Assays for lysosomal storage diseases

Abstract

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 effectiveness of anti-lysosomal enzyme antibody assays (e.g., improving assay sensitivity and circulating lysosomal enzyme resistance), the methods comprising pretreating a biological sample from the subject with a base at a pH of about 11 or higher and measuring the presence of anti-lysosomal enzyme antibodies in the biological sample from the subject.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of the filing date of U.S. Provisional Application No. 63 / 379,597, filed October 14, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Technical Field 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 effectiveness of anti-lysosomal enzyme antibody assays (e.g., improving assay sensitivity and circulating lysosomal enzyme resistance), the methods comprising pretreating a biological sample from the subject with a base at a pH of about 11 or higher and measuring the presence of anti-lysosomal enzyme antibodies in the biological sample from the subject. [Background technology]

[0003] Current treatments for lysosomal storage disorders (LSDs) are highly immunogenic, and therefore it is important to measure anti-lysosomal enzyme antibodies in patients to be able to understand how and whether new drugs will work in these patients. In the case of lysosomal storage disorders with currently approved enzyme replacement therapies (ERTs), there is a significant incidence of anti-ERT antibodies, including anti-ERT neutralizing antibodies.

[0004] Current methods for accurately measuring anti-lysosomal enzyme antibodies may require blood sampling at trough drug levels to ensure there are no residual lysosomal enzymes in the blood, as residual enzymes can interfere with circulating antibodies by forming complexes that limit detection. In the case of treatments such as gene therapy, treatment and administration cannot be discontinued, and therefore anti-lysosomal enzyme antibodies cannot be accurately measured.

[0005] Therefore, there is a need for improved methods to accurately measure anti-lysosomal enzyme antibodies, thereby improving the customization of treatment for patients diagnosed with lysosomal storage disorders. Summary of the Invention

[0006] In some aspects, the present disclosure relates to a method for detecting anti-lysosomal enzyme antibodies in a human subject, comprising measuring the presence of anti-lysosomal enzyme antibodies in a biological sample from the subject, wherein the biological sample is pretreated with a base at a pH of about 11 or greater.

[0007] In some aspects, the methods of the present disclosure comprise detecting anti-lysosomal enzyme antibodies in the presence of circulating lysosomal enzymes.

[0008] In some aspects, the present disclosure relates to a method for improving the efficacy of an anti-lysosomal enzyme antibody assay, the method comprising pretreating a biological sample from a human subject with a base at a pH of about 11 or greater, and further comprising measuring the presence of the anti-lysosomal enzyme antibody in the biological sample. In some aspects, improving the efficacy of the anti-lysosomal enzyme antibody assay of the present disclosure comprises improving assay sensitivity and circulating lysosomal enzyme resistance.

[0009] In some embodiments, the base is a non-buffering base. In some embodiments, the non-buffering base is NaOH or Ca(OH). In some embodiments, the NaOH or Ca(OH) 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. In some embodiments, the NaOH or Ca(OH) is about 0.02 M.

[0010] In some embodiments, the biological sample is a serum sample or a plasma sample.

[0011] In some embodiments, the biological sample is diluted at a minimum required dilution (MRD) of about 2-fold or greater.

[0012] In some embodiments, the biological sample is about 1x, 2x, about 3x, about 4x, about 5x, about 6x, about 7x, about 8x, about 9x, about 10x, about 11x, about 12x, about 13x, about 14x, about 15x, about 16x, about 17x, about 18x, about 19x, about 20x, about 21x, about 22x, 23x, about 24x, about 25x, about 26x, about 27x, about 28x, about 29x, about 30x, about 31x, about 32x, about 33x, about 34x, about 35x, about 36x, about 37x, about 38x, about 39x, about 40x, about 41x, about 42x, about 43x, about 44x, about 45x, about 46x, about 47x, about 48x, about 49x, about 50x, about 51x, about 52x, about 53x, about 54x, about 55x, about 56x, about 57x, about 58x, about 59x, about 60x, about 61x, about 62x, about 63x, about 64x, about 65x, about 66x, about 67x, about 68x, about 69x, about 70x, about 71x, about 72x, about 73x, about 74x, about 75x, about 76x, about 77x, about 78x, about 79x, about 80x, about 81x, about 82x, about 83x, about 84x, about 85x, about 86 times, approximately 43 times, approximately 44 times, approximately 45 times, approximately 46 times, approximately 47 times, 48 ​​times, approximately 49 times, approximately 50 times, approximately 51 times, approximately 52 times, approximately 53 times, approximately 54 times, approximately 55 times, approximately 56 times, approximately 57 times, approximately 58 times, approximately 59 times, approximately 60 times, approximately 61 times, approximately 62 times, approximately 63 times, approximately 64x, 65x, 66x, 67x, 68x, 69x, 70x, 71x, 72x, 73x, 74x, 75x, 76x, 77x, 78x, 79x, 80x, 81x, 82x, 83x, 84x, 85x 86x, 87x, 88x, 89x, 90x, 91x, 92x, 93x, 94x, 95x, 96x, 97x, 98x, 99x, 100x, 200x, 300x, 400x, 500x, 600x , about 700 times, about 800 times, about 900 times, about 1000 times, about 1100 times, about 1200 times, about 1300 times, about 1400 times, about 1500 times, about 1600 times, about 1700 times, about 1800 times, about 1900 times, about 2000 times, about 2100 times, about 220 The antibody is diluted to an MRD of 0x, about 2300x, about 2400x, about 2500x, about 2600x, about 2700x, about 2800x, about 2900x, about 3000x, about 3100x, about 3200x, about 3300x, about 3400x, about 3500x, about 3600x, about 3700x, about 3800x, about 3900x, about 4000x, about 4100x, about 4200x, about 4300x, about 4400x, about 4500x, about 4600x, about 4700x, about 4800x, about 4900x, or about 5000x.

[0013] In some embodiments, 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 2.5, 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.

[0014] In some embodiments, the pH is 12.45.

[0015] In some embodiments, 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.

[0016] In some embodiments, the subject is suffering from a lysosomal storage disease, hi some embodiments, the lysosomal storage disease is selected from the group consisting of Fabry disease, Gaucher disease, Pompe disease, mucopolysaccharidosis (MPS) I disease, MPS II, MPS III, MPS IV, MPS VI, MPS VII, mucolipidosis (ML), Niemann-Pick disease, Tay-Sachs disease, and Batten disease.

[0017] In some embodiments, the presence of anti-lysosomal enzyme neutralizing antibodies (NAb) is measured. In some embodiments, the presence of anti-lysosomal enzyme total antibodies (TAb) is measured.

[0018] In some embodiments, the methods of the present disclosure further include mixing the pretreated biological sample with a lysosomal enzyme. In some embodiments, the lysosomal enzyme is selected from the group consisting of α-galactosidase A (α-Gal A), glucocerebrosidase, alpha-glucosidase, alpha-L-iduronidase, iduronate 2-sulfatase, sulfamidase, galactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, beta-glucuronidase, N-acetylglucosamine-1-phosphotransferase, Niemann-Pick C1 protein, hexosaminidase A, and tripeptidyl peptidase 1. In some embodiments, the lysosomal enzyme is α-galactosidase A.

[0019] In some embodiments, 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-beta-glucuronidase antibody, anti-N-acetylglucosamine-1-phosphotransferase antibody, anti-Niemann-Pick C1 protein antibody, anti-hexosaminidase A, and anti-tripeptidyl peptidase 1. In some embodiments, the neutralizing anti-lysosomal enzyme antibody is an anti-α-galactosidase A antibody.

[0020] In some embodiments, the pretreated biological sample and lysosomal enzyme mixture are incubated for at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, or at least about 24 hours.

[0021] In some aspects, the methods of the present disclosure further comprise combining the pre-treated biological sample and the lysosomal enzyme with a reaction mixture.

[0022] In some embodiments, the lysosomal enzyme is at a concentration of 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.

[0023] In some embodiments, the pretreated biological sample and lysosomal enzyme mixture are incubated for at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, or at least about 24 hours.

[0024] In some embodiments, the pretreated biological sample and lysosomal enzyme mixture are incubated for a period 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.

[0025] In some embodiments, the reaction mixture includes a substrate and / or an inhibitor. In some embodiments, the substrate is 4-methylumbelliferyl-α-D-galactopyranoside, 4-methylumbelliferyl-β-D-glucopyranoside, 4-methylumbelliferyl α-D-glucopyranoside, 4-methylumbelliferyl α-L-idopyranoside, 4-methylumbelliferyl-α-L-iduronide 2-sulfate, 4-methylumbelliferyl-2-sulfamino-2-deoxy-α-D-glucopyranoside, 4-methylumbelliferyl α-L-iduronide 2-sulfate, 4-methylumbelliferyl ... 4-methylumbelliferyl-β-D-galactose-6-sulfate, 4-methylumbelliferyl-N-acetyl-α-d-galactoseamidin-4-sulfate, 4-methylumbelliferyl-β-d-glucuronide, 4-methylumbelliferyl-α-D-mannopyrronoside, 4-methylumbelliferyl N-acetyl-β-D-glucosamide, and Ala-Ala-Phe-7-amido-4-methyl-coumarin.

[0026] In some embodiments, 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 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.

[0027] In some embodiments, the inhibitor comprises N-acetylgalactosamine (GALNAc).

[0028] In some embodiments, the inhibitor is at a concentration of 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.

[0029] In some embodiments, the reaction mixture and the biological sample pretreated with the lysosomal enzyme mixture are combined in a high-throughput plate.

[0030] In some embodiments, the reaction mixture and the biological sample pretreated with the lysosomal enzyme mixture are incubated at room temperature at 300, 400, 500, or 600 revolutions per minute (RPM).

[0031] In some embodiments, the disclosed methods further comprise adding a stop buffer to the mixture after incubation, hi some embodiments, 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.

[0032] In some embodiments, the stop buffer comprises glycine, hi some embodiments, the stop buffer is in a volume of less than about 1 mL, less than about 900 uL, less than about 800 uL, less than about 700 uL, less than about 600 uL, less than about 500 uL, less than about 400 uL, less than about 300 uL, less than about 200 uL, or less than about 100 uL.

[0033] In some embodiments, the methods of the present disclosure further comprise neutralizing the pretreated biological sample with an acid. In some embodiments, the acid is acetic acid or hydrochloric acid. In some embodiments, 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 embodiments, the acetic acid is about 30 mM.

[0034] In some aspects, the methods of the present disclosure further comprise mixing the neutralized pre-treated biological sample with a lysosomal enzyme.

[0035] In some embodiments, the neutralized pretreated biological sample and lysosomal enzyme mixture are added to a high-throughput plate coated with a lysosomal enzyme antigen. In some embodiments, 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, beta-glucuronidase antigen, N-acetylglucosamine-1-phosphotransferase antigen, Niemann-Pick C1 protein antigen, hexosaminidase A antigen, and tripeptidyl peptidase 1 antigen. In some embodiments, the lysosomal enzyme antigen is α-Gal A antigen.

[0036] In some embodiments, an enzyme-conjugated detection antibody or an enzyme-conjugated lysosomal enzyme is added to the plate to generate a signal.

[0037] In some embodiments, a detection antibody or lysosomal enzyme with a ruthenylated label is added to the plate to generate a signal.

[0038] In some embodiments, the detection antibody is an anti-human IgG antibody.

[0039] In some embodiments, the method of the present disclosure further comprises adding a substrate. In some embodiments, the substrate is converted by an enzyme on the detection antibody to produce a colored reaction product. In some embodiments, the plate is read with a plate reader that detects the colored reaction product and outputs an optical density (OD) value. In some embodiments, the OD value represents the total level of anti-lysosomal neutralizing antibodies.

[0040] In some embodiments, the plate is read on a plate reader to detect luminescence from the ruthenylated label and output electrochemiluminescence units (ECLu), which in some embodiments represent the total level of anti-lysosomal total antibodies. [Brief explanation of the drawings]

[0041] [Figure 1] 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 mucopolysaccharide sulfation syndrome (MPS) type I disease, MPS type II disease, MPS type IV disease, and MPS type VI disease). Antibody incidence values ​​were obtained from approved drug labels. [Figure 2] Lysosomal enzyme neutralizing (NAb) antibody assay format is shown. [Figure 3] Figures 3A-3B show the effect of circulating levels of lysosomal enzymes (on-board lysosomal enzyme concentrations (ng / ml) [0-2000 ng / ml (Figure 3A)] and [0 ng / ml-500 ng / ml (Figure 3B)]) on the enzyme neutralization ability (% inhibition of lysosomal enzyme activity (signal inhibition)) of polyclonal RP-01 antibody (a neutralizing anti-lysosomal enzyme antibody) at different concentrations (50 ug / ml, 100 ug / ml, and 150 ug / ml). [Figure 4] Figures 4A-4B show the effect of heat pretreatment of biological samples on the enzyme neutralization ability (% inhibition of lysosomal enzyme activity (signal inhibition)) of RP-01 antibody at different concentrations (0 μg, 50 μg, 100 μg, and 150 μg). Figure 4A shows the results without sample pretreatment, and Figure 4B shows the results after pretreatment of serum samples at 56°C. [Figure 5] Shown are various pretreatment methods, pH 11, Melon Gel, pH 2, and standard assay diluent (AD) conditions, that were evaluated to test the ability to detect anti-lysosomal enzyme neutralizing antibody NAb (RP-01) in the presence of on-board lysosomal enzymes (concentration (ng / ml)). [Figure 6] This shows the effect of alkaline (base) pretreatment of biological samples on the ability to detect anti-lysosomal enzyme neutralizing antibody NAb (RP-01) at different concentrations (50 μg / ml, 100 μg / ml, and 150 μg / ml) in the presence of on-board lysosomal enzymes (concentration (ng / ml)). [Figure 7]This shows the effect of alkaline (base) pretreatment of biological samples on the ability to detect anti-lysosomal enzyme neutralizing antibody NAb (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 on-board lysosomal enzymes (concentration (ng / ml)). [Figure 8] This shows the effect of high molar base pretreatment of biological samples on the ability to detect anti-lysosomal enzyme neutralizing antibody NAb (monoclonal positive control anti-lysosomal enzyme NAb (7H11)) at different concentrations (50 μg / ml, 100 μg / ml, and 150 μg / ml) in the presence of on-board lysosomal enzymes (concentration (ng / ml)). [Figure 9] Shown is the ability to detect anti-lysosomal enzyme neutralizing antibody NAbs at different concentrations (50 μg / ml, 100 μg / ml, and 150 μg / ml) in the presence of on-board lysosomal enzymes (concentration (ng / ml)) as measured by a standard anti-lysosomal neutralizing antibody assay: a polyclonal positive control anti-lysosomal enzyme NAb (RP-01), and monoclonal positive control anti-lysosomal enzyme NAbs (7H11 and 19D6) at different concentrations (125 ng / ml, 250 ng / ml, and 1000 ng / ml). [Figure 10] This shows the effect of alkaline (base) pretreatment of biological samples on the ability to detect anti-lysosomal enzyme neutralizing antibody NAbs: polyclonal positive control anti-lysosomal enzyme NAb, (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 μg / ml, 250 μg / ml, and 1000 μg / ml) in the presence of on-board lysosomal enzymes (concentrations (ng / ml)). [Figure 11] Lysosomal enzyme total antibody (TAb) assay format is shown. [Figure 12]Figures 12A-12B show the effect of various acidic pretreatments (with or without heat pretreatment (Figure 12A) or with various base neutralization treatments (Figure 12B)) on the ability to detect serum anti-lysosomal antibodies (as measured by optical density (OD) at 450 nm) in the presence of on-board lysosomal enzymes. Samples tested were 1 μg / ml 19D6 Ab + 2 μg / ml α-Gal A; 1 μg / ml 19D6 Ab; and 2 μg / ml α-Gal A. [Figure 13] The effect of various alkaline pretreatments (pH 12, pH 11 + heat, and pH 12 + heat) on the ability to detect serum anti-lysosomal antibodies in the presence of on-board lysosomal enzymes (as measured by optical density (OD) at 450 nm) is shown. Samples tested were 1 μg / ml 19D6 Ab + 2 μg / ml α-Gal A; 1 μg / ml 19D6 Ab; and 2 μg / ml α-Gal A. [Figure 14] Figures 14A-14B show the effect of neutral and basic pH pretreatment on the ability to detect serum anti-lysosomal antibodies (measured by optical density (OD) at 450 nm) in the absence (Figure 14A) or presence (Figure 14B) of on-board lysosomal enzymes. [Figure 15] Shown are measurements of total anti-lysosomal enzyme antibodies (as measured by optical density (OD) at 450 nm) in serum samples from an individual without Fabry disease (negative serum); a serum sample from individual No. 3 with Fabry disease in the presence of 4 μg / ml of on-board lysosomal enzymes (Fabry serum #3 + 4 μg / ml α-Gal A); a serum sample from individual No. 3 with Fabry disease (Fabry serum #3); and a serum sample from individual No. 2 with Fabry disease (Fabry serum #2), either untreated or after alkaline pretreatment. [Figure 16]Figures 16A-16B show measurements of total anti-lysosomal enzyme antibodies (as measured by optical density (OD) at 450 nm) in serum samples from individual #1 with Fabry disease in the absence or presence of on-board lysosomal enzymes (Fabry serum #1 and Fabry serum #1 + α-Gal A, respectively) (Figure 16A), and individual #4 with Fabry disease in the absence and presence of on-board lysosomal enzymes (Fabry serum #4 and Fabry serum #4 + α-Gal A, respectively) (Figure 16B), after untreated or alkaline pretreatment. DETAILED DESCRIPTION OF THE INVENTION

[0042] 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 effectiveness of anti-lysosomal enzyme antibody assays (e.g., improving assay sensitivity and circulating lysosomal enzyme resistance), the methods comprising pretreating a biological sample from the subject with a base at a pH of about 11 or higher and measuring the presence of anti-lysosomal enzyme antibodies in the biological sample from the subject.

[0043] I. Terminology In order that this disclosure may be more readily understood, certain terms are first defined. As used in this application, unless expressly defined otherwise herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout this application.

[0044] Where an embodiment is described herein using the term "comprising," it is understood that similar embodiments described using the terms "consisting of" and / or "consisting essentially of" are also provided.

[0045] 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. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press provide those skilled in the art with a general dictionary of many of the terms used in this disclosure.

[0046] Units, prefixes, and symbols are shown in the format accepted by the International System of Units (SI). Numerical ranges are inclusive of the numbers defining the range. Unless otherwise specified, nucleotide sequences are written left to right in a 5' to 3' orientation. Amino acid sequences are written left to right in an amino to carboxy orientation. The headings provided herein are not intended to limit the various aspects of the disclosure, which may be had by reference to the specification in its entirety. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0047] A "polypeptide" refers to a chain comprising at least two consecutively linked amino acid residues, with no upper limit to the length of the chain. One or more amino acid residues of the protein may contain modifications, including, but not limited to, glycosylation, phosphorylation, or disulfide bond formation. A "protein" may include one or more polypeptides.

[0048] The term "lysosomal enzyme" refers to proteins with enzymatic activity, such as glycosidases, proteases, and sulfatases. Mammalian lysosomal enzymes are synthesized in the cytosol and traverse the endoplasmic reticulum (ER), where they are glycosylated with N-linked high-mannose carbohydrates. In the Golgi apparatus, high-mannose carbohydrates are modified on lysosomal proteins by the addition of mannose-6-phosphate (M6P), which targets these proteins to lysosomes. M6P-modified proteins are delivered to lysosomes via interaction with one of two M6P receptors. In some embodiments, lysosomal enzymes include, but are not limited to, α-galactosidase A (α-Gal A), glucocerebrosidase, alpha-glucosidase, alpha-L-iduronidase, iduronate 2-sulfatase, sulfamidase, galactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, beta-glucuronidase, N-acetylglucosamine-1-phosphotransferase, Niemann-Pick C1 protein, hexosaminidase A, and tripeptidyl peptidase 1.

[0049] The terms "α-galactosidase A," "α-Gal A," and "GAL" are used interchangeably and refer to proteins having an enzymatic activity that involves hydrolyzing the terminal, non-reducing α-D-galactose residues of α-D-galactosides, including galactose oligosaccharides, galactomannans, and galactolipids. In some embodiments, α-Gal A includes enzymes described in IUBMB Enzyme Nomenclature EC 3.2.1.22 (e.g., Suzuki et al., J. Biol. Chem. 245:781-786 (1970); Wiederschain, G. and Beyer, E., Dokl. Akad. Nauk SSSR 231:486-488 (1976)). In some embodiments, α-Gal A includes proteins encoded by nucleic acids comprising the human GLA gene, e.g., the human α-Gal A gene defined by GenBank Accession No. NM_000169. In some embodiments, α-Gal A comprises a protein comprising the amino acid sequence defined by GenBank Accession No. NP_000160. In some embodiments, α-Gal A is agalsidase alfa produced by genetic engineering in a human cell line. Agalsidase alfa is available as Replagal® from Shire Plc (Dublin, Ireland). In some embodiments, α-Gal A is agalsidase beta produced by recombinant DNA technology in a Chinese hamster ovary (CHO) cell line. Agalsidase beta is available as Fabrazyme® from Sanofi Genzyme (Cambridge, Mass.). In some embodiments, 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)) (identified as JR-051).

[0050] The terms "glucocerebrosidase," "GCase," "β-glucocerebrosidase," "β-glucosidase," "β-glucocerebrosidase," "β-glucosidase," "D-glucosyl-N-acylfingosin glucohydrolase," "glucosylceramidase," and "glucosylceramidase beta 1 (GBA1)" are used interchangeably and refer to enzymes with glucosylceramidase activity required for hydrolytic cleavage of the beta-glycosidic bond of glucocerebroside, a chemical intermediate in glycolipid metabolism that is abundant in cell membranes (especially skin cells). Glucocerebrosidase is localized in lysosomes and remains bound to the lysosomal membrane. (Rijnboutt et al., J. Biol. Chem. 266(8):4862-8 (1991)). Glucocerebrosidase is 497 amino acids long and has a molecular weight of 59,700 daltons. In some embodiments, the glucocerebrosidase comprises an enzyme described by 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 embodiments, the glucocerebrosidase comprises a protein encoded by a nucleic acid comprising a human glucocerebrosidase gene, e.g., the human glucocerebrosidase gene defined by GenBank Accession No. NM_000157, NM_001005741, or NM_001005742. In some embodiments, the glucocerebrosidase comprises a protein comprising the amino acid sequence defined by GenBank Accession No. NP_000148, NP_001005741, NP_001005742, NP_001165282, or NP_001165283.

[0051] The terms "alpha-glucosidase," "α-glucosidase," "acid α-glucosidase," "GAA," "lysosomal α-glucosidase," "maltase," "glucoinvertase," "glucosidosucrase," "maltase-glucoamylase," "α-glucopyranosidase," "glucoside invertase," "α-D-glucosidase," "α-glucoside hydrolase," "α-1,4-glucosidase," and "α-D-glucoside glucohydrolase" are used interchangeably and refer to enzymes essential for the breakdown of glycogen to glucose in lysosomes. Alpha-glucosidase has the highest activity toward α-1,4-linked glycosidic bonds, but can also hydrolyze α-1,6-linked glucans. In some embodiments, the alpha-glucosidase comprises an enzyme described by 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 embodiments, the alpha-glucosidase comprises a protein encoded by a nucleic acid comprising a human alpha-glucosidase gene, e.g., a human alpha-glucosidase gene defined by GenBank Accession No. NM_000152, NM_001079803, or NM_001079804. In some embodiments, the alpha-glucosidase comprises a protein comprising an amino acid sequence defined by GenBank Accession No. NP_000143, NP_001073271, or NP_001073272.

[0052] The terms "alpha-L-iduronidase," "L-iduronidase," "laronidase," "α-L-iduronidase," and "glycosaminoglycan α-L-iduronohydrolase" are used interchangeably and refer to enzymes involved in the degradation of glycosaminoglycans such as dermatan sulfate and heparan sulfate. The enzyme acts by hydrolyzing the terminal α-L-iduronic acid residues of these molecules, degrading them. Alpha-L-iduronidase is a glycoprotein enzyme found in lysosomes of cells and is reported to have a mass of approximately 83 kilodaltons. In some embodiments, alpha-L-iduronidase includes an enzyme described by IUBMB Enzyme Nomenclature EC3.2.1.76 (e.g., as described in Rome et al., Arch Biochem Biophys 189:344-53 (1978); Scott et al. Proc Natl Acad Sci US A. 88(21):9695-9 (1991)). In some embodiments, alpha-L-iduronidase includes a protein encoded by a nucleic acid comprising a human alpha-L-iduronidase gene, e.g., the human alpha-L-iduronidase gene defined by GenBank Accession No. NM_000203 or NM_001363576. In some embodiments, alpha-L-iduronidase includes a protein comprising the amino acid sequence defined by GenBank Accession No. NP_000194 or NP_001350505.

[0053] The terms "iduronate 2-sulfatase," "IDS," "chondroitin sulfatase," "iduronide-2-sulfate sulfatase," "L-idurone sulfatase," "L-idurone sulfate sulfatase," "iduronate sulfatase," "sulfo-L-iduronate sulfatase," "L-iduronate 2-sulfate sulfatase," "sulfoiduronate sulfohydrolase," "2-sulfo-L-iduronate 2-sulfatase," "iduronate-2-sulfate sulfatase," "iduronate sulfate sulfatase," and "L-iduronate-2-sulfate 2-sulfohydrolase" are used interchangeably and refer to sulfatase enzymes that catalyze the hydrolysis of the 2-sulfate group of the L-iduronate 2-sulfate unit of dermatan sulfate, heparan sulfate, and heparin. In some embodiments, the iduronate 2-sulfatase comprises an enzyme described by 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 embodiments, the iduronate 2-sulfatase comprises 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 No. NM_000202, NM_001166550, or NM_006123. In some embodiments, the iduronate 2-sulfatase comprises a protein comprising the amino acid sequence defined by GenBank Accession No. NP_000193, NP_001160022, or NP_006114.

[0054] The terms "sulfamidase," "sulfamidase," "N-sulfoglucosamine sulfohydrolase," "SGSH," "N-sulfo-D-glucosamine sulfohydrolase," "sulfoglucosamine sulfamidase," "heparin sulfamidase," "2-desoxy-D-glucoside-2-sulfamate sulfohydrolase," "sulfamate," and "sulfohydrolase" are used interchangeably and refer to enzymes involved in the lysosomal degradation of heparan sulfate. This enzyme belongs to the family of hydrolases, specifically those that act on sulfur-nitrogen bonds. Some embodiments include enzymes described by IUBMB Enzyme Nomenclature EC3.10.1.1 (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 embodiments, the sulfamidase comprises a protein encoded by a nucleic acid comprising a human sulfamidase gene, e.g., a human sulfamidase gene defined in GenBank Accession No. NM_000199, NM_001352921, or NM_001352922. In some embodiments, the sulfamidase comprises a protein comprising an amino acid sequence defined by GenBank Accession No. NP_000190, NP_001339850, or NP_001339851.

[0055] "Galactosamine-6-sulfatase", "N-acetylgalactosamine-6-sulfatase", "GALNS", "Galactosamine (N-acetyl)-6-sulfatase", "Chondroitin sulfatase", "Chondroitinase", "Galactose-6-sulfate sulfatase", "Acetylgalactosamine 6-sulfatase", The terms "galactosamine-6-sulfate sulfatase" and "N-acetylgalactosamine-6-sulfate sulfatase" are used interchangeably and refer to a lysosomal exohydrolase required for the degradation of the glycosaminoglycans keratan sulfate and chondroitin 6-sulfate. In some embodiments, galactosamine-6-sulfatase includes an enzyme described by IUBMB Enzyme Nomenclature EC 3.1.6.4 (e.g., as described in [End Page 110] et al., (1991)). In some embodiments, galactosamine-6-sulfatase includes a protein encoded by a nucleic acid comprising a human galactosamine-6-sulfatase gene, e.g., the human galactosamine-6-sulfatase gene defined by GenBank Accession Nos. NM_000512, NM_001323543, or NM_001323544. In some embodiments, the galactosamine-6-sulfatase comprises a protein comprising an amino acid sequence defined by GenBank Accession No. NP_000503, NP_001310472, or NP_001310473.

[0056] The terms "N-acetylgalactosamine-4-sulfatase," "N-acetylgalactosamine-4-sulfatase," "chondroitin sulfatase," "chondroitinase," "arylsulfatase B," "acetylgalactosamine 4-sulfatase," "N-acetylgalactosamine 4-sulfate sulfohydrolase," and "N-acetyl-D-galactosamine-4-sulfate 4-sulfohydrolase" are used interchangeably and refer to arylsulfatases that catalyze the hydrolysis of the 4-sulfate group of the N-acetyl-D-galactosamine 4-sulfate unit of chondroitin sulfate and dermatan sulfate. In some embodiments, e includes enzymes described by IUBMB Enzyme Nomenclature EC 3.1.6.12 (e.g., as described in Farooqui et al., Experientia 32:1242-1244 (1976)). In some embodiments, the N-acetylgalactosamine-4 sulfatase comprises a protein encoded by a nucleic acid comprising a human N-acetylgalactosamine-4 sulfatase gene, e.g., the human N-acetylgalactosamine-4 sulfatase gene defined by GenBank Accession No. NM_00046. In some embodiments, the N-acetylgalactosamine-4 sulfatase comprises a protein comprising the amino acid sequence defined by GenBank Accession No. NC_000005 or NM_198709.

[0057] The terms "beta-glucuronidase," "β-glucuronidase," "β-glucuronide glucuronohydrolase glucuronidase," "β-D-glucuronoside glucuronosohydrolase," "exo-β-D-glucuronidase," and "ketidase" are used interchangeably and refer to enzymes that catalyze the hydrolysis of β-D-glucuronic acid residues from the non-reducing ends of mucopolysaccharides (also called glycosaminoglycans), such as heparan sulfate. In some embodiments, beta-glucuronidase includes enzymes described by 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 embodiments, the beta-glucuronidase comprises a protein encoded by a nucleic acid comprising a beta-glucuronidase gene, e.g., a human beta-glucuronidase gene defined by GenBank Accession No. NM_000181, NM_001284290, NM_001293104, or NM_001293105. In some embodiments, the beta-glucuronidase comprises a protein comprising an amino acid sequence defined by GenBank Accession No. NP_000172, NP_001271219, or NP_001280034.

[0058] The terms "N-acetylglucosamine-1-phosphotransferase" and "N-acetylglucosamine-1-phosphate transferase" are used interchangeably and refer to an enzyme that catalyzes the first step in the synthesis of the mannose-6-phosphate lysosomal recognition marker. In some embodiments, N-acetylglucosamine-1-phosphotransferase includes enzymes described by IUBMB Enzyme Nomenclature EC 2.7.8.17 (e.g., as described in Nishikawa, A. Lysosomal Enzyme GrcNAc-1-Phosphotransferase. In Handbook of Glycosyltransferases. Springer, Tokyo (2002)). In some embodiments, the N-acetylglucosamine-1-phosphotransferase comprises 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 No. NM_032520, NM_024312, XM_011538731, or XM_006719593. In some embodiments, the N-acetylglucosamine-1-phosphotransferase comprises a protein comprising an amino acid sequence defined by GenBank Accession No. NP_115909, NP_077288, XP_011537033, or XP_006719656.

[0059] The terms "Niemann-Pick C1 protein," "NPC intracellular cholesterol transporter 1," and "NPC1" are used interchangeably to refer to an enzyme present in the limiting membranes of endosomes and lysosomes that mediates intracellular cholesterol transport via binding of cholesterol to its N-terminal domain (Xiaochun et al., Proc Natl Acad Sci. 113(29):8212-7(2016)). In some embodiments, the Niemann-Pick C1 protein comprises a protein encoded by a nucleic acid comprising a human Niemann-Pick C1 protein gene, e.g., a human Niemann-Pick C1 protein gene defined by GenBank Accession Nos. NM_000271, XM_005258279, XM_005258277, XM_017025787, XM_006722479, XM_047437539, XM_017025786, XM_017025785, XM_017025784, or XM_005258278. In some embodiments, a Niemann-Pick C1 protein includes 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.

[0060] The terms "hexosaminidase A," "beta-acetylaminodeoxyhexosidase," "N-acetyl-beta-D-hexosaminidase," "N-acetyl-beta-hexosaminidase," "N-acetylhexosaminidase," "beta-hexosaminidase," "beta-acetylhexosaminidase," "beta-DN-acetylhexosaminidase," "beta-N-acetyl-D-hexosaminidase," "beta-N-acetylglucosaminidase," "hexosaminidase A," "N-acetylhexosaminidase," "beta-D-hexosaminidase," "β-N-acetylhexosaminidase," and "HEXA" are used interchangeably and refer to the enzyme responsible for the hydrolysis of the terminal N-acetyl-D-hexosamine residue in N-acetyl-β-D-hexosamide. In some embodiments, hexosaminidase A includes an enzyme described by IUBMB Enzyme Nomenclature EC 3.2.1.52 (e.g., as described in Tse et al., Biochemistry. 35(23):7599-607 (1996)). In some embodiments, hexosaminidase A includes a protein encoded by a nucleic acid comprising a human hexosaminidase A gene, e.g., the human hexosaminidase A gene defined by GenBank Accession No. NM_000520 or NM_001318825. In some embodiments, hexosaminidase A includes a protein comprising the amino acid sequence defined by GenBank Accession No. NP_000511 or NP_001305754.

[0061] The terms "tripeptidyl peptidase 1," "TPP1," and "lysosomal pepstatin-insensitive protease" are used interchangeably and refer to lysosomal serine proteases. In some embodiments, tripeptidyl peptidase 1 includes an enzyme described by IUBMB Enzyme Nomenclature EC 3.4.14.9 (e.g., as described in Esaki et al., J. Neurochem. 72:2573-2582 (1999)). In some embodiments, tripeptidyl peptidase 1 includes a protein encoded by a nucleic acid comprising a human tripeptidyl peptidase 1 gene, e.g., the human tripeptidyl peptidase 1 gene defined by GenBank Accession No. NM_000391. In some embodiments, tripeptidyl peptidase 1 includes a protein comprising the amino acid sequence defined by GenBank Accession No. NP_000382.

[0062] In some embodiments, the lysosomal enzyme (e.g., α-Gal A, glucocerebrosidase, alpha-glucosidase, alpha-L-iduronidase, iduronate 2-sulfatase, sulfamidase, galactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, beta-glucuronidase, N-acetylglucosamine-1-phosphotransferase, Niemann-Pick C1 protein, hexosaminidase A, or tripeptidyl peptidase 1) is a lysosomal enzyme, or a lysosomal enzyme (e.g., α-Gal A, glucocerebrosidase, alpha-glucosidase, alpha-L-iduronidase, iduronate 2-sulfatase, sulfamidase, galactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, beta-glucuronidase, N-acetylglucosamine-1-phosphotransferase, Niemann-Pick C1 protein, hexosaminidase A, or tripeptidyl peptidase 1). A, glucocerebrosidase, alpha-glucosidase, alpha-L-iduronidase, iduronate 2-sulfatase, sulfamidase, galactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, beta-glucuronidase, N-acetylglucosamine-1-phosphotransferase, Niemann-Pick C1 protein, hexosaminidase A, or tripeptidyl peptidase 1), or as described herein, may be obtained from cells endogenously expressing recombinant human lysosomal enzymes (e.g., α-Gal A, glucocerebrosidase, alpha-glucosidase, alpha-L-iduronidase, iduronate 2-sulfatase, sulfamidase, galactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, beta-glucuronidase, N-acetylglucosamine-1-phosphotransferase, Niemann-Pick C1 protein, hexosaminidase A, or tripeptidyl peptidase 1).In some embodiments, the recombinant human lysosomal enzyme (e.g., α-Gal A, glucocerebrosidase, alpha-glucosidase, alpha-L-iduronidase, iduronate 2-sulfatase, sulfamidase, galactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, beta-glucuronidase, N-acetylglucosamine-1-phosphotransferase, Niemann-Pick C1 protein, hexosaminidase A, or tripeptidyl peptidase 1) is a full-length wild-type ...). A, glucocerebrosidase, alpha-glucosidase, alpha-L-iduronidase, iduronate 2-sulfatase, sulfamidase, galactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, beta-glucuronidase, N-acetylglucosamine-1-phosphotransferase, Niemann-Pick C1 protein, hexosaminidase A, or tripeptidyl peptidase 1).In some embodiments, the recombinant human lysosomal enzyme (e.g., α-Gal A, glucocerebrosidase, alpha-glucosidase, alpha-L-iduronidase, iduronate 2-sulfatase, sulfamidase, galactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, beta-glucuronidase, N-acetylglucosamine-1-phosphotransferase, Niemann-Pick C1 protein, hexosaminidase A, or tripeptidyl peptidase 1) is a wild-type lysosomal enzyme (e.g., α-Gal A, glucocerebrosidase, alpha-glucosidase, alpha-L-iduronidase, iduronate 2-sulfatase, sulfamidase, galactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, beta-glucuronidase, N-acetylglucosamine-1-phosphotransferase, Niemann-Pick C1 protein, hexosaminidase A, or tripeptidyl peptidase 1). A, glucocerebrosidase, alpha-glucosidase, alpha-L-iduronidase, iduronate 2-sulfatase, sulfamidase, galactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, beta-glucuronidase, N-acetylglucosamine-1-phosphotransferase, Niemann-Pick C1 protein, hexosaminidase A, or tripeptidase 1), wherein the subset comprises a subset of amino acid residues present in a wild-type lysosomal enzyme (e.g., α-Gal A, glucocerebrosidase, alpha-glucosidase, alpha-L-iduronidase, iduronate 2-sulfatase, sulfamidase, galactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, beta-glucuronidase, N-acetylglucosamine-1-phosphotransferase, Niemann-Pick C1 protein, hexosaminidase A, or tripeptidase 1), which form the active site for substrate binding and / or substrate reduction. A, glucocerebrosidase, alpha-glucosidase, alpha-L-iduronidase, iduronate 2-sulfatase, sulfamidase, galactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, beta-glucuronidase, N-acetylglucosamine-1-phosphotransferase, Niemann-Pick C1 protein, hexosaminidase A, or tripeptidyl peptidase 1).

[0063] In some embodiments, recombinant human lysosomal enzymes (e.g., α-Gal A, glucocerebrosidase, alpha-glucosidase, alpha-L-iduronidase, iduronate 2-sulfatase, sulfamidase, galactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, beta-glucuronidase, N-acetylglucosamine-1-phosphotransferase, Niemann-Pick C1 protein, hexosaminidase A, or tripeptidyl peptidase 1) are used in place of wild-type lysosomal enzymes (e.g., α-Gal A, glucocerebrosidase, alpha-glucosidase, alpha-L-iduronidase, iduronate 2-sulfatase, sulfamidase, galactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, beta-glucuronidase, N-acetylglucosamine-1-phosphotransferase, Niemann-Pick C1 protein, hexosaminidase A, or tripeptidyl peptidase 1). A, glucocerebrosidase, alpha-glucosidase, alpha-L-iduronidase, iduronate 2-sulfatase, sulfamidase, galactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, beta-glucuronidase, N-acetylglucosamine-1-phosphotransferase, Niemann-Pick C1 protein, hexosaminidase A, or tripeptidyl peptidase 1) active site (for substrate binding and / or substrate reduction), as well as those that may be present in wild-type recombinant human lysosomal enzymes. Other amino acid residues that may or may not be present (e.g., α-GalA, glucocerebrosidase, alpha-glucosidase, alpha-L-iduronidase, iduronate 2-sulfatase, sulfamidase, galactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, beta-glucuronidase, N-acetylglucosamine-1-phosphotransferase, Niemann-Pick C1 protein, hexosaminidase A, or tripeptidyl peptidase 1) recombinant human lysosomal enzymes (e.g., α-Gal A fusion protein comprising: glucocerebrosidase, alpha-glucosidase, alpha-L-iduronidase, iduronate 2-sulfatase, sulfamidase, galactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, beta-glucuronidase, N-acetylglucosamine-1-phosphotransferase, Niemann-Pick C1 protein, hexosaminidase A, or peptidyl peptidase 1).

[0064] Lysosomal enzymes (e.g., α-Gal A, glucocerebrosidase, alpha-glucosidase, alpha-L-iduronidase, iduronate 2-sulfatase, sulfamidase, galactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, beta-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 well known to those skilled in the art. The wild-type enzyme can be purified from recombinant cell expression systems (e.g., mammalian cells such as CHO cells, or insect cells; see, e.g., U.S. Pat. Nos. 5,580,757, 6,395,884, 6,458,574, 6,461,609, 6,210,666, 6,083,725), human placenta, or animal milk.

[0065] Other synthetic techniques for obtaining lysosomal enzymes suitable for pharmaceutical use (e.g., α-Gal A, glucocerebrosidase, alpha-glucosidase, alpha-L-iduronidase, iduronate 2-sulfatase, sulfamidase, galactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, beta-glucuronidase, N-acetylglucosamine-1-phosphotransferase, Niemann-Pick C1 protein, hexosaminidase A, or tripeptidyl peptidase 1) are described, for example, in U.S. Pat. No. 7,560,060. ,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.

[0066] In addition to proteins containing amino acid sequences identical to the human lysosomal enzymes described herein (e.g., α-Gal A, glucocerebrosidase, alpha-glucosidase, alpha-L-iduronidase, iduronate 2-sulfatase, sulfamidase, galactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, beta-glucuronidase, N-acetylglucosamine-1-phosphotransferase, Niemann-Pick C1 protein, hexosaminidase A, or tripeptidyl peptidase 1), the present disclosure also provides ... which are "substantially similar" thereto (e.g., α-Gal A, A, glucocerebrosidase, alpha-glucosidase, alpha-L-iduronidase, iduronate 2-sulfatase, sulfamidase, galactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, beta-glucuronidase, N-acetylglucosamine-1-phosphotransferase, Niemann-Pick C1 protein, hexosaminidase A, or peptidyl 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, yet differ from the native amino acid sequence at one or more amino acid positions (i.e., by amino acid substitution).

[0067] Proteins that are altered from the native sequence can be prepared by substituting amino acid residues in the native protein and selecting proteins with the desired activity. For example, amino acid residues in a lysosomal enzyme, such as α-Gal A protein, can be systematically substituted with other residues, and the substituted proteins tested in standard assays to assess the effect of such substitutions on the protein's ability to hydrolyze terminal non-reducing α-D-galactose residues in α-D-galactosides, including galactose oligosaccharides, galactomannans, and galactolipids, and / or to treat or prevent Fabry disease.

[0068] In some embodiments, conservative amino acid substitutions are made 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 with similar side chains have been defined in the art. These families include amino acids with 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), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In some embodiments, a predicted non-essential amino acid residue in the α-Gal A protein is replaced with another amino acid residue from the same side chain family. Methods for identifying conservative nucleotide and amino acid substitutions that do not eliminate 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)).

[0069] In some embodiments, the lysosomal enzymes of the present disclosure (e.g., α-Gal A, glucocerebrosidase, alpha-glucosidase, alpha-L-iduronidase, iduronate 2-sulfatase, sulfamidase, galactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, beta-glucuronidase, N-acetylglucosamine-1-phosphotransferase, Niemann-Pick C1 protein, hexosaminidase A, or tripeptidyl peptidase 1) can be synthesized using any of the lysosomal enzymes described herein or known in the art (e.g., α-Gal A, A, glucocerebrosidase, alpha-glucosidase, alpha-L-iduronidase, iduronate 2-sulfatase, sulfamidase, galactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, beta-glucuronidase, N-acetylglucosamine-1-phosphotransferase, Niemann-Pick C1 protein, hexosaminidase The amino acid sequence of the tripeptidyl peptidase A or tripeptidyl peptidase 1) 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 tripeptidyl peptidase A or tripeptidyl peptidase 1).

[0070] The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that needs to be introduced for optimal alignment of the two sequences (i.e., % homology = number of identical positions / total number of positions x 100). The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.

[0071] The percent identity between two nucleotide sequences may be determined using the GAP program in the GCG software package (available at worldwideweb.gcg.com) using the NWSgapdna.CMP matrix and gap weights of 40, 50, 60, 70, or 80, and length weights of 1, 2, 3, 4, 5, or 6. The percent identity between two nucleotide or amino acid sequences may also be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Furthermore, percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) incorporated into the GAP program of the GCG software package (available at http: / / www.gcg.com), using either a Blossum62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0072] The nucleic acid and protein sequences described herein may further be used as "query sequences" to search public databases, for example, to identify related sequences. Such searches may 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 may be performed using the NBLAST program, score = 100, word length = 12, to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches may be performed using the XBLAST program, score = 50, word length = 3, to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST may be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing 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.

[0073] "Antibody" (Ab) includes, but is not limited to, a glycoprotein immunoglobulin comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds that specifically binds to an antigen, or an antigen-binding portion thereof. Each H chain comprises a heavy chain variable region (referred to herein as V H The heavy chain constant region comprises three constant domains, C H1 , C H2 , and C H3 Each light chain comprises a light chain variable region (referred to herein as V L The light chain constant region comprises one constant domain, C L Includes V H and V LThe regions can be further subdivided into regions of hypervariability, termed complementarity-determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). H and V L contains three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, such as various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The term "anti-lysosomal enzyme antibody" includes, for example, a complete antibody having two heavy chains and two light chains that specifically bind to a lysosomal enzyme (such as α-Gal A, glucocerebrosidase, alpha-glucosidase, alpha-L-iduronidase, iduronate 2-sulfatase, sulfamidase, galactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, beta-glucuronidase, N-acetylglucosamine-1-phosphotransferase, Niemann-Pick C1 protein, hexosaminidase A, or tripeptidyl peptidase 1), and an antigen-binding portion of the complete antibody.

[0074] Immunoglobulins can be derived from any commonly known isotype, 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 (e.g., IgM or IgG1) encoded by heavy chain constant region genes. The term "antibody" includes, by way of example, both 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 may be humanized by recombinant methods to reduce immunogenicity in humans. Unless explicitly stated and unless the context dictates otherwise, the term "antibody" also includes antigen-binding fragments or portions of any of the foregoing immunoglobulins, including monovalent and bivalent fragments or portions, and single-chain antibodies.

[0075] An "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds α-Gal A is substantially free of antibodies that specifically bind antigens other than α-Gal A). However, an isolated antibody that specifically binds α-Gal A may exhibit cross-reactivity to other antigens, such as α-Gal A molecules from different species. Furthermore, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0076] The term "monoclonal antibody" (mAb) refers to a non-naturally occurring preparation of antibody molecules of single molecular composition, i.e., antibody molecules which are essentially identical in primary sequence and which display a single binding specificity and affinity for a particular epitope. Monoclonal antibodies are examples of isolated antibodies. Monoclonal antibodies may be produced by hybridoma, recombinant, transgenic, or other techniques known to those skilled in the art.

[0077] The term "polyclonal antibody" (pAb) refers to a mixture of heterogeneous antibodies, usually produced by different B-cell clones in the body. They can recognize and bind to many different epitopes of a single antigen. In some embodiments, the RP-01 antibody described herein is a polyclonal antibody.

[0078] A "human antibody" (HuMAb) refers to an antibody having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. The human antibodies of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. The terms "human antibody" and "fully human antibody" are used interchangeably.

[0079] A "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 from a human immunoglobulin. In one embodiment 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 a human immunoglobulin, 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 permissible as long as they do not interfere with the antibody's ability to bind to a specific antigen. A "humanized antibody" retains the same antigen specificity as the original antibody.

[0080] By "chimeric antibody" is meant an antibody whose variable region is derived from one species and whose constant region is derived from another species, such as an antibody whose variable region is derived from a mouse antibody and whose constant region is derived from a human antibody.

[0081] "Anti-antigen antibody" refers to an antibody that specifically binds to an antigen. For example, an anti-GAL antibody specifically binds to GAL; an anti-glucocerebrosidase antibody specifically binds to glucocerebrosidase; an anti-alpha-glucosidase antibody specifically binds to alpha-glucosidase; an anti-alpha-L-iduronidase antibody specifically binds to alpha-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-6-sulfatase antibody specifically binds to N-acetylgalactosamine-6-sulfatase; The anti-galactosamine-4 sulfatase antibody specifically binds to N-acetylgalactosamine-4 sulfatase, the anti-beta-glucuronidase antibody specifically binds to beta-glucuronidase, the anti-N-acetylglucosamine-1-phosphotransferase antibody specifically binds to N-acetylglucosamine-1-phosphotransferase, the anti-Niemann-Pick C1 protein antibody specifically binds to Niemann-Pick C1 protein, the anti-hexosaminidase A antibody specifically binds to hexosaminidase A, and the anti-tripeptidyl peptidase 1 antibody specifically binds to peptidyl peptidase 1.

[0082] 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, alpha-glucosidase, alpha-L-iduronidase, iduronate 2-sulfatase, sulfamidase, galactosamine-6-sulfatase, N-acetylgalactosamine-4 sulfatase, beta-glucuronidase, N-acetylglucosamine-1-phosphotransferase, Niemann-Pick C1 protein, hexosaminidase A, or peptidyl peptidase 1). For example, in the presence of anti-lysosomal enzyme neutralizing antibodies (e.g., anti-α-galactosidase A, anti-glucocerebrosidase, anti-α-glucosidase, anti-α-L-iduronidase, anti-iduronate 2-sulfatase, anti-sulfamidase, anti-galactosamine-6-sulfatase, anti-N-acetylgalactosamine-4-sulfatase, anti-β-glucuronidase, anti-N-acetylglucosamine-1-phosphotransferase, anti-Niemann-Pick C1 protein, anti-hexosaminidase A, or anti-tripeptidyl peptidase 1), enzyme replacement therapy may be directly inactivated (neutralized) by the anti-lysosomal enzyme neutralizing antibodies in the plasma, which may inhibit cellular uptake or enzyme activity within the lysosomes. In some embodiments, when anti-lysosomal neutralizing antibodies are present, they can neutralize ERT activity by binding to an enzyme (eg, a recombinant lysosomal enzyme).

[0083] In some embodiments, the anti-lysosomal enzyme neutralizing antibody is an IgG antibody. In some embodiments, the anti-lysosomal enzyme neutralizing antibody is an IgG4 antibody. In some embodiments, the anti-lysosomal enzyme neutralizing antibody is an IgG2 antibody. In some embodiments, the anti-lysosomal enzyme neutralizing antibody is an IgG1 antibody.

[0084] In some embodiments, anti-lysosomal enzyme neutralizing antibodies may appear within about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months after initiation of enzyme replacement therapy.

[0085] The terms "total anti-lysosomal enzyme antibodies," "anti-lysosomal enzyme TAbs," or "bound anti-lysosomal enzyme antibodies" refer to pre-existing anti-lysosomal enzyme antibodies (e.g., anti-lysosomal enzyme antibodies present in a subject prior to treatment (e.g., treatment for a lysosomal storage disease) (or prior to the initiation of said clinical trial)), and antibodies boosted by said treatment (e.g., pre-existing anti-lysosomal enzyme antibodies boosted to higher levels after administration of treatment for a lysosomal storage disease)). As used herein, the terms "pre-existing anti-lysosomal enzyme antibodies" and "treatment-boosted anti-lysosomal enzyme antibodies" refer to, for example, total anti-drug antibodies (ADA) against a lysosomal enzyme (e.g., α-Gal A, glucocerebrosidase, alpha-glucosidase, alpha-L-iduronidase, iduronate 2-sulfatase, sulfamidase, galactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, beta-glucuronidase, N-acetylglucosamine-1-phosphotransferase, Niemann-Pick C1 protein, hexosaminidase A, or tripeptidyl peptidase 1) in human serum, where the total ADA antibodies can be detected by, for example, an enzyme-linked immunosorbent assay (ELISA) as described in Partridge, MA et al., J Immunol Res. 2016:6262383. (2016), or by other methods, such as those described in Partridge, MA et al., J Immunol Res. 2016:6262383. (2016) refers to antibodies detected using an electrochemiluminescence (ECL) type assay (e.g., Meso Scale Discovery (MSD)).

[0086] An "antigen-binding portion" (also called an "antigen-binding fragment") of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to the antigen to which the whole antibody binds. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody, such as an anti-GLA 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-β-glucuronidase antibody, anti-N-acetylglucosamine-1-phosphotransferase antibody, anti-Niemann-Pick C1 protein antibody, anti-hexosaminidase A, or anti-tripeptidyl peptidase 1 described herein, include (i) an Fab fragment (a fragment from papain cleavage), or a V ... L , V H (ii) F(ab')2 fragments (fragments from papain cleavage) or similar bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) V H and an Fd fragment consisting of the CH1 domain; (iv) a V of a single arm of an antibody L and V H Fv fragment consisting of domains; (v) V H (vi) isolated complementarity-determining regions (CDRs); and (vii) combinations of two or more isolated CDRs, which may optionally be joined by a synthetic linker. Furthermore, the two domains V of the Fv fragment are L and V H Although the V are encoded by separate genes, they can be joined by a synthetic linker that allows them to be produced as a single protein chain using recombinant methods. L and V HThe domains pair to form a monovalent molecule known as a 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 by the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies. Antigen-binding portions may be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins.

[0087] "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, "binding affinity" as used herein refers to the intrinsic binding affinity, which 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 (KD). Affinity is determined by the equilibrium dissociation constant (K D ), and the equilibrium binding constant (K A K D is k off / k on is calculated from the quotient of A is k on / k off It is calculated from the quotient of k on refers to the binding rate constant between an antibody and an antigen, and k off refers to, for example, the dissociation of an antibody and an antigen. on and k off can be determined by techniques known to those skilled in the art, such as immunoassays (e.g., enzyme-linked immunosorbent assays (ELISAs)), BIAcore®, BLI (biolayer interferometry), or kinetic exclusion assays (KinExA®).

[0088] As used herein, the terms "specifically bind," "specifically recognize," "specific binding," "selective binding," and "selectively bind" are similar terms in the context of antibodies and refer to a molecule (e.g., an antibody) that binds to an antigen (e.g., an epitope or immune complex) according to binding understood by those of skill in the art. For example, a molecule that specifically binds to an antigen may bind to other peptides or polypeptides, generally with lower affinity, as determined by, for example, immunoassays, BIAcore®, KinExA® 3000 instrument (Sapidyne Instruments, Boise, ID), or other assays known in the art. In certain embodiments, a molecule that specifically binds to an antigen has a K A At least 2, 2.5, 3, or 4 logs higher than K A binds to the antigen.

[0089] Antibodies are usually 10 -5 ~10 -11 The dissociation constant (K D ) specifically binds to its cognate antigen with high affinity, reflected by approximately 10 -4 K over M D As used herein, an antibody that "specifically binds" to an antigen is an antibody that binds with high affinity to the antigen and a substantially identical antigen, as measured, for example, by immunoassay (e.g., ELISA) surface plasmon resonance (SPR) technology in a BIACORE™ 2000 instrument using a given antigen, or BLI (biolayer interferometry), with a binding affinity of 10 or more. -7 M or less, preferably 10 -8 M or less, and even more preferably 10 -9 M or less, most preferably 10 -8 M to 10 -10 K below M D This refers to an antibody that has high affinity, meaning that it does not bind with high affinity to unrelated antigens.

[0090] As used herein, the term "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. A nucleic acid molecule may be single-stranded or double-stranded, and may be cDNA.

[0091] Nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. Nucleic acids are "isolated" or "substantially pure" when they have been purified from other cellular components or other contaminants, such as other cellular nucleic acids (e.g., other parts of chromosomes) or proteins, by standard techniques such as alkali / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, or other techniques well known in the art. See F. Ausubel, et al., ed., Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987).

[0092] Nucleic acids, e.g., cDNA, can be mutated according to standard techniques to provide gene sequences. In the case of 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 naturally occurring V, D, J, constant, switch, and other such sequences described herein are contemplated ("derived" indicates that one sequence is identical to or modified from another sequence).

[0093] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Some vectors are capable of autonomous replication in a host cell 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 be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Furthermore, some vectors can direct the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. As used herein, "plasmid" and "vector" may be used interchangeably as the plasmid is the most commonly used form of vector. However, other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses, adeno-associated viruses ("AAV"), and lentiviruses), which serve equivalent functions are also included.

[0094] As used herein, the term "recombinant host cell" (or simply "host cell") is intended to refer to a cell containing a nucleic acid that is not naturally occurring within the cell, and may be a cell into which a recombinant expression vector has been introduced. It is understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because some modifications may occur in successive generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0095] As used herein, the term "conjugation" refers to the attachment of two or more molecules. The attachment can be covalent or non-covalent. The attachment can also be genetic (i.e., recombinant fusion). Such attachment can be achieved using a variety of art-recognized techniques, including chemical conjugation and recombinant protein production.

[0096] The terms "lysosomal storage disease," "lysosomal storage disorder," "LSD," "lysosomal storage diseases," "lysosomal storage disorders," and "LSD" are used interchangeably and refer to a group of over 40 disorders that result from defects in genes encoding enzymes that break down glycolipid or polysaccharide waste products within cellular lysosomes. The enzyme products, e.g., sugars and lipids, are then recycled into new products. Each of these disorders results from an inherited autosomal or X-linked recessive trait that affects enzyme levels in lysosomes. Generally, affected individuals have no biological or functional activity of the affected enzymes in their cells and tissues. In some embodiments, lysosomal storage diseases include, but are not limited to, Fabry disease, Gaucher disease, Pompe disease, mucopolysaccharidosis (MPS) type I disease, MPS type II, MPS type III, MPS type IV, MPS type VI, MPS type VII, mucolipidosis (ML), Niemann-Pick disease, Taye-Sachs disease, and Batten disease.

[0097] The term "Fabry disease" refers to classic Fabry disease, late-onset Fabry disease, and hemizygous females with a mutation in the gene encoding α-Gal A. As used herein, the term "Fabry disease" further includes any condition in which a subject exhibits lower-than-normal endogenous α-Gal A activity. Fabry disease is also known by many other names, including alpha-galactosidase A deficiency, Anderson-Fabry disease, angiokeratoma corporis, angiokeratoma diffuse, ceramide trihexosidase deficiency, Fabry disease, GLA deficiency, and hereditary ectopic lipidosis. In some embodiments, Fabry disease is the classic type 1 phenotype or the late-onset type 2 phenotype.

[0098] The term "Gaucher disease" refers to a rare inherited metabolic disorder in which a deficiency of the enzyme glucocerebrosidase leads to the accumulation of harmful amounts of certain fats (lipids), particularly the glycolipid glucocerebroside, throughout the body, particularly in the bone marrow, spleen, and liver. Gaucher disease has many other names, including cerebroside lipidosis syndrome, Gaucher splenomegaly, glucocerebrosidase deficiency, glucocerebrosidosis, glucosylceramidase deficiency, glucosylcerebroside lipidosis, keratin lipoidosis, keratin tesaurimatosis, lipid histiocytosis (keratin type), and sphingolipidosis type 1.

[0099] The term "Pompe disease" refers to a rare, multisystem genetic disorder in which a deficiency in the enzyme alpha-glucosidase prevents the breakdown of glycogen into glucose. Consequently, glycogen begins to accumulate in all types of tissue, but primarily in skeletal, smooth, and cardiac muscle, damaging tissue structure and function. Pompe disease has also been referred to by many other names, including glycogen storage disease type II (GSDII), acid maltase deficiency (AMD), and acid alpha-glucosidase (GAA) deficiency.

[0100] The term "mucopolysaccharidosis" or "MPS" refers to a group of inherited lysosomal storage disorders.

[0101] "MPS type I disease" is characterized by a deficiency of the enzyme alpha-L-iduronidase, which leads to the accumulation of dermatan and / or heparan sulfate. There are three types of MPS type I disease: Hurler syndrome (mucopolysaccharidosis type 1-H; MPS 1-H), Skeier syndrome (mucopolysaccharidosis type IS; MPS 1-S), and Hurler-Skeier syndrome (mucopolysaccharidosis type IH / S; MPS-IH / S).

[0102] "MPS 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 leads to the accumulation of heparan sulfate and dermatan sulfate in all body tissues. This is the only type of MPS disorder that is inherited as an X-linked trait. MPS II disease is also known by other names, such as Hanta syndrome, mucopolysaccharidosis type II, and MPS II.

[0103] "MPS type III disease" has four subtypes (A, B, C, and D) distinguished by four different enzyme deficiencies: heparan N-sulfatase, alpha-N-acetylglucosaminidase, acetyl-CoA:alpha-glucosamide N-acetyltransferase, and N-acetylglucosamine-6-sulfatase. MPS type II disease is also known by other names, such as Sanfilippo syndrome, mucopolysaccharidosis type III, and MPS III.

[0104] "MPS IV disease" is a rare metabolic disorder in which the body is unable to process glycosaminoglycans (also known as GAGs, or mucopolysaccharides). It is also known by other names, such as Morquio syndrome, mucopolysaccharidosis type IV, and MPS IV. Morquio syndromes A and B are caused by deficiencies of the enzymes N-acetyl-galactosamine-6-sulfatase and beta-galactosidase, respectively, resulting in the accumulation of keratan and chondroitin sulfate in type A and keratan sulfate in type B.

[0105] "MPS VI disease" is characterized by a deficiency of the enzyme N-acetylgalactosamine-4-sulfatase, which leads to the accumulation of dermatan sulfate. It is also known by other names, such as Malraux-Lamy syndrome, mucopolysaccharidosis type VI, and MPS type VI.

[0106] "MPS VII disease" is characterized by a deficiency of the enzyme beta-glucuronidase, which leads to the accumulation of three glycosaminoglycans: dermatan sulfate, heparan sulfate, and chondroitin sulfate. It is also known by other names, such as Sly syndrome, mucopolysaccharidosis type VII, and MPS VII.

[0107] The term "mucolipidosis (ML)" refers to a group of inherited metabolic disorders that affect the body's ability to perform normal metabolic turnover of various substances within cells. Four conditions (types I, II, III, and IV) were historically classified as mucolipids. However, type I (sialidosis) is now classified as a glycoproteinosis, and type IV (mucolipidosis type IV) is now classified as a gangliosidosis. Mucolipidosis types II and III (ML II and ML III) are caused by a deficiency of the enzyme N-acetylglucosamine-1-phosphotransferase, which phosphorylates target carbohydrate residues on N-linked glycoproteins. Without this phosphorylation, glycoproteins are exported from the cell rather than transported to the lysosome.

[0108] "Niemann-Pick disease," "Niemann-Pick disease type C," "NPC," or "NPD-C" is a rare, progressive genetic disorder characterized by the body's inability to transport cholesterol and other fatty substances (lipids) into cells. 95% of cases are caused by loss of function of the Niemann-Pick C1 protein (NPC1).

[0109] "Tay-Sachs disease" is a rare neurodegenerative disorder caused by a deficiency of the enzyme hexosaminidase A, which leads to excessive accumulation of gangliosides in the brain and nerve cells. It has also been called many other names, including HEXA deficiency, hexosaminidase A deficiency, TSD, GM2 gangliosidosis type 1, hexosaminidase alpha subunit deficiency (variant B), B-variant GM2 gangliosidosis, sphingolipidosis, and Tay-Sachs disease.

[0110] "Batten disease" is the general name for a broad class of rare, fatal, inherited disorders of the nervous system, also known as neuronal ceroid lipofuscinosis or NCL. This disorder occurs when brain cells lack an enzyme called tripeptidyl peptidase 1 (TPP1), causing waste products to accumulate in the neurons. It has also been referred to by many other names, including juvenile CLN3 disease, CLN3, CLN3-NCL, JNCL, juvenile Batten disease, juvenile neuronal ceroid lipofuscinosis, neuronal ceroid lipofuscinosis 3, Spillmeyer-Sjögren's disease, Vogt-Spielmeyer disease, and Vogt-Spielmeyer-Sjögren's disease.

[0111] The term "enzyme replacement therapy" or "ERT" refers to the introduction of a non-naturally occurring purified enzyme into an individual deficient in such an enzyme (e.g., a lysosomal enzyme described herein). The administered enzyme can be obtained from a natural source or by recombinant expression. The term also refers to the introduction of a purified enzyme into an individual who requires or would benefit from administration of the purified enzyme, e.g., who suffers from a protein deficiency. The introduced enzyme can be a purified recombinant enzyme produced in vitro, or an enzyme purified from an isolated tissue or fluid, e.g., placenta or animal milk, or a plant. Figure 1 shows examples of currently approved enzyme replacement therapies (ERT) for lysosomal storage disorders, such as Fabry disease, Pompe disease, Gaucher disease, MPS I, MPS II, MPS IV, and MPS VI.

[0112] The term "non-enzyme replacement therapy" refers to a therapy that is not an enzyme replacement therapy (e.g., a lysosomal storage disease therapy). Non-enzyme replacement therapy can include small molecule therapy. Some emerging drug development strategies for small molecule therapy of lysosomal storage diseases include, but are not limited to, substrate reduction therapy (SRT), residual enzyme activation, protein homeostasis modulation (proteostasis), and pharmacological chaperone therapy (CCT).

[0113] The term "stabilizing proper conformation" refers to the ability of a compound, peptide, or other molecule to bind to a wild-type protein or a mutant protein capable of wild-type function in vitro and in vivo, and maintain the structure of the wild-type or mutant protein in its native or proper form. This effect may be manifested in practice through one or more of the following: (i) an extended shelf life of the protein; (ii) higher activity per unit / amount of protein; or (iii) greater in vivo efficacy. This may be observed experimentally by similar means, such as increased yield from the ER during expression, increased resistance to unfolding at elevated temperatures (e.g., as determined by thermostability assays), or the presence of chaotropic agents.

[0114] As used herein, the term "active site" refers to a region of a protein that has a specific biological activity. For example, this region may be a site that binds a substrate or other binding partner and provides amino acid residues directly involved in chemical bond formation and cleavage. Active sites in this application may include the catalytic site of an enzyme, the antigen-binding site of an antibody, the ligand-binding domain of a receptor, the binding domain of a regulatory factor, or the receptor-binding domain of a secreted protein. Active sites may also include transcriptional activation, protein-protein interaction, or DNA-binding domains of transcription factors and regulatory factors.

[0115] As used herein, the term "active site-specific chaperone" refers to any molecule, including proteins, peptides, nucleic acids, carbohydrates, etc., that specifically and reversibly interacts with the active site of a protein and promotes the formation of a stable molecular structure. As used herein, "active site-specific chaperone" does not include endogenous general chaperones present in the endothelium of cells, such as Bip, calnexin, and calreticulin, or general non-specific chemical chaperones, such as deuterated water, DMSO, and TMAO.

[0116] The term "immunotherapy" refers to the treatment of a subject suffering from a disease or at risk of developing or experiencing a recurrence of a disease by methods that involve inducing, enhancing, suppressing, or otherwise modifying the immune response. "Treatment" or "therapy" of a subject refers to any type of intervention or process performed on a subject, or the administration of an active agent to a subject, with the intent of reversing, mitigating, ameliorating, inhibiting, delaying, or preventing the onset, progression, development, severity, or recurrence of symptoms, complications, conditions, or biochemical manifestations associated with a disease.

[0117] A "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 embodiments, the subject is a human. The terms "subject" and "patient" are used interchangeably herein.

[0118] The term "fixed dose," when used with respect to the methods and dosages of the present disclosure, refers to a dosage administered to a patient without regard to the patient's weight or body surface area (BSA). Thus, a fixed dose is provided not as a mg / kg dose, but rather as an absolute amount of agent (e.g., recombinant α-Gal A protein). For example, a person weighing 60 kg and a person weighing 100 kg would receive the same amount of antibody (e.g., 12 mg of recombinant α-Gal A protein).

[0119] 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, if a 60 kg patient requires 0.2 mg / kg of recombinant α-Gal A protein, then the appropriate amount of recombinant α-Gal A protein (i.e., 12 mg) can be calculated and used for administration.

[0120] A "therapeutically effective amount" or "therapeutically effective dose" of a drug or therapeutic agent is that amount of any drug that, when used alone or in combination with another therapeutic agent, protects a subject from developing a disease or promotes regression of the disease as evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or prevention of disability or disability due to the affliction of the disease. The ability of a therapeutic agent to promote regression of a disease can be evaluated using a variety of methods known to the skilled practitioner, such as by assaying the activity of the therapeutic agent in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or in in vitro assays.

[0121] As used herein, the terms "treat," "treating," and "treatment" refer to any type of intervention or process performed on a subject, or administering an active agent to a subject, for the purpose of reversing, alleviating, ameliorating, inhibiting, or slowing or preventing the progression, onset, severity, or recurrence of symptoms, complications, conditions, or biochemical manifestations associated with a disease, or improving overall survival. Treatment can be administered to subjects with a disease or to subjects without a disease (e.g., prophylaxis).

[0122] The term "effective amount" or "effective dosage" is defined as an amount sufficient to achieve, or at least partially achieve, a desired effect. A "therapeutically effective amount" or "therapeutically effective dosage" of a drug or therapeutic agent is an amount of drug that, when used alone or in combination with another therapeutic agent, promotes regression of a disease as evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, an increase in overall survival (the length of time from the date of diagnosis of a disease, such as cancer, or the start of treatment that a patient diagnosed with the disease is still alive), or prevention of disability or disabling illness caused by the disease. A therapeutically effective amount or dosage of a drug includes a "prophylactically effective amount" or "prophylactically effective dosage," which is an amount of drug that, when administered alone or in combination with another therapeutic agent to a subject at risk of developing a disease or at risk of disease recurrence, prevents the onset or recurrence of the disease. The ability of a therapeutic agent to promote regression of disease or inhibit the onset or recurrence of disease may be assessed using a variety of methods known to the skilled practitioner, such as assaying the activity of the therapeutic agent in human subjects in clinical trials, in animal model systems predictive of efficacy in humans, or in in vitro assays.

[0123] A "sample" or "biological sample" of the present disclosure, in some embodiments, is of biological origin, such as from a eukaryotic organism. In some embodiments, the sample is a human sample, although animal samples may also be used. Non-limiting sources of samples for use in the present disclosure include, for example, solid tissue, biopsy aspirate, ascites, fluid extract, blood, plasma, serum, spinal fluid, lymphatic fluid, external portions of the skin, respiratory tract, intestinal tract, genitourinary tract, tears, saliva, milk, tumor, organ, cell culture, and / or cell culture components.

[0124] "Administering" refers to the physical introduction of a composition containing a therapeutic agent into a subject using any of a variety of methods and delivery systems known to those of skill in the art. Preferred routes of administration for recombinant α-Gal A protein or genetically expressed α-Gal A include intravenous administration, for example, by injection or infusion, or other parenteral routes of administration. The term "parenteral administration," as used herein, refers to methods of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intrathecal, epidural, intrasternal, and in vivo electroporation. Other parenteral routes include oral, topical, epidermal or mucosal routes of administration, e.g., intranasal, intravaginal, rectal, sublingual or topical. Administration can also be, for example, single, multiple and / or over one or more extended periods of time.

[0125] As used herein, terms such as "about once per week," "about once per two weeks," or other similar terms relating to administration intervals refer to approximate numbers. "About once per week" can include every 7 days ± 1 day, i.e., every 6 to 8 days. "About once per two weeks" can include every 14 days ± 3 days, i.e., every 11 to 17 days. Similar approximations apply, for example, to about once per 3 weeks, about once per 4 weeks, about once per 5 weeks, about once per 6 weeks, and about once per 12 weeks. In some embodiments, an administration interval of about once per 6 weeks or about once per 12 weeks means that the first administration may be administered on any day in the first week, followed by the next administration on any day in the sixth or twelfth week, respectively. In other embodiments, a dosing interval of about once every 6 weeks or about once every 12 weeks means that the first dose is administered on a particular day of the week (e.g., Monday) in week 1, and then the next dose is administered on the same day of the week (i.e., Monday) in week 6 or week 12, respectively.

[0126] The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives. As used herein, the indefinite article "a" or "an" should be understood to refer to "one or more" of any listed or listed members.

[0127] The terms "about" or "essentially comprising" refer to a value or composition that is within an acceptable error range for a particular value or composition, as determined by one of ordinary skill in the art, and depend in part on the method by which the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "essentially comprising" can mean within one standard deviation or more than one standard deviation, as practiced in the art. Alternatively, "about" or "essentially comprising" can mean a range of up to 10%. Furthermore, particularly with respect to biological systems or processes, the term can mean up to an order of magnitude or up to five times the value. When a particular value or composition is provided in this application and claims, unless otherwise specified, the meaning of "about" or "essentially comprising" should be considered to be within an acceptable error range for the particular value or composition.

[0128] As described herein, any concentration range, percentage range, ratio range, or integer range is understood to include any integer value within the recited range, and, where appropriate, fractions thereof (such as 1 / 10 and 1 / 100 of an integer), unless otherwise specified.

[0129] Various aspects of the disclosure are described in further detail in the following subsections.

[0130] II. Methods of the Disclosure Provided herein are methods for detecting anti-lysosomal enzyme antibodies in a human subject, as well as methods for improving the effectiveness of anti-lysosomal enzyme antibody assays, the methods comprising pretreating a biological sample from the subject with a base at a pH of about 11 or higher, and measuring the presence of the anti-lysosomal enzyme antibodies in the biological sample from the subject.

[0131] In some aspects, the present disclosure relates to a method for detecting anti-lysosomal enzyme antibodies (e.g., in the presence of circulating lysosomal enzymes) in a human subject, comprising measuring the presence of anti-lysosomal enzyme antibodies in a biological sample from the subject, wherein the biological sample is pretreated with a base at a pH of about 11 or higher.

[0132] In some aspects, the present disclosure relates to a method for improving the effectiveness of an anti-lysosomal enzyme antibody assay (e.g., improving assay sensitivity and circulating lysosomal enzyme resistance), the method comprising pretreating a biological sample from a human subject with a base at a pH of about 11 or greater, and further comprising determining the presence of the anti-lysosomal enzyme antibody in the biological sample.

[0133] As used herein, the term "improving the effectiveness of an anti-lysosomal enzyme antibody assay" refers to any improvement resulting from optimizing various assay conditions (e.g., pretreatment of the biological sample with high or low molar base at a pH of about 11 or higher, heat pretreatment of the biological sample, varying on-board lysosomal enzyme concentrations (ng / ml), neutralizing the base-pretreated biological sample with acid, or any combination thereof) that may result in advantageous results (e.g., improved assay sensitivity and circulating lysosomal enzyme resistance) compared to results achieved with an anti-lysosomal enzyme antibody assay when performed without the same assay conditions (e.g., including heat pretreatment of the biological sample with high or low molar base at a pH of about 11 or higher, heat pretreatment of the biological sample, varying on-board lysosomal enzyme concentrations (ng / ml), neutralizing the base-pretreated biological sample with acid, or any combination thereof). Enhanced efficacy and the determination of enhanced efficacy can be measured by various parameters, such as, but not limited to, (i) improved ability to detect anti-lysosomal enzyme neutralizing antibodies (NAb) and / or total anti-lysosomal antibodies (TAb) in the presence or absence of on-board lysosomal enzymes, and / or (ii) improved assay sensitivity as measured by improved resistance to circulating lysosomal enzymes, as measured by, for example, increased resistance of on-board lysosomal enzymes in detecting anti-lysosomal enzyme NAb, as described in Examples 2-4 below.

[0134] IIA. Anti-lysosomal enzyme neutralizing antibody (NAb) assay In some aspects, the present disclosure provides a method for detecting anti-lysosomal enzyme antibodies (e.g., anti-α-galactosidase A antibodies, anti-glucocerebrosidase antibodies, anti-alpha-glucosidase antibodies, anti-α-L-iduronidase antibodies, anti-iduronate 2-sulfatase antibodies, anti-sulfamidase antibodies, anti-galactosamine-6-sulfatase antibodies, anti-N-acetylgalactosamine-4-sulfatase antibodies, anti-beta-glucuronidase antibodies, anti-N-acetylglucosamine-1-phosphotransferase antibodies) in a human subject. and a method for detecting an anti-lysosomal enzyme antibody (anti-Niemann-Pick C1 protein antibody, anti-hexosaminidase A, or anti-tripeptidyl peptidase 1 antibody), and a method for improving the effectiveness of an anti-lysosomal enzyme antibody assay, comprising pretreating the biological sample from the subject with a base at a pH of about 11 or higher, and measuring the presence of the anti-lysosomal enzyme antibody in the biological sample from the subject with the anti-lysosomal enzyme neutralizing antibody as described herein.

[0135] In some embodiments, as described in Example 2 below, an anti-lysosomal antibody neutralizing antibody assay determines the presence of anti-lysosomal enzyme neutralizing antibodies (e.g., anti-α-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, or anti-tripeptidyl peptidase 1 antibody) by assessing the neutralizing ability of human serum against lysosomal enzyme activity.

[0136] In some embodiments, the results are comparable to those obtained with artificial substrates, such as 4-methylumbelliferyl-α-D-galactopyranoside, 4-methylumbelliferyl-β-D-glucopyranoside, 4-methylumbelliferyl α-D-glucopyranoside, 4-methylumbelliferyl α-L-idopyranoside, 4-methylumbelliferyl-α-L-iduronide 2-sulfate, 4-methylumbelliferyl-2-sulfamino-2-deoxy-α-D-glucopyranoside, 4-methylumbelliferyl α-L-iduronide 2-sulfate, 4-methylumbelliferyl ... It is determined by measuring the products resulting from the cleavage of tilumbelliferyl-β-D-galactose-6-sulfate, 4-methylumbelliferyl-N-acetyl-α-d-galactoseamidin-4-sulfate, 4-methylumbelliferyl-β-d-glucuronide, 4-methylumbelliferyl-α-D-mannopyrronoside, 4-methylumbelliferyl N-acetyl-β-D-glucosamide, or Ala-Ala-Phe-7-amido-4-methyl-coumarin.

[0137] In some embodiments, the results are determined by measuring the 4-methylumbelliferone (4-MU) product resulting from cleavage of the artificial substrate.

[0138] Any anti-lysosomal enzyme-neutralizing antibodies present in human serum bind to the lysosomal enzyme and prevent cleavage of the product from the substrate. This decrease in relative fluorescence unit (RFU) signal is directly proportional to the amount of anti-lysosomal enzyme-neutralizing antibodies (e.g., anti-α-galactosidase A, anti-glucocerebrosidase, anti-α-glucosidase, anti-α-L-iduronidase, anti-iduronate 2-sulfatase, anti-sulfamidase, anti-galactosamine-6-sulfatase, anti-N-acetylgalactosamine-4-sulfatase, anti-β-glucuronidase, anti-N-acetylglucosamine-1-phosphotransferase, anti-Niemann-Pick C1 protein, anti-hexosaminidase A, or anti-tripeptidyl peptidase 1) present in human serum.

[0139] In some embodiments, the biological sample (e.g., a serum or plasma sample) is diluted to a minimum required dilution (MRD) of about 2-fold or greater. In some embodiments, the biological sample is diluted 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, 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 43x, 44x, 45x, 46x, 47x, 48x, 49x, 50x, 51x, 52x, 53x, 54x, 55x, 56x, 57x, 58x, 59x, 60x, 61x, 62x, 63x, 64x times, approximately 65 times, approximately 66 times, approximately 67 times, approximately 68 times, approximately 69 times, approximately 70 times, approximately 71 times, approximately 72 times, approximately 73 times, approximately 74 times, approximately 75 times, approximately 76 times, approximately 77 times, approximately 78 times, approximately 79 times, approximately 80 times, approximately 81 times, approximately 82 times, approximately 83 times, approximately 84 times, approximately 85 times, Approximately 86 times, approximately 87 times, approximately 88 times, approximately 89 times, approximately 90 times, approximately 91 times, approximately 92 times, approximately 93 times, approximately 94 times, approximately 95 times, approximately 96 times, approximately 97 times, approximately 98 times, approximately 99 times, approximately 100 times, approximately 200 times, approximately 300 times, approximately 400 times, approximately 500 times, approximately 600 times, 700x, 800x, 900x, 1000x, 1100x, 1200x, 1300x, 1400x, 1500x, 1600x, 1700x, 1800x, 1900x, 2000x, 2100x, 2200x 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.

[0140] In some embodiments, serum samples are diluted in MRD10 (1:5 dilution with assay buffer (e.g., 0.1 M citric acid, 0.2 M sodium phosphate, and 0.05% Triton X-100, pH 4.6±0.1) followed by a 1:2 dilution with 2X drug diluent (e.g., 40 ng / mL reconstituted GLA protein in assay buffer)).

[0141] In some embodiments, lysosomal enzymes (e.g., α-galactosidase A (α-Gal A), glucocerebrosidase, alpha-glucosidase, alpha-L-iduronidase, iduronate 2-sulfatase, sulfamidase, galactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, beta-glucuronidase, N-acetylglucosamine-1-phosphotransferase, Niemann-Pick C1 protein, hexosaminidase A, or tripeptidyl peptidase 1) are 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 The concentration is less than 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.

[0142] In some embodiments, human lysosomal proteins were diluted to 40 ng / mL in an acidic sample buffer containing 0.1 M citric acid, 0.2 M sodium phosphate, and 0.05% Triton X-100 at pH 4.6.

[0143] In some embodiments, the biological sample (eg, a serum or plasma sample) is pretreated with a base at a pH of about 11 or higher.

[0144] In some embodiments, the base is a non-buffering base. In some embodiments, the non-buffering base is NaOH or Ca(OH).

[0145] In some embodiments, 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. In some embodiments, the NaOH or Ca(OH)2 is about 0.02 M.

[0146] In some embodiments, 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 2.5, 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.

[0147] In some embodiments, 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.

[0148] In some embodiments, human serum samples were diluted to 20% serum in alkaline sample buffer at pH 12.45 and then mixed 1:1 with diluted acidic enzyme buffer. In some embodiments, the final sample incubation concentration contained 20 ng / mL lysosomal enzymes in 10% serum with a final pH of 4.9.

[0149] In some embodiments, the pre-treated biological sample is mixed with a lysosomal enzyme.

[0150] In some embodiments, the pretreated biological sample and lysosomal enzyme mixture are incubated for at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, or at least about 24 hours.

[0151] In some embodiments, the pretreated biological sample and lysosomal enzyme mixture are incubated for a period 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.

[0152] In some embodiments, the pre-treated biological sample and the lysosomal enzyme are combined with a reaction mixture, hi some embodiments, the reaction mixture includes a substrate and / or an inhibitor.

[0153] In some embodiments, the substrate is 4-methylumbelliferyl-α-D-galactopyranoside, 4-methylumbelliferyl-β-D-glucopyranoside, 4-methylumbelliferyl α-D-glucopyranoside, 4-methylumbelliferyl α-L-idopyranoside, 4-methylumbelliferyl-α-L-iduronide 2-sulfate, 4-methylumbelliferyl-2-sulfamino-2-deoxy-α-D-glucopyranoside, 4 4-methylumbelliferyl-β-D-galactose-6-sulfate, 4-methylumbelliferyl-N-acetyl-α-d-galactoseamidin-4-sulfate, 4-methylumbelliferyl-β-d-glucuronide, 4-methylumbelliferyl-α-D-mannopyrronoside, 4-methylumbelliferyl N-acetyl-β-D-glucosamide, and Ala-Ala-Phe-7-amido-4-methyl-coumarin.

[0154] In some embodiments, the concentration of 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 In some embodiments, the substrate is at a concentration of at least about 2.5 mM.

[0155] In some embodiments, the inhibitor comprises N-acetylgalactosamine (GALNAc).

[0156] In some embodiments, the inhibitor is at a concentration of 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 embodiments, the inhibitor is at a concentration of at least about 125 mM.

[0157] In some embodiments, the reaction mixture and the biological sample pretreated with the lysosomal enzyme mixture are combined and mixed in a high-throughput plate. In some embodiments, the reaction mixture and the biological sample pretreated with the lysosomal enzyme mixture are incubated at room temperature at 400 revolutions per minute (RPM).

[0158] In some embodiments, the methods disclosed herein further comprise adding a stop buffer to the mixture after incubation. In some embodiments, 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 embodiments, the incubation period is at least about 60 minutes.

[0159] In some embodiments, the stop buffer comprises glycine. In some embodiments, the stop buffer has a volume of less than about 1 mL, less than about 900 uL, less than about 800 uL, less than about 700 uL, less than about 600 uL, less than about 500 uL, less than about 400 uL, less than about 300 uL, less than about 200 uL, or less than about 100 uL. In some embodiments, the stop buffer has a volume of about 100 uL.

[0160] In some embodiments, human biological samples with a percent inhibition (%) equal to or greater than the cutoff point are identified as positive for anti-lysosomal enzyme neutralizing antibodies, while those below the cutoff point are considered negative for anti-lysosomal enzyme neutralizing antibodies.

[0161] In some embodiments, an anti-lysosomal enzyme neutralizing antibody-negative subject described herein has a biological sample in which inhibition of a lysosomal enzyme (e.g., α-Gal A, glucocerebrosidase, alpha-glucosidase, alpha-L-iduronidase, iduronate 2-sulfatase, sulfamidase, galactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, beta-glucuronidase, N-acetylglucosamine-1-phosphotransferase, Niemann-Pick C1 protein, hexosaminidase A, or tripeptidyl peptidase 1) is less than about 50% as measured by an anti-lysosomal enzyme neutralizing antibody assay described herein. In some embodiments, an anti-lysosomal enzyme neutralizing antibody-negative sample described herein has 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% inhibition of lysosomal enzyme (e.g., α-Gal A, glucocerebrosidase, alpha-glucosidase, alpha-L-iduronidase, iduronate 2-sulfatase, sulfamidase, galactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, beta-glucuronidase, N-acetylglucosamine-1-phosphotransferase, Niemann-Pick C1 protein, hexosaminidase A, or tripeptidyl peptidase 1) activity.

[0162] In some embodiments, a subject who is positive for an anti-lysosomal enzyme neutralizing antibody described herein has a biological sample that exhibits greater than about 10% inhibition of lysosomal enzyme (e.g., α-Gal A, glucocerebrosidase, alpha-glucosidase, alpha-L-iduronidase, iduronate 2-sulfatase, sulfamidase, galactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, beta-glucuronidase, N-acetylglucosamine-1-phosphotransferase, Niemann-Pick C1 protein, hexosaminidase A, or tripeptidyl peptidase 1) activity as measured by an anti-lysosomal enzyme neutralizing antibody assay described herein. In some embodiments, the anti-lysosomal enzyme neutralizing antibody positive samples described herein have signal inhibition of about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%.

[0163] IIB. Standardization of anti-lysosomal enzyme neutralizing antibody (NAb) assays In some aspects, the present disclosure relates to methods for standardizing anti-lysosomal enzyme neutralizing antibody assays, comprising determining lysosomal drug diluent concentrations by measuring the effect of lysosomal enzyme (e.g., α-Gal A) drug levels on the inhibitory effect of a positive control antibody (e.g., antibodies designated RP-01, 7H11, or 19D6), e.g., as described in Examples 2 and 3 below.

[0164] In some embodiments, the positive control antibody includes, but is not limited to, the RP-01 antibody. In some embodiments, RP-01 is a polyclonal antibody.

[0165] In some embodiments, the positive control antibody includes, but is not limited to, the 7H11 antibody. In some embodiments, 7H11 is a monoclonal antibody.

[0166] In some aspects, the positive control antibody includes, but is not limited to, the 19D6 antibody. In some embodiments, 19D6 is a monoclonal antibody.

[0167] In some embodiments, normalization involves determining the lysosomal enzyme drug dilution concentration by measuring the effect of lysosomal enzyme (e.g., α-Gal A) drug levels on the inhibitory effect of RP-01 antibody (anti-α-Gal antibody).

[0168] In some embodiments, normalization involves determining the lysosomal enzyme drug dilution concentration by measuring the effect of lysosomal enzyme (e.g., α-Gal A) drug levels on the inhibitory effect of the 7H11 antibody (anti-α-Gal antibody).

[0169] In some embodiments, normalization involves determining a lysosomal enzyme drug dilution concentration by measuring the effect of lysosomal enzyme (e.g., α-Gal A) drug levels on the inhibitory effect of 19D6 antibody (anti-α-Gal antibody).

[0170] In some embodiments, 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 embodiments, the lysosomal enzyme (e.g., α-Gal A) drug diluent concentration is less than about 125 ng / ml. In some embodiments, the lysosomal enzyme (e.g., α-Gal A) drug dilution concentration is about 40 ng / ml. In some embodiments, the lysosomal enzyme (e.g., α-Gal A) drug dilution concentration is about 20 ng / ml.

[0171] In some embodiments, 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.

[0172] In some embodiments, the GLA drug is about 1x, 2x, about 3x, about 4x, about 5x, about 6x, about 7x, about 8x, about 9x, about 10x, about 11x, about 12x, about 13x, about 14x, about 15x, about 16x, about 17x, about 18x, about 19x, about 20x, about 21x, about 22x, 23x, about 24x, about 25x, about 26x, about 27x, about 28x, about 29x, about 30x, about 31x, about 32x, about 33x, about 34x, about 35x, about 36x, about 37x, about 38x, about 39x, about 40x, about 41x, about 42x , approx. 43x, approx. 44x, approx. 45x, approx. 46x, approx. 47x, 48x, approx. 49x, approx. 50x, approx. 51x, approx. 52x, approx. 53x, approx. times, approximately 65 times, approximately 66 times, approximately 67 times, approximately 68 times, approximately 69 times, approximately 70 times, approximately 71 times, approximately 72 times, approximately 73 times, approximately 74 times, approximately 75 times, approximately 76 times, approximately 77 times, approximately 78 times, approximately 79 times, approximately 80 times, approximately 81 times, approximately 82 times, approximately 83 times, approximately 84 times, approximately 85 times, approximately 86 times, approximately 87 times, approximately 88 times, approximately 89 times, approximately 90 times, approximately 91 times, approximately 92 times, approximately 93 times, approximately 94 times, approximately 95 times, approximately 96 times, approximately 97 times, approximately 98 times, approximately 99 times, approximately 100 times, approximately 200 times, approximately 300 times, approximately 400 times, approximately 500 times, approximately 600 times, approximately 7 00x, approx. 800x, approx. 900x, approx. 1000x, approx. 1100x, approx. 1200x, approx. 1300x, approx. 1400x, approx. 1500x, approx. 1600x, approx. and a minimum required dilution (MRD) of 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.

[0173] In some embodiments, the RP-01 antibody, 7H11 antibody, or 19D6 antibody has a specific antigen binding activity of 2.6×10 as determined by immunoassay (e.g., ELISA) surface plasmon resonance (SPR) technology using a given antigen or BLI (biolayer interferometry), e.g., on a BIACORE™ 2000 instrument. -10 Less than M, 2.5 x 10 -10 Less than M, 2.0 x 10 -10 Less than M, 1.5 x 10 -10 Less than M, 1.0 x 10 -10 Less than M, 9 x 10 -11 Less than M, 8 x 10 -11 Less than M, 7 x 10 -11 Less than M, 6 x 10 -11 Less than M, 5 x 10 -11 Less than M, 4 x 10 -11 Less than M, 3 x 10 -11 Less than M, 2 x 10 -11 Less than M, 1 x 10 -11 Less than M, 9 x 10 -12 Less than M, 8 x 10 -12 Less than M, 7 x 10 -12 Less than M, 6 x 10 -12 Less than M, 5 x 10 -12 Less than M, 4 x 10 -12 Less than M, 3 x 10 -12 Less than M, 2 x 10 -12 Less than M, 1 x 10 -12 Less than M, 9 x 10 -13 Less than M or 8 x 10 -13 The binding affinity (K D )

[0174] IIC. Anti-lysosomal enzyme total antibody (TAb) assay In some aspects, the present disclosure provides a method for detecting an anti-lysosomal enzyme antibody (e.g., an anti-α-galactosidase A antibody, an anti-glucocerebrosidase antibody, an anti-alpha-glucosidase antibody, an anti-alpha-L-iduronidase antibody, an anti-iduronate 2-sulfatase antibody, an anti-sulfamidase antibody, an anti-galactosamine-6-sulfatase antibody, an anti-N-acetylgalactosamine-4-sulfatase antibody, an anti-beta-glucuronidase antibody, an anti-N-acetylglucosamine-1-phosphotransferase antibody, an anti-β-glucuronidase antibody, an anti-β-glucuronidase antibody, an anti-β-glucosamine-1-phosphotransferase ... The present invention relates to a method for detecting antibodies (anti-Niemann-Pick C1 protein antibodies, anti-hexosaminidase A, or anti-tripeptidyl peptidase 1 antibodies) and a method for improving the effectiveness of an anti-lysosomal enzyme antibody assay (e.g., improving assay sensitivity and circulating lysosomal enzyme resistance), the method comprising pretreating the biological sample from the subject with a base at a pH of about 11 or higher, and measuring the presence of the anti-lysosomal enzyme antibodies in the biological sample from the subject by the anti-lysosomal enzyme total antibody assay as described herein (e.g., in Example 5 below).

[0175] In some embodiments, as described in Example 5 below, an anti-lysosomal antibody total antibody assay determines the presence of anti-lysosomal enzyme neutralizing antibodies (e.g., anti-α-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, or anti-tripeptidyl peptidase 1 antibody) by assessing the ability to detect serum anti-lysosomal antibodies in the presence of onboard lysosomal enzyme levels.

[0176] In some embodiments, the methods disclosed herein further comprise neutralizing the pretreated biological sample with an acid. In some embodiments, the acid is acetic acid or hydrochloric acid. In some embodiments, 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 embodiments, the acetic acid is about 30 mM. In some embodiments, the hydrochloric acid is about 120 mM.

[0177] In some embodiments, the serum sample is pretreated with base at pH 11 for about 1 hour at room temperature and then neutralized with 30 mM acetic acid.

[0178] In some aspects, the methods disclosed herein further comprise mixing the neutralized pre-treated biological sample with a lysosomal enzyme.

[0179] In some embodiments, the neutralized pre-treated biological sample and the lysosomal enzyme mixture are added to a high-throughput plate coated with a lysosomal enzyme antigen, and in some embodiments, the neutralized pre-treated biological sample and the lysosomal enzyme mixture are incubated with shaking at room temperature for about 1 hour to about 2 hours.

[0180] In some embodiments, the lysosomal enzyme antigen is selected from the group consisting of α-Gal A antigen, glucocerebrosidase antigen, alpha-glucosidase antigen, alpha-L-iduronidase antigen, iduronate 2-sulfatase antigen, sulfamidase antigen, galactosamine-6-sulfatase antigen, N-acetylgalactosamine-4-sulfatase antigen, beta-glucuronidase antigen, N-acetylglucosamine-1-phosphotransferase antigen, Niemann-Pick C1 protein antigen, hexosaminidase A antigen, and tripeptidyl peptidase 1 antigen. In some embodiments, the lysosomal enzyme antigen is α-Gal A antigen.

[0181] In some embodiments, the detection antibody or lysosomal enzyme with ruthenylated label is added to the plate to generate a signal.In some embodiments, the detection antibody is an anti-human IgG antibody.In some embodiments, the ruthenylated anti-human IgG is added to the plate and incubated at room temperature for 1 hour by shaking.

[0182] In some embodiments, the plate is read on a plate reader to detect light emitted by the ruthenylated label and output electrochemiluminescence units (ECLu), which in some embodiments represents the total level of anti-lysosomal total antibody.

[0183] III. Kit Also within the scope of the present disclosure is a kit comprising the anti-lysosomal enzyme neutralizing antibody assay described herein, comprising: The kit comprises: (a) an assay buffer; (b) a substrate; (c) a GALNAc inhibitor; (d) a stop solution; and (e) a package insert containing instructions for use of the kit.

[0184] Kits typically include labels and instructions indicating the intended use of the contents of the kit. The term label includes any writing, or recorded material supplied on or with the kit, or which otherwise accompanies the kit.

[0185] All references cited above and throughout this specification are incorporated herein by reference in their entirety.

[0186] The following examples are offered by way of illustration and not by way of limitation. [Example]

[0187] Example 1 Anti-lysosomal enzyme antibody assay Current treatments for lysosomal storage disorders (LSDs) are highly immunogenic, making it important to measure anti-lysosomal enzyme antibodies in patients to understand how and whether new drugs will work in these patients. Figure 1 shows that there is a significant incidence of anti-ERT antibodies, including anti-ERT neutralizing antibodies, for lysosomal storage disorders (e.g., Fabry disease, Pompe disease, Gaucher disease, and mucopolysaccharide saccharification (MPS) I, II, IV, and VI diseases) with currently approved ERT treatments. The method shown in Figures 2 and 11 allows for the detection of anti-lysosomal neutralizing antibodies (Figure 2) and total anti-lysosomal antibodies (Figure 11) in the presence of circulating lysosomal enzymes. This is achieved by optimizing the assay, including a novel high-pH alkaline treatment that dissociates and specifically denatures lysosomal enzymes, enabling accurate detection of anti-lysosomal enzyme antibodies even in the presence of supraphysiological levels of lysosomal enzymes in matrices such as serum and plasma. This alkaline assay allows for the measurement of anti-lysosomal enzyme antibodies in human subjects while they are being treated, for example, with ERT or gene therapy, allowing for subject stratification 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.

[0188] Example 2 Anti-lysosomal enzyme neutralizing antibody (NAb) assay The anti-lysosomal enzyme neutralizing antibody (NAb) assay utilizes artificial fluorogenic substrates, such as 4-methylumbelliferyl α-D-galactopyranoside (4-MU-α-Gal), to measure lysosomal enzyme activity and the inhibitory effects of neutralizing antibodies in human serum. Serum is treated with a high-pH base treatment to dissociate lysosomal enzyme and anti-lysosomal enzyme antibody complexes and further denature the lysosomal enzymes, eliminating catalytic enzymatic activity. Alkaline-treated serum samples containing a constant low level of lysosomal enzymes are then incubated overnight with human serum in an acidic assay buffer. After incubation, the samples are mixed with the fluorogenic substrate in a plate format. In the presence of catalytically active lysosomal enzymes, the substrate is cleaved, releasing the fluorescent 4-methylumbelliferone (4-MU), which can be quantified at basic pH with excitation at 365 nm and emission at 450 nm.

[0189] The presence of neutralizing antibodies in serum limits substrate cleavage and reduces the relative fluorescence unit (RFU) signal. Each sample was normalized to the fluorescence observed in negative wells to determine the % relative inhibition of the sample. ( TIFF2025535757000001.tif538). If the inhibition (% inhibition of α-lysosomal enzyme activity) of a sample is greater than the calculated cutoff threshold, the sample is considered to be positive for anti-lysosomal neutralizing antibodies.

[0190] Human lysosomal protein (α-galactosidase A) was diluted to 40 ng / mL in an acidic sample buffer (pH 4.6) containing 0.1 M citric acid, 0.2 M sodium phosphate, and 0.05% Triton X-100 (mimicking lysosomal-like conditions). Human serum samples were diluted to 20% serum in an alkaline sample buffer (0.02 M Ca(OH)2 (calcium hydroxide) buffer) at pH 12.45 and then mixed 1:1 with the diluted acidic enzyme buffer. The final sample incubation concentration was 20 ng / mL lysosomal enzyme in 10% serum, with a final pH of 4.9. Positive controls were prepared by utilizing human serum containing known concentrations of anti-lysosomal enzyme neutralizing antibodies (e.g., RP-01, 7H11, or 19D6), and negative controls utilized human serum without anti-lysosomal enzyme antibodies. Add the sample mixture in duplicate to a non-binding 96-well plate, seal, and incubate overnight at 2-8 °C.

[0191] After incubation, the samples were transferred to another 96-well plate and diluted 5-fold with a reaction buffer containing specific lysosomal enzyme fluorescent substrates and nonspecific enzyme inhibitors, e.g., 2.5 mM 4-MU-α-Gal and 0.125 M GALNAc, respectively. These components were prepared in an acidic buffer at pH 4.6 containing 0.1 M citric acid, 0.2 M sodium phosphate, and 0.05% Triton X-100. This reaction buffer continued to mimic lysosomal conditions but also contained fluorogenic substrates for specific lysosomal enzymes. The reaction was carried out at room temperature for 1 hour. The reaction was terminated by the addition of a basic glycine solution at pH 10.7 containing 0.25 M glycine. The plate was then analyzed in a spectrophotometer at 365 nm excitation and 450 nm emission wavelengths. All samples were normalized and evaluated against negative control wells containing negative serum.

[0192] Figures 3A and 3B show that the anti-lysosomal enzyme NAb assay cannot accurately detect antibodies when 125 ng / mL of circulating lysosomal enzyme (α-galactosidase A) is present in serum. The assay completely fails to detect any anti-lysosomal enzyme NAb at lysosomal enzyme (α-galactosidase A) concentrations of 500 ng / mL or higher, demonstrating significant negative signal inhibition as enzyme concentration increases (Figure A3). Figures 3A and 3B show that polyclonal lysosomal enzyme NAb cannot be detected in the presence of onboard lysosomal enzymes in serum.

[0193] Example 3 Sample pretreatment for detecting anti-lysosomal enzyme neutralizing antibodies To improve the ability to detect anti-lysosomal enzyme-neutralizing antibodies in the presence of onboard lysosomal enzymes, various treatments were evaluated. Heat pretreatment was evaluated by heating serum to attempt to dissociate and denature onboard lysosomal enzymes. As shown in Figure 4A, the standard assay detects less NAb inhibition as onboard lysosomal enzyme (α-galactosidase A) levels are increased to 200 ng / ml. Figure 4B shows that in the absence of NAb, heat pretreatment at 56°C was able to denature onboard lysosomal enzyme (α-galactosidase A) but failed to reduce interference at any NAb level (50 μg, 100 μg, or 150 μg).

[0194] Figure 5 shows other pretreatment methods evaluated to improve the ability to detect anti-lysosomal enzyme NAbs in the presence of onboard lysosomal enzymes (α-galactosidase A). Acidic pH 2 pretreatment failed to increase the resistance of onboard lysosomal enzymes (α-galactosidase A) in detecting NAbs and was less effective than standard assay diluent (AD) conditions (McIlvaine buffer, pH 4.5: 0.1 M citric acid + 0.2 M disodium hydrogen phosphate + 0.05% Triton X). 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 approach failed to increase lysosomal enzyme resistance and completely lost the ability to detect NAbs, even in the absence of onboard lysosomal enzymes. Alkaline pH 11 treatment showed some increase in resistance to lysosomal enzymes in detecting NAbs and was the most effective of all treatments evaluated in this experiment. Although pH 11 pretreatment showed an improvement over the standard assay diluent, it was not sufficient to detect all NAb inhibition when on-board lysosomal enzymes were increased.

[0195] Example 4 Optimized alkaline sample pretreatment for detecting anti-lysosomal enzyme neutralizing antibodies The anti-lysosomal enzyme neutralizing antibody assay was optimized for lysosomal enzyme (α-galactosidase A) resistance by testing the enzyme neutralizing capacity (% inhibition of lysosomal enzyme activity) of positive control antibodies (RP-01, 7H11, and 19D6) under various assay conditions.

[0196] The polyclonal RP-01 positive control antibody was produced as described in International Publication No. WO 2022 / 072706. The monoclonal 7H11 and 19D6 rabbit monoclonal antibodies were produced by Yurogen Biosystems according to the following protocol: Rabbits were first immunized with α-Gal A protein. Spleens were isolated, and the resulting splenocytes were fused with partner cells to generate immortal cell lines expressing the antibodies (hybridoma technology). Hybridomas were screened for the most effective binding to α-Gal A protein. Clones with the best binding affinity were expanded and purified. To screen for the best clones, plates were coated with α-Gal A, and the purified antibodies were diluted and plated on the plates (7-point, 3-fold serial dilutions of 1 μg / mL), with HRP-conjugated goat anti-rabbit IgG used for final detection (1:5K).

[0197] Figure 6 shows that alkaline sample pretreatment was able to remove onboard lysosomal enzyme interference and dramatically increase the detection of anti-lysosomal enzyme neutralizing antibodies. The NAb assay was able to tolerate up to 2000 ng / ml of onboard lysosomal enzyme (α-galactosidase A) at RP-01 polyclonal positive control levels of 150 μg / mL, 100 μg / mL, and 50 μg / mL. Figure 7 shows that alkaline sample pretreatment increased the tolerance of onboard lysosomal enzyme (α-galactosidase A) up to 2000 ng / mL at 7H11 monoclonal positive control levels of 125 ng / mL, 250 ng / mL, and 1000 ng / mL. The alkaline sample pretreatment used was a low-molarity, pH 12.45 solution (0.02 M Ca(OH)2 buffer) with limited buffering capacity. FIG. 8 shows that a more molarity buffered pH 12.45 solution (2 M NaOH buffer) adversely affected the assay, and no NAb inhibition could be detected.

[0198] Figure 9 shows that a standard anti-lysosomal neutralizing antibody assay (without the low molarity alkaline solution pretreatment step) is not tolerant to high levels of serum lysosomal enzyme (α-galactosidase A) for any of the three positive controls evaluated. Monoclonal antibodies 19D6 and 7H11 showed a loss of NAb inhibition similar to polyclonal antibody RP-01 as on-board lysosomal enzyme increased above 125 ng / mL.

[0199] Figure 10 shows 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).

[0200] Example 5 Anti-lysosomal enzyme total antibody (TAb) assay The anti-lysosomal enzyme total antibody assay was optimized for lysosomal enzyme resistance by testing the binding ability of the 19D6 positive control antibody to plate-bound lysosomal enzyme (α-galactosidase A) in an enzyme-linked immunosorbent assay (ELISA) format, as shown in Figure 11. Polystyrene ELISA plates were coated with 1 μg / mL of lysosomal enzyme in 1X phosphate-buffered saline (PBS) and incubated overnight at 2–8°C. Serum samples were pretreated with an alkaline solution at pH 11 (10 μL sample + 30 μL NaOH for 1 hour at room temperature), neutralized with an acidic solution (30 mM acetic acid), and further diluted to an MRD of 40 in a neutral pH solution (assay buffer: 1% BSA, 0.35 M NaCl, 0.25% CHAPS, 5 mM EDTA, 0.05% Tween 100, or PBS containing <5% BSA). These serum samples are then added to an ELISA plate that has been incubated overnight to allow the anti-lysosomal enzyme antibodies to bind to the lysosomal enzymes on the plate. After incubation and washing the plate, a horseradish peroxidase (HRP)-conjugated anti-human IgG detection antibody is then added to the plate, which, in the presence of TMB substrate, can produce a color signal relative to the amount of anti-lysosomal enzyme antibodies present in the serum.

[0201] Figures 12A and 12B show various unsuccessful acidic pretreatments that did not increase the ability to detect serum anti-lysosomal antibodies in the presence of on-board lysosomal enzyme (α-galactosidase A) levels. Acidic treatments with and without heat did not show any specific signal in the anti-lysosomal enzyme TAb assay (Figure 12A). Acidic treatments with various alkaline neutralizations also failed to alleviate on-board lysosomal enzyme interference, showing high signals in serum samples without any specific anti-lysosomal enzyme antibodies on-board (Figure 12B).

[0202] Figure 13 shows various unsuccessful alkaline pretreatments that did not increase the ability to detect serum anti-lysosomal antibodies in the presence of on-board lysosomal enzyme (α-galactosidase A) levels. Alkaline treatment at pH 12.45 with or without heat showed high nonspecific signals in serum without any on-board anti-lysosomal enzymes. Alkaline treatment at pH 11 with heat treatment eliminated all signal in the assay, limiting the ability to detect any anti-lysosomal enzyme antibodies.

[0203] Figures 14A and 14B show that specific alkaline buffer sample pretreatment (NaOH at pH 11) increases the detection of anti-lysosomal antibodies from positive control antibody levels of 39.0625 ng / mL to 2500 ng / mL using 5 μg / mL of lysosomal enzyme onboard (α-galactosidase A). Standard assay diluent at neutral pH shows a significant loss of 450 nm optical density (OD) when lysosomal enzymes are onboard. This is dramatically improved after pretreatment of serum with alkaline buffer at pH 11, restoring the ability to detect anti-lysosomal enzyme antibody levels in the presence of onboard serum lysosomal enzymes (5 μg / mL of α-galactosidase A) (Figure 14B).

[0204] Figure 15 shows that alkaline pretreatment at pH 11 increases the detection of anti-lysosomal enzyme antibodies in the serum of Fabry patients in the presence of 4 μg / ml of on-board lysosomal enzyme (α-galactosidase A). Alkaline pretreatment at pH 11 does not significantly alter the sensitivity or detection of anti-lysosomal enzyme antibodies in the absence of on-board lysosomal enzyme.

[0205] Figures 16A and 16B show how alkaline base treatment (low molar, (approximately 0.0001 M) NaOH, pH 11) improved total antibody detection in Fabry patient serum, allowing assessment of antibody levels in the presence of on-board lysosomal enzymes (α-galactosidase A). In both Fabry subject #1 and Fabry subject #4, alkaline treatment increased the optical density signal of samples with on-board lysosomal enzymes to a level similar to untreated serum without on-board lysosomal enzymes, demonstrating the successful use of alkaline treatment to increase detection of anti-lysosomal enzyme antibodies in serum.

Claims

1. A method for detecting an anti-lysosomal enzyme antibody in a human subject, comprising measuring the presence of the anti-lysosomal enzyme antibody in a biological sample from the subject, the biological sample being pretreated with a base at a pH of about 11 or higher.

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

3. 3. The method of claim 1 or 2, wherein the base is a non-buffering base.

4. The non-buffering base is NaOH or Ca(OH) ２ The method of claim 3, wherein

5. NaOH or Ca(OH) ２ 5. The method of claim 4, wherein the pH 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. NaOH or Ca(OH) ２ The method of claim 5, wherein is about 0.02M.

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 of any one of claims 1 to 7, wherein the biological sample is diluted by about 2-fold or more of the minimum required dilution (MRD).

9. The biological sample is 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, 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, 4 times, approximately 45 times, approximately 46 times, approximately 47 times, 48 ​​times, approximately 49 times, approximately 50 times, approximately 51 times, approximately 52 times, approximately 53 times, approximately 54 times, approximately 55 times, approximately 56 times, approximately 57 times, approximately 58 times, approximately 59 times, approximately 60 times, approximately 61 times, approximately 62 times, approximately 63 times, approximately 64 times, approximately 65 times, Approx. 66 times, approx. 67 times, approx. 68 times, approx. 69 times, approx. 70 times, approx. 71 times, approx. 72 times, approx. 73 times, approx. 74 times, approx. 75 times, approx. 76 times, approx. 77 times, approx. times, approximately 88 times, approximately 89 times, approximately 90 times, approximately 91 times, approximately 92 times, approximately 93 times, approximately 94 times, approximately 95 times, approximately 96 times, approximately 97 times, approximately 98 times, approximately 99 times, approximately 100 times, approximately 200 times, approximately 300 times, approximately 400 times, approximately 500 times, approximately 600 times, approximately 700 times, approximately 80 times 0 times, approximately 900 times, approximately 1000 times, approximately 1100 times, approximately 1200 times, approximately 1300 times, approximately 1400 times, approximately 1500 times, approximately 1600 times, approximately 1700 times, approximately 1800 times, approximately 1900 times, approximately 2000 times, approximately 2100 times, approximately 2200 times, approximately 2300 times, 9. The method of claim 8, wherein the antibody is diluted by an MRD of 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.

10. 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.

10. The method of claim 1, wherein the pH is 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. 11. The method of claim 10, wherein the pH is 12.

45.

12. 10. The method of 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 of any one of claims 1 to 12, wherein the subject is suffering from a lysosomal storage disease.

14. 14. The method of claim 13, wherein the lysosomal storage disease is selected from the group consisting of Fabry disease, Gaucher disease, Pompe disease, mucopolysaccharidosis (MPS) 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 determined.

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

17. 16. The method of claim 15, further comprising mixing the pre-treated biological sample with a lysosomal enzyme.

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

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

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

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

22. 22. The method of any one of claims 17-21, wherein the pretreated biological sample and the lysosomal enzyme mixture are incubated for at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, or at least about 24 hours.

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

24. 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, 24. The method of any one of claims 16 to 23, wherein the concentration is less than 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.

25. 25. The method of any one of claims 17-24, wherein the pretreated biological sample and the lysosomal enzyme mixture are incubated for at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, or at least about 24 hours.

26. 25. The method of any one of claims 17 to 24, wherein the pretreated biological sample and the lysosomal enzyme mixture are incubated for a period 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. 24. The method of claim 23, wherein the reaction mixture comprises a substrate and / or an inhibitor.

28. The substrate is 4-methylumbelliferyl-α-D-galactopyranoside, 4-methylumbelliferyl-β-D-glucopyranoside, 4-methylumbelliferyl α-D-glucopyranoside, 4-methylumbelliferyl α-L-idopyranoside, 4-methylumbelliferyl-α-L-iduronide 2-sulfate, 4-methylumbelliferyl-2-sulfamino-2-deoxy-α-D-glucopyranoside, 4-methylumbelliferyl 28. The method of claim 27, wherein the 4-methylumbelliferyl-β-D-galactose-6-sulfate, 4-methylumbelliferyl-N-acetyl-α-d-galactoseamid-4-sulfate, 4-methylumbelliferyl-β-d-glucuronide, 4-methylumbelliferyl-α-D-mannopyrronoside, 4-methylumbelliferyl N-acetyl-β-D-glucosamide, and Ala-Ala-Phe-7-amido-4-methyl-coumarin.

29. 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, 29. The method of claim 27 or 28, wherein the ATP is at a concentration of 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. 28. The method of claim 27, wherein the inhibitor comprises N-acetylgalactosamine (GALNAc).

31. 28. The method of claim 27, wherein the inhibitor is at a concentration of 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 of any one of claims 23 to 31, wherein the reaction mixture and the biological sample pretreated with the lysosomal enzyme mixture are mixed in a high-throughput plate.

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

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

35. 35. The method of any one of claims 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. 36. The method of claim 34 or 35, wherein the stop buffer comprises glycine.

37. 37. The method of any one of claims 34-36, wherein the stop buffer is in a volume of less than about 1 mL, less than about 900 uL, less than about 800 uL, less than about 700 uL, less than about 600 uL, less than about 500 uL, less than about 400 uL, less than about 300 uL, less than about 200 uL, or less than about 100 uL.

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

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

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

42. 39. The method of claim 38, further comprising mixing the neutralized pre-treated biological sample with a lysosomal enzyme.

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

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

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

46. The method of 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 of any one of claims 43 to 45, wherein a detection antibody or a lysosomal enzyme with a ruthenylated tag is added to the plate to generate a signal.

48. 48. The method of any one of claims 46 or 47, wherein the detection antibody is an anti-human IgG antibody.

49. 49. The method of any one of claims 46 or 48, further comprising adding a substrate.

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

51. 51. The method of claim 50, wherein the plate is read with a plate reader that detects the colored reaction product and outputs an optical density (OD) value.

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

53. 48. The method of claim 47, wherein the plate is read in a plate reader that detects luminescence from the ruthenylated label and outputs electrochemiluminescence units (ECLu).

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

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

Images