Immunosuppression therapy to mitigate immune responses against soluble alkaline phosphatase

Abstract

The present disclosure features methods for treating neutralizing antibodies that reduce the efficacy of soluble alkaline phosphatase therapy (e.g., asfoenzyme alpha), which is an enzyme replacement therapy, such as for the treatment of bone mineralization disorders, e.g., hypophosphoesterase (HPP). The methods include diagnosing the subject for the presence of a neutralizing antibody that affects the efficacy of the treatment, administering a treatment suitable for reducing the deleterious effects of the neutralizing antibody, and continuing an alkaline phosphatase treatment.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 531,955, filed August 10, 2023, the contents of which are incorporated herein by reference in their entirety. Background Technology

[0003] Hypophosphatase syndrome (HPP) is a rare, inherited disorder of bone mineralization, with the most severe form occurring in approximately 1 in 100,000 newborns. This condition is caused by a loss-of-function mutation in the gene encoding tissue-specific alkaline phosphatase (TNALP). HPP manifests with a wide range of symptoms and severity, from rickets to almost complete absence of bone mineralization in utero.

[0004] STRENSIQ ® Asfozyme Alpha (ASA) has been approved as the first enzyme replacement therapy (ERT) for the treatment of HPP. Enzyme replacement therapy may cause the production of neutralizing antibodies against the enzyme in some subjects. Methods are needed to determine whether neutralizing antibodies lead to loss of enzyme efficacy, and methods are needed to treat antibody-mediated loss of efficacy when it occurs in subjects with bone mineralization disorders. Summary of the Invention

[0005] A first aspect of this disclosure is characterized by a method for reducing the amount of components of cellular and humoral immune responses, thereby reducing the risk of forming sALP-specific antibodies or T cells or attenuating the effect of such antibodies or T cells in subjects receiving soluble alkaline phosphatase (sALP) therapy. The method comprises administering to the subject a therapy containing one or more of a dihydrofolate reductase (DHFR) inhibitor and an anti-CD20 antibody or an antigen-binding fragment thereof, thereby reducing the amount of anti-sALP immune response, reducing the formation of an anti-sALP immune response, or attenuating the effect of an anti-sALP immune response.

[0006] On the other hand, a method is characterized by reducing the amount of sALP-specific antibodies, decreasing the risk of antibody formation, or attenuating the effect of the antibody in a subject receiving sALP treatment. The method includes administering to the subject a therapy comprising one or both of bortezomib and a DHFR inhibitor and an anti-CD20 antibody or an antigen-binding fragment thereof, thereby reducing the amount of the antibody, decreasing antibody formation, or attenuating the antibody's effect. The method may include administering bortezomib and a DHFR inhibitor. The method may include administering bortezomib and an anti-CD20 antibody or an antigen-binding fragment thereof.

[0007] In some embodiments, the method includes administering a DHFR inhibitor. In some embodiments, the DHFR inhibitor is methotrexate. Methotrexate may be administered every 5 to 10 days (e.g., every 5, 6, 7, 8, 9, or 10 days, e.g., every 7 days). For example, methotrexate may be administered to a subject in need every 7 days. It may be administered at a dose of approximately 10 mg / m². 2 Approximately 20 mg / m 2 (For example, about 10 mg / m²) 2 11mg / m 2 12mg / m 2 13mg / m 2 14mg / m 2 15mg / m 2 16mg / m 2 17mg / m 2 18mg / m 2 19mg / m 2 Or 20mg / m 2 For example, approximately 15 mg / m³ 2 Methotrexate can be administered at a dose of approximately 15 mg / m². 2 Methotrexate was administered at the prescribed dosage.

[0008] In some embodiments, the method includes administering an anti-CD20 antibody or an antigen-binding fragment thereof. In some embodiments, the anti-CD20 antibody is rituximab or an antigen-binding fragment thereof. Rituximab can be administered every 5 to 10 days (e.g., every 5, 6, 7, 8, 9, or 10 days, e.g., every 7 days). For example, rituximab can be administered to a subject in need every 7 days. It can be administered at a dose of approximately 100 mg / m². 2 Approximately 500 mg / m 2 (For example, approximately 100 mg / m²) 2 125mg / m 2 150mg / m 2 175mg / m 2 200mg / m 2 225mg / m 2 250mg / m 2 275mg / m 2 300mg / m 2 325mg / m 2 350mg / m 2 375mg / m 2 400mg / m 2 425mg / m 2 450mg / m 2 475mg / m2 Or 500mg / m 2 For example, approximately 375 mg / m³ 2 Rituximab or its antigen-binding fragment can be administered at a dose of approximately 375 mg / m². 2 Rituximab is administered at the prescribed dose. Rituximab or its antigen-binding fragment may be administered intravenously. In some embodiments, rituximab or its antigen-binding fragment is discontinued while the subject is receiving methotrexate. In other embodiments, methotrexate and rituximab or its antigen-binding fragment are administered in combination or alone.

[0009] In some implementations, the method further includes administering a proteasome inhibitor, such as bortezomib, to the subject in need. Bortezomib can specifically target plasma cells via at least one mechanism of action, thus offering an advantage over other treatment modalities that control certain pre-B cells and / or B cells but fail to target plasma cells, resulting in circulating antibodies and therapy failure. Bortezomib can be administered every 5 to 10 days (e.g., every 5, 6, 7, 8, 9, or 10 days, e.g., every 7 days). For example, bortezomib can be administered every 7 days. It can be administered at a dose of approximately 0.2 mg / m². 2 Approximately 2 mg / m 2 (For example, approximately 0.2 mg / m²) 2 0.3 mg / m 2 0.4 mg / m 2 0.5 mg / m 2 0.6 mg / m 2 0.7 mg / m 2 0.8 mg / m 2 0.9 mg / m 2 1mg / m 2 1.1 mg / m 2 1.2 mg / m 2 1.3 mg / m 2 1.4 mg / m 2 1.5mg / m 2 1.6 mg / m 2 1.7 mg / m 2 1.8 mg / m 2 1.9 mg / m 2 Or 2mg / m 2 Bortezomib can be administered at a dose of approximately 0.5 mg / m². 2 Approximately 0.7 mg / m 2 Approximately 1 mg / m 2 Or approximately 1.3 mg / m 2Bortezomib can be administered at the prescribed dose. It can be administered intravenously or subcutaneously. Intravenous administration of bortezomib may be done as a bolus injection. Bortezomib may be administered after a subject demonstrates a worsening of their rickets severity score (RSS) relative to baseline (e.g., corresponding to a value prior to treatment with sALP or prior to treatment with the therapy described herein) by one or more points. In a specific implementation, the RSS is calculated during study screening prior to initiation of therapy, and this score is considered the baseline RSS score. Bortezomib may be discontinued if the subject is receiving methotrexate and / or rituximab.

[0010] In some embodiments, anti-sALP antibody therapy (e.g., methotrexate, rituximab, and / or proteasome inhibitors, such as bortezomib) is administered for a duration of at least 6 months. For example, the therapy may be administered for a duration of at least 12, 18, or 24 months. In some embodiments, DHFR inhibitors (e.g., methotrexate) are administered for a duration of at least 6, 12, 18, or 24 months. In some embodiments, anti-CD20 antibodies or antigen-binding fragments (e.g., rituximab) are administered for a duration of at least 6, 12, 18, or 24 months. In some embodiments, the therapy is discontinued, for example, after the subject exhibits a decrease in antibody titer relative to baseline by at least two titer steps or becomes negative (e.g., undetectable) and / or exhibits an increase in RSS by at least one or more points relative to baseline. For example, the therapy may be discontinued when the amount of sALP-specific antibodies, their formation risk, or their effect is reduced relative to pre-treatment levels.

[0011] Anti-sALP antibody therapy may also include the administration of immunoglobulin. Immunoglobulin may be administered every 5 to 10 days (e.g., every 5, 6, 7, 8, 9, or 10 days, or, for example, every 7 days). For example, immunoglobulin may be administered to a subject in need every 7 days. Immunoglobulin may be administered at a dose of about 300 mg / kg to about 700 mg / kg (e.g., about 300 mg / kg, 350 mg / kg, 400 mg / kg, 450 mg / kg, 500 mg / kg, 550 mg / kg, 600 mg / kg, 650 mg / kg, or 700 mg / kg, or, for example, about 500 mg / kg). For example, immunoglobulin may be administered at a dose of about 500 mg / kg. Immunoglobulin may be administered intravenously. Immunoglobulin can be administered intravenously once a month at a dose of approximately 500 mg / kg, while receiving methotrexate and / or rituximab, with or without a proteasome inhibitor (e.g., bortezomib).

[0012] Anti-sALP antibody therapy may also include folic acid administration. Folic acid may be administered, for example, once daily. It can be administered at a dose of 1 mg. Folic acid may not be administered on the same day as methotrexate. Folic acid can be taken orally.

[0013] The methods described herein can be combined with sALP therapy (e.g., administration of sALP). This therapy can be administered before or after sALP therapy. sALP can be administered 1 to 7 times per week or every two weeks (e.g., 1, 2, 3, 4, 5, 6, or 7 times), for example, 2, 3, or 6 times per week. For example, sALP can be administered 3 or 6 times per week. sALP can be administered at doses from 1 mg / kg / week to 10 mg / kg / week, for example, from 3 mg / kg / week to 9 mg / kg / week (e.g., 1 mg / kg / week, 2 mg / kg / week, 3 mg / kg / week, 4 mg / kg / week, 5 mg / kg / week, 6 mg / kg / week, 7 mg / kg / week, 8 mg / kg / week, 9 mg / kg / week, or 10 mg / kg / week). For example, sALP can be administered at a dose of 6 mg / kg / week. Specifically, the sALP administered according to the above dosage may have at least 85% sequence identity with SEQ ID NO:1 or its sequence (e.g., at least 90%, 95%, or 99% sequence identity).

[0014] sALP may have at least 85% sequence identity with any of SEQ ID NO: 1-3. For example, sALP may have at least 85%, 90%, 95%, 97%, 99%, or 100% sequence identity with SEQ ID NO: 1. sALP may contain or consist of the sequence of SEQ ID NO: 1 (e.g., asforase α). sALP may have at least 85%, 90%, 95%, 97%, 99%, or 100% sequence identity with SEQ ID NO: 2. sALP may contain or consist of the sequence of SEQ ID NO: 2. sALP may have at least 85%, 90%, 95%, 97%, 99%, or 100% sequence identity with SEQ ID NO: 3. sALP may contain or consist of the sequence of SEQ ID NO: 3.

[0015] The methods described herein may include treating a subject who has been administered two different sALPs. In some embodiments, the subject has received treatment with a first sALP and a second sALP different from the first sALP. The first sALP may have the amino acid sequence of SEQ ID NO:1. The second sALP may have the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:3 (e.g., SEQ ID NO:2). For example, the initial therapy administered to the subject may include a dose of the sALP of SEQ ID NO:1. The subject may be administered a weekly dose of the sALP for days, weeks, months, or years. Subsequently, the subject may switch to a different sALP (e.g., the sALP of SEQ ID NO:2 or 3). The subject may receive treatment with different sALPs for days, weeks, months, or years. Alternatively, the sALPs administered to the subject may be alternated between any of SEQ ID NO:1-3. When treated with sALP of SEQ ID NO: 1, the subject may be administered a dose ranging from 1 mg / kg / week to 10 mg / kg / week, for example, from 3 mg / kg / week to 9 mg / kg / week (e.g., 1 mg / kg / week, 2 mg / kg / week, 3 mg / kg / week, 4 mg / kg / week, 5 mg / kg / week, 6 mg / kg / week, 7 mg / kg / week, 8 mg / kg / week, 9 mg / kg / week, or 10 mg / kg / week). For example, administration of sALP of SEQ ID NO: 1 may be 1 mg / kg six times a week, 2 mg / kg three times a week, or 3 mg / kg twice a week. In a specific embodiment, administration is 3 mg / kg three times a week, or increased from 3 mg / kg twice a week to 3 mg / kg three times a week. Vials are prepared at concentrations up to 100 mg / ml, such as 40 mg / ml or 100 mg / ml. When treated with sALP containing SEQ ID NO: 2 or 3, a dose of 20 mg, 35 mg, or 50 mg may be administered to the subject once every 1 or 2 weeks (e.g., once every 2 weeks). In some embodiments, the pharmaceutical composition containing sALP containing SEQ ID NO: 2 or 3 may be administered subcutaneously or intravenously. The composition may be administered in volumes of, for example, about 5 mL or less (e.g., 4.0 mL, 3.0 mL, 2.0 mL, 1.0 mL, 0.9 mL, 0.8 mL, 0.7 mL, 0.6 mL, 0.5 mL, 0.4 mL, 0.3 mL, 0.2 mL, or 0.1 mL, or in volumes ranging from about 5 mL to about 0.1 mL). sALP may be discontinued during administration of anti-sALP antibody therapy.For example, sALP administration may be discontinued for about 1, 2, 3, 4 weeks or longer (e.g., until the measure used to assess the effect of anti-sALP antibodies (e.g., the subject's RSS) improves) and / or until the level of anti-sALP antibodies in the subject (e.g., in the subject's biological fluids, such as blood, serum or urine) decreases (e.g., to an undetectable level).

[0016] In some embodiments, the second sALP treatment involves administration at a lower frequency than the treatment containing the first sALP. In some embodiments, the sALP is administered to the subject receiving sALP treatment at a dose of 1 mg / kg / week to 10 mg / kg / week, and the subject also receives treatment with a second sALP that is different from the first sALP, and the second sALP is administered every 2 weeks at a dose of 20 mg, 35 mg, or 50 mg.

[0017] In some implementations, the method described herein also includes monitoring antibody levels by detecting the presence of antibodies in biological samples (e.g., blood, plasma, or urine) from a subject.

[0018] The bioanalytical methods described herein may also include determining whether a subject has experienced a reduction in sALP efficacy. A reduction in efficacy can be determined, for example, by measuring a metric, wherein an increase or decrease in the metric relative to baseline indicates a reduction in sALP efficacy. For example, a reduction in efficacy can be determined by measuring one or more of the blood and / or urine levels of inorganic pyrophosphate (PPi) and pyridoxal 5'-phosphate (PLP) (e.g., relative to baseline). For example, PPi and / or PLP levels may be elevated relative to baseline (e.g., an increase of 25% or more, such as 30%, 35%, 40%, 45%, 50%, 55%, 60%, or more than 60%, relative to the concentrations of PPi and / or PLP in a plasma sample from the subject before the observed reduction in sALP efficacy).

[0019] Decreased efficacy can be determined, for example, by measuring one or more symptoms selected from a group consisting of: incomplete mineralization, hypercalciuria, skeletal deformities, waddling gait, bone pain, fractures, calcium pyrophosphate dihydrate crystal deposition, arthritis, pyrophosphate arthropathy, chondrocalcinosis, calcific periarthritis, pseudofractures, skeletal deformities, hypotonia, muscle weakness, rheumatoid complications, arthritis, pseudogout, dysmobility, pain, premature tooth loss, pulmonary hypoplasia, respiratory failure, seizures, body shape, growth, rickets, and immunogenicity. Subjects may exhibit a worsening of one or more of these symptoms relative to baseline (e.g., increased frequency or duration). This determination may include confirming that the subject does not exhibit poor treatment adherence or improper injection technique and / or does not have vitamin D deficiency, malnutrition, or comorbidities.

[0020] A decline in efficacy can be identified, for example, by conducting a quality of life assessment. The quality of life assessment can be selected from one or more of the following: the EuroQol 5-Dimensional Questionnaire, the Child Health Assessment Questionnaire, the Pediatric Outcomes Data Collection Scale, the Child Health Utility Index-9D, the Pediatric Quality of Life Scale, the Mini-Health Survey 36, and the Mini-Health Survey 12. Participants may show a decrease in their quality of life assessment score relative to baseline.

[0021] A decline in effectiveness can be identified through performance evaluation. Performance evaluation can be selected from one or more of the following: the Six-Minute Walk Test (6MWT), the Bunnier Test of Motor Skills, Version 2 (BOT-2), the Bailey Scales of Infant Development, Version 3 (BSID-III), gait analysis, use of a walking aid, the Peabody Developmental Motor Scale 2 (PDMS-2), or X-ray. Subjects may show a decline in performance evaluation scores relative to baseline.

[0022] In some implementations, baselines are calculated based on metrics in subjects before a decrease in sALP efficacy occurs, in reference subjects treated with sALP and in whom no neutralizing antibodies against sALP are detected, or in reference subjects not treated with sALP.

[0023] The method may also include determining whether the antibody is a neutralizing antibody (e.g., an anti-sALP antibody). The method may also include testing the effect of the neutralizing antibody on one or both of sALP catalytic activity and bone targeting in the presence of a neutralizing antibody. Subjects may exhibit reduced sALP catalytic activity and / or reduced bone targeting. The test may include one or more assays, such as screening assays, confirmatory assays, titration assays, and / or neutralizing antibody assays.

[0024] In any of the above-described embodiments, the subject may have a bone mineralization disorder, such as hypophospholipase syndrome (HPP). The subject may be an adolescent, adult, infant, or newborn. The subject may have a fracture, osteoporosis, sclerotic ossification, chondrocalcinosis, hypotonia, Duchenne muscular dystrophy, tracheobronchomalacia, seizures, neurofibromatosis 1 (NF-1), craniosynostosis, or a myasthenic disorder such as calcium pyrophosphate deposition (CPPD) or familial hypophosphatemia.

[0025] In some implementations, the subject has a defined immunogenic status, such as positive anti-drug antibody (ADA+), negative anti-drug antibody (ADA-), positive neutralizing antibody (NAb+), and / or negative neutralizing antibody (NAb-). Subjects can be ADA+ and Nab-.

[0026] definition

[0027] As used in this article, the term “about” refers to a value within ±10% of the reference value.

[0028] As used herein, when a polypeptide or nucleic acid sequence is said to have “at least X% sequence identity” with a reference sequence, where “X” is a real number, it means that when the sequence is in optimal alignment, at least X% of the amino acid residues or nucleotides in the polypeptide or nucleic acid are identical to those amino acid residues or nucleotides in the reference sequence. Optimal sequence alignment can be determined in various ways within the scope of the art, such as the Smith Waterman alignment algorithm (Smith et al., J. Mol. Biol. 147:195-7, 1981) and BLAST (Basic Local Alignment Search Tool; Altschul et al., J. Mol. Biol. 215: 403-10, 1990). These alignment algorithms, and others, can be computed using publicly available computer software, such as “Best Fit” incorporated into GeneMatcher Plus (Schwarz and Dayhoff, Atlas of Protein Sequence and Structure, Dayhoff, MO, Ed pp 353-358, 1979) (Smith and Waterman, Advances in Applied Mathematics, 482-489, 1981), BLAST, BLAST-2, BLAST-P, BLAST-N, BLAST-X, WU-BLAST-2, ALIGN, ALIGN-2, CLUSTAL, Megalign (DNASTAR), or other software / hardware for alignment. Furthermore, those skilled in the art can determine the appropriate parameters for measuring alignment, including any algorithms required to achieve optimal alignment in terms of the length of the sequences being compared.

[0029] The terms “patient” or “subject” refer to mammals, including but not limited to humans or non-human mammals such as cattle, horses, dogs, sheep, or cats. “Patient” specifically refers to humans.

[0030] The terms “sALP,” “soluble alkaline phosphatase,” and “extracellular domain of alkaline phosphatase” are used interchangeably (unless the context otherwise indicates) to refer to a soluble, non-membrane-bound alkaline phosphatase or a biologically active fragment or variant thereof. sALP includes, for example, alkaline phosphatases lacking a C-terminal GPI signaling sequence and additional variants and analogs that retain alkaline phosphatase activity (e.g., the ability to hydrolyze PPi or other natural or artificial substrates). Unless otherwise specified, this includes soluble fragments and biologically active fragments or variants corresponding to the extracellular domains of TNSALP, PALP, GLALP, and IALP. Mature sALP lacks both the GPI membrane anchor and the signal peptide, which is cleaved during processing. Attached Figure Description

[0031] Figure 1 This is an example flowchart of the determination of various implementation schemes as described in this article.

[0032] Figure 2 This is a schematic diagram illustrating the overall research design and treatment process of immunosuppressive therapy. The Treatment and Surveillance Committee (TMB) will consist of HPP clinical advisors, immunology experts, field investigators, and medical monitors (sponsor); TMB: Treatment and Surveillance Committee; MTX: Methotrexate; RTX: Rituximab; BTZ: Bortezomib; E: Enrollment; B: Baseline; EOS: Study End.

[0033] Figure 3 This is a schematic diagram illustrating the overall research design and treatment process of immunosuppressive therapy. Administer intravenous immunoglobulin (IVIG) 500 mg / kg once a month, while simultaneously receiving rituximab + / - bortezomib; Laboratory, radiography; TMB: Treatment and Monitoring Committee).

[0034] Figure 4 This is a schematic diagram of a decision tree that can be used to assess whether to continue, modify, or discontinue immunosuppressive therapy, such as one or more of methotrexate, rituximab, and / or proteasome inhibitors (e.g., bortezomib). Detailed Implementation

[0035] Subjects with bone mineralization disorders, such as hypophosphatase syndrome (HPP), may develop antibodies that bind to sALP while receiving enzyme replacement therapy (ERT) for soluble alkaline phosphatase (sALP). In some subjects, these antibodies are neutralizing antibodies and may reduce the efficacy of sALP administered during ERT. Therefore, methods for treating subjects with bone mineralization disorders, such as HPP, who develop neutralizing antibodies are disclosed. These treatments are referred to as immune tolerance induction (ITI), immune tolerance therapy (ITT), immune tolerance regimens (ITR), or immunosuppressive therapy (IST) (collectively, “IST”). IST methods involve reducing neutralizing antibody titers and / or preventing or reducing the formation of neutralizing antibodies.

[0036] The presence of neutralizing antibodies in subjects can be assessed using assays as described herein. Not all antibodies that bind to sALP therapeutic agents (such as asfortase alfa) reduce the efficacy of sALP in subjects receiving ERT (e.g., HPP subjects). For example, these antibodies may bind to domains of sALP (e.g., asfortase alfa), which may not affect its therapeutic function (e.g., its ability to promote bone mineralization). Therefore, as described herein, one or more additional assays can be used to determine whether these antibodies both neutralize and reduce the efficacy of sALP.

[0037] The method is further characterized by its ability to identify subjects receiving sALP ERT as requiring IST therapy by using one or more assays to determine the presence of anti-sALP antibodies and, where necessary, by using one or more assays to determine whether these antibodies have a neutralizing effect and reduce the efficacy of sALP. IST therapy can also be administered to treatment-naïve subjects who have not yet received sALP treatment, provided there is a reasonable expectation that such subjects will develop neutralizing anti-sALP antibodies. Furthermore, subjects may include those who may have had normal or non-elevated antibody titers prior to IST therapy but are still at risk of developing high or high neutralizing antibody titers in the future.

[0038] The methods described herein can be used to treat subjects receiving sALP therapy. Subjects may have bone mineralization disorders such as HPP, or may have fractures, osteoporosis, sclerotic ossification, chondrocalcinosis, hypotonia, Duchenne muscular dystrophy, tracheobronchomalacia, seizures, neurofibromatosis 1 (NF-1), craniosynostosis, or myasthenic disorders such as calcium pyrophosphate deposition (CPPD) or familial hypophosphatemia.

[0039] IST treatment agents

[0040] Helper T cells are crucial in the formation of most ADAs, while memory B cells and long-lived plasma cells amplify and maintain responses that form the basis for the use of combined IST when efficacy is lost due to the presence of ADA / NAbs. Therapeutic proteins, such as asforase α, may be endocytosed by antigen-presenting cells after administration and processed into their component peptides. A portion of these peptides can then be presented to helper T cells specific for that peptide in the context of human leukocyte antigen (HLA) molecules. The helper T cells signal and activate Ag-specific B cells, causing them to proliferate and differentiate into memory B cells and Ab-secreting plasma cells (including short-lived and long-lived types).

[0041] The ISTs described in this article include one or more of methotrexate, rituximab, and bortezomib, which can be used based on their effects on helper T cells, plasma cells, and memory B cells. Methotrexate is a dihydrofolate reductase inhibitor that affects rapidly dividing T cells and B cells by preventing the reduction of dihydrobiopterin (BH2) to tetrahydrobiopterin (BH4), leading to the uncoupling of nitric oxide synthase and increasing the sensitivity of T cells to apoptosis, thereby weakening the immune response.

[0042] Rituximab is a chimeric monoclonal antibody that targets the human B lymphocyte surface antigen (CD20). This transmembrane protein is present on almost all B cells and is downregulated from the stage of B cell directed development until B cells differentiate into antibody-secreting plasma cells. Rituximab causes rapid and almost complete depletion of peripheral mature B cells and pre-B cells; in most participants, these cells cease to appear within approximately 6 months. CD20 is lost when B cells differentiate into antibody-producing plasma cells. Therefore, once B cells differentiate into plasma cells, replacement and / or adjunctive agents must be used to address ADA produced by circulating plasma cells.

[0043] Bortezomib is a proteasome inhibitor. Proteasome inhibitors are a potent and specific way to target plasma cells, which are highly dependent on proteasome activity. Bortezomib is a reversible proteasome inhibitor that can target both short-lived and long-lived plasma cells simultaneously because these plasma cells have a high rate of immunoglobulin production.

[0044] This document describes a method for administering IST to a subject. In a particular implementation, IST is administered prophylactically to avoid generating an immune response, such as one, two, or more days (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or more) prior to sALP administration. In other implementations, IST is administered to the subject, for example, after it has been determined that the subject has anti-sALP antibodies that interfere with ERT. IST therapy may be administered to subjects who exhibit reduced or lost sALP efficacy or are at risk of reduced or lost efficacy after a diagnosis of anti-sALP antibodies in the subject and verification that the antibodies have a neutralizing effect. IST therapy involves reducing the presence or negative effects of neutralizing antibodies against sALP therapeutic agents (e.g., asforase alfa). Additionally, the safety and tolerability of IST may be assessed by the subject. This may optionally be achieved by administering a small starting dose before administering the full course of treatment. IST therapy may be discontinued if the initial IST therapy is unsafe and / or not tolerated by the subject.

[0045] IST therapy involves identifying appropriate cellular and molecular targets for tolerance-inducing therapies, such as the cellular and molecular mechanisms that generate neutralizing antibodies. Given the predominance of neutralizing antibodies of the IgG1 and IgG4 isotypes, the immunoglobulin response to protein therapeutics may originate from a classic T helper cell-dependent B cell mechanism. Therefore, appropriate cellular targets for tolerance induction include antigen-specific T and B cells, as well as antigen-presenting cells. The antibody response may also require T cell helper involvement after the initial response.

[0046] IST may include, for example, administering to a subject a therapy comprising a dihydrofolate reductase (DHFR) inhibitor and an anti-CD20 antibody or an antigen-binding fragment thereof, thereby reducing the amount of the antibody, reducing its formation, or attenuating its effect. The DHFR inhibitor may be, for example, methotrexate. The anti-CD20 antibody may be, for example, rituximab or an antigen-binding fragment thereof. The method may also include administering to the subject a proteasome inhibitor, such as bortezomib.

[0047] DHFR inhibitors can be methotrexate. Methotrexate can be administered every 5 to 10 days (e.g., every 5, 6, 7, 8, 9, or 10 days, or every 7 days). For example, methotrexate can be administered every 7 days. It can be given at a dose of approximately 10 mg / m². 2 Approximately 20 mg / m 2 (For example, 10 mg / m²) 2 11mg / m 2 12mg / m 2 13mg / m 2 14mg / m 2 15mg / m 2 16mg / m 217mg / m 2 18mg / m 2 19mg / m 2 Or 20mg / m 2 For example, approximately 15 mg / m³ 2 Methotrexate can be administered at a dose of 15 mg / m². For example, it can be administered at 15 mg / m². 2 Methotrexate was administered at the prescribed dosage.

[0048] The anti-CD20 antibody may be rituximab or its antigen-binding fragment. Rituximab or its antigen-binding fragment can be administered every 5 to 10 days (e.g., every 5, 6, 7, 8, 9, or 10 days, or, for example, every 7 days). For example, rituximab can be administered every 7 days. It can be administered at a dose of approximately 100 mg / m². 2 Approximately 500 mg / m 2 (For example, 100mg / m²) 2 125mg / m 2 150mg / m 2 175mg / m 2 200mg / m 2 225mg / m 2 250mg / m 2 275mg / m 2 300mg / m 2 325mg / m 2 350mg / m 2 375mg / m 2 400mg / m 2 425mg / m 2 450mg / m 2 475mg / m 2 Or 500mg / m 2 For example, approximately 375 mg / m³ 2 Rituximab or its antigen-binding fragment can be administered at a dose of 375 mg / m². For example, it can be administered at 375 mg / m². 2 Rituximab is administered at the prescribed dose. Rituximab or its antigen-binding fragment may be administered intravenously. In some embodiments, rituximab or its antigen-binding fragment may be discontinued while the subject continues to receive methotrexate and / or rituximab or its antigen-binding fragment.

[0049] IST therapy can be administered for a predetermined duration (e.g., 1 day, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 6 months, 12 months, 18 months, 24 months, or longer) depending on the observed reduction in sALP efficacy. Alternatively, IST therapy can continue until successful treatment or reduction of neutralizing antibody titers has been established (e.g., a reduction of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%). This article describes IST therapy, and IST therapy includes the administration of therapeutic agents, such as immunomodulators or optionally proteasome inhibitors.

[0050] One subject may be selected to receive IST treatment. The subject may begin receiving methotrexate and rituximab, optionally in combination with bortezomib, for an initial duration of, for example, 24 weeks. Following this initial duration (e.g., the 24-week period), the subject's treatment status and ADA or NAb status may be determined (using assessment metrics as described herein). sALP therapy may continue concurrently with IST or not. The subject may continue with subsequent treatment durations (e.g., selected durations, such as 24 weeks), and the subject's treatment status and ADA or NAb status may be assessed, for example, to determine the benefit or risk of continuing the IST treatment regimen.

[0051] Although undetectable anti-sALP antibody titers alone are sufficient to stop IST, a second measure can be used to determine whether and when to withdraw IST if no recovery of sALP ERT efficacy is observed when the subject's anti-sALP antibody titer is undetectable.

[0052] In some implementations, such as after a subject exhibits a decrease in anti-sALP antibody titers, the therapy may be discontinued.

[0053] Proteasome inhibitors

[0054] IST treatment may optionally include the administration of one or more proteasome inhibitors, such as bortezomib. Proteasome inhibitors can be used to induce plasma cell exhaustion. Proteasome inhibitors may optionally be administered in a certain amount and under such conditions to achieve a reduction in antibodies specific to sALP (e.g., neutralizing anti-sALP antibodies that reduce the efficacy of sALP ERT).

[0055] Proteasome inhibitors that can be used in the methods described herein are disclosed, for example, in U.S. Patent No. 7,531,526, the disclosure of which is incorporated herein by reference in its entirety. Examples of suitable proteasome inhibitors include, but are not limited to, the following compounds and their pharmaceutically acceptable salts and boronic esters: N-(4-morpholine)carbonyl-β-(1-naphthyl)-L-alanine-L-leucine boronic acid, N-(8-quinoline)sulfonyl-β-(1-naphthyl)-L-alanine-L-leucine boronic acid, N-(2-pyrazine)carbonyl-L-phenylalanine-L-leucine boronic acid, L-proline-L-leucine boronic acid, N-(2-quinoline)carbonyl-L-homophenylalanine-L-leucine boronic acid, N-(3-pyridine)carbonyl-L-phenylalanine-L-leucine boronic acid, N-(3-phenylpropionyl)-L-phenylalanine-L-leucine boronic acid, N-(4-morpholine)carbonyl-L-phenylalanine-L-leucine boronic acid, N-(4-morpholine)carbonyl-(O-benzyl)-L-tyrosine-L-leucine boronic acid, N-(4-morpholine)carbonyl-L-tyrosine-L-leucine boronic acid, and N-(4-morpholine)carbonyl-[O-(2-pyridylmethyl)]-L-tyrosine-L-leucine boronic acid.

[0056] Proteasome inhibitors can be used in vitro or in vivo. For example, proteasome inhibitors can be used in vitro in conjunction with assays to determine whether they are a safe and effective component of IST therapy. Furthermore, proteasome inhibitors can be used in vivo for IST therapy in subjects (e.g., humans) in need. Proteasome inhibitors can be administered via any number of known routes, including oral, intravenous, intramuscular, subcutaneous, intrathecal, local, and by infusion (Platt et al., U.S. Patent No. 4,510,130; Badalamente et al., Proc. Natl. Acad. Sci. USA 86:5983-5987, 1989; Staubli et al., Brain Research 444:153-158 (1988)), and are generally administered in combination with physiologically acceptable carriers (e.g., saline). The effective amount of inhibitor administered can be determined empirically and can be based on considerations such as the specific proteasome inhibitor used, the individual's condition, and the individual's body size and weight.

[0057] Proteasome inhibitors can be bortezomib (e.g., VELCADE) ® ), namely, the monomeric boric acid [(1R)-3-methyl-1-[[(2S)-1-oxo-3-phenyl-2-[(pyrazinylcarbonyl)amino]propyl]amino]butyl]boronic acid with the following structure:

[0058]

[0059] Bortezomib can be administered every 5 to 10 days (e.g., every 5, 6, 7, 8, 9, or 10 days, or every 7 days). For example, bortezomib can be administered every 7 days. It can be administered at a dose of approximately 0.2 mg / m². 2 Approximately 2 mg / m 2 (For example, 0.2 mg / m²) 2 0.3 mg / m 2 0.4 mg / m 2 0.5 mg / m 2 0.6 mg / m 2 0.7 mg / m 2 0.8 mg / m 2 0.9 mg / m 2 1mg / m 2 1.1 mg / m 2 1.2 mg / m 2 1.3 mg / m 2 1.4 mg / m 2 1.5mg / m 2 1.6 mg / m 2 1.7 mg / m 2 1.8 mg / m 2 1.9 mg / m 2 Or 2mg / m 2 For example, it can be administered at approximately 0.5 mg / m³. 2 Approximately 0.7 mg / m 2 Approximately 1 mg / m 2 Or approximately 1.3 mg / m 2 Bortezomib can be administered at the recommended dose. It can be administered intravenously or subcutaneously. Intravenous administration of bortezomib may be given as a bolus injection. Bortezomib may be administered after a subject demonstrates a worsening of their rickets severity score (RSS) by 1 or more points relative to baseline. Bortezomib may be discontinued if the subject continues to receive methotrexate and / or rituximab.

[0060] Other proteasome inhibitors suitable for the treatments described herein include those currently being evaluated in preclinical and clinical trials for the treatment of a variety of conditions, such as MLN4924, NEDD-8 inhibitors, or E3 ligase inhibitors. Combinations of proteasome inhibitors belonging to the same or different classes or subclasses may be administered (e.g., a combination of two, three, four, or more proteasome inhibitors).

[0061] The optimal dosing regimen for proteasome inhibitors can be determined by those skilled in the art and may vary depending on the proteasome inhibitor, the subject, and the desired effect. For example, it may be administered at approximately 0.01 mg / m². 2 Approximately 200 mg / m 2For example, approximately 0.01 mg / m³ 2 Approximately 2 mg / m 2 For example, approximately 0.2 mg / m³ 2 Approximately 2 mg / m 2 Inhibitors can be administered intravenously in doses and various dosing schedules may be used, which may involve, for example, dosing twice weekly. It can also be administered at a typical range of about 0.01 mg / m². 2 Approximately 200 mg / m 2 For example, approximately 0.01 mg / m³ 2 Approximately 2 mg / m 2 For example, approximately 0.6 mg / m³ 2 Approximately 1.5 mg / m² 2 The optimal dose of a proteasome inhibitor should be administered. The optimal dose of a proteasome inhibitor should take into account factors such as the desired dosing schedule and the bioavailability of the drug at a given route of administration, and should be guided by the subject's clinical and laboratory parameters at any given time.

[0062] Considerations regarding antibody titers are described, for example, in the minutes of a meeting dated July 15, 1999, entitled “Immune Reactions Against Therapeutic and Diagnostic Biological Products,” where the FDA Biological Response Modifiers Advisory Committee stated that “the most important criterion for assessing the importance of antibodies against therapeutic proteins is the correlation between antibody incidence and quantity and clinical pharmacokinetics, pharmacodynamics, efficacy, and safety” (reference: Center for Biologics Evaluation and Review (fda.gov / ohrms / dockets / ac / cber99.htm)). Qualitatively, a “high titer” can be described as a level at which clinical decline typically occurs in the subject. It should be noted that, for example, not 100% of subjects receiving sALPERT will develop a “high antibody titer,” but for any given disease or symptom, a subset of subjects may have, or could potentially develop, high titers that affect the efficacy of sALP. Assays performed to detect antibodies (neutralizing or non-neutralizing) can sometimes have limitations, potentially leading to undetectable or underestimated antibody titers. In such cases, even undetectable or low titers can still result in reduced sALP efficacy during sALP ERT (such as ERT using asfortase α). While undetectable anti-sALP antibody titers alone are sufficient to stop IST, a second metric can be used to determine whether and when to withdraw IST if no recovery of sALP ERT efficacy is observed when the subject's anti-sALP antibody titer is undetectable.

[0063] Proteasome inhibitors (such as bortezomib) can cause adverse side effects, such as hematologic or non-hematologic toxicities. For example, bortezomib can cause neutropenia (low platelet count), cardiotoxicity, neuropathic pain, peripheral neuropathy, and / or liver dysfunction. In the event of one or more of these side effects, the dose of bortezomib may be reduced (e.g., by 25% or more, for example, from 1.3 mg / m²). 2 Reduced to 1 mg / m 2 or from 1 mg / m 2 Reduced to 0.7 mg / m³ 2 If the risks outweigh the benefits, bortezomib may be discontinued. For example, if a subject develops heart failure (e.g., based on echocardiographic findings or elevated troponin levels), bortezomib should not be continued.

[0064] Other immunomodulators and combination therapies

[0065] During IST therapy, other immunomodulatory agents may be administered to the subject. These agents may be administered alone or optionally in combination with proteasome inhibitors (such as those described above), administered at, before, or after the administration of the proteasome inhibitor to reduce / prevent antibody titer formation. Examples of immunomodulatory agents include, but are not limited to, belimumab, anti-CD3 antibodies, anti-CD19 antibodies, and anti-CD22 antibodies, corticosteroids (e.g., prednisolone), rapamycin, methotrexate, WIG, cyclophosphamide, cyclosporine A, azathioprine, and mycophenolate mofetil, and their derivatives. These immunomodulatory agents or their derivatives include agents that target / alter antigen presentation and / or humoral or cell-mediated immune responses.

[0066] IST therapies can be used that trigger the expression of inhibitory FcRs on B cells and antigen-presenting cells and target B cell survival and activating factors such as B cell activating factors and B lymphocyte stimulating factors. The depletion approach using rituximab (a chimeric monoclonal antibody with a constant human IgG1 domain that depletes mature B cells expressing the CD20 molecule) may be particularly useful for prophylaxis, potentially allowing the introduction of the enzyme at a stage where immature, enzyme-specific pre-B cells and pro-B cells may be cleared or become unresponsive.

[0067] A combination of rituximab and B-cell activating factor antibody may also be used. Treatment methods for IST following ERT are described in, for example, U.S. Patent Nos. 8,809,282, 9,050,333, 9,592,247, 9,850,474, and 10,028,993, the disclosures of which are incorporated herein by reference in their entirety.

[0068] IST therapy may also include immunoadsorption alone or in combination with a therapeutic agent (e.g., rituximab). Immunoadsorbents (such as THERASORB) can be used. ® Immunosorbents can be used to deplete neutralizing antibodies. They can be applied, for example, daily, weekly, bi-weekly, or monthly. Immunosorbents can be applied for a duration of at least one month (e.g., at least two, three, four, five, six, twelve, eighteen, twenty-four months, or more).

[0069] Immunoglobulins can also be administered as part of IST therapy (e.g., at doses of approximately 100 mg / kg, 200 mg / kg, 300 mg / kg, 400 mg / kg, 500 mg / kg, 600 mg / kg, 700 mg / kg, 800 mg / kg, 900 mg / kg, or 1000 mg / kg) alone or in combination with other agents. Gamma globulin can also be administered as part of IST therapy.

[0070] Immunoglobulin can be administered every 5 to 10 days (e.g., every 5, 6, 7, 8, 9, or 10 days, or every 7 days). For example, immunoglobulin can be administered every 7 days. Immunoglobulin can be administered at a dose of about 300 mg / kg to about 700 mg / kg (e.g., about 300 mg / kg, 350 mg / kg, 400 mg / kg, 450 mg / kg, 500 mg / kg, 550 mg / kg, 600 mg / kg, 650 mg / kg, or 700 mg / kg, or about 500 mg / kg). For example, immunoglobulin can be administered at a dose of about 500 mg / kg. Immunoglobulin can be administered intravenously. Immunoglobulin can be administered intravenously once a month at a dose of about 500 mg / kg, concurrently with rituximab, with or without bortezomib. Immunoglobulin can be administered for a duration of at least 1 month (e.g., at least 2 months, 3 months, 4 months, 5 months, 6 months, 12 months, 18 months, 24 months or more).

[0071] In some specific implementations, rituximab may be administered during IST treatment. Rituximab may be administered alone or as a combination therapy with methotrexate. The two treatment agents may be administered substantially simultaneously or sequentially. IST treatment, including rituximab or a combination of rituximab and methotrexate, may be administered for a period of approximately one month. During this treatment, sALP ERT may be discontinued or may be continued during IST treatment. Rituximab may also be used in combination with immunoglobulins, gamma globulins, and / or immunoadsorption (e.g., THERASORB). ® Simultaneous administration. In some specific embodiments, the therapy may include one or more therapeutic agents administered alone or in combination (e.g., simultaneously). One or more therapeutic agents (e.g., any of the therapeutic agents described herein) may be administered as a single composition mixed together or as a single composition. The various combination therapies described herein may be administered as a first course of treatment (e.g., rituximab, immunoglobulin, and an immunoadsorbent) or a second subsequent course of treatment (e.g., rituximab, immunoglobulin, dexamethasone, and an immunoadsorbent, and optionally a proteasome inhibitor). In some embodiments, rituximab, immunoglobulin, dexamethasone, and an immunoadsorbent, and optionally a proteasome inhibitor, are administered as a first course of treatment.

[0072] This therapy may also include the administration of folic acid. Folic acid may be administered, for example, once daily. It may be administered at doses of approximately 0.1 mg to approximately 10 mg, such as approximately 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, or 10 mg, such as approximately 1 mg. Folic acid may be administered on a different day than the day methotrexate is administered. Folic acid can be taken orally. Methotrexate is a folic acid antagonist; therefore, in this particular implementation, folic acid is administered as a supplement.

[0073] Termination of IST

[0074] Following IST therapy, the reduction in the presence of neutralizing antibodies in the subject can be monitored. If IST therapy has not resulted in improvement in the treated subject, the subject may still experience worsening or reduced relief of symptoms of bone mineralization disorders (e.g., HPP) and / or persistent neutralizing antibodies that reduce sALP efficacy. In such cases, the dose of the IST treatment agent may be increased (e.g., the dose of one or more IST agents may be changed, for example, if the subject is receiving a combination of agents as IST therapy), the administered IST agent may be changed, or a different IST therapy may be administered to the subject until the presence of neutralizing antibodies is reduced and / or sALP efficacy is restored. sALP ERT may be discontinued during IST therapy, or it may be continued during IST treatment. For example, IST therapy can last for one week or more, one month or more, or one year or more (e.g., 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 3 years, or longer), during which sALPERT is terminated for all or part of that time period. Repeatable assays (such as those described above) can be performed to assess the presence and / or titer of neutralizing antibodies after IST therapy. sALPERT can be resumed upon successful completion of IST therapy.

[0075] Evaluation of therapeutic efficacy

[0076] The following describes assays used to assess the health status of subjects receiving sALP ERT. If the assays show a decline in the subject's health status, the subject may be further tested for the presence of neutralizing anti-sALP antibodies. The onset of signs of a decreased response to sALP ERT in a subject may indicate the need to test for neutralizing antibodies. If a decrease in sALP efficacy is suspected, the subject may be tested for neutralizing antibodies. This test is particularly useful in subjects who have received sALP treatment and have also been observed to have improved symptoms of bone mineralization disorders (e.g., HPP) over time periods such as approximately 3 months, 6 months, 1 year, or longer (e.g., 2, 3, 4, 5, 6, 7, 8 years, or more). Subjects may be tested to determine whether neutralizing antibodies (if present) are contributing to the decrease in sALP efficacy during ERT, for example using the assays described herein. If a subject is found to have neutralizing antibodies that decrease sALP efficacy during ERT, the IST therapy procedure may be initiated.

[0077] Several metrics can be used to evaluate the therapeutic efficacy of sALP administered to subjects during ERT. These metrics can also be used as benchmarks to assess whether subjects with neutralizing antibodies experience a loss of sALP efficacy due to neutralizing antibodies. Furthermore, these metrics can be used in conjunction with IST therapy to monitor changes in sALP efficacy (e.g., recovery of sALP efficacy). Specifically, these metrics can be used in conjunction with neutralizing antibody assays as described herein, both before and after IST therapy to indicate whether IST therapy is needed. For example, these metrics can be used to measure a decrease in sALP efficacy (e.g., a decrease in efficacy of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%), thereby indicating the need for IST therapy. A decrease in efficacy can be measured relative to: baseline (e.g., a subject's value in one or more metrics prior to any decrease in efficacy, such as during a predetermined time period after the initiation of sALP ERT, e.g., 1 month, 3 months, 6 months, 1 year, 2 years, or longer after the initiation of sALP ERT), the mean of reference subjects with bone mineralization disorders (e.g., HPP) receiving sALP ERT, or reference subjects who have received sALP ERT. A decrease in sALP efficacy exceeding a predetermined threshold (e.g., a decrease in efficacy of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, e.g., relative to the time prior to the decrease in efficacy) based on one or more of the metrics described herein may indicate the need for IST therapy. When IST therapy is determined to be needed to treat reduced sALP efficacy due to the presence of neutralizing antibodies, one or more of these measures may also be used to track the recovery or improvement of sALP efficacy (e.g., an improvement of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% relative to, for example, the time after the observed efficacy reduction but before the initiation of IST therapy). The improvement in efficacy may be measured relative to: baseline (e.g., the subject's value before the improvement or recovery of efficacy), the mean of reference subjects with bone mineralization disorders (e.g., HPP), or reference subjects who experienced reduced sALP efficacy during ERT or had received IST therapy. IST therapy may be discontinued and considered successful when a subject's efficacy improvement after IST therapy exceeds a predetermined threshold (e.g., an improvement of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, for example, relative to the time after the observed efficacy reduction but before the initiation of IST therapy) or when the subject demonstrates a recovery in sALP ERT efficacy levels relative to the time before the efficacy reduction.

[0078] Exemplary measures that can be used to evaluate the efficacy of treatment with sALP ERT (e.g., asforase alfa) are bone healing, bone mineralization, bone mineral density, and plasma PPi and / or PLP concentrations. These methods may include using one or more of the described measures (e.g., bone healing, mineralization, bone mineral density, or plasma PPi and / or PLP concentrations), alone or in combination, to assess a reduction in sALP efficacy that may indicate that neutralizing antibodies are suppressing a subject's response to sALP ERT. Alternatively, these methods may be used to demonstrate that sALP treatment efficacy has been restored after IST therapy.

[0079] Bone healing and mineralization

[0080] Decreased bone healing and mineralization can be used as a measure to diagnose whether a patient has or may have neutralizing antibodies that bind to sALP and reduce its efficacy. In subjects with bone mineralization disorders (e.g., HPP) who have not yet developed neutralizing anti-sALP antibodies, administration of sALP (e.g., asforase alfa) can improve bone healing after successful treatment. Therefore, decreased bone healing and mineralization in subjects with bone mineralization disorders (e.g., HPP) after sALP treatment may be due to the presence of neutralizing antibodies.

[0081] Decreased bone healing leads to bone loss and may be accompanied by decreased mineralization, resulting in nonunion of two or more bones. Decreased bone healing and mineralization can be compared to reference bone (e.g., bone of a healthy subject (e.g., without bone mineralization disorders) or bone of a subject without anti-sALP antibodies). Methods used to identify decreased bone healing and mineralization are routine and include non-invasive techniques such as radiographic imaging and computed tomography (CT). Typically, images of relevant areas of the subject are taken at one or more time points before and after sALP treatment, and the images can be compared to assess treatment efficacy. Decreased bone healing and / or mineralization can be identified as reduced 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 decreased efficacy is suspected. Decreased bone healing and / or mineralization in subjects may not be detectable for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks or months after the start of sALP ERT treatment. In some cases, decreased bone healing and / or mineralization may 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. Therefore, decreased bone healing and mineralization after sALP treatment may trigger the need for IST therapy to treat neutralizing antibodies and restore sALP efficacy. Following IST therapy, the same metrics can be used to monitor the recovery of sALP efficacy. In this case, improved bone mineralization and healing can be used to determine that sALP efficacy has been restored and that bone mineralization disorders (e.g., HPP) have been effectively treated with sALP ERT after IST therapy.

[0082] Bone mineral density (BMD)

[0083] A decrease in BMD can be used as a measure to diagnose whether a patient has or may have neutralizing antibodies that bind to sALP and reduce its efficacy. In subjects with bone mineralization disorders (e.g., HPP) who have not yet developed neutralizing anti-sALP antibodies, administration of sALP (e.g., asforase alfa) can lead to an increase in BMD after successful treatment. Therefore, a decrease in BMD in subjects with bone mineralization disorders (e.g., HPP) after sALP treatment may be due to the presence of neutralizing antibodies.

[0084] A decrease in BMD can be used to monitor the efficacy of sALP (e.g., asforazyme A) during ERT. Methods for measuring BMD are known in the art and include, for example, bone biopsy, dual-energy X-ray absorptiometry (DXA or DEXA), peripheral quantitative CT (pQCT), high-resolution pQCT (HR-pQCT), and quantitative ultrasound (QUS). Measurements can be performed using any routine method, including CT Henle 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. The BMD value at the post-treatment reference point may decrease by, for example, 0.01%, 0.05%, 0.1%, 0.5%, or 1%. The decrease in BMD value at the reference point after the start of sALP treatment may also remain unchanged, or the change may be undetectable. In some cases, a decrease in BMD in a subject may persist for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks, months, or years after the initiation of sALP ERT treatment. Therefore, a decrease in BMD following sALP treatment may trigger the need for IST therapy to treat the neutralizing antibodies and restore sALP efficacy. Following IST therapy, the same metric can be used to monitor the recovery of sALP efficacy. In this case, an increase in BMD (such as an increase of, for example, about 0.01%, 0.05%, 0.1%, 0.5%, or 1% or more) can be used to determine that sALP efficacy has been restored after IST therapy and that bone mineralization disorders (e.g., HPP) are being effectively treated.

[0085] PPi and PLP levels

[0086] Elevated levels of alkaline phosphatase substrates (such as PPi and / or PLP) can be used as a measure to diagnose whether a patient has or may have neutralizing antibodies that bind to sALP and reduce its efficacy. In subjects with bone mineralization disorders (e.g., HPP) who have not yet developed neutralizing anti-sALP antibodies, administration of sALP (e.g., asforase alpha) will result in a decrease in PPi and / or PLP after successful treatment because sALP has enzymatic activity over these substrates. Therefore, the elevation of PPi and / or PLP in subjects with bone mineralization disorders (e.g., HPP) after sALP treatment may be due to the presence of neutralizing antibodies that reduce the catalytic activity of sALP, leading to excessive accumulation of these substrates.

[0087] Elevated amounts or concentrations of substrates (such as PPi or PLP) can be used to monitor the efficacy of sALP (e.g., asforase α) before and during ERT treatment, to monitor for a decrease in sALP efficacy during ERT. Normal and abnormal concentrations of these substrates are described, for example, in PCT Publications WO 2016 / 123342 and WO 2017 / 171871, the disclosures of which are incorporated herein by reference in their entirety.

[0088] The average increase in PPi concentration in plasma samples from subjects receiving ERT using sALP (e.g., asforamzyme alfa) relative to the concentration observed before a decrease in sALP efficacy was observed can be about 25% or more (e.g., 30%, 35%, 40%, 45%, 50%, 55%, 60%, or more than 60%). In some cases, the average increase in PPi concentration in plasma samples from subjects can persist for at least one week, six months, or up to one year or longer during treatment. This increase can persist throughout the duration of treatment with sALP (e.g., asforamzyme alfa). If the average PPi concentration, based on typical values ​​during sALP ERT treatment, increases above a predetermined threshold (e.g., an increase of 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or more), IST therapy can be determined to be necessary. Conversely, if the average PPi concentration decreases below a predetermined threshold (e.g., a decrease of 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60% or more) or to a normal level compared to a reference subject (e.g., a subject who has successfully received IST therapy), it can be determined that IST therapy is no longer needed or can be discontinued.

[0089] Elevated PLP levels can be used to monitor the efficacy of sALP (e.g., asforamase alfa) before and during ERT treatment to monitor for a decrease in sALP efficacy during ERT. The mean increase in PLP concentration in plasma samples from subjects receiving ERT using sALP (e.g., asforamase alfa) relative to the concentration in plasma samples from which a decrease in sALP efficacy was observed can be approximately 50% or greater (e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%). In some cases, the mean increase in PLP concentration in plasma samples from subjects can persist for at least one week and up to one year or longer during treatment. This increase can persist throughout the duration of treatment with sALP (e.g., asforamase alfa). If the mean PLP concentration, based on the value during sALP ERT treatment, increases above a predetermined threshold (e.g., an increase of 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or more), then IST therapy is deemed necessary. Conversely, if, after IST therapy, the mean PLP concentration decreases below a predetermined threshold (e.g., a decrease of 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or more) or returns to normal PLP levels (e.g., compared to a reference subject or a subject who has successfully received IST therapy), this indicates that IST therapy is no longer necessary and can be discontinued.

[0090] Any method known to those skilled in the art can be used to quantify the concentrations of PPi and PLP in plasma samples or alternatively, urine samples, for example, as Whyte et al. (JJ. Clin Invest. 95(4): 1440-1445, The literature described in detail in 1995 is incorporated herein by reference in its entirety. Specifically, the concentrations of PPi and PLP in plasma samples can be used to evaluate sALP activity in subjects. Subjects with HPP typically exhibit elevated plasma PPi and PLP concentrations compared to healthy subjects, such as PPi concentrations of approximately 5 μM or higher and / or PLP concentrations of approximately 50 ng / ml or higher. The lower limit of normal for plasma PPi concentration in healthy adults is approximately 1 μM, while the upper limit is approximately 5.9 μM. The lower limit of normal for plasma PPi concentration in adolescent adults is approximately 0.8 μM, while the upper limit is approximately 4.9 μM. The lower limit of normal for plasma PLP concentration in healthy adults is less than approximately 10 ng / ml, while the upper limit is approximately 60 ng / ml. The lower limit of normal for plasma PLP concentration in adolescent adults is less than approximately 10 ng / ml, while the upper limit is less than approximately 25 ng / ml. Therefore, these boundaries of normal PPi and PLP concentrations can be used as predetermined thresholds to trigger the initiation or termination of IST therapy.

[0091] Physical measurements and quality of life assessment

[0092] A decrease in scores from performance or quality of life assessments can be used as a measure to diagnose whether a patient has or may have neutralizing antibodies that bind to sALP and reduce its efficacy. In subjects with bone mineralization disorders (e.g., HPP), administration of sALP (e.g., asforase alfa) leads to improved scores after successful sALP treatment (e.g., associated with positive performance metrics or higher quality of life). Therefore, a decrease in scores in subjects with bone mineralization disorders (e.g., HPP) after sALP treatment (e.g., associated with negative performance metrics or lower quality of life) may be due to the presence of neutralizing antibodies that reduce the efficacy of sALP.

[0093] Physical fitness and quality of life measures can be used to measure the efficacy of neutralizing sALP antibodies during ERT (e.g., sALP, such as asforazyme A) and / or IST treatment. For example, a negative assessment using any of the following measures may indicate that the efficacy of sALP has decreased during ERT and that IST treatment may be necessary. Conversely, a positive assessment using any of the following measures after IST treatment may indicate that IST treatment has been successful and that IST treatment can be discontinued. Non-limiting examples of physical fitness measures that can be used to evaluate the efficacy of treatment with sALP (e.g., SEQ ID NO: 1) are, for example, the Six-Minute Walk Test (6MWT), the Bunie Test of Motor Skills Version 2 (BOT-2), the Bayley Scales of Infant Development Version 3 (BSID-III), and gait analysis. Additional tests and measures may include the Tinetti Performance-Oriented Action Assessment (POMA; optional modification) and spatiotemporal gait analysis software and hardware, such as GAITRITE. ®(Clinical Image Retrieval System Inc.). Exemplary quality of life assessments include the EuroQol 5D Questionnaire (EQ-5D), the Child Health Assessment Questionnaire (CHAQ), the Pediatric Outcomes Data Collection Scale (PODCI), the Child Health Utility Index-9D (CHU-9D), the Pediatric Quality of Life Scale (PedsQL), the Short Form Health Survey 36 (SF-36), and the Short Form Health Survey 12 (SF-12). The methods described herein may include using one or more of the aforementioned measures or assessments, alone or in combination, to evaluate the therapeutic efficacy of sALP (e.g., asforase α) in subjects with bone mineralization disorders such as HPP, wherein improvement relative to a specific score or value indicates that sALP is effective in treating HPP. Furthermore, the activity level of sALP in samples (e.g., plasma samples) from subjects with HPP may also be used alone or in combination with one or each of these measures to evaluate the therapeutic efficacy of sALP (e.g., sALP of any of SEQ ID NO: 1-3) in subjects. Measurements used to measure physical fitness and assess quality of life associated with bone mineralization disorders such as HPP are described, for example, in PCT applications PCT / US2018 / 025206 and PCT / US2018 / 026868, the disclosures of which are incorporated herein by reference in their entirety. These measurements can be used to monitor the efficacy or changes in efficacy of sALP during ERT and before, during, or after IST therapy, and can therefore be used to guide the initiation or termination of IST therapy.

[0094] Assay for detecting and characterizing neutralizing antibodies

[0095] Following the identification of reduced sALP efficacy during ERT, subjects may undergo further testing to detect and characterize the presence of neutralizing antibodies. For example, the presence and / or titer of neutralizing antibodies against sALP (such as asfortase α or other alkaline phosphatases as described herein) may be tested in the subject's sample (e.g., blood, serum). Various assays may be used to characterize antibodies, such as screening assays, confirmatory assays, titration assays, and / or neutralizing antibody assays. Sensitive screening assays for binding antibodies may be used as a first step. Furthermore, antibody levels or titers may be measured relative to controls or baseline values, such as the amount of antibody before the observed reduction in efficacy or the amount of antibody in a reference subject with bone mineralization disorders (e.g., HPP) but without reduced sALP (e.g., asfortase α) efficacy. A confirmatory assay may then be performed after this step to confirm that the positive response is antibody-mediated. The neutralizing activity of the antibody in positive samples may then be tested in enzyme activity and bone targeting assays, and antibody titers and isotypes may be further evaluated.

[0096] Specifically, the assessment of the immune response to sALP (e.g., asforase α or other alkaline phosphatases described herein) may involve one or more immunoassays to detect the sensitivity, specificity, and / or robustness of antibody / therapeutic agent interactions. Given the complex biology of sALP ERT, the assessment of antibody neutralization may particularly involve developing at least two types of neutralizing antibody assays: one to assess the neutralizing effect of enzymes targeting bone (e.g., osteoblasts present in the mineral phase of bone), and another to measure the ability of neutralizing antibodies to inhibit the catalytic activity of sALP. These distinct functions are mediated by different regions in sALP fusion proteins (e.g., asforase α or other alkaline phosphatases described herein). For example, the targeting of asforase α to osteoblasts (e.g., the hydroxyapatite mineral phase of bone) is mediated by a 10-residue polyaspartic acid region, while enzyme activity (including substrate binding and catalysis) is mediated by the sALP region. Therefore, antibodies that neutralize sALP efficacy during ERT may be specific for one or two of the various functional domains of the sALP fusion protein (e.g., the enzymatic portion and / or the bone-targeting portion). Antibodies targeting other conserved elements of alkaline phosphatases (such as asforase α) (e.g., the Fc domain) may not be neutralizing, but when present at high titers, they may inhibit efficacy by diverting the enzyme to a more restricted cell population (e.g., to cells carrying Fc receptors or FcRs) or by altering pharmacokinetics. Assays of antibodies targeting neutralizing or catalytic activity can be performed side-by-side using the same subject sample or duplicates of the subject sample, and the results can be analyzed in conjunction with information about the subject's clinical outcomes.

[0097] The neutralization of catalytic activity can be assessed by mixing serially diluted subject serum into a validated potency assay that measures the activity of sALP against a specific substrate (e.g., PPi). This assay can be validated according to recommended standards. An antibody is determined to interfere with the efficacy of sALP if it is present in sufficient titers to block targeting of osteocytes, significantly alters the pharmacokinetics of sALP, or inactivates sALP. Furthermore, antibody binding to sALP can alter enzyme conformation, making sALP susceptible to proteolytic degradation. Assays for monitoring the bone-targeting and catalytic activity of sALP are disclosed, for example, in PCT Publication WO 2005 / 103263, the entire disclosure of which is incorporated herein by reference.

[0098] One or more of these assays can be used to monitor the efficacy or changes in efficacy of sALP during, before, during, or after IST therapy, and thus can be used to guide the initiation or termination of IST therapy.

[0099] ADA assessment via a multi-layered approach

[0100] In specific embodiments, the development of anti-drug antibody (ADA) assays is at least partially based on a multi-tiered approach. For example, the first tier may include a screening assay, and the second tier may include a confirmatory assay. Screened and confirmed positive samples may then be further characterized in titration and neutralizing antibody assays. In specific embodiments, a screening assay, a confirmatory assay, a titration assay, and a neutralization assay are performed. In other embodiments, at least two or at least three of the screening assay, confirmatory assay, titration assay, and neutralization assay are performed.

[0101] The assays described herein may include electrochemiluminescence (ECL) bridging assays, which may include a sample acidification step to dissociate the drug-antibody complex and prolong drug tolerance by allowing unbound anti-drug antibodies to compete freely in the assay. Positive controls may be used, such as polyclonal antibodies purified from rabbits hyperimmunized with sALP (e.g., asforase α). ADA assays may use an anti-sALP antibody bridge with a fixed antigen and a reporter antigen. The fixed antigen may be biotin-labeled sALP (B-sALP), and the reporter antigen may be ruthenium-labeled sALP (Ru-sALP). The measurement output can then be generated by the chemiluminescent signal produced by the electrical stimulation of the ruthenium-labeled sALP when captured by a streptavidin plate.

[0102] Screening assay

[0103] Screening assays (also known as binding antibody assays) can be used to detect antibodies that bind to therapeutic protein products. These assays can be deliberately designed to be sensitive enough to detect both low-affinity and high-affinity ADA in clinical samples. Binding specificity of samples that test positive in the screening assay is further evaluated in a second-tier confirmatory assay.

[0104] The cutoff point for a screening assay can be a response threshold in the assay, defining whether a given sample responds positively or negatively. The cutoff point for this assay can be affected by numerous interfering products or matrix components. These components are considered early in assay development when defining the cutoff point. In a specific embodiment, approximately 25 to approximately 50 individual samples are used to estimate the cutoff point. In a specific embodiment, the initial screening assay has a low but well-defined false positive rate of approximately 5% to maximize the detection of true positives. In a specific embodiment, the well-defined false positive rate is approximately 1% to approximately 5%, or less than approximately 5%, less than approximately 3%, or less than approximately 1%. Because samples from different target populations and disease states may contain components that can cause variations in the background signal from the assay, different cutoff points may be required for individual target patient populations.

[0105] In a specific implementation, a cutoff point is determined statistically using samples from treatment-naïve subjects. The variability of the assay can be estimated by performing repeated assay runs (such as 2, 3, 4, or 5 runs) with these samples. Statistical methods for determining the cutoff point may include removing statistical outliers and taking into account pre-existing antibodies. Each sample may be tested in an ADA screening assay by at least two analysts on at least three different days, for a total of at least six individual measurements. One method allowing for a higher assurance of a 5% false positive rate is to apply a 90% one-sided confidence interval lower limit to the 95th percentile of the negative control group. This ensures a false positive rate of at least 5% at a 90% confidence level. This method increases the probability that the assay identifies all subjects who may have produced antibodies. Statistical methods for determining the cutoff point may be based on the statistical distribution of the data. For example, in a specific implementation, the 95th percentile of a normal distribution is estimated by the mean plus a standard deviation of 1.645.

[0106] In a specific implementation plan, when determining the true incidence of immunogenicity, subsequent confirmatory assays or other assays can be used to exclude false positive results.

[0107] Confirmatory determination

[0108] The confirmatory assay used in this specific implementation is a competitive assay that confirms the specific binding of ADA to the therapeutic protein of interest. The purpose of this assay is to eliminate potential false positives from the initial screening assay. ADA-confirmed samples can be further characterized in subsequent titer and neutralization assays.

[0109] Confirmatory assays can take the form of competitive assays, where competing substances, such as unlabeled therapeutic protein products, are used. In specific implementation plans, implementing appropriate confirmatory assays ensures that data from false-positive ADA subjects do not interfere with the analysis of the impact of ADA on safety and efficacy.

[0110] An exemplary method for determining a cutoff point in a confirmatory assay uses data from signals generated from antibody-negative treatment-naïve subjects in the presence of a competing drug. In this case, the amount of the therapeutic protein product can be used to establish the cutoff point. In a specific embodiment, the amount of the therapeutic protein product used in this assay is the same as the amount of the competitive inhibitor.

[0111] In a specific implementation of the confirmatory assay, sALP (e.g., 100 μg / mL, 150 μg / mL, 200 μg / mL, 250 μg / mL, 300 μg / mL, 350 μg / mL, 400 μg / mL, 450 μg / mL, or 500 μg / mL) is used as a competitive inhibitor of the binding of B-sALP and Ru-sALP to ADA. After the incubation period, the B-sALP / ADA / Ru-sALP complex captured by the streptavidin plate can be measured, for example, using an MSD Sector Imager 6000 plate reader. In a specific implementation, the percentage of inhibition by the sALP assay inhibitor for each sample can be calculated as follows:

[0112]

[0113] In a specific implementation of the confirmatory assay, the cutoff point can be determined through statistical analysis of data from treatment-naïve normal donors measured in the presence and absence of free sALP. The confirmatory assay cutoff point (e.g., 5%, 10%, 15%, 20%, 25%, or 30% inhibition) can be selected using, for example, the 99.9th upper percentile of the statistical analysis. If the percentage of inhibition is equal to or greater than the inhibitory cutoff point for the confirmatory assay, the sample can be confirmed as anti-sALP positive.

[0114] In the specific implementation plan, samples that test positive in the confirmatory assay are reported as positive and further evaluated in a titration assay. Samples below the confirmatory assay inhibition cutoff point may be reported as negative and do not require further testing.

[0115] Titration determination

[0116] In a specific implementation, titration may be used to characterize the magnitude of the ADA response. Titration is used to characterize the magnitude because the effects of ADA on pharmacokinetics, pharmacodynamics, safety, and efficacy may be related to ADA titer and persistence, rather than incidence.

[0117] In a specific implementation, a titration-specific cutoff point is determined using a false positive rate of approximately 0.1%, or a screening cutoff point is used as the titration cutoff point. In a specific implementation, the titration cutoff point is determined to be approximately 0.01% to approximately 0.1% or less than approximately 0.1%.

[0118] Neutralization determination

[0119] In a specific implementation, a neutralizing antibody (NAb) is a specific ADA capable of interfering with the interaction between a therapeutic protein product and its target. A neutralization assay can assess the neutralizing activity of ADA. In a specific implementation, the neutralizing activity of ADA is characterized by partially assessing the effects of ADA on pharmacokinetics, pharmacodynamics, safety, and efficacy, which may be related to NAb activity rather than the incidence of ADA.

[0120] In the specific implementation plan, the test method selected to assess the neutralization potential of ADA-positive samples is based on the mechanism of action of the therapeutic protein product. In vitro neutralization assays can indicate the potential of ADA to inhibit the therapeutic activity of the product. NAbs can interfere with the clinical activity of a therapeutic protein product by preventing it from reaching its target or by interfering with its pharmacological activities, such as receptor-ligand interactions.

[0121] Figure 1 This is an example flowchart of the determination of various embodiments as described herein. The methods applied to each substance associated with SEQ ID NO: 1-3 may be the same or different in the application of the flowcharts or processes described herein.

[0122] In a specific implementation, the NAb assay for detecting whether an anti-sALP antibody is a neutralizing antibody uses an enzyme-catalyzed steady-state kinetic assay to determine the effect of the NAb on sALP efficacy. For example, this assay can measure the hydrolysis of pNPP, which is proportional to enzyme activity. The NAb assay may include acid treatment (e.g., 0.1 M glycine-HCl; 15% v / v) of the negative control, positive control, and study samples prior to the enzyme activity assay. Acidification of the samples can be used to dissociate the antibody bound to sALP and can also be used to denature and inactivate sALP. After the neutralization step, the sample can be added to the activity assay. In the activity assay, a small aliquot (e.g., 20 μL) of a fixed concentration of sALP (e.g., 95.0 ng / mL) can be added to each well of the assay. After incubation, the reaction can be initiated by adding pNPP (e.g., a final concentration of, for example, 1 mM, 2 mM, 3 mM, 4 mM, or 5 mM or higher) to a suitable reaction buffer (e.g., 20 mM bis-tris propane, 50 mM NaCl, 0.5 mM MgCl2, 50 μM ZnCl2, 0.5 mg / mL BSA, and pH 9.0 at 37°C). Study samples containing NAb will inhibit pNPP turnover compared to the negative control. The antibody activity against ascites α in each sample (V0) can be calculated using the following formula. max Inhibition % of:

[0123]

[0124] The neutralization assay cutoff point can be determined by analyzing, for example, 50 single-drug-naïve human serum samples evaluated in two or more individual runs (e.g., 3, 4, 5, or 6 runs). The assay cutoff point can then be obtained using statistical methods, and this cutoff point is determined, for example, by the reaction rate (V0). max The 99.9th percentile of inhibition is used to indicate the NAb level. Samples with an inhibition percentage below this cutoff point are considered NAb negative, while samples with an inhibition percentage equal to or higher than this cutoff point are reported as NAb positive.

[0125] Results from the above assays can be validated using positive controls, such as polyclonal anti-sALP antibodies. Parameters such as sensitivity (e.g., the concentration of a positive control that produces a positive response in all assays), selectivity (e.g., requiring 60%, 70%, 80%, 90%, or more of the tested sALP batches to meet the criteria), precision and accuracy (e.g., signal-to-noise ratio of concentration variability <5%, 15%, 20%, or 25%), specificity, drug tolerance / interference (e.g., the highest drug concentration that produces a positive response), and sample stability (e.g., recovery within 100 ± 20%) can also be evaluated using predetermined thresholds for acceptance criteria.

[0126] Define immunogenicity subgroups

[0127] Analysis of PK measurements can be used to determine whether sALP efficacy is reduced during ERT. Specific aspects of the immunogenic response that can be investigated include, for example, the time-varying nature of immunogenicity, immunogenic status (e.g., ADA+ vs. ADA- and NAb+ vs. NAb-), and magnitude (e.g., high vs. low ADA and NAb titers). To assess the impact of antibody production, subjects can be assigned to subgroups based on immunogenicity information regarding seroconversion in their serum (e.g., production of sALP, ADA, and NAb). Various PK parameters (e.g., PPi and / or PLP concentrations, C...) can then be examined between these subgroups. avg C max and V max The difference in PK parameters is used to assess the impact of immunogenicity on PK. Exemplary PK parameters are described, for example, in PCT application PCT / US2018 / 025206, which is incorporated herein by reference in its entirety.

[0128] To assess immunogenicity, one approach is to assign subjects to one of two groups, such as antibody-positive or antibody-negative. An antibody-positive subject can be defined, for example, by a single event of testing positive for ADA at any time after treatment with sALP (e.g., asforase alfa). This subject can then be assigned to the ADA+ group (ADA-positive throughout treatment). If a subject consistently fails to produce measurable antibodies, they are assigned to the ADA- group (ADA-negative throughout treatment). Each subject can be further assigned to one of three possible subgroups: ADA- (antidrug-negative, NAb not measurable), ADA+ / NAb+ (ADA+ and neutralizing antibody positive), and ADA+ / NAb- (ADA+ but neutralizing antibody negative). Example 2 provides additional details regarding the assessment of immunogenicity grouping.

[0129] alkaline phosphatase

[0130] Alkaline phosphatases, administered as part of an ERT (Extracorporeal Therapy) to subjects with bone mineralization disorders such as HPP, comprise a group of enzymes that catalyze the partial cleavage of phosphate (e.g., hydrolysis of pyrophosphate PPi). Four known mammalian alkaline phosphatase (ALP) isoenzymes exist: tissue-nonspecific alkaline phosphatase (TNALP; further described below), placental alkaline phosphatase (PLALP) (e.g., accession numbers P05187, NP_112603, and NP_001623), germ cell alkaline phosphatase (GALP) (e.g., accession number P10696), and intestinal alkaline phosphatase (IALP) (e.g., accession numbers P09923 and NP_001622). In addition to the exemplary ALPs discussed herein, any polypeptide having the same or similar catalytic site structure and / or enzymatic activity as ALPs may be used in the methods disclosed herein (e.g., as sALP or sALP fusion polypeptides as described herein).

[0131] sALP can be a soluble form of, for example, human tissue-nonspecific alkaline phosphatase (human TNALP (hTNALP)). An exemplary soluble alkaline phosphatase (sALP) is asforase α, which is a human TNALP fusion polypeptide. Specifically, a polypeptide of any of SEQ ID NO:1-3 or a variant thereof having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity may be used for administration to treat bone mineralization disorders such as HPP. sALP may comprise or consist of the amino acid sequence of SEQ ID NO:1. sALP may comprise or consist of the amino acid sequence of SEQ ID NO:2. sALP may comprise or consist of the amino acid sequence of SEQ ID NO:3.

[0132] Alkaline phosphatase may contain the sequence shown in SEQ ID NO: 1: LVPEKEKDPKYWRDQAQETLKYALELQKLNTNVAKNVIMFLGDGMGVSTVTAARILKGQLHHNPGEETRLEMDKFPFVALSKTYNTNAQVPDSAGTATAYLCGVKANEGTVGVSAATERSRCNTTQGNEVTSILRWAKDAGKSVGIVTTTRVNHATPSAAYAHSADRDWYSDNEMPPEALSQGCKDIAYQLMHNIRDIDVIMGGGRKYMYPKNKTDVEYESDEKARGTRLDGLDLVDTWKSFKPRYKHSHFIWNRTELLTLDPHNVDYLLGLFEPGDMQYELNRNNVTDPSLSEMVVVAIQILRKNPKGFFLLVEGGRIDHGHHEGKAKQALHEAVEMDRAIGQAGSLTSSEDTLTVVTADHSHVFTFGGYTPRGNSIFGLAPMLSDTDKKPFTAILYGNGPGYKVVGGERENVSMVDYAHNNYQAQSAVPLRHETHGGEDVAVFSKGPMAHLLHGVHEQNYVPHVMAYAACIGANLGHCAPASSLKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKDIDDDDDDDDDD (SEQ ID NO: 1)

[0133] Alkaline phosphatase may contain the sequence shown in SEQ ID NO: 2:

[0134] LVPEKEKDPKYWRDQAQETLKYALELQKLNTNVAKNVIMFLGDGMGVSTVTAARILKGQLHHNPGEETRLEMDKFPFVALSKTYNTNAQVPDSAGTATAYLCGVKANMGTVGVSAATERSRCNTTQGNEVTSILRWAKDAGKSVGIVTTTRVNHATPSAAYAHSADRDWYSDNEMPPEALSQGCKDIAYQLMHNIRDIDVIMGGGRKYMYPKQKTDVEYESDEKARGTRLDGLDLVDTWKSFKPRYKHSHFIWNRTELLTLDPHNVDYLLGLFEPGDMQYELNRNQVTDPSLSEMVVVAIQILRKNPKGFFLLVEGGRIDHGHHEGKAKQALHEAVEMDRAIGQAGSLTSSEDTLTVVTADHSHVFTFGGYTPRGNSIFGLAPMLSDTDKKPFTAILYGNGPGYKVVGGERENVSMVDYAHNNYQAQSAVPLRHETHGGEDVAVFSKGPMAHLLHGVHEQNYVPHVMAYAACIGANLGHCAPASSAGSLAAVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKDDDDDDDDDD (SEQ ID NO: 2).

[0135] The alkaline phosphatase may comprise the sequence shown in SEQ ID NO: 3:

[0136] (SEQ ID NO: 3).

[0137] Exemplary sALPs are those that are physiologically active, such as phosphoethanolamine (PEA), inorganic pyrophosphate (PPi), and pyridoxal 5'-phosphate (PLP). Specifically, sALPs are those with catalytic activity that can improve bone mineralization in bone. Examples of mutations that can be introduced into the ALP sequence are described in U.S. Publication No. 2013 / 0323244.

[0138] The sALP (such as TNALP, for example, the sALP fusion peptide of any of SEQ ID NO: 1-3 or a peptide variant having at least 95% sequence identity with any of SEQ ID NO: 1-3, such as asforase α), linker, spacer region (e.g., Fc region), and bone-targeting portion described herein may be in the form of a fusion peptide having the structure Z-sALP-Y-spacer-X-Wn-V, Z-Wn-X-spacer-Y-sALP-V, Z-sALP-Y-Wn-X-spacer-V, or Z-Wn-X-sALP-Y-spacer-V. Specifically, the sALP fusion peptide may have the structure Z-sALP-Y-spacer-X-Wn-V or Z-Wn-X-spacer-Y-sALP-V. The sALP in sALP fusion peptides can be full-length ALP or functional fragments of ALP, such as soluble extracellular domains of ALP, as described herein (e.g., TNALP, PALP, GCALP, and IALP). Bone delivery conjugates including sALP are further described in PCT publications: WO 2005 / 103263 and WO 2008 / 138131, the contents of which are each incorporated herein by reference in their entirety.

[0139] Any one of X, Y, Z, and V and / or the spacer region may be absent, or it may be a linker region containing an amino acid sequence of at least one amino acid. For example, X, Y, Z, and V may be dipeptide sequences (e.g., leucine-lysine or aspartic-isoleucine), such as a two-residue linker at the Y position (e.g., leucine-lysine) or at the X position (e.g., aspartic-isoleucine). For example, the sALP fusion polypeptide may have the structure hTNALP-Fc-D10 (e.g., a sALP fusion polypeptide containing the amino acid sequence of any one of SEQ ID NO: 1-3 or a polypeptide variant having at least 85% (e.g., at least 90%, 95%, 97%, or 99%) sequence identity with any one of SEQ ID NO: 1-3, such as asforase α).

[0140] Wn can be a bone-targeting moiety, for example, having a series of consecutive aspartic (D) or glutamic (E) residues, where n = 1 to 50, e.g., n = 3 to 30, e.g., 5 to 15, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 36, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. The bone-targeting moiety (if present) can be located anywhere on the fusion polypeptide, e.g., at or near the N-terminus or C-terminus, and / or in the linker region. For example, the bone-targeting moiety can be present at the C-terminus of the sALP fusion polypeptide. sALP and fusion peptides may also lack the bone-targeting component.

[0141] The methods described herein can be combined with sALP therapy (e.g., administration of sALP). This therapy can be administered before or after sALP therapy. sALP (e.g., any of the sALPs in SEQ ID NO: 1-3, such as the sALP of SEQ ID NO: 1) can be administered 1 to 7 times per week or every two weeks (e.g., 2, 3, 4, 5, 6, or 7 times), for example, 2, 3, or 6 times per week. sALP can be administered once per week, once every two weeks, once every three weeks, or once every four weeks. For example, sALP can be administered 3 or 6 times per week. sALP (e.g., sALP of SEQ ID NO: 1) can be administered at doses from 1 mg / kg / week to 10 mg / kg / week (e.g., 3 mg / kg / week to 9 mg / kg / week, e.g., 1 mg / kg / week, 2 mg / kg / week, 3 mg / kg / week, 4 mg / kg / week, 5 mg / kg / week, 6 mg / kg / week, 7 mg / kg / week, 8 mg / kg / week, 9 mg / kg / week, or 10 mg / kg / week). sALP can be administered at a dose of 1 mg / kg / week. sALP can be administered at a dose of 2 mg / kg / week. sALP can be administered at a dose of 3 mg / kg / week. sALP can be administered at a dose of 6 mg / kg / week. sALP can be administered at a dose of 9 mg / kg / week. sALP can be administered at a dose of 1 mg / kg six times a week, 2 mg / kg three times a week, or 3 mg / kg twice a week. In some implementations, the dosage is 3 mg / kg three times a week, or increased from 3 mg / kg twice a week to 3 mg / kg three times a week.

[0142] In one embodiment, sALP (e.g., the sALP of any of SEQ ID NO: 1-3, such as the sALP of SEQ ID NO: 2 or 3) is administered at a dose of about 10 mg to 50 mg, for example, once a week or every two weeks. For example, sALP (e.g., the sALP of SEQ ID NO: 2 or 3, such as the sALP of SEQ ID NO: 2) can be administered at a dose of 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg or 50 mg (e.g., 20 mg, 35 mg or 50 mg), for example, once a week or every two weeks.

[0143] Vials containing sALP (e.g., sALP of SEQ ID NO: 1) can be prepared at concentrations up to 100 mg / mL, such as 40 mg / mL or 100 mg / mL (e.g., 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL or 100 mg / mL).

[0144] In some embodiments, the pharmaceutical composition is administered subcutaneously or intravenously. For example, for sALP (e.g., the sALP of SEQ ID NO: 2 or 3, such as the sALP of SEQ ID NO: 2), about 10 mg to about 100 mg (e.g., about 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg or 100 mg, such as about 15 mg, 45 mg or 90 mg, such as about 25 mg, 35 mg or 50 mg) may be administered subcutaneously to the subject, such as once or twice a week, such as for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks or more or longer (e.g., the subject's lifetime). The composition can be applied in volumes of, for example, about 5 mL or less (e.g., 5.0 mL, 4.0 mL, 3.0 mL, 2.0 mL, 1.0 mL, 0.9 mL, 0.8 mL, 0.7 mL, 0.6 mL, 0.5 mL, 0.4 mL, 0.3 mL, 0.2 mL or 0.1 mL, or in volumes ranging from about 5 mL to about 0.1 mL).

[0145] In some embodiments, the second sALP treatment involves administration at a lower frequency than the treatment containing the first sALP. In some embodiments, the sALP is administered to the subject receiving sALP treatment at a dose of 1 mg / kg / week to 10 mg / kg / week, and the subject also receives treatment with a second sALP that is different from the first sALP, and the second sALP is administered every 2 weeks at a dose of 20 mg, 35 mg, or 50 mg.

[0146] The methods described herein may include treating a subject who has been administered two different sALPs. For example, in some embodiments, the subject has been treated with a first sALP (e.g., the sALP of SEQ ID NO: 1) for a period of time (e.g., one or more days, one or more weeks, one or more months, or one or more years), and is subsequently administered a second sALP different from the first sALP (e.g., the sALP of SEQ ID NO: 2 or 3, such as the sALP of SEQ ID NO: 2). The subject's treatment may alternate between the first sALP and the second sALP, or the subject may receive the first sALP and then switch entirely to the second sALP.

[0147] sALP can be discontinued during treatment. For example, sALP can be discontinued for about 1 week, 2 weeks, 3 weeks, 4 weeks or longer, for example, until a reduction in neutralizing antibodies is achieved.

[0148] The above-mentioned sALP can be administered to subjects in need to treat mineralization disorders, such as HPP, or to treat their symptoms. Symptoms of bone mineralization disorders (such as HPP) include, for example, incomplete mineralization, skeletal deformities, fractures, and bone pain. Symptoms of adult HPP as defined herein include, for example, elevated blood and / or urine levels of phosphoethanolamine (PEA), incomplete mineralization, hypercalciuria, skeletal deformities, waddling gait, bone pain, fractures, calcium pyrophosphate dihydrate crystal deposition, arthritis, pyrophosphate arthropathy, chondrocalcinosis, calcific periarthritis, pseudofractures, skeletal deformities, hypotonia, muscle weakness, rheumatoid complications, arthritis, pseudogout, difficulty walking, pain, premature tooth loss, pulmonary hypoplasia, respiratory failure, seizures, body size, growth, rickets, and immunogenicity. Symptoms of adolescent HPP as defined in this article include, for example, elevated blood or urine levels of PPi, PEA, or PLP; osteomalacia; one or more skeletal deformities; hypotonia; muscle weakness; rheumatoid complications; arthritis; pseudogout; waddling gait; difficulty walking; bone pain; pain; premature tooth loss; incomplete mineralization; pulmonary hypoplasia; respiratory failure; seizures; hypercalciuria; short stature; and growth retardation. Changes in any of these symptoms can be used to monitor sALP efficacy during ERT (e.g., with asifola frondole alfa), before and after ERT treatment, and / or before or during IST therapy to monitor for decreases or increases in sALP efficacy during ERT.

[0149] For subjects at risk of developing neutralizing antibodies against sALP after administration or experiencing loss or reduction in sALP efficacy during sALP ERT, treatment with IST therapy can be performed using the following methods. IST therapy includes one or more of the following steps: (a) identifying and / or diagnosing that a subject with reduced sALP efficacy requires IST therapy; (b) testing for neutralizing antibodies against the sALP treatment agent in a sample from the subject; (c) determining whether the neutralizing antibodies are the cause of the reduced sALP efficacy; (d) treating the subject with IST therapy; and (e) determining when to discontinue IST therapy.

[0150] Example

[0151] The following examples are intended to illustrate, not limit, this disclosure. It should be understood that the specific embodiments, materials, quantities, and procedures are to be interpreted broadly within the scope and spirit of this disclosure set forth herein.

[0152] Example 1. An immunosuppressive therapy to reduce asftase α (STRENSIQ) ® Treatment of hypophosphatase syndrome An interventional, prospective, open-label study (RESTORE) on immune-mediated loss of therapeutic response.

[0153] Basic principles :

[0154] Administering biological (protein) drugs to patients, especially with continuous exposure for the treatment of chronic conditions, carries the risk of inducing anti-drug antibodies (ADAs). This risk is greater if the biological therapeutic agent has a potential "endogenous counterpart," such as the protein administered as an alternative therapy for diseases like hypophospholipase syndrome (HPP). Neutralizing antibodies (NAbs) can negate the clinical benefits of exogenous biological therapeutic agents. Furthermore, non-NAbs can also reduce efficacy by accelerating drug clearance (CL), pharmacodynamics (PD), and pharmacokinetics (PK).

[0155] In postmarketing safety surveillance, some patients treated with asforase alfa exhibited initial treatment response but subsequently experienced relapse and progression of disease-related clinical, laboratory, and radiological markers. Because some of these events occurred in the presence of documented positive ADA and Nab, immune-mediated effects on the pharmacological action of asforase alfa may contribute to disease progression. Based on this information, the U.S. FDA has requested a Postmarketing Requirements (PMR) study to assess and characterize the potential serious risk of immune-mediated loss of effectiveness (LoE) and to evaluate strategies to mitigate this risk (PMR#2949-7, BLA 125513).

[0156] This prospective, open-label clinical study will evaluate the ability of three immunosuppressive therapies (ISTs) (i.e., methotrexate, rituximab, and bortezomib) to reduce immune-mediated LoE in pediatric HPP patients receiving long-term asforase alfa therapy. The study will also investigate whether long-term ISTs are necessary to maintain the desired efficacy of asforase alfa in HPP participants.

[0157] Table 1. Goals and Endpoints :

[0158]

[0159] a ADA will be the primary outcome for both ADA-positive and NAb-negative patients. NAb will be the primary outcome for both ADA-positive and NAb-positive patients.

[0160] Abbreviations: ADA = Anti-drug antibody; CD19 = Differentiation group 19 (B lymphocyte antigen); ECG = Electrocardiogram; HPP = Hypophosphatase syndrome; IST = Immunosuppressive therapy; LoE = Loss of efficacy; NAb = Neutralizing antibody; PD = Pharmacodynamics; PK = Pharmacokinetics; PLP = Pyridoxal 5'-phosphate (Vitamin B6); PPi = Inorganic pyrophosphate; RSS = Rickets severity score; TEAE = Treatment-related adverse events; TESAE = Treatment-related serious adverse events; TNSALP = Tissue nonspecific alkaline phosphatase

[0161] Overall Design :

[0162] This is a phase 4, prospective, open-label study designed to evaluate the impact of IST on the clinical presentation of HPP in pediatric participants who received asforase α therapy and exhibited immune-mediated LoE.

[0163] The identification of immune-mediated LoE requires radiographic evidence of rickets in pediatric patients aged 2 to <18 years with an open epiphyseal growth plate and ADA and / or NAb:

[0164] 1. Children who have shown an initial effective response after at least 6 months of continuous treatment with asforamin alfa and are currently receiving asforamin alfa, and whose rickets has relapsed or worsened within the past 3 months. The Rickets Severity Scale (RSS) will be used to determine the severity at baseline.

[0165] 2. ADA is present during screening, regardless of the presence or titer of NAb.

[0166] 3. Confirm the existence of both clinical evidence and immunogenicity-mediated association.

[0167] Participants will be identified from the HPP Global Registry (ALX-HPP-501) and HPP Registry Sub-studies (ALX-HPP-501s). Additionally, patients outside the registry will be identified via an ADA test available in the United States, or, if the participant is outside the United States, via a pre-screening portion of the study. All potential participants must also have a documented clinical LoE.

[0168] Participants will be followed for 104 weeks after their first IST dose. At the start of the study, an initial course of methotrexate and rituximab will be administered from day 1 for 24 weeks. After this initial 24-week period, the schedule will be repeated as follows: weeks 25–26; weeks 27–50 (Treatment Assessment [TA] phase 2); weeks 51–52; weeks 53–76 (TA phase 3); weeks 77–78; weeks 79–104 (TA phase 4). Throughout the study, asforokinase α therapy for HPP will continue in parallel with IST. At the end of each 24-week treatment period, radiographic outcomes of rickets will be assessed using RSS and ADA and / or NAb status and titers to determine the benefit / risk of adopting or continuing the IST treatment regimen in the subsequent 24 weeks. It should be noted that methotrexate will be used continuously throughout the study, and rituximab will be administered uninterruptedly for at least the first 18 months (approximately 74 weeks) unless safety concerns arise. Intravenous immunoglobulin (IVIG) 500 mg / kg will be co-administered monthly with rituximab, with or without bortezomib. The decision to add bortezomib to the IST regimen will be based on a radiographic assessment of rickets with a 1 or more improvement in RSS score in the presence of ADA or NAb levels relative to baseline. If a participant demonstrates a complete response at 18 months and is receiving both methotrexate and rituximab, rituximab will be discontinued, and methotrexate will continue until the end of the study. If a participant demonstrates a complete response at 18 months and is receiving methotrexate, rituximab, and bortezomib, bortezomib will be discontinued, and methotrexate and rituximab will continue until the end of the study. The dosing regimen is described in [details omitted]. Figure 3 And as shown in Tables 4 and 5. During the study, participants will continue to receive asforase α treatment according to the same subcutaneous (SC) dosing regimen as at enrollment.

[0169] The study completion (EoS) assessment will be conducted at the 100-week visit or early termination (ED; [if applicable]).

[0170] Disclosure Statement :

[0171] This was a single-arm, non-blinded study designed to treat HPP participants exhibiting immune-mediated LoE.

[0172] Intervention group and duration :

[0173] During the 104-week IST treatment period in the study, all participants continued to receive asforokinase alfa therapy uninterruptedly according to the standard treatment regimen prescribed by their attending physician. IST treatment will follow the Schedule of Activities (SoA). EoS assessment will be performed at the 100-week visit or ED (if applicable).

[0174] All participants will receive a safety follow-up call in week 104, and blood and urine samples will be collected for safety testing. The global end of the study is defined as the date the last participant completes their final safety follow-up call.

[0175]

[0176]

[0177]

[0178]

[0179]

[0180]

[0181]

[0182]

[0183]

[0184]

[0185] Background Technology

[0186] Clinical experience with the immunogenicity of asforokinase Alpha therapy

[0187] In clinical studies, the majority of participants treated with asforase α for HPP were ADA-positive at some point after baseline (97 / 109; 89%). Typically, ADA titers were low, with a median peak titer of 32.0 (range: 0 to 2048). The median time to first ADA positivity was 43.0 days (range: 14 to 2374 days). Similar to ADA results, participants who tested positive for NAbs at some point after baseline did not remain positive after their first positive result. Of the 97 participants who were ADA-positive after baseline, 55 (56.7%) were NAb-positive at some point after baseline. The median peak NAb inhibition percentage was 9.910 (range: 4.54 to 95.7). No correlation was observed between ADA titer and NAb (inhibition %) values.

[0188] Overall, these ADA responses had minimal impact on the asfetoprotein α (API) drug concentration profile. Population pharmacokinetic (Pop-PK) modeling (N=58) results indicated that ADA status had no significant effect on API CL (≤20% increase). Simulations evaluating the effect of immunogenicity on CL revealed a 90% confidence interval (CI) for steady-state mean exposure, supporting the conclusion that low-titer antibody responses have a small overall impact on API PK. Given the small impact of immunogenicity on PK, and considering that participants receiving a 6 mg / kg / week dose are nearing the exposure-efficacy plateau, immunogenicity is not expected to affect the efficacy and safety of most HPP participants receiving API. This conclusion has been confirmed based on a review of comprehensive data from individual participants, focusing on the relationship between immunogenicity and efficacy / safety.

[0189] In postmarketing safety surveillance, some patients treated with asforase alfa showed an initial treatment response but subsequently experienced relapse and progression of disease-related clinical, laboratory, and radiological markers. Because some of these events occurred in the presence of documented positive ADA and NAb, immune-mediated effects on the pharmacological action of asforase alfa may contribute to disease progression.

[0190] The proposed IST agent will be administered during the initial 24-week course, followed by administration over 2-week intervals to evaluate clinical, laboratory, and radiological changes to determine if the IST regimen needs to be repeated. If participants do not receive IST treatment, they will continue to be observed, or the IST regimen will be modified for the next 24 weeks. Unless there are any safety concerns, methotrexate and rituximab will be administered for at least 18 months (approximately 74 weeks). IVIG 500 mg / kg will be co-administered monthly, concurrently with rituximab, with or without bortezomib.

[0191] Throughout the study, all data from each participant will be reviewed every 24 weeks to assess the impact of IST on immune-mediated LoE. This cycle will be repeated for all participants until the study is completed or ED is reached. During the study, HPP asforase α treatment will continue concurrently with IST, and if a participant is enrolled in the HPP global registry at the end of the study, they will continue to be followed according to standard treatment. Participants will be treated as case studies, and the impact of the IST regimen on ADA / NAb response and the corresponding impact on efficacy improvement will be assessed through intra-participant analyses.

[0192] Risk / Benefit Assessment

[0193] risk assessment

[0194] In this open-label study, participants will continue to receive a subcutaneous administration of asforase alfa at enrollment and during the study, prescribed by their attending physician according to the country’s approved label.

[0195] Because there are no controlled studies using these agents in patients with HPP, the toxicity of this group receiving asforase alfa is unknown. Furthermore, the proposed combination of asforase alfa with methotrexate, rituximab, and / or bortezomib has not been used, and the combination of agents may have a wider range of side effects than those observed with the individual agents. During this study, participants should be closely monitored for any signs of toxicity, including any changes in serum creatinine, blood urea nitrogen, platelet count, white blood cell count, alanine aminotransferase (ALT), aspartate aminotransferase (AST), bilirubin, and the presence of red blood cells in the urine.

[0196] Immunosuppression can also lead to opportunistic infections, such as bacterial, fungal, and viral infections. Participants should be closely monitored for any signs of infection during the study, and aggressive treatment and management should be initiated if any signs of infection occur. When selecting antibiotics to treat infections, potential drug interactions between the investigational agent and certain antibiotics should be considered. Consultation with an infectious disease specialist is also recommended before administering any antimicrobial agent to participants receiving the IST regimen for active infection. Routine administration of IVIG has been incorporated into the treatment protocol as a measure to reduce the risk of infection. Furthermore, folic acid deficiency may increase methotrexate-related adverse reactions. Therefore, folic acid should be administered to participants receiving methotrexate.

[0197] Information regarding the known and anticipated benefits and risks of rituximab, methotrexate, and bortezomib, as well as reasonably anticipated adverse events (AEs), can be found in the Reference Safety Information (RSI) for each IST. The current EU SmPC is most suitable for the corresponding active substance, route of administration, and dosage regimen, and will therefore be used as the RSI for the IST. The IST regimen for each participant may be added to or removed based on a review of their clinical, radiological, and experimental data.

[0198] Benefit assessment

[0199] Because this study aims to improve participants' immune-mediated LoE with asforase alfa treatment, participation can lead to improved health and quality of life for participants diagnosed with HPP. If participants demonstrate a benefit from the IST regimen during the study, they can continue asforase alfa treatment for IST after EoS.

[0200] In addition, tests used in clinical trials but not yet available in most countries (inorganic pyrophosphate [PPi], pyridoxal 5'-phosphate [{vitamin B6} PLP] combined with levamisole and asforase α concentrations) will be available to attending physicians and analyzed in a central laboratory. These recommended but not yet widely available blood tests pose no additional risk, and participants can benefit from undergoing these tests to aid in treatment monitoring.

[0201] Overall Benefits: Risk Conclusion

[0202] Given the measures taken in this study to minimize the risks to participants, and considering that rituximab, methotrexate, and bortezomib are expected to provide benefits to HPP participants who received asforase α treatment and developed immune-mediated LoE, identifying the potential risks associated with rituximab, methotrexate, and bortezomib is worthwhile.

[0203] Immune response

[0204] Helper T cells are crucial in the formation of most ADAs, while memory B cells and long-lived plasma cells amplify and maintain responses that form the basis for the use of combined IST when LoE occurs due to the presence of ADAs. Therapeutic proteins, such as asforase α, may be endocytosed by antigen-presenting cells after administration and processed therein into their component peptides. A portion of these peptides can then be presented to helper T cells specific to that peptide in the context of human leukocyte antigen (HLA) molecules. Helper T cells signal and activate Ag-specific B cells, causing them to proliferate and differentiate into memory B cells and Ab-secreting plasma cells (including short-lived and long-lived types).

[0205] Immunosuppressive therapy

[0206] Based on the effects of the following ISTs on helper T cells, plasma cells, and memory B cells, their use in this study will be considered. Since there are no controlled studies using these agents in HPP patients, toxicity is unknown.

[0207] Rituximab

[0208] Rituximab is a chimeric monoclonal antibody approved for use in a variety of malignancies and autoimmune diseases. Its target is the human B lymphocyte surface antigen (CD20), a transmembrane protein present on almost all B cells. This antigen is downregulated from the stage of B cell directed development until B cells differentiate into antibody-secreting plasma cells. Rituximab induces rapid and almost complete depletion of peripheral B cells; in most participants, these cells no longer appear within approximately 6 months.

[0209] However, CD20 is lost when B cells differentiate into antibody-producing plasma cells. Therefore, once B cells differentiate into plasma cells, alternative and / or adjunctive agents must be used to address ADA produced by circulating plasma cells.

[0210] Methotrexate

[0211] Methotrexate is a dihydrofolate reductase inhibitor that affects rapidly dividing T cells and B cells by preventing the reduction of dihydrobiopterin (BH2) to tetrahydrobiopterin (BH4), which leads to the uncoupling of nitric oxide synthase and increases the sensitivity of T cells to apoptosis, thereby weakening the immune response.

[0212] Bortezomib

[0213] Bortezomib is a proteasome inhibitor. Proteasome inhibitors are a potent and specific way to target plasma cells, which are highly dependent on proteasome activity. Bortezomib is a reversible proteasome inhibitor that targets both short-lived and long-lived plasma cells simultaneously because these plasma cells have a high rate of immunoglobulin production. Although bortezomib was initially approved for multiple myeloma, animal studies have shown that proteasome inhibition can also kill normal plasma cells. Bortezomib has improved the HSAT response to ERT in some patients with IPD and has also shown benefit in several case reports of thrombotic thrombocytopenic purpura and autoimmune cytopenia that are refractory to standard therapy, with only mild and tolerable adverse events in children.

[0214] Participants recruited in this study who exhibited loss of efficacy were all ADA-positive at baseline and had “refractory” ADA responses. Rituximab depletes B cells, but plasma cells (which produce ADA) are not affected. Therefore, patients receiving methotrexate and rituximab with refractory ADA responses may have an inadequate response to asfort enzyme αERT. Bortezomib will be considered in cases of disease progression with elevated ADA and NAb levels to deplete ADA-producing plasma cells and memory B cells.

[0215] Dosage rationalization

[0216] Asforase α

[0217] Asifolium-α, the NIMP used in this study, will be administered at enrollment at the recommended dosage prescribed by the attending physician according to the approved label in that country.

[0218] Immunosuppressive therapy (IST)

[0219] This study will use standard doses of rituximab, methotrexate, and bortezomib. Clinical, laboratory, and radiological data will be collected to determine IST treatment and monitoring.

[0220] End of study definition

[0221] Participants who complete the final scheduled procedure shown in the SoA will be considered to have completed the study. If a participant withdraws / is withdrawn from the study before completing the EoS visit, they will be considered to have terminated the study early. The global end of the study is defined as the date the last participant completes their final security follow-up call. Participants who complete the study will be eligible for registration in the HPP Global Register.

[0222] research group

[0223] During the eight-week screening period, the participants' medical history, laboratory results, and physical examinations will be reviewed, and a consensus decision on whether to continue with IST will be made after obtaining informed consent.

[0224] Anticipated approval of protocol deviations (also known as protocol waivers or exemptions) from recruitment and enrollment criteria is not permitted.

[0225] Research Intervention

[0226] A research intervention is defined as any research intervention, commercially available product, placebo, or medical device intended to be administered to research participants in accordance with the research protocol.

[0227] Research interventions applied

[0228] All research interventions (investigational medicine products [IMP] and non-investigational medicine products [NIMP]) will be recorded as follows:

[0229] •start date

[0230] • Dosing regimen (e.g., dosage [mg / kg], frequency, missed doses, treatment interruptions)

[0231] For details on recommended dosing regimens for rituximab, methotrexate, and bortezomib, please refer to Table 6.

[0232] The body surface area (BSA) for the dosing regimen will be determined monthly. The BSA will be calculated using the Dubois method.

[0233] BSA(m) 2 = Height (0.725) × Weight (0.425) × 0.007184, where height is in meters (m) and weight is in kilograms (kg).

[0234] Asforase alfa (ASA) administered as part of the NIMP in this study will be given at the recommended dosage prescribed by the attending physician according to the approved label in the country. However, once participants are enrolled, the ASA dosage regimen must remain stable throughout the study. ASA will not be provided within the scope of this study.

[0235] Table 6: Research Intervention Information

[0236]

[0237]

[0238] Note: The definitions of IMP and NIMP are based on guidance issued by the European Commission.

[0239] a For IST, dosage modifications (increasing, removing, and / or changing the methotrexate dose) are permitted in cases of drug toxicity.

[0240] b IVIG should be administered whenever rituximab is being given.

[0241] c Folic acid will be procured through commercial channels, while IVIG and IMP will be supplied.

[0242] d An experimental immune tolerance therapy for LoE caused by an immune response to asforokinase alpha (ADA, with or without NAb).

[0243] Abbreviations: ADA = Anti-drug antibody; EU = European Union; HPP = Hypophosphatase syndrome; LoE = Loss of efficacy; IMP = Investigational drug; IST = Immunosuppressive therapy; IV = Intravenous; IVIG = Intravenous immunoglobulin; MTX = Methotrexate; n / a = Not applicable; NAb = Neutralizing antibody; NIMP = Non-investigational drug; SC = Subcutaneous; TA = Treatment evaluation (period); w / out = Without

[0244] Companion therapy

[0245] Any medications (including over-the-counter or prescription drugs, vitamins, herbal supplements, and / or prophylaxis medications used prior to IST administration), vaccines, or other specific categories of interest that participants received at enrollment or during the study must be documented along with the following information:

[0246] • Reasons for use

[0247] • Application date, including start and end dates

[0248] • Dosage information including dosage and frequency

[0249] Dosage modification

[0250] After each participant completes each 24-week treatment period, clinical, laboratory, and radiological changes will be evaluated to determine whether each participant needs to repeat the current IST, continue observation only, or add / remove the IST regimen for the next 24-week treatment period.

[0251] It should be noted that unless there are any safety concerns, methotrexate should be administered for at least 18 months. This article describes dosage modifications for potential toxicity.

[0252] Methotrexate dosage modification

[0253] Methotrexate may be associated with multi-organ toxicities, including hematologic, renal, hepatic, gastrointestinal, pulmonary, skin, and nervous system toxicities. Depending on the nature and severity of the toxicity, dose interruption, dose reduction, or discontinuation may be necessary. For any grade 3 or higher toxicity, methotrexate should be discontinued, and the patient should be evaluated immediately. Depending on the nature and severity of the adverse reaction, restarting methotrexate at the same or lower dose may be considered.

[0254] In patients with renal impairment, methotrexate elimination is reduced; for patients with moderate renal impairment (creatinine clearance >30 to 59 mL / min), the methotrexate dose should be reduced by 50%; while for patients with severe renal impairment (creatinine clearance <30 mL / min), methotrexate should not be administered.

[0255] Rituximab Dosage Modification

[0256] For rituximab-related toxicities that may affect multiple organ systems and are life-threatening or fatal (e.g., severe mucocutaneous reactions, severe infections, toxicities affecting the heart, kidneys, and gastrointestinal system), dose reduction is not recommended to reduce risk. For any grade 3 or higher toxicity, rituximab infusion should be suspended until the toxicity resolves or further diagnostic evaluation is performed. Rituximab should not be administered to patients with active, serious infections (e.g., tuberculosis, sepsis, opportunistic infections, or severe viral infections, including hepatitis B or other viral reactivation). Permanent discontinuation of rituximab should be considered if toxicity does not resolve or cannot be adequately controlled with standard treatment interventions, or if adverse reactions recur upon re-administration.

[0257] Bortezomib dosage modification

[0258] In addition to neuropathy, bortezomib treatment must be discontinued in the event of any grade 3 or higher toxicity (see Table 7). Bortezomib can be restarted at a lower dose once the toxicity has improved, according to the criteria provided below. If the toxicity does not subside or recurs at the lowest dose, bortezomib should be discontinued unless the treatment benefit significantly outweighs the risks.

[0259] Table 7: Bortezomib Dosage Modifications for Hematologic and Non-Hematologic Toxicity

[0260]

[0261] Source: Bortezomib SmPC

[0262] Abbreviations: ANC = Absolute Neutrophil Count; AV-Block = Atrioventricular Block; ECG = Electrocardiogram; GLS = Global Longitudinal Strain; LVEF = Left Ventricular Ejection Fraction; NT-proBNP = N-terminal Pro-Brain Natriuretic Peptide; SmPC = Product Features Summary

[0263] Dosage modification and treatment continuity criteria for peripheral neuropathy in patients receiving bortezomib treatment

[0264] Participants with pre-existing severe neuropathy may receive bortezomib treatment only after a careful risk-benefit assessment.

[0265] • Grade 1 (asymptomatic; loss of deep tendon reflexes or paresthesia) and no pain or loss of function: No intervention required.

[0266] • Grade 1 with pain or Grade 2: Reduce the dose to 1 mg / m²

[0267] • Grade 2 pain or Grade 3 pain: Discontinue use until symptoms of toxicity subside; may restart dosing weekly at 0.7 mg / m².

[0268] • Level 4: Discontinue bortezomib

[0269] Dosage modification of bortezomib in cases of liver dysfunction

[0270] For participants with mild hepatic impairment, do not adjust the starting dose. For participants with moderate or severe hepatic impairment, the dose should initially be reduced to 0.7 mg / m² per injection during the first cycle. 2 Furthermore, the subsequent dose can be increased to 1 mg / m² depending on patient tolerance. 2 Or further reduce the dosage to 0.5 mg / m² 2 (See Table 8).

[0271] Table 8: Modified Recommended Starting Dose of Bortezomib in Patients with Hepatic Impairment

[0272]

[0273] Source: Bortezomib SmPC

[0274] a Based on the NCI Organ Dysfunction Working Group's classification criteria for liver function impairment (mild, moderate, severe).

[0275] Abbreviations: AST = Aspartate aminotransferase; NCI = National Cancer Institute; SGOT = Serum aspartate aminotransferase; SmPC = Product Characteristics Summary; ULN = Upper Normal Limit.

[0276] Managing infusion response with IST therapy

[0277] A prophylactic medication consisting of an antipyretic and an antihistamine (e.g., acetaminophen and diphenhydramine) should always be administered before each rituximab infusion. The prophylactic medication should begin with the initial administration of rituximab.

[0278] According to the institution's protocol, a prophylactic drug is administered before infusion or injection of other ISTs.

[0279] If an infusion response is observed, the following guidelines for participant treatment and dose modification or delay are provided and may be modified based on research center guidelines.

[0280] Grade 1 infusion response :

[0281] Grade 1 infusion reactions are characterized by mild symptoms, indicating that neither intervention nor interruption of the study infusion is necessary. For Grade 1 infusion reactions:

[0282] • Remain at the bedside and monitor the participant until they recover from symptoms (or return to baseline).

[0283] • Administer a prophylactic medication to participants at least 30 minutes prior to the subsequent study drug infusion. Recommended medication:

[0284] ○ Diphenhydramine 50mg (or equivalent) and

[0285] ○ Acetaminophen / paracetamol 325mg to 1000mg

[0286] Level 2 infusion response :

[0287] Grade 2 infusion reactions are characterized by moderate symptoms requiring therapy or interruption of the study drug infusion, but with a rapid response to treatment (e.g., antihistamines, nonsteroidal anti-inflammatory drugs (NSAIDs), anesthetics, corticosteroids, bronchodilators, IV fluids). Prophylactic medications are applicable for ≤24 hours. For Grade 2 infusion reactions:

[0288] • Stop the infusion of the study drug and start IV infusion of 0.9% sodium chloride.

[0289] • Administer diphenhydramine 50 mg IV (or equivalent) and / or acetaminophen / paracetamol 325 mg to 1000 mg

[0290] • Remain at the bedside and monitor the patient until they recover from symptoms (or return to baseline).

[0291] • When appropriate, administer corticosteroids and / or bronchodilators.

[0292] When symptoms subside, restart the study drug infusion at 50% of the original infusion rate. If the patient does not experience further complications after 30 minutes, the infusion rate can be increased to the original infusion rate.

[0293] Administer a prophylactic medication to participants at least 30 minutes prior to the subsequent study drug infusion. Recommended medication:

[0294] • Diphenhydramine 50mg (or equivalent) and

[0295] • Acetaminophen / paracetamol 325mg to 1000mg

[0296] • Corticosteroids (up to 25 mg of hydrocortisone or an equivalent drug) may also be given.

[0297] Grade 3 or 4 infusion response :

[0298] Grade 3 or 4 infusion reactions are serious. Grade 3 reactions are characterized by their prolonged duration (i.e., no rapid response to treatment and / or brief interruption of the study drug infusion) and recurrence of symptoms after initial improvement. Hospitalization may be required for other clinical sequelae (e.g., renal impairment, pulmonary infiltration). Grade 4 reactions are life-threatening and require vasopressors or ventilation support. For Grade 3 or 4 infusion reactions:

[0299] • Immediately stop the study drug infusion and begin an IV infusion of 0.9% sodium chloride.

[0300] • Administer bronchodilators, subcutaneous or intravenous epinephrine and / or diphenhydramine combined with steroids, and other supportive treatments as needed.

[0301] • Remain at the bedside and monitor the patient until their symptoms have subsided (or returned to baseline) and do not recur.

[0302] • Follow the institutional guidelines for treating allergic reactions.

[0303] Post-study intervention

[0304] After completing their final study visit, participants will return to their primary physician for disease management and may continue to receive commercially available IST.

[0305] Intervention interruptions and terminations, as well as participant discontinuation / withdrawal from the study intervention.

[0306] Research Intervention Interruption and Suspension

[0307] • During the study, participants will be carefully monitored for any signs of infection or other potential IST-related toxicities. In the event of drug-related toxicities, participants may need to permanently discontinue the study intervention. If the study intervention is discontinued, participants should remain in the study for safety evaluations and other assessments as specified in the SoA (Table 3).

[0308] • Participants should permanently discontinue the study intervention if any of the following occurs:

[0309] • Confirmed based on the average of three electrocardiogram (ECG) readings, there is an unexplained grade 3 or higher QT interval prolongation (QTc > 500 ms or uncorrected QT > 600 ms or QTc change relative to baseline > 60 ms).

[0310] • Grade 4 infusion reaction or other severe attributable hypersensitivity reaction (including anaphylactic reaction), Grade 4 IST attributable mucocutaneous reaction or any confirmed Stevens-Johnson syndrome, or toxic epidermal necrolysis.

[0311] •Pregnant or planning pregnancy

[0312] • New or recurrent malignant tumors other than superficial cancers that can be surgically removed.

[0313] • If any of the following criteria are met, or any other clinically significant outcome is observed, the IST should be paused / postponed to allow for further evaluation of the participant:

[0314] • Any Level 4 SAE that may be related to a research intervention or procedure: The decision on whether to reuse the suspected IST on the participant must be discussed and agreed upon in advance.

[0315] • Kidneys: Grade 2 or higher elevation of serum creatinine (>2×ULN or >2×baseline, if baseline>ULN), oliguria, or a decrease in serum creatinine clearance >50%.

[0316] • Liver: ALT or AST elevated by grade 2 or higher (>3×ULN or >3×baseline, if baseline>ULN) or total bilirubin elevated by grade 2 (>2×ULN or >2×baseline, if baseline>ULN).

[0317] • Participants showed a significant worsening of their lung symptoms.

[0318] • Severe infection: IST should be suspended / postponed, and an infectious disease specialist should be consulted.

[0319] • Unresponsive to standard therapy or with significant neutropenia (<1000 / mm³) 3 Any grade 3 infection or any grade 2 infection that is suspected of being a viral reactivation syndrome (e.g., hepatitis B, cytomegalovirus, EB virus, shingles).

[0320] • If viral reactivation is confirmed, IST may not be administered.

[0321] • Hematology: Grade 3 or higher neutropenia (<1000 / mm) 3 ), thrombocytopenia (<50,000 / mm3) or hemoglobin deficiency (<8g / dl).

[0322] • Grade 2 neutropenia (<1500 / mm3); thrombocytopenia (<75000 / mm3) 3 ), decreased hemoglobin (<10g / dl), or a grade 2 decrease in any hematological parameter accompanied by clinically significant symptoms (e.g., decreased neutrophil count with fever, menorrhagia).

[0323] • Gastrointestinal tract: Symptoms suggestive of intestinal obstruction or blockage (e.g., severe constipation, intractable constipation).

[0324] • Cardiopulmonary: New or worsening symptoms suggestive of cardiac or pulmonary toxicity (e.g., grade 2 or higher (e.g., dyspnea, peripheral edema, palpitations, cough, wheezing, high or low blood pressure)).

[0325] • If pulmonary or cardiac toxicity associated with IST is diagnosed (e.g., pneumonia, drug-related left ventricular dysfunction, or life-threatening arrhythmia), suspected IST should be permanently discontinued.

[0326] • Nervous system: Patients presenting with symptoms suggestive of progressive multifocal leukoencephalopathy (PML) or posterior reversible encephalopathy syndrome / reversible posterior leukoencephalopathy syndrome (PRES / RPLS).

[0327] Data collected at the time of intervention termination and follow-up, as well as any data required for further evaluation, are provided in the SoA.

[0328] Efficacy evaluation

[0329] As described in this article, an efficacy evaluation will be conducted.

[0330] Rickets severity score

[0331] • This is a quantitative method for assessing the severity of wrist and knee rickets based on the degree of epiphyseal wear, indentation, and the proportion of growth plates affected.

[0332] • A 10-point scale, where 10 points represent the most severe degree of rickets, and 0 points represent the absence of radiographic changes associated with rickets.

[0333] • Radiographic response following treatment for nutritional rickets can be assessed using the RSS.

[0334] • RSS values ​​are correlated with serum alkaline phosphatase (ALP) values, which are a biochemical indicator of rickets activity.

[0335] • A score of 0 indicates that rickets has completely disappeared.

[0336] Peabody Developmental Motor Scales - Second Edition (PDMS-2)

[0337] The PDMS-2 is a revised version of the original Peabody Developmental Motor Scales from 1983. The PDMS-2 consists of six subtests (reflexes, rest, movement, object manipulation, grasping, and visual-motor integration), which measure the interrelated motor abilities of children from birth to 5 years of age.

[0338] • Reflexes (8 subtests that measure a child’s ability to respond to environmental events; only measured in children from birth to 11 months of age, as reflexes are usually fully integrated by 12 months of age)

[0339] • Static (30 subtests that measure a child’s ability to keep their body within their center of gravity and maintain balance)

[0340] • Motor skills (89 subtests that measure a child’s ability to move from one place to another by crawling, walking, running, hopping on one foot and jumping forward)

[0341] • Object manipulation (24 subtests that measure a child’s ability to manipulate a ball by catching, throwing and kicking it, and are only measured in children 12 months and older)

[0342] • Grasping (26 sub-tests that measure a child’s ability to use both hands, starting with grasping objects and gradually developing into the controlled use of the fingers of both hands)

[0343] • Visual-motor integration (72 subtests that measure a child’s ability to use his or her visual perception skills to perform complex eye-hand coordination tasks, such as reaching and grasping objects, building blocks, and copying patterns.)

[0344] BOT-2 (Bunny's Proficiency Test - Version 2) - Summary Table

[0345] • A series of goal-oriented activities are conducted on participants according to a standardized procedure to measure fine or gross motor function.

[0346] • The BOT-2 consists of eight different subtests, which are divided into four motor domains for comprehensive scoring: (i) fine hand control, (ii) hand coordination, (iii) body coordination and (iv) strength and agility. These eight subtests contribute in a balanced manner to the overall motor ability score.

[0347] • The overall composite score is correlated with other athletic performance indicators (including PDMS-2).

[0348] Use of motivating agents

[0349] HPP is associated with high fracture and orthopedic / dental surgery burden, pain, mobility impairment, need for assistive walking devices, decreased functional status, and impaired activities of daily living (ADL) in children and adults. This study will assess the reasons for assistive device use, including pain, weakness, fatigue, balance, fear of falling, or fear of fracture. Information on the type of device used will also be collected (e.g., crutches, wheelchair-dependent [full-time], wheelchair-dependent [partial-time], walkers, canes, leg braces, escalators, bathroom / shower modifications, handrails / rails, or other assistive devices). Assistive device use will be continuously monitored throughout the study.

[0350] X-rays

[0351] A chest X-ray should be performed before starting methotrexate administration so that it can be used for subsequent comparisons should respiratory complications occur during the study.

[0352] Peripheral neuropathy

[0353] It can monitor patients receiving bortezomib for the development or worsening of symptoms of neuropathy, such as burning sensation, hyperesthesia, hypoesthesia, paresthesia, malaise, neuropathic pain, or weakness. Furthermore, peripheral neuropathy in participants receiving bortezomib can be assessed before and at the end of each bortezomib cycle using the Pediatric Modified Total Neuropathic Variables Scale (ped-mTNS) assessment tool.

[0354] Echocardiography and cardiac biomarkers

[0355] Acute development or exacerbation of congestive heart failure and new-onset reduction in left ventricular ejection fraction have occurred during bortezomib treatment, and have also been reported in patients without risk factors for reduced left ventricular ejection fraction. Patients with risk factors for heart disease or those who already have heart disease should be monitored frequently.

[0356] Patients receiving rituximab may experience adverse cardiac reactions, including ventricular fibrillation, myocardial infarction, and cardiogenic shock. Cardiac monitoring should be performed during and after all rituximab infusions in patients with clinically significant arrhythmias or a history of arrhythmias or angina.

[0357] Cardiotoxicity

[0358] Left ventricular ejection fraction (LVEF, based on left ventricular end-diastolic [LVED] and left ventricular end-systolic [LVES] volumes estimated using a modified biplane Simpson's rule technique) and GLS were measured using ECG and transthoracic 2D echocardiography. Drug-related cardiotoxicity in participants receiving bortezomib was monitored at the time points indicated in Table 3. In addition, cardiac safety biomarkers, namely cardiac troponin (T or I, preferably high sensitivity) and N-terminal pro-brain natriuretic peptide (NT-proBNP), were evaluated prior to bortezomib treatment and were repeated when clinically indicated (see Table 7).

[0359] Pharmacokinetics

[0360] • Blood samples will be collected to measure serum asfetoprotein alpha concentration. Samples may be collected at additional time points during the study if necessary.

[0361] • Serum samples will be used to evaluate the PK characteristics of the enzyme activity of asforase α, and may also be used to evaluate safety or efficacy aspects related to problems that arise during or after the study.

[0362] Immunogenicity assessment

[0363] • Serum samples will be collected at specific time points for ADA and NAb analysis.

[0364] • Samples will be collected prior to administration for the analysis of ADA and NAb.

[0365] Drug resistance antibody variables

[0366] ADA variables included ADA response category, incidence, and titer during the study period, as detailed below. Samples that were positive in the ADA assay were further analyzed for the presence of neutralizing activity in the NAb assay. ADA response category definitions and titer thresholds are as defined herein. The effects of ADA and / or NAb on asfetoprotein α PK / PD / biomarkers can be assessed, as well as the effects of IST on immunogenicity characteristics (e.g., ADA and / or NAb) and their corresponding impact on asfetoprotein α PK / PD / biomarkers and clinical efficacy.

[0367] ADA Response Categories

[0368] • ADA negative

[0369] • ADA positive

[0370] •NAb negative

[0371] • NAb positive

[0372] Participants who test positive for ADA will be categorized as follows:

[0373] •Impaired ADA response to IST treatment

[0374] • IST treatment enhances ADA response

[0375] Based on the duration of these responses, ADA responses that are enhanced or weakened by IST treatment will be further categorized as follows:

[0376] • Sustained response during treatment

[0377] • Uncertain responses during treatment

[0378] • Transient response during treatment

[0379] Pharmacodynamics

[0380] • Blood samples will be collected for time-point measurement of plasma PPi and PLP concentrations (containing levamisole) and ALP.

[0381] • Blood samples will be used to evaluate asifolium α PD, which may also be used to evaluate safety or efficacy aspects related to problems that arise during or after the study.

[0382] • Plasma (levamisole-containing) PLP and PPi concentrations (PD biomarkers for HPP). At pre-specified visits, observed plasma PLP and PPi concentrations and changes relative to baseline (CFB) will be summarized. Participant-level data will be presented chronologically and plotted. The same analysis will be performed on other biochemical biomarkers.

[0383] Monitoring changes in rickets and immunogenicity

[0384] Monitoring of rickets :

[0385] Radiographic evidence of rickets will be assessed using the RSS (Radiological Sequence of Surgery) at the end of each 24-week treatment period. Complete remission of rickets is defined as an RSS of 0. Worsening of rickets is defined as an increase in RSS of 1 or more points relative to baseline. Patients are considered stable if their RSS remains unchanged or decreases relative to baseline.

[0386] Monitoring of immunogenicity :

[0387] The aim of this study was to restore the pharmacological effects of asforase α on participants diagnosed with HPP and exhibiting immune-mediated LoE (related to ADA and NAb). Therefore, ADA was assessed in each participant at predetermined time points throughout the study period and analyzed using a validated assay. ADA variables included ADA response category, incidence, and titer during the study period. The ADA titer of ADA-positive samples and its impact on STRESIQ will be further characterized. ® The presence of neutralizing activity. A positive ADA response will be classified as either attenuated or enhanced IST treatment and will be further classified based on the level of response (titer threshold) and duration (continuous / transient / variable).

[0388] Participants are considered to have a significant attenuation of ADA response due to IST when ADA and / or NAb titers decrease by at least two titer steps from baseline or become negative.

[0389] Standards for IST modification (continue or stop)

[0390] In the RESTORE study, a risk-based, personalized approach was used as the basis for developing IST usage criteria. This approach evaluated the risks of untreated HPP or partially effective therapies versus the risks of using IST to improve effectiveness.

[0391] In this study, participants will receive the IST combination (methotrexate and rituximab, and / or bortezomib) for up to 104 weeks. After the initial 24 weeks, the schedule will be repeated as follows for a total of 104 weeks: Weeks 25–26 (Treatment Monitoring Board (period for reviewing clinical data and treatment decisions); Weeks 27–50 (Treatment Assessment (TA) Phase 2); Weeks 51–52 (Phase); Weeks 53–76 (TA Phase 3); Weeks 77–78 (Phase 3); Weeks 79–102 (TA Phase 4); Weeks 103–104 (Phase). Participants will continue to receive asforokinase alfa as per their initial subcutaneous administration regimen. All participants will receive folic acid concurrently to reduce potential side effects from methotrexate and intravenous immunoglobulin (IVIG) therapy, thereby providing passive immunity and minimizing the risk of infection.

[0392] According to the study design, it is anticipated that most participants recruited in this study will have a "refractory ADA response." Therefore, in these participants, downregulation of T cells (methotrexate) and B cells (rituximab) may not be sufficient to demonstrate a positive impact on efficacy. Additional ISTs such as bortezomib can help downregulate plasma cells and memory B cells and reduce ADA levels. If rickets worsens during rituximab and methotrexate treatment with an increase of 1 or more points relative to baseline and elevated ADA / NAb levels, bortezomib will be considered, and bortezomib will be added to the regimen in addition to rituximab and methotrexate. Figure 4 ).

[0393] Definition of "IST Full Response"

[0394] Regardless of the scheme used, the following criteria have been established to define “IST full response” in week 100.

[0395] • ADA or NAb titers decreased by at least two titer steps relative to baseline or became negative. ,

[0396] as well as

[0397] • Radiographic evidence of an improvement in RSS score of at least 1 or more relative to baseline.

[0398] ADA will be the primary outcome for both ADA-positive and NAb-negative patients. NAb will be the primary outcome for both ADA-positive and NAb-positive patients.

[0399] Standards for IST modification (continue or stop)

[0400] Decision trees will be used ( Figure 4 A comprehensive assessment will be conducted to determine whether to continue, modify, or discontinue the IST program.

[0401] Because methotrexate and rituximab will be administered for at least 18 months, there is no specific standard of care for methotrexate and rituximab treatment unless any safety issues are observed. Methotrexate and rituximab should be discontinued if any grade 3 or higher toxicity is reported in a participant.

[0402] If a participant demonstrates a complete response at 18 months and is receiving methotrexate and rituximab, rituximab will be discontinued, and methotrexate will continue until the end of the study. If a participant demonstrates a complete response at 18 months and is receiving methotrexate, rituximab, and bortezomib, bortezomib will be discontinued, and methotrexate and rituximab will continue until the end of the study.

[0403] Standard of treatment with bortezomib :

[0404] Participants recruited in this study who exhibited loss of efficacy were all ADA-positive at baseline and had “refractory” ADA responses. Rituximab depletes B cells, but plasma cells (which produce ADA) are not affected. Therefore, participants receiving methotrexate and rituximab with refractory ADA responses may have an inadequate response to asfort enzyme α replacement therapy (ERT). Bortezomib (Banugaria et al., PLOS One, 8: e67052, 2013) will be considered in cases of disease progression with elevated ADA and NAb levels to deplete ADA-producing plasma cells and memory B cells.

[0405] In the RESTORE study, bortezomib will only be considered after the first 24-week cycle of methotrexate + rituximab has been completed. Methotrexate + rituximab will continue to be used in combination with bortezomib.

[0406] During this study, the following decision trees and criteria were used every 24 weeks ( Figure 4 This information is used to decide whether to add bortezomib, continue using bortezomib, or stop using bortezomib.

[0407] • Bortezomib treatment should be discontinued in the event of any suspected bortezomib-related toxicity. In addition to neuropathy, bortezomib treatment must be paused in the event of any grade 3 or higher toxicity (see section 6.6.3 of HPP-407) or complete IST response.

[0408] In cases where ADA or NAb improves by at least two titer steps relative to baseline and radiographic RSS improves by one or more points relative to baseline, bortezomib will be added or continued in addition to rituximab and methotrexate.

[0409] IST Complete Response in Week 100 - Key Endpoint Components

[0410] The definition of a complete IST response in week 100 is as follows:

[0411] • ADA or NAb titers decreased by at least two titer steps relative to baseline or became negative. ,as well as

[0412] • Radiographic evidence of an RSS increase of at least one or more points relative to baseline

[0413] ADA will be the primary outcome for both ADA-positive and NAb-negative patients. NAb will be the primary outcome for both ADA-positive and NAb-positive patients.

[0414] Immunogenicity analysis

[0415] All ADA analyses will be performed using the ADA variable on the immunogenicity analysis set.

[0416] The incidence of ADA response categories will be listed and summarized in terms of absolute occurrences (n) and percentage of all participants (%). ADA and NAb titer levels will be listed, and the highest ADA and NAb titer levels for ADA-positive participants will be summarized. NAb-positive and NAb-negative participants will be grouped into the ADA-positive category and summarized in terms of absolute occurrences (n) and percentage of all participants (%).

[0417] The association between ADA response category and systemic IST exposure can be explored in all treated participants to analyze the potential impact of IST on the incidence and titer of immunogenicity and its potential impact on individual PK characteristics.

[0418] The association between ADA response categories and TEAEs and TESAEs can be explored, including TESAEs such as systemic hypersensitivity, allergic reactions, infusion / injection site reactions (ISRs) lasting more than 24 hours, and other immune-related SAEs.

[0419] The association between ADA response category and PD biomarkers can be explored in all participants to assess the potential impact of IST on the incidence and titer of immunogenicity and its potential impact on individual PD biomarker characteristics and efficacy.

[0420] Table 9: Definitions and Procedures for AE: Recording, Evaluation, Follow-up and Reporting

[0421] Definition of AE

[0422]

[0423]

[0424]

[0425] Table 10: Definition of SAE

[0426] If an event is not an AE as defined above, it cannot be an SAE even if it meets the criteria for a serious illness (e.g., hospitalization due to signs / symptoms of the disease being studied, or death due to the progression of the disease).

[0427]

[0428]

[0429] Table 11: Abbreviations and Technical Terms

[0430]

[0431]

[0432] Other implementation plans

[0433] The foregoing specific embodiments and examples are provided for clarity only. Unnecessary limitations should not be construed as derived from them. This disclosure is not limited to the exact details shown and described, as obvious variations to those skilled in the art will be included within the scope of this disclosure as defined by the claims.

[0434] Unless otherwise stated, all figures representing amounts of components, molecular weights, etc., used in the specification and claims should be understood to be modified by the term "about" in all cases. Therefore, unless otherwise stated, the numerical parameters set forth in the specification and claims are approximate values ​​that may vary depending on the desired characteristics sought to be obtained in this disclosure. At the very least, and not in an attempt to limit the doctrine of equivalence to the scope of the claims, each numerical parameter should be interpreted based on the number of significant figures reported and the application of conventional rounding techniques.

[0435] Although the numerical ranges and parameters described in this disclosure are approximate, the values ​​illustrated in the specific examples are reported as precisely as possible. However, all values ​​inherently contain ranges that are necessarily generated by the standard deviation present in their respective test measurements.

[0436] All headings are for the reader's convenience and should not limit the meaning of the text following them unless otherwise specified.

[0437] All patents, patent applications (including provisional patent applications), publications (including patent publications and non-patent publications) cited herein, including their full disclosures and electronically available materials (including nucleotide sequence submissions in, for example, GenBank and RefSeq, and amino acid sequence submissions in, for example, SwissProt, PIR, PRF, PDB, and translations of annotated coding regions from GenBank and RefSeq) are incorporated herein by reference.

Claims

1. A method of reducing the amount of an antibody specific for soluble alkaline phosphatase (sALP), reducing the risk of formation of the antibody, or attenuating the effect of the antibody in a subject receiving treatment with the sALP, the method comprising administering to the subject a therapy comprising a dihydrofolate reductase (DHFR) inhibitor and an anti-CD20 antibody or antigen-binding fragment thereof, thereby reducing the amount of the antibody, reducing the formation of the antibody, or attenuating the effect of the antibody.

2. The method of claim 1, wherein the DHFR inhibitor is methotrexate.

3. The method of claim 2, wherein the methotrexate is administered once every 5 to 10 days.

4. The method of claim 3, wherein the methotrexate is administered once every 7 days.

5. The method of any one of claims 2-4, wherein the methotrexate is administered at a dose of about 10 mg / m 2 to about 20 mg / m 2 .

6. The method of claim 5, wherein the methotrexate is administered at a dose of about 15 mg / m 2 2.

7. The method of any one of claims 1 to 6, wherein the anti-CD20 antibody is rituximab or an antigen-binding fragment thereof.

8. The method of claim 7, wherein the rituximab or antigen-binding fragment thereof is administered once every 5 to 10 days.

9. The method of claim 8, wherein the rituximab or antigen-binding fragment thereof is administered once every 7 days.

10. The method of any one of claims 7-9, wherein the rituximab or antigen-binding fragment thereof is administered at a dose of about 100 mg / m 2 to about 500 mg / m 2 .

11. The method of claim 10, wherein the rituximab or antigen-binding fragment thereof is administered at a dose of about 375 mg / m 2 2.

12. The method of any one of claims 7 to 11, wherein the rituximab or antigen-binding fragment thereof is administered intravenously.

13. The method of any one of claims 7 to 12, wherein the rituximab or antigen-binding fragment thereof is discontinued while the subject continues to receive methotrexate.

14. The method of any one of claims 1 to 13, further comprising administering a proteasome inhibitor.

15. The method of claim 14, wherein the proteasome inhibitor is bortezomib.

16. A method of reducing the amount of an antibody specific for soluble alkaline phosphatase (sALP), reducing the risk of formation of the antibody, or attenuating the effect of the antibody in a subject receiving treatment with the sALP, the method comprising administering to the subject a therapy comprising bortezomib and one or both of a dihydrofolate reductase (DHFR) inhibitor and an anti-CD20 antibody or antigen-binding fragment thereof, thereby reducing the amount of the antibody, reducing the formation of the antibody, or attenuating the effect of the antibody.

17. The method of claim 16, wherein the method comprises administering bortezomib and the DHFR inhibitor.

18. The method of claim 17, wherein the DHFR inhibitor is methotrexate.

19. The method of claim 16, wherein the method comprises administering bortezomib and the anti-CD20 antibody or antigen-binding fragment thereof.

20. The method of claim 19, wherein the anti-CD20 antibody is rituximab or an antigen-binding fragment thereof.

21. The method of any one of claims 15 to 20, wherein the bortezomib is administered twice every 5 to 10 days.

22. The method of claim 21, wherein the bortezomib is administered twice every 7 days.

23. The method of any one of claims 15 to 22, wherein the bortezomib is administered at a dose of about 0.2 mg / m 2 to about 2 mg / m 2 .

24. The method of claim 23, wherein the bortezomib is administered at a dose of about 0.5 mg / m 2 , about 0.7 mg / m 2 , about 1 mg / m 2 , or about 1.3 mg / m 2 .

25. The method of any one of claims 15-24, wherein the bortezomib is administered intravenously or subcutaneously.

26. The method of claim 25, wherein the bortezomib is administered intravenously in a bolus.

27. The method of any one of claims 15-26, wherein the bortezomib is administered after the subject exhibits a 1 or more point worsening in rickets severity score (RSS) relative to baseline.

28. The method of any one of claims 15-27, wherein the bortezomib is discontinued while the subject continues to receive the methotrexate and / or the rituximab or antigen binding fragment thereof.

29. The method of claim 28, wherein the bortezomib is discontinued after it is determined that the subject has received treatment with the antibody specific for the sALP.

30. The method of claim 29, wherein the amount of, risk of formation of, or effect of the antibody specific for the sALP has been reduced relative to prior to the treatment.

31. The method of any one of claims 1-30, wherein the therapy is administered for a duration of at least 6 months.

32. The method of claim 31, wherein the therapy is administered for a duration of at least 12, 18, or 24 months.

33. The method of any one of claims 1-32, wherein the therapy is discontinued.

34. The method of claim 33, wherein the therapy is discontinued after the subject exhibits a decrease in antibody titer of at least 2 titer steps or becomes negative and exhibits a 1 or more point increase in RSS relative to baseline.

35. The method of any one of claims 1-34, wherein the therapy further comprises administration of immunoglobulin.

36. The method of claim 35, wherein the immunoglobulin is administered once every 5 to 10 days.

37. The method of claim 36, wherein the immunoglobulin is administered once every 7 days.

38. The method of any one of claims 35-37, wherein the immunoglobulin is administered at a dose of about 300 mg / kg to about 700 mg / kg.

39. The method of claim 38, wherein the immunoglobulin is administered at a dose of about 500 mg / kg.

40. The method of any one of claims 35-39, wherein the immunoglobulin is administered intravenously.

41. The method of any one of claims 35-40, wherein the immunoglobulin is administered intravenously once a month at a dose of about 500 mg / kg, with or without rituximab, with or without bortezomib.

42. The method of any one of claims 1-41, wherein the therapy further comprises administration of folic acid.

43. The method of claim 42, wherein the folic acid is administered once a day.

44. The method of claim 42 or 43, wherein the folic acid is administered at a dose of 1 mg.

45. The method of any one of claims 42-44, wherein the folate is not administered on the same day as methotrexate.

46. The method of any one of claims 42-45, wherein the folate is administered orally.

47. The method of any one of claims 1-46, wherein the sALP is administered 1-7 times per week or once every two weeks.

48. The method of claim 47, wherein the sALP is administered 2, 3, or 6 times per week.

49. The method of any one of claims 1-48, wherein the sALP is administered at a dose of 1-10 mg / kg / week.

50. The method of claim 49, wherein the sALP is administered at a dose of 6 mg / kg / week.

51. The method of any one of claims 1-50, wherein the sALP has at least 85% sequence identity to any one of SEQ ID NOs: 1-3.

52. The method of claim 51, wherein the sALP has at least 85% sequence identity to SEQ ID NO:

1.

53. The method of claim 52, wherein the sALP has the amino acid sequence of SEQ ID NO:

1.

54. The method of claim 51, wherein the sALP has at least 85% sequence identity to SEQ ID NO:

2.

55. The method of claim 54, wherein the sALP has the amino acid sequence of SEQ ID NO:

2.

56. The method of claim 51, wherein the sALP has at least 85% sequence identity to SEQ ID NO:

3.

57. The method of claim 56, wherein the sALP has the amino acid sequence of SEQ ID NO:

3.

58. The method of any one of claims 1-57, wherein the subject has received treatment with a first sALP and a second sALP that is different from the first sALP.

59. The method of claim 58, wherein the second sALP treatment comprises administration less frequently than treatment comprising the first sALP.

60. The method of claim 58 or 59, wherein the sALP is administered to the subject receiving treatment with the sALP at a dose of 1-10 mg / kg / week, and wherein the subject also receives treatment with a second sALP that is different from the first sALP, and the second sALP is administered at 20 mg, 35 mg, or 50 mg once every 2 weeks.

61. The method of any one of claims 58-60, wherein the first sALP has the amino acid sequence of SEQ ID NO:

1.

62. The method of any one of claims 58-61, wherein the second sALP has the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO:

3.

63. The method of claim 62, wherein the second sALP has the amino acid sequence of SEQ ID NO:

2.

64. The method of any one of claims 1-63, wherein administration of the sALP is discontinued during administration of the therapy.

65. The method of claim 64, wherein administration of the sALP is discontinued for about 4 weeks.

66. The method of any one of claims 1-65, further comprising monitoring the level of the antibody by detecting the presence of the antibody in a biological sample from the subject.

67. The method of claim 66, wherein the biological sample is blood, plasma, or urine.

68. The method of any one of claims 1-67, further comprising determining whether the subject exhibits a decrease in efficacy of the sALP.

69. The method of claim 68, wherein the determination of a decrease in efficacy comprises measuring a metric, wherein an increase or decrease in the metric relative to a baseline indicates the decrease in efficacy of the sALP.

70. The method of claim 68 or 69, wherein the determination of a decrease in efficacy comprises measuring one or more of blood and / or urine levels of inorganic pyrophosphate (PPi) and 5'-phosphopyridoxyl (PLP).

71. The method of claim 70, wherein the levels of PPi and / or PLP are elevated relative to the baseline.

72. The method of any one of claims 68-71, wherein the determination of a decrease in efficacy comprises measuring one or more symptoms selected from the group consisting of: rickets, hypercalciuria, skeletal deformities, waddling gait, bone pain, bone fractures, calcium phosphate dihydrate crystal deposition, arthritis, pyrophosphate arthropathy, chondrocalcinosis, calcific periarthritis, pseudofracture, skeletal deformities, hypotonia, muscle weakness, rheumatoid complications, arthritis, pseudogout, difficulty walking, pain, premature tooth loss, pulmonary hypoplasia, respiratory insufficiency, seizures, stature, growth, rickets, and immunogenicity.

73. The method of claim 72, wherein the subject exhibits a worsening of one or more of the symptoms relative to the baseline.

74. The method of any one of claims 68-73, wherein the determination of a decrease in efficacy comprises confirming that the subject does not exhibit poor treatment adherence or improper injection technique or does not have a vitamin D deficiency, malnutrition, or a concomitant illness.

75. The method of any one of claims 68-74, wherein the determination of a decrease in efficacy comprises performing a quality of life assessment.

76. The method of claim 75, wherein the quality of life assessment is selected from one or more of the following: EuroQol five-dimensional questionnaire, Pediatric Quality of Life Inventory, Pediatric Outcomes Data Collection Instrument, Child Health Utility Index-9D, Pediatric Quality of Life Inventory, Short Form Health Survey 36, and Short Form Health Survey 12.

77. The method of claim 76, wherein the subject exhibits a decreased quality of life assessment score relative to the baseline.

78. The method of any one of claims 68-77, wherein the determining of decreased efficacy comprises performing a physical performance measure assessment.

79. The method of claim 78, wherein the physical performance measure assessment is selected from one or more of the following: a six-minute walk test (6MWT), the Bruininks-Oseretsky Test of Motor Proficiency, Second Edition (BOT-2), the Bayley Scales of Infant and Toddler Development, Third Edition (BSID-III), gait analysis, use of a walking aid, the Peabody Developmental Motor Scales 2 (PDMS-2), or x-ray.

80. The method of claim 79, wherein the subject exhibits a decreased physical performance measure assessment score relative to the baseline.

81. The method of any one of claims 69-80, wherein the baseline is calculated based on a value of the measure in the subject prior to the occurrence of decreased efficacy of the sALP, in a reference subject who receives sALP treatment but does not have a neutralizing antibody to sALP, or in a reference subject who is not administered sALP.

82. The method of any one of claims 1-81, wherein the method further comprises determining whether the antibody is a neutralizing antibody.

83. The method of claim 82, wherein the method further comprises testing the neutralizing antibody for an effect on one or both of catalytic activity and bone targeting of the sALP in the presence of the neutralizing antibody.

84. The method of claim 83, wherein the subject exhibits decreased catalytic activity and / or bone targeting of the sALP.

85. The method of claim 83 or 84, wherein the testing comprises one or more of a screening assay, a confirmatory assay, a titration assay, and a neutralizing antibody assay.

86. The method of any one of claims 1-85, wherein the subject has a bone mineralization disorder.

87. The method of claim 86, wherein the bone mineralization disorder is hypophosphatasia (HPP).

88. The method of any one of claims 1-87, wherein the subject is a juvenile, an adult, an infant, or a neonate.

89. The method of any one of claims 1-88, wherein the subject has a bone fracture, osteoporosis, osteopetrosis, chondrocalcinosis, hypotonia, Duchenne muscular dystrophy, tracheobronchial cartilage malacia, seizures, neurofibromatosis 1 (NF-1), craniosynostosis, or a myasthenic disease.

90. The method of claim 89, wherein the myasthenic disease is calcium pyrophosphate deposition (CPPD) or familial hypophosphatemia.

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