP1 polypeptide lacks a nuclease domain. In other embodiments, the ENPP1 polypeptide is shortened so that the nuclease domain is removed. In yet another embodiment, the ENPP1 polypeptide is shortened so that the nuclease domain, consisting of approximately 524 to 885 residues related to SEQ ID NO:1, is removed, leaving only the catalytic domain, consisting of approximately 186 to 586 residues related to SEQ ID NO:1, so that the catalytic activity of the protein is preserved.

[0178] In certain embodiments, the ENPP1 polypeptide is modified, compared to SEQ ID NO:1, by a segment of the extracellular region of ENPP1 that includes a peptidase cleavage site after the signal peptide and between the transmembrane domain and the extracellular domain.

[0179] In certain embodiments, the ENPP1 polypeptide is modified with a segment of the extracellular region of ENPP1 that includes a fulin cleavage site between the transmembrane domain and the extracellular domain, compared to SEQ ID NO:1. In other embodiments, the ENPP1 polypeptide is not modified with a segment of the extracellular region of ENPP1 that includes a fulin cleavage site between the transmembrane domain and the extracellular domain, compared to SEQ ID NO:1.

[0180] In certain embodiments, the ENPP1 polypeptide is modified in the extracellular region segment of ENPP2, which includes the signal peptidase cleavage site, compared to SEQ ID NO:1. In other embodiments, the ENPP1 polypeptide is not modified in the extracellular region segment of ENPP2, which includes the signal peptidase cleavage site, compared to SEQ ID NO:1.

[0181] In another aspect, the present disclosure provides ENPP1 mutant polypeptides, ENPP1-containing polypeptides, or fusions expressed from CHO cell lines stably transfected with human ST6 beta-galactoside alpha-2,6-sialyltransferase (also known as ST6GAL1).

[0182] In yet another aspect, the present disclosure provides ENPP1 mutant polypeptides, ENPP1-containing polypeptides, or fusions that are grown in cell cultures supplemented with sialic acid and / or N-acetylmannosamine (also known as 1,3,4-O-Bu3ManNAc).

[0183] Pharmaceutical compositions comprising any one of the ENPP1 mutant polypeptides, ENPP1 mutant polypeptide fusions, conjugates or other polypeptides and proteins described herein, as well as a pharmaceutically acceptable carrier, are also provided herein.

[0184] In certain embodiments, the polypeptide is soluble. In other embodiments, the polypeptide is a recombinant polypeptide. In yet another embodiment, the polypeptide comprises an ENPP1 polypeptide lacking the ENPP1 transmembrane domain. In yet another embodiment, 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, in 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 a proteolytic process to produce a polypeptide containing the ENPP1 polypeptide. In other embodiments, the signal peptide is selected from the group consisting of the signal peptides of ENPP2, ENPP5, and ENPP7. In yet another embodiment, the polypeptide comprises ENPP1 and another polypeptide, for example, an ENPP1 polypeptide containing the transmembrane domain of ENPP2 as a non-limiting example. In yet another embodiment, the ENPP1 polypeptide comprises the cleavage product of a precursor ENPP1 polypeptide containing the transmembrane domain of ENPP2. In yet another embodiment, the ENPP2 transmembrane domain contains residues 12-30 of SEQ ID NO:7 corresponding to IISLFTFAVGVNICLGFTA.

[0186] In certain embodiments, the ENPP1 polypeptide is C-terminus-fused 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 N-terminus-fused 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 another embodiment, the presence of the IgFc domain improves the half-life and solubility of the ENPP1 polypeptide, reduces immunogenicity, and increases activity.

[0187] In certain embodiments, the ENPP1 polypeptide is fused to human serum albumin at its C-terminus. Human serum albumin may be conjugated to the ENPP1 protein via chemical linkers, including but not limited to naturally occurring or modified disulfide bonds, or by genetic fusion to ENPP1 or its fragments and / or variants.

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

[0189] In certain embodiments, the polypeptide has a k -1 enzyme -1 value for substrate ATP of about 3.4 (±0.4) s cat where k cat is determined by measuring the hydrolysis rate of ATP for the polypeptide.

[0190] In certain embodiments, the polypeptide has a K M value for substrate ATP of less than about 2 μM or about 2 μM, where K M is determined by measuring the hydrolysis rate of ATP for the polypeptide.

[0191] In certain embodiments, the polypeptide is formulated as a liquid formulation. In other embodiments, the disclosure provides a pharmaceutical composition in the form of a dry product comprising a therapeutically effective amount of the polypeptide of the disclosure, which dry product is reconstitutable in a solution of the compound in liquid form.

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

[0193] In certain embodiments, the polypeptide lacks a negatively charged bone targeting sequence. In still other embodiments, a polyaspartic acid domain (about 2 to about 20 or more contiguous aspartic acid residues) is a non-limiting example of a negatively charged bone targeting sequence. In other embodiments, the polypeptide has a negatively charged bone targeting sequence.

[0194] It is understood that the ENPP1 polypeptides in accordance with this disclosure include not only the native human protein but also any fragments, derivatives, fusions, conjugates, or variants having ATP hydrolysis activity of the native protein. Where used herein, the phrase “ENPP1 polypeptide, variant, or its variant fragment” also includes any compound or polypeptide (e.g., fusion proteins) containing the ENPP1 polypeptide, variant, or its variant fragment. Fusion proteins in accordance with this disclosure are considered bioequivalents of ENPP1 but are intended to provide a stronger potency, either through a longer half-life as determined by the “area under the curve” (AUC) or an extended half-life in pharmacokinetic experiments, or through increased in vivo biological exposure.

[0195] Vectors and cells Nucleic acids encoding any one of the ENPP1 mutant polypeptides, ENPP1-containing polypeptides, or fusions described herein are also provided herein. The disclosure further provides vectors containing such nucleic acids, such as expression vectors. Cells, cells, or a group of cells (e.g., mammalian cells) containing any one of the nucleic acids, vectors, or expression vectors described herein are also provided. Methods for producing a protein (e.g., any one of the ENPP1 mutant polypeptides, ENPP1-containing polypeptides, or fusions described herein) are also provided, which in a particular embodiment include the step of culturing the cell, cells, or group of cells under conditions suitable for the expression of the protein by the cell or cells from the nucleic acid, vector, or expression vector. The method may also include the step of purifying the protein from the cell, cells, or group of cells, or from the medium in which the cell, cells, or group of cells were cultured. In addition, the disclosure provides proteins purified by any such method.

[0196] The Disclosure further provides autonomously replicating or embedded mammalian cell vectors comprising recombinant nucleic acids encoding the polypeptide of the 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 another embodiment, the vector further comprises at least one nucleic acid sequence that directs and / or controls the expression of the polypeptide. In yet another embodiment, the recombinant nucleic acid encodes a polypeptide comprising the ENPP1 polypeptide and a signal peptide, which is proteolytically processed upon secretion from a cell to produce the ENPP1 polypeptide of the Disclosure.

[0197] In yet another aspect, the Disclosure provides isolated host cells containing the vector of the Disclosure. In certain embodiments, the cells are non-human cells. In other embodiments, the cells are mammalian. In yet another embodiment, the vector of the Disclosure comprises recombinant nucleic acids encoding polypeptides including the ENPP1 polypeptide and signal peptide of the Disclosure. In yet another embodiment, the polypeptide is proteolytically processed upon secretion from the cell to produce the ENPP1 polypeptide of the Disclosure.

[0198] Cloning and expression of ENPP1 ENPP1 or the ENPP1 polypeptide is prepared as described in US 2015 / 0359858 A1, which is incorporated herein by reference in its entirety. ENPP1 is a transmembrane protein that localizes to the cell surface and has different intramembrane domains. To express ENPP1 as a soluble extracellular protein, the transmembrane domain of ENPP1 may be replaced with the transmembrane domain of ENPP2, thereby accumulating soluble recombinant ENPP1 in the extracellular fluid of baculovirus cultures.

[0199] Signal sequences of any other known proteins, such as, non-limitingly, signal sequences of immunoglobulin kappa and lambda light chain proteins, may also be used to secrete the extracellular domain of ENPP1. Furthermore, this disclosure should not be construed as being limited to the polypeptides described herein, but also includes polypeptides comprising any enzymatically active truncated form of the ENPP1 extracellular domain.

[0200] ENPP1 is made soluble by removing its 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 having a TEV protease cleavage site followed by a C-terminal 9-F1IS tag. This vector was then cloned and expressed in insect cells, and both proteins were expressed in a baculovirus system as previously described (Albright, et al., 2012, Blood 120:4432-4440; Saunders, et al., 2011, J. Biol. Chem. 18:994-1004; Saunders, et al., 2008, Mol. Cancer Ther. 7:3352-3362), allowing the soluble recombinant proteins to accumulate in the extracellular fluid.

[0201] Production and purification of ENPP1 and ENPP1 fusion proteins In certain embodiments, soluble ENPP1 polypeptides comprising an IgG Fc domain or an enzymatically / biologically active fragment thereof are effective for treating, mitigating, and / or preventing the progression of diseases or disorders envisioned herein. In other embodiments, soluble ENPP1 polypeptides do not contain bone-targeting domains, such as 2 to 20 consecutive polyaspartic acid residues or 2 to 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 this fusion protein was expressed in a stable CHO cell line. This protein can also be expressed from HEK293 cells, baculovirus insect cell systems, or CHO cells or Pichia yeast expression systems using a suitable vector. This protein can be produced in either adherent or suspension cells. Preferably, this 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 those in bacteria (e.g., Escherichia coli and Bacillus subtilis), yeasts (e.g., Saccharomyces cerevisiae, Kluyveronmyces lactis, and Pichia pastoris), filamentous fungi (e.g., Aspergillus), plant cells, animal cells, and insect cells. The desired protein can be produced by conventional methods, for example, from a coding sequence inserted into a host chromosome or on a free plasmid.

[0204] Yeast can be transformed with the coding sequence of a desired protein by any conventional method, such as electroporation. A method for transforming yeast by electroporation is disclosed in Becker & Guarente, 1990, Methods Enzymol. 194: 182. Successfully transformed cells, i.e., cells containing the DNA constructs of this disclosure, can be identified by well-known techniques. For example, cells obtained by introducing an expression construct can be grown to produce the desired polypeptide. The cells can be collected and lysed, and their DNA content can be tested for the presence of 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 protein in the supernatant can be detected using an antibody.

[0205] Useful yeast plasmid vectors, including pRS403-406 and pRS413-416, are typically available from Strat:1.gene Cloning Systems, La Jolla, CA, USA. Plasmids pRS403, pRS404, pRS405, and pRS406 are yeast integration plasmids (Y1ps) containing yeast selection markers I-llS3, TRP1, LEU2, and lJRA3. Plasmids pRS413-416 are yeast centromere plasmids (YCps).

[0206] Various methods have been developed to functionally link DNA to a vector via complementary sticky ends. For example, a complementary homopolymer tract can be added to the DNA segment to be inserted into the vector DNA. The vector and DNA segment are then connected by hydrogen bonds between the complementary homopolymer tails to form a recombinant DNA molecule.

[0207] Synthetic linkers containing one or more restriction sites provide an alternative method for linking DNA segments to vectors. DNA segments produced by endonuclease restriction digestion are treated with bacteriophage T4 DNA polymerase or E. coli DNA polymerase I, enzymes that remove the protruding 3' single-stranded ends by their 3'-5' exonuclease activity and fill in the inverted 3' ends by their polymerization activity.

[0208] The combination of these activities, therefore, generates 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 product is DNA segments having polymer linker sequences at their ends. These DNA segments are then ligated into expression vectors that have been cleaved with appropriate restriction enzymes and then cleaved with enzymes that produce ends that fit those of the DNA segments.

[0209] Next, a single, stably transfected cell clone is established and screened for high-expression clones of the 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 the synthase substrate pNP-TMP, as previously described (Albright, et al., 2015, Nat. Commun. 6:10006). Following the identification of high-expression clones through screening, protein production can be achieved in a shaking flask or bioreactor, as previously described in Albright, et al., 2015, Nat. Commun. 6:10006.

[0210] The purification of ENPP1 can be achieved using a combination of standard purification techniques known in the art. An example of this is described above with respect to the production of ENPP1 protein. After purification, ENPP1-Fc is Zn2+ and Mg 2+ Dialyze the solution into PBS (PBSplus) supplemented with the substance to concentrate it to 5-7 mg / ml, and freeze it at -80°C in 200-500 μl aliquots. Thaw the aliquots immediately before use 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 useful polypeptides in this disclosure may be used in gene therapy protocols for the treatment of diseases or disorders envisioned herein. Improved constructs encoding such polypeptides may 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, are used in the art to introduce genes into a wide variety of different target cells. Typically, a vector is exposed to target cells so that transformation occurs in a sufficient proportion of cells to provide a useful therapeutic or prophylactic effect from the expression of a desired polypeptide (e.g., a receptor). The transfected nucleic acid may be permanently integrated into the genome of each target cell to provide a long-lasting effect, or the treatment may need to be repeated periodically. In a particular embodiment, a (viral) vector is transfected in vivo into liver cells with genetic material encoding the polypeptide of the present disclosure.

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

[0214] Gene therapy via AAV AAV, a parvovirus belonging to the genus Dependvirus, possesses several 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. Furthermore, AAV can infect cells of various species. Importantly, AAV is not associated with any human or animal disease and does not appear to alter the physiological properties of host cells upon incorporation. Finally, AAV is stable under a wide range of physical and chemical conditions, which makes it suitable for manufacturing, storage, and transport requirements.

[0215] The AAV genome, a linear, single-stranded DNA molecule containing approximately 4,700 nucleotides (the AAV-2 genome consists of 4,681 nucleotides, and the AAV-4 genome consists of 4,767 nucleotides), typically contains internal non-repeat segments adjacent to inverted end repeats (ITRs) at each end. ITRs are approximately 145 nucleotides long (AAV-1 has an ITR of 143 nucleotides) and have multiple functions, including acting as an origin of replication and as a packaging signal for the viral genome.

[0216] The internal non-repeat region of this genome contains two large open reading frames (ORFs) known as the AAV replication (rep) and capsid (cap) regions. These ORFs encode replication and capsid gene products, enabling the replication, assembly, and packaging of the complete AAV virion. More specifically, a family of at least four viral proteins, Rep 78, Rep 68, Rep 52, and Rep 40, all named so from their apparent molecular weights, are expressed from the AAV rep region. The AAV cap region encodes at least three proteins, VP1, VP2, and VP3.

[0217] AAV is a helper-dependent virus, meaning it requires co-infection with a helper virus (e.g., adenovirus, herpesvirus, or vacciniavirus) to form a functionally complete AAV virion. Without helper virus co-infection, AAV enters a latent state where the viral genome is inserted into the host cell's 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 a viral capsid, thereby reconstructing an infectious virion. AAV can infect cells of different species, but the helper virus must be of the same species as the host cell. Therefore, for example, human AAV replicates in canine cells co-infected with canine adenovirus.

[0218] To generate infectious recombinant AAV (rAAV) containing heterologous nucleic acid sequences, an AAV vector containing heterologous nucleic acid sequences but lacking AAV helper functional genes, rep, and cap can be transfected into a suitable host cell line. Then, the AAV helper functional genes can be provided in a separate vector. Alternatively, instead of providing a replicating helper virus (e.g., adenovirus, herpesvirus, or vaccinia), only the helper virus genes required for AAV generation (i.e., accessory functional genes) can be provided in the vector.

[0219] In summary, AAV helper functional genes (i.e., rep and cap) and accessory functional genes can be provided in one or more vectors. The helper and accessory functional gene products can then be expressed in host cells, where they can act in trans on rAAV vectors containing heterologous nucleic acid sequences. The rAAV vector containing heterologous nucleic acid sequences can then be replicated and packaged to form a recombinant virion, similar to a wild-type (wt) AAV genome. When the resulting rAAV virion infects a patient's cells, the heterologous nucleic acid sequences enter and are expressed within the patient's cells. Since the patient's cells lack rep and cap genes and accessory functional genes, the rAAV cannot further replicate and package their genomes. Furthermore, without a source of rep and cap genes, wtAAV cannot be formed within the patient's cells.

[0220] There are 11 known AAV serotypes, from AAV-1 to 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 monkey isolates, but both serotypes can be transduced into human cells (Chiorini, et al., 1997, J Virol 71:6823-6833; Chou, et al., 2000, Mol Ther 2:619-623). Of these six known serotypes, AAV-2 is the most distinctive. For example, AAV-2 has been used in a wide range of in vitro transduction experiments, including mice (US Patent No. 5,858,351; US ​​Patent No. 6,093,392), canine 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); and mouse heart (Su, et al., 2000, Proc. Natl. Acad. Sci. USA). It has been shown to transduce many different tissue types, including 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 utilized to deliver tissue-specific transgenes. For example, AAV-2 vectors have been used to deliver the following genes: a cystic fibrosis membrane conductance regulator to rabbit lung (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. The transgenes (16:270-276; U.S. Patent No. 6,093,392) have been introduced into mouse liver, dog and mouse muscle (U.S. Patent No. 6,093,392); the erythropoietin gene into mouse muscle (U.S. Patent No. 5,858,351); the vascular endothelial growth factor (VEGF) gene into mouse heart (Su, et al., 2000, Proc. Natl. Acad. Sci. USA 97:13801-13806); and the aromatic 1-amino acid decarboxylase gene into monkey nerve cells. The expression of transgenes delivered by specific rAAVs has shown therapeutic effects in experimental animals; for example, the expression of factor IX has been reported to restore phenotypic normality in a canine model of hemophilia B (U.S. Patent No. 6,093,392). Furthermore, the expression of NEGF delivered to mouse myocardium by rAAV resulted in cardiovascular formation (Su, et al., 2000, Proc. Natl. Acad. Sci. USA 97:13801-13806), and the expression of AADC delivered to the brains of Parkinson's disease monkeys by rAAV resulted in the restoration of dopaminergic function.

[0222] The delivery of a protein of interest to mammalian cells is achieved by first constructing an AAV vector containing DNA encoding the protein of interest, and then administering that vector to the mammal. Therefore, this disclosure should be considered to include an AAV vector containing DNA encoding the polypeptide of interest. Upon reading this disclosure, the construction of this / these AAV vectors containing DNA encoding these polypeptides will be obvious to those skilled in the art.

[0223] In certain embodiments, the rAAV vectors of the present disclosure include various essential DNA elements. In certain embodiments, these DNA elements include at least two copies of an AAV ITR sequence, a promoter / enhancer element, a transcription termination signal, and any required 5' or 3' untranslated region adjacent to the DNA encoding the protein of interest or a bioactive fragment thereof. The rAAV vectors of the present disclosure may also include a portion of the introns of the protein of interest. Optionally, the rAAV vectors of the present disclosure may also include DNA encoding the mutant polypeptide of interest.

[0224] In certain embodiments, the vector comprises a promoter / regulatory sequence containing an indiscriminate promoter capable of inducing high levels of heterologous gene expression in many different cell types. Such promoters include, but are not limited to, cytomegalovirus (CMV) early promoter / enhancer sequences and Roussarcoma virus promoter / enhancer sequences. In certain embodiments, the promoter / regulatory sequence in the rAAV vector of this disclosure is the CMV early promoter / enhancer. However, the promoter sequence used to induce heterologous gene expression may also be an inducible promoter, e.g., a steroid-inducible promoter, or a tissue-specific promoter, e.g., a skeletal α-actin promoter and a muscle creatine kinase promoter / enhancer, which are not limited to muscle tissue.

[0225] In certain embodiments, the rAAV vectors of the Disclosure include a transcription termination signal. Any transcription termination signal may be included in the vectors of the Disclosure, but in certain embodiments, the transcription termination signal is the SV40 transcription termination signal.

[0226] In certain embodiments, the rAAV vectors of this disclosure comprise isolated DNA encoding a polypeptide of interest or a biologically active fragment of a polypeptide of interest. This disclosure should be interpreted as encompassing any mammalian sequence of the polypeptide of interest, whether known or unknown. Accordingly, this disclosure should be interpreted as encompassing a gene of non-human mammalian origin in which the polypeptide functions substantially similarly to a human polypeptide. Preferably, the nucleotide sequence containing the gene encoding the polypeptide of interest is about 50% homologous, more preferably about 70% homologous, even more preferably about 80% homologous, and most preferably about 90% homologous to the gene encoding the polypeptide of interest.

[0227] Furthermore, this disclosure should be interpreted as encompassing naturally occurring variants or recombinant variants of wild-type protein sequences that are as therapeutically effective as, or even more therapeutically effective than, the full-length polypeptide in the gene therapies of this disclosure.

[0228] This disclosure should also be interpreted as encompassing DNA encoding variants that preserve 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, for example, variants that confer enhanced stability to the protein in plasma or enhanced specific activity to the protein. Analogs may differ from naturally occurring proteins or peptides by conserved amino acid sequence differences, by sequence-independent modifications, or both. For example, there may be conserved amino acid changes that alter the primary sequence of the protein or peptide but do not typically alter its function.

[0229] This disclosure is not limited to the specific rAAV vectors illustrated in the experimental examples, and should be interpreted as encompassing any suitable AAV vector, including but not limited to vectors based on AAV-1, AAV-3, AAV-4, and AAV-6.

[0230] Methods for treating a mammal with a disease or disorder in an amount effective to provide a therapeutic effect are also included in this disclosure. These methods include administering an rAAV vector encoding a polypeptide of interest to the mammal. Preferably, the mammal is human.

[0231] Typically, the number of viral vector genomes per mammal administered in a single injection is approximately 1 × 10⁶. 8 ~Approx. 5×10 16 This is within the range of a few individuals. Preferably, the number of viral vector genomes / mammal administered in a single injection is about 1 × 10⁶. 10 ~Approx. 1×10 15 The number of viral vector genomes per mammal administered in a single injection is approximately 5 × 10⁶. 10 ~Approx. 5×10 15 The number of viral vector genomes administered to a mammal in a single injection is, most preferably, approximately 5 × 10⁶. 11~Approx. 5×10 14 It is an individual.

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

[0233] For the administration of the rAAV vector of this disclosure by single-site injection, in certain embodiments, the virus-containing composition is injected directly into the organ of interest (e.g., the liver of interest).

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

[0235] The rAAV vectors of this disclosure may also be provided in kit form, which may include, for example, a lyophilized preparation of the vector in a dry salt formulation, sterile water for suspending the vector / salt composition, and instructions for suspending the vector and administering it to a mammal.

[0236] array SEQ ID NO:1:hENPP1 amino acid sequence TIFF0007874896000004.tif98149

[0237] SEQ ID NO:2:ENPP2 amino acid sequence TIFF0007874896000005.tif98149

[0238] SEQ ID NO:3:hIgG Fc domain, Fc TIFF0007874896000006.tif24148

[0239] SEQ ID NO:4:hENPP5 protein transport signal sequence TIFF0007874896000007.tif3128 Here, Xaa 23 It does not exist or is L. Here, Xaa 23 If Xaa does not exist, 24 It does not exist, Xaa 23 If L, then Xaa 24 It either does not exist or is Q.

[0240] SEQ ID NO: 5: hENPP7 protein transport signal sequence TIFF0007874896000008.tif3128

[0241] SEQ ID NO: 6: hENPP7 protein transport signal sequence TIFF0007874896000009.tif3128

[0242] SEQ ID NO:7:ENPP1-Fc TIFF0007874896000010.tif118149 Bold: Signal array Standard: ENPP1 extracellular domain Underlined: Linker array Italics: Fc domain

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

[0244] SEQ ID NO: 9: Exemplary amino acid linker sequence (GGGGS) n n is an integer between 1 and 10, including 1 and 10, 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 TIFF0007874896000011.tif91148

[0246] SEQ ID NO:11: Exemplary ENPP1 mutant polypeptide (substitutions for wild-type human ENPP1 are shown in bold / underlined) TIFF0007874896000012.tif91148

[0247] SEQ ID NO:12: Exemplary ENPP1 mutant polypeptide (substitutions for wild-type human ENPP1 are shown in bold / underlined) TIFF0007874896000013.tif91148

[0248] SEQ ID NO: 13: Exemplary human IgG1 Fc region TIFF0007874896000014.tif24148

[0249] SEQ ID NO: 14: Exemplary variant human IgG1 Fc region (including MST / YTE substitution (bold / underlined)) TIFF0007874896000015.tif24148

[0250] SEQ ID NO: 15: Exemplary ENPP1-containing fusion: The extracellular domain of (GGGGS)1 (SEQ ID NO: 9 (n=1); double underlined) fused at its C-terminus to the variant human IgG Fc region (SEQ ID NO: 14; unmodified text). TIFF0007874896000016.tif118149 Italics: ENPP1 extracellular domain Double underline: Linker array Standard: IgG Fc region

[0251] SEQ ID NO: 16: Exemplary ENPP1-containing fusion: The amino acid sequence LIN (SEQ ID NO: 8; double underlined) fused at its C-terminus to the variant human IgG Fc region (SEQ ID NO: 14; unmodified text), and the ENPP1 extracellular domain (SEQ ID NO: 10; italicized) fused at its C-terminus to the LIN amino acid sequence (SEQ ID NO: 8; double underlined). TIFF0007874896000017.tif118149 Italics: ENPP1 extracellular domain Double underline: Linker array Standard: IgG Fc region

[0252] SEQ ID NO: 17: Exemplary ENPP1 mutant polypeptide fusion: ENPP1 mutant polypeptide (SEQ ID NO: 11; italics) fused at the C-terminus of LIN (SEQ ID NO: 8; double underlined) fused at the C-terminus of the variant human IgG Fc region (SEQ ID NO: 14; unmodified text) TIFF0007874896000018.tif118149

[0253] SEQ ID NO: 18: Exemplary ENPP1 mutant polypeptide fusion: ENPP1 mutant polypeptide (SEQ ID NO: 11; italics) fused at its C-terminus to (GGGGS)1 (SEQ ID NO: 9, n=1; double underlined) fused at its C-terminus to the variant human IgG Fc region (SEQ ID NO: 14; unmodified text). TIFF0007874896000019.tif121150

[0254] method This disclosure includes a method for reducing or preventing the progression of pathological calcification in a subject where such reduction is needed, the method comprising the step of administering a therapeutically effective amount of the polypeptide of this disclosure to the subject.

[0255] The Disclosure further includes a method for reducing or preventing the progression of pathological ossification in a subject where such reduction is needed, comprising the step of administering a therapeutically effective amount of the polypeptide of the Disclosure to the subject.

[0256] The Disclosure further includes a method for reducing or preventing the progression of ectopic calcification of soft tissue, including reducing, improving, or preventing vascular calcification, in a subject where such reduction is needed, comprising the step of administering a therapeutically effective amount of the polypeptide of the Disclosure to a subject.

[0257] The disclosure further includes methods for mitigating or preventing the progression of diseases caused by ENPP1 deficiency. ENPP1 deficiency is characterized by a reduced level of ENPP1 activity and / or deficient ENPP1 expression in a subject in need (compared to that of a normal healthy subject, either ENPP1 activity level or ENPP1 expression level), and the methods include administering a therapeutically effective dose of the polypeptide of the disclosure to the subject.

[0258] The Disclosure further includes a method for mitigating or preventing the progression of a disease caused by low plasma PPi levels in a subject in need, comprising the step of administering a therapeutically effective dose of the polypeptide of the Disclosure to the subject to increase the subject's plasma PPi to a normal (1–3 μM) or above-normal (30–50% higher) level, and then maintain the plasma PPi at a constant normal or above-normal level. The Method further includes the step of administering additional therapeutically effective doses at intervals of 2 days, 3 days, 1 week, or 1 month to maintain the subject's plasma PPi at a constant normal or above-normal level to reduce or prevent the progression of pathological calcification or ossification.

[0259] The disclosure further includes a method for treating, improving, or preventing the progression of ossification of the posterior longitudinal ligament (OPLL) in a subject in need thereof, the method comprising the step of administering a therapeutically effective dose of the polypeptide of the disclosure to the subject.

[0260] The Disclosure further includes a method for treating, reversing or preventing the progression of hypophosphatemic rickets in a subject in need thereof, the method comprising administering a therapeutically effective dose of the polypeptide of the Disclosure to the subject.

[0261] The Disclosure further includes a method for alleviating or preventing the progression of at least one disease selected from the group consisting of chronic kidney disease (CKD), end-stage renal disease (ESRD), uremic arteriolar calcification (CUA), calciphylaxis, ossification of the posterior longitudinal ligament (OPLL), hypophosphatemic rickets, osteoarthritis, age-related arteriosclerosis, idiopathic infantile arteriosclerosis (IIAC), generalized infantile arteriosclerosis (GACI), and atherosclerotic plaque calcification in a subject diagnosed with said at least one disease, the method comprising administering a therapeutically effective dose of the polypeptide of the Disclosure to the subject.

[0262] The Disclosure further includes 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 a therapeutically effective amount of the polypeptide of the Disclosure to the subject.

[0263] The Disclosure further includes a method for mitigating or preventing the progression of a disease caused by ENPP1 deficiency (e.g., a decrease in the level of ENPP1 activity and / or ENPP1 expression compared to that of a normal healthy subject, respectively), comprising the step of administering a therapeutically effective dose of the polypeptide of the Disclosure to a subject.

[0264] The Disclosure further includes a method for mitigating or preventing the progression of a disease caused by lower-than-normal plasma PPi levels in a subject in need thereof, comprising the step of administering to a subject a therapeutically effective dose of the polypeptide of the Disclosure to increase and / or maintain the plasma PPi of the subject to a level of approximately 90%, 95%, 100%, 105%, 110%, 120%, 130%, 140%, or 150% of a normal PPi level (approximately 1–3 μM). In certain embodiments, the Method further includes further administration of the polypeptide of the Disclosure every two days, three days, one week, or one month to maintain the plasma PPi level at approximately 90%, 95%, 100%, 105%, 110%, 120%, 130%, 140%, or 150% of a normal PPi level, thereby preventing the progression of pathological calcification or ossification.

[0265] The Disclosure further includes a method for treating, improving, or preventing the progression of pseudoxanthoma elasticum (PXE) in a subject in need thereof, the method comprising the step of administering a therapeutically effective dose of the polypeptide of the Disclosure to the subject.

[0266] The Disclosure further includes a method for treating, reversing, or preventing the progression of calcification of atherosclerotic plaque in arterial vessels in a subject where such treatment is needed, the method comprising administering a therapeutically effective dose of the polypeptide of the Disclosure to the subject.

[0267] The disclosure further includes a method for treating, improving or preventing the progression of osteoarthritis in a subject in need thereof, comprising the step of administering a therapeutically effective amount of the polypeptide of the disclosure to the subject.

[0268] The Disclosure further includes a method for treating, reversing, or preventing the progression of arteriosclerosis caused by progeria in a subject in need thereof, the method comprising the step of administering a therapeutically effective amount of the polypeptide of the Disclosure to the subject.

[0269] The Disclosure further includes methods for treating, improving, or preventing the progression of X-linked hypophosphatemic rickets (XLH), hereditary hypophosphatemic rickets (HHRH), hypophosphatemic osteopathy (HBD), autosomal dominant hypophosphatemic rickets (ADHR), and / or autosomal recessive hypophosphatemic rickets in a subject where such treatment is needed, comprising the step of administering a therapeutically effective dose of the polypeptide of the Disclosure to the subject.

[0270] The Disclosure further includes a method for treating, improving, or preventing the progression of age-related osteopenia in a subject in need thereof, the method comprising the step of administering a therapeutically effective amount of the polypeptide of the Disclosure to the subject.

[0271] The Disclosure further includes a method for treating, improving, or preventing the progression of ankylosing spondylitis in a subject in need thereof, the method comprising the step of administering a therapeutically effective amount of the polypeptide of the Disclosure to the subject.

[0272] The Disclosure further includes a method for treating, improving, or preventing the progression of a pediatric sickle cell anemia in a subject in need thereof, the method comprising administering a therapeutically effective dose of the polypeptide of the Disclosure to the subject.

[0273] The Disclosure further includes a method for reducing or preventing the progression of pathological calcification in a subject in need thereof, comprising the step of administering to a subject a therapeutically effective dose 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 for reducing or preventing the progression of pathological ossification in a subject in need thereof, comprising the step of administering to a subject a therapeutically effective dose 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 for reducing or preventing the progression of ectopic calcification of soft tissue in a subject in need thereof, comprising the step of administering to a subject in a therapeutically effective dose 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 disclosure further includes a method for treating, improving or preventing the progression of ossification of the posterior longitudinal ligament (OPLL) in a subject in need thereof, comprising the step of administering to the subject a therapeutically effective dose of any one of the ENPP1 mutant polypeptides, fusions, ENPP-1-containing polypeptides or conjugates described herein, thereby reducing, improving or preventing ossification of the posterior longitudinal ligament (OPLL) in the subject.

[0277] The disclosure further includes 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 dose of any one of the ENPP1 mutant polypeptides, fusions, ENPP-1-containing polypeptides or conjugates described herein, thereby reducing, improving or preventing the progression of hypophosphatemic rickets in the subject.

[0278] The disclosure further includes 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 arteriosclerosis (IIAC), systemic infantile arteriosclerosis (GACI), and arteriosclerotic plaque calcification in a subject diagnosed with said at least one disease, comprising the step of administering to the subject in a therapeutically effective dose any one of the ENPP1 mutant polypeptides, fusions, ENPP-1-containing polypeptides or conjugates described herein, thereby reducing or preventing the progression of said disease.

[0279] The disclosure further includes a method for reducing or preventing the progression of age-related arteriosclerosis in a subject in need thereof, comprising the step of administering to a subject in a therapeutically effective dose 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 arteriosclerosis in the subject.

[0280] The Disclosure further includes a method for increasing the PPi level in a subject having a PPi level lower than the normal level, comprising administering to the subject a therapeutically effective dose of any one of the ENPP1 mutant polypeptides, fusions, ENPP-1-containing polypeptides or conjugates described herein, wherein the administration raises the PPi level in the subject to at least 2 μM, and maintains it at substantially the same level.

[0281] The Disclosure further includes a method for reducing or preventing the progression of pathological calcification or ossification in a subject having a PPi level lower than a normal level, comprising the step of administering to the subject a therapeutically effective dose 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 or ossification in the subject.

[0282] The disclosure further includes a method for treating an ENPP1 defect that manifests as a decrease in extracellular pyrophosphate (PPi) concentration in a subject requiring such treatment, comprising administering to the subject a therapeutically effective dose of any one of the ENPP1 mutant polypeptides, fusions, ENPP-1-containing polypeptides or conjugates described herein, thereby increasing the PPi level in the subject.

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

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

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

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

[0287] In a specific manner, the subjects are individuals diagnosed with progeria.

[0288] In certain embodiments of any method described herein, the ENPP1 mutant polypeptide, fusion, or ENPP1-containing polypeptide is a secretion product of an ENPP1 precursor protein expressed in mammalian cells, the ENPP1 precursor protein comprising a signal peptide sequence and an ENPP1 polypeptide, and the ENPP1 precursor protein undergoes a proteolytic process to produce the ENPP1 polypeptide. In certain embodiments, the polypeptide of the Disclosure is a secretion product of an ENPP1 precursor protein expressed in mammalian cells. In other embodiments, the ENPP1 precursor protein comprises a signal peptide sequence and an ENPP1 polypeptide, and the ENPP1 precursor protein undergoes a proteolytic process to become the polypeptide of the Disclosure. In yet another embodiment, in the ENPP1 precursor protein, the signal peptide sequence is ligated to the N-terminus of the ENPP1 polypeptide. Proteolytic cleavage of the signal peptide sequence from the ENPP1 precursor protein provides the ENPP1 polypeptide. In certain embodiments, the signal peptide sequence is selected from the group consisting of the ENPP1 signal peptide sequence, the ENPP2 signal peptide sequence, the ENPP7 signal peptide sequence, and the ENPP5 signal peptide sequence.

[0289] In certain embodiments, polypeptides are administered to subjects over a short or long term. In other embodiments, polypeptides are administered to subjects topically, regionally, parenterally, or systemically.

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

[0291] In certain embodiments, the ENPP1 mutant polypeptide, ENPP1-containing polypeptide or fusion, or its precursor protein, is administered by at least one route selected from the group consisting of subcutaneous, oral, aerosol, inhalation, rectal, vaginal, transdermal, subcutaneous, intranasal, oral, sublingual, parenteral, intrathecal, gastric, ocular, pulmonary, and topical administration. In other embodiments, the ENPP1 mutant polypeptide, ENPP1-containing polypeptide or fusion, or its precursor protein, is administered to a subject as a pharmaceutical composition further comprising at least one pharmaceutically acceptable carrier.

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

[0293] With this disclosure, including the methods detailed herein, it will be understood by those skilled in the art that this disclosure is not limited to the treatment of an established disease or disorder. In particular, the symptoms of the disease or disorder do not need to be manifest to a degree that is harmful to the subject, and in fact, the disease or disorder does not need to be detectable in the subject before treatment is performed. That is, the substantial symptoms of the disease or disorder do not need to have occurred before this disclosure can provide any benefit.

[0294] Accordingly, the Disclosure includes methods for preventing diseases and disorders in a subject, as fully described herein, in that the polypeptides of the Disclosure may be administered to a subject before the onset of a disease or disorder, as discussed elsewhere herein, thereby preventing the progression of the disease or disorder. In particular, if the symptoms of the disease or disorder have not manifested to a point of harm to the subject, the disease or disorder does not actually need to be detected in the subject before treatment is performed. That is, the substantial pathological condition from the disease or disorder does not need to have occurred before the Disclosure can provide a benefit. Accordingly, the Disclosure includes methods for preventing or delaying the onset of a disease or disorder, or reducing the progression or growth of a disease or disorder, in that the polypeptides of the Disclosure may be administered to a subject before the detection of a disease or disorder. In certain embodiments, the polypeptides of the Disclosure are administered to a subject with a strong family history of a disease or disorder, thereby preventing or delaying the onset or progression of the disease or disorder.

[0295] Therefore, a person skilled in the art, with the disclosures herein, will understand that the prevention of disease or impairment in a subject includes the administration of the polypeptides of this disclosure to the subject as a preventive measure against such disease or impairment.

[0296] Pharmaceutical compositions and formulations This disclosure provides pharmaceutical compositions comprising the polypeptides of this disclosure, as included in the methods described herein.

[0297] Such pharmaceutical compositions are in a form suitable for administration to a subject, or the pharmaceutical composition may further include one or more pharmaceutically acceptable carriers, one or more additional components, or some combination thereof. Various components of the pharmaceutical composition may be prepared in the form of physiologically acceptable salts, for example, in combination with physiologically acceptable cations or anions, as is well known in the art.

[0298] In one embodiment, a pharmaceutical composition useful for carrying out the method of the present disclosure may be administered to deliver a dose of 1 ng / kg / day to 100 mg / kg / day. In another embodiment, a pharmaceutical composition useful for carrying out the method 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 the active ingredient, pharmaceutically acceptable carrier, and any additional ingredients in the pharmaceutical compositions of this disclosure may vary depending on the specificity, size, and state of the object being treated, and further depending on the route through which the composition is administered. For example, a composition may contain about 0.1% to about 100% (w / w) of the active ingredient.

[0300] Pharmaceutical compositions useful in the methods disclosed herein can be appropriately developed for inhalation, oral, rectal, vaginal, parenteral, topical, transdermal, pulmonary, intranasal, buccal, ophthalmic, intrathecal, intravenous, or other routes of administration. Other conceivable formulations include projected nanoparticles, liposomal preparations, resealed erythrocytes containing active ingredients, and immuno-based formulations. The route of administration will be immediately apparent to those skilled in the art and will depend on any many factors, including the type and severity of the disease being treated, the type and age of the animal or human patient being treated, and so on.

[0301] Formulations of the pharmaceutical compositions described herein may be prepared by any method known or subsequently developed in the pharmaceutical field. Generally, such preparations include the steps of combining the active ingredient with a carrier or one or more other auxiliary ingredients, and subsequently, if necessary or desirable, forming or packaging the product into desired single or multi-dose units.

[0302] As used herein, “unit dose” refers to a specific amount of a pharmaceutical composition containing a predetermined amount of the active ingredient. The amount of the active ingredient is usually equal to the dose of the active ingredient administered to the subject or a convenient portion of such a dose, for example, half or one-third of such a dose. A unit dosage form may be for one of two doses: once daily or multiple daily doses (e.g., about 1 to 4 times or more per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose.

[0303] Administration / Medication The administration regimen may influence what constitutes an effective dose. For example, divided doses and alternating doses may be administered daily or continuously, or the dose may be administered by continuous infusion or bolus injection. Furthermore, the administration of the therapeutic formulation may be increased or decreased in proportion to the urgency of the treatment or prevention situation. In certain embodiments, administration of the compounds of this disclosure to a subject raises the subject's plasma PPi to a near-normal level, where the normal level of PPi in mammals is 1–3 μM. "Near-normal" means 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] The administration of the compounds of this disclosure to patients, e.g., mammals, e.g., humans, may be carried out using known methods in doses and for durations effective in treating the disease or disorder in that patient. The effective amount of the therapeutic compound required to achieve a therapeutic effect may vary depending on factors such as the activity of the individual compound used; the time of administration; the elimination 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, overall health, and previous treatment history of the patient being treated, as well as similar factors well known in the medical field. The dosing regimen may be adjusted to provide an optimal therapeutic response. The dose is determined based on the biological activity of the therapeutic compound, which depends on the half-life and the area under the plasma time curve of the therapeutic compound. Polypeptides according to this disclosure are administered at appropriate time intervals, every two days, every four days, weekly, or monthly, to achieve a sustained level of plasma PPi that is either close to the normal (1–3 μM) level of PPi or above the normal level (30–50% higher). The therapeutic dose of the polypeptides of this disclosure may also be determined based on the half-life or the rate at which the therapeutic polypeptide is removed from the body. Polypeptides in accordance with this disclosure are administered at appropriate time intervals every two days, every four days, weekly, or monthly to achieve a certain 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 urgency of the treatment situation. A non-limiting example of the effective dose range of the therapeutic compounds disclosed herein is about 0.01 to 50 mg / kg body weight / day. In certain embodiments, the effective dose range of the therapeutic compounds disclosed herein is about 50 ng to 500 ng / kg, preferably 100 ng to 300 ng / kg body weight. Those skilled in the art will likely be able to study and determine the relevant factors relating to the effective amount of the therapeutic compound without excessive experimentation.

[0306] The compound may be administered to the patient several times a day, or it may be administered less frequently, for example, once a day, once a week, once every two weeks, once a month, or even less frequently, for example, once every few months or once a year or less. The amount of the compound administered per day is understood to be, in non-limiting examples, daily, every other day, every two days, every three days, every four days, or every five days. For example, in the case of administration every other day, a dose of 5 mg per day may be started on Monday, a first subsequent dose of 5 mg per day may be administered on Wednesday, and a second subsequent dose of 5 mg per day may be administered on Friday. The frequency of administration will be immediately apparent to those skilled in the art and will depend on any many factors, such as the type and severity of the disease being treated, as well as the type and age of the patient.

[0307] The actual dose levels of the active ingredient in the pharmaceutical compositions of this disclosure may be modified to obtain an amount of the active ingredient that is effective in achieving the desired therapeutic response in individual patients, compositions, and modes of administration without causing toxicity to patients.

[0308] A physician with ordinary skill in the art, such as a doctor, can immediately determine and prescribe the effective amount of the required pharmaceutical composition. For example, a doctor or veterinarian may start the dose 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 dose until the desired effect is achieved.

[0309] In certain embodiments, the compositions of the Disclosure are administered to a patient in doses ranging from one to five times per day or more. In other embodiments, the compositions of the Disclosure are administered to a patient in a range of doses including, but not limited to, once daily, every two days, every three days, once a week, and once every two weeks. The frequency of administration of the various combination compositions of the Disclosure will vary from patient to patient depending on many factors, including, but not limited to, age, the disease or disorder being treated, sex, overall health, and other factors. Therefore, the Disclosure should not be construed as being limited to any particular medication regimen, and the exact dose and composition to be administered to any patient should be determined by the attending physician, taking into account all other factors concerning that patient.

[0310] In certain embodiments, the Disclosure relates to a packaged pharmaceutical composition comprising a container for holding a therapeutically effective amount of the 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 compound of the present disclosure include inhalation, oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, ​​vagina (e.g., transvaginal and perivaginal), nasal (intra) and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastric, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.

[0312] Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, lozenges, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magma, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powders or aerosol formulations for inhalation, intravesical compositions and formulations, etc. Formulations and compositions that may be useful in this 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 route of administration characterized by the administration of the pharmaceutical composition through physical rupture and fissures in the tissue of interest. Parenteral administration therefore includes, but is not limited to, the administration of a pharmaceutical composition by injection, application of the composition through surgical incision, application of the composition through a non-surgical wound penetrating the tissue, etc. In particular, parenteral administration is assumed to include, but is not limited to, subcutaneous, intravenous, intraperitoneal, intramuscular, intrasternal injection, and renal dialysis infusion techniques.

[0314] Further administration methods Further forms of dosing described herein include the forms of dosing 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. Further forms of dosing described herein also include the forms of dosing described in U.S. Patent Applications Nos. 20030147952, 20030104062, 20030104053, 20030044466, 20030039688 and 20020051820. Further drug formulations of this disclosure also include those described in PCT applications 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 Controlled or sustained-release formulations of the pharmaceutical compositions of this disclosure can be prepared using prior art. In some examples, the dosage forms used may be provided as sustained or controlled release of one or more active ingredients, using, for example, hydroxypropyl methylcellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes or microspheres, or combinations thereof, in various proportions to provide a desired release profile. Orally administered single-unit dosage forms suitable for controlled release, such as tablets, capsules, gel caps and caplets, are included in this disclosure.

[0316] In certain embodiments, the formulations of the present disclosure may be short-release, rapid-offset, and controlled-release formulations, such as sustained-release, delayed-release, and pulsed-release formulations, but are not limited thereto.

[0317] The term sustained-release, used in its conventional sense, refers to a drug formulation that provides a gradual release of the drug over a long period, resulting in a substantially constant blood level of the drug over a long period, although this is not necessarily required. The period can be as long as one month or longer, and should be longer than the release of the same amount of drug administered in bolus form. For sustained-release, the compound may be formulated with a suitable polymer or hydrophobic substance that provides the compound with sustained-release properties. In this case, the compound used in the method of the present disclosure may be administered, for example, by injection in the form of microparticles, or by implantation in the form of wafers or discs. In certain embodiments of the present disclosure, the compound of the present disclosure is administered to a patient, alone or in combination with another drug, using a sustained-release formulation.

[0318] The term "delayed release" is used herein in its conventional sense to describe a drug formulation that provides an initial release of the drug after a certain delay following drug administration, which may include delays of approximately 10 minutes to up to approximately 12 hours, although this delay is not required. The term "pulsed release" is used herein in its conventional sense to describe a drug formulation that provides a release of the drug in a manner that generates a pulsed plasma profile of the drug after drug administration. The term "immediate release" is used herein in its conventional sense to describe a drug formulation that provides a release of the drug immediately after drug administration.

[0319] As used herein, short term refers to any period of time after drug administration that includes 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, and any period of any or all of those full or partial increments.

[0320] As used herein, rapid elimination refers to any period of time after drug administration that includes approximately 8 hours, approximately 7 hours, approximately 6 hours, approximately 5 hours, approximately 4 hours, approximately 3 hours, approximately 2 hours, approximately 1 hour, approximately 40 minutes, approximately 20 minutes, or approximately 10 minutes, and any period of any and all of those full or partial increments.

[0321] It is expected that those skilled in the art will be able to recognize and confirm many equivalents of the individual procedures, embodiments, claims, and examples described herein using only conventional experiments. Such equivalents are deemed to be within the scope of this disclosure and included in the appended claims. For example, modifications of reaction and preparation conditions using only conventional experiments with substitutes known in the art should be understood to be within the scope of this application.

[0322] Whatever values ​​and ranges may be provided herein, it should be understood that all values ​​and ranges encompassed within those values ​​and ranges are included within the scope of this disclosure. Furthermore, the upper or lower limits of all values ​​and ranges encompassed within those limits are also intended by this application.

[0323] The following embodiments further illustrate aspects of the present disclosure; however, they do not constitute in any way an limitation of the teachings or disclosures of the present disclosure as set forth herein. [Examples]

[0324] This disclosure is described herein by reference to the following examples. These examples are provided for illustrative purposes only, and this disclosure is not limited to these examples, but encompasses all variations that become apparent as a result of the teachings provided herein.

[0325] Methods and materials Unless otherwise specified, expression of the construct in CHO cells or modified CHO cells with and without supplementation, V max Assay, K m / K cat Assays, AUC assays, and half-life assays were performed using protocols described elsewhere in this specification.

[0326] Construction of ENPP1-Fc mutant constructs Human NPP1 (human: NCBI accession NP 006199) was modified to express soluble recombinant proteins fused to IgGl by subcloning into either pFUSE-hlgGl-Fcl or pFUSE-mlgGl-Fcl plasmids (InvivoGen, San Diego CA). These constructs were prepared from SEQ ID NO:7 using site-directed mutagenesis with a commercially available kit (Q5® Site-Directed Mutagenesis Kit / New England Biolabs). The constructed constructs were sequenced for nucleic acid sequence validation and subsequently used for protein expression.

[0327] Expression of ENPP1-Fc mutant constructs Stable transfection of the ENPPl-Fc construct was established in CHO K1 cells (Sigma Aldrich, 85051005) under zeosin / gentamicin selectivity and adapted for suspension growth. Using the adapted cells, liquid culture growth was performed in CD FortiCHO® medium (A1148301, Thermo Fischer) or PEPROGROW® AF-CHO (PeproTech AF-CHO) in a shaking flask at 37°C, 5% CO2, 120 rpm, and high humidity. This culture was gradually expanded to the desired target volume and then maintained for an additional 2 days to accumulate extracellular proteins.

[0328] Expression of ENPP1-Fc mutant constructs in modified CHO cells CHO-K1 cells were modified to generate CHO-K1-MOD cells that stably express the human α-2,6-sialyltransferase (α-2,6-ST) enzyme. Stable transfection of the ENPPL-Fc construct was established in CHO K1-MOD cells, and the protein was expressed according to the same protocol as above. Where necessary, for some constructs, the culture medium for 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 to promote high levels of glycosylation during protein production.

[0329] Purification of ENPP1-Fc mutant constructs This liquid culture was centrifuged at 4300 × g for 5 minutes, and the supernatant was filtered through a 0.2 μm membrane. Pellicon® 3 0. 0.11 m 2 The supernatant was concentrated via 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. By using any one of these steps in sequence, those skilled in the art of protein chemistry can purify the composition of interest described to a homogeneity that does not show any contaminating protein bands on a silver-stained gel. The resulting protein samples were then tested using the Pierce LAL Chromogenic Endotoxin Quantitation Kit (catalog no. 88282) to confirm that all were endotoxin-free.

[0330] To quantify the biological effects of clonal optimization, the pharmacokinetic effects of selected ENPP1-Fc isoforms were quantified by measuring plasma PPi concentrations at multiple time points after a single subcutaneous dose of each isoform.

[0331] K m / K cat decision The steady-state hydrolysis of ATP by the ENPP1 construct was measured by HPLC. Briefly, the enzymatic reaction was initiated by adding 10 nM PPi to various concentrations of ATP in a reaction buffer containing 20 mM Tris, pH 7.4, 150 mM NaCl, 4.5 nM KCl, 14 mM ZnCl2, 1 mM MgCl2, and 1 mM CaCl2. At various time points, 50 μl of the reaction solution was taken out and the reaction was stopped with an equal volume of 3 M formic acid. The stopped 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) and eluted with a 0% to 20% methanol gradient. The substrate and product 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 Phosphodiesterase activity was analyzed for each of the prepared mutants 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 over time, also known as the area under the curve (AUC), can be used as a measure to assess the volume of distribution (V), total elimination clearance (CL), and bioavailability (F) for extravascular drug delivery. The plasma time area under the curve for each expressed and purified ENPP1-Fc construct was calculated using a standard equation, such as that shown in Equation 1, to determine the half-life and bioavailability of a single biomaterial after subcutaneous injection.

[0334] Half-life determination Drug half-life (t 1 / 2 Half-life ) is the time required for the plasma concentration or volume of a drug or biosubstance in the body to decrease by 50%. The half-life values ​​for each expressed and purified ENPP1-Fc construct were determined according to protocols described in the Art and / or herein, e.g., Equation 1, which enable the determination of half-life and bioavailability after a single subcutaneous injection of a biosubstance.

[0335] The drug half-life can be calculated using Equation 1, which corresponds to the relationship between the fraction of systemic concentration of a drug administered to a subcutaneous injection site in a single injection and time. Plotting this data as the fraction of absorbed drug (F) against time (t) allows for the elimination (k) by fitting this data to the equation for total systemic absorption of the drug administered to the subcutaneous injection site at time t=0. e ) and absorption (k a ) Enables the determination of constants. TIFF0007874896000020.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 Fc to the neonatal orphan receptor (FcRn). The mutations tested are shown elsewhere in this specification, and 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 to enhance pH-dependent recycling of the fusion protein. To guide the selection of these additional sites, we used electron density maps obtained from X-ray diffraction of mouse Enpp1 crystals, which revealed four glycosylation sites in Enpp1. These sites are presumed to be present in highly homologous human ENPP1, and in addition, human ENPP1 contains four further N-linked glycosylation consensus sequences whose glycosylation state is unknown (Figure 7B).

[0338] To identify regions of ENPP1 capable of superglycosylation without adversely affecting catalytic activity, a combination of structural modeling, clinical data, and genetic data from ENPP1 in GACI patients was used. First, N-linked glycosylation consensus sequences were identified in ENPP2–7, and sequences that could be easily introduced through a single adjacent residue change were evaluated. Next, ENPP2–7 were structurally modeled using standard software, and the sequences were threaded through the mouse Enpp1 structure (PDB ID code 4GTW). The locations of proposed glycosylation sites were compared with the locations of known inactivating ENPP1 mutations in GACI (Figures 7A–7B) and the locations of disulfide bonds within this enzyme. If the spatial location of a proposed glycosylation site was expected to interfere with any of these, that site was rejected. These modeling studies identified several putative sites for additional N-linked glycosylation procedures that could be readily introduced into ENPP1 without interfering with its protein folding or enzymatic activity (Figures 8A–8D, 16A–16B, and 17).

[0339] Next, an additional N-linked glycosylation consensus sequence was introduced into human ENPP1-Fc (hENPP1-Fc, construct #770) via site-directed mutagenesis. This protein was transiently expressed in CHO cells in a 96-well plate, and the enzymatic activity of the extracellular supernatant from each clone was screened in a triplicate high-throughput assay using pNP-TMP as the chromogenic substrate, as described in the methods (Figures 7A-7D). The rates of pNP-TMP hydrolysis in the 10 ENPP1-Fc isoforms were equivalent to or greater than those of construct #770 (Figures 7A-7D), and these 10 glycoforms were selected for combinatorial optimization with the IgG1 Fc domain, as described elsewhere in this specification.

[0340] FcRn is the primary homeostatic regulator of the human IgG1 Fc serum half-life, and mutations within the Fc domain that enhance the pH-dependent interaction of Fc with FcRn prolonged the circulating half-life of the biological antibody. Here, we tested the effects of two Fc mutations reported to enhance pH-dependent recycling: H433K / N434F, hereafter referred to as the HN variant, and M242Y / S254T / T246E, hereafter referred to as the MST variant (Figures 9A-9B). Twelve further ENPP1-Fc clones were generated by randomly combining one or more of the two variants of the Fc domain with one or more of the ten ENPP1-Fc glycoforms that exhibited acceptable hydrolysis rates (Table 3). Some of these clones were selected to test the effects of multiple glycoforms on the pharmacokinetics of ENPP1-Fc, and two spatially distinct putative glycosylation sites on different protein domains were selected to enhance the potential glycan shielding effect on the protein surface region (Table 3; constructs #1057, #1064, #1014, #1040, #1101). Other clones tested only the effect of Fc mutations on pK properties, either alone or in the presence of one additional putative glycosylation (Table 3; constructs #981 and #1051, respectively).

[0341] Example 2: Expression and proliferation conditions using the CHO cell line Non-human Chinese hamster ovary (CHO) cells are widely used for biomaterial production due to the similarity in glycosylation patterns between CHO and human proteins 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 links, while CHO cells contain only α-2,3 links.

[0342] To test whether differences in terminal sialylation between CHO and human cells affect PK and bioavailability in the system of the present invention, a CHO cell line stably expressing human α-2,6-sialyltransferase (α-2,6-ST) was established as the host, and this clone was used to produce seven ENPP1 isoforms to compare the effects of α-2,6 binding on PK and bioavailability in various constructs (Table 5; construct numbers ending in "-ST"). To investigate the effects of growth conditions on PK and bioavailability, cells stably transfected with selected ENPP1-Fc isoforms (both CHO K1 cells and CHO K1 cells stably transfected with human α-2,6-ST) were supplemented with a "high-flux" precursor of sialic acid called 1,3,4-O-Bu3ManNAc during protein production (Table 5).

[0343] The ENPP1-Fc isoforms were purified to homogeneity using the same purification scheme, and the Michaelis-Menten enzyme rate constants and pharmacokinetic properties were determined as described elsewhere herein. Finally, to quantify the biological effects of clonal optimization, the pharmacodynamic effects of the selected ENPP1-Fc isoforms were determined by determining plasma PPi concentrations at multiple time points after a single subcutaneous administration of each isoform. The half-life and area under the curve were determined by plotting the fraction of absorbed drug per unit time (F) and its elimination (k). e ) and absorption (k aThe constant was obtained by fitting this data to Equation 1, which represents the total systemic absorption of the drug administered to the 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 showing a half-life of 34 hours and an area under the curve (AUC) of 3,027 (construction #770, table) is shown in Figure 2B. The addition of the N-linked glycosylation site using the in silico prediction and HTS method described above significantly increased mouse in vivo exposure to ENPP1-Fc in two glycoforms—quadruple in construct #1020 and 7.7 times in construct #922 (Figure 10 and Table 2). The I256T mutation introduced into construct #922 further increased the half-life by 160%. Residue 256 is close to the catalytic threonine of human ENPP1, which is responsible for nucleophilic addition to phosphate anhydride substrates. While not limited by any theory, this sequence variation exists in a similar location in human ENPP3. This may be a substrate preference modulator: ENPP2 lacks this loop and can accommodate larger lipid substrates in its catalytic pocket; both ENPP1 and ENPP3 have this loop, but only ENPP3 has the N-glycan consensus sequence (N-GCS).

[0345] To determine which sequence variations increased glycosylation and resulted in a corresponding increase in molecular weight, the sizes of the ENPP1-Fc isoforms in Table 2 were compared using SDS-PAGE gels. MALDI-TOF was used to determine if the sequence changes in construct #1020 successfully introduced glycosylation, further confirming the presence of glycosylation at these sites.

[0346] Not all N-GCSs are actually glycosylated in one phase: steric hindrance related to the position of the N-GCS can occur, such as when an Asn residue cannot accept glycan due to a specific adjacent amino acid. Therefore, unless the purified protein is analyzed for glycan content, it is impossible to determine whether the Pk effect of a particular N-GCS is due to the shielding of a new glycan, or whether amino acid changes alter the enzyme kinetics, or both. For this reason, the effect of N-GCSs on PK should be investigated by glycan analysis. Using mass spectrometry, digested peptide fragments are analyzed. We confirmed that ENPP1-Fc clone 19, which possesses the I256T mutation located within TIFF0007874896000021.tif5128, is indeed glycosylated at the Asn254 position, as indicated by the abundance of sialocylopeptide peaks (Figure 2D), compared to the parental ENPP1-Fc clone lacking the I256T mutation.

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

[0348] Example 4: Pharmacokinetic effects of Fc IgG1 mutations (Figures 11-12) Antibodies containing mutations within the Fc domain that enhance their affinity for FcRn and increase pH-dependent antibody recycling have not been used in therapeutic enzymes fused to the Fc domain to date. Some Fc mutations successfully increased the affinity of the Fc domain to the FcRn receptor but resulted in undesirable PK properties in in vivo antibody PK, while others were shown to enhance PK properties in vivo.

[0349] FcRn is a major homeostatic regulator of the serum half-life of human IgG1 Fc. To determine whether similar Fc alterations enhance the PK properties of enzyme fusion proteins, we investigated two specific IgG1 mutations in Fc previously used in biological antibodies: H433K / N434F and M242Y / S254T / T246E. Generally, the M242Y / S254T / T246E mutation was found to be superior to H433K / N434F in terms of improving the properties of ENPP1-Fc. For example, compared to construct #770, construct #981, which had only the M242Y / S254T / T246E mutation, increased the half-life by 3.3 times and the AUC by 5.8 times. In contrast, constructs with the H433K / N434F mutation achieved smaller half-life increases of 1.2 to 1.7 times for various ENPP1 mutations.

[0350] Overall, Fc MST mutations increased biological exposure more significantly than Fc HN mutations (Table 3, Figures 14A-14E). For example, the addition of an MST mutation to the parental isoform increased AUC 6-fold and half-life approximately 2.5-fold compared to the HN mutation, which increased AUC 4.5-fold in the presence of the additional glycan (comparison of clone 14 with clones 9-12 in Table 3 and Figures 14A-14E). However, in some cases, certain N-GCS mutations actually reduced bioavailability with respect to certain Fc mutations; namely, the N-GCS mutation at residue 766 reduced the AUC of MST-Fc-containing constructs (comparison of clone 8 and clone 14, Table 3, Figure 14A) and HN-Fc-containing constructs (comparison of clone 9 and clone 11, Table 3, Figure 14A). To demonstrate the experimental design and reproducibility, two independent CHO cell clones with the same mutation were constructed, and little variability was observed in their respective pharmacodynamic properties (comparison of clones 11 and 12 in Table 3). Note that the improvement in PK induced by the Fc mutation did not reach the level of improvement achieved by the addition of N-glycan at residue 256, thus clearly highlighting the importance of selective glycosylation for pharmacokinetics. To compare the PK effect of the MST Fc mutation with the effect of the additional glycan at position 256 (via the I256T mutation), the plasma activity of the ENPP1-Fc isoform was plotted against time (Figure 14B). The figure shows that the Fc mutation enhances PK by increasing the half-life of this biomolecule in plasma, as indicated by the decrease in the slope of the activity-time curve in clone 14 versus clone 7. In contrast, the addition of I256T glycosylation increases drug absorption into plasma, i.e., C maxPK is enhanced by increasing (Figure 14B). Although the combination of the MST Fc mutation and I256T hyperglycosylation increased overall biological exposure (AUC) by only 16% compared to the effect of the I256T mutation alone, the net effect on the parent isoform was substantially 11.5 times, thus supporting the combined use of both methods to maximize bioavailability (comparison of clones 7 and 17, Table 3 and Figure 14A).

[0351] Example 5: Effects of host cells and proliferation conditions Protein expression in CHO cells stably transfected with human α-2,6-ST, which produces recombinant biomaterial containing terminal sialic acid residues with both alpha-2,3 and alpha-2,6 linkages, was achieved to varying degrees of success, and increased and decreased PK properties depending on the biomaterial were reported. There are differences in glycosylation between hamsters and humans; in particular, human N-linked glycans contain terminal sialic acid residues with both α-2,3 and α-2,6 linkages, while 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-lives of seven ENPP1-Fc isoforms produced in either CHOK1 cells or CHOK1 cells stably transfected with human α-2,6-ST were directly compared (Table 4). An overall trend was observed in the production of the biomaterial in CHOK1 cells stably transfected with human α-2,6-ST, indicating a beneficial effect. The strongest effect was observed in the biomaterial's exposure to the drug (AUC), with responsive isoforms (constructs #1057, #1028, #951, #930, and #981) showing a 1.7–4.6-fold increase in AUC. Another trend was that isoforms with lower initial AUCs (constructs #951 and #1057) showed a greater impact on AUC. However, the longer-acting isoforms (constructs #1028 and #981) also showed significant effects, exhibiting AUC values ​​8 to 10 times higher than their parent polypeptides produced in CHOK1 cells.

[0353] The effect of α-2,6 binding on half-life was smaller, showing a 20–30% increase in the responsive construct. To understand the differences in the effects of α-2,6 binding on AUC and protein activity half-life versus time, isoforms produced in CHOk1 and 1078 cells were compared.

[0354] Example 6: Pharmacokinetic effects of proliferation using a high-flux sialic acid precursor To determine the effect of growth conditions on PK properties, the culture medium of selected clones was supplemented with 1,3,4-O-Bu3ManNAc, a "high-flux" precursor of sialic acid, or sialic acid itself. Supplementation of CHOK1 cells with 1,3,4-O-Bu3ManNAc showed little improvement in the PK properties of ENPP1-Fc. However, when this biomaterial was produced in CHOK1 cells stably transfected with human α-2,6-ST, its effect on half-life and AUC was significant (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 (C2C in clones 7 and 14). max Please note the difference (Figure 14B).

[0355] For example, supplementing the cell culture medium of CHOK1 cells producing construct #1014 (clone 15) with 1,3,4-O-Bu3ManNAc did not significantly enhance the AUC and appeared to decrease the half-life of the isoform.

[0356] In contrast, the effect was more dramatic when 1,3,4-O-Bu3ManNAc was added to the cell culture medium of construct #1057 (clone 9, which has two additional glycosylations in the signal sequence and nuclease domain, as well as an HN Fc mutation) produced in CHOK1 cells stably transfected with α-2,6-ST. Biological exposure to this clone was increased 2.6-fold by expressing this clone in CHO cells containing human α-2,6-ST, and could be further increased 1.4-fold by supplementing the growth medium of these cells with a sialic acid precursor. These effects resulted in a net increase of 4-fold and 2-fold AUC and half-life compared to the same isoform produced in CHOK1 grown in medium without 1,3,4-O-Bu3ManNAc supplementation (Figure 13 and Table 4). The percentage of N-acetylneuraminic acid content in ENPP1-Fc gradually increased when expressed in CHO K1 cells that were stably transfected with human α-2,6-ST and grown in the presence of the sialic acid precursor 1,3,4-O-Bu3ManNAc (Figure 14E), which is consistent with the view that these methods can increase the sialic acid content in the enzyme biomaterial, thereby having a favorable effect on pharmacokinetics.

[0357] The effect of expressing the biomaterial in CHO cells containing only human α-2,6-ST ranges from a 4.5-fold increase in the least potent isoform (clone 1 vs. clone 1-ST, Table 5 and Figure 14C) to a milder effect in the most potent construct. However, these milder effects resulted in a substantial overall increase, as demonstrated by clone 17. Expression of clone 17 in CHO cells containing human α-2,6-ST resulted in a mild 28% increase in AUC, which is still three times the absolute value of the AUC of the starting clone due to the enhanced properties of these optimized biomaterials. The final enhancements beyond clone 770 were 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 clone (Figure 15A, clones 17-ST and 19-ST). Expression of clone 19-ST in culture medium containing the sialic acid precursor 1,3,4-O-Bu3ManNAc resulted in a highly active polypeptide. The increase derived from the expression of this protein in medium containing 1,3,4-O-Bu3ManNAc is represented by the dark gray shaded area in Figure 15A, resulting in a net increase of nearly 18-fold in bioavailability compared to the parent construct (Figure 15B). Mass spectrometry of sialylated content in the parent clone and the final product revealed that only 78.4% of the available sites (glycans with at least one galactose for sialic acid transport) in the parent clone were sialylated, compared to 99.2% in clone 19-ST-A (Figure 15C). The combined findings demonstrate the importance of sialic acid capping in glycosylation in enzymatic biomaterials and the ability of the described method to enhance the bioavailability of glycan-optimized enzymatic biomaterials.

[0358] Example 6: Pharmacokinetic effects ENPP1-Fc is the only enzyme in mammals capable of producing plasma PPi, and plasma PPi is therefore a biomarker that predicts the efficacy of ENPP1 enzyme replacement therapy under ENPP1 deficiency. To determine the pharmacodynamic effects of optimized ENPP1-Fc isoforms, Enpp1 asj / asj Mice were administered a subcutaneous dose of either construct #770 or clone 19-ST at 0.3 mg / kg, and plasma PPi and enzyme levels were measured in plasma for 263 hours (Figure 15D). In mice administered construct #770, plasma PPi increased to the normal range 24 hours after administration but returned to baseline at 48 hours, whereas clone 19-ST increased plasma PPi to approximately twice the normal range and maintained elevated levels above or within the normal range for approximately 250 hours. These experiments demonstrate that the pharmacokinetic effects observed in the optimized ENPP1-Fc isoform are directly translated into enhanced pharmacodynamic activity.

[0359] (Table 1) TIFF0007874896000022.tif218156TIFF0007874896000023.tif104156

[0360] (Table 2) Effect of additional N-linked glycosylation on pharmacokinetics (PK) TIFF0007874896000024.tif100143

[0361] (Table 3) Effects of Fc mutations on pharmacokinetics (PK) TIFF0007874896000025.tif163149

[0362] (Table 4) Effects of cell lines 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 enhanced the amount of sialylation of the fusion protein compared to the fusion protein expressed in normal CHO cell lines. The enhanced sialylation of the constructs improved the AUC and half-life values. TIFF0007874896000026.tif228154

[0363] (Table 5) Effects 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 the fusion protein expressed in a normal CHO cell line. Constructs marked "-A" were prepared in cells grown in culture medium supplemented with 1,3,4-O-Bu3ManNAc, a "high-flux" precursor of sialic acid, during protein production. TIFF0007874896000027.tif132154

[0364] (Table 6) List of polypeptides and corresponding mutations TIFF0007874896000028.tif234151

[0365] (Table 7) List of mutations in the ENPP1 polypeptide TIFF0007874896000029.tif143128

[0366] List of types The following exemplary embodiments are provided, but their numbering should not be considered to indicate a level of importance.

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

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

[0369] Embodiment 3 provides a polypeptide fusion of Embodiment 1, wherein the Fc region contains at least one mutation selected from the group consisting of S885N, M883Y, M883Y / S885T / T887E, and H1064K / N1065F with respect to SEQ ID NO:7.

[0370] Embodiment 4 provides a 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 with respect to SEQ ID NO:7.

[0371] Embodiment 5 provides a polypeptide fusion of any of Embodiments 1 to 4, wherein the ENPP1 polypeptide contains at least one mutation selected from the group consisting of C25N / K27T and V29N with respect to SEQ ID NO:7.

[0372] Embodiment 6 provides a 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 relating to SEQ ID NO:7.

[0373] Embodiment 7 provides a polypeptide fusion of any of Embodiments 1 to 6, wherein the ENPP1 polypeptide contains the mutation K369N / I371T related to SEQ ID NO:7.

[0374] Embodiment 8 provides a 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 relating to SEQ ID NO:7.

[0375] Embodiment 9 provides a polypeptide fusion according to any of Embodiments 1 to 8, wherein the ENPP1 polypeptide contains 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.

[0376] Embodiment 10 provides a 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 relating to SEQ ID NO:7.

[0377] Embodiment 11 provides a polypeptide fusion of any of Embodiments 1 to 10, wherein the ENPP1 polypeptide contains at least the E864N / L866T mutation relating to SEQ ID NO:7.

[0378] Appearance 12 is SEQ ID The present invention provides polypeptide fusions according to any of embodiments 1 to 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, and H1064K / N1065F for NO:7.

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

[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 relating 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 with respect 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 immunoglobulin Fc region, comprising mutations I256T, M883Y, S885T, and T887E relating to SEQ ID NO:7.

[0384] Embodiment 18 provides an ENPP1 polypeptide fusion comprising an ENPP1 polypeptide and an immunoglobulin Fc region, comprising mutations I256T, P534N, V536T, M883Y, S885T, and T887E relating to SEQ ID NO:7.

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

[0386] Embodiment 20 is an ENPP1 mutant polypeptide containing amino acids 23-849 of SEQ ID NO:7, The mutation I256T related to SEQ ID NO:7 is included, and further includes mutations selected from the group consisting of S766N, P534N, V536T, P554L, R545T, and E592N. This provides an ENPP1 mutant polypeptide.

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

[0388] Embodiment 22 provides a mutant polypeptide according to any of Embodiments 20 to 21, which contains 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 relating to SEQ ID NO:7.

[0390] Embodiment 24 provides a mutant polypeptide according to any of Embodiments 21 to 23, which includes 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 includes the S885N mutation relating to SEQ ID NO:7.

[0392] Embodiment 26 provides a mutant polypeptide of any of Embodiments 20 to 25, which includes the S766N mutation relating to SEQ ID NO:7.

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

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

[0395] Embodiment 29 provides a mutant polypeptide of any of Embodiments 20 to 28, comprising mutations P554L and R545T related to SEQ ID NO:7.

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

[0397] Embodiment 31 provides a mutant polypeptide of any of Embodiments 21 to 30, comprising mutations E592N, H1064K, and N1065F related to SEQ ID NO:7.

[0398] Embodiment 32 provides a mutant polypeptide of any of Embodiments 21 to 31, comprising mutations P534N, V536T, M883Y, S885T, and T887E relating 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, which is expressed from a CHO cell line stably transfected with human ST6 beta-galactoside alpha-2,6-sialyltransferase (also known as ST6GAL1).

[0400] Embodiment 34 provides a polypeptide fusion of any of Embodiments 1 to 19 or a mutant polypeptide of any of Embodiments 20 to 32, which is grown in a cell culture supplemented with sialic acid and / or N-acetylmannosamine (also known as 1,3,4-O-Bu3ManNAc).

[0401] Embodiment 35 provides a method for reducing or preventing the progression of pathological calcification in a subject requiring such reduction, comprising the step of administering to the subject a therapeutically effective dose of a polypeptide fusion of any of Embodiments 1-19 and 33-34 or a mutant polypeptide of any of Embodiments 20-34.

[0402] Embodiment 36 provides a method for reducing or preventing the progression of pathological ossification in a subject requiring it, comprising the step of administering to the subject a therapeutically effective dose 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 requiring such reduction, comprising the step of administering to the subject a therapeutically effective dose 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.

[0404] Embodiment 38 provides a method for treating, improving, or preventing the progression of ossification of the posterior longitudinal ligament (OPLL) in a subject in need thereof, comprising the step of administering to the subject a therapeutically effective dose 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.

[0405] Embodiment 39 provides a method for treating, reversing, or preventing the progression of hypophosphatemic rickets in a subject in need thereof, comprising the step of administering to the subject a therapeutically effective dose of a polypeptide fusion of any of Embodiments 1-19 and 33-34 or a mutant polypeptide of any of Embodiments 20-34.

[0406] Embodiment 40 is 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 arteriosclerosis (CUA), calciphylaxis, ossification of the posterior longitudinal ligament (OPLL), hypophosphatemic rickets, osteoarthritis, age-related arteriosclerosis, idiopathic infantile arteriosclerosis (IIAC), generalized infantile arteriosclerosis (GACI), and arteriosclerotic plaque calcification in a subject diagnosed with said at least one disease, The present invention provides a method comprising the step of administering to a subject a therapeutically effective dose 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 requiring it, comprising the step of administering to the subject 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 in a therapeutically effective amount.

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

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

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

[0411] Embodiment 45 provides the method of Embodiment 37, wherein the soft tissue is selected from the group consisting of arteriosclerotic plaque, muscular arteries, joints, vertebrae, articular cartilage, intervertebral disc cartilage, blood vessels, and connective tissue.

[0412] Embodiment 46 is a method for increasing the PPi level in a subject having a PPi level lower than the normal level of pyrophosphate (PPi), A step of administering to a subject a polypeptide 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 in a therapeutically effective dose, wherein the administration raises the level of PPi in the subject to a normal level of at least 2 μM and maintains it at substantially the same level. This provides a method that includes [something].

[0413] Embodiment 47 is a method for reducing or preventing the progression of pathological calcification or ossification in subjects having pyrophosphate (PPi) levels lower than normal levels, A step of administering 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 to a subject in a therapeutically effective dose, wherein pathological calcification or ossification in the subject is reduced or the progression of pathological calcification or ossification in the subject is prevented. This provides a method that includes [something].

[0414] Embodiment 48 is a method for treating an ENPP1 defect that becomes apparent due to a decrease in extracellular pyrophosphate (PPi) concentration in a subject requiring it, A step of administering a polypeptide fusion of any of embodiments 1-19 and 33-34 or a mutant polypeptide of any of embodiments 20-34 to a subject in a therapeutically effective dose, wherein the level of PPi in the subject increases. This provides a method that includes [something].

[0415] Embodiment 49 provides a method according to any one of Embodiments 35 to 48, wherein the polypeptide fusion or mutant polypeptide is a secretion product of an ENPP1 precursor protein expressed in mammalian cells, the ENPP1 precursor protein comprises a signal peptide sequence and an ENPP1 polypeptide, and the ENPP1 precursor protein undergoes proteolytic processing to produce an ENPP1 polypeptide.

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

[0417] Embodiment 51 provides a method according to any one of Embodiments 49 to 50, wherein the signal peptide sequence is selected from the group consisting of ENPP1 signal peptide sequence, ENPP2 signal peptide sequence, ENPP7 signal peptide sequence, and ENPP5 signal peptide sequence.

[0418] Embodiment 52 provides a method according to any of Embodiments 35 to 51, wherein a polypeptide fusion or mutant polypeptide is administered to a subject in the short term or long term.

[0419] Embodiment 53 provides a method according to any of Embodiments 35 to 52, wherein a polypeptide fusion or mutant polypeptide is administered to a subject locally, regionally, parenterally, or systemically.

[0420] Embodiment 54 provides a method according to any of Embodiments 35 to 53, wherein the polypeptide fusion or mutant polypeptide is administered to a subject by at least one route selected from the group consisting of subcutaneous, oral, aerosol, inhalation, rectal, vaginal, transdermal, subcutaneous, intranasal, oral, sublingual, parenteral, intrathecal, gastric, ocular, pulmonary, and topical.

[0421] Embodiment 55 provides a method according to any of embodiments 35 to 54, wherein a polypeptide fusion or mutant polypeptide is administered to a subject as a pharmaceutical composition further comprising at least one pharmaceutically acceptable carrier.

[0422] Embodiment 56 provides any of the methods 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 relating to SEQ ID NO:7, wherein the amino acid substitution is located at position 256 with respect to SEQ ID NO:7.

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

[0426] Embodiment 60 is an ENPP1 mutant polypeptide containing amino acids 23-849 of SEQ ID NO:7, There are 10 or fewer amino acid substitutions between amino acids 23-849 of SEQ ID NO:7. SEQ ID NO:7 contains an amino acid substitution at position 256. This provides an ENPP1 mutant polypeptide.

[0427] Embodiment 61 provides one of the ENPP1 mutant polypeptides 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 serine (S) with isoleucine (I) at position 256 relative to SEQ ID NO:7.

[0429] Embodiment 63 is an ENPP1 mutant polypeptide comprising an amino acid sequence that is at least 90% identical to amino acids 23-849 of SEQ ID NO:7, Includes mutation I256T related to SEQ ID NO:7, Further including mutations selected from the group consisting of S766N, P534N, V536T, P554L, R545T, and E592N related to SEQ ID NO:7, This provides an ENPP1 mutant polypeptide.

[0430] Embodiment 64 provides the 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 with respect to SEQ ID NO:7.

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

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

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

[0434] Embodiment 68 provides an 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 according to any one of Embodiments 67 to 68, wherein the heterologous protein is located on the carboxyl terminal side of the ENPP1 mutant polypeptide of the fusion.

[0436] Embodiment 70 provides an ENPP1 mutant polypeptide fusion according to any one of Embodiments 67 to 68, wherein the heterologous protein is located at the amino-terminal end of the ENPP1 mutant polypeptide of the fusion.

[0437] Embodiment 71 provides an 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 an 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 an ENPP1 mutant polypeptide fusion of Embodiment 72, wherein the immunoglobulin molecule is IgG1.

[0440] Embodiment 74 provides an 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 68-70 and 72-74, wherein the FcRn binding domain is the Fc portion of a human IgG1 molecule and comprises the following amino acid substitutions for SEQ ID NO:7: M883Y, S885T, and T887E, respectively.

[0442] Embodiment 76 provides an ENPP1 mutant polypeptide fusion of any one of embodiments 68-70 and 72-75, 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.

[0443] Embodiment 77 provides an ENPP1 mutant polypeptide fusion of any one of Embodiments 68-70 and 72-76, which contains the S885N mutation relating to SEQ ID NO:7.

[0444] Embodiment 78 provides an ENPP1 mutant polypeptide fusion of any one of embodiments 68-70 and 72-77, which includes the S766N mutation relating to SEQ ID NO:7.

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

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

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

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

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

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

[0451] Embodiment 85 provides an ENPP1 mutant polypeptide fusion of any one of embodiments 68-70 and 72-84, wherein the Fc region contains at least one mutation selected from the group consisting of M883Y, S885N, S885T, T887E, H1064K, and N1065F relating to SEQ ID NO:7.

[0452] Embodiment 86 provides an ENPP1 mutant polypeptide fusion of any one of embodiments 68-70 and 72-85, wherein the Fc region contains at least one mutation selected from the group consisting of S885N, M883Y, M883Y / S885T / T887E, and H1064K / N1065F relating to SEQ ID NO:7.

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

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

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

[0456] Embodiment 90 provides an ENPP1 mutant polypeptide fusion from any one of Embodiments 68-70 and 72-89 or an ENPP1 mutant polypeptide from any one of Embodiments 58-65 and 87-89, containing the mutation K369N / I371T related to SEQ ID NO:7.

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

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

[0459] Appearance 93 is 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, S885 The present invention provides an ENPP1 mutant polypeptide fusion according to any one of embodiments 68-70 and 72-92, or an ENPP1 mutant polypeptide according to any one of embodiments 58-65 and 87-92, comprising at least one mutation selected from the group consisting of N, S885T, T887E, H1064K, N1065F, M883Y / S885T / T887E, and H1064K / N1065F.

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

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

[0462] Embodiment 96 provides an ENPP1 mutant polypeptide fusion from any one of Embodiments 68-70 and 72-95 or an ENPP1 mutant polypeptide from any one of Embodiments 58-65 and 87-95, 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 68-70 and 72-96, comprising mutations I256T, M883Y, S885T, and T887E relating to SEQ ID NO:7.

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

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

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

[0467] Embodiment 101 provides a fusion of Embodiment 100 in which the linker amino acid sequence connects the ENPP1 mutant polypeptide portion of the fusion to a heterologous protein.

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

[0469] Embodiment 103 provides a nucleic acid encoding any one of the ENPP1 mutant polypeptides from Embodiments 58 to 66 or any one of the fusions from 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 group 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 the cells or a plurality of cells of Embodiment 106, which are CHO cells and / or NS0 cells.

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

[0475] Embodiment 109 provides a method for producing an ENPP1 mutant polypeptide or fusion, comprising the step of culturing one or more cells from any of Embodiments 106 to 108 under conditions suitable for the expression of the ENPP1 mutant polypeptide or fusion by said cells or multiple cells.

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

[0477] Embodiment 111 provides one of the methods of Embodiments 109 to 110, further comprising the step of purifying an ENPP1 mutant polypeptide or fusion from the cells, the plurality of cells, or a culture medium in which the cells or the plurality of cells are cultured.

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

[0479] Embodiment 113 is, (i) any one ENPP1 mutant polypeptide of embodiments 58-66, 87-96 and 112, and / or any one ENPP1 mutant polypeptide fusion of embodiments 67-102 and 112, and (ii) dissimilar parts; It provides a conjugate that includes [this].

[0480] Embodiment 114 provides the conjugate of Embodiment 113, wherein the dissimilar portion is polyethylene glycol.

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

[0482] Embodiment 116 provides a method for reducing or preventing the progression of pathological calcification in a subject in need thereof, comprising the steps of administering to a subject in a therapeutically effective amount (a) any one ENPP1 mutant polypeptide of Embodiments 58-66, 87-96 and 112; (b) any one fusion of Embodiments 67-102 and 112; (c) any one conjugate 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 the steps of administering to a subject in a therapeutically effective amount (a) any one ENPP1 mutant polypeptide of Embodiments 58-66, 87-96 and 112; (b) any one fusion of Embodiments 67-102 and 112; (c) any one conjugate 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 requiring such reduction, comprising the steps of administering to a subject in a therapeutically effective amount (a) any one ENPP1 mutant polypeptide of Embodiments 58-66, 87-96, and 112; (b) any one fusion of Embodiments 67-102 and 112; (c) any one conjugate 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 for treating, improving or preventing the progression of ossification of the posterior longitudinal ligament (OPLL) in a subject in need thereof, comprising the steps of administering to a subject in a therapeutically effective amount (a) any one ENPP1 mutant polypeptide of Embodiments 58-66, 87-96 and 112; (b) any one fusion of Embodiments 67-102 and 112; (c) any one conjugate of Embodiments 113-114; and / or (d) a pharmaceutical composition of Embodiment 115, thereby reducing, improving or preventing ossification of the posterior longitudinal ligament (OPLL) in the subject.

[0486] Embodiment 120 provides a method for treating, reversing or preventing the progression of hypophosphatemic rickets in a subject in need thereof, comprising the steps of administering to a subject in a therapeutically effective amount (a) any one ENPP1 mutant polypeptide of Embodiments 58-66, 87-96 and 112; (b) any one fusion of Embodiments 67-102 and 112; (c) any one conjugate 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] Embodiment 121 reduces at least one disease selected from the group consisting of chronic kidney disease (CKD), end-stage renal disease (ESRD), uremic arteriosclerosis (CUA), calciphylaxis, ossification of the posterior longitudinal ligament (OPLL), hypophosphatemic rickets, osteoarthritis, age-related arteriosclerosis, idiopathic infantile arteriosclerosis (IIAC), generalized arteriosclerosis of infants (GACI), and arteriosclerotic plaque calcification in subjects diagnosed with at least one of these diseases. The present invention provides a method for reducing or preventing the progression of a disease, comprising the steps of administering to a subject in a therapeutically effective amount (a) any one ENPP1 mutant polypeptide of embodiments 58-66, 87-96, and 112; (b) any one fusion of embodiments 67-102 and 112; (c) any one conjugate of embodiments 113-114; 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 steps of administering to a subject in a therapeutically effective amount (a) any one ENPP1 mutant polypeptide of Embodiments 58-66, 87-96 and 112; (b) any one fusion of Embodiments 67-102 and 112; (c) any one conjugate of Embodiments 113-114; 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 the PPi level in a subject having a PPi level lower than the normal level, comprising the steps of administering to the subject in a therapeutically effective dose of (a) any one ENPP1 mutant polypeptide of Embodiments 58-66, 87-96 and 112; (b) any one fusion of Embodiments 67-102 and 112; (c) any one conjugate of Embodiments 113-114; and / or (d) a pharmaceutical composition of Embodiment 115, wherein the administration raises the PPi level in the subject to at least the normal level of 2 μM and maintains it at substantially 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 PPi level lower than a normal level, comprising the steps of administering to the subject in a therapeutically effective amount (a) any one ENPP1 mutant polypeptide of Embodiments 58-66, 87-96 and 112; (b) any one fusion of Embodiments 67-102 and 112; (c) any one conjugate of Embodiments 113-114; and / or (d) a pharmaceutical composition of Embodiment 115, thereby reducing or preventing the progression of pathological calcification or ossification in the subject.

[0491] Embodiment 125 provides a method for treating an ENPP1 defect that manifests as a decrease in extracellular pyrophosphate (PPi) concentration in a subject requiring such treatment, comprising the steps of administering to a subject in a therapeutically effective amount (a) any one ENPP1 mutant polypeptide from Embodiments 58-66, 87-96, and 112; (b) any one fusion from Embodiments 67-102 and 112; (c) any one conjugate from Embodiments 113-114; and / or (d) a pharmaceutical composition of Embodiment 115, thereby increasing the PPi level in the subject.

[0492] Embodiment 126 provides one of the methods described in Embodiments 116 to 125, wherein a mutant polypeptide, fusion, conjugate, or pharmaceutical composition is administered to a subject over a short or long period of time.

[0493] Embodiment 127 provides one of the methods described in Embodiments 116 to 126 for administering a mutant polypeptide, fusion, conjugate, or pharmaceutical composition to a subject topically, regionally, parenterally, or systemically.

[0494] Embodiment 128 provides one of the methods from Embodiments 116 to 127, wherein the subject is a human.

[0495] Each and every disclosure of patents, patent applications and publications cited herein is incorporated herein by reference in their entirety. While this disclosure is made with reference to specific embodiments, it will be apparent that other embodiments and variations of this disclosure can be derived by those skilled in the art without departing from the true spirit and scope of this disclosure. The appended claims are intended to be considered to encompass all such embodiments and equivalent variations.

[0496] Sequence information SEQUENCE LISTING <110> Yale Plaza <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 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> 14 <211> 227 <212> PRT <213> Artificial Sequence <220> <223> Recombinantly synthesized <400> 14 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 Tyr 20 25 30 Ile Thr Arg Glu 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> 15 <211> 1059 <212> PRT <213> Artificial Sequence <220> <223> Recombinantly synthesized <400> 15 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 ...

Claims

1. An ENPP1 polypeptide fusion comprising an ENPP1 polypeptide fused to the Fc region of an immunoglobulin, The ENPP1 polypeptide contains an amino acid sequence that is at least 90% identical to amino acid residues 23-849 of SEQ ID NO:

7. The ENPP1 polypeptide contains the mutation I256T in SEQ ID NO:7, and The ENPP1 polypeptide fusion has enzymatic activity. The aforementioned ENPP1 polypeptide fusion.

2. (i) The Fc region contains at least one mutation selected from the group consisting of M883Y, S885T, T887E, H1064K, and N1065F in SEQ ID NO:7; (ii) The Fc region contains at least one mutation selected from the group consisting of M883Y, (M883Y and S885T and T887E), and (H1064K and N1065F) in SEQ ID NO:7; (iii) Whether the ENPP1 polypeptide further contains the V29N mutation in SEQ ID NO:7; (iv) The ENPP1 polypeptide further contains at least one mutation selected from the group consisting of P534N and V536T in SEQ ID NO:7; (v) The ENPP1 polypeptide further contains mutations in SEQ ID NO:7 (P534N and V536T); or (vi) The ENPP1 polypeptide fusion contains at least one mutation selected from the group consisting of V29N, P534N, V536T, (P534N and V536T), M883Y, S885T, T887E, (H1064K and N1065F and M883Y and S885T and T887E), (H1064K and N1065F) in SEQ ID NO:

7. The ENPP1 polypeptide fusion according to claim 1.

3. An ENPP1 polypeptide fusion comprising an ENPP1 polypeptide and an immunoglobulin Fc region, The ENPP1 polypeptide contains an amino acid sequence that is at least 90% identical to amino acid residues 23-849 of SEQ ID NO:

7. The ENPP1 polypeptide fusion has enzymatic activity, and (i) The ENPP1 polypeptide fusion contains mutations I256T, M883Y, S885T, and T887E in SEQ ID NO:7; or (ii) The ENPP1 polypeptide fusion includes mutations I256T, P534N, V536T, M883Y, S885T, and T887E in SEQ ID NO:7, The aforementioned ENPP1 polypeptide fusion.

4. An ENPP1 polypeptide fusion according to any one of claims 1 to 3, wherein the Fc region is the Fc region of immunoglobulin G (IgG).

5. An ENPP1 polypeptide fusion according to any one of claims 1 to 3, wherein the Fc region contains the amino acid sequence of SEQ ID NO:

14.

6. An ENPP1 polypeptide fusion according to any one of claims 1 to 3, comprising the amino acid sequence of SEQ ID NO:

17.

7. An ENPP1 mutant polypeptide comprising an ENPP1 polypeptide fused to the Fc region of an immunoglobulin, The ENPP1 polypeptide contains an amino acid sequence that is at least 90% identical to amino acid residues 23-849 of SEQ ID NO:

7. The ENPP1 mutant polypeptide contains the mutation I256T in SEQ ID NO:7, and further contains at least one mutation selected from the group consisting of P534N and V536T in SEQ ID NO:

7. The aforementioned ENPP1 mutant polypeptide.

8. comprising mutations in SEQ ID NO:7 (P534N and V536T), The ENPP1 mutant polypeptide according to claim 7.

9. (i) containing at least one mutation selected from the group consisting of (M883Y and S885T and T887E), (P534N and V536T and H1064K and N1065F), and (P534N and V536T and M883Y and S885T and T887E) in SEQ ID NO:7; (ii) Does it contain mutations M883Y, S885T, and T887E in SEQ ID NO:7? (iii) containing mutations P534N, V536T, H1064K, and N1065F in SEQ ID NO:7; or (iv) Including mutations P534N, V536T, M883Y, S885T, and T887E in SEQ ID NO:7, The ENPP1 mutant polypeptide according to claim 7.

10. An ENPP1 polypeptide fusion according to any one of claims 1 to 3 or an ENPP1 mutant polypeptide according to any one of claims 7 to 9, expressed from a CHO cell line stably transfected with human ST6 beta-galactoside alpha-2,6-sialyltransferase (also known as ST6GAL1).

11. Sialic acid and / or N-acetylmannosamine (1,3,4-O-Bu) 3 A polypeptide fusion according to any one of claims 1 to 3 or an ENPP1 mutant polypeptide according to any one of claims 7 to 9, which is grown in a cell culture supplemented with ManNAc (also known as ManNAc).

12. A pharmaceutical composition comprising an ENPP1 polypeptide fusion according to any one of claims 1 to 3 or an ENPP1 mutant polypeptide according to any one of claims 7 to 9, and optionally further comprising at least one pharmaceutically acceptable carrier.

13. A pharmaceutical composition comprising an ENPP1 polypeptide fusion according to any one of claims 1 to 3 or an ENPP1 mutant polypeptide according to any one of claims 7 to 9, (i) to reduce or prevent the progression of pathological calcification in subjects in need; (ii) to reduce or prevent the progression of pathological ossification in subjects who require it; (iii) to reduce or prevent the progression of ectopic calcification of soft tissue in subjects in need; (iv) To treat, improve, or prevent the progression of ossification of the posterior longitudinal ligament (OPLL) in patients who require it; (v) To treat, reverse, or prevent the progression of hypophosphatemic rickets in those who need it; (vi) 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 arteriosclerosis (CUA), calciphylaxis, ossification of the posterior longitudinal ligament (OPLL), hypophosphatemic rickets, osteoarthritis, age-related arteriosclerosis, idiopathic infantile arteriosclerosis (IIAC), generalized infantile arteriosclerosis (GACI), and arteriosclerotic plaque calcification in subjects diagnosed with at least one of the diseases; or (vii) Reduce or prevent the progression of age-related arteriosclerosis in those who need it. The aforementioned pharmaceutical composition.

14. (i) Pathological calcification is selected from the group consisting of idiopathic infant arterial calcification (IIAC) and atherosclerotic plaque calcification; (ii) Pathological ossification is selected from the group consisting of ossification of the posterior longitudinal ligament (OPLL), hypophosphatemic rickets, and osteoarthritis; (iii) Soft tissue calcification is selected from the group consisting of IIAC and osteoarthritis; or (iv) The soft tissue is selected from the group consisting of arteriosclerotic plaque, muscular arteries, joints, spine, articular cartilage, intervertebral disc cartilage, blood vessels, and connective tissue. The pharmaceutical composition according to claim 13.

15. (i) A pharmaceutical composition for raising the PPi level in a subject having a PPi level lower than the normal level, wherein administration of the pharmaceutical composition to the subject raises the PPi level in the subject to at least 2 μM, and maintains it at substantially the same level; (ii) A pharmaceutical composition for reducing or preventing the progression of pathological calcification or ossification in a subject having a PPi level lower than normal, wherein administration of the pharmaceutical composition to the subject reduces or prevents the progression of pathological calcification or ossification in the subject; or (iii) A pharmaceutical composition for treating an ENPP1 deficiency that manifests as a decrease in extracellular pyrophosphate (PPi) concentration in a subject requiring such treatment, wherein administration of the pharmaceutical composition to the subject causes an increase in the level of PPi in the subject. The pharmaceutical composition according to claim 13.

16. The pharmaceutical composition according to any one of claims 13 to 15, wherein an ENPP1 polypeptide fusion or an ENPP1 mutant polypeptide is obtained from an ENPP1 precursor protein expressed in mammalian cells, the ENPP1 precursor protein comprises a signal peptide sequence and an ENPP1 polypeptide, and the ENPP1 precursor protein undergoes proteolytic processing to produce an ENPP1 polypeptide.

17. The pharmaceutical composition according to claim 16, wherein the signal peptide sequence in the ENPP1 precursor protein is ligated to the N-terminus of the ENPP1 polypeptide.

18. The pharmaceutical composition according to claim 16, wherein the signal peptide sequence is selected from the group consisting of the ENPP1 signal peptide sequence, the ENPP2 signal peptide sequence, the ENPP7 signal peptide sequence, and the ENPP5 signal peptide sequence.

19. (i) administered to the target in the short term or long term; or (ii) administered to the subject locally, regionally, parenterally, or systemically; or (iii) Administered to the subject by at least one route selected from the group consisting of subcutaneous, oral, aerosol, inhalation, rectal, vaginal, transdermal, subcutaneous, intranasal, oral, sublingual, parenteral, intrathecal, gastric, ocular, pulmonary, and topical. A pharmaceutical composition according to any one of claims 13 to 15.

20. A pharmaceutical composition according to any one of claims 13 to 15, further comprising at least one pharmaceutically acceptable carrier.

21. A pharmaceutical composition according to any one of claims 13 to 15, wherein the target is a mammal.

22. The pharmaceutical composition according to claim 21, wherein the mammal is a human.

23. A nucleic acid encoding an ENPP1 mutant polypeptide according to any one of claims 7 to 9 or an ENPP1 polypeptide fusion according to any one of claims 1 to 3.

24. A vector comprising the nucleic acid described in claim 23.

25. An expression vector comprising the nucleic acid according to claim 23.

26. A cell or a plurality of cells, each comprising the nucleic acid according to claim 23, the vector according to claim 24, and / or the expression vector according to claim 25.

27. The cell or plurality of cells according to claim 26, which are CHO cells and / or NS0 cells.

28. The cell or plurality of cells according to claim 27, wherein CHO cells are stably transfected with human ST6 beta-galactoside alpha-2,6-sialyltransferase.

29. A method for producing an ENPP1 mutant polypeptide or an ENPP1 polypeptide fusion, comprising the step of culturing the cells or a plurality of cells described in any one of claims 26 to 28 under conditions suitable for the expression of an ENPP1 mutant polypeptide or an ENPP1 polypeptide fusion by said cells or a plurality of cells.

30. The method according to claim 29, wherein the cells are cultured in a medium supplemented with sialic acid and / or N-acetylmannosamine.

31. The method according to claim 29 or 30, further comprising the step of purifying an ENPP1 mutant polypeptide or a plurality of cell fusions from the aforementioned cells, the plurality of cells, or a culture medium in which the aforementioned cells or the plurality of cells are cultured.

32. An ENPP1 mutant polypeptide or fusion product purified by the method described in any one of claims 29 to 30.

33. (i) an ENPP1 mutant polypeptide according to any one of claims 7 to 9, and / or an ENPP1 polypeptide fusion according to any one of claims 1 to 3, (ii) heterogeneous parts; A conjugate that includes this.

34. The conjugate according to claim 33, wherein the dissimilar portion is polyethylene glycol.

35. Pharmacologically acceptable carriers, and At least one of the following: an ENPP1 mutant polypeptide according to any one of claims 7 to 9, an ENPP1 polypeptide fusion according to any one of claims 1 to 3, a nucleic acid according to claim 23, a vector according to claim 24, an expression vector according to claim 25, and / or a conjugate according to claim 33 or 34. A pharmaceutical composition containing [the specified substance].

36. (i) for use in reducing or preventing the progression of pathological calcification in subjects in need; (ii) For use in reducing or preventing the progression of pathological ossification in subjects in need; (iii) For use in reducing or preventing the progression of ectopic calcification of soft tissue in subjects where it is needed; (iv) For use in treating, improving or preventing the progression of ossification of the posterior longitudinal ligament (OPLL) in patients who require it; (v) for use in treating, reversing or preventing the progression of hypophosphatemic rickets in persons who need it; (vi) For use in 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 arteriosclerosis (CUA), calciphylaxis, ossification of the posterior longitudinal ligament (OPLL), hypophosphatemic rickets, osteoarthritis, age-related arteriosclerosis, idiopathic infantile arteriosclerosis (IIAC), generalized infantile arteriosclerosis (GACI), and arteriosclerotic plaque calcification, in subjects diagnosed with at least one of the said diseases; (vii) For use in reducing or preventing the progression of age-related arteriosclerosis in those who need it; (viii) For use in raising the PPi level in subjects having a PPi level lower than the normal level, wherein administration of the pharmaceutical composition to the subject raises the PPi level in the subject to at least 2 μM, and maintains it at substantially the same level; (ix) For use in reducing or preventing the progression of pathological calcification or ossification in subjects having PPi levels lower than normal levels, wherein administration of the pharmaceutical composition to the subject reduces pathological calcification or ossification in the subject or prevents the progression of pathological calcification or ossification in the subject; (x) For use in treating ENPP1 deficiencies that manifest due to a decrease in extracellular pyrophosphate (PPi) concentration in subjects requiring it, wherein administration of the pharmaceutical composition to the subject increases the PPi level in the subject, The pharmaceutical composition according to claim 35.

37. The pharmaceutical composition according to claim 36, administered to a subject in the short term or long term.

38. The pharmaceutical composition according to claim 36 or 37, which is administered to a subject locally, regionally, parenterally, or systemically.

39. A pharmaceutical composition according to any one of claims 36 to 37, wherein the subject is a human.

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