Immunosuppressive therapy to reduce the anti-soluble alkaline phosphatase immune response

Abstract

This disclosure features a method for treating neutralizing antibodies that reduce the effectiveness of enzyme replacement therapy for soluble alkaline phosphatase therapy (e.g., asfotase alfa), such as for the treatment of osteocalcinosis disorders, such as hypophosphatasia (HPP). The method comprises diagnosing a subject with the presence of neutralizing antibodies that affect the effectiveness of the therapy, administering a suitable treatment to reduce the adverse effects of the neutralizing antibodies, and continuing alkaline phosphatase therapy.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims priority to U.S. Provisional Application No. 63 / 531,955, filed on 10 August 2023, the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Hypophosphatasia (HPP) is a rare hereditary disorder of bone mineralization, with an incidence of 1 in 100,000 births for the most severe forms of the disorder. This disorder arises from loss-of-function mutations in the gene encoding tissue-nonspecific alkaline phosphatase (TNALP). HPP presents with a marked range of symptoms and severity, from rickets to near-complete absence of bone mineralization in utero.

[0003] STRENSIQ® (asfotase alfa) is the first enzyme replacement therapy (ERT) approved for the treatment of HPP. Enzyme replacement therapy can lead to the development of neutralizing antibodies against the enzyme in some subjects. Methods are needed to determine whether neutralizing antibodies lead to a loss of enzyme efficacy, and similarly, methods are needed to treat antibody-mediated loss of efficacy in subjects who develop impaired bone mineralization. [Overview of the project]

[0004] The first aspect of the present disclosure features a method of reducing the amount of cellular and humoral immune response components that result in a decrease in the risk of formation or a reduction in the effect of antibodies or T cells specific to soluble alkaline phosphatase (sALP) in a subject treated with sALP. The method includes administering to the subject a therapy comprising one or more of a dihydrofolate reductase (DHFR) inhibitor and an anti-CD20 antibody or an antigen-binding fragment thereof, which reduces the amount of the anti-sALP immune response, reduces its formation, or reduces its effect.

[0005] In another aspect, a method is characterized by reducing the amount of an antibody specific to sALP, reducing the risk of its formation, or reducing its effect in a subject treated with sALP. The method includes administering to the subject a therapy comprising bortezomib and one or both of a DHFR inhibitor and an anti-CD20 antibody or an antigen-binding fragment thereof, which reduces the amount of the antibody, reduces its formation, or reduces the effect of the antibody. The method may include administering bortezomib and a DHFR inhibitor. The method may include administering bortezomib and an anti-CD20 antibody or an antigen-binding fragment thereof.

[0006] In some embodiments, the method includes administering a DHFR inhibitor. In some embodiments, the DHFR inhibitor is methotrexate. Methotrexate can 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 can be administered once every 7 days to a subject who needs it. Methotrexate is from about 10 mg / m 2 , 2 ~ about 20 mg / m 2 (e.g., about 10 mg / m<unk>0000003, 11 mg / m 2 , 12 mg / m 2 , 13 mg / m 2 , 14 mg / m 2 , 15 mg / m 2 , 16 mg / m 2 , 17 mg / m 2, 18 mg / m² 2 , 19 mg / m² 2 , or 20 mg / m² 2 For example, approximately 15 mg / m² 2 It can be administered in doses of approximately 15 mg / m². For example, methotrexate may be administered in doses of approximately 15 mg / m². 2 It can be administered in the following dosage.

[0007] In some embodiments, the method involves administering an anti-CD20 antibody or its antigen-binding fragment. In some embodiments, the anti-CD20 antibody is rituximab or its antigen-binding fragment. 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 subjects requiring it. The rituximab or its antigen-binding fragment may be administered at approximately 100 mg / m². 2 ~about 500mg / m 2 (For example, approximately 100 mg / m²) 2 , 125 mg / m² 2 , 150 mg / m² 2 , 175 mg / m² 2 , 200 mg / m² 2 , 225 mg / m² 2 , 250 mg / m² 2 , 275 mg / m² 2 , 300 mg / m² 2 , 325 mg / m² 2 , 350 mg / m² 2 , 375 mg / m² 2 , 400 mg / m² 2 , 425 mg / m² 2 , 450 mg / m² 2 , 475 mg / m² 2 , or 500 mg / m² 2 For example, approximately 375 mg / m² 2 It can be administered in doses of approximately 375 mg / m². For example, rituximab can be administered at a dose of approximately 375 mg / m². 2It may be administered in the following dosage. Rituximab or its antigen-binding fragment may be administered intravenously. In some embodiments, rituximab or its antigen-binding fragment is discontinued while the subject is being treated with methotrexate. In other embodiments, methotrexate and rituximab or its antigen-binding fragment are administered together or separately.

[0008] In some embodiments, the method further includes administering a proteasome inhibitor, such as bortezomib, to a target requiring it. Plasma cells may be specifically targeted via at least one mechanism of action of bortezomib, thereby providing benefits over other modes of treatment that control certain pre-B cells and / or B cells but fail to target plasma cells, thereby resulting in circulating antibodies and treatment failure. 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. Bortezomib is administered at approximately 0.2 mg / m². 2 ~about 2mg / m 2 (For example, approximately 0.2 mg / m²) 2 , 0.3 mg / m² 2 , 0.4 mg / m² 2 , 0.5 mg / m² 2 , 0.6 mg / m² 2 , 0.7 mg / m² 2 , 0.8 mg / m² 2 , 0.9 mg / m² 2 , 1 mg / m² 2 , 1.1 mg / m² 2 , 1.2 mg / m² 2 , 1.3 mg / m² 2 , 1.4 mg / m² 2 , 1.5 mg / m² 2 , 1.6 mg / m² 2 , 1.7 mg / m² 2 , 1.8 mg / m² 2 , 1.9 mg / m² 2 , or 2 mg / m² 2 It can be administered in doses of approximately 0.5 mg / m². For example, bortezomib can be administered at approximately 0.5 mg / m². 2 , about 0.7mg / m 2 , about 1mg / m 2, or approximately 1.3 mg / m² 2 It may be administered in the following doses. Bortezomib may be administered intravenously or subcutaneously. Bortezomib may be administered intravenously as a bolus. Bortezomib may be administered after a subject has shown a worsening of rickets severity score (RSS) of 1 point or more from baseline (e.g., corresponding to the subject's value before treatment with sALP or therapies described herein). In certain embodiments, the RSS is calculated during screening for the study before initiating therapy, and this is considered the baseline score of the RSS. Bortezomib may be discontinued while the subject is being treated with methotrexate and / or rituximab.

[0009] In some embodiments, anti-sALP antibody therapy (e.g., methotrexate, rituximab, and / or proteasome inhibitors, e.g., bortezomib) is administered for a period of at least 6 months. For example, therapy may be administered for a period of at least 12, 18, or 24 months. In some embodiments, DHFR inhibitors (e.g., methotrexate) are administered for a period of at least 6, 12, 18, or 24 months. In some embodiments, anti-CD20 antibodies or antigen-binding fragments (e.g., rituximab) are administered for a period of at least 6, 12, 18, or 24 months. In some embodiments, therapy is discontinued, for example, after the subject has shown a decrease in antibody titer of at least two titer steps, or has become negative (e.g., undetectable), and / or has shown an improvement of at least one point in RSS from baseline. For example, therapy may be discontinued if the amount of sALP-specific antibodies, the risk of their formation, or their effect has decreased compared to pre-treatment levels.

[0010] Anti-sALP antibody therapy may further include the administration of immunoglobulin. 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, immunoglobulin may be administered once every 7 days to a subject who needs it. Immunoglobulin may be administered in doses of approximately 300 to approximately 700 mg / kg (e.g., approximately 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., approximately 500 mg / kg). For example, immunoglobulin may be administered in doses of approximately 500 mg / kg. Immunoglobulin may be administered intravenously. Immunoglobulins can be administered intravenously once monthly at a dose of approximately 500 mg / kg, either with or without a proteasome inhibitor (e.g., bortezomib), while receiving methotrexate and / or rituximab.

[0011] Anti-sALP antibody therapy may further include the administration of folic acid. Folic acid may be administered, for example, once daily. Folic acid may be administered in doses of 1 mg. Folic acid does not have to be administered on the same day as methotrexate. Folic acid may be administered orally.

[0012] The methods described herein may be carried out in conjunction with sALP therapy, for example, with the administration of sALP. The therapy may be carried out before or after the administration of sALP therapy. sALP may be administered 1 to 7 times per week or every two weeks (e.g., 1, 2, 3, 4, 5, 6, or 7 times), for example, 2, 3, or 6 times per week. For example, sALP may be administered 3 or 6 times per week. sALP may be administered in doses of 1 mg / kg / week to 10 mg / kg / week, for example, 3 mg / kg / week to 9 mg / kg / week (e.g., 1 mg / kg / week, 2 mg / kg / week, 3 mg / kg / week, 4 mg / kg / week, 5 mg / kg / week, 6 mg / kg / week, 7 mg / kg / week, 8 mg / kg / week, 9 mg / kg / week, or 10 mg / kg / week). For example, sALP may be administered at a dose of 6 mg / kg / week. In particular, sALP administered according to the above dose may have at least 85% sequence identity with respect to the sequence of SEQ ID NO: 1 (e.g., at least 90%, 95%, or 99% sequence identity).

[0013] sALP may have at least 85% sequence identity to any one of sequence numbers 1-3. For example, sALP may have at least 85%, 90%, 95%, 97%, 99%, or 100% sequence identity to sequence number 1. sALP may contain or consist of the sequence of sequence number 1 (e.g., asfotase alpha). sALP may have at least 85%, 90%, 95%, 97%, 99%, or 100% sequence identity to sequence number 2. sALP may contain or consist of the sequence of sequence number 2. sALP may have at least 85%, 90%, 95%, 97%, 99%, or 100% sequence identity to sequence number 3. sALP may contain or consist of the sequence of sequence number 3.

[0014] The methods described herein may include treating a subject administered with two different sALPs. In some embodiments, the subject is treated with a first sALP and a second sALP different from the first sALP. The first sALP may have the amino acid sequence of SEQ ID NO: 1. The second sALP may have the amino acid sequence of SEQ ID NO: 2 or 3, for example, SEQ ID NO: 2. For example, the initial therapy administered to the subject may include a dose of the sALP of SEQ ID NO: 1. The subject may be administered weekly doses of sALP for several days, weeks, months, or years. Thereafter, the subject may be switched to a different sALP (for example, the sALP of SEQ ID NO: 2 or 3). The subject may be treated with different sALPs for several days, weeks, months, or years. Alternatively, the sALPs administered to the subject may alternate between any one of SEQ ID NOs: 1 to 3. When treated with sALP of SEQ ID NO: 1, the patient may be administered a dose of 1 mg / kg / week to 10 mg / kg / week, for example, 3 mg / kg / week to 9 mg / kg / week (e.g., 1 mg / kg / week, 2 mg / kg / week, 3 mg / kg / week, 4 mg / kg / week, 5 mg / kg / week, 6 mg / kg / week, 7 mg / kg / week, 8 mg / kg / week, 9 mg / kg / week, or 10 mg / kg / week). For example, administration of sALP of SEQ ID NO: 1 may be 1 mg / kg six times a week, 2 mg / kg three times a week, or 3 mg / kg twice a week. In specific embodiments, the dosage may be 3 mg / kg three times a week, or increased from 3 mg / kg twice a week to 3 mg / kg three times a week. Vials are prepared at a maximum of 100 mg / ml, for example, 40 mg / ml or 100 mg / ml. When treated with 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 one or two weeks (for example, once every two weeks). In some embodiments, the pharmaceutical composition containing sALP of SEQ ID NO: 2 or 3 is administered subcutaneously or intravenously. The composition may be administered in a volume of about 5 mL or less (for example, 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 a volume in the range of about 5 mL to about 0.1 mL).sALP may be discontinued during administration of anti-sALP antibody therapy. For example, sALP administration may be discontinued for about one, two, three, four weeks, or longer (e.g., until there is an improvement in the metrics used to evaluate the effectiveness of the anti-sALP antibody (e.g., the subject's RSS)) and / or until there is a reduction in the level of anti-sALP antibody in the subject's biological fluids, such as blood, serum, or urine (e.g., a reduction to an undetectable level).

[0015] In some embodiments, the second sALP treatment involved less frequent administration than the treatment involving the first sALP. In some embodiments, subjects treated with sALP were administered sALP at doses ranging from 1 mg / kg / week to 10 mg / kg / week, and subjects were also treated with a second sALP distinct from the first sALP, which was administered once every two weeks at doses of 20 mg, 35 mg, or 50 mg.

[0016] In some embodiments, the methods described herein further include monitoring antibody levels by testing a biological sample (e.g., blood, plasma, or urine) from a subject for the presence of antibodies.

[0017] The bioanalytical methods described herein may further include determining whether a subject is suffering from reduced efficacy of sALP. Reduced efficacy may be determined, for example, by measuring a metric, where an increase or decrease in the metric relative to baseline indicates reduced efficacy of sALP. For example, reduced efficacy may be determined by measuring one or more blood and / or urine levels of inorganic pyrophosphate (PPi) and pyridoxal 5'-phosphate (PLP) (for example, relative to baseline). For example, levels of PPi and / or PLP may increase relative to baseline (for example, by 25% or more relative to the PPi and / or PLP concentration in a plasma sample from the subject prior to the observed reduction in sALP efficacy, e.g., 30%, 35%, 40%, 45%, 50%, 55%, 60%, or more than 60%).

[0018] Reduced efficacy may be determined by measuring one or more symptoms selected from the group consisting of, for example, hypocalcification, hypercalciuria, skeletal deformity, duck gait, bone pain, fracture, calcium pyrophosphate dihydrate crystal deposition, arthritis, pyrophosphate arthropathy, chondrocalcification, calcifying periarthritis, pseudofracture, skeletal deformity, hypotonia, muscle weakness, rheumatic complications, arthritis, pseudogout, dysphagia, pain, premature tooth loss, pulmonary dysplasia, respiratory failure, seizures, height, growth, rickets, and immunogenicity. Subjects may exhibit worsening of one or more symptoms (e.g., increased frequency or duration) compared to baseline. The determination may include establishing that subjects do not exhibit poor treatment adherence or inappropriate injection technique, and / or are not suffering from vitamin D deficiency, malnutrition, or associated diseases.

[0019] A reduction in effectiveness may be determined, for example, by conducting a quality of life assessment. The quality of life assessment may be selected from one or more of the following: the EuroQol 5D Questionnaire, the Pediatric Health Assessment Questionnaire, the Pediatric Outcome Data Collection Tool, the Pediatric Health Utility Index-9D, the Pediatric Quality of Life Inventory, the Short Health Survey36, and the Short Health Survey12. Participants may exhibit reduced quality of life assessment scores compared to baseline.

[0020] Reduced effectiveness can be determined by performing physical metric assessments. These assessments may be selected from one or more of the following: the Six Minute Walk Test (6MWT), the Bruininks-Oseretsky Test of Motor Proficiency 2nd Edition (BOT-2), the Bayley Scales of Infant and Toddler Development, 3rd Edition (BSID-III), gait analysis, use of mobility aids, the Peabody Developmental Motor Scales 2 (PDMS-2), or radiographic examination. Participants may exhibit reduced physical metric scores compared to baseline.

[0021] In some embodiments, baseline is calculated based on metric values ​​in a subject before suffering reduced efficacy of sALP, a reference subject treated with sALP and in which no neutralizing antibodies against sALP are detected, or a reference subject not administered sALP.

[0022] This method may further include determining whether the antibody is a neutralizing antibody (e.g., an anti-sALP antibody). This method may further include testing the effect of the neutralizing antibody on one or both of the catalytic activity and / or bone targeting of sALP in the presence of the neutralizing antibody. The subject may exhibit a reduction in the catalytic activity and / or bone targeting of sALP. The test may include one or more assays, such as a screening assay, a confirmatory assay, a titration assay, and / or a neutralizing antibody assay.

[0023] In any of the embodiments described above, the subject may have a bone mineralization disorder such as hypophosphatasia (HPP). The subject may be a young person, an adult, an infant, or a newborn. The subject may have a muscle weakness disorder such as fracture, osteoporosis, indurative ossification, chondrocalcinosis, hypotonia, Duchenne muscular dystrophy, tracheobronchomalacia, seizures, neurofibromatosis 1 (NF-1), craniosynostosis, or calcium pyrophosphate deposition (CPPD) or familial hypophosphatemia.

[0024] In some embodiments, the subject has a defined immunogenicity status, such as 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-.

[0025] definition As used herein, the term “approximately” refers to a value that is ±10% of the reference value.

[0026] As used herein, when a polypeptide or nucleic acid sequence is referred to as having "at least X% sequence identity" with respect to a reference sequence (where "X" is a real number), it means 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. Optimal sequence alignment can be determined by various methods within the skill of those skilled in the art, for example, by 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 readily available computer software such as GeneMatcher Plus (Schwarz and Dayhoff, Atlas of Protein Sequence and Structure, Dayhoff, MO, Ed pp 353-358, 1979), BLAST, BLAST-2, BLAST-P, BLAST-N, BLAST-X, WU-BLAST-2, ALIGN, ALIGN-2, CLUSTAL, Megalign (DNASTAR), or "Best Fit" (Smith and Waterman, Advances in Applied Mathematics, 482-489, 1981), which are incorporated into other software / hardware for alignment. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm necessary to achieve optimal alignment across the entire length of the sequences being compared.

[0027] The terms “patient” or “subject” refer to human or non-human mammals, including but not limited to cattle, horses, dogs, sheep, or cats. “Patient” specifically refers to humans.

[0028] The terms “sALP,” “soluble alkaline phosphatase,” and “extracellular domain of alkaline phosphatase” are used interchangeably (unless the context otherwise indicates) to mean soluble non-membrane-bound alkaline phosphatase or its biologically active fragments or variants. sALP includes, for example, alkaline phosphatases lacking the C-terminal GPI signal sequence, as well as further variants and analogues thereof that retain alkaline phosphatase activity, such as the ability to hydrolyze PPi or other natural or artificial substrates. This includes, unless otherwise specified, soluble fragments corresponding to the extracellular domains of TNSALP, PALP, GLALP, and IALP, as well as their biologically active fragments or variants. Mature sALP lacks the GPI membrane anchor and signal peptide, which are cleaved during processing. [Brief explanation of the drawing]

[0029] [Figure 1] This is an illustrative flowchart of an assay in various embodiments as described herein. [Figure 2] This is a schematic diagram showing the overall study design and treatment algorithm for immunosuppressive therapy. (*The Treatment and Monitoring Board (TMB) consists of HPP clinical advisors, immunology specialists, site investigators, and medical monitors (sponsors). TMB: Treatment and Monitoring Board, MTX: Methotrexate, RTX: Rituximab, BTZ: Bortezomib, E: Registration, B: Baseline, EOS: End of Study). [Figure 3] This is a schematic diagram showing the overall research design and treatment algorithm for immunosuppressive therapy. (*Concurrent administration of 500 mg / kg of intravenous immunoglobulin (IVIG) once a month while receiving rituximab + / - bortezomib; **Laboratory, X-ray; TMB: Treatment and Monitoring Committee). [Figure 4]This is a schematic diagram showing a decision tree that can be used to evaluate whether to continue, modify, or discontinue immunosuppressive therapy, such as methotrexate, rituximab, and / or proteasome inhibitors, such as bortezomib. [Modes for carrying out the invention]

[0030] Subjects with osteocalcinosis disorders such as hypophosphatasia (HPP) receiving enzyme replacement therapy (ERT) with soluble alkaline phosphatase (sALP) may develop antibodies that bind to sALP. In certain subjects, these antibodies are neutralizing antibodies that can reduce the effectiveness of sALP administered during ERT. Disclosed is a method for treating subjects with osteocalcinosis disorders such as HPP that develop neutralizing antibodies. The treatment method is known as immune tolerance induction (ITI), immune tolerance therapy (ITT), immune tolerance regimen (ITR), or immunosuppressive therapy (IST) (hereinafter collectively referred to as "IST"). The IST method includes reducing the titer of neutralizing antibodies and / or preventing or reducing the formation of neutralizing antibodies.

[0031] The presence of neutralizing antibodies in a subject can be assessed using the assays described herein. Not all antibodies that bind to sALP therapeutic agents, such as asfotase alfa, reduce the efficacy of sALP in subjects treated with ERT (e.g., HPP subjects). For example, antibodies may bind to domains of sALP (e.g., asfotase alfa) that do not affect its therapeutic function (e.g., its ability to promote bone mineralization). Therefore, one or more additional assays may be used, as described herein, to determine whether an antibody neutralizes and reduces the efficacy of sALP.

[0032] The method also features a method for identifying subjects undergoing sALP ERT as requiring IST therapy by using one or more assays to determine the presence of anti-sALP antibodies, and, if necessary, one or more assays to determine whether the antibodies neutralize and reduce the effectiveness of sALP. IST therapy may also be administered to untreated subjects who have not received sALP therapeutics, if there is reason to expect such subjects to produce neutralizing anti-sALP antibodies. Furthermore, subjects may include those who have normal or non-elevated antibody titers prior to IST therapy, but who are nevertheless at risk of developing high neutralizing antibody titers in the future.

[0033] The methods described herein may be used to treat subjects receiving sALP therapy. Subjects may have osteocalcinosis disorders such as HPP, or may have muscle weakness disorders such as fractures, osteoporosis, indurative ossification, chondrocalcinosis, hypotonia, Duchenne muscular dystrophy, tracheobronchomalacia, seizures, neurofibromatosis 1 (NF-1), craniosynostosis, or calcium pyrophosphate deposition (CPPD) or familial hypophosphatemia.

[0034] IST treatment agent Helper T cells are essential for most ADA development, while memory B cells and long-lived plasma cells amplify and maintain the response that constitutes the basis for using combined ISTs when there is a loss of efficacy due to the presence of ADA / NAb. Therapeutic proteins such as asfotase alfa can be endocytized by antigen-presenting cells after administration, where they are processed into their component peptides. Subsets of these peptides can then be presented to peptide-specific helper T cells in the context of human leukocyte antigen (HLA) molecules. Helper T cells signal to Ag-specific B cells, which are activated, proliferate, and differentiate into memory B cells and Ab-secreting plasma cells (both short-lived and long-lived).

[0035] The ISTs described herein include one or more methotrexate, rituximab, and bortezomib, which may be used based on their effects on helper T cells, plasma cells, and memory B cells. Methotrexate, a dihydrofolate reductase inhibitor, affects rapidly dividing T and B cells by preventing the reduction of dihydrobiopterin (BH2) to tetrahydrobiopterin (BH4), resulting in increased T cell sensitivity to nitric oxide synthase uncoupling and apoptosis, thereby reducing the immune response.

[0036] Rituximab is a chimeric monoclonal antibody that targets the cell surface antigen (CD20) of human B lymphocytes, a transmembrane protein present on virtually all B cells from the stage in which they become involved in B cell development until it is downregulated when they differentiate into antibody-secreting plasma cells. Rituximab causes rapid and almost complete depletion of peripheral mature B cells 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. Therefore, once B cells differentiate into plasma cells, alternative and / or additional drugs must be used to address the ADA generated by circulating plasma cells.

[0037] Bortezomib is a proteasome inhibitor. Proteasome inhibitors are a potent and specific method for targeting plasma cells that are highly dependent on proteasome activity. Bortezomib is a reversible proteasome inhibitor that targets both short-lived and long-lived plasma cells due to their high immunoglobulin production rates.

[0038] This specification describes methods for administering ISTs to subjects. In certain embodiments, ISTs are administered prophylactically to avoid the onset of an immune response, such as one, two, or more days (e.g., three, four, five, six, seven, eight, nine, ten, or more) before administration of sALP. In other embodiments, ISTs are administered to a subject, for example, following a determination that the subject has anti-sALP antibodies that interfere with ERT. After the diagnosis of a subject having anti-sALP antibodies and verification that the antibodies are neutralized, IST therapy can be administered to subjects who are experiencing reduced or lost efficacy of sALP, or who are at risk of reduced or lost efficacy. IST therapy involves reducing the presence or negative effect of neutralizing antibodies produced by the subject against sALP therapeutic agents (e.g., asfotase alfa). Furthermore, the safety and tolerability of ISTs can be evaluated by the subject. This can be achieved, optionally, by administering a small initial dose before administering the entire course of treatment. If the initial IST therapy is unsafe and / or unacceptable to the patient, IST therapy may be discontinued.

[0039] IST therapy involves identifying appropriate cellular and molecular targets for tolerance induction therapy, such as the cellular and molecular mechanisms that produce neutralizing antibodies. Given the prevalence of neutralizing antibodies of IgG1 and IgG4 isotypes, the immunoglobulin response to protein-based therapeutics may arise from a classical T-helper-dependent B-cell mechanism. Therefore, appropriate cellular targets for tolerance induction include antigen-specific T cells and B cells, as well as antigen-presenting cells. The antibody response may also require the involvement of T-cell assistance after the initiation of the response.

[0040] The IST may include administering a therapy comprising a dihydrofolate reductase (DHFR) inhibitor and an anti-CD20 antibody or its antigen-binding fragment, for example, reducing the amount of antibody, decreasing its formation, or reducing its effect. The DHFR inhibitor may be, for example, methotrexate. The anti-CD20 antibody may be, for example, rituximab or its antigen-binding fragment. The method may further include administering a proteasome inhibitor such as bortezomib.

[0041] The DHFR inhibitor may be methotrexate. 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. Methotrexate may be administered at a dose of approximately 10 mg / m². 2 ~about 20mg / m 2 (For example, 10 mg / m²) 2 , 11 mg / m² 2 , 12 mg / m² 2 , 13 mg / m² 2 , 14 mg / m² 2 , 15 mg / m² 2 , 16 mg / m² 2 , 17 mg / m² 2 , 18 mg / m² 2 , 19 mg / m² 2 , or 20 mg / m² 2 For example, approximately 15 mg / m² 2 It may be administered in doses of 15 mg / m². For example, methotrexate may be administered at 15 mg / m². 2 It can be administered in the following dosage.

[0042] The anti-CD20 antibody may be rituximab or its antigen-binding fragment. Rituximab or its antigen-binding fragment 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 dosage of rituximab or its antigen-binding fragment is approximately 100 mg / m². 2 ~about 500mg / m 2 (For example, 100 mg / m²) 2 , 125 mg / m² 2 , 150 mg / m²2 、 175 mg / m 2 、 200 mg / m 2 、 225 mg / m 2 、 250 mg / m 2 、 275 mg / m 2 、 300 mg / m 2 、 325 mg / m 2 、 350 mg / m 2 、 375 mg / m 2 、 400 mg / m 2 、 425 mg / m 2 、 450 mg / m 2 、 475 mg / m 2 、 or 500 mg / m 2 、 For example, at a dose of about 375 mg / m 2 ) can be administered. For example, rituximab can be administered at a dose of 375 mg / m 2 . Rituximab or an antigen-binding fragment thereof can be administered intravenously. In some embodiments, rituximab or an antigen-binding fragment thereof is discontinued while the subject continues to receive methotrexate and / or rituximab or an antigen-binding fragment thereof.

[0043] IST therapy can be administered for a predetermined period 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) in response to a reduction in efficacy observed for sALP. Alternatively, IST therapy can continue until it is determined that IST therapy has successfully treated or reduced the titer of neutralizing antibodies (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction). IST therapy is described herein and includes administration of an immunomodulatory agent or a therapeutic agent such as an optionally proteasome inhibitor.

[0044] Subjects may be selected for treatment with IST. Subjects may begin receiving methotrexate and rituximab, for example, in addition to bortezomib, for example, optionally, during an initial time course of 24 weeks. After this initial time course (e.g., 24 weeks), the subject's treatment status and ADA or NAb status may be determined (using the evaluation metrics described herein). sALP therapy may or may not be continued in parallel with IST. Subjects may continue to receive subsequent treatment courses (e.g., selected periods such as 24 weeks), and their treatment status and ADA or NAb status may be evaluated, for example, to determine the benefits or risks of continuing the IST treatment regimen.

[0045] While an undetectable titer of anti-sALP antibodies alone may be sufficient to discontinue IST, if recovery of sALP ERT efficacy is not observed in subjects with undetectable anti-sALP antibodies, a second criterion can be used to determine whether and when to discontinue IST.

[0046] In some embodiments, for example, therapy may be discontinued after the subject exhibits a decrease in anti-sALP antibody titer.

[0047] Proteasome inhibitors IST treatment may optionally include the administration of one or more proteasome inhibitors, such as bortezomib. Proteasome inhibitors may be used to induce plasma cell depletion. Proteasome inhibitors may optionally be administered in amounts and under conditions that enable the achievement of a reduction in sALP-specific antibodies (e.g., neutralizing anti-sALP antibodies that reduce the effectiveness of sALP ERT).

[0048] Proteasome inhibitors that may be used in conjunction with the methods described herein are disclosed, for example, in U.S. Patent No. 7,531,526, the entirety of which is incorporated herein by reference. Examples of suitable proteasome inhibitors include, but are not limited to, the following compounds, as well as their pharmaceutically acceptable salts and boronic acid esters: N-(4-morpholine)carbonyl-β-(1-naphthyl)-L-alanine-L-leucineboronic acid, N-(8-quinoline)sulfonyl-β-(1-naphthyl)-L-alanine-L-leucineboronic acid, N-(2-pyrazine)carbonyl-L-phenylalanine-L-leucineboronic acid, L-proline-L-leucineboronic acid, N-(2-quinoline)carbonyl-L-homophenylalanine-L-leucineboronic acid N-(3-pyridine)carbonyl-L-phenylalanine-L-leucineboronic acid, N-(3-phenylpropionyl)-L-phenylalanine-L-leucineboronic acid, N-(4-morpholine)carbonyl-L-phenylalanine-L-leucineboronic acid, N-(4-morpholine)carbonyl-(O-benzyl)-L-tyrosine-L-leucineboronic acid, N-(4-morpholine)carbonyl-L-tyrosine-L-leucineboronic acid, and N-(4-morpholine)carbonyl-[O-(2-pyridylmethyl)]-L-tyrosine-L-leucineboronic acid.

[0049] Proteasome inhibitors can be used in vitro or in vivo. For example, proteasome inhibitors can be used in vitro in combination with assays to determine whether they are a safe and effective IST therapeutic component. Furthermore, proteasome inhibitors can be used in vivo for IST therapy in subjects that require it (e.g., humans). Proteasome inhibitors can be administered by any number of known routes, including orally, intravenously, intramuscularly, subcutaneously, intrathecally, topically, and by infusion (Platt et al., U.S. Patent No. 4,510,130; Badalamente et al., Proc. Natl. Acad. Sci. USA 86:5983-5987, 1989; Staubli et al., Brain Research 444:153-158 (1988)), and can generally be administered in combination with a physiologically acceptable carrier (e.g., saline). The effective dose of the inhibitor to be administered can be determined empirically, based on considerations such as the specific proteasome inhibitor used, the individual's condition, and the individual's size and weight.

[0050] Proteasome inhibitors may include bortezomib (e.g., VELCADE®) and monomeric boronic acid [(1R)-3-methyl-1-[[(2S)-1-oxo-3-phenyl-2-[(pyradinylcarbonyl)amino]propyl]amino]butyl]boronic acid having the following structure.

[0051] [ka]

[0052] 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 dosage of bortezomib is approximately 0.2 mg / m². 2 ~about 2mg / m 2 (For example, 0.2 mg / m²) 2 , 0.3 mg / m² 2 , 0.4 mg / m²2 , 0.5 mg / m² 2 , 0.6 mg / m² 2 , 0.7 mg / m² 2 , 0.8 mg / m² 2 , 0.9 mg / m² 2 , 1 mg / m² 2 , 1.1 mg / m² 2 , 1.2 mg / m² 2 , 1.3 mg / m² 2 , 1.4 mg / m² 2 , 1.5 mg / m² 2 , 1.6 mg / m² 2 , 1.7 mg / m² 2 , 1.8 mg / m² 2 , 1.9 mg / m² 2 , or 2 mg / m² 2 It can be administered in doses of approximately 0.5 mg / m². For example, bortezomib can be administered at approximately 0.5 mg / m². 2 , about 0.7mg / m 2 , about 1mg / m 2 , or approximately 1.3 mg / m² 2 It may be administered in the following doses. Bortezomib may be administered intravenously or subcutaneously. Bortezomib may be administered intravenously as a bolus. Bortezomib may be administered after the patient has experienced a worsening of rickets severity score (RSS) of 1 point or more from baseline. Bortezomib may be discontinued while the patient continues to receive methotrexate and / or rituximab.

[0053] Other proteasome inhibitors suitable for use in the therapies described herein include MLN4924, NEDD-8 inhibitors, or E3 ligase inhibitors, which are currently evaluated in preclinical and clinical trials for the treatment of numerous conditions. Combinations of proteasome inhibitors belonging to the same or different classes or subclasses may be administered (e.g., combinations of two, three, four, or more proteasome inhibitors).

[0054] The optimal dosing regimen for proteasome inhibitors can be determined by those skilled in the art and may vary depending on the proteasome inhibitor, the target, and the desired effect. For example, the inhibitor may be administered at approximately 0.01 mg / m². 2 ~about 200mg / m2 For example, approximately 0.01 mg / m² 2 ~about 2mg / m 2 For example, approximately 0.2 mg / m² 2 ~about 2mg / m 2 This amount can be administered intravenously in various dosing schedules, such as twice-weekly doses. Proteasome inhibitors are also commonly administered at approximately 0.01 mg / m². 2 ~about 200mg / m 2 For example, approximately 0.01 mg / m² 2 ~about 2mg / m 2 For example, approximately 0.6 mg / m² 2 ~about 1.5mg / m 2 It can be administered in doses within this range. Optimal dosing of proteasome inhibitors can be guided by the clinical and laboratory parameters of the subject at any given time, taking into account considerations such as the desired dosing schedule and the bioavailability of the drug for a given route of administration.

[0055] Considerations regarding antibody titers are described, for example, in the document entitled "Immune Reactions Against Therapeutic and Diagnostic Biological Products" from the July 15, 1999 meeting, in which the FDA Biological Response Modifiers Advisory Committee states that "the most important criteria for evaluating the significance of antibodies against therapeutic proteins are the correlation between the incidence and quantity of antibodies and their clinical pharmacokinetics, pharmacodynamics, efficacy, and safety" (Reference: Center for Biologics Evaluation and Review (fda.gov / ohrms / dockets / ac / cber99.htm)). Qualitatively, "high titer" can be described as an antibody titer (or an antibody titer above) at which subjects would generally experience clinical decline. It should be noted that "high antibody titers" do not necessarily occur in 100% of subjects receiving sALP ERT, but there may be a subset of subjects who have, or are potentially capable of developing, high titers that affect sALP efficacy for any given disease or condition. Assays performed to detect antibodies (neutralizing or non-neutralizing) may have limitations that can lead to undetected or underestimated antibody titers. In this situation, even if the titer is undetectable or at a low level, it can still result in a decrease in sALP efficacy during sALP ERT, such as ERT using asfotase alfa. While undetectable titers of anti-sALP antibodies alone may be sufficient to discontinue IST, if recovery of sALP ERT efficacy is not observed in subjects with undetectable anti-sALP antibodies, a second criterion can be used to determine whether and when to discontinue IST.

[0056] Proteasome inhibitors such as bortezomib may cause undesirable side effects, including hematological or non-hematological toxicity. For example, bortezomib may cause neutropenia (low platelet count), cardiotoxicity, neuropathic pain, peripheral neuropathy, and / or hepatic impairment. If one or more of these side effects occur, the dose of bortezomib may be reduced (e.g., by 25% or more, e.g., 1.3 mg / m²). 2 From 1 mg / m² 2 , or 1 mg / m² 2 From 0.7 mg / m² 2 ), or if the risks outweigh the benefits, the treatment may be discontinued. For example, if a patient experiences cardiac dysfunction, for example, based on the results of echocardiography or elevated troponin levels, bortezomib should not be continued in the patient.

[0057] Other immunomodulators and combination therapies Other immunomodulators may be administered to the patient during the course of IST therapy. Immunomodulators may be administered alone or, optionally, in combination with proteasome inhibitors for reducing / preventing antibody titer formation, such as those mentioned above, at the time of administration, before administration, or after administration. Examples of immunomodulators include, but are not limited to, belimumab, anti-CD3 antibodies, anti-CD19 antibodies, and anti-CD22 antibodies, corticosteroids (e.g., prednisolone), rapamycin, methotrexate, WIG, cyclophosphamide, cyclosporine A, azathioprine, and mycophenolate mofetil, and their derivatives. These immunomodulators or their derivatives include agents that target / alter antigen presentation and / or fluid-mediated or cell-mediated immune responses.

[0058] IST therapies that induce inhibitory FcR expressed on B cells and antigen-presenting cells and target B cell survival and activation factors (such as B cell activators and B lymphocyte stimulators) may be used. A depletion approach using rituximab, a chimeric monoclonal antibody with a human IgG1 constant domain that depletes mature B cells expressing the CD20 molecule, may be particularly useful in prophylactic settings, potentially allowing for enzyme introduction at a stage where immature enzyme-specific pre-B cells and pro-B cells may be deleted or rendered unresponsive.

[0059] Combinations of rituximab with antibodies against B-cell activators may also be used. Therapeutic methods including IST following ERT are described, for example, in U.S. Patents 8,809,282, 9,050,333, 9,592,247, 9,850,474, and 10,028,993, the disclosures of which are incorporated herein by reference in their entirety.

[0060] IST therapy may also include immunoadsorption, either alone or in combination with a therapeutic agent (e.g., rituximab). Immunoadsorbents 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 period of at least one month (e.g., at least two, three, four, five, six, twelve, eighteen, twenty-four months, or longer).

[0061] Immunoglobulins may also be administered as part of IST therapy (for example, in doses of approximately 100 mg / kg, 200 mg / kg, 300 mg / kg, 400 mg / kg, 500 mg / kg, 600 mg / kg, 700 mg / kg, 800 mg / kg, 900 mg / kg, or 1000 mg / kg), either alone or in combination with other drugs. Gamma globulin may also be administered as part of IST therapy.

[0062] 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, immunoglobulin may be administered once every 7 days. Immunoglobulin may be administered in doses of approximately 300 to approximately 700 mg / kg (e.g., approximately 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., approximately 500 mg / kg). For example, immunoglobulin may be administered in doses of approximately 500 mg / kg. Immunoglobulin may be administered intravenously. Immunoglobulin may be administered intravenously once a month in doses of approximately 500 mg / kg while receiving rituximab, either with or without bortezomib. Immunoglobulin may be administered for a period of at least one month (for example, at least two, three, four, five, six, twelve, eighteen, twenty-four months, or longer).

[0063] In some specific embodiments, rituximab may be administered during IST therapy. Rituximab may be administered alone or in combination with methotrexate. The two therapeutic agents may be administered substantially simultaneously or sequentially. IST therapy, comprising rituximab or a combination of rituximab and methotrexate, may be administered for a period of about one month. During this treatment period, sALP ERT may be discontinued, or sALP ERT may continue to be administered during IST therapy. Rituximab may also be administered concurrently with immunoglobulins, gamma globulins, and / or immunoadsorbents (e.g., THERASORB®). In some specific embodiments, the therapy may comprise one or more therapeutic agents administered separately or in combination (e.g., simultaneously). One or more therapeutic agents (e.g., any of the therapeutic agents described herein) may be administered as a single composition mixed together or as separate compositions. The various combination therapies described herein may be administered as a first course of treatment (e.g., rituximab, immunoglobulin, and immunoadsorbent) or as a second subsequent course of treatment (e.g., rituximab, immunoglobulin, dexamethasone, and immunoadsorbent, and optionally a proteasome inhibitor). In some embodiments, rituximab, immunoglobulin, dexamethasone, and immunoadsorbent, and optionally a proteasome inhibitor, are administered as the first course of treatment.

[0064] Treatment may further include the administration of folic acid. Folic acid may be administered, for example, once daily. Folic acid may be administered in doses of about 0.1 mg to about 10 mg, for example, 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, for example, about 1 mg. Folic acid may be administered on a day different from the day on which methotrexate is administered. Folic acid may be administered orally. Since methotrexate is a folic acid antagonist, in certain embodiments, folate may be administered as a supplement.

[0065] IST termination Following the course of IST therapy, the subject can be monitored for a reduction in the presence of neutralizing antibodies. If IST therapy has not yet achieved improvement in the treated subject, the subject may continue to experience the continued presence of neutralizing antibodies, which can worsen or reduce the relief of symptoms of osteocalcinosis disorders (e.g., HPP) and / or reduce the effectiveness of sALP. In such situations, the dosage of the IST medication can be increased (e.g., if the subject is receiving a combination of drugs as IST therapy, the dosage of one or more of the IST drugs can be changed), the administered IST drug can be changed, or the subject can be administered a different IST therapy. During IST therapy, sALP ERT may be terminated, or sALP ERT may be continued during IST treatment. For example, IST therapy may be continued for a period of more than one week, several months, or several years (e.g., two weeks, three weeks, four weeks, five weeks, six weeks, six months, three months, one year, two years, three years, or longer), during which time sALP ERT may be terminated for all or part of this period. The above assays may be repeated to evaluate the presence and / or titer of neutralizing antibodies after IST therapy. After successful IST therapy, sALP ERT may be restarted.

[0066] Evaluation of treatment effectiveness Assays for evaluating the health status of subjects receiving sALP ERT are described below. If the assay indicates a decline in the subject's health status, the subject may be further tested for the presence of neutralizing anti-sALP antibodies. Signs that the subject is beginning to show a decline in responsiveness to sALP ERT may indicate that the subject should be tested for the presence of neutralizing antibodies. If a reduction in the effectiveness of sALP is suspected, the subject may be tested for the presence of neutralizing antibodies. This test can be performed particularly in subjects who are being treated with sALP and who have been observed to show improvement in symptoms of osteocalcinosis disorders (e.g., HPP) over a period of time, for example, about 3 months, 6 months, 1 year, or longer (e.g., 2, 3, 4, 5, 6, 7, 8 years, or longer). Subjects may be tested using, for example, the assays described herein, to determine whether neutralizing antibodies, if present, are causing a reduction in the effectiveness of sALP during ERT. If it is determined that a subject has neutralizing antibodies that reduce the effectiveness of sALP during ERT, the course of IST therapy may be initiated.

[0067] Several metrics can be used to evaluate the therapeutic effectiveness of sALP administered to a subject during ERT. These metrics can also be used as benchmarks to assess whether a subject with neutralizing antibodies is experiencing a loss of sALP effectiveness caused by the neutralizing antibodies. Furthermore, these metrics can be used in combination with IST therapy to monitor changes in sALP effectiveness (e.g., recovery of sALP effectiveness). In particular, these metrics can be used in combination with the neutralizing antibody assays described herein, both to indicate the need for IST therapy before it is administered and to indicate the success of IST therapy after it is administered. For example, the metrics can be used to measure a reduction in sALP effectiveness (e.g., a reduction of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of effectiveness), thereby indicating the need for IST therapy. A reduction in efficacy may be measured against baseline (e.g., before any reduction in efficacy, e.g., within a predetermined period after the initiation of sALP ERT, e.g., within 1 month, 3 months, 6 months, 1 year, 2 years, or longer after the initiation of sALP ERT) in one or more subjects, a reference subject with bone mineralization disorder (e.g., HPP) receiving sALP ERT, or the mean of a reference subject who has received sALP ERT. A reduction in sALP efficacy exceeding a predetermined threshold (e.g., a reduction of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of efficacy relative to the time before the reduction in efficacy) according to one or more of the metrics described herein may indicate the need for IST therapy. If it is determined that IST therapy is necessary to treat the reduced sALP efficacy caused by the presence of neutralizing antibodies, recovery or increase in sALP efficacy (e.g., an increase of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of efficacy relative to the time before the initiation of IST therapy, after the reduction in efficacy has been observed) may be tracked using one or more of these metrics.Increased efficacy may be measured relative to baseline (e.g., the subject's value before the increase or recovery of efficacy), a reference subject with impaired bone mineralization (e.g., HPP), or the mean of a reference subject who experienced a reduction in sALP efficacy during ERT or who received IST therapy. IST therapy may be discontinued and considered successful if the subject experiences an improvement in efficacy after IST therapy that exceeds a predetermined threshold (e.g., an increase of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of efficacy relative to the time before the start of IST therapy, even after a reduction in efficacy has been observed), or if the subject exhibits a recovery in the level of efficacy of sALP ERT relative to the time before the reduction in efficacy.

[0068] Examples of useful metrics for evaluating the effectiveness of sALP ERT (e.g., asfotase alfa) therapy include bone healing, bone mineralization, bone mineral density, and plasma PPi and / or PLP concentrations. This method may include evaluating a reduction in sALP effectiveness using one or more of the described metrics (e.g., bone healing, mineralization, bone mineral density, or plasma PPi and / or PLP concentrations) alone or in combination, where a reduction may indicate that neutralizing antibodies are inhibiting the subject's response to sALP ERT. Alternatively, these methods may be used to demonstrate that the effectiveness of sALP therapy has been restored after IST therapy.

[0069] Bone healing and calcification Reduced bone healing and calcification can be used as a criterion for diagnosing a patient who has, or may have, neutralizing antibodies that bind to sALP and reduce its effectiveness. In subjects with osteocalcinosis (e.g., HPP) who do not develop neutralizing anti-sALP antibodies, administration of sALP (e.g., asfotase alfa) results in increased bone healing after successful treatment. Therefore, subjects with osteocalcinosis (e.g., HPP) treated with sALP who experience reduced bone healing and calcification may experience this reduction due to the presence of neutralizing antibodies.

[0070] Reduced bone healing may include reduced calcification resulting in bone loss and separation of two or more bones. Reduced bone healing and calcification may be compared to reference bone (e.g., bone of a healthy subject, e.g., a subject without impaired bone mineralization, or a subject without anti-sALP antibodies). Methods for identifying reduced bone healing and calcification are routine and include non-invasive techniques such as radiography and computed tomography (CT). Typically, images of the relevant region of the subject can be taken before and at one or more time points after sALP treatment, and the images can be compared to assess treatment effectiveness. Reduced bone healing and / or calcification may be identified as a decrease in opacity. Images can be taken at any time during sALP treatment, for example, 1, 2, 3, 4, 5, or 6 days, weeks, months, or years after the start of sALP ERT treatment, or at a point in time when a decrease in effectiveness is suspected. A decrease in bone healing and / or calcification in a subject may become detectable at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks or 1 month after the start of the sALP ERT treatment period. In some cases, the decrease in bone healing and / or calcification in a subject may persist for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks, months, or years after the start of the sALP ERT treatment period. Therefore, a decrease in bone healing and calcification after sALP treatment may trigger the need for IST therapy to treat neutralizing antibodies and restore the effectiveness of sALP. After IST therapy, the same metrics may be used to monitor the recovery of sALP effectiveness. In this case, an increase in bone mineralization and healing may be used to determine that the effectiveness of sALP has been restored and that bone mineralization disorders (e.g., HPP) are effectively treated with sALP ERT after IST therapy.

[0071] Bone mineral density (BMD) A reduction in BMD can be used as a criterion to diagnose a patient who has, or may have, a neutralizing antibody that binds to sALP and reduces its effectiveness. In subjects with osteocalcinosis (e.g., HPP) who have not developed neutralizing anti-sALP antibodies, administration of sALP (e.g., asfotase alfa) results in an increase in BMD after successful treatment. Therefore, subjects with osteocalcinosis (e.g., HPP) treated with sALP who experience a reduction in BMD may experience this reduction due to the presence of neutralizing antibodies.

[0072] The effectiveness of sALP (e.g., asfotase alfa) during ERT can be monitored using the decrease in BMD. Methods for measuring BMD are known in the art and include, for example, bone biopsy, dual-energy absorptiometry (DXA or DEXA), peripheral quantitative CT (pQCT), high-resolution pQCT (HR-pQCT), and quantitative ultrasound (QUS). Measurements can be performed by any routine method, including CT Hounsfield measurement, and the results can be compared with a standard database or control group. BMD may also be reported as a Z-score or T-score. Pre-treatment BMD values ​​can be measured at any point during sALP ERT treatment and can be timed to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days, weeks, months, or years after the start of sALP ERT. Post-treatment baseline BMD values ​​may decrease by, for example, 0.01%, 0.05%, 0.1%, 0.5%, or 1%. The decrease in baseline BMD values ​​after the initiation of sALP treatment may also remain unchanged or be undetectable. The decrease in BMD in a subject may, in some cases, persist for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks, months, or years after the initiation of sALP ERT treatment. Therefore, a reduction in BMD after sALP treatment may trigger the need for IST therapy to treat neutralizing antibodies and restore the effectiveness of sALP. After IST therapy, the restoration of sALP effectiveness may be monitored using the same metrics. In this case, an increase in BMD of, for example, approximately 0.01%, 0.05%, 0.1%, 0.5%, or 1% or more may be used to determine that the effectiveness of sALP has been restored and that bone mineralization disorders (e.g., HPP) have been effectively treated after IST therapy.

[0073] PPi and PLP levels Increased levels of alkaline phosphatase substrates such as PPi and / or PLP can be used as a criterion for diagnosing a patient who has, or is likely to have, neutralizing antibodies that bind to sALP and reduce its effectiveness. In subjects with osteocalcinosis disorders (e.g., HPP) who have 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 after successful treatment, because sALP is enzymatically active against these substrates. Therefore, subjects with osteocalcinosis disorders (e.g., HPP) treated with sALP and experiencing an increase in PPi and / or PLP may experience this increase due to the presence of neutralizing antibodies that reduce the catalytic activity of sALP, leading to an excessive accumulation of these substrates.

[0074] The efficacy of sALP (e.g., asfotase alfa) can be monitored both before and during ERT treatment to monitor a decrease in the efficacy of sALP during ERT by increasing the amount or concentration of substrates such as PPi or PLP. Normal and abnormal concentrations of these substrates are described, for example, in PCT Publications WO2016 / 123342 and WO2017 / 171871, and these disclosures are incorporated herein by reference in their entirety.

[0075] The mean increase in PPi concentration in plasma samples from subjects receiving ERT with sALP (e.g., asfotase alfa) may be approximately 25% or more (e.g., 30%, 35%, 40%, 45%, 50%, 55%, 60%, or greater than 60%) compared to the PPi concentration in plasma samples from subjects prior to the observed reduction in the effectiveness of sALP. In some cases, the mean increase in PPi concentration in plasma samples from subjects may persist for at least one week, six months, or up to one year or more of treatment duration. This increase may persist for the duration of treatment with sALP (e.g., asfotase alfa). If the mean PPi concentration increases beyond a predetermined threshold based on typical values ​​during treatment with sALP ERT (e.g., an increase of 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or more), IST therapy may be deemed necessary. In contrast, if the mean PPi concentration decreases below a predetermined threshold (e.g., by 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or more) or to a normal level compared to a reference group (e.g., a group successfully treated with IST therapy), it may be determined that IST therapy is no longer necessary or can be discontinued.

[0076] The effectiveness of sALP (e.g., asfotase alfa) can be monitored both before and during ERT treatment to monitor the decrease in the effectiveness of sALP during ERT by using the increase in PLP levels. The mean increase in PLP concentration in plasma samples from subjects receiving ERT with sALP (e.g., asfotase alfa) may be approximately 50% or more (e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or greater than 95%) compared to the PLP concentration in plasma samples from subjects before the observed decrease in the effectiveness of sALP. In some cases, the mean increase in PLP concentration in plasma samples from subjects may persist for at least one week and up to one year or more during the treatment period. This increase may persist over the duration of treatment with sALP (e.g., asfotase alfa). If the mean PLP concentration increases beyond a predetermined threshold based on the value during treatment with sALP ERT (e.g., an increase of 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or more), it may be determined that IST therapy is necessary. In contrast, after IST therapy, a decrease in mean PLP concentration below a predetermined threshold (e.g., a decrease of 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or more) or below normal PLP levels, compared to, for example, a reference subject or a subject successfully treated with IST therapy, indicates that IST therapy is no longer necessary and can be discontinued.

[0077] Using any method known to those skilled in the art, PPi and PLP concentrations in plasma or urine samples can be quantified, for example, as described in Whyte et al., (J. Clin Invest. 95(4):1440-1445, 1995) (which is incorporated herein by reference in its entirety). In particular, PPi and PLP concentrations in plasma samples can be used to evaluate the sALP activity of a subject. Subjects with HPP typically exhibit higher plasma concentrations of PPi and PLP than healthy subjects, e.g., PPi concentrations of approximately 5 μM or higher and / or PLP concentrations of approximately 50 ng / ml or higher. The lower limit of normal plasma PPi concentration in healthy adults is approximately 1 μM, while the upper limit is approximately 5.9 μM. The lower limit of normal plasma PPi concentration in adolescent adults is approximately 0.8 μM, while the upper limit is approximately 4.9 μM. In healthy adults, the lower limit of normal plasma PLP concentration is less than approximately 10 ng / ml, while the upper limit is approximately 60 ng / ml. In adolescent adults, the lower limit of normal plasma PLP concentration is less than approximately 10 ng / ml, while the upper limit is approximately 25 ng / ml. Therefore, these boundaries for normal PPi and PLP concentrations can be used as predetermined thresholds to trigger the initiation or termination of IST therapy.

[0078] Physical measurements and quality of life assessment A reduction in scores from physical assessments or quality of life assessments can be used as a criterion to diagnose a patient having, or potentially having, neutralizing antibodies that bind to sALP and reduce its effectiveness. In subjects with osteocalcinosis disorders (e.g., HPP), administration of sALP (e.g., asfotase alfa) results in an increase in scores (e.g., correlated with positive physical fitness metrics or higher quality of life) after successful sALP treatment. Therefore, subjects with osteocalcinosis disorders (e.g., HPP) treated with sALP who experience a decrease in scores (e.g., correlated with negative physical fitness metrics or lower quality of life) may experience this result due to the presence of neutralizing antibodies that reduce the effectiveness of sALP.

[0079] Physical and quality of life metrics may be used to measure the effectiveness of neutralizing sALP antibody ERT (e.g., sALP, e.g., asfotase alfa) and / or IST therapy. For example, a negative evaluation using any of the metrics described below may indicate reduced effectiveness of sALP during ERT and that IST therapy may be necessary. In contrast, a positive evaluation using any of the metrics described below after IST therapy may indicate that IST therapy was successful and may be discontinued. Non-limiting examples of physical metrics useful for evaluating the effectiveness of therapy using sALP (e.g., SEQ ID NO: 1) include, for example, the 6-minute walk test (6MWT), the Bruininks-Oseretsky Motor Skills Scale, 2nd edition (BOT-2), the Bayley Infant and Toddler Growth Scale, 3rd edition (BSID-III), and gait analysis. Additional tests and metrics may include the Tinetti Performance Oriented Mobility Assessment (POMA; optionally modified), and spatiotemporal gait analysis software and hardware such as GAITRITE® (Clinical Image Retrieval System Inc.). Exemplary quality of life assessments include the EuroQol Five Dimension Questionnaire (EQ-5D), the Childhood Health Assessment Questionnaire (CHAQ), the Pediatric Outcomes Data Collection Instrument (PODCI), the Child Health Utility Index-9D (CHU-9D), the Pediatric Quality of Life Inventory (PedsQL), the Short Form Health Survey 36 (SF-36), and the Short Form Health Survey 12 (SF-12).The methods described herein may include using one or more of the described metrics or evaluations, individually or in combination, to evaluate the therapeutic efficacy of sALP (e.g., asfotase alfa) in subjects with osteocalcinosis disorders such as HPP, where improvement to a particular score or value demonstrates that sALP is effective in treating HPP. Furthermore, the activity level of sALP in a sample from a subject with HPP (e.g., a plasma sample) may also be used individually or in combination with one or each of these metrics to evaluate the therapeutic efficacy of sALP (e.g., any one of sALP from SEQ ID NOs: 1-3) in a subject. Assays for measuring physical metrics related to osteocalcinosis disorders such as HPP and for conducting quality of life assessments are described, for example, in PCT applications PCT / US2018 / 025206 and PCT / US2018 / 026868, the disclosures of which are incorporated herein by reference in their entirety. These metrics may be used to monitor the effectiveness or changes in effectiveness of sALP during ERT and before, during, or after IST therapy, and therefore may be used to guide the initiation or termination of IST therapy.

[0080] Assay for detecting and characterizing neutralizing antibodies Following the identification of reduced efficacy of sALP during ERT, subjects may undergo further testing to detect and characterize the presence of neutralizing antibodies. For example, a sample of the subject (e.g., blood, serum) may be tested for the presence and / or titer of neutralizing antibodies against sALP, e.g., asfotase alfa, or other alkaline phosphatases described herein. Antibodies may be characterized using various assays, such as screening assays, confirmatory assays, titration assays, and / or neutralizing antibody assays. A highly sensitive screening assay for conjugated antibodies can be used as a first step. Furthermore, the level or titer of the antibody can be measured against a control or baseline value, e.g., the amount of antibody before the observed reduction in efficacy, or the amount of a reference subject with impaired bone mineralization (e.g., HPP) that has not experienced reduced efficacy of sALP (e.g., asfotase alfa). Following this step, a confirmatory assay can be performed to confirm that the positive response is antibody-mediated. The positive sample can then be tested for the neutralizing activity of the antibody in both enzyme activity assays and bone targeting assays, and the antibody may be further evaluated for titer and isotype.

[0081] In particular, the evaluation of the immune response to sALP (e.g., asfotase alpha or other alkaline phosphatases as described herein) may involve one or more immunoassays to detect the sensitivity, specificity, and / or robustness of the antibody / therapeutic interaction. Given the complex biology of sALP ERT, the evaluation of antibody neutralization may involve, in particular, the development of at least two types of neutralizing antibody assays: one to evaluate the neutralization of the enzyme targeting bone (e.g., osteoblasts present in the mineral phase of bone), and another to measure the ability of a neutralizing antibody to inhibit the catalytic activity of sALP. These different functions are mediated by different regions in the sALP fusion protein (e.g., asfotase alpha or other alkaline phosphatases as described herein). For example, the targeting of asfotase alpha to osteoblasts (e.g., the hydroxyapatite mineral phase of bone) is mediated by a 10-residue polyaspartate region, while the enzyme activity, including substrate binding and catalytic activity, is mediated by the sALP region. Therefore, antibodies that neutralize the efficacy of sALP during ERT may be specific to one or both of the various functional domains of the sALP fusion protein (e.g., the enzymatic portion and / or the bone-targeting portion). Antibodies against other conserved elements of alkaline phosphatases, such as asfotase alpha, e.g., the Fc domain, may not neutralize, but if present at high titers, they may inhibit the efficacy of the enzyme by redirecting it to more restricted cell populations (e.g., cells with Fc receptors or FcRs) or by altering its pharmacokinetics. Assays for antibodies that neutralize targeting or catalytic activity can be performed in parallel using the same subject sample or a replica of the subject sample, and the results can be analyzed in conjunction with information on clinical outcomes in the subject.

[0082] Neutralization of catalytic activity can be evaluated by mixing serially diluted target serum with a validated potency assay that measures the activity of sALP against a specific substrate (e.g., PPi). This assay can be validated according to recommended standards. Neutralizing antibodies specific to the bone-targeting or catalytic domain of sALP are determined to interfere with the efficacy of sALP if the antibody is present at a titer sufficient to prevent targeting to osteocytes, if the antibody significantly alters the pharmacokinetics of sALP, or if the antibody inactivates sALP. Furthermore, antibody binding to sALP can alter the conformation of the enzyme, thereby making sALP sensitive to proteolysis. Assays that can be used to monitor the bone-targeting and catalytic activity of sALP are disclosed, for example, in PCT Publication WO2005 / 103263, the entirety of which is incorporated herein by reference.

[0083] One or more of these assays can be used to monitor the efficacy or changes in efficacy of sALP during ERT and before, during, or after IST therapy, and thus guide the initiation or termination of IST therapy.

[0084] Multilayer approach to ADA evaluation The development of anti-drug antibody (ADA) assays in certain embodiments is based, at least in part, on a multilayer approach. For example, the first layer may include a screening assay, and the second layer may include a confirmation assay. The screened and confirmed positive samples can then be further characterized in titration assays and neutralizing antibody assays. In certain embodiments, a screening assay is performed, a confirmation 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 the screening assay, confirmation assay, titration assay, and neutralizing assay are performed.

[0085] The assays described herein may include electrochemiluminescent (ECL) crosslinking assays, which may include a sample acidification step to extend drug resistance by dissociating drug-antibody complexes and allowing unbound anti-drug antibodies to freely compete in the assay. Positive controls, such as polyclonal antibodies purified from rabbits overimmunized with sALP such as asfotase alfa, may be used. The ADA assay may also use anti-sALP antibody crosslinking between an immobilized antigen and a reporting antigen. The immobilized antigen may be biotin-labeled sALP (B-sALP), and the reporting antigen may be ruthenium-labeled sALP (Ru-sALP). The measurement output may then be generated by a chemiluminescent signal produced by electrical stimulation of ruthenium-labeled sALP when captured by a streptavidin plate.

[0086] Screening assay Screening assays, also known as binding antibody assays, can be used to detect antibodies that bind to therapeutic protein products. Such assays can be intentionally made highly sensitive to detect low-affinity and high-affinity ADA in clinical samples. Samples positive in the screening assay are further evaluated for the specificity of their binding in a second step of a confirmatory assay.

[0087] The cutpoint of a screening assay can be the response threshold in the assay that defines whether a given sample response is positive or negative. The assay cutpoint can be influenced by a multitude of interfering products or matrix components. These components are considered early in assay development when defining the cutpoint. In certain embodiments, the cutpoint is estimated using approximately 25 to 50 individual samples. In certain embodiments, the initial screening assay has a low but defined false positive rate of approximately 5% to maximize the detection of true positives. In certain embodiments, the defined false positive rate is approximately 1 to 5%, or less than 5%, less than 3%, or less than 1%. Since samples from different target populations and disease states may have components that can alter the background signal from the assay, different cutpoints may be required for individual target patient populations.

[0088] In certain embodiments, the cutpoint is statistically determined using samples from subjects who have not previously received treatment. The variability of the assay can be estimated by performing repeated assay runs (e.g., 2, 3, 4, or 5 runs) using these samples. Statistical approaches used to determine the cutpoint may include removing statistical outliers and considering existing antibodies. Each sample can be tested in the ADA screening assay for at least six individual measurements in total, on at least three different days, by at least two analysts. One approach that allows for a high guarantee of a 5% false-positive rate may be to apply a lower one-sided confidence interval of 90% to the 95th percentile of the negative control population. This can guarantee a false-positive rate of at least 5% at a 90% confidence level. This approach can improve the probability of the assay identifying all subjects who may develop antibodies. The statistical method used to determine the cutpoint may be based on the statistical distribution of the data. For example, in certain embodiments, the 95th percentile of a normal distribution is estimated by the mean + 1.645 standard deviation.

[0089] In certain embodiments, when determining the true incidence of immunogenicity, a subsequent confirmatory assay or other assay may be used to eliminate false-positive results.

[0090] Confirmation assay The confirmatory assay used in a particular embodiment is a competitive assay that can confirm the specific binding of ADA to the therapeutic protein of interest. The purpose of this assay is to eliminate potential false-positive samples from the initial screening assay. ADA confirmatory samples can be further characterized in subsequent titer assays and neutralization assays.

[0091] The confirmation assay format can be a competitive assay in which competitors, such as unlabeled therapeutic protein products, are used. In certain embodiments, performing a suitable confirmation assay ensures that data on ADA false-positive subjects does not confuse the analysis of the impact of ADA on safety and efficacy.

[0092] An exemplary approach to determining the cutpoint in a confirmatory assay uses data from signals generated by antibody-negative, treatment-naive control samples in the presence of a competing drug. In this case, the amount of therapeutic protein product can be used to establish the cutpoint. In certain embodiments, the amount of therapeutic protein product used is the same as the amount of the competing inhibitor in the assay.

[0093] In certain embodiments of the confirmation assay, the assay uses sALP (e.g., 100 μg / mL, 150 μg / mL, 200 μg / mL, 250 μg / mL, 300 μg / mL, 350 μg / mL, 400 μg / mL, 450 μg / mL, or 500 μg / mL) as a competitive inhibitor of B-sALP and Ru-sALP binding to ADA. Following the incubation period, the B-sALP / ADA / Ru-sALP complex captured by streptavidin plates can be measured, for example, by an MSDSectorImager6000 plate reader. In certain embodiments, for each sample, the inhibition percentage by the sALP assay inhibitor can be calculated as follows:

[0094]

number

[0095] In certain embodiments of the confirmatory assay, the cut-off point can be established from a statistical analysis of data from assayed drug-naive normal donors, with or without free sALP. For example, the top 99.9th percentile of the statistical analysis may be used to select the confirmatory assay cut-off point (e.g., 5%, 10%, 15%, 20%, 25%, or 30% inhibition). If the inhibition percentage is greater than or equal to the confirmatory assay cut-off point at inhibition, the sample can be confirmed as anti-sALP positive.

[0096] In certain embodiments, samples confirmed positive in the confirmatory assay are reported as positive and further evaluated in the titration assay. Samples below the confirmatory assay inhibition cutoff point can be reported as negative and no further testing is required.

[0097] Titration assay Titration assays can be used in certain embodiments to characterize the magnitude of the ADA response. In certain embodiments, the magnitude is characterized by titration assays because the effects of ADA on pharmacokinetics, pharmacodynamics, safety, and efficacy may correlate with ADA titer and persistence rather than incidence.

[0098] In certain embodiments, a titration assay-specific cutpoint is established using a false-positive rate of approximately 0.1%, or a screening cutpoint is used as the titration cutpoint. In certain embodiments, the titration assay cutpoint is established at approximately 0.01% to approximately 0.1% or less than approximately 0.1%.

[0099] Neutralization assay A neutralizing antibody (NAb), in certain embodiments, refers to a specific ADA that has the ability to interfere with the interaction between a therapeutic protein product and its target. A neutralization assay can evaluate the neutralizing activity of an ADA. In certain embodiments, the neutralizing activity of an ADA is characterized by partially evaluating the effect of the ADA on pharmacokinetics, pharmacodynamics, safety, and efficacy, which may correlate with NAb activity rather than ADA incidence.

[0100] In certain embodiments, the test method selected to evaluate the neutralizing ability of ADA-positive samples is based on the mechanism of action of the therapeutic protein product. In vitro neutralization assays can demonstrate the potential of ADA to inhibit the therapeutic activity of a product. NAbs can interfere with the clinical activity of therapeutic protein products by preventing them from reaching their target or by interfering with pharmacological activities such as receptor-ligand interactions.

[0101] Figure 1 is an illustrative flowchart of an assay in various embodiments as described herein. The methods applied to each of the matters related to Sequence IDs 1-3 may be the same or different in the application of the flowcharts or parts of the processes described herein.

[0102] In certain embodiments, a NAb assay for detecting whether an anti-sALP antibody is a neutralizing antibody uses an enzyme-catalyzed steady-state kinetic assay to determine the effect of NAb on sALP efficacy. For example, the assay may measure the hydrolysis of pNPP proportional to the enzyme activity. The NAb assay may include a negative control, a positive control, and acid treatment (e.g., 0.1 M glycine-HCl, 15% v / v) of the study sample prior to the enzyme activity assay. Acidification of the sample may be used to dissociate the antibody bound to sALP and to denature and inactivate sALP. Following the neutralization step, the sample may be added to the activity assay. In the activity assay, small aliquots (e.g., 20 μL) of a fixed concentration of sALP (e.g., 95.0 ng / mL) may be added to each well of the assay. After incubation, the reaction can be initiated by adding pNPP (e.g., final concentration of, for example, 1 mM, 2 mM, 3 mM, 4 mM, or 5 mM or higher) to a suitable reaction buffer (e.g., 20 mM bis-trispropane, 50 mM NaCl, 0.5 mM MgCl2, 50 μM ZnCl2, 0.5 mg / mL BSA, and pH 9.0 at 37°C). Research samples containing NAb inhibit the turnover of pNPP compared to assay-negative controls. Inhibition of asfotase alpha activity by antibodies in each sample (% (V)) max ) can be calculated using the following formula:

[0103]

number

[0104] The cut-off point for the neutralization assay can be determined, for example, from the analysis of 50 individual drug-naïve human serum samples evaluated in two or more separate runs (e.g., 3, 4, 5, or 6). The assay cut-off point is then statistically determined, for example, by the top 99.9 percentile inhibition (V) of the reaction rate. maxThis can be expressed as follows: Samples with an inhibition percentage below this cutoff point may be considered NAb-negative, while samples with an inhibition percentage above this cutoff point may be reported as NAb-positive.

[0105] The results from the above assays can be validated using a positive control, such as a polyclonal anti-sALP antibody. Parameters such as sensitivity (e.g., the positive control concentration that elicits a positive reaction in all assays), selectivity (e.g., 60%, 70%, 80%, 90% or more of the tested sALP lots required to meet the criteria), precision and accuracy (e.g., signal-to-noise ratio of concentration variability <5%, 15%, 20%, or 25%), specificity, drug tolerance / interference (e.g., the highest drug concentration that elicits a positive reaction), and sample stability (e.g., recovery rate within 100±20%) can also be evaluated using predetermined thresholds used for acceptance criteria.

[0106] Definition of immunogenicity subgroups Analysis of PK measurements may be used to determine whether sALP efficacy is reduced during ERT. Specific aspects of the immunogenic response that can be investigated include, for example, the temporal variability of immunogenicity, the immunogenic state (e.g., ADA+ vs. ADA- and NAb+ vs. NAb-), and the magnitude (e.g., high vs. low for ADA and NAb titers). To assess the impact of antibody development, subjects may be assigned to subgroups according to their seroconversion, e.g., immunogenicity for sALP ADA and NAb development. The impact of immunogenicity on PK is then assessed based on the various PK parameters (e.g., PPi and / or PLP concentrations, C) between these subgroups. avg , C max , and V max The differences in ) can be evaluated by testing. Exemplary PK parameters are described, for example, in PCT application PCT / US2018 / 025206, which is incorporated herein by reference in whole.

[0107] To assess immunogenicity, one approach may be to assign subjects to one of two groups, such as positive or negative for antibodies. Defining a subject positive for antibodies can be done, for example, using a single occurrence of a subject showing a positive test result for ADA at any point after treatment with sALP (e.g., asfotase alfa). The subject may then be assigned to the ADA+ group (ADA-positive throughout the entire treatment period). If a subject never produces a measurable antibody, the subject is assigned to the ADA- group (ADA-negative throughout the entire treatment period). Each subject may be further assigned to one of three possible subgroups: ADA- (negative for anti-drug antibodies, NAb not measurable), ADA+ / NAb+ (positive for ADA+ and neutralizing antibodies), and ADA+ / NAb- (ADA+ but negative for neutralizing antibodies). Example 2 provides further details regarding the assessment of immunogenicity grouping.

[0108] Alkaline phosphatase Alkaline phosphatases that can be administered as part of ERT to subjects with bone mineralization disorders such as HPP include a group of enzymes that catalyze the cleavage of phosphate moieties (e.g., hydrolysis of pyrophosphate, PPi). There are four known mammalian alkaline phosphatase (ALP) isozymes: tissue-nonspecific alkaline phosphatase (TNALP; further described below), placental alkaline phosphatase (PLALP) (e.g., accession numbers P05187, NP_112603, and NP_001623), germ cell alkaline phosphatase (GALP) (e.g., accession number P10696), and intestinal alkaline phosphatase (IALP) (e.g., accession numbers P09923 and NP_001622). In addition to the exemplary ALPs discussed herein, any polypeptide having the same or similar catalytic site structure and / or enzymatic activity as ALP may be used in the methods disclosed herein (for example, as sALP or sALP fusion polypeptide as described herein).

[0109] sALP can be, for example, a soluble form of human tissue-nonspecific alkaline phosphatase (human TNALP (hTNALP)). An example of soluble alkaline phosphatase (sALP) is asfotase alpha, a human TNALP fusion polypeptide. In particular, any one polypeptide 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 administered and used to treat osteocalcinosis disorders such as HPP. sALP may contain or consist of the amino acid sequence of SEQ ID NOs. sALP may contain or consist of the amino acid sequence of SEQ ID NOs. sALP may contain or consist of the amino acid sequence of SEQ ID NOs. sALP may contain or consist of the amino acid sequence of SEQ ID NOs. 3

[0110] Alkaline phosphatase may contain the sequence described in SEQ ID NO: 1 (Sequence ID 1)

[0111] Alkaline phosphatase may contain the sequence described in Sequence ID No. 2: (Sequence ID 2).

[0112] Alkaline phosphatase may contain the sequence described in SEQ ID NO: 3: (Sequence ID 3).

[0113] Exemplary sALPs are physiologically active against, for example, phosphoethanolamine (PEA), inorganic pyrophosphate (PPi), and pyridoxal 5'-phosphate (PLP). In particular, sALPs are catalytically suitable for improving skeletal calcification in bone. Examples of mutations that can be introduced into ALP sequences are described in U.S. Patent Application Publication 2013 / 0323244.

[0114] The sALP (TNALP, for example, an sALP fusion polypeptide of any one of SEQ ID NOs: 1-3, or a polypeptide variant having at least 95% sequence identity to any one of SEQ ID NOs: 1-3, for example, asfotase alpha), linker, spacer (for example, Fc region), and bone targeting moiety described herein may be in the form of a fusion polypeptide having the structure Z-sALP-Y-spacer-X-Wn-V, Z-Wn-X-spacer-Y-sALP-V, Z-sALP-Y-Wn-X-spacer-V, or Z-Wn-X-sALP-Y-spacer-V. In particular, the structure of the sALP fusion polypeptide may be Z-sALP-Y-spacer-X-Wn-V or Z-Wn-X-spacer-Y-sALP-V. The sALP in the sALP fusion polypeptide may be full-length ALP or a functional fragment of ALP, such as the soluble extracellular domain of ALP (e.g., TNALP, PALP, GCALP, and IALP), as described herein. Bone delivery conjugates containing sALP are further described in PCT Publications WO2005 / 103263 and WO2008 / 138131, the disclosures of which are incorporated herein by reference in their entirety.

[0115] Any one of X, Y, Z, and V and / or a spacer may be absent or a linker region containing the 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 aspartate-isoleucine), a two-residue linker at position Y (e.g., leucine-lysine), or a two-residue linker at position X (e.g., aspartate-isoleucine). For example, the sALP fusion polypeptide may have the structure hTNALP-Fc-D10 (e.g., an sALP fusion polypeptide containing 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 with any one of SEQ ID NOs: 1-3, e.g., asfotase alpha).

[0116] Wn can be, for example, a bone-targeting moiety having a series of consecutive aspartate (D) or glutamate (E) residues, where n=1 to 50, for example n=3 to 30, for example 5 to 15, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 36, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. If present, the bone-targeting moiety can be located anywhere in the fusion polypeptide, for example, at the N-terminus or C-terminus, or in its vicinity, and / or in the linker region. For example, the bone-targeting moiety can be located at the C-terminus of the sALP fusion polypeptide. Both sALP and the fusion polypeptide may also lack a bone-targeting moiety.

[0117] The methods described herein may be performed in conjunction with sALP therapy, for example, administration of sALP. The therapy may be performed before or after administration of sALP therapy. sALP (for example, any one of sALPs from SEQ ID NOs. 1 to 3, such as sALP of SEQ ID NO: 1) may be administered 1 to 7 times per week or every two weeks (for example, 2, 3, 4, 5, 6, or 7 times), for example, 2, 3, or 6 times per week. sALP may be administered once a week, once every two weeks, once every three weeks, or once every four weeks. For example, sALP may be administered 3 or 6 times per week. sALP (for example, sALP of SEQ ID NO: 1) may be administered in doses of 1 mg / kg / week to 10 mg / kg / week (for example, 3 mg / kg / week to 9 mg / kg / week, e.g., 1 mg / kg / week, 2 mg / kg / week, 3 mg / kg / week, 4 mg / kg / week, 5 mg / kg / week, 6 mg / kg / week, 7 mg / kg / week, 8 mg / kg / week, 9 mg / kg / week, or 10 mg / kg / week). sALP may be administered at a dose of 1 mg / kg / week. sALP may be administered at a dose of 2 mg / kg / week. sALP may be administered at a dose of 3 mg / kg / week. sALP may be administered at a dose of 6 mg / kg / week. sALP may be administered at a dose of 9 mg / kg / week. sALP may be administered at a dose of 1 mg / kg six times a week, 2 mg / kg three times a week, or 3 mg / kg twice a week. In some embodiments, the dosage is 3 mg / kg three times a week, or increased from 3 mg / kg twice a week to 3 mg / kg three times a week.

[0118] In one embodiment, sALP (for example, sALP of any one of sequence numbers 1 to 3, such as sALP of sequence number 2 or 3) is administered in a dose of about 10 mg to 50 mg (for example, once per week or once every two weeks). For example, sALP (for example, sALP of sequence number 2 or 3, such as sALP of sequence number 2) may be administered in doses of 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, or 50 mg, for example, 20 mg, 35 mg, or 50 mg, for example, once per week or once every two weeks.

[0119] Vials containing sALP (e.g., sALP of Sequence ID No. 1) can be prepared at concentrations up to 100 mg / mL, for example, 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).

[0120] In some embodiments, the pharmaceutical composition is administered subcutaneously or intravenously. For example, 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, for example, about 15 mg, 45 mg, or 90 mg, for example, about 25 mg, 35 mg, or 50 mg) of sALP (e.g., sALP of SEQ ID NO: 2, or 3) may be administered subcutaneously to the subject, for example once or twice per week, for example, for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks, or longer (e.g., over the lifetime of the subject). The composition may be administered in amounts of approximately 5 mL or less (for example, 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 amounts in the range of approximately 5 mL to approximately 0.1 mL).

[0121] In some embodiments, the second sALP treatment involved less frequent administration than the treatment involving the first sALP. In some embodiments, subjects treated with sALP were administered sALP at doses ranging from 1 mg / kg / week to 10 mg / kg / week, and subjects were also treated with a second sALP distinct from the first sALP, which was administered once every two weeks at doses of 20 mg, 35 mg, or 50 mg.

[0122] The methods described herein may include treating a subject administered with two different sALPs. For example, in some embodiments, a subject is treated with a first sALP (e.g., sALP of SEQ ID NO: 1) for a period of time (e.g., one day or more, one week or more, one month or more, or one year or more), and then administered a second sALP different from the first sALP (e.g., sALP of SEQ ID NO: 2, or sALP of SEQ ID NO: 2 or 3). The treatment of the subject may be performed alternately between the first and second sALPs, or the subject may receive the first sALP and then transition to the second sALP only.

[0123] sALP may be discontinued during the administration of therapy. For example, sALP may be discontinued for about one, two, three, four weeks, or longer, for example, until a reduction in neutralizing antibodies is achieved.

[0124] The above-mentioned sALP can be administered to subjects in need for the treatment of calcification disorders such as HPP, or for the treatment of their symptoms. Symptoms of bone calcification disorders such as HPP include, for example, hypocalcification, skeletal deformities, fractures, and bone pain. Symptoms of adult HPP as defined herein include, for example, elevated blood and / or urinary levels of phosphoethanolamine (PEA), hypocalcification, hypercalciuria, skeletal deformities, duck gait, bone pain, fractures, calcium pyrophosphate dihydrate crystal deposition, arthritis, pyrophosphate arthralgia, chondrocalcification, calcifying periarthritis, pseudofractures, skeletal deformities, hypotonia, muscle weakness, rheumatic complications, arthritis, pseudogout, difficulty walking, pain, premature tooth loss, pulmonary dysplasia, respiratory failure, seizures, height, growth, rickets, and immunogenicity. Symptoms of adolescent HPP as defined herein include, for example, elevated blood or urinary levels of PPi, PEA, or PLP; osteomalacia; one or more skeletal deformities; hypotonia; muscle weakness; rheumatic complications; arthritis; pseudogout; duck gait; dysphagia; bone pain; pain; premature tooth loss; hypocalcification; pulmonary dysplasia; respiratory failure; seizures; hypercalciuria; short stature; and growth retardation. Changes in any of these symptoms can be used to monitor the effectiveness of sALP during ERT (e.g., using asfotase alfa) before and after ERT treatment and / or before or during IST therapy to monitor a decrease or increase in the effectiveness of sALP during ERT.

[0125] Subjects who are at risk of developing neutralizing antibodies against sALP, or who have experienced loss or reduction in the effectiveness of sALP during sALP ERT, and who have been administered sALP, may be treated with IST therapy using the method described below. IST therapy includes one or more of the following steps: (a) identifying and / or diagnosing a subject who has experienced reduced effectiveness of sALP and who requires IST therapy; (b) testing a sample from the subject for neutralizing antibodies against sALP therapeutic agents; (c) determining whether the neutralizing antibodies are the cause of the reduced effectiveness of sALP; (d) treating the subject with IST therapy; and (e) determining when to discontinue IST therapy. [Examples]

[0126] The following examples are intended to be illustrative, not limiting, of this disclosure. It should be understood that specific examples, materials, quantities, and procedures should be interpreted broadly in accordance with the scope and spirit of this disclosure as described herein.

[0127] Example 1. Interventional prospective open-label study (RESTORE) of immunosuppressive therapy to mitigate immune-mediated loss of treatment response to asfotase alfa (STRENSIQ®) for hypophosphatasia. Theoretical basis: The administration of biological (protein) drugs to patients, particularly through continuous exposure to treat chronic conditions, carries the risk of inducing anti-drug antibodies (ADAs). This risk is greater when the biotherapy drug has a potential "endogenous counterpart," such as when proteins are administered for replacement therapy to treat conditions like hypophosphatasia (HPP). Neutralizing antibodies (NAbs) can neutralize the clinical benefits of exogenous biotherapy agents. At the same time, non-NAbs can also reduce efficacy by accelerating drug clearance (CL), pharmacodynamics (PD), and pharmacokinetics (PK).

[0128] In post-marketing safety studies, some patients treated with asfotase alfa showed an initial therapeutic response but subsequently experienced disease relapse and progression associated with clinical, laboratory, and radiographic markers. Since some of these events occurred in conjunction with the presence of recorded positive ADA and Nab, an immune-mediated effect on the pharmacological action of asfotase alfa may have contributed to disease progression. Based on this information, the U.S. FDA requested a post-marketing requirement (PMR) study to assess and characterize the potential significant risk of immune-mediated loss of effectiveness (LoE) and to evaluate strategies to mitigate it (PMR#2949-7, BLA 125513).

[0129] This prospective, open-label clinical study will evaluate the ability of three immunosuppressive therapies (ISTs)—methotrexate, rituximab, and bortezomib—to reduce immune-mediated LoE in pediatric patients with HPP receiving long-term asfotase alfa therapy. The study will also investigate whether long-term ISTs are necessary to maintain the desired efficacy of asfotase alfa in participants with HPP.

[0130] [Table 1] a ADA is the primary outcome for patients who are ADA-positive and NAb-negative. NAb is the primary outcome for patients who are ADA-positive and NAb-positive. Abbreviations: ADA = anti-drug antibody, CD19 = differentiated cluster 19 (B lymphocyte antigen), ECG = electrocardiogram, HPP = hypophosphatasia, IST = immunosuppressive therapy, LoE = loss of efficacy, NAb = neutralizing antibody, PD = pharmacokinetics, PK = pharmacokinetics, PLP = pyridoxal-5'-phosphate (vitamin B6), PPi = inorganic pyrophosphate, RSS = rickets severity score, TEAE = adverse events occurring under treatment, TESAE = serious adverse events occurring under treatment, TNSALP = tissue-nonspecific alkaline phosphatase

[0131] Overall design: This is a phase 4 prospective, open-label trial designed to evaluate the effect of IST on the clinical symptoms of HPP in pediatric participants receiving treatment with asfotase alfa exhibiting immune-mediated LoE.

[0132] Identification of immune-mediated LoE requires radiographic evidence of rickets in pediatric patients aged 2 to under 18 years with open epiphyseal plates in the presence of ADA and / or NAb, based on the following criteria: 1. Relapse or exacerbation of rickets in the past 3 months in children who have shown an initial effective response to asfotase alfa after at least 6 months of continuous treatment and are currently receiving asfotase alfa. Baseline severity is determined using the Rickets Severity Score (RSS). 2. Regardless of the presence of ADA, NAb, or their titers at the time of screening. 3. Confirmation of the existence of both clinical evidence and immunogenicity-mediated associations.

[0133] Participants will be identified from the HPP Global Registry (ALX-HPP-501) and HPP Registry Substudies (ALX-HPP-501s). Furthermore, patients outside the registry will be identified through the ADA trial offered in the United States, or through a preliminary screening portion of the study if the participant is outside the United States. All potential participants must also have a documented clinical LoE.

[0134] Participants will be followed for 104 weeks after the initial IST administration. At the start of the study, methotrexate and rituximab will be administered for an initial 24-week period starting from day 1. Following this initial 24-week period, the schedule will be repeated as follows: weeks 25-26, weeks 27-50 (TA period 2), weeks 51-52, weeks 53-76 (TA period 3), weeks 77-78, and weeks 79-104 (TA period 4). Asfotase alfa therapy for HPP will be continued in parallel with IST throughout the study. After each 24-week treatment period, the radiographic findings of rickets will be assessed using RSS and ADA and / or NAb status and titers to determine the benefits / risks of using or continuing the IST treatment plan over the following 24 weeks. Unless there are safety concerns, it should be noted that methotrexate will be continued throughout the study, and rituximab will be administered without interruption for at least the first 18 months (approximately 74 weeks). While administering rituximab, 500 mg / kg of intravenous immunoglobulin (IVIG) will be administered concurrently once monthly, either with or without bortezomib. The decision to add bortezomib to the IST regimen will be based on radiographic assessment of rickets with an increase of one or more points on the RSS in the presence of an increase in ADA or NAb levels from baseline. At 18 months, if a participant has demonstrated a complete response and is receiving methotrexate and rituximab, rituximab will be discontinued and methotrexate will be continued until the end of the study. At 18 months, if a participant has achieved a complete response and is receiving methotrexate, rituximab, and bortezomib, bortezomib will be discontinued, and methotrexate and rituximab will be continued until the end of the study. The drug regimens are shown in Figure 3 and Tables 4 and 5. Participants will continue to be treated with the same subcutaneous (SC) asfotase alfa regimen as enrollment during the study.

[0135] The end-of-study (EoS) evaluation will be conducted at the 100-week visit or in the event of early discontinuation (ED, if applicable).

[0136] Disclosure statement: This is an unblinded, single-arm study to treat participants with HPP exhibiting immune-mediated LoE.

[0137] Intervention group and duration: All participants will continue receiving asfotase alfa therapy without interruption throughout the 104 weeks of IST treatment in the study, in accordance with the standard treatment prescribed by their treating physician. IST treatment will follow the Schedule of Activity (SoA). End of Study (EoS) assessments will be performed at the 100-week visit or at the End of Study (ED) (if applicable).

[0138] All participants will receive a safety follow-up call, and safety blood and urine tests will be collected at week 104. The global end of the study will be defined as the date of the last completed safety follow-up call for the last participant.

[0139] [Table 2-1]

[0140] [Table 2-2]

[0141] [Table 2-3]

[0142] [Table 2-4] a For assessments performed between screening and baseline (i.e., on day 1 of week 1). For assessments not performed again on day 1, the screening will be used as their baseline. b The first TA period begins with the first dose of IST on day 1. cA signed and dated IRB / IEC approved ICF must be obtained before any study-specific screening procedure is performed. d After reviewing each participant's HPP history, a decision will be made regarding each participant's eligibility to enroll in the study. e ADA samples should be collected before asfotase alfa administration (i.e., before drug trough administration). Only ADA-positive samples should be analyzed by NAb assay. f This review will determine the eligibility of participants. g If available, the historical results are acceptable and no confirmation test is required. h Postmenopausal women who are not using hormonal contraception or hormone replacement therapy (HRT). i Vitamin D levels should be >20 ng / mL. If levels are <20 ng / mL at the time of screening, rescreening is possible after vitamin D supplementation. j Serum pregnancy tests should be performed at each point in time for all female participants who may be pregnant. Urine pregnancy tests should be performed monthly at home by female participants if facility visits are not available. k We will evaluate new and temporal changes in the use of mobility aids (e.g., canes, walkers, wheelchairs) across all age groups. l If an X-ray is available to the participant and was taken within the past two weeks prior to the indicated clinic visit, it does not need to be repeated. m If these tests are conducted within three months of the screening visit, they do not need to be repeated. nIf a fracture is present at screening, follow-up X-rays will be performed at weeks 12, 22, 48, 74, and 100, or earlier if the fracture has healed. If a fracture develops after screening, X-rays will be performed at least every three months during study participation, or immediately if the fracture has healed. o IVIG 500 mg / kg is administered monthly, either with or without bortezomib, 24 to 48 hours after the initial dose of a series of rituximab treatments. Abbreviations: 25-OH = 25-hydroxy, ADA = anti-drug antibody, ALP = alkaline phosphatase, ALPL = gene encoding tissue-nonspecific alkaline phosphatase (TNSALP) isoenzyme, βHCG = β-human chorionic gonadotropin, BOT-2 = Bruininks-Oseretsky Exercise Scale, 2nd Edition, BPI-SF = Shortened Pain Inventory, CD19 = Differentiation Cluster 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 drug, IRB = Institutional Review Board, ISR = Injection / Injection Site Statistical response, IST = immunosuppressive therapy, IVIG = intravenous immunoglobulin, LFT = liver function test, mIgM = immunoglobulin M, N / A = not applicable, NAb = neutralizing antibody, NIMP = non-investigational drug, PE = physical examination, PEA = phenylethylamine, PDMS-2 = Peabody Developmental Motor Scale II, PK = pharmacokinetics, PLP = pyridoxal-5'-phosphate (vitamin B6), PPi = inorganic pyrophosphate, PTH = parathyroid hormone, RSS = rickets severity score, SAE = serious adverse event, TA = treatment evaluation, TEAE = adverse event occurring under treatment, TESAE = serious adverse event occurring under treatment, Wk = week

[0143] [Table 3-1]

[0144] [Table 3-2] a The treatment regimen will be determined by considering all available participant data during the 2-week intervals between each 24-week TA period. b Scheduled repetitions 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 have blood drawn at week 104 for safety assessment. c Facility staff should pay attention to each participant's decisions in their CRF (Clinical Research Form). d ADA samples should be collected before asfotase alfa administration (i.e., before drug trough administration). Only ADA-positive samples should be analyzed by NAb assay. e The screening and study participation tests were conducted only once a year. f The assessment tool used is the Pediatric-modified Total Neuropathy Scale (ped-mTNS). Assessments should be performed before the first dose and at the end of each cycle in which the participant receives bortezomib. g The evaluation was conducted only on participants receiving bortezomib. hIf participants receive bortezomib, echocardiography and ECG evaluation should be performed before the first dose of bortezomib. Subsequently, evaluations should be performed at 3, 6, and 12 months, after the first dose, and at the end of the study (e.g., for participants starting bortezomib at week 27, evaluations should be performed at weeks 27, 38, 53, 79, and 100), or whenever clinically required. In addition, cardiac safety biomarkers, cardiac troponin (T or I, preferably high sensitivity, where applicable) and NT-pro BNP should be measured before the first dose and repeated as clinically indicated. See Table 7. i Participants receiving bortezomib alone will have samples collected before bortezomib administration on day 1 and day 29. j IgG testing is performed before every rituximab infusion cycle. k Serum pregnancy tests should be performed at each point in time for all female participants who may be pregnant. Urine pregnancy tests should be performed monthly at home by female participants if facility visits are not available. l New and evolving uses of mobility aids (e.g., canes, walkers, wheelchairs) for all age groups. m If an X-ray is available to the participant and was taken within the past two weeks prior to the indicated clinic visit, it does not need to be performed again. n If a fracture is present at the time of screening, follow-up X-rays will be performed at weeks 12, 22, 48, 74, and 100, or earlier if the fracture has healed. If a fracture develops after screening, X-rays will be performed at least every three months during study participation, or immediately if the fracture has healed. o Twice a week during each indicated week, following the medication schedule shown in Table 5. p IVIG 500 mg / kg is administered monthly, either with or without bortezomib, 24 or 48 hours after the first dose of a series of rituximab doses. Abbreviations: ADA = Anti-drug antibody, ALP = Alkaline phosphatase, βHCG = β-Human chorionic gonadotropin, BOT-2 = Bruininks-Oseretsky Exercise Scale, 2nd Edition, CD19 = Differentiation Cluster 19 (B lymphocyte antigen), CRF = Case report form, ECG = Electrocardiogram, ED = Early termination, EoS = End of study, ICF = Informed consent form, Ig = Immunoglobulin, IMP = Investigational drug; ISR = Injection site reaction, IST = Immunosuppressive therapy, IVIG = Intravenous immunoglobulin, LFT = Liver function test, N / A = Not applicable, NAb = Neutralizing antibody, NIMP = Non-investigational drug, NT-pro BNP = N-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 evaluation, TEAE = adverse event occurring under treatment, TESAE = serious adverse event occurring under treatment IST treatment schedule and medication regimen: Methotrexate: Methotrexate is administered starting on day 1 and once every 7 days. The treatment schedule and dosing regimen are within the approved labeling for methotrexate (Table 7). Folic acid deficiency may increase the risk of methotrexate-related adverse reactions. Therefore, folic acid should be administered to participants while they are receiving methotrexate.

[0145] [Table 4] Note: 375 mg / m² per 24-week period. 2 ×4 doses. * Align the treatment schedule and medication regimen with the approved labeling for rituximab. Abbreviations: RTX = Rituximab, ND = No medication, TA = Treatment evaluation (duration)

[0146] [Table 5] Note: 1.3 mg / m² per dose 2 This is the starting level. * Bortezomib is not currently approved for use in children. Abbreviations: BTZ = Bortezomib, ND = No medication, TA = Treatment evaluation (duration)

[0147] background Clinical immunogenicity experiments using asfotase alfa therapy In the clinical study, most participants treated with asfotase alfa for HPP were positive for ADA at some point after baseline (97 / 109, 89%). Generally, ADA titers were low, with a median peak titer of 32.0 (range: 0–2048). The median time to the first ADA positive result was 43.0 days (range: 14–2374 days). Similar to the ADA results, participants who tested positive for NAb at some point after baseline were not consistently positive after the initial positive result. Of the 97 participants who were positive for ADA after baseline, 55 (56.7%) were positive for NAb at some point after baseline. The median peak NAb inhibition percentage was 9.910 (range: 4.54–95.7). No correlation was observed between ADA titer and NAb (% inhibition) values.

[0148] Overall, these ADA responses had a very small impact on the asfotase alfa drug concentration profile. Population-pharmacokinetic (Pop-PK) model results (N=58) demonstrated that the effect of ADA status on asfotase alfa CL was not significant (≤20% increase). Simulations evaluating the effect of immunogenicity on CL revealed a 90% confidence interval (CI) for mean exposure at steady state, supporting the finding that the overall impact of low-titer antibody responses on asfotase alfa PK was small. Considering the small impact of immunogenicity on PK and the fact that participants administered 6 mg / kg / week were close to a plateau in the exposure-efficacy relationship, immunogenicity is not expected to affect efficacy and safety in the majority of HPP participants treated with asfotase alfa. This was confirmed by examining composite data for individual participants focusing on the relationship between immunogenicity and efficacy / safety.

[0149] In post-marketing safety studies, some patients treated with asfotase alfa showed an initial therapeutic response but subsequently experienced disease relapse and progression associated with clinical, laboratory, and radiographic markers. Some of these events occurred in conjunction with the presence of recorded positive ADA and NAb, suggesting that an immunomediated effect on the pharmacological action of asfotase alfa may have contributed to disease progression.

[0150] The proposed IST agent will be administered for an initial 24-week course, followed by a 2-week period to assess clinical, laboratory, and radiological changes to determine the need for repeating the IST regimen. If the participant is not treated with IST, observation will continue or the IST regimen will be modified for the next 24 weeks. Methotrexate and rituximab will be administered for at least 18 months (approximately 74 weeks) unless there are safety concerns. IVIG 500 mg / kg will be administered monthly concurrently with rituximab, either with or without bortezomib.

[0151] A comprehensive review of data will be conducted every 24 weeks for each participant throughout the study to assess the impact of IST on immune-mediated LoE. This cycle will be repeated for all participants until study completion or ED. Asfotase alfa therapy for HPP will continue in parallel with IST throughout the study, and participants will continue to be followed according to standard treatment if they are enrolled in the HPP global registry after the completion of this study. Participants will be treated as a case study, with within-participant analyses used to evaluate the impact of the IST regimen on the ADA / NAb response and the corresponding effects on improvement in efficacy.

[0152] Risk / Benefit Assessment Risk assessment In this open-label study, participants will continue to be treated with a subcutaneous asfotase alfa regimen at enrollment and throughout the study, as prescribed by a physician treating them according to nationally approved labels.

[0153] Since there are no controlled studies using these drugs in patients with HPP, the toxicity in this population receiving asfotase alfa is unknown. Furthermore, the proposed combination of asfotase alfa with methotrexate, rituximab, and / or bortezomib has not been used, and drug combinations may have a broader range of side effects than those observed with the individual drugs. 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 blood cell count, alanine aminotransferase (ALT), aspartate aminotransferase (AST), bilirubin, and the presence of red blood cells in the urine.

[0154] Immunosuppression can also lead to opportunistic infections, such as bacterial, fungal, and viral infections. Participants should be closely monitored for any signs of infection during the study, and if this occurs, it will result in aggressive treatment and management. When selecting antibiotics to treat infections, attention should be paid to potential drug interactions between the investigational drug and certain antibiotics. Whenever any antimicrobial agent is administered to participants receiving an IST regimen for the treatment of an active infection, it is also recommended to consult an infectious disease specialist. Routine administration of IVIG is incorporated into the treatment algorithm as a way to reduce the risk of infection. Furthermore, folic acid deficiency may increase methotrexate-related adverse reactions. Therefore, folic acid should be administered to participants while they are receiving methotrexate.

[0155] Information regarding the known and expected benefits and risks, as well as reasonably expected adverse events (AEs), of rituximab, methotrexate, and bortezomib can be found in the reference safety information (RSI) for each IST. The current EU SmPC most applicable to each active substance, route of administration, and drug regimen will be used as the RSI for the IST. Each participant's IST regimen may be added to or removed based on a review of clinical, radiological, and laboratory data.

[0156] Profit evaluation This study aims to improve participants' immune-mediated LoE in response to asfotase alfa therapy; therefore, participation in this study may provide improved health and quality of life for these participants diagnosed with HPP. If participants demonstrate benefit with the IST regimen during the study, they may continue IST with asfotase alfa therapy after EoS.

[0157] Furthermore, tests used in clinical studies but not yet commercially available in most countries (concentrations of inorganic pyrophosphate [PPi], pyridoxal-5'-phosphate [{vitamin B6}PLP] and levamisole, and asfotase alfa) are provided for the treating physician to obtain and analyzed in a central laboratory. These recommended but not generally available blood tests pose no additional risk, and participants may benefit from access to these tests to aid in treatment monitoring.

[0158] Overall Benefits: Conclusion of Risks Considering the measures taken to minimize risk to participants in this study, the potential risks identified in relation to rituximab, methotrexate, and bortezomib are justified by the expected benefits that may be given to participants treated with asfotase alfa in HPP experiencing immune-mediated LoE.

[0159] Immune response Helper T cells are essential for most ADA development, while memory B cells and long-lived plasma cells amplify and maintain the response that constitutes the basis for the use of combined ISTs when LoE is present due to the presence of ADA. Therapeutic proteins such as asfotase alfa can be endocytized by antigen-presenting cells after administration, where they are processed into their component peptides. Subsets of these peptides can then be presented to peptide-specific helper T cells in the context of human leukocyte antigen (HLA) molecules. Helper T cells signal to Ag-specific B cells, which are activated, proliferate, and differentiate into memory B cells and Ab-secreting plasma cells (both short-lived and long-lived).

[0160] immunosuppressive therapy The following ISTs are considered for use in this study based on their effects on helper T cells, plasma cells, and memory B cells. Since there are no controlled trials using these drugs in patients with HPP, their toxicity is unknown.

[0161] Rituximab Rituximab is a chimeric monoclonal antibody approved for multiple types of malignant and autoimmune diseases. It targets the cell surface antigen (CD20) of human B lymphocytes, a transmembrane protein present on virtually all B cells from the stage in which they become involved in B cell development until it is downregulated when they differentiate into antibody-secreting plasma cells. Rituximab causes rapid and almost complete depletion of peripheral B cells, which does not relapse for approximately 6 months in most participants.

[0162] However, CD20 is lost when B cells differentiate into antibody-producing plasma cells. Therefore, once B cells differentiate into plasma cells, alternative and / or additional agents must be used to address the ADA generated by circulating plasma cells.

[0163] Methotrexate Methotrexate, a dihydrofolate reductase inhibitor, affects rapidly dividing T cells and B cells by preventing the reduction of dihydrobiopterin (BH2) to tetrahydrobiopterin (BH4), leading to increased T cell sensitivity to nitric oxide synthase uncoupling and apoptosis, thereby reducing the immune response.

[0164] Bortezomib Bortezomib is a proteasome inhibitor. Proteasome inhibitors are a potent and specific method for targeting plasma cells that are highly dependent on proteasome activity. Bortezomib is a reversible proteasome inhibitor that targets both short-lived and long-lived plasma cells due to their high immunoglobulin production rates. Bortezomib was initially approved for multiple myeloma, but animal studies have shown that proteasome inhibition can also kill normal plasma cells. Bortezomib has also been shown to improve the HSAT response to ERT in some IPD patients and has been beneficial in several case reports of thrombotic thrombocytopenic purpura and autoimmune cytopenia refractory to standard therapy with minimal and tolerable adverse events in children.

[0165] All participants recruited in this study who showed loss of efficacy were ADA-positive at baseline and had an "established" ADA response. Rituximab depletes B cells but does not affect plasma cells (which produce ADA). Therefore, patients receiving methotrexate and rituximab therapy with an established ADA response may not respond well to asfotase alfa-ERT treatment. Bortezomib should be considered in cases of disease progression and simultaneous increases in ADA and NAb to deplete ADA-producing plasma and memory B cells.

[0166] Justification for dosage Asfotase alpha The dosage of asfotase alfa (NIMP in this study) at the start of the study will follow the dosage prescribed by the treating physician, in accordance with recommendations and national approval labels.

[0167] Immunosuppressive therapy (IST) This study will use standard dosing regimens of rituximab, methotrexate, and bortezomib. Clinical, laboratory, and radiological data will be collected to determine IST treatment and monitoring.

[0168] End of research definition Participants who complete the last scheduled procedure outlined in the Statement of Action (SoA) are considered to have completed the study. Participants who withdraw from / are withdrawn from the study before completing the End of Study (EoS) visit are considered to have terminated the study early. 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 have completed the study are eligible to be registered in the HPP global registry.

[0169] research group Participants' medical history, laboratory findings, and physical examinations will be reviewed during a screening period of up to eight weeks, and the decision to proceed to IST will be made through consensus and after informed consent has been obtained.

[0170] Prior approval (also known as protocol abandonment or exemption) of protocol deviations from recruitment and inclusion criteria is not permitted.

[0171] research intervention A research intervention is defined as any clinical trial intervention, marketed product, placebo, or medical device intended to be administered to research participants in accordance with a research protocol.

[0172] The administered research intervention All research interventions (investigational medicinal products, IMPs) and non-investigational medicinal products, NIMPs) are recorded as follows: ·start date • Medication regimen (e.g., dosage [mg / kg], frequency, administration failure, treatment interruption)

[0173] For details on recommended drug regimens for rituximab, methotrexate, and bortezomib, please refer to Table 6.

[0174] The body surface area (BSA) for the medication regimen is determined monthly. The BSA is calculated using the Du Bois method.

[0175] BSA(m 2 ) = height (0.725) × weight (0.425) × 0.007184, where height is in meters (m) and weight is in kilograms (kg).

[0176] In this study, the administration of asfotase alfa, the NIMP (non-invasive pain management) drug, will follow the dosage prescribed by the treating physician, in accordance with recommendations and national approval labels. However, once participants are enrolled, the asfotase alfa dosing regimen must be maintained stable throughout the study. Asfotase alfa will not be provided within the scope of this study.

[0177] [Table 6] Note: The definitions of IMP and NIMP are based on guidance issued by the European Commission. a For ISTs, dose modifications (addition, removal, and / or change of methotrexate dose) may be permitted in the presence of drug toxicity. b IVIG is administered as long as rituximab is being administered. c Folic acid is commercially available, and IVIG and IMP are also supplied. d Experimental immune tolerance therapy for LoE via immune response to asfotase alfa (ADA with or without NAb). Abbreviations: ADA = anti-drug antibody, EU = European Union, HPP = hypophosphatasia, LoE = loss of efficacy, IMP = investigational drug, IST = immunosuppressive therapy, IV = intravenous, IVIG = intravenous immunoglobulin, MTX = methotrexate, n / a = not applicable, NAb = neutralizing antibody, NIMP = non-investigational drug, SC = subcutaneous (intracutaneous), TA = treatment evaluation (duration), w / out = none

[0178] Combination therapy Any medications (including over-the-counter or prescription drugs, vitamins, herbal supplements, and / or premedications used before IST administration), vaccines, or other categories for specific purposes that participants receive at registration or during the study must be recorded together with the following. · Reason for use · Date of administration, including start and end dates · Dosage information, including dosage and frequency

[0179] Dosage change After each participant completes the 24-week treatment period, clinical, laboratory, and radiological changes are evaluated to determine whether each participant repeats the current IST, continues with observation only, or the need to add / remove an IST regimen for the next 24-week treatment period.

[0180] Note that methotrexate should be administered for at least 18 months unless there are safety concerns. Modifications to the dosage for toxicity are described herein.

[0181] Modification of methotrexate dosage Methotrexate can be associated with multiple organ toxicities (e.g., hematological, renal, hepatic, gastrointestinal, pulmonary, dermal, and neurological). Interruption of administration, dosage reduction, or discontinuation can be warranted based on the nature and severity of the toxicity. Methotrexate should be withheld for any toxicity of grade 3 or higher, and the patient should be evaluated promptly. Depending on the nature and severity of the adverse reaction, resuming methotrexate at the same or a lower dosage can be considered.

[0182] The excretion of methotrexate is reduced in patients with renal dysfunction. The dosage of methotrexate should be reduced by 50% for patients with moderate renal dysfunction (creatinine clearance > 30 - 59 mL / min), and methotrexate should not be administered to patients with severe renal dysfunction (creatinine clearance < 30 mL / min).

[0183] Rituximab dose modification Dose reduction is not a recommended risk reduction for rituximab-related toxicities that affect multiple organ systems and may be life-threatening or fatal (e.g., severe mucocutaneous reactions, severe infections, and toxicities affecting the heart, kidneys, and gastrointestinal system). Rituximab infusion should be withheld until any grade 3 or higher toxicity is resolved or further diagnostic evaluation is performed. Rituximab should not be administered to patients with active, severe infections (e.g., tuberculosis, sepsis, opportunistic infections, or severe viral infections including hepatitis B or other viral reactivation). Permanent discontinuation of rituximab should be considered if toxicity does not resolve or cannot be adequately managed with standard therapeutic interventions, or if adverse reactions recur upon readmission.

[0184] Bortezomib dose modification Bortezomib treatment should be withheld in the event of any grade 3 or higher toxicity, excluding neuropathy (see Table 7). Bortezomib may be restarted at a lower dose after toxicity has improved according to the criteria provided below. If toxicity does not resolve or recurs at the lowest dose, discontinuation of bortezomib should be considered unless the therapeutic benefits clearly outweigh the risks.

[0185] [Table 7] Source: Bortezomib SmPC Abbreviations: ANC = Absolute Neutrophil Count, AV Block = Atrioventricular Block, ECG = Electrocardiogram, GLS = Overall Longitudinal Distortion, LVEF = Left Ventricular Ejection Fraction, NT-pro BNP = N-Terminal Pro-Brain Natriuretic Peptide, SmPC = Product Characteristics Summary Dose adjustment and continuation criteria for peripheral neuropathy in patients treated with bortezomib

[0186] Participants with pre-existing severe neurological disorders should only be treated with bortezomib after a careful risk-benefit assessment. • Grade 1 (asymptomatic, loss of deep tendon reflexes, or paresthesia): No pain or loss of function: No effect • Grade 1 or Grade 2 pain: Dosage 1 mg / m² 2 Reduce • Grade 2 or Grade 3 pain: Withhold the drug until the toxic symptoms subside. 0.7 mg / m² per week. 2 It can be resumed. • Grade 4: Discontinue bortezomib

[0187] Modification of bortezomib dosage in hepatic impairment For participants with mild hepatic impairment, the starting dose will not be adjusted. For participants with moderate or severe hepatic impairment, the starting dose will be 0.7 mg / m² per injection during the first cycle. 2 Start with a reduced dose of 1 mg / m² and, based on the patient's tolerance, reduce to 1 mg / m². 2 Subsequent dose escalation to 0.5 mg / m² 2 Further dose reduction should be considered (see Table 8).

[0188] [Table 8] Source: Bortezomib SmPC a Based on the NCI Organ Dysfunction Working Group classification for classifying liver damage (mild, moderate, severe) Abbreviations: AST = Aspartate aminotransferase, NCI = National Cancer Institute, SGOT = Serum glutamate-oxaloacetate transaminase, SmPC = Summary of product characteristics, ULN = Upper limit of normal

[0189] Management of infusion reactions with IST therapy Premedication consisting of antipyretics and antihistamines, such as paracetamol and diphenhydramine, should always be administered before each dose of rituximab. Premedication prior to rituximab infusion should be initiated with the first dose.

[0190] Premedicate before infusion or injection of other ISTs according to the facility's protocol.

[0191] If an infusion reaction is observed, the following guidelines for participant treatment and dose modification or delay are provided and may be modified based on the investigational site guidelines.

[0192] Grade 1 infusion reaction: Grade 1 infusion reactions are characterized by mild reaction symptoms that do not indicate interruption of the study intervention infusion. For Grade 1 infusion reactions: <o000965>● Stay at the bedside and monitor the participant until recovery from symptoms (or return to baseline). ● Premedicate the participant at least 30 minutes prior to subsequent study drug infusions. Recommended dosing: [[ID=十四]] 〇 Diphenhydramine 50 mg (or equivalent) and 〇 Acetaminophen / paracetamol 325 mg - 1000 mg

[0193] Grade 2 infusion reaction: Grade 2 infusion reactions are characterized by moderate reaction symptoms that require interruption of therapy or study drug infusion but respond promptly to treatment (e.g., antihistamines, non-steroidal anti-inflammatory drugs (NSAIDs), narcotics, corticosteroids, bronchodilators, IV fluids). Prophylactic dosing is indicated for ≤24 hours. For Grade 2 infusion reactions: · Stop the study drug infusion and initiate an IV infusion of 0.9% sodium chloride · Administer diphenhydramine 50 mg IV (or equivalent) and / or acetaminophen / paracetamol 325 mg - 1000 mg · Stay at the bedside and monitor the patient until recovery from symptoms (or return to baseline). · Administer corticosteroids and / or bronchodilator therapy as appropriate.

[0194] When the symptoms subside, resume infusion of the investigational drug at 50% of the original infusion rate. If the patient does not experience any further complications after 30 minutes, the infusion rate may be increased back to the original rate.

[0195] Premedicate participants at least 30 minutes before subsequent investigational drug infusion. Recommended medication: • Diphenhydramine 50 mg (or equivalent) and Acetaminophen / paracetamol 325mg~1000mg Corticosteroids (hydrocortisone or equivalent up to 25 mg) may also be administered.

[0196] Grade 3 or Grade 4 injection reaction: Grade 3 or 4 infusion reactions are severe. Grade 3 reactions are characterized as prolonged (i.e., failure to respond quickly to brief interruptions of treatment and / or investigational drug infusion) with relapses of symptoms after initial improvement. Hospitalization may be indicated for other clinical complications (e.g., renal impairment, pulmonary infiltration). Grade 4 reactions are life-threatening and require vasopressor or ventilatory support. Regarding Grade 3 or Grade 4 infusion reactions: • Immediately discontinue the investigational drug infusion and begin intravenous infusion of 0.9% sodium chloride. Administer bronchodilators, epinephrine subcutaneously or intravenously, and / or diphenhydramine with steroids, and provide other supportive care as needed. • Remain at the bedside and monitor the patient until their symptoms resolve (or return to baseline) and do not recur. • Follow facility guidelines for the treatment of anaphylaxis.

[0197] Post-study interventions Once the final research visit is complete, participants can return to the care of their treating physician for disease management and continue to receive commercial supplies of IST.

[0198] Interruption and discontinuation of research interventions, as well as interruption / withdrawal of participants. Interruption and termination of research interventions Participants will be closely monitored for any signs of infection or other potential IST-related toxicity during the study. In the event of drug-related toxicity, it may be necessary for the 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 Statement of Acknowledgments (Table 3). Participants should permanently discontinue the research intervention if any of the following occurs: • Confirmed, unexplained grade 3 or higher QT prolongation (QTc > 500 msec or uncorrected QT > 600 msec or change from baseline QTc > 60 msec) based on the mean of three electrocardiogram (ECG) readings. • Grade 4 injection reaction or other severe hypersensitivity reactions, including anaphylaxis. Grade 4 mucocutaneous reactions or any confirmed cases resulting from IST, such as Stevens-Johnson syndrome or toxic epidermal necrolysis. • Pregnancy or planned pregnancy New or recurrent malignant tumors, excluding superficial cancers that can be cured by surgical removal. • If any of the following criteria are met, or if any other clinically significant findings are present that would allow for further evaluation of the participant, the IST should be withheld / postponed. The decision to rechallenge a participant who has any Grade 4 SAE: Suspected IST potentially related to any of the research interventions or procedures must be discussed and agreed upon in advance. • Kidney: Serum creatinine (if baseline > ULN, >2 × ULN or >2 × baseline), grade 2 or higher increase in oliglia, or >50% decrease in serum creatinine clearance. • Liver: Grade 2 or higher increase in ALT and AST (if baseline > ULN, >3 × ULN or 3 × baseline) or grade 2 increase in total bilirubin (if baseline > ULN, >2 × ULN or >2 × baseline). • Participants exhibit a significant worsening of pulmonary symptoms. • Severe infection: IST should be postponed / delayed, and a specialist in infectious diseases should be consulted. • Unresponsive to standard therapy or accompanied by significant neutropenia (<1000 / mm³) 3 Any grade 3 infection or any grade 2 infection suspected of being a viral reactivation syndrome (e.g., hepatitis B, cytomegalovirus, Epstein-Barr virus, herpes zoster). • If viral reactivation is confirmed, IST may not be administered. • Blood: Grade 3 or higher with decreased neutrophil count (<1000 / mm³) 3 ), decreased platelet count (<50,000 / mm3), or decreased hemoglobin (<8 g / dl). Grade 2 decreased neutrophils (<1500 / mm3), platelets (<75000 / mm3), and hemoglobin (<10 g / dl), and a Grade 2 decrease in any one of the hematological parameters was accompanied by clinically significant symptoms (e.g., decreased neutrophil count with fever and menorrhagia). • Gastrointestinal: Symptoms suggestive of intestinal 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, hypertension, or hypotension)). If pulmonary or cardiac toxicity associated with an IST is confirmed (e.g., pneumonia, drug-related cardiac LV failure, or life-threatening arrhythmia), the suspected IST should be permanently discontinued. • Neurology: Patients with symptoms suggestive of progressive multifocal leukoencephalopathy (PML) or posterior reversible encephalopathy syndrome / reversible posterior leukoencephalopathy syndrome (PRES / RPLS).

[0199] Data collected at the time of discontinuation of the study intervention and follow-up, as well as for any further evaluations that need to be completed, will be provided to the SoA.

[0200] Effectiveness evaluation The efficacy evaluation will be carried out as described herein.

[0201] Rickets severity score This is a quantitative method for assessing the severity of rickets in the wrist and knee based on the degree of fraying and concavity of the metaphysis and the percentage of the growth plate affected. • This is a 10-point scale, where 10 represents the most extreme degree of rickets severity, and 0 represents no changes in rickets on X-ray. • The radiographic response after treatment for nutritional deficiencies can be evaluated by RSS. • RSS values ​​correlate with serum alkaline phosphatase (ALP), which is a biochemical measure of rickets activity. A score of 0 represents the complete disappearance of rickets.

[0202] Peabody Developmental Motor Scale, 2nd Edition (PDMS-2) The PDMS-2 is a revised version of the original Peabody Developmental Motor Scale from 1983. The PDMS-2 consists of six subtests (reflexes, static, locomotion, object manipulation, grasping, and visual-motor integration) that measure the interrelated motor skills of children from birth to 5 years of age. • Reflexes (an 8-item subtest measuring a child's ability to react to environmental events; reflexes are typically integrated by 12 months of age, so they are only measured from birth to 11 months of age.) • Static (A 30-item subtest measuring a child's ability to control their body within their center of gravity and maintain balance) • Mobility (89 subtests measuring a child's ability to move from one place to another through crawling, walking, running, jumping, and leaping forward) • Object manipulation (a 24-item subtest that measures a child's ability to manipulate a ball by catching, throwing, and kicking it; this test is only administered to children 12 months of age and older.) • Grasping (A 26-item subtest measuring a child's ability to use their hands, starting with holding an object and progressing through controlled use of the fingers of both hands.) • Vision-Motor Integration (a 72-item subtest that measures a child's ability to use visual perception skills to perform complex eye-hand coordination tasks, such as reaching for and grasping objects to build blocks and copy designs).

[0203] Bruininks-Oseretsky Motor Skills Test, 2nd Edition (BOT-2), Short Version • A measure of fine or overall motor function through a series of goal-oriented activities administered to participants according to a uniform procedure. BOT-2 consists of eight different subtests organized into four motor domain complexes: (i) precision manual control, (ii) manual coordination, (iii) physical coordination, and (iv) strength and agility. The eight subtests provide a balanced contribution to the total motor composite score. • The total composite score correlates with other measures of motor ability, including PDMS-2.

[0204] Use of mobility aids HPP is associated with a high fracture and orthopedic / dental surgical burden, pain, motor impairment, need for walking aids, decreased functional status, and impairment of activities of daily living (ADL) in children and adults. This study will assess the reasons for using assistive devices, including pain, weakness, fatigue, balance, risk of falling, or risk of fracture. Information on the type of device used (e.g., crutches, wheelchair dependent [full-time], wheelchair dependent [part-time], walker, cane, leg brace, ramp, bath / shower modification, grab bar / handrail, or other assistive device) will also be collected. Assistive device use will be continuously monitored throughout the study.

[0205] x-ray Chest X-rays should be obtained before initiating methotrexate administration so that they can be used later for comparison in case respiratory complications occur during the study.

[0206] Peripheral neuropathy Patients receiving bortezomib can be monitored for new onset or worsening neurological symptoms such as burning, hyperesthesia, hypoesthesia, paresthesia, discomfort, neuropathic pain, or weakness. Furthermore, participants receiving bortezomib can be assessed for peripheral neuropathy using the Modified Pediatric Whole Neuropathy Scale (ped-mTNS) assessment tool before and at the end of each bortezomib cycle.

[0207] Echocardiography and cardiac biomarkers This includes reports of acute onset or exacerbation of congestive heart failure and new onset of decreased left ventricular ejection fraction occurring during treatment with bortezomib, in patients without risk factors for decreased left ventricular ejection fraction. Patients with risk factors for cardiovascular disease, or who have cardiovascular disease, should be monitored frequently.

[0208] Cardiac adverse reactions, including ventricular fibrillation, myocardial infarction, and cardiogenic shock, may occur in patients receiving rituximab. Cardiac monitoring should be performed during and after all rituximab infusions in patients who develop clinically significant arrhythmias or who have a history of arrhythmias or angina.

[0209] Cardiotoxicity Participants receiving bortezomib will be monitored for drug-related cardiotoxicity at the time points shown in Table 3, using ECG and transthoracic 2D echocardiography to measure left ventricular ejection fraction (LVEF based on estimated left ventricular end diastolic (LVED) and left ventricular end systolic (LVES) volumes using the modified biplane-Simpson 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 necessary (see Table 7).

[0210] Pharmacokinetics • Collect blood samples to measure serum asfotase alpha concentration. If guaranteed, samples may be collected at further points during the study. • The PK profile of asfotase alfa enzyme activity may be evaluated using serum samples, and this may be used to assess safety or efficacy aspects related to concerns arising during or after the study.

[0211] Immunogenicity evaluation • Serum samples for ADA and NAb analysis are collected at the specified time. • Samples collected for ADA and NAb analysis are collected before drug administration.

[0212] Antidrug antibody variables The ADA variables include the ADA response category, incidence, and titer over the duration of the study, as follows. Samples positive in the ADA assay are further analyzed for the presence of neutralizing activity in the NAb assay. The definitions of the ADA response categories and titer thresholds are as defined herein. The evaluation of the effects of ADA and / or NAb on asfotase alpha PK / PD / biomarkers may be carried out in conjunction with the effects of IST on the immunogenicity profile (e.g., ADA and / or NAb), and their corresponding effects on the asfotase alpha PK / PD / biomarkers and clinical efficacy.

[0213] ADA response categories ·ADA negative ·ADA positive ·NAb negative ·NAb positive

[0214] Participants who are ADA-positive are classified as follows: • ADA response attenuated by IST treatment • ADA response enhanced by IST treatment

[0215] Based on the duration of these responses, ADA responses enhanced by IST treatment and ADA responses attenuated by IST treatment are further classified as follows: • Responses observed under sustained treatment • Responses observed under uncertain treatment conditions • Responses observed during transient treatment

[0216] Pharmacodynamics • Blood samples are collected at each time point to measure plasma PPi and PLP concentrations (including levamisole) and ALP. Plasma samples may be used to evaluate asfotase alfa-PD, and these samples may be used to assess safety or efficacy aspects related to concerns arising during or after the study. • Plasma (including levamisole) PLP and PPi concentrations (PD biomarkers for HPP). Observations and changes from baseline (CFB) in plasma concentrations of PLP and PPi will be summarized at pre-specified visits. Participant-level data will be listed and plotted over time. The same analysis will be performed for other biochemical biomarkers.

[0217] Monitoring of rickets and immunogenicity changes Monitoring for rickets: After each 24-week treatment period, the RSS is used to assess radiographic evidence of rickets. Complete resolution of rickets is defined as an RSS of 0. Exacerbation of rickets is defined as an increase of 1 or more points in the RSS from baseline. Patients are considered stable if there is either no change in the RSS or a decrease from baseline.

[0218] Immunogenicity monitoring: The objective of this study is to restore the pharmacological effects of asfotase alfa in participants diagnosed with HPP exhibiting immunomediated (ADA and NAb-related) LoE. Therefore, ADA assessments are performed in each participant at predefined time points throughout the study and analyzed using validated assays. ADA variables include the incidence and titer of ADA response categories over the study period. ADA-positive samples are further characterized for ADA titer and the presence of neutralizing antibodies against STRENSIQ®. ADA-positive responses are classified based on the level (titer threshold) and duration (persistent / transient / uncertain) of the response, whether attenuated or enhanced by IST treatment.

[0219] Participants are considered to have a significant attenuation of the ADA response due to IST if their ADA and / or NAb titers decrease by at least two titer steps compared to baseline, or become negative.

[0220] Criteria for IST modifications (continuation or suspension) The RESTORE study suggests that an individualized risk-based approach, which assesses the risks of untreated HPP disease or partially effective therapies compared to the risks of using IST regimens that lead to improved efficacy, serves as a basis for devising criteria for the use of ISTs.

[0221] 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 be repeated over a total of 104 weeks as follows: weeks 25-26 (Treatment Monitoring Committee (period for reviewing clinical data and treatment decisions), weeks 27-50 (Treatment Assessment (TA) period 2), weeks 51-52 (period), weeks 53-76 (TA period 3), weeks 77-78 (period 3), weeks 79-102 (TA period 4), and weeks 103-104 (period). Participants will continue the same subcutaneous asfotase alfa regimen as at enrollment. All participants will receive folic acid to reduce potential side effects of methotrexate, and concurrently receive intravenous immunoglobulin (IVIG) therapy to provide passive immunity and minimize the risk of infection.

[0222] In accordance with the study design, most participants recruited in this study are expected to have an "established ADA response." Therefore, downregulation of T cells (methotrexate) and B cells (rituximab) may not be sufficient to demonstrate a positive effect on efficacy in these participants. Further ISTs, such as bortezomib, may help downregulate plasma and memory B cells and reduce ADA levels. Bortezomib should be considered and added to the regimen in addition to rituximab and methotrexate if rickets worsen with an increase of 1 point or more from baseline, in the presence of elevated ADA / NAb levels during treatment with rituximab and methotrexate (Figure 4).

[0223] Definition of "IST complete response" Regardless of the regimen used, the following criteria were established to define "complete IST response" at week 100. • ADA or NAb titers decrease by at least two titer steps from baseline, or become negative. * , and • X-ray evidence of improvement in RSS by at least 1 point from baseline. * ADA is the primary outcome for patients who are ADA-positive and NAb-negative. NAb is the primary outcome for patients who are ADA-positive and NAb-positive.

[0224] Criteria for IST modifications (continuation or suspension) A decision tree (Figure 4) is used to perform an overall evaluation and decide whether to continue, modify, or discontinue the IST regimen.

[0225] Since methotrexate and rituximab are administered for at least 18 months unless safety concerns are observed, there are 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.

[0226] If a participant demonstrates a complete response at 18 months and is receiving methotrexate and rituximab, rituximab will be discontinued and methotrexate will be continued until the end of the study. If a participant demonstrates a complete response at 18 months and is receiving methotrexate, rituximab, and bortezomib, bortezomib will be discontinued and methotrexate and rituximab will be continued until the end of the study.

[0227] Criteria for bortezomib treatment: All participants recruited in this study who showed loss of efficacy were ADA-positive at baseline and had an "established" ADA response. Rituximab depletes B cells but does not affect plasma cells (which produce ADA). Therefore, participants in methotrexate and rituximab treatment with an established ADA response may not respond well to asfotase alpha enzyme replacement therapy (ERT). Bortezomib should be considered in cases of disease progression and concurrent increases in ADA and NAb to deplete ADA-producing plasma and memory B cells (Banugaria et al, PLOS One, 8:e67052, 2013).

[0228] In the RESTORE study, the addition of bortezomib is only considered after completion of the first 24-week cycle with methotrexate plus rituximab. Methotrexate plus rituximab is continued alongside bortezomib treatment.

[0229] The following decision tree and criteria (Figure 4) can be used to decide at 24-week intervals throughout the duration of this study whether to add bortezomib, continue bortezomib, or discontinue bortezomib.

[0230] • If any bortezomib-related toxicity is suspected, discontinue bortezomib treatment. Bortezomib treatment should be withheld in the event of any grade 3 or higher toxicity other than neuropathy (see Sec 6.6.3 of the HPP-407 protocol) or in the event of a complete response to IST.

[0231] Bortezomib should be added to or continued in addition to rituximab and methotrexate, in the presence of at least two titer steps of ADA or NAb from baseline, and an increase of 1 or more in the radiographic RSS score from baseline.

[0232] Complete IST response at week 100 - primary endpoint component The definition of complete IST response at week 100 is as follows: • ADA or NAb titers decrease by at least two titer steps from baseline, or become negative. * , and • X-ray evidence of improvement in RSS score by at least 1 point from baseline. * ADA is the primary outcome for patients who are ADA-positive and NAb-negative. NAb is the primary outcome for patients who are ADA-positive and NAb-positive.

[0233] Immunogenicity analysis All ADA analyses will be performed using the ADA variable in an immunogenicity analysis set.

[0234] The incidence of each ADA response category is listed and summarized as absolute incidence (n) and percentage of all participants (%). ADA and NAb titer levels are listed, and the maximum ADA and NAb titer levels are summarized for ADA-positive participants. NAb-positive and NAb-negative participants are summarized under the ADA-positive category as absolute incidence (n) and percentage (%) of all participants.

[0235] The association between ADA response categories and systemic exposure to IST can be explored for all treated participants to analyze the potential impact of IST on immunogenicity, titer, and their potential impact on individual PK profiles.

[0236] 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 longer than 24 hours, and other immune-related SAEs.

[0237] The association between ADA response categories and PD markers may be explored for all participants to assess the potential impact of IST on immunogenicity, titer, and their potential impact on individual PD marker profiles and efficacy.

[0238] [Table 9]

[0239] Table 10: Definition of SAE If an event is not an AE according to the above definition, it may not be a SAE even if serious conditions are met (e.g., hospitalization due to signs / symptoms of the disease under study, death due to disease progression).

[0240] [Table 10]

[0241] [Table 11-1]

[0242] [Table 11-2]

[0243] [Table 11-3]

[0244] Other Embodiments The detailed descriptions and examples provided above are for the purpose of clarifying understanding. No unnecessary limitations should be inferred therefrom. This disclosure is not limited to the exact details shown and described, and variations that are obvious to those skilled in the art are included within the scope of this disclosure as defined by the claims.

[0245] Unless otherwise specified, all figures representing quantities, molecular weights, etc., of components used herein and in the claims should be understood in all cases to be modified by the term "approximately." Therefore, unless otherwise indicated, the numerical parameters described herein and in the claims are approximations that may vary depending on the desired properties to be obtained by this disclosure. Each numerical parameter should be interpreted by applying ordinary rounding techniques, at least taking into account the number of significant figures reported, and not as an attempt to limit the doctrine of equivalents to the claims.

[0246] Although the numerical ranges and parameters described in this disclosure are approximations, the numerical values ​​described in specific examples are reported as accurately as possible. However, all numerical values ​​inherently include a range that inevitably arises from the standard deviation found in their respective test measurements.

[0247] All headings are for the reader's convenience and should not be used to limit the meaning of the text that follows them unless otherwise specified.

[0248] The complete disclosure of all patents, patent applications including provisional patent applications, publications including patent and non-patent publications, and electronically available materials (including, for example, nucleotide sequence submissions in GenBank and RefSeq, as well as amino acid sequence submissions in SwissProt, PIR, PRF, PDB, as well as translations derived from annotated coding regions in GenBank and RefSeq) cited herein is incorporated by reference.

Claims

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

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

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

4. The method according to claim 3, wherein the methotrexate is administered once every seven days.

5. The aforementioned methotrexate is approximately 10 mg / m² ２ ~About 20mg / m ２ The method according to any one of claims 2 to 4, administered in the specified dose.

6. The aforementioned methotrexate is approximately 15 mg / m². ２ The method according to claim 5, administered in the specified dose.

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

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

9. The method according to claim 8, wherein the rituximab or its antigen-binding fragment is administered once every seven days.

10. The rituximab or its antigen-binding fragment is approximately 100 mg / m² ２ ~Approx. 500mg / m ２ The method according to any one of claims 7 to 9, administered in the specified dose.

11. The rituximab or its antigen-binding fragment is approximately 375 mg / m² ２ The method according to claim 10, administered in the specified dose.

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

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

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

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

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

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

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

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

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

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

22. The method according to claim 21, wherein the bortezomib is administered twice, every seven days.

23. The aforementioned bortezomib is approximately 0.2 mg / m² ２ ~Approx. 2mg / m ２ The method according to any one of claims 15 to 22, administered in the specified dose.

24. The bortezomib is administered at a dosage of about 0.5 mg / m ２ , about 0.7 mg / m ２ , about 1 mg / m ２ , or about 1. N3 mg / m ２ The method according to claim 23, wherein the method is administered at a dosage of

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

26. The method according to claim 25, wherein the bortezomib is administered intravenously as a bolus.

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

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

29. The method according to claim 28, wherein the bortezomib is discontinued after it is determined that the subject has been treated for the antibody specific to the sALP.

30. The method according to claim 29, wherein the amount of the antibody specific to sALP, the risk of its formation, or its effect is reduced compared to before treatment.

31. The method according to any one of claims 1 to 30, wherein the therapy is administered for a period of at least six months.

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

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

34. The method according to claim 33, wherein the therapy is discontinued after the subject has shown a decrease in antibody titer of at least two titer steps or has become negative and has shown an improvement of at least one point in RSS from baseline.

35. The method according to any one of claims 1 to 34, wherein the therapy further comprises administering immunoglobulin.

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

37. The method according to claim 36, wherein the immunoglobulin is administered once every seven days.

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

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

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

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

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

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

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

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

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

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

48. The method according to claim 47, wherein the sALP is administered two, three, or six times per week.

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

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

51. The method according to any one of claims 1 to 50, wherein the sALP has at least 85% sequence identity with respect to any one of sequence numbers 1 to 3.

52. The method according to claim 51, wherein the sALP has a minimum of 85% sequence identity with respect to sequence number 1.

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

1.

54. The method according to claim 51, wherein the sALP has a minimum of 85% sequence identity with respect to sequence number 2.

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

2.

56. The method according to claim 51, wherein the sALP has a minimum of 85% sequence identity with respect to sequence number 3.

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

3.

58. The method according to any one of claims 1 to 57, wherein the subject is treated with a first sALP and a second sALP different from the first sALP.

59. The method according to claim 58, wherein the second sALP treatment includes administration at a lower frequency than the treatment comprising the first sALP.

60. The method according to claim 58 or 59, wherein the subject treated with the sALP is administered the sALP at a dose of 1 mg / kg / week to 10 mg / kg / week, and the subject is also treated with a second sALP different from the first sALP, the second sALP being administered once every two weeks at a dose of 20 mg, 35 mg, or 50 mg.

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

1.

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

3.

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

2.

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

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

66. The method according to any one of claims 1 to 65, further comprising monitoring the level of the antibody by testing a biological sample from the subject for the presence of the antibody.

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

68. The method according to any one of claims 1 to 67, further comprising determining whether the subject is suffering from a reduction in the effectiveness of the sALP.

69. The method according to claim 68, wherein determining a reduction in effectiveness includes measuring a metric, and an increase or decrease in the metric relative to a baseline indicates a reduction in the effectiveness of the sALP.

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

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

72. The method according to any one of claims 68 to 71, wherein determining the reduction in effectiveness includes measuring one or more symptoms selected from the group consisting of hypocalcification, hypercalciuria, skeletal deformity, duck gait, bone pain, fracture, calcium pyrophosphate dihydrate crystal deposition, arthritis, pyrophosphate arthropathy, chondrocalcification, calcifying periarthritis, pseudofracture, skeletal deformity, hypotonia, muscle weakness, rheumatic complications, arthritis, pseudogout, difficulty walking, pain, premature tooth loss, pulmonary dysplasia, respiratory failure, seizures, height, growth, rickets, and immunogenicity.

73. The method according to claim 72, wherein the subject exhibits an exacerbation of one or more of the symptoms compared to the baseline.

74. The method according to any one of claims 68 to 73, wherein determining the reduction in effectiveness includes establishing that the subject does not exhibit insufficient treatment adherence or improper injection technique, or is not suffering from vitamin D deficiency, nutritional deficiency, or associated disease.

75. The method according to any one of claims 68 to 74, wherein determining the reduction in effectiveness includes conducting a quality of life assessment.

76. The method according to claim 75, wherein the quality of life assessment is selected from one or more of the EuroQol 5D questionnaire, pediatric health assessment questionnaire, pediatric outcome data collection tool, pediatric health utility index-9D, pediatric quality of life inventory, abbreviated health survey 36, and abbreviated health survey 12.

77. The method according to claim 76, wherein the subject exhibits a reduced quality of life assessment score compared to the baseline.

78. The method according to any one of claims 68 to 77, wherein determining the reduction in effectiveness includes performing a physical measurement evaluation.

79. The method according to claim 78, wherein the physical measurement criteria evaluation is selected from one or more of the following: the 6-minute walk test (6MWT), the Bruininks-Oseretsky Motor Skills Test, second edition (BOT-2), the Bayley Scale for Infant and Toddler Development, third edition (BSID-III), gait analysis, use of mobility aids, the Peabody Developmental Motor Scale II (PDMS-2), or X-ray.

80. The method according to claim 79, wherein the subject exhibits a reduced physical measurement evaluation score compared to the baseline.

81. The method according to any one of claims 69 to 80, wherein the baseline is calculated based on the value of the metric in a reference subject treated with sALP and without neutralizing antibodies against sALP, or in a reference subject that has not been administered sALP, before the subject suffered from reduced efficacy of sALP.

82. The method according to any one of claims 1 to 81, further comprising determining whether the antibody is a neutralizing antibody.

83. The method according to claim 82, further comprising testing the effect of the neutralizing antibody on either or both of the catalytic activity and / or bone targeting of the sALP in the presence of the neutralizing antibody.

84. The method according to claim 83, wherein the subject exhibits a reduction in the catalytic activity and / or bone targeting of the sALP.

85. The method according to claim 83 or 84, wherein the test comprises one or more of the following: a screening assay, a confirmation assay, a titration assay, and a neutralizing antibody assay.

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

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

88. The method according to any one of claims 1 to 87, wherein the subject is an adolescent, an adult, an infant, or a newborn.

89. The method according to any one of claims 1 to 88, wherein the subject has a fracture, osteoporosis, indurative ossification, chondrocalcification, hypotonia, Duchenne muscular dystrophy, tracheobronchomalacia, seizure, neurofibromatosis 1 (NF-1), craniosynostosis, or muscle weakness disorder.

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

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