Alkaline phosphatase polypeptides and methods of use thereof

Abstract

The present invention features polypeptides comprising soluble alkaline phosphatase, mutants, fragments, fusion proteins thereof, and methods of use thereof, for the treatment of bone mineralization disorders, such as hypophosphatasia (HPP) and symptoms thereof. These polypeptides include soluble alkaline phosphatase (sALP) or fragments thereof, derived from naturally occurring alkaline phosphatase (ALP).

Description

Background Technology

[0001] Hypophosphatase disorder (HPP) is a rare, inherited skeletal disorder, with the most severe form occurring in 1 in 100,000 newborns. This disorder is typically caused by a loss-of-function mutation in the gene encoding tissue-specific alkaline phosphatase (TNSALP). Symptoms and severity of HPP vary widely, ranging from premature tooth loss to almost complete lack of bone mineralization in utero. The presentation of HPP differs significantly between patients and between patients of different ages. Many patients with HPP exhibit skeletal changes, short stature, chronic pain, lower extremity pain, muscle weakness, gait disturbances, and premature, non-traumatic tooth loss.

[0002] Asfotase alfa (STRENSIQ) ® Alexion Pharmaceuticals, Inc., a recombinant enzyme replacement therapy (ERT) comprising a soluble fragment of TNSALP, is the first ERT available for patients with HPP. Asfor enzyme α has shown transformative effects on the most severe forms of HPP, as evidenced by improvements in bone mineralization and density, as well as respiratory and motor function, cognitive development, and muscle strength (Whyte et al.). New Engl. J. Med [New England Journal of Medicine] 366:904-913, 2012. While the safety and therapeutic benefits of asfozyme alfa have been established, adherence to multiple injection regimens can be challenging, especially in pediatric patients. The most common side effects of subcutaneous administration three to six times per week are injection site reactions (i.e., itching, pain, and erythema). Therefore, improvements to enhance quality of life are desired, for example, by reducing the volume and frequency of administration and increasing adherence. Consequently, novel compositions and methods for treating HPP and related bone mineralization disorders would be beneficial.

[0003] Summary of the invention.

[0004] In one aspect, the feature is a polypeptide comprising a recombinant alkaline phosphatase having at least one mutation relative to a naturally occurring alkaline phosphatase. Compared to a naturally occurring alkaline phosphatase without the at least one mutation, the mutation may improve at least one activity or pharmacokinetic (PK) property.

[0005] Naturally occurring alkaline phosphatases can be tissue-nonspecific alkaline phosphatase (TNSALP), placental alkaline phosphatase (PALP), reproductive alkaline phosphatase (GALP), or intestinal alkaline phosphatase (IALP). 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 with amino acids 1-491 of SEQ ID NO: 1.

[0006] 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, recombinant alkaline phosphatase may have the E108M, N213Q, and N286Q mutations relative to SEQ ID NO: 1. Recombinant alkaline phosphatase may not have the E108A mutation relative to SEQ ID NO: 1.

[0007] The at least one mutation in the ALP described herein that improves at least one activity or pharmacokinetic (PK) property may be present in a specific region of the enzyme. This mutation may be present in the extracellular domain of the alkaline phosphatase. For example, the mutation may be present in the crown domain, catalytic domain, or dimerization domain. The mutation may be present in the GPI anchoring domain (if included). Based on alignment and / or sequence homology, the polypeptide may include mutations present in amino acids 1-491 or 1-486 of human TNSALP or similar ALP positions. For example, the polypeptide may have mutations at 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 may have mutations at 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 may have mutations between positions 108, 213, 286, and / or 429 relative to SEQ ID NO: 1. In some embodiments, the mutation is within the sequence of ALP, and the mutation is not a non-natural amino acid segment present at the N- or C-terminal domain of ALP.

[0008] Naturally occurring alkaline phosphatases can be IALPs (e.g., mammalian IALPs such as those of gorillas, chimpanzees, cynomolgus monkeys, rhesus monkeys, rats, cattle, goats, llamas, or humans). The polypeptide can have at least 85% (e.g., 90%, 95%, 97%, 99%, or 100%) sequence identity with amino acids 1-486 of SEQ ID NO: 4. Recombinant alkaline phosphatases can have a W245X mutation relative to SEQ ID NO: 4, where X is any naturally occurring conserved amino acid from a different species. Recombinant alkaline phosphatases can have a W245R mutation relative to SEQ ID NO: 4. Recombinant alkaline phosphatases can have a C481X mutation relative to SEQ ID NO: 4, where X is any amino acid that does not contain thiols. Recombinant alkaline phosphatases can have a C481G mutation relative to SEQ ID NO: 4.

[0009] Recombinant alkaline phosphatases can have mutations at a common N-linked glycosylation site. This common N-linked glycosylation site comprises a motif with the sequence asparagine-XZ, where X is any amino acid other than P, and Z is any amino acid other than S or T. The asparagine site can be mutated to a glutamine residue.

[0010] Recombinant alkaline phosphatase may contain mutations selected from the group consisting of S429Q, S429H, S429E, and S429D relative to SEQ ID NO: 4. Recombinant alkaline phosphatase may also contain mutations selected from the group consisting of S428R, S428Q, and S428D relative to SEQ ID NO: 4.

[0011] The recombinant alkaline phosphatase may have at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with at least 50 amino acids (e.g., at least 100, 150, 200, 250, 300, 350, 400, 450 or more) of the sequence of any one of SEQ ID NO: 7-223, 247 and 262-264.

[0012] At least one activity improved by mutation can 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 5.0–7.5, reduced dimerization, reduced aggregation, and increased manufacturability. Increased catalytic activity may include increased hydrolysis of pyridoxal 5-phosphate and / or pyrophosphate. The increased catalytic activity may be about 2 to about 30 times better than the activity of naturally occurring alkaline phosphatases. This at least one PK property improved by mutation can be selected from the group consisting of: increased substrate specificity, increased activity to natural substrates, increased activity to artificial substrates, decreased Km to natural substrates, and increased area under the curve (AUC) per dose. Exemplary artificial substrates are 4-methylumbelliferyl phosphate (4-MUP), umbelliferyl ketone phosphate, and p-nitrophenyl phosphate (pNPP), and exemplary natural substrates are pyridoxal-5'-phosphate, PLP, and PEA.

[0013] In some embodiments, the polypeptide may further include region Y, wherein Y is an amino acid sequence of at least one amino acid. Y may be a fragment crystallizable region (Fc). The Fc region may include IgG1, IgG2, IgG3, or IgG4, or a chimera thereof. For example, the Fc region may include an IgG2 / 4 chimera. The Fc region may include the sequence of SEQ ID NO: 253 or have at least 85% (e.g., 90%, 95%, 97%, 99%, or 100%) sequence identity with it.

[0014] The polypeptide may further include a bone-targeting moiety. A polypeptide comprising 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 has an amino acid sequence of at least one amino acid; Xn is the bone-targeting moiety selected from the group consisting of: polyaspartic acid (Dn), polyglutamic acid (En), poly(aspartic-alanine-aspartic acid)(DAD)n, poly(aspartic-aspartic-serine)(DDS)n, poly(aspartic-serine-serine)(DSS)n, poly(glutamic acid-glutamic acid-serine)(EES)n, and VHH, and n = 1 to 50; and ALP is a recombinant alkaline phosphatase.

[0015] The polypeptide may have the structure Y-ALP-Z-Xn, or any of its topological arrangements (e.g., Xn-Y-ALP-Z and Y-Xn-ALP-Z).

[0016] The polypeptide may include or consist of an amino acid sequence having at least 85% (e.g., 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NO: 7-223, 247, and 262-264 (e.g., any one of SEQ ID NO: 72, 123, 155, or 177). For example, the polypeptide may include or consist of a sequence of any one of SEQ ID NO: 72, 123, 155, or 177. The polypeptide may include any sALP catalytic domain, Fc IgG isotype, or bone-targeting motif listed in Table 1, and their topological arrangements. The polypeptide may include or consist of an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with SEQ ID NO: 123. The polypeptide may include or consist of SEQ ID NO: 123.

[0017] The polypeptide may include or consist of an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with SEQ ID NO: 177. The polypeptide may include or consist of SEQ ID NO: 177.

[0018] The polypeptide may include or consist of an amino acid sequence having at least 85% (e.g., 90%, 95%, 97%, 99%, or 100%) sequence identity with SEQ ID NO: 260 or 261. The polypeptide may include or consist of the sequence of SEQ ID NO: 260. The polypeptide may include or consist of the sequence of SEQ ID NO: 261.

[0019] The polypeptide may include a secretion signal peptide. The polypeptide may include, or consist of, an amino acid sequence having at least 85% (e.g., 90%, 95%, 97%, 99%, or 100%) sequence identity with SEQ ID NO: 263 or 264. The polypeptide may include, or consist of, the sequence of SEQ ID NO: 263. The polypeptide may include, or consist of, the sequence of SEQ ID NO: 264.

[0020] In some embodiments, the polypeptide is a dimer. Alternatively, the polypeptide may be a monomer.

[0021] The product is further characterized by 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 with it. 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 vicuña alkaline phosphatase). The recombinant alkaline phosphatase may comprise the sequence of any one of SEQ ID NO: 1-6 or a fragment thereof. For example, the recombinant alkaline phosphatase may comprise amino acids 1-491 of SEQ ID NO: 1 or amino acids 1-486 of SEQ ID NO: 4.

[0022] In some embodiments of any of the foregoing aspects, the polypeptide further comprises a bone-targeting portion Xn, selected from the group consisting of Dn, En, (DAD)n, (DDS)n, (DSS)n, (EES)n, and VHH, wherein n = 1 to 50 (e.g., 1 to 30). In some specific embodiments, the bone-targeting portion may be Dn, wherein n = 7 to 10; En, wherein n = 10 to 15; (DAD)n, wherein n = 2 to 4; (DDS)n, wherein n = 2 to 4; (DSS)n, wherein n = 3; or (EES)n, wherein n = 3 to 4. For example, the bone-targeting portion may include (DAD)3 or (DDS)3.

[0023] The bone-targeting portion of the VHH may include one or more substitutions. For example, at least one (e.g., two or three) complementarity-determining regions (CDRs) of the VHH may be substituted with at least one (e.g., 2 to 30, such as 5 or 7) glutamic acid or aspartic acid residues.

[0024] In some embodiments, the polypeptides described herein are post-translational modified (e.g., glycosylated or sialylated).

[0025] The feature further comprises a polynucleotide encoding a polypeptide of any of the above aspects, a carrier containing the polynucleotide, and a cell (e.g., a mammalian cell, such as a CHO cell or a HEK293 cell) containing the polynucleotide or the carrier. The polynucleotide may encode an amino acid sequence having at least 85% (e.g., 90%, 95%, 97%, 99%, or 100%) sequence identity with any of SEQ ID NO: 7-223, 247, and 262-264 (e.g., any of SEQ ID NO: 72, 123, 155, or 177). The polynucleotide may have at least 85% (e.g., 90%, 95%, 97%, 99%, or 100%) sequence identity with any of SEQ ID NO: 265-268. The polynucleotide may include or consist of any of SEQ ID NO: 265-268. The polynucleotide may include or consist of SEQ ID NO: 265. The polynucleotide may include or consist of SEQ ID NO: 266. The polynucleotide may include or consist of SEQ ID NO: 267. The polynucleotide may include or consist of SEQ ID NO: 268.

[0026] In some embodiments, the polynucleotide is not or does not include the sequence of SEQ ID NO: 269.

[0027] In some embodiments, the alkaline phosphatase does not include alkaline phosphatase of Asforase α (e.g., amino acids 1-485) of SEQ ID NO: 269.

[0028] In some embodiments, the polypeptide is not a polypeptide having the amino acid sequence of SEQ ID NO: 269, and does not include alkaline phosphatase of Asforase α (e.g., amino acids 1-485) of SEQ ID NO: 269.

[0029] The feature also includes a method for producing a polypeptide of any of the above aspects by: providing cells (e.g., mammalian cells, such as CHO cells or HEK293 cells) transformed with a polynucleotide or carrier encoding the polypeptide, such that the polynucleotide is localized in the cells for expression; culturing the transformed cells under conditions suitable for expressing the polynucleotide, wherein the culture results in the expression of the polypeptide; and isolating the polypeptide.

[0030] A further feature is a pharmaceutical composition comprising a polypeptide of any one of the foregoing aspects and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may include sodium chloride and / or sodium phosphate. The composition may include about 150 mM sodium chloride and / or about 25 mM sodium phosphate at a pH of about 7.4.

[0031] The composition can be formulated at a dose of about 0.1 mg / mL to about 10 mg / mL. The composition can be formulated into a volume of about 0.1 mL to about 50 mL (e.g., about 0.1 to about 10 mL).

[0032] On the other hand, it is characterized by a method of treating a disease or one or more symptoms thereof selected from the group consisting of: hypophospholipase syndrome (HPP), fracture, osteoporosis, ossifying sclerosis, chondrocalcinosis, hypotonia, Duchenne muscular dystrophy, tracheobronchomalacia, seizures, neurofibromatosis (e.g., NF-1), and craniosynostosis. The polypeptide may be administered in an amount and for a duration sufficient to treat the disease or alleviate one or more of its symptoms. This treatment may enhance bone formation in the subject. The polypeptide may be used to treat myasthenia gravis.

[0033] The peptide can be administered at a dose of about 0.01 mg / kg to about 60 mg / kg (e.g., about 0.1 mg / kg to about 50 mg / kg, or about 0.1 mg / kg to about 20 mg / kg, or about 0.1 mg / kg to about 10 mg / kg). The peptide can be administered once daily, weekly, monthly, or annually (e.g., once weekly). The peptide can be administered at a dose 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 peptide can be administered for at least one day, one week, one month, one year, or longer.

[0034] This polypeptide can be administered subcutaneously, intravenously, intramuscularly, sublingually, intrathecally, or intradermally. In particular, the polypeptide or a composition containing the polypeptide can be administered subcutaneously.

[0035] Subjects can be human subjects, such as newborns, children, adolescents, or adults.

[0036] Prior to administration of the recombinant peptide, subjects could be characterized as having an average walking distance of approximately 350 meters or less in 6 minutes. Administration of the recombinant peptide could promote an increase in the average walking distance in 6 minutes of at least 100 meters or more. After administration of the recombinant peptide (e.g., after a treatment period of 1 month to 1 year), subjects could exhibit an average walking distance of approximately 500 meters or more in 6 minutes.

[0037] After administration of the recombinant peptide, subjects may exhibit reduced dependence on assistive mobility devices (e.g., walkers, wheelchairs, braces, canes, and orthotics).

[0038] Prior to administration of the recombinant peptide, subjects could be characterized with a plasma PPi concentration of approximately 4.5 μM or greater. Administration of the recombinant peptide promoted a median reduction in PPi concentration of at least approximately 1 μM in plasma samples from subjects. Following administration of the recombinant peptide, subjects could exhibit plasma PPi concentrations of approximately 2 μM to approximately 5 μM.

[0039] In some embodiments, the subject is aged 0 to 14 days and is characterized by having a plasma ALP concentration of about 90 U / L or less prior to administration of the recombinant peptide; the subject is aged 15 days to less than 1 year and is characterized by having a plasma ALP concentration of about 134 U / L or less prior to administration of the recombinant peptide; the subject is aged about 1 year to less than 10 years and is characterized by having a plasma ALP concentration of about 156 U / L or less prior to administration of the recombinant peptide; the subject is aged about 10 to about 13 years and is characterized by having a plasma ALP concentration of about 141 U / L or less prior to administration of the recombinant peptide; the subject is female and aged about 13 to about 15 years and is characterized by having a plasma ALP concentration of about 62 U / L or less prior to administration of the recombinant peptide; the subject is male and aged about 13 to about 15 years and is characterized by having a plasma ALP concentration of about 127 U / L or less prior to administration of the recombinant peptide; the subject is female and aged about 15 to about 17 years and is characterized by having a plasma ALP concentration of about 54 U / L or less prior to administration of the recombinant peptide. The subjects are male and aged from about 15 to about 17 years, and are characterized by having a plasma ALP concentration of about 89 U / L or less prior to administration of the recombinant peptide; the subjects are about 17 years or older, and are characterized by having a plasma ALP concentration of about 48 U / L or less prior to administration of the recombinant peptide; or the subjects are about 17 years or older, and are characterized by having a plasma ALP concentration of about 59 U / L or less prior to administration of the recombinant peptide.

[0040] Administration of the recombinant peptide can promote a median increase in ALP concentration of at least about 100 U / L or greater in plasma samples from subjects.

[0041] In some embodiments, the subject is aged 0 to 14 days and, after administration of the recombinant peptide, is characterized by having a plasma ALP concentration of about 273 U / L or greater; the subject is aged 15 days to less than 1 year and, after administration of the recombinant peptide, is characterized by having a plasma ALP concentration of about 518 U / L or greater; the subject is aged about 1 year to less than about 10 years and, after administration of the recombinant peptide, is characterized by having a plasma ALP concentration of about 369 U / L or greater; the subject is aged about 10 to about 13 years and, after administration of the recombinant peptide, is characterized by having a plasma ALP concentration of about 460 U / L or greater; the subject is female and aged about 13 to about 15 years and, after administration of the recombinant peptide, is characterized by having a plasma ALP concentration of about 280 U / L or greater; the subject is male and aged about 13 to about 15 years and, after administration of the recombinant peptide, is characterized by having a plasma ALP concentration of about 517 U / L or greater; the subject is female and aged about 15 to about 17 years and, after administration of the recombinant peptide, is characterized by having a plasma ALP concentration of about 128 U / L or greater. The subject is male and aged from about 15 to about 17 years, and is characterized by having a plasma ALP concentration of about 365 U / L or greater after administration of the recombinant peptide; the subject is female and aged from about 17 years or older, and is characterized by having a plasma ALP concentration of about 95 U / L or greater after administration of the recombinant peptide; or the subject is male and aged from about 17 years or older, and is characterized by having a plasma ALP concentration of about 164 U / L or greater after administration of the recombinant peptide.

[0042] Prior to administration of the recombinant peptide, subjects could be characterized as having a mean Bunisk Test of Motor Skills Version 2 (BOT-2) strength score of approximately 10 or lower. Prior to administration of the recombinant peptide, subjects could be characterized as having a mean BOT-2 running speed and agility score of approximately 5 or lower. Administration of the recombinant peptide resulted in subjects having a mean BOT-2 strength score of approximately 10 or higher. Administration of the recombinant peptide resulted in subjects having a mean BOT-2 running speed and agility score of approximately 5 or higher.

[0043] Prior to administration of the recombinant peptide, subjects could be characterized as having a mean Childhood Health Assessment Questionnaire (CHAQ) score of approximately 0.8 or higher. Administration of the recombinant peptide could result in a mean CHAQ score of approximately 0.5 or lower for the subjects.

[0044] Prior to administration of the recombinant peptide, subjects could be characterized as having a mean Pediatric Outcome Data Collection Tool (PODCI) score of approximately 40 or lower. Administration of the recombinant peptide resulted in a mean PODCI score of approximately 40 or higher for the subjects.

[0045] Prior to administration of the recombinant peptide, subjects could be characterized as having an average muscle strength grade of less than approximately 5. Administration of the recombinant peptide resulted in an average increase in the subjects' muscle strength grade of approximately 1 grade or more.

[0046] Prior to administration of the recombinant peptide, subjects could be characterized as having an average hand dynamometer (HHD) value that was approximately 80% lower than the expected HHD value. Administration of the recombinant peptide resulted in subjects having an average HHD value that was approximately 80% or higher of the expected HHD value. HHD values ​​can represent a subject's grip strength, knee flexion, knee extension, hip flexion, hip extension, or hip abduction.

[0047] In another aspect, the present invention is characterized by a method for determining the activity (e.g., binding activity) of a polypeptide described herein, the polypeptide comprising sALP or a sALP fusion polypeptide (e.g., a polypeptide having the sequence of any one of SEQ ID NO: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity with and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein). The method may include providing a bone homogenate, for example, derived from bone (e.g., femur, such as bone from, for example, mammals (e.g., mice or humans)). A bone homogenate can be obtained by acquiring bone tissue and processing the tissue, for example, by removing connective tissue with collagenase. The bone can be purified, for example, by removing bone marrow and / or other contaminants. Once the bone is dried, the bone homogenate can be homogenized, for example, by grinding, crushing, and / or slicing the bone, for example, until a bone tissue homogenate is obtained. The bone homogenate can then be resuspended in a liquid (e.g., PBS) for subsequent determination. The peptides 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 longer. The sample can then be vortexed and / or centrifuged to obtain protein fractions in the supernatant or bound to the bone homogenate. The ratio of bound protein to unbound protein can then be measured and quantified to determine the binding affinity of the peptide to the homogenate.

[0048] definition The terms "one" or "more" preceding a noun represent one or more of the specific noun. For example, the phrase "mammalian cell" means "one or more mammalian cells".

[0049] The term “about” is intended to describe the variation due to experimental error; in some embodiments, “about” represents ±10% of the value. All measurements reported herein should be understood to be modified by the term “about”, whether or not the term is explicitly used, unless otherwise explicitly stated.

[0050] The term "bone-targeting fraction" refers to an amino acid sequence of at least 3 amino acid residues that has sufficient affinity for the bone matrix, such that when used alone, the bone-targeting fraction exhibits an affinity for the bone matrix of at least approximately 1 x 10⁻⁶. -5 M or better (e.g., about 10) -6 M, approximately 10 -7 M, approximately 10 -8 M, approximately 10 -9 In vivo binding affinity (M or better).

[0051] As used interchangeably in this article, the terms "Brief Pain Inventory-ShortForm" and "BPI-SF" refer to a method for measuring pain in patients (especially those with HPP, e.g., patients aged approximately 13 years or older). BPI-SF was developed by Cleeland and Ryan (…). Ann Acad Med Singapore The self-reported pain measurement described in [Singapore Medical College Yearbook], 23(2), 129-138; 1994], is hereby incorporated by reference in its entirety. The BPI-SF is a questionnaire designed to assess the severity of pain and its impact on daily functioning. The BPI-SF consists of 11 items and uses a numerical rating scale to assess pain severity (4 items) and pain interference (7 items) in the 24 hours prior to questionnaire administration. The BPI-SF questionnaire provides information on the intensity of pain and the extent to which pain interferes with daily functioning in patients (e.g., HPP patients aged approximately 13 years or older) on a numerical rating scale from 0 (no pain) to 10 (severe pain or significant interference caused by pain); lower scores indicate better quality of life outcomes and reduced pain. For example, the BPI-SF score for HPP adolescents and adults is a combination of the 11 pain assessments.

[0052] As used herein, the terms “Bruininks Test of Motor Skills, Version 2” and “BOT-2” refer to the second version of a standardized test for gross and fine motor skills in individuals with HPP (e.g., children with HPP aged approximately 5 to approximately 12 years, adolescents with HPP aged approximately 13 to approximately 17 years, or adults with HPP aged approximately 18 years or older). See Bruininks, RH (2005). Bruininks-Oseretsky Test of Motor Proficiency[Bruni's Motor Skills Test] (BOT-2) Minneapolis, MN: Pearson Assessment, Inc., which is incorporated herein by reference in its entirety. The BOT-2 is administered alone to assess gross and fine motor skills in a range of patients. For example, the BOT-2 can be used to assess physical impairment and mobility limitations in patients with HPP (e.g., children with HPP aged approximately 5 to approximately 12 years, adolescents with HPP aged approximately 13 to approximately 17 years, or adults with HPP aged approximately 18 years or older). The BOT-2 provides a comprehensive BOT-2 score in the following exemplary areas: strength, running speed and agility, fine motor precision, fine motor integration, hand dexterity, bilateral coordination, balance, and upper limb coordination. For example, the total BOT-2 strength score can be determined by having the patient perform sit-ups, V-ups, standing long jumps, wall sits, and push-ups. Running speed and agility scores can be determined by having patients step over a balance beam or perform shuttle runs, two-legged side jumps, or one-legged side jumps. Both the BOT-2 total intensity and the BOT-2 running speed and agility scores range from 0 to 25, with scores of approximately 10 to 25 considered representative of healthy subjects.

[0053] As used herein, the term "catalytically active" refers to sALPs that hydrolyze the bone mineralization inhibitor inorganic pyrophosphate (PPi) to provide inorganic phosphate (Pi), thereby reducing the extracellular concentration of PPi. Therefore, catalytically active sALPs improve bone mineralization by modulating PPi concentration.

[0054] As used herein, the terms “Childhood 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 the incidence of pain) of patients aged 1 to 19 years (such as children, adolescents, and some adults with HPP). For a description of the CHAQ index, see Bruce and Fries (…). J. Rheumatol [Journal of Rheumatology] 30(1): 167-178, 2003), which is hereby incorporated in its entirety by reference. For children older than 8 years, the CHAQ can be administered via interview or self-report. The CHAQ consists of eight subscales for dressing / grooming, getting up, eating, walking, hygiene, stretching, grasping, and activity. Scores within each category range from 0 to 3, where 0 indicates no difficulty; 1 indicates some difficulty; 2 indicates very difficult; and 3 indicates the patient cannot perform the activity. The CHAQ index can also be used to determine the presence and severity of pain.

[0055] As used herein, the terms “EuroQol Five-Dimensional Questionnaire” and “EQ-5D” refer to a questionnaire used to assess the health status (e.g., mobility, self-care, ability to perform daily activities at school, work, or household chores, ability to perform ADLs (e.g., dressing, toileting, and cooking), experience of pain or discomfort, and anxiety or depression) of patients (e.g., children with HPP aged approximately 5 to 12 years, adolescents with HPP aged approximately 13 to 17 years, or adults with HPP aged approximately 18 years or older). For a description of the EQ-5D index, see Reenan and 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 used in an interview. The EQ-5D questionnaire comprises 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 in this article, the EQ-5D can be used in combination with at least one physical assessment (such as the 6MWT) to classify HPP patients into a Grade I health status indicating no problems with physical condition, a Grade II health status indicating some problems with physical condition, a Grade III health status indicating extreme problems with physical condition, or a Grade IV health status indicating the most extreme problems with physical condition. 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 health status IV to III, IV to II, IV to 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 [Applied Health Economics and Health Policy] 9(3): 157-69, 2011) and PCT Publication No. WO 2018 / 191254, which are hereby incorporated in their entirety by reference.

[0056] The term "efficacy" refers to the energy expenditure (E) of a compound in a dose-response assay. max value.

[0057] The term "Fc" refers to a fragment crystallizable region of an immunoglobulin, such as IgG1, IgG2, IgG3, or IgG4, including 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 can be derived from any mammal, including humans, and can be post-translational modified (e.g., by glycosylation or sialylation). In a non-limiting example, 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 having SEQ ID NO: 253.

[0058] "Fragment" means a portion of a polypeptide or polynucleotide, preferably containing at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the full length of a reference polynucleotide or polypeptide. The fragment can contain, 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, 1,000, 1100, 1200, 1300, 1 400, 1500, 1600, 1700, 1800, 1900, 2000, 2100 or more nucleotides, up to the full length of a 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 a polypeptide.

[0059] As used interchangeably in this document, the terms "handheld dynamometer" and "HHD" refer to a method for measuring grip strength and muscle strength in a subject (particularly, a subject with HPP aged approximately 13 years or older). The dynamometer can be used to assess grip strength, knee flexion, knee extension, hip flexion, hip extension, and hip abduction in a subject with HPP. For example, a MICROFET2™ dynamometer can be used to measure knee flexion and knee extension, as well as hip flexion, hip extension, and hip abduction in a subject with HPP aged approximately 13 years or older, while a JAMAR® grip dynamometer can be used to measure the subject's grip strength. Specifically, the applicator keeps the dynamometer stationary, and the subject applies maximum force to the dynamometer. Peak force data are collected in pounds and then converted to Newtons (N). Torque values ​​are then calculated using limb length in N meters. The torque values ​​can then be compared to values ​​of, for example, normal subjects of approximately the same age, sex, and / or height, and expressed as a percentage to produce the subject's HHD score.

[0060] As used herein, the term "health status" refers to the characteristic physical condition of a patient with HPP (e.g., a child with HPP aged approximately 5 to approximately 12 years, an adolescent with HPP aged approximately 13 to approximately 17 years, or an adult with HPP aged approximately 18 years or older). The health status of a patient with HPP can be characterized by at least one physical assessment selected from one or more of the following measures: 6MWT, BOT-2, BSID-III, and gait analysis; and by at least one quality of life assessment selected from one or more of the following measures: EQ-5D, CHAQ, PODCI, CHU-9D, SF-36, SF-12, and PedsQL. In particular, the health status of a patient with HPP is characterized by, for example, 6MWT in combination with EQ-5D. After obtaining the results of at least one physical assessment and at least one quality of life assessment selected from the above measures, HPP patients can be identified as having a Grade I health status indicating no problems with their physical condition, a Grade II health status indicating some problems with their physical condition, a Grade III health status indicating extreme problems with their physical condition, or a Grade IV health status indicating the most extreme problems with their physical condition. These one or more measures can be used to assess the transition of HPP patients from one health status to another after treatment with sALP as described herein, such as a transition from health status IV to III, IV to II, IV to I, III to II, III to I, or II to I after sALP administration.

[0061] As used herein, the terms “hypophosphatase disorder” and “HPP” refer to a rare, heritable skeletal disorder caused by one or more loss-of-function mutations in genes such as ALPL (alkaline phosphatase, liver / bone / kidney) that encode tissue-nonspecific alkaline phosphatase (TNSALP). HPP can be further characterized as infantile HPP, childhood HPP, perinatal HPP (e.g., benign or lethal perinatal HPP), dental HPP, adolescent HPP, or adult HPP. For example, “childhood HPP” describes patients with HPP aged approximately 5 to approximately 12 years, “adolescent HPP” describes patients with HPP aged approximately 13 to approximately 17 years, and “adult HPP” describes patients with HPP aged approximately 18 years or older. As used herein, “adult HPP” refers to a condition or phenotype characterized by the presence of one or more of the following symptoms: elevated blood and / or urine levels of inorganic pyrophosphate (PPi), hypomineralization, 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. As used herein, “adolescent HPP” refers to a condition or phenotype characterized by the presence of one or more of the following symptoms: elevated blood or urine levels of PPi, PEA, or PLP; osteomalacia, one or more skeletal deformities, hypotonia, muscle weakness, rheumatoid complications, arthritis, pseudogout, unsteady gait, difficulty walking, bone pain, pain, premature tooth loss, hypomineralization, pulmonary hypoplasia, respiratory insufficiency, seizures, hypercalciuria, short stature, and growth retardation. As used in this article, “childhood HPP” refers to a condition or phenotype characterized by the presence of one or more of the following symptoms: elevated blood or urine levels of PPi, PEA, or PLP; rickets, rickets-related ribs, one or more skeletal deformities, hypotonia, myasthenia gravis, rheumatoid complications, arthritis, pseudogout, unsteady gait, difficulty walking, bone pain, pain, premature tooth loss, hypomineralization, delayed motor development, seizures, hypercalciuria, short stature, fractures, pseudofractures, and growth retardation.

[0062] In this article, the terms “Lower Extremity Functional Scale” and “LEFS” are used interchangeably to refer to a method for measuring lower extremity functional disability in patients (particularly, patients with HPP, e.g., patients aged approximately 13 years or older). LEFS was developed by Binkley et al. ( Phys Ther.The self-reported measurement described in [Physical Therapy] 79:371-83, 1999, is hereby incorporated by reference in its entirety. Total LEFS scores range from 0 to 80, with higher scores indicating better lower limb function. A change in LEFS score of approximately 9 points is considered clinically significant. Licensed physical therapists may administer LEFS to HPP patients (e.g., HPP patients approximately 13 years of age or older) via interview. Higher LEFS scores indicate improved lower limb function, including transitional movements (e.g., getting out of the bathtub or rolling in bed), movements (e.g., walking or running on uneven surfaces), climbing stairs, and squatting. LEFS can be used to assess functional impairment in one or both lower limbs in HPP patients, including the ability to monitor patients long-term and evaluate the effectiveness of Asfor enzyme α treatment.

[0063] The terms “nucleic acid,” “nucleic acid molecule,” and “polynucleotide” refer to polymeric molecules, such as RNA or DNA, having a sequence of two or more covalently bonded, naturally occurring or modified nucleotides. Nucleic acid molecules can be, for example, single-stranded or double-stranded, and can include modified or unmodified nucleotides, or mixtures or combinations thereof. Various salts, mixed salts, and free acid forms of nucleic acid molecules are also included.

[0064] As used in this article, the terms “Pediatric Outcome Data Collection Tool” and “PODCI” refer to a questionnaire used to assess the overall health, pain incidence, and ability to perform activities of daily living (ADL) in patients under the age of 19, particularly those with chronic health disorders such as HPP. For a description of the PODCI, see Plat et al. (…). J. Pediatr. Orthop.[Pediatric Orthopaedics Journal] 23(6): 788-790, 2003), which is hereby incorporated in its entirety by reference. The questionnaire can be completed by the patient or by the patient's parents / guardians who are familiar with the patient's condition. The eight scales generated from the PODCI include: 1) the Upper Limb and Physical Function Scale, which measures difficulties encountered in performing daily personal care and school activities; 2) the Transfer and Basic Mobility Scale, which measures difficulties experienced in performing routine movements and motor activities in daily activities; 3) the Motor / Physical Function Scale, which measures difficulties or limitations encountered in participating in more active activities or motor activities; 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 of treatment; 6) the Well-being Scale, which measures overall satisfaction with one's appearance and similarity with 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 General Function Scale, which is a general combination scale calculated from the first four scales listed above. Standardized scores are generated from a series of questions in the PODCI and converted into a scale from 0 to 100, where 0 represents severe disability and 100 represents mild disability.

[0065] The term "recombinant protein" is known in the art. A recombinant protein can be a glycoprotein. For example, a recombinant protein or recombinant protein variant prepared in CHO cells is glycosylated, wherein the sugar moiety is covalently linked to the protein, and is a glycoprotein. In short, the term "recombinant protein" can refer to a protein that can be manufactured using a cell culture system. Cells in a cell culture system can be derived, for example, mammalian cells, including human cells, CHO cells, insect cells, yeast cells, or bacterial cells. Typically, the cells in the cell culture contain an introduced polynucleotide encoding the recombinant protein of interest (this polynucleotide can be carried on a vector, such as a plasmid vector). The polynucleotide encoding the recombinant protein may also contain a heterologous promoter operatively linked to the polynucleotide encoding the protein.

[0066] As used herein, the “6-minute walk test” and “6MWT” refer to a physical assessment that is a standardized test used to evaluate the walking ability of patients with HPP (e.g., children with HPP aged approximately 5 to approximately 12 years, adolescents with HPP aged approximately 13 to approximately 17 years, or adults with HPP aged approximately 18 years or older). Specifically, walking ability refers to the patient’s ability to lift and lower each foot alternately. See American Thoracic Society statement: guidelines for the six-minute walk test. Amer. J. of Respiratory and Critical Care Medicine [American Journal of Respiratory and Critical Care Medicine], 166(1):111-7, 2002, which is hereby incorporated in its entirety by reference. 6MWT is determined by the distance (e.g., in meters) a patient walks on a flat, hard surface within 6 minutes. The 6MWT distance can then be compared to the patient's 6MWT distance at baseline, the 6MWT distance of untreated subjects (e.g., untreated subjects of approximately the same age, height, and / or sex), or the 6MWT distance of healthy subjects (e.g., healthy subjects of approximately the same age, height, and / or sex), and expressed as a percentage, to determine the 6MWT value.

[0067] "Treatment" refers to the medical management of a patient with the aim of curing, improving, stabilizing, reducing the likelihood of or preventing a disease condition (such as HPP (e.g., HPP in children, adolescents, or adults)) or one or more symptoms thereof, and / or the management of patients who exhibit or may have a disease condition (such as HPP), for example by administering a pharmaceutical composition (e.g., sALP as described herein). This term includes active treatment, i.e., treatment specifically targeting the improvement of a disease, pathological condition, disorder, or event, or associated with its cure, and also includes etiological treatment, i.e., treatment targeting the removal of the cause of the associated disease, pathological condition, disorder, or event. In addition, this term includes palliative care, which is treatment designed to alleviate or improve at least one symptom rather than cure a disease, pathology, disorder, or event; symptomatic treatment, which is treatment of systemic symptoms related to a disease, pathology, disorder, or event; preventive treatment, which is treatment designed to minimize or partially or completely suppress the development of a disease, pathology, disorder, or event, for example, in patients who are not yet ill but are susceptible to a particular disease, pathology, disorder, or event or otherwise at risk of it; and supportive treatment, which is treatment used to complement another specific therapy aimed at improving a related disease, pathology, disorder, or event.

[0068] 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, without regard to post-translational modifications (e.g., glycosylation, sialylation, or phosphorylation), which, as described herein, constitute all or part of a naturally occurring or non-natural polypeptide or peptide.

[0069] The terms “ALP,” “sALP,” “soluble alkaline phosphatase,” “alkaline phosphatase,” and “extracellular domain of alkaline phosphatase” are used interchangeably (unless the context otherwise requires) and refer to a soluble, non-membrane-bound alkaline phosphatase or its domain, bioactive fragment, or bioactive variant thereof. ALP includes, for example, alkaline phosphatases lacking a C-terminal GPI signaling sequence, and other variants and analogs that retain alkaline phosphatase activity (e.g., the ability to hydrolyze PPi or other natural or artificial substrates). This includes the TNSALP, PALP, GALP, and IALP domains, and their bioactive fragments or bioactive variants, unless otherwise specified. Mature sALP lacks a GPI membrane anchor and a signal peptide that is cleaved during processing.

[0070] The term "ALP polypeptide" means any sequence, including ALP sequences as defined herein. Exemplary ALP polypeptides include those having the structure A-ALP-B, where neither A nor B is present, or an amino acid sequence of at least one amino acid (e.g., any ALP fusion polypeptide described herein).

[0071] The terms "isolated" or "purified" mean isolated from other naturally occurring associated components. Typically, a compound (e.g., protein, polypeptide, polynucleotide, or small molecule), factor, cell, or other component is considered isolated when it is at least, for example, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or even 99% free of proteins, antibodies, naturally occurring organic molecules, and other naturally associated components by weight. In some cases, the component is at least 75%, 90%, or even 99% pure by weight. An isolated component can be obtained by chemical synthesis, isolation of the factor from a natural source, or generation of the component in a recombinant host cell that does not naturally produce the component. Those skilled in the art can purify proteins and small molecules using standard techniques, such as those described by Ausubel et al. (…). Current Protocols in Molecular Biology The techniques described in *Modern Methods in Molecular Biology*, John Wiley & Sons, New York, 2000, are preferred. The component is preferably at least 2, 5, or 10 times pure than the starting material, as measured using, for example, polyacrylamide gel electrophoresis, column chromatography, densitometrics, HPLC analysis, or Western spectroscopy (Ausubel et al., 2000). Exemplary purification methods include column chromatography, filtration, immunoprecipitation, and magnetic bead immunoaffinity purification.

[0072] The terms "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" refer to a carrier or excipient that is physiologically acceptable to the treated patient while retaining the therapeutic properties of the compound administered with it. An exemplary pharmaceutically acceptable carrier substance is physiological saline. Other physiologically acceptable carriers and formulations thereof are known to those skilled in the art, and for example in... Remington (As described in The Science and Practice of Pharmacy, 22nd edition, edited by Allen, 2012).

[0073] The term "pharmaceutical composition" means a composition containing a polypeptide or polynucleotide as described herein, formulated with pharmaceutically acceptable excipients, and including those compositions manufactured or sold as part of a treatment regimen for the treatment or prevention of a patient's disease or event, subject to approval by a government regulatory authority. Pharmaceutical compositions may be formulated for subcutaneous administration, intravenous administration (e.g., as a sterile solution without emboli and in a solvent system suitable for intravenous use), oral administration (e.g., tablets, capsules, gelcapsule tablets, or syrups), or any other formulation described herein, for example, in unit dosage forms.

[0074] The terms “subject” or “patient” refer to mammals, including but not limited to humans or non-human mammals such as cattle, horses, dogs, sheep, or cats.

[0075] The term "mammalian cell" is known in the art and can refer to any cell derived from or originating from any mammal, including, for example, human, hamster, mouse, green monkey, rat, pig, cow, hamster, or rabbit. Mammalian cells can be immortalized cells, differentiated cells, or undifferentiated cells.

[0076] The term "therapeutic effective amount" means the amount of the polypeptide or polynucleotide described herein that is sufficient to adequately treat, prevent, delay, inhibit, or stop any symptoms of the disease or condition described herein (particularly HPP). The therapeutic effective amount of the compositions described herein may depend on the severity of the disorder being treated and the subject's condition, weight, and general status, and can be determined by a person skilled in the art taking such factors into account. The therapeutic effective amount of the compositions described herein may be administered to the subject as a single dose or as multiple doses over a period of time.

[0077] As used herein, when a polypeptide or nucleic acid sequence is described as having "at least X% sequence identity" with a reference sequence, it means that when these sequences are optimally aligned, at least X% of the amino acid residues or nucleotides in the polypeptide or nucleic acid are identical to the reference sequence. Optimal sequence alignment can be determined using various methods within the scope of the art, such as the Smith-Waterman alignment algorithm (Smith et al.). J. Mol. Biol [Journal of Molecular Biology] 147:195-7, 1981) and BLAST (basic local alignment search tool; Altschul et al.), J. Mol. Biol [Journal of Molecular Biology] 215:403-10, 1990. These and other alignment algorithms can be obtained using publicly available computer software, such as those incorporated into GeneMatcher Plus™ (Schwarz and Dayhof, Atlas of Protein Sequence and Structure [Protein Sequence and Structure Maps], Dayhoff, MO (ed.), pp. 353-358, 1979, "Best Fit" (Smith and Waterman, Advances in Applied Mathematics [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). Furthermore, those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to achieve optimal alignment across the lengths of the sequences being compared.

[0078] The terms "preferred" and "ideally" refer to embodiments in which the disclosed compounds, compositions, and methods may provide certain benefits under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the description of one or more preferred embodiments does not imply that other embodiments are useless, and is not intended to exclude other embodiments from the scope of this disclosure.

[0079] For any method disclosed herein that includes discontinuous steps, these steps may be performed in any feasible order. Additionally, any combination of two or more steps may be performed simultaneously, if appropriate.

[0080] The above overview is not intended to describe every disclosed embodiment or every implementation of the disclosed compounds, compositions, and methods. The following description provides more specific examples of illustrative embodiments. Throughout the application, guidance may be provided by a list of examples, which may be used in various combinations. In each case, the list is intended only as a representative group and should not be construed as an exclusive list.

[0081] All headings are intended to facilitate the reader and should not be used to limit the meaning of the text following the heading, unless otherwise stated.

[0082] Attached image description.

[0083] This application contains at least one color drawing. Upon request and payment of the necessary fees, the official authority will provide a copy of this patent or patent application with the color drawing.

[0084] Figure 1A-1D This is a diagram showing the binding of fluorescent Fc-fusion protein (a dimer fusion protein consisting of an N-terminal HA-binding sequence fused with human IgG1 Fc-Katushka 2s fluorescent protein) to hydroxyapatite (HA) or bone homogenate. Figure 1A It shows the relationship with CAPTAL ® The fluorescence intensity of the HA-bound fluorescent Fc-fusion protein was normalized to the non-targeted Fc-Katushka2s sample (FLU002). Figure 1B The fluorescence intensity of the protein suspension after 2 hours of incubation with HA is shown, representing the total amount of unbound protein after incubation; Figure 1C (Combined) and Figure 1D (Unbound) shows similar quantification of the fluorescent Fc-fusion bound to mouse C56BL / 6 femoral homogenate. N = 2 ± maximum / minimum (*p < 0.05 compared to FLU002, one-way ANOVA, Dunnet post-hoc analysis).

[0085] Figure 2A-2B This demonstrates bone targeting (ALP-Fc-D) bound to mouse bone homogenate as measured by a fluorescent probe. 10 Image of the fusion of (ALP031; SEQ ID NO: 31) and non-targeted (ALP-Fc) (ALP086; SEQ ID NO: 222). Bone homogenate-bound (ALP031; SEQ ID NO: 31) Figure 2A ) and unbound ( Figure 2B The fluorescence intensity of the protein fraction was normalized to that of the non-targeted negative control (ALP-Fc). N = 3 ± SD.

[0086] Figures 3A-3B This diagram shows the binding of fluorescently labeled VHH protein to mouse bone homogenate. (Bone homogenate binding...) Figure 3A ) and unbound ( Figure 3B The fluorescence intensity of the protein fraction was normalized to the non-targeted negative control VHH. N = 3 ± SD.

[0087] Figure 4 It is ALP-Fc-D 10A set of representative raw fluorescence images of J:NU mice treated with (ALP031; SEQ ID NO: 31) and ALP-Fc (ALP086; SEQ ID NO: 222) over time (24 to 432 hours) using longitudinal whole-body in vivo imaging spectroscopy (IVIS).

[0088] Figures 5A-5B This shows the use of ALP-Fc-D 10 A graph of quantitative data of longitudinal whole-body IVIS in vivo animal imaging of mice treated with (ALP031; SEQ ID NO: 31) or ALP-Fc (ALP086; SEQ ID NO: 222). Figure 5A The plotted whole-body regions of interest and the quantified total radiation efficiency are shown. Figure 5B The total radioefficiency within the spinal region of interest plotted throughout the 18-day study is shown, along with quantified and longitudinally plotted radioefficiencies. N = 6 ± SD, *p < 0.05, **p < 0.01. Mann-Whitney unpaired t-test, two-tailed.

[0089] Figures 6A-6B This is a graph showing the in vitro fluorescence quantification of protein accumulation in selected tissues (spine, skull, femur, liver, kidney, and spleen) after 18 days. Figure 6A The results showed that 18 days after a single dose, the levels of ALP-Fc-D... 10 Raw total radiation efficiency of tissue specimens from mice treated with (ALP031; SEQ ID NO: 31) and ALP-Fc (ALP086; SEQ ID NO: 222). Figure 6B The image shows the result from ALP-Fc-D 10 (ALP031; SEQ ID NO: 31) Volume-normalized total radiative efficiency of isolated bone tissue from treated mice. N = 6 ± SD, **p < 0.01 Mann-Whitney unpaired t-test, two-tailed.

[0090] Figure 7 This is a diagram showing representative 2D fluorescent IVIS images from longitudinal studies conducted in mice that were administered a single IV dose of a fluorescently labeled compound with non-targeted VHH (SEQ ID NO: 248), VHH001 (SEQ ID NO: 249), or VHH002 (SEQ ID NO: 250).

[0091] Figure 8This is a set of raw fluorescence images illustrating quantitative data from a longitudinal study of total radiative efficiency in mice administered a single IV dose of a fluorescently labeled compound with untargeted VHH (SEQ ID NO: 248), VHH001 (SEQ ID NO: 249), or VHH002 (SEQ ID NO: 250). Radiation efficiency was obtained from the entire region of interest (ROI) in the mice. N = 5 ± SD, **** p < 0.0001, one-way ANOVA, Tukey post-hoc analysis.

[0092] Figures 9A-9C This is a graph showing the quantification of isolated bone tissue from mice administered with fluorescently labeled compounds having non-targeted VHH (SEQ ID NO: 248), VHH001 (SEQ ID NO: 249), and VHH002 (SEQ ID NO: 250). Figure 9A The spine is shown. Figure 9B The hind limbs were shown, and Figure 9C Skulls are shown, harvested from all mice in all treatment groups 7 days (168 hours) after a single dose. Equivalent regions of interest (ROIs) were plotted around similar tissues, and total radiation efficiency was normalized to tissue volume. N = 5 ± SD, *** p < 0.001, one-way ANOVA, Tukey post-hoc analysis.

[0093] Figures 10A-10B This is a diagram showing the binding and dissociation of bone-targeting proteins at multiple doses in vitro. Figure 10A VHH001 (SEQ ID NO: 249), VHH002 (SEQ ID NO: 250), and ALP-Fc-D are shown. 10 (ALP031; SEQ ID NO: 31) bone homogenate binding protein, and Figure 10B VHH001 (SEQ ID NO: 249), VHH002 (SEQ ID NO: 250), and ALP-Fc-D are shown. 10 Unbound protein of (ALP031; SEQ ID NO: 31).

[0094] Figure 11 This is a graph showing the change in MUP fluorescence intensity over time generated by 16 bone marker fusion proteins bound to bone homogenate (ALP031 (ALP-Fc-D10), ALP086 (ALP-Fc), and ALP202-ALP216, corresponding to SEQ ID NO:31, 222, and 124-138, respectively).

[0095] Figure 12 These are a series of two-dimensional 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.

[0096] Figure 13 These are a series of two-dimensional IVIS images of mice treated with the ALP-Fc fusion protein (untagged, ALP086, Example 8, N = 6), showing in vivo bone integration over time.

[0097] Figure 14 This graph shows the binding activity in the supernatant, comparing the binding and unbound fractions of various labeled constructs (ALP230-ALP239, ALP242, ALP243, ALP247, ALP248, ALP250, ALP2521, ALP253, and ALP254). The dashed line represents baseline bone binding as a control. Constructs with D5 or D6 bone-targeting fractions showed nearly equal binding (B) and unbound (U) fractions.

[0098] Figures 15A-15B This is a graph showing pK data, which illustrates the data transmitted via the IV pathway ( Figure 15A or subcutaneous route ( Figure 15B The relationship between plasma protein levels (expressed as mg / L / dose [(mg / L) / (mg / kg)]) and hours after administration to healthy male C57BL / 6 mice.

[0099] Figure 16 This is a graph showing the Kaplan-Meier survival curves of HPP mice treated subcutaneously with PBS or ALP201 daily in daily, every other day, and weekly dosing regimens. The median survival time for HOM PBS QD animals was 21.5 days.

[0100] Figure 17 This is a graph showing the change in average body weight over time (36-day study) of HPP mice treated with ALP201 or PBS.

[0101] Figure 18 This is a graph showing the hind paw mineralization index of HPP mice treated with ALP201 or PBS on day 11.

[0102] Figure 19 This is a graph showing exemplary process curves for the hydrolysis determination of 4-methylumbelliferyl phosphate (MUP) using different concentrations of ALP023, where fluorescence is plotted against time.

[0103] Figure 20This is a schematic diagram illustrating an exemplary VHH structural domain. F represents the VHH framework region, and the gray areas represent CDRH1, H2, and H3, as well as the terminal segments, which can be used to locate or space bone-targeting sequences.

[0104] Figure 21 This is a schematic diagram illustrating an exemplary ALP-Fc-bone tag molecule, in which a single structural domain is labeled.

[0105] Figure 22 This is a set of graphs showing saturation curves, illustrating the relationship between increased pyrophosphate (PPi) levels and the pyrophosphate hydrolysis rate of the selected constructs, where the reaction rate is plotted against the pyrophosphate concentrations of ALP201, ALP259, and Asforase α (SEQ ID NO: 269).

[0106] Figure 23 This is a graph showing an exemplary process curve for the determination of PLP hydrolysis coupled with ALP / TPLDH, where fluorescence is plotted against time for 10 ng / ml ALP201.

[0107] Figure 24 This is a set of graphs showing saturation curves, illustrating the relationship between increased pyridoxine 5'-phosphate (PLP) levels and PLP hydrolysis rates in the ALP / TPLDH coupling assay using selected constructs (ALP201, ALP259, and Asforase α; 10 ng / mL, 10 ng / mL, and 12.5 ng / mL, respectively), where the reaction rate is plotted against PLP concentrations expressed in μM.

[0108] Figure 25 This is a graph showing the hind paw mineralization index of HPP mice treated with ALP201, ALP259, Asforase α, or PBS on day 36 / 37.

[0109] Figure 26 This is a graph showing the tibia length of HPP mice treated with ALP201, Asforase α, or PBS on day 36 / 37.

[0110] Figure 27 This is a graph showing the femur length of HPP mice treated with ALP201, Asforase α, or PBS on day 36 / 37.

[0111] Figure 28 This is a graph showing the bone alkaline phosphatase activity levels in HPP mice treated with ALP201, Asforase α, or PBS on days 36 / 37.

[0112] Figure 29This is a graph showing the bone alkaline phosphatase activity levels in Akp2GW HPP mice treated with ALP201 or ALP259 on days 36 / 37.

[0113] Figure 30 This is a graph showing an exemplary process curve of a 4-MUP hydrolysis assay performed on femoral tissue of HPP mice, in which fluorescence (RFU) is plotted relative to time (seconds).

[0114] Figure 31 This is a graph showing the body weight of HPP mice treated with ALP201, Asforase α, or PBS on day 36 / 37.

[0115] Detailed implementation method.

[0116] This invention is characterized by polypeptides comprising soluble alkaline phosphatases, fragments thereof, fusion proteins, and methods of use thereof for the treatment of bone mineralization disorders, such as hypophosphatase syndrome (HPP) and its symptoms. These polypeptides comprise soluble alkaline phosphatase (sALP) or fragments thereof derived from naturally occurring alkaline phosphatases (ALP). Alkaline phosphatases include various isoenzymes differentially expressed in different tissues. The four major ALP isoenzymes include tissue nonspecific alkaline phosphatase (TNSALP), placental alkaline phosphatase (PALP), germline alkaline phosphatase (GALP), and intestinal alkaline phosphatase (IALP). Therefore, the invention is characterized by proteins derived from these ALP isoenzymes.

[0117] HPP is a rare inherited skeletal disorder, with the most severe form occurring in 1 in 100,000 newborns. This disorder is typically caused by a loss-of-function mutation in the gene encoding TNSALP. Symptoms and severity of HPP vary widely, ranging from premature tooth loss to almost complete lack of bone mineralization in utero. 。 The manifestations of HPP varied significantly among subjects and across different age groups. Many subjects with HPP exhibited skeletal changes, short stature, chronic pain, lower extremity pain, 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 peptides described in this article to restore native ALP activity and provide normal bone matrix mineralization.

[0118] We systematically examined the protein activity, protein stability, and pharmacokinetic properties of alkaline phosphatase peptides and their fusion proteins through site-directed mutagenesis. Variations in the alkaline phosphatase peptides and their fusion proteins described herein include changes in amino acid residues located in and around the active site of alkaline phosphatase, at one or more shared sites of N-linked glycosylation, in the bone-targeting portion (if included), and / or in the fragment crystallizable (Fc) region (if included). This paper discloses alkaline phosphatase peptides and their fusion proteins containing one or more of these variations, along with data showing that such variations improve one or more of the protein activity, protein stability, and pharmacokinetic properties. The alkaline phosphatase peptides and their fusion proteins are described in more detail herein.

[0119] Soluble alkaline phosphatase The feature is a polypeptide containing ALP, including naturally occurring ALP and fragments thereof, and ALP having one or more mutations, which, relative to naturally occurring ALP without the at least one mutation, improve at least one activity or pharmacokinetic (PK) property.

[0120] 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 and can be, for example, a mammalian ALP. Mammal ALPs include, for example, human, gorilla, mouse, rabbit, chimpanzee, cynomolgus monkey, rhesus monkey, orangutan, baboon, rat, cow, goat, and llama ALPs. In some specific 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 with any one of SEQ ID NO: 1-6 (e.g., at least 85% sequence identity with residues 1-491 of SEQ ID NO: 1 or residues 1-486 of SEQ ID NO: 4). ALP may have at least 70% (e.g., 75%, 80%, 85%, 90%, 95%, 97%, 99% or 100%) sequence identity with a region of at least 50 amino acids (e.g., at least 100, 150, 200, 250, 300, 350, 400, 450 or more) of any one of SEQ ID NO: 1-6.

[0121] TNSALP is a membrane-bound protein (Swiss-Prot, P05186) with a glycolipid moiety anchored at its C-terminus. The glycolipid anchor (GPI) is added post-translationally after the removal of the hydrophobic C-terminus, serving both as a temporary membrane anchor and as a signal for GPI addition. While the GPI anchor resides within the cell membrane, the remainder of TNSALP is extracellular. 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, resulting in engineered soluble TNSALP containing all the amino acid residues of the native anchored form of TNSALP but lacking the GPI membrane anchor. Those skilled in the art will understand that the location of the GPI membrane anchor will vary in different ALPs and may 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. Therefore, the C-terminus of native ALPs can be truncated by certain amino acids without affecting ALP activity.

[0122] 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 at the time of synthesis, but is cleaved from TNSALP after transport into the endoplasmic reticulum. An exemplary N-terminal signal peptide is MISPFLVLAIGTCLTNS (SEQ ID NO: 251).

[0123] The sALP described herein includes both its secreted form (i.e., lacking an N-terminal signal) and its non-secreted form (i.e., possessing an N-terminal signal). Those skilled in the art will understand that the position of the N-terminal signal peptide will vary among different alkaline phosphatases and may 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, for example, using appropriate computer algorithms, such as those described by Bendtsen et al. (…). J. Mol. Biol. The algorithm is described in [Molecular Biology Journal] 340(4):783-795, 2004 and is available at www.cbs.dtu.dk / services / SignalP / .

[0124] ALP may include one or more mutations, such as mutations that are not naturally occurring. These one or more mutations preferably enhance the therapeutic characteristics of alkaline phosphatase and / or bone-targeting conjugates. For example, the one or more mutations may improve the clearance, activity, efficacy, and / or solubility of the bone-targeting conjugate. The mutation may be an amino acid substitution, or the insertion or deletion of one or more amino acids. The one or more mutations may improve at least one activity of the pharmacokinetic (PK) properties relative to naturally occurring ALP without the at least one mutation. An ALP (e.g., human TNSALP) may have at least one mutation selected from the group consisting of E108X, M384X, L385X, N213X, and N286X of SEQ ID NO: 1, where X is any amino acid. These one or more mutations may include, for example, E108S, E108T, E108Q, E108M, E108K, E108L, M384R, L385T, N213Q, and N286Q of SEQ ID NO: 1. An ALP can include more than one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) mutation at different amino acid positions. For example, an ALP can include the E108M, N213Q, and N286Q mutations relative to SEQ ID NO: 1. An ALP (e.g., human IALP) can have the W245X mutation relative to SEQ ID NO: 4, where X is any amino acid. An ALP can have the W245R mutation relative to SEQ ID NO: 4. An ALP can have the C481X mutation relative to SEQ ID NO: 4, where X is any amino acid that does not contain a thiol (e.g., C481G).

[0125] The polypeptide described herein may contain an ALP containing a mutation relative to naturally occurring ALP located in a specific region of the ALP. This mutation may be present in the extracellular domain of the ALP. For example, the mutation may be present in the crown domain, catalytic domain, or dimerization domain of the ALP. The mutation may be present in the GPI anchoring domain (if included). Based on alignment and / or sequence homology, the polypeptide may include mutations present in amino acids 1-491 or 1-486 of human TNSALP or similar ALP positions. For example, the polypeptide may have mutations at 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 may have mutations at 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 may have mutations between positions 108, 213, 286, and / or 429 relative to SEQ ID NO: 1. In some embodiments, the mutation is within the sequence of ALP, and the mutation is not a non-natural amino acid segment present at the N- or C-terminal domain of ALP.

[0126] ALP can have mutations at common N-linked glycosylation sites. These common N-linked glycosylation sites include the asparagine-XZ motif, where X is any amino acid other than P, and Z is any amino acid other than S or T. Asparagine can be mutated to glutamine.

[0127] ALP can have an S429X mutation relative to SEQ ID NO: 4, where X is any amino acid. For example, ALP can have S429Q, S429H, S429E, or S429D mutations relative to SEQ ID NO: 4.

[0128] ALP may have at least 70% (e.g., 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity with a region of at least 50 amino acids (e.g., 100, 150, 200, 250, 350, 400, or more) of the amino acid regions of any one of SEQ ID NO: 7-223, 247, and 262-264, and / or may include one or more of the mutations discussed above.

[0129] Mutated ALPs, compared to naturally occurring ALPs, can provide at least one improvement in PK properties. For example, mutations can provide one or more of the following: increased catalytic activity of the ALP, increased temperature stability, increased zinc binding, maintenance of activity in zinc-depleted buffers, maintenance of activity at pH 5.0–7.5, reduced dimerization, reduced aggregation, and / or increased manufacturability. Increased catalytic activity may include increased hydrolysis of pyridoxal 5-phosphate and / or pyrophosphate. The increased catalytic activity may be at least 2-fold better than that of naturally occurring ALPs (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). Improved PK properties may include one or more of the following: increased substrate specificity, increased activity to natural substrates, increased activity to artificial substrates, decreased Km to natural substrates, and increased area under the curve (AUC). Artificial ALP substrates include, for example, umbelliferous phosphates (e.g., 4-methylumbelliferous phosphate (4-MUP)), umbelliferous ketone phosphates, and p-nitrophenyl phosphate (pNPP). Natural substrates include, for example, pyridoxal-5'-phosphate, PLP, and PEA.

[0130] The ALP described herein can be in dimer form. Alternatively, ALP can be in monomer form.

[0131] Bone-targeted portion The peptides described herein may further include a bone-targeting moiety. The bone-targeting moiety is any sequence of amino acids with sufficient affinity for bone (e.g., the hydroxyapatite mineral phase of bone). The mineral phase of bone contains positively charged regions. Therefore, negatively charged amino acids, such as glutamic acid or aspartic acid, can target the peptide to bone. The bone-targeting moiety allows sALP to localize to bone tissue and remain bound until the activity of sALP diminishes. This allows sALP to catalyze hydroxyapatite bone formation at the site of native TNSALP.

[0132] The bone-targeting portion can be located at the N-terminus or C-terminus of ALP. The bone-targeting portion may 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, and poly(glutamic acid-glutamic acid-serine) (EES)n. n can be any integer from 1 to 50 (e.g., 1 to 30, 3 to 30, 3 to 20, 5 to 16, 10 to 16, such as 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 portion may include structure Dn, where n = 7 to 10; En, where n = 10 to 15; (DAD)n, where n = 2 to 4; (DDS)n, where n = 2 to 4; (DSS)n, where n = 3; or (EES)n, where n = 3 to 4.

[0133] The targeting sequence may include Dn, where n is, for example, 3-9, 17-30, 10-16, or 3-30. The targeting sequence may include En, where n is, for example, 3-9, 17-30, 10-16, or 3-30. The bone-targeting portion may include a sequence containing both aspartic acid residues and glutamic acid residues.

[0134] Optionally, the Dn or En bone-targeting sequence may include one or more other amino acids, such as Thr, Gly, Gln, Asn, Lys, Ser, and / or Ala. As a non-limiting example, the targeting portion may include (DDS)n, where n is 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0135] The bone-targeting portion may include all or part of a bone-targeting sequence derived from casein (a salivary protein known to bind bone minerals). An example of a casein-rich bone-targeting sequence is DDSEEKFLRRIGRFG (SEQ ID NO: 252). The bone-targeting sequence may include a Dn or En sequence conjugated to a bone-targeting sequence derived from casein.

[0136] Optionally, for any of the bone-targeting conjugate examples described herein, the targeting sequence may be side-attached with a cysteine ​​residue at the N-terminus, C-terminus, or both. The cysteine ​​residue can be used for conjugation of the peptide to another chemical moiety.

[0137] The bone-targeting portion may include a VHH antibody or a fragment thereof containing a bone-targeting sequence. The bone-targeting sequence may be located within and / or at the end of the VHH antibody fragment to produce a bone-targeting VHH construct. The VHH antibody fragment includes all or part of the variable domain (VHH) of a heavy chain homodimer IgG (such as those found in camels, e.g., llamas). Homodimer antibodies lack the light chains (VL and CL) and the first heavy chain constant region (CH1) characteristic of conventional antibodies. Like the variable domains of conventional antibodies, the variable domains of these heavy chain homodimers (VHHs) have three complementarity-determining regions (CDRs), sometimes referred to as CDRH1, CDR H2, and CDR H3. The CDR H3 from llamas can be three times longer than the equivalent mouse CDR3. The VHH sequence may include multiple frame (F) regions flanking the three CDR regions. VHH sequences include those that are naturally occurring and those identified using phage display (such as MA10, MG6, and MG7) (Emelie D. Rodrigues, Single Domain Antibodies in Tissue Engineering [Single-domain antibodies in tissue engineering], University of Twente, Netherlands, 2014. Bone-targeting VHH constructs can be prepared to include at least one bone-targeting sequence, such as Dn or En, where n = 1 to 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 (Illustrative diagram). One, two, or all three CDRs or portions thereof in a VHH can be replaced with a bone-targeting sequence. Parts of the VHH fragment that do not contain CDRs, such as the frame (F) region, can function as spacers within the bone-targeting portion. Optionally, the bone-targeting sequence can be located within two or all three CDR sequences. Figure 20 As illustrated, F represents the VHH framework region, and the gray area includes CDRs H1, H2, and H3, as well as the terminal segments, which can be used to locate or space bone-targeting sequences.

[0138] Fc district The polypeptides described herein may further include an Fc region. For example, sALP may be a fusion polypeptide containing, for example, the Fc region of an immunoglobulin at the N-terminus or C-terminus of the polypeptide. Immunoglobulin molecules have structures well known in the art. They comprise two light chains (approximately 23 kD each) and two heavy chains (approximately 50-70 kD each) linked by interchain disulfide bonds. Immunoglobulins are readily cleaved by proteolytic hydrolysis (e.g., by papain) into Fab (containing the light chain and VH and CH1 domains of the heavy chain) and Fc (containing the CH2 and CH3 domains of the heavy chain, as well as adjacent sequences). Useful Fc fragments as described herein include any immunoglobulin molecule from any mammal (e.g., human), including Fc fragments of IgG, IgM, IgA, IgD, or IgE, and their various subclasses (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2). For example, an Fc fragment may be human IgG1.

[0139] The Fc region may include all or part of the Fc fragment of IgG. 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 PCT Publication No. WO 2007 / 106585, which is hereby incorporated by reference. The Fc fragment may increase the half-life and cycling time of the peptide after administration to a subject. For example, the half-life of the peptide may be from about 10 to about 100 hours (e.g., about 20, 30, 40, 50, 60, 70, 80, or 90 hours). The half-life may vary based on the administration method. 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 optionally be glycosylated at any suitable one or more amino acid residues known to those skilled in the art. Specifically, 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 with SEQ ID NO: 253 or SEQ ID NO: 259. Engineered, for example, non-naturally occurring Fc regions may also be used (see, for example, International Application Publication No. WO 2005 / 007809, which is hereby incorporated by reference). Relative to any of the Fc segments described herein, the Fc segments described herein may 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.

[0140] Connectors and spacers The polypeptides described herein may include one or more linkers or spacers of one or more amino acids. These polypeptides may also include one or more terminal residues located at the N- or C-terminus of the polypeptide. Linkers or spacers may include a sequence of one or more 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). Linkers or spacers may be omitted from the polypeptides described herein.

[0141] Linkers can include any suitable amino acid to promote flexibility between adjacent domains or eliminate stereointerference between domains. Furthermore, linkers or spacers can be used to enhance protein folding, thermal stability, and / or recombinant expression of the polypeptide.

[0142] One or more additional amino acids may be located at the N-terminus, C-terminus, and / or serve as spacer sequences between any or all components of the polypeptide described herein. For example, additional amino acids may be included between the Fc region and the bone-targeting portion. Exemplary spacer sequences include one or more glycine or serine residues, such as GGGGS (SEQ ID NO: 254), which may be located between the Fc region and the bone-targeting portion sequence. In some embodiments, longer spacer sequences may be employed to provide additional flexibility.

[0143] In some embodiments, the connector includes 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 connector does not include 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).

[0144] Topological structure of peptides The peptides described herein may include one or more of sALP, bone-targeting moieties, Fc regions, spacers, and linkers. The components of the peptides described herein may include any suitable topological arrangement from the N-terminus to the C-terminus, providing appropriate function. Peptides containing bone-targeting moieties may be referred to as bone-targeting conjugates or bone-targeting Fc conjugates.

[0145] For example, a polypeptide containing sALP and Fc regions may have the structure N-sALP-Fc-C or N-Fc-sALP-C. Optionally, the polypeptide may further include 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.

[0146] The polypeptide may include sALP and a bone-targeting portion (BTM) and has the structure N-sALP-BTM-C or N-BTM-sALP-C. Optionally, the polypeptide may further include 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.

[0147] The polypeptide may include sALP, an Fc region, and a BTM. A polypeptide containing all three components may have the structures 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 include 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.

[0148] 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 with any one of SEQ ID NO: 7-223, 247, and 262-264. For example, the polypeptide may comprise or consist of a sequence of any one of SEQ ID NO: 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 with SEQ ID NO: 123, wherein, optionally, the polypeptide contains 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 with SEQ ID NO: 177. The polypeptide may include or be composed of SEQ ID NO:177. The polypeptide may include or be composed of an amino acid sequence having at least 85% (e.g., 90%, 95%, 97%, 99%, or 100%) sequence identity with or composed of SEQ ID NO:260 or 261. The polypeptide may include or be composed of the sequence of SEQ ID NO:260. The polypeptide may include or be composed of the sequence of SEQ ID NO:261. The polypeptide may include a secretion signal peptide. The polypeptide may include or be composed of an amino acid sequence having at least 85% (e.g., 90%, 95%, 97%, 99%, or 100%) sequence identity with or composed of SEQ ID NO:263 or 264. The polypeptide may include or be composed of the sequence of SEQ ID NO:263. The polypeptide may include or be composed of the sequence of SEQ ID NO:264. The polypeptide may include any sALP catalytic domain, Fc IgG isotype (e.g., Fc of IgG2 / 4), or bone-targeting moiety listed in Table 1, and their topological arrangement. The polypeptide may include any single domain listed in Table 1.

[0149] Table 1: Exemplary sALP Constructions Adjustable integration with bone tissue The bone-targeting conjugates described herein can exhibit variable binding to bone tissue. For example, bone-targeting conjugates can exhibit differences associated with bone (binding) and / or dissociation behavior. For instance, bone-targeting Fc conjugates including polyD and polyE targeting sequences exhibit high binding affinity to bone tissue, while some bone-targeting Fc conjugates using casein-rich or VHH sequences as targeting sequences can exhibit moderate binding affinity to bone tissue (see, for example, Figures 1 and 14).

[0150] Bone-targeting conjugates may exhibit differences in dissociation behavior, even if their binding affinity is similar. For example, bone-targeting Fc conjugates that bind to bone tissue, such as ALP-Fc-D... 10(SEQ ID NO: 31) may not readily exchange with unbound conjugates in solution, while single-domain bone-targeting VHH constructs containing polyD or polyE sequences within one or more of their CDRs (e.g., SEQ ID NO: 249 and 250) can exhibit greater mobility in the presence of unbound conjugates to bind to and dissociate from bone tissue (see, for example, Figures 10A-10B ).

[0151] The bone-targeting conjugates described herein can be tuned by selecting the target sequence and other components to meet specific therapeutic needs, thereby altering binding affinity, dissociation rate, residence time, and migration. For example, balanced binding with the target activity ensures that the peptide does not block the active site on bone like the strongest binding molecule. In particular, constructs that bind too strongly to bone will dissociate more slowly. Because the catalytic activity of ALP depends on zinc and magnesium ions, ALP may become inactive over time due to the loss of these ions. 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. Options can be selected for the target sequence (e.g., poly D, poly E, phage display-derived sequences, complete or partial casein-rich sequences, complete or partial VHH sequences, or combinations thereof) and different topological options (e.g., the bone-targeting portion at the N-terminus or C-terminus of the conjugate). This ensures favorable binding kinetics to establish bone-bonding residence time, which increases efficacy and reduces long-term binding of inactive enzymes. Bone-bonding residence time can range from approximately 1 second to approximately 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).

[0152] Methods for testing sALP constructs The peptides described herein (e.g., peptides having the sequence of any one of SEQ ID NO: 7-223, 247 and 262-264, or variants thereof having at least 85% sequence identity with and / or 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.

[0153] Hydroxyapatite binding determination Synthetic HA can be used to detect the binding of the peptide described herein to HA. The bone-targeting peptide can be diluted in separate centrifuge tubes, for example, in bovine serum albumin (BSA) in phosphate-buffered saline (PBS) at pH 7.4 (1 mL suspension per tube). 1 mg of synthetic HA can be added to each tube containing the peptide, and the tubes can be incubated at room temperature for 2 hours to prevent HA precipitation. After incubation, the sample 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 with PBS, for example, three times, and the final HA fraction can be resuspended in 100 µL of PBS. The resuspended HA-bound fraction and 100 µL of unbound protein suspension are transferred to a 96-well black plate (one fraction per well), and the relative protein concentration can be determined using a fluorescence plate reader (Spectramax i3x) via fluorescence excitation / emission at 488 nm / 585 nm.

[0154] Bone homogenate assay Before use, femurs from male C57BL / 6 mice can be stored at -80°C. Femurs can be transferred to 2 mL centrifuge tubes (2 femurs per tube) containing 1 mL of 0.2% (w / w) type 2 collagenase in BS (Worthington) and 1x of a mixture of EDTA-free serine and cysteine ​​protease inhibitors (COMPLETE™, Roche). The femurs can be briefly vortexed and incubated at 37°C with shaking (800 rpm) for 1 hour. Any remaining connective tissue can be removed, and the femurs can be placed in a culture dish on ice. The bone marrow can be rinsed with PBS using a needle and syringe. The dry bone can be aggregated (typically 30–50 mg per femur) and placed in pre-chilled hard tissue, then ground with 0.75 mL PBS containing 1x protease inhibitor using a single disposable stirrer (PRECELLYS). ® (Bertin Instruments). Femur homogenization can be performed using a high-throughput bead mill homogenizer (bullet blender, 4°C, maximum speed, 4 cycles for 30 seconds). The homogenate can be transferred to 1.5 mL centrifuge tubes and centrifuged at 12,000 xg for 15 minutes at 4°C to separate the bone homogenate from the released protein / cell debris. Other methods for separating the 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 binding assays.

[0155] The bone-targeting peptide can be diluted to 50 nM in PBS containing 0.1% BSA and incubated with 3 mg of bone homogenate in a single 1.5 mL centrifuge tube (1 mL per tube). The sample can be mixed at room temperature for 2 hours, followed by centrifugation to separate the bound and unbound fractions of the bone homogenate. The bone homogenate fractions can be washed three times with PBS, and the final homogenate precipitate can be resuspended in 100 µL of PBS. The relative fluorescent Fc-fusion concentrations of the resuspended bone homogenate and the 100 µL unbound protein suspension can be quantified using a fluorescent plate reader.

[0156] Determination of relative protein affinity of bone homogenate in vitro A multi-dose assay can be developed to classify the relative affinity of bone-binding proteins. For proteins that effectively bind to bone homogenate, the relative dissociation rate is determined by this kinetic bone-protein exchange assay. Proteins can be evaluated individually (e.g., one protein type per tube) by incubating saturated concentrations (1 μM) of unlabeled bone-binding protein and 5 mg of bone homogenate in 1.5 mL Eppendorf tubes. After 24 hours of incubation with the unlabeled protein, the bone homogenate can be centrifuged (16,000 rcf, 5 min) to remove excess unbound protein. The bone homogenate saturated with a given bone-binding protein can be resuspended in ALEXA FLUOR. ® Bone homogenates were incubated for 1, 2, 4, 8, and 24 hours in a 0.5 μM solution of the same bone-binding protein labeled with a fluorescent probe. The homogenates were then centrifuged, washed three times with PBS, and transferred to a 96-well black plate. The supernatant from the first centrifugation could also be collected to quantify the amount of remaining (unbound) fluorescent protein in the suspension. A fluorescent plate reader could 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.

[0157] Metabolic assay of ALP activity (MUP) 4-Methylumbelliferyl phosphate (4-MUP) can be used as an artificial substrate to determine the ALP activity of protein samples (supernatant, partially purified, or column-purified samples) in solution. Hydrolysis of the phosphate ester bond in 4-MUP releases the fluorescent compound 4-methylumbelliferone, which is readily detectable by a fluorometer. Product quantification can be performed using a standard curve of 4-methylumbelliferone (4-MU) measured on the same plate, with 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 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 can be prepared as a 0.1 mg / mL solution in assay buffer and serially diluted to an appropriate final concentration (e.g., 1 nM) for assay in assay buffer. A 4-MUP stock solution was prepared in the assay buffer. All solutions were brought to 37°C before the assay by adding 10 μM of 4-MUP to the protein sample. 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 in a plate reader maintained at 37°C for a total of 20 minutes. The reaction rate was calculated in units of activity using linear regression, where 1 U = 1 μmol of 4-MUP hydrolysis / min. Specific activity was calculated in units / mg of protein as determined.

[0158] Plate bone measurement ALP activity in bone homogenate fractions can be determined. The bone homogenate fraction can be suspended in 100 µL of PBS and transferred to a 96-well plate. Alternatively, 100 µL of unbound protein suspension can be transferred to individual wells of the 96-well plate. 100 µL of ALP detection solution (10 µM 4-MUP, 1% BSA) can be added to each well, and kinetic fluorescence readings (at 360 / 465) can be initiated immediately and run for 20 minutes, during which fluorescence intensity emission is collected every 30 seconds. The slope of fluorescence intensity relative to time represents the ALP concentration in each sample fraction.

[0159] 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 to maintain linearity. The MUP activity ratio for each fusion protein can be determined as ratio = (bound activity) / (unbound activity).

[0160] Protein activity in serum assays Serum samples can be diluted 100-fold 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 pK. Diluted samples can be quantified, and standard curves can be generated based on the known activity and concentration of Asforase α. The slope of fluorescence intensity relative to time represents the rate of 4-MU production, corresponding to ALP activity, as a function of units / mL serum in each sample fraction.

[0161] In vivo fluorescence imaging in mice You can use ALEXA FLUOR ® Semi-quantitative biodistribution studies of 750-labeled bone-targeting proteins and protein fragments (e.g., VHH) were performed in nude mice. The biodistribution can be achieved using the Invitrogen SAIVI kit (via covalent conjugation with activated succinimide ester) with ALEXA FLUOR. ® Bone-targeting ALP-Fc fusion protein and bone-targeting VHH were fluorescently labeled with 750 and purified in an exclusion resin to remove unconjugated fluorophores. The purified proteins (suspended in PBS) could be injected into nude mice via tail vein at a dose of approximately 3 mg / kg.

[0162] Female J:NU distantly bred mice (Jackson Laboratories, Bar Harbor, ME) were administered 3 mg / kg of the test sample via a volume-normalized 100 µL intravenous tail vein injection. For in vivo imaging, subjects were maintained under 2%–3% isoflurane anesthesia on an imaging platform (IVIS spectral imaging system, PerkinElmer Inc., Waltham, MA). Automated exposure settings with a field of view (FOV) C, F / Stop 2, medium binning, and an 800 nm emission / 750 nm excitation filter were used for both 2D epi-illumination and 3D transmitted illumination acquisition. In addition to the ability to acquire all samples of each tissue type simultaneously in a single image, 2D epi-illumination fluorescence imaging of ex vivo tissue samples was acquired under the same conditions. All animal studies were conducted in accordance with the Animal Welfare Act and the principles of the Laboratory Animal Care and Use Guidelines.

[0163] Fluorescence imaging analysis can be performed using the manufacturer's 2D / 3D software (Living Image 4.5.1, PerkinElmer). Manual localization of regions of interest (ROIs) of uniform area applied to each group of subjects addresses variability in subject localization. Longitudinal in vivo image color scale ranges can be normalized across all subjects and time points; color scales for ex vivo samples can be individually determined to best represent the fluorescence signal of each tissue group.

[0164] Determination of pyrophosphate hydrolysis The activity of the peptides described herein against the natural substrate pyrophosphate can be determined. Pyrophosphate hydrolysis can be measured using the PiBlue assay reagent (BioAssay Systems, Bosch), which turns bright green upon phosphate binding. The phosphate level in each well can be quantified using a standard curve of the phosphate solution prepared in assay buffer, which can be read on the assay plate. A 10 mM stock solution of sodium pyrophosphate decahydrate (Sigma Chemicals) can be prepared in pure water. The purified fusion protein sample is prepared as a 0.1 mg / mL solution 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. The pyrophosphate sample for assay can be prepared by diluting the stock solution in assay buffer. All solutions are brought to 37°C before the reaction begins. Add the protein solution to a clean 96-well plate and place the plate in a Jitterbug plate shaker maintained at 37°C. The reaction can be initiated by adding pyrophosphate solution to the protein solution. Typically, pyrophosphate hydrolysis can be 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. Set up 8 reaction wells at each pyrophosphate concentration, and terminate the reaction by adding PiBlue reagent (in an amount equal to the final reaction volume) after 0, 1, 2, 3, 4, 5, 6, and 7 minutes after adding pyrophosphate to the plate. Adding PiBlue reagent stops any further reaction by lowering the pH of the assay reagent and inactivating the enzyme. Allow the color 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 process curves from various time points. Using Michaelis-Menten enzyme kinetic fitting, the reaction rate at each concentration can be used to calculate the Km and Vmax values ​​in GraphPad prism. The kcat value is calculated as follows: Vmax / (number of moles of protein measured) = kcat.

[0165] Determination of pyridoxine 5'-phosphate hydrolysis.

[0166] The second natural substrate of alkaline phosphatase is pyridoxine 5'-phosphate (PLP). The activity of the peptide for PLP can be determined by a coupled assay, in which pyridoxine, a product of PLP hydrolysis, is detected via Rhizobium loureirii (…). M. loti Tetrapyridoxine dehydrogenase (tPLDH, SEQ ID NO: 246) is converted to fluorescent pyridoxine. The 6xHis-labeled tPLDH gene can be synthesized using standard methods and cloned into a bacterial expression plasmid controlled by the T7 promoter. 6xHis-labeled tPLDH is expressed in BL21(DE3) cells using a standard protocol and purified by standard affinity chromatography. The protein can be concentrated to 1900 uM using centrifugation (i.e., an Amicon Ultra15 spin concentrator) and frozen at -80°C until ready for assay. The purified fusion protein sample can be prepared as a 0.1 mg / mL solution 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 black 96-well plates with pyridoxine standards (prepared in assay buffer). NAD+ can be added to the protein sample. + Solutions of tPLDH and PLP (prepared in assay buffer) were prepared to achieve final concentrations of 3 mM NAD+, 4 uM tPLDH, and 3 uM PLP, and mixed. All solutions were brought to 37°C before the reaction began, and the reaction plate was incubated at 37°C, with fluorescence detected by excitation at 355 nm and emission measured at 445 nm. The amount of pyridoxine product produced could be calculated using a standard curve of measured fluorescence from the pyridoxine wells in the plate. The reaction rate could be calculated by linear regression of the progress curve in micromoles / minutes of pyridoxine produced. Specific activity could be calculated by dividing the reaction rate by the protein concentration used in the assay reaction.

[0167] Pharmacokinetic analysis in mouse models Sample protein in 11-12 week old male C57BL / 6 mice (Jackson Laboratory) can be administered via a single injection of 4-7 mg / kg into the tail vein or subcutaneously, with follow-up for 14-21 days. Two intermediate and one final blood draws (cardiac puncture, CO2 anesthesia) can be performed per mouse, staggered in the cohort (4 mice per molecule and per administration type, 4 groups per cohort). Blood samples (100 µL, yielding 50 µL plasma after centrifugation) can be collected in 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 flash-frozen in liquid N2 and stored at -80°C for further quantification.

[0168] In vivo mouse HPP model Efficacy can be measured in an in vivo mouse HPP model. In this prophylactic study, treatment can begin at birth and continue for 35 days. The peptide or its carrier (PBS) can be administered subcutaneously. Akp2GW - / - Within the scapular region of mice. Efficacy endpoints that can be assessed at the end of treatment are survival, bone mineralization defects, and growth (body weight). Body weight can also be assessed daily as an indicator of overall animal health. Age- and fetal-matched PBS-treated WT mice can serve as a reference control. Animals can receive daily subcutaneous injections of the peptide until day 24. On day 25, the daily subcutaneous dose of the peptide can be reduced to half the initial dose and maintained until the final treatment dose on day 35.

[0169] Pharmaceutical compositions, formulations and administration The polypeptides described herein, including sALP or sALP fusion polypeptides (e.g., polypeptides having the sequence of any one of SEQ ID NO: 7-223, 247, and 262-264, or variants thereof having at least 85% sequence identity with and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein), can be formulated into pharmaceutical compositions by a variety of methods known in the art. As those skilled in the art will understand, the route of administration and / or mode of administration can be used to specify the formulation. The route of administration can depend on a variety of factors, such as environment and therapeutic goals. In particular, the polypeptides and fusion polypeptides described herein can be formulations administered via any route known in the art (e.g., subcutaneous (e.g., by subcutaneous injection), intravenous, oral, intranasal, intramuscular, sublingual, intrathecal, or intradermal). For example, the pharmaceutical composition can be in the form of a liquid, solution, suspension, pill, capsule, tablet, capsule tablet, powder, gel, ointment, cream, spray, mist, atomized vapor, aerosol, or phytosome.

[0170] Preparations Compositions comprising sALP and sALP fusion peptides (e.g., peptides having the sequence of any one of SEQ ID NO: 7-223, 247, and 262-264, or variants thereof having at least 85% sequence identity with and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein) can be formulated according to standard methods. Pharmaceutical formulation is a well-established technique and, for example... Remington: The Science and Practice of Pharmacy[Remington: The Science and Practice of Pharmacy], 22nd ed., Allen, 2012; further described in Ansel et al. (1999) “Pharmaceutical Dosage Forms and Drug Delivery Systems,” 7th ed., Lippincott Williams & Wilkins Publishers (ISBN: 0683305727) and Kibbe (2000) “Handbook of Pharmaceutical Excipients of the American Pharmaceutical Association,” 3rd ed. (ISBN: 091733096X). For example, sALP compositions (e.g., peptides having the sequence of any one of SEQ ID NO: 7-223, 247, and 262-264, or variants thereof having at least 85% sequence identity and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein) can be formulated into buffer solutions of, for example, suitable concentrations and adapted for storage at 2°C–8°C (e.g., 4°C). The compositions can also be formulated for storage at temperatures below 0°C (e.g., -20°C or -80°C). The compositions can be further formulated for storage at 2°C–8°C (e.g., 4°C) for up to 2 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). Therefore, the compositions described herein can be stored stably at 2°C–8°C (e.g., 4°C) for at least 1 year. The composition can be formulated to a suitable volume, for example, from about 0.1 mL to about 10 mL.

[0171] Compositions comprising sALP and sALP fusion peptides (e.g., peptides having the sequence of any one of SEQ ID NO: 7-223, 247, and 262-264, or variants thereof having at least 85% sequence identity with and / or having 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 insoluble solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. Preferred forms depend in part on the intended mode of administration and therapeutic application.

[0172] For example, compositions intended for systemic or local delivery may be in the form of injectable or insoluble solutions. Thus, compositions (e.g., polypeptides having the sequence of any one of SEQ ID NO: 7-223, 247, and 262-264, or variants thereof having at least 85% sequence identity and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein) can be formulated for administration via parenteral routes (e.g., subcutaneous, intravenous, intraperitoneal, or intramuscular injection). As used herein, “parenteral administration,” “via parenteral,” and other grammatically equivalent phrases refer to routes of administration other than enteral and local administration, typically by injection, and include, but are not limited to, subcutaneous, intradermal, intravenous, intranasal, intraocular, pulmonary, intramuscular, intraarterial, intrathecal, intracapsular, intra-abdominal, intratracheal, subcutaneous, intra-articular, subcapsular, subarachnoid, spinal, epidural, intracerebral, intracranial, carotid, and intrasternal injections and infusions. Specific routes of administration include intravenous and subcutaneous administration.

[0173] Compositions comprising sALP and sALP fusion peptides (e.g., peptides having the sequence of any one of SEQ ID NO: 7-223, 247, and 262-264, or variants thereof having at least 85% sequence identity and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein) can be formulated into solutions, microemulsions, dispersions, liposomes, or other ordered structures suitable for stable storage at high concentrations. Sterile injectable solutions can be prepared by incorporating the desired amount of the composition described herein with one or more of the components listed above into a suitable solvent, followed by filtration sterilization. Typically, dispersions are prepared by incorporating the composition described herein into a sterile medium containing a base dispersion medium and other desired components from those listed above. In the case of sterile powders used to prepare sterile injectable solutions, preparation methods include vacuum drying and freeze-drying, which produce powders of the composition described herein plus any additional desired components from their previous sterile-filtered solutions (see below). Proper fluidity of the solution can be maintained, for example, by using coatings such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using surfactants. Extended absorption of injectable compositions can be achieved by including, for example, delayed-absorption agents such as monostearate and gelatin in the composition.

[0174] 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 by Epstein et al. (1985). Proc Natl Acad Sci USA [Proceedings of the National Academy of Sciences of the United States of America] 82:3688; Hwang et al. (1980) Proc Natl Acad Sci USAThe methods described in [Proceedings of the National Academy of Sciences] 77:4030 and U.S. Patent Nos. 4,485,045 and 4,544,545. Liposomes with extended cycle times are disclosed, for example, in U.S. Patent No. 5,013,556.

[0175] Compositions comprising sALP and sALP fusion peptides (e.g., peptides having the sequence of any one of SEQ ID NO: 7-223, 247, and 262-264, or variants thereof having at least 85% sequence identity with and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein) can also be formulated with a carrier that protects the composition (e.g., sALP peptides or sALP fusion peptides) 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.

[0176] Compositions containing sALP or sALP fusion peptides (e.g., peptides having the sequence of any one of SEQ ID NO: 7-223, 247, and 262-264, or variants thereof having at least 85% sequence identity with and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein) can be formulated into injectable solutions that are clear, colorless to slightly yellow aqueous solutions at pH 7.4. sALP or sALP peptides (e.g., peptides having the sequence of any one of SEQ ID NO: 7-223, 247, and 262-264, or variants thereof having at least 85% sequence identity with and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein) 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 peptides can be formulated using carriers such as sodium chloride and / or sodium phosphate (e.g., about 150 mM NaCl and / or about 25 mM sodium phosphate, especially at a pH of about 7.4). Specifically, the composition can be formulated into an injectable solution of 40 mg / ml, wherein each ml of solution contains 40 mg sALP or sALP peptide (e.g., each vial contains 0.3 ml of solution and 12 mg sALP (40 mg / ml), each vial contains 0.45 ml of solution and 18 mg sALP (40 mg / ml), each vial contains 0.7 ml of solution and 28 mg sALP (40 mg / ml), or each vial contains 1.0 ml of solution and 40 mg sALP or sALP peptide (40 mg / ml)). sALP or sALP peptides (e.g., peptides having the sequence of any one of SEQ ID NO: 7-223, 247, and 262-264, or variants thereof having at least 85% sequence identity with and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein) can be formulated into an injectable solution at a concentration of 100 mg / ml, wherein each 1 ml solution contains 100 mg of sALP or sALP peptide (e.g., each vial contains 0.8 ml solution and 80 mg of aspherase α (100 mg / ml)). sALP can be formulated such that about 0.1 mg to about 500 mg is present in a volume of about 0.01 mL to about 10 mL.

[0177] When the composition is used in combination with a second active agent, the composition can be formulated together 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, for example, before administration, and administered together, or they can be administered separately, for example, at the same time or at different times.

[0178] Compositions comprising sALP and sALP fusion peptides (e.g., peptides having the sequence of any one of SEQ ID NO: 7-223, 247 and 262-264 or variants thereof having at least 85% sequence identity with and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein) can be formulated for administration to a subject together with intravenous immunoglobulin therapy (IVIG), plasma removal, plasma exchange or plasma exchange, or, in the case of administration to a fetus, to a woman carrying the fetus.

[0179] carrier / medium Formulations containing sALP or sALP fusion peptides (e.g., peptides having the sequence of any one of SEQ ID NO: 7-223, 247, and 262-264, or variants thereof having at least 85% sequence identity with and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein) may be administered to subjects with or susceptible to bone mineralization disorders (such as HPP) in combination with pharmaceutically acceptable sterile aqueous or non-aqueous solvents, suspensions, or emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils, fish oils, and injectable organic esters. Aqueous carriers include water, water-alcohol solutions, emulsions, or suspensions, including saline and buffered medical parenteral media, including sodium chloride solutions, Ringer's dextran solutions, glucose plus sodium chloride solutions, lactose-containing Ringer's solutions, or non-volatile oils. For example, a pharmaceutically acceptable carrier may include sodium chloride and / or sodium phosphate, wherein the composition comprises, for example, about 150 mM sodium chloride and / or about 25 mM sodium phosphate, pH 7.4.

[0180] Intravenous mediators can include fluids and nutritional supplements, electrolyte supplements, such as those based on Ringer's dextran, etc. These can include pharmaceutically acceptable salts, such as inorganic acid salts like hydrochloride, hydrobromide, phosphate, sulfate, etc.; and salts of organic acids, such as acetate, propionate, malonate, benzoate, etc. Additionally, excipients, such as wetting agents or emulsifiers, pH buffers, etc., may be present in these mediators. A detailed discussion of pharmaceutically acceptable carriers can be found in [link to relevant literature]. Remington: The Science and Practice of Pharmacy [Remington: The Science and Practice of Pharmaceutical Sciences], 22nd edition, edited by Allen, 2012.

[0181] dose It can be administered in a single dose range, such as 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 50 mg / kg, 0.5 mg / kg to 25 mg / kg, 1.0 mg / kg to 10 mg / kg, 1.5 mg / kg to 5 mg / kg, or 2.0 mg / kg to 3.0 mg / kg) or 1 μg / kg to 1,000 μg / kg (e.g., 5 μg / kg to 1,000 μg / kg, 1 μg / kg to 750 μg / kg, 5 The sALP peptide described herein (e.g., a peptide having the sequence of any one of SEQ ID NO: 7-223, 247 and 262-264 or a variant thereof having at least 85% sequence identity with it and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein) is administered to subjects who have or are susceptible to bone mineralization disorders (such as HPP).

[0182] 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, or 500 mg / kg; or 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” may be used to modify these doses by ±10% of the endpoints of the values ​​or ranges. Specifically, the composition according to this disclosure (e.g., a polypeptide having the sequence of any one of SEQ ID NO: 7-223, 247 and 262-264 or a variant thereof having at least 85% sequence identity with it and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein) may be administered to the subject at a dose ranging from about 0.001 mg / kg / day to about 500 mg / kg / day, about 0.01 mg / kg / day to about 100 mg / kg / day, or about 0.01 mg / kg / day to about 20 mg / kg / day. For example, a sALP composition (e.g., a polypeptide having the sequence of any one of SEQ ID NO: 7-223, 247 and 262-264 or a variant thereof having at least 85% sequence identity with it and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein) may be administered to a subject at a weekly dose ranging from, for example, from about 0.5 mg / kg / week to about 140 mg / kg / week, from, for example, from about 0.8 mg / kg / week to about 50 mg / kg / week, or from about 1 mg / kg / week to about 10 mg / kg / week (e.g., about 6 or about 9 mg / kg / week)). Specifically, sALP can be administered once or more per week (e.g., 1, 2, 3, 4, 5, 6, 7 or more per week), once or more every other week, or once or more per month (e.g., every 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days).

[0183] Specifically, sALP (e.g., a polypeptide having the sequence of any one of SEQ ID NO: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity with it and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein) can be administered at doses of 2 mg / kg three times a week (total dose 6 mg / kg / week), 1 mg / kg six times a week (total dose 6 mg / kg / week), 3 mg / kg three times a week (total dose 9 mg / kg / week), 0.5 mg / kg three times a week (total dose 1.5 mg / kg / week), or 9.3 mg / kg three times a week (total dose 28 mg / kg / week). This dosage can be adjusted by the clinician based on routine factors such as the severity of the disease and different parameters from subjects with or predisposed to bone mineralization disorders (such as HPP). Alternatively, it can be administered once weekly from 0.1 mg / kg to 9 mg / kg.

[0184] Doses of compositions comprising sALP and sALP fusion peptides (e.g., peptides having the sequence of any one of SEQ ID NO: 7-223, 247, and 262-264, or variants thereof having at least 85% sequence identity with and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein) can be provided in single-dose or multi-dose regimens. Doses can be administered, for example, hourly, every two hours, daily, every two days, twice a week, three times a week, four times a week, five times a week, six times a week, weekly, every two weeks, monthly, every two months, or annually. Alternatively, doses can be administered, for example, twice, three, four, five, six, seven, eight, nine, ten, eleven, or twelve times daily, weekly, or monthly. In particular, the dosing regimen is once 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 even the remaining lifespan of subjects with or predisposed to bone mineralization disorders (such as HPP). The dosage, frequency, and duration will be adjusted by the clinician based on routine factors such as the severity of the disease and different parameters from subjects with or predisposed to bone mineralization disorders (such as HPP).

[0185] For example, the dosage of sALP or sALP fusion peptides (e.g., peptides having the sequence of any one of SEQ ID NO: 7-223, 247 and 262-264 or variants thereof having at least 85% sequence identity with and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein) may be about 0.1 mg / kg body weight to about 10 mg / kg body weight, administered subcutaneously or intravenously once or more per week (e.g., 2, 3, 4, 5, 6 or 7 times).

[0186] Production of nucleic acids and peptides Polynucleotides encoding sALP and sALP fusion peptides (e.g., peptides having the sequence of any one of SEQ ID NO: 7-223, 247, and 262-264, or variants thereof having at least 85% sequence identity with and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein) can be generated by any method known in the art. The polynucleotide may encode an amino acid sequence having at least 85% (e.g., 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NO: 7-223, 247, and 262-264 (e.g., any one of SEQ ID NO: 72, 123, 155, or 177). The polynucleotide may have at least 85% (e.g., 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NO: 265-268. The polynucleotide may comprise or consist of any one of SEQ ID NO: 265-268. The polynucleotide may include or be composed of SEQ ID NO: 265. The polynucleotide may include or be composed of SEQ ID NO: 266. The polynucleotide may include or be composed of SEQ ID NO: 267. The polynucleotide may include or be composed of SEQ ID NO: 268.

[0187] Typically, molecular cloning methods are used to generate polynucleotides encoding the desired fusion polypeptide, and these are usually placed in a vector (such as a plasmid or virus). The vector is used to transform the polynucleotide into a host cell suitable for expressing the fusion polypeptide. Representative methods are disclosed, for example, by Maniatis et al. (Cold Springs Harbor Laboratory, 1989). Many cell types can be used as suitable host cells, although mammalian cells are preferred because they are capable of conferring appropriate post-translational modifications (e.g., glycosylation or sialylation). Host cells disclosed herein may 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, host cells may be Chinese hamster ovary (CHO) cells (e.g., CHO-DG44 cells) or HEK293 cells.

[0188] sALP and sALP fusion peptides (e.g., peptides having the sequence of any one of SEQ ID NO: 7-223, 247, and 262-264, or variants thereof having at least 85% sequence identity and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein) can be generated under any conditions suitable for achieving sALP peptide expression in host cells. Such conditions include appropriately selected culture media prepared with components such as buffers, bicarbonate and / or HEPES, ions (e.g., chloride, phosphate, calcium, sodium, potassium, magnesium, iron), carbon sources (e.g., monosaccharides, amino acids, potential lipids, nucleotides, vitamins), and growth factors (e.g., insulin); commonly available commercially available media such as α-MEM, DMEM, Ham's-F12, and IMDM supplemented with 2-4 mM L-glutamine and 5% fetal bovine serum; and commonly available commercially available animal protein-free media such as HYCLONE™ SFM4CHO and Sigma CHO DHFR. - Cambrex POWER™ CHO CD supplemented with 2-4 mM L-glutamine. These media are ideally prepared in the absence of thymidine, hypoxanthine, and L-glycine to maintain selection pressure, thereby allowing stable protein product expression.

[0189] Large-scale methods for producing bulk proteins are described, for example, in PCT Publications WO 2017 / 031114 and WO2017 / 214130, the disclosures of which are hereby incorporated by reference in their entirety.

[0190] Treatment This article provides methods for treating or improving at least one symptom in subjects with bone mineralization disorders such as HPP. Other diseases or disorders, such as fractures, osteoporosis, sclerotic ossification, chondrocalcinosis, hypotonia, Duchenne muscular dystrophy, tracheobronchomalacia, seizures, neurofibromatosis 1 (NF-1), and craniosynostosis, may also be treated by the compositions and methods described herein. Subjects may have myasthenia gravis. Subjects may have myasthenic disorders such as calcium pyrophosphate deposition (CPPD) or familial hypophosphatemia. Such treatment may include administration of alkaline phosphatase or a polypeptide having alkaline phosphatase activity to reduce elevated PPi concentrations in such subjects. For example, soluble alkaline phosphatase (sALP, e.g., a polypeptide having the sequence of any one of SEQ ID NO: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity with and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein) may be administered to neonates, children, adolescents, or adults.

[0191] Subjects may be diagnosed with bone mineralization disorders (e.g., HPP) prior to administration of alkaline phosphatase or a polypeptide with alkaline phosphatase activity (e.g., sALP, such as a polypeptide having a sequence of any one of SEQ ID NO: 7-223, 247, and 262-264 or a variant thereof having at least 85% sequence identity with it and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein). Additionally, subjects with or predisposed to bone mineralization disorders (such as HPP) may be initial subjects who have not previously been treated with sALP (e.g., a polypeptide having a sequence of any one of SEQ ID NO: 7-223, 247, and 262-264 or a variant thereof having at least 85% sequence identity with it and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein).

[0192] The method comprises administering alkaline phosphatase or a polypeptide with alkaline phosphatase activity (e.g., sALP, such as a polypeptide having the sequence of any one of SEQ ID NO: 7-223, 247 and 262-264 or a variant thereof having at least 85% sequence identity with it and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein) in single or multiple doses over a period of time to a subject who has or is susceptible to bone mineralization disorders (such as HPP). Specifically, sALP (such as peptides having the sequence of any one of SEQ ID NO: 7-223, 247 and 262-264) can be administered to subjects previously identified as having elevated inorganic pyrophosphate (PPi) concentrations or HPP symptoms (e.g., myasthenia gravis) with at least one pre-determined biomarker / score (e.g., mean BOT-2 strength score less than 10, mean BOT-2 running speed and agility score less than 5, mean CHAQ index score greater than about 0.8, and / or mean PODCI score less than about 40, mean 6MWT less than about 80% of the predicted 6MWT value, muscle strength grade less than 5, and / or mean HHD value (e.g., mean HHD muscle strength or grip strength value) less than about 80% of the predicted HHD value. For example, sALP can be administered to subjects previously identified as having the following PPi concentrations in a sample (e.g., a plasma sample): greater than about 5.71 μM for infants or children (e.g., subjects younger than about 12 years of age); greater than about 4.78 μM for adolescents (e.g., subjects aged from about 13 to about 18 years of age); or greater than about 5.82 μM for adults (e.g., subjects older than about 18 years of age). In other embodiments, the bone mineralization disorders described herein (such as HPP) are caused by elevated concentrations of at least one alkaline phosphatase substrate (e.g., PPi, PLP, PEA, etc.). Alternatively, alkaline phosphatase or a polypeptide with alkaline phosphatase activity (e.g., sALP, such as a polypeptide having the sequence of any one of SEQ ID NO: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity with it and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein) may be administered to subjects with or susceptible to bone mineralization disorders (such as HPP) before determining muscle weakness scores (e.g., using BOT-2 strength score, BOT-2 running speed and agility score, CHAQ index score, BSID-III scale score, PDMS-2 standard score, muscle strength score, 6MWT value, and / or HHD value).

[0193] In addition, each of the scores described herein for subjects with or susceptible to bone mineralization disorders (such as HPP) (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 described in Phillips et al. 2015 Bone Abstracts 4: P136), or HHD value) can be used alone or in any combination to evaluate the therapeutic efficacy of sALP (e.g., peptides having sequences of any one of SEQ ID NO: 7-223, 247, and 262-264 or variants thereof having at least 85% sequence identity with and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein), wherein improvement relative to a certain test score demonstrates that sALP is effective for treating bone mineralization disorders (such as HPP).

[0194] For example, when administration of alkaline phosphatase or a peptide with alkaline phosphatase activity (e.g., sALP, such as a peptide having the sequence of any one of SEQ ID NO: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity with it and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein) to a subject with or susceptible to bone mineralization disorders (such as HPP) results in an average increase in BOT-2 strength score to about 10 or greater than about 10, wherein the subject previously had an average BOT-2 strength score of less than about 10, then treatment with alkaline phosphatase or a peptide with alkaline phosphatase activity is effective in treating, for example, physical damage associated with bone mineralization disorders (such as HPP). Alternatively, when administration of sALP does not result in an average increase in BOT-2 strength score to about 10 or greater than about 10, the dose and / or frequency of administration of alkaline phosphatase or a peptide with alkaline phosphatase activity may be varied to determine the effective amount of alkaline phosphatase or a peptide with alkaline phosphatase activity for the subject. For example, the dose of sALP (e.g., a polypeptide having the sequence of any one of SEQ ID NO: 7-223, 247 and 262-264 or a variant thereof having at least 85% sequence identity with it and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein) can be increased from, 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.

[0195] Additionally, when alkaline phosphatase or a peptide with alkaline phosphatase activity (e.g., sALP, such as a peptide having the sequence of any one of SEQ ID NO: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity with it and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein) is administered to a subject with or susceptible to a bone mineralization disorder (such as HPP) resulting in an improvement in one or more of the subject's muscle strength class classification (e.g., from a previously lower muscle strength class to a muscle strength class of 1, 2, 3, 4, or 5), wherein the subject previously had an average muscle strength class of less than about 5, then alkaline phosphatase or a peptide with alkaline phosphatase activity is effective in treating, for example, physical injuries associated with a bone mineralization disorder (such as HPP). Alternatively, when administration of sALP does not result in an improvement of one or more muscle strength class classifications in a subject from a previously lower muscle strength class, the dose and / or frequency of administration of alkaline phosphatase or a peptide with alkaline phosphatase activity may be altered (e.g., increased) to determine the effective amount of alkaline phosphatase or a peptide with alkaline phosphatase activity for the subject. For example, the dose of sALP (e.g., a peptide having the sequence of any one of SEQ ID NO: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity with it and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein) may be increased from, 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.

[0196] Biomarkers / endpoints for diagnosis and / or treatment Bone mineralization disorders (such as HPP, including, for example, perinatal HPP, infant HPP, childhood HPP, and dental hypoalkaline phosphatase syndrome, HPP-like disease, CPPD, and familial hypophosphatemia, as described herein) can be treated with alkaline phosphatase or peptides with alkaline phosphatase activity (e.g., sALP, such as peptides having the sequence of any one of SEQ ID NO: 7-223, 247, and 262-264, or variants thereof having at least 85% sequence identity with it and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein). The methods described herein can also be used to diagnose subjects with or predisposed to bone mineralization disorders (such as HPP), identify subjects with or predisposed to bone mineralization disorders (such as HPP), or test the efficacy of treatments for bone mineralization disorders (such as HPP). For example, a subject can be diagnosed with or predisposed to bone mineralization disorders (such as HPP) if they are characterized by certain characteristic biomarkers or metric scores. Subjects may be treated with alkaline phosphatase or peptides with alkaline phosphatase activity (e.g., sALP, such as peptides having the sequence of any one of SEQ ID NO: 7-223, 247, and 262-264, or variants thereof having at least 85% sequence identity with and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein), and treatment efficacy or effect may be analyzed using characteristic biomarkers or metric scores. Such biomarkers may include, for example, elevated concentrations of inorganic pyrophosphate (PPi) and / or decreased alkaline phosphatase (ALP) in the subject's serum, bone or muscle tissue, or urine. Exemplary measures useful in the methods described herein for determining the efficacy of myasthenia gravis treatment may include: (1) the Bunie Test of Motor Skills, Version 2 (BOT-2), (2) the Childhood Health Assessment Questionnaire (CHAQ), (3) the Pediatric Outcomes Data Collection Tool (PODCI), (4) the Bailey Scales of Infant and Toddler Development, Version 3 (BSID-III), (5) the Peabody Developmental Motor Scales, Version 2 (PDMS-2), (6) the 6-Minute Walk Test (6MWT), (7) muscle strength ratings, and (8) handheld dynamometers (HHD), which will be described in further detail below.

[0197] Bone healing and mineralization Subjects with or susceptible to bone mineralization disorders (such as HPP) can be identified based on their bone mineralization levels for treatment with alkaline phosphatase or peptides with alkaline phosphatase activity (e.g., sALP, such as peptides having the sequence of any one of SEQ ID NO: 7-223, 247, and 262-264, or variants thereof having at least 85% sequence identity with and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein). For example, bone mineralization can be used as a diagnostic measure of a patient having a bone mineralization disorder (such as HPP) or to test the efficacy of the peptides described herein. In subjects with bone mineralization disorders (e.g., HPP), administration of sALP resulted in increased bone healing in subjects following successful treatment.

[0198] Reduced bone healing leads to bone loss and includes reduced mineralization resulting in nonunion of two or more bones. Bone healing and reduced mineralization can be compared to reference bone. Methods used to identify reduced bone healing and reduced mineralization are routine and include non-invasive techniques such as radiography and computed tomography (CT). Typically, images of relevant areas of the subject can be taken at one or more time points before and after sALP treatment, and these images can be compared to assess treatment efficacy. Reduced bone healing and / or reduced mineralization can be identified as a reduction in opacity. Images can be taken at any time during sALP treatment and can be timed, for example, 1, 2, 3, 4, 5, or 6 days, weeks, months, or years after the start of sALP ERT treatment, or when a decline in efficacy is suspected. Reduced bone healing and / or reduced mineralization in the subject can become detectable at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks or months after the start of sALP treatment. In some cases, the reduction in bone healing and / or mineralization in subjects can 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 sALP ERT treatment. Increased bone healing and mineralization can be used to determine the recovery of sALP efficacy and the effective treatment of bone mineralization disorders (e.g., HPP) after therapy.

[0199] Bone mineral density (BMD) Subjects with or predisposed to bone mineralization disorders (such as HPP) can be identified based on BMD levels for treatment with alkaline phosphatase or peptides with alkaline phosphatase activity (e.g., sALP, such as peptides having the sequence of any one of SEQ ID NO: 7-223, 247, and 262-264, or variants thereof having at least 85% sequence identity with and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein). A decrease in BMD (e.g., relative to normal subjects) can be used as a measure to diagnose a patient with a bone mineralization disorder (such as HPP).

[0200] During ERT, a decrease in BMD can be used to monitor the efficacy of sALP (e.g., peptides of any one of SEQ ID NO: 7-223, 247, and 262-264). 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 using any conventional method, including CT Hounsfield measurements, and results can be compared with normative databases or control subjects. BMD is sometimes reported as a Z-score or T-score. Pretreatment BMD values ​​can be measured at any time during sALP ERT treatment and can be timed at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days, weeks, months, or years after the start of sALP ERT. Post-treatment reference BMD values ​​may decrease by, for example, 0.01%, 0.05%, 0.1%, 0.5%, or 1%. After sALP treatment begins, the decrease in BMD at the reference point may remain unchanged or the change may be undetectable. An increase in BMD can be used to determine the recovery of sALP efficacy and whether bone mineralization disorders (e.g., HPP) have been effectively treated after therapy.

[0201] Plasma inorganic pyrophosphate (PPi) and alkaline phosphatase (ALP) concentrations Subjects with or predisposing to bone mineralization disorders (such as HPP) can be identified by measuring the concentrations of inorganic pyrophosphate (PPi) and / or alkaline phosphatase (ALP) in samples from subjects (such as plasma or urine samples) for treatment with alkaline phosphatase or a polypeptide with alkaline phosphatase activity (e.g., sALP, for example, a polypeptide having the sequence of any one of SEQ ID NO: 7-223, 247, and 262-264 or a variant thereof having at least 85% sequence identity with it and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein). Any method known to those skilled in the art can be used to quantify PPi and / or ALP concentrations in plasma or urine samples, as described in detail in Whyte et al., 1995 (J. Clin. Invest. [Journal of Clinical Research] 95(4): 1440-1445), which is hereby incorporated by reference in its entirety. Methods for quantifying PPi concentrations in plasma or urine samples are also described in Cheung et al., 1977 (Anal. Biochem. [Analytical Biochemistry] 83: 61-63), Cook et al., 1978 (Anal. Biochem. [Analytical Biochemistry] 91: 557-565), and Johnson et al., 1968 (Anal. Biochem. [Analytical Biochemistry] 26: 137-145), each of which is incorporated herein by reference in its entirety.

[0202] Specifically, alkaline phosphatase or a polypeptide with alkaline phosphatase activity (e.g., sALP, such as a polypeptide having the sequence of any one of SEQ ID NO: 7-223, 247 and 262-264 or a variant thereof having at least 85% sequence identity with it and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein) may be administered to subjects previously identified as having or susceptible to bone mineralization disorders (such as HPP) with plasma PPi concentrations up to about 6 µM (e.g., about 4.5 µM, about 5 µM, or about 5.5 µM or in the range of about 4.5 µM to about 6 µM). For example, alkaline phosphatase or a polypeptide with alkaline phosphatase activity may be administered to infants or children (e.g., subjects younger than about 12 years of age) with plasma PPI concentrations of about 5.71 μM or higher; adolescents (e.g., subjects aged from about 13 to about 18 years of age) with plasma PPI concentrations of about 4.78 μM or higher; or adults (e.g., subjects older than about 18 years of age) with plasma PPI concentrations of about 5.82 μM or higher. Additionally, alkaline phosphatase or peptides with alkaline phosphatase activity can be administered to subjects (e.g., humans) who have or are susceptible to bone mineralization disorders (such as HPP) and who have been previously identified as having the following plasma ALP concentrations: for example, approximately 90 U / L or less for subjects aged 0 to 14 days; approximately 134 U / L or less for subjects aged 15 days to less than 1 year; approximately 156 U / L or less for subjects aged approximately 1 year to less than 10 years; approximately 141 U / L or less for subjects aged approximately 10 years to less than approximately 13 years; approximately 62 U / L or less for female subjects aged approximately 13 years to less than approximately 15 years; approximately 127 U / L or less for male subjects aged approximately 13 years to less than approximately 15 years; and approximately 54 U / L or less for female subjects aged approximately 15 years to less than approximately 17 years. U / L or lower; for male subjects aged approximately 15 years to less than approximately 17 years, approximately 89 U / L or lower; for female subjects aged approximately 17 years or older, approximately 48 U / L or lower; or for male subjects aged approximately 17 years or older, approximately 59 U / L or lower.

[0203] Plasma PPi and / or plasma ALP concentrations in subjects (e.g., humans) with or susceptible to bone mineralization disorders (such as HPP) can be compared to those in normal subjects to determine the therapeutic effect of administering alkaline phosphatase or a polypeptide with alkaline phosphatase activity (e.g., sALP, such as a polypeptide having the sequence of any one of SEQ ID NO: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity with it and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein). Specifically, alkaline phosphatase or a polypeptide with alkaline phosphatase activity may be administered for a treatment period of at least one year (e.g., at least two, three, four, five, six, seven, eight, nine, ten, or longer than ten years, such as the subject's lifetime). Alternatively, these methods may include measuring plasma PPi and / or plasma ALP concentrations prior to administration of alkaline phosphatase or a peptide with alkaline phosphatase activity to assess the efficacy of treatment of subjects with alkaline phosphatase or a peptide with alkaline phosphatase activity.

[0204] These methods result in a decrease in PPi and / or an increase in ALP concentration in samples (e.g., plasma samples) from subjects who have or are susceptible to bone mineralization disorders (such as HPP) (e.g., human subjects). For example, treatment with alkaline phosphatase or a polypeptide with alkaline phosphatase activity (e.g., sALP, such as a polypeptide having the sequence of any one of SEQ ID NO: 7-223, 247, and 262-264 or a variant thereof having at least 85% sequence identity with it and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein) results in a decrease in PPi concentration 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%) in samples (e.g., plasma samples) from subjects. Therefore, after administration of alkaline phosphatase or a peptide with alkaline phosphatase activity, subjects exhibit plasma PPi concentrations 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.

[0205] Similarly, treatment with alkaline phosphatase or a peptide with alkaline phosphatase activity resulted in an increase of 30%, 35%, 40%, 45%, 50%, 55%, 60%, or more in ALP concentrations in samples (e.g., plasma samples) from subjects (e.g., humans) with or susceptible to bone mineralization disorders (such as HPP) compared to subjects before administration of alkaline phosphatase or a peptide with alkaline phosphatase activity. For example, administration of alkaline phosphatase or a peptide with alkaline phosphatase activity increases the concentration of ALP in samples (e.g., plasma samples) from subjects to the following levels: approximately 273 U / L or greater for subjects aged 0 to 14 days; approximately 518 U / L or greater for subjects aged 15 days to less than 1 year; approximately 369 U / L or greater for subjects aged approximately 1 year to less than 10 years; approximately 460 U / L or greater for subjects aged approximately 10 years to less than approximately 13 years; approximately 280 U / L or greater for female subjects aged approximately 13 years to less than approximately 15 years; approximately 517 U / L or greater for male subjects aged approximately 13 years to less than approximately 15 years; approximately 128 U / L or greater for female subjects aged approximately 15 years to less than approximately 17 years; and approximately 365 U / L or greater for male subjects aged approximately 15 years to less than approximately 17 years. U / L or greater; for female subjects aged approximately 17 years or older, approximately 95 U / L or greater; or for male subjects aged approximately 17 years or older, approximately 164 U / L or greater.

[0206] Decreased plasma PPi and / or increased ALP concentrations in subjects (e.g., humans) with or susceptible to bone mineralization disorders (such as HPP) can persist throughout administration of alkaline phosphatase or a polypeptide with alkaline phosphatase activity (e.g., sALP, such as a polypeptide having the sequence of any one of SEQ ID NO: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity with it and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein). For example, during treatment with sALP, plasma PPi concentrations decrease by about 25% and remain at ±10% of the decreased plasma PPi concentration, and / or during treatment with alkaline phosphatase or a polypeptide with alkaline phosphatase activity, plasma ALP concentrations increase by about 50% and remain at ±10% of the increased plasma ALP concentration.

[0207] Alternatively, when administration of alkaline phosphatase or a polypeptide with alkaline phosphatase activity (e.g., sALP, such as a polypeptide having the sequence of any one of SEQ ID NO: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity with and / or 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 the PPi concentration in plasma samples from subjects (e.g., humans) with or susceptible to bone mineralization disorders (such as HPP), the dose and / or frequency of sALP administration may be varied to determine the effective amount of sALP for the subject. Similarly, when administration of alkaline phosphatase or a polypeptide with alkaline phosphatase activity does not result in an average increase of about 50% or more in the ALP concentration in plasma samples from the subject, the dose and / or frequency of alkaline phosphatase or a polypeptide with alkaline phosphatase activity may be varied to determine the effective amount of alkaline phosphatase or a polypeptide with alkaline phosphatase activity for the subject. For example, the dose of alkaline phosphatase or a polypeptide with alkaline phosphatase activity can be increased from, for example, about 0.5 mg / kg / week or about 3.5 mg / kg / week to about 3-6 mg / kg / week or about 6-9 mg / kg / week.

[0208] BOT-2 (Boot-2) The exemplary Bruininks Test of Motor Skills Version 2 (BOT-2) is described in Bruininks, RH (2005). Bruininks-Oseretsky Test of Motor Proficiency [Bruni's Motor Skills Test] (BOT-2) Minneapolis, Minnesota: Pearson Assessment, Inc., which is incorporated herein by reference in its entirety. In particular, BOT-2 can be used to assess physical impairment and mobility limitations in subjects who have or are susceptible to bone mineralization disorders (e.g., HPP) to generate a subject's BOT-2 score.

[0209] BOT-2 includes a series of tests to assess a subject's physical impairment, which can be performed using kits that include these tests. BOT-2 provides a comprehensive BOT-2 score in the following areas: strength, running speed and agility, fine motor precision, fine motor integration, hand dexterity, bilateral coordination, balance, and upper limb coordination. For example, subjects with or predisposed to bone mineralization disorders (such as HPP) can perform sit-ups, V-ups, standing long jumps, wall sits, and / or push-ups to determine their BOT-2 strength score. Subjects with or predisposed to bone mineralization disorders (such as HPP) can perform a balance beam and / or shuttle runs, two-legged lateral jumps, and / or one-legged lateral jumps to determine their BOT-2 running speed and agility score. Subjects with or predisposed to bone mineralization disorders (such as HPP) can cut a circle and / or connect dots to determine their BOT-2 fine motor precision score. Subjects with or predisposed to bone mineralization disorders (such as HPP) can replicate stars and / or squares to determine their BOT-2 fine motor integration score. Subjects with or predisposed to bone mineralization disorders (such as HPP) can transfer coins, sorting cards, and / or blocks to determine their hand dexterity score. Subjects with or predisposed to bone mineralization disorders (such as HPP) can tap their feet and fingers and / or perform jumping jacks to determine their BOT-2 bilateral coordination score. Subjects with or predisposed to bone mineralization disorders (such as HPP) can walk forward in a line and / or stand on one leg on a balance beam to determine their BOT-2 balance score. Subjects with or predisposed to bone mineralization disorders (such as HPP) can throw a ball at a target and / or catch the thrown ball to determine their BOT-2 upper limb coordination score.

[0210] Subjects with or predisposed to bone mineralization disorders (such as HPP) may be tested in one or more described domains (strength, running speed and agility, fine motor precision, fine motor integration, hand dexterity, bilateral coordination, balance, and upper limb coordination) to generate a BOT-2 score indicative of physical impairment. Within each BOT-2 domain (strength, running speed and agility, fine motor precision, fine motor integration, hand dexterity, bilateral coordination, balance, and upper limb coordination), such subjects may perform one or more tests to determine their BOT-2 score. For example, subjects may perform one or more of sit-ups, V-ups, standing long jumps, wall sits, and push-ups to determine their BOT-2 strength score. Therefore, only one test (e.g., a test selected from the group of sit-ups, V-ups, standing long jumps, wall sits, and push-ups) may be performed to determine the BOT-2 score (e.g., BOT-2 strength score) of subjects with or predisposed to bone mineralization disorders (such as HPP (e.g., HPP-like disease)).

[0211] Each BOT-2 score (intensity, running speed and agility, fine motor precision, fine motor integration, hand dexterity, bilateral coordination, balance, and upper limb coordination) of a subject with or susceptible to bone mineralization disorders (such as HPP) can be compared to the BOT-2 score of a subject without bone mineralization disorders (such as HPP) to, for example, determine a baseline comparison of BOT-2 scores. Each BOT-2 score (e.g., intensity, running speed and agility, fine motor precision, fine motor integration, hand dexterity, bilateral coordination, balance, and upper limb coordination) of a subject with or susceptible to bone mineralization disorders (such as HPP) can be compared to the BOT-2 score of other subjects with or susceptible to bone mineralization disorders (such as HPP) to, for example, provide a relative BOT-2 score for the subject.

[0212] BOT-2 scores (e.g., strength, running speed and agility, fine motor precision, fine motor integration, hand dexterity, bilateral coordination, balance, and upper limb coordination) range from about 0 to equal to or less than about 25, where scores of about 10 to about 20 are considered representative of healthy subjects (e.g., subjects without bone mineralization disorders such as HPP). Subjects with average BOT-2 scores (e.g., strength, running speed and agility, fine motor precision, fine motor integration, hand dexterity, bilateral coordination, balance, and upper limb coordination) less than about 10 can be treated with alkaline phosphatase or a peptide with alkaline phosphatase activity (e.g., sALP, e.g., a peptide having a sequence of any one of SEQ ID NO: 7-223, 247, and 262-264 or a variant thereof having at least 85% sequence identity with it and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein).

[0213] For example, subjects with or predisposing to bone mineralization disorders (such as HPP) can be treated with sALP (e.g., a polypeptide having a sequence of any one of SEQ ID NO: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity with it 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 lifetime of the subject. Similarly, subjects with or predisposing to bone mineralization disorders (such as HPP) who have 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 then be treated with sALP (e.g., a polypeptide having a sequence of any one of SEQ ID NO: 7-223, 247, and 262-264 or a variant thereof having at least 85% sequence identity with it 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 lifetime of the subject.

[0214] These methods can lead to improvements in BOT-2 scores (e.g., strength, running speed and agility, fine motor precision, fine motor integration, hand dexterity, bilateral coordination, balance, and / or upper limb coordination scores) in subjects with or predisposed to bone mineralization disorders (such as HPP). For example, treatment with alkaline phosphatase or peptides with alkaline phosphatase activity (such as sALP (e.g., peptides having the sequence of any one of SEQ ID NO: 7-223, 247, and 262-264 or variants thereof having at least 85% sequence identity with it and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein)) 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). Additionally, treatment with sALP (e.g., a polypeptide having a sequence of any one of SEQ ID NO: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity with it and / or having 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).

[0215] Increases in BOT-2 scores (e.g., scores for strength, running speed and agility, fine motor precision, fine motor integration, hand dexterity, bilateral coordination, balance, and / or upper limb coordination) can persist for a period of time, for example, throughout the administration of alkaline phosphatase or peptides with alkaline phosphatase activity (such as sALP (e.g., peptides having the sequence of any one of SEQ ID NO: 7-223, 247, and 262-264, or variants thereof having at least 85% sequence identity with it and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein)). Similarly, reductions in muscle physical damage following administration of alkaline phosphatase or peptides with alkaline phosphatase activity can persist throughout the administration of alkaline phosphatase or peptides with alkaline phosphatase activity.

[0216] BOT-2 scores (strength, running speed and agility, fine motor precision, fine motor integration, hand dexterity, bilateral coordination, balance, and upper limb coordination) in subjects with or predisposed to bone mineralization disorders (such as HPP) can be used alone or in combination with other measures to assess the efficacy of treatment with alkaline phosphatase or peptides with alkaline phosphatase activity (such as sALP (e.g., peptides having the sequence of any one of SEQ ID NO: 7-223, 247, and 262-264 or variants thereof having at least 85% sequence identity with it and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein)), wherein improvement relative to a test score demonstrates that alkaline phosphatase or peptides with alkaline phosphatase activity are effective for treating muscle damage associated with bone mineralization disorders (such as HPP). For example, when administration of the sALP described herein to subjects with or susceptible to bone mineralization disorders (such as HPP) results in an average increase in BOT-2 running speed and agility scores of about 5 or greater than about 5, where the subjects previously had an average BOT-2 running speed and agility score of less than about 5, then the sALP is considered effective, for example, in treating physical injuries associated with bone mineralization disorders (such as HPP).

[0217] In addition, within each BOT-2 domain (intensity, running speed and agility, fine motor precision, fine motor integration, hand dexterity, bilateral coordination, balance, and upper limb coordination), subjects with or predisposed to bone mineralization disorders (such as HPP) may undergo one or more tests to determine their BOT-2 score.

[0218] Alternatively, when administration of alkaline phosphatase or a peptide with alkaline phosphatase activity (such as sALP as described herein) does not result in an average increase in BOT-2 running speed and agility scores to greater than about 5, the dosage and / or frequency of administration may be varied to determine the effective amount of alkaline phosphatase or a peptide with alkaline phosphatase activity for subjects with or susceptible to bone mineralization disorders (such as HPP). For example, the dosage of sALP (e.g., a peptide having the sequence of any one of SEQ ID NO: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity with it and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein) may be increased from, for example, 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.

[0219] Childhood Health Assessment Questionnaire (CHAQ) The Childhood Health Assessment Questionnaire (CHAQ) can be used to assess the health status of children with bone mineralization disorders (such as HPP) to generate a CHAQ score for the child, as used by Bruce and Fries. J. Rheumatol. [Journal of Rheumatology] 30(1): 167-178, 2003) and Klepper ( Arthritis & Rheumatism The CHAQ, described in [Arthritis and Rheumatology], 49:S5-S14, 2003, is hereby incorporated by reference in its entirety. The CHAQ comprises eight categories of questions on dressing / grooming, getting up, eating, walking, hygiene, stretching, grasping, and activity, in which parents or guardians record the degree of difficulty a child with bone mineralization disorders (such as HPP) experiences in performing the corresponding activities. Scores within each category range from 0 to 3, where 0 indicates no difficulty; 1 indicates some difficulty; 2 indicates very difficult; and 3 indicates the child cannot perform the activity.

[0220] Children with or predisposing to bone mineralization disorders (such as HPP) who have an average CHAQ score (e.g., an indicator of disability and / or pain in activities of daily living (ADL)) 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 administration of alkaline phosphatase or a polypeptide with alkaline phosphatase activity (e.g., sALP, having a sequence of any one of SEQ ID NO: 7-223, 247, and 262-264 or a variant thereof having at least 85% sequence identity with it and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein)). For example, children with a mean CHAQ score greater than about 0.8 can be treated by administering alkaline phosphatase or a polypeptide with alkaline phosphatase activity (such as sALP (e.g., a polypeptide having the sequence of any one of SEQ ID NO: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity with it 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 lifetime of the subject. Furthermore, children with or susceptible to bone mineralization disorders (such as HPP) disclosed herein can be asked one or more questions in one or more of eight categories (dressing / grooming, getting up, eating, walking, hygiene, stretching, grasping, and activity) to obtain a mean CHAQ score, and if the mean CHAQ score is greater than about 0.8, the child can be treated by administering alkaline phosphatase or a polypeptide with alkaline phosphatase activity (such as sALP).

[0221] The CHAQ score of children who have or are susceptible to the bone mineralization disorders disclosed herein (such as HPP) can be compared with the CHAQ score of children who do not have such bone mineralization disorders (such as HPP) to, for example, determine the standard deviation of the CHAQ score. Additionally, the CHAQ score of children who have or are susceptible to the bone mineralization disorders disclosed herein (such as HPP) can be compared with the CHAQ score of other children who have or are susceptible to the bone mineralization disorders disclosed herein (such as HPP) to, for example, determine the standard deviation of the CHAQ score.

[0222] These methods can lead to improvements in CHAQ score (e.g., an indicator of disability and / or pain in ADL) in children who have or are susceptible to the bone mineralization disorders (such as HPP) disclosed herein. For example, treatment with sALP (e.g., a polypeptide having a sequence of any one of SEQ ID NO: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity with it 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, up to the child's lifetime, can result in a mean reduction in CHAQ score in children with HPP-like disorders to about 0 to equal to or less than about 0.5 (e.g., about 0, about 0.1, about 0.2, about 0.4, or about 0.5).

[0223] The decrease in CHAQ score in children with or predisposed to bone mineralization disorders (such as HPP) can persist throughout the course of administration of alkaline phosphatase or peptides with alkaline phosphatase activity (such as sALP (e.g., peptides having the sequence of any one of SEQ ID NO: 7-223, 247, and 262-264 or variants thereof having at least 85% sequence identity with it and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein)), for example, for a period of time, even into the child's life. Similarly, an increase in ADL and / or a reduction in pain in children can persist for a period of time, even into the child's life, throughout the course of administration of sALP (e.g., peptides having the sequence of any one of SEQ ID NO: 7-223, 247, and 262-264 or variants thereof having at least 85% sequence identity with it and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein).

[0224] The CHAQ score of children with or susceptible to bone mineralization disorders (such as HPP) can be used to assess the efficacy of treatment with alkaline phosphatase or peptides with alkaline phosphatase activity (such as sALP (e.g., peptides having the sequence of any one of SEQ ID NO: 7-223, 247, and 262-264 or variants thereof having at least 85% sequence identity with and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein)). Improvement relative to a particular test score demonstrates the effectiveness of alkaline phosphatase or peptides with alkaline phosphatase activity in treating, for example, disabilities in activities of daily living (ADL) and pain associated with bone mineralization disorders (such as HPP). Specifically, children with or susceptible to bone mineralization disorders (such as HPP) can be asked one or more questions in one or more of eight categories (dressing / grooming, getting up, eating, walking, hygiene, stretching, grasping, and activity) to obtain an average CHAQ score and assess the efficacy of sALP administration. For example, when administration of the sALP described herein to a child with or predisposed to bone mineralization disorders (such as HPP) results in a mean reduction in CHAQ score to or less than about 0.5, where the child previously had a mean CHAQ score greater than about 0.8, then the sALP is effective in treating, for example, disabilities in activities of daily living (ADL) and pain associated with bone mineralization disorders (such as HPP). Alternatively, when administration of the sALP does not result in a mean reduction in CHAQ score to or less than about 0.5, the dosage and / or frequency of the sALP administration may be varied to determine the effective amount of the sALP for a child with or predisposed to bone mineralization disorders (such as HPP). For example, the dose of sALP (e.g., a polypeptide having the sequence of any one of SEQ ID NO: 7-223, 247 and 262-264 or a variant thereof having at least 85% sequence identity with it and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein) can be increased from, 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.

[0225] Pediatric Outcomes Data Collection Tool (PODCI) The Pediatric Outcome Data Collection Tool (PODCI) can be used to identify certain subjects with or predisposed to bone mineralization disorders (such as HPP) for treatment with alkaline phosphatase or peptides with alkaline phosphatase activity, such as sALP (e.g., peptides having the sequence of any one of SEQ ID NO: 7-223, 247, and 262-264 or variants thereof having at least 85% sequence identity with and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein). The PODCI can be administered to assess a child's health status to generate a PODCI score for the subject, as described by Plat et al. (…). J. Pediatr. Orthop. [Journal of Pediatric Orthopedics] 23 As described in (6): 788-790, 2003). PODCI includes eight categories of questions that can be completed by subjects who have or are susceptible to bone mineralization disorders (such as HPP) or by the subjects' parents / guardians. Categories of PODCIs that can be used to identify subjects with or predisposed to bone mineralization disorders (such as HPP) include the following: 1) Upper limb and physical function scales, used to measure difficulties encountered in performing daily personal care and school activities; 2) Transfer and basic mobility scales, used to measure difficulties experienced in performing routine movements and motor activities in daily activities; 3) Motor / physical function scales, used to measure difficulties or limitations encountered in participating in more active activities or sports; 4) Pain / comfort scales, used to measure the level of pain experienced in the past week; 5) Treatment expectation scales, used to measure long-term expectations of treatment; 6) Well-being scales, used to measure overall satisfaction with one's personal appearance and similarity with friends and peers; 7) Symptom satisfaction scales, used to measure a subject's acceptance of current limitations (if this is a lifelong condition); and 8) General functioning scales, which is a general combination scale calculated from the first four scales listed above. In each category, standardized scores were assigned to subjects who had or were susceptible to bone mineralization disorders (such as HPP), and then converted to a scale of 0 to 100, where 0 represents significant disability and 100 represents mild disability.

[0226] Subjects with or predisposing to bone mineralization disorders (such as HPP) whose mean PODCI score (e.g., indicating disability and / or pain in ADL) is 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 administration of alkaline phosphatase or a polypeptide with alkaline phosphatase activity (e.g., sALP, having at least 85% sequence identity with it and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein)). For example, subjects with a mean PODCI score less than 40 can be treated by administration of sALP for a period of time, up to the lifetime of the subject. In addition, subjects with or predisposed to bone mineralization disorders (such as HPP) can be asked one or more questions from one or more of the above eight scales (e.g., transfer and basic mobility, motor / physical function and pain / comfort scales) to obtain an average PODCI score, and if the average PODCI score is greater than or less than 40, the subject can be treated by administering sALP.

[0227] The methods described herein can result in an increase in PODCI scores (e.g., indicative of disability and / or pain in ADL) in subjects who have or are susceptible to bone mineralization disorders (such as HPP). For example, treatment with alkaline phosphatase or a polypeptide with alkaline phosphatase activity (such as sALP (e.g., a polypeptide having a sequence of any one of SEQ ID NO: 7-223, 247, and 262-264 or a variant thereof having at least 85% sequence identity with it 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 lifetime of the subject, can result in an average increase in PODCI scores of 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).

[0228] An increase in PODCI score can persist for some time throughout the course of administration of alkaline phosphatase or a polypeptide with alkaline phosphatase activity (such as sALP (e.g., a polypeptide having the sequence of any one of SEQ ID NO: 7-223, 247, and 262-264 or a variant thereof having at least 85% sequence identity with it and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein)) and may continue for some time throughout the subject's life. Similarly, an increase in ADL and / or a reduction in pain can persist for some time throughout the course of administration of sALP (e.g., a polypeptide having the sequence of any one of SEQ ID NO: 7-223, 247, and 262-264 or a variant thereof having at least 85% sequence identity with it and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein) and may continue for some time throughout the subject's life.

[0229] The PODCI score of subjects with or predisposed to bone mineralization disorders (such as HPP) can be used to assess the therapeutic efficacy of alkaline phosphatase or peptides with alkaline phosphatase activity (such as sALP (e.g., peptides having the sequence of any one of SEQ ID NO: 7-223, 247 and 262-264 or variants thereof having at least 85% sequence identity with and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein)), wherein improvement relative to a certain test score demonstrates that alkaline phosphatase or peptides with alkaline phosphatase activity are effective for treating, for example, disabilities in activities of daily living (ADL) and pain associated with bone mineralization disorders (such as HPP). Specifically, subjects with or predisposed to bone mineralization disorders (such as HPP) can be asked one or more questions from one of eight scales (Upper Limb and Body Function Scale, Transfer and Basic Mobility Scale, Motor / Body Function Scale, Pain / Comfort Scale, Treatment Expectations Scale, Well-being Scale, Symptom Satisfaction Scale, and Gross Function Scale) to obtain an average PODCI score and assess the therapeutic efficacy of sALP administration.

[0230] For example, when administration of the sALP described herein to subjects with or predisposed to bone mineralization disorders (such as HPP) results in an average increase in PODCI score to about 40 or greater than about 40, where the subjects previously had an average PODCI score of less than about 40, then the sALP is effective in treating, for example, disabilities in activities of daily living (ADL) and pain associated with bone mineralization disorders (such as HPP). Alternatively, when administration of the sALP described herein does not result in an average increase in PODCI score to about 40 or greater than about 40, the dose and frequency of sALP administration may be varied to determine the effective amount of sALP for subjects with or predisposed to bone mineralization disorders (such as HPP). For example, the dose of sALP (e.g., a polypeptide having the sequence of any one of SEQ ID NO: 7-223, 247 and 262-264 or a variant thereof having at least 85% sequence identity with it and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein) can be increased from, 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.

[0231] Bailey Scales of Infant and Toddler Development, 3rd Edition (BSID-III) Another endpoint, the Bayley Infant Development Scale, 3rd Edition (BSID-III), can be applied to assess the health status of subjects with or predisposed to bone mineralization disorders (such as HPP) from birth to generate a subject’s BSID-III score, as Bayley (2006). Bayley scales of infant and toddler development: administration manualAs described in [Bayley Infant and Toddler Developmental Scales: Management Manual] San Antonio, TX: Harcourt Assessment. The BSID-III includes a series of developmental play tasks that can be administered to subjects to determine raw BSID-III scores. For example, categories used to determine BSID-III scores for subjects with or predisposed to bone mineralization disorders (such as HPP) (e.g., infants with HPP aged approximately three years or younger) could include grasping, sensorimotor integration, motor planning and speed, visual tracking, touch, object grasping, object manipulation, functional hand skills, response to tactile information, limb and trunk movement, static positioning, dynamic movement, balance, and motor planning. BSID-III measurements are then converted into BSID-III scale scores, which can be used to determine a subject's performance compared to healthy, age-adjusted subjects. Subjects with or predisposed to bone mineralization disorders (such as HPP) (e.g., subjects with HPP) may have BSID-III scores ranging from 0 to 14, with scores of approximately 7 to approximately 13 considered to be within the normal range for healthy subjects.

[0232] Subjects with or predisposed to bone mineralization disorders (such as HPP) can be tested in one or more of the categories (grasping, sensorimotor integration, motor planning and speed, visual tracking, touch, object grasping, object manipulation, functional hand skills, response to tactile information, limb and trunk movement, static positioning, dynamic movement, balance, and motor planning) at an infant age (e.g., around 3 years or younger) to produce a BSID-III score indicating motor developmental delay. Subjects with or predisposing to bone mineralization disorders (such as HPP) who have a mean BSID-III score of less than about 2 in one or more of the categories described herein (grasping, sensorimotor integration, motor planning and speed, visual tracking, touch, object grasping, object manipulation, functional hand skills, response to tactile information, limb and trunk movement, static positioning, dynamic movement, balance, and motor planning) can be treated by administration of sALP (e.g., a polypeptide having a sequence of any one of SEQ ID NO: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity with it and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein).

[0233] These methods can lead to an improvement in the mean BSID-III score (e.g., an indicator of motor developmental delay) in subjects who have or are susceptible to bone mineralization disorders (such as HPP). For example, treatment with sALP (e.g., a polypeptide having a sequence of any one of SEQ ID NO: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity with it 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 a subject's lifetime, 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).

[0234] An increase in BSID-III scores can persist for some time throughout the course of administration of alkaline phosphatase or a polypeptide with alkaline phosphatase activity (such as sALP (e.g., a polypeptide having the sequence of any one of SEQ ID NO: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity with it and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein)) and into the lifetime of the subject with or predisposing to bone mineralization disorders (such as HPP). Similarly, an increase in motor development can persist for some time throughout the course of administration of alkaline phosphatase or a polypeptide with alkaline phosphatase activity (such as sALP (e.g., a polypeptide having the sequence of any one of SEQ ID NO: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity with it and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein)) and into the lifetime of the subject.

[0235] The BSID-III score of subjects with or predisposed to bone mineralization disorders (such as HPP) can be used to assess the efficacy of treatment with alkaline phosphatase or peptides with alkaline phosphatase activity (such as sALP (e.g., peptides having the sequence of any one of SEQ ID NO: 7-223, 247 and 262-264 or variants thereof having at least 85% sequence identity with and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein)), wherein improvement relative to a certain test score demonstrates that alkaline phosphatase or peptides with alkaline phosphatase activity are effective for treating, for example, motor developmental delay associated with bone mineralization disorders (such as HPP). Specifically, subjects with or predisposed to bone mineralization disorders (such as HPP) can be tested in one or more of the categories (grasping, sensorimotor integration, motor planning and speed, visual tracking, touch, object grasping, object manipulation, functional hand skills, response to tactile information, limb and trunk movement, static positioning, dynamic movement, balance, and motor planning) in infancy (e.g., at approximately 3 years of age or younger, with HPP) to obtain a mean BSID-III score and assess the therapeutic efficacy of sALP administration.

[0236] For example, when administration of sALP to a child with or predisposing to a bone mineralization disorder (such as HPP) results in an average increase in BSID-III score to greater than about 5, where the child previously had an average BSID-III score of less than about 2 as an infant (e.g., at about 3 years of age or less), then sALP is effective in treating, for example, motor developmental delay associated with HPP-like disorders. Alternatively, when administration of sALP does not result in an average increase in BSID-III score to greater than about 5, the dose and / or frequency of sALP administration can be varied to determine the effective amount of sALP for a child with or predisposing to a bone mineralization disorder (such as HPP). For example, the dose of sALP (e.g., a polypeptide having the sequence of any one of SEQ ID NO: 7-223, 247 and 262-264 or a variant thereof having at least 85% sequence identity with it and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein) can be increased from, 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.

[0237] Peabody Developmental Motor Scales, 2nd Edition (PDMS-2) Another endpoint, the Peabody Developmental Motor Scale, 2nd Edition (PDMS-2), can be applied to assess the health status of subjects with or predisposed to bone mineralization disorders (such as HPP) from birth to generate the subjects' PDMS-2 scores, as described by van Hartingsveldt et al. ( Occup. Ther. Int. As described in [International Journal of Occupational Therapy] 12(1): 1-13, 2005. PDMS-2 includes six subtests to measure the motor skills of subjects (such as those with HPP).

[0238] Specifically, PDMS-2 measurements can be determined by the following subtests: 1) a motor subtest to measure a subject’s ability to move from one place to another (measurements include crawling, walking, running, jumping, and forward jumping); 2) a reflex subtest to measure a subject’s ability to react automatically to environmental events; 3) a stationary subtest to measure a subject’s ability to maintain body control and balance within their center of gravity; 4) an object manipulation subtest to measure a subject’s ability to manipulate objects, such as catching, throwing, and kicking a ball; 5) a grasping subtest to measure a subject’s ability to use his or her hands, such as the ability to grasp an object with one hand and actions involving the use of both hands’ finger control; and 6) a visual-motor integration subtest to measure a subject’s ability to use his or her visual perception skills to perform complex eye-hand coordination tasks, such as reaching out and grasping an object, building with blocks, and replicating designs. For subjects who have or are susceptible to bone mineralization disorders (such as HPP), PDMS-2 measurements for one or more of these categories can be determined and then converted into PDMS-2 scores, such as the PDMS-2 exercise standard score ranging from 0 to 13, where the range for healthy subjects (e.g., subjects without bone mineralization disorders (such as HPP)) is approximately 7 to approximately 13.

[0239] Subjects with or predisposed to bone mineralization disorders (such as HPP) who have a mean PDMS score (e.g., indicating developmental delay of motor function) can be treated by administration of sALP (e.g., a polypeptide having a sequence of any one of SEQ ID NO: 7-223, 247 and 262-264 or a variant thereof having at least 85% sequence identity with it and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein).

[0240] The methods described herein can lead to improvements in PDMS-2 scores (e.g., indicators of motor developmental delay) in subjects with or predisposed to bone mineralization disorders (such as HPP). For example, treatment with alkaline phosphatase or peptides with alkaline phosphatase activity (such as sALP (e.g., peptides having the sequence of any one of SEQ ID NO: 7-223, 247, and 262-264 or variants thereof having at least 85% sequence identity with it and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein)) can result in an average increase in PDMS-2 scores of about 7 to about 13 (e.g., about 7, about 8, about 9, about 10, about 11, about 12, or about 13).

[0241] An increase in PDMS-2 scores can persist for a prolonged period of time throughout the administration of alkaline phosphatase or peptides with alkaline phosphatase activity (such as sALP (e.g., peptides having the sequence of any one of SEQ ID NO: 7-223, 247, and 262-264 or variants thereof having at least 85% sequence identity with it and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein)), for a period of time, extending into the lifetime of a subject with or predisposing to bone mineralization disorders (such as HPP). Similarly, an increase in motor development can persist for a period of time throughout the administration of sALP (e.g., peptides having the sequence of any one of SEQ ID NO: 7-223, 247, and 262-264 or variants thereof having at least 85% sequence identity with it and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein) extending into the lifetime of a subject with or predisposing to bone mineralization disorders (such as HPP).

[0242] The PDMS-2 score of subjects with or predisposed to bone mineralization disorders (such as HPP) can be used to assess the efficacy of treatment with alkaline phosphatase or peptides with alkaline phosphatase activity (such as sALP (e.g., peptides having a sequence of any one of SEQ ID NO: 7-223, 247, and 262-264 or variants thereof having at least 85% sequence identity with and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein)), where improvement relative to a particular test score demonstrates the effectiveness of alkaline phosphatase or peptides with alkaline phosphatase activity in treating, for example, motor developmental delay associated with bone mineralization disorders (such as HPP). For example, children with or predisposed to bone mineralization disorders (such as HPP) can be tested in one or more of the categories (motor, reflex, rest, object manipulation, grasping, and visual-motor) at approximately 5 years of age or less to obtain an average PDMS-2 score and assess the efficacy of sALP administration.

[0243] For example, when administration of sALP to a child with or susceptible to bone mineralization disorders (such as HPP) results in an average increase in the PDMS-2 standard score to about 7, where the child previously had an average PDMS-2 standard score of about 5, then sALP is effective in treating, for example, motor developmental delay associated with HPP-like disorders. Alternatively, when administration of sALP does not result in an average increase in the PDMS-2 standard score to about 7, the dose and / or frequency of sALP administration can be varied to determine the effective amount of sALP for the child. For example, the dose of sALP (e.g., a polypeptide having the sequence of any one of SEQ ID NO: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity with it and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein) can be increased from, 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.

[0244] 6-minute walk test (6MWT) The 6MWT can be used to identify subjects with bone mineralization disorders (such as HPP) who have been treated with alkaline phosphatase or a polypeptide with alkaline phosphatase activity (e.g., sALP, for example, a polypeptide having the sequence of any one of SEQ ID NO: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity with it and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein). Specifically, the 6MWT can be used to assess the walking ability of adults with bone mineralization disorders (such as HPP) to generate a 6MWT value for that adult. 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., meters) walked by the subject with bone mineralization disorders (such as HPP). For example, the length of the corridor can be marked every three meters to determine the number of meters walked by a subject with a bone mineralization disorder (such as HPP), with a turnaround point marked at 30 meters and a starting line also marked. The distance walked by the subject with HPP in 6 minutes can then be compared to the predicted number of meters walked by a normal subject, for example, of approximately the same age, sex, and / or height, and expressed as a percentage to generate the subject's 6MWT value. The 6MWT value of the subject with HPP can be compared to the subject's 6MWT value at baseline. Additionally, the 6MWT value of the subject with HPP can be compared to the 6MWT value of a normal subject.

[0245] Alkaline phosphatase or peptides with alkaline phosphatase activity (e.g., sALP, for example, having SEQ ID NO: ) can be used. A polypeptide of any one of the sequences 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein, is used to treat subjects with bone mineralization disorders (such as HPT) whose mean 6MWT is less than approximately 80% of the predicted 6MWT value (e.g., relative to normal subjects of roughly the same age, sex, and / or height), such as by administering alkaline phosphatase or a polypeptide with alkaline phosphatase activity for at least two weeks (e.g., at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least ten weeks, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least one year, at least two years, at least three years, at least four years, at least five years, at least six years, at least seven years, at least eight years, at least nine years, or at least ten years, or the lifetime of the subject; particularly at least six weeks). For example, alkaline phosphatase or peptides with alkaline phosphatase activity can be used to treat subjects with bone mineralization disorders (such as HPT) whose average 6MWT is less than about 80% of the predicted 6MWT value (e.g., about 50%, 55%, 60%, 65%, 70%, or 75% of the predicted 6MWT value) for a period of at least two weeks (e.g., at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least ten weeks, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least one year, at least two years, at least three years, at least four years, at least five years, at least six years, at least seven years, at least eight years, at least nine years, or at least ten years, or the lifetime of the subject; particularly at least six weeks).

[0246] These methods can lead to improvements in 6MWT values ​​in subjects with bone mineralization disorders (such as HPP). For example, treatment with alkaline phosphatase or a peptide with alkaline phosphatase activity (e.g., sALP, such as a peptide having the sequence of any one of SEQ ID NO: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity with it and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein) for at least two weeks (e.g., at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least ten weeks, at least three months, at least four months, at least five months, at least six months, at least seven weeks) can lead to improvements in 6MWT values ​​in subjects with bone mineralization disorders (such as HPP). Treatment periods of at least 1 month, 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) can result in an average increase in 6MWT values ​​to approximately 80% or more of the subject's predicted 6MWT values ​​(e.g., approximately 82%, approximately 84%, approximately 86%, approximately 88%, approximately 90%, approximately 92%, approximately 94%, approximately 96%, approximately 98% or more of the predicted 6MWT values).

[0247] An increase in the 6MWT value in subjects with bone mineralization disorders (such as HPP) can persist throughout the course of treatment with alkaline phosphatase or a polypeptide with alkaline phosphatase activity (e.g., sALP, such as a polypeptide having a sequence of any one of SEQ ID NO: 7-223, 247 and 262-264 or a variant thereof having at least 85% sequence identity with it and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein), for example, for at least two weeks (e.g., at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least ten weeks, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least one year, at least two years, at least three years, at least four years, at least five years, at least six years, at least seven years, at least eight years, at least nine years, or at least ten years, or the lifetime of the subject; particularly at least six weeks). For example, during treatment with alkaline phosphatase or peptides with alkaline phosphatase activity, the 6MWT value increased to approximately 80% greater than the predicted 6MWT value for subjects with bone mineralization disorders (such as HPP), and remained at ± 10% of the increased 6MWT value.

[0248] Similarly, improvements in walking ability in subjects with bone mineralization disorders (such as HPP) can persist throughout the course of treatment with alkaline phosphatase or peptides with alkaline phosphatase activity, for example, for at least two weeks (e.g., at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least ten weeks, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least one year, at least two years, at least three years, at least four years, at least five years, at least six years, at least seven years, at least eight years, at least nine years, or at least ten years, or the subject's lifetime; particularly at least six weeks). For example, during treatment with sALP, subjects with bone mineralization disorders (such as HPP) showed reduced dependence on assistive mobility devices (such as walkers, wheelchairs, braces, canes, or orthotics).

[0249] Alternatively, when administration of alkaline phosphatase or a peptide with alkaline phosphatase activity (e.g., sALP, such as a peptide having the sequence of any one of SEQ ID NO: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity with it and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein) does not result in an average increase in 6MWT values ​​to greater than 80% of the predicted 6MWT values ​​(e.g., in normal subjects of roughly the same age, sex, and / or height), the dose and / or frequency of administration of alkaline phosphatase or a peptide with alkaline phosphatase activity may be varied to determine the effective amount of alkaline phosphatase or a peptide with alkaline phosphatase activity for subjects with bone mineralization disorders (such as HPP). For example, the dose of alkaline phosphatase or a peptide with alkaline phosphatase activity may be increased from, for example, 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.

[0250] Handheld force gauge (HHD) Handheld dynamometers (HHDs) can be used to assess grip strength and muscle strength in subjects with or predisposed to bone mineralization disorders such as HPP. For example, the MICROFET2 can be used. TMA dynamometer is used to measure knee flexion and extension, as well as hip flexion, extension, and abduction, in subjects with or predisposed to bone mineralization disorders (such as HPP). Simultaneously, a grip strength tester, such as a Jamar grip strength tester, can be used to measure the subject's grip strength. Specifically, the applicant keeps the dynamometer stationary, and the subject applies maximum force to the dynamometer. Peak force data are collected in pounds and then converted to Newtons (N). Torque values ​​are then calculated using limb length in N-meters. The torque values ​​can then be compared to those of normal subjects, for example, of approximately the same age, sex, and / or height, and expressed as a percentage to produce the subject's HHD value.

[0251] Alkaline phosphatase or peptides with alkaline phosphatase activity (e.g., sALP, for example, having SEQ ID NO: ) can be used. A polypeptide of any one of the sequences 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein, is used to treat subjects with bone mineralization disorders (such as HPP) whose mean HHD value is less than approximately 80% of the predicted HHD value (e.g., relative to normal subjects of approximately the same age, sex, and / or height), such as by administering alkaline phosphatase or a polypeptide with alkaline phosphatase activity for a period of at least two weeks (e.g., at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least ten weeks, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least one year, at least two years, at least three years, at least four years, at least five years, at least six years, at least seven years, at least eight years, at least nine years, or at least ten years, or the lifetime of the subject; particularly at least six weeks). For example, alkaline phosphatase or peptides with alkaline phosphatase activity can be used to treat subjects with bone mineralization disorders (such as HPP) whose average HHD is 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) for a period of at least two weeks (e.g., at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least ten weeks, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least one year, at least two years, at least three years, at least four years, at least five years, at least six years, at least seven years, at least eight years, at least nine years, or at least ten years, or the lifetime of the subject; particularly at least six weeks).

[0252] These methods can lead to improvements in HHD values ​​in subjects with bone mineralization disorders (such as HPP). For example, treatment with alkaline phosphatase or a peptide with alkaline phosphatase activity (e.g., sALP, such as a peptide having the sequence of any one of SEQ ID NO: 7-223, 247, and 262-264, or a variant thereof having at least 85% sequence identity with it and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein) for at least two weeks (e.g., at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least ten weeks, at least three months, at least four months, at least five months, at least six months, at least...) can lead to improvements in HHD values ​​in subjects with bone mineralization disorders (such as HPP). Treatment periods of seven months, at least eight months, at least nine months, at least one year, at least two years, at least three years, at least four years, at least five years, at least six years, at least seven years, at least eight years, at least nine years, or at least ten years, or the lifetime of the subject; particularly at least six weeks) can result in an average increase in HHD values ​​to about 80% or more of the subject's predicted HHD values ​​(e.g., about 83%, about 85%, about 87%, about 90%, about 93%, about 95%, about 97%, or about 100%, or about 100% of the predicted HHD values).

[0253] An increase in HHD values ​​in subjects with bone mineralization disorders (such as HPP) can persist throughout the course of treatment with alkaline phosphatase or a polypeptide with alkaline phosphatase activity (e.g., sALP, for example, a polypeptide having a sequence of any one of SEQ ID NO: 7-223, 247 and 262-264 or a variant thereof having at least 85% sequence identity with it and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein), for example, for at least two weeks (e.g., at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least ten weeks, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least one year, at least two years, at least three years, at least four years, at least five years, at least six years, at least seven years, at least eight years, at least nine years, or at least ten years, or the lifetime of the subject; particularly at least six weeks). For example, during treatment with alkaline phosphatase or peptides with alkaline phosphatase activity, HHD values ​​increased to approximately 80% greater than the predicted HHD values ​​of subjects with bone mineralization disorders (such as HPP), and remained within ± 10% of the increased HHD values.

[0254] Alternatively, when administration of alkaline phosphatase or a polypeptide with alkaline phosphatase activity (e.g., sALP, such as a polypeptide having the sequence of any one of SEQ ID NO: 7-223, 247 and 262-264 or a variant thereof having at least 85% sequence identity with it and / or having at least one amino acid mutation relative to naturally occurring ALP, as described herein) does not result in an average increase in HHD values ​​to greater than 80% of the predicted HHD values ​​(e.g., in subjects with bone mineralization disorders (such as HPP) of approximately the same age, sex and / or height), the dose and / or frequency of administration of alkaline phosphatase or a polypeptide with alkaline phosphatase activity may be varied in order to determine the effective amount of alkaline phosphatase or a polypeptide with alkaline phosphatase activity for subjects with bone mineralization disorders (such as HPP). For example, the dose of alkaline phosphatase or a polypeptide with alkaline phosphatase activity can be increased from, for example, 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.

[0255] Example This disclosure is illustrated by the following examples. It should be understood that specific examples, materials, quantities, and procedures should be interpreted broadly in accordance with the scope and spirit of this disclosure as set forth herein.

[0256] Example 1 Protein design and expression Alkaline phosphatase fusion protein.

[0257] To systematically examine the protein activity, stability, and pharmacokinetic properties of alkaline phosphatase fusion proteins, several constructs were designed and tested (see Table 1). Some constructs were generated using standard DNA synthesis techniques, and their sequences were validated by DNA sequencing of the final constructed plasmids. Additional constructs were generated using standard site-directed mutagenesis of existing plasmids. Mutation of the alkaline phosphatase fusion protein was tested for residue variations in isoforms located in and around the enzyme active site, at shared N-linked glycosylation sites, in the bone-targeting region, and in the crystallizable (Fc) region of the protein-linked fragment. These constructs were expressed via transient transfection of Expi293F and ExpiCHO cells, as detailed below.

[0258] Bone-targeted fluorescent-Fc fusion compound.

[0259] To systematically compare the HA and bone-binding capabilities of peptides and VHH sequences, protein fusions (SEQ ID NO: 224-245) containing (N-terminal bone-targeting - human IgG1 crystallization region (Fc) - Katushka2s fluorescent protein - C-terminal His tag) were designed. A list of fluorescent fusion proteins for targeting is shown in Table 2. The fluorescent fusion proteins were transiently transfected into Expi293F cells, as detailed below.

[0260] Table 2: Overview of the targeting sequence of the bone-targeting fluorescent Fc-fusion protein Biologically inactive ERT substitute molecule (ALP-Fc).

[0261] To mimic the bone-targeting capabilities of clinically available ERTs, ALP-Fc-(Asp) was used. 10 The fusions were compared with ALP-Fc fusions. The ALPs used in these studies were specifically engineered to target the enzymatic activity of the synthetic substrate 4-methylumbelliferyl phosphate (4-MUP). Figure 21 The diagram shows ALP-Fc-(Asp). 10 The structural model is described in detail below, using ExpiCHO cells transiently transfected with ALP-Fc (SEQ ID NO: 222) and ALP-Fc-(Asp). 10 (SEQ ID NO: 31) protein.

[0262] Single-domain variable heavy chain of heavy chain (VHH) antibody fragment.

[0263] Bone-targeting VHH proteins were designed by replacing the complementarity-determining region (CDR) of non-targeting VHH molecules with polyaspartic acid sequences. Table 3 shows a list of bone-bound VHH proteins for evaluation. The transient transfection of VHH proteins into Expi293F cells is described in detail below.

[0264] Table 3: Overview of VHH sequences. .

[0265] Transient transfection of cell culture All Expi293 transfections were performed at a ratio of 1.0 µg plasmid DNA / mL transfection culture volume, and all ExpiCHO transfections were performed at a ratio of 0.8 µg plasmid DNA / mL transfection culture volume. Cultures were maintained at 120 RPM on a 25.4 mm oscillator.

[0266] Expi293F cells were cultured at 37°C in Expi293 expression medium (Thermo Fisher Scientific) in a humidified 8% CO2 incubator. The day before transfection, cells were divided into 2.5 x 102 divisions. 6 Cells / mL. After 24 hours, the stock solution contained approximately 4 x 10⁻⁶ cells / mL. 6 Cells / mL, and diluted to 2.5 x 10⁻⁶. 6 Cells / mL. Perform Expi293 transfection according to the manufacturer's protocol. Use the Expi293 Expression System Kit (Thermo Fisher Scientific) containing the transfection enhancer and ExpiFectamine 293 transfection reagent for all Expi293 transfections. In summary, dilute the ExpiFectamine 293 transfection reagent and plasmid DNA separately in OptiMEM medium (Thermo Fisher Scientific). Combine the ExpiFectamine 293 and DNA mixture and incubate for another 10–20 minutes. Then add the ExpiFectamine 293-DNA-OptiMEM mixture to the cells. 16–18 hours post-transfection, add enhancer 1 and enhancer 2 to the transfected culture.

[0267] ExpiCHO-S cells were cultured at 37°C in ExpiCHO expression medium (Thermo Fisher Scientific) in a humidified 8% CO2 incubator. One day before transfection, cells were divided into 3-4 x 102 divisions. 6 Cells / mL. After 24 hours, the stock solution was 7-10 x 10⁻⁶ cells / mL. 6 Cells / mL, and diluted to 6 x 10⁻⁶ cells / mL by adding fresh culture medium. 6 Cells / mL. ExpiCHO transfection was performed using the ExpiCHO Expression System Kit (Thermo Fisher Scientific) according to the manufacturer's standard protocol. In summary, the ExpiFectamine CHO transfection reagent and plasmid DNA were diluted separately in OptiPRO SFM (Thermo Fisher Scientific). The ExpiFectamine CHO and DNA mixture was immediately combined and incubated for 1–5 minutes. The ExpiFectamine CHO-DNA-OptiPRO mixture was then added to the cells. 18–22 hours post-transfection, enhancers and 30% v / v feed were added.

[0268] Cell Culture Harvest On day 4, Expi293 transfections were harvested; on day 5, ExpiCHO transfections were harvested. Cell cultures were centrifuged at 1900 xg for 15 minutes and the supernatant was aseptically filtered through a 0.2 µm disposable membrane filter (polyethersulfone (PES) filter, Fisher Scientific).

[0269] Large-scale protein expression ALP201 and ALP213 were expressed in a stable cell culture (CHO KS) according to standard procedures and grown for 11 days in a 5 L bioreactor (APPLIKON), starting from an initial expression rate of 5.0 x 10⁻⁶ cells / mL. 5 Starting with 100 cells / mL seeding, with temperature (36.5°C, changing to 33°C on day 5) and stirring (80 W / m). 2 Compare with O2 (on demand, 0 to 0.6 VVM).

[0270] Protein purification.

[0271] VHH and fluorescent Fc-fusion proteins were purified 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 for 48 hours at 4°C using dialysis flasks (Slide-A-Lyzer™, Thermo Fisher Scientific) with Ni-NTA binding / wash buffer (2.5 M sodium chloride, 0.1 M sodium phosphate, 0.1 M imidazole, buffered with sodium hydroxide to pH 7.4). The Ni-NTA Superflow resin was washed and resuspended in Ni-NTA binding / wash buffer, then incubated with dialysis supernatant (approximately 8 mg protein / mL resin) at 4°C with shaking for 90 minutes. The gravity purification column was sterilized with 0.1 N sodium hydroxide for 1 hour, followed by washing with Ni-NTA binding / wash buffer before protein loading / elution. The dialysis supernatant containing Ni resin was poured into 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 / washing 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 the buffer was incubated with the resin for 5 minutes before being transferred to the conical tubes. The fractions were then centrifuged using a 30 kD MWCO filter (Amicon). ®The eluted protein buffer was exchanged into PBS at pH 7.4, and the final protein was concentrated by filtration. Protein concentration was quantified using UV / VIS absorbance at 280 nm and 558 nm (maximum Katushka 2s absorbance), and then stored in the dark at 4°C.

[0272] The ALP-Fc fusion was purified using protein A resin.

[0273] Use protein A resin (MABSELECT) TM SURE TM GE Life Sciences purified the ALP-Fc fusion in batches. The resin was thoroughly washed and resuspended in protein loading buffer (50 mM sodium phosphate, pH 7.5, 100 mM sodium chloride), then incubated overnight (at 4°C) with cell supernatant containing the expressed target protein (approximately 10 mg protein / mL resin). The column was sterilized with sodium hydroxide, washed with loading buffer, and protein A resin / supernatant was loaded. Before elution with 50 mM Tris-HCl pH 11, the resin-bound protein was washed with 40 column volumes (approximately 200 mL) of loading buffer. Protein was eluted in 3 mL fractions and immediately neutralized with loading buffer at a 1:1 dilution. The fusion was then purified by strong anion exchange chromatography (CAPTO). TM 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) for 12 column volumes, starting with 25% buffer B and ending with 100% buffer B. Additional samples were purified. Fractions containing ALP-Fc fusions were pooled and dialyzed to PBS at pH 7.4, or buffer-exchanged to PBS at pH 7.4 using a 30 kD MWCO centrifuge filter. Protein concentration was determined by absorbance at 280 nm, and purity was assessed by SDS-PAGE gel electrophoresis. Proteins were filtered and stored at 4°C. Large-scale protein purification In addition to using POROS TM In addition to strong anion exchange chromatography on a 50HQ column, use 13-15 column volumes of the same buffer as the previous sample purification of proteins expressed from stable cell line bioreactors, employing a gradient buffer B of 0% to 70%-75%. Concentrate or dialyze the sample into PBS via UF / DF, filter sterilely, and store at 4°C (sample concentrations are typically approximately 4-8 mg / mL).

[0274] Example 2 Assay of the activity of 4-methylumbelliferyl phosphate hydrolysate protein Alkaline phosphatase activity was routinely measured using the artificial substrate 4-methylumbelliferyl phosphate (4-MUP). Cleavage of the phosphate ester bond on 4-MUP produces a fluorescent product, 4-methylumbelliferone (4-MU), which can be detected at 360 nm / 465 nm excitation / emission.

[0275] ALP activity in protein samples (supernatant, partially purified, or column-purified samples) was determined in solution using 4-methylumbelliferyl phosphate (4-MUP) as an artificial substrate. Hydrolysis of the phosphate ester bond in 4-MUP releases the fluorescent compound 4-methylumbelliferone, which is readily detectable by a fluorometer. Product quantification was performed using a standard curve of 4-methylumbelliferone (4-MU) measured on the same plate, with 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 to 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 appropriate final concentrations (typically approximately 1 nM) for assay in assay buffer. A 4-MUP stock solution was prepared in assay buffer. All solutions were brought to 37°C before the assay by adding 10 μM of 4-MUP to the protein samples. The production of 4-MUP was measured at an excitation wavelength of 360 nm and an emission wavelength of 465 nm. Data were collected in a plate reader maintained at 37°C every 40 seconds for a total of 20 minutes. The reaction rate was calculated in activity units by linear regression, where 1 U = 1 μmol of 4-MUP hydrolysis / min. Specific activity was calculated in units / mg of protein determined. The progress curve of the 4-MUP hydrolysis assay is shown in [the figure]. Figure 19 The specific activities are shown in Table 4 as follows: < 2 U / mg (-), 2-9.9 U / mg (+), 10-30 U / mg (++) and > 30 U / mg (+++).

[0276] Table 4: 4-MUP hydrolysis specific activity of exemplary alkaline phosphatase fusion proteins Construct MUP activity ALP035, ALP039, ALP133, ALP134, ALP136, ALP137, ALP138, ALP139, ALP142, ALP143, ALP187, ALP201, ALP235, ALP259 +++ ALP001, ALP004, ALP011, ALP012, ALP023, ALP031, ALP043, ALP047, ALP048, ALP049, ALP050, ALP051, ALP052, ALP053, ALP054, ALP055, ALP056, ALP057. ALP058, ALP058, ALP059, ALP060, ALP062, ALP063, ALP089, ALP132, ALP144, ALP147, ALP148, ALP151, ALP 155, ALP156, ALP157, ALP158, ALP170, ALP173, ALP174, ALP185, ALP186, ALP188, ALP213, ALP214, ALP215 ++ ALP002, ALP044, ALP045, ALP130, ALP135, ALP140, ALP145, ALP146, ALP149, ALP150, ALP152, ALP163, ALP169, + ALP003, ALP013, ALP040, ALP041, ALP164, ALP165, ALP166, ALP168 - Plate bone measurement ALP activity in bone homogenate fractions was determined. The bone homogenate fraction was resuspended in 100 µL of PBS and transferred to a 96-well black plate. 100 µL of unbound protein suspension was also transferred to individual wells of the 96-well plate. 100 µL of ALP detection solution (10 µM 4-MUP, 1% BSA) was added to each well, and kinetic fluorescence readings were immediately initiated (at 360 / 465) and run for 20 minutes, during which fluorescence intensity emission was collected every 30 seconds. The slope of fluorescence intensity relative to time represents the ALP concentration in each sample fraction.

[0277] The MUP activities of the bound and unbound fractions of the 17 constructs were measured by the initial activity slope over the first 5 minutes to maintain linearity. The MUP activity ratio for each fusion protein was determined as ratio = (bound activity) / (unbound activity), and the results are shown in Table 5. Figure 14 The exemplary progress curves from MUP hydrolysis assays of bone homogenates using various constructs are shown in [the figure]. Figure 11 The values ​​are shown in the table below, and the binding activities are listed in Table 5. The MUP activity quantifications in Table 5 are as follows: < 0.2 (-); 0.2 to 0.49 (+); 0.5 to 5.0 (++); > 5.0 (+++).

[0278] Table 5: Combination with bone homogenate Determine the MUP activity ratio for each fusion protein, ratio = (bound activity) / (unbound activity). Legend: <0.2 (-); 0.2 to 0.49 (+); 0.5 to 5.0 (++); >5.0 (+++) Example 3 The activity of alkaline phosphatase constructs against natural substrates The improved properties of the protein fusion molecules described in this article were validated in various enzyme assays measuring the hydrolysis of natural and artificial substrates. Artificial substrates used in the activity assays included 4-MUP and p-nitrophenyl phosphate (pNPP). Natural substrates used in the activity assays included pyrophosphate (PPi) and pyridoxine 5'-phosphate (PLP). Wild-type ALP activity ranged from negative (decreased enzyme activity) to up to a 9.5-fold increase (850% increase in activity).

[0279] Determination of pyrophosphate hydrolysis.

[0280] The activity of certain purified alkaline phosphatase fusion proteins on the natural substrate pyrophosphate was determined. Pyrophosphate hydrolysis was measured by detecting the phosphate anion in the product using the PiBlue assay reagent (Bosch Biotech Ltd.), which turns bright green upon phosphate binding. Phosphate levels in each well were quantified using a standard curve of phosphate solutions prepared in assay buffer, which was read from the assay plate. A 10 mM stock solution of sodium pyrophosphate decahydrate (Sigma Chemicals Ltd.) was prepared 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 were prepared by diluting the stock solutions in assay buffer. All solutions were brought to 37°C before the reaction began. Add the protein solution to a clean 96-well plate and place the plate in a Gitbar plate shaker maintained at 37°C. Initiate the reaction by adding pyrophosphate solution to the protein solution.

[0281] Typically, pyrophosphate hydrolysis was performed simultaneously in the same plate at pyrophosphate concentrations of 0 uM, 1.56 uM, 3.12 uM, 6.25 uM, 12.5 uM, 25 uM, 50 uM, 100 uM, 200 uM, and 400 uM. Eight wells were set up for each pyrophosphate concentration, and the reaction was terminated by adding PiBlue reagent (in a volume equal to the final reaction volume) after 0, 1, 2, 3, 4, 5, 6, and 7 minutes following the addition of pyrophosphate to the plate. Adding PiBlue reagent stopped any further reaction because the low pH of the assay reagent inactivated the enzyme. The 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 was calculated by constructing progress curves from each time point. The reaction rate at each concentration was used to calculate the Km and Vmax values ​​in the GraphPad Prism using Michaelis-Menten enzyme kinetic fitting. The kcat value is calculated from the Vmax value as follows: Vmax / (number of moles of protein measured) = kcat.

[0282] Catalytic efficiency is defined as: Catalytic efficiency = kcat / Km.

[0283] Michaelis-Menten plots detailing the pyrophosphate hydrolysis activity of the selected compounds are shown in [the original text]. Figure 22The following concentrations are shown: 10 ng / mL ALP201, 10 ng / mL ALP250, and 25 ng / mL Aspherase α (SEQ ID NO: 269). Standard deviations (bars) are shown, N = 3.

[0284] Table 6: Pyrophosphate hydrolysis parameters of the tested alkaline phosphatase fusion protein For kcat parameters: (++) > 130 s⁻¹, (++): 130 - 65 s⁻¹, (+): 65 - 33 s⁻¹, (-) < 33 s⁻¹ For catalytic efficiency parameters: (++) > 20 * 10^6 M-1 s-1, (++): 20 * 10^6 - 6 * 10^6 M-1 s-1, (+): 6 * 10^6 - 3 * 10^6 M-1 s-1, (-) < 3 * 10^6 M-1 s-1 Determination of pyridoxine 5'-phosphate hydrolysis.

[0285] The second natural substrate of alkaline phosphatase is pyridoxine 5'-phosphate (PLP). The anti-PLP activity of certain purified alkaline phosphatase fusion proteins was determined by a coupled assay in which pyridoxine, a product of PLP hydrolysis, was converted to fluorescent pyridoxine by *Rhizobium loureirii* tetrapyridoxine dehydrogenase (tPLDH, SEQ ID NO: 246). A 6xHis-labeled tPLDH gene was synthesized using standard methods and cloned into a bacterial expression plasmid controlled by the T7 promoter. The 6xHis-labeled tPLDH was expressed in BL21(DE3) cells using a standard protocol and purified by standard affinity chromatography. The protein was concentrated to 1900 μM using an Amicon Ultra15 spin concentrator and frozen at -80°C until ready for assay. The purified fusion protein sample was prepared into a 0.1 mg / mL solution 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 then placed in black 96-well plates along with pyridoxine standards (prepared in assay buffer). NAD+ was added to the protein sample. +tPLDH and PLP solutions (prepared in assay buffer) were mixed to achieve a final concentration of 3 mM NAD+, 4 uM tPLDH, and 3 uM PLP. All solutions were brought to 37°C before the reaction began, and the reaction plate was incubated at 37°C, with fluorescence detected by excitation at 355 nm and emission measured at 445 nm. The amount of pyridoxine product produced was calculated using a standard curve generated from the measured fluorescence from the pyridoxine wells in the plate. The reaction rate was calculated by linear regression of the progress curve in micromoles / minute of pyridoxine produced. Specific activity was calculated by dividing the reaction rate by the protein concentration used in the assay reaction. An exemplary progress curve for 10 ng / mL ALP201, based on data generated by this assay, is shown in [reference needed]. Figure 23 The Michaelis-Menten figure, detailing the hydrolytic activities of pyridoxal-5'-phosphate at 10 ng / mL ALP201, 10 ng / mL ALP250, and 12.5 ng / mL Asforase α, is shown in [the figure]. Figure 24 As shown in the image.

[0286] Table 7: Measured PLP hydrolytic activity of alkaline phosphatase fusion protein Legend: Activity < 1 μmol / min / mg (-), 1-2 μmol / min / mg (+), > 2 μmol / min / mg (++) Example 4 Hydroxyapatite binding determination Characterization of the binding of fluorescent Fc-fusions to hydroxyapatite (HA).

[0287] Synthetic HA (CAPTAL) ®The HA was purchased from Plasma-Biotal Ltd. (Derbyshire, UK). The bone-targeting fluorescent Fc-fusion was diluted to 50 nM (1 mL suspension per tube) with 0.1% (w / w) bovine serum albumin (BSA) in phosphate-buffered saline (PBS) at pH 7.4 in separate 1.5 mL centrifuge tubes. 1 mg of synthetic HA was added to each tube containing the fluorescent Fc-fusion, and the tubes were incubated at room temperature for 2 hours to prevent HA precipitation. After incubation, the samples were centrifuged at 16,000 rcf for 5 min 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 resuspended in 100 µL PBS. The suspended HA-bound fraction and 100 µL of unbound protein suspension were transferred to a 96-well black plate (one fraction per well), and the relative protein concentration was determined by fluorescence excitation / emission at 488 nm / 585 nm using a Spectramax i3x fluorescent plate reader.

[0288] Twenty fluorescent Fc-fusion proteins were transiently expressed in HEK cells and purified using Ni-NTA resin (sequence details are shown in Table 1). The fluorescent Fc-fusion proteins differed only in their N-terminal HA-binding sequences to allow for systematic evaluation of bone-targeting moieties in vitro. The fluorescent Fc-fusion proteins were incubated with HA in suspension in the presence of BSA in separate tubes to prevent nonspecific binding. Figure 1A The fluorescence intensity of the HA-binding fraction for each fluorescent Fc-fusion sample relative to the untargeted fluorescent Fc-fusion (FLU002) is shown. Furthermore, the unbound protein fraction was collected immediately after incubation with HA (before the first wash of the HA precipitate). These fluorescence intensities were also plotted against FLU002. Figure 1B As shown in the figure. For FLU001, 003, 004, 016, 027, and 031, 100% of the proteins were bound to HA, as these proteins were not detected above the background in the unbound protein fraction. Conversely, FLU002 showed 4-fold less binding to HA compared to FLU001, and a significant unbound fluorescence signal (100-fold greater than FLU001 or the background). FLU005-009, 011-014, and 020 showed similar HA binding characteristics to FLU002. Interestingly, two proteins (FLU010 and FLU015) showed significant fluorescence (above the negative control) in both the HA-bound and unbound fractions.

[0289] Hydroxyapatite (HA) has been used interchangeably with bone as a binding substrate for in vitro bone-targeting assays; however, significant differences have been noted between ceramic HA and natural bone, particularly regarding the role of collagen and other extracellular matrix proteins in controlled crystal nucleation in vivo (Zhai, Y. et al.). J. Crystal Growth [Journal of Crystal Growth], 202-206, 2006. CAPTAL was specifically used in this study. ® The brand HA is named as such because it is a bone mineral analog based on its Ca:PO4 ratio (1.67), iron content (100 ppm), magnesium content (800-1100 ppm), and lattice structure. However, in CAPTAL... ® Significant differences in total protein binding were observed between HA and bone homogenate. Most importantly, nonspecific binding was observed to all fluorescent Fc-fusion proteins incubated with HA, even in the presence of a BSA blocking agent. When incubated with bone homogenate, the untargeted fluorescent Fc-fusion control protein (FLU002) did not show a fluorescence signal higher than the background (bone homogenate alone). Therefore, bone homogenate is a more tightly bound substrate compared to HA, which behaves more like a typical ion exchange resin. However, when normalized to the appropriate control protein (e.g., ...), the binding signal was significantly different. Figure 1A-1D As shown, HA binding data can be used to identify effective bone-binding proteins, although the dynamic range of fluorescent Fc-fusion protein detection is significantly depleted (approximately 5-fold) in HA binding assays. ALP-Fc proteins exhibit nonspecific binding to HA and even larger amounts, most likely due to the negative charge of the ALP catalytic domain. In some cases, this has even led to some ALP-Fc-X proteins being incorrectly identified as having HA-binding properties, which was not observed when incubated with bone homogenate. For this reason, bone homogenate is considered an excellent substrate for screening bone-binding proteins and is used in all subsequent in vitro assays.

[0290] Example 5 Bone homogenate assay Previous literature studies have relied on bone slices or other three-dimensionally physically constrained substrates. A more accurate, rapid, and efficient assay is needed to differentiate and quantify protein efficacy and the percentage of binding.

[0291] Preparation of bone homogenate .

[0292] Prior to use, femurs from male C57BL / 6 mice were stored at -80°C. The femurs were transferred to 2 mL centrifuge tubes (2 femurs per tube) containing 1 mL of 0.2% (w / w) type 2 collagenase (Worthington) in BS and 1x a mixture of EDTA-free serine and cysteine ​​protease inhibitors (COMPLETE™, Roche). The femurs were briefly vortexed and incubated at 37°C with shaking (800 rpm) for 1 hour. Residual connective tissue was removed, and the femurs were placed in culture dishes on ice. The bone marrow was flushed with PBS using a needle and syringe. The dry bone was aggregated (typically 30–50 mg per femur) and placed in pre-chilled, hardened tissue, then ground with 0.75 mL PBS containing 1x protease inhibitor using a single disposable stirrer (PRECELLYS). ® (Beltan Instruments, Inc.). The femur was then homogenized using a high-throughput bead mill homogenizer (bullet mixer, 4°C, maximum speed, 4 cycles for 30 seconds). The homogenate was transferred to 1.5 mL centrifuge tubes and centrifuged at 12,000 xg for 15 minutes at 4°C to separate the bone homogenate from the released protein / cell debris. The bone homogenate was resuspended in 0.1% BSA in PBS for binding assays.

[0293] In vitro binding of fluorescent Fc-fusions to bone homogenates.

[0294] The fluorescent Fc-fusion protein was 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). The samples were mixed at room temperature for 2 hours, followed by centrifugation to separate the bound and unbound fractions of the bone homogenate. The bone homogenate fractions were washed three times with PBS, and the final homogenate precipitate was resuspended in 100 µL of PBS. The relative concentrations of the fluorescent Fc-fusion protein in the resuspended bone homogenate and the 100 µL unbound protein suspension were quantified using a fluorescent plate reader.

[0295] For all fluorescent Fc-fusion proteins, binding to bone homogenate follows a similar trend to HA binding; however, there are greater numerical differences between effectively bone-binding proteins (e.g., FLU001) and non-binding proteins (e.g., FLU002). Figure 1CAs shown. In the bone-bonded fraction, FLU001 showed an 8-fold increase in fluorescence compared to the negative control (FLU002), while its binding to HA was observed to be 4-fold increased. FLU010 did not show significantly higher binding to bone homogenate than the negative control (FLU002); however, FLU015 still showed effective binding to bone homogenate and was significantly present in the unbound fraction. To ensure these results were not statistically abnormal, the experiment was repeated with n = 4, and the same results were observed for FLU010 and FLU015.

[0296] Importantly, raw fluorescence data quantifying the total amount of protein bound to HA (Example 2) and bone homogenate showed that non-target proteins bound significantly more to an equivalent mass of HA compared to bone homogenate (in some cases, binding to HA increased by up to 75-fold), indicating that proteins exhibit some non-specific binding to HA even in the absence of an effective bone-targeting fraction. This non-specific binding to HA was further amplified when the blocking agent was removed from protein incubation, demonstrating HA's ability to bind to a variety of proteins, including those without a specific HA-binding fraction. While HA is not very useful in distinguishing between moderately HA-bound fusions and non-targeted fusions, it is a suitable substrate for determining highly efficient bone-binding proteins. Because bone homogenate is a more robust binding substrate and does not show any non-specific binding to non-target proteins (see Figure 1), all subsequent bone-binding screening assays were performed using mouse bone homogenate as the binding substrate. Figure 2A-2B ).

[0297] The fluorescence intensities of bound and unbound fractions were normalized to non-targeted fluorescent Fc-fusion proteins for comparison between different target moieties. Figures 3A-3B The in vitro binding experiment was conducted in duplicate, with ± standard deviation.

[0298] Characterization of constructs bound to bone homogenate .

[0299] Prior to in vivo administration, an in vitro model was used to characterize bone binding of ERT-like molecules. To utilize hydroxyapatite-based fluorescence-based binding assays, ALP031 (ALP-Fc-D10) and ALP086 (ALP-Fc) proteins were fluorescently labeled using a commercially available antibody labeling kit (Ingenieur / Thermo Fisher Scientific). The Alexa Fluor dye was previously activated with succinimide esters or tetrafluorophenyl esters that react with primary amines on the proteins. Therefore, the fluorescent labeling was not site-specific, and the degree of labeling ranged from 3–5 moles of fluorophores per mole of antibody. Once labeled and purified to remove free dye, a 30 nM protein solution was prepared in 0.1% BSA, and bone homogenate binding assays were performed on the FFC constructs as described above. Exemplary data on constructs with different affinities to bone homogenates are available in […]. Figure 1C , Figure 1D As shown in Figures 2 and 3.

[0300] Plate bone ALP activity assay ALP activity in bone homogenate fractions was determined using 4-MUP. The bone homogenate fractions were resuspended in 100 µL of PBS and transferred to a 96-well black plate. 100 µL of unbound protein suspension was also transferred to individual wells of the 96-well plate. 100 µL of ALP detection solution (10 µM 4-MUP, 1% BSA) was added to each well, and kinetic fluorescence readings were immediately initiated (at 360 / 465) and run for 20 minutes, during which fluorescence intensity emission was collected every 30 seconds. The slope of fluorescence intensity relative to time represents the ALP concentration in each sample fraction.

[0301] The MUP activity of the bound and unbound fractions of various constructs was measured via the initial activity slope over the first 5 minutes to maintain linearity. The MUP activity ratio for each fusion protein was determined as (bound activity) / (unbound activity), and the results are shown below. Figure 14 As shown, this ratio demonstrates an exponential response with increasing length of the polyacid bone-targeting tag, resulting in significantly stronger bone homogenate binding as individual residues are added to the polyacid peptide.

[0302] Determination of relative protein affinity of bone homogenate in vitro A multi-dose assay was developed to classify the relative affinity of bone-binding proteins. For proteins that effectively bind to bone homogenate, the relative dissociation rate was determined by this kinetic bone protein exchange assay.

[0303] Proteins were evaluated individually (e.g., one protein type per tube) by incubating saturated concentrations (1 μM) of unlabeled bone-binding protein and 5 mg of bone homogenate in 1.5 mL Eppendorf tubes. After incubation with the unlabeled protein for 24 hours, the bone homogenate was centrifuged (16,000 rcf, 5 min) to remove excess unbound protein. The bone homogenate saturated with the given bone-binding protein was resuspended in ALEXA FLUOR. ® Bone homogenates were incubated for 1, 2, 4, 8, and 24 hours in a 0.5 μM solution of the same bone-binding protein labeled with the fluorescent probe. The homogenates were then 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 remaining (unbound) fluorescent protein in the 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.

[0304] Bone homogenates were initially 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 after protein exchange. Fluorescence signals were normalized to samples not pretreated with saturated concentrations of unlabeled protein for 24 hours, representing the maximum potential binding of the fluorescently labeled protein. VHH001 (circle) shows an immediate equilibrium between bound and unbound protein concentrations, indicating a rapid exchange rate or low-affinity bone homogenate binder. VHH002 (square) shows a more progressive substitution of the fluorescently labeled protein for the bound protein in solution. ALP031 (triangle) shows minimal substitution of the fluorescently labeled protein for the bound protein, indicating high affinity for bone once bound.

[0305] The kinetic curve of fluorescent protein accumulation on presaturated bone shows that unlabeled proteins on bone are replaced by labeled proteins. Figures 10A-10B ).

[0306] ALP-Fc-(Asp) 10 The characteristics of ALP-Fc binding to bone homogenate.

[0307] Prior to in vivo administration, an in vitro model was used to characterize bone binding of ERT-like molecules. To utilize a hydroxyapatite fluorescence-based binding assay, ALP-Fc-(Asp) was labeled using a commercially available antibody labeling kit (Ingenieur / Thermo Fisher Scientific). 10 Fluorescent labeling with ALP-Fc fusion protein. ALEXA FLUOR ® The dye was previously activated with succinimide esters or tetrafluorophenyl esters that react with primary amines on the protein. Therefore, the fluorescent labeling was not site-specific, and the degree of labeling was between 3 and 5 moles of fluorophore per mole of antibody. Once labeled and purified 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.

[0308] The bound / unbound fraction changes according to an approximate Gaussian distribution that is a function of bone marker length.

[0309] result In vitro characterization of bone homogenate binding indicates in vivo bone targeting (see Example 5); however, studying the association (binding) alone cannot distinguish between two highly effective bone-targeting fractions. For example, ALP-Fc-D 10 Both (SEQ ID NO: 31) and VHH002 (SEQ ID NO: 250) demonstrated quantitative binding to bone homogenate in vitro and effective binding and retention to bone in vivo. 。Although these proteins are almost indistinguishable from these experiments, they exhibit very different affinities for bone based on their dissociation behavior. Figures 10A-10B As shown, bone-bound VHH002 can exchange with excess soluble fluorescently labeled VHH002 in solution. In contrast, once bound, ALP-Fc-D... 10 It showed almost no dissociation from bone homogenate, even in the presence of excess soluble fluorescently labeled ALP-Fc-D. 10 In this case, therefore, compared to VHH002, ALP-Fc-D 10 They exhibited significantly greater bone affinity, despite having nearly equal association (binding) rates. VHH002 and ALP-Fc-D 10 They are effectively localized to bone and remain in the bone chamber for more than 7 days, but their localization mechanisms may be quite different. VHH002 has demonstrated the ability to dissociate from bone, while ALP-Fc-D 10 It exhibits significantly slower dissociation after binding. This is likely due to ALP-Fc-D. 10 The molecule remains closer to the initial binding site, while VHH002 has greater fluidity to bind to and dissociate throughout the bone tissue.

[0310] A proper understanding of the mechanisms of bone localization and the impact of novel targeting domains on therapeutic efficacy will be crucial for developing optimal and effective enzyme replacement therapies. Furthermore, modulating the duration and fluidity of the therapy on bone can be used to optimize sALP constructs for use in the treatment of diseases such as HPP.

[0311] Example 6 Pharmacokinetic analysis in mouse models Male C57BL / 6 mice (Jackson Laboratory) aged 11–12 weeks were administered a single injection of 4–7 mg / kg via tail vein or subcutaneous injection of 1 mg / mL sample protein in sterile PBS (calcium or magnesium-free) and followed up for 14–21 days. Two intermediate and one final blood draws (cardiac puncture, CO2 anesthesia) were performed on each mouse, staggered in the cohort (4 mice per molecule and per administration type, 4 groups per cohort). Blood samples (100 µL, yielding 50 µL plasma after centrifugation) were collected into Li / heparinized tubes at 0.25, 1, 6, 24, 48, 72, 96, 120, 192, 264, 336, and 480 hours. Blood samples were stored at 4°C until processed into plasma; subsequently, plasma samples were flash-frozen in liquid N2 and stored at -80°C.

[0312] Quantification of enzyme activity in mouse plasma samples: A slight modification was made to the 4-MUP assay used in Example 2 to determine specific enzyme activity in collected samples with unknown alkaline phosphatase concentrations. Serum samples were diluted 100-fold 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 the concentration of active alkaline phosphatase. Except that the standard curve was generated based on the observed activity of a reference standard alkaline phosphatase sample with known activity and concentration, the diluted samples were quantified as described in Example 2. The slope of fluorescence intensity relative to time represents the rate of 4-MU production, which corresponds to ALP activity, as a function in each sample fraction but here expressed in units of / mL serum. Exemplary mouse PK curves for the selected compounds after IV and subcutaneous administration are available in [reference missing]. Figure 15A (IV) and Figure 15B (Subcutaneous) and Table 8.

[0313] Table 8: Mouse plasma pK values ​​of the fusion protein Example 7 Testing of alkaline phosphatase constructs in HPP mouse efficacy model Preclinical efficacy studies of the alkaline phosphatase construct were conducted using the Akp2GW(- / -) mouse model with HPP. The Akp2GW(- / -) mice share the same HPP-induced TNSALP mutation used in Akp2(- / -) mice (Narisawa et al. 1997), which was previously used for preclinical evaluation of Asfor enzyme α. In these studies, multiple doses of the test product (ALP201 or the mediator (PBS)) were administered subcutaneously to the scapular region from day 1 to day 35 postnatally. Reported results included overall survival, weight gain rate, bone mineralization of the hind paw bone at day 36 (or at death in the case of study end [EOS]), and EOS glutar plasma enzyme activity levels (collected on day 36 in the daily [QD] and weekly [Q1W] groups, and on day 37 in the every 2 days [Q2D] dose group). In some studies, femoral and tibial lengths and mouse femoral alkaline phosphatase activity were measured.

[0314] Animal weight was assessed daily as an indicator of overall health. Age- and fetal-matched PBS-treated WT mice served as a reference control. Group 7 animals received the same daily subcutaneous injections of ALP201 as groups 3 through 5 until day 24. On day 25, the daily subcutaneous dose of ALP201 was reduced to half the initial dose and maintained until the final treatment dose on day 35. Compared to the negative (PBS) control, ALP201 showed statistically significant improvements in survival and bone mineralization.

[0315] Survival curve at Figure 16 As shown in the figure, the average weight is at Figure 17 The figure in the middle shows the correction of bone mineralization phenotype by the hind paw mineralization index on day 11. Figure 18 The dosing groups for these studies are listed in Table 9.

[0316] Table 9: Dosage grouping study plan for efficacy studies of Akp2GW (- / -) Abbreviations: PBS = Phosphate-buffered saline; Q1W = Once a week; Q2D = Every 2 days; QD = Once a day; UMUP = Active unit in the hydrolysis of 4-methylumbelliferyl phosphate.

[0317] Bone mineralization results Bone mineralization at day 36 / 37 was determined by X-ray analysis of the hind paws of treated Akp2GW(- / -) mice. X-ray visualizations of hind paw bone mineralization were compared to baseline X-ray images from day 36, illustrating four classification categories: unaffected, slightly deficient, moderately deficient, and severely deficient. Detailed descriptions of these categories are shown in Table 10. Images were assigned to each mouse in a blinded manner, and scoring was performed within individual dose groups once completed.

[0318] Table 10: Classification of hind claw bone mineralization index scores on day 36 1 Severe The deformity is severe, with no middle or distal phalanges of the fingers and no secondary ossification centers whatsoever. 2 moderate The finger is fully formed, but there are still no obvious secondary ossification centers. 3 Mild A fully formed finger has variable but incomplete secondary ossification centers. 4 Unaffected The fully formed finger and all secondary ossification centers are present. The distribution of bone mineralization index in the efficacy study is listed in Table 11, and... Figure 25 The diagram is shown graphically.

[0319] Table 11: Distribution of bone mineralization index of hind claw bone in the treated Akp2GW (- / -) mouse group at the end of the study. Dosage group Number in the group Number of unaffected (score = 4) Number of minor deficiencies (score = 3) Number of moderately deficient (score = 2) The number of severely deficient (score = 1) WT PBS 20 20（100%） 0 0 0 ALP201 4.8 mg / kg QD 25 25（100%） 0 0 0 ALP201 4.8 / 1.5mg / kg QD 17 17（100%） 0 0 0 ALP201 4.8 mg / kg Q2D 24 24（100%） 0 0 0 ALP201 2.0 mg / kg Q2D 31 29（93.5%） 2（6.5%） 0 0 ALP201 0.8 mg / kg Q2D 29 18（62.1%） 11（37.9%） 0 0 ALP201 0.3 mg / kg Q2D 31 18（58.1%） 10（32.3%） 1（3.2%） 2（6.5%） ALP201 0.15mg / kg Q2D 29 14（48.3%） 9（31.0%） 6（20.7%） 0 ALP201 4.8 mg / kg Q1W 19 14（73.7%） 2（10.5%） 3（15.8%） 0 Asfozyme Alpha 9.8 mg / kg QD 16 16（100%） 0 0 0 Asfozyme Alpha 2.5 mg / kg QD 26 22（84.6%） 4（15.4%） 0 0 HOM PBS (at the time of death) 50 7（14.0%） 12（24.0%） 24（48.0%） 7（14.0%） ALP259 4.8 mg / kg Q2D 15 15（100%） 0 0 0 Abbreviations: HOM = homozygous knockout, PBS = phosphate-buffered saline; QD = daily; Q2D = every 2 days; Q1W = once a week; WT = wild type.

[0320] Survival results All PBS-treated Akp2GW (- / -) mice died on or before day 26 of the study, with a mean survival of 20 days. Treatment with ALP201 and ALP259 significantly improved 36-day survival in all dose groups compared to the PBS-mediated control. Figure 16All ALP201 dosing groups achieved an overall survival rate of at least 69%, with all QD and Q2D interval dosing groups at doses higher than 0.15 mg / kg / day achieving an overall survival rate of 88% or higher. Survival curves for the ALP201 4.8 mg / kg Q1W group and the Asfor enzyme α 9.8 mg / kg QD group showed highly similar results (Table 12 and...). Figure 16 ).

[0321] Table 12: Summary of survival rates in the Akp2GW (- / -) mouse group treated in the 36 / 37-day efficacy study ALP201 4.8 qd 100 ALP201 4.8 / 1.5 qd 100 ALP201 4.8 q2d 96 ALP201 2 q2d 94 ALP201 0.8 q2d 97 ALP201 0.3 q2d 88 ALP201 0.15 q2d 69 ALP201 4.8 q1w 83 ALP259 4.8 q2d 100 Asforase α 9.8 qd 81 Asforase α 2.5 qd 96 PBS NA NA 0 Plasma alkaline phosphatase activity assay and results at the end of the study 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 groups), mouse plasma samples were collected and prepared. Alkaline phosphatase activity in the plasma samples was determined using the method outlined in Example 6. The measurement rate of the reaction in the plasma samples was calculated using a standard curve of known enzyme activity to obtain a value of plasma alkaline phosphatase activity in U / mL. Samples were run twice in duplicate to collect data for each independent sampling point. The values ​​in U / mL were converted to mg / L using the following relationship: Concentration in mg / L = (Measured U / mL activity value / Specific activity of test sample in U / mg) * 1000.

[0322] At the end of the study, the distribution of plasma activity levels was as follows: Figure 28 The average values ​​are shown in Table 13.

[0323] Table 13: Average plasma active enzyme concentration at the end of the study in dose groups Construct Dosage (mg / kg) interval Dosage quantity in the study The average concentration of active enzymes (ug / mL) at the end of the study. Collection Date ALP201 4.8 qd 35 48.7 36 ALP201 4.8 / 1.5 qd 35 21.8 36 ALP201 4.8 q2d 18 19.7 37 ALP201 2 q2d 18 6.4 37 ALP201 0.8 q2d 18 3.6 37 ALP201 0.3 q2d 18 1.5 37 ALP201 0.15 q2d 18 0.6 37 ALP201 4.8 q1w 5 0.6 36 Asforase α 9.8 qd 35 6.2 36 Asforase α 2.5 qd 35 1.0 36 ALP259 4.8 q2d 18 51.8 37 The study on alkaline phosphatase activity assay in bone tissue has been completed. Mouse femoral samples were harvested on days 36 / 37 of this study. Excess tissue was removed, the samples were rapidly frozen, and stored at -80°C. Prior to analysis, the femoral samples were transferred to dry ice, placed on sterile cold culture dishes, and rinsed to remove any residual connective tissue. The distal end of the femur was cut off with bone shears, and the bone shaft was transferred to a 0.5 mL collection tube (prepared by punching a hole in the bottom with a 20-gauge syringe needle) on wet ice. The collection tube was then placed into a 1.5 mL centrifuge tube and centrifuged at 4000 rpm for 30 seconds in a benchtop microcentrifuge to effectively remove bone marrow from the mouse femur. The prepared mouse femoral shaft was weighed and transferred to 90 μL 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 the mouse is too long to fit into the measuring hole, the femoral shaft is cut in half and both halves are placed into the same hole.

[0324] On each assay plate, the control consisted of femoral shafts from untreated wild-type and AKP2GW(+ / -) heterozygous mice, which were kept at 90°C for 60 minutes prior to assay to effectively heat-kill alkaline phosphatase activity on these bone samples, serving as a negative control for the assay.

[0325] A standard curve was generated on each plate using a series of dilutions of an alkaline phosphatase activity reference standard with known specific activity, and the plate was transferred to the assay plate at final assay concentrations of 0, 1.25, 2.5, 5, 10, 20, 35, and 50 ng / mL.

[0326] Prior to the assay, the plate was sealed and heated to 37°C for 15 minutes on a Gitbar heated plate shaker (without shaking). Once heated 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 250 μL volume. The process curve of 4-methylumbelliferone production was measured at 37°C using a Molecular Devices i3x plate reader (set to excitation λ = 365 nm and emission λ = 445 nm) or a Molecular Devices Paradigm plate reader (set to excitation λ = 360 nm filter and emission λ = 445 nm filter).

[0327] The activity slope for all samples was calculated using linear regression in the commercial spreadsheet program (Microsoft Excel). Where the activity slope became significantly non-linear during the experiment, linear regression was performed using the initial linear portion of the sample progression curve to capture the initial reaction rate. The slope of each individual progression curve was calculated in relative fluorescence units (RFU) / minute. An example of the determined progression curve is shown in [example missing]. Figure 30 The diagram shows the distribution of values ​​from individual subjects in... Figure 28 and Figure 29 Chinese standard drawing.

[0328] The treated AkpGW (- / -) femurs showed a significant increase in alkaline phosphatase activity (Table 14), demonstrating the ability of ALP201 and ALP259 to deliver enzyme activity to bone tissue.

[0329] Table 14: Alkaline phosphatase activity at the end of the study, measured on the femoral shaft of treated Akp2GW(- / -) mice. Construct dose Collection Date Bone alkaline phosphatase activity (% units / mg wild-type bone) ALP201 2 mg / kg q2d 37 44 ALP201 0.8 mg / kg q2d 37 34 ALP201 0.3 mg / kg q2d 37 31 ALP201 0.15 mg / kg q2d 37 21 Asforase α 2.5 mg / kg qd 36 24 ALP201 4.8 mg / kg q2d 37 48 ALP259 4.8 mg / kg q2d 37 67 Tibial and femoral 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 Faxitron X-rays, so that the length of the longest part of the bone (ImageJ length) was captured by a straight line connecting the analysis points.

[0330] Calculate bone length in millimeters (mm) using the following formula: In a one-way ANOVA:Dunnett multiple comparison test of asforase α, AKP2GW (- / -) mice treated with ALP201 daily and Q2D showed statistically significant improvements in both femoral and tibia lengths after 36 / 37 days of treatment compared to asforase α administered daily at 9.8 mg / kg, specifically p = 0.0212 and p = 0.0032 for femoral length and p = 0.0030 and p = 0.0088 for tibia length, respectively. Figure 26 tibia, and Figure 27 (femur).

[0331] Weight results In the efficacy study, the body weight of all mice was monitored. ALP201 and ALP259 treated Akp2GW(- / -) mice consistently had slightly lower body weights than their wild-type littermates treated with PBS, but there was no statistically significant difference between any two groups at any time point during the study period. Figure 17).

[0332] Following IV administration to C57BL / 6 mice, ALP259 showed twice the enzymatic activity of Asforase α in pyrophosphate hydrolysis and a 4-5 times longer in vivo half-life. In the HPP-induced AKP2GW(- / -) mouse model, ALP259 demonstrated approximately 50 times greater plasma accumulation at study end than Asforase α, and bone activity accumulation in AKP2GW(- / -) mice at study end was 140% that of ALP201. ALP259 demonstrated clear efficacy for bone mineralization in the AKP2GW(- / -) mouse model, with <20% of mice exhibiting a normal bone phenotype relative to untreated mice, and 100% of mice treated with 4.8 mg / kg at q2d dose intervals exhibiting normal bone mineralization.

[0333] Example 8 In vivo characterization of bone targeting: in vivo fluorescence imaging in mice Semi-quantitative biodistribution studies were conducted in nude mice using ALEXA FLUOR® 750-labeled bone-targeting proteins and protein fragments (VHH). Bone-targeting ALP-Fc fusion protein and bone-targeting VHH were fluorescently labeled with ALEXA FLUOR® 750 using an Invitrogen SAIVI kit (via activated succinimide covalent conjugation), and purified in exclusion resin to remove unconjugated fluorophores. The purified proteins (suspended in PBS) were injected into nude mice via tail vein at a dose of approximately 3 mg / kg.

[0334] Female J:NU distantly bred mice (Jackson Laboratory, Bar Harbor, Maine) received 3 mg / kg of test sample via a volume-normalized 100 µL intravenous tail vein injection. For in vivo imaging, subjects were maintained under 2%–3% isoflurane anesthesia on an imaging platform (IVIS® Spectroscopic Imaging System, PerkinElmer, Waltham, MA). Automated exposure settings with field of view (FOV) C, F / Stop 2, intermediate facets, and 800 nm emission / 750 nm excitation filters were used for both 2D epi-illumination and 3D transmitted illumination acquisition. Ex vivo tissue samples were acquired under identical conditions for 2D epi-illumination fluorescence imaging, except that all samples of each tissue type were acquired simultaneously in a single image. All animal studies were conducted in accordance with the Animal Welfare Act and the Guidelines for the Care and Use of Laboratory Animals.

[0335] Fluorescence imaging analysis was performed using 2D / 3D software provided by the manufacturer (Living Image 4.5.1, PerkinElmer). Regions of interest (ROIs) of uniform area were manually located for each group of subjects to address variability in subject localization. Longitudinal in vivo image color scale ranges were normalized across all subjects and time points; color scales for ex vivo samples were determined individually to best represent the fluorescence signal of each tissue group. Figure 4 , Figure 8 , Figure 12 and Figure 13 All images were represented using the following parameters: Opacity = 80%, Color Table = "Blue Hot", Smoothing = None, Logarithmic Scale. Readout bias subtraction, adaptive FL background subtraction, and flat field, cosmic, and lens distortion corrections were also applied. The ALEXA FLUOR® 750 signal is represented as radiometric efficiency (p / sec / cm²). 2 / sr) / (µW / cm 2 ).

[0336] Ex vivo bone samples were individually imaged on a miniature computed tomography X-ray system (Quantum FX µCT, PerkinElmer, Waltham, MA). The following parameters were used for all acquisitions: voltage = 90 kV, current = 180 µA, FOV = 40 mm, and acquisition time = 17 seconds. Using the bone growth workflow, the total bone volume (Tt.BV, mm²) was quantified using the manufacturer's software (AccuCT 1.0 Advanced Analysis Software, PerkinElmer, Waltham, MA). 3 The normalized representation of bone volume using ALEXA FLUOR® 750 signals is expressed as (p / sec / cm). 2 / sr) / (µW / cm 2 )) / mm 3 Statistical analysis was performed using commercially available statistical software (GraphPad Prism 7, La Jolla, CA).

[0337] Because ALP-Fc-D 10 (SEQ ID NO: 31) maximizes the potential signal of bone-targeting proteins, and ALP-Fc (SEQ ID NO: 222) shows no binding to bone homogenate; therefore, these proteins were used as positive and negative control proteins in in vivo bone-targeting experiments. ALEXA FLUOR ® 750 marked ALF-Fc or ALP-Fc-D 10Protein biodistribution was tracked in vivo in naked adolescent mice via intravenous injection throughout the 18-day study. Two-dimensional IVIS in vivo 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, kidneys, spleen, femur, skull, and spine. Figure 4 and 5A -5B shows the total radioactivity of fluorescent proteins detected within the whole-body imaging gate. Quantitatively, throughout the first 4 days post-administration, the radioactivity of fluorescently labeled bone-targeting ALP-Fc-D was measured. 10 The mean total radiation efficiency of the treated mice was similar to that of mice treated with untargeted ALP-Fc. However, on day 7 and beyond, ALP-Fc-D... 10 It showed significantly higher radiation efficiency than ALP-Fc, due to the accumulation of target proteins on bone and the clearance of non-target proteins. Figure 5B A comparison of the total radiation efficiency of the same treated mice within the spinal region of interest is shown. Within the spinal region of interest, a statistically significant difference between targeted and untargeted ALP-Fc fusions was observed within 4 days, and this difference persisted throughout the entire 18-day study period. Figure 4 Representative images from each processing group at each imaging time point are shown, where color scales represent radiometric efficiency.

[0338] The newly developed bone homogenate screening assay was validated in vivo. Through the developed screening assay, ALP-Fc-D... 10 ALP-Fc and ALP-Fc represent two extremes of bone integration characteristics: quantitative binding of the protein to bone homogenate and nonspecific binding to bone homogenate. In vivo biodistribution studies showed that after a single intravenous injection, ALP031 remained in bone tissue for more than two weeks. Figure 12 (Radiation efficiency shown by color scale). In contrast, ALP086 (non-targeted ALP-Fc) was undetectable in vivo 4 to 7 days after injection. Figure 13 (Radiation efficiency shown by color scale). Furthermore, intravenous administration of ALP031 resulted in uniform distribution of the drug throughout the mineralized tissues of the mouse spine, skull, and femur. Figures 6A-6B These results confirm that the bone homogenate screening assay distinguishes between bone-targeting and non-targeting proteins.

[0339] Further in vivo studies of the VHH construct distinguished between highly efficient bone-binding proteins and intermediate bone-binding proteins with lower affinity. Figure 7 , 8(9A-9C and 10A-10B). Therefore, it is advantageous to study VHH proteins in vivo because non-target VHH proteins are rapidly cleared from systemic circulation via renal excretion, which limits background signaling and allows for early differentiation of bone localization. Figure 8 The study showed increased whole-body fluorescence signals in mice treated with VHH001 and VHH002 at 24 hours post-injection compared to the non-targeted control, with VHH002 showing a significantly enhanced signal compared to VHH001. Over time, the signal from VHH001-treated mice approached baseline levels of the non-targeted control, while the signal from VHH002-treated mice remained significantly increased throughout the 7-day study. This bone-targeting behavior was observed in a developed in vitro screening assay that distinguished the intermediate binding of the non-targeted VHH control protein to VHH001 and the efficient binding to VHH002, demonstrating a verifiable correlation between screening and in vivo bone targeting.

[0340] Example 9 One adult male subject presented with elevated inorganic pyrophosphate (PPi) levels of approximately 5.82 μM and a mean BOT-2 intensity score of less than 10. The subject was 24 years old and experiencing lower extremity pain and gait disturbance. The subject could be diagnosed with HPP and selected for treatment. The subject could undergo X-ray and bone mineral density testing, both of which showed reduced bone mineralization in the legs.

[0341] A pharmaceutical formulation containing the peptide of SEQ ID NO: 72 can be formulated to a concentration of 0.1 mg / mL. The formulation can be administered subcutaneously to subjects once weekly at a dose of 0.1 mg / kg / week for 8 weeks. Treatment efficacy can be assessed after the 8-week treatment regimen. Subjects may notice reduction in bone pain and normalization of gait. Subjects can undergo subsequent X-ray and bone mineral density tests, which can demonstrate normalization of bone mineralization relative to pre-treatment levels. A reduction in the subject's PPi concentration to below 5 μM and an improvement in their BOT-2 intensity score to 12 indicate therapeutic efficacy of the peptide.

[0342] Example 10 An adolescent female subject presented with elevated inorganic pyrophosphate (PPi) levels of approximately 4.78 μM and a mean 6MWT less than approximately 70% of the predicted 6MWT value. The subject was 14 years old and experiencing tooth loss and chronic pain. The subject could be diagnosed with HPP and selected for treatment. The subject could undergo X-ray and bone mineral density testing, both of which could show bone mineralization in her teeth and femur.

[0343] A pharmaceutical formulation containing the peptide of SEQ ID NO: 123 can be formulated to a concentration of 0.5 mg / mL. The formulation can be administered subcutaneously to the subject once weekly for 4 weeks at a dose of 0.5 mg / kg / week. Treatment efficacy can be assessed after the 4-week treatment regimen. The subject may notice a reduction in chronic pain. The subject can undergo subsequent X-ray and bone mineral density tests, which can show normalization of bone mineralization in the femur relative to pre-treatment levels. A reduction in the subject's PPi concentration to below 4 μM and an improvement in her 6MWT score to approximately 85% of the predicted value indicate therapeutic efficacy of the peptide.

[0344] Example 11 An adolescent female subject presented with elevated inorganic pyrophosphate (PPi) concentrations of approximately 4.85 μM and a mean 6MWT less than approximately 65% ​​of the predicted 6MWT value. The subject was 15 years old and experiencing tooth loss and chronic pain. The subject could be diagnosed with HPP and selected for treatment. The subject could undergo X-ray and bone mineral density testing, both of which could show bone mineralization in her teeth and femur.

[0345] A pharmaceutical formulation containing the peptide of SEQ ID NO: 177 can be formulated to a concentration of 0.7 mg / mL. The formulation can be administered subcutaneously to subjects once weekly for 6 weeks at a dose of 0.7 mg / kg / week. Treatment efficacy can be assessed after the 6-week treatment regimen. Subjects may notice a reduction in chronic pain. Subjects can undergo subsequent X-ray and bone mineral density tests, which can show normalization of bone mineralization in their femur relative to pre-treatment levels. A reduction in the subject's PPi concentration to below 4 μM and an improvement in her 6MWT score to approximately 88% of the predicted value indicate therapeutic efficacy of the peptide.

[0346] Other embodiments The above detailed descriptions and examples are provided for clarity only and should not be construed as unnecessary limitations. The invention is not limited to the precise details shown and described, as variations that will be apparent to those skilled in the art are included within the scope of the invention as defined in the claims.

[0347] Unless otherwise specified, in all cases, all figures indicating the amount, molecular weight, etc., of components used in the specification and claims shall be understood to be modified by the term “about.” Therefore, unless otherwise indicated, the numerical parameters set forth in the specification and claims are approximate values ​​and may vary according to the desired characteristics sought to be obtained according to the invention. At least, it is not an attempt to limit the doctrine of equivalence to the scope of the claims; each numerical parameter should be interpreted at least according to the number of significant digits reported and by applying ordinary rounding techniques.

[0348] While the numerical ranges and parameters illustrating the broad scope of the invention are approximate, the values ​​described in specific examples are reported as precisely as possible. However, all values ​​inherently contain a range, which necessarily arises from the standard deviation present in their respective test measurements.

[0349] All patents, patent applications (including provisional patent applications), and publications (including patent and non-patent publications) and electronically available materials (including, for example, nucleotide sequences submitted to, for example, GenBank and RefSeq, and amino acid sequences submitted to, for example, SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are fully disclosed by reference. The detailed descriptions and examples above are given only for clarity and should not be construed as unnecessarily limiting. This disclosure is not limited to the precise details shown and described, and variations that will be apparent to those skilled in the art will be included within the embodiments defined by the claims.

Claims

1. A polypeptide comprising a recombinant alkaline phosphatase having three mutations relative to a naturally occurring alkaline phosphatase, wherein the three mutations improve at least one activity or pharmacokinetic property relative to a naturally occurring alkaline phosphatase without the three mutations, wherein the three mutations are E108M, N213Q and N286Q relative to SEQ ID NO:

1.

2. The polypeptide of claim 1, wherein the naturally occurring alkaline phosphatase is a tissue nonspecific alkaline phosphatase (TNSALP).

3. The polypeptide of claim 2, wherein the TNSALP is a mammalian TNSALP.

4. The polypeptide of claim 3, wherein the mammalian TNSALP is a human, gorilla, mouse, rabbit, chimpanzee, cynomolgus monkey, rhesus monkey, orangutan, baboon, rat, cow, goat or llama TNSALP.

5. The polypeptide of claim 4, wherein the recombinant alkaline phosphatase is a human tissue-nonspecific alkaline phosphatase.

6. The polypeptide of claim 5, wherein the polypeptide comprises amino acids 1-491 of SEQ ID NO:

123.

7. The polypeptide of claim 1, further comprising a bone-targeting portion comprising 3 to 30 consecutive aspartic or glutamic acid residues.

8. The polypeptide of claim 1, 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 buffers, maintenance of activity at pH 5.0–7.5, reduced dimerization, reduced aggregation, and increased manufacturability.

9. The polypeptide of claim 8, wherein the increased catalytic activity comprises increased hydrolysis of pyridoxal 5-phosphate and / or pyrophosphate.

10. The polypeptide of claim 8, wherein the increased catalytic activity is 4 to 30 times better than the activity of the naturally occurring alkaline phosphatase.

11. The polypeptide of claim 1, wherein the at least one pharmacokinetic property is selected from the group consisting of: increased substrate specificity, increased activity against natural substrates, increased activity against artificial substrates, decreased Km against natural substrates, and increased area under the curve (AUC) per dose.

12. The polypeptide of claim 11, wherein the artificial substrate is selected from the group consisting of: umbrella phosphate, p-nitrophenyl phosphate (pNPP) and MUP; and / or wherein the natural substrate is selected from the group consisting of pyridoxal-5'-phosphate, PLP and PEA.

13. The polypeptide of claim 1, further comprising region Y, wherein Y is an amino acid sequence of at least one amino acid.

14. The polypeptide of claim 1, wherein the polypeptide comprises the following structure Z-ALP-Y-Xn, where: Y is an amino acid sequence of at least one amino acid; Z either does not exist or has an amino acid sequence of at least one amino acid; Xn is the bone-targeting fraction, selected from groups composed of polyaspartic acid (Dn) and polyglutamic acid (En), where n = 3 to 30; and ALP is the recombinant alkaline phosphatase.

15. The polypeptide of claim 13, wherein Y is the crystallizable region of the fragment (Fc).

16. The polypeptide of claim 15, wherein the Fc region comprises IgG1, IgG2, IgG3, or IgG4, or a chimera thereof.

17. The polypeptide of claim 16, wherein the Fc region comprises an IgG2 / 4 chimera.

18. The polypeptide of claim 17, wherein the Fc region comprises the sequence of SEQ ID NO:

253.

19. The polypeptide of claim 7, wherein the bone-targeting portion is: (a) Dn and n = 7 to 10; or (b) En and n = 10 to 15.

20. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

155.

21. The polypeptide of claim 1, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:

123.

22. A polypeptide comprising: (1) a recombinant alkaline phosphatase; (2) a fragment crystallizable (Fc) region, wherein the Fc region is an IgG2 / 4 chimera; wherein the recombinant alkaline phosphatase has three mutations relative to naturally occurring alkaline phosphatase, wherein the three mutations improve at least one activity or pharmacokinetic property relative to naturally occurring alkaline phosphatase without the three mutations, wherein the three mutations are E108M, N213Q and N286Q relative to SEQ ID NO: 1; and (3) a bone-targeting portion comprising 3 to 30 consecutive aspartic acid or glutamic acid residues.

23. The polypeptide of claim 22, wherein the Fc region comprises the sequence of SEQ ID NO:

253.

24. The polypeptide of claim 22, wherein the recombinant alkaline phosphatase is TNSALP.

25. The polypeptide of claim 24, wherein the recombinant alkaline phosphatase is a mammalian alkaline phosphatase.

26. The polypeptide of claim 25, wherein the mammalian alkaline phosphatase is a human, gorilla, mouse, rabbit, chimpanzee, cynomolgus monkey, rhesus monkey, orangutan, baboon, rat, cow, goat, or llama alkaline phosphatase.

27. The polypeptide of claim 22, wherein the bone-targeting portion is: (a) Dn and n = 7 to 10; or (b) En and n = 10 to 15.

28. A polynucleotide encoding a polypeptide as described in any one of claims 1-27.

29. A vector comprising the polynucleotide as described in claim 28.

30. A cell comprising the polynucleotide of claim 28 or the carrier of claim 29.

31. A method for producing a polypeptide as described in any one of claims 1-27, the method comprising: (a) Providing cells transformed with the polynucleotide of claim 28 or the vector of claim 29, wherein the polynucleotide is targeted for expression in the cells; (b) The transformed cells are cultured under conditions suitable for the expression of the polynucleotide, wherein the culture results in the expression of the polypeptide; and (c) Isolate the polypeptide.

32. A pharmaceutical composition comprising a polypeptide as described in any one of claims 1-27 and a pharmaceutically acceptable carrier.

33. The pharmaceutical composition of claim 32, wherein the pharmaceutically acceptable carrier comprises sodium chloride and / or sodium phosphate.

34. The pharmaceutical composition of claim 33, wherein the composition comprises 150 mM sodium chloride and / or 25 mM sodium phosphate at pH 7.

4.

35. The pharmaceutical composition of claim 32, wherein the composition is formulated at a dose of 0.1 mg / mL to 10 mg / mL.

36. The pharmaceutical composition of claim 32, wherein the composition is formulated to a volume of 0.1 mL to 50 mL.

37. Use of the pharmaceutical composition of claim 32 in the preparation of a medicament for treating hypophosphatase syndrome in subjects in need.

38. The application as described in claim 37, wherein the polypeptide is formulated for administration in an amount and duration sufficient to treat the hypophosphodiesterase syndrome.

39. The application as described in claim 37, wherein the treatment enhances bone formation in the subject.

40. The application as described in claim 37, wherein the polypeptide is formulated for administration at a dose of 0.01 mg / kg to 20 mg / kg.

41. The application as described in claim 40, wherein the polypeptide is formulated for administration at a dose of 0.1 mg / kg to 10 mg / kg.

42. The application as described in claim 37, wherein the polypeptide is formulated for daily, weekly, monthly or annual administration.

43. The application as described in claim 37, wherein the polypeptide is formulated for application for at least one day, one week, one month, one year, or longer.

44. The application as described in claim 37, wherein the composition is formulated for subcutaneous, intravenous, intramuscular, sublingual, intrathecal, or intradermal administration.

45. The application as described in claim 44, wherein the composition is formulated for subcutaneous or intravenous administration.

46. ​​The application as described in claim 37, wherein the subject is a human being.

47. The application as described in claim 46, wherein the person is a newborn, child, adolescent, or adult.

48. The application as described in claim 37, wherein, Prior to administration of the peptide, the subject was characterized as having an average walking distance of 350 meters or less in 6 minutes.

49. The application as described in claim 37, wherein administration of the polypeptide promotes an increase in the subject's average walking distance of at least 100 meters or more within 6 minutes.

50. The application as described in claim 37, wherein after administration of the peptide, the subject exhibits an average walking distance of 500 meters or more within 6 minutes.

51. The application as described in claim 37, wherein the subject exhibits reduced dependence on assistive mobility devices after administration of the peptide.

52. The application as described in claim 51, wherein the assistive mobility device is at least one device selected from the group consisting of a walker, wheelchair, brace, cane, and orthosis.

53. The application as described in claim 37, wherein, Prior to administration of the peptide, the subject was characterized as having a plasma PPi concentration of 4.5 μM or greater.

54. The application of claim 37, wherein administration of the polypeptide promotes a median reduction of at least 1 μM in the PPi concentration in a plasma sample from the subject.

55. The application as described in claim 37, wherein after administration of the polypeptide, the subject exhibits a plasma PPi concentration of 2 μM to 5 μM.

56. The application as described in claim 37, wherein: i) The subject is aged 0 to 14 days and is characterized by having a plasma ALP concentration of 90 U / L or lower prior to administration of the peptide; ii) The subject is 15 days to less than 1 year old and is characterized by having a plasma ALP concentration of 134 U / L or lower prior to administration of the peptide; iii) The subject is aged 1 year to less than 10 years and is characterized by having a plasma ALP concentration of 156 U / L or lower prior to administration of the polypeptide; iv) The subject was aged between 10 and 13 years and was characterized by having a plasma ALP concentration of 141 U / L or lower prior to administration of the peptide; v) The subject was female and aged 13 to 15 years, and was characterized by having a plasma ALP concentration of 62 U / L or lower prior to administration of the peptide; vi) The subject was male and aged 13 to 15 years, and was characterized by having a plasma ALP concentration of 127 U / L or lower prior to administration of the peptide; vii) The subject was female and aged 15 to 17 years, and was characterized by having a plasma ALP concentration of 54 U / L or lower prior to administration of the peptide; viii) The subject was male and aged 15 to 17 years, and was characterized by having a plasma ALP concentration of 89 U / L or lower prior to administration of the peptide; ix) The subject is 17 years of age or older and is characterized by having a plasma ALP concentration of 48 U / L or lower prior to administration of the peptide; or x) The subject is 17 years of age or older and is characterized by having a plasma ALP concentration of 59 U / L or lower prior to administration of the peptide.

57. The application of claim 37, wherein administration of the polypeptide promotes a median increase in ALP concentration of at least 100 U / L or greater in a plasma sample from the subject.

58. The application as described in claim 37, wherein: i) The subject is aged 0 to 14 days and is characterized by having a plasma ALP concentration of 273 U / L or greater after administration of the polypeptide; ii) The subject is 15 days to less than 1 year old and is characterized by having a plasma ALP concentration of 518 U / L or greater after administration of the polypeptide; iii) The subject is aged 1 year to less than 10 years and is characterized by having a plasma ALP concentration of 369 U / L or greater after administration of the polypeptide; iv) The subject was aged between 10 and 13 years and was characterized by having a plasma ALP concentration of 460 U / L or greater after administration of the polypeptide; v) The subject was female and aged 13 to 15 years, and was characterized by having a plasma ALP concentration of 280 U / L or greater after administration of the polypeptide; vi) The subject was male and aged 13 to 15 years, and was characterized by having a plasma ALP concentration of 517 U / L or greater after administration of the polypeptide; vii) The subject was female and aged 15 to 17 years, and was characterized by having a plasma ALP concentration of 128 U / L or greater after administration of the polypeptide; viii) The subject was male and aged 15 to 17 years, and was characterized by having a plasma ALP concentration of 365 U / L or greater after administration of the polypeptide; ix) The subject is female and 17 years of age or older, and is characterized by having a plasma ALP concentration of 95 U / L or greater after administration of the peptide; or x) The subject is male and 17 years of age or older, and is characterized by having a plasma ALP concentration of 164 U / L or greater after administration of the polypeptide.

59. The application as described in claim 37, wherein, Prior to administration of the peptide, the subject was characterized as having a mean Bunies Proficiency Test Version 2 (BOT-2) strength score of 10 or lower.

60. The application as described in claim 59, wherein, Prior to administration of the peptide, the subject was characterized as having an average BOT-2 running speed and agility score of 5 or lower.

61. The application as described in claim 37, wherein administration of the peptide results in the subject having an average BOT-2 strength score of 10 or higher.

62. The application as described in claim 37, wherein administration of the peptide results in the subject having an average BOT-2 running speed and agility score of 5 or higher.

63. The application as described in claim 37, wherein, Prior to administration of the peptide, the subject was characterized as having a mean Childhood Health Assessment Questionnaire (CHAQ) score of 0.8 or higher.

64. The application as described in claim 37, wherein administration of the polypeptide results in the subject having an average CHAQ score of 0.5 or lower.

65. The application as described in claim 37, wherein, Prior to administration of the peptide, the subject was characterized as having a mean Pediatric Outcome Data Collection Tool (PODCI) score of 40 or lower.

66. The application as described in claim 37, wherein administration of the polypeptide results in the subject having an average PODCI score of 40 or higher.

67. The application as described in claim 37, wherein, Prior to administration of the peptide, the subject was characterized as having an average muscle strength grade of less than 5.

68. The application as described in claim 37, wherein administration of the polypeptide results in an average increase of 1 or more in the subject's muscle strength grade.

69. The application as described in claim 37, wherein, Prior to administration of the peptide, the subject was characterized as having an average handheld force gauge (HHD) value that was less than 80% of the expected HHD value.

70. The application as described in claim 37, wherein administration of the polypeptide results in the subject having an average HHD value of 80% or more of the expected HHD value.

71. The application as described in claim 37, wherein the HHD value represents the subject's grip strength, knee flexion, knee extension, hip flexion, hip extension, or hip abduction.

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