Alkaline phosphatase polypeptides and methods of use thereof

Abstract

Alkaline phosphatase polypeptides and methods of use thereof are provided. [Solution] Polypeptides, including soluble alkaline phosphatase, variants, fragments, and fusion proteins thereof, and methods of use thereof for treating bone mineralization disorders and symptoms thereof, such as hypophosphatasia (HPP), are described. The polypeptides include soluble alkaline phosphatase (sALP) or fragments thereof derived from naturally occurring alkaline phosphatase (ALP). In one aspect, polypeptides are described that include recombinant alkaline phosphatase having at least one mutation relative to naturally occurring alkaline phosphatase. The mutation may improve at least one activity or pharmacokinetic (PK) property relative to naturally occurring alkaline phosphatase lacking the at least one mutation.

Description

[Background technology]

[0001] Hypophosphatasia (HPP) is a rare, inherited skeletal disorder, with a prevalence of 1 in 100,000 live births in its most severe form. The disorder is typically caused by loss-of-function mutations in the gene encoding tissue-nonspecific alkaline phosphatase (TNSALP). HPP manifests in a marked range of symptoms and severity, from premature tooth loss to a near-complete lack of bone mineralization in utero. Symptoms of HPP vary significantly among patients and by patient age. Many patients with HPP present with skeletal changes, short stature, chronic pain, painful lower extremities, muscle weakness, gait disturbances, and early, non-traumatic tooth loss. Summary of the Invention [Problem to be solved by the invention]

[0002] Asfotase alfa (STRENSIQ®, Alexion Pharmaceuticals, Inc.), a recombinant enzyme replacement therapy (ERT) containing a soluble fragment of TNSALP, is the first ERT available for HPP patients. Asfotase alfa has shown transformative effects in the most severe forms of HPP, as evidenced by improvements in bone mineralization and density, respiratory and motor function, cognitive development, and muscle strength (Whyte et al., New Engl. J. Med. 366:904-913, 2012). While the safety and therapeutic benefits of asfotase alfa have been documented, compliance with the multiple injection regimen can be challenging, particularly among pediatric patients. The most common side effects of subcutaneous administration three to six times weekly are injection site reactions (i.e., pruritus, pain, and erythema). Therefore, improvements to improve quality of life, for example, by reducing dosage and frequency and increasing compliance, would be desirable. Therefore, new compositions and methods for treating HPP and related bone mineralization disorders would be beneficial. [Means for solving the problem]

[0003] In one aspect, a polypeptide is described that includes a recombinant alkaline phosphatase having at least one mutation relative to a naturally occurring alkaline phosphatase, wherein the mutation can improve at least one activity or pharmacokinetic (PK) property relative to a naturally occurring alkaline phosphatase lacking the at least one mutation.

[0004] The naturally occurring alkaline phosphatase can be tissue-nonspecific alkaline phosphatase (TNSALP), placental alkaline phosphatase (PALP), germline alkaline phosphatase (GALP), or intestinal alkaline phosphatase (IALP). The TNSALP can be mammalian TNSALP (e.g., human, gorilla, mouse, rabbit, chimpanzee, cynomolgus monkey, rhesus monkey, orangutan, baboon, rat, cow, goat, or llama TNSALP). The polypeptide can have at least 85% (e.g., 90%, 95%, 97%, 99%, or 100%) sequence identity to amino acids 1-491 of SEQ ID NO:1.

[0005] The at least one mutation may be selected from the group consisting of E108X, M384X, L385X, N213X, and N286X relative to SEQ ID NO: 1, and X is any amino acid. The at least one mutation may be selected from the group consisting of E108S, E108T, E108Q, E108M, E108K, E108L, M384R, L385T, N213Q, and N286Q relative to SEQ ID NO: 1. The recombinant alkaline phosphatase may have at least two, three, four, or five mutations selected from the group consisting of E108S, E108T, E108Q, E108M, E108K, E108L, M384R, L385T, N213Q, and N286Q relative to SEQ ID NO: 1. For example, the recombinant alkaline phosphatase may have E108M, N213Q, and N286Q mutations relative to SEQ ID NO: 1. The recombinant alkaline phosphatase may not have an E108A mutation relative to SEQ ID NO: 1.

[0006] At least one mutation in the alkaline phosphatase described herein that improves at least one activity or pharmacokinetic (PK) property can be present within a specific region of the enzyme. The mutation can be present in the ectodomain of alkaline phosphatase. For example, the mutation can be present in the crown domain, catalytic domain, or dimerization domain. The mutation, if included, can be present in the GPI anchor domain. The polypeptide can include a mutation present within amino acids 1-491 or 1-486 of human TNSALP or an analogous ALP position based on alignment and / or sequence homology. For example, the polypeptide can have a mutation within positions 1-491, 1-486, 25-475, 25-240, 50-400, 50-350, and / or 100-300 relative to SEQ ID NO: 1. The polypeptide can have a mutation within positions 100-125, 100-110, 200-225, 210-220, 275-300, 280-290, 425-450, and / or 425-435 relative to SEQ ID NO: 1. For example, the polypeptide can have a mutation between positions 108, 213, 286, and / or 429 relative to SEQ ID NO: 1. In certain embodiments, the mutation is within the sequence of ALP, and the mutation is not a stretch of unnatural amino acids present in the N-terminal or C-terminal domain of ALP.

[0007] The naturally occurring alkaline phosphatase can be an IALP (e.g., a mammalian IALP such as a gorilla, chimpanzee, cynomolgus monkey, rhesus monkey, rat, cow, goat, llama, or human IALP). The polypeptide can have at least 85% (e.g., 90%, 95%, 97%, 99%, or 100%) sequence identity to amino acids 1-486 of SEQ ID NO:4. The recombinant alkaline phosphatase can have a W245X mutation relative to SEQ ID NO:4, where X is any naturally occurring conserved amino acid from different species. The recombinant alkaline phosphatase can have a W245R mutation relative to SEQ ID NO:4. The recombinant alkaline phosphatase can have a C481X mutation relative to SEQ ID NO:4, where X is any non-thiol-containing amino acid. The recombinant alkaline phosphatase can have a C481G mutation relative to SEQ ID NO:4.

[0008] The recombinant alkaline phosphatase can have a mutation at a consensus N-linked glycosylation site. The consensus N-linked glycosylation site comprises a motif having the sequence asparagine-XZ, where X is any amino acid except P and Z is any amino acid except S or T. The asparagine site can be mutated to a glutamine residue.

[0009] The recombinant alkaline phosphatase may have a mutation selected from the group consisting of S429Q, S429H, S429E and S429D relative to SEQ ID NO: 4. The recombinant alkaline phosphatase may have a mutation selected from the group consisting of S428R, S428Q and S428D relative to SEQ ID NO: 4.

[0010] The recombinant alkaline phosphatase may have at least 80%, 85%, 90%, 95%, 99% or 100% sequence identity to at least 50 (e.g., at least 100, 150, 200, 250, 300, 350, 400, 450 or more) amino acids of any one of SEQ ID NOs: 7-223, 247 and 262-264.

[0011] The at least one activity improved by the mutation may be selected from the group consisting of increased catalytic activity, increased temperature stability, increased zinc binding, maintenance of activity in zinc-depleted buffers, maintenance of activity at pH 5.0 to 7.5, reduced dimerization, reduced aggregation, and increased manufacturability. The increased catalytic activity may include increased hydrolysis of pyridoxal 5-phosphate and / or pyrophosphate. The increased catalytic activity may be about 2-fold to about 30-fold better than the activity of naturally occurring alkaline phosphatase. The at least one PK characteristic improved by the mutation may be selected from the group consisting of increased substrate specificity, increased activity for natural substrates, increased activity for artificial substrates, decreased Km for natural substrates, and increased area under the curve (AUC) per dose. Exemplary artificial substrates are 4-methylumbelliferyl phosphate (4-MUP), umbelliferone phosphate, and para-nitrophenyl phosphate (pNPP), and exemplary natural substrates are pyridoxal 5'-phosphate, PLP, and PEA.

[0012] In some embodiments, the polypeptide may further comprise a region Y, where Y is an amino acid sequence of at least one amino acid. Y may be a fragment crystallizable region (Fc). The Fc region may comprise IgG1, IgG2, IgG3, or IgG4, or a chimera thereof. For example, the Fc region may comprise an IgG2 / 4 chimera. The Fc region may comprise the sequence of SEQ ID NO: 253, or may have at least 85% (e.g., 90%, 95%, 97%, 99%, or 100%) sequence identity thereto.

[0013] The polypeptide may further comprise a bone-targeting moiety. A polypeptide comprising an ALP, a bone-targeting moiety, and a Y region may have the structure Z-ALP-Y-Xn, where Y is an amino acid sequence of at least one amino acid; Z is absent or is an amino acid sequence of at least one amino acid; Xn is a bone-targeting moiety selected from the group consisting of polyaspartic acid (Dn), polyglutamic acid (En), poly(aspartic acid-alanine-aspartic acid) (DAD)n, poly(aspartic acid-aspartic acid-serine) (DDS)n, poly(aspartic acid-serine-serine (DSS)n, poly(glutamic acid-glutamic acid-serine) (EES)n, and VHH, and n=1 to 50; and ALP is recombinant alkaline phosphatase.

[0014] The polypeptide can have the structure Y-ALP-Z-Xn or any topological arrangement thereof (e.g., Xn-Y-ALP-Z and Y-Xn-ALP-Z).

[0015] The polypeptide may comprise or consist of an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 7-223, 247, and 262-264 (e.g., any one of SEQ ID NOs: 72, 123, 155, or 177). For example, the polypeptide may comprise or consist of the sequence of any one of SEQ ID NOs: 72, 123, 155, or 177. The polypeptide may comprise any sALP catalytic domain, Fc IgG isotype, or bone-targeting moiety listed in Table 1 and their topological sequences. The polypeptide may comprise or consist of an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 123. The polypeptide may comprise or consist of SEQ ID NO: 123.

[0016] The polypeptide may comprise or consist of an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99% or 100%) sequence identity to SEQ ID NO: 177. The polypeptide may comprise or consist of SEQ ID NO: 177.

[0017] The polypeptide may comprise or consist of an amino acid sequence having at least 85% (e.g., 90%, 95%, 97%, 99% or 100%) sequence identity to SEQ ID NO: 260 or 261. The polypeptide may comprise or consist of the sequence of SEQ ID NO: 260. The polypeptide may comprise or consist of the sequence of SEQ ID NO: 261.

[0018] The polypeptide may comprise a secretory signal peptide. The polypeptide may comprise or consist of an amino acid sequence having at least 85% (e.g., 90%, 95%, 97%, 99% or 100%) sequence identity to SEQ ID NO: 263 or 264. The polypeptide may comprise or consist of the sequence of SEQ ID NO: 263. The polypeptide may comprise or consist of the sequence of SEQ ID NO: 264.

[0019] In some embodiments, the polypeptide is a dimer. Alternatively, the polypeptide may be a monomer.

[0020] Also described is a polypeptide comprising an alkaline phosphatase and a fragment crystallizable (Fc) region, wherein the Fc region is an IgG2 / 4 chimera. The Fc region may comprise the sequence of SEQ ID NO: 253 or have at least 85% (e.g., 90%, 95%, 97%, 99%, or 100%) sequence identity thereto. The recombinant alkaline phosphatase may be selected from the group consisting of TNSALP, IALP, placental alkaline phosphatase (PALP), and germline alkaline phosphatase (GALP). The recombinant alkaline phosphatase may be a mammalian alkaline phosphatase (e.g., human, gorilla, mouse, rabbit, chimpanzee, cynomolgus monkey, rhesus monkey, orangutan, baboon, rat, bovine, goat, or llama alkaline phosphatase). The recombinant alkaline phosphatase may comprise the sequence of any one of SEQ ID NOs: 1-6 or a fragment thereof. For example, the recombinant alkaline phosphatase can comprise amino acids 1 to 491 of SEQ ID NO:1 or amino acids 1 to 486 of SEQ ID NO:4.

[0021] In some embodiments of any of the above aspects, the polypeptide further comprises a bone-targeting moiety Xn selected from the group consisting of Dn, En, (DAD)n, (DDS)n, (DSS)n, (EES)n, and VHH, where n=1-50 (e.g., 1-30). In some particular embodiments, the bone-targeting moiety can be Dn when n=7-10; En when n=10-15; (DAD)n when n=2-4; (DDS)n when n=2-4; (DSS)n when n=3; or (EES)n when n=3-4. For example, the bone-targeting moiety can comprise (DAD)3 or (DDS)3.

[0022] The VHH bone-targeting moiety may comprise one or more substitutions, for example at least one (e.g., two or three) complementarity determining region (CDR) of the VHH may be substituted with at least one (e.g., 2 to 30, e.g., 5 or 7) glutamic acid or aspartic acid residue.

[0023] In some embodiments, the polypeptides described herein are post-translationally modified (eg, glycosylated or sialylated).

[0024] Also described are polynucleotides encoding the polypeptides of any of the above aspects, vectors comprising the polynucleotides, and cells (e.g., mammalian cells such as CHO cells or HEK293 cells) comprising the polynucleotides or vectors. The polynucleotides may encode an amino acid sequence having at least 85% (e.g., 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 7-223, 247, and 262-264 (e.g., any one of SEQ ID NOs: 72, 123, 155, or 177). The polynucleotides may have at least 85% (e.g., 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 265-268. The polynucleotides may comprise or consist of any one of SEQ ID NOs: 265-268. The polynucleotides may comprise or consist of SEQ ID NO: 265. The polynucleotides may comprise or consist of SEQ ID NO: 266. The polynucleotide may comprise or consist of SEQ ID NO: 267. The polynucleotide may comprise or consist of SEQ ID NO: 268.

[0025] In some embodiments, the polypeptide is not or does not include the sequence of SEQ ID NO:269.

[0026] In some embodiments, the alkaline phosphatase does not include the alkaline phosphatase alfa of SEQ ID NO: 269 (eg, amino acids 1-485).

[0027] In some embodiments, the polypeptide is not a polypeptide having the amino acid sequence of SEQ ID NO: 269 and does not include the alkaline phosphatase alfa of SEQ ID NO: 269 (eg, amino acids 1-485).

[0028] Also described are methods of producing the polypeptide of any of the above aspects by providing a cell (e.g., a mammalian cell such as a CHO cell or a HEK293 cell) transformed with the polynucleotide or a vector encoding the polynucleotide, such that the polynucleotide is positioned for expression in the cell; culturing the transformed cell under conditions suitable for expression of the polynucleotide, resulting in expression of the polypeptide; and isolating the polypeptide.

[0029] Also described is a pharmaceutical composition comprising the polypeptide of any of the above embodiments and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may comprise sodium chloride and / or sodium phosphate. The composition may comprise about 150 mM sodium chloride and / or about 25 mM sodium phosphate at a pH of about 7.4.

[0030] The composition may be formulated at a dosage of about 0.1 mg / mL to about 10 mg / mL. The composition may be formulated at a volume of about 0.1 mL to about 50 mL (e.g., about 0.1 to about 10 mL).

[0031] In another aspect, methods are described for treating a disease selected from the group consisting of hypophosphatasia (HPP), bone fracture, osteoporosis, sclerosteosis, chondrocalcinosis, hypotonia, Duchenne muscular dystrophy, tracheobronchomalacia, stroke, neurofibromatosis (e.g., NF-1), craniosynostosis, or one or more symptoms thereof, in a subject (e.g., a human subject) in need thereof by administering to the subject a polypeptide or pharmaceutical composition of any of the above aspects. The polypeptide can be administered in an amount and for a period sufficient to treat the disease or alleviate one or more symptoms thereof. The treatment can enhance bone formation in the subject. The polypeptide can be used to treat muscle weakness.

[0032] The polypeptide may be administered at a dosage of about 0.01 mg / kg to about 60 mg / kg (e.g., about 0.1 mg / kg to about 50 mg / kg, e.g., about 0.1 mg / kg to about 20 mg / kg, e.g., about 0.1 mg / kg to about 10 mg / kg). The polypeptide may be administered once per day, week, month, or year (e.g., once per week). The polypeptide may be administered at a dosage of about 0.01 mg / kg / week to about 20 mg / kg / week (e.g., about 0.1 mg / kg / week to about 10 mg / kg / week). The polypeptide may be administered for at least one day, one week, one month, one year, or more.

[0033] The polypeptide may be administered subcutaneously, intravenously, intramuscularly, sublingually, intrathecally or intradermally. In particular, the polypeptide or polypeptide-containing composition may be administered by subcutaneous administration.

[0034] The subject can be a human subject, such as a neonate, child, adolescent, or adult.

[0035] Prior to administration of the recombinant polypeptide, the subject may be characterized as having an average six-minute walking distance of about 350 meters or less. Administration of the recombinant polypeptide may promote an increase in the subject's average six-minute walking distance of at least 100 meters or more. After administration of the recombinant polypeptide (e.g., after a one-month to one-year treatment period), the subject may exhibit an average six-minute walking distance of about 500 meters or more.

[0036] Subjects may exhibit decreased dependency on assistive mobility devices (eg, walkers, wheelchairs, braces, crutches, and orthotics) after administration of the recombinant polypeptide.

[0037] Prior to administration of the recombinant polypeptide, the subject may be characterized as having a plasma PPi concentration of about 4.5 μM or greater. Administration of the recombinant polypeptide may promote a median decrease in PPi concentration in plasma samples from the subject of at least about 1 μM. After administration of the recombinant polypeptide, the subject may exhibit a plasma PPi concentration of about 2 μM to about 5 μM.

[0038] In some embodiments, the subject is between 0 and 14 days old and is characterized as having a plasma ALP concentration of about 90 U / L or less prior to administration of the recombinant polypeptide; the subject is between 15 days old and less than 1 year old and is characterized as having a plasma ALP concentration of about 134 U / L or less prior to administration of the recombinant polypeptide; the subject is between about 1 year old and less than 10 years old and is characterized as having a plasma ALP concentration of about 156 U / L or less prior to administration of the recombinant polypeptide; the subject is between about 10 years old and about 13 years old and is characterized as having a plasma ALP concentration of about 141 U / L or less prior to administration of the recombinant polypeptide; or the subject is female and between about 13 years old and about 15 years old and is characterized as having a plasma ALP concentration of about 62 U / L or less prior to administration of the recombinant polypeptide. the subject is male and about 13 to about 15 years of age and is characterized as having a plasma ALP concentration of about 127 U / L or less before administration of the recombinant polypeptide; the subject is female and about 15 to about 17 years of age and is characterized as having a plasma ALP concentration of about 54 U / L or less before administration of the recombinant polypeptide; the subject is male and about 15 to about 17 years of age and is characterized as having a plasma ALP concentration of about 89 U / L or less before administration of the recombinant polypeptide; the subject is about 17 years of age or older and is characterized as having a plasma ALP concentration of about 48 U / L or less before administration of the recombinant polypeptide; or the subject is about 17 years of age or older and is characterized as having a plasma ALP concentration of about 59 U / L or less before administration of the recombinant polypeptide.

[0039] Administration of the recombinant polypeptide can promote a median increase in ALP concentration in plasma samples from the subject of at least about 100 U / L or more.

[0040] In some embodiments, the subject is between 0 and 14 days old and is characterized as having a plasma ALP concentration of about 273 U / L or greater after administration of the recombinant polypeptide; the subject is between 15 days old and less than 1 year old and is characterized as having a plasma ALP concentration of about 518 U / L or greater after administration of the recombinant polypeptide; the subject is between about 1 year old and less than about 10 years old and is characterized as having a plasma ALP concentration of about 369 U / L or greater after administration of the recombinant polypeptide; the subject is between about 10 years old and about 13 years old and is characterized as having a plasma ALP concentration of about 460 U / L or greater after administration of the recombinant polypeptide; the subject is female and between about 13 years old and about 15 years old and is characterized as having a plasma ALP concentration of about 280 U / L or greater after administration of the recombinant polypeptide; the subject is male and the subject is female and about 15 to about 17 years old and is characterized as having a plasma ALP concentration of about 128 U / L or greater after administration of the recombinant polypeptide; the subject is male and about 15 to about 17 years old and is characterized as having a plasma ALP concentration of about 365 U / L or greater after administration of the recombinant polypeptide; the subject is female and about 17 years old or older and is characterized as having a plasma ALP concentration of about 95 U / L or greater after administration of the recombinant polypeptide; or the subject is male and about 17 years old or older and is characterized as having a plasma ALP concentration of about 164 U / L or greater after administration of the recombinant polypeptide.

[0041] Prior to administration of the recombinant polypeptide, the subject may be characterized as having an average Bruininks-Oseretsky Test of Motor Proficiency, Second Edition (BOT-2) strength score of about 10 or less. Prior to administration of the recombinant polypeptide, the subject may be characterized as having an average BOT-2 running speed and agility score of about 5 or less. Administration of the recombinant polypeptide may result in the subject having an average BOT-2 strength score of about 10 or greater. Administration of the recombinant polypeptide may result in the subject having an average BOT-2 running speed and agility score of about 5 or greater.

[0042] Prior to administration of the recombinant polypeptide, the subject may be characterized as having an average Childhood Health Assessment Questionnaire (CHAQ) index score of about 0.8 or greater. Administration of the recombinant polypeptide may result in the subject having an average CHAQ index score of about 0.5 or less.

[0043] Prior to administration of the recombinant polypeptide, the subject may be characterized as having an average Pediatric Outcomes Data Collection Instrument (PODCI) score of about 40 or less. Administration of the recombinant polypeptide may result in the subject having an average PODCI score of about 40 or greater.

[0044] Prior to administration of the recombinant polypeptide, the subject may be characterized as having an average muscle strength grade of less than about 5. Administration of the recombinant polypeptide results in an average increase in the subject's muscle strength grade of about 1 or greater.

[0045] Prior to administration of the recombinant polypeptide, the subject may be characterized as having a mean handheld dynamometry (HHD) value less than about 80% of the predicted HHD value. Administration of the recombinant polypeptide may result in the subject having a mean HHD value of about 80% or greater of the predicted HHD value. The HHD value may represent the subject's grip strength, knee flexion, knee extension, hip flexion, hip extension, or hip abduction.

[0046] In another aspect, the invention features a method for determining the activity (e.g., binding activity) of a polypeptide described herein, including sALP or sALP fusion polypeptides (e.g., a polypeptide having at least one amino acid mutation relative to naturally occurring ALP, as described herein, having a sequence of any one of SEQ ID NOS: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, as described herein). The method can include, for example, providing a bone homogenate derived from bone (e.g., a femur, such as a bone from a mammal (e.g., a mouse or a human)). The bone homogenate can be obtained by harvesting bone tissue and treating the tissue, for example, with collagenase, to remove connective tissue. The bone can be purified, for example, by removing bone marrow and / or other contaminants. Once the bone is dry, it can be homogenized, for example, by grinding, crushing, and / or slicing the bone until the bone tissue is homogenized. The bone homogenate can then be resuspended in a liquid, for example, PBS, for use in subsequent assays. The polypeptides described herein can be incubated with the homogenate for, for example, at least 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, or more. The sample can then be vortexed and / or centrifuged to obtain the supernatant or the fraction of protein bound to the bone homogenate. The ratio of bound to unbound protein can then be measured and quantified to determine the binding affinity of the polypeptide to the homogenate.

[0047] definition The words "a" or "plurality" before a noun refer to one or more of that particular noun. For example, the phrase "mammalian cells" refers to "one or more mammalian cells."

[0048] The term "about" is meant to account for variations due to experimental error, and in some embodiments, "about" represents ±10% of the stated value. All measurements reported herein are understood to be modified by the term "about," whether expressly used or not, unless otherwise indicated.

[0049] The term "bone-targeting moiety" refers to a bone-targeting moiety that alone targets at least about 1×10 -5 M or better (e.g., about 10 -6 M, about 10 -7 M, about 10 -8 M, about 10 -9 By "bone matrix" is meant an amino acid sequence of at least three amino acid residues in length that has sufficient affinity for bone matrix so as to have an in vivo binding affinity (M or better).

[0050] The terms "Brief Pain Inventory-Short Form" and "BPI-SF," used interchangeably herein, refer to a method for measuring pain in patients, particularly those with HPP (e.g., patients approximately 13 years of age or older). The BPI-SF is a self-report pain measure described in Cleeland & Ryan (Ann Acad Med Singapore, 23(2), 129-138; 1994), the entire contents of which are incorporated herein by reference. The BPI-SF is a questionnaire designed to assess pain severity and its impact on daily functioning. The BPI-SF consists of 11 items that utilize a numeric rating scale to assess pain severity (4 items) and pain interference (7 items) in the 24 hours prior to administration of the questionnaire. The BPI-SF questionnaire provides information on pain intensity and the degree to which pain interferes with a patient's (e.g., HPP patients approximately 13 years of age or older) daily functioning on a numeric rating scale ranging from 0 (no pain) to 10 (severe pain or significant interference caused by pain). Lower scores indicate better quality of life and less pain. For example, the BPI-SF score for adolescents and adults with HPP is a composite of 11 pain assessments.

[0051] As used herein, the terms "Bruininks-Oseretsky Motor Skills Test, Second Edition" and "BOT-2" refer to the second edition of a standardized test of gross and fine motor skills in patients with HPP (e.g., children with HPP between about 5 and about 12 years of age, adolescents with HPP between about 13 and about 17 years of age, or adults with HPP aged about 18 years or older). See Bruininks, RH (2005). Bruininks-Oseretsky Test of Motor Proficiency, (BOT-2). Minneapolis, MN: Pearson Assessment, the entire contents of which are incorporated herein by reference. The BOT-2 is administered individually to assess the gross and fine motor skills of various patients. For example, the BOT-2 can be used to assess physical disabilities and mobility limitations in patients with HPP (e.g., children with HPP between about 5 and about 12 years of age, adolescents with HPP between about 13 and about 17 years of age, or adults with HPP aged about 18 years or older). The BOT-2 provides a composite BOT-2 score in the following exemplary domains: strength, running speed and agility, fine motor precision, fine motor integration, manual dexterity, bilateral coordination, balance, and upper limb coordination. For example, a BOT-2 strength total score can be determined by having the patient perform sit-ups, V-sits, standing broad jumps, wall jumps, air chairs, and push-ups. A running speed and agility total score can be determined by having the patient straddle a balance beam or perform a shuttle run, a two-footed side jump, or a one-footed side jump. BOT-2 total strength and BOT-2 running speed and agility total scores range from 0 to 25, with a score of approximately 10 to 25 being considered representative of a healthy subject.

[0052] As used herein, the term "catalytically competent" refers to sALP that hydrolyzes the bone mineralization inhibitor inorganic pyrophosphate (PPi) to provide inorganic phosphate (Pi), thereby reducing the extracellular concentration of PPi. Thus, catalytically competent sALP improves bone skeletal mineralization by regulating the concentration of PPi.

[0053] As used herein, the terms "Child Health Assessment Questionnaire" and "CHAQ" refer to a questionnaire used to assess the health status (e.g., ability to perform activities of daily living (ADL) and incidence of pain) of patients aged 1 to 19 years, including children, adolescents, and some adults with HPP. For a description of the CHAQ index, see Bruce & Fries (J. Rheumatol. 30(1):167-178, 2003), which is incorporated herein by reference in its entirety. The CHAQ can be administered to children over 8 years of age via interview or self-report. The CHAQ includes eight subscales: dressing / grooming, getting up, eating, walking, hygiene, reaching, gripping, and movement. The range of scores within each category is 0 to 3, where a score of 0 indicates no difficulty; a score of 1 indicates some difficulty; a score of 2 indicates great difficulty; and a score of 3 indicates the patient is unable to perform the activity. The CHAQ index can also be used to determine the presence and severity of pain.

[0054] As used herein, the terms "EuroQol 5-Dimensional Questionnaire" and "EQ-5D" refer to a questionnaire used to assess the health status (e.g., mobility, self-care, ability to perform usual activities of school, work, or household chores, ability to perform ADLs (e.g., dressing, toileting, cooking), experience of pain or discomfort, and anxiety or depression) of patients, such as children with HPP aged about 5 to about 12 years, adolescents with HPP aged about 13 to about 17 years, or adults with HPP aged about 18 years or older. For a description of the EQ-5D index, see Reenan & Oppe (EQ-5D-3L User Guide Version 5.1, 2015), which is incorporated herein by reference in its entirety. The EQ-5D can be self-administered, administered by a clinician, or administered via interview. The EQ-5D questionnaire includes five dimensions that characterize the health status of patients with HPP: mobility, self-care, ability to perform ADLs, incidence of pain or discomfort, and anxiety or depression. As described herein, the EQ-5D can be used in combination with at least one physical assessment, such as the 6MWT, to classify HPP patients as having a health status of Level I, indicating no physiological problems, Level II, indicating some physiological problems, Level III, indicating extreme physiological problems, or Level IV, indicating the most extreme physiological problems. The EQ-5D can also be used as part of an analysis to assess the transition of HPP patients from one health status to another, such as from Level IV to Level III, IV to Level II, IV to Level I, III to II, III to I, or II to I. The Child Health Utility Index-9D (CHU-9D) can also be used to assess the health status of HPP patients. For a description of the CHU-9D and EQ-5D indices, see Stevens (Appl Health Econ Health Policy. 9(3):157-69, 2011) and International Publication No. WO 2018 / 191254, both of which are incorporated herein by reference in their entireties.

[0055] The term "efficacy" refers to the E of a compound in a dose-response assay. maxmeans the value.

[0056] The term "Fc" refers to a fragment crystallizable region of an immunoglobulin, e.g., IgG1, IgG2, IgG3, or IgG4, comprising the CH2 and CH3 domains of the immunoglobulin heavy chain. Fc may also include any portion of the hinge region connecting the Fab and Fc regions. Fc may be derived from any mammal, including humans, and may be post-translationally modified (e.g., by glycosylation or sialylation). In non-limiting examples, Fc may be a fragment crystallizable region of human IgG1 having the amino acid sequence of SEQ ID NO: 259, or Fc may be a fragment crystallizable region of human IgG2 / 4 of SEQ ID NO: 253.

[0057] By "fragment" is meant a portion of a polypeptide or polynucleotide that preferably comprises at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the entire length of a reference polynucleotide or polypeptide. Fragments may be, for example, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 160 1700, 1800, 1900, 2000, 2100 or more nucleotides, up to the full length of the polynucleotide or 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 400, 500, 600, 700 or more amino acid residues, up to the full length of the polypeptide.

[0058] The terms "handheld dynamometry" and "HHD," used interchangeably herein, refer to a method for measuring grip and muscle strength in a subject, particularly a subject with HPP aged approximately 13 years or older. A dynamometer can be used to assess grip strength, knee flexion, knee extension, hip flexion, hip extension, and hip abduction in a subject with HPP aged approximately 13 years or older. For example, knee flexion and extension and hip flexion, extension, and abduction in a subject with HPP aged approximately 13 years or older can be measured using, for example, a MICROFET2™ dynamometer, and the subject's grip strength can be measured using, for example, a JAMAR® Grip Dynamometer. Specifically, an administrator holds the dynamometer stationary while the subject exerts maximum force against the dynamometer. Peak force data is collected in pounds and converted to Newtons (N). Torque values ​​are then calculated using the limb length in N meters. The torque values ​​can then be compared to those of normal subjects, e.g., of approximately the same age, sex, and / or height, and expressed as a percentage to generate the subject's HHD score.

[0059] The term "health status" as used herein refers to the characterized physiological status of a patient with HPP, such as a child with HPP aged about 5 to about 12 years, an adolescent with HPP aged about 13 to about 17 years, or an adult with HPP aged about 18 years or older. The health status of an HPP patient can be characterized by at least one physical assessment selected from one or more metrics of the 6MWT, BOT-2, BSID-III, and gait analysis, and at least one quality of life assessment selected from one or more indices of the EQ-5D, CHAQ, PODCI, CHU-9D, SF-36, SF-12, and PedsQL. In particular, the health status of an HPP patient can be characterized, for example, by the 6MWT in combination with the EQ-5D. After obtaining the results of at least one physical assessment and at least one quality of life assessment selected from the above metrics, an HPP patient can be identified as having a health status of Level I, indicating no problems with the physiological status; Level II, indicating some problems with the physiological status; Level III, indicating extreme problems with the physiological status; or Level IV, indicating the most extreme problems with the physiological status. The metrics can be used, for example, to assess the transition of an HPP patient from one health state to another health state (e.g., transition from health state IV to III, IV to II, IV to I, III to II, III to I, or II to I after administration of sALP) following treatment with sALP as described herein.

[0060] As used herein, the terms "hypophosphatasia" and "HPP" refer to a rare inherited skeletal disorder caused by one or more loss-of-function mutations in the ALPL (alkaline phosphatase, liver / bone / kidney) gene, which encodes, for example, tissue-nonspecific alkaline phosphatase (TNSALP). HPP can be further characterized as infantile HPP, childhood HPP, perinatal HPP (e.g., benign perinatal HPP or lethal perinatal HPP), dentate HPP, adolescent HPP, or adult HPP. For example, "childhood HPP" refers to patients with HPP between about 5 and about 12 years of age, "adolescent HPP" refers to patients with HPP between about 13 and about 17 years of age, and "adult HPP" refers to patients with HPP aged about 18 years or older. As used herein, the term "adult HPP" refers to a condition or phenotype characterized by the presence of one or more of the following symptoms: elevated blood and / or urinary levels of inorganic pyrophosphate (PPi), hypocalcification, hypercalciuria, one or more skeletal deformities, hypotonia, muscle weakness, rheumatoid complications, unsteady gait, difficulty walking, bone pain, pain, fractures, calcium pyrophosphate dihydrate crystal deposition, pseudogout, arthritis, pyrophosphate arthropathy, chondrocalcinosis, calcific periarthritis, and pseudofractures. The term "adolescent HPP" as used herein refers to a condition or phenotype characterized by the presence of one or more of the following symptoms: elevated blood or urinary levels of PPi, PEA, or PLP; osteomalacia, one or more skeletal deformities, hypotonia, muscle weakness, rheumatic complications, arthritis, pseudogout, unsteady gait, difficulty walking, bone pain, pain, premature tooth loss, hypocalcification, pulmonary hypoplasia, respiratory insufficiency, seizures, hypercalciuria, short stature, and growth retardation. The term "pediatric HPP" as used herein refers to a condition or phenotype characterized by the presence of one or more of the following symptoms: elevated blood or urinary levels of PPi, PEA, or PLP; rickets, rachitic ribs, one or more skeletal deformities, hypotonia, muscle weakness, rheumatic complications, arthritis, pseudogout, unsteady gait, difficulty walking, bone pain, pain, premature tooth loss, hypocalcification, delayed motor development, seizures, hypercalciuria, short stature, fractures, pseudofractures, and growth retardation.

[0061] The terms "Lower Extremity Function Scale" and "LEFS," used interchangeably herein, refer to a method for measuring lower extremity impairment in patients, particularly those with HPP (e.g., patients approximately 13 years of age or older). The LEFS is a self-report measure described in Binkley et al. (Phys Ther. 79:371-83, 1999), the entire contents of which are incorporated herein by reference. The total LEFS score ranges from 0 to 80, with higher scores indicating better lower extremity function. A change in LEFS score of approximately 9 points is considered clinically meaningful. A qualified physical therapist can administer the LEFS to HPP patients (e.g., those approximately 13 years of age or older) via an interview. A higher LEFS score indicates improved lower extremity function, including transfer activities (e.g., getting out of the bath, rolling over in bed), mobility (e.g., walking on uneven ground, running), stair climbing, and squatting. The LEFS can be used to assess functional impairment of one or both lower extremities in patients with HPP, including the ability to monitor patients over time and assess the effectiveness of asfotase alfa treatment.

[0062] The terms "nucleic acid," "nucleic acid molecule," and "polynucleotide" refer to a polymeric material, e.g., RNA or DNA, having a sequence of two or more covalently linked, naturally occurring or modified nucleotides. Nucleic acid molecules can be, for example, single-stranded or double-stranded and can contain modified or unmodified nucleotides, or mixtures or combinations thereof. Various salts, mixed salts, and free acid forms of nucleic acid molecules are also included.

[0063] As used herein, the terms "Pediatric Outcomes Data Collection Tool" and "PODCI" refer to a questionnaire used to assess the overall health status, incidence of pain, and ability to perform ADLs in patients under the age of 19, particularly those with chronic health conditions such as those with HPP. For a description of the PODCI index, see Plint et al. (J. Pediatr. Orthop. 23(6):788-790, 2003), which is incorporated herein by reference in its entirety. The questionnaire can be completed by the patient or the patient's parent / guardian who is aware of the patient's condition. The eight scales generated from the PODCI include: The PODCI consists of three scales: 1) the Upper Limb and Physical Function Scale, which measures the difficulties encountered when performing daily personal care and student activities; 2) the Transfer and Basic Mobility Scale, which measures the difficulties experienced when performing routine and athletic activities; 3) the Sports / Physical Function Scale, which measures the difficulties or limitations encountered when participating in more vigorous activities or sports; 4) the Pain / Comfort Scale, which measures the level of pain experienced in the past week; 5) the Treatment Expectations Scale, which measures long-term expectations for treatment; 6) the Well-Being Scale, which measures overall satisfaction with personal appearance and similarity to friends and peers; 7) the Symptom Satisfaction Scale, which measures the patient's acceptance of current limitations if this is a lifelong condition; and 8) the Global Function Scale, a common composite scale calculated from the first four scales above. Standardized scores are generated from the PODCI series and converted to a 0-100 scale, where 0 represents significant disability and 100 represents minimal disability.

[0064] The term "recombinant protein" is known in the art. A recombinant protein may be a glycoprotein. For example, a recombinant protein or recombinant protein variant made in CHO cells is glycosylated, with sugar moieties covalently attached to the protein, and is a glycoprotein. Briefly, the term "recombinant protein" may refer to a protein that can be produced using a cell culture system. The cells in the cell culture system may be derived from mammalian cells, including, for example, human cells, CHO cells, insect cells, yeast cells, or bacterial cells. Generally, the cells in the cell culture contain an introduced polynucleotide encoding a recombinant protein of interest (this polynucleotide may be carried on a vector, such as a plasmid vector). The polynucleotide encoding the recombinant protein may also contain a heterologous promoter operably linked to the polynucleotide encoding the protein.

[0065] As used herein, "6-minute walk test" and "6MWT" refer to a physical assessment that is a standardized test to assess the walking ability of a patient with HPP (e.g., a child with HPP between about 5 and about 12 years of age, an adolescent with HPP between about 13 and about 17 years of age, or an adult with HPP who is about 18 years of age or older). In particular, walking ability refers to the patient's ability to lift and lower each leg in turn. See the American Thoracic Society statement: guidelines for the six-minute walk test (Amer. J. of Respiratory and Critical Care Medicine, 166(1):111-7, 2002, incorporated herein by reference in its entirety). The 6MWT is determined from the distance (e.g., in meters) that a patient walks on a flat, hard surface in 6 minutes. The 6MWT distance can then be compared with the patient's 6MWT distance at baseline, the 6MWT distance of an untreated subject (e.g., an untreated subject of approximately the same age, height and / or sex), or the 6MWT distance of a healthy subject (e.g., a healthy subject of approximately the same age, height and / or sex) and expressed as a percentage to determine the 6MWT value.

[0066] "Treating," "treat," and "treatment" refer to the medical management of a patient and / or the management of a patient exhibiting or likely to have a condition, such as HPP, for the purpose of curing, ameliorating, stabilizing, reducing the likelihood of, or preventing a condition, such as HPP (e.g., pediatric, adolescent, or adult HPP), or one or more symptoms thereof, by, for example, administering a pharmaceutical composition (e.g., an sALP as described herein). The term includes active treatment, i.e., treatment specifically directed toward ameliorating or associated with a cure, and also includes causal treatment, i.e., treatment directed toward removing the cause of the associated disease, condition, disorder, or event. The term also includes palliative treatment, i.e., treatment designed to relieve or ameliorate at least one symptom of the disease, pathological condition, disorder or event rather than to cure it; symptomatic treatment, i.e., treatment directed to the systemic manifestations of the relevant disease, pathological condition, disorder or event; prophylactic treatment, i.e., treatment aimed at minimizing or partially or completely inhibiting the onset of the relevant disease, pathological condition, disorder or event, for example, in patients who are not yet ill but who are susceptible to or otherwise at risk of the particular disease, pathological condition, disorder or event; and supportive treatment, i.e., treatment employed to supplement another specific therapy directed at ameliorating the relevant disease, pathological condition, disorder or event.

[0067] The terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to any chain of two or more natural or non-natural amino acid residues that constitutes all or part of a naturally occurring or non-naturally occurring polypeptide or peptide, as described herein, regardless of post-translational modification (e.g., glycosylation, sialylation, or phosphorylation).

[0068] The terms "ALP," "sALP," "soluble alkaline phosphatase," "alkaline phosphatase," and "extracellular domain of alkaline phosphatase" are used interchangeably (unless the context indicates otherwise) and refer to a soluble, non-membrane-bound alkaline phosphatase or domain, biologically active fragment, or biologically active variant thereof. ALP includes, for example, alkaline phosphatases lacking a C-terminal GPI signal sequence and further variants and analogs thereof that retain alkaline phosphatase activity, e.g., the ability to hydrolyze PPi or other natural or artificial substrates. This includes TNSALP, PALP, GALP, and IALP domains, and biologically active fragments or biologically active variants thereof, unless otherwise specified. Mature sALP lacks the GPI membrane anchor and signal peptide, which are cleaved during processing.

[0069] The term "ALP polypeptide" refers to any sequence that includes an ALP sequence, as defined herein. Exemplary ALP polypeptides include those having the structure A-ALP-B, where A and B are each absent or are an amino acid sequence of at least one amino acid (e.g., any ALP fusion polypeptide described herein).

[0070] The terms "isolated" or "purified" mean separated from other naturally associated components. Typically, a compound (e.g., a protein, polypeptide, polynucleotide, or small molecule), factor, cell, or other component is considered isolated when it is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or even 99% free by weight from, for example, proteins, antibodies, naturally occurring organic molecules, and other components with which it is naturally associated. In some examples, the component is at least 75%, 90%, or even 99% pure by weight. Isolated components can be obtained by chemical synthesis, separation of factors from natural sources, or production of the component in a recombinant host cell that does not naturally produce the component. Proteins and small molecules can be purified by one of skill in the art using standard techniques, such as those described by Ausubel et al. (Current Protocols in Molecular Biology, John Wiley & Sons, New York, 2000). Components are preferably at least, for example, 2-, 5-, or 10-fold purer than the starting material, as measured, for example, using polyacrylamide gel electrophoresis, column chromatography, optical density, HPLC analysis, or Western analysis (Ausubel et al., 2000). Exemplary methods of purification are column chromatography, filtration, immunoprecipitation, and magnetic bead immunoaffinity purification.

[0071] The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" refers to a carrier or excipient that is physiologically acceptable to the patient being treated while retaining the therapeutic properties of the compound with which it is administered. One exemplary pharmaceutically acceptable carrier material is physiological saline. Other physiologically acceptable carriers and their formulations are known to those skilled in the art and are described, for example, in Remington (The Science and Practice of Pharmacy, 22nd Ed., Allen, Ed. 2012).

[0072] The term "pharmaceutical composition" refers to a composition comprising a polypeptide or polynucleotide described herein formulated in a pharmaceutically acceptable excipient, including those manufactured or sold with the approval of a government regulatory agency as part of a treatment regimen for the treatment or prevention of a disease or condition in a patient. Pharmaceutical compositions can be formulated, for example, for subcutaneous administration, intravenous administration (e.g., as a sterile solution in a solvent system suitable for intravenous use that is free of particulate emboli), oral administration (e.g., as a tablet, capsule, caplet, gelcap, or syrup), or any other formulation described herein, e.g., unit dosage form.

[0073] The term "subject" or "patient" means a mammal, including, but not limited to, a human or non-human mammal (such as a cow, horse, dog, sheep, or cat).

[0074] The term "mammalian cell" is known in the art and can refer to any cell from or derived from any mammal, including, for example, a human, hamster, mouse, green monkey, rat, pig, cow, hamster, or rabbit. Mammalian cells can be immortalized, differentiated, or undifferentiated.

[0075] The term "therapeutically effective amount" refers to an amount of a polypeptide or polynucleotide described herein that is sufficient to substantially treat, prevent, delay, suppress, or arrest any symptoms of a disease or condition described herein, particularly HPP. A therapeutically effective amount of a composition described herein may depend on the severity of the disorder being treated and the condition, weight, and general condition of the subject, and can be determined by one of skill in the art taking such factors into consideration. A therapeutically effective amount of a composition described herein can be administered to a subject in a single dose or in multiple doses administered over a period of time.

[0076] As used herein, when a polypeptide or nucleic acid sequence is said to have "at least X% sequence identity" to a reference sequence, it means that at least X percent of the amino acid residues or nucleotides in the polypeptide or nucleic acid are identical to those in the reference sequence when the sequences are optimally aligned. Optimal alignment of sequences can be determined by a variety of methods within the skill of the art, such as the Smith Waterman alignment algorithm (Smith et al., J. Mol. Biol. 147:195-7, 1981) and BLAST (Basic Local Alignment Search Tool; Altschul et al., J. Mol. Biol. 215:403-10, 1990). These and other alignment algorithms are described in detail in GeneMatcher Plus™ (Schwarz and Alignment can be accessed using publicly available computer software such as "BestFit" (Smith and Waterman, Advances in Applied Mathematics, 482-489, 1981), BLAST, BLAST-2, BLAST-P, BLAST-N, BLAST-X, WU-BLAST-2, ALIGN, ALIGN-2, CLUSTAL, or Megalign (DNASTAR), as incorporated in "Dayhoff, Atlas of Protein Sequence and Structure," Dayhoff, MO, Ed., pp 353-358, 1979). Furthermore, those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve optimal alignment over the length of the sequences being compared.

[0077] The words "preferred" and "preferably" refer to embodiments of the disclosed compounds, compositions, and methods that may offer certain benefits, under particular circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the present disclosure.

[0078] For any method disclosed herein that includes discrete steps, the steps can be performed in any practicable order, and any combination of two or more steps can be performed simultaneously, if desired.

[0079] The above summary is not intended to describe each disclosed embodiment or every implementation of the disclosed compounds, compositions, and methods. The following description more particularly exemplifies exemplary embodiments. In several places throughout this application, guidance may be provided through lists of examples, which examples can be used in various combinations. In each case, the recited list serves only as a representative group and should not be interpreted as an exclusive list.

[0080] All headings are for the convenience of the reader and should not be used to limit the meaning of the document that follows the heading, unless specifically stated.

[0081] The application file contains at least one drawing executed in color. Copies of this patent or patent application with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0082] [Figure 1A][Figures 1A-1D] Graphs showing fluorescent Fc fusion protein (dimeric fusion protein consisting of an N-terminal HA-binding sequence fused to human IgG1 Fc-Katushka 2s fluorescent protein) binding to hydroxyapatite (HA) or bone homogenate. Figure 1A shows the fluorescence intensity of fluorescent Fc fusion protein bound to CAPTAL® HA, normalized to a non-target Fc-Katushka 2s sample (FLU002); Figure 1B shows the fluorescence intensity of the protein suspension after 2 hours of incubation with HA, representing the total amount of unbound protein after incubation; Figure 1C (bound) and Figure 1D (unbound) show similar quantification of fluorescent Fc fusion binding to mouse C56BL / 6 femur homogenate. N = 2 ± max / min (*p < 0.05 vs. FLU002, one-way ANOVA, Dunnett's post-hoc analysis). [Figure 1B] Same as above. [Figure 1C] Same as above. [Figure 1D] Same as above. [Figure 2A-2B] 2A and 2B are graphs showing bone-targeted (ALP-Fc-D10) (ALP031; SEQ ID NO: 31) and non-targeted (ALP-Fc) (ALP086; SEQ ID NO: 222) fusion binding to mouse bone homogenate as measured via a fluorescent probe. Fluorescence intensities of bone homogenate-bound (FIG. 2A) and non-bound (FIG. 2B) protein fractions were normalized to the non-targeted negative control (ALP-Fc). N=3±SD. [Figure 3A-3B] 3A and 3B are graphs showing fluorescently labeled VHH protein binding to mouse bone homogenate. Fluorescence intensities of bone homogenate-bound (FIG. 3A) and non-bound (FIG. 3B) protein fractions were normalized to a non-targeting negative control VHH. N=3±SD. [Figure 4] A representative set of raw fluorescence images from longitudinal whole-body in vivo imaging spectrum (IVIS) live animal imaging over time (24-432 hours) of J:NU mice treated with ALP-Fc-D10 (ALP031; SEQ ID NO: 31) and ALP-Fc (ALP086; SEQ ID NO: 222). [Figure 5A][Figures 5A-5B] Graphs showing quantitative data from longitudinal whole-body IVIS live-animal imaging of mice treated with ALP-Fc-D10 (ALP031; SEQ ID NO: 31) or ALP-Fc (ALP086; SEQ ID NO: 222). Figure 5A shows the whole-body region of interest and quantified total radiant efficiency. Figure 5B shows the total and quantified radiant efficiency within the spinal region of interest, plotted longitudinally throughout the 18-day study. N=6±SD, *p<0.05, **p<0.01 Mann-Whitney unpaired t-test, two-tailed. [Figure 5B] Same as above. [Figure 6A] [Figures 6A-6B] Graphs showing ex vivo fluorescence quantification of protein accumulation in selected tissues (spine, skull, femur, liver, kidney, and spleen) after 18 days. Figure 6A shows the raw total radiant efficiency of tissue specimens 18 days after a single dose from mice treated with ALP-Fc-D10 (ALP031; SEQ ID NO: 31) and ALP-Fc (ALP086; SEQ ID NO: 222). Figure 6B shows the volume-normalized total radiant efficiency of ex vivo bone tissue from mice treated with ALP-Fc-D10 (ALP031; SEQ ID NO: 31). N=6±SD, **p<0.01 Mann-Whitney unpaired t-test, two-tailed. [Figure 6B] Same as above. [Figure 7] Graph showing representative 2D fluorescence IVIS images from a longitudinal study in mice administered a single intravenous dose of fluorescently labeled compound using non-targeting VHH (sequence number 248), VHH001 (sequence number 249) or VHH002 (sequence number 250). [Figure 8]

[0023] Figure 1 is a set of raw fluorescent images showing quantitative data from a longitudinal study of total radiant efficiency in mice administered a single intravenous dose of fluorescently labeled compounds using non-targeted VHH (SEQ ID NO: 248), VHH001 (SEQ ID NO: 249), or VHH002 (SEQ ID NO: 250) from 2D IVIS images. Radiant efficiency was obtained from the entire mouse region of interest (ROI). N=5±SD, ****p<0.0001, one-way ANOVA, Tukey's post hoc analysis. [Figure 9A][Figures 9A-9C] Graphs showing quantification of ex vivo bone tissue from mice administered fluorescently labeled compounds using non-targeting VHH (SEQ ID NO: 248), VHH001 (SEQ ID NO: 249), and VHH002 (SEQ ID NO: 250). Figure 9A shows the spine, Figure 9B shows the hind limb, and Figure 9C shows the skull, which were collected from all mice from all treatment groups 7 days (168 hours) after single-dose injection. Equivalent regions of interest (ROIs) were drawn around similar tissues, and total radiant efficiency was normalized to tissue volume. N=5±SD, ***p<0.001, one-way ANOVA, Tukey's post-hoc analysis. [Figure 9B] Same as above. [Figure 9C] Same as above. [Figure 10A] [Figures 10A-10B] Graphs showing in vitro binding and dissociation of multiple doses of bone-targeting proteins. Figure 10A shows bone homogenate binding proteins of VHH001 (SEQ ID NO: 249), VHH002 (SEQ ID NO: 250), and ALP-Fc-D10 (ALP031; SEQ ID NO: 31), and Figure 10B shows non-binding proteins of VHH001 (SEQ ID NO: 249), VHH002 (SEQ ID NO: 250), and ALP-Fc-D10 (ALP031; SEQ ID NO: 31). [Figure 10B] Same as above. [Figure 11] FIG. 1 is a graph showing MUP fluorescence intensity produced by 16 bone-tagged fusion proteins bound to bone homogenate as a function of time (ALP031 (ALP-Fc-D10), ALP086 (ALP-Fc), and ALP202-ALP216, corresponding to SEQ ID NOs: 31, 222, and 124-138, respectively). [Figure 12] 1 is a series of 2D IVIS images of mice treated with ALP-Fc-D10, ALP031 (SEQ ID NO: 31) (Example 8, N=6), showing in vivo bone integration over time. [Figure 13] FIG. 1 is a series of 2D IVIS images of mice treated with ALP-Fc fusion protein, untagged, ALP086, Example 8, N=6, showing in vivo bone integration over time. [Figure 14]Figure 1 shows the binding activity in the supernatants comparing the bound and unbound fractions for various tagged constructs (ALP230-ALP239, ALP242, ALP243, ALP247, ALP248, ALP250, ALP2521, ALP253, and ALP254). The dashed line indicates baseline bone binding as a control. Constructs with D5 or D6 bone-targeting moieties showed approximately equal bound (B) and unbound (U) components. [Figure 15A] [Figures 15A-15B] Graphs showing pK data showing plasma protein levels in mg / L / dose [(mg / L) / (mg / kg)] versus time after dose administration to healthy male C57BL / 6 mice via the iV route (Figure 15A) or subcutaneous route (Figure 15B). [Figure 15B] Same as above. [Figure 16] 1 is a graph showing Kaplan-Meier survival curves for HPP mice treated subcutaneously with daily PBS or ALP201 on daily, every other day, and weekly dosing schedules. Median survival time for HOM PBS QD animals was 21.5 days. [Figure 17] FIG. 1 is a graph showing the mean body weight as a function of time (36 day study) of HPP mice treated with either ALP201 or PBS. [Figure 18] 1 is a graph showing the mineralization index of the hind paws on day 11 of HPP mice treated with either ALP201 or PBS. [Figure 19] 1 is a graph showing exemplary progress curves for a 4-methylumbelliferyl phosphate (MUP) hydrolysis assay, in which fluorescence is plotted against time, using ALP023 at various concentrations. [Figure 20] Schematic diagram showing an exemplary VHH domain. F represents the VHH framework regions and the grey regions represent end segments that may be used for positioning or spacing of the CDRs H1, H2, H3 and bone-targeting sequences. [Figure 21] FIG. 1 is a schematic diagram showing an exemplary ALP-Fc bone-tag molecule in which individual domains are labeled. [Figure 22]FIG. 10 is a series of graphs showing saturation curves illustrating the relationship between the rate of pyrophosphate hydrolysis and increasing pyrophosphate (PPi) levels by selected constructs with reaction rates plotted against pyrophosphate concentration for ALP201, ALP259, and asphatase alpha (SEQ ID NO: 269). [Figure 23] 1 is a graph showing an exemplary progress curve for an ALP / TPLDH-coupled PLP hydrolysis assay, where fluorescence is plotted against time for ALP201 at 10 ng / ml. [Figure 24] 1 is a series of graphs showing saturation curves illustrating the relationship between increasing pyridoxyl-5' phosphate (PLP) levels and the rate of PLP hydrolysis in an ALP / TPLDH binding assay with selected constructs (ALP201, ALP259, and asphaltase alpha; 10 ng / mL, 10 ng / mL, and 12.5 ng / mL, respectively), with reaction rate plotted against PLP concentration in uM. [Figure 25] 1 is a graph showing the mineralization index of the hind paws on day 36 / 37 of HPP mice treated with either ALP201, ALP259, asfotase alfa, or PBS. [Figure 26] FIG. 1 is a graph showing tibia length at day 36 / 37 in HPP mice treated with either ALP201, asfotase alfa, or PBS. [Figure 27] FIG. 1 is a graph showing femur length at day 36 / 37 in HPP mice treated with either ALP201, asfotase alfa, or PBS. [Figure 28] FIG. 1 is a graph showing mouse bone alkaline phosphatase activity levels on day 36 / 37 in HPP mice treated with either ALP201, asphaltase alfa, or PBS. [Figure 29] 1 is a graph showing mouse bone alkaline phosphatase activity levels on day 36 / 37 in Akp2GW HPP mice treated with either ALP201 or ALP259. [Figure 30]FIG. 1 is a graph showing an exemplary progress curve of a 4-MUP hydrolysis assay performed on HPP mouse femur tissue, where fluorescence (RFU) is plotted against time (seconds). [Figure 31] FIG. 1 is a graph showing the body weights of HPP mice treated with either ALP201, asfotase alfa, or PBS on day 36 / 37. DETAILED DESCRIPTION OF THE INVENTION

[0083] Polypeptides, including soluble alkaline phosphatase, fragments thereof, and fusion proteins, and methods of using the same for treating bone mineralization disorders and symptoms thereof, such as hypophosphatasia (HPP), are described. The polypeptides include soluble alkaline phosphatase (sALP) or fragments thereof, which are derived from naturally occurring alkaline phosphatase (ALP). Alkaline phosphatase includes various isozymes that are differentially expressed in different tissues. Four major ALP isozymes include tissue-nonspecific alkaline phosphatase (TNSALP), placental alkaline phosphatase (PALP), germline alkaline phosphatase (GALP), and intestinal alkaline phosphatase (IALP). Accordingly, proteins derived from these ALP isozymes are described.

[0084] HPP is a rare genetic skeletal disease, with the most severe form occurring in 1 in 100,000 live births. The disorder is typically caused by loss-of-function mutations in the gene encoding TNSALP. HPP exhibits a marked range of symptoms and severity, from premature tooth loss to a near-complete lack of bone mineralization in utero. Symptoms of HPP vary significantly among subjects and by age. Many subjects with HPP exhibit skeletal changes, short stature, chronic pain, painful lower extremities, gait disturbances, and premature, non-traumatic tooth loss. Due to loss-of-function mutations in endogenous TNSALP, subjects with HPP require functional ALP activity of the polypeptides described herein to restore native ALP activity and provide normal bone matrix mineralization.

[0085] Site-directed mutagenesis was performed to systematically investigate the protein activity, protein stability, and pharmacokinetic properties of alkaline phosphatase polypeptides and their fusion proteins. The mutations in the alkaline phosphatase polypeptides and their fusion proteins described herein include changes to amino acid residues within and around the active site of the alkaline phosphatase enzyme, one or more consensus sites for N-linked glycosylation, a bone-targeting moiety, if included, and / or a fragment-crystallizable (Fc) region, if included. Disclosed herein are alkaline phosphatase polypeptides and their fusion proteins containing one or more of these changes, as well as data demonstrating that the changes improve one or more of the protein activity, protein stability, and pharmacokinetic properties. Alkaline phosphatase polypeptides and their fusion proteins are described in more detail herein.

[0086] soluble alkaline phosphatase Polypeptides comprising ALP are described, including naturally occurring ALP and fragments thereof, and ALP with one or more mutations that improve at least one activity or pharmacokinetic (PK) property compared to naturally occurring ALP without that at least one mutation.

[0087] The ALP can be a soluble fragment of TNSALP, PALP, GALP, or IALP, or a chimera thereof. The ALP can be derived from any suitable organism, for example, a mammalian ALP. Mammalian ALPs include, for example, human, gorilla, mouse, rabbit, chimpanzee, cynomolgus monkey, rhesus monkey, orangutan, baboon, rat, cow, goat, and llama ALPs. In certain embodiments, the ALP is a human ALP, such as human TNSALP or human IALP. The ALP can have at least 70% (e.g., 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOS: 1-6 (e.g., at least 85% sequence identity to residues 1-491 of SEQ ID NOS: 1 or residues 1-486 of SEQ ID NOS: 4). The ALP can have at least 70% (e.g., 75%, 80%, 85%, 90%, 95%, 97%, 99% or 100%) sequence identity to a region of at least 50 (e.g., at least 100, 150, 200, 250, 300, 350, 400, 450 or more) amino acids of any one of SEQ ID NOs: 1-6.

[0088] TNSALP is a membrane-bound protein anchored by a glycolipid moiety at its C-terminus (Swiss-Prot, P05186). The glycolipid anchor (GPI) is added post-translationally after removal of the hydrophobic C-terminus. This functions as both a temporary membrane anchor and a signal for GPI addition. The GPI anchor is located in the plasma membrane, while the remainder of TNSALP is located extracellularly. In particular, TNSALP (e.g., human TNSALP (hTNSALP)) can be engineered to replace the first amino acid (alanine) of the hydrophobic C-terminal sequence with a stop codon, thereby producing an engineered soluble TNSALP that contains all amino acid residues of the native anchored form of TNSALP but lacks the GPI membrane anchor. Those skilled in the art will understand that the location of the GPI membrane anchor varies among ALPs and can include, for example, the last 10, 12, 14, 16, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 34, 36, 38, 40, 45, 50 or more amino acid residues at the C-terminus of the polypeptide. Thus, the C-terminus of native ALP can be truncated by specific amino acids without affecting ALP activity.

[0089] In addition to the C-terminal GPI anchor, TNSALP also has an N-terminal signal peptide sequence. The N-terminal signal peptide is present on the protein during synthesis, but is cleaved from TNSALP after transport into the endoplasmic reticulum. An exemplary N-terminal signal peptide is MISPFLVLAIGTCLTNS (SEQ ID NO: 251).

[0090] The sALPs described herein include both secreted (i.e., lacking an N-terminal signal) and non-secreted (i.e., having an N-terminal signal) forms thereof. Those skilled in the art will understand that the location of the N-terminal signal peptide varies among alkaline phosphatases and can include, for example, the first 5, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 30, or more amino acid residues at the N-terminus of the polypeptide. Those skilled in the art can predict the location of the signal sequence cleavage site using a suitable computer algorithm, such as that described in Bendtsen et al. (J. Mol. Biol. 340(4):783-795, 2004) and available at www.cbs.dtu.dk / services / SignalP / .

[0091] The ALP may contain one or more mutations, for example, mutations that are not naturally occurring. The one or more mutations preferably enhance the therapeutic properties of alkaline phosphatase and / or the bone-targeting conjugate. For example, the one or more mutations may improve the clearance rate, activity, efficacy, and / or solubility of the bone-targeting conjugate. The mutations may be amino acid substitutions or insertions or deletions of one or more amino acids. The one or more mutations may improve the activity of at least one pharmacokinetic (PK) property compared to a naturally occurring ALP without the at least one mutation. The ALP (e.g., human TNSALP) may have at least one mutation selected from the group consisting of E108X, M384X, L385X, N213X, and N286X relative to SEQ ID NO: 1, where X is any amino acid. The one or more mutations can include, for example, E108S, E108T, E108Q, E108M, E108K, E108L, M384R, L385T, N213Q, and N286Q relative to SEQ ID NO: 1. The ALP can include two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) mutations at different amino acid positions. For example, the ALP can include E108M, N213Q, and N286Q mutations relative to SEQ ID NO: 1. The ALP (e.g., human IALP) can have a W245X mutation relative to SEQ ID NO: 4, where X is any amino acid. The ALP can have a W245R mutation relative to SEQ ID NO: 4. The ALP can have a C481X mutation relative to SEQ ID NO: 4, where X is any non-thiol-containing amino acid (e.g., C481G).

[0092] The polypeptides described herein may include ALPs containing mutations relative to naturally occurring ALPs that occur within specific regions of the ALP. The mutations may occur in the ectodomain of the ALP. For example, the mutations may occur in the crown domain, catalytic domain, or dimerization domain of the ALP. If present, the mutations may occur in the GPI anchor domain. The polypeptides may contain mutations within amino acids 1-491 or 1-486 of human TNSALP or analogous ALP positions based on alignment and / or sequence homology. For example, the polypeptides may have mutations within positions 1-491, 1-486, 25-475, 25-240, 50-400, 50-350, and / or 100-300 relative to SEQ ID NO: 1. The polypeptide can have a mutation within positions 100-125, 100-110, 200-225, 210-220, 275-300, 280-290, 425-450, and / or 425-435 relative to SEQ ID NO: 1. For example, the polypeptide can have a mutation between positions 108, 213, 286, and / or 429 relative to SEQ ID NO: 1. In certain embodiments, the mutation is within the sequence of ALP, and the mutation is not a stretch of unnatural amino acids present in the N-terminal or C-terminal domain of ALP.

[0093] The ALP can have a mutation at a consensus N-linked glycosylation site. The consensus N-linked glycosylation site comprises an asparagine-XZ motif, where X is any amino acid except P and Z is any amino acid except S or T. The asparagine can be mutated to glutamine.

[0094] The ALP can have a S429X mutation relative to SEQ ID NO:4, where X is any amino acid. For example, the ALP can have a S429Q, S429H, S429E, or S429D mutation relative to SEQ ID NO:4.

[0095] The ALP may have at least 70% (e.g., 75%, 80%, 85%, 90%, 95%, 97%, 99% or 100%) sequence identity to a region of at least 50 (e.g., 100, 150, 200, 250, 350, 400 or more) amino acids of any one of SEQ ID NOs: 7-223, 247 and 262-264, and / or may contain one or more of the mutations described above.

[0096] The mutant ALP may have at least one improved PK characteristic relative to naturally occurring ALP. For example, the mutation may provide one or more of increased catalytic activity, increased temperature stability, increased zinc binding, maintenance of activity in zinc-depleted buffers, maintenance of activity at pH 5.0 to 7.5, reduced dimerization, reduced aggregation, and / or increased producibility of ALP. Increased catalytic activity may include increased hydrolysis of pyridoxal 5-phosphate and / or pyrophosphate. Increased catalytic activity may be at least two-fold (e.g., 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, e.g., about 2-fold to about 30-fold) better than the activity of naturally occurring ALP. Improved PK characteristics may include one or more of increased substrate specificity, increased activity for natural substrates, increased activity for artificial substrates, decreased Km for natural substrates, and increased area under the curve (AUC) per dose. Artificial ALP substrates include, for example, umbelliferyl phosphate (e.g., 4-methylumbelliferyl phosphate (4-MUP)), umbelliferone phosphate, and paranitrophenyl phosphate (pNPP). Natural substrates include, for example, pyridoxal-5'-phosphate, PLP, and PEA.

[0097] The ALPs described herein may be in the form of dimers. Alternatively, the ALPs may be in the form of monomers.

[0098] Bone targeting part The polypeptides described herein may further comprise a bone-targeting moiety. The bone-targeting moiety is any sequence of amino acids that has sufficient affinity for bone (e.g., the hydroxyapatite mineral phase of bone). The mineral phase of bone contains positively charged regions. Thus, negatively charged amino acids such as glutamic acid or aspartic acid can target the polypeptide to bone. The bone-targeting moiety allows sALP to localize in bone tissue and remain bound until the activity of sALP decreases. This allows sALP to catalyze hydroxyapatite bone formation at the site of native TNSALP.

[0099] The bone-targeting moiety can be located at the N-terminus or C-terminus of ALP. The bone-targeting moiety can include polyaspartic acid (Dn), polyglutamic acid (En), poly(aspartic acid-alanine-aspartic acid) (DAD)n, poly(aspartic acid-aspartic acid-serine) (DDS)n, poly(aspartic acid-serine-serine (DSS)n, poly(glutamic acid-glutamic acid-serine) (EES)n, where n is any integer from 1 to 50 (e.g., 1 to 30, 3 to 30, 3 to 20, 5 to 16, 10 to 16, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, , 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 24, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50). In some embodiments, the bone-targeting moiety can comprise the structure Dn when n=7-10; En when n=10-15; (DAD)n when n=2-4; (DDS)n when n=2-4; (DSS)n when n=3; or (EES)n when n=3-4.

[0100] The targeting sequence can include Dn, where n is, for example, 3 to 9, 17 to 30, 10 to 16, or 3 to 30. The targeting sequence can include En, where n is, for example, 3 to 9, 17 to 30, 10 to 16, or 3 to 30. The bone-targeting moiety can include a sequence that includes both aspartic acid and glutamic acid residues.

[0101] Optionally, other amino acids, such as one or more of Thr, Gly, Gln, Asn, Lys, Ser, and / or Ala, can be included within the Dn or En bone-targeting sequence. As a non-limiting example, the targeting moiety can include (DDS)n, where n is 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0102] The bone-targeting moiety may comprise all or a portion of the bone-targeting sequence from statherin, a salivary protein known to bind to bone mineral. An example of a statherin bone-targeting sequence is DDSEEKFLRRIGRFG (SEQ ID NO: 252). The bone-targeting sequence may comprise a Dn or En sequence conjugated to the bone-targeting sequence from statherin.

[0103] Optionally, for any of the embodiments of the bone-targeting conjugates described herein, the targeting sequence may be flanked at the N-terminus, C-terminus, or both by cysteine ​​residues, which can be used to conjugate the polypeptide to another chemical moiety.

[0104] The bone-targeting moiety may comprise a VHH antibody or fragment thereof comprising a bone-targeting sequence. The bone-targeting sequence may be located within and / or at the end of a VHH antibody fragment to generate a bone-targeting VHH construct. The VHH antibody fragment comprises all or part of the variable domain of a heavy chain homodimer IgG (VHH), as found in camelids (e.g., llamas). Homodimeric antibodies lack the light chains (VL and CL) and the first heavy chain constant region (CH1) characteristic of conventional antibodies. Similar to the variable domains of conventional antibodies, the variable domains of these heavy chain homodimers (VHH) comprise CDR H1, CDR H2, and CDR H3. VHHs have three complementarity-determining regions (CDRs), sometimes referred to as H3s. CDR H3s from llama antibodies can be up to three times longer than the equivalent mouse CDR3. VHH sequences can contain multiple framework (F) regions flanking the three CDR regions. VHH sequences include naturally occurring sequences and sequences identified using phage display, such as MA10, MG6, and MG7 (Emelie D. Rodrigues, Single Domain Antibodies in Tissue Engineering, University of Twente, Netherlands, 2014). Bone-targeting VHH constructs can be prepared containing at least one bone-targeting sequence, such as Dn or En (n = 1-50). The bone-targeting sequence can be located within the CDR sequence of the VHH and / or at one or both ends of the VHH sequence (Figure 20, schematic diagram). One, two, or all three CDRs of a VHH, or portions thereof, can be replaced with a bone-targeting sequence. Portions of the VHH fragment that do not contain the CDRs, such as the framework (F) region, can function as spacer regions in the bone-targeting moiety. Optionally, bone-targeting sequences can be located within two or all three of the CDR sequences. As shown schematically in Figure 20, F represents the VHH framework region, and the grey region includes the CDRs H1, H2, H3 and end segments that can be used for positioning or spacing the bone-targeting sequences.

[0105] Fc area The polypeptides described herein may further comprise an Fc region. For example, sALP may be a fusion polypeptide containing an immunoglobulin Fc region, e.g., at the N- or C-terminus of the polypeptide. Immunoglobulin molecules have a structure well known in the art. They include two light chains (approximately 23 kD each) and two heavy chains (approximately 50-70 kD each) connected by interchain disulfide bonds. Immunoglobulins are readily proteolytically cleaved (e.g., by papain cleavage) into Fab (containing the light chain and the VH and CH1 domains of the heavy chain) and Fc (containing the CH2 and CH3 domains of the heavy chain along with adjacent sequences). Useful Fc fragments described herein include Fc fragments of any immunoglobulin molecule, including IgG, IgM, IgA, IgD, or IgE, from any mammal (e.g., human), and their various subclasses (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2). For example, the Fc fragment may be human IgG1.

[0106] The Fc region may comprise all or a portion of the Fc fragment of an IgG. The IgG may be IgG1, IgG2, IgG3, or IgG4, or a chimera of two or more IgGs, such as IgG2 / 4. An IgG2 / 4 chimera is described, for example, in International Publication No. WO 2007 / 106585, which is incorporated herein by reference. The Fc fragment may increase the half-life and circulation time of the polypeptide after administration to a subject. For example, the half-life of the polypeptide may be about 10 to about 100 hours (e.g., about 20, 30, 40, 50, 60, 70, 80, or 90 hours). The half-life may vary depending on the administration form. The Fc fragment may include, for example, the CH2 and CH3 domains of the heavy chain and any portion of the hinge region. The Fc region may be optionally glycosylated at any suitable one or more amino acid residues known to those skilled in the art. In particular, the Fc fragment of the fusion polypeptide has the amino acid sequence of SEQ ID NO: 253 or SEQ ID NO: 259, or has at least 50% (e.g., 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO: 253 or SEQ ID NO: 259. Engineered, e.g., non-naturally occurring, Fc regions can also be used (see, e.g., WO 2005 / 007809, incorporated herein by reference). The Fc fragments described herein can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50 or more additions, deletions or substitutions relative to any of the Fc fragments described herein.

[0107] Linkers and Spacers The polypeptides described herein can include one or more linkers or spacers of one or more amino acids. The polypeptides can also include one or more terminal residues located at the N-terminus or C-terminus of the polypeptide. The linker or spacer can include one or more sequences of amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, or more). The linker or spacer can be omitted from the polypeptides described herein.

[0108] The linker can comprise any suitable amino acid to promote flexibility between adjacent domains or to eliminate steric hindrance between the domains. Additionally, linkers or spacers can be used to enhance protein folding, thermostability, and / or recombinant expression of the polypeptide.

[0109] One or more additional amino acids can be positioned at the N-terminus, the C-terminus, and / or as an intervening sequence between any or all of the components of the polypeptides described herein. For example, additional amino acids can be included between the Fc region and the bone-targeting moiety. Exemplary intervening sequences include one or more glycines or serines, such as GGGGS (SEQ ID NO: 254), which can be positioned between the Fc region and the bone-targeting moiety sequence. In some embodiments, a longer intervening sequence can be used to provide additional flexibility.

[0110] In some embodiments, the linker comprises the sequence (GGGGA)2GGGGS (SEQ ID NO:255), (GGGGQ)2GGGGS (SEQ ID NO:256), (GGGPS)2GGGGS (SEQ ID NO:257), or GGGGS(PGGGS)2 (SEQ ID NO:258). In some embodiments, the linker does not comprise the sequence (GGGGA)2GGGGS (SEQ ID NO:255), (GGGGQ)2GGGGS (SEQ ID NO:256), (GGGPS)2GGGGS (SEQ ID NO:257), or GGGGS(PGGGS)2 (SEQ ID NO:258).

[0111] Polypeptide topology The polypeptides described herein may comprise one or more of sALP, a bone-targeting moiety, an Fc region, a spacer, and a linker. The components of the polypeptides described herein may comprise any suitable topology arranged from N-terminus to C-terminus that provides the appropriate function. A polypeptide comprising a bone-targeting moiety may be referred to as a bone-targeting conjugate or a bone-targeting Fc conjugate.

[0112] For example, a polypeptide comprising sALP and an Fc region can have the structure N-sALP-Fc-C or N-Fc-sALP-C. Optionally, the polypeptide can further comprise a linker or spacer of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acids between one or more of these domains.

[0113] The polypeptide may comprise a sALP and a bone-targeting moiety (BTM) and may have the structure N-sALP-BTM-C or N-BTM-sALP-C. Optionally, the polypeptide may further comprise a linker or spacer of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acids between one or more of these domains.

[0114] The polypeptide may comprise an sALP, an Fc region, and a BTM. A polypeptide comprising all three components may have the structure N-sALP-Fc-BTM-C, N-Fc-BTM-sALP-C, N-BTM-sALP-Fc-C, N-sALP-BTM-Fc-C, N-Fc-sALP-BTM-C, or N-BTM-Fc-sALP-C. Optionally, the polypeptide may further comprise one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid linkers or spacers between one or more of these domains.

[0115] The polypeptide may comprise or consist of an amino acid sequence having at least 85% (e.g., 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 7-223, 247, and 262-264. For example, the polypeptide may comprise or consist of the sequence of any one of SEQ ID NOs: 72, 123, 155, or 177. The polypeptide may comprise or consist of an amino acid sequence having at least 85% (e.g., 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 123, wherein, optionally, the polypeptide includes one or more of the following modifications: E108M, N213Q, and N286Q. The polypeptide may comprise or consist of SEQ ID NO: 123. The polypeptide may comprise or consist of an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99% or 100%) sequence identity to SEQ ID NO: 177. The polypeptide may comprise or consist of SEQ ID NO: 177. The polypeptide may comprise or consist of an amino acid sequence having at least 85% (e.g., 90%, 95%, 97%, 99% or 100%) sequence identity to SEQ ID NO: 260 or 261. The polypeptide may comprise or consist of the sequence of SEQ ID NO: 260. The polypeptide may comprise or consist of the sequence of SEQ ID NO: 261. The polypeptide may include a secretory signal peptide. The polypeptide may comprise or consist of an amino acid sequence having at least 85% (e.g., 90%, 95%, 97%, 99% or 100%) sequence identity to SEQ ID NO: 263 or 264. The polypeptide may comprise or consist of the sequence of SEQ ID NO: 263. The polypeptide may comprise or consist of the sequence of SEQ ID NO: 264. The polypeptide may comprise any sALP catalytic domain, Fc IgG isotype (e.g., Fc of IgG2 / 4), or bone-targeting moiety and their topological sequences listed in Table 1. The polypeptide may comprise any single domain listed in Table 1.

[0116] [Table 1-1]

[0117] Table 1-2

[0118] Table 1-3

[0119] Table 1-4

[0120] Table 1-5

[0121] Table 1-6

[0122] Table 1-7

[0123] Table 1-8

[0124] Table 1-9

[0125] Table 1-10

[0126] Table 1-11

[0127] [Table 1-12]

[0128] [Table 1-13]

[0129] [Table 1-14]

[0130] Controllable binding to bone tissue The bone-targeting conjugates described herein can exhibit various binding properties to bone tissue. For example, bone-targeting conjugates can exhibit differences in terms of bone (binding) and / or dissociation behavior. Bone-targeting Fc conjugates comprising polyD and polyE targeting sequences, for example, can exhibit high binding affinity to bone tissue, while certain bone-targeting Fc conjugates using statherin-derived or VHH sequences as targeting sequences can exhibit moderate binding affinity to bone tissue (see, e.g., Figures 1 and 14).

[0131] Bone-targeting conjugates may exhibit differences in dissociation behavior even when their binding affinities are similar. For example, ALP-Fc-D bound to bone tissue 10 While bone-targeted Fc conjugates such as (SEQ ID NO: 31) may not be able to easily exchange with unbound conjugates in solution, single-domain bone-targeted VHH constructs containing polyD or polyE sequences within one or more of their CDRs (e.g., SEQ ID NOs: 249 and 250) may exhibit more mobility to bind and dissociate from bone tissue in the presence of unbound conjugates (see, e.g., Figures 10A-10B).

[0132] The bone-targeting conjugates described herein can be tailored to meet specific therapeutic needs by selecting targeting sequences and other components to alter binding affinity, dissociation rate, residence time, and mobility. For example, active equilibrium binding to the target can ensure that the polypeptide does not block active sites on bone, as would be the case with the most strongly binding molecules. In particular, constructs that bind too strongly to bone dissociate more slowly. Because ALP catalytic activity depends on zinc and magnesium ions, loss of these ions can inactivate ALP over time. If inactive ALP constructs remain bound to bone, they prevent the recruitment of active ALP. Conversely, rapid dissociation of active ALP from bone reduces efficacy by preventing ALP from maximizing its enzymatic activity. Therefore, a balance of binding is desirable. Selection can be made from the described options for targeting sequences (e.g., polyD, polyE, phage display derived sequences, full or partial statherin sequences, full or partial VHH sequences, or combinations thereof) and different topological options (e.g., bone-targeting moieties at the N- or C-terminus of the conjugate) to ensure favorable binding kinetics to establish bone-binding residence times that enhance efficacy and reduce prolonged binding of inactive enzyme. The bone bonding residence time may range from about 1 second to about 1 month (e.g., 2 seconds, 3 seconds, 4 seconds, 5 seconds, 6 seconds, 7 seconds, 8 seconds, 9 seconds, 10 seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds, 1 minute, 2 minutes, 2 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month).

[0133] Methods for testing sALP constructs The polypeptides described herein (e.g., any one of SEQ ID NOs: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or a polypeptide having at least one amino acid mutation relative to naturally occurring ALP, as described herein) can be tested using one or more assays described in more detail below to test catalytic activity, bone-targeting ability, and PK profile.

[0134] Hydroxyapatite binding assay Synthetic HA can be used to probe HA binding by the polypeptides described herein. In separate centrifuge tubes, bone-targeting polypeptides can be diluted with, for example, bovine serum albumin (BSA) in phosphate-buffered saline (PBS) pH 7.4 (1 mL of suspension per tube). 1 mg of synthetic HA can be added to each polypeptide-containing tube, and the tubes can be incubated for 2 hours at room temperature with orbital mixing to prevent HA from settling. After incubation, the samples can be centrifuged at 16,000 rcf for 5 minutes to separate the solid HA-bound fraction from the unbound protein suspension. The HA-bound fraction can then be washed, for example, three times with PBS, and the final HA fraction can be suspended in 100 μL of PBS. The suspended HA-bound fraction and 100 μL of the unbound protein suspension can be transferred to a 96-well black plate (one fraction per well), and relative protein concentrations can be measured by fluorescence excitation / emission at 488 nm / 585 nm using a fluorescence plate reader (Spectramax i3x).

[0135] Bone homogenate assay Femurs from male C57BL / 6 mice can be stored at -80°C before use. The femurs can be transferred to 2 mL centrifuge tubes (two femurs per tube) containing 1 mL 0.2% (w / w) collagenase type 2 (Worthington) in PBS containing 1x EDTA-free serine and cysteine ​​protease inhibitor cocktail (COMPLETE™, Roche). The femurs can be vortexed briefly and incubated at 37°C for 1 hour with shaking (800 rpm). Remaining connective tissue can be removed, and the femurs can be placed in a Petri dish on ice. The bone marrow can be flushed out with PBS using a needle and syringe. The dried bones are weighed (typically 30-50 mg per femur) and placed in a pre-chilled individual disposable beater (PRECELLYS®, Bertin Instruments) with 0.75 mL PBS containing 1x protease inhibitors. Femurs can be homogenized using a high-throughput bead mill homogenizer (Bullet Blender, 4°C, maximum speed, 4 cycles of 30 seconds). The homogenate can be transferred to a 1.5 mL centrifuge tube and centrifuged at 12,000 x g for 15 minutes at 4°C to separate the bone homogenate from released protein / cellular debris. Other methods of isolating bone homogenate can be used, including slicing and other grinding mechanisms. The bone homogenate can then be resuspended in 0.1% BSA in PBS for use in binding assays.

[0136] Bone-targeting polypeptides can be diluted to 50 nM in PBS containing 0.1% BSA and incubated with 3 mg of bone homogenate in individual 1.5 mL centrifuge tubes (1 mL per tube). After mixing the samples for 2 hours at room temperature, the bone homogenate-bound and unbound fractions can be separated by centrifugation. The bone homogenate fraction can be washed three times with PBS, and the final homogenate pellet can be suspended in 100 μL of PBS. The suspended bone homogenate and 100 μL of unbound protein suspension can be quantified for relative fluorescent Fc fusion concentration using a fluorescent plate reader.

[0137] Determination of relative protein affinity to bone homogenate in vitro A multiple-dose assay can be developed to rank the relative affinity of bone-binding proteins. For proteins that efficiently bind to bone homogenate, the relative dissociation rates were determined by this kinetic protein-to-bone exchange assay. Proteins can be evaluated individually (e.g., one protein type per tube) by incubating a saturating concentration (1 μM) of unlabeled bone-binding protein with 5 mg of bone homogenate in a 1.5 mL Eppendorf tube. After 24 hours of incubation with the unlabeled protein, the bone homogenate can be centrifuged (16,000 rcf, 5 minutes) to remove excess unbound protein. Bone homogenate saturated with a given bone-binding protein can be resuspended in a 0.5 μM solution of the same bone-binding protein labeled with an ALEXA FLUOR® fluorescent probe. After 1, 2, 4, 8, and 24 hours of incubation, the bone homogenate can be centrifuged, washed three times with PBS, and transferred to a 96-well black plate. The supernatant from the initial centrifugation can also be collected to quantify the amount of (unbound) fluorescent protein remaining in suspension. A fluorescent plate reader can be used to quantify the amount of bound and unbound fluorescently labeled protein at each time point, allowing for a kinetic representation of the dissociation rate of the unlabeled protein.

[0138] Metabolic assay of ALP activity (MUP) Protein samples (supernatant, partially purified, or column-purified samples) can be assayed for ALP activity in solution using 4-methylumbelliferyl phosphate (4-MUP) as an artificial substrate. Hydrolysis of the phosphoester bond of 4-MUP releases the fluorescent compound 4-methylumbelliferone, which can be easily detected with a fluorometer. Quantitation of the product can be performed using a 4-methylumbelliferone (4-MU) standard curve measured on the same plate at standard concentrations of 0, 1.25 μM, 2.5 μM, 5 μM, 10 μM, and 20 μM. A 10 mM stock solution of 4-MU can be prepared in ethanol and diluted with assay buffer [50 mM HEPES pH 7.4, 150 mM NaCl, 1 mM MgCl2, 1 mg / mL bovine serum albumin]. Purified fusion protein samples can be prepared as 0.1 mg / mL solutions in assay buffer and serially diluted to the appropriate final concentration for the assay (e.g., 1 nM) in assay buffer. A 4-MUP stock solution was prepared in assay buffer. Prior to starting the assay, 4-MUP was added to the protein sample at a final concentration of 10 μM, and all solutions were brought to 37°C. 4-MU production was measured at an excitation wavelength of 360 nm and an emission wavelength of 465 nm. Data was collected every 40 seconds for a total of 20 minutes on a plate reader maintained at 37°C. Reaction rates were calculated by linear regression in units of activity, where 1 U = 1 μmole of 4-MUP hydrolyzed / min. Specific activity was calculated in units / mg of protein assayed.

[0139] Plate bone assay Bone homogenate fractions can be assayed for ALP activity. The bone homogenate fractions can be suspended in 100 μL of PBS and transferred to a 96-well black plate. 100 μL of unbound protein suspension can also be transferred to separate wells of the 96-well plate. To each well, 100 μL of ALP detection solution (10 μM 4-MUP, 1% BSA) can be added, and a dynamic fluorescence reading (360 / 465) can be initiated immediately and run for 20 minutes, collecting fluorescence intensity readings every 30 seconds. The slope of the fluorescence intensity versus time can represent the ALP concentration in each sample fraction.

[0140] To maintain linearity, the MUP activity of the bound and unbound fractions of the tested constructs can be measured by the initial activity slope over the first 5 minutes. The ratio of MUP activity can be determined for each fusion protein as ratio = (bound activity) / (unbound activity).

[0141] Protein activity in serum assays Serum samples can be diluted 100- to 6,000-fold in assay buffer (50 mM HEPES, 150 mM NaCl, 1 mM MgCl2, pH 7.4, and 1 mg / mL BSA) to measure pK. Diluted samples can be quantified, and a standard curve can be constructed based on known activity and concentration of asphaltase alfa. The slope of the fluorescence intensity versus time can represent the rate of 4-MU production, corresponding to ALP activity, in each sample fraction as a function of units / mL serum.

[0142] In vivo fluorescence imaging in mice Semiquantitative biodistribution studies can be performed in nude mice using ALEXA FLUOR® 750-labeled bone-targeted proteins and protein fragments (e.g., VHHs). Bone-targeted ALP-Fc fusion proteins and bone-targeted VHHs can be fluorescently labeled with ALEXA FLUOR® 750 using the Invitrogen SAIVI kit (covalent coupling via activated succinimidyl ester) and purified with gel exclusion resin to remove unconjugated fluorophore. Purified proteins (suspended in PBS) can be injected into nude mice via the tail vein at a dose of approximately 3 mg / kg.

[0143] Female J:NU outbred mice (Jackson Laboratories, Bar Harbor, ME) were administered 3 mg / kg of test article via tail vein injection in a normalized 100 μL volume. For in vivo image acquisition, subjects were placed on an imaging platform (IVIS Spectrum Imaging System) Animals can be maintained under 2-3% isoflurane anesthesia using a 3D microscope (PerkinElmer Inc., Waltham, MA). An autoexposure setting with a field of view (FOV) of C, F / Stop 2, medium binning, and an 800 nm emission / 750 nm excitation filter was used for both 2D epi-illumination and 3D trans-illumination acquisition. 2D epi-illumination fluorescence imaging of ex vivo tissue specimens can be acquired under identical conditions, except that all specimens of each tissue type can be acquired simultaneously in a single image. All animal experiments are conducted in accordance with the provisions of the Animal Welfare Act and the principles of the Guide for the Care and Use of Laboratory Animals.

[0144] Fluorescence image analysis can be performed using the manufacturer's 2D / 3D software (Living Image 4.5.1, Perkin Elmer). Regions of interest (ROIs) of uniform regions for each set of subjects were manually positioned to account for differences in subject position. The longitudinal in vivo image color scale range can be normalized across all subjects and time points. The color scale for ex vivo specimens can be individually determined to best represent the fluorescent signal of each tissue set.

[0145] Pyrophosphate hydrolysis assay The polypeptides described herein can be assayed for activity toward the natural substrate, pyrophosphate. Pyrophosphate hydrolysis can be measured by detecting the product phosphate anion using PiBlue assay reagent (BioAssay Systems), which turns bright green upon phosphate binding. Quantification of phosphate levels in each well can be performed using a standard curve of phosphate solutions made in assay buffer, which can be read on the assay plate. A stock solution of sodium pyrophosphate decahydrate (Sigma Chemicals) can be prepared at a concentration of 10 mM in pure water. Purified fusion protein samples were prepared as 0.1 mg / mL solutions in assay buffer [50 mM HEPES pH 7.4, 150 mM NaCl, 1 mM MgCl2, 1 mg / mL bovine serum albumin] and serially diluted to the appropriate final concentration for assay in assay buffer. Pyrophosphate samples for assay can be prepared by diluting the stock solution with assay buffer. All solutions were brought to 37°C before starting the reaction. The protein solution was added to a clear 96-well plate, which was then placed in a Jitterbug plate shaker maintained at 37°C. The reaction was initiated by adding pyrophosphate solution to the protein solution. Typically, pyrophosphate hydrolysis reactions were performed simultaneously in the same plate at pyrophosphate concentrations of 0 μM, 1.56 μM, 3.12 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM, 200 μM, and 400 μM. Eight reaction wells were set up for each pyrophosphate concentration, and the reaction was stopped by adding PiBlue reagent (added in a volume equal to the final reaction volume) 0, 1, 2, 3, 4, 5, 6, and 7 minutes after pyrophosphate was added to the plate. The addition of PiBlue reagent lowered the pH of the detection reagent, inactivating the enzyme and thereby halting further reaction. Color was allowed to develop in the plate for 30 minutes before reading the absorbance at 620 nm. The reaction rate at each pyrophosphate concentration can be calculated by constructing a progress curve from the individual time points. Using a Michaelis-Menten enzyme kinetic fit, the reaction rate at each concentration can be used to calculate Km and Vmax values ​​in GraphPad Prism.The kcat value was calculated as follows: Vmax / (molar protein analyzed)=kcat.

[0146] Pyridoxyl-5'-phosphate hydrolysis assay. The second natural substrate of alkaline phosphatase is pyridoxyl-5'-phosphate (PLP). Polypeptides can be assayed for activity against PLP in a binding assay format, in which pyridoxyl, the product of PLP hydrolysis, is converted to fluorescent pyridoxolactone by M. loti tetrametric pyridoxyl dehydrogenase (tPLDH, SEQ ID NO: 246). The gene for 6xHis-tagged tPLDH can be synthesized using standard methods and cloned into a bacterial expression plasmid under the control of a T7 promoter. 6xHis-tagged tPLDH was expressed in BL21(DE3) cells using standard protocols and purified by standard affinity chromatography. Protein can be concentrated to a concentration of 1900 μM by centrifugation, i.e., using an Amicon Ultra15 spin concentrator, and frozen at -80°C until use in the assay. Purified fusion protein samples can be prepared as 0.1 mg / mL solutions in assay buffer [50 mM HEPES pH 7.4, 150 mM NaCl, 1 mM MgCl2, 1 mg / mL bovine serum albumin]. The final serially diluted samples can be placed in a black 96-well plate along with pyridoxolactone standards (made in assay buffer). Protein samples can be enriched for NAD +tPLDH and PLP solutions (made in assay buffer) can be added to give final concentrations of 3 mM NAD+, 4 μM tPLDH, and 3 μM PLP and mixed. Prior to initiating the reaction, all solutions can be brought to 37°C, and the reaction plate can be incubated at 37°C, with fluorescence detected by excitation at 355 nm and emission measured at 445 nm. The amount of pyridoxolactone product produced can be calculated using a standard curve generated from the fluorescence measured from the pyridoxolactone wells of the plate. The reaction rate can be calculated by linear regression of the progress curve in μmoles of pyridoxyl produced / min. Specific activity can be calculated by dividing the reaction rate by the protein concentration used in the assay reaction.

[0147] Pharmacokinetic analysis in mouse models Male C57BL / 6 mice (Jackson Labs) aged 11–12 weeks can be administered a single 4–7 mg / kg injection of 1 mg / mL sample protein in sterile PBS (without calcium or magnesium) via the tail vein or subcutaneously and followed for 14–21 days. Two interim and one terminal blood draw (cardiac puncture, CO2 anesthesia) can be performed on each mouse, staggered within the cohort (four mice in each of four groups, per molecule, per dose type). Blood samples (100 μL, generating 50 μL of plasma after centrifugation) can be collected into Li / heparinized tubes at 0.25, 1, 6, 24, 48, 72, 96, 120, 192, 264, 336, and 480 hours. Blood samples can be stored at 4°C until processed into plasma. Plasma samples can be snap frozen in liquid nitrogen and stored at -80°C for further quantification.

[0148] In vivo mouse HPP model Efficacy can be measured in an in vivo mouse HPP model. In this preventative study, treatment can begin at birth and continue for 35 days. The polypeptide or vehicle (PBS) was administered to mice containing Akp2GW. - / -Mice can be administered subcutaneously in the scapular region. Efficacy endpoints that can be assessed at the end of treatment include survival, bone mineralization impairment, and growth (body weight). Body weight can also be assessed daily as an indicator of the animals' general well-being. Age- and litter-matched PBS-treated WT mice can be used as a reference control. Animals can receive daily subcutaneous injections of the polypeptide until day 24. On day 25, the daily subcutaneous dose of the polypeptide can be reduced by half-log of the initial dose and maintained until the final treatment dose on day 35.

[0149] Pharmaceutical Compositions, Formulations and Administration The polypeptides described herein, including sALP or sALP fusion polypeptides (e.g., a polypeptide having a sequence of any one of SEQ ID NOS: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein), can be formulated as pharmaceutical compositions by various methods known in the art. As will be appreciated by those skilled in the art, the route and / or mode of administration can be used to define the formulation. The route of administration can vary depending on various factors, such as the environment and therapeutic goals. In particular, the polypeptides and fusion polypeptides described herein can be formulated for administration by any route known in the art, for example, subcutaneous (e.g., via subcutaneous injection), intravenous, oral, intranasal, intramuscular, sublingual, intrathecal, or intradermal. By way of example, the pharmaceutical composition can be in the form of a liquid, solution, suspension, pill, capsule, tablet, gelcap, powder, gel, ointment, cream, spray, mist, vapor spray, aerosol, or phytosome.

[0150] formulation Compositions comprising sALP and sALP fusion polypeptides (e.g., as described herein, a polypeptide having any one of the sequences of SEQ ID NOS: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or at least one amino acid mutation relative to naturally occurring ALP) can be formulated according to standard methods. Pharmaceutical formulation is a well-established technique, and is described, for example, in Remington: The Science and Practice of Pharmacy, 22nd Ed., Allen, Ed. 2012; Ansel et al. (1999) "Pharmaceutical Dosage Forms and Drug Delivery Systems,”7 th Edition, Lippincott Williams & Wilkins Publishers (ISBN:0683305727); and Kibbe (2000) “Handbook of Pharmaceutical Excipients American Pharmaceutical Association,” 3 rd This is further described in the International Standards and Regulation Agency (ISBN: 091733096X). For example, an sALP composition (e.g., a polypeptide having any one of SEQ ID NOS: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein) can be formulated, for example, at an appropriate concentration in a buffer suitable for storage at 2-8°C (e.g., 4°C). The composition can also be formulated for storage at temperatures below 0°C (e.g., -20°C or -80°C). The composition can further be formulated for storage at 2-8°C for up to two years (e.g., 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 1.5 years, or 2 years). Thus, the compositions described herein can be stable upon storage at 2-8°C (e.g., 4°C) for at least one year. The composition can be formulated in an appropriate volume, for example, from about 0.1 mL to about 10 mL.

[0151] Compositions comprising sALP and sALP fusion polypeptides (e.g., polypeptides having any one of the sequences of SEQ ID NOS: 7-223, 247, and 262-264, or variants thereof having at least 85% sequence identity thereto, and / or at least one amino acid mutation relative to naturally occurring ALP, as described herein) can be in a variety of forms. These forms include, for example, liquid, semi-solid, and solid dosage forms such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. The preferred form will depend, in part, on the intended mode of administration and therapeutic application.

[0152] For example, compositions intended for systemic or local delivery may be in the form of an injectable or infusible solution. Thus, compositions (e.g., a polypeptide having any one of SEQ ID NOs: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or at least one amino acid mutation relative to naturally occurring ALP, as described herein) can be formulated for parenteral administration (e.g., subcutaneous, intravenous, intraperitoneal, or intramuscular injection). "Parenteral administration," "parenterally administered," and other grammatically equivalent phrases used herein refer to modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, subcutaneous, intradermal, intravenous, intranasal, intraocular, pulmonary, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intrapulmonary, intraperitoneal, transtracheal, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intracerebral, intracranial, intracarotid, and intrasternal injection and infusion. Specific routes of administration include intravenous and subcutaneous administration.

[0153] Compositions containing sALP and sALP fusion polypeptides (e.g., as described herein, any one of SEQ ID NOS: 7-223, 247, and 262-264, or variants thereof having at least 85% sequence identity thereto, and / or polypeptides having at least one amino acid mutation relative to naturally occurring ALP) can be formulated as solutions, microemulsions, dispersions, liposomes, or other ordered structures suitable for stable storage at high concentrations. Sterile injectable solutions can be prepared by incorporating the compositions described herein in the required amount in an appropriate solvent with one or a combination of ingredients listed above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the compositions described herein into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation include vacuum drying and freeze-drying, which yield a powder of the compositions described herein plus any additional desired ingredients (see below) from a previously sterile-filtered solution. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin.

[0154] The compositions described herein can also be formulated into immunoliposome compositions. Such formulations can be prepared by methods known in the art, such as those described in Epstein et al. (1985) Proc Natl Acad Sci USA 82:3688; Hwang et al. (1980) Proc Natl Acad Sci USA 77:4030; and U.S. Patent Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed, for example, in U.S. Patent No. 5,013,556.

[0155] Compositions comprising sALP and sALP fusion polypeptides (e.g., polypeptides having any one of the sequences of SEQ ID NOS: 7-223, 247, and 262-264, or variants thereof having at least 85% sequence identity thereto, and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein) can also be formulated with carriers that protect the composition (e.g., sALP polypeptide or sALP fusion polypeptide) from rapid release, such as controlled-release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, can be used. Many methods for preparing such formulations are known in the art. See, for example, JR Robinson (1978) "Sustained and Controlled Release Drug Delivery Systems," Marcel Dekker, Inc., New York.

[0156] Compositions containing sALP or sALP fusion polypeptides (e.g., as described herein, any one of SEQ ID NOS: 7-223, 247, and 262-264, or variants thereof having at least 85% sequence identity thereto, and / or polypeptides having at least one amino acid mutation relative to naturally occurring ALP) can be formulated as clear, colorless to slightly yellow aqueous solutions with a pH of 7.4 for injection. sALP or sALP polypeptides (e.g., as described herein, any one of SEQ ID NOS: 7-223, 247, and 262-264, or variants thereof having at least 85% sequence identity thereto, and / or polypeptides having at least one amino acid mutation relative to naturally occurring ALP) can be formulated at concentrations of 12 mg / 0.3 mL, 18 mg / 0.45 mL, 28 mg / 0.7 mL, 40 mg / 1 mL, or 80 mg / 0.8 mL. sALP or sALP polypeptides (e.g., formulated at concentrations of 12 mg / 0.3 mL, 18 mg / 0.45 mL, 28 mg / 0.7 mL, 40 mg / 1 mL, or 80 mg / 0.8 mL) may be formulated with a carrier such as sodium chloride and / or sodium phosphate (e.g., about 150 mM NaCl and / or about 25 mM sodium phosphate, particularly at a pH of about 7.4). In particular, the composition can be formulated as a 40 mg / ml solution for injection, where each ml of solution contains 40 mg of sALP or sALP polypeptide (e.g., each vial contains 0.3 ml of solution and 12 mg of sALP (40 mg / ml), or each vial contains 0.45 ml of solution and 18 mg of sALP (40 mg / ml), or each vial contains 0.7 ml of solution and 28 mg of sALP (40 mg / ml), or each vial contains 1.0 ml of solution and 40 mg of sALP or sALP polypeptide (40 mg / ml)).sALP or sALP polypeptide (e.g., as described herein, any one of SEQ ID NOS: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or a polypeptide having at least one amino acid mutation relative to naturally occurring sALP) can be formulated as an injectable solution at a concentration of 100 mg / ml, where each 1 ml of solution contains 100 mg of sALP or sALP polypeptide (e.g., each vial contains 0.8 ml of solution and 80 mg of asphaltase alfa (100 mg / ml)). sALP can be formulated so that from about 0.1 mg to about 500 mg is present in a volume of from about 0.01 mL to about 10 mL.

[0157] When the composition is used in combination with a second active agent, the composition can be formulated with the second agent, or the composition can be formulated separately from the second agent formulation. For example, the respective pharmaceutical compositions can be mixed, e.g., immediately prior to administration, and administered together, or can be administered separately, e.g., at the same time or at different times.

[0158] Compositions comprising sALP and sALP fusion polypeptides (e.g., as described herein, a polypeptide having the sequence of any one of SEQ ID NOs: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or at least one amino acid mutation relative to naturally occurring ALP) can be formulated for administration to a subject or, if administered to a fetus, to a woman pregnant with such a fetus in conjunction with intravenous gamma globulin therapy (IVIG), plasmapheresis, plasma replacement, or plasma exchange.

[0159] Carrier / Vehicle Preparations containing sALP or sALP fusion polypeptides (e.g., as described herein, any one of SEQ ID NOS: 7-223, 247, and 262-264, or variants thereof having at least 85% sequence identity thereto, and / or polypeptides having at least one amino acid mutation relative to naturally occurring ALP) can be combined with a pharmaceutically acceptable sterile aqueous or non-aqueous solvent, suspension, or emulsion and provided to subjects with or susceptible to developing bone mineralization disorders such as HPP. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil, fish oil, and injectable organic esters. Aqueous carriers include water, water-alcohol solutions, emulsions or suspensions containing saline, and buffered medical parenteral vehicles including sodium chloride solution, Ringer's dextrose solution, dextrose and sodium chloride solution, Ringer's solution containing lactose, or fixed oils. For example, the pharmaceutically acceptable carrier can include sodium chloride and / or sodium phosphate, the composition comprising, for example, about 150 mM sodium chloride and / or about 25 mM sodium phosphate, pH 7.4.

[0160] Intravenous vehicles may include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Pharmaceutically acceptable salts may be included therein, for example, mineral acid salts such as hydrochloride, hydrobromide, phosphate, sulfate, and the like; and salts of organic acids such as acetate, propionate, malonate, benzoate, and the like. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. A thorough discussion of pharmaceutically acceptable carriers can be found in Remington: The Science and Practice of Pharmacy, 22nd Ed., Allen, Ed. 2012.

[0161] Dosage The sALP polypeptide described herein (e.g., as described herein, any one of the sequences of SEQ ID NOs: 7 to 223, 247, and 262 to 264, or a variant thereof having at least 85% sequence identity thereto, and / or a polypeptide having at least one amino acid mutation relative to naturally occurring ALP) may be administered to a subject having or prone to developing a bone mineralization disorder such as HPP at a dose of, for example, 0.01 mg / kg to 500 mg / kg (e.g., 0.05 mg / kg to 500 mg / kg, 0.1 mg / kg to 60 mg / kg, 0.1 mg / kg to 50 mg / kg, 0.1 mg / kg to 20 mg / kg, 5 mg / kg to 500 mg / kg, 0.1 mg / kg to 100 mg / kg, 10 mg / kg to 100 mg / kg, 0.1 mg / kg to 50mg / kg, 0.5mg / kg~25mg / kg, 1.0mg / kg~10mg / kg, 1.5mg / kg~5mg / kg or 2.0mg / kg~3.0mg / kg) or 1μg / kg~1,000μg / kg (e.g., 5μg / kg~1,000μg / kg, 1μg / kg~750μg / kg, 5μg / kg~750μg / kg, 10μg / kg~750μg / k g, 1 μg / kg to 500 μg / kg, 5 μg / kg to 500 μg / kg, 10 μg / kg to 500 μg / kg, 1 μg / kg to 100 μg / kg, 5 μg / kg to 100 μg / kg, 10 μg / kg to 100 μg / kg, 1 μg / kg to 50 μg / kg, 5 μg / kg to 50 μg / kg, or 10 μg / kg to 50 μg / kg).

[0162] Exemplary doses of sALP include, for example, 0.01, 0.05, 0.1, 0.5, 1, 2, 2.5, 5, 10, 20, 25, 50, 100, 125, 150, 200, 250, 500, 750, 900, or 1,000 μg / kg. For all doses or ranges described herein, the term "about" can be used to modify these doses by ±10% of the stated value or range endpoint. In particular, a composition according to the present disclosure (e.g., a polypeptide having any one of SEQ ID NOs: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein) can be administered to a subject at a dose ranging from about 0.001 mg / kg / day to about 500 mg / kg / day, from about 0.01 mg / kg / day to about 100 mg / kg / day, or from about 0.01 mg / kg / day to about 20 mg / kg / day. For example, an sALP composition (e.g., as described herein, any one of SEQ ID NOs: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or a polypeptide having at least one amino acid mutation relative to naturally occurring ALP) can be administered to a subject at a weekly dose ranging from, for example, about 0.5 mg / kg / week to about 140 mg / kg / week, e.g., about 0.8 mg / kg / week to about 50 mg / kg / week, or about 1 mg / kg / week to about 10 mg / kg / week (e.g., about 6 or about 9 mg / kg / week). In particular, sALP can be administered once or more weekly (e.g., 1, 2, 3, 4, 5, 6, 7 or more times per week), once or more every other week, or once or more monthly (e.g., once every 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days).

[0163] In particular, sALP (e.g., a polypeptide having at least one amino acid mutation relative to a naturally occurring ALP, such as any one of SEQ ID NOS: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, as described herein) can be administered at a dose of 2 mg / kg three times a week (total dose of 6 mg / kg / week), 1 mg / kg six times a week (total dose of 6 mg / kg / week), 3 mg / kg three times a week (total dose of 9 mg / kg / week), 0.5 mg / kg three times a week (total dose of 1.5 mg / kg / week), or 9.3 mg / kg three times a week (total dose of 28 mg / kg / week). The dosage can be adapted by the clinician according to conventional factors, such as the extent of disease and parameters different from those for subjects with or prone to develop bone mineralization disorders such as HPP. Alternatively, 0.1 mg / kg to 9 mg / kg can be administered once a week.

[0164] Doses of compositions comprising sALP and sALP fusion polypeptides (e.g., as described herein, any one of SEQ ID NOS: 7-223, 247, and 262-264, or variants thereof having at least 85% sequence identity thereto, and / or polypeptides having at least one amino acid mutation relative to naturally occurring ALP) can be provided in either single or multiple dosing regimens. Doses can be administered, for example, hourly, every other hour, daily, every other day, twice a week, three times a week, four times a week, five times a week, six times a week, weekly, biweekly, monthly, bimonthly, or yearly. Alternatively, doses can be administered, for example, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve times daily, weekly, weekly, or monthly. In particular, the dosing regimen is weekly. The duration of the dosing regimen can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days, weeks, or months, or the remaining lifespan of a subject having or susceptible to developing a bone mineralization disorder such as HPP. The dosage, frequency, and duration of administration will be adapted by the clinician according to conventional factors, such as the extent of the disease and parameters that differ from those of subjects having or susceptible to developing a bone mineralization disorder such as HPP.

[0165] For example, the dosage of sALP or sALP fusion polypeptide (e.g., any one of SEQ ID NOS: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or a polypeptide having at least one amino acid mutation relative to naturally occurring ALP, as described herein) can be about 0.1 mg / kg body weight to about 10 mg / kg body weight, administered subcutaneously or intravenously one or more times per week (e.g., 2, 3, 4, 5, 6, or 7 times).

[0166] Production of nucleic acids and polypeptides Polynucleotides encoding sALP and sALP fusion polypeptides (e.g., as described herein, any one of SEQ ID NOS: 7-223, 247, and 262-264, or variants thereof having at least 85% sequence identity thereto, and / or polypeptides having at least one amino acid mutation relative to naturally occurring ALP) can be produced by any method known in the art. The polynucleotides can encode an amino acid sequence having at least 85% (e.g., 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOS: 7-223, 247, and 262-264 (e.g., any one of SEQ ID NOS: 72, 123, 155, or 177). The polynucleotides can have at least 85% (e.g., 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOS: 265-268. The polynucleotides can comprise or consist of any one of SEQ ID NOS: 265-268. The polynucleotide may comprise or consist of SEQ ID NO: 265. The polynucleotide may comprise or consist of SEQ ID NO: 266. The polynucleotide may comprise or consist of SEQ ID NO: 267. The polynucleotide may comprise or consist of SEQ ID NO: 268.

[0167] Typically, a polynucleotide encoding the desired fusion polypeptide is generated using molecular cloning techniques and generally placed into a vector, such as a plasmid or virus. The vector is used to transform the polynucleotide into a host cell suitable for expression of the fusion polypeptide. Representative methods are disclosed, for example, in Maniatis et al. (Cold Springs Harbor Laboratory, 1989). While many cell types can be used as suitable host cells, mammalian cells are preferred because they can impart appropriate post-translational modifications (e.g., glycosylation or sialylation). Host cells of the present disclosure can include, for example, Chinese hamster ovary (CHO) cells, L cells, C127 cells, 3T3 cells, BHK cells, COS-7 cells, or any other suitable host cell known in the art. For example, the host cell is a Chinese hamster ovary (CHO) cell (e.g., CHO-DG44 cell) or a HEK293 cell.

[0168] sALP and sALP fusion polypeptides (e.g., a polypeptide having at least one amino acid mutation relative to a naturally occurring sALP, as described herein, or a sequence of any one of SEQ ID NOS: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, as described herein) can be produced under any conditions suitable for resulting in expression of the sALP polypeptide in a host cell. Such conditions may include buffers, bicarbonate and / or HEPES, ions such as chloride, phosphate, calcium, sodium, potassium, magnesium, and iron, monosaccharides, amino acids, potentially lipids, nucleotides, vitamins, and carbon sources such as insulin-like growth factors; conventional commercially available media such as alphaMEM, DMEM, Ham's-F12, and IMDM supplemented with 2-4 mM L-glutamine and 5% fetal bovine serum; HYCLONE™ SFM4CHO, Sigma-Aldrich CHO, or DHFR supplemented with 2-4 mM L-glutamine; -Suitable media include a suitable selection of conventional commercially available animal protein-free media such as Cambrex POWER™ CHO CD, etc. These media are preferably formulated without thymidine, hypoxanthine, and L-glycine to maintain selective pressure and allow stable expression of the protein product.

[0169] Large-scale methods for producing bulk proteins are described, for example, in WO 2017 / 031114 and WO 2017 / 214130, the disclosures of which are incorporated herein by reference in their entireties.

[0170] Treatment method Provided herein are methods for treating or ameliorating at least one symptom of a subject with a bone mineralization disorder, such as HPP. Other diseases or disorders, such as bone fractures, osteoporosis, sclerosteosis, chondrocalcinosis, hypotonia, Duchenne muscular dystrophy, tracheobronchomalacia, stroke, neurofibromatosis 1 (NF-1), and craniosynostosis, can also be treated with the compositions and methods described herein. The subject may have muscle weakness. The subject may have a muscle weakness disorder, such as calcium pyrophosphate deposition disorder (CPPD) or familial hypophosphatemia. Such treatment may include administering alkaline phosphatase or a polypeptide with alkaline phosphatase activity to reduce elevated PPi levels in such subjects. For example, soluble alkaline phosphatase (sALP, e.g., a polypeptide having any one of the sequences of SEQ ID NOs: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or at least one amino acid mutation relative to naturally occurring ALP, as described herein, can be administered to a neonate, child, adolescent, or adult.

[0171] A subject may be diagnosed with a bone mineralization disorder (e.g., HPP) prior to administration of alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, e.g., a polypeptide having the sequence of any one of SEQ ID NOS: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein). Furthermore, a subject having or susceptible to developing a bone mineralization disorder such as HPP may be a naive subject who has not previously been treated with sALP (e.g., a polypeptide having the sequence of any one of SEQ ID NOS: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein).

[0172] The method includes administering alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, e.g., a sequence of any one of SEQ ID NOS: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or a polypeptide having at least one amino acid mutation relative to a naturally occurring ALP, as described herein) in single or multiple doses over a period of time to a subject having or susceptible to developing a bone mineralization disorder, such as HPP. In particular, sALP, such as a polypeptide having the sequence of any one of SEQ ID NOS: 7-223, 247, and 262-264, can be administered to subjects previously determined to have elevated inorganic pyrophosphate (PPi) levels or at least one predetermined biomarker / score for HPP symptoms (e.g., muscle weakness) (such as an average BOT-2 strength score of less than 10, an average BOT-2 running speed and agility score of less than 5, an average CHAQ index score of greater than about 0.8, and / or an average PODCI score of less than about 40, and an average 6MWT of less than about 80% of the predicted 6MWT value, a muscle strength grade of less than 5, and / or an average HHD value (e.g., average HHD muscle strength or grip strength value) of less than about 80% of the predicted HHD value). For example, sALP can be administered to a subject previously determined to have a concentration of PPi in a sample (e.g., a plasma sample) greater than about 5.71 μM for an infant or child (e.g., a subject under about 12 years of age), greater than about 4.78 μM for an adolescent (e.g., a subject between about 13 and about 18 years of age), or greater than about 5.82 μM for an adult (e.g., a subject over about 18 years of age). In other embodiments, a bone mineralization disorder, such as HPP described herein, is caused by an elevated concentration of at least one alkaline phosphatase substrate (e.g., PPi, PLP, PEA, etc.).Alternatively, alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, e.g., a sequence of any one of SEQ ID NOS: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or a polypeptide having at least one amino acid mutation relative to a naturally occurring ALP, as described herein) can be administered to a subject with or susceptible to developing a bone mineralization disorder, such as HPP, prior to determining a muscle weakness score (e.g., using a BOT-2 strength score, a BOT-2 running speed and agility score, a CHAQ index score, a BSID-III scale score, a PDMS-2 standard score, a muscle strength score, a 6MWT score, and / or an HHD score). Treatment with ALP according to the methods described herein can promote, for example, increased ADL activity, decreased pain, and / or improved motor development.

[0173] Furthermore, each of the described scores (e.g., BOT-2 strength score, BOT-2 running speed and agility score, CHAQ index score, BSID-III scale score, PDMS-2 standard score, 6MWT, 12-POMA-G, modified Performance-Oriented Mobility Assessment (mPOMA-G as set forth in Phillips et al. 2015 Bone Abstracts 4:136), or HHD score) of a subject having or susceptible to developing a bone mineralization disorder, such as HPP, as described herein, can be used alone or in any combination to evaluate the effect of treatment with sALP (e.g., a sequence of any one of SEQ ID NOS: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or a polypeptide having at least one amino acid mutation relative to naturally occurring ALP, as described herein), wherein an improvement compared to the particular test score indicates that sALP is effective in treating a bone mineralization disorder, such as HPP.

[0174] For example, if administration of alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, e.g., a sequence of any one of SEQ ID NOs: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or a polypeptide having at least one amino acid mutation relative to a naturally occurring ALP, as described herein) to a subject having or prone to developing a bone mineralization disorder, such as HPP, results in an average increase in the BOT-2 intensity score of about 10 or greater (the subject's previous average BOT-2 intensity score was less than about 10), then alkaline phosphatase or a polypeptide having alkaline phosphatase activity treatment is effective in treating a physical disorder associated with a bone mineralization disorder, such as HPP. Alternatively, if administration of sALP does not result in a mean increase in the BOT-2 intensity score to about 10 or greater, the dosage and / or frequency of administration of alkaline phosphatase or a polypeptide having alkaline phosphatase activity can be modified to determine an effective amount of alkaline phosphatase or a polypeptide having alkaline phosphatase activity for the subject. For example, the dosage of sALP (e.g., a sequence of any one of SEQ ID NOS: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or a polypeptide having at least one amino acid mutation relative to naturally occurring ALP, as described herein) can be increased, for example, from about 0.5-3 mg / kg / week to about 3-6 mg / kg / week or from about 3-6 mg / kg / week to about 6-9 mg / kg / week.

[0175] Alternatively, if administration of alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, e.g., a sequence of any one of SEQ ID NOs: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or a polypeptide having at least one amino acid mutation relative to a naturally occurring ALP, as described herein) to a subject having or prone to developing a bone mineralization disorder, such as HPP, results in an improvement in one or more of the subject's muscle strength grade classifications (e.g., an improvement from a previous lower strength grade to a strength grade of 1, 2, 3, 4, or 5) (the subject's previous average muscle strength grade was less than about 5), then alkaline phosphatase or a polypeptide having alkaline phosphatase activity treatment is effective in treating a physical disorder associated with a bone mineralization disorder, such as HPP. Alternatively, if administration of sALP does not result in an improvement in one or more subject muscle strength grade classifications from a previous lower muscle strength grade, the dosage and / or frequency of administration of alkaline phosphatase or a polypeptide having alkaline phosphatase activity can be altered (e.g., increased) to determine an effective amount of alkaline phosphatase or a polypeptide having alkaline phosphatase activity for the subject. For example, the dosage of sALP (e.g., a polypeptide having any one of the sequences of SEQ ID NOS: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or a polypeptide having at least one amino acid mutation relative to naturally occurring ALP, as described herein) can be increased, for example, from about 0.5-3 mg / kg / week to about 3-6 mg / kg / week or from about 3-6 mg / kg / week to about 6-9 mg / kg / week.

[0176] Biomarkers / Endpoints for Diagnosis and / or Treatment Bone mineralization (e.g., perinatal HPP, infantile HPP, childhood HPP, and HPP, including odontohypophosphatasia, HPP-like disorders, CPPD, and familial hypophosphatemia, as described herein) can be treated with alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, e.g., a polypeptide having the sequence of any one of SEQ ID NOS: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or at least one amino acid mutation relative to naturally occurring ALP, as described herein). The methods described herein are also useful for diagnosing subjects with or susceptible to developing bone mineralization disorders such as HPP, identifying subjects with or susceptible to developing bone mineralization disorders such as HPP, or testing the efficacy of treatments for bone mineralization disorders such as HPP. For example, a subject can be diagnosed as having or susceptible to developing a bone mineralization disorder such as HPP if the subject is characterized as having a particular characteristic biomarker or metric score. A subject can be treated with alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, e.g., a polypeptide having a sequence of any one of SEQ ID NOS: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or at least one amino acid mutation relative to a naturally occurring ALP, as described herein), and the efficacy or effect of the treatment can be analyzed using a characteristic biomarker or metric score. Such biomarkers can include, for example, an increase in inorganic pyrophosphate (PPi) concentration and / or a decrease in alkaline phosphatase (ALP) in serum, bone or muscle tissue, or the subject's urine. Exemplary metrics useful in the methods described herein for determining the efficacy of muscle weakness treatment include: (1) the Bruininks-Oseretsky Test of Motor Proficiency, Second Edition (BOT-2); (2) the Childhood Health Assessment Questionnaire (CHA); (3) Pediatric Outcomes Data Collection Instrument (PODCI), (4) Bayley Scales of Infant and Toddler Development, Third Edition (BSID-III), (5) Peabody Developmental Motor Scales, Second Edition (PDMS-2), (6) 6-Minute Walk Test (6MWT), (7) muscle strength grade, and (8) handheld dynamometry (HHD), which are described in more detail below.

[0177] Bone healing and mineralization Subjects with or susceptible to developing a bone mineralization disorder, such as HPP, can be identified for treatment with alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, e.g., a polypeptide having any one of the sequences of SEQ ID NOS: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or at least one amino acid mutation relative to naturally occurring ALP, as described herein) based on the level of bone mineralization. For example, bone mineralization can be used as a metric for diagnosing a patient as having a bone mineralization disorder, such as HPP, or for testing the effectiveness of the polypeptides described herein. In subjects with a bone mineralization disorder (e.g., HPP), administration of sALP results in increased bone healing in the subject following successful treatment.

[0178] Decreased bone healing results in bone loss, decreased mineralization, and separation of two or more bones. The decrease in bone healing and mineralization can be compared to a reference bone. Methods for identifying decreased bone healing and mineralization are routine and include non-invasive techniques such as radiography and computed tomography (CT). Typically, images of the relevant area of ​​the subject can be taken before and at one or more time points after sALP treatment, and the images can be compared to assess treatment efficacy. Decreased bone healing and / or mineralization can be identified as a decrease in opacity. Images can be taken at any time during sALP treatment, or if a decrease in timing or efficacy is suspected, for example, 1, 2, 3, 4, 5, 6 days, weeks, months, or years after initiation of sALP ERT treatment. Decreased bone healing and / or mineralization in a subject can be detectable at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks or months after initiation of the sALP treatment period. The reduction in bone healing and / or mineralization in a subject can optionally persist for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks, months, or years after the start of the sALP ERT treatment period. The increase in bone healing and mineralization can be used to confirm that the effectiveness of sALP has been restored and that the bone mineralization disorder (such as HPP) is being effectively treated after treatment.

[0179] bone mineral density (BMD) Subjects with or susceptible to developing a bone mineralization disorder, such as HPP, can be identified for treatment with alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, e.g., a sequence of any one of SEQ ID NOS: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or a polypeptide having at least one amino acid mutation relative to naturally occurring ALP, as described herein) based on BMD level. A decrease in BMD (e.g., compared to a normal subject) can be used as a metric for diagnosing a patient as having a bone mineralization disorder, such as HPP.

[0180] The reduction in BMD can be used to monitor the effectiveness of sALP (e.g., a polypeptide of any one of SEQ ID NOS: 7-223, 247, and 262-264) during ERT. Methods for measuring BMD are known in the art and include, for example, bone biopsy, dual-energy X-ray absorptiometry (DXA or DEXA), peripheral quantitative CT (pQCT), high-resolution pQCT (HR-pQCT), and quantitative ultrasound (QUS). Measurements can be performed by any conventional method, including CT Hounsfield measurements, and results can be compared to normative databases or control subjects. BMD may be reported as a Z-score or T-score. Pre-treatment BMD values ​​can be measured at any time during sALP ERT treatment, and can be taken 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days, weeks, months, or years after initiation of sALP ERT treatment. The baseline BMD value after treatment may decrease by, for example, 0.01%, 0.05%, 0.1%, 0.5%, or 1%. The decrease in baseline BMD value after initiation of sALP treatment may also be unchanged or undetectable. The increase in BMD may be used to confirm that the effectiveness of sALP has been restored and that a bone mineralization disorder (such as HPP) is being effectively treated after treatment.

[0181] Plasma inorganic pyrophosphate (PPi) and alkaline phosphatase (ALP) concentrations Subjects with or susceptible to developing a bone mineralization disorder, such as HPP, can be identified for treatment with alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, e.g., a polypeptide having the sequence of any one of SEQ ID NOS: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or at least one amino acid mutation relative to naturally occurring ALP, as described herein) by measuring inorganic pyrophosphate (PPi) and / or alkaline phosphatase (ALP) concentrations in a sample, such as a plasma or urine sample, from the subject. As described in detail in Whyte et al., 1995 (J. Clin. Invest. 95(4):1440-1445), which is incorporated herein by reference in its entirety, any method known to those skilled in the art can be used to quantify PPi and / or ALP concentrations in a plasma sample or, alternatively, a urine sample. Methods for quantifying PPi concentrations in plasma or urine samples are also described in Cheung et al., 1977 (Anal. Biochem. 83:61-63), Cook et al., 1978 (Anal. Biochem. 91:557-565), and Johnson et al., 1968 (Anal. Biochem. 26:137-145), each of which is incorporated herein by reference in its entirety.

[0182] In particular, alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, e.g., a sequence of any one of SEQ ID NOS: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or a polypeptide having at least one amino acid mutation relative to a naturally occurring ALP, as described herein) can be administered to a subject having or susceptible to developing a bone mineralization disorder, such as HPP, who has previously been determined to have a plasma PPi concentration of up to about 6 μM (e.g., about 4.5 μM, about 5 μM, or about 5.5 μM, or a plasma PPi concentration in the range of about 4.5 μM to about 6 μM). For example, alkaline phosphatase or a polypeptide having alkaline phosphatase activity is administered to, for example, an infant or child (e.g., a subject under about 12 years of age) having a plasma PPi concentration of about 5.71 μM or greater; an adolescent (e.g., a subject between about 13 and about 18 years of age) having a plasma PPi concentration of about 4.78 μM or greater; or an adult (e.g., a subject over about 18 years of age) having a plasma PPi concentration of about 5.82 μM or greater. Furthermore, alkaline phosphatase or a polypeptide having alkaline phosphatase activity can be administered to a subject (e.g., a human) having or susceptible to developing a bone mineralization disorder, such as HPP, whose plasma ALP concentration has previously been determined to be, for example, about 90 U / L or less in subjects aged 0 to 14 days; about 134 U / L or less in subjects aged 15 days to less than 1 year; about 156 U / L or less in subjects aged about 1 year to less than 10 years; about 141 U / L or less in subjects aged about 10 years to less than 13 years; about 62 U / L or less in female subjects aged about 13 years to less than 15 years; about 127 U / L or less in male subjects aged about 13 years to less than 15 years; about 54 U / L or less in female subjects aged about 15 years to less than 17 years; about 89 U / L or less in male subjects aged about 15 years to less than 17 years; about 48 U / L or less in female subjects aged about 17 years or older; or about 59 U / L or less in male subjects aged about 17 years or older.

[0183] The plasma PPi and / or ALP concentrations of a subject (e.g., a human) having or susceptible to developing a bone mineralization disorder such as HPP can be compared to the plasma PPi and / or ALP concentrations of a normal subject to determine the efficacy of treatment of the subject administered alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, e.g., a polypeptide having a sequence of any one of SEQ ID NOS: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or a polypeptide having at least one amino acid mutation relative to naturally occurring ALP, as described herein. In particular, the alkaline phosphatase or polypeptide having alkaline phosphatase activity can be administered for a treatment period of at least 1 year (e.g., at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 6 years, at least 7 years, at least 8 years, at least 9 years, at least 10 years, or more than 10 years, such as for the lifetime of the subject). Alternatively, the method may include determining the plasma PPi concentration and / or plasma ALP concentration before administering alkaline phosphatase or a polypeptide having alkaline phosphatase activity to assess the effect of treatment with alkaline phosphatase or a polypeptide having alkaline phosphatase activity in the subject.

[0184] The method results in a decrease in PPi and / or an increase in ALP concentration in a sample (e.g., a plasma sample) from a subject (e.g., a human subject) having or susceptible to developing a bone mineralization disorder such as HPP. For example, treatment with alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, e.g., a sequence of any one of SEQ ID NOS: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or a polypeptide having at least one amino acid mutation relative to naturally occurring ALP, as described herein) results in a decrease in PPi concentration in a sample (e.g., a plasma sample) from the subject of about 1 μM, about 1.5 μM, about 2 μM, about 2.5 μM, or about 3 μM, or 25% or more (e.g., 30%, 35%, 40%, 45%, 50%, 55%, 60%, or more than 60%). Thus, after administration of alkaline phosphatase or a polypeptide having alkaline phosphatase activity, the subject exhibits a plasma PPi concentration of, for example, about 2 μM to about 5 μM, about 3 μM to about 5 μM, about 2 μM to about 4 μM, or about 2 μM to about 3 μM.

[0185] Similarly, treatment with alkaline phosphatase or a polypeptide having alkaline phosphatase activity results in a 30%, 35%, 40%, 45%, 50%, 55%, 60% or greater increase in ALP concentration in a sample (e.g., a plasma sample) from a subject (e.g., a human) having or susceptible to developing a bone mineralization disorder such as HPP, compared to the subject prior to administration of the alkaline phosphatase or polypeptide having alkaline phosphatase activity. For example, administration of alkaline phosphatase or a polypeptide having alkaline phosphatase activity increases the ALP concentration in a sample (e.g., a plasma sample) from a subject to, for example, about 273 U / L or more in a subject aged 0 to 14 days; about 518 U / L or more in a subject aged 15 days to less than 1 year; about 369 U / L or more in a subject aged about 1 year to less than 10 years; about 460 U / L or more in a subject aged about 10 years to less than 13 years; about 280 U / L or more in a female subject aged about 13 years to less than 15 years; about 517 U / L or more in a male subject aged about 13 years to less than 15 years; about 128 U / L or more in a female subject aged about 15 years to less than 17 years; about 365 U / L or more in a male subject aged about 15 years to less than 17 years; about 95 U / L or more in a female subject aged about 17 years or older; or about 164 U / L or more in a male subject aged about 17 years or older.

[0186] The reduction in plasma PPi and / or increase in ALP concentration in a subject (e.g., a human) with or susceptible to developing a bone mineralization disorder such as HPP can be sustained through administration of alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, e.g., a polypeptide having any one of the sequences of SEQ ID NOS: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or a polypeptide having at least one amino acid mutation relative to naturally occurring ALP, as described herein). For example, the plasma PPi concentration can be reduced by about 25%, remaining ±10% of the plasma PPi concentration reduced during treatment with sALP, and / or the plasma ALP concentration can be increased by about 50%, remaining ±10% of the plasma ALP concentration increased during treatment with alkaline phosphatase or a polypeptide having alkaline phosphatase activity.

[0187] Alternatively, if administration of alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, e.g., a sequence of any one of SEQ ID NOS: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or a polypeptide having at least one amino acid mutation relative to naturally occurring ALP, as described herein) does not result in an average decrease of about 25% or more in PPi concentrations in plasma samples from subjects (e.g., humans) having or susceptible to developing a bone mineralization disorder such as HPP, the dosage and / or frequency of sALP administration can be modified to determine an effective amount of sALP for the subject. Similarly, if administration of alkaline phosphatase or a polypeptide having alkaline phosphatase activity does not result in an increase of about 50% or more in ALP concentrations in plasma samples from the subject, the dosage and / or frequency of alkaline phosphatase or a polypeptide having alkaline phosphatase activity can be modified to determine an effective amount of alkaline phosphatase or a polypeptide having alkaline phosphatase activity for the subject. For example, the dosage of alkaline phosphatase or a polypeptide having alkaline phosphatase activity can be increased from, for example, about 0.5 mg / kg / week or about 3.5 mg / kg / week to about 3 to 6 mg / kg / week or about 6 to 9 mg / kg / week.

[0188] Bruininks-Oseretsky Test of Motor Proficiency 2nd Edition (BOT-2) An exemplary Bruininks-Oseretsky Test of Motor Proficiency, Second Edition (BOT-2) is described in Bruininks, RH (2005). Bruininks-Oseretsky Test of Motor Proficiency, (BOT-2), Minneapolis, MN: Pearson Assessment, which is incorporated herein by reference in its entirety. In particular, the BOT-2 can be used to assess physical disability and mobility limitations in subjects with or susceptible to developing bone mineralization disorders (e.g., HPP) to generate a BOT-2 score for the subject.

[0189] The BOT-2 includes a series of tests for assessing a subject's physical disability, which can be performed, for example, using a kit containing the tests. The BOT-2 provides a composite BOT-2 score in the following areas: strength, running speed and agility, fine motor precision, fine motor integration, manual dexterity, bilateral coordination, balance, and upper limb coordination. For example, a subject with or at risk for developing a bone mineralization disorder such as HPP can perform sit-ups, V-sits, standing long jumps, air chairs, and / or push-ups to determine a BOT-2 strength score. A subject with or at risk for developing a bone mineralization disorder such as HPP can perform balance beam jumps, shuttle runs, two-footed side jumps, and / or one-footed side jumps to determine a BOT-2 running speed and agility score. A subject with or at risk for developing a bone mineralization disorder such as HPP can perform cut-outs and / or connect-the-dots to determine a BOT-2 fine motor precision score. Subjects with or at risk for bone mineralization disorders such as HPP can copy stars and / or copy squares to determine their BOT-2 fine motor integration score. Subjects with or at risk for bone mineralization disorders such as HPP can perform coin flipping, card sorting, and / or block threading to determine their manual dexterity score. Subjects with or at risk for bone mineralization disorders such as HPP can perform foot and finger tapping and / or hand jumping exercises to determine their BOT-2 bilateral coordination score. Subjects with or at risk for bone mineralization disorders such as HPP can step forward on a balance beam and / or stand on one foot to determine their BOT-2 balance score. Subjects with or at risk for bone mineralization disorders such as HPP can throw a ball at a target and / or catch a thrown ball to determine their BOT-2 upper extremity coordination score.

[0190] A subject with or at risk for developing a bone mineralization disorder, such as HPP, can perform tests in one or more of the described domains (strength, running speed and agility, fine motor accuracy, fine motor integration, manual dexterity, bilateral coordination, balance, and upper limb coordination) to generate a BOT-2 score indicative of the subject's physical impairment. Within each BOT-2 domain (strength, running speed and agility, fine motor accuracy, fine motor integration, manual dexterity, bilateral coordination, balance, and upper limb coordination), such a subject can perform one or more tests to determine the subject's BOT-2 score; for example, a subject can perform one or more of sit-ups, V-sits, standing broad jumps, wall jumps, air chairs, and push-ups to determine a BOT-2 strength score. Thus, only one test (e.g., one test selected from the group of sit-ups, V-sits, standing long jump, wall hang, air chair, and push-ups) can be performed to determine the BOT-2 score (e.g., BOT-2 strength score) of a subject having or susceptible to developing a bone mineralization disorder such as HPP (e.g., an HPP-like disease).

[0191] Each BOT-2 score (e.g., strength, running speed and agility, fine motor accuracy, fine motor integration, manual dexterity, bilateral coordination, balance, and upper limb coordination) of a subject with or prone to developing a bone mineralization disorder such as HPP can be compared with the BOT-2 scores of subjects without a bone mineralization disorder such as HPP to, for example, determine a baseline comparison of BOT-2 scores. Each BOT-2 score (e.g., strength, running speed and agility, fine motor accuracy, fine motor integration, manual dexterity, bilateral coordination, balance, and upper limb coordination) of a subject with or prone to developing a bone mineralization disorder such as HPP can be compared with the BOT-2 scores of other subjects with or prone to developing a bone mineralization disorder such as HPP to, for example, provide the subject's relative BOT-2 score.

[0192] BOT-2 scores (e.g., strength, running speed and agility, fine motor accuracy, fine motor integration, manual dexterity, bilateral coordination, balance, and upper limb coordination scores) range from about 0 to about 25 or less, with a score of about 10-20 being considered representative of a healthy subject (e.g., a subject without a bone mineralization disorder such as HPP). Subjects with an average BOT-2 score (e.g., strength, running speed and agility, fine motor accuracy, fine motor integration, manual dexterity, bilateral coordination, balance, and upper limb coordination scores) of less than about 10 can be treated with alkaline phosphatase or a polypeptide having alkaline phosphatase activity, e.g., sALP, e.g., a sequence of any one of SEQ ID NOS: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or a polypeptide having at least one amino acid mutation relative to naturally occurring ALP, as described herein.

[0193] For example, a subject having or susceptible to developing a bone mineralization disorder, such as HPP, who has a BOT-2 intensity score of less than 10 (e.g., about 0, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10) can be treated with sALP (e.g., a polypeptide having the sequence of any one of SEQ ID NOs: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein) for a period up to the subject's lifetime. Similarly, a subject having or susceptible to developing a bone mineralization disorder, such as HPP, who has a BOT-2 running speed and agility score of less than 10 (e.g., about 0, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10) can be treated with sALP (e.g., a polypeptide having the sequence of any one of SEQ ID NOS: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein) for a period up to the subject's lifetime.

[0194] The method can result in an improvement in BOT-2 scores (e.g., strength, running speed and agility, fine motor precision, fine motor integration, manual dexterity, bilateral coordination, balance, and / or upper limb coordination scores) in subjects with or susceptible to developing a bone mineralization disorder, such as HPP. For example, treatment with a polypeptide having alkaline phosphatase activity, such as alkaline phosphatase or sALP (e.g., a polypeptide having any one of the sequences of SEQ ID NOS: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or a polypeptide having at least one amino acid mutation relative to naturally occurring ALP, as described herein), such as treatment with sALP for a period of time, can result in an average increase in BOT-2 strength scores of about 10 to about 20 (e.g., about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20). Furthermore, treatment with sALP (e.g., a polypeptide having a sequence of any one of SEQ ID NOS: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or at least one amino acid mutation relative to naturally occurring ALP, as described herein) can result in an average increase in BOT-2 running speed and agility scores of about 5 to about 20 (e.g., about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20).

[0195] An increase in BOT-2 scores (e.g., strength, running speed and agility, fine motor precision, fine motor integration, manual dexterity, bilateral coordination, balance, and / or upper limb coordination scores) can be sustained, e.g., through administration of alkaline phosphatase or a polypeptide having alkaline phosphatase activity, such as sALP (e.g., a sequence of any one of SEQ ID NOS: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or a polypeptide having at least one amino acid mutation relative to naturally occurring ALP, as described herein), for a period of time. Similarly, a decrease in muscle disability following administration of alkaline phosphatase or a polypeptide having alkaline phosphatase activity can be sustained through administration of alkaline phosphatase or a polypeptide having alkaline phosphatase activity.

[0196] The BOT-2 scores (strength, running speed and agility, fine motor precision, fine motor integration, manual dexterity, bilateral coordination, balance, and upper limb coordination scores) of subjects with or at risk for developing a bone mineralization disorder such as HPP can be used alone or in combination with other metrics to assess the efficacy of treatment with alkaline phosphatase or a polypeptide having alkaline phosphatase activity, such as sALP (e.g., a sequence of any one of SEQ ID NOS: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or a polypeptide having at least one amino acid mutation relative to naturally occurring ALP, as described herein), where an improvement compared to the specified test score indicates that the alkaline phosphatase or a polypeptide having alkaline phosphatase activity is effective in treating a muscle disorder associated with a bone mineralization disorder such as HPP. For example, if administration of a sALP as described herein to a subject having or susceptible to developing a bone mineralization disorder, such as HPP, results in a mean increase in BOT-2 running speed and agility score of about 5 or greater (the subject's previous mean BOT-2 running speed and agility score was less than about 5), the sALP is considered to be effective in treating a disability associated with a bone mineralization disorder, such as, for example, HPP.

[0197] Additionally, within each BOT-2 domain (strength, running speed and agility, fine motor precision, fine motor integration, manual dexterity, bilateral coordination, balance and upper limb coordination), a subject with or susceptible to developing a bone mineralization disorder such as HPP may perform one or more tests to determine the subject's BOT-2 score.

[0198] Alternatively, if administration of an alkaline phosphatase or a polypeptide having alkaline phosphatase activity described herein, such as sALP, does not result in a mean increase in BOT-2 running speed and agility scores of greater than about 5, the dosage and / or frequency of administration can be modified to determine an effective amount of alkaline phosphatase or a polypeptide having alkaline phosphatase activity for a subject with or susceptible to developing a bone mineralization disorder, such as HPP. For example, the dosage of sALP (e.g., a polypeptide having a sequence of any one of SEQ ID NOS: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or a polypeptide having at least one amino acid mutation relative to naturally occurring ALP, as described herein) can be increased, for example, from about 0.5-3 mg / kg / week to about 3-6 mg / kg / week or from about 3-6 mg / kg / week to about 6-9 mg / kg / week.

[0199] Childhood Health Assessment Questionnaire (CHAQ) The Childhood Health Assessment Questionnaire (CHAQ), as described by Bruce & Fries (J. Rheumatol. 30(1):167-178, 2003) and Klepper (Arthritis & Rheumatism, 49:S5-S14, 2003), the entire contents of which are incorporated herein by reference, can be administered to assess the health status of children with bone mineralization disorders such as HPP and generate a CHAQ index score for the child. The CHAQ includes questions in eight categories: dressing / grooming, getting up, eating, walking, hygiene, reaching, gripping, and movement. Parents or guardians record the degree of difficulty their child with a bone mineralization disorder such as HPP has performing each activity. Scores within each category range from 0 to 3, with a score of 0 indicating no difficulty; a score of 1 indicating some difficulty; a score of 2 indicating great difficulty; and a score of 3 indicating the child is unable to perform the activity.

[0200] Children who have or are susceptible to developing a bone mineralization disorder, such as HPP, who have a mean CHAQ index score (e.g., indicating impairment in activities of daily living (ADL) and / or pain) of greater than about 0.8 (e.g., about 0.8, about 1, about 1.2, about 1.4, about 1.6, about 1.8, about 2.0, about 2.2, about 2.4, about 2.6, about 2.8, or about 3.0) can be treated by administering a polypeptide having alkaline phosphatase activity, such as alkaline phosphatase or sALP (e.g., a sequence of any one of SEQ ID NOs: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or a polypeptide having at least one amino acid mutation relative to naturally occurring ALP, as described herein). For example, children with an average CHAQ index score greater than about 0.8 can be treated over a period of time, up to the subject's lifetime, by administering alkaline phosphatase or a polypeptide having alkaline phosphatase activity, such as sALP (e.g., a sequence of any one of SEQ ID NOS: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or a polypeptide having at least one amino acid mutation relative to naturally occurring ALP, as described herein). Additionally, children with or susceptible to developing bone mineralization disorders, such as HPP, as disclosed herein, can be asked one or more questions in one or more of eight categories (dressing / grooming, getting up, eating, walking, hygiene, reaching, gripping, and movement) to arrive at an average CHAQ index score, and if the average CHAQ index score is greater than about 0.8, the child can be treated by administering alkaline phosphatase or a polypeptide having alkaline phosphatase activity, such as sALP.

[0201] The CHAQ index scores of children who have or are prone to developing a bone mineralization disorder, such as HPP, as disclosed herein can be compared with the CHAQ index scores of children who do not have such a bone mineralization disorder, such as HPP, to determine, for example, the standard deviation of the CHAQ index scores. Furthermore, the CHAQ index scores of children who have or are prone to developing a bone mineralization disorder, such as HPP, as disclosed herein can be compared with the CHAQ index scores of other children who have or are prone to developing a bone mineralization disorder, such as HPP, as disclosed herein, to determine, for example, the standard deviation of the CHAQ index scores.

[0202] The methods can result in an improvement in CHAQ index scores (e.g., indicating ADL impairment and / or pain) in children with or susceptible to developing a bone mineralization disorder, such as HPP, as disclosed herein. For example, treatment with sALP (e.g., a polypeptide having any one of SEQ ID NOS: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein, for a period of time up to the child's lifespan, can result in a mean reduction in CHAQ index scores to about 0 to about 0.5 or less (e.g., about 0, about 0.1, about 0.2, about 0.4, or about 0.5) in children with HPP-like disease.

[0203] A reduction in CHAQ index score in a child with or susceptible to developing a bone mineralization disorder, such as HPP, can be sustained, for example, over the course of the child's life, through administration of alkaline phosphatase or a polypeptide having alkaline phosphatase activity, such as sALP (e.g., a polypeptide having the sequence of any one of SEQ ID NOS: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or at least one amino acid mutation relative to naturally occurring ALP, as described herein). Similarly, an increase in ADL and / or a reduction in pain in a child can be sustained, for example, over the course of the child's life, through administration of sALP (e.g., a polypeptide having the sequence of any one of SEQ ID NOS: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or at least one amino acid mutation relative to naturally occurring ALP, as described herein).

[0204] The CHAQ index scores of children with or prone to developing bone mineralization disorders such as HPP can be used to assess the efficacy of treatment with alkaline phosphatase or a polypeptide having alkaline phosphatase activity, such as sALP (e.g., a sequence of any one of SEQ ID NOS: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or a polypeptide having at least one amino acid mutation relative to naturally occurring ALP, as described herein), where improvement relative to a specific test score indicates that the alkaline phosphatase or polypeptide having alkaline phosphatase activity is effective in treating, for example, impairment in activities of daily living (ADL) and pain associated with bone mineralization disorders such as HPP. In particular, children with or prone to developing bone mineralization disorders such as HPP can be asked one or more questions in one or more of eight categories (dressing / grooming, rising, eating, walking, hygiene, reaching, gripping, and movement) to arrive at an average CHAQ index score and assess the treatment efficacy of sALP administration. For example, if administration of a sALP as described herein to a child having or susceptible to developing a bone mineralization disorder, such as HPP, results in a mean reduction in CHAQ index score to about 0.5 or less (the child's previous mean CHAQ index score was greater than about 0.8), the sALP is effective in treating the impairment in activities of daily living (ADL) and pain associated with a bone mineralization disorder, such as HPP. Alternatively, if administration of sALP does not result in a mean reduction in CHAQ index score to about 0.5 or less, the dosage and / or frequency of sALP administration can be modified to determine an effective amount of sALP for a child having or susceptible to developing a bone mineralization disorder, such as HPP. For example, the dosage of sALP (e.g., as described herein, any one of the sequences of SEQ ID NOs: 7 to 223, 247, and 262 to 264, or a variant thereof having at least 85% sequence identity thereto, and / or a polypeptide having at least one amino acid mutation relative to naturally occurring ALP) can be increased, for example, from about 0.5 to 3 mg / kg / week to about 3 to 6 mg / kg / week or from about 3 to 6 mg / kg / week to about 6 to 9 mg / kg / week.

[0205] Pediatric Outcomes Data Collection Instrument (PODCI) Certain subjects with or susceptible to developing bone mineralization disorders, such as HPP, can be identified for treatment with a polypeptide having alkaline phosphatase activity, such as alkaline phosphatase or sALP (e.g., a polypeptide having at least one amino acid mutation relative to naturally occurring ALP, or a sequence of any one of SEQ ID NOS: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, as described herein, using the Pediatric Outcomes Data Collection Instrument (PODCI). As described by Plint et al. (J. Pediatr. Orthop. 23(6):788-790, 2003), the PODCI can be administered to assess a child's health status to generate a PODCI score for the subject. The PODCI includes eight categories of questions that can be completed by a subject with or susceptible to developing bone mineralization disorders, such as HPP, or the subject's parent / guardian. Categories that can be used to determine the PODCI of subjects with or prone to developing bone mineralization disorders such as HPP include: 1) the Upper Limb and Physical Function Scale to measure the difficulty encountered when performing daily personal care and student activities; 2) the Transfer and Basic Mobility Scale to measure the difficulty experienced when performing routine movement and athletic activities in daily activities; 3) the Sports / Physical Function Scale to measure the difficulty or limitation encountered when participating in more vigorous activities or sports; 4) the Pain / Comfort Scale to measure the level of pain experienced in the past week; 5) the Treatment Expectations Scale to measure long-term expectations of treatment; 6) the Well-Being Scale to measure overall satisfaction with personal appearance and similarity to friends and others of the same age; 7) the Symptom Satisfaction Scale to measure the subject's acceptance of current limitations if this is a lifelong condition; and 8) the Global Functioning Scale, a general composite scale calculated from the first four scales above.In each category, a standardized score is determined for subjects with or susceptible to developing a bone mineralization disorder such as HPP and converted to a scale of 0 to 100, where 0 represents severe impairment and 100 represents minimal impairment.

[0206] Subjects with or susceptible to developing a bone mineralization disorder, such as HPP, who have an average PODCI score (e.g., indicating ADL impairment and / or pain) of less than about 40 (e.g., about 5, about 10, about 15, about 20, about 25, about 30, about 35, or about 39) can be treated by administering alkaline phosphatase or a polypeptide having alkaline phosphatase activity, such as sALP (e.g., a sequence of any one of SEQ ID NOS: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or a polypeptide having at least one amino acid mutation relative to naturally occurring ALP, as described herein). For example, subjects with an average PODCI score of less than 40 can be treated by administering sALP for a period of time up to the subject's lifetime. Additionally, subjects with or susceptible to developing a bone mineralization disorder such as HPP can be asked one or more questions on one or more of the eight scales described above (e.g., the mobility and basic mobility, sports / physical function, and pain / comfort scales) to arrive at an average PODCI score, and if the average PODCI score is less than 40, the subject can be treated by administering sALP.

[0207] The methods described herein can result in an increase in the PODCI score (e.g., indicating impairment in ADL and / or pain) in a subject with or susceptible to developing a bone mineralization disorder such as HPP. For example, treatment with a polypeptide having alkaline phosphatase activity, such as alkaline phosphatase or sALP (e.g., a sequence of any one of SEQ ID NOS: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or a polypeptide having at least one amino acid mutation relative to naturally occurring ALP, as described herein), such as treatment with sALP for a period of time over the subject's lifetime, can result in an average increase in the PODCI score to about 40 to about 50 (e.g., about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, or about 50).

[0208] An increase in PODCI score can be sustained for a period of time, for example, over the lifespan of a subject having or prone to developing a bone mineralization disorder, such as HPP, through administration of alkaline phosphatase or a polypeptide having alkaline phosphatase activity, such as sALP (e.g., a polypeptide having the sequence of any one of SEQ ID NOS: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or at least one amino acid mutation relative to naturally occurring ALP, as described herein). Similarly, an increase in ADL and / or a decrease in pain can be sustained for a period of time, for example, over the lifespan of a subject through administration of sALP (e.g., a polypeptide having the sequence of any one of SEQ ID NOS: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or at least one amino acid mutation relative to naturally occurring ALP, as described herein).

[0209] The PODCI score of a subject having or susceptible to developing a bone mineralization disorder such as HPP is used to evaluate the efficacy of treatment with alkaline phosphatase or a polypeptide having alkaline phosphatase activity such as sALP (e.g., a sequence of any one of SEQ ID NOs: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or a polypeptide having at least one amino acid mutation relative to naturally occurring ALP, as described herein), wherein an improvement compared to a particular test score indicates that the alkaline phosphatase or a polypeptide having alkaline phosphatase activity is effective in treating, for example, impairment in activities of daily living (ADL) and pain associated with a bone mineralization disorder such as HPP. In particular, subjects with or susceptible to developing a bone mineralization disorder such as HPP may be asked one or more questions on one or more of eight scales (Upper Limb and Physical Function Scale, Transfer and Basic Mobility Scale, Sports / Physical Function Scale, Pain / Comfort Scale, Treatment Expectations Scale, Well-Being Scale, Symptom Satisfaction Scale, and Global Function Scale) to arrive at an average PODCI score and assess the treatment effect of sALP administration.

[0210] For example, if administration of a sALP described herein to a subject having or susceptible to developing a bone mineralization disorder, such as HPP, results in a mean increase in the PODCI score to about 40 or greater (the subject's previous mean PODCI score was less than about 40), the sALP is effective in treating the impairment in activities of daily living (ADL) and pain associated with a bone mineralization disorder, such as HPP. Alternatively, if administration of a sALP described herein does not result in a mean increase in the PODCI score to about 40 or greater, the dosage and frequency of sALP administration can be modified to determine an effective amount of sALP for a subject having or susceptible to developing a bone mineralization disorder, such as HPP. For example, the dosage of sALP (e.g., as described herein, any one of the sequences of SEQ ID NOs: 7 to 223, 247, and 262 to 264, or a variant thereof having at least 85% sequence identity thereto, and / or a polypeptide having at least one amino acid mutation relative to naturally occurring ALP) can be increased, for example, from about 0.5 to 3 mg / kg / week to about 3 to 6 mg / kg / week or from about 3 to 6 mg / kg / week to about 6 to 9 mg / kg / week.

[0211] Bayley Scales of Infant Development and Toddler Development), 3rd Edition (BSID-III) Another endpoint, the Bayley Scales of Infant and Toddler Development, Third Edition (BSID-III), as described in Bayley. (2006). Bayley scales of infant and toddler development: administration manual. San Antonio, TX: Harcourt Assessment, can be administered to assess the health status of subjects who have or are at risk for developing bone mineralization disorders such as HPP from birth, generating a BSID-III score for the subject. The BSID-III includes a series of developmental play tasks that can be administered to the subject to determine a raw BSID-III score. For example, categories for determining a BSID-III score for subjects who have or are at risk for developing bone mineralization disorders such as HPP (e.g., infants under about 3 years of age with HPP) can include grasping, perceptual-motor integration, motor planning and speed, visual tracking, reaching, object grasping, object manipulation, functional manual skills, response to tactile information, limb and trunk movements, static positioning, dynamic movement, balance, and motor planning. The BSID-III measurements are then converted to a scaled BSID-III score, which can be used to determine a subject's performance relative to healthy, age-adjusted subjects. The BSID-III scale score for subjects with or prone to developing a bone mineralization disorder such as HPP (e.g., subjects with HPP) can range from 0 to 14, with a score of about 7 to about 13 being considered the normal range for healthy subjects.

[0212] A subject with or susceptible to developing a bone mineralization disorder such as HPP may be tested as an infant (e.g., about 3 years of age or younger) in one or more of the listed categories (grasping, perceptual-motor integration, motor planning and speed, visual tracking, reaching, object grasping, object manipulation, functional manual skills, response to tactile information, limb and trunk movements, static positioning, dynamic movement, balance and motor planning) to generate a BSID-III score indicative of delayed motor development. Subjects who have or are susceptible to developing a bone mineralization disorder, such as HPP, and who have an average BSID-III score of less than about 2 as infants in one or more of the listed categories (grasping, sensorimotor integration, motor planning and speed, visual tracking, reaching, object grasping, object manipulation, functional manual skills, response to tactile information, limb and trunk movements, static positioning, dynamic movement, balance and motor planning) can be treated by administering sALP (e.g., a polypeptide having a sequence of any one of SEQ ID NOS: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein). In particular, subjects who have or are susceptible to developing a bone mineralization disorder, such as HPP, and who have an average BSID-III score of less than about 2 as infants can be treated by administering sALP over a period of time over the course of the subject's life.

[0213] This method can result in an improvement in the mean BSID-III score (e.g., indicative of delayed motor development) of subjects with or susceptible to developing a bone mineralization disorder such as HPP. For example, treatment with sALP (e.g., a polypeptide having any one of SEQ ID NOS: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein), such as treatment with sALP for a period of time over the subject's lifespan, can result in a mean increase in the BSID-III score to greater than about 5 (e.g., about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, or about 13).

[0214] Increased BSID-III scores can be sustained over the lifespan of a subject with or prone to developing a bone mineralization disorder, such as HPP, through administration of alkaline phosphatase or a polypeptide having alkaline phosphatase activity, such as sALP (e.g., a polypeptide having the sequence of any one of SEQ ID NOS: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or a polypeptide having at least one amino acid mutation relative to naturally occurring ALP, as described herein). Similarly, increased motor development can be sustained over the lifespan of a subject through administration of alkaline phosphatase or a polypeptide having alkaline phosphatase activity, such as sALP (e.g., a polypeptide having the sequence of any one of SEQ ID NOS: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or a polypeptide having at least one amino acid mutation relative to naturally occurring ALP, as described herein).

[0215] The BSID-III score of a subject having or susceptible to developing a bone mineralization disorder such as HPP is used to evaluate the efficacy of treatment with alkaline phosphatase or a polypeptide having alkaline phosphatase activity, such as sALP (e.g., a sequence of any one of SEQ ID NOS: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or a polypeptide having at least one amino acid mutation relative to naturally occurring ALP, as described herein), wherein an improvement compared to a particular test score indicates that the alkaline phosphatase or a polypeptide having alkaline phosphatase activity is effective in treating, for example, motor development delays associated with a bone mineralization disorder such as HPP. In particular, subjects with or susceptible to developing bone mineralization disorders such as HPP may perform tests in one or more of the listed categories (grasping, perceptual-motor integration, motor planning and speed, visual tracking, reaching, object grasping, object manipulation, functional manual skills, response to tactile information, limb and trunk movements, static positioning, dynamic movement, balance and motor planning) as infants (e.g., those about 3 years of age or younger with HPP) to reach an average BSID-III score and assess the treatment effect of sALP administration.

[0216] For example, if administration of sALP to a child having or prone to developing a bone mineralization disorder such as HPP results in a mean increase in BSID-III scale score of greater than about 5 (where the child's previous mean BSID-III scale score was less than about 2 as an infant (e.g., about or under 3 years of age)), the sALP is effective in treating, e.g., delayed motor development, associated with an HPP-like disease. Alternatively, if administration of sALP does not result in a mean increase in BSID-III scale score of greater than about 5, the dosage and / or frequency of sALP administration can be modified to determine an effective amount of sALP for a child having or prone to developing a bone mineralization disorder such as HPP. For example, the dosage of sALP (e.g., as described herein, any one of the sequences of SEQ ID NOs: 7 to 223, 247, and 262 to 264, or a variant thereof having at least 85% sequence identity thereto, and / or a polypeptide having at least one amino acid mutation relative to naturally occurring ALP) can be increased, for example, from about 0.5 to 3 mg / kg / week to about 3 to 6 mg / kg / week or from about 3 to 6 mg / kg / week to about 6 to 9 mg / kg / week.

[0217] Peabody Developmental Motor Scales, Second Edition (PDMS-2) Another endpoint, the Peabody Developmental Motor Scales, Second Edition (PDMS-2), as described by van Hartingsveldt et al. (Occup. Ther. Int. 12(1):1-13, 2005), can be administered to assess the health status of subjects who have had or are predisposed to developing bone mineralization disorders, such as HPP, since birth to generate a PDMS-2 score for the subject. The PDMS-2 includes six categories of subtests to measure the motor skills of subjects, such as those with HPP.

[0218] Specifically, the PDMS-2 measures can be determined from the following subtests: 1) Locomotor Activity subtest to measure the subject's ability to move from one place to another (measures include crawling, walking, running, hopping, jumping forward, etc.); 2) Reflexes subtest to measure the subject's ability to automatically respond to environmental events; 3) Static subtest to measure the subject's ability to maintain body control and balance within the center of gravity; 4) Object Manipulation subtest to measure the subject's ability to manipulate objects, such as catching, throwing, and kicking a ball; 5) Grasping subtest to measure the subject's ability to use their hands, including the ability to hold an object in one hand and perform actions involving the controlled use of the fingers of both hands; and 6) Visual-Motor Integration subtest to measure the subject's ability to use visual perceptual skills to perform complex eye-hand coordination tasks, such as reaching and grasping objects, building with blocks, and copying designs. PDMS-2 measurements can be determined for one or more of these categories of subjects having or susceptible to developing a bone mineralization disorder, such as HPP, and then converted to a PDMS-2 score, such as a PDMS-2 motor standard score, ranging from 0 to 13, where healthy subjects (e.g., subjects without a bone mineralization disorder, such as HPP) range from about 7 to about 13.

[0219] Subjects having or susceptible to developing a bone mineralization disorder, such as HPP, who have an average PDMS score (e.g., indicative of delayed motor development) can be treated by administering sALP (e.g., a polypeptide having the sequence of any one of SEQ ID NOS: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein).

[0220] The methods described herein can result in an improvement in PDMS-2I scores (e.g., indicating delayed motor development) in subjects with or susceptible to bone mineralization disorders such as HPP. For example, treatment with a polypeptide having alkaline phosphatase activity, such as alkaline phosphatase or sALP (e.g., a polypeptide having any one of the sequences of SEQ ID NOS: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or a polypeptide having at least one amino acid mutation relative to naturally occurring ALP, as described herein), can result in an average increase in PDMS-2I scores from about 7 to about 13 (e.g., about 7, about 8, about 9, about 10, about 11, about 12, or about 13).

[0221] Increased PDMS-2 scores can be sustained over time, for example, over the lifespan of a subject with or prone to developing a bone mineralization disorder, such as HPP, through administration of alkaline phosphatase or a polypeptide having alkaline phosphatase activity, such as sALP (e.g., a polypeptide having the sequence of any one of SEQ ID NOS: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or at least one amino acid mutation relative to naturally occurring ALP, as described herein). Similarly, increased motor development can be sustained over the lifespan of a subject with or prone to developing a bone mineralization disorder, such as HPP, through administration of sALP (e.g., a polypeptide having the sequence of any one of SEQ ID NOS: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or at least one amino acid mutation relative to naturally occurring ALP, as described herein).

[0222] The PDMS-2 scores of subjects with or prone to developing bone mineralization disorders such as HPP can be used to assess the efficacy of treatment with alkaline phosphatase or a polypeptide having alkaline phosphatase activity, such as sALP (e.g., a sequence of any one of SEQ ID NOS: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or a polypeptide having at least one amino acid mutation relative to naturally occurring ALP, as described herein), where improvement relative to a specific test score indicates that the alkaline phosphatase or polypeptide having alkaline phosphatase activity is effective in treating, for example, motor developmental delays associated with bone mineralization disorders such as HPP. For example, children with or prone to developing bone mineralization disorders such as HPP can achieve an average PDMS-2 score and perform tests in one or more of the listed categories (movement, reflexes, immobility, object manipulation, grasping, and visual-motor) at about or under the age of 5 years to assess the treatment efficacy of sALP administration.

[0223] For example, if administration of sALP to children with or susceptible to developing a bone mineralization disorder such as HPP results in a mean increase in PDMS-2 standard score to about 7 (the children's previous mean PDMS-2 standard score was about 5), then sALP is effective in treating, for example, delayed motor development, associated with HPP-like disorders. Alternatively, if administration of sALP does not result in a mean increase in PDMS-2 standard score to about 7, the dosage and / or frequency of sALP administration can be modified to determine an effective amount of sALP for the child. For example, the dosage of sALP (e.g., a sequence of any one of SEQ ID NOS: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or a polypeptide having at least one amino acid mutation relative to naturally occurring sALP, as described herein) can be increased, for example, from about 0.5-3 mg / kg / week to about 3-6 mg / kg / week or from about 3-6 mg / kg / week to about 6-9 mg / kg / week.

[0224] 6-minute walk test (6MWT) Subjects with bone mineralization disorders, such as HPP, can be identified for treatment with alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, e.g., a polypeptide having at least one amino acid mutation relative to a naturally occurring ALP, e.g., a sequence of any one of SEQ ID NOS: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, as described herein). In particular, the 6MWT can be used to assess the walking ability of adults with bone mineralization disorders, such as HPP, and generate a 6MWT score for adults. The 6MWT can be performed indoors or outdoors using a flat, straight, enclosed corridor (e.g., approximately 30 meters in length) with a hard surface. A stopwatch or other timer can be used to track time, and a mechanical counter or other device can be used to determine the distance (e.g., in meters) walked by a subject with a bone mineralization disorder, such as HPP. For example, the length of the corridor can be marked every 3 meters to determine the number of meters walked by a subject with a bone mineralization disorder, such as HPP. The turning point is 30 meters, and the starting line is also marked.The distance walked by the subject with bone mineralization disorder such as HPP in 6 minutes can then be compared with, for example, the number of meters predicted by a normal subject with approximately the same age, sex and / or height, and expressed as a percentage value to generate the subject's 6MWT value.The 6MWT value of the subject with bone mineralization disorder such as HPP can be compared with the subject's 6MWT value at baseline.Furthermore, the 6MWT value of the subject with bone mineralization disorder such as HPP can be compared with the 6MWT value of a normal subject.

[0225] A subject with a bone mineralization disorder, such as HPP, having a mean 6MWT of less than about 80% of the predicted 6MWT value (e.g., relative to a normal subject of approximately the same age, sex, and / or height) may be treated with steroids for at least 2 weeks (e.g., at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 6 years, at least The subject can be treated with alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, e.g., as described herein, any one of the sequences of SEQ ID NOs: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or a polypeptide having at least one amino acid mutation relative to naturally occurring ALP), such as by administering alkaline phosphatase or a polypeptide having alkaline phosphatase activity for a treatment period of at least 7 years, at least 8 years, at least 9 years, or at least 10 years, or the subject's lifetime, particularly at least 6 weeks. For example, a subject with a bone mineralization disorder, such as HPP, who has a mean 6MWT of less than about 80% of the predicted 6MWT value (e.g., about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% of the predicted 6MWT value) can be treated with alkaline phosphatase or a polypeptide having alkaline phosphatase activity for a treatment period of at least 2 weeks (e.g., at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 6 years, at least 7 years, at least 8 years, at least 9 years, or at least 10 years, or the subject's lifetime, particularly at least 6 weeks).

[0226] The method can result in an improvement in 6MWT values ​​in a subject with a bone mineralization disorder, such as HPP. For example, the alkaline phosphatase or polypeptides having alkaline phosphatase activity can be increased over a treatment period of at least 2 weeks (e.g., at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 6 years, at least 7 years, at least 8 years, at least 9 years, or at least 10 years, or the subject's lifetime, particularly at least 6 weeks). Treatment with alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, e.g., a sequence of any one of SEQ ID NOs: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, as described herein, and / or a polypeptide having at least one amino acid mutation relative to a naturally occurring ALP), such as by treatment with a peptide, can result in an average increase in the subject's 6MWT value to about 80% or more of the predicted 6MWT value (e.g., about 82%, about 84%, about 86%, about 88%, about 90%, about 92%, about 94%, about 96%, about 98% or more of the predicted 6MWT value).

[0227] An increase in 6MWT values ​​in a subject with a bone mineralization disorder, such as HPP, can be sustained through administration of alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, e.g., a sequence of any one of SEQ ID NOs: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or a polypeptide having at least one amino acid mutation relative to naturally occurring ALP, as described herein) over a treatment period of at least 2 weeks (e.g., at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 6 years, at least 7 years, at least 8 years, at least 9 years, or at least 10 years, or the subject's lifetime, particularly at least 6 weeks). For example, the 6MWT value increases to greater than about 80% of the predicted 6MWT value in a subject with a bone mineralization disorder, such as HPP, and remains within ±10% of the 6MWT value increased during treatment with alkaline phosphatase or a polypeptide having alkaline phosphatase activity.

[0228] Similarly, improvement in walking ability in a subject with a bone mineralization disorder such as HPP can be sustained through administration of alkaline phosphatase or a polypeptide having alkaline phosphatase activity for a treatment period of, for example, at least 2 weeks (e.g., at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 6 years, at least 7 years, at least 8 years, at least 9 years, or at least 10 years, or the subject's lifetime, particularly at least 6 weeks). For example, a subject with a bone mineralization disorder such as HPP exhibits decreased dependency on an assistive mobility device, such as a walker, wheelchair, brace, crutches, or orthotic, during treatment with sALP.

[0229] Alternatively, if administration of alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, e.g., a sequence of any one of SEQ ID NOS: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or a polypeptide having at least one amino acid mutation relative to naturally occurring ALP, as described herein) does not result in a mean increase in 6MWT value of more than 80% of the predicted 6MWT value (e.g., for a normal subject of approximately the same age, sex, and / or height), the dosage and / or frequency of administration of alkaline phosphatase or a polypeptide having alkaline phosphatase activity can be modified to determine an effective amount of alkaline phosphatase or a polypeptide having alkaline phosphatase activity for a subject with a bone mineralization disorder, such as HPP. For example, the dosage of alkaline phosphatase or a polypeptide having alkaline phosphatase activity can be increased, for example, from about 0.1-3 mg / kg / week or about 3-6 mg / kg / week to about 3-6 mg / kg / week or about 6-9 mg / kg / week.

[0230] Handheld Dynamometry (HHD) The grip and muscle strength of a subject with or prone to developing a bone mineralization disorder such as HPP can be assessed using handheld dynamometry (HHD). For example, knee flexion and extension and hip flexion, extension, and abduction of a subject with or prone to developing a bone mineralization disorder such as HPP can be measured, for example, using a MICROFET2™ dynamometer, and the subject's grip strength can be measured, for example, using a Jamar® grip dynamometer. Specifically, an administrator holds the dynamometer stationary while the subject exerts maximum force against the dynamometer. Peak force data is collected in pounds and converted to Newtons (N). Torque values ​​are then calculated using the limb length in N meters. The torque values ​​can then be compared, for example, to those of a normal subject of approximately the same age, sex, and / or height and expressed as a percentage to generate the subject's HHD value.

[0231] A subject with a bone mineralization disorder, such as HPP, having a mean HHD value that is less than about 80% of the predicted HHD value (e.g., for a normal subject of approximately the same age, sex, and / or height) may be at least 2 weeks (e.g., at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 6 years, at least The subject can be treated with alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, e.g., as described herein, any one of SEQ ID NOS: 7-223, 247, and 262-264 or a variant thereof having at least 85% sequence identity thereto and / or a polypeptide having at least one amino acid mutation relative to naturally occurring ALP), such as by administering alkaline phosphatase or a polypeptide having alkaline phosphatase activity for a treatment period of at least 7 years, at least 8 years, at least 9 years, or at least 10 years, or the subject's lifetime, particularly at least 6 weeks.For example, a subject with a bone mineralization disorder, such as HPP, who has an average HHD of less than about 80% of the predicted HHD value (e.g., about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% of the predicted HHD value) can be treated with alkaline phosphatase or a polypeptide having alkaline phosphatase activity for a treatment period of at least 2 weeks (e.g., at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 6 years, at least 7 years, at least 8 years, at least 9 years, or at least 10 years, or the subject's lifetime, particularly at least 6 weeks).

[0232] This method can result in an improvement in HHD scores in subjects with bone mineralization disorders such as HPP. For example, over a treatment period of at least 2 weeks (e.g., at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 6 years, at least 7 years, at least 8 years, at least 9 years, or at least 10 years, or the subject's lifetime, particularly at least 6 weeks), alkaline phosphatase or alkaline phosphatase activity can be improved. Treatment with alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, e.g., a sequence of any one of SEQ ID NOS: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, as described herein, and / or a polypeptide having at least one amino acid mutation relative to a naturally occurring ALP), such as by treatment with a polypeptide, can result in an average increase in the subject's HHD value to about 80% or more of the predicted HHD value (e.g., about 83%, about 85%, about 87%, about 90%, about 93%, about 95%, about 97%, or about 100% or 100% of the predicted HHD value).

[0233] Increases in HHD values ​​in a subject with a bone mineralization disorder, such as HPP, can be sustained through administration of alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, e.g., a sequence of any one of SEQ ID NOS: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or a polypeptide having at least one amino acid mutation relative to a naturally occurring ALP, as described herein) over a treatment period of, for example, at least 2 weeks (e.g., at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 6 years, at least 7 years, at least 8 years, at least 9 years, or at least 10 years, or the subject's lifetime, particularly at least 6 weeks). For example, the HHD value increases to greater than about 80% of the predicted HHD value in a subject with a bone mineralization disorder, such as HPP, and remains within ±10% of the HHD value increased during treatment with alkaline phosphatase or a polypeptide having alkaline phosphatase activity.

[0234] Alternatively, if administration of alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, e.g., a sequence of any one of SEQ ID NOs: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity thereto, and / or a polypeptide having at least one amino acid mutation relative to a naturally occurring ALP, as described herein) does not result in a mean increase in HHD value of more than 80% of the predicted HHD value (e.g., in subjects of approximately the same age, sex, and / or height with a bone mineralization disorder, such as HPP), the dosage and / or frequency of administration of alkaline phosphatase or a polypeptide having alkaline phosphatase activity can be modified to determine an effective amount of alkaline phosphatase or a polypeptide having alkaline phosphatase activity for a subject with a bone mineralization disorder, such as HPP. For example, the dosage of alkaline phosphatase or a polypeptide having alkaline phosphatase activity can be increased from, for example, about 0.1 to 3 mg / kg / week or about 3 to 6 mg / kg / week to about 3 to 6 mg / kg / week or about 6 to 9 mg / kg / week. [Example]

[0235] The present disclosure is illustrated by the following examples, with the understanding that the specific examples, materials, amounts and procedures are to be interpreted broadly in accordance with the scope and spirit of the disclosure set forth herein.

[0236] Example 1 Protein Design and Expression Alkaline phosphatase fusion protein. To systematically examine the protein activity, protein stability, and pharmacokinetic properties of alkaline phosphatase fusion proteins, several constructs were designed and tested (see Table 1). Some constructs were generated by standard DNA synthesis techniques, and their sequences were verified by DNA sequencing of the final constructed plasmid. Additional constructs were generated by performing standard site-directed mutagenesis techniques on existing plasmids. The alkaline phosphatase fusion protein mutations tested varied in residues located within and around the active site of the enzyme, consensus sites for N-linked glycosylation, bone-targeting moieties, and the isotype of the fragment-crystallizable (Fc) region attached to the protein. These constructs were expressed by transiently transfecting Expi293F and ExpiCHO cells, as detailed below.

[0237] Bone-targeted fluorescent-Fc fusion. To systematically compare the HA- and bone-binding abilities of polypeptides and VHH sequences, protein fusions containing (N-terminal bone-targeting, human IgG1 crystal domain (Fc), Katushka2s fluorescent protein, and C-terminal His tag) were designed (SEQ ID NOs: 224-245). A list of the fluorescent fusion proteins used for targeting is shown in Table 2. The fluorescent fusion proteins were transiently transfected into Expi293F cells as detailed below.

[0238] [Table 2]

[0239] A biologically inactive ERT surrogate molecule (ALP-Fc). To model the bone-targeting function of clinically available ERTs, ALP-Fc-(Asp) 10 The ALP-Fc-(ASP) fusion was compared with the ALP-Fc fusion. The ALP used in these studies was specifically engineered for enzymatic activity against the synthetic substrate, 4-methylumbelliferyl phosphate (4-MUP). Figure 21 shows the ALP-Fc-(ASP) fusion.10 The structural models of ALP-Fc (SEQ ID NO: 222) and ALP-Fc-(Asp) are shown. 10 (SEQ ID NO: 31) protein was transiently transfected using ExpiCHO cells as detailed below.

[0240] Heavy chain only single domain variable heavy chain (VHH) antibody fragment. Bone-targeting VHH proteins were designed by replacing the complementarity-determining regions (CDRs) of non-targeting VHH molecules with polyaspartic acid sequences. Table 3 shows a list of VHH proteins that were evaluated for bone binding. VHH proteins were transiently transfected using Expi293F cells as detailed below.

[0241] [Table 3]

[0242] Transient transfection of cell cultures A ratio of 1.0 μg plasmid DNA / mL transfection culture was used for all Expi293 transfections, and a ratio of 0.8 μg plasmid DNA / ml transfection culture was used for all ExpiCHO transfections. Cultures were maintained at 120 RPM on a shaker with a throw of 25.4 mm.

[0243] Expi293F cells were cultured in Expi293 expression medium (Thermo Fisher) in a humidified 8% CO2 incubator at 37°C. The day before transfection, cells were cultured at 2.5 × 10 6 After 24 hours, the stock was approximately 4 x 10 cells / mL. 6 cells / mL, 2.5 x 10 6The Expi293 transfections were performed according to the manufacturer's protocol. The Expi293 Expression System Kit (Thermo Fisher), which contains a transfection enhancer and ExpiFectamine 293 transfection reagent, was used for all Expi293 transfections. Briefly, ExpiFectamine The 293 transfection reagent and plasmid DNA were separately diluted in OptiMEM medium (Thermo Fisher). The ExpiFectamine 293 and DNA mixtures were combined and incubated for an additional 10–20 minutes. The ExpiFectamine 293-DNA-OptiMEM mixture was then added to the cells. Enhancer 1 and Enhancer 2 were added to the transfected cultures 16–18 hours after transfection.

[0244] ExpiCHO-S cells were cultured in ExpiCHO expression medium (Thermo Fisher) in a humidified 8% CO2 incubator at 37°C. The day before transfection, cells were cultured at 3–4 × 10 6 After 24 hours, the stock was 7–10 × 10 cells / mL. 6 cells / mL and add fresh medium to obtain 6 x 10 6 The cells were diluted to 1000 cells / mL. ExpiCHO transfection was performed using the ExpiCHO Expression System Kit (Thermo Fisher) according to the manufacturer's standard protocol. Briefly, ExpiFectamine CHO transfection reagent and plasmid DNA were separately diluted in OptiPRO SFM (Thermo Fisher). The ExpiFectamine CHO and DNA mixtures were immediately combined and incubated for 1–5 min. The ExpiFectamine CHO-DNA-OptiPRO mixture was then added to the cells. Enhancer and 30% v / v feed were added 18–22 h after transfection.

[0245] Cell culture harvest Expi293 transfectants were harvested on day 4, and ExpiCHO transfectants were harvested on day 5. Cell cultures were centrifuged at 1900 × g for 15 min, and the supernatants were sterile filtered through 0.2 μm disposable membrane filters (polyethersulfone (PES) filters, Fisher Scientific).

[0246] Large-scale protein expression ALP201 and ALP213 were expressed in stable cell culture (CHO KS) at 5.0 × 10 5 Growth was performed in a 5 L bioreactor (APPLIKON) from an initial seeding of 100 cells / mL and controlled by temperature (36.5 °C, shifted to 33 °C on day 5), agitation (80 W / m 2 ) and O2 (0-0.6 VVM, as needed) are controlled for 11 days according to standard procedures.

[0247] Protein purification. Purification of VHH and fluorescent Fc fusion proteins using Ni-NTA resin. Fluorescent Fc fusion proteins with a C-terminal His6 tag were purified in batches using Ni-NTA Superflow resin (Qiagen). Cell supernatants were dialyzed against Ni-NTA binding / wash buffer (2.5 M sodium chloride, 0.1 M sodium phosphate, 0.1 M imidazole, buffered to pH 7.4 with sodium hydroxide) using dialysis flasks (Slide-A-Lyzer™, ThermoFisher) for 48 hours at 4°C. The Ni-NTA Superflow resin was washed, resuspended in Ni-NTA binding / wash buffer, and then incubated with the dialyzed supernatant (approximately 8 mg of protein per mL of resin) for 90 minutes with shaking at 4°C. The gravity purification column was sanitized with 0.1 N sodium hydroxide for 1 hour and then rinsed with Ni-NTA binding / wash buffer before protein loading and elution. The dialyzed supernatant containing the Ni resin was poured onto the column, allowing the unbound protein suspension to flow through the resin. The resin-bound protein was washed with 40 column volumes (approximately 200 mL) of Ni-NTA binding / wash buffer. The fluorescent Fc-fusion protein was eluted into three fractions by adding 5 mL of elution buffer (20 mM sodium phosphate pH 7.0, 500 mM imidazole pH 7.5, 500 mM sodium chloride) and incubating with the resin for 5 minutes before elution into a conical tube. The eluted protein was buffer exchanged into PBS pH 7.4 using a 30 kD MWCO centrifugal filter (Amicon®), and the final concentrated protein was filtered. Protein concentration was quantified using UV / VIS absorbance at 280 nm and 558 nm (maximum Katushka 2s absorbance) and then stored at 4°C protected from light.

[0248] Purification of ALP-Fc fusions with protein A resin. The ALP-Fc fusion was purified in batches using Protein A resin (MABSELECT™ SURE™, GE Life Sciences). The resin was thoroughly washed and suspended in protein loading buffer (50 mM sodium phosphate, pH 7.5, 100 mM sodium chloride), then incubated overnight (4°C) with cell supernatant containing the expressed target protein (approximately 10 mg of protein per mL of resin). A gravity column was sanitized with sodium hydroxide, rinsed with loading buffer, and loaded with the Protein A resin / supernatant. The resin-bound protein was washed with 40 column volumes (approximately 200 mL) of loading buffer before elution with 50 mM Tris-HCl pH 11. The protein was eluted in 3 mL fractions and immediately neutralized by diluting 1:1 with loading buffer. Certain samples were further purified by strong anion exchange chromatography (CAPTO™ Q IMPRES, 5 mL column; column wash buffer 20 mM sodium phosphate buffer, pH 7.4, buffer A; gradient elution between buffer A and buffer B (20 mM sodium phosphate, 1 M NaCl, pH 7.4) over 12 column volumes, starting with 25% buffer B and progressing to 100% buffer B). Fractions containing the ALP-Fc fusion were pooled and either dialyzed against PBS pH 7.4 or buffer exchanged into PBS pH 7.4 using a 30 kD MWCO centrifugal filter. Protein concentration was determined via absorbance at 280 nm, and purity was assessed by SDS-PAGE gel. The protein was filtered and stored at 4°C.

[0249] Large-scale protein purification Protein from stable cell line bioreactor expression was purified similarly to the previous sample, except that strong anion exchange chromatography was performed using a POROS™ 50HQ column with the same buffer, over 13-15 column volumes, with a gradient from 0% to 70-75% buffer B. Samples were concentrated by UF / DF or dialyzed against PBS, sterile filtered, and stored at 4°C (sample concentrations were typically approximately 4-8 mg / mL).

[0250] Example 2 4-Methylumbelliferyl phosphate hydrolyzing protein activity assay Alkaline phosphatase activity was routinely measured using the artificial substrate 4-methylumbelliferyl phosphate (4-MUP). Cleavage of the phosphoester bond in 4-MUP generates the fluorescent product 4-methylumbelliferone (4-MU), which can be detected at excitation / emission of 360 nm / 465 nm.

[0251] Protein samples (supernatant, partially purified, or column-purified samples) were assayed for ALP activity in solution using 4-methylumbelliferyl phosphate (4-MUP) as an artificial substrate. Hydrolysis of the phosphoester bond of 4-MUP releases the fluorescent compound 4-methylumbelliferone, which can be easily detected with a fluorometer. Quantitation of the product was performed using a 4-methylumbelliferone (4-MU) standard curve measured on the same plate at standard concentrations of 0, 1.25 μM, 2.5 μM, 5 μM, 10 μM, and 20 μM. A 10 mM stock solution of 4-MU was prepared in ethanol and diluted in assay buffer [50 mM HEPES pH 7.4, 150 mM NaCl, 1 mM MgCl2, 1 mg / mL bovine serum albumin]. Purified fusion protein samples were prepared as 0.1 mg / mL solutions in assay buffer and serially diluted to the appropriate final concentration for the assay (usually approximately 1 nM) in assay buffer. A 4-MUP stock solution was prepared in assay buffer. Prior to starting the assay, 4-MUP was added to the protein sample at a final concentration of 10 μM, and all solutions were brought to 37°C. 4-MU production was measured at an excitation wavelength of 360 nm and an emission wavelength of 465 nm. Data were collected every 40 seconds for a total of 20 minutes on a plate reader maintained at 37°C. Reaction rates were calculated by linear regression in units of activity, where 1 U = 1 μmole of 4-MUP hydrolyzed / min. Specific activity was calculated in units / mg of assayed protein. The progress curve for the 4-MUP hydrolysis assay is shown in Figure 19. Specific activities in Table 4 are as follows: <2 U / mg (-), 2-9.9 U / mg (+), 10-30 U / mg (++), and >30 U / mg (+++).

[0252] [Table 4]

[0253] Plate bone assay Bone homogenate fractions were assayed for ALP activity. The bone homogenate fractions were suspended in 100 μL of PBS and transferred to a 96-well black plate. 100 μL of unbound protein suspension was also transferred to separate wells of the 96-well plate. 100 μL of ALP detection solution (10 μM 4-MUP, 1% BSA) was added to each well, and a dynamic fluorescence reading (360 / 465) was initiated immediately and run for 20 minutes, collecting fluorescence intensity emissions every 30 seconds. The slope of the fluorescence intensity versus time represented the ALP concentration in each sample fraction.

[0254] To maintain linearity, the MUP activity of the bound and unbound fractions of the 17 constructs was measured by the initial activity slope over the first 5 minutes. The ratio of MUP activity was determined for each fusion protein as the ratio = (bound activity) / (unbound activity). The results are shown in Table 5 and Figure 14. Exemplary progress curves from MUP hydrolysis assays performed on bone homogenates using various constructs are shown in Figure 11, and the bound activity is tabulated in Table 5. The MUP activity in Table 5 was quantified as follows: <0.2 (-); 0.2-0.49 (+); 0.5-5.0 (++); >5.0 (+++).

[0255] [Table 5]

[0256] Example 3 Activity of alkaline phosphatase constructs against natural substrates The improved properties of the protein fusion molecules described herein were verified in different enzyme assays measuring the hydrolysis of natural and artificial substrates. Artificial substrates used in the activity assays included 4-MUP and paranitrophenyl phosphate (pNPP). Natural substrates used in the activity assays included pyrophosphate (PPi) and pyridoxyl-5'-phosphate (PLP). The change in activity relative to wild-type ALP ranged from negative (decreased enzyme activity) to a maximum 9.5-fold increase in enzyme activity (850% increase in activity).

[0257] Pyrophosphate hydrolysis assay. Specific purified alkaline phosphatase fusion proteins were assayed for activity toward the natural substrate, pyrophosphate. Pyrophosphate hydrolysis was measured by detecting the product phosphate anion using PiBlue assay reagent (BioAssay Systems), which turns bright green upon phosphate binding. Quantification of phosphate levels in each well was performed using a standard curve of phosphate solutions made in assay buffer read on the assay plate. A stock solution of sodium pyrophosphate decahydrate (Sigma Chemicals) was prepared at a concentration of 10 mM in pure water. Purified fusion protein samples were prepared as 0.1 mg / mL solutions in assay buffer [50 mM HEPES pH 7.4, 150 mM NaCl, 1 mM MgCl2, 1 mg / mL bovine serum albumin] and serially diluted to the appropriate final concentration for assay in assay buffer. Pyrophosphate samples for the assay were prepared by diluting the stock solution in assay buffer. All solutions were brought to 37°C before starting the reaction. The protein solution was added to a clear 96-well plate and the plate was placed in a Jitterbug plate shaker maintained at 37° C. The reaction was initiated by adding the pyrophosphate solution to the protein solution.

[0258] Typically, pyrophosphate hydrolysis reactions were performed simultaneously in the same plate at pyrophosphate concentrations of 0 μM, 1.56 μM, 3.12 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM, 200 μM, and 400 μM. Eight reaction wells were set up at each pyrophosphate concentration, and the reaction was stopped by adding PiBlue reagent (added in a volume equal to the final reaction volume) 0, 1, 2, 3, 4, 5, 6, and 7 minutes after pyrophosphate was added to the plate. The low pH of the detection reagent inactivates the enzyme, so the addition of PiBlue reagent stops further reaction. The plate was allowed to develop for 30 minutes before reading the absorbance at 620 nm. The reaction rate at each pyrophosphate concentration was calculated by constructing a progress curve from the individual time points. Using a Michaelis-Menten enzyme kinetic fit, the reaction rate at each concentration was used to calculate Km and Vmax values ​​in GraphPad Prism. The kcat value was calculated from the Vmax value as follows: Vmax / (moles of protein analyzed)=kcat. Catalytic efficiency is defined as follows: Catalytic efficiency = kcat / Km.

[0259] Michaelis-Menten plots detailing the pyrophosphate hydrolysis activity of selected compounds are shown in Figure 22. 10 ng / mL ALP201, 10 ng / mL ALP250, and 25 ng / mL asphaltase alpha (SEQ ID NO: 269) are all shown. Standard deviations are shown (bars), N=3.

[0260] [Table 6]

[0261] Pyridoxyl-5'-phosphate hydrolysis assay. The second natural substrate of alkaline phosphatase is pyridoxyl-5'-phosphate (PLP). Specific purified alkaline phosphatase fusion proteins were assayed for activity against PLP in a binding assay format in which pyridoxyl, the product of PLP hydrolysis, is converted to fluorescent pyridoxolactone by M. loti tetrametric pyridoxyl dehydrogenase (tPLDH, SEQ ID NO: 246). The gene for 6xHis-tagged tPLDH was synthesized using standard methods and cloned into a bacterial expression plasmid under the control of a T7 promoter. 6xHis-tagged tPLDH was expressed in BL21(DE3) cells using standard protocols and purified by standard affinity chromatography methods. The protein was concentrated to a concentration of 1900 μM using an Amicon Ultra15 spin concentrator and frozen at -80°C until use in the assay. Purified fusion protein samples were prepared as 0.1 mg / mL solutions in assay buffer [50 mM HEPES pH 7.4, 150 mM NaCl, 1 mM MgCl2, 1 mg / mL bovine serum albumin]. The final serially diluted samples were placed in a black 96-well plate along with pyridoxolactone standards (made in assay buffer). Protein samples were enriched for NAD. +Add tPLDH and PLP solutions (made in assay buffer) to a final concentration of 3 mM NAD+, 4 μM tPLDH, and 3 μM PLP and mix. Prior to initiating the reaction, all solutions are brought to 37°C. The reaction plate is incubated at 37°C, and fluorescence is detected by excitation at 355 nm and emission at 445 nm. The amount of pyridoxolactone product produced is calculated using a standard curve generated from the fluorescence measured from the pyridoxolactone wells of the plate. The reaction rate is calculated by linear regression of the progress curve in μmoles of pyridoxyl produced / min. Specific activity is calculated by dividing the reaction rate by the protein concentration used in the assay reaction. An exemplary progress curve for data generated by this assay for ALP201, 10 ng / mL, is shown in Figure 23. A Michaelis-Menten plot detailing the pyridoxal-5'-phosphate hydrolysis activity of 10 ng / mL ALP201, 10 ng / mL ALP250, and 12.5 ng / mL asphaltase alpha is shown in Figure 24.

[0262] [Table 7]

[0263] Example 4 Hydroxyapatite binding assay Characterization of fluorescent Fc fusion binding to hydroxyapatite (HA). Synthetic HA (CAPTAL®) was purchased from Plasma-Biotal Ltd. (Derbyshire, UK). In separate 1.5 mL centrifuge tubes, bone-targeting fluorescent Fc fusions were diluted to 50 nM with 0.1% (w / w) bovine serum albumin (BSA) in phosphate-buffered saline (PBS) pH 7.4 (1 mL of suspension per tube). To each fluorescent Fc fusion-containing tube, 1 mg of synthetic HA was added, and the tubes were incubated at room temperature for 2 hours with orbital mixing to prevent HA from settling. After incubation, the samples were centrifuged at 16,000 rcf for 5 minutes to separate the solid HA-bound fraction from the unbound protein suspension. The HA-bound fraction was then washed three times with PBS, and the final HA fraction was suspended in 100 μL of PBS. The suspended HA-bound fraction and 100 μL of the unbound protein suspension were transferred to a 96-well black plate (one fraction per well), and relative protein concentrations were measured by fluorescence excitation / emission at 488 nm / 585 nm using a fluorescence plate reader (Spectramax i3x).

[0264] Twenty fluorescent Fc fusion proteins were transiently expressed in HEK cells and purified using Ni-NTA resin (see Table 1 for sequence details). The fluorescent Fc fusion proteins differed only in their N-terminal HA-binding sequence to systematically evaluate bone-targeting moieties in vitro. In separate tubes, the fluorescent Fc fusion proteins were incubated in suspension with HA in the presence of BSA to prevent nonspecific binding. Figure 1A shows the fluorescence intensity of the HA-bound fraction of each fluorescent Fc fusion sample compared to that of a nontargeting fluorescent Fc fusion (FLU002). Additionally, the unbound protein fraction was collected immediately after incubation with HA (before the first wash of the HA pellet). These fluorescence intensities are also plotted relative to FLU002 and are shown in Figure 1B. For FLU001, 003, 004, 016, 027, and 031, 100% of the protein bound to HA, as none of these proteins were detected above background in the unbound protein fraction. Conversely, FLU002 showed 4-fold less binding to HA than FLU001 and a significant unbound fluorescent signal (100-fold above FLU001 or background). FLU005-009, 011-014, and 020 exhibited HA-binding properties similar to FLU002. Interestingly, two proteins (FLU010 and FLU015) showed significant fluorescence (above the negative control) in both the HA-bound and unbound fractions.

[0265] Although hydroxyapatite (HA) has been used interchangeably with bone as a binding substrate in in vitro bone-targeting assays, significant differences have been observed between ceramic HA and natural bone, particularly with regard to the role of collagen and other extracellular matrix proteins in controlled crystal nucleation in vivo (Zhai, Y., et al., J. Crystal Growth, 202-206, 2006). CAPTAL® brand HA was specifically used in this study because it was manufactured as a bone mineral analog based on its Ca:PO4 ratio (1.67), iron content (100 ppm), magnesium content (800-1100 ppm), and crystal lattice. Still, significant differences in total protein binding were observed between CAPTAL® HA and bone homogenate. Most importantly, nonspecific binding was observed for all fluorescent Fc-fusion proteins incubated with HA, even in the presence of BSA blocking agent. When incubated with bone homogenate, a nontargeting fluorescent Fc-fusion control protein (FLU002) did not exhibit a fluorescent signal above background (bone homogenate alone). Thus, bone homogenate was a more stringent binding substrate compared to HA, which behaves more like a typical ion-exchange resin. However, when normalized to an appropriate control protein (as in Figures 1A-1D), the HA-binding data were useful for identifying efficient bone-binding proteins, but the dynamic range of fluorescent Fc fusion protein detection was significantly reduced (approximately 5-fold) in the HA-binding assay. The ALP-Fc protein exhibited even greater nonspecific binding to HA. This is likely due to the negative charge of the ALP catalytic domain. In some cases, this led to the false identification of some ALP-Fc-X proteins as having HA-binding properties, which was not observed when they were incubated with bone homogenate. For this reason, bone homogenate was considered an excellent substrate for screening bone-binding proteins and was used for all subsequent in vitro assays.

[0266] Example 5 Bone homogenate assay Previous literature assays relied on bone slices or other three-dimensionally physically constrained substrates. A more accurate, faster, and more efficient assay was needed to identify and quantify protein availability and quantify percentage binding.

[0267] Preparation of bone homogenate. Femurs from male C57BL / 6 mice were stored at -80°C before use. The femurs were transferred to 2 mL centrifuge tubes (two femurs per tube) containing 1 mL 0.2% (w / w) collagenase type 2 (Worthington) in PBS containing 1x EDTA-free serine and cysteine ​​protease inhibitor cocktail (COMPLETE™, Roche). The femurs were briefly vortexed and incubated at 37°C for 1 hour with shaking (800 rpm). Remaining connective tissue was removed, and the femurs were placed in a Petri dish on ice. The bone marrow was flushed out with PBS using a needle and syringe. The dried bones were weighed (typically 30–50 mg per femur) and placed in individual pre-chilled disposable beaters (PRECELLYS®, Bertin Instruments) with 0.75 mL PBS containing 1x protease inhibitors. The femurs were then homogenized using a high-throughput bead mill homogenizer (Bullet Blender, 4°C, maximum speed, 4 cycles of 30 seconds). The homogenate was transferred to a 1.5 mL centrifuge tube and centrifuged at 12,000 x g for 15 minutes at 4°C to separate the bone homogenate from released protein / cellular debris. The bone homogenate was resuspended in 0.1% BSA in PBS for use in binding assays.

[0268] Binding of fluorescent Fc fusions to bone homogenates in vitro. Fluorescent Fc-fusion proteins were diluted to 50 nM in PBS containing 0.1% BSA and incubated with 3 mg of bone homogenate in individual 1.5 mL centrifuge tubes (1 mL per tube). After mixing the samples for 2 hours at room temperature, the bone homogenate-bound and unbound fractions were separated by centrifugation. The bone homogenate fraction was washed three times with PBS, and the final homogenate pellet was suspended in 100 μL of PBS. The suspended bone homogenate and 100 μL of the unbound protein suspension were quantified for relative fluorescent Fc-fusion concentration using a fluorescent plate reader.

[0269] For all fluorescent Fc fusion proteins, binding to bone homogenate showed a similar trend as binding to HA; however, as shown in Figure 1C, there was a large numerical difference between efficient bone-binding proteins (e.g., FLU001) and non-binders (e.g., FLU002). In the bone-binding fraction, an 8-fold increase in fluorescence was observed with FLU001 over the negative control (FLU002), compared to the 4-fold increase observed for binding to HA. While binding of FLU010 to bone homogenate was not significantly greater than the negative control (FLU002), FLU015 still showed efficient binding to bone homogenate and was significantly present in the non-binding fraction. To confirm that these results were not statistical anomalies, the experiment was repeated four times, and the same results were observed for FLU010 and FLU015.

[0270] Importantly, raw fluorescence data quantifying the total amount of protein bound to HA (Example 2) and bone homogenate showed significantly greater binding of non-targeted proteins to equivalent masses of HA compared to bone homogenate (as much as a 75-fold increase in binding to HA in some cases), indicating that some non-specific binding of proteins to HA exists even in the absence of an efficient bone-targeting moiety. Non-specific binding to HA was exacerbated when the blocking agent was removed from the protein incubation. This demonstrates HA's ability to bind to a variety of proteins, including those without specific HA-binding moieties. While HA was less useful for distinguishing moderately HA-binding fusions from non-targeted fusions, it was a suitable substrate for determining highly efficient bone-binding proteins. Because bone homogenate is a more stringent binding substrate and did not exhibit non-specific binding of non-targeted proteins (see Figure 1), all subsequent bone-binding screening assays were performed using mouse bone homogenate as the binding substrate (Figures 2A-2B).

[0271] The fluorescence intensities of the bound and unbound fractions were normalized to a non-targeting fluorescent Fc fusion protein to compare different targeting moieties (Figures 3A-3B). In vitro binding experiments were performed in duplicate ± standard deviation.

[0272] Characterization of constructs binding to bone homogenate An in vitro model was used to characterize the bone binding of ERT-like molecules prior to in vivo administration. To utilize a hydroxyapatite fluorescence-based binding assay, ALP031 (ALP-Fc-D10) and ALP086 (ALP-Fc) proteins were fluorescently labeled using a commercially available antibody labeling kit (Invitrogen / ThermoFisher). Alexa Fluor dyes were preactivated with succinimidyl esters or tetrafluorophenyl esters, which react with primary amines of proteins. Therefore, fluorescent labeling was not site-specific, and the degree of labeling was 3–5 moles of fluorophore per mole of antibody. After labeling and purification to remove free dye, a 30 nM protein solution was prepared in 0.1% BSA, and bone homogenate binding assays were performed as described above for the FFC constructs. Exemplary data for constructs with different affinities for bone homogenate are shown in Figures 1C, 1D, 2, and 3.

[0273] Plate bone ALP activity assay Bone homogenate fractions were assayed for ALP activity using 4-MUP. Bone homogenate fractions were suspended in 100 μL of PBS and transferred to a 96-well black plate. 100 μL of unbound protein suspension was also transferred to separate wells of the 96-well plate. To each well, 100 μL of ALP detection solution (10 μM 4-MUP, 1% BSA) was added, and a dynamic fluorescence reading (360 / 465) was initiated immediately and run for 20 minutes, collecting fluorescence intensity emissions every 30 seconds. The slope of the fluorescence intensity versus time represented the ALP concentration in each sample fraction.

[0274] To maintain linearity, the MUP activity of the bound and unbound fractions of multiple constructs was measured by an initial activity slope over the first 5 minutes. The ratio of MUP activity was determined for each fusion protein as the ratio = (bound activity) / (unbound activity). The results are shown in Figure 14. This ratio showed an exponential response as the length of the polyacidic bone-targeting tag increased, with bone homogenate binding significantly enhanced as individual residues were added to the polyacidic peptide.

[0275] Determination of relative protein affinity to bone homogenate in vitro A multiple-dose assay was developed to rank the relative affinities of bone-binding proteins. For proteins that bind efficiently to bone homogenate, the relative dissociation rates were determined by this kinetic protein-to-bone exchange assay.

[0276] Proteins were individually assessed by incubating a saturating concentration (1 μM) of unlabeled bone-binding protein with 5 mg of bone homogenate in 1.5 mL Eppendorf tubes (e.g., one protein type per tube). After 24 h of incubation with unlabeled protein, the bone homogenate was centrifuged (16,000 rcf, 5 min) to remove excess unbound protein. Bone homogenate saturated with a given bone-binding protein was resuspended in a 0.5 μM solution of the same bone-binding protein labeled with an ALEXA FLUOR® fluorescent probe. After 1, 2, 4, 8, and 24 h of incubation, the bone homogenate was centrifuged, washed three times with PBS, and transferred to a 96-well black plate. The supernatant from the first centrifugation was also collected to quantify the amount of (unbound) fluorescent protein remaining in suspension. A fluorescent plate reader was used to quantify the amount of bound and unbound fluorescently labeled protein at each time point, allowing for a kinetic representation of the dissociation rate of the unlabeled protein.

[0277] Bone homogenate was first saturated with a given protein for 24 hours, thoroughly washed to remove excess unlabeled protein, and finally incubated with the same protein containing a fluorescent label to saturate the surface of the bone homogenate with soluble (fluorescently labeled) protein and track protein exchange. Fluorescent signals were normalized to samples not pretreated with a saturating concentration of unlabeled protein for 24 hours, representing maximum potential binding of the fluorescently labeled protein. VHH001 (circles) showed immediate equilibrium between bound and unbound protein concentrations, indicating a rapid exchange rate or low affinity bone homogenate binder. VHH002 (squares) showed more gradual displacement of the bound protein by the fluorescently labeled protein in solution. ALP031 (triangles) showed minimal displacement of the bound protein by the fluorescently labeled protein, indicating high affinity for bone upon binding.

[0278] The kinetic curve of fluorescent protein accumulation on pre-saturated bone showed that unlabeled protein on bone was replaced by labeled protein (Figures 10A-10B).

[0279] ALP-Fc-(Asp) to bone homogenate 10 and characterization of ALP-Fc binding. An in vitro model was used to characterize the bone binding of ERT-like molecules prior to in vivo administration. To utilize a hydroxyapatite fluorescence-based binding assay, ALP-Fc-(Asp) 10 The ALP-Fc fusion proteins were fluorescently labeled using a commercially available antibody labeling kit (Invitrogen / ThermoFisher). ALEXA FLUOR® dyes were preactivated with succinimidyl esters or tetrafluorophenyl esters, which react with primary amines of proteins. Therefore, fluorescent labeling was not site-specific, and the degree of labeling was 3–5 moles of fluorophore per mole of antibody. After labeling and purification to remove free dye, a 30 nM protein solution was prepared in 0.1% BSA, and bone homogenate binding assays were performed as described.

[0280] The bound / unbound fraction varied according to an approximate Gaussian distribution as a function of bone tag length.

[0281] result Although in vitro characterization of bone homogenate binding indicated in vivo bone targeting (see Example 5), association (binding) only studies could not distinguish between two highly efficient bone-targeting moieties. For example, ALP-Fc-D 10 Both ALP-Fc-D (SEQ ID NO: 31) and VHH002 (SEQ ID NO: 250) quantitatively bound to bone homogenates in vitro and were shown to efficiently bind and be retained in bone in vivo. While these proteins are largely indistinguishable in these experiments, they have distinct affinities for bone based on their dissociation behavior. As shown in Figures 10A-10B, bone-bound VHH002 can be exchanged with excess soluble fluorescently labeled VHH002 in solution. In contrast, ALP-Fc-D 10 excess soluble fluorescently labeled ALP-Fc-D 10 Once bound, ALP-Fc-D shows little dissociation from bone homogenate, even in the presence of 10 showed much higher affinity for bone compared to VHH002, despite having almost the same association (binding) rate. 10 VHH002 efficiently localized to bone and remained in the bone compartment for over 7 days, although their localization mechanisms were likely quite different. VHH002 showed the ability to dissociate from bone, whereas ALP-Fc-D 10 showed significantly slower dissociation after binding. 10 The molecule may remain close to the initial binding site, while VHH002 has more mobility to bind and dissociate throughout the bone tissue.

[0282] A proper understanding of the mechanisms of bone localization and the impact of novel targeting domains on therapeutic efficacy is crucial for developing optimally effective enzyme replacement therapies. Furthermore, tailoring the residence time and dynamics of therapeutic agents in bone can optimize sALP constructs for use in treating diseases such as HPP.

[0283] Example 6 Pharmacokinetic analysis in mouse models Male C57BL / 6 mice (Jackson Labs) aged 11–12 weeks were administered a single 4–7 mg / kg injection of 1 mg / mL sample protein in sterile PBS (without calcium or magnesium) via the tail vein or subcutaneously and followed for 14–21 days. Two interim and one terminal blood draw (cardiac puncture, CO2 anesthesia) were performed on each mouse, staggered within cohorts (four mice in each of four groups, per molecule, per dose type). Blood samples (100 μL, generating 50 μL of plasma after centrifugation) were collected into Li / heparinized tubes at 0.25, 1, 6, 24, 48, 72, 96, 120, 192, 264, 336, and 480 h. Blood samples were stored at 4°C until processed to plasma. Plasma samples were then flash-frozen in liquid nitrogen and stored at -80°C.

[0284] Quantification of enzyme activity in mouse plasma samples: The 4-MU assay used to determine enzyme specific activity in Example 2 was slightly modified to measure activity in samples collected with unknown alkaline phosphatase concentrations. Serum samples were diluted 100- to 6,000-fold in assay buffer (50 mM HEPES, 150 mM NaCl, 1 mM MgCl2, pH 7.4, and 1 mg / mL BSA) to determine active alkaline phosphatase concentrations. Diluted samples were quantified as described in Example 2, except that a standard curve was constructed based on the observed activity of reference standard alkaline phosphatase samples of known activity and concentration. The slope of the fluorescence intensity versus time represented the rate of 4-MU production, which corresponds to the ALP activity in each sample fraction as a function (herein expressed as units / mL serum). Exemplary mouse PK curves for selected compounds after intravenous and subcutaneous administration can be found in Figure 15A (IV) and Figure 15B (subcutaneous) and Table 8.

[0285] [Table 8]

[0286] Example 7 Testing alkaline phosphatase constructs in a murine efficacy model of HPP Preclinical efficacy studies of alkaline phosphatase constructs were conducted using the Akp2GW(- / -) mouse model of HPP. Akp2GW(- / -) mice share the same HPP-inducing TNSALP mutation as the Akp2(- / -) mice previously used in the preclinical evaluation of asphaltase alfa (Narisawa et al. 1997). In these studies, test articles (ALP201 or vehicle (PBS)) were administered subcutaneously in the scapular region from postnatal day 1 to postnatal day 35. Reported outcomes included overall survival, weight gain, hindfoot bone mineralization at day 36 (or at time of death if before the end of study [EOS]), and EOS trough plasma enzyme activity levels (obtained on day 36 for the daily [QD] and weekly [Q1W] groups and day 37 for the every 2-day [Q2D] dose group). Several studies have measured EOS femur and tibia length and mouse femur alkaline phosphatase activity.

[0287] Body weight was also assessed daily as an indicator of the animals' general well-being. Age- and litter-matched PBS-treated WT mice served as the reference control. Group 7 animals received the same daily subcutaneous injections of ALP201 as Groups 3-5 until Day 24. On Day 25, the daily subcutaneous dose of ALP201 was reduced by half-log of the initial dose and maintained until the final treatment dose on Day 35. ALP201 demonstrated statistically significant improvements in survival and bone mineralization compared to the negative (PBS) control.

[0288] Survival curves are shown in Figure 16 and mean body weights are graphed in Figure 17. Correction of bone mineralization phenotype by hind paw mineralization index on day 11 is shown in Figure 18. A tabulation of dose groups for these studies is provided in Table 9.

[0289] [Table 9]

[0290] Bone mineralization results Bone mineralization outcomes on days 36 / 37 were determined by X-ray analysis of the hind paws of treated Akp2GW(- / -) mice. X-ray visualization of hind paw bone mineralization was compared to benchmark X-ray images on day 36, which illustrate four classification categories: no effect, mild defect, moderate defect, and severe defect. A detailed description of these classifications can be found in Table 10. A blinded individual assigned a score to each mouse image, and upon completion, scores were accumulated within individual dose groups.

[0291] [Table 10]

[0292] The distribution of bone mineralization indices in the efficacy studies is shown in Table 11 and graphically in FIG.

[0293] [Table 11]

[0294] survival outcome All PBS-treated Akp2GW(- / -) mice died before day 26 of the study, with a median survival of 20 days. Treatment of Akp2GW(- / -) mice with ALP201 and ALP259 significantly improved 36-day survival in all dose groups compared with PBS vehicle controls (Figure 16). All ALP201-treated groups achieved at least a 69% EOS survival rate, and all QD and Q2D interval-treated groups above the 0.15 mg / kg / day dose recorded overall survival rates of 88% or greater. The survival curves for the ALP201 4.8 mg / kg Q1W group and the asfotase alfa 9.8 mg / kg QD group were very similar (Table 12 and Figure 16).

[0295] [Table 12]

[0296] Measurement of plasma alkaline phosphatase activity and results at the end of the study Mouse plasma samples were collected and prepared at the end of the study (day 36 for the daily (QD) and weekly (Q1W) dosing groups, and day 37 for the every 2 days (Q2D) dosing group). Alkaline phosphatase activity in the plasma samples was assayed using the method outlined in Example 6. The measured reaction rate in the plasma samples was calculated using a standard curve of known enzyme activity to obtain values ​​for plasma alkaline phosphatase activity in units of U / mL. Samples were run in duplicate to compile data for each independent sampling point. The U / mL values ​​were converted to units of mg / L by the following relationship: Concentration in mg / L = (measured activity in U / mL / specific activity of test substance in U / mg) * 1000.

[0297] The distribution of plasma activity levels at the end of the study is shown in FIG. 28 and the mean values ​​are shown in Table 13.

[0298] [Table 13]

[0299] Measurement of alkaline phosphatase activity in bone tissue at the end of the study On study days 36 / 37, mouse femur samples were collected, trimmed of excess tissue, flash-frozen, and stored at -80°C. Prior to analysis, femur samples were transferred to dry ice, placed in a sterile, cold Petri dish, and washed to remove any residual connective tissue. Using osteosynthesis scissors, the ends of the femurs were cut off, and the shafts were transferred to 0.5 mL collection tubes (prepared by puncturing a single hole in the bottom with a 20-gauge syringe needle) on wet ice. The collection tubes were loaded into 1.5 mL centrifuge tubes and spun at 4000 rpm for 30 seconds in a benchtop microcentrifuge to effectively drain the bone marrow from the mouse femurs. The prepared mouse femur shafts were weighed and transferred to 90 microliters of dilution buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 1 mM MgCl2, 1 mg / mL BSA) in a 96-well black opaque assay plate. If the femoral shaft of a mouse was too long to fit into the assay well, the femoral shaft was cut in half and both halves were placed in the same well.

[0300] In each assay plate, controls included femoral shafts from untreated wild-type and AKP2GW(+ / -) heterozygous mice, which were kept at 90°C for 60 minutes before assay, effectively heat-killing alkaline phosphatase activity in these bone samples, which served as negative controls for the assay.

[0301] A standard curve was generated on each plate using a dilution series of alkaline phosphatase activity reference standards with known specific activities and transferred to the assay plates at final assay concentrations of 0, 1.25, 2.5, 5, 10, 20, 35 and 50 ng / mL.

[0302] Prior to the assay, plates were sealed and heated to 37°C for 15 minutes on a Jitterbug heated plate shaker (no shaking). Once warmed to 37°C, 160 mL of substrate solution (50 mM HEPES pH 7.4, 150 mM NaCl, 1 mM MgCl2, 1 mg / mL BSA, 15.625–156.25 mM 4-MUP) was added. The final concentration of 4-MUP in the wells was 10–100 μM in a volume of 250 μL. Progress curves of 4-methylumbelliferone production were measured at 37°C using either a Molecular Devices i3x plate reader (excitation λ = 365 nm and emission λ = 445 nm) or a Molecular Devices Paradigm plate reader (excitation λ = 360 nm filter and emission λ = 445 nm filter).

[0303] The activity slopes for all samples were calculated using linear regression in a commercially available spreadsheet program (Microsoft Excel). If the activity slope became visibly nonlinear over the course of the experiment, the initial linear portion of the sample's progress curve was used to perform a linear regression to obtain the initial reaction rate. The slope of each progress curve was calculated in relative fluorescence units (RFU) / min. An example progress curve for this assay is shown in Figure 30, with the distribution of values ​​from individual subjects plotted in Figures 28 and 29.

[0304] Treated AkpGW(- / -) femurs showed a clear increase in alkaline phosphatase activity (Table 14), demonstrating the ability of ALP201 and ALP259 to deliver enzymatic activity to bone tissue.

[0305] [Table 14]

[0306] Tibia and femur length results At the end of the study, the lengths of the tibia and femur were measured using ImageJ software to obtain analysis points, one at each end of the bone image captured by the Faxitron X-Ray, such that the length of the longest part of the bone was captured by a straight line connecting the analysis points (ImageJ length).

[0307] Bone length in millimeters (mm) was calculated using the following formula:

number

[0308] Compared with daily administration of asfotase alfa at 9.8 mg / kg, AKP2GW(- / -) mice treated with ALP201 daily and Q2D showed statistically significant improvements in both femur and tibia length: femur length p=0.0212 and p=0.0032, respectively, and tibia length p=0.0030 and p=0.0088, respectively, one-way ANOVA, Dunnett's multiple comparisons test for asfotase alfa after 36 / 37 days of treatment (Figure 26, tibia and Figure 27, femur).

[0309] Weight Results Body weights of all mice were monitored during efficacy studies. Across all treatment groups, the body weights of Akp2GW(- / -) mice treated with ALP201 and ALP259 were consistently slightly lower than those of wild-type littermates treated with PBS, but there were no statistically significant differences at any time point during the study or in any group (Figure 17).

[0310] ALP259 demonstrated twofold longer enzymatic activity for pyrophosphate hydrolysis and a 4-5-fold longer in vivo half-life than asfotase alfa after IV administration to C57BL / 6 mice. ALP259 accumulated approximately 50-fold more in plasma than asfotase alfa at the end of the study in the AKP2GW(- / -) mouse model of HPP, and AKP2GW(- / -) bone activity accumulation at the end of the study was 140% of that of ALP201. ALP259 demonstrated clear efficacy on bone mineralization in the AKP2GW(- / -) mouse model, with 100% of mice treated at 4.8 mg / kg q2d dose intervals having normal bone mineralization compared with less than 20% of untreated mice with a normal bone phenotype.

[0311] Example 8 In vivo characterization of bone targeting: In vivo fluorescence imaging in mice Semiquantitative biodistribution studies were performed in nude mice using ALEXA FLUOR® 750-labeled bone-targeted proteins and protein fragments (VHHs). Bone-targeted ALP-Fc fusion proteins and bone-targeted VHHs were fluorescently labeled with ALEXA FLUOR® 750 using the Invitrogen SAIVI kit (covalent coupling via activated succinimidyl ester) and purified with gel exclusion resin to remove unconjugated fluorophores. The purified proteins (suspended in PBS) were injected into nude mice via the tail vein at a dose of approximately 3 mg / kg.

[0312] Female J:NU outbred mice (Jackson Laboratories, Bar Harbor, ME) were administered 3 mg / kg of test article via a 100 μL tail vein injection, normalized to a normalized volume. For in vivo image acquisition, subjects were maintained under 2-3% isoflurane anesthesia on an imaging platform (IVIS® Spectrum Imaging System, PerkinElmer Inc., Waltham, MA). An autoexposure setting with a field of view (FOV) of C, F / Stop 2, medium binning, and an 800 nm emission / 750 nm excitation filter was used for both 2D epi-illumination and 3D trans-illumination acquisition. 2D epi-illumination fluorescence imaging of ex vivo tissue specimens was acquired under identical conditions, except that all specimens of each tissue type were simultaneously acquired in a single image. All animal experiments were conducted in accordance with the provisions of the Animal Welfare Act and the principles of the Guide for the Care and Use of Laboratory Animals.

[0313] Fluorescence image analysis was performed using the manufacturer's 2D / 3D software (Living Image 4.5.1, Perkin Elmer). Regions of interest (ROIs) of uniform area applied to each set of subjects were manually positioned to account for differences in subject position. The longitudinal in vivo image color scale range was normalized across all subjects and time points. The color scale of the ex vivo specimens was determined individually to best represent the fluorescence signal of each tissue set. All images in Figures 4, 8, 12, and 13 are presented applying the following parameters: Opacity = 80, Color Table = "Blue Hot", Smoothing = None, Logarithmic Scale. Read Bias Subtraction, Adaptive FL Background Subtraction, and Flat Field, Cosmic, and Lens Distortion Correct were also applied. The ALEXA FLUOR® 750 signal was calculated as radiant efficiency (p / sec / cm). 2 / sr) / (μW / cm 2)

[0314] Ex vivo bone specimens were individually imaged with a micro-computed tomography X-ray system (Quantum FX μCT, PerkinElmer, Inc., Waltham, MA). The following parameters were used for all acquisitions: voltage = 90 kV, current = 180 μA, FOV = 40 mm, and acquisition time = 17 seconds. Total bone volume (Tt.BV, mm 3 Quantification of bone volume (p / sec / cm) was calculated using the manufacturer-provided software (AccuCT 1.0 Advanced Analysis Software, PerkinElmer, Inc., Waltham, MA) using the bone growth workflow. Normalization of ALEXA FLUOR® 750 signal to bone volume was calculated as (p / sec / cm). 2 / sr) / (μW / cm 2 )) / mm 3 Statistical analyses were performed using commercially available statistical software (GraphPad Prism 7, La Jolla, CA).

[0315] ALP-Fc-D 10 Because ALP-Fc (SEQ ID NO: 31) maximized the possible signal of the bone-targeting protein, and ALP-Fc (SEQ ID NO: 222) showed no binding to bone homogenate, these proteins were used as positive and negative control proteins for in vivo bone-targeting experiments. 10 Intravenous injections of ALP-Fc-D were administered to adolescent nude mice, and the biodistribution of the protein was tracked in vivo throughout the 18-day study. Two-dimensional IVIS live-animal fluorescence imaging was performed on all mice on days 1, 4, 7, 11, 15, and 18. At the end of the 18-day study, mice were sacrificed, and ex vivo specific fluorescence imaging was performed on the liver, kidney, spleen, femur, skull, and spine. Figures 4 and 5A-5B show the total radiative efficiency of the fluorescent protein detected within the whole-body imaging gate. Quantitatively, the fluorescently labeled bone-targeted ALP-Fc-D 10The mean total radiative efficiency of mice treated with ALP-Fc-D was similar to that of mice treated with untargeted ALP-Fc throughout the first 4 days after administration. However, from day 7 onwards, the mean total radiative efficiency of mice treated with ALP-Fc-D was significantly higher than that of mice treated with untargeted ALP-Fc-D. 10 showed significantly higher radiative efficiency than ALP-Fc due to accumulation of targeted proteins in bone and clearance of non-targeted proteins. Figure 5B shows a comparison of the total radiative efficiency of the same treated mice within the spinal cord region of interest. In the spinal cord region of interest, a statistical difference was observed between targeted and non-targeted ALP-Fc fusions within 4 days, and this difference was maintained throughout the 18-day study period. Figure 4 shows representative images from each treatment group at each imaging time point, with the color scale indicating radiative efficiency.

[0316] The newly developed bone homogenate screening assay was validated in vivo. 10 ALP031 and ALP-Fc exhibited two extremes of bone-binding properties: quantitative binding of the protein to bone homogenate and no specific binding to bone homogenate, respectively. In vivo biodistribution studies demonstrated prolonged retention of ALP031 in bone tissue beyond 2 weeks after a single intravenous injection (Figure 12, radiative efficiency indicated by the color scale). In contrast, ALP086 (untargeted ALP-Fc) was undetectable in vivo for 4 to 7 days after injection (Figure 13, radiative efficiency indicated by the color scale). Furthermore, intravenous administration of ALP031 evenly distributed the drug throughout the mineralized tissues of the spine, skull, and femur of mice (Figures 6A-6B). These results confirmed that the bone homogenate screening assay distinguished bone-targeted proteins from non-targeted proteins.

[0317] Further in vivo studies of VHH constructs distinguished between highly efficient bone-binding agents and bone-binding protein intermediates with lower affinity (Figures 7, 8, 9A-9C, and 10A-10B). To this end, it was advantageous to study VHH proteins in vivo because non-targeted VHH proteins were rapidly cleared from the systemic circulation via renal excretion, limiting background signal and allowing early differentiation of bone localization. Figure 8 shows increased whole-body fluorescence signal in mice treated with VHH001 and VHH002 at 24 h post-injection compared to non-targeted controls, as well as enhanced signal for VHH002 compared to VHH001. Over time, signal from mice treated with VHH001 approached baseline levels of non-targeted controls, while signal from mice treated with VHH002 remained significantly increased throughout the 7-day study. This bone-targeting behavior was observed in the developed in vitro screening assay, distinguishing non-targeting VHH control proteins from the intermediate binding of VHH001 and the highly efficient binding of VHH002, demonstrating a verifiable correlation between screening and in vivo bone targeting.

[0318] Example 9 An adult male subject exhibits elevated inorganic pyrophosphate (PPi) levels of approximately 5.82 μM and a mean BOT-2 intensity score of less than 10. The subject is 24 years old and experiences painful lower limbs and difficulty walking. The subject may be diagnosed with HPP and selected for treatment. The subject may undergo x-rays and bone mineral density tests, both of which may show decreased bone mineralization in the legs.

[0319] The formulation containing the polypeptide of SEQ ID NO: 72 can be formulated at 0.1 mg / mL. The formulation can be subcutaneously injected into the subject once a week for 8 weeks at a dose of 0.1 mg / kg / week. The subject can be evaluated for treatment efficacy after 8 weeks of treatment regimen. The subject can observe a reduction in bone pain and normalization of gait. The subject can undergo follow-up X-ray examination and bone mineral density examination, which can show normalization of bone mineralization compared to before treatment. The subject's PPi concentration can be reduced to less than 5 μM, and the BOT-2 intensity score can improve to 12, indicating the effectiveness of treatment with the polypeptide.

[0320] Example 10 An adolescent female subject exhibits elevated inorganic pyrophosphate (PPi) levels of approximately 4.78 μM and a mean 6MWT of less than approximately 70% of the predicted 6MWT value. The subject is 14 years old and is experiencing tooth loss and chronic pain. The subject may be diagnosed with HPP and selected for treatment. The subject may undergo x-rays and bone mineral density tests, both of which may reveal bone mineralization in the subject's teeth and femur.

[0321] A formulation comprising the polypeptide of SEQ ID NO: 123 can be formulated at 0.5 mg / mL. The formulation can be subcutaneously injected into a subject once a week for four weeks at a dose of 0.5 mg / kg / week. The subject can be evaluated for treatment efficacy after four weeks of treatment. The subject can experience relief from chronic pain. The subject can undergo follow-up X-ray and bone mineral density tests, which can show normalization of bone mineralization in the subject's femur compared to pre-treatment. The subject's PPi concentration can decrease to less than 4 μM, and the subject's 6MWT score improves to approximately 85% of predicted value, indicating treatment efficacy with the polypeptide.

[0322] Example 11 An adolescent female subject exhibits elevated inorganic pyrophosphate (PPi) levels of approximately 4.85 μM and a mean 6MWT of less than approximately 65% ​​of the predicted 6MWT value. The subject is 15 years old and is experiencing tooth loss and chronic pain. The subject may be diagnosed with HPP and selected for treatment. The subject may undergo x-rays and bone mineral density tests, both of which may reveal bone mineralization in the subject's teeth and femur.

[0323] A formulation comprising the polypeptide of SEQ ID NO: 177 can be formulated at 0.7 mg / mL. The formulation can be subcutaneously injected into a subject once a week for 6 weeks at a dose of 0.7 mg / kg / week. The subject can be evaluated for treatment efficacy after 6 weeks of treatment regimen. The subject can experience relief from chronic pain. The subject can undergo follow-up X-ray and bone mineral density tests, which can show normalization of bone mineralization in the subject's femur compared to pre-treatment. The subject's PPi concentration can decrease to less than 4 μM, and the subject's 6MWT score improves to about 88% of predicted value, indicating treatment efficacy with the polypeptide.

[0324] Other embodiments The foregoing detailed description and examples have been given for clarity of understanding only, and no unnecessary limitations should be understood therefrom. The invention is not limited to the exact details shown and described, since variations obvious to those skilled in the art will be included within the invention as defined by the claims.

[0325] Unless otherwise noted, all numbers expressing quantities of ingredients, molecular weights, and the like used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0326] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible, however, all numerical values ​​inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.

[0327] All patents, patent applications, including provisional patent applications, published documents, including patent and non-patent publications, and electronically available materials (including, for example, nucleotide sequence submissions in GenBank and RefSeq, and amino acid sequence submissions in, for example, SwissProt, PIR, PRF, PDB, and translations from the annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference. The foregoing detailed description and examples are provided for clarity of understanding only. No unnecessary limitations should be understood therefrom. The disclosure is not limited to the exact details shown and described, since variations obvious to those skilled in the art will be included in the embodiments defined by the claims. The present invention provides, for example, the following items. (Item 1) A polypeptide comprising a recombinant alkaline phosphatase having at least one mutation relative to a naturally occurring alkaline phosphatase, wherein the mutation improves at least one activity or pharmacokinetic property relative to the naturally occurring alkaline phosphatase lacking the at least one mutation. (Item 2) 2. The polypeptide according to item 1, wherein the naturally occurring alkaline phosphatase is tissue non-specific alkaline phosphatase (TNSALP), placental alkaline phosphatase (PALP), germline alkaline phosphatase (GALP) or intestinal alkaline phosphatase (IALP). (Item 3) 3. The polypeptide according to item 2, wherein the TNSALP is a mammalian TNSALP. (Item 4) 4. The polypeptide according to item 3, wherein the mammalian TNSALP is human, gorilla, mouse, rabbit, chimpanzee, cynomolgus monkey, rhesus monkey, orangutan, baboon, rat, bovine, goat or llama TNSALP. (Item 5) 5. The polypeptide according to item 4, wherein the mammalian TNSALP is human TNSALP. (Item 6) 6. The polypeptide according to item 5, having at least 85% sequence identity to amino acids 1 to 491 of SEQ ID NO: 1. (Item 7) 7. The polypeptide of any one of items 1 to 6, wherein the at least one mutation is selected from the group consisting of E108X, M384X, L385X, N213X, and N286X relative to SEQ ID NO: 1, wherein X is any amino acid. (Item 8) 8. The polypeptide of item 7, wherein the at least one mutation is selected from the group consisting of E108S, E108T, E108Q, E108M, E108K, E108L, M384R, L385T, N213Q, and N286Q relative to SEQ ID NO: 1. (Item 9) 9. The polypeptide of item 8, wherein the recombinant alkaline phosphatase has at least one mutation selected from the group consisting of E108T, E108M, and E108L relative to SEQ ID NO: 1. (Item 10) 10. The polypeptide of item 9, wherein the mutation is E108M relative to SEQ ID NO: 1. (Item 11) 11. The polypeptide according to any one of Items 7 to 10, wherein the recombinant alkaline phosphatase has at least two, three, four, or five mutations selected from the group consisting of E108S, E108T, E108Q, E108M, E108K, E108L, M384R, L385T, N213Q, and N286Q relative to SEQ ID NO: 1. (Item 12) 12. The polypeptide of item 11, wherein the recombinant alkaline phosphatase has E108M, N213Q, and N286Q mutations relative to SEQ ID NO: 1. (Item 13) 13. The polypeptide according to any one of items 1 to 12, wherein the recombinant alkaline phosphatase does not have an E108A mutation relative to SEQ ID NO: 1. (Item 14) 3. The polypeptide according to item 2, wherein the naturally occurring alkaline phosphatase is IALP. (Item 15) 15. The polypeptide according to item 14, wherein the IALP is a mammalian IALP. (Item 16) 16. The polypeptide according to item 15, wherein the mammalian IALP is a gorilla, chimpanzee, cynomolgus monkey, rhesus monkey, rat, cow, goat, llama or human IALP. (Item 17) 17. The polypeptide according to item 16, wherein the mammalian IALP is human IALP. (Item 18) 18. The polypeptide of item 17, having at least 85% sequence identity to amino acids 1 to 486 of SEQ ID NO: 4. (Item 19) 19. The polypeptide of any one of items 14 to 18, wherein the recombinant alkaline phosphatase has a W245X mutation relative to SEQ ID NO: 4, where X is any naturally occurring conserved amino acid from different species. (Item 20) 20. The polypeptide of item 19, wherein the recombinant alkaline phosphatase has a W245R mutation relative to SEQ ID NO: 4. (Item 21) 21. The polypeptide of any one of items 14 to 20, wherein the recombinant alkaline phosphatase has a C481X mutation relative to SEQ ID NO: 4, where X is any non-thiol-containing amino acid. (Item 22) 22. The polypeptide of item 21, wherein the recombinant alkaline phosphatase has a C481G mutation relative to SEQ ID NO: 4. (Item 23) 23. The polypeptide of any one of Items 14 to 22, wherein the recombinant alkaline phosphatase has a mutation at a consensus N-linked glycosylation site comprising the sequence asparagine-XZ, where X is any amino acid except P and Z is any amino acid except S or T. (Item 24) 24. The polypeptide of item 23, wherein the asparagine site is mutated to a glutamine residue. (Item 25) 25. The polypeptide according to any one of items 14 to 24, wherein the recombinant alkaline phosphatase has a mutation selected from the group consisting of S429Q, S429H, S429E, and S429D relative to SEQ ID NO:4. (Item 26) 26. The polypeptide of item 25, wherein the recombinant alkaline phosphatase has a S429H mutation relative to SEQ ID NO: 4. (Item 27) 27. The polypeptide according to any one of items 14 to 26, wherein the recombinant alkaline phosphatase has a mutation selected from the group consisting of S428R, S428Q, and S428D relative to SEQ ID NO:4. (Item 28) 28. The polypeptide of item 27, wherein the recombinant alkaline phosphatase has an S428Q or S428D mutation relative to SEQ ID NO: 4. (Item 29) 29. The polypeptide according to any one of Items 1 to 28, wherein the recombinant alkaline phosphatase has at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to at least 50 amino acids of any one of SEQ ID NOs: 7 to 223, 247, and 262 to 264. (Item 30) 30. The polypeptide of any one of items 1 to 29, wherein the at least one activity is selected from the group consisting of increased catalytic activity, increased temperature stability, increased zinc binding, maintenance of activity in zinc-depleted buffer, maintenance of activity at pH 5.0 to 7.5, reduced dimerization, reduced aggregation, and increased manufacturability. (Item 31) 31. The polypeptide of item 30, wherein the increased catalytic activity comprises increased hydrolysis of pyridoxal 5-phosphate and / or pyrophosphate. (Item 32) 32. The polypeptide according to item 30 or 31, wherein the increased catalytic activity is about 4 to about 30 times better than the activity of the naturally occurring alkaline phosphatase. (Item 33) 33. The polypeptide of any one of items 1 to 32, wherein the at least one pharmacokinetic property is selected from the group consisting of increased substrate specificity, increased activity for natural substrates, increased activity for artificial substrates, decreased Km for natural substrates, and increased area under the curve (AUC) per dose. (Item 34) The polypeptide according to Item 33, wherein the artificial substrate is selected from the group consisting of umbelliferyl phosphate, paranitrophenyl phosphate (pNPP), and MUP; and / or the natural substrate is selected from the group consisting of pyridoxal-5'-phosphate, PLP, and PEA. 35. The polypeptide according to any one of items 1 to 34, further comprising a region Y, wherein Y is an amino acid sequence of at least one amino acid. (Item 36) structure Z-ALP-Y-Xn (In the formula, Y is an amino acid sequence of at least one amino acid; Z is absent or is an amino acid sequence of at least one amino acid; Xn is a bone-targeting moiety selected from the group consisting of polyaspartic acid (Dn), polyglutamic acid (En), poly(aspartic acid-alanine-aspartic acid) (DAD)n, poly(aspartic acid-aspartic acid-serine) (DDS)n, poly(aspartic acid-serine-serine (DSS)n, poly(glutamic acid-glutamic acid-serine) (EES)n and VHH, and n=1 to 50; and ALP is the recombinant alkaline phosphatase 36. The polypeptide according to any one of items 1 to 35, comprising: (Item 37) 37. The polypeptide according to item 35 or 36, wherein Y is a fragment crystallizable region (Fc). (Item 38) 38. The polypeptide according to item 37, wherein the Fc region comprises IgG1, IgG2, IgG3, or IgG4, or a chimera thereof. (Item 39) 39. The polypeptide of item 38, wherein the Fc region comprises an IgG2 / 4 chimera. (Item 40) 40. The polypeptide of item 39, wherein the Fc region comprises the sequence of SEQ ID NO: 253 or has at least 85% sequence identity thereto. (Item 41) 41. The polypeptide according to Item 40, comprising or consisting of an amino acid sequence having at least 85%, 90%, 95%, 97%, or 99% sequence identity to any one of SEQ ID NOs: 7 to 223, 247, and 262 to 264. (Item 42) 42. The polypeptide according to Item 41, comprising or consisting of an amino acid sequence having any one of SEQ ID NOs: 7 to 223, 247 and 262 to 264. (Item 43) 42. The polypeptide of item 41, comprising or consisting of an amino acid sequence having at least 85%, 90%, 95%, 97% or 99% sequence identity to any one of SEQ ID NOs: 72, 123, 155 or 177. (Item 44) 44. The polypeptide according to item 43, comprising or consisting of any one of the sequences of SEQ ID NOs: 72, 123, 155 or 177. (Item 45) 44. The polypeptide according to item 43, comprising or consisting of an amino acid sequence having at least 85%, 90%, 95%, 97% or 99% sequence identity to SEQ ID NO: 123. (Item 46) 46. ​​The polypeptide according to item 45, comprising or consisting of the amino acid sequence of SEQ ID NO: 123. (Item 47) 47. The polypeptide according to item 46, consisting of the amino acid sequence of SEQ ID NO: 123. (Item 48) 48. The polypeptide according to any one of items 1 to 47, which is a dimer. (Item 49) A polypeptide comprising alkaline phosphatase and a fragment crystallizable (Fc) region, wherein the Fc region is an IgG2 / 4 chimera. (Item 50) 50. The polypeptide of item 49, wherein the Fc region comprises the sequence of SEQ ID NO: 253 or has at least 85% sequence identity thereto. (Item 51) 51. The polypeptide according to item 49 or 50, wherein the recombinant alkaline phosphatase is selected from the group consisting of TNSALP, IALP, placental alkaline phosphatase (PALP) and germline alkaline phosphatase (GALP). (Item 52) 52. The polypeptide of item 51, wherein the recombinant alkaline phosphatase is a mammalian alkaline phosphatase. (Item 53) 53. The polypeptide according to item 52, wherein the mammalian alkaline phosphatase is human, gorilla, mouse, rabbit, chimpanzee, cynomolgus monkey, rhesus monkey, orangutan, baboon, rat, bovine, goat or llama alkaline phosphatase. (Item 54) 54. The polypeptide according to Item 53, wherein the recombinant alkaline phosphatase comprises any one of the sequences set forth in SEQ ID NOs: 1 to 6 or a fragment thereof. (Item 55) 55. The polypeptide according to Item 54, wherein the recombinant alkaline phosphatase comprises amino acids 1 to 491 of SEQ ID NO: 1 or amino acids 1 to 486 of SEQ ID NO: 4. (Item 56) 56. The polypeptide of any one of items 1 to 38 or 49 to 55, further comprising a bone-targeting moiety Xn selected from the group consisting of Dn, En, (DAD)n, (DDS)n, (DSS)n, (EES)n and VHH, wherein n=1 to 50. (Item 57) 57. The polypeptide according to item 56, wherein n=1 to 30. (Item 58) The bone-targeting moiety comprises: (a) Dn and n = 7 to 10; (b) En and n = 10 to 15; (c) (DAD)n and n = 2 to 4; (d) (DDS) n, and n = 2 to 4; (e) (DSS)n and n=3; or (f) The polypeptide according to Item 57, wherein (EES)n and n=3 to 4. (Item 59) 59. The polypeptide of item 58, wherein the bone-targeting moiety comprises (DAD)3 or (DDS)3. (Item 60) 60. The polypeptide according to any one of Items 56 to 59, wherein at least one complementarity determining region (CDR) of the VHH is substituted with at least one glutamic acid or aspartic acid residue. (Item 61) 61. The polypeptide according to item 60, wherein the at least one CDR is substituted with 2 to 30 glutamic acid or aspartic acid residues. (Item 62) 62. The polypeptide according to item 60 or 61, wherein two or three CDRs are substituted with 2 to 30 glutamic acid or aspartic acid residues. (Item 63) 63. The polypeptide of item 61 or 62, wherein the at least one CDR is substituted with five or seven glutamic or aspartic acid residues. (Item 64) 64. A polynucleotide encoding the polypeptide according to any one of items 1 to 63. (Item 65) 65. A vector comprising the polynucleotide of item 64. (Item 66) 66. A cell comprising the polynucleotide of item 64 or the vector of item 65. (Item 67) A method for producing the polypeptide according to any one of Items 1 to 63, comprising: (a) providing a cell transformed with the polynucleotide of item 64 or the vector of item 65, wherein the polynucleotide is positioned for expression in the cell; (b) culturing the transformed cells under conditions suitable for expression of the polynucleotide, resulting in expression of the polypeptide; and (c) isolating the polypeptide A method comprising: (Item 68) 64. A pharmaceutical composition comprising the polypeptide according to any one of items 1 to 63 and a pharmaceutically acceptable carrier. (Item 69) 69. The pharmaceutical composition according to item 68, wherein the pharmaceutically acceptable carrier comprises sodium chloride and / or sodium phosphate. (Item 70) 70. The pharmaceutical composition of item 69, comprising about 150 mM sodium chloride and / or about 25 mM sodium phosphate at a pH of about 7.4. (Item 71) 71. The pharmaceutical composition according to any one of items 68 to 70, formulated at a dosage of about 0.1 mg / mL to about 10 mg / mL. (Item 72) 72. The pharmaceutical composition according to any one of items 68 to 71, formulated in a volume of about 0.1 mL to about 50 mL. (Item 73) 74. A method for treating a disease selected from hypophosphatasia (HPP), bone fracture, osteoporosis, sclerosteosis, chondrocalcinosis, hypotonia, Duchenne muscular dystrophy, tracheobronchomalacia, stroke, neurofibromatosis and craniosynostosis in a subject in need thereof, the method comprising administering to the subject the polypeptide of any one of items 1 to 63 or the pharmaceutical composition of any one of items 68 to 73. (Item 74) 74. The method of claim 73, wherein the polypeptide is administered in an amount and for a period sufficient to treat the disease. (Item 75) 75. The method of claim 73 or 74, wherein the treatment enhances bone formation in the subject. (Item 76) 76. The method of any one of items 73 to 75, wherein the polypeptide is administered at a dosage of about 0.01 mg / kg to about 20 mg / kg. (Item 77) Item 77. The method of item 76, wherein the polypeptide is administered at a dosage of about 0.1 mg / kg to about 10 mg / kg. (Item 78) 78. The method of any one of items 73 to 77, wherein the polypeptide is administered once per day, week, month or year. (Item 79) 79. The method of any one of items 73 to 78, wherein the polypeptide is administered for at least one day, one week, one month, one year or more. (Item 80) 80. The method according to any one of items 73 to 79, wherein the composition is administered subcutaneously, intravenously, intramuscularly, sublingually, intrathecally or intradermally. (Item 81) 81. The method of claim 80, wherein the composition is administered subcutaneously or intravenously. (Item 82) 82. The method according to any one of items 73 to 81, wherein the subject is a human. (Item 83) 83. The method of item 82, wherein the human is a neonate, a child, an adolescent, or an adult. (Item 84) 84. The method of any one of items 73 to 83, wherein prior to administration of the recombinant polypeptide, the subject is characterized as having an average 6-minute walking distance of about 350 meters or less. (Item 85) 85. The method of any one of items 73 to 84, wherein administration of the recombinant polypeptide promotes an increase in the average 6-minute walking distance by the subject of at least 100 meters or more. (Item 86) 86. The method of any one of items 73 to 85, wherein the subject exhibits an average walking distance of about 500 meters or more in 6 minutes after administration of the recombinant polypeptide. (Item 87) 87. The method of any one of items 73 to 86, wherein the subject exhibits decreased dependency on an assisted mobility device after administration of the recombinant polypeptide. (Item 88) Item 88. The method of item 87, wherein the assistive mobility device is at least one device selected from the group consisting of a walker, a wheelchair, a brace, a crutch, and an orthotic. (Item 89) 89. The method of any one of items 73 to 88, wherein prior to administration of the recombinant polypeptide, the subject is characterized as having a plasma PPi concentration of about 4.5 μM or greater. (Item 90) Item 91. The method of any one of Items 73 to 89, wherein administration of the recombinant polypeptide promotes a median decrease of at least about 1 μM in PPi concentration in a plasma sample from the subject. 91. The method according to any one of items 73 to 90, wherein the subject exhibits a plasma PPi concentration of about 2 μM to about 5 μM after administration of the recombinant polypeptide. (Item 92) i) the subject is characterized as being 0-14 days old and having a plasma ALP concentration of about 90 U / L or less prior to administration of the recombinant polypeptide; ii) the subject is characterized as being between 15 days and less than 1 year of age and having a plasma ALP concentration of about 134 U / L or less prior to administration of the recombinant polypeptide; iii) the subject is characterized as being between about 1 and less than 10 years of age and having a plasma ALP concentration of about 156 U / L or less prior to administration of the recombinant polypeptide; iv) the subject is characterized as being about 10 to about 13 years of age and having a plasma ALP concentration of about 141 U / L or less prior to administration of the recombinant polypeptide; v) the subject is female and about 13 to about 15 years of age and is characterized as having a plasma ALP concentration of about 62 U / L or less prior to administration of the recombinant polypeptide; vi) the subject is male and about 13 to about 15 years of age and is characterized as having a plasma ALP concentration of about 127 U / L or less prior to administration of the recombinant polypeptide; vii) the subject is characterized as being female and about 15 to about 17 years of age and having a plasma ALP concentration of about 54 U / L or less prior to administration of the recombinant polypeptide; viii) the subject is characterized as being male and about 15 to about 17 years of age, and having a plasma ALP concentration of about 89 U / L or less prior to administration of the recombinant polypeptide; ix) the subject is characterized as being about 17 years of age or older and having a plasma ALP concentration of about 48 U / L or less prior to administration of the recombinant polypeptide; or x) The method of any one of items 73 to 91, wherein the subject is about 17 years of age or older and is characterized as having a plasma ALP concentration of about 59 U / L or less prior to administration of the recombinant polypeptide. (Item 93) 93. The method of any one of items 73 to 92, wherein administration of the recombinant polypeptide promotes a median increase in ALP concentration in plasma samples from the subject of at least about 100 U / L or more. (Item 94) i) the subject is characterized as being 0-14 days old and having a plasma ALP concentration of about 273 U / L or greater after administration of the recombinant polypeptide; ii) the subject is characterized as being between 15 days and less than 1 year of age and having a plasma ALP concentration of about 518 U / L or greater after administration of the recombinant polypeptide; iii) the subject is characterized as being about 1 year old to less than about 10 years old and having a plasma ALP concentration of about 369 U / L or greater after administration of the recombinant polypeptide; iv) the subject is characterized as being about 10 to about 13 years of age and having a plasma ALP concentration of about 460 U / L or greater after administration of the recombinant polypeptide; v) the subject is female and about 13 to about 15 years of age, and is characterized as having a plasma ALP concentration of about 280 U / L or greater after administration of the recombinant polypeptide; vi) the subject is male and about 13 to about 15 years of age, and is characterized as having a plasma ALP concentration of about 517 U / L or greater after administration of the recombinant polypeptide; vii) the subject is characterized as being female and about 15 to about 17 years of age, and having a plasma ALP concentration of about 128 U / L or greater after administration of the recombinant polypeptide; viii) the subject is characterized as being male and about 15 to about 17 years of age, and having a plasma ALP concentration of about 365 U / L or greater after administration of the recombinant polypeptide; ix) the subject is characterized as being female and about 17 years of age or older and having a plasma ALP concentration of about 95 U / L or greater after administration of the recombinant polypeptide; or x) The method of any one of items 73 to 93, wherein the subject is male, is about 17 years of age or older, and is characterized as having a plasma ALP concentration of about 164 U / L or greater after administration of the recombinant polypeptide. (Item 95) 95. The method of any one of items 73 to 94, wherein prior to administration of the recombinant polypeptide, the subject is characterized as having an average Bruininks-Oseretsky Motor Skills Test, Second Edition (BOT-2) strength score of about 10 or less. (Item 96) 96. The method of claim 95, wherein prior to administration of the recombinant polypeptide, the subject is characterized as having an average BOT-2 running speed and agility score of about 5 or less. (Item 97) 97. The method of any one of items 73 to 96, wherein administration of the recombinant polypeptide results in a mean BOT-2 intensity score of about 10 or greater in the subject. (Item 98) 98. The method of any one of items 73 to 97, wherein administration of the recombinant polypeptide results in a mean BOT-2 running speed and agility score of about 5 or greater in the subject. (Item 99) 99. The method of any one of items 73 to 98, wherein prior to administration of the recombinant polypeptide, the subject is characterized as having an average Child Health Assessment Questionnaire (CHAQ) index score of about 0.8 or greater. (Item 100) 99. The method of any one of items 73 to 99, wherein administration of the recombinant polypeptide results in a mean CHAQ index score of about 0.5 or less in the subject. (Item 101) 101. The method of any one of items 73 to 100, wherein prior to administration of the recombinant polypeptide, the subject is characterized as having an average Pediatric Outcomes Data Collection Instrument (PODCI) score of about 40 or less. (Item 102) 102. The method of any one of items 3 to 101, wherein administration of the recombinant polypeptide results in a mean PODCI score of about 40 or greater in the subject. (Item 103) 103. The method of any one of items 73 to 102, wherein prior to administration of the recombinant polypeptide, the subject is characterized as having an average muscle strength grade of less than about 5. (Item 104) 104. The method of any one of items 73 to 103, wherein administration of the recombinant polypeptide results in an average increase of about 1 or more in muscle strength grade of the subject. (Item 105) 105. The method of any one of items 73 to 104, wherein prior to administration of the recombinant polypeptide, the subject is characterized as having a mean handheld dynamometry (HHD) value that is less than about 80% of the predicted HHD value. (Item 106) 106. The method of any one of items 73 to 105, wherein administration of the recombinant polypeptide results in a mean HHD value in the subject that is about 80% or greater than the predicted HHD value. (Item 107) 107. The method of claim 105 or 106, wherein the HHD value represents the subject's grip strength, knee flexion, knee extension, hip flexion, hip extension or hip abduction.

Claims

[Claim 1] The invention as set forth in the drawings.

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