Assay of lysosomal storage disease

Abstract

The present disclosure provides a method of detecting an anti-lysosomal enzyme antibody in a human subject (e.g., in the presence of a circulating lysosomal enzyme) and a method of increasing the efficacy of an anti-lysosomal enzyme antibody assay (e.g., increasing assay sensitivity and circulating lysosomal enzyme tolerance), comprising pre-treating the biological sample of the subject with a base having a pH of about 11 or greater, and measuring the presence of the anti-lysosomal enzyme antibody in the biological sample of the subject.

Description

[0001] Cross - reference to related applications

[0002] This application claims the benefit of the filing date of U.S. Provisional Patent Application No. 63 / 379,597, filed on October 14, 2022, the content of which is incorporated herein by reference in its entirety. Field of the invention

[0003] The present disclosure provides methods for detecting anti - lysosomal enzyme antibodies in a human subject (e.g., in the presence of circulating lysosomal enzymes) and methods for improving the efficacy of anti - lysosomal enzyme antibody assays (e.g., improving assay sensitivity and circulating lysosomal enzyme tolerance), including pretreating a biological sample of the subject with a base having a pH of about 11 or higher and measuring the presence of anti - lysosomal enzyme antibodies in the biological sample of the subject. Background of the invention

[0005] Current treatments for lysosomal storage diseases (LSDs) are significantly immunogenic. Therefore, it is important to measure anti - lysosomal enzyme antibodies in patients to understand whether and how new drugs will work for these patients. For lysosomal storage diseases currently approved for enzyme replacement therapy (ERT), the incidence of anti - ERT antibodies (including anti - ERT neutralizing antibodies) is high.

[0006] Current methods for accurately measuring anti - lysosomal enzyme antibodies may require blood sampling at drug trough levels to ensure that there are no residual lysosomal enzymes in the blood, because residual enzymes can interfere with circulating antibodies by forming complexes that limit detection. For treatments such as gene therapy, it is not possible to stop treatment and dosing, and thus it is not possible to accurately measure anti - lysosomal enzyme antibodies.

[0007] Therefore, there is a need to improve methods for accurately measuring anti - lysosomal enzyme antibodies, thereby improving personalized treatment for patients diagnosed with lysosomal storage diseases. Summary of the invention

[0008] In some aspects, the present disclosure relates to a method for detecting anti - lysosomal enzyme antibodies in a human subject, which includes measuring the presence of anti - lysosomal enzyme antibodies in a biological sample of the subject, wherein the biological sample is pretreated with a base having a pH of about 11 or higher.

[0009] In some aspects, the methods of the present disclosure include detecting anti - lysosomal enzyme antibodies in the presence of circulating lysosomal enzymes.

[0010] In some aspects, the present disclosure relates to a method of enhancing the efficacy of an anti - lysosomal enzyme antibody assay, which includes pretreating a biological sample from a human subject with a base having a pH of about 11 or higher, and further includes measuring the presence of anti - lysosomal enzyme antibodies in the biological sample. In some aspects, enhancing the efficacy of the anti - lysosomal enzyme antibody assay of the present disclosure includes enhancing assay sensitivity and circulating lysosomal enzyme tolerance.

[0011] In some aspects, the base is a non - buffered base. In some aspects, the non - buffered base is NaOH or Ca(OH)2. In some aspects, the NaOH or Ca(OH)2 is about 0.01M, about 0.02M, about 0.03M, about 0.04M, about 0.05M, about 0.06M, about 0.07M, about 0.08M, about 0.09M or about 0.1M. In some aspects, the NaOH or Ca(OH)2 is about 0.02M.

[0012] In some aspects, the biological sample is a serum sample or a plasma sample. In some aspects,

[0013] In some aspects, the biological sample is diluted to a minimum required dilution (MRD) of about 2 - fold or higher.

[0014] In some aspects, the biological sample is diluted to an MRD of about 1-fold, 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 11-fold, about 12-fold, about 13-fold, about 14-fold, about 15-fold, about 16-fold, about 17-fold, about 18-fold, about 19-fold, about 20-fold, about 21-fold, about 22-fold, about 23-fold, about 24-fold, about 25-fold, about 26-fold, about 27-fold, about 28-fold, about 29-fold, about 30-fold, about 31-fold, about 32-fold, about 33-fold, about 34-fold, about 35-fold, about 36-fold, about 37-fold, about 38-fold, about 39-fold, about 40-fold, about 41-fold, about 42-fold, about 43-fold, about 44-fold, about 45-fold, about 46-fold, about 47-fold, about 48-fold, about 49-fold, about 50-fold, about 51-fold, about 52-fold, about 53-fold, about 54-fold, about 55-fold, about 56-fold, about 57-fold, about 58-fold, about 59-fold, about 60-fold, about 61-fold, about 62-fold, about 63-fold, about 64-fold, about 65-fold, about 66-fold, about 67-fold, about 68-fold, about 69-fold, about 70-fold, about 71-fold, about 72-fold, about 73-fold, about 74-fold, about 75-fold, about 76-fold, about 77-fold, about 78-fold, about 79-fold, about 80-fold, about 81-fold, about 82-fold, about 83-fold, about 84-fold, about 85-fold, about 86-fold, about 87-fold, about 88-fold, about 89-fold, about 90-fold, about 91-fold, about 92-fold, about 93-fold, about 94-fold, about 95-fold, about 96-fold, about 97-fold, about 98-fold, about 99-fold, about 100-fold, about 200-fold, about 300-fold, about 400-fold, about 500-fold, about 600-fold, about 700-fold, about 800-fold, about 900-fold, about 1000-fold, about 1100-fold, about 1200-fold, about 1300-fold, about 1400-fold, about 1500-fold, about 1600-fold, about 1700-fold, about 1800-fold, about 1900-fold, about 2000-fold, about 2100-fold, about 2200-fold, about 2300-fold, about 2400-fold, about 2500-fold, about 2600-fold, about 2700-fold, about 2800-fold, about 2900-fold, about 3000-fold, about 3100-fold, about 3200-fold, about 3300-fold, about 3400-fold, about 3500-fold, about 3600-fold, about 3700-fold, about 3800-fold, about 3900-fold, about 4000-fold, about 4100-fold, about 4200-fold, about 4300-fold, about 4400-fold, about 4500-fold, about 4600-fold, about 4700-fold, about 4800-fold, about 4900-fold or about 5000-fold.

[0015] In some aspects, the pH is about 11, about 11.1, about 11.15, about 11.2, about 11.25, about 11.3, about 11.35, about 11.4, about 11.45, about 11.5, about 11.55, about 11.6, about 11.65, about 11.7, about 11.75, about 11.8, about 11.85, about 11.9, about 11.95, about 12, 12.1, about 12.15, about 12.2, about 12.25, about 12.3, about 12.35, about 12.4, about 12.45, about 12.5, about 12.55, about 12.6, about 12.65, about 12.7, about 12.75, about 12.8, about 12.85, about 12.9, about 12.95, about 13, 13.1, about 13.15, about 13.2, about 13.25, about 13.3, about 13.35, about 13.4, about 13.45, about 13.5, about 13.55, about 13.6, about 13.65, about 13.7, about 13.75, about 13.8, about 13.85, about 13.9, about 13.95, or about 14.

[0016] In some aspects, the pH is 12.45.

[0017] In some aspects, the pH is greater than 11 and less than 12, greater than 11 and less than 13, or greater than 11 and less than 14.

[0018] In some aspects, the subject has a lysosomal storage disease. In some aspects, the lysosomal storage disease is selected from the group consisting of: Fabry disease, Gaucher disease, Pompe disease, mucopolysaccharidosis (MPS) type I disease, MPS II, MPS III, MPS IV, MPS VI, MPS VII, mucolipidosis (ML), Niemann-Pick disease, Tay-Sachs disease, and Batten disease.

[0019] In some aspects, the presence of anti-lysosomal enzyme neutralizing antibodies (NAb) is measured. In some aspects, the presence of anti-lysosomal enzyme total antibodies (TAb) is measured.

[0020] In some aspects, the method of the present disclosure further includes mixing the pretreated biological sample with a lysosomal enzyme. In some aspects, the lysosomal enzyme is selected from the group consisting of: α-galactosidase A (α-GalA), glucocerebrosidase, α-glucosidase, α-L-iduronidase, iduronate 2-sulfatase, sulfamidase, galactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, β-glucuronidase, N-acetylglucosamine-1-phosphotransferase, Niemann-Pick C1 protein, hexosaminidase A, and tripeptidyl peptidase 1. In some aspects, the lysosomal enzyme is α-galactosidase A.

[0021] In some aspects, the neutralizing anti - lysosomal enzyme antibody is selected from the group consisting of: anti - alpha - galactosidase A antibody, anti - glucocerebrosidase antibody, anti - alpha - glucosidase antibody, anti - alpha - L - iduronidase antibody, anti - iduronate 2 - sulfatase antibody, anti - sulfamidase antibody, anti - galactosamine - 6 - sulfatase antibody, anti - N - acetylgalactosamine - 4 - sulfatase antibody, anti - beta - glucuronidase antibody, anti - N - acetylglucosamine - 1 - phosphotransferase antibody, anti - Niemann - Pick C1 protein antibody, anti - hexosaminidase A, and anti - tripeptidyl peptidase 1 antibody. In some aspects, the neutralizing anti - lysosomal enzyme antibody is an anti - alpha - galactosidase A antibody.

[0022] In some aspects, the pretreated biological sample and the lysosomal enzyme mixture are incubated for at least about one hour, at least about two hours, at least about three hours, at least about four hours, at least about five hours, at least about six hours, at least about seven hours, at least about eight hours, at least about nine hours, at least about ten hours, at least about eleven hours, at least about twelve hours, at least about thirteen hours, at least about fourteen hours, at least about fifteen hours, at least about sixteen hours, at least about seventeen hours, at least about eighteen hours, at least about nineteen hours, at least about twenty hours, at least about twenty - one hours, at least about twenty - two hours, at least about twenty - three hours, or at least about twenty - four hours.

[0023] In some aspects, the method of the present disclosure further comprises mixing the pretreated biological sample and the lysosomal enzyme with a reaction mixture.

[0024] In some aspects, the concentration of the lysosomal enzyme is less than about 10 ug / mL, less than about 9 ug / mL, less than about 8 ug / mL, less than about 7 ug / mL, less than about 6 ug / mL, less than about 5 ug / mL, less than about 4 ug / mL, less than about 3 ug / mL, less than about 2 ug / mL, less than about 1 ug / mL, less than about 0.9 ug / mL, less than about 0.8 ug / mL, less than about 0.7 ug / mL, less than about 0.6 ug / mL, less than about 0.5 ug / mL, less than about 0.4 ug / mL, less than about 0.3 ug / mL, less than about 0.2 ug / mL, less than about 100 ng / ml, less than about 90 ng / ml, less than about 80 ng / ml, less than about 70 ng / ml, less than about 60 ng / ml, less than about 50 ng / ml, less than about 40 ng / ml, less than about 30 ng / ml, less than about 20 ng / ml, or less than about 10 ng / ml.

[0025] In some aspects, the pretreated biological sample and the lysosomal enzyme mixture are incubated for at least about one hour, at least about two hours, at least about three hours, at least about four hours, at least about five hours, at least about six hours, at least about seven hours, at least about eight hours, at least about nine hours, at least about ten hours, at least about eleven hours, at least about twelve hours, at least about thirteen hours, at least about fourteen hours, at least about fifteen hours, at least about sixteen hours, at least about seventeen hours, at least about eighteen hours, at least about nineteen hours, at least about twenty hours, at least about twenty-one hours, at least about twenty-two hours, at least about twenty-three hours, or at least about twenty-four hours.

[0026] In some aspects, the pretreated biological sample and the lysosomal enzyme mixture are incubated for a duration of from about 1 to about 15 hours, from about 2 to about 14 hours, from about 3 to about 13 hours, from about 4 to about 12 hours, from about 5 to about 11 hours, from about 6 to about 10 hours, or from about 7 to about 9 hours.

[0027] In some aspects, the reaction mixture includes a substrate and / or an inhibitor. In some aspects, the substrate is selected from the group consisting of: 4-methylumbelliferyl-α-D-galactopyranoside, 4-methylumbelliferyl-β-D-glucopyranoside, 4-methylumbelliferyl a-D-glucopyranoside, 4-methylumbelliferyl α-L-iduronide, 4-methylumbelliferyl-α-L-iduronide 2-sulfate, 4-methylumbelliferyl-2-sulfoamino-2-deoxy-α-D-glucopyranoside, 4-methylumbelliferyl-β-D-galactose-6-sulfate, 4-methylumbelliferyl-N-acetyl-α-d-galactosamine-4-sulfate, 4-methylumbelliferyl-β-d-glucuronide, 4-methylumbelliferyl-α-D-mannopyranoside, 4-methylumbelliferyl N-acetyl-β-D-glucosaminide, and Ala-Ala-Phe-7-amido-4-methylcoumarin.

[0028] In some aspects, the concentration of the substrate is at least about 1.1 mM, at least about 1.2 mM, at least about 1.3 mM, at least about 1.4 mM, at least about 1.5 mM, at least about 1.6 mM, at least about 1.7 mM, at least about 1.8 mM, at least about 1.9 mM, at least about 2 mM, at least about 2.1 mM, at least about 2.2 mM, at least about 2.3 mM, at least about 2.4 mM, at least about 2.5 mM, at least about 2.6 mM, at least about 2.7 mM, at least about 2.8 mM, at least about 2.9 mM, at least about 3 mM, at least about 3.1 mM, at least about 3.2 mM, at least about 3.3 mM, at least about 3.4 mM, at least about 3.5 mM, at least about 3.6 mM, at least about 3.7 mM, at least about 3.8 mM, at least about 3.9 mM, at least about 4 mM, at least about 4.1 mM, at least about 4.2 mM, at least about 4.3 mM, at least about 4.4 mM, at least about 4.5 mM, at least about 4.6 mM, at least about 4.7 mM, at least about 4.8 mM, at least about 4.9 mM or at least about 5 mM.

[0029] In some aspects, the inhibitor includes N-acetylgalactosamine (GALNAc).

[0030] In some aspects, the concentration of the inhibitor is less than about 200 mM, less than about 195 mM, less than about 190 mM, less than about 185 mM, less than about 180 mM, less than about 175 mM, less than about 170 mM, less than about 165 mM, less than about 160 mM, less than about 155 mM, less than about 150 mM, less than about 145 mM, less than about 140 mM, less than about 135 mM, less than about 130 mM, less than about 125 mM, less than about 120 mM, less than about 115 mM or less than about 110 mM.

[0031] In some aspects, the reaction mixture and the pretreated biological sample are combined with the lysosomal enzyme mixture in a high-throughput plate.

[0032] In some aspects, the reaction mixture and the pretreated biological sample are incubated with the lysosomal enzyme mixture at 300, 400, 500 or 600 revolutions per minute (RPM) at room temperature.

[0033] In some aspects, the method of the present disclosure further includes adding a termination buffer to the mixture after incubation. In some aspects, the incubation period is at least about 30 minutes, at least about 35 minutes, at least about 40 minutes, at least about 45 minutes, at least about 50 minutes, at least about 55 minutes, at least about 60 minutes, at least about 65 minutes, at least about 70 minutes, at least about 75 minutes or at least about 80 minutes.

[0034] In some aspects, the termination buffer contains glycine. In some aspects, the volume of the termination buffer is less than about 1 mL, less than about 900 μL, less than about 800 μL, less than about 700 μL, less than about 600 μL, less than about 500 μL, less than about 400 μL, less than about 300 μL, less than about 200 μL, or less than about 100 μL.

[0035] In some aspects, the methods of the present disclosure further include neutralizing the pretreated biological sample with an acid. In some aspects, the acid is acetic acid or hydrochloric acid. In some aspects, the acetic acid or hydrochloric acid is about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 200 mM, about 300 mM, about 400 mM, about 500 mM, or about 600 mM. In some aspects, the acetic acid is about 30 mM.

[0036] In some aspects, the methods of the present disclosure further include mixing the neutralized pretreated biological sample with lysosomal enzymes.

[0037] In some aspects, the neutralized pretreated biological sample and lysosomal enzyme mixture are added to a high-throughput plate coated with a lysosomal enzyme antigen. In some aspects, the lysosomal enzyme antigen is selected from the group consisting of: α-Gal A antigen, glucocerebrosidase antigen, α-glucosidase antigen, α-L-iduronidase antigen, iduronate 2-sulfatase antigen, sulfamidase antigen, galactosamine-6-sulfatase antigen, N-acetylgalactosamine-4-sulfatase antigen, β-glucuronidase antigen, N-acetylglucosamine-1-phosphotransferase antigen, Niemann-Pick C1 protein antigen, hexosaminidase A antigen, and tripeptidyl peptidase 1 antigen. In some aspects, the lysosomal enzyme antigen is the α-Gal A antigen.

[0038] In some aspects, an enzyme-conjugated detection antibody or an enzyme-conjugated lysosomal enzyme is added to the plate to generate a signal.

[0039] In some aspects, a detection antibody or lysosomal enzyme labeled with ruthenium is added to the plate to generate a signal.

[0040] In some aspects, the detection antibody is an anti-human IgG antibody.

[0041] In some aspects, the methods of the present disclosure further include adding a substrate. In some aspects, the substrate is converted by the enzyme on the detection antibody to produce a chromogenic reaction product. In some aspects, the plate is read in a plate reader that detects the chromogenic reaction product and outputs an optical density (OD) value. In some aspects, the OD value represents the total level of anti-lysosomal neutralizing antibody.

[0042] In some aspects, a plate is read in a plate reader that detects the light emission of ruthenium-labeled and outputs an electrochemiluminescence unit (ECLu). In some aspects, the ECLu represents the total level of anti-lysosomal total antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 Shows the incidence of anti-enzyme replacement therapy (ERT) antibodies, including anti-ERT neutralizing antibodies for lysosomal storage diseases (Fabry disease, Pompe disease, Gaucher disease, and mucopolysaccharidosis (MPS) type I disease, MPS type II disease, MPS type IV disease, and MPS type VI disease). The antibody incidence values were obtained from approved drug labels.

[0044] Figure 2 Shows the form of the lysosomal enzyme neutralization (NAb) antibody assay.

[0045] Figures 3A - 3B Shows the circulating lysosomal enzyme levels (airborne lysosomal enzyme concentration (ng / ml) [0 to 2000 ng / ml ( Figure 3A )] and [0 ng / ml to 500 ng / ml ( Figure 3B )] on the enzyme neutralization ability (lysosomal enzyme activity inhibition % (signal inhibition)) of polyclonal RP-01 antibody (neutralizing anti-lysosomal enzyme antibody) at different concentrations (50 ug / ml, 100 ug / ml, and 150 ug / ml).

[0046] Figures 4A - 4B Shows the effect of heat-pretreated biological samples on the enzyme neutralization ability (lysosomal enzyme activity inhibition % (signal inhibition)) of RP-01 antibody at different concentrations (0 ug, 50 ug, 100 ug, and 150 ug). Figure 4A Shows the results without sample pretreatment, Figure 4B Shows the results of 56 °C-pretreated serum samples.

[0047] Figure 5 Shows the various pretreatment methods evaluated (pH 11, Melon Gel, pH 2, and standard assay diluent (AD) conditions) to test the ability to detect anti-lysosomal enzyme neutralizing antibodies NAbs (RP-01) in the presence of airborne lysosomal enzymes (concentration (ng / ml)).

[0048] Figure 6 Shows the effect of alkaline (base) pretreated biological samples on the ability to detect anti-lysosomal enzyme neutralizing antibodies NAbs (RP-01) at different concentrations (50 ug / ml, 100 ug / ml, and 150 ug / ml) in the presence of airborne lysosomal enzymes (concentration (ng / ml)).

[0049] Figure 7 Shows the effect of alkaline (base) pre-treated biological samples on the ability to detect anti-lysosomal enzyme neutralizing antibodies NAbs (monoclonal positive control anti-lysosomal enzyme NAb, 7H11) at different concentrations (125 ng / ml, 250 ng / ml, and 1000 ng / ml) in the presence of airborne lysosomal enzyme (concentration (ng / ml)).

[0050] Figure 8 Shows the effect of high molarity base pre-treated biological samples on the ability to detect anti-lysosomal enzyme neutralizing antibodies NAbs (monoclonal positive control anti-lysosomal enzyme NAb (7H11)) at different concentrations (50 ng / ml, 100 ng / ml, and 150 ng / ml) in the presence of airborne lysosomal enzyme (concentration (ng / ml)).

[0051] Figure 9 Shows the ability to detect anti-lysosomal enzyme neutralizing antibodies NAbs: polyclonal positive control anti-lysosomal enzyme NAbs (RP-01) at different concentrations (50 μg / ml, 100 μg / ml, and 150 μg / ml) and monoclonal positive control anti-lysosomal enzyme NAbs (7H11 and 19D6) at different concentrations (125 ng / ml, 250 ng / ml, and 1000 ng / ml) in the presence of airborne lysosomal enzyme (concentration (ng / ml)), as measured by the standard anti-lysosomal neutralizing antibody assay.

[0052] Figure 10 Shows the effect of alkaline (base) pre-treated biological samples on the ability to detect anti-lysosomal enzyme neutralizing antibodies NAbs: polyclonal positive control anti-lysosomal enzyme NAbs (RP-01) at different concentrations (50 μg / ml, 100 μg / ml, and 150 μg / ml) and monoclonal positive control anti-lysosomal enzyme NAbs (7H11 and 19D6) at different concentrations (125 ng / ml, 250 ng / ml, and 1000 ng / ml) in the presence of airborne lysosomal enzyme (concentration (ng / ml)).

[0053] Figure 11 Shows the form of the total lysosomal enzyme antibody (TAb) assay.

[0054] Figures 12A - 12B Shows that in the presence of airborne lysosomal enzyme, various acidic pre-treatments (with or without heat pre-treatment ( Figure 12A )) or various base neutralization treatments ( Figure 12B) Influence on the ability to detect serum anti - lysosomal antibodies (measured by optical density (OD) at 450 nm). Test samples were 1 μg / ml 19D6 Ab + 2 μg / ml α - Gal A; 1 μg / ml 19D6 antibody; and 2 μg / ml α - Gal A.

[0055] Figure 13 Shows the influence on the ability to detect serum anti - lysosomal antibodies (measured by optical density (OD) at 450 nm) of various alkaline pre - treatments (pH 12, pH 11 + heating, and pH 12 + heating) in the presence of airborne lysosomal enzymes. Test samples were 1 μg / ml 19D6 Ab + 2 μg / ml α - Gal A; 1 μg / ml 19D6 antibody; and 2 μg / ml α - Gal A.

[0056] Figures 14A - 14B Shows the influence of neutral pH and alkaline pH pre - treatments on the ability to detect serum anti - lysosomal antibodies ( Figure 14A ) in the absence ( Figure 14B ) or presence (

[0057] Figure 15 ) of airborne lysosomal enzymes (measured by optical density (OD) at 450 nm).

[0058] Figures 16A - 16B Shows the measurement results of total anti - lysosomal enzyme antibodies in serum samples after untreated or alkaline pre - treatment with the following: serum samples from individuals without Fabry disease (negative serum); serum samples from individual #3 with Fabry disease in the presence of 4 μg / ml airborne lysosomal enzymes (Fabry serum #3 + 4 μg / ml α - Gal A); serum samples from individual #3 with Fabry disease (Fabry serum #3); and serum samples from individual #2 with Fabry disease (Fabry serum #2). (measured by optical density (OD) at 450 nm) Figure 16A ) and serum samples from individual #4 with Fabry disease in the absence or presence of airborne lysosomal enzymes (Fabry serum #4 and Fabry serum #4 + α - Gal A respectively) ( Figure 16B ). (measured by optical density (OD) at 450 nm) Detailed Description

[0059] The present disclosure provides methods for detecting anti - lysosomal enzyme antibodies in a human subject (e.g., in the presence of circulating lysosomal enzymes) and methods for enhancing the efficacy of anti - lysosomal enzyme antibody assays (e.g., enhancing assay sensitivity and circulating lysosomal enzyme tolerance), including pretreating a biological sample of the subject with a base having a pH of about 11 or higher and measuring the presence of anti - lysosomal enzyme antibodies in the biological sample of the subject.

[0060] I. Terms

[0061] To facilitate a more ready understanding of the present disclosure, some terms are first defined. As used in this application, unless expressly provided otherwise herein, each of the following terms shall have the meaning set forth below. Other definitions are set forth in this application.

[0062] It should be understood that wherever the term "comprising" is used to describe an aspect herein, aspects that are otherwise similar described in terms of "consisting of" and / or "consisting essentially of" are also provided.

[0063] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. By way of example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei - Show, 2nd Edition, 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd Edition, 1999, Academic Press; and Oxford Dictionary of Biochemistry And Molecular Biology, Revised Edition, 2000, Oxford University Press provide comprehensive dictionaries of many of the terms used in this disclosure for those skilled in the art.

[0064] Units, prefixes, and symbols are expressed in their accepted form of the International System of Units (Système International de Unites, SI). Numerical ranges include the numbers defining the range. Unless otherwise indicated, nucleotide sequences are written from left to right in a 5' to 3' orientation. Amino acid sequences are written from left to right in an amino - to - carboxyl orientation. The headings provided herein are not limitations on the various aspects of the disclosure and may be referred to in connection with the entire specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the entire specification as a whole.

[0065] "Polypeptide" refers to a chain containing at least two continuously linked amino acid residues, and there is no upper limit to the length of the chain. One or more amino acid residues in a protein may contain modifications, such as but not limited to glycosylation, phosphorylation, or disulfide bond formation. "Protein" may include one or more polypeptides.

[0066] The term "lysosomal enzyme" refers to a protein having enzymatic activity, such as glycosidase, protease, and sulfatase. Mammalian lysosomal enzymes are synthesized in the cytoplasm and pass through the endoplasmic reticulum (ER), where they are glycosylated with N-linked high-mannose carbohydrates. In the Golgi apparatus, the high-mannose carbohydrates are modified on lysosomal proteins by the addition of mannose-6-phosphate (M6P), thereby targeting these proteins to the lysosome. M6P-modified proteins are delivered to the lysosome by interacting with one of two M6P receptors. In some aspects, the lysosomal enzymes include but are not limited to α-galactosidase A (α-Gal A), glucocerebrosidase, α-glucosidase, α-L-iduronidase, iduronate 2-sulfatase, sulfamidase, galactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, β-glucuronidase, N-acetylglucosamine-1-phosphotransferase, Niemann-Pick C1 protein, hexosaminidase A, and tripeptidyl peptidase 1.

[0067] The terms "α-galactosidase A", "α-Gal A", and "GAL" are used interchangeably and refer to a protein having enzymatic activity that includes hydrolysis of the terminal non-reducing α-D-galactose residue in α-D-galactosides including galactooligosaccharides, galactomannans, and galactolipids. In some aspects, α-Gal A comprises the enzyme described by the IUBMB Enzyme Nomenclature EC 3.2.1.22 (as described, for example, in Suzuki et al., J. Biol. Chem. 245:781-786 (1970); Wiederschain, G. and Beyer, E. Dokl. Akad. Nauk S.S.S.R. 231:486-488 (1976)). In some aspects, α-Gal A includes a protein encoded by a nucleic acid comprising the human GLA gene (such as the human α-Gal A gene defined by GenBank accession number NM_000169). In some aspects, α-Gal A includes a protein comprising the amino acid sequence defined by GenBank accession number NP_000160. In some aspects, α-Gal A is galactosidase α produced by genetic engineering techniques in a human cell line. Galactosidase α may be obtained from Shire Plc. (Dublin, Ireland). In some aspects, α-Gal A is galactosidase β produced by recombinant DNA techniques in a Chinese hamster ovary (CHO) cell line. Galactosidase β may be obtained from Obtained from Sanofi Genzyme (Cambridge, Mass.). In some aspects, α-Gal A is recombinant human α-Gal A produced in CHO cells transformed with an expression vector encoding the human α-Gal A gene (JCR Pharmaceuticals Co., Ltd., (Japan)) and is identified as JR-051.

[0068] The terms “glucocerebrosidase,” “GCase,” “β-glucocerebrosidase,” “β-glucosidase,” “β-glucosidase,” “β-glucosidase,” “D-glucosyl-N-acylsphingosine glucohydrolase,” “glucosylceramidase,” and “glucosylceramidase β1 (GBA1)” are used interchangeably and refer to an enzyme having glucosylceramidase activity that requires hydrolysis to cleave the β-glycosidic bond of the chemical glucocerebroside, which is an intermediate in glycolipid metabolism and is present in large amounts in cell membranes (particularly skin cells). Glucocerebrosidase is located in lysosomes, where it remains bound to the lysosomal membrane. (Rijnboutt et al., J. Biol. Chem. 266(8):4862-8 (1991)). Glucocerebrosidase is 497 amino acids in length and has a molecular weight of 59,700 daltons. In some aspects, glucocerebrosidase includes the enzyme described by the IUBMB Enzyme Nomenclature EC 3.2.1.45 (e.g., as described in Boer et al., J. Clin. Med. 9(3):736 (2020)). In some aspects, glucocerebrosidase includes a protein encoded by a nucleic acid comprising a human glucocerebrosidase gene (e.g., the human glucocerebrosidase gene defined by GenBank accession number NM_000157, NM_001005741, or NM_001005742). In some aspects, glucocerebrosidase includes a protein comprising an amino acid sequence defined by GenBank accession number NP_000148, NP_001005741, NP_001005742, NP_001165282, or NP_001165283.

[0069] The terms "α-glucosidase", "alpha-glucosidase", "acid alpha-glucosidase", "GAA", "lysosomal alpha-glucosidase", "maltase", "glucoamylase", "glucosucrase", "maltase-glucoamylase", "alpha-pyranoglucosidase", "glucoside-converting enzyme", "alpha-D-glucosidase", "alpha-glucoside hydrolase", "alpha-1,4-glucosidase", and "alpha-D-glucosylglucose hydrolase" are used interchangeably and refer to the enzyme necessary for the degradation of glycogen to glucose in lysosomes. Alpha-glucosidase has the highest activity towards alpha-1,4-linked glycosidic bonds but can also hydrolyze alpha-1,6-linked glucans. In some aspects, alpha-glucosidase includes the enzyme described by the IUBMB Enzyme Nomenclature EC 3.2.1.20 (e.g., as described in Bruni et al., Biochim. Biophys. Acta 212:470-477 (1970)). In some aspects, alpha-glucosidase includes the protein encoded by a nucleic acid comprising a human alpha-glucosidase gene (e.g., the human alpha-glucosidase gene defined by GenBank accession number NM_000152, NM_001079803, or NM_001079804). In some aspects, alpha-glucosidase includes the protein comprising the amino acid sequence defined by GenBank accession number NP_000143, NP_001073271, or NP_001073272.

[0070] The terms "α-L-iduronidase", "L-iduronidase", "laronidase", "α-L-iduronosidase", and "glycosaminoglycan α-L-iduronohydrolase" are used interchangeably and refer to an enzyme involved in the degradation of glycosaminoglycans (such as dermatan sulfate and heparin sulfate). The enzyme acts by hydrolyzing the α-L-iduronic acid residues at the ends of these molecules, thereby degrading them. α-L-iduronidase is a glycoprotein present in cell lysosomes and has been reported to have a mass of approximately 83 kilodaltons. In some aspects, α-L-iduronidase includes the enzyme described by the IUBMB Enzyme Nomenclature EC 3.2.1.76 (e.g., as described in Rome et al., Arch Biochem Biophys 189:344-53 (1978); Scott et al., Proc Natl Acad Sci U S A. 88(21):9695-9 (1991)). In some aspects, α-L-iduronidase includes a protein encoded by a nucleic acid comprising the human α-L-iduronidase gene (e.g., the human α-L-iduronidase gene defined by GenBank accession number NM_000203 or NM_001363576). In some aspects, α-L-iduronidase includes a protein comprising the amino acid sequence defined by GenBank accession number NP_000194 or NP_001350505.

[0071] The terms "iduronate 2-sulfatase", "IDS", "chondroitin sulfatase", "iduronic acid-2-sulfatase", "L-iduronate sulfatase", "L-iduronate sulfatase", "iduronate sulfatase", "sulfonyl-L-iduronate sulfatase", "L-iduronate 2-sulfatase", "sulfogalactosylsulfohydrolase", "2-sulfo-L-iduronate 2-sulfatase", "iduronic acid-2-sulfatase", "iduronate sulfatase", and "L-iduronate-2-sulfate 2-sulfohydrolase" are used interchangeably and refer to a sulfatase that catalyzes the hydrolysis of the 2-sulfate group of the L-iduronate 2-sulfate units of dermatan sulfate, heparan sulfate, and heparin. In some aspects, iduronic acid-2-sulfatase includes the enzyme described by the IUBMB Enzyme Nomenclature EC 3.1.6.13 (e.g., as described in Archer et al., Biochim Biophys Acta 708:134-40 (1982)). In some aspects, iduronate 2-sulfatase includes a protein encoded by a nucleic acid comprising a human iduronate 2-sulfatase gene (e.g., the human iduronate 2-sulfatase gene defined by GenBank accession number NM_000202, NM_001166550, or NM_006123). In some aspects, iduronic acid-2-sulfatase includes a protein comprising an amino acid sequence defined by GenBank accession number NP_000193, NP_001160022, or NP_006114.

[0072] The terms "sulfamidase", "sulfamidase", "N-sulfo-glucosamine sulfohydrolase", "SGSH", "N-sulfo-D-glucosamine sulfohydrolase", "sulfoglucosamine sulfamidase", "heparan sulfamidase", "2-deoxy-D-glucoside-2-sulfamide sulfohydrolase", "sulfamide", and "sulfamide hydrolase" are used interchangeably and refer to an enzyme involved in the lysosomal degradation of heparan sulfate. This enzyme belongs to the hydrolase family and, specifically, is an enzyme that acts on sulfur-nitrogen bonds. In some aspects, it includes the enzyme described by the IUBMB Enzyme Nomenclature EC 3.10.1.1 (e.g., as described in Dietrich CP. Biochem J 111:91-5 (1969); Mahuran et al., Biochim Biophys Acta 757:359-65 (1983)). In some aspects, sulfamidase includes a protein encoded by a nucleic acid comprising a human sulfamidase gene (e.g., a human sulfamidase gene defined by GenBank accession number NM_000199, NM_001352921, or NM_001352922). In some aspects, sulfamidase includes a protein comprising an amino acid sequence defined by GenBank accession number NP_000190, NP_001339850, or NP_001339851.

[0073] The terms "galactosamine-6-sulfatase", "N-acetylgalactosamine-6-sulfatase", "GALNS", "galactosamine (N-acetyl)-6-sulfatase", "chondroitin sulfatase", "chondroitinase", "galactose-6-sulfate sulfatase", "acetylgalactosamine 6-sulfatase", and "N-acetylgalactosamine-6-sulfate sulfatase" are used interchangeably and refer to an exolysosomal hydrolase required for the degradation of the glycosaminoglycans keratan sulfate and chondroitin-6-sulfate. In some aspects, galactosamine-6-sulfatase includes the enzyme described by the IUBMB Enzyme Nomenclature EC 3.1.6.4 (e.g., as described in et al., (1991)). In some aspects, galactosamine-6-sulfatase includes a protein encoded by a nucleic acid comprising a human galactosamine-6-sulfatase gene (e.g., a human galactosamine-6-sulfatase gene defined by GenBank accession number NM_000512, NM_001323543, or NM_001323544). In some aspects, galactosamine-6-sulfatase includes a protein comprising an amino acid sequence defined by GenBank accession number NP_000503, NP_001310472, or NP_001310473.

[0074] The terms "N-acetylgalactosamine-4-sulfatase", "N-acetylgalactosamine-4-sulphatase", "chondroitin sulfatase", "chondroitinase", "arylsulfatase B", "acetylgalactosamine 4-sulfatase", "N-acetylgalactosamine 4-sulphate sulfohydrolase", and "N-acetyl-D-galactosamine-4-sulphate 4-sulphohydrolase" are used interchangeably and refer to an arylsulfatase that catalyzes the hydrolysis of the 4-sulphate group of the N-acetyl-D-galactosamine 4-sulphate unit of chondroitin sulfate and dermatan sulfate. In some aspects, e includes the enzyme described by the IUBMB Enzyme Nomenclature EC 3.1.6.12 (e.g., as described in Farooqui et al., Experientia 32:1242-1244 (1976)). In some aspects, N-acetylgalactosamine-4-sulfatase includes a protein encoded by a nucleic acid comprising the human N-acetylgalactosamine-4-sulfatase gene (e.g., the human N-acetylgalactosamine-4-sulfatase gene defined by GenBank accession number NM_00046). In some aspects, N-acetylgalactosamine-4-sulfatase includes a protein comprising the amino acid sequence defined by GenBank accession number NC_000005 or NM_198709.

[0075] The terms "β-glucuronidase", "beta-glucuronidase", "β-glucuronosyl glucuronohydrolase glucuronidase", "β-D-glucuronosyl glucuronohydrolase", "exo-β-D-glucuronidase", and "ketonase" are used interchangeably and refer to an enzyme that catalyzes the hydrolysis of β-D-glucuronic acid residues (e.g., heparan sulfate) at the non-reducing end of mucopolysaccharides (also known as glycosaminoglycans). In some aspects, β-glucuronidase includes the enzyme described by the IUBMB Enzyme Nomenclature EC 3.2.1.31 (e.g., as described in Diez et al., L.Eur.J.Biochem. 93:301–311 (1978)). In some aspects, β-glucuronidase includes a protein encoded by a nucleic acid comprising the β-glucuronidase gene (e.g., the human β-glucuronidase gene defined by GenBank accession numbers NM_000181, NM_001284290, NM_001293104, or NM_001293105). In some aspects, β-glucuronidase includes a protein comprising the amino acid sequence defined by GenBank accession numbers NP_000172, NP_001271219, or NP_001280034.

[0076] The terms "N-acetylglucosamine-1-phosphotransferase" and "N-acetylglucosamine-1-phosphotransferase" are used interchangeably and refer to the enzyme that catalyzes the first step in the synthesis of the mannose-6-phosphate lysosomal recognition marker. In some aspects, N-acetylglucosamine-1-phosphotransferase includes the enzyme described by the IUBMB Enzyme Nomenclature EC 2.7.8.17 (e.g., as described in Nishikawa, A. Lysosomal Enzyme GlcNAc-1-Phosphotransferase. Handbook of Glycosyltransferases and Related Genes. Springer, Tokyo (2002)). In some aspects, N-acetylglucosamine-1-phosphotransferase includes a protein encoded by a nucleic acid comprising a human N-acetylglucosamine-1-phosphotransferase gene (e.g., a human N-acetylglucosamine-1-phosphotransferase gene defined by GenBank accession number NM_032520, NM_024312, XM_011538731, or XM_006719593). In some aspects, N-acetylglucosamine-1-phosphotransferase includes a protein comprising an amino acid sequence defined by GenBank accession number NP_115909, NP_077288, XP_011537033, or XP_006719656.

[0077] The terms "Niemann-Pick C1 protein", "NPC intracellular cholesterol transporter 1", and "NPC1" are used interchangeably and refer to an enzyme located in the limiting membranes of endosomes and lysosomes that mediates intracellular cholesterol transport through the binding of cholesterol to its N-terminal domain (Xiaochun et al., Proc Natl Acad Sci. 113(29):8212-7 (2016)). In some aspects, the Niemann-Pick C1 protein comprises a protein encoded by a nucleic acid comprising the human Niemann-Pick C1 protein gene (e.g., the human Niemann-Pick C1 protein gene defined by GenBank accession numbers NM_000271, XM_005258279, XM_005258277, XM_017025787, XM_006722479, XM_047437539, XM_017025786, XM_017025785, XM_017025784, or XM_005258278). In some aspects, the Niemann-Pick C1 protein comprises a protein comprising an amino acid sequence defined by GenBank accession numbers NP_000262, XP_005258336, XP_005258334, XP_016881276, XP_006722542, XP_047293495, XP_016881275, XP_016881274, XP_016881273, or XP_005258335.

[0078] The terms "hexosaminidase A", "beta-acetylaminodeoxyhexosaminidase", "N-acetyl-beta-D-hexosaminidase", "N-acetyl-beta-hexosaminidase", "N-acetylhexosaminidase", "beta-hexosaminidase", "beta-acetylhexosaminidase", "beta-D-N-acetylhexosaminidase", "beta-N-acetyl-D-hexosaminidase", "beta-N-acetylglucosaminidase", "hexosaminidase A", "N-acetylhexosaminidase", "beta-D-hexosaminidase", "beta-N-acetylglucosaminidase", "hexosaminidase A", "N-acetylhexosaminidase", "beta-D-hexosaminidase", "beta-N-acetylhexosaminidase", and "HEXA" are used interchangeably and refer to an enzyme that participates in the hydrolysis of the terminal N-acetyl-D-hexosamine residue in N-acetyl-beta-D-hexosamine. In some aspects, hexosaminidase A includes the enzyme described by the IUBMB Enzyme Nomenclature EC 3.2.1.52 (e.g., as described in Tse et al., Biochemistry. 35(23):7599-607 (1996)). In some aspects, hexosaminidase A comprises a protein encoded by a nucleic acid comprising the human hexosaminidase A gene (e.g., the human hexosaminidase A gene defined by GenBank accession number NM_000520 or NM_001318825). In some aspects, hexosaminidase A includes a protein comprising the amino acid sequence defined by GenBank accession number NP_000511 or NP_001305754.

[0079] The terms "tripeptidyl peptidase 1", "TPP1", and "lysosomal pepstatin-insensitive protease" are used interchangeably and refer to a lysosomal serine protease. In some aspects, tripeptidyl peptidase 1 includes the enzyme described by the IUBMB Enzyme Nomenclature EC 3.4.14.9 (e.g., as described in Ezaki et al., J. Neurochem. 72:2573–2582 (1999)). In some aspects, tripeptidyl peptidase 1 includes a protein encoded by a nucleic acid comprising the human tripeptidyl peptidase 1 gene (e.g., the human tripeptidyl peptidase 1 gene defined by GenBank accession number NM_000391). In some aspects, tripeptidyl peptidase 1 includes a protein comprising the amino acid sequence defined by GenBank accession number NP_000382.

[0080] In some aspects, lysosomal enzymes (e.g., α-GalA, glucocerebrosidase, α-glucosidase, α-L-iduronidase, iduronate 2-sulfatase, sulfamidase, galactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, β-glucuronidase, N-acetylglucosamine-1-phosphotransferase, Niemann-Pick C1 protein, hexosaminidase A, or tripeptidyl peptidase 1) can be obtained from cells that endogenously express the lysosomal enzyme, or the lysosomal enzyme (e.g., α-GalA, glucocerebrosidase, α-glucosidase, α-L-iduronidase, iduronate 2-sulfatase, sulfamidase, galactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, β-glucuronidase, N-acetylglucosamine-1-phosphotransferase, Niemann-Pick C1 protein, hexosaminidase A or tripeptidyl peptidase 1) can be a recombinant human lysosomal enzyme (e.g., α-Gal A, glucocerebrosidase, α-glucosidase, α-L-iduronidase, iduronate 2-sulfatase, sulfamidase, galactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, β-glucuronidase, N-acetylglucosamine-1-phosphotransferase, Niemann-Pick C1 protein, hexosaminidase A or tripeptidyl peptidase 1), as described herein. In some aspects, the recombinant human lysosomal enzyme (e.g., α-Gal A, glucocerebrosidase, α-glucosidase, α-L-iduronidase, iduronate 2-sulfatase, sulfamidase, galactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, β-glucuronidase, N-acetylglucosamine-1-phosphotransferase, Niemann-Pick C1 protein, hexosaminidase A or tripeptidyl peptidase 1) is a full-length wild-type lysosomal enzyme (e.g., α-Gal A, glucocerebrosidase, α-glucosidase, α-L-iduronidase, iduronate 2-sulfatase, sulfamidase, galactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, β-glucuronidase, N-acetylglucosamine-1-phosphotransferase, Niemann-Pick C1 protein, hexosaminidase A or tripeptidyl peptidase 1).In some aspects, a recombinant human lysosomal enzyme (e.g., α-GalA, glucocerebrosidase, α-glucosidase, α-L-iduronidase, iduronate 2-sulfatase, arylsulfatase, galactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, β-glucuronidase, N-acetylglucosamine-1-phosphotransferase, Niemann-Pick C1 protein, hexosaminidase A, or tripeptidyl peptidase 1) includes a subset of the amino acid residues present in the wild-type lysosomal enzyme (e.g., α-GalA, glucocerebrosidase, α-glucosidase, α-L-iduronidase, iduronate 2-sulfatase, arylsulfatase, galactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, β-glucuronidase, N-acetylglucosamine-1-phosphotransferase, Niemann-Pick C1 protein, hexosaminidase A, or tripeptidyl peptidase 1), wherein the subset includes the amino acid residues that form the active site of the wild-type lysosomal enzyme (e.g., α-Gal A, glucocerebrosidase, α-glucosidase, α-L-iduronidase, iduronate 2-sulfatase, arylsulfatase, galactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, β-glucuronidase, N-acetylglucosamine-1-phosphotransferase, Niemann-Pick C1 protein, hexosaminidase A, or tripeptidyl peptidase 1) that binds and / or reduces a substrate.

[0081] In some aspects, the recombinant human lysosomal enzyme (e.g., α-GalA, glucocerebrosidase, α-glucosidase, α-L-iduronidase, iduronate 2-sulfatase, sulfamidase, galactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, β-glucuronidase, N-acetylglucosamine-1-phosphotransferase, Niemann-Pick C1 protein, hexosaminidase A or tripeptidyl peptidase 1) is a fusion protein that includes the active site of the wild-type lysosomal enzyme (e.g., α-Gal A, glucocerebrosidase, α-glucosidase, α-L-iduronidase, iduronate 2-sulfatase, sulfamidase, galactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, β-glucuronidase, N-acetylglucosamine-1-phosphotransferase, Niemann-Pick C1 protein, hexosaminidase A or tripeptidyl peptidase 1) for substrate binding and / or substrate reduction, and other amino acid residues that may or may not be present in the wild-type recombinant human lysosomal enzyme (e.g., α-GalA, glucocerebrosidase, α-glucosidase, α-L-iduronidase, iduronate 2-sulfatase, sulfamidase, galactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, β-glucuronidase, N-acetylglucosamine-1-phosphotransferase, Niemann-Pick C1 protein, hexosaminidase A or tripeptidyl peptidase 1) recombinant human lysosomal enzyme (e.g., α-GalA, glucocerebrosidase, α-glucosidase, α-L-iduronidase, iduronate 2-sulfatase, sulfamidase, galactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, β-glucuronidase, N-acetylglucosamine-1-phosphotransferase, Niemann-Pick C1 protein, hexosaminidase A or tripeptidyl peptidase 1).

[0082] Lysosomal enzymes (e.g., α-Gal A, glucocerebrosidase, α-glucosidase, α-L-iduronidase, iduronate 2-sulfatase, sulfamidase, galactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, β-glucuronidase, N-acetylglucosamine-1-phosphotransferase, Niemann-Pick C1 protein, hexosaminidase A or tripeptidyl peptidase 1) can be obtained from commercial sources or by synthetic techniques known to those skilled in the art. Wild-type enzymes can be purified from recombinant cell expression systems (e.g., mammalian cells such as CHO cells, or insect cells, see for example U.S. Patent Nos. 5,580,757; 6,395,884; 6,458,574; 6,461,609; 6,210,666; 6,083,725), human placenta or animal milk.

[0083] Other synthetic techniques for obtaining lysosomal enzymes suitable for pharmaceutical use (e.g., α-GalA, glucocerebrosidase, α-glucosidase, α-L-iduronidase, iduronate 2-sulfatase, sulfamidase, galactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, β-glucuronidase, N-acetylglucosamine-1-phosphotransferase, Niemann-Pick C1 protein, hexosaminidase A or tripeptidyl peptidase 1) can be found, for example, in U.S. Patent Nos. 7,560,424, 7,396,811, 423,135, 6,534,300 and 6,537,785; U.S. Published Application Nos. 2009 / 0203575; 2009 / 0029467; 2008 / 0299640; 2008 / 0241118; 2006 / 0121018; 2005 / 0244400; 2007 / 0280925; and 2004 / 0029779, as well as International Published Application No. 2005 / 077093.

[0084] In addition to proteins that contain the same amino acid sequence as the human lysosomal enzymes described herein (e.g., α-Gal A, glucocerebrosidase, α-glucosidase, α-L-iduronidase, iduronate 2-sulfatase, sulfamidase, galactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, β-glucuronidase, N-acetylglucosamine-1-phosphotransferase, Niemann-Pick C1 protein, hexosaminidase A or tripeptidyl peptidase 1), the present disclosure also encompasses lysosomal enzymes that are "substantially similar" thereto (e.g., α-GalA, glucocerebrosidase, α-glucosidase, α-L-iduronidase, iduronate 2-sulfatase, sulfamidase, galactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, β-glucuronidase, N-acetylglucosamine-1-phosphotransferase, Niemann-Pick C1 protein, hexosaminidase A or tripeptidyl peptidase 1). Proteins described herein as "substantially similar" to a reference protein include proteins that retain some of the structural and functional characteristics of the native protein and differ from the native amino acid sequence at one or more amino acid positions (i.e., by amino acid substitution).

[0085] Proteins that are altered relative to the native sequence can be prepared by substituting amino acid residues within the native protein and selecting the protein with the desired activity. For example, the amino acid residues of a lysosomal enzyme, such as the α-Gal A protein, can be systematically substituted with other residues, and then the substituted protein can be tested in standard assays to evaluate the effect of such substitutions on the ability of the protein to hydrolyze terminal non-reducing α-D-galactose residues in α-D-galactosides including galactooligosaccharides, galactomannans, and galactolipids, and / or on the ability to treat or prevent Fabry disease.

[0086] In some aspects, conservative amino acid substitutions are made in order to retain functional activity. As used herein, "conservative amino acid substitution" refers to the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been identified in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In some aspects, an expected non-essential amino acid residue in the α-GalA protein is replaced with another amino acid residue from the same side chain family. Methods for identifying nucleotide and amino acid conservative substitutions that do not abolish antigen binding are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).

[0087] In some aspects, the lysosomal enzymes of the present disclosure (e.g., α-galactosidase A, glucocerebrosidase, α-glucosidase, α-L-iduronidase, iduronate 2-sulfatase, sulfamidase, galactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, β-glucuronidase, N-acetylglucosamine-1-phosphotransferase, Niemann-Pick C1 protein, hexosaminidase A, or tripeptidyl peptidase 1) have an amino acid sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of a lysosomal enzyme described herein or known in the art (e.g., α-Gal A, glucocerebrosidase, α-glucosidase, α-L-iduronidase, iduronate 2-sulfatase, sulfamidase, galactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, β-glucuronidase, N-acetylglucosamine-1-phosphotransferase, Niemann-Pick C1 protein, hexosaminidase A, or tripeptidyl peptidase 1).

[0088] The percent identity between two sequences is a function of the number of positions at which the sequences differ (i.e., % homology = number of different positions / total number of positions × 100), taking into account the number of gaps and the length of each gap, which are introduced to obtain the best alignment of the two sequences. The sequence comparison between two sequences and the determination of the percent identity can be achieved using the mathematical algorithms described in the following non-limiting examples.

[0089] The percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at worldwideweb.gcg.com), using the NWSgapdna.CMP matrix, and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. The percent identity between two nucleotide or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)), which has been incorporated into the ALIGN program (version 2.0), using the PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at http: / / www.gcg.com), using the Blossum 62 matrix or the PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0090] The nucleic acid and protein sequences described herein can further be used as "query sequences" to search public databases to identify, for example, related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed using the NBLAST program with a score = 100 and wordlength = 12 to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed using the XBLAST program with a score = 50 and wordlength = 3 to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison purposes, gapped BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When using the BLAST and gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See worldwideweb.ncbi.nlm.nih.gov.

[0091] "Antibody" (Ab) includes, but is not limited to, a glycoprotein immunoglobulin that specifically binds to an antigen and comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. Each H chain comprises a heavy chain variable region (abbreviated herein as V H ) and a heavy chain constant region. The heavy chain constant region comprises three constant domains: C H1 , C H2 , and C H3 . Each L chain comprises a light chain variable region (abbreviated herein as V L ) and a light chain constant region. The light chain constant region comprises one constant domain C L . The V H and V L regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each V H and V LIt contains three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain the binding domains that interact with the antigen. The constant regions of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and the first component of the classical complement system (C1q). For example, the term "anti-lysosomal enzyme antibody" includes intact antibodies having two heavy chains and two light chains that specifically bind to lysosomal enzymes (α-Gal A, glucocerebrosidase, α-glucosidase, α-L-iduronidase, iduronate 2-sulfatase, sulfamidase, galactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, β-glucuronidase, N-acetylglucosamine-1-phosphotransferase, Niemann-Pick C1 protein, hexosaminidase A, or tripeptidyl peptidase 1) and the antigen-binding portions of the intact antibodies.

[0092] The immunoglobulin can be derived from any of the commonly known isotypes, including but not limited to IgA, secretory IgA, IgG, and IgM. IgG subclasses are also well known to those skilled in the art and include but are not limited to human IgG1, IgG2, IgG3, and IgG4. "Isotype" refers to the class or subclass of antibody encoded by the heavy chain constant region gene (such as IgM or IgG1). The term "antibody" includes, for example, naturally occurring and non-naturally occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human or non-human antibodies; fully synthetic antibodies; and single-chain antibodies. Non-human antibodies can be humanized by recombinant methods to reduce their immunogenicity in humans. Unless otherwise stated and unless the context indicates otherwise, the term "antibody" also includes the antigen-binding fragments or antigen-binding portions of any of the above immunoglobulins, and includes monovalent and divalent fragments or portions and single-chain antibodies.

[0093] An "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to α-Gal A is substantially free of antibodies that specifically bind to antigens other than α-Gal A). However, an isolated antibody that specifically binds to α-Gal A may have cross-reactivity with other antigens, such as α-Gal A molecules from different species. In addition, an isolated antibody may be substantially free of other cellular materials and / or chemicals.

[0094] The term "monoclonal antibody" (mAb) refers to a preparation of antibody molecules that are not naturally occurring and are composed of a single molecule, i.e., the primary sequence of the antibody molecules is substantially the same and exhibits a single binding specificity and affinity for a particular epitope. Monoclonal antibodies are an example of isolated antibodies. Monoclonal antibodies can be produced by hybridoma, recombinant, transgenic, or other techniques known to those skilled in the art.

[0095] The term "polyclonal antibody" (pAb) refers to a heterogeneous mixture of antibodies that are typically produced by different B cell clones in vivo. It can recognize and bind to many different epitopes of a single antigen. In some aspects, the RP-01 antibody described herein is a polyclonal antibody.

[0096] "Human antibody" (HuMAb) refers to an antibody having variable regions in which both the framework regions and the CDR regions are derived from human germline immunoglobulin sequences. In addition, if the antibody contains constant regions, the constant regions are also derived from human germline immunoglobulin sequences. The human antibodies of the present disclosure may include amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-specific mutagenesis or by in vivo somatic mutation). However, the term "human antibody" as used herein is not intended to include antibodies in which the CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been grafted onto human framework sequences. The terms "human antibody" and "fully human antibody" are used synonymously.

[0097] "Humanized antibody" refers to an antibody in which some, most, or all of the amino acids outside the CDRs of a non-human antibody have been replaced with the corresponding amino acids derived from human immunoglobulins. In one aspect of a humanized form of an antibody, some, most, or all of the amino acids outside the CDRs have been replaced with amino acids from human immunoglobulins, while some, most, or all of the amino acids within one or more CDRs remain unchanged. Minor additions, deletions, insertions, substitutions, or modifications of amino acids are permitted as long as they do not eliminate the ability of the antibody to bind to a particular antigen. A "humanized antibody" retains antigen specificity similar to that of the original antibody.

[0098] "Chimeric antibody" refers to an antibody in which the variable region is from one species and the constant region is from another species, e.g., an antibody in which the variable region is from a mouse antibody and the constant region is from a human antibody.

[0099] "Anti-antigen antibody" refers to an antibody that specifically binds to the antigen. For example, an anti-GAL antibody specifically binds to GAL; an anti-glucocerebrosidase antibody specifically binds to glucocerebrosidase, an anti-α-glucosidase antibody specifically binds to α-glucosidase, an anti-α-L-iduronidase antibody specifically binds to α-L-iduronidase, an anti-iduronate 2-sulfatase antibody specifically binds to iduronate 2-sulfatase, an anti-sulfamidase antibody specifically binds to sulfamidase, an anti-galactosamine-6-sulfatase antibody specifically binds to galactosamine-6-sulfatase, an anti-N-acetylgalactosamine-4-sulfatase antibody specifically binds to N-acetylgalactosamine-4-sulfatase, an anti-β-glucuronidase antibody specifically binds to β-glucuronidase, an anti-N-acetylglucosamine-1-phosphotransferase antibody specifically binds to N-acetylglucosamine-1-phosphotransferase, an anti-Niemann-Pick C1 protein antibody specifically binds to Niemann-Pick C1 protein, an anti-hexosaminidase A specifically binds to hexosaminidase A, and an anti-tripeptidyl peptidase 1 specifically binds to tripeptidyl peptidase 1.

[0100] The terms "neutralizing anti-lysosomal enzyme antibody", "anti-lysosomal enzyme NAb", "neutralizing anti-drug antibody", or "neutralizing ADA" refer to antibodies that bind to and inactivate (neutralize) lysosomal enzymes (e.g., α-Gal A, glucocerebrosidase, α-glucosidase, α-L-iduronidase, iduronate 2-sulfatase, sulfamidase, galactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, β-glucuronidase, N-acetylglucosamine-1-phosphotransferase, Niemann-Pick C1 protein, hexosaminidase A, or tripeptidyl peptidase 1). For example, if neutralizing anti-lysosomal enzyme antibodies are present (e.g., anti-α-galactosidase A antibody, anti-glucocerebrosidase antibody, anti-α-glucosidase antibody, anti-α-L-iduronidase antibody, anti-iduronate 2-sulfatase antibody, anti-sulfamidase antibody, anti-galactosamine-6-sulfatase antibody, anti-N-acetylgalactosamine-4-sulfatase antibody, anti-β-glucuronidase antibody, anti-N-acetylglucosamine-1-phosphotransferase antibody, anti-Niemann-Pick C1 protein antibody, anti-hexosaminidase A, or anti-tripeptidyl peptidase 1 antibody), the enzyme replacement therapy is directly inactivated (neutralized) by the neutralizing anti-lysosomal enzyme antibodies in the plasma, which can inhibit cellular uptake or enzyme activity within the lysosome. In some aspects, if neutralizing anti-lysosomal enzyme antibodies are present, they can neutralize ERT activity by binding to the enzyme (e.g., recombinant lysosomal enzyme).

[0101] In some aspects, the anti - lysosomal enzyme neutralizing antibody is an IgG antibody. In some aspects, the anti - lysosomal enzyme neutralizing antibody is an IgG4 antibody. In some aspects, the anti - lysosomal enzyme neutralizing antibody is an IgG2 antibody. In some aspects, the anti - lysosomal enzyme neutralizing antibody is an IgG1 antibody.

[0102] In some aspects, the anti - lysosomal enzyme neutralizing antibody can be produced within about one month, about two months, about three months, about four months, about five months, about six months, about seven months, about eight months, about nine months, about ten months, about eleven months, or about twelve months after the start of enzyme replacement therapy.

[0103] The terms "total anti - lysosomal enzyme antibody", "anti - lysosomal enzyme TAb", or "binding anti - lysosomal enzyme antibody" refer to pre - existing anti - lysosomal enzyme antibodies (e.g., anti - lysosomal enzyme antibodies present in a subject prior to treatment (e.g., prior to lysosomal storage disease treatment) or prior to the start of a clinical study) and treatment - enhanced antibodies (e.g., pre - existing anti - lysosomal enzyme antibodies that are enhanced to higher levels after administration of a lysosomal storage disease treatment). As used herein, the terms "pre - existing anti - lysosomal enzyme antibody" and "treatment - enhanced anti - lysosomal enzyme antibody" refer to total anti - drug antibodies (ADA) against lysosomal enzymes (e.g., α - GalA, glucocerebrosidase, α - glucosidase, α - L - iduronidase, iduronate - 2 - sulfatase, arylsulfatase, galactosamine - 6 - sulfate sulfatase, N - acetylgalactosamine - 4 - sulfate sulfatase, β - glucuronidase, N - acetylglucosamine - 1 - phosphotransferase, Niemann - Pick C1 protein, hexosaminidase A, or tripeptidyl peptidase 1), where the total ADA antibodies are detected using: an enzyme - linked immunosorbent assay (ELISA), e.g., as described in Partridge, M.A. et al., J Immunol Res.; 2016:6262383. (2016), or an electrochemiluminescence (ECL) - type assay (e.g., MesoScale Discovery (MSD)), e.g., as described in Partridge, M.A. et al., J Immunol Res.; 2016:6262383. (2016).

[0104] The "antigen-binding portion" (also referred to as "antigen-binding fragment") of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen bound by the intact antibody. It has been demonstrated that the antigen-binding function of an antibody can be performed by fragments of the full-length antibody. The term antibody (e.g., anti-GLA antibody, anti-glucocerebrosidase antibody, anti-α-glucosidase antibody, anti-α-L-iduronidase antibody, anti-iduronate-2-sulfatase antibody, anti-sulfamidase antibody, anti-galactosamine-6-sulfatase antibody, anti-N-acetylgalactosamine-4-sulfatase antibody, anti-β-glucuronidase antibody, anti-N-acetylglucosamine-1-phosphotransferase antibody, anti-Niemann-Pick C1 protein antibody, anti-hexosaminidase A or anti-tripeptidyl peptidase 1 as described herein) "antigen-binding portion" encompasses examples of binding fragments including (i) Fab fragments (fragments from papain cleavage) consisting of V L 、V H 、LC and CH1 domains or similar monovalent fragments; (ii) F(ab')2 fragments (fragments from pepsin cleavage) containing two Fab fragments linked by disulfide bonds in the hinge region or similar divalent fragments; (iii) Fd fragments consisting of V H and CH1 domains; (iv) Fv fragments consisting of V L and V H domains of a single arm of the antibody, (v) dAb fragments consisting of V H domains (Ward et al., (1989) Nature 341:544-546); (vi) isolated complementarity-determining regions (CDRs) and (vii) combinations of two or more isolated CDRs optionally linked by a synthetic linker. In addition, although the two domains V L and V H of the Fv fragment are encoded by separate genes, they can be joined by recombinant methods using a synthetic linker so that they can be prepared as a single protein chain, in which the V L and V H regions pair to form a monovalent molecule (referred to as single-chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be encompassed within the term antibody "antigen-binding portion". These antibody fragments are obtained using conventional techniques known to those skilled in the art and are screened for efficacy in the same manner as intact antibodies. The antigen-binding portion can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

[0105] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed by the dissociation constant (K D Affinity can be measured and / or expressed in many ways known in the art, including but not limited to the equilibrium dissociation constant (K D ) and equilibrium association constant (K A ). D By k 解离 / k 缔合 The quotient of is calculated and expressed as molar concentration (M), while K A By k 缔合 / k 解离 k 缔合 refers to, for example, the rate constant for the association of an antibody with an antigen, and k 解离 Refers to, for example, the dissociation of antibodies and antigens. 缔合 and k 解离 The determination can be made by techniques known to those of ordinary skill in the art, such as immunoassays (e.g., enzyme-linked immunosorbent assay (ELISA)), BLI (Bio-Layer Interferometry) or Kinetic Exclusion Analysis

[0106] As used herein, the terms "specific binding", "specific recognition", "specific binding", "selective binding", and "selective binding" are analogous terms in the context of antibodies and refer to the binding of a molecule (e.g., an antibody) to an antigen (e.g., an epitope or an immune complex), as such binding is understood by those skilled in the art. For example, a molecule that specifically binds to an antigen can be typically detected, for example, by immunoassays, 3000 instrument (Sapidyne Instruments, Boise, ID) or other assays known in the art. In a particular aspect, a molecule that specifically binds to an antigen binds to another antigen with a lower affinity than when the molecule binds to the antigen. A K at least 2log, 2.5log, 3log, 4log or more A binds to the antigen.

[0107] Antibodies are usually -5 Up to 10 -11 M or less dissociation constant (K D ) reflects high affinity specific binding to its cognate antigen. Greater than about 10 -4Any K of M D is generally considered to indicate non-specific binding. As used herein, an antibody that "specifically binds" to an antigen means that the antibody binds to the antigen and substantially the same antigen with high affinity, meaning that when using a predetermined antigen or BLI (Biolayer Interferometry) assay in a BIACORE TM 2000 instrument by, for example, immunoassay (e.g., ELISA), surface plasmon resonance (SPR) technology, it has a K -7 of 10 M or less, preferably 10 -8 M or less, even more preferably 10 -9 M or less, and most preferably between 10 -8 M and 10 -10 M or less, but does not bind to unrelated antigens with high affinity. D

[0108] As used herein, the term "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. The nucleic acid molecule can be single-stranded or double-stranded and can be cDNA.

[0109] The nucleic acid can be present in whole cells, cell lysates, or in a partially purified or substantially pure form. The nucleic acid is "isolated" or "becomes substantially pure" when purified away from other cellular components or other contaminants (such as other cellular nucleic acids (e.g., other parts of the chromosome) or proteins) by standard techniques, including alkaline / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other techniques well known in the art. See F. Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987).

[0110] Nucleic acids (e.g., cDNA) can be mutated according to standard techniques to provide gene sequences. For coding sequences, these mutations can affect the amino acid sequence as needed. In particular, DNA sequences that are substantially homologous to or derived from the native V, D, J constant switch, and other such sequences described herein are encompassed (where "derived" indicates that the sequence is the same as or modified from another sequence).

[0111] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting other nucleic acids to which it has been linked. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop to which other DNA segments can be ligated. Another type of vector is a viral vector, into which other DNA segments can be ligated within the viral genome. Some vectors are capable of autonomous replication in the host cells into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can integrate into the genome of the host cell after introduction into the host cell and thereby replicate with the host genome. In addition, some vectors are capable of directing the expression of genes operably linked thereto. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Generally, expression vectors used in recombinant DNA techniques often take the form of plasmids. In this specification, the terms "plasmid" and "vector" are used interchangeably because plasmids are the most commonly used form of vector. However, other forms of expression vectors that provide equivalent functions are also included, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, adeno-associated viruses ("AAV"), and lentiviruses).

[0112] As used herein, the term "recombinant host cell" (or simply "host cell") is intended to refer to a cell that contains nucleic acids not naturally present in the cell and can be a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to a particular subject cell but also to the progeny of such a cell. Since some modifications may occur in the progeny due to mutations or environmental influences, such progeny will not actually be identical to the parental cell, but are still included within the scope of the term "host cell" as used herein.

[0113] As used herein, the term "linked" refers to the association of two or more molecules. The linkage can be covalent or non-covalent. The linkage can also be genetic (i.e., recombinantly fused). Such linkages can be achieved using a variety of techniques recognized in the art, such as chemical conjugation and the production of recombinant proteins.

[0114] The terms "lysosomal storage disease", "lysosomal storage disorder", "LSD", "lysosomal storage diseases", "lysosomal storage disorders", and "LSDs" are used interchangeably and refer to a group of more than 40 disorders that are caused by genetic defects in genes encoding enzymes that break down glycolipids or polysaccharide waste products within the lysosomes of cells. The enzyme products (e.g., sugars and lipids) are then recycled into new products. These disorders are caused by inherited autosomal or X-linked recessive genetic traits and affect the levels of enzymes in the lysosomes. Generally, the affected enzymes in the cells and tissues of affected individuals are not biologically or functionally active. In some aspects, lysosomal storage diseases include, but are not limited to, Fabry disease, Gaucher disease, Pompe disease, mucopolysaccharidosis (MPS) type I disease, MPS II, MPS III, MPS IV, MPS VI, MPS VII, mucolipidosis (ML), Niemann-Pick disease, Tay-Sachs disease, or Batten disease.

[0115] The term "Fabry disease" refers to classical Fabry disease, late-onset Fabry disease, and hemizygous females with mutations in the gene encoding α-GalA. As used herein, the term "Fabry disease" also includes any disorder in which a subject exhibits lower than normal endogenous α-Gal A activity. Fabry disease is referred to by many other names, such as α-galactosidase A deficiency, Anderson-Fabry disease, angiokeratoma corporis diffusum, diffuse angiokeratoma, ceramidetrihexosidase deficiency, Fabry's disease, GLA deficiency, and hereditary ectopic lipidosis. In some aspects, Fabry disease is of the type 1 classical phenotype or the type 2 late-onset phenotype.

[0116] "Gaucher disease" is a rare genetic metabolic disorder that results from a deficiency of glucocerebrosidase, leading to the accumulation of harmful amounts of certain fats (lipids), particularly the glycolipid glucocerebroside, throughout the body, especially in the bone marrow, spleen, and liver. Gaucher disease has many other names, such as, cerebrosidelipidosis syndrome, Gaucher splenomegaly, glucocerebrosidase deficiency, glucocerebroside deposition disease, glucocerebrosidase deficiency, glucocerebrosidelipidosis, kerasin lipidosis, kerasin storage disease, lipid histiocytosis (kerasin type), and sphingolipidosis 1.

[0117] "Pompe disease" is a rare multi-system genetic disorder in which, due to a deficiency of alpha-glucosidase, glycogen cannot be degraded into glucose. As a result, glycogen begins to accumulate in various tissues, but mainly in skeletal, smooth, and cardiac muscle, and causes damage to tissue structure and function. Pompe disease has many other names, such as glycogen storage disease type II (GSDII), acid maltase deficiency (AMD), and acid alpha-glucosidase (GAA) deficiency.

[0118] The term "mucopolysaccharidosis" or "MPS" refers to a group of inherited lysosomal storage disorders.

[0119] "MPS type I disease" is characterized by a deficiency of the enzyme alpha-L-iduronidase, resulting in the accumulation of dermatan sulfate and / or heparan sulfate. There are 3 types of MPS I disease: Hurler syndrome (mucopolysaccharidosis 1-H type; MPS1-H), Scheie syndrome (mucopolysaccharidosis IS type; MPS1-S), and Hurler-Scheie syndrome (mucopolysaccharidosis IH / S type; MPS-IH / S).

[0120] "MPS type II disease" is a rare genetic disorder in which glycosaminoglycans (or GAGs or mucopolysaccharides) accumulate in body tissues. It is caused by a deficiency of the lysosomal enzyme iduronate-2-sulfatase (I2S). The lack of this enzyme causes heparan sulfate and dermatan sulfate to accumulate in all tissues of the body. It is the only MPS disorder that is inherited as an X-linked trait. MPS type II disease has other names, such as Hunter syndrome, mucopolysaccharidosis type II, and MPS II.

[0121] "MPS type III disease" has four subtypes (A, B, C, and D), which are distinguished by four different enzyme deficiencies: heparan N-sulfatase, alpha-N-acetylglucosaminidase, acetyl-CoA:alpha-glucosaminide N-acetyltransferase, and N-acetylglucosamine-6-sulfatase. MPS type III disease has other names, such as Sanfilippo syndrome, mucopolysaccharidosis type III, and MPS III.

[0122] "MPS type IV disease" is a rare metabolic disorder in which the body is unable to process glycosaminoglycans (also known as GAGs, or mucopolysaccharides). It has other names, such as Morquio syndrome, mucopolysaccharidosis type IV, and MPS IV. Morquio syndrome A and B occur due to a deficiency of N-acetylgalactosamine-6-sulfatase and beta-galactosidase, respectively, resulting in the accumulation of keratan sulfate and chondroitin sulfate in type A patients, while type B patients accumulate keratan sulfate.

[0123] "MPS VI disease" is characterized by a deficiency of N-acetylgalactosamine-4-sulfatase, resulting in the accumulation of dermatan sulfate. It has other names, such as Maroteaux-Lamy syndrome, mucopolysaccharidosis VI, and MPS VI.

[0124] "MPS VII disease" is characterized by a deficiency of the enzyme β-glucuronidase, resulting in the accumulation of three glycosaminoglycans: dermatan sulfate, heparan sulfate, and chondroitin sulfate. It has other names, such as Sly syndrome, mucopolysaccharidosis VII, and MPS VII.

[0125] The term "mucolipidosis (ML)" refers to a group of inherited metabolic disorders that affect the body's ability to carry out normal intracellular turnover of various substances. Historically, four conditions (types I, II, III, and IV) were labeled as mucolipidosis. However, type I (sialidosis) is now classified as a glycoproteinosis, while type IV (mucolipidosis IV) is now classified as a gangliosidosis. Mucolipidosis II and III (ML II and ML III) are due to a deficiency of N-acetylglucosamine-1-phosphotransferase, an enzyme that phosphorylates target carbohydrate residues on N-linked glycoproteins. Without this phosphorylation, glycoproteins do not enter lysosomes but instead escape to the extracellular space.

[0126] "Niemann-Pick disease", "Niemann-Pick disease, type C", "NPC", or "NPD-C" is a rare, progressive, inherited disorder characterized by the body's inability to transport cholesterol and other fat substances (lipids) within cells. 95% of cases are caused by loss of function of the Niemann-Pick C1 protein (NPC1).

[0127] "Tay-Sachs disease" is a rare neurodegenerative disorder in which patients have an excessive accumulation of gangliosides in the brain and nerve cells due to a deficiency of hexosaminidase A. It has many other names, such as, HEXA deficiency, hexosaminidase A deficiency, TSD, GM2 gangliosidosis, type 1, hexosaminidase alpha subunit deficiency (variant B), variant B GM2 gangliosidosis, sphingolipidosis, and Tay-Sachs.

[0128] "Batten disease" is the common name for a large group of rare and fatal genetic neurological disorders, also known as neuronal ceroid lipofuscinosis, or NCL. It is a disorder in which brain cells lack an enzyme called tripeptidyl peptidase 1 (TPP1), and the disorder causes waste to accumulate in the cell neurons. It has many other names, for example, juvenile CLN3 disease, CLN3, CLN3-NCL, JNCL, juvenile Batten disease, juvenile neuronal ceroid lipofuscinosis, neuronal ceroid lipofuscinosis 3, Spielmeyer-Sjogren disease, Vogt-Spielmeyer disease, and Vogt-Spielmeyer-Sjogren disease.

[0129] The term "enzyme replacement therapy" or "ERT" refers to the introduction of a non-native purified enzyme into an individual who lacks such an enzyme (such as a lysosomal enzyme as described herein). The enzyme administered can be obtained from natural sources or by recombinant expression. The term also refers to the introduction of a purified enzyme into an individual who otherwise requires or benefits from the administration of a purified enzyme (such as suffering from protein deficiency). The introduced enzyme can be a purified recombinant enzyme produced in vitro, or an enzyme purified from isolated tissues or fluids such as placenta or animal milk or from plants. Figure 1 Examples of enzyme replacement therapy (ERT) currently approved for the treatment of lysosomal storage diseases such as Fabry disease, Pompe disease, Gaucher disease, MPSI, MPSII, MPSIV, and MPS VI are shown.

[0130] The term "non-enzyme replacement therapy" refers to a therapy that is not enzyme replacement therapy (such as a lysosomal storage disease therapy). Non-enzyme replacement therapies can include small molecule therapies. Some emerging drug development strategies for small molecule therapies for lysosomal storage diseases include, but are not limited to, substrate reduction therapy (SRT), residual enzyme activation, protein homeostasis regulation (protein homeostasis), and pharmacological chaperone therapy (PCT).

[0131] The term "stable appropriate conformation" refers to the ability of a compound or peptide or other molecule to associate with a wild-type protein or with a mutant protein in such a way that the structure of the wild-type or mutant protein can maintain its native or appropriate form and thus can perform its wild-type function in vitro and in vivo. This effect itself can actually manifest through one or more of the following: (i) increased protein shelf life; (ii) higher activity per unit or per unit amount of protein; or (iii) greater in vivo efficacy. This effect can be experimentally observed by increased yield from the ER during expression; greater resistance to unfolding due to increased temperature (such as as determined in a thermal stability analysis) or the presence of a chaotropic agent, and by similar means.

[0132] As used herein, the term "active site" refers to a region in a protein that has a particular biological activity. For example, it can be a site that binds a substrate or other binding partner and contributes amino acid residues that are directly involved in the formation and breakage of chemical bonds. The active sites in the present application can encompass the catalytic sites of enzymes, the antigen-binding sites of antibodies, the ligand-binding domains of receptors, the binding domains of modulators, or the receptor-binding domains of secreted proteins. Active sites can also encompass the transactivation, protein-protein interaction, or DNA-binding domains of transcription factors and modulators.

[0133] As used herein, the term "active site-specific chaperone" refers to any molecule that reversibly and specifically interacts with the active site of a protein and enhances the formation of a stable molecular conformation, including proteins, peptides, nucleic acids, carbohydrates, and the like. As used herein, "active site-specific chaperone" does not include endogenous general chaperones present in the ER of cells, such as Bip, calnexin, or calreticulin; or general non-specific chemical chaperones, such as deuterated water, DMSO, or TMAO.

[0134] The term "immunotherapy" refers to the treatment of a subject suffering from a disease or at risk of contracting or suffering a recurrence of a disease by methods that include inducing, enhancing, suppressing, or otherwise altering an immune response. "Treatment" or "therapy" of a subject refers to any type of intervention or treatment of the subject or the administration of an active agent to the subject, with the goal of reversing, alleviating, ameliorating, suppressing, slowing, or preventing the onset, progression, development, severity, or recurrence of symptoms, complications, or afflictions associated with the disease or biochemical markers associated with the disease.

[0135] "Subject" includes any human or non-human animal. The term "non-human animal" includes, but is not limited to, vertebrates such as non-human primates, sheep, dogs, and rodents such as mice, rats, and guinea pigs. In some aspects, the subject is a human. The terms "subject" and "patient" are used interchangeably herein.

[0136] The use of the term "fixed dose" with respect to the methods and dosages of the present disclosure means a dose that is administered to a patient without regard to the patient's body weight or body surface area (BSA). Thus, a fixed dose is provided not as a mg / kg dose, but as an absolute amount of the agent (e.g., recombinant α-Gal A protein). For example, a 60 kg person and a 100 kg person will receive the same dose of an antibody (e.g., 12 mg of recombinant α-Gal A protein).

[0137] As used herein, the term "weight-based dose" means that the dose administered to a patient is calculated based on the patient's weight. For example, when a patient with a body weight of 60 kg requires 0.2 mg / kg of recombinant α-Gal A protein, we can calculate and use an appropriate amount of recombinant α-Gal A protein (i.e., 12 mg) for administration.

[0138] A "therapeutically effective amount" or "therapeutically effective dose" of a drug or therapeutic agent is any amount that, when used alone or in combination with another therapeutic agent, prevents the onset of disease or causes the regression of disease in a subject, as evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of periods of freedom from disease symptoms, or the prevention of damage or disability caused by the disease. The ability of a therapeutic agent to cause the regression of disease can be evaluated using a variety of methods known to a skilled practitioner, such as evaluation in human subjects during clinical trials, evaluation in animal model systems that predict efficacy in humans, or analysis of the activity of the agent in in vitro assays.

[0139] As used herein, the term "treat / treating / treatment" refers to any type of intervention or process performed on a subject or the administration of an active agent to a subject, with the goal of reversing, alleviating, improving, inhibiting, or slowing or preventing the progression, development, severity, or recurrence of symptoms, complications, disorders, or biochemical markers associated with a disease or enhancing overall survival. Treatment can be for a subject with a disease or a subject without a disease (e.g., for prevention).

[0140] The term "effective dose" is defined as the amount sufficient to achieve or at least partially achieve the desired effect. A "therapeutically effective amount" or "therapeutically effective dose" of a drug or therapeutic agent is any amount that, when used alone or in combination with another therapeutic agent, promotes the regression of disease, as evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of periods of freedom from disease symptoms, an increase in overall survival (the length of time that a patient diagnosed with the disease remains alive from the date of diagnosis or initiation of treatment for the disease, such as cancer), or the prevention of damage or disability caused by the disease. The therapeutically effective amount or dose of a drug includes a "prophylactically effective amount" or "prophylactically effective dose", which is any amount that, when administered alone or in combination with another therapeutic agent to a subject at risk of developing a disease or suffering a recurrence of the disease, inhibits the development or recurrence of the disease. The ability of a therapeutic agent to cause the regression of disease or inhibit the development or recurrence of disease can be evaluated using a variety of methods known to a skilled practitioner, such as evaluation in human subjects during clinical trials, evaluation in animal model systems that predict efficacy in humans, or analysis of the activity of the agent in in vitro assays.

[0141] A "sample" or "biological sample" of the present disclosure is of biological origin and, in some aspects, such as from eukaryotes. In some aspects, the sample is a human sample, but animal samples can also be used. Non-limiting sources of samples for use in the present disclosure include, for example, solid tissues, biopsies, ascites, fluid extracts, blood, plasma, serum, cerebrospinal fluid, lymphatic fluid, external portions of the skin, respiratory tract, intestine, and urogenital tract, tears, saliva, milk, tumors, organs, cell cultures, and / or cell culture components.

[0142] "Administer" means physically introducing a composition comprising a therapeutic agent into a subject using any of a variety of methods and delivery systems known to those skilled in the art. Preferred routes of administration of the recombinant α-Gal A protein or the gene expressing α-GalA include intravenous or other parenteral routes of administration, such as by injection or infusion. As used herein, the phrase "parenteral administration" means a mode of administration other than enteral and topical administration, typically by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, and in vivo electroporation. Other non-parenteral routes include oral, topical, transdermal, or transmucosal routes of administration, such as intranasal, vaginal, rectal, sublingual, or topical. Administration can also be carried out, for example, once, multiple times, and / or over one or more extended periods of time.

[0143] The terms "about once a week", "about once every two weeks", or any other similar dosing interval term as used herein mean approximate numbers. "About once a week" can include every seven days ± one day, i.e., every six days to every eight days. "About once every two weeks" can include every fourteen days ± three days, i.e., every eleven days to every seventeen days. Similar approximations apply to, for example, about once every three weeks, about once every four weeks, about once every five weeks, about once every six weeks, and about once every twelve weeks. In some aspects, a dosing interval of about once every six weeks or about once every twelve weeks means that the first dose can be administered on any day of the first week, and then the next dose can be administered on any day of the sixth week or the twelfth week, respectively. In other aspects, a dosing interval of about once every six weeks or about once every twelve weeks means that the first dose is administered on a specific day of the first week (e.g., Monday), and then the next dose is administered on the same day of the sixth week or the twelfth week (i.e., Monday).

[0144] The use of alternatives (e.g., "or") should be understood to mean one, both, or any combination of the alternatives. As used herein, the indefinite article "a" or "an" should be understood to refer to "one or more" of any of the components listed or enumerated.

[0145] The terms "about" or "substantially comprises" means that a value or a composition is within an acceptable error range of a particular value or composition as determined by a person of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "substantially comprises" can mean within 1 or more standard deviations according to the practice in the art. Alternatively, "about" or "substantially comprises" can mean a range of up to 10%. In addition, especially with respect to biological systems or methods, the term can mean up to one order of magnitude or up to 5-fold of a value. When a particular value or composition is provided in the present application and the claims, unless otherwise stated, the meaning of "about" or "substantially comprises" should be assumed to be within the acceptable error range of the particular value or composition.

[0146] Unless otherwise indicated, as described herein, any concentration range, percentage range, ratio range or integer range should be understood to include any integer within the listed range and (where appropriate) values of its fractions (such as one-tenth and one-hundredth of an integer).

[0147] The following subsections further describe various aspects of the present disclosure in detail.

[0148] II. Methods of the Invention

[0149] Methods for detecting anti-lysosomal enzyme antibodies in a human subject and methods for improving the efficacy of an anti-lysosomal enzyme antibody assay are provided herein, which include pretreating a biological sample of the subject with a base having a pH of about 11 or higher and measuring the presence of anti-lysosomal enzyme antibodies in the biological sample of the subject.

[0150] In some aspects, the present disclosure relates to a method for detecting anti-lysosomal enzyme antibodies in a human subject (e.g., in the presence of circulating lysosomal enzymes), which includes measuring the presence of anti-lysosomal enzyme antibodies in a biological sample of the subject, wherein the biological sample is pretreated with a base having a pH of about 11 or higher.

[0151] In some aspects, the present disclosure relates to a method for improving the efficacy of an anti-lysosomal enzyme antibody assay (e.g., improving assay sensitivity and circulating lysosomal enzyme tolerance), which includes pretreating a biological sample of a human subject with a base having a pH of about 11 or higher, and further includes measuring the presence of anti-lysosomal enzyme antibodies in the biological sample.

[0152] As used herein, the term "enhancing the efficacy of anti - lysosomal enzyme antibody assays" refers to any improvement resulting from optimizing various assay conditions (e.g., pretreatment of a biological sample with a base at a high or low molar concentration at a pH of about 11 or higher, heat pretreatment of the biological sample, various levels of endogenous lysosomal enzyme concentration (ng / ml), neutralizing the base - pretreated biological sample with an acid, or any combination thereof), which can produce favorable results (e.g., increased assay sensitivity and circulating lysosomal enzyme tolerance) compared to the results obtained from performing anti - lysosomal enzyme antibody assays without the same assay conditions (e.g., pretreatment of a biological sample with a base at a high or low molar concentration at a pH of about 11 or higher, heat pretreatment of the biological sample, various levels of endogenous lysosomal enzyme concentration (ng / ml), neutralizing the base - pretreated biological sample with an acid, or any combination thereof). Enhancement and determination of enhancement can be measured by various parameters, such as but not limited to: increased assay sensitivity, e.g., measured by (i) an increased ability to detect anti - lysosomal enzyme neutralizing antibodies (NAbs) and / or total anti - lysosomal antibodies (TAbs) in the presence or absence of endogenous lysosomal enzymes, and / or (ii) increased circulating lysosomal enzyme tolerance, e.g., measured by increased tolerance to endogenous lysosomal enzymes when detecting anti - lysosomal enzyme NAbs, as described in Examples 2 - 4 below.

[0153] IIA. Detection of Anti - Lysosomal Enzyme Neutralizing Antibody (NAb)

[0154] In some aspects, the present disclosure relates to methods for detecting anti - lysosomal enzyme antibodies (e.g., anti - α - galactosidase A antibody, anti - glucocerebrosidase antibody, anti - α - glucosidase antibody, anti - α - L - iduronidase antibody, anti - iduronate - 2 - sulfatase antibody, anti - sulfamidase antibody, anti - galactosamine - 6 - sulfatase antibody, anti - N - acetylgalactosamine - 4 - sulfatase antibody, anti - β - glucuronidase antibody, anti - N - acetylglucosamine - 1 - phosphotransferase antibody, anti - Niemann - Pick C1 protein antibody, anti - hexosaminidase A or anti - tripeptidyl peptidase 1 antibody) in a human subject, and methods for improving the efficacy of anti - lysosomal enzyme antibody assays, which include pretreating a biological sample of the subject with a base having a pH of about 11 or higher and measuring the presence of anti - lysosomal enzyme antibodies in the biological sample of the subject by the anti - lysosomal enzyme neutralizing antibody assay described herein.

[0155] In some aspects, the anti - lysosomal antibody neutralizing antibody assay, as described in Example 2 below, determines the presence of anti - lysosomal enzyme neutralizing antibodies (such as anti - α - galactosidase A antibody, anti - glucocerebrosidase antibody, anti - α - glucosidase antibody, anti - α - L - iduronidase antibody, anti - iduronate - 2 - sulfatase antibody, anti - sulfamidase antibody, anti - galactosamine - 6 - sulfate sulfatase antibody, anti - N - acetylgalactosamine - 4 - sulfatase antibody, anti - β - glucuronidase antibody, anti - N - acetylglucosamine - 1 - phosphotransferase antibody, anti - Niemann - Pick C1 protein antibody, anti - hexosaminidase A or anti - tripeptidyl peptidase 1 antibody) by assessing the ability of human serum to neutralize lysosomal enzyme activity.

[0156] In some aspects, the results are determined by measuring the products generated from the cleavage of artificial substrates such as 4 - methylumbelliferyl - α - D - galactopyranoside, 4 - methylumbelliferyl - β - D - glucopyranoside, 4 - methylumbelliferyl α - D - glucopyranoside, 4 - methylumbelliferyl α - L - iduronide, 4 - methylumbelliferyl - α - L - iduronide 2 - sulfate, 4 - methylumbelliferyl - 2 - sulfamido - 2 - deoxy - α - D - glucopyranoside, 4 - methylumbelliferyl - β - D - galactose - 6 - sulfate, 4 - methylumbelliferyl - N - acetyl - α - d - galactosamine - 4 - sulfate, 4 - methylumbelliferyl - β - d - glucuronide, 4 - methylumbelliferyl - α - D - mannapyranoside, 4 - methylumbelliferyl N - acetyl - β - D - glucosaminide or Ala - Ala - Phe - 7 - amide - 4 - methylcoumarin.

[0157] In some aspects, the results are determined by measuring the 4 - methylumbelliferone (4 - MU) product generated from the cleavage of the artificial substrate.

[0158] Any anti - lysosomal enzyme neutralizing antibodies present in human serum will bind to the lysosomal enzyme and prevent the cleavage of the product from the substrate. The decrease in the relative fluorescence unit (RFU) signal is proportional to the amount of anti - lysosomal enzyme neutralizing antibodies (such as anti - α - galactosidase A antibody, anti - glucocerebrosidase antibody, anti - α - glucosidase antibody, anti - α - L - iduronidase antibody, anti - iduronate - 2 - sulfatase antibody, anti - sulfamidase antibody, anti - galactosamine - 6 - sulfate sulfatase antibody, anti - N - acetylgalactosamine - 4 - sulfatase antibody, anti - β - glucuronidase antibody, anti - N - acetylglucosamine - 1 - phosphotransferase antibody, anti - Niemann - Pick C1 protein antibody, anti - hexosaminidase A or anti - tripeptidyl peptidase 1 antibody) present in human serum.

[0159] In some aspects, a biological sample (e.g., a serum or plasma sample) is diluted to a minimum required dilution (MRD) of about 2-fold or higher. In some aspects, the biological sample is diluted to an MRD of about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 11-fold, about 12-fold, about 13-fold, about 14-fold, about 15-fold, about 16-fold, about 17-fold, about 18-fold, about 19-fold, about 20-fold, about 21-fold, about 22-fold, about 23-fold, about 24-fold, about 25-fold, about 26-fold, about 27-fold, about 28-fold, about 29-fold, about 30-fold, about 31-fold, about 32-fold, about 33-fold, about 34-fold, about 35-fold, about 36-fold, about 37-fold, about 38-fold, about 39-fold, about 40-fold, about 41-fold, about 42-fold, about 43-fold, about 44-fold, about 45-fold, about 46-fold, about 47-fold, about 48-fold, about 49-fold, about 50-fold, about 51-fold, about 52-fold, about 53-fold, about 54-fold, about 55-fold, about 56-fold, about 57-fold, about 58-fold, about 59-fold, about 60-fold, about 61-fold, about 62-fold, about 63-fold, about 64-fold, about 65-fold, about 66-fold, about 67-fold, about 68-fold, about 69-fold, about 70-fold, about 71-fold, about 72-fold, about 73-fold, about 74-fold, about 75-fold, about 76-fold, about 77-fold, about 78-fold, about 79-fold, about 80-fold, about 81-fold, about 82-fold, about 83-fold, about 84-fold, about 85-fold, about 86-fold, about 87-fold, about 88-fold, about 89-fold, about 90-fold, about 91-fold, about 92-fold, about 93-fold, about 94-fold, about 95-fold, about 96-fold, about 97-fold, about 98-fold, about 99-fold, about 100-fold, about 200-fold, about 300-fold, about 400-fold, about 500-fold, about 600-fold, about 700-fold, about 800-fold, about 900-fold, about 1000-fold, about 1100-fold, about 1200-fold, about 1300-fold, about 1400-fold, about 1500-fold, about 1600-fold, about 1700-fold, about 1800-fold, about 1900-fold, about 2000-fold, about 2100-fold, about 2200-fold, about 2300-fold, about 2400-fold, about 2500-fold, about 2600-fold, about 2700-fold, about 2800-fold, about 2900-fold, about 3000-fold, about 3100-fold, about 3200-fold, about 3300-fold, about 3400-fold, about 3500-fold, about 3600-fold, about 3700-fold, about 3800-fold, about 3900-fold, about 4000-fold, about 4100-fold, about 4200-fold, about 4300-fold, about 4400-fold, about 4500-fold, about 4600-fold, about 4700-fold, about 4800-fold, about 4900-fold or about 5000-fold.

[0160] In some aspects, the serum sample is diluted at MRD10 (diluted 1:5 in assay buffer (e.g., 0.1M citric acid, 0.2M sodium phosphate, and 0.05% Triton X-100, pH 4.6 ± 0.1), and then diluted 1:2 in 2X drug diluent (e.g., 40 ng / mL reconstituted GLA protein in assay buffer)).

[0161] In some aspects, the concentration of lysosomal enzymes (e.g., α-galactosidase A (α-Gal A), glucocerebrosidase, α-glucosidase, α-L-iduronidase, iduronate 2-sulfatase, sulfamidase, galactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, β-glucuronidase, N-acetylglucosamine-1-phosphotransferase, Niemann-Pick C1 protein, hexosaminidase A, or tripeptidyl peptidase 1) is less than about 10 ug / mL, less than about 9 ug / mL, less than about 8 ug / mL, less than about 7 ug / mL, less than about 6 ug / mL, less than about 5 ug / mL, less than about 4 ug / mL, less than about 3 ug / mL, less than about 2 ug / mL, less than about 1 ug / mL, less than about 0.9 ug / mL, less than about 0.8 ug / mL, less than about 0.7 ug / mL, less than about 0.6 ug / mL, less than about 0.5 ug / mL, less than about 0.4 ug / mL, less than about 0.3 ug / mL, less than about 0.2 ug / mL, less than about 100 ng / ml, less than about 90 ng / ml, less than about 80 ng / ml, less than about 70 ng / ml, less than about 60 ng / ml, less than about 50 ng / ml, less than about 40 ng / ml, less than about 30 ng / ml, less than about 20 ng / ml, or less than about 10 ng / ml.

[0162] In some aspects, the human lysosomal protein is diluted to 40 ng / mL in acidic sample buffer containing 0.1M citric acid, 0.2M sodium phosphate, and 0.05% Triton X-100 (pH 4.6).

[0163] In some aspects, the biological sample (e.g., serum or plasma sample) is pretreated with a base having a pH of about 11 or higher.

[0164] In some aspects, the base is a non-buffered base. In some aspects, the non-buffered base is NaOH or Ca(OH)2.

[0165] In some aspects, NaOH or Ca(OH)2 is about 0.01M, about 0.02M, about 0.03M, about 0.04M, about 0.05M, about 0.06M, about 0.07M, about 0.08M, about 0.09M, or about 0.1M. In some aspects, NaOH or Ca(OH)2 is about 0.02M.

[0166] In some aspects, the pH is about 11, about 11.1, about 11.15, about 11.2, about 11.25, about 11.3, about 11.35, about 11.4, about 11.45, about 11.5, about 11.55, about 11.6, about 11.65, about 11.7, about 11.75, about 11.8, about 11.85, about 11.9, about 11.95, about 12, 12.1, about 12.15, about 12.2, about 12.25, about 12.3, about 12.35, about 12.4, about 12.45, about 12.5, about 12.55, about 12.6, about 12.65, about 12.7, about 12.75, about 12.8, about 12.85, about 12.9, about 12.95, about 13, 13.1, about 13.15, about 13.2, about 13.25, about 13.3, about 13.35, about 13.4, about 13.45, about 13.5, about 13.55, about 13.6, about 13.65, about 13.7, about 13.75, about 13.8, about 13.85, about 13.9, about 13.95 or about 14.

[0167] In some aspects, the pH is greater than 11 and less than 12, greater than 11 and less than 13, or greater than 11 and less than 14.

[0168] In some aspects, a human serum sample is diluted to 20% serum in a pH 12.45 alkaline sample buffer and then mixed 1:1 with a diluted acidic enzyme buffer. In some aspects, the final sample incubation concentration contains 20 ng / mL lysosomal enzyme at 10% serum and the final pH is 4.9.

[0169] In some aspects, a pretreated biological sample is mixed with a lysosomal enzyme.

[0170] In some aspects, the pretreated biological sample and the lysosomal enzyme mixture are incubated for at least about one hour, at least about two hours, at least about three hours, at least about four hours, at least about five hours, at least about six hours, at least about seven hours, at least about eight hours, at least about nine hours, at least about ten hours, at least about eleven hours, at least about twelve hours, at least about thirteen hours, at least about fourteen hours, at least about fifteen hours, at least about sixteen hours, at least about seventeen hours, at least about eighteen hours, at least about nineteen hours, at least about twenty hours, at least about twenty - one hours, at least about twenty - two hours, at least about twenty - three hours or at least about twenty - four hours.

[0171] In some aspects, the pretreated biological sample and the lysosomal enzyme mixture are incubated for a duration of about 1 to about 15 hours, about 2 to about 14 hours, about 3 to about 13 hours, about 4 to about 12 hours, about 5 to about 11 hours, about 6 to about 10 hours, or about 7 to about 9 hours.

[0172] In some aspects, a pretreated biological sample and a lysosomal enzyme are mixed with a reaction mixture. In some aspects, the reaction mixture includes a substrate and / or an inhibitor.

[0173] In some aspects, the substrate is selected from the group consisting of: 4-methylumbelliferyl-α-D-galactopyranoside, 4-methylumbelliferyl-β-D-glucopyranoside, 4-methylumbelliferyl a-D-glucopyranoside, 4-methylumbelliferyl α-L-iduronide, 4-methylumbelliferyl-α-L-iduronide 2-sulfate, 4-methylumbelliferyl-2-sulfoamino-2-deoxy-α-D-glucopyranoside, 4-methylumbelliferyl-β-D-galactose-6-sulfate, 4-methylumbelliferyl-N-acetyl-α-d-galactosamine-4-sulfate, 4-methylumbelliferyl-β-d-glucuronide, 4-methylumbelliferyl-α-D-mannopyranoside, 4-methylumbelliferyl N-acetyl-β-D-glucosaminide, and Ala-Ala-Phe-7-amide-4-methylcoumarin.

[0174] In some aspects, the concentration of the substrate is at least about 1.1 mM, at least about 1.2 mM, at least about 1.3 mM, at least about 1.4 mM, at least about 1.5 mM, at least about 1.6 mM, at least about 1.7 mM, at least about 1.8 mM, at least about 1.9 mM, at least about 2 mM, at least about 2.1 mM, at least about 2.2 mM, at least about 2.3 mM, at least about 2.4 mM, at least about 2.5 mM, at least about 2.6 mM, at least about 2.7 mM, at least about 2.8 mM, at least about 2.9 mM, at least about 3 mM, at least about 3.1 mM, at least about 3.2 mM, at least about 3.3 mM, at least about 3.4 mM, at least about 3.5 mM, at least about 3.6 mM, at least about 3.7 mM, at least about 3.8 mM, at least about 3.9 mM, at least about 4 mM, at least about 4.1 mM, at least about 4.2 mM, at least about 4.3 mM, at least about 4.4 mM, at least about 4.5 mM, at least about 4.6 mM, at least about 4.7 mM, at least about 4.8 mM, at least about 4.9 mM, or at least about 5 mM. In some aspects, the concentration of the substrate is at least about 2.5 mM.

[0175] In some aspects, the inhibitor includes N-acetylgalactosamine (GALNAc).

[0176] In some aspects, the concentration of the inhibitor is less than about 200 mM, less than about 195 mM, less than about 190 mM, less than about 185 mM, less than about 180 mM, less than about 175 mM, less than about 170 mM, less than about 165 mM, less than about 160 mM, less than about 155 mM, less than about 150 mM, less than about 145 mM, less than about 140 mM, less than about 135 mM, less than about 130 mM, less than about 125 mM, less than about 120 mM, less than about 115 mM, or less than about 110 mM. In some aspects, the concentration of the inhibitor is at least about 125 mM.

[0177] In some aspects, the reaction mixture and the pretreated biological sample are combined and mixed with the lysosomal enzyme mixture in a high-throughput plate. In some aspects, the reaction mixture and the pretreated biological sample are incubated with the lysosomal enzyme mixture at 400 revolutions per minute (RPM) at room temperature.

[0178] In some aspects, the methods disclosed herein further include adding a stop buffer to the mixture after incubation. In some aspects, the incubation period is at least about 30 minutes, at least about 35 minutes, at least about 40 minutes, at least about 45 minutes, at least about 50 minutes, at least about 55 minutes, at least about 60 minutes, at least about 65 minutes, at least about 70 minutes, at least about 75 minutes, or at least about 80 minutes. In some aspects, the incubation period is at least about 60 minutes.

[0179] In some aspects, the stop buffer contains glycine. In some aspects, the volume of the stop buffer is less than about 1 mL, less than about 900 μL, less than about 800 μL, less than about 700 μL, less than about 600 μL, less than about 500 μL, less than about 400 μL, less than about 300 μL, less than about 200 μL, or less than about 100 μL. In some aspects, the volume of the stop buffer is about 100 μL.

[0180] In some aspects, a human biological sample with a percentage (%) inhibition equal to or greater than the cut-off point is identified as positive for anti-lysosomal enzyme neutralizing antibodies, while a human biological sample below the cut-off point is considered negative for anti-lysosomal enzyme neutralizing antibodies.

[0181] In some aspects, anti-lysosomal enzyme neutralizing antibody-negative subjects as described herein have a biological sample that inhibits the activity of a lysosomal enzyme (e.g., α-Gal A, glucocerebrosidase, α-glucosidase, α-L-iduronidase, iduronate 2-sulfatase, sulfamidase, galactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, β-glucuronidase, N-acetylglucosamine-1-phosphotransferase, Niemann-Pick C1 protein, hexosaminidase A, or tripeptidyl peptidase 1) by less than about 50%, as measured by the anti-lysosomal enzyme neutralizing antibody assay described herein. In some aspects, an anti-lysosomal enzyme neutralizing antibody-negative sample as described herein inhibits the activity of a lysosomal enzyme (e.g., α-Gal A, glucocerebrosidase, α-glucosidase, α-L-iduronidase, iduronate 2-sulfatase, sulfamidase, galactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, β-glucuronidase, N-acetylglucosamine-1-phosphotransferase, Niemann-Pick C1 protein, hexosaminidase A, or tripeptidyl peptidase 1) by less than about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50%.

[0182] In some aspects, anti-lysosomal enzyme neutralizing antibody-positive subjects as described herein have a biological sample that inhibits the activity of a lysosomal enzyme (e.g., α-Gal A, glucocerebrosidase, α-glucosidase, α-L-iduronidase, iduronate 2-sulfatase, sulfamidase, galactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, β-glucuronidase, N-acetylglucosamine-1-phosphotransferase, Niemann-Pick C1 protein, hexosaminidase A, or tripeptidyl peptidase 1) by more than less than about 10%, as measured by the anti-lysosomal enzyme neutralizing antibody assay described herein. In some aspects, an anti-lysosomal enzyme neutralizing antibody-positive sample as described herein has a signal inhibition of about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%.

[0183] IIB. Standardization of Anti-Lysosomal Enzyme Neutralizing Antibody (NAb) Assays

[0184] In some aspects, the present disclosure relates to a method for standardizing an anti-lysosomal enzyme neutralizing antibody assay, which includes determining a lysosomal drug diluent concentration by measuring the effect of a lysosomal enzyme (e.g., α-Gal A) drug level on the inhibition of a positive control antibody (e.g., an antibody designated as RP-01, 7H11, or 19D6), as described in Examples 2 and 3 below.

[0185] In some aspects, the positive control antibody includes, but is not limited to, the RP-01 antibody. In some aspects, RP-01 is a polyclonal antibody.

[0186] In some aspects, the positive control antibody includes, but is not limited to, the 7H11 antibody. In some aspects, 7H11 is a monoclonal antibody.

[0187] In some aspects, the positive control antibody includes, but is not limited to, the 19D6 antibody. In some aspects, 19D6 is a monoclonal antibody.

[0188] In some aspects, standardization includes determining a lysosomal enzyme drug diluent concentration by measuring the effect of a lysosomal enzyme (e.g., α-Gal A) drug level on the inhibition of the RP-01 antibody (anti-α-Gal antibody).

[0189] In some aspects, standardization includes determining a lysosomal enzyme drug diluent concentration by measuring the effect of a lysosomal enzyme (e.g., α-Gal A) drug level on the inhibition of the 7H11 antibody (anti-α-Gal antibody).

[0190] In some aspects, standardization includes determining a lysosomal enzyme drug diluent concentration by measuring the effect of a lysosomal enzyme (e.g., α-Gal A) drug level on the inhibition of the 19D6 antibody (anti-α-Gal antibody).

[0191] In some aspects, the lysosomal enzyme drug diluent concentration is less than about 500 ng / ml, less than about 400 ng / ml, less than about 300 ng / ml, less than about 200 ng / ml, less than about 150 ng / ml, less than about 145 ng / ml, less than about 140 ng / ml, less than about 135 ng / ml, less than about 130 ng / ml, less than about 125 ng / ml, less than about 120 ng / ml, less than about 110 ng / ml, less than about 105 ng / ml, less than about 100 ng / ml, less than about 90 ng / ml, less than about 80 ng / ml, less than about 70 ng / ml, less than about 60 ng / ml, less than about 50 ng / ml, less than about 40 ng / ml, less than about 30 ng / ml, less than about 20 ng / ml, or less than about 10 ng / ml. In some aspects, the lysosomal enzyme (e.g., α-Gal A) drug diluent concentration is less than about 125 ng / ml. In some aspects, the lysosomal enzyme (e.g., α-Gal A) drug diluent concentration is less than about 40 ng / ml. In some aspects, the lysosomal enzyme (e.g., α-Gal A) drug diluent concentration is less than about 20 ng / ml.

[0192] In some aspects, the inhibitory effect of the positive control antibody (RP-01, 7H11, or 19D6) is expressed as about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% inhibition of α-galactosidase A activity.

[0193] In some aspects, the minimum required dilution (MRD) of the GLA drug is about 1-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 11-fold, about 12-fold, about 13-fold, about 14-fold, about 15-fold, about 16-fold, about 17-fold, about 18-fold, about 19-fold, about 20-fold, about 21-fold, about 22-fold, about 23-fold, about 24-fold, about 25-fold, about 26-fold, about 27-fold, about 28-fold, about 29-fold, about 30-fold, about 31-fold, about 32-fold, about 33-fold, about 34-fold, about 35-fold, about 36-fold, about 37-fold, about 38-fold, about 39-fold, about 40-fold, about 41-fold, about 42-fold, about 43-fold, about 44-fold, about 45-fold, about 46-fold, about 47-fold, about 48-fold, about 49-fold, about 50-fold, about 51-fold, about 52-fold, about 53-fold, about 54-fold, about 55-fold, about 56-fold, about 57-fold, about 58-fold, about 59-fold, about 60-fold, about 61-fold, about 62-fold, about 63-fold, about 64-fold, about 65-fold, about 66-fold, about 67-fold, about 68-fold, about 69-fold, about 70-fold, about 71-fold, about 72-fold, about 73-fold, about 74-fold, about 75-fold, about 76-fold, about 77-fold, about 78-fold, about 79-fold, about 80-fold, about 81-fold, about 82-fold, about 83-fold, about 84-fold, about 85-fold, about 86-fold, about 87-fold, about 88-fold, about 89-fold, about 90-fold, about 91-fold, about 92-fold, about 93-fold, about 94-fold, about 95-fold, about 96-fold, about 97-fold, about 98-fold, about 99-fold, about 100-fold, about 200-fold, about 300-fold, about 400-fold, about 500-fold, about 600-fold, about 700-fold, about 800-fold, about 900-fold, about 1000-fold, about 1100-fold, about 1200-fold, about 1300-fold, about 1400-fold, about 1500-fold, about 1600-fold, about 1700-fold, about 1800-fold, about 1900-fold, about 2000-fold, about 2100-fold, about 2200-fold, about 2300-fold, about 2400-fold, about 2500-fold, about 2600-fold, about 2700-fold, about 2800-fold, about 2900-fold, about 3000-fold, about 3100-fold, about 3200-fold, about 3300-fold, about 3400-fold, about 3500-fold, about 3600-fold, about 3700-fold, about 3800-fold, about 3900-fold, about 4000-fold, about 4100-fold, about 4200-fold, about 4300-fold, about 4400-fold, about 4500-fold, about 4600-fold, about 4700-fold, about 4800-fold, about 4900-fold or about 5000-fold.

[0194] In some aspects, when using a predetermined antigen or BLI (Biolayer Interferometry) assay in a BIACORE 2000 instrument by, for example, immunoassay (e.g., ELISA) surface plasmon resonance (SPR) technology, the (K TM of the RP-01 antibody, 7H11 antibody or 19D6 antibodyD ) is less than 2.6×10 -10 M, less than 2.5×10 -10 M, less than 2.0×10 -10 M, less than 1.5×10 -10 M, less than 1.0×10 -10 M, less than 9×10 -11 M, less than 8×10 -11 M, less than 7×10 -11 M, less than 6×10 -11 M, less than 5×10 -11 M, less than 4×10 -11 M, less than 3×10 -11 M, less than 2×10 -11 M, less than 1×10 -11 M, less than 9×10 -12 M, less than 8×10 -12 M, less than 7×10 -12 M, less than 6×10 -12 M, less than 5×10 -12 M, less than 4×10 -12 M, less than 3×10 -12 M, less than 2×10 -12 M, less than 1×10 -12 M, less than 9×10 -13 M or less than 8×10 -13 M.

[0195] IIC. Determination of total anti - lysosomal enzyme antibody (TAb)

[0196] In some aspects, the present disclosure relates to methods for detecting anti - lysosomal enzyme antibodies (e.g., anti - α - galactosidase A antibody, anti - glucocerebrosidase antibody, anti - α - glucosidase antibody, anti - α - L - iduronidase antibody, anti - iduronate - 2 - sulfatase antibody, anti - sulfamidase antibody, anti - galactosamine - 6 - sulfatase antibody, anti - N - acetylgalactosamine - 4 - sulfatase antibody, anti - β - glucuronidase antibody, anti - N - acetylglucosamine - 1 - phosphotransferase antibody, anti - Niemann - Pick C1 protein antibody, anti - hexosaminidase A or anti - tripeptidyl peptidase 1 antibody) in a human subject (e.g., in the presence of circulating lysosomal enzymes), and methods for improving the efficacy of anti - lysosomal enzyme antibody assays (e.g., increasing assay sensitivity and circulating lysosomal enzyme tolerance), which include pretreating a biological sample of the subject with a base having a pH of about 11 or higher and measuring the presence of anti - lysosomal enzyme antibodies in the biological sample of the subject by the total anti - lysosomal enzyme antibody assay described herein (e.g., in Example 5 below).

[0197] In some aspects, the total anti-lysosomal antibody assay, as described in Example 5 below, determines anti-lysosomal enzyme neutralizing antibodies (such as anti-α-galactosidase A antibody, anti-glucocerebrosidase antibody, anti-α-glucosidase antibody, anti-α-L-iduronidase antibody, anti-iduronate 2-sulfatase antibody, anti-sulfamidase antibody, anti-galactosamine-6-sulfatase antibody, anti-N-acetylgalactosamine-4-sulfatase antibody, anti-β-glucuronidase antibody, anti-N-acetylglucosamine-1-phosphotransferase antibody, anti-Niemann-Pick C1 protein antibody, anti-hexosaminidase A or anti-tripeptidyl peptidase 1 antibody) by assessing the ability to detect serum anti-lysosomal antibodies in the presence of airborne lysosomal enzyme levels.

[0198] In some aspects, the methods disclosed herein further include pretreating the biological sample with acid neutralization. In some aspects, the acid is acetic acid or hydrochloric acid. In some aspects, the acetic acid or hydrochloric acid is about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 200 mM, about 300 mM, about 400 mM, about 500 mM or about 600 mM. In some aspects, the acetic acid is about 30 mM. In some aspects, the hydrochloric acid is about 120 mM.

[0199] In some aspects, the serum sample is pretreated with a base at pH 11 at room temperature for about 1 hour and then neutralized with 30 mM acetic acid.

[0200] In some aspects, the methods disclosed herein further include mixing the neutralized pretreated biological sample with lysosomal enzyme.

[0201] In some aspects, the neutralized pretreated biological sample and lysosomal enzyme mixture are added to a high-throughput plate coated with lysosomal enzyme antigen. In some aspects, the neutralized pretreated biological sample and lysosomal enzyme mixture are incubated with shaking at room temperature for about 1 hour to about 2 hours.

[0202] In some aspects, the lysosomal enzyme antigen is selected from the group consisting of: α-GalA antigen, glucocerebrosidase antigen, α-glucosidase antigen, α-L-iduronidase antigen, iduronate 2-sulfatase antigen, sulfamidase antigen, galactosamine-6-sulfatase antigen, N-acetylgalactosamine-4-sulfatase antigen, β-glucuronidase antigen, N-acetylglucosamine-1-phosphotransferase antigen, Niemann-Pick C1 protein antigen, hexosaminidase A antigen and tripeptidyl peptidase 1 antigen. In some aspects, the lysosomal enzyme antigen is α-GalA antigen.

[0203] In some aspects, a detection antibody or lysosomal enzyme with a ruthenium label is added to the plate to generate a signal. In some aspects, the detection antibody is an anti-human IgG antibody. In some aspects, ruthenated anti-human IgG is added to the plate and incubated with shaking at room temperature for one hour.

[0204] In some aspects, the plate is read in a plate reader that detects the light emission of the ruthenium label and outputs an electrochemiluminescence unit (ECLu). In some aspects, the ECLu represents the total level of anti-lysosomal total antibodies.

[0205] III. Kits

[0206] Also within the scope of the present disclosure are kits comprising the anti-lysosomal enzyme neutralizing antibody assay described herein, wherein the kits comprise:

[0207] (a) an assay buffer; (b) a substrate; (c) a GALNAc inhibitor; (d) a termination solution; and (e) an instruction manual containing instructions for using the kit.

[0208] Kits generally include a label that indicates the intended use and instructions for using the kit contents. The term "label" includes any written or recorded material provided on or with the kit, or additional material attached to the kit.

[0209] All references cited above and all references cited herein are incorporated herein by reference in their entirety.

[0210] The following examples are provided by way of illustration and are not limited thereto.

[0211] Examples

[0212] Example 1

[0213] Anti-lysosomal enzyme antibody assay

[0214] Current treatments for lysosomal storage diseases (LSDs) are significantly immunogenic. Therefore, it is important to measure anti-lysosomal enzyme antibodies in patients to understand whether and how new drugs will work for these patients. Figure 1 It has been shown that for lysosomal storage diseases (such as Fabry disease, Pompe disease, Gaucher disease, and mucopolysaccharidosis (MPS) type I disease, MPS type II disease, MPS type IV disease, and MPS type VI disease) treated with currently approved enzyme replacement therapy (ERT), the incidence of anti-ERT antibodies (including anti-ERT neutralizing antibodies) is high. As Figure 2 and Figure 11 The method shown allows the detection of anti-lysosomal neutralizing antibodies ( Figure 2 ) and total anti-lysosomal antibodies ( Figure 11) This is achieved through assay optimization, including a novel high-pH alkaline treatment that dissociates and specifically denatures lysosomal enzymes, allowing accurate detection of anti-lysosomal enzyme antibodies even in the presence of supra-physiological lysosomal enzyme levels in matrices such as serum and plasma. This alkaline assay allows measurement of anti-lysosomal enzyme antibodies in human subjects, for example, when receiving ERT or gene therapy, thereby allowing stratification of subjects and understanding the impact of antibody levels on the safety and efficacy of treatment for lysosomal enzyme disorders, including, for example, Fabry disease, Gaucher disease, Pompe disease, MPS I disease, MPS II disease, MPS VI disease, and Batten disease.

[0215] Example 2

[0216] Anti-lysosomal enzyme neutralizing antibody (NAb) detection

[0217] The anti-lysosomal enzyme neutralizing antibody (NAb) assay utilizes an artificial fluorescent substrate, such as 4-methylumbelliferyl α-D-galactopyranoside (4-MU-α-Gal), to measure lysosomal enzyme activity and the inhibition by neutralizing antibodies in human serum. Serum is treated with high-pH base to dissociate lysosomal enzyme and anti-lysosomal enzyme antibody complexes and further denature lysosomal enzymes to eliminate catalytic enzyme activity. The base-treated serum sample is transferred to a constant low level of lysosomal enzyme, which is incubated with human serum in an acidic assay buffer overnight. After incubation, the sample is mixed with the fluorescent substrate in plate form. In the presence of catalytically active lysosomal enzyme, the substrate is cleaved and releases fluorescent 4-methylumbelliferone (4-MU), which can be quantified at 365 nm excitation and 450 nm emission at alkaline pH.

[0218] The presence of neutralizing antibodies in serum limits substrate cleavage and attenuates the relative fluorescence unit (RFU) signal. Each sample is normalized to the fluorescence observed in the negative wells to determine the relative percent inhibition of the sample If the sample inhibition rate (percent inhibition of α-lysosomal enzyme activity) is greater than the calculated cut-off threshold, the sample is considered positive for anti-lysosomal neutralizing antibody.

[0219] Dilute human lysosomal protein (α-galactosidase A) to 40 ng / mL in an acidic sample buffer (simulating lysosome-like conditions) containing 0.1 M citric acid, 0.2 M sodium phosphate, and 0.05% Triton X-100 (pH 4.6). Dilute human serum samples to 20% serum in an alkaline sample buffer at pH 12.45 (0.02 M Ca(OH)2 buffer), and then mix 1:1 with the diluted acidic enzyme buffer. The final sample incubation concentration contains 20 ng / mL lysosomal enzyme at 10% serum, and the final pH is 4.9. Prepare positive controls using human serum containing known concentrations of anti-lysosomal enzyme neutralizing antibodies (e.g., RP-01, 7H11, or 19D6), and negative controls using human serum without anti-lysosomal enzyme antibodies. Add the sample mixtures in duplicate to non-binding 96-well plates, seal, and incubate overnight at 2 - 8°C.

[0220] After incubation, transfer the samples to separate 96-well plates and dilute five-fold with a reaction buffer containing a specific lysosomal enzyme fluorescent substrate and a non-specific enzyme inhibitor, namely 2.5 mM 4-MU-α-Gal and 0.125 M GALNAc, respectively. These components are made in an acidic buffer containing 0.1 M citric acid, 0.2 M sodium phosphate, and 0.05% Triton X-100 (pH 4.6). This reaction buffer continues to simulate lysosome-like conditions while also containing a specific lysosomal enzyme fluorescent substrate. The reaction is carried out at room temperature for one hour. Terminate the reaction by adding an alkaline glycine solution containing 0.25 M glycine (pH 10.7). Then analyze the plates using a spectrophotometer at an excitation wavelength of 365 nm and an emission wavelength of 450 nm. All samples are normalized and evaluated according to the negative control wells containing negative serum.

[0221] Figure 3A and Figure 3B Show that when there is 125 ng / mL lysosomal enzyme (circulating α-galactosidase A) in the serum, the anti-lysosomal enzyme NAb assay for women cannot accurately detect antibodies. The assay indicates that when the concentration of lysosomal enzyme (α-galactosidase A) is 500 ng / ml or higher, no anti-lysosomal enzyme NAbs can be detected at all, and as the enzyme concentration increases, the negative signal inhibition is significant ( Figure 3A ). Figure 3A and Figure 3B Depict that polyclonal lysosomal enzyme NAbs cannot be detected in the presence of airborne lysosomal enzymes in the serum.

[0222] Example 3

[0223] Sample Pretreatment for Detecting Anti-Lysosomal Enzyme Neutralizing Antibodies

[0224] Different treatments were evaluated to improve the ability to detect anti - lysosomal enzyme neutralizing antibodies in the presence of airborne lysosomal enzymes. Heat pretreatment was evaluated by heating serum to attempt dissociation and denaturation of airborne lysosomal enzymes. As Figure 4A shown, as the airborne lysosomal enzyme (α - galactosidase A) increased to 200 ng / ml, less NAb inhibition was detected by the standard assay. In Figure 4B , when no NAb was present, heat pretreatment at 56 °C was able to denature the airborne lysosomal enzyme (α - galactosidase A), but could not alleviate the interference at any NAb level (50 μg, 100 μg, or 150 μg).

[0225] Figure 5 Other pretreatment methods were shown, which were evaluated to improve the ability to detect anti - lysosomal enzyme NAbs in the presence of airborne lysosomal enzyme (α - galactosidase A). Acidic pH 2 pretreatment could not improve the tolerance of airborne lysosomal enzyme (α - galactosidase A) in detecting NAbs and performed worse than the standard assay diluent (AD) condition (pH 4.5 McIlvaine buffer: 0.1 M citric acid + 0.2 M disodium hydrogen phosphate + 0.05% TritonX). Melon Gel (Thermo Fisher catalog number 45206) treatment was evaluated as a method to purify IgG from serum samples and remove lysosomal enzymes. The Melon Gel method failed to successfully improve lysosomal enzyme tolerance and completely lost the ability to detect any NAbs even in the absence of airborne lysosomal enzymes. Alkaline pH 11 treatment showed an increase in tolerance to lysosomal enzymes in detecting NAbs and performed the best among all evaluated treatments in this experiment. pH 11 pretreatment showed an improvement over the standard assay diluent, but was not sufficient to detect all NAb inhibitions when the airborne lysosomal enzyme increased.

[0226] Example 4

[0227] Optimizing Alkaline Sample Pretreatment for the Detection of Anti - lysosomal Enzyme Neutralizing Antibodies

[0228] The tolerance of the anti - lysosomal enzyme neutralizing antibody detection to lysosomal enzyme (α - galactosidase A) was optimized by testing the enzyme neutralizing ability (percentage of inhibition of lysosomal enzyme activity) of positive control antibodies (RP - 01, 7H11, and 19D6) under various detection conditions.

[0229] The polyclonal RP-01 positive control antibody was generated as described in International Publication No. WO 2022 / 072706. The monoclonal 7H11 and 19D6 rabbit monoclonal antibodies were generated by Yurogen Biosystems according to the following protocol. First, rabbits were immunized with α-Gal A protein. Spleens were isolated and the resulting splenocytes were fused with partner cells to generate immortal cell lines expressing antibodies (hybridoma technology). Hybridomas that bound most effectively to the α-Gal A protein were screened out. Clones with the best binding affinity were amplified and purified. To screen for the best clones, plates were coated with α-Gal A, the purified antibodies were diluted and plated onto the plates (1 μg / mL serial dilutions in triplicate, 7 points in total), and a final detection was performed using HRP-conjugated goat anti-rabbit IgG (1:5K).

[0230] Figure 6 It is shown that pretreatment of alkaline samples can eliminate the interference of airborne lysosomal enzymes and significantly improve the detection rate of anti-lysosomal enzyme neutralizing antibodies. At the polyclonal positive control levels of RP-01 at 150 μg / mL, 100 μg / mL, and 50 μg / mL, NAb detection can tolerate up to 2000 ng / ml of airborne lysosomal enzyme (α-galactosidase A). Figure 7 It is shown that at the monoclonal positive control levels of 7H11 at 125 ng / mL, 250 ng / mL, and 1000 ng / mL, pretreatment of alkaline samples can increase the tolerance to airborne lysosomal enzyme (α-galactosidase A) to up to 2000 ng / ml. The alkaline sample pretreatment used was a low molarity pH 12.45 solution (0.02M Ca(OH)2 buffer) with limited buffering capacity. Figure 8 It is shown that a higher molarity buffered pH 12.45 solution (2M NaOH buffer) has a negative impact on the assay and no NAb inhibition can be detected.

[0231] Figure 9 It is shown that the standard anti-lysosomal neutralizing antibody assay (excluding the low molarity alkaline solution pretreatment step) cannot tolerate high levels of serum lysosomal enzyme (α-galactosidase A) for any of the three positive controls evaluated. As the airborne lysosomal enzyme increased above 125 ng / mL, monoclonal antibodies 19D6 and 7H11 showed a loss of NAb inhibition similar to that of the polyclonal antibody RP-01.

[0232] Figure 10 It is shown that the anti-lysosomal neutralizing antibody assay as described herein is tolerant to high levels of serum lysosomal enzyme (α-galactosidase A) and can accurately measure NAb inhibition from three different positive controls (RP-01, 7H11, and 19D6).

[0233] Example 5

[0234] Total anti - lysosomal enzyme antibody (TAb) assay

[0235] As Figure 11 shown, the total anti - lysosomal enzyme antibody assay was optimized for lysosomal enzyme tolerance by testing the binding ability of the 19D6 positive control antibody to plate - bound lysosomal enzyme (α - galactosidase A) in the form of an enzyme - linked immunosorbent assay (ELISA). The polystyrene ELISA plates were coated with 1X phosphate - buffered saline (PBS) containing 1 μg / mL lysosomal enzyme and incubated overnight at 2 - 8°C. Serum samples were pretreated with an alkaline solution at pH 11 (10 μL sample + 30 μL NaOH, 1 hour at room temperature), neutralized with an acidic solution (30 mM acetic acid), and further diluted to MRD40 with a neutral pH solution (assay buffer: 1% BSA, 0.35 M NaCl, 0.25% CHAPS, 5 mM EDTA, 0.05% Tween x - 100 or PBS containing <5% BSA). These serum samples were then added to the ELISA plates incubated overnight to allow the anti - lysosomal enzyme antibodies to bind to the lysosomal enzyme on the plate. After incubation and plate washing, a horseradish peroxidase (HRP) - conjugated anti - human IgG detection antibody was then added to the plate, which in the presence of TMB substrate was able to generate a colorimetric signal relative to the amount of anti - lysosomal enzyme antibody present in the serum.

[0236] Figure 12A and 12B show various unsuccessful acidic pretreatments that did not improve the ability to detect serum anti - lysosomal antibodies in the presence of airborne lysosomal enzyme (α - galactosidase A) levels. Whether heated or not, the acidic treatment did not show any specific signal in the anti - lysosomal enzyme TAb assay ( Figure 12A ). Acidic treatment with various alkaline neutralization methods also failed to successfully mitigate the interference of airborne lysosomal enzyme and showed high signals in serum samples without any specific airborne anti - lysosomal enzyme antibodies ( Figure 12B ).

[0237] Figure 13 show various unsuccessful alkaline pretreatments that did not improve the ability to detect serum anti - lysosomal antibodies in the presence of airborne lysosomal enzyme (α - galactosidase A) levels. Alkaline treatment at pH 12.45, whether heated or not, showed a high non - specific signal in the serum in the absence of any airborne anti - lysosomal enzyme. Alkaline treatment and heat treatment at pH 11 eliminated all signals in the assay and limited the ability to detect any anti - lysosomal enzyme antibodies.

[0238] Figure 14A and 14BShowing that pre-treatment of specific alkaline buffer samples (NaOH at pH 11) increased the detection rate of anti-lysosomal antibodies against 5 μg / mL of lysosomal enzyme (α-galactosidase A) on-board from the positive control antibody level of 39.0625 ng / mL to 2500 ng / ml. When lysosomal enzyme was on-board, the standard assay diluent at neutral pH showed a significant loss of optical density (OD) at 450 nm. This was significantly improved after serum pre-treatment with the pH 11 alkaline buffer, restoring the ability to detect anti-lysosomal enzyme antibody levels in the presence of serum lysosomal enzyme (5 μg / ml α-galactosidase A). Figure 14B )

[0239] Figure 15 Showing that in the presence of 4 μg / ml of lysosomal enzyme (α-galactosidase A) on-board, pH 11 alkaline pre-treatment increased the detection of anti-lysosomal enzyme antibodies in the sera of Fabry disease patients. When no lysosomal enzyme was on-board, pH 11 alkaline treatment did not significantly alter the sensitivity or detection rate of anti-lysosomal enzyme antibodies.

[0240] Figure 16A and Figure 16B Showing how alkaline base treatment (low molar concentration, (~0.0001M) NaOH, pH 11) improved the total antibody detection in the sera of Fabry disease patients, thus allowing the assessment of antibody levels in the presence of lysosomal enzyme (α-galactosidase A) on-board. For Fabry disease individual #1 and Fabry disease individual #4, alkaline treatment increased the optical density signal of samples containing lysosomal enzyme on-board to levels similar to those of untreated sera without lysosomal enzyme on-board. This indicates the successful use of alkaline treatment to increase the detection rate of anti-lysosomal enzyme antibodies in serum.

Claims

1. A method for detecting anti - lysosomal enzyme antibodies in a human subject, the method comprising measuring the presence of the anti - lysosomal enzyme antibodies in a biological sample of the subject, wherein the biological sample is pretreated with a base having a pH of about 11 or higher.

2. A method for enhancing the efficacy of an anti - lysosomal enzyme antibody assay, the method comprising pretreating a biological sample of a human subject with a base having a pH of about 11 or higher, and further comprising measuring the presence of the anti - lysosomal enzyme antibodies in the biological sample.

3. The method according to claim 1 or 2, wherein the base is a non - buffered base.

4. The method according to claim 3, wherein the non - buffered base is NaOH or Ca(OH)2.

5. The method according to claim 4, wherein the NaOH or Ca(OH)2 is about 0.01 M, about 0.02 M, about 0.03 M, about 0.04 M, about 0.05 M, about 0.06 M, about 0.07 M, about 0.08 M, about 0.09 M or about 0.1 M.

6. The method according to claim 5, wherein the NaOH or Ca(OH)2 is about 0.02 M.

7. The method according to any one of claims 1 to 6, wherein the biological sample is a serum sample or a plasma sample.

8. The method according to any one of claims 1 to 7, wherein the biological sample is diluted to a minimum required dilution (MRD) of about 2 - fold or higher.

9. The method according to claim 8, wherein the biological sample is diluted to about 1-fold, 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 11-fold, about 12-fold, about 13-fold, about 14-fold, about 15-fold, about 16-fold, about 17-fold, about 18-fold, about 19-fold, about 20-fold, about 21-fold, about 22-fold, about 23-fold, about 24-fold, about 25-fold, about 26-fold, about 27-fold, about 28-fold, about 29-fold, about 30-fold, about 31-fold, about 32-fold, about 33-fold, about 34-fold, about 35-fold, about 36-fold, about 37-fold, about 38-fold, about 39-fold, about 40-fold, about 41-fold, about 42-fold, about 43-fold, about 44-fold, about 45-fold, about 46-fold, about 47-fold, about 48-fold, about 49-fold, about 50-fold, about 51-fold, about 52-fold, about 53-fold, about 54-fold, about 55-fold, about 56-fold, about 57-fold, about 58-fold, about 59-fold, about 60-fold, about 61-fold, about 62-fold, about 63-fold, about 64-fold, about 65-fold, about 66-fold, about 67-fold, about 68-fold, about 69-fold, about 70-fold, about 71-fold, about 72-fold, about 73-fold, about 74-fold, about 75-fold, about 76-fold, about 77-fold, about 78-fold, about 79-fold, about 80-fold, about 81-fold, about 82-fold, about 83-fold, about 84-fold, about 85-fold, about 86-fold, about 87-fold, about 88-fold, about 89-fold, about 90-fold, about 91-fold, about 92-fold, about 93-fold, about 94-fold, about 95-fold, about 96-fold, about 97-fold, about 98-fold, about 99-fold, about 100-fold, about 200-fold, about 300-fold, about 400-fold, about 500-fold, about 600-fold, about 700-fold, about 800-fold, about 900-fold, about 1000-fold, about 1100-fold, about 1200-fold, about 1300-fold, about 1400-fold, about 1500-fold, about 1600-fold, about 1700-fold, about 1800-fold, about 1900-fold, about 2000-fold, about 2100-fold, about 2200-fold, about 2300-fold, about 2400-fold, about 2500-fold, about 2600-fold, about 2700-fold, about 2800-fold, about 2900-fold, about 3000-fold, about 3100-fold, about 3200-fold, about 3300-fold, about 3400-fold, about 3500-fold, about 3600-fold, about 3700-fold, about 3800-fold, about 3900-fold, about 4000-fold, about 4100-fold, about 4200-fold, about 4300-fold, about 4400-fold, about 4500-fold, about 4600-fold, about 4700-fold, about 4800-fold, about 4900-fold or about 5000-fold of MRD.

10. The method according to any one of claims 1 to 9, wherein the pH is about 11, about 11.1, about 11.15, about 11.2, about 11.25, about 11.3, about 11.35, about 11.4, about 11.45, about 11.5, about 11.55, about 11.6, about 11.65, about 11.7, about 11.75, about 11.8, about 11.85, about 11.9, about 11.95, about 12, 12.1, about 12.15, about 12.2, about 12.25, about 12.3, about 12.35, about 12.4, about 12.45, about 12.5, about 12.55, about 12.6, about 12.65, about 12.7, about 12.75, about 12.8, about 12.85, about 12.9, about 12.95, about 13, 13.1, about 13.15, about 13.2, about 13.25, about 13.3, about 13.35, about 13.4, about 13.45, about 13.5, about 13.55, about 13.6, about 13.65, about 13.7, about 13.75, about 13.8, about 13.85, about 13.9, about 13.95 or about 14.

11. The method according to claim 10, wherein the pH is 12.

45.

12. The method according to any one of claims 1 to 9, wherein the pH is greater than 11 and less than 12, greater than 11 and less than 13, or greater than 11 and less than 14.

13. The method according to any one of claims 1 to 12, wherein the subject has a lysosomal storage disease.

14. The method according to claim 13, wherein the lysosomal storage disease is selected from the group consisting of: Fabry disease, Gaucher disease, Pompe disease, mucopolysaccharidosis (MPS) type I disease, MPS II, MPS III, MPS IV, MPS VI, MPS VII, mucolipidosis (ML), Niemann-Pick disease, Tay-Sachs disease, and Batten disease.

15. The method according to any one of claims 1 to 14, wherein the presence of anti-lysosomal enzyme neutralizing antibodies is measured.

16. The method according to any one of claims 1 to 14, wherein the presence of anti-lysosomal enzyme total antibodies is measured.

17. The method according to claim 15, the method further comprising mixing a pretreated biological sample with a lysosomal enzyme.

18. The method according to claim 17, wherein the lysosomal enzyme is selected from the group consisting of: α-galactosidase A (α-GalA), glucocerebrosidase, α-glucosidase, α-L-iduronidase, iduronate 2-sulfatase, sulfamidase, galactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, β-glucuronidase, N-acetylglucosamine-1-phosphotransferase, Niemann-Pick C1 protein, hexosaminidase A, and tripeptidyl peptidase 1.

19. The method according to claim 18, wherein the lysosomal enzyme is α-galactosidase A.

20. The method according to any one of claims 17 to 19, wherein the neutralizing anti - lysosomal enzyme antibody is selected from the group consisting of: anti - α - galactosidase A antibody, anti - glucocerebrosidase antibody, anti - α - glucosidase antibody, anti - α - L - iduronidase antibody, anti - iduronate - 2 - sulfatase antibody, anti - sulfamidase antibody, anti - galactosamine - 6 - sulfatase antibody, anti - N - acetylgalactosamine - 4 - sulfatase antibody, anti - β - glucuronidase antibody, anti - N - acetylglucosamine - 1 - phosphotransferase antibody, anti - Niemann - Pick C1 protein antibody, anti - hexosaminidase A, and anti - tripeptidyl peptidase 1 antibody.

21. The method according to claim 20, wherein the neutralizing anti - lysosomal enzyme antibody is an anti - α - galactosidase A antibody.

22. The method according to any one of claims 17 to 21, wherein the pre - treated biological sample and the lysosomal enzyme mixture are incubated for at least about one hour, at least about two hours, at least about three hours, at least about four hours, at least about five hours, at least about six hours, at least about seven hours, at least about eight hours, at least about nine hours, at least about ten hours, at least about eleven hours, at least about twelve hours, at least about thirteen hours, at least about fourteen hours, at least about fifteen hours, at least about sixteen hours, at least about seventeen hours, at least about eighteen hours, at least about nineteen hours, at least about twenty hours, at least about twenty - one hours, at least about twenty - two hours, at least about twenty - three hours, or at least about twenty - four hours.

23. The method according to any one of claims 17 to 22, the method further comprising mixing the pre - treated biological sample and the lysosomal enzyme with a reaction mixture.

24. The method according to any one of claims 16 to 23, wherein the concentration of the lysosomal enzyme is less than about 10 μg / mL, less than about 9 μg / mL, less than about 8 μg / mL, less than about 7 μg / mL, less than about 6 μg / mL, less than about 5 μg / mL, less than about 4 μg / mL, less than about 3 μg / mL, less than about 2 μg / mL, less than about 1 μg / mL, less than about 0.9 μg / mL, less than about 0.8 μg / mL, less than about 0.7 μg / mL, less than about 0.6 μg / mL, less than about 0.5 μg / mL, less than about 0.4 μg / mL, less than about 0.3 μg / mL, less than about 0.2 μg / mL, less than about 100 ng / ml, less than about 90 ng / ml, less than about 80 ng / ml, less than about 70 ng / ml, less than about 60 ng / ml, less than about 50 ng / ml, less than about 40 ng / ml, less than about 30 ng / ml, less than about 20 ng / ml, or less than about 10 ng / ml.

25. The method according to any one of claims 17 to 24, wherein the pretreated biological sample and the lysosomal enzyme mixture are incubated for at least about one hour, at least about two hours, at least about three hours, at least about four hours, at least about five hours, at least about six hours, at least about seven hours, at least about eight hours, at least about nine hours, at least about ten hours, at least about eleven hours, at least about twelve hours, at least about thirteen hours, at least about fourteen hours, at least about fifteen hours, at least about sixteen hours, at least about seventeen hours, at least about eighteen hours, at least about nineteen hours, at least about twenty hours, at least about twenty-one hours, at least about twenty-two hours, at least about twenty-three hours or at least about twenty-four hours.

26. The method according to any one of claims 17 to 24, wherein the pretreated biological sample and the lysosomal enzyme mixture are incubated for a duration of about 1 to about 15 hours, about 2 to about 14 hours, about 3 to about 13 hours, about 4 to about 12 hours, about 5 to about 11 hours, about 6 to about 10 hours, or about 7 to about 9 hours.

27. The method according to claim 23, wherein the reaction mixture comprises a substrate and / or an inhibitor.

28. The method according to claim 27, wherein the substrate is selected from the group consisting of: 4-methylumbelliferyl-α-D-galactopyranoside, 4-methylumbelliferyl-β-D-glucopyranoside, 4-methylumbelliferyl α-D-glucopyranoside, 4-methylumbelliferyl α-L-iduronide, 4-methylumbelliferyl-α-L-iduronide 2-sulfate, 4-methylumbelliferyl-2-sulfoamino-2-deoxy-α-D-glucopyranoside, 4-methylumbelliferyl-β-D-galactose-6-sulfate, 4-methylumbelliferyl-N-acetyl-α-d-galactosamine-4-sulfate, 4-methylumbelliferyl-β-d-glucuronide, 4-methylumbelliferyl-α-D-mannopyranoside, 4-methylumbelliferyl N-acetyl-β-D-glucosaminide and Ala-Ala-Phe-7-amido-4-methylcoumarin.

29. The method according to claim 27 or 28, wherein the concentration of the substrate is at least about 1.1 mM, at least about 1.2 mM, at least about 1.3 mM, at least about 1.4 mM, at least about 1.5 mM, at least about 1.6 mM, at least about 1.7 mM, at least about 1.8 mM, at least about 1.9 mM, at least about 2 mM, at least about 2.1 mM, at least about 2.2 mM, at least about 2.3 mM, at least about 2.4 mM, at least about 2.5 mM, at least about 2.6 mM, at least about 2.7 mM, at least about 2.8 mM, at least about 2.9 mM, at least about 3 mM, at least about 3.1 mM, at least about 3.2 mM, at least about 3.3 mM, at least about 3.4 mM, at least about 3.5 mM, at least about 3.6 mM, at least about 3.7 mM, at least about 3.8 mM, at least about 3.9 mM, at least about 4 mM, at least about 4.1 mM, at least about 4.2 mM, at least about 4.3 mM, at least about 4.4 mM, at least about 4.5 mM, at least about 4.6 mM, at least about 4.7 mM, at least about 4.8 mM, at least about 4.9 mM or at least about 5 mM.

30. The method according to claim 27, wherein the inhibitor comprises N-acetylgalactosamine (GALNAc).

31. The method according to claim 27, wherein the concentration of the inhibitor is less than about 200 mM, less than about 195 mM, less than about 190 mM, less than about 185 mM, less than about 180 mM, less than about 175 mM, less than about 170 mM, less than about 165 mM, less than about 160 mM, less than about 155 mM, less than about 150 mM, less than about 145 mM, less than about 140 mM, less than about 135 mM, less than about 130 mM, less than about 125 mM, less than about 120 mM, less than about 115 mM or less than about 110 mM.

32. The method according to any one of claims 23 to 31, wherein the reaction mixture and the pretreated biological sample are combined with the lysosomal enzyme mixture in a high-throughput plate.

33. The method according to claim 32, wherein the reaction mixture and the pretreated biological sample are incubated with the lysosomal enzyme mixture at 300, 400, 500 or 600 revolutions per minute (RPM) at room temperature.

34. The method according to claim 33, further comprising adding a termination buffer to the mixture after incubation.

35. The method according to claim 33 or 34, wherein the incubation period is at least about 30 minutes, at least about 35 minutes, at least about 40 minutes, at least about 45 minutes, at least about 50 minutes, at least about 55 minutes, at least about 60 minutes, at least about 65 minutes, at least about 70 minutes, at least about 75 minutes or at least about 80 minutes.

36. The method according to claim 34 or 35, wherein the termination buffer contains glycine.

37. The method according to any one of claims 34 to 36, wherein the volume of the termination buffer is less than about 1 mL, less than about 900 μL, less than about 800 μL, less than about 700 μL, less than about 600 μL, less than about 500 μL, less than about 400 μL, less than about 300 μL, less than about 200 μL or less than about 100 μL.

38. The method according to claim 16, the method further comprising neutralizing the pretreated biological sample with an acid.

39. The method according to claim 38, wherein the acid is acetic acid or hydrochloric acid.

40. The method according to claim 39, wherein the acetic acid or hydrochloric acid is about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 200 mM, about 300 mM, about 400 mM, about 500 mM or about 600 mM.

41. The method according to claim 40, wherein the acetic acid is about 30 mM.

42. The method according to claim 38, the method further comprising mixing the neutralized pretreated biological sample with lysosomal enzymes.

43. The method according to claim 42, wherein the neutralized pretreated biological sample and the lysosomal enzyme mixture are added to a high-throughput plate coated with a lysosomal enzyme antigen.

44. The method according to claim 43, wherein the lysosomal enzyme antigen is selected from the group consisting of: α-Gal A antigen, glucocerebrosidase antigen, α-glucosidase antigen, α-L-iduronidase antigen, iduronate 2-sulfatase antigen, sulfamidase antigen, galactosamine-6-sulfatase antigen, N-acetylgalactosamine-4-sulfatase antigen, β-glucuronidase antigen, N-acetylglucosamine-1-phosphotransferase antigen, Niemann-Pick C1 protein antigen, hexosaminidase A antigen and tripeptidyl peptidase 1 antigen.

45. The method according to claim 44, wherein the lysosomal enzyme antigen is α-Gal A antigen.

46. The method according to any one of claims 43 to 45, wherein an enzyme-conjugated detection antibody or an enzyme-conjugated lysosomal enzyme is added to the plate to generate a signal.

47. The method according to any one of claims 43 to 45, wherein a detection antibody or a lysosomal enzyme with a ruthenium label is added to the plate to generate a signal.

48. The method according to claim 46 or 47, wherein the detection antibody is an anti-human IgG antibody.

49. The method according to claim 46 or 48, the method further comprising adding a substrate.

50. The method according to claim 49, wherein the substrate is converted by the enzyme on the detection antibody to produce a chromogenic reaction product.

51. The method according to claim 50, wherein the plate is read in a plate reader, and the plate reader detects the chromogenic reaction product and outputs an optical density (OD) value.

52. The method according to claim 51, wherein the OD value represents the total level of the anti-lysosomal neutralizing antibody.

53. The method according to claim 47, wherein the plate is read in a plate reader that detects the light emission of the ruthenium label and outputs an electrochemiluminescence unit (ECLu).

54. The method according to claim 53, wherein the ECLu represents the total level of the anti - lysosomal total antibody.

55. The method according to any one of claims 1 to 54, wherein the presence of the anti - lysosomal enzyme antibody is measured in the presence of circulating lysosomal enzymes in the biological sample.

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