EP4757810A1 - Immunosuppressive therapy to mitigate anti-soluble alkaline phosphatase immune response - Google Patents

Abstract

The disclosure features methods for treating neutralizing antibodies that reduce the efficacy of soluble alkaline phosphatase treatment (e.g., asfotase alfa), an enzyme replacement therapy, such as for the treatment of bone mineralization disorders, e.g., hypophosphatasia (HPP). The methods include diagnosing a subject as having the presence of neutralizing antibodies that affect the efficacy of treatment, administering a treatment suitable for reducing the deleterious effects of neutralizing antibodies, and continuing alkaline phosphatase treatment.

Description

IMMUNOSUPPRESSIVE THERAPY TO MITIGATE ANTI-SOLUBLE ALKALINE PHOSPHATASE IMMUNE RESPONSE

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 63/531 ,955, filed August 10, 2023, the contents of which is incorporated by reference herein in its entirety.

BACKGROUND

Hypophosphatasia (HPP) is a rare, heritable bone mineralization disorder with an incidence of 1 per 100,000 births for the most severe forms of the disease. The disorder results from loss-of- function mutations in the gene coding for tissue-nonspecific alkaline phosphatase (TNALP) HPP exhibits a remarkable range of symptoms and severity, from rickets to almost complete absence of bone mineralization in utero.

STRENSIQ® (asfotase alfa) has been approved as the first enzyme replacement therapy (ERT) to treat HPP. Enzyme replacement therapies can result in the development in some subjects of neutralizing antibodies to the enzyme. Methods for determining whether neutralizing antibodies result in a loss of efficacy of the enzyme are needed, as are methods for treating antibody-mediated loss of efficacy in subjects with bone mineralization disorders when it occurs

SUMMARY OF THE DISCLOSURE

A first aspect of the disclosure features a method of reducing an amount of cellular and humoral immune response components that will lead to decreasing the risk of formation or reducing an effect of an antibody or T cells specific for soluble alkaline phosphatase (sALP) in a subject treated with the sALP. The method includes administering a therapy that includes one or more of a dihydrofolate reductase (DHFR) inhibitor and an anti-CD20 antibody or antigen-binding fragment thereof to the subject that reduces the amount, decreases the formation, or reduces the effect of the anti-sALP immune response.

In another aspect, featured is a method of reducing an amount, decreasing the risk of formation, or reducing an effect, of an antibody specific for a sALP in a subject treated with the sALP. The method includes administering a therapy that includes bortezomib and one or both of a DHFR inhibitor and an anti-CD20 antibody or antigen-binding fragment thereof to the subject that reduces the amount, decreases the formation, or reduces the effect, of the antibody The method may include administering bortezomib and the DHFR inhibitor. The method may include administering bortezomib and the anti-CD20 antibody or antigen bonding fragment thereof.

In some embodiments, the method includes administration of a DHFR inhibitor. In some embodiments, the DHFR inhibitor is methotrexate. The methotrexate may be administered once every 5 to 10 days (e g., once every 5, 6, 7, 8, 9, or 10 days, e g., once every 7 days). For example, methotrexate may be administered once every 7 days to a subject in need thereof. The methotrexate may be administered at a dosage from about 10 mg/m² to about 20 mg/m² (e g , about 10 mg/m², 11 mg/m², 12 mg/m², 13 mg/m², 14 mg/m², 15 mg/m², 16 mg/m², 17 mg/m², 18 mg/m², 19 mg/m², or 20 mg/m², e.g., about 15 mg/m²). For example, methotrexate may be administered at a dosage of about 15 mg/m²

In some embodiments, the method includes administration of an anti-CD20 antibody or antigen-binding fragment thereof In some embodiments, the anti-CD20 antibody is rituximab or an antigen-binding fragment thereof. Rituximab may be administered once every 5 to 10 days (e.g. , once every 5, 6, 7, 8, 9, or 10 days, e.g , once every 7 days) For example, rituximab may be administered once every 7 days to a subject in need thereof. The rituximab or antigen-binding fragment thereof may be administered at a dosage from about 100 mg/m² to about 500 mg/m² (e.g., about 100 mg/m², 125 mg/m², 150 mg/m², 175 mg/m², 200 mg/m², 225 mg/m², 250 mg/m², 275 mg/m², 300 mg/m², 325 mg/m², 350 mg/m², 375 mg/m², 400 mg/m², 425 mg/m², 450 mg/m², 475 mg/m², or 500 mg/m², e g , about 375 mg/m²) For example, rituximab may be administered at a dosage of about 375 mg/m². The rituximab or antigen-binding fragment thereof may be administered intravenously. In some embodiments, the rituximab or antigen-binding fragment thereof is discontinued while the subject is being treated with methotrexate. In other embodiments, the methotrexate and the rituximab or antigen-binding fragment thereof are administered in combination or are administered separately.

In some embodiments, the method further includes administering a proteasome inhibitor, such as bortezomib, to a subject in need thereof Plasma cells may be specifically targeted via at least one mechanism of action of bortezomib, thereby offering a benefit versus other treatment modalities which control certain pre-B cells and/or B-cells but fail to target plasma cells, thereby resulting in circulating antibodies and therapy failure. The bortezomib may be administered once every 5 to 10 days (e.g., once every 5, 6, 7, 8, 9, or 10 days, e.g., once every 7 days). For example, bortezomib may be administered once every 7 days The bortezomib may be administered at a dosage of about 0 2 mg/m² to about 2 mg/m² (e.g., about 0.2 mg/m², 0.3 mg/m², 0.4 mg/m², 0. 5 mg/m², 0 6 mg/m², 0 7 mg/m², 0 8 mg/m², 0 9 mg/m², 1 mg/m², 1 1 mg/m², 1 2 mg/m², 1 3 mg/m², 1 4 mg/m², 1.5 mg/m², 1.6 mg/m², 1.7 mg/m², 1 8 mg/m², 1.9 mg/m², or 2 mg/m². For example, bortezomib may be administered at a dosage of about 0.5 mg/m², about 0.7 mg/m², about 1 mg/m², or about 1.3 mg/m². The bortezomib may be administered intravenously or subcutaneously. The bortezomib may be administered intravenously in a bolus. The bortezomib may be administered after the subject exhibits a worsening of a rickets severity score (RSS) of 1 or more points from baseline (e.g., corresponding to a value of the subject prior to treatment with a sALP or prior to treatment with a therapy as described herein) In specific embodiments, the RSS is calculated during screening for the study, prior to commencing therapy, and this is considered a baseline score for RSS. The bortezomib may be discontinued while the subject is being treated with methotrexate and/or rituximab.

In some embodiments, the anti-sALP antibody therapy (e.g., the methotrexate, rituximab, and/or the proteasome inhibitor, e.g., bortezomib) is administered for 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, the DHFR inhibitor (e.g., methotrexate) is administered for a duration of at least 6, 12, 18, or 24 months In some embodiments, the anti-CD20 antibody or antigen-binding fragment (e.g., rituximab) is administered for a duration of at least 6, 12, 18, or 24 months. In some embodiments, the therapy is discontinued, e.g., after the subject exhibits a decrease in antibody titer by at least 2 titer steps or becomes negative (e.g., undetectable) and/or exhibits an improvement in an RSS of at least 1 or more points from baseline. For example, the therapy may be discontinued when the amount of the antibody specific for the sALP, its risk of formation, or its effect has been reduced relative to prior to the treatment.

The anti-sALP antibody therapy may further include administering immunoglobulin. The immunoglobulin may be administered once every 5 to 10 days (e.g , once every 5, 6, 7, 8, 9, or 10 days, e.g., once every 7 days). For example, the immunoglobulin may be administered once every 7 days to a subject in need thereof. The immunoglobulin may be administered at a dosage from about 300 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, e g , about 500 mg/kg) For example, the immunoglobulin may be administered at a dosage of about 500 mg/kg. The immunoglobulin may be administered intravenously. The immunoglobulin may be administered intravenously at a dosage of about 500 mg/kg once a month while receiving methotrexate and/or rituximab with or without a proteasome inhibitor (e.g., bortezomib).

The anti-sALP antibody therapy may further include administering folic acid. The folic acid may be administered, e.g., once per day. The folic acid may be administered at a dosage of 1 mg. The folic acid may not be administered on the same day as methotrexate The folic acid may be administered orally.

The methods described herein may be performed in conjunction with sALP therapy, e.g., administration of a sALP The therapy may be performed prior to administration of a sALP therapy or after administration of a sALP therapy. The sALP may be administered 1 to 7 (e.g., 1 , 2, 3, 4, 5, 6, or 7) times per week or every two weeks, e.g., 2, 3, or 6 times per week. For example, the sALP may be administered 3 or 6 times per week. The sALP may be administered at a dosage of 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). For example, the sALP may be administered at a dosage of 6 mg/kg/week. In particular, the sALP administered according to the doses noted above may have at least 85% sequence identity to (e.g., at least 90%, 95%, 99% sequence identity to), or the sequence of, SEQ ID NO: 1.

The sALP may have at least 85% sequence identity to any one of SEQ ID NOS: 1-3. For example, the sALP may have at least 85%, 90%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO: 1. The sALP may include or consist of the sequence of SEQ ID NO: 1 (e.g., asfotase alfa). The sALP may have at least 85%, 90%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO: 2. The sALP may include or consist of the sequence of SEQ ID NO: 2. The sALP may have at least 85%, 90%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO: 3. The sALP may include or consist of the sequence of SEQ ID NO: 3.

The methods described herein may include treating a subject that has been administered two different sALPs In some embodiments, the subject has been treated with a first sALP and a second sALP that is 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, an initial therapy administered to the subject may include doses of an sALP of SEQ ID NO: 1 . The subject may be administered weekly doses of the sALP of several days, weeks, months, or years Subsequently, the subject may be transitioned to a different sALP (e.g., an sALP of SEQ ID NO: 2 or 3). The subject may be treated with the different sALP for several days, weeks, months, or years. Alternatively, the sALP administered to the subject may alternate between any one of SEQ ID NOs: 1-3. When treated with the sALP of SEQ ID NO: 1 , the subject may be administered a dose of 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) For example, administration of the sALP of SEQ ID NO: 1 may be at 1 mg/kg 6 times a week, 2 mg/kg 3 times a week, or 3 mg/kg 2 times a week. In specific embodiments, dosing is 3 mg/kg 3 times a week or is increased from 3 mg/kg 2 times a week to 3 mg/kg 3 times a week. Vials are prepared at up to 100 mg/ml such as 40 mg/ml or 100 mg/ml. When treated with the sALP of SEQ ID NO: 2 or 3, the subject may be administered a dose of 20 mg, 35 mg, or 50 mg once every 1 or 2 weeks (e.g , once every 2 weeks). In some embodiments, the pharmaceutical composition containing the sALP of SEQ ID NO: 2 or 3 is administered subcutaneously or intravenously. The composition may be administered in a volume of, e g , 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, 04 mL, 0.3 mL, 0.2 mL, or 0.1 mL, or in a volume in a range of from about 5 mL to about 0.1 mL). The sALP may be discontinued during administration of the anti-sALP antibody therapy. For example, administration of the sALP may be discontinued for about 1 week, 2 weeks, 3 weeks, 4 weeks, or longer (e.g., until there is an improvement in a metric used to assess an effect of an anti- sALP antibody (e.g., an RSS of the subject) and/or until there is a reduction in the level of the anti- sALP antibody in the subject (e.g., a reduction to an undetectable level), e.g., in a biological fluid of the subject, such as blood, serum, or urine

In some embodiments, the second sALP treatment comprised administration that was less frequent than the treatment comprising the first sALP. In some embodiments, the subject treated with the sALP was administered the sALP at a dosage of 1 mg/kg/week to 10 mg/kg/week, and wherein the subject was also treated with a second sALP, different from the first sALP, and the second sALP was administered at 20 mg, 35 mg, or 50 mg once every 2 weeks.

In some embodiments, the methods described herein further include monitoring a level of the antibody by testing a biological sample (e g , blood, plasma, or urine) from the subject for the presence of the antibody.

The bioanalytical methods described herein may further include determining if the subject is suffering from a reduction in efficacy of the sALP. A reduction in efficacy may be determined by, e g., measuring a metric, in which an increase or decrease in the metric relative to a baseline indicates that the efficacy of the sALP is reduced. For example, a reduction in efficacy may be determined by measuring one or more of blood and/or urine levels of inorganic pyrophosphate (PPi) and pyridoxal 5'- phosphate (PLP) (e g , relative to the baseline) For example, the levels of PPi and/or PLP may be increased relative to the baseline (e.g., by 25% or more, e.g., 30%, 35%, 40%, 45%, 50%, 55%, 60%, or more than 60%) relative to PPi and/or PLP concentrations in a plasma sample from the subject prior to the observed reduction in efficacy of the sALP).

A reduction in efficacy may be determined by, e.g., measuring one or more symptoms selected from the group consisting of hypomineralization, hypercalcinuria, skeletal deformity, waddling gait, bone pain, bone fracture, calcium pyrophosphate dihydrate crystal deposition, arthritis, pyrophosphate arthropathy, chondrocalcinosis, calcific periarthritis, pseudofracture, skeletal deformities, hypotonia, muscle weakness, rheumatoid complications, arthritis, pseudogout, ambulatory difficulties, pain, premature loss of teeth, pulmonary hypoplasia, respiratory insufficiency, seizures, stature, growth, rickets, and immunogenicity. The subject may exhibit a worsening (e.g., an increase in frequency or duration) of one or more of the symptoms relative to the baseline. The determination may include establishing that the subject does not exhibit poor treatment adherence or inappropriate injection technique and/or does not suffer from a vitamin D deficiency, undernutrition, or concomitant disease.

A reduction in efficacy may be determined by, e.g., performing a quality-of-life assessment. The quality-of-life assessment may be selected from one or more of EuroQol Five Dimension Questionnaire, Childhood Health Assessment Questionnaire, Pediatric Outcomes Data Collection Instrument, Child Health Utility lndex-9D, Pediatric Quality of Life Inventory, Short Form Health Survey 36, and Short Form Health Survey 12 The subject may exhibit a reduced quality-of-life assessment score relative to the baseline

A reduction in efficacy may be determined by performing a physical metric assessment. The physical metric assessment may be selected from one or more of a Six Minute Walk Test (6MWT), Bruininks-Oseretsky Test of Motor Proficiency 2nd Edition (BOT-2), Bayley Scales of Infant and Toddler Development, 3rd Edition (BSID-III), gait analysis, use of a mobility aid, Peabody Developmental Motor Scales 2 (PDMS-2), or x-ray. The subject may exhibit a reduced physical metric assessment score relative to the baseline

In some embodiments, the baseline is calculated based on a value of the metric in the subject prior to suffering from a reduction in efficacy of the sALP, in a reference subject treated with a sALP and in whom no neutralizing antibody against a sALP is detected, or in a reference subject not administered a sALP.

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

In any of the above embodiments, the subject may have a bone mineralization disorder, such as hypophosphatasia (HPP). The subject may be a juvenile, adult, infant, or neonate. The subject may have bone fracture, osteoporosis, sclerosteosis, chondrocalcinosis, hypotonia, Duchenne's muscular dystrophy, tracheobronchomalacia, seizure, neurofibromatosis 1 (NF-1), craniosynostosis or a muscle weakness disease, such as calcium pyrophosphate deposition (CPPD) or familial hypophosphatemia. In some embodiments, the subject has a defined immunogenicity status, e.g., positive for antidrug antibody (ADA+), negative for antidrug antibody (ADA-), positive for neutralizing antibody (NAb+), and/or negative for neutralizing antibody (NAb-) The subject may be ADA+ and Nab-

DEFINITIONS

The term “about,” as used herein refers to a value that is ± 10% of a reference value.

As used herein, when a polypeptide or nucleic acid sequence is referred to as having “at least X% sequence identity” to a reference sequence, wherein “X” is a real number, it is meant that at least X percent of the amino acid residues or nucleotides in the polypeptide or nucleic acid are identical to those of the reference sequence when the sequences are optimally aligned An optimal alignment of sequences can be determined in various ways that are within the skill in the art, for instance, the Smith Waterman alignment algorithm (Smith et al., J. Mol. Biol. 147:195-7, 1981 ) and BLAST (Basic Local Alignment Search Tool; Altschul et al., J. Mol. Biol. 215: 403-10, 1990). These and other alignment algorithms are accessible using publicly available computer software such as “Best Fit” (Smith and Waterman, Advances in Applied Mathematics, 482-489, 1981) as incorporated into GeneMatcher Plus (Schwarz and Dayhoff, Atlas of Protein Sequence and Structure, Dayhoff, M.O., Ed pp 353-358, 1979), BLAST, BLAST-2, BLAST-P, BLAST-N, BLAST-X, WU-BLAST-2, ALIGN, ALIGN-2, CLUSTAL, Megalign (DNASTAR), or other software/hardware for alignment. In addition, those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve optimal alignment over the length of the sequences being compared

The terms “patient” or “subject” refer to a mammal, including, but not limited to, a human or a non-human mammal, such as a bovine, equine, canine, ovine, or feline. “Patient” in particular refers to a human.

The terms “sALP,” “soluble alkaline phosphatase,” and “extracellular domain of an alkaline phosphatase” are used interchangeably (unless the context indicates otherwise) to mean a soluble, non-membrane-bound alkaline phosphatase or a biologically active fragment or variant thereof. sALPs include, for example, an alkaline phosphatase lacking a C-terminal GPI signal sequence, and additional variants and analogs thereof which retain alkaline phosphatase activity, e.g., the ability to hydrolyze PPi or other natural or artificial substrate(s). This includes soluble fragments corresponding to the extracellular domains of TNSALP, PALP, GLALP, and IALP and biologically active fragments or variants thereof, unless specified otherwise A mature sALP lacks the GPI membrane anchor and the signal peptide, which is cleaved during processing.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an example flow-chart of assays of various embodiments, as described herein.

FIG. 2 is a schematic drawing showing the overall study design and treatment algorithm for immunosuppressive therapy. (*The Treatment and Monitoring Board (TMB) will consist of HPP clinical advisor(s), immunology expert(s), site investigator(s), and medical monitor (sponsor); TMB: Treatment and Monitoring Board; MTX: methotrexate; RTX: rituximab; BTZ: bortezomib; E: enrollment; B: baseline: EOS: End of study). FIG. 3 is a schematic drawing showing the overall study design and treatment algorithm for immunosuppressive therapy. (*Co-administration of intravenous immunoglobulin (IVIG) 500 mg/kg once monthly while receiving rituximab +/- bortezomib; **Laboratory, radiography; TMB: Treatment and Monitoring Board)

FIG. 4 is a schematic drawing showing a decision tree that can be used to assess whether to continue, modify, or stop immunosuppressive therapy, e g., therapy with one or more of methotrexate, rituximab, and/or a proteasome inhibitor, e.g., bortezomib.

DETAILED DESCRIPTION

Subjects with bone mineralization disorders, such as hypophosphatasia (HPP), undergoing enzyme replacement therapy (ERT) treatment with soluble alkaline phosphatase (sALP) may develop antibodies that bind to the sALP. In certain subjects, these antibodies are neutralizing antibodies that may reduce the efficacy of the sALP that is administered during ERT. Accordingly, disclosed are methods of treating subjects with bone mineralization disorders, such as HPP, that develop neutralizing antibodies. The methods of treatment are known as immune tolerance induction (ITI), immune tolerance therapy (ITT), immune tolerance regimen (ITR), or immunosuppressive therapy (1ST) (collectively referred to hereafter as “1ST”) Methods of 1ST include reducing neutralizing antibody titers and/or preventing or reducing the formation of neutralizing antibodies.

The presence of neutralizing antibodies in a subject can be assessed by using an assay as described herein. Not all antibodies that bind to a sALP therapeutic, such as asfotase alfa, reduce the efficacy of the sALP in a subject (e.g., an HPP subject) treated with ERT. For example, the antibodies can bind to a domain of the sALP (e.g., asfotase alfa) that may not affect its therapeutic function (e.g., the ability to promote bone mineralization). Therefore, as described herein, one or more additional assays may be used to determine if the antibodies are both neutralizing and reduce the efficacy of the sALP.

Also featured are methods for identifying a subject receiving sALP ERT as being in need of 1ST therapy by using one or more assays to determine the presence of anti-sALP antibodies and, if necessary, one or more assays to determine if the antibodies are neutralizing and reduce the efficacy of the sALP. The 1ST therapy can also be administered to naive subjects that have not been administered a sALP therapeutic if there is a reason to expect that such a subject would generate neutralizing anti-sALP antibodies Furthermore, the subjects may include those who may have normal or non-elevated antibody titers prior to the 1ST therapy, but who may, nonetheless, be at risk of developing high neutralizing antibody titers in the future.

The methods described herein may be used to treat a subject undergoing sALP therapy. The subject may have a bone mineralization disorder, such as HPP, or may have bone fracture, osteoporosis, sclerosteosis, chondrocalcinosis, hypotonia, Duchenne’s muscular dystrophy, tracheobronchomalacia, seizure, neurofibromatosis 1 (NF-1 ), craniosynostosis or a muscle weakness disease, such as calcium pyrophosphate deposition (CPPD) or familial hypophosphatemia 1ST therapeutic agents

Helper T-cells are essential in the development of most ADAs, while memory B-cells and long-lived plasma cells amplify and maintain the responses that constitute the basis for using combination ISTs when there is loss of efficacy due to the presence of ADAs/NAbs. Therapeutic proteins, such as asfotase alfa, may be endocytosed by antigen-presenting cells following administration, where they are processed into their component peptides. A subset of these peptides may then be presented to helper T-cells specific for the peptide in the context of human leukocyte antigen (HLA) molecules. Helper T-cells signal and activate Ag specific B-cells to proliferate and differentiate into memory B-cells and Ab secreting plasma cells (both short- and long-lived)

The ISTs described herein include one or more of methotrexate, rituximab, and bortezomib, which may be used based on their action on helper T cells, plasma cells, and memory B cells. Methotrexate, an inhibitor of dihydrofolate reductase, impacts rapidly dividing T and B cells by preventing the reduction of dihydrobiopterin (BH2) to tetrahydrobiopterin (BH4), leading to nitric oxide synthase uncoupling and increased sensitivity of T cells to apoptosis, thereby diminishing immune responses.

Rituximab is a chimeric monoclonal antibody that targets cell-surface antigen of human B lymphocytes (CD20), a transmembrane protein present on virtually all B cells from the stage at which they become committed to B-cell development until it is down regulated when they differentiate into antibody-secreting plasma cells. Rituximab causes prompt and nearly complete depletion of peripheral mature B and pre-B cells, which do not reappear for approximately 6 months in most participants. CD20 is lost when B cells differentiate into antibody-producing plasma cells. Thus, once B cells differentiate into plasma cells, alternative and/or additional agents must be utilized to address ADAs generated by circulating plasma cells

Bortezomib is a proteasome inhibitor Proteasome inhibitors are a powerful and specific way to target plasma cells, which are highly dependent on proteasome activity. Bortezomib is a reversible proteasome inhibitor that targets both short- and long-lived plasma cells due to their high rates of immunoglobulin production.

Described herein are methods of administering an 1ST to a subject. In a specific embodiment, 1ST is administered prophylactically to avoid the generation of an immune response, such as one, two, or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or more) days prior to administration of a sALP. In other embodiments, 1ST is administered to a subject, e g , following a determination that the subject has anti-sALP antibodies that are interfering with ERT. Following the diagnosis of a subject as having anti-sALP antibodies and validation that the antibodies are neutralizing, 1ST therapy can be administered to a subject experiencing a reduction in or loss of efficacy of sALP or are at risk of a reduction in or loss of efficacy. 1ST therapy involves reducing the presence, or the negative impact, of neutralizing antibodies produced by the subject against a sALP therapeutic (e.g., asfotase alfa). Additionally, 1ST can be assessed for its safety and tolerability by the subject. This may, optionally, be accomplished by administering small starting doses before the full course of treatment is administered. If the initial 1ST therapy is not safe and/or tolerable by the subject, the 1ST therapy may be discontinued. 1ST therapy involves identifying the appropriate cellular and molecular targets of tolerance inducing therapies, such as the cellular and molecular mechanisms that generate the neutralizing antibody Given the preponderance of neutralizing antibodies of the lgG1 and lgG4 isotypes, immunoglobulin responses to protein therapeutics may arise from classic T-helper-dependent B-cell mechanisms. Thus, appropriate cellular targets for tolerance induction include antigen-specific T and B cells and antigen-presenting cells. The antibody response may also require the participation of T- cell help after the initiation of a response.

The 1ST may include, for example, administering a therapy that includes a dihydrofolate reductase (DHFR) inhibitor and an anti-CD20 antibody or antigen-binding fragment thereof to the subject that reduces the amount, decreases the formation, or reduces the effect, of the antibody The DHFR inhibitor may be, e.g., methotrexate. The anti-CD20 antibody may be, e.g., rituximab or an antigen-binding fragment thereof. The method may further include administering a proteasome inhibitor, such as bortezomib, to the subject

The DHFR inhibitor may be methotrexate. The methotrexate may be administered once every 5 to 10 days (e.g., once every 5, 6, 7, 8, 9, or 10 days, e.g., once every 7 days). For example, methotrexate may be administered once every 7 days. The methotrexate may be administered at a dosage of about 10 mg/m² to about 20 mg/m² (e g , 10 mg/m², 11 mg/m², 12 mg/m², 13 mg/m², 14 mg/m², 15 mg/m², 16 mg/m², 17 mg/m², 18 mg/m², 19 mg/m², or 20 mg/m², e.g., about 15 mg/m²). For example, methotrexate may be administered at a dosage of 15 mg/m².

The anti-CD20 antibody may be rituximab or an antigen-binding fragment thereof. The rituximab or antigen-binding fragment thereof may be administered once every 5 to 10 days (e.g., once every 5, 6, 7, 8, 9, or 10 days, e.g , once every 7 days) For example, rituximab may be administered once every 7 days. The rituximab or antigen-binding fragment thereof may be administered at a dosage from about 100 mg/m² to about 500 mg/m² (e g , 100 mg/m², 125 mg/m², 150 mg/m², 175 mg/m², 200 mg/m², 225 mg/m², 250 mg/m², 275 mg/m², 300 mg/m², 325 mg/m², 350 mg/m², 375 mg/m², 400 mg/m², 425 mg/m², 450 mg/m², 475 mg/m², or 500 mg/m², e.g., about 375 mg/m²). For example, rituximab may be administered at a dosage of 375 mg/m². The rituximab or antigen-binding fragment thereof may be administered intravenously. In some embodiments, the rituximab or antigen-binding fragment thereof is discontinued while the subject continues to receive the methotrexate and/or the rituximab or antigen-binding fragment thereof.

1ST therapy may be administered for a predetermined amount of time (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 reduction in efficacy observed for the sALP. Alternatively, the 1ST therapy may continue until it has been determined that the 1ST therapy is successful in treating or reducing the titer of neutralizing antibodies (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction). 1ST therapies are described herein and include administration of therapeutic agents, such as an immunomodulatory agent or optionally, a proteasome inhibitor.

A subject may be selected for treatment with 1ST The subject may commence receiving methotrexate and rituximab, e.g., optionally with bortezomib, for an initial time course, e.g., of 24 weeks. Following this initial time course (e.g., 24-week period), the treatment status and ADA or NAb status of the subject may be determined (using an assessment metric as described herein). sALP therapy may or may not continue in parallel with 1ST. The subject may continue to undergo subsequent time courses of treatment (e g , of a selected duration, such as for 24 weeks) and may be assessed for treatment status and ADA or NAb status, e.g., to determine the benefit or risk of continuing the 1ST treatment regimen.

While undetectable titers alone of anti-sALP antibodies may be sufficient to cease the 1ST, if no return of sALP ERT efficacy is observed in a subject with undetectable anti-sALP antibodies, a second metric can be used to ascertain if and when to withdraw the 1ST.

In some embodiments, the therapy may be discontinued, e.g , after the subject exhibits a decrease in an anti-sALP antibody titer

Proteasome inhibitors

1ST therapeutic agents may optionally include the administration of one or more proteasome inhibitors, such as bortezomib. Proteasome inhibitors may be used to induce plasma cell depletion. The proteasome inhibitor may optionally be administered in an amount and under such conditions to make it possible to achieve a reduction in antibodies specific for the sALP (e.g., neutralizing anti-sALP antibodies that reduce the efficacy of sALP ERT)

Proteasome inhibitors that may be used with the methods described herein are disclosed, for example, in U.S. Pat. No. 7,531 ,526, the disclosure of which is hereby incorporated by reference in its entirety. Examples of suitable proteasome inhibitors include without limitation the following compounds, as well as pharmaceutically acceptable salts and boronate esters thereof: N-(4- morpholine)carbonyl-p-(1-naphthyl)-L-alanine-L-leucine boronic acid, N-(8-quinoline)sulfonyl-p-(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-(0-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.

A proteasome inhibitor may be used in vitro or in vivo. For example, a proteasome inhibitor can be used in vitro in conjunction with an assay to determine whether it is a safe and effective 1ST therapy component. Furthermore, a proteasome inhibitor can be used in vivo for 1ST therapy in a subject in need thereof (e.g., a human). A proteasome inhibitor can be administered by any number of known routes, including orally, intravenously, intramuscularly, subcutaneously, intrathecally, topically, and by infusion (Platt et al., U.S. Pat. No. 4,510,130; Badalamente et al., Proc. Natl. Acad. Sci. U.S.A. 86:5983-5987, 1989; Staubli et al., Brain Research 444:153-158 (1988)) and can generally be administered in combination with a physiologically acceptable carrier (e.g., saline). The effective quantity of inhibitor given can be determined empirically and can be based on such considerations as the particular proteasome inhibitor used, the condition of the individual, and the size and weight of the individual. The proteasome inhibitor may be bortezomib (e g., VELCADE®), the monomeric boronic acid [(1 R)-3-methyl-1-[[(2S)-1-oxo-3-phenyl-2-[(pyrazinylcarbonyl)amino]propyl]amino]butyl]boronic acid having the structure:

The bortezomib may be administered once every 5 to 10 days (e.g., once every 5, 6, 7, 8, 9, or 10 days, e.g., once every 7 days). For example, bortezomib may be administered once every 7 days The bortezomib may be administered at a dosage of about 0 2 mg/m² to about 2 mg/m² (e g , 0.2 mg/m², 0.3 mg/m², 0.4 mg/m², 0. 5 mg/m², 0.6 mg/m², 0.7 mg/m², 0.8 mg/m², 0.9 mg/m², 1 mg/m², 1.1 mg/m², 1.2 mg/m², 1.3 mg/m², 1.4 mg/m², 1.5 mg/m², 1.6 mg/m², 1.7 mg/m², 1.8 mg/m², 1.9 mg/m², or 2 mg/m². For example, bortezomib may be administered at a dosage of about 0.5 mg/m², about 0.7 mg/m², about 1 mg/m², or about 1.3 mg/m². The bortezomib may be administered intravenously or subcutaneously. The bortezomib may be administered intravenously in a bolus. The bortezomib may be administered after the subject exhibits a worsening of a rickets severity score (RSS) of 1 or more points from baseline The bortezomib may be discontinued while the subject continues to receive methotrexate and/or rituximab.

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

Optimum dosing regimens of a proteasome inhibitor can be determined by one skilled in the art and can vary with the proteasome inhibitor, the subject, and the effect sought. For example, the inhibitor can be administered intravenously in an amount of about 0.01 mg/m²to about 200 mg/m², e.g., about 0.01 mg/m² to about 2 mg/m², e.g., about 0.2 mg/m² to about 2 mg/m², with various dosing schedules which may involve, for example, twice weekly dosing. A proteasome inhibitor can also be administered in doses generally ranging from about 0.01 mg/m² to about 200 mg/m², e.g., about 0.01 mg/m² to about 2 mg/m², e g., about 0.6 mg/m² to about 1.5 mg/m². Optimal dosing of a proteasome inhibitor may take into account such considerations as the desired dosing schedule(s) and the bioavailability of the drug for a given route(s) of administration and guided by the clinical and laboratory parameters of the subject at any particular time.

Considerations pertaining to antibody titers are described, for example, in the Jul. 15, 1999 meeting transcript entitled “Immune Reactions Against Therapeutic and Diagnostic Biological Products”, in which the FDA Biological Response Modifiers Advisory Committee stated, “the most important criterion for assessing the significance of antibodies against therapeutic proteins is the correlation of antibody incidence and amount with clinical pharmacokinetics, pharmacodynamics, efficacy, and safety” (Reference: Center for Biologies Evaluation and Review (fda gov/ohrms/dockets/ac/cber99 htm) Qualitatively, “high titers” can be described as an antibody titer at which (or above which) a subject would generally experience clinical decline. It should be noted that “high antibody titers” do not necessarily develop in 100% of subjects receiving sALP ERT, for instance, but, for any given disease or condition, there may be a subset of subjects who have or could potentially develop high titers that affect sALP efficacy. Assays performed to detect antibodies (neutralizing or non-neutralizing) sometimes have limitations which may result in non-detection or under estimation of antibody titers. In this situation, even if the titers are undetectable or at the low level, it can still result in decreased efficacy of sALP during sALP ERT, such as ERT using asfotase alfa. While undetectable titers of anti-sALP antibodies alone may be sufficient to cease the 1ST, if no return of sALP ERT efficacy is observed in a subject with undetectable anti-sALP antibodies, a second metric can be used to ascertain if and when to withdraw the 1ST.

Proteasome inhibitors, such as bortezomib, may cause undesired side effects, such as hematological or nonhematological toxicity. For example, bortezomib may cause neutropenia (low platelet count), cardiac toxicity, neuropathic pain, peripheral neuropathy, and/or hepatic impairment. Upon presentation with one or more of these side effects, the dosage of bortezomib may be reduced (e.g., by 25% or more, e.g., from 1.3 mg/m² to 1 mg/m², or from 1 mg/m² to 0.7 mg/m²) or may be discontinued if the risk outweighs the benefit. For example, if a subject experiences cardiac dysfunction, e g., based on results from echocardiography or elevated troponin levels, bortezomib should not continue to be administered to the subject.

Other immunomodulating agents and combination therapies

Other immunomodulating agents may be administered to a subject in a course of 1ST therapy The immunomodulating agents may be administered alone or optionally, in combination with a proteasome inhibitor, such as those described above, at the time, prior to, or after the administration of a proteasome inhibitor for reduction/prevention of formation of antibody titers. Examples of immunomodulating agents include, but are not limited, to the following: belimumab, anti-CD3 antibodies, anti-CD19 antibody, and anti-CD22 antibody, corticosteroids (e.g., Prednisolone), rapamycin, methotrexate, WIG, cyclophosphamide, cyclosporine A, azathioprine, and mycophenolate mofetil, and their derivatives These immunomodulating agents or their derivatives include agents targeting/altering antigen presentation and/or humoral or cell-mediated immune response.

1ST therapies that trigger inhibitory FcR expressed on B cells and antigen-presenting cells and that target B-cell survival and activation factors (such as B-cell activating factor and B-lymphocyte stimulator) may be used. Depleting approaches using rituximab, a chimeric monoclonal antibody with human lgG1 constant domains that depletes mature B cells expressing the CD20 molecule, may be of particular use in the prophylaxis setting, potentially allowing introduction of enzyme at a stage when immature enzyme specific pre- and pro-B cells may be deleted or rendered nonresponsive

Combinations of rituximab and antibodies to B-cell activating factor may also be used. Methods of treatment including 1ST following ERT are described, for example, in US Pat. Nos. 8,809,282, 9,050,333, 9,592,247, 9,850,474, and 10,028,993, the disclosures of which are hereby incorporated by reference in their entirety.

1ST therapy may also include immunoadsorption alone or in combination with the therapeutic agent (e.g., rituximab). An immunoadsorption agent, such as THERASORB® may be used to immunodeplete neutralizing antibodies. Immunoadsorption may be administered, for example, daily, weekly, bi-weekly, or monthly. Immunoadsorption may be administered for a duration of at least 1 month (e.g., at least 2, 3, 4, 5, 6, 12, 18, 24, or more months).

Immunoglobulin may also be administered as part of 1ST therapy (e.g., at a dosage of about 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 may also be administered as part of 1ST therapy.

The immunoglobulin may be administered once every 5 to 10 days (e.g., once every 5, 6, 7, 8, 9, or 10 days, e g., once every 7 days). For example, the immunoglobulin may be administered once every 7 days. The immunoglobulin may be administered at a dosage from about 300 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, e.g., about 500 mg/kg). For example, the immunoglobulin may be administered at a dosage of about 500 mg/kg The immunoglobulin may be administered intravenously. The immunoglobulin may be administered intravenously at a dosage of about 500 mg/kg one a month while receiving rituximab with or without bortezomib. Immunoglobulin may be administered for a duration of at least 1 month (e.g., at least 2, 3, 4, 5, 6, 12, 18, 24, or more months).

In some specific embodiments, rituximab may be administered during 1ST therapy. Rituximab may be administered alone, or as a combination therapy with methotrexate. The two therapeutic agents may be administered together at substantially the same time or sequentially. The 1ST therapy including rituximab or the combination of rituximab and methotrexate may be administered for a time period of about a month. During this treatment period, sALP ERT may be halted, or the sALP ERT may continue to be administered during 1ST therapy. Rituximab may also be administered concomitant with immunoglobulin, gamma globulin, and/or immunoadsorption (e.g., THERASORB®). In some specific embodiments, the therapy may include one or more therapeutic agents that are administered separately or in combination (e.g., concomitantly). The one or more therapeutic agents (e.g., any of the therapeutics described herein) may be administered as a single composition that is admixed together or as separate compositions The various combination therapies described herein may be administered as a first course of treatment (e.g., rituximab, immunoglobulin, and immunoadsorption agent) or a second, follow-on course of treatment (e.g., rituximab, immunoglobulin, dexamethasone, and an immunoadsorption agent, and optionally, a proteasome inhibitor). In some embodiments, rituximab, immunoglobulin, dexamethasone, and an immunoadsorption agent, and optionally, a proteasome inhibitor, are administered as a first course of treatment.

The therapy may further include administering folic acid. The folic acid may be administered, e g , once per day The folic acid may be administered at a dosage of about 0 1 mg to about 10 mg, e.g., about 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, e.g., about 1 mg The folic acid may be administered on a day that is different from the day methotrexate is administered. The folic acid may be administered orally. Methotrexate is a folate antagonist, so in specific embodiments, folic acid is administered as a supplement

Termination of 1ST

Following the course of 1ST therapy, the subject can be monitored for a reduction in the presence of the neutralizing antibodies. If the 1ST therapy has not yet achieved an improvement in the treated subject, the subject may continue to experience a worsening in, or a reduction in the alleviation of, the symptoms of the bone mineralization disorder (e.g., HPP) and/or the continued presence of neutralizing antibodies that can reduce the efficacy of the sALP In such a situation, the dosage of the 1ST therapeutic can be increased (e.g., the dosage of one or more of the 1ST agents can be changed, e.g., if the subject is receiving a combination of agents as the 1ST therapy), a change in the 1ST agents being administered can be implemented, or the subject can be administered a different 1ST therapy until the presence of neutralizing antibodies diminishes and/or sALP efficacy is restored. During the 1ST therapy, the sALP ERT may be terminated or the sALP ERT may be continued during 1ST treatment. For example, the 1ST therapy may last for a duration of 1 or more weeks, months, or years (e g., 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 3 months, 4 months, 5 months, 6 month, 1 year, 2 year, 3 years, or more) during which time the sALP ERT is terminated during all or a portion of this period. Assays, such as those described above, may be repeated in order to assess the presence and/or titer of neutralizing antibodies following 1ST therapy. After successful 1ST therapy, sALP ERT may resume.

Evaluation of treatment efficacy

Assays for assessing the health state of a subject receiving sALP ERT are described below If the assays(s) shows a reduction in the health state of the subject, the subject can be further tested for the presence of neutralizing anti-sALP antibodies. Signs that the subject is beginning to show a decline in responsiveness to the sALP ERT may signal a need to test the subject for the presence of neutralizing antibodies. If a reduction in efficacy of the sALP is suspected, the subject can be tested for the presence of neutralizing antibodies. This test can be performed especially in a subject that has been undergoing treatment with a sALP and has otherwise been observed to exhibit an improvement in symptoms of the bone mineralization disorder (e g , HPP) over a period of, e g , about 3 months, 6 months, 1 year, or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more years). The subject can be tested to determine whether the neutralizing antibodies, if present, are responsible for the reduction of efficacy of the sALP during ERT, e.g., using assays described herein. If it is determined that the subject has neutralizing antibodies that reduce the efficacy of sALP during ERT, a course of 1ST therapy can be initiated.

Several metrics can be used to evaluate the treatment efficacy of a sALP that is administered to a subject during ERT These metrics may also be used as a benchmark to assess whether a subject with neutralizing antibodies is experiencing a loss of sALP efficacy caused by the neutralizing antibodies. Furthermore, these metrics can be used in combination with 1ST therapy to monitor a change in sALP efficacy (e.g , a restoration of sALP efficacy). In particular, these metrics may be used in combination with neutralizing antibody assays as described herein both before 1ST therapy, to signal the need for 1ST therapy, and after 1ST therapy, to signal successful 1ST therapy For example, the metrics may be used to measure a reduction in efficacy (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in efficacy) of the sALP, thereby signaling a need for 1ST therapy. The reduction in efficacy may be measured with respect to a baseline (e.g., a subject’s value(s) in one or more of the metrics before any reduction in efficacy, e.g., within a predetermined time period after initiation of sALP ERT, e g., 1 month, 3 months, 6 months, 1 year, 2 years, or more after the initiation of sALP ERT), a reference subject with a bone mineralization disorder (e.g., HPP) undergoing sALP ERT, or an average of reference subjects who have received sALP ERT A reduction in efficacy of sALP that surpasses a predetermined threshold (e.g., a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in efficacy, e.g , relative to a time prior to reduction in efficacy), according to one or more of the metrics described herein, may signal a need for 1ST therapy. When it is determined that 1ST therapy is needed to treat a reduction in sALP efficacy due to the presence of neutralizing antibodies, one or more of these metrics may also be used to track a restoration of, or increase in, sALP efficacy (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% increase in efficacy, e g , relative to a time after a reduction in efficacy is observed but prior to the initiation of 1ST therapy). An increase in efficacy may be measured with respect to a baseline (e.g., the subject’s values before increase or restoration of efficacy), a reference subject with a bone mineralization disorder (e.g., HPP), or an average of reference subjects who have experienced a reduction in efficacy of sALP during ERT or who have received 1ST therapy. When a subject experiences an improvement in efficacy following 1ST therapy that surpasses a predetermined threshold (e.g., a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% increase in efficacy, e g., relative to a time after a reduction in efficacy is observed but prior to the initiation of 1ST therapy) or the subject exhibits a recovery in a level of efficacy of the sALP ERT relative to a time prior to the reduction in efficacy, 1ST therapy may be halted and may be deemed successful.

Exemplary metrics useful for evaluating the efficacy of treatment using sALP ERT (e.g., asfotase alfa) are bone healing, bone mineralization, bone mineral density, and plasma PPi and/or PLP concentrations. The methods may include the use of one or more of the described metrics (e g., bone healing, mineralization, bone mineral density, or plasma PPi and/or PLP concentrations), singly or in combination, to assess a reduction in sALP efficacy, which reduction may indicate that neutralizing antibodies are inhibiting the subject’s response to sALP ERT. Alternatively, these methods may be used to demonstrate that sALP treatment efficacy has been restored following 1ST therapy.

Bone healing and mineralization

A reduction in bone healing and mineralization may be used as a metric to diagnose a patient as having, or likely to have, neutralizing antibodies that bind to, and reduce the efficacy of, the sALP In a subject with a bone mineralization disorder (e g., HPP) that has not developed neutralizing anti- sALP antibodies, administration of sALP (e.g., asfotase alfa) results in an increase in bone healing in the subject following successful treatment. Thus, a subject with a bone mineralization disorder (e g., HPP) who is treated with a sALP who experiences a reduction in bone healing and mineralization may experience this reduction due to the presence of neutralizing antibodies

A decrease in bone healing results in loss of bone and may include decreased mineralization that results in the disunion of two or more bones. Bone healing and decreased mineralization may be compared to a reference bone (e.g., a bone of a healthy subject, e.g., without a bone mineralization disorder, or of a subject that does not have anti-sALP antibodies). Methods for identifying a decrease in bone healing and mineralization are routine and include non-invasive techniques such as radiography and computed tomography (CT). Typically, images of the relevant area of the subject can be taken before and at one or more time points following the sALP treatment, and the images can be compared to assess treatment efficacy. Decreased bone healing and/or mineralization can be identified as decreased opacity. Images can be taken at any time during sALP treatment, and can be timed to be, e.g., 1, 2, 3, 4, 5, or 6 day(s), week(s), or month(s), or year(s) following initiation of the sALP ERT treatment or at the time a decrease in efficacy is suspected. The decrease in bone healing and/or mineralization in the subject may become detectable at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12 week(s) or month(s) following initiation of the sALP ERT treatment period. The decrease in bone healing and/or mineralization in the subject may in some cases be sustained for at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 week(s), month(s), or year(s) following initiation of the sALP ERT treatment period. Thus, a reduction in bone healing and mineralization following sALP treatment may trigger the need for 1ST therapy in order to treat the neutralizing antibodies and restore the efficacy of the sALP. Following 1ST therapy, the same metrics may be used to monitor a restoration of sALP efficacy. In this case, an increase in bone mineralization and healing may be used to determine that the sALP efficacy has been restored and that the bone mineralization disorder (e g., HPP) is being efficaciously treated with the sALP ERT following 1ST therapy

Bone mineral density (BMD)

A reduction in BMD may be used as a metric to diagnose a patient as having, or likely to have, neutralizing antibodies that bind to, and reduce the efficacy of, the sALP. In a subject with a bone mineralization disorder (e.g , HPP) that has not developed neutralizing anti-sALP antibodies, administration of sALP (e.g., asfotase alfa) results in an increase in BMD in the subject following successful treatment Thus, a subject with a bone mineralization disorder (e g , HPP) who is treated with a sALP and experiences a reduction in BMD may experience this reduction due to the presence of neutralizing antibodies.

A decrease in BMD can be used to monitor the efficacy of the sALP (e.g., asfotase alfa) during the ERT. Methods for measuring BMD are known in the art and include, for instance, 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 made by any routine method, including CT Hounsfield measurement, and can use comparison of results to a normative database or control subject. BMD is sometimes reported as a Z-score or a T-score. Pretreatment BMD values can be measured at any time during sALP ERT treatment, and can be timed to be 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12 day(s), week(s), month(s), or year(s) following initiation of sALP ERT. The BMD value of a reference point after treatment may decrease by, e.g., 0.01%, 0 05%, 0 1%, 0 5%, or 1% The decrease in a BMD value of a reference point after initiation of sALP treatment may also be unchanged or a change is undetectable. The decrease in BMD in the subject may in some cases be sustained for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 week(s), month(s), or year(s) following initiation of the sALP ERT treatment period. Thus, a reduction in BMD following sALP treatment may trigger the need for 1ST therapy in order to treat the neutralizing antibodies and restore the efficacy of the sALP. Following 1ST therapy, the same metrics may be used to monitor a restoration of sALP efficacy. In this case, an increase in BMD, such as by, e.g., about 0.01%, 0.05%, 0 1%, 0 5%, or 1% or more, may be used to determine that the sALP efficacy is restored and the bone mineralization disorder (e.g , HPP) is being efficaciously treated following 1ST therapy.

PPi and PLP levels

An increase in alkaline phosphatase substrate levels, such as PPi and/or PLP, may be used as a metric to diagnose a patient as having, or likely to have, neutralizing antibodies that bind to, and reduce the efficacy of, the sALP. In a subject with a bone mineralization disorder (e.g , HPP) that has not developed neutralizing anti-sALP antibodies, administration of sALP (e g., asfotase alfa) results in a decrease in PPi and/or PLP in the subject following successful treatment, as sALP is enzymatically active towards these substrates. Thus, a subject with a bone mineralization disorder (e.g., HPP) who is treated with a sALP and experiences an increase in PPi and/or PLP may experience this increase due to the presence of neutralizing antibodies that reduce the catalytic activity of the sALP, leading to an excessive buildup of these substrates.

An increase in the amount or concentration of substrates such as PPi or PLP can be used to monitor the efficacy of the sALP (e g , asfotase alfa) both before and during ERT treatment to monitor the reduction in efficacy of the sALP during the ERT Normal and abnormal concentrations of these substrates are described, for example, in PCT Publication Nos WO 2016/123342 and WO 2017/171871, the disclosures of which are hereby incorporated by reference in their entirety

The average increase in PPi concentrations in a plasma sample from the subject receiving ERT with a sALP (e.g., asfotase alfa) may be about 25% or more (e.g., 30%, 35%, 40%, 45%, 50%, 55%, 60%, or more than 60%) relative to PPi concentrations in a plasma sample from the subject prior to the observed reduction in efficacy of the sALP The average increase in PPi concentrations in a plasma sample from the subject may in some cases be sustained during a treatment period of at least one week, six months, or up to one year or more The increase may be sustained for the duration of the treatment period with the sALP (e.g., asfotase alfa). If the average PPi concentration increases above a predetermined threshold (e.g., 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60% increase, or more) based on typical values during treatment with sALP ERT, it may be determined that 1ST therapy is needed. In contrast, if the average PPi concentration decreases below a predetermined threshold (e g , 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60% decrease, or more) or to normal levels compared to a reference subject (e.g , one who has been successfully treated with 1ST therapy), it may be determined that 1ST therapy is no longer needed or may be halted. An increase in the level of PLP can be used to monitor the efficacy of the sALP (e.g., asfotase alfa) both before and during ERT treatment to monitor the reduction in efficacy of the sALP during the ERT The average increase in PLP concentrations in a plasma sample from the subject receiving ERT with a sALP (e.g., asfotase alfa) may be about 50% or greater (e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%) relative to PLP concentrations in a plasma sample from the subject prior to the observed reduction in efficacy of the sALP. The average increase in PLP concentrations in a plasma sample from the subject may in some cases be sustained during a treatment period of at least one week and up to one year or more. The increase may be sustained for the duration of the treatment period with the sALP (e.g., asfotase alfa). If the average PLP concentration increases above a predetermined threshold (e g , 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60% increase, or more) based on values during treatment with the sALP ERT, it may be determined that 1ST therapy is needed. In contrast, after 1ST therapy, a decrease in the average PLP concentration below a predetermined threshold (e g., 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60% decrease, or more) or a normal level of PLP, e.g., as compared to a reference subject or one who has been successfully treated with 1ST therapy indicates that 1ST therapy is no longer needed and may be halted.

Any method known to those of skill in the art can be used to quantify the PPi and PLP concentrations in a plasma sample or, alternatively, in a urine sample, e.g., as described in detail in, e.g., Whyte et al., (J. Clin Invest. 95(4): 1440-1445, 1995), which is hereby incorporated by reference in its entirety. In particular, PPi and PLP concentrations in a plasma sample can be used to evaluate sALP activity for the subject. Subjects with HPP typically exhibit elevated plasma concentrations of PPi and PLP, such as a PPi concentration of about 5 pM or greater and/or a PLP concentration of about 50 ng/ml or greater than a healthy subject. The lower normal limit for plasma PPi concentrations of healthy adults is about 1 M, while the upper normal limit is about 5 9 pM The lower normal limit for plasma PPi concentrations of adolescent adults is about 0.8 pM, while the upper normal limit is about 4.9 pM. The lower normal limit for plasma PLP concentrations of healthy adults is less than about 10 ng/ml, while the upper normal limit is about 60 ng/ml. The lower normal limit for plasma PLP concentrations of adolescent adults is less than about 10 ng/ml, while the upper normal limit is less than about 25 ng/ml. Thus, these boundaries for normal concentrations of PPi and PLP can be used as predetermined thresholds to trigger onset or termination of 1ST therapy.

Physical metrics and quality-of-life assessments

A reduction in a score from a physical or quality-of-life assessment may be used as a metric to diagnose a patient as having, or likely to have, neutralizing antibodies that bind to, and reduce the efficacy of, the sALP. In subjects with a bone mineralization disorder (e.g., HPP), administration of sALP (e.g., asfotase alfa) results in an increased score (e.g., correlating with a positive physical performance metric or higher quality-of-life) in the subject following successful sALP treatment. Thus, a subject with a bone mineralization disorder (e g , HPP) who is treated with a sALP and experiences a decreased score (e.g., correlating with a negative physical performance metric or lower quality-of- life) may experience this result due to the presence of neutralizing antibodies that reduce the efficacy of the sALP.

Physical and quality-of-life metrics may be used to measure the efficacy of ERT (e.g., sALP, e g , asfotase alfa) and/or 1ST treatment of neutralizing sALP antibodies For example, a negative assessment using any of the metrics described below may indicate that the efficacy of the sALP during ERT has been reduced and that 1ST therapy may be required. In contrast, after 1ST therapy, a positive assessment using any of the metrics described below may indicate that the 1ST therapy has been successful and may be halted. Non-limiting examples of physical metrics useful for evaluating the efficacy of treatment using a sALP (e.g., SEQ ID NO: 1) are, e.g., the Six Minute Walk Test (6MWT), Bruininks-Oseretsky Test of Motor Proficiency 2^nd Edition (BOT-2), Bayley Scales of Infant and Toddler Development, 3^rd Edition (BSID-111), and gait analysis Additional testing and metrics may include the Tinetti Performance Oriented Mobility Assessment (POMA; optionally modified) and temporo-spatial gait analysis software and hardware, such as GAITRITE® (Clinical Image Retrieval System Inc.). Exemplary quality-of-life assessments are the EuroQol Five Dimension Questionnaire (EQ-5D), Childhood Health Assessment Questionnaire (CHAQ), Pediatric Outcomes Data Collection Instrument (PODCI), Child Health Utility lndex-9D (CHU-9D), Pediatric Quality of Life Inventory (PedsQL), Short Form Health Survey 36 (SF-36), and Short Form Health Survey 12 (SF-12) The methods described herein may include the use of one or more of the described metrics or assessments singly, or in combination, to assess treatment efficacy using an sALP (e.g., asfotase alfa) for a subject having a bone mineralization disorder, such as HPP, in which improvements relative to a certain score or value demonstrate that the sALP is effective for treating HPP. In addition, the activity level of sALP in a sample (e.g., a plasma sample) from the subject having HPP may also be used singly or in combination with one or each of these metrics to assess treatment efficacy of the sALP (e.g , the sALP of any one of SEQ ID NOs: 1-3) for the subject. Assays to measure physical metrics and perform quality-of-life assessments associated with bone mineralization disorders, such as HPP, are described, for example, in PCT Application Nos. PCT/US2018/025206 and PCT/US2018/026868, the disclosures of which are hereby incorporated by reference in their entirety. These metrics may be used to monitor the efficacy of, or a change in efficacy of, sALP during ERT and before, during, or after 1ST therapy, and therefore, may be used to guide the onset or termination of 1ST therapy.

Assays for detecting and characterizing neutralizing antibodies

Following the identification of a reduction in efficacy of a sALP during ERT, a subject can undergo further tests to detect and characterize the presence of neutralizing antibodies. For example, a subject’s sample (e.g., blood, serum) can be tested for the presence and/or titer of neutralizing antibodies against a sALP, such as asfotase alfa or other alkaline phosphatase described herein. Various assays may be used to characterize the antibodies, such as a screening assay, a confirmatory assay, a titration assay, and/or a neutralizing antibody assay. A sensitive screening assay for binding antibodies can be used as a first step Furthermore, the level or titer of antibody can be measured relative to a control or a baseline value, such as the amount of the antibody prior to the observed reduction in efficacy or of a reference subject with a bone mineralization disorder (e.g., HPP) not experiencing a reduction in efficacy of the sALP (e.g., asfotase alfa). This step can be followed by a confirmatory assay that confirms that a positive response is antibody-mediated. Positive samples can then be tested for the neutralizing activity of the antibody in both enzyme activity and bone-targeting assays, and the antibody may be further assessed for titer and isotype.

In particular, an evaluation of an immune response to a sALP (e.g., asfotase alfa or other alkaline phosphatase described herein) can involve one or more immunoassays that detect sensitivity, specificity, and/or robustness of the antibody/therapeutic interaction. Given the complex biology of sALP ERT, assessment of antibody neutralization, in particular, can involve development of at least two types of neutralizing antibody assays: one that assesses neutralization of enzyme targeting to bone (e g , osteoblasts present in the mineral phase of bone) and one that measures the ability of neutralizing antibodies to inhibit catalytic activity of the sALP. These distinct functions are mediated by different regions in the sALP fusion protein (e g., asfotase alfa or other alkaline phosphatase described herein). For example, targeting of asfotase alfa to osteoblasts (e.g., the hydroxyapatite mineral phase of bone) is mediated by a 10 residue polyaspartate region, while enzymatic activity, including substrate binding and catalysis, is mediated by the sALP region. Antibodies that neutralize the efficacy of sALP during ERT may, accordingly, have specificity for one or both of the various functional domains of a sALP fusion protein (e g , the enzyme portion and/or the bone-targeting portion). Antibodies directed to other conserved elements of an alkaline phosphatase, such as asfotase alfa, for example the Fc domain, may not neutralize, but, when present in high titer, may inhibit efficacy by diverting the enzyme into a more restricted cell population (e.g., into cells bearing Fc receptor, or FcR) or by altering pharmacokinetics Assays for antibodies that neutralize targeting or catalytic activity can be carried out side by side using the same subject samples or replicates of the subject sample, and the results can be analyzed in conjunction with information on clinical outcome in the subject

Neutralization of catalytic activity can be assessed by mixing serially diluted subject sera into a validated potency assay that measures the activity of the sALP toward the specific substrate (e.g., PPi). This assay can be validated according to recommended standards. Neutralizing antibodies specific for the bone targeting domain or the catalytic domain of a sALP are determined to interfere with the efficacy of the sALP if the antibody is present in sufficient titer to prevent targeting to bone cells, the antibody significantly alters pharmacokinetics of the sALP, or the antibody inactivates the sALP Furthermore, the binding of an antibody to a sALP may alter enzyme conformation, thereby rendering the sALP susceptible to proteolytic degradation. Assays that may be used to monitor bone targeting and catalytic activity of the sALP are disclosed, for example, in PCT Publication No. WO 2005/103263, the disclosure of which is hereby incorporated by reference in its entirety.

One or more of these assays may be used to monitor the efficacy of, or a change in efficacy of, sALP during ERT and before, during, or after 1ST therapy, and therefore, may be used to guide the onset or termination of 1ST therapy.

ADA assessment via a multi-tiered approach Development of an anti-drug antibody (ADA) assay in specific embodiments is based at least in part on a multi-tiered approach. For example, a first tier may include a screening assay, and a second tier may include a confirmatory assay Screened and confirmed positive samples can then be further characterized in a titration assay and a neutralization antibody assay. In specific embodiments, a screening assay is performed, a confirmatory assay is performed, a titration assay is performed, and a neutralizing assay is performed. In other embodiments, at least two or at least three of a screening assay, a confirmatory assay, a titration assay, and a neutralizing assay are performed.

Assays described herein can include an electrochemiluminescent (ECL) bridging assay, which may include a sample acidification step to dissociate drug-antibody complexes and extend the drug tolerance by allowing unbound anti-drug antibodies to compete freely in the assay A positive control can be used, such as a polyclonal antibody purified from a rabbit hyperimmunized with a sALP, such as asfotase alfa. An ADA assay may use an anti-sALP antibody bridge of immobilizing antigen and reporting antigen. The immobilizing antigen may be a biotin-labeled sALP (B-sALP) and the reporting antigen may be ruthenium-labeled sALP (Ru-sALP). The measurement output then may be produced by a chemiluminescent signal produced by electrical stimulation of the ruthenium- labelled sALP when captured by a streptavidin plate.

Screening assay

A screening assay, also known as a binding antibody assay, can be used to detect antibodies that bind to a therapeutic protein product. Such assays can be deliberately made sensitive to detect low- and high- affinity ADAs within the clinical sample. Samples positive in the screen assay are further evaluated for the specificity of their binding in the second tier of confirmatory assay.

A cut-point of the screening assay can be a threshold of response in the assay that defines if a given sample response is positive or negative A cut-point of the assay can be influenced by a myriad of interfering product or matrix components. These components are considered early in assay development when defining a cut-point. In a specific embodiment, about 25 to about 50 individual samples are used to estimate a cut-point. In specific embodiments, there is a low but defined falsepositive rate of about 5% for the initial screening assay to maximize the detection of true positives. In specific embodiments, the defined false positive rate is from about 1 to about 5%, or less than about 5%, less than about 3%, or less than about 1%. As samples from different target populations and disease states may have components that can cause the background signal from the assay to vary, different cut-points may be needed for individual target patient populations.

In specific embodiments, a cut-point is statistically determined using samples from treatment naive subjects. By performing replicate assay runs (such as 2, 3, 4, or 5 runs) with these samples, the variability of the assay can be estimated. The statistical approach employed to determine the cut-point can include removing statistical outliers and accounting for pre-existing antibodies. Each sample can be tested in the ADA screen assay by at least two analysts on at least three different days for a total of at least six individual measurements One approach that allows for high assurance of a 5% falsepositive rate can be to apply a 90% one-sided lower confidence interval for the 95th percentile of the negative control population. This can ensure at least a 5% false-positive rate with a 90% confidence level. This approach can improve the probability of the assay identifying all subjects who may develop antibodies. A statistical method used to determine a cut-point can be based on a statistical distribution of the data For example, in a specific embodiment, the 95th percentile of the normal distribution is estimated by the mean plus 1.645 standard deviation.

In specific embodiments, a subsequent confirmatory assay or other assays can be employed to exclude false-positive results when determining the true incidence of immunogenicity.

Confirmatory Assay

A confirmatory assay, used in specific embodiments, is a competition assay that can confirm specific binding of ADA to the therapeutic protein of interest The objective of this assay is to eliminate potential false-positive samples from the initial screening assay. ADA-confirmed samples can be further characterized in a subsequent titer assay and a neutralizing assay.

A confirmatory assay format can be a competition assay in which a competitor, such as an unlabeled therapeutic protein product is used. In specific embodiments, implementation of a suitable confirmatory assay ensures prevention of data on ADA false-positive subjects from confounding the analyses of the impact of ADA on safety and efficacy.

An exemplary approach to determining a cut-point in a confirmatory assay uses the data from the signal generated by antibody-negative treatment-naive subject samples in the presence of the competitor drug. In this case, the amount of therapeutic protein product can be used to establish the cut-point. In specific embodiments, the amount of therapeutic protein product that is used is the same that of a competitive inhibitor in the assay.

In a specific embodiment of a confirmatory assay, the assay uses sALP (e g., 100 pg/mL, 150 pg/mL, 200 pg/mL, 250 pg/mL, 300 pg/mL, 350 pg/mL, 400 pg/mL, 450 pg/mL, or 500 pg/mL) as a competitive inhibitor of B-sALP and Ru-sALP binding to ADA Following an incubation period, the B- sALP/ADA/Ru-sALP complex captured by a streptavidin plate may be measured, for example, by an MSD Sector Imager 6000 Plate Reader. In specific embodiments, for each sample, percent inhibition by the sALP assay inhibitor may be calculated as follows: sample response inhibited

% inhibition X 100 mean sample response uninhibite d

In specific embodiments of the confirmatory assay, the cut-point can be established from the statistical analysis of data from drug-naive normal donors assayed with and without free sALP. Using, for example, an upper 99.9^th percentile of the statistical analysis, a confirmatory assay cut-point may be selected (e.g , 5%, 10%, 15%, 20%, 25%, or 30% inhibition). A sample may be confirmed as anti- sALP positive if the percent inhibition is equal to or greater than the confirmatory assay cut-point at inhibition.

In specific embodiments, samples that are confirmed positive in the confirmatory assay are reported as positive and further evaluated in the titration assay. Samples below the confirmatory assay inhibition cut-point can be reported as negative and no further testing is needed.

Titration Assay Titration assays can be used in specific embodiments to characterize the magnitude of an ADA response. In specific embodiments, magnitude is characterized with a titration assay, because the impact of ADA on pharmacokinetics, pharmacodynamics, safety, and efficacy may correlate with ADA titer and persistence rather than incidence.

In specific embodiments, a titration assay specific cut-point is established using about a 0.1% false-positive rate or the screen cut-point is used as the titration cut-point. In specific embodiments, the titration assay cut-point is established from about 0.01% to about 0.1% or less than about 0.1%.

Neutralizing Assay

Neutralizing antibodies (NAbs) in specific embodiments refer to specific ADA with an ability to interfere with interactions between a therapeutic protein product and a target. Neutralization assays can assess ADA for neutralizing activity. In specific embodiments, neutralizing activity of ADA is characterized to, in part, assess the impact of ADA on pharmacokinetics, pharmacodynamics, safety, and efficacy, which may correlate with NAb activity rather than ADA incidence.

The testing method selected to assess neutralizing potential for ADA-positive samples in specific embodiments is based on a mechanism of action of a therapeutic protein product. In vitro neutralization assays can indicate potential of ADA to inhibit therapeutic activity of a product NAb can interfere with the clinical activity of a therapeutic protein product by preventing the product from reaching a target or by interfering with pharmacologic activity such as receptor-ligand interactions.

FIG. 1 is an example flow-chart of assays of various embodiments, as described herein. Methods applying to each of matters related to SEQ ID NOS: 1-3 may be the same or may be different in application of portions of the flowchart or processes as herein described.

In specific embodiments, a NAb assay for detecting whether the anti-sALP antibodies are neutralizing antibodies use enzyme catalyzed steady-state kinetic assays to determine the effect of the NAbs on sALP efficacy. For example, the assay may measure hydrolysis of pNPP, which is proportional to enzymatic activity. The NAb assay may include an acid treatment (e.g., 0.1 M glycine- HOI; 15% v/v) to the negative control, positive control, and study samples prior to the enzymatic activity assay. Acidification of samples may be used to dissociate antibodies bound with sALP and also to denature and inactivate the sALP. Following a neutralization step, the samples may be added to an activity assay. In an activity assay, a small aliquot (e.g , 20 pL) of a fixed concentration of sALP (e g , 95 0 ng/mL) may be added to each well in the assay Following an incubation, the reaction may be started with the addition of pNPP (e.g., final concentration of e.g., 1 mM, 2 mM, 3 mM, 4 mM, or 5 mM, or higher) in a suitable reaction buffer (e.g., 20mM bis-tris Propane, 50mM NaCI, 0.5mM MgCh, 50pM ZnCL, 0 5 mg/mL BSA and a pH 9.0 at 37°C). Study samples containing NAbs will inhibit the turnover of pNPP relative to the assay negative control. The % inhibition of asfotase alfa activity (Vmax) by the antibodies contained in each sample may be calculated using the following formula: sample Vmax

% inhibition = X 100 mean negative control Vmax

A neutralizing assay cut-point may be determined from the analysis of, e.g., 50 individual drug naive human serum samples evaluated in 2 or more (e.g., 3, 4, 5, or 6) separate runs. The assay cut-point may then be obtained statistically and represented by, for example, the upper 99.9^th percentile inhibition of the rate of reaction (Vmax). Samples with percent inhibition below this cut-point may be considered NAb negative, whereas samples with percent inhibition equal to or above this cut-point may be reported NAb positive.

The results from assays described above may be validated using a positive control, such as a polyclonal anti-sALP antibody. Parameters, such as sensitivity (e.g. , positive control concentration yielding a positive response in all assays), selectivity (e.g., 60%, 70%, 80%, 90% or more of the sALP lots tested required to meet criteria), precision and accuracy (e.g , signal: noise ratio of concentration variability < 5%, 15%, 20%, or 25%), specificity, drug tolerance/interference (e.g., highest drug concentration yielding a positive response), and sample stability (e g , within 100 + 20% recovery), can also be assessed with a predetermined threshold used for acceptance criteria.

Defining immunogenic subgroups

An analysis of PK measurements may be used to determine if sALP efficacy is reduced during ERT. Specific aspects of immunogenicity response that can be investigated include, for example, time-varying nature of immunogenicity, immunogenicity status (e.g., ADA+ versus ADA- and NAb+ versus NAb-), and magnitude (e g , high versus low for ADA and NAb titers) To assess the impact of antibody development, a subject may be assigned to a subgroup according to immunogenicity information about their seroconversion, e.g., development of sALP ADA and NAbs. The impact of immunogenicity on PK may then be assessed by examining differences in various PK parameters (e.g., PPi and/or PLP concentrations, Cavg, Cmax, and Vmax) among these subgroups. Exemplary PK parameters are described, e.g., in PCT Application No. PCT/US2018/025206, which is hereby incorporated by reference in its entirety.

For assessing immunogenicity, one approach may be to assign a subject to one of 2 groups, such as positive or negative for antibody. Defining a subject positive for antibodies can be done, for example, using a single occurrence of the subject testing positive for ADA at any time posttreatment with sALP (e.g., asfotase alfa). The subject may then be assigned to the ADA+ group (ADA positive status for the whole treatment period). If the subject never develops measurable antibodies, then the subject would be assigned to the ADA- group (ADA negative status for the whole treatment period). Each subject may be further assigned to one of 3 possible subgroups: ADA- (negative for anti-drug antibody, no NAb measured), ADA+/NAb+ (ADA+ and positive for neutralizing antibody), and ADA+/NAb-(ADA+ but negative for neutralizing antibody). Example 2 provides additional details on assessment of immunogenicity groupings.

Alkaline Phosphatase

Alkaline phosphatases, which may be administered as part of ERT to subjects with bone mineralization disorders, such as HPP, encompass a group of enzymes that catalyze the cleavage of a phosphate moiety (e g , hydrolysis of pyrophosphate, PPi) There are four known mammalian alkaline phosphatase (ALP) isozymes: tissue nonspecific alkaline phosphatase (TNALP; described further below), placental alkaline phosphatase (PLALP) (e.g., Accession Nos. P05187, NP_112603, and NP_001623), germ cell alkaline phosphatase (GALP) (e.g., Accession No. P10696), and intestinal alkaline phosphatase (IALP) (e.g., Accession Nos. P09923 and NP_001622). In addition to the exemplary ALPs discussed herein, any polypeptide having the identical or similar catalytic site structure and/or enzymatic activity of ALP can be used (e.g., as a sALP or a sALP fusion polypeptide as described herein) in a method disclosed herein.

The sALP can be, for example, a soluble form of human tissue non-specific alkaline phosphatase (human TNALP (hTNALP)). An exemplary soluble alkaline phosphatase (sALP) is asfotase alfa, which is a human TNALP fusion polypeptide. In particular, the polypeptides of any one of SEQ ID NOs: 1-3 or a variant thereof having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity thereto can be used administered to treat bone mineralization disorders, such as HPP. The sALP may include or consist of the amino acid sequence of SEQ ID NO: 1. The sALP may include or consist of the amino acid sequence of SEQ ID NO: 2. The sALP may include or consist of the amino acid sequence of SEQ ID NO: 3.

The alkaline phosphatase may include the sequence set forth in SEQ ID NO: 1 : LVPEKEKDPKYWRDQAQETLKYALELQKLNTNVAKNVIMFLGDGMGVSTVTAARILKGQLHHNPGEE TRLEMDKFPFVALSKTYNTNAQVPDSAGTATAYLCGVKANEGTVGVSAATERSRCNTTQGNEVTSIL RWAKDAGKSVGIVTTTRVNHATPSAAYAHSADRDWYSDNEMPPEALSQGCKDIAYQLMHNIRDIDVI MGGGRKYMYPKNKTDVEYESDEKARGTRLDGLDLVDTWKSFKPRYKHSHFIWNRTELLTLDPHNVD YLLGLFEPGDMQYELNRNNVTDPSLSEMVVVAIQILRKNPKGFFLLVEGGRIDHGHHEGKAKQALHE AVEMDRAIGQAGSLTSSEDTLTWTADHSHVFTFGGYTPRGNSIFGLAPMLSDTDKKPFTAILYGNGP GYKVVGGERENVSMVDYAHNNYQAQSAVPLRHETHGGEDVAVFSKGPMAHLLHGVHEQNYVPHV MAYAACIGANLGHCAPASSLKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKDIDDDDDDDDDD (SEQ ID NO: 1 )

The alkaline phosphatase may include the sequence set forth in SEQ ID NO: 2:

LVPEKEKDPKYWRDQAQETLKYALELQKLNTNVAKNVIMFLGDGMGVSTVTAARILKGQLHH NPGEETRLEMDKFPFVALSKTYNTNAQVPDSAGTATAYLCGVKANMGTVGVSAATERSRCNTTQGN EVTSILRWAKDAGKSVGIVTTTRVNHATPSAAYAHSADRDWYSDNEMPPEALSQGCKDIAYQLMHNI RDIDVIMGGGRKYMYPKQKTDVEYESDEKARGTRLDGLDLVDTWKSFKPRYKHSHFIWNRTELLTLD PHNVDYLLGLFEPGDMQYELNRNQVTDPSLSEMVWAIQILRKNPKGFFLLVEGGRIDHGHHEGKAK QALHEAVEMDRAIGQAGSLTSSEDTLTVVTADHSHVFTFGGYTPRGNSIFGLAPMLSDTDKKPFTAIL YGNGPGYKVVGGERENVSMVDYAHNNYQAQSAVPLRHETHGGEDVAVFSKGPMAHLLHGVHEQN YVPHVMAYAACIGANLGHCAPASSAGSLAAVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK GLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKDDDDDDDDDD (SEQ ID NO: 2).

The alkaline phosphatase may include the sequence set forth in SEQ ID NO: 3: LVPEKEKDPKYWRDQAQETLKYALELQKLNTNVAKNVIMFLGDGMGVSTVTAARILKGQLHH NPGEETRLEMDKFPFVALSKTYNTNAQVPDSAGTATAYLCGVKANMGTVGVSAATERSRCNTTQGN EVTSILRWAKDAGKSVGIVTTTRVNHATPSAAYAHSADRDWYSDNEMPPEALSQGCKDIAYQLMHNI RDIDVIMGGGRKYMYPKNKTDVEYESDEKARGTRLDGLDLVDTWKSFKPRYKHSHFIWNRTELLTLD PHNVDYLLGLFEPGDMQYELNRNNVTDPSLSEMVVVAIQILRKNPKGFFLLVEGGRIDHGHHEGKAK QALHEAVEMDRAIGQAGSLTSSEDTLTVVTADHSHVFTFGGYTPRGNSIFGLAPMLSDTDKKPFTAIL YGNGPGYKVVGGERENVSMVDYAHNNYQAQSAVPLRHETHGGEDVAVFSKGPMAHLLHGVHEQN YVPHVMAYAACIGANLGHCAPASSAGSLAAVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK GLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKDDDDDDD (SEQ ID NO: 3).

An exemplary sALP is one that is physiologically active toward, e.g , phosphoethanolamine (PEA), inorganic pyrophosphate (PPi), and pyridoxal 5’-phosphate (PLP). In particular, a sALP is one that is catalytically competent to improve skeletal mineralization in bone. Examples of mutations that can be introduced into an ALP sequence are described in US Publication No. 2013/0323244.

A sALP (such as TNALP, for example the sALP fusion polypeptide of any one of SEQ ID NOs: 1-3 or a polypeptide variant having at least 95% sequence identity to the sequence of any one of SEQ ID NOs: 1-3, e g., asfotase alfa), linkers, spacers (e.g., Fc regions), and bone-targeting moieties described herein can be in the form of a fusion polypeptide having the structure of 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. In particular, the structure of the sALP fusion polypeptide can be Z-sALP-Y-spacer-X-Wn-V or Z-Wn- X-spacer-Y-sALP-V. The sALP of the sALP fusion polypeptide can be the full-length ALP or functional fragments of ALPs, such as the soluble, extracellular domain of the ALP, as is described herein (e.g., TNALP, PALP, GCALP and IALP). Bone delivery conjugates including sALP are further described in PCT Publication Nos: WO 2005/103263 and WO 2008/138131, the disclosures of which are each hereby incorporated by reference in their entirety

Any one of X, Y, Z, and V and/or the spacer can be absent or a linker region including an amino acid sequence of at least one amino acid. For example, X, Y, Z, and V may be a dipeptide sequence (e.g., leucine-lysine or aspartic acid-isoleucine), such as a two-residue linker at the Y position (e g , leucine-lysine) or a two residue linker at the X position (e g , aspartic acid-isoleucine) For example, sALP fusion polypeptides can have the structure hTNALP-Fc-D10 (e.g., a sALP fusion polypeptide including the amino acid sequence of any one of SEQ ID NOs: 1-3 or a polypeptide variant having at least 85% (e.g., at least 90%, 95%, 97%, or 99%) sequence identity to the sequence of any one of SEQ ID NOs: 1-3, e.g., asfotase alfa).

Wn can be a bone-targeting moiety, e.g., having a series of consecutive aspartate (D) or glutamate (E) residues, in which n = 1 to 50, e.g., n = 3-30, e.g., 5-15, 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 positioned anywhere in the fusion polypeptide, e.g. , at or near the N-terminal or C-terminal end, and/or in the linker region. For instance, the bone-targeting moiety can be present at the C-terminal end of a sALP fusion polypeptide. sALPs and fusion polypeptides can also lack a bone-targeting moiety

The methods described herein may be performed in conjunction with sALP therapy, e.g., administration of a sALP The therapy may be performed prior to administration of a sALP therapy or after administration of a sALP therapy. The sALP (e.g., the sALP of any one of SEQ ID NOs: 1-3, such as the sALP of SEQ ID NO: 1 ) may be administered 1-7 times (e.g., 2, 3, 4, 5, 6, or 7 times) per week or every two weeks, e.g., 2, 3, or 6 times per week. The sALP may be administered once every week, once every two weeks, once every three weeks, or once every four weeks. For example, the sALP may be administered 3 or 6 times per week The sALP (e g , the sALP of SEQ ID NO: 1 ) may be administered at a dosage of 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). The sALP may be administered at a dosage of 1 mg/kg week. The sALP may be administered at a dosage of 2 mg/kg/week. The sALP may be administered at a dosage of 3 mg/kg/week The sALP may be administered at a dosage of 6 mg/kg/week. The sALP may be administered at a dosage of 9 mg/kg/week The sALP may be administered at a dosage of 1 m/kg 6 times a week, 2 mg/kg 3 times a week, or 3 mg/kg 2 times a week. In some embodiments, dosing is 3 mg/kg 3 times a week or is increased from 3 mg/kg 2 times a week to 3 mg/kg 3 times a week.

In one embodiment, the sALP (e.g., the sALP of any one of SEQ ID NOs: 1-3, such as the sALP of SEQ ID NO: 2 or 3) is administered at a dosage from about 10 mg to 50 mg (e.g., once per week or once every two weeks For example, the sALP (e.g , the sALP of SEQ ID NO: 2 or 3, such as the sALP of SEQ ID NO: 2) may be administered at a dosage 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, e g, once per week or once every two weeks.

A vial containing the sALP (e g, the sALP of SEQ ID NO: 1 ) may be prepared at a concentration of 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).

In some embodiments, the pharmaceutical composition is administered subcutaneously or intravenously. For example, for an sALP (e g, an sALP of SEQ ID NO: 2 or 3, such as the sALP of SEQ ID NO: 2), from 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, e.g, about 15 mg, 45 mg, or 90 mg, such as about 25 mg, 35 mg, or 50 mg) may be administered to the subject subcutaneously, e.g, once or twice per week, e.g, for at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more weeks, or for longer (e.g, for the life of the subject). The composition may be administered in a volume of, e.g, 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 a volume in a range of from about 5 mL to about 0 1 mL)

In some embodiments, the second sALP treatment comprised administration that was less frequent than the treatment comprising the first sALP. In some embodiments, the subject treated with the sALP was administered the sALP at a dosage of 1 mg/kg/week to 10 mg/kg/week, and wherein the subject was also treated with a second sALP, different from the first sALP, and the second sALP was administered at 20 mg, 35 mg, or 50 mg once every 2 weeks

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

The sALP may be discontinued during administration of the therapy. For example, the sALP may be discontinued for about 1 week, 2 weeks, 3 weeks, 4 weeks, or longer, e.g , until reduction of neutralizing antibodies is achieved.

The sALPs described above can be administered to a subject in need thereof for the treatment of a mineralization disorder, such as HPP, or for the treatment of a symptom thereof. Symptoms of bone mineralization disorders, such as HPP, include, for example, hypomineralization, skeletal deformity, bone fracture, and bone pain Symptoms of adult HPP as defined herein include, e.g., elevated blood and/or urine levels of phosphoethanolamine (PEA), hypomineralization, hypercalcinuria, skeletal deformity, waddling gait, bone pain, bone fracture, calcium pyrophosphate dihydrate crystal deposition, arthritis, pyrophosphate arthropathy, chondrocalcinosis, calcific periarthritis, pseudofracture, skeletal deformities, hypotonia, muscle weakness, rheumatoid complications, arthritis, pseudogout, ambulatory difficulties, pain, premature loss of teeth, pulmonary hypoplasia, respiratory insufficiency, seizures, stature, growth, rickets, and immunogenicity. Symptoms of adolescent HPP as defined herein include, e g , 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, ambulatory difficulties, bone pain, pain, premature loss of teeth, hypomineralization, pulmonary hypoplasia, respiratory insufficiency, seizures, hypercalcinuria, short stature, and growth delay. A change of any of these symptoms can be used to monitor the efficacy of sALP during ERT (e g., using asfotase alfa) both before and after ERT treatment and/or before or during 1ST therapy to monitor a reduction of or increase in efficacy of sALP during ERT

A subject administered a sALP that is at risk for the generation of neutralizing antibodies against the sALP or that has experienced a loss of or reduction in the efficacy of the sALP during sALP ERT can be treated by 1ST therapy using the methods described below 1ST therapy includes one or more of the following steps: (a) identifying and/or diagnosing subjects experiencing a reduction in efficacy of a sALP as in need of 1ST therapy, (b) testing a sample from the subject for neutralizing antibodies against the sALP therapeutic, (c) determining whether the neutralizing antibodies are responsible for the reduction in efficacy of the sALP, (d) treating the subject with 1ST therapy, and (e) determining when to terminate 1ST therapy. EXAMPLES

The following examples are intended to illustrate, rather than limit, the disclosure. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the disclosure as set forth herein.

Example 1. An Interventional, Prospective Open-Label Study of Immunosuppressive Therapies to Mitigate Immune-Mediated Loss of Therapeutic Response to Asfotase Alfa (STRENSIQ®) for Hypophosphatasia (RESTORE)

Rationale:

The administration of biological (protein) drugs to patients, especially serial exposures to treat chronic conditions, carries a risk of eliciting antidrug antibodies (ADAs). This risk is greater if the biotherapeutic has a potential “endogenous-counterpart" as in the case of proteins administered for replacement therapy to treat diseases, such as hypophosphatasia (HPP). Neutralizing antibodies (NAbs) can neutralize the clinical benefit of the exogenous biotherapeutic agent. At the same time, non-NAbs can also reduce effectiveness, by accelerating the drug clearance (CL), pharmacodynamics (PD), and pharmacokinetics (PK)

In post-marketing safety surveillance, some patients treated with asfotase alfa demonstrated an initial therapeutic response, but subsequently experienced recurrence and progression of disease associated clinical, laboratory, and radiographic markers. As some of these events occurred with the presence of documented positive ADAs and Nabs, it is possible that immune-mediated effects on the pharmacologic action of asfotase alfa resulted in disease progression. Based on this information, the US FDA requested a post-marketing requirement (PMR) study to assess and characterize a potential serious risk of immune-mediated loss of effectiveness (LoE) and evaluate strategies to mitigate it (PMR#2949-7, BLA 125513).

This prospective, open-label clinical study will evaluate 3 immunosuppressive therapies (ISTs), methotrexate, rituximab, and bortezomib, for their ability to mitigate immune-mediated LoE in pediatric patients with HPP undergoing long-term asfotase alfa treatment. The study will also explore if long-term 1ST is required to maintain the desired efficacy of asfotase alfa in participants with HPP.

Table 1. Objectives and Endpoints: ^a ADA will be the primary outcome for patients who are ADA positive and NAb negative. NAb will be the primary outcome for patients who are ADA positive and NAb positive.

Abbreviations: ADA = antidrug antibody; CD19 = cluster of differentiation 19 (B lymphocyte antigen); ECG = electrocardiogram; HPP = hypophosphatasia; 1ST = immunosuppressive therapy; LoE = loss of effectiveness; NAb = neutralizing antibody; PD = pharmacodynamic; PK = pharmacokinetic;

PLP = pyridoxal-5 -phosphate (vitamin B6); PPi = inorganic pyrophosphate; RSS = Rickets Severity Score; TEAE = treatment-emergent adverse event; TESAE = treatment-emergent serious adverse event; TNSALP = tissue-nonspecific alkaline phosphatase Overall Design:

This is a Phase 4, prospective, open-label study designed to evaluate the effect of ISTs on the clinical manifestations of HPP in pediatric participants receiving treatment with asfotase alfa who demonstrate immune-mediated LoE.

Identification of immune-mediated LoE requires radiographic evidence of rickets in pediatric patients with open epiphyseal growth plates between the age of 2 years and < 18 years in the presence of ADA and/or NAb, based on the following criteria:

1 . Re-occurrence or worsening of rickets for at least the past 3 months in children who showed an initial effective response to asfotase alfa after at least 6 months of continuous treatment and currently receiving asfotase alfa. Rickets Severity Score (RSS) will be used to determine severity at Baseline.

2. Presence of ADAs at Screening, with or without NAbs, irrespective of their titers.

3. Confirmation that both the clinical evidence and immunogenicity-mediated association are present.

Participants will be identified from the HPP Global Registry (ALX-HPP-501) and the HPP Registry substudy (ALX-HPP-501 s) In addition, patients outside of the Registry will be identified via ADA testing provided in the US or via the pre-screening portion of the study if participants are outside of the US, and all potential participants must also have a documented clinical LoE.

Participants will be followed for 104 weeks following their first 1ST dose. At the study start, methotrexate and rituximab will be administered from Day 1 for an initial course of 24 weeks Following this initial 24-week period, the schedule will repeat as follows: Weeks 25-26; Weeks 27-50 (Treatment Assessment [TA] Period 2); Weeks 51-52; Weeks 53-76 (TA Period 3); Weeks 77-78; Weeks 79-104 (TA Period 4) Asfotase alfa treatment for HPP will continue in parallel to 1ST throughout the study. After each 24-week treatment period, assess radiographic findings of rickets using RSS and ADA and/or NAb status and titers to determine the benefit/risk of employing or continuing the 1ST treatment regimen(s) for the subsequent 24 weeks. Note that methotrexate will be continued for the duration of the study and rituximab will be administered without interruptions for at least the first 18 months (approximately 74 weeks) unless there are safety concerns lntravenous immunoglobulin (IVIG) 500 mg/kg will be coadministered once monthly while receiving rituximab, with or without bortezomib The decision to add bortezomib to the 1ST regimen will be based on radiographic rickets assessment of an increase in 1 or more points on RSS in the presence of increasing ADA or NAb levels from Baseline. If at 18 months, the participant has shown a complete response and is receiving methotrexate and rituximab, then rituximab will be discontinued, and methotrexate will be continued through the end of the study. If at 18 months, the participant has shown a complete response and is receiving methotrexate, rituximab, and bortezomib, then bortezomib will be discontinued, and methotrexate and rituximab will be continued through the end of the study The dosing regimens are described in FIG. 3 and Tables 4 and 5 Participants will continue to be treated with the same subcutaneous (SC) dosing regimen of asfotase alfa during the study at the time of enrollment. End of study (EoS) assessments will be performed at the Week 100 Visit or early discontinuation (ED; [if applicable]).

Disclosure Statement: This is a single-group study without blinding to treat participants with HPP who demonstrate immune-mediated LoE.

Intervention Groups and Duration:

All participants will continue to receive asfotase alfa treatment per standard of care as prescribed by their treating physician for 104 weeks of 1ST treatment in the study, without interruption 1ST treatment will follow the Schedule of Activities (SoA). EoS assessments will be performed at the Week 100 Visit or at ED (if applicable).

All participants will receive a safety follow-up call and have safety blood and urine tests collected at Week 104. The global end of the study is defined as the date of the last completed safety follow-up call for the last participant.

Schedule of Activities

a For assessments performed during both Screening and Baseline (i.e., Day 1 of Week 1). Assessments not performed again on Day 1 will use Screening as their Baseline. b The first TA Period will begin at the first dose of 1ST on Day 1 . c Signed and dated IRB/IEC approved ICF must be obtained before any study-specific Screening procedures are performed.

5 ^d Includes a decision regarding each participant’s eligibility to enroll in the study after reviewing their HPP history. e ADA samples should be collected prior to asfotase alfa administration (i.e., predose at drug trough). Only ADA-positive samples will be analyzed in the NAb assay. f The review will determine participant eligibility.

⁹ If available, historical results are acceptable and a confirmatory test is not required.

10 ^h In postmenopausal women not using hormonal contraception or hormone replacement therapy (HRT).

' Vitamin D level should be > 20 ng/mL. If < 20 ng/mL at Screening, rescreening is allowed after vitamin D supplementation. j A serum pregnancy test will be performed at each time point for all female participants of childbearing potential. Urine pregnancy test should be done by female participants at home, every month, when there are no site visits. k New use and change in use over time in mobility aids (e.g., cane, walker, wheelchair) will be assessed for all ages.

15 ¹ X-rays do not have to be performed again if available for the participant and taken within the past 2 weeks of the indicated clinic visit. m If these tests are done within 3 months of Screening Visit, they need not be repeated. n If fracture is present at Screening, follow up x-rays will be performed at Weeks 12, 22, 48, 74 and 100 or will end sooner if fracture is healing. If fracture occurs after Screening, x-rays will be performed at least every 3 months during study participation or end sooner if the fracture is healing. ° I VI G 500 mq/kg will be co-administered once monthly 24 to 48 hours after the first dose of rituximab in the series with or without bortezomib.

20 Abbreviations: 25-OH = 25-hydroxy; ADA = anti-drug antibody; ALP = alkaline phosphatase; ALPL = gene encoding the tissue-nonspecific alkaline phosphatase [TNSALP] isoenzyme; pHCG = p-human chorionic gonadotropin; BOT-2 = Bruininks-Oseretsky Test of Motor Proficiency, second edition; BPI-SF = Brief Pain Inventory, Short Form; CD19 = cluster of differentiation 19 (B lymphocyte antigen); CRF = case report form; D = day; ECG = electrocardiogram; FSH = follicle-stimulating hormone; HIV = human immunodeficiency virus; HPP = hypophosphatasia; ICF = Informed Consent Form; IEC = Independent Ethics Committee; Ig = immunoglobulin; IMP = investigational medicinal product; IRB = Institutional Review

Board; ISR = infusion/injection site reaction; 1ST = immunosuppressive therapy; MG = intravenous immunoglobulin; LFT = liver function test; mlgM = immunoglobulin M; N/A = not applicable; NAb = neutralizing antibody; NIMP = non-investigational medicinal product; PE = physical examination; PEA = phenylethylamine; PDMS-2 = Peabody Developmental Motor Scale II; PK = pharmacokinetic; PLP = pyridoxal-5 -phosphate (vitamin B6); PPi = inorganic pyrophosphate; PTH = parathyroid hormone; RSS = Rickets Severity Score; SAE = serious adverse event;

5 TA = Treatment Assessment; TEAE = treatment-emergent adverse event; TESAE = treatment-emergent serious adverse event; Wk = week w co

a review period of all available participant data during a 2-week period between each 24-week TA Period to make dosing regimen decisions. b Schedule repeats for each TA Period (2-4 only): Weeks 25-26 (Period); Weeks 27-50 (TA Period 2); Weeks 51-52 (Period); Weeks 53-76 (TA Period 3); Weeks 77-78 (Period); Weeks 79-104 (TA Period 4). Participants will receive a safety follow-up call and will have blood drawn for safety assessments at Week 104.

⁰ Site staff should note the decisions in each participant’s CRF. d ADA samples should be collected prior to asfotase alfa administration (i.e., predose at drug trough). Only ADA-positive samples will be analyzed in the NAb assay. e Only performed at Screening and annually during study participation. f The assessment tool, Pediatric-modified Total Neuropathy Scale (ped-mTNS) will be used. The assessment should be performed prior to first

10 dose and at the end of each cycle for which the participant received bortezomib.

⁹ Assessments performed only for participants receiving bortezomib. h If a participant receives bortezomib, the echocardiography and ECG assessments should be conducted prior to first dose of bortezomib. Thereafter at 3, 6 and 12 months, post first dose, and at end of study (e.g., assessments to be conducted on Weeks 27, 38, 53, 79 and 100 for participants who begin bortezomib at Week 27) or whenever clinically needed. In addition, cardiac safety biomarkers, cardiac troponin (T or I as applicable, preferably high sensitivity) and NT-pro BNP are to be measured prior to the first dose and repeated as clinically indicated, see Table 7. ' Participants on bortezomib only, samples to be collected prior to bortezomib dose on Days 1 and 29.

^J IgG testing will be done prior to all rituximab infusion cycles. k A serum pregnancy test will be performed at each time point for all female participants of childbearing potential. Urine pregnancy test should be done by female participants at home, every month, when there are no site visits.

¹ New use and change in use over time in mobility aids (e.g., cane, walker, wheelchair), all ages.

5 ^m X-rays do not have to be performed again if available for the participant and taken within the past 2 weeks of the indicated clinic visit. n If fracture is present at Screening, follow up x-rays will be performed at Weeks 12, 22, 48, 74 and 100 or will end sooner if fracture is healing. If fracture occurs after Screening, x-rays will be performed at least every 3 months during study participation or end sooner if the fracture is healing. 0 Twice weekly during each week indicated. Follow dosing days shown in Table 5. p I VI G 500 mg/kg will be co administered once monthly 24 48 hours after the first dose of rituximab in the series with or without bortezomib.

10 Abbreviations: ADA = anti-drug antibody; ALP = alkaline phosphatase; pHCG = p-human chorionic gonadotropin; BOT-2 = Bruininks-Oseretsky Test of Motor Proficiency, second edition; CD19 = cluster of differentiation 19 (B lymphocyte antigen); CRF = case report form;

ECG - electrocardiogram; ED = early discontinuation; EoS = end of study; ICF = Informed Consent Form; Ig = immunoglobulin; IMP = investigational medicinal product; ISR = infusion/injection site reaction; 1ST = immunosuppressive therapy; MG = intravenous immunoglobulin; LFT = liver function test; N/A = not applicable; NAb - neutralizing antibody; NIMP = non-investigational medicinal product; NT-pro BNP = N- ^M 15 terminal pro brain natriuretic peptide; PE = physical examination; PEA = phenylethylamine; PDMS-2 = Peabody Developmental Motor Scale II;

PLP = pyridoxal-5 -phosphate (vitamin B6); PPi = inorganic pyrophosphate; RSS = Rickets Severity Score; SAE = serious adverse event;

TA = Treatment Assessment; TEAE= treatment-emergent adverse event; TESAE = treatment-emergent serious adverse event

Treatment Schedule and Dosing Regimens of ISTs:

Methotrexate:

Methotrexate is administered starting on Day 1 and is dosed once every 7 days. The treatment schedule and dosing regimen are within the

5 approved labeling for methotrexate (Table 7). Folate deficiency may increase methotrexate-related adverse reactions. Therefore, folic acid should be administered to participants while receiving methotrexate.

Table 4: Rituximab Treatment Schedule - 1 Dose/Week

Note: 375 mg/m² x 4 doses in each 24-week period.

10 *The treatment schedules and dosing regimens are aligned with approved labelling for rituximab.

Abbreviations: RTX = rituximab; ND = non-dosing; TA = Treatment Assessment (Period)

Note: 1 .3 mg/m² per dose is the starting level.

* Bortezomib is currently not approved for pediatric use.

15 Abbreviations: BTZ = bortezomib; ND = non-dosing; TA = Treatment Assessment (Period)

Background

Clinical Immunogenicity Experience with Asfotase Alfa Treatment

In clinical studies, most participants treated with asfotase alfa for HPP were positive for ADAs at some point post-baseline (97/109; 89%). Generally, ADA titers were low, with a median peak titer of 32.0 (range: O to 2048). Median time to the first ADA-positive result was 43.0 days (range: 14 to 2374 days). As with the results for ADAs, participants who tested positive for NAbs at some point post-baseline were not consistently positive after the first positive result. Of the 97 participants who were ever positive for ADAs post-baseline, 55 (56.7%) participants were positive for NAbs at some time point post-baseline. The median peak NAb percent inhibition was 9.910 (range: 4.54 to 95.7). No correlation was observed between the ADA titer and NAb (% inhibition) values.

Overall, these ADA responses had a very small impact on the drug concentration profiles of asfotase alfa. The results of a population-pharmacokinetics (Pop-PK) model (N = 58) demonstrated that the effect of ADA status on asfotase alfa CL was insignificant (increased < 20%). Simulations evaluating the effect of immunogenicity on CL revealed 90% confidence interval (Cl) for average exposure at steady state, supporting the finding that overall impact of low titer antibody response on asfotase alfa PK is small. Given the small effect of immunogenicity on PK and given that participants dosed 6 mg/kg/week were near the plateau of the exposure-efficacy relationship, immunogenicity is not expected to impact efficacy and safety in the majority of the HPP participants treated with asfotase alfa. This has been confirmed based on the review of the composite data for individual participants with a focus on the relationships between immunogenicity and efficacy/safety.

In post-marketing safety surveillance, some patients treated with asfotase alfa demonstrated an initial therapeutic response, but subsequently experienced recurrence and progression of disease associated clinical, laboratory, and radiographic markers. As some of these events occurred with the presence of documented positive ADAs and NAbs, it is possible that immune-mediated effects on the pharmacologic action of asfotase alfa resulted in disease progression.

The proposed 1ST agents will be administered for an initial course of 24 weeks, followed by a 2-week period to evaluate clinical, laboratory, and radiological changes that will determine the need to repeat 1ST regimens, continue observation if the participant is not treated with ISTs, or modify 1ST regimen for the next 24 weeks. Methotrexate and rituximab will be administered for at least 18 months (approximately 74 weeks) unless there are any safety concerns. MG 500 mg/kg will be co-administered once monthly while receiving rituximab with or without bortezomib.

A review of the totality of the data every 24 weeks for each participant will occur throughout the study to assess the impact of 1ST on immune-mediated LoE. This cycle will repeat for all participants until study completion or ED. Asfotase alfa treatment for HPP will continue in parallel to 1ST during the study, and participants will be continued to be followed per standard of care if they enroll in the HPP Global Registry after this study ends. Participants will be treated as case studies, with intra-participant analyses for the assessment of impact of 1ST regimens on ADA/NAb responses and the corresponding effect on improvement in efficacy.

Risk/Benefit Assessment

Risk Assessment

In this open-label study, participants will continue to be treated with the subcutaneous dosing regimen of asfotase alfa at the time of enrollment and during the study, as prescribed by the treating physician as per the country’s approved label.

As there are no controlled studies using these agents in patients with HPP, the toxicities in this population receiving asfotase alfa are unknown. Furthermore, the proposed treatment combinations of asfotase alfa with methotrexate, rituximab, and/or bortezomib have not been used, and the combination of agents could have broader side effects than those observed with the individual agents. During this study, participants should be closely monitored for any signs of toxicity, including monitoring for any changes in serum creatinine, blood urea nitrogen, platelets, white cell count, alanine aminotransferase (ALT), aspartate aminotransferase (AST), bilirubin, and the presence of red blood cells in the urine.

Immunosuppression can also result in opportunistic infections such as bacterial, fungal, and viral infections. Participants should be monitored closely for any sign of infection during the study, with consequent aggressive treatment and management if this should occur. In selecting an antibiotic to treat the infection, attention should be given to potential drug interactions among the investigative agents and certain antibiotics. It is also recommended that an infectious disease specialist be consulted whenever any antimicrobial agent is to be administered for the treatment of active infection in participants receiving the 1ST regimen. Routine administration of I VIG is incorporated into the treatment algorithms as an infection risk mitigation. In addition, folate deficiency may increase methotrexate-related adverse reactions. Therefore, folic acid should be administered to participants while receiving methotrexate.

Information about the known and expected benefits and risks and reasonably expected AEs of rituximab, methotrexate, and bortezomib may be found in the reference safety information (RSI) for each 1ST. The current EU SmPC, which is most applicable for the respective active substance, route of administration, and dosage regimen, will be used as RSI for the ISTs. 1ST regimens for each participant may be added or removed based upon review of their clinical, radiological, and laboratory data.

Benefit Assessment

As this study aims to ameliorate participant’s immune-mediated LoE to asfotase alfa treatment, participation in this study may provide increased health and quality-of-life for these participants diagnosed with HPP. If a participant shows benefit with 1ST regimens during the study, they may continue 1ST with their asfotase alfa treatment following EoS. In addition, tests that were used in clinical studies, but are not yet commercially available in most countries (inorganic pyrophosphate [PPi], pyridoxal-5 -phosphate [{vitamin B6} PLP] with levamisole, and asfotase alfa concentrations), will be offered for treating Physicians to obtain and will be analyzed in a central laboratory. These recommended, but not generally available blood tests will not cause any additional risk, and participants may benefit from access to these tests to aid with treatment monitoring.

Overall Benefit: Risk Conclusion

Considering the measures taken to minimize risk to participants in this study, the potential risks identified in association with rituximab, methotrexate, and bortezomib are justified by the anticipated benefits that may be afforded to asfotase alfa-treated participants with HPP who are experiencing immune-mediated LoE.

Immune Response

Helper T-cells are essential in the development of most ADAs, while memory B-cells and long lived-plasma cells amplify and maintain the responses that constitute the basis for using combination ISTs when there is LoE due to the presence of ADAs. Therapeutic proteins, such as asfotase alfa, may be endocytosed by antigen-presenting cells following administration, where they are processed into their component peptides. A subset of these peptides may then be presented to helper T-cells specific for the peptide in the context of human leukocyte antigen (HLA) molecules. Helper T-cells signal and activate Ag-specific B-cells to proliferate and differentiate into memory B-cells and Ab-secreting plasma cells (both short- and long-lived).

Immunosuppression Therapies

The following ISTs will be considered for use in this study based on their action on helper T cells, plasma cells, and memory B cells. As there are no controlled studies using these agents in patients with HPP, the toxicities are unknown.

Rituximab

Rituximab is a chimeric monoclonal antibody that has been approved for multiple types of malignancy and autoimmune diseases. It targets cell-surface antigen of human B lymphocytes (CD20), a transmembrane protein present on virtually all B cells from the stage at which they become committed to B-cell development until it is down-regulated when they differentiate into antibody-secreting plasma cells. Rituximab causes prompt and nearly complete depletion of peripheral B cells, which do not reappear for approximately 6 months in most participants.

However, CD20 is lost when B cells differentiate into antibody-producing plasma cells. Thus, once B cells differentiate into plasma cells, alternative and/or additional agents must be utilized to address ADAs generated by circulating plasma cells. Methotrexate

Methotrexate, an inhibitor of dihydrofolate reductase, impacts rapidly dividing T and B cells by preventing the reduction of dihydrobiopterin (BH2) to tetrahydrobiopterin (BH4), leading to nitric oxide synthase uncoupling and increased sensitivity of T cells to apoptosis, thereby diminishing immune responses.

Bortezomib

Bortezomib is a proteasome inhibitor. Proteasome inhibitors are a powerful and specific way to target plasma cells, which are highly dependent on proteasome activity. Bortezomib is a reversible proteasome inhibitor that targets both short- and long-lived plasma cells due to their high rates of immunoglobulin production. While bortezomib was initially approved for multiple myeloma, animal studies proved proteasome inhibition could kill normal plasma cells also. Bortezomib ameliorated HSAT responses to ERT in some IPD patients and was also shown to be beneficial in several case reports of thrombotic thrombocytopenia purpura and autoimmune cytopenias refractory to standard therapies with minimal and tolerable AEs in children.

Participants recruited in this study and showing loss of effectiveness will all be ADA positive at Baseline with an “entrenched” ADA response. Rituximab depletes B cells, but plasma cells (producing ADAs) will not be impacted. Therefore, patients on methotrexate and rituximab treatment with entrenched ADA response may respond inadequately to the asfotase alfa ERT treatment. In order to deplete the ADA producing plasma and memory B cells, bortezomib will be considered in case of worsening of disease and concomitant increase in ADAs & NAbs.

Justification for Dose

Asfotase Alfa

Dosing of asfotase alfa, which is an NIMP in this study, upon study entry will be as per recommendation and according to the prescribed dose by the treating physician as per the country’s approved label.

Immuno Suppresive Therapies (ISTs)

This study will use standard dosing for rituximab, methotrexate, and bortezomib. Clinical, laboratory, and radiological data will be performed to decide 1ST treatment and monitoring.

End of Study Definition

Participants who complete the last scheduled procedure shown in the SoA will be considered to have completed the study. Participants will be considered early terminated from the study if they withdraw/are withdrawn from the study prior to completion of the EoS Visit. The global end of the study is defined as the date of the last completed safety follow-up call for the last participant. Participants who complete the study will be eligible to enroll in the HPP Global Registry.

STUDY POPULATION

Assessment of participant history, laboratory findings, and physical examination will be reviewed during the up to 8-week Screening Period, and the decision to proceed to 1ST will be made through consensus and after informed consent has been obtained.

Prospective approval of protocol deviations to recruitment and enrollment criteria, also known as protocol waivers or exemptions, is not permitted.

STUDY INTERVENTION

Study intervention is defined as any investigational intervention(s), marketed product(s), placebo, or medical device(s) intended to be administered to a study participant according to the study protocol.

Study Intervention(s) Administered

All study interventions (investigational medicinal product [IMP] and non-investigational medicinal product [NIMP]) will be recorded as follows:

• Start date

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

Please refer to Table 6 for details of the recommended dosing regimen for rituximab, methotrexate, and bortezomib.

The body surface area (BSA) for dosing regimen will be determined monthly. BSA will be calculated using the Du Bois method.

BSA (m²) = Height (0.725) * Weight (0.425) x 0.007184, where height is in meters (m) and weight is in kilograms (kg).

Dosing of asfotase alfa, an NIMP in this study, will be per recommendation and according to the dose prescribed by the treating physician as per the country’s approved label. Once a participant is enrolled, however, asfotase alfa dose regimen must remain stable throughout the study. Asfotase alfa will not be provided in the scope of this study. Table 6: Study Intervention Information

Note: Definition of IMP and NIMP are based on guidance issued by the European Commission. ^a For the ISTs, dose modification (addition, removal, and/or change in methotrexate dose) may be permitted in the presence of drug toxicity. ^b IVIG to be administered as long as rituximab is being dosed.

⁰ Folic acid will be sourced commercially, IVIG and IMPs will be supplied. ^d Experimental immune tolerance therapy for LoE due to immune response to asfotase alfa (ADAs with or w/out NAbs).

Abbreviations: ADA = anti-drug antibody; EU = European Union; HPP = hypophosphatasia; LoE = loss of effectiveness; IMP = investigational medicinal product; 1ST = immunosuppressive therapy;

IV - intravenous; IVIG = intravenous immunoglobulin; MTX = methotrexate; n/a - not applicable; NAb = neutralizing antibody; NIMP - non-investigational medicinal product; SC = subcutaneous(ly); TA = Treatment Assessment (Period); w/out = without Concomitant Therapy

Any medication (including over-the-counter or prescription medicines, vitamins, herbal supplements, and/or pre-medication used before ISTs are administered), vaccine, or other specific categories of interest that the participant is receiving at the time of enrollment or receives during the study must be recorded along with:

• Reason for use

• Dates of administration, including start and end dates

• Dosage information including dose and frequency

Dose Modification

After each participant completes each 24-week treatment period, clinical, laboratory, and radiological changes will be evaluated to determine the need for each participant to repeat current 1ST, continue observation only, or add/remove an 1ST regimen(s) for the next 24-week treatment period. Note that methotrexate will be administered for at least 18 months unless there are any safety concerns. Dosing modifications for toxicity are described herein.

Methotrexate Dose Modifications

Methotrexate may be associated with multiple organ toxicities (e.g., hematologic, renal, hepatic, gastrointestinal, pulmonary, skin, and neurologic systems). Dose interruption, dose reduction or discontinuation may be warranted based on the nature and severity of the toxicity. Methotrexate should be held for any Grade 3 or higher toxicity and the patient promptly evaluated. Depending on the nature and severity of the adverse reaction, reinitiating methotrexate at the same or a lower dose may be considered.

Methotrexate elimination is reduced in patients with renal impairment; methotrexate dose should be decreased by 50% dose for patients with moderate renal impairment (creatinine clearance > 30 to 59 mL/min) and methotrexate should not be administered to patients with severe renal impairment (creatinine clearance < 30 mL/min).

Rituximab Dose Modifications

Dose reductions are not a recommended risk mitigation for rituximab associated toxicities which can affect multiple organ systems and be life-threatening or fatal (e.g., severe mucocutaneous reactions, severe infections, toxicities affecting the cardiac, renal, and gastrointestinal systems). Rituximab infusions should be held pending resolution or further diagnostic evaluation for any Grade 3 or higher toxicity. Rituximab should not be administered to patients with an active, severe infection (e.g., tuberculosis, sepsis, opportunistic infections, or severe viral infections including Hepatitis B or other viral reactivations). If the toxicity does not resolve or cannot be appropriately managed with standard of care interventions or if the adverse reaction recurs upon re-administration, permanent discontinuation of rituximab should be considered.

Bortezomib Dose Modifications Bortezomib treatment must be withheld at the onset of any Grade 3 or higher toxicity except neuropathy (see Table 7). Bortezomib may be reinstituted at a lower dose once the toxicity has improved according to criteria provided below. If the toxicity is not resolved or if it recurs at the lowest dose, discontinuation of bortezomib must be considered unless the benefit of treatment clearly outweighs the risk.

Table 7: Bortezomib Dose Modifications for Hematological and Nonhematological Toxicities

Source: Bortezomib SmPC

Abbreviations: ANC = absolute neutrophil count; AV-block = atrioventricular block;

ECG = electrocardiogram; GLS = global longitudinal strain; LVEF = left ventricular ejection fraction; NT- pro BNP = N-terminal pro brain natriuretic peptide; SmPC = summary of product characteristics

Dose Modification and Continuation Criteria for Peripheral Neuropathy for Patients Treated with Bortezomib

Participants with pre-existing severe neuropathy should be treated with bortezomib only after careful risk-benefit assessment, • Grade 1 (asymptomatic; loss of deep tendon reflexes or paresthesia) without pain or loss of function: No action

• Grade 1 with pain or Grade 2: Reduce dose to 1 mg/m² • Grade 2 with pain or Grade 3: Withhold drug until toxicity symptoms resolve; may reinitiate at 0.7 mg/m² every week

• Grade 4: Discontinue bortezomib

Bortezomib Dose Modification in Hepatic Impairment

Do not adjust the starting dose for participants with mild hepatic impairment. Start participants with moderate or severe hepatic impairment at a reduced dose of 0.7 mg/m² per injection during the first cycle and consider subsequent dose escalation to 1 mg/m² or further dose reduction to 0.5 mg/m² based on patient tolerance (see Table 8).

Table 8: Recommended Starting Dose Modification for Bortezomib in Patients with Hepatic Impairment

Source: Bortezomib SmPC ^aBased on NCI Organ Dysfunction Working Group classification for categorizing hepatic impairment (mild, moderate, severe)

Abbreviations: AST = aspartate aminotransferase; NCI = National Cancer Institute; SGOT = serum glutamic-oxaloacetic transaminase; SmPC = summary of product characteristics ULN = upper limit of normal

Management of Infusion Reactions with 1ST therapy

Premedication consisting of an antipyretic and an antihistaminic, e.g., paracetamol and diphenhydramine, should always be given before each administration of rituximab. Premedication prior to infusion of rituximab should commence with the initial administration. Premedicate prior to infusion or injection of other ISTs according to institutional protocols.

If an infusion reaction is observed, the following guidelines for treatment of the participant and dose modifications or delays are provided and may be modified based on investigational site guidelines.

Grade 1 infusion reaction:

A Grade 1 infusion reaction is characterized by mild reaction symptoms where neither intervention nor interruption of the study intervention infusion are indicated. For Grade 1 infusion reactions:

• Remain at the bedside and monitor the participant until they recover from symptoms (or return to Baseline).

• Premedicate the participant at least 30 minutes prior to subsequent study drug infusions. Recommended medications: o diphenhydramine 50 mg (or equivalent) and o acetaminophen/paracetamol 325 mg to 1000 mg

Grade 2 infusion reaction:

A Grade 2 infusion reaction is characterized by moderate reaction symptoms which require therapy or interruption of the study drug infusion but responds promptly to treatment (e.g., antihistamines, nonsteroidal anti-inflammatory drugs (NSAID), narcotics, corticosteroids, bronchodilators, IV fluids). Prophylactic medications are indicated for < 24 hours. For Grade 2 infusion reactions:

• Stop the study drug infusion and begin an IV infusion of 0.9% sodium chloride

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

• Remain at the bedside and monitor the patient until they recover from symptoms (or return to Baseline)

• Administer corticosteroid and/or bronchodilator therapy as appropriate.

When symptoms resolve, restart the study drug infusion at 50% of the original infusion rate. If the patient has no further complications after 30 minutes, the infusion rate may be increased to the original infusion rate.

Premedicate the participant at least 30 minutes prior to subsequent study drug infusions. Recommended medications:

• diphenhydramine 50 mg (or equivalent) and

• acetaminophen/paracetamol 325 mg to 1000 mg

• corticosteroids (up to 25 mg hydrocortisone or equivalent) may also be given. Grade 3 or Grade 4 infusion reaction:

A Grade 3 or 4 infusion reaction is a severe reaction. A Grade 3 reaction is characterized as prolonged (i.e., not rapidly responsive to treatment and/or brief interruption of the study drug infusion) with recurrence of symptoms following initial improvement. Hospitalization may be indicated for other clinical sequalae (e.g., renal impairment, pulmonary infiltrates). A Grade 4 reaction is life-threatening requiring pressor or ventilatory support. For Grade 3 or Grade 4 infusion reactions:

• Immediately discontinue the study drug infusion and begin an IV infusion of 0.9% sodium chloride

• Administer bronchodilators, epinephrine subcutaneously or IV and/or diphenhydramine with steroids, and other supportive care as needed

• Remain at the bedside and monitor the patient until the patient’s symptoms have resolved (or returned to Baseline) and, the symptoms will not recur

• Follow institutional guidelines for treatment of anaphylaxis.

Intervention After the End of the Study

Upon completion of the last study visit, participants will return to the care of their treating physician for management of their disease and may continue to receive commercial supply of ISTs.

INTERRUPTION AND DISCONTINUATION OF STUDY INTERVENTION AND PARTICIPANT DISCONTINUATION/WITHDRAWAL

Interruption and Discontinuation of Study Intervention

• Participants will be carefully monitored for any signs of infection or other potential 1ST -related toxicities during the study. In the event of drug related toxicity, it may be necessary for a participant to permanently discontinue the study intervention. If the study intervention is discontinued, the participant should remain in the study to be evaluated for safety and other assessments as specified in the SoA (Table 3).

• Participants should be permanently discontinued from study intervention if any of the following occur:

• Confirmed, unexplained Grade 3 or higher QT prolongation (QTc >500 msec OR uncorrected QT >600 msec or change from Baseline of QTc > 60 msec) based on the average of triplicate electrocardiogram (ECG) readings

• Grade 4 infusion reaction or other severe attributable hypersensitivity reaction including anaphylaxis, Grade 4 1ST- attributable mucocutaneous reactions or any confirmed cases Steven’s Johnson Syndrome, Toxic Epidermal Necrolysis

• Pregnancy or planned pregnancy

• New or recurrent malignancy except superficial cancers curative with surgical extirpation • The 1ST should be held/delayed if any of the criteria below are met or for any other clinically significant finding to permit further evaluation of the participant:

• Any Grade 4 SAE potentially related to any of the study interventions or procedures: A decision to rechallenge the participant with the suspect 1ST must be discussed and agreed in advance.

• Renal: Grade 2 or higher increase in serum creatinine (> 2 * ULN or > 2 x Baseline if Baseline > ULN), oliguria, or decrease serum creatinine clearance > 50%.

• Hepatic: Grade 2 or higher increase in ALT, AST (> 3 x ULN or > 3 x Baseline if Baseline > ULN) or Grade 2 increased total bilirubin (> 2 x ULN or > 2 x Baseline if Baseline > ULN).

• The participant exhibits significant worsening of pulmonary symptoms.

• Severe infections: 1ST should be held/delayed, and an Infectious Disease specialist consulted.

• Any Grade 3 infections or any Grade 2 infections not responsive to a course of standard therapy or with significant neutropenia (< 1000/mm³), or suspected viral reactivation syndromes (e.g., hepatitis B, cytomegalovirus, Epstein Barr virus, herpes zoster).

• 1ST may not be administered if viral reactivation is confirmed.

• Hematologic: Grade 3 or higher decreased neutrophils (< 1000/mm³), decreased platelets (< 50 000/mm3) or decreased hemoglobin (< 8 g/dl).

• Grade 2 decreased neutrophils (< 1500/ mm3;), platelets (< 75 000/mm³), and hemoglobin (< 10 g/dl) or a Grade 2 decrease in any one hematologic parameter accompanied by clinically significant symptoms (e.g., decreased neutrophil counts with fever, menorrhagia).

• Gastrointestinal: Symptoms suggestive of ileus or bowel obstruction (e.g., severe constipation, obstipation).

• Cardiopulmonary: New onset or worsening symptoms suggestive of cardiac or pulmonary toxicity (e.g., Grade 2 or higher (e.g., dyspnea, peripheral edema, palpitations, cough, wheezing, hyper- or hypotension, etc.).

• If 1ST related pulmonary or cardiac toxicity is confirmed (e.g., pneumonitis, drug related cardiac LV dysfunction or life threatening arrythmia), the suspect 1ST should be permanently discontinued.

• Neurological: Patients with symptoms suggestive of progressive multifocal leukoencephalopathy (PML) or posterior reversible encephalopathy syndrome/reversible posterior leukoencephalopathy syndrome (PRES/RPLS)

Data collected at the time of study intervention discontinuation and follow-up and for any further evaluations that need to be completed is provided in the SoA. Efficacy Assessments

Efficacy assessments will be performed as described herein.

Rickets Severity Score

• This is a quantitative method to assess severity of rickets in the wrists and knees based on the degree of metaphyseal fraying, concavity, and the proportion of the growth plate affected.

• A 10-point scale, where 10 represents the most extreme degree of rickets severity and 0 represents the absence of radiographic changes of rickets.

• The radiographic response following treatment of nutritional rickets can be assessed by the RSS.

• RSS values correlate with values of serum alkaline phosphatase (ALP), a biochemical measure of rachitic activity.

• A score of 0 represents the complete resolution of rickets.

Peabody Developmental Motor Scales - Second Edition (PDMS-2)

PDMS-2 is a revision of the original Peabody Developmental Motor Scales from 1983. PDMS-2 is composed of 6 subtests (reflexes, stationary, locomotion, object manipulation, grasping, visual-motor integration) that measure interrelated motor abilities of children from birth through age 5 years of age.

• Reflexes (8-item subtest that measures child’s ability to react to environmental events; only measured from birth through 1 1 months as reflexes are typically integrated by 12 months of age)

• Stationary (30-item subtest that measures child’s ability to control body within center of gravity and retain equilibrium)

• Locomotion (89-item subtest that measures child’s ability to move from one place to another through crawling, walking, running, hopping, and jumping forward)

• Object manipulation (24-item subtest that measures child’s ability to manipulate balls through catching, throwing, and kicking balls and is only measured in children 12 months of age and older)

• Grasping (26-item subtest that measures child’s ability to use his or her hands starting with holding an object and progressing through controlled use of the fingers of both hands)

• Visual-motor integration (72-item subtest that measures child’s ability to use his or her visual perceptual skills to perform complex eye-hand coordination tasks, such as reaching and grasping for an object to building with blocks and copying designs). Bruininks-Oseretsky Test of Motor Proficiency - Second Edition (BOT-2), Short Form

• A measure of fine or gross motor function through a series of goal-orientated activities administered to participants in accordance with uniform procedures.

• The BOT-2 consists of 8 different subtests organized into 4 motor area composites: (i) Fine Manual Control, (ii) Manual Coordination, (iii) Body Coordination and (iv) Strength & Agility, with the 8 subtests providing a balanced contribution to the total motor composite score.

• The total composite score correlates with other measures of motor performance including the PDMS-2.

Use of Mobility Aids

HPP is associated with a high fracture and orthopedic/dental surgical burden, pain, impaired mobility, need for assistive walking devices, decreased functional status, and impairments in activities of daily living (ADL) in children and adults. This study will assess the reasons for use of assistive devices including pain, weakness, fatigue, balance, fear of falling, or fear of fracture. The information of types of devices used (e.g., crutches, wheelchair dependent [full time], wheelchair dependent [part time], walker, cane, leg braces, ramps, modification to bath/shower, grab bar/railings, or other assistive devices) will also be collected. The use of assistive devices will be monitored continuously throughout the study.

X-ray

A chest x-ray should be obtained prior to beginning of methotrexate dosing so that it may be used at a later time for comparison should respiratory complications arise during the study.

Peripheral Neuropathy

Patients receiving bortezomib can be monitored for symptoms of new onset or worsening neuropathy, such as a burning sensation, hyperesthesia, hypoesthesia, paresthesia, discomfort, neuropathic pain or weakness. In addition, participants receiving bortezomib can be assessed for peripheral neuropathy utilizing the Pediatric-modified Total Neuropathy Scale (ped-mTNS) assessment tool prior to and at the end of each bortezomib cycle.

Echocardiography and Cardiac biomarkers

Acute development or exacerbation of congestive heart failure and new onset of decreased left ventricular ejection fraction have occurred during treatment with bortezomib, including reports in patients with no risk factors for decreased left ventricular ejection fraction. Patients with risk factors for, or existing heart disease should be frequently monitored.

Cardiac adverse reactions, including ventricular fibrillation, myocardial infarction, and cardiogenic shock may occur in patients receiving rituximab. Perform cardiac monitoring during and after all infusions of rituximab for patients who develop clinically significant arrhythmias, or who have a history of arrhythmia or angina.

Cardiac toxicity

Participants receiving bortezomib will be monitored for drug-related cardiac toxicity at the timepoints indicated in Table 3, using ECGs and transthoracic 2D-echocardiography for measurement of left ventricular ejection fraction (LVEF based on the estimated left ventricular end diastolic [LVED] and left ventricular end systolic [LVES] volume, by using the modified biplane Simpson’s rule technique) and GLS. In addition, cardiac safety biomarkers, cardiac troponin (T or I, preferably high sensitivity) and N- terminal pro brain natriuretic peptide (NT-pro BNP), will be assessed prior to bortezomib and repeated if clinically indicated (see Table 7).

Pharmacokinetics

• Blood samples will be collected for the measurement of serum asfotase alfa concentrations. Samples may be collected at additional time points during the study if warranted.

• Serum samples will be used to evaluate PK profile of the enzymatic activity of asfotase alfa, which may also be used to evaluate safety or efficacy aspects relating to concerns arising during or after the study.

Immunogenicity Assessments

• Serum samples for ADA and NAb analysis will be collected at time points.

• Samples collected for ADA and NAb analysis will be collected prior to dosing.

Antidrug Antibody Variables

ADA variables include ADA response category, incidence and titer over the duration of the study as follows. Samples that are positive in the ADA assay will be further analyzed for the presence of neutralizing activity in the NAb assay. ADA response category definitions and titer thresholds are as defined herein. Assessment of the impact of ADAs and/or NAbs on asfotase alfa PK/PD/biomarker may be performed, along with impact of ISTs on immunogenicity profile (e.g., ADAs and/or NAbs) and its corresponding impact on asfotase alfa PK/PD/biomarkers and clinical efficacy.

ADA response categories

• ADA negative

• ADA positive

• NAb negative

• NAb positive Participants who are ADA positive will be categorized as follows:

• 1ST treatment-attenuated ADA responses

• 1ST treatment-boosted ADA responses

Based on the duration of these responses, the 1ST treatment-boosted and 1ST treatment-attenuated ADA responses will be further categorized as follows:

• Persistent treatment-emergent responses

• Indeterminant treatment-emergent responses

• Transient treatment-emergent responses

Pharmacodynamics

• Blood samples will be collected for measuring plasma PPi and PLP concentrations (with levamisole) and ALP at timepoints.

• Plasma samples will be used to evaluate asfotase alfa PD which may also be used to evaluate safety or efficacy aspects related to concerns arising during or after the study.

• Plasma (with levamisole) PLP and PPi concentrations (PD biomarkers of HPP). The observed and change from Baseline (CFB) in plasma concentrations of PLP and PPi will be summarized at prespecified visits. Participant level- data will be listed and plotted over time. The same analyses will be performed on other biochemical biomarkers.

MONITORING FOR CHANGE IN RICKETS AND IMMUNOGENICITY

Monitoring of Rickets:

After each 24-week treatment period, radiographic evidence of rickets will be assessed using the RSS. A complete resolution of rickets is defined as an RSS of 0. A worsening of rickets is defined as an increase in RSS by a score of 1 or more points from baseline. Patients are considered stable if there is either no change or a decrease in RSS from baseline.

Monitoring of Immunogenicity:

The aim of this study is to restore the pharmacological effect of asfotase alfa in participants diagnosed with HPP who demonstrate immune-mediated (associated with ADA and NAbs) LoE. Therefore, ADA assessments are conducted in each participant at pre-defined time points throughout the duration of the study and analyzed using validated assays. ADA variables include ADA response categories, incidence and titer over the duration of the study. ADA positive samples will be further characterized for ADA titers and the presence of neutralizing activity to STRENSIQ®. ADA positive responses will be categorized as 1ST treatment-attenuated or 1ST treatment-boosted and based on the level (titer threshold) and duration (persistent/transient/indeterminant) of the response. Participants will be considered to have a significant attenuation of ADA response due to 1ST when ADA and/or NAb titer is decreased by at least 2 titer steps or becomes negative as compared to baseline.

Criteria for 1ST Modification (Continuation or Stopping)

In the RESTORE study, an individualized risk-based approach that evaluates the risk of an untreated HPP disease or partially effective therapy versus the risk of use of an 1ST regimen resulting in improvement of effectiveness serves as the basis for devising criteria for the use of 1ST.

In this study, participants will receive a combination of ISTs (methotrexate and rituximab, and/or bortezomib) for up to 104 weeks. Following the initial 24 week period, the schedule will repeat as follows for a total of 104 Weeks: Weeks 25-26 (Treatment Monitoring Board (Period for review of clinical data and treatment decision; Weeks 27-50 (Treatment Assessment (TA) Period 2); Weeks 51-52 (Period); Weeks 53-76 (TA Period 3); Weeks 77-78 (Period3); Weeks 79-102 (TA Period 4), Weeks 103-104 (Period). Participants will continue their subcutaneous dosing regimen of asfotase alfa as was at the time of enrollment. All participants will receive concomitant folic acid in order to reduce the potential sideeffects of methotrexate and intravenous immunoglobulin (IVIG) treatment in order to provide these participants passive immunity and minimize risk of infection.

As per the study design, most of the participants recruited in this study are expected to have “entrenched ADA responses.” Therefore, downregulation of the T cells (methotrexate) and the B cells (rituximab) may not be sufficient in these participants to demonstrate a positive impact on effectiveness. An additional 1ST like bortezomib may help downregulate the plasma and memory B cells and lower the ADA levels. During treatment with rituximab and methotrexate, if there is worsening of rickets by an increase of 1 or more points from baseline, in the presence of increasing levels of ADAs/NAbs, then bortezomib will be considered and added to the regimen on top of rituximab and methotrexate (FIG. 4)

Definition of the ‘1ST Complete Response’

Irrespective of the regimen used, the following criteria have been determined to define the “1ST complete response” at Week 100:

• ADA or NAb titer is decreased from baseline by at least 2 titer steps or becomes negative*, AND

• Radiographic evidence of improvement on RSS by a score of at least 1 or more points from baseline.

*ADA will be the primary outcome for patients who are ADA positive and NAb negative. NAb will be the primary outcome for patients who are ADA positive and NAb positive.

Criteria for 1ST Modification (Continuation or Stopping)

A decision tree (FIG. 4) will be used to make the overall assessments and to decide whether to continue, modify, or stop the 1ST regimen. As methotrexate and rituximab will be administered for at least 18 months unless any safety concerns are observed, there will be no specific criteria for methotrexate and rituximab treatment. Methotrexate and rituximab should be withheld if any Grade 3 or higher toxicity is reported in a participant.

If at 18 months, the participant has shown a complete response and is receiving methotrexate and rituximab, then rituximab will be discontinued, and methotrexate will be continued through the end of the study. If at 18 months, the participant has shown a complete response and is receiving methotrexate, rituximab, and bortezomib, then bortezomib will be discontinued, and methotrexate and rituximab will be continued through the end of the study.

Criteria for bortezomib treatment:

Participants recruited in this study and showing loss of effectiveness will all be ADA positive at baseline with an “entrenched” ADA response. Rituximab depletes B cells, but plasma cells (producing ADAs) will not be impacted. Therefore, participants on methotrexate and rituximab treatment with entrenched ADA response may respond inadequately to the asfotase alfa enzyme replacement therapy (ERT). In order to deplete the ADA producing plasma and memory B cells, bortezomib will be considered in case of worsening of disease and concomitant increase in ADAs and NAbs (Banugaria et al, PLOS One, 8: e67052, 2013).

In the RESTORE study, consideration for the addition of bortezomib will occur only after the completion of the first 24-week cycle on methotrexate + rituximab. Methotrexate + rituximab will be continued along with bortezomib treatment.

The following decision tree and criteria (FIG. 4) can be used to decide addition of bortezomib, continuation of bortezomib, or stopping of bortezomib, at every 24-week interval through the duration of this study.

• Bortezomib treatment will be stopped in the event of any suspected bortezomib associated toxicity Bortezomib treatment must be withheld at the onset of any Grade 3 or higher toxicity except neuropathy” (ref: Sec 6.6.3 of HPP-407 protocol) or 1ST complete response.

Bortezomib will be added or continued, in addition to rituximab and methotrexate, in the presence of an increase of ADA or NAb from baseline by at least 2 titer steps, and an increase in radiographic RSS by a score 1 or more points from baseline.

1ST Complete Response at Week 100 - Key Endpoint Components

The definition of 1ST complete response at Week 100 is as follows:

• ADA or NAb titer is decreased from baseline by at least 2 titer steps or becomes negative* and

• Radiographic evidence of improvement on RSS by a score of at least 1 or more points from baseline *ADA will be the primary outcome for patients who are ADA positive and NAb negative. NAb will be the primary outcome for patients who are ADA positive and NAb positive.

Immunogenicity Analyses All ADA analyses will be performed on the Immunogenicity Analysis Set using ADA variables.

The incidence of ADA response categories will be listed and summarized as absolute occurrence (n) and percent of all participants (%). ADA and NAb titer levels will be listed, and maximum ADA and NAb titer levels will be summarized for ADA-positive participants. NAb positive and NAb negative participants will be summarized under ADA positive categories as absolute occurrence (n) and percentage (%) of all participants.

Associations between ADA response categories and systemic exposure to ISTs may be explored for all treated participants, for analyzing the potential impact of ISTs on immunogenicity incidence, titer, and their potential impact on individual PK profiles.

Associations between ADA response categories and TEAEs and TESAEs may be explored, including TESAEs such as systemic hypersensitivity, anaphylaxis, infusion/injection site reactions (ISRs) lasting more than 24 hours, and other immune-related SAEs.

Associations between ADA response categories and PD markers may be explored for all participants to assess the potential impact of ISTs on immunogenicity incidence, titer, and their potential impact on individual PD marker profiles and efficacy.

Table 9: AEs: Definitions and Procedures for Recording, Evaluating, Follow-up, and Reporting Definition of AE

Table 10: Definition of SAE

If an event is not an AE per definition above, then it cannot be an SAE even if serious conditions are met (e.g., hospitalization for signs/symptoms of the disease under study, death due to progression of disease).

Table 11 : Abbreviations and Specialist Terms

OTHER EMBODIMENTS

The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The disclosure is not limited to the exact details shown and described, for variations apparent to one skilled in the art will be included within the disclosure defined by the claims.

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

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

All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

The complete disclosures of all patents, patent applications including provisional patent applications, publications including patent publications and non-patent publications, and electronically available material (including, for example, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference.

Claims

1 . A method of reducing an amount, decreasing the risk of formation, or reducing an effect, of an antibody specific for a soluble alkaline phosphatase (sALP) in a subject treated with the sALP, the method comprising administering a therapy comprising a dihydrofolate reductase (DHFR) inhibitor and an anti-CD20 antibody or antigen-binding fragment thereof to the subject that reduces the amount, decreases the formation, or reduces 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 dosage of about

10 mg/m² to about 20 mg/m².

6. The method of claim 5, wherein the methotrexate is administered at a dosage of about 15 mg/m².

7. The method of any one of claims 1 -6, wherein the anti-CD20 antibody is rituximab or an antigenbinding 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 claim any one of claims 7-9, wherein the rituximab or antigen-binding fragment thereof is administered at a dosage from about 100 mg/m² to about 500 mg/m².

11 . The method of claim 10, wherein the rituximab or antigen-binding fragment thereof is administered at a dosage of about 375 mg/m².

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

13. The method of any one of claims 7-12, 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 1-13, further comprising administering a proteasome inhibitor.

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

16. A method of reducing an amount, decreasing the risk of formation, or reducing an effect, of an antibody specific for a soluble alkaline phosphatase (sALP) in a subject treated with the sALP, the method comprising administering a therapy comprising bortezomib and one or both of a dihydrofolate reductase (DHFR) inhibitor and an anti-CD20 antibody or antigen-binding fragment thereof to the subject that reduces the amount, decreases the formation, or reduces 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 15-20, 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 15-22, wherein the bortezomib is administered at a dosage from about 0.2 mg/m² to about 2 mg/m².

24. The method of claim 23, wherein the bortezomib is administered at a dosage of about 0.5 mg/m², about 0.7 mg/m², about 1 mg/m², or about 1.3 mg/m².

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 worsening of a rickets severity score (RSS) of 1 or more points from 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 determining that the subject has been treated for the antibody specific for the sALP.

30. The method of claim 29, wherein the amount of the antibody specific for the sALP, its risk of formation, or its effect 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 duration of at least 6 months.

32. The method of claim 31 , wherein the therapy is administered for 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 by at least 2 titer steps or becomes negative and exhibits an improvement in an RSS of at least 1 or more points from baseline.

35. The method of any one of claims 1 -34, wherein the therapy further comprises administering 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 dosage from about 300 to about 700 mg/kg.

39. The method of claim 38, wherein the immunoglobulin is administered at a dosage 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 at a dosage of about 500 mg/kg one a month while receiving rituximab with or without bortezomib.

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

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

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

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

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

47. The method of any one of claims 1 -46, wherein the sALP is administered 1 -7 times per week or 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 dosage of 1 mg/kg/week to 10 mg/kg/week.

50. The method of claim 49, wherein the sALP is administered at a dosage 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 the 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 the 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 the 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 been treated 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 comprised administration that was less frequent than the treatment comprising the first sALP.

60. The method of claim 58 or 59, wherein the subject treated with the sALP was administered the sALP at a dosage of 1 mg/kg/week to 10 mg/kg/week, and wherein the subject was also treated with a second sALP, different from the first sALP, and the second sALP was 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 a level of the antibody by testing a biological sample from the subject for the presence of the antibody.

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 if the subject is suffering from a reduction in efficacy of the sALP.

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

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

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

72. The method of any one of claims 68-71 , wherein the determining of reduction in efficacy comprises measuring one or more symptoms selected from the group consisting of hypomineralization, hypercalcinuria, skeletal deformity, waddling gait, bone pain, bone fracture, calcium pyrophosphate dihydrate crystal deposition, arthritis, pyrophosphate arthropathy, chondrocalcinosis, calcific periarthritis, pseudofracture, skeletal deformities, hypotonia, muscle weakness, rheumatoid complications, arthritis, pseudogout, ambulatory difficulties, pain, premature loss of teeth, 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 determining of reduction in efficacy comprises establishing that the subject does not exhibit poor treatment adherence or inappropriate injection technique, or does not suffer from a vitamin D deficiency, undernutrition, or concomitant disease.

75. The method of any one of claims 68-74, wherein the determining of reduction 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 EuroQol Five Dimension Questionnaire, Childhood Health Assessment Questionnaire, Pediatric Outcomes Data Collection Instrument, Child Health Utility lndex-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 reduced quality-of-life assessment score relative to the baseline.

78. The method of any one of claims 68-77, wherein the determining of reduction in efficacy comprises performing a physical metric assessment.

79. The method of claim 78, wherein the physical metric assessment is selected from one or more of a Six Minute Walk Test (6MWT), Bruininks-Oseretsky Test of Motor Proficiency 2nd Edition (BOT-2), Bayley Scales of Infant and Toddler Development, 3rd Edition (BSID-II I), gait analysis, use of a mobility aid, Peabody Developmental Motor Scales 2 (PDMS-2), or x-ray.

80. The method of claim 79, wherein the subject exhibits a reduced physical metric 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 metric in the subject prior to suffering from a reduction in efficacy of the sALP, in a reference subject treated with a sALP and without a neutralizing antibody against a sALP, or in a reference subject not administered a sALP.

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

83. The method claim 82, wherein the method further comprises testing an effect of the neutralizing antibody 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 reduced 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, adult, infant, or neonate.

89. The method of any one of claims 1-88, wherein he subject has a bone fracture, osteoporosis, sclerosteosis, chondrocalcinosis, hypotonia, Duchenne’s muscular dystrophy, tracheobronchomalacia, seizure, neurofibromatosis 1 (NF-1), craniosynostosis, or a muscle weakness disease.

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