Methods and compositions for treating arginase 1 deficiency

Abstract

The present invention provides a method and composition for treating a subject suffering from arginase 1 (ARG1) deficiency (ARG1-D) and for rapidly reducing the level of at least one of arginine and / or guanidino compounds in the subject.

Description

[0001] This application is a divisional application of Chinese national phase application No. 201880079119.9 of international application No. PCT / US2018 / 063982, with a filing date of December 5, 2018.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of priority to U.S. Provisional Application No. 62 / 594,747, filed December 5, 2017, U.S. Provisional Application No. 62 / 725,612, filed August 31, 2018, and U.S. Provisional Application No. 62 / 745,000, filed October 12, 2018, the entire contents of which are incorporated herein by reference.

[0004] References to sequence listings

[0005] The sequence listing associated with this application is submitted electronically via EFS-Web and is hereby incorporated by reference into this specification in its entirety. The text file containing the sequence listing is named 218107-0011-00-WO_ST25. The text file is 6,247 bytes in size and was created on December 4, 2018. Background Art

[0006] Disclosed are methods of treating arginase 1 deficiency in a patient and compositions for treating arginase 1 deficiency (ARG1-D) in a patient.

[0007] Arginase 1 deficiency (also known as hyperargininemia or argininemia) is caused by a deficiency or inactivity of the arginase 1 protein, such as due to mutations in the arginase 1 (ARG1) gene. ARG1 is a urea cycle enzyme that converts arginine to ornithine. ARG1-D is a rare, progressive disorder estimated to occur in 1 in 300,000 to 1,000,000 individuals. ARG1-D is an autosomal recessive urea cycle disorder that causes the toxic accumulation of arginine and other guanidino compounds (GCs). Clinical features of ARG1-D typically manifest in early childhood but may develop shortly after birth in some affected infants. Manifestations include seizures, developmental delay, seizures, protein avoidance, occasional hyperammonemia, nausea, and vomiting. As the disease progresses during adolescence, patients may become immobile, unable to speak or comprehend, suffer from malnutrition, and develop vitamin D deficiency. The disease and its associated symptoms progress to include liver damage evidenced by elevated transaminases and fibrosis, severe spasticity and muscle contractures, intellectual disability, and a limited lifespan. Unfortunately, dietary restrictions and nitrogen scavengers are insufficient to prevent disease progression due to the chronic, marked elevations in arginine and other guanidine compounds and occasional hyperammonemia.

[0008] When patients with ARG1-D are given a low-protein diet, arginine levels can be reduced to about 265-300 μmol / L (Schlune et al., "Hyperargininemia due to arginase 1 deficiency: the original patients and their natural history, and a review of the literature", AminoAcids 47: 1751-1762, 2015). Arginine de novo synthesis is affected by dietary protein intake, accounting for about 5-15% of plasma arginine synthesis, and the main source of arginine is human tissue conversion (Wu, G. et al., "Argininemetabolism: nitric oxide and beyond", Biochem J, 336 (Pt 1), 1-17, 1998). Therefore, the effect of dietary protein restriction on plasma arginine levels is limited, and due to the inability to degrade excess arginine, the circulating levels of arginine in subjects remain high.

[0009] Nonclinical studies of most therapies in ARG1-D animal models have not shown a substantial effect on disease outcome, even with reduced plasma arginine levels. Given that most of these approaches do not address the root cause of the disease, namely, correcting the cytoplasmic deficiency of ARG1 in the liver, the U.S. Food and Drug Administration (FDA) has questioned whether increasing plasma arginase levels can achieve the same overall effect as correcting intracellular ARG1 activity (including reducing arginine-derived guanidino compounds, which may play a role in disease pathogenesis). Although there is literature evidence that dietary arginine restriction can reduce plasma arginine levels, the beneficial effect of dietary approaches on disease manifestations is limited due to the chronic and significant elevation of plasma arginine levels, which are well above the physiological normal range and medical indicators. This is supported by a study group that found that despite dietary treatment and reduction of plasma arginine, 4 of 11 patients still experienced progressive spasticity (Prasad et al., "Argininemia: Atreatable genetic cause of progressive spastic diplegia simulating cerebral palsy - case reports and literature review", J. Child Neurol. 12:301-309, 1997).Based on this principle, it has also been suggested that high levels of arginine metabolites, such as argininic acid, guanidinoacetic acid, β-guanidinopropionic acid, β-guanidinobutyric acid and N-α-acetylarginine, may play an important role in causing the neurological sequelae in patients with ARG1 deficiency (Deignan et al., "Increased plasma and tissue guanidine compounds in a mouse model of hyperargininemia", Mol. Genet. Metab. 93:172-178, 2008; Segawa et al., "Along-term survival case of arginase deficiency with severe multicystic white matter and compound mutations", Brain Dev. 33:45-48, 2011; Wyse et al., "Invitro stimulation of oxidative stress in cerebral cortex of rats by theguanidino compounds accumulating in hyperargininemia", Brain Res. 2001, 923(1-2):50-7). Given the potential importance of guanidine compounds in ARG1-D, a low-arginine diet has been studied for its ability to reduce these potentially neurotoxic metabolites (Lambert et al., "Hyperargininemia: intellectual and motor improvement related to changes in biochemical data", J. Pediatr., 1991, 118(3):420-4). Although Lambert et al. were able to demonstrate that a low-arginine diet reduced blood arginine levels within approximately one month of strict dietary restriction, levels of guanidinoacetate and α-keto-δ-guanidinovaleric acid (GVA) did not change, indicating that the diet did not demonstrate potential as a therapeutic intervention to rapidly control GC levels. Given the condition and the complex underlying mechanisms, new treatments are needed to rapidly reduce and control high arginine levels in patients to address this unmet medical need. Summary of the Invention

[0010] Newly disclosed herein are methods, compounds, and compositions for treating patients with ARG1-D, which preferably achieve a rapid response in the patient, wherein the patient is a human and can be an adult, child, or infant.

[0011] A method of treating arginase 1 (ARG1) deficiency (ARG1-D) in a subject is provided, the method comprising administering to the subject an amount of arginase sufficient to reduce the subject's plasma level of arginine to less than 200 μmol / L within about 2 to about 4 days after the initial administration of the arginase. After the initial administration of the arginase, the subject's plasma level of arginine can be reduced to a range of 40 μmol / L to 115 μmol / L. The subject can be a human adult, human child, or human infant (e.g., less than 12 months old) with ARG1-D.

[0012] The arginase can be wild-type arginase I (e.g., SEQ ID NO: 2) or arginase II (e.g., SEQ ID NO: 1). The arginase can be PEGylated arginase 1, and it can have a cobalt metal cofactor instead of a manganese metal cofactor. The PEGylated arginase 1 can be a PEGylated arginase.

[0013] The disclosed methods of treatment may further include administering arginase such that the plasma level of at least one of N-α-acetylarginine (NAArg), argininic acid (ArgA), GVA, guanidinoacetic acid (GAA), and arginine in the subject is reduced to normal levels at least once within less than 7 days, 3 days, 2 days, and / or 1 day after the initial administration of arginase. The methods of treatment using arginase may result in the plasma level of GAA reaching normal levels after administration. A method wherein an amount sufficient to reduce the plasma level of arginine in a subject improves one or more characteristics such as resting spasticity, leg cramps associated with spasticity, adaptive behavior, and Patient-Reported Outcomes Measurement Information System (PROMIS) physical function scores.

[0014] A method of treatment contemplates administering an intravenous dose of about 0.005 to about 1.00 mg / kg of patient body weight. Other ranges or uses for intravenous administration may include 0.01 to 0.5 mg / kg, 0.01 to 0.2 mg / kg, 0.015 to 0.25 mg / kg, and 0.015 to 0.075 mg / kg, with each 0.005 increment between the recited ranges also contemplated.

[0015] A method of treatment contemplates administering a subcutaneous dose of about 0.01 to about 1.50 mg / kg of patient body weight. Other ranges or uses for intravenous administration may include 0.015 to 0.75 mg / kg, 0.015 to 0.30 mg / kg, 0.015 to 0.25 mg / kg, and 0.015 to 0.075 mg / kg, with each 0.005 increment between the recited ranges also contemplated.

[0016] Another method contemplates administering arginase to a subject at a dose sufficient to reduce the plasma level of at least one compound in the subject by at least 2-fold, the at least one compound selected from the group consisting of ArgA, NAArg, GVA, GAA, and arginine, wherein the plasma level is determined approximately 24 to 48 hours after administration. Another method contemplates administering a dose that reduces the plasma level of NAArg by at least 3-fold. Another method contemplates administering a dose that reduces the plasma level of GAA by at least 2-fold. The arginase of the disclosed methods of treatment can be administered to the patient intravenously or subcutaneously, or a combination of the two. The arginase can be administered to the subject daily, weekly, twice monthly, or monthly.

[0017] A nitrogen scavenger may also be administered to the subject. The disclosed compositions containing arginase may comprise a nitrogen scavenger. The disclosed treatment methods using arginase may be administered to a subject together with a nitrogen scavenger, and optionally, the subject may further be on a low-arginine diet. Another method of treating a subject with arginase comprises administering a nucleic acid operably linked to an adenoviral vector for delivery to the subject, and the arginase produces arginase in the subject after administration to the subject. The amount of arginase produced by adenoviral vector delivery is therapeutically effective to reduce the plasma level of at least one compound in the subject by at least 2-fold, the at least one compound selected from the group consisting of: ArgA, NAArg, GVA, GAA, and arginine, wherein the plasma level is measured approximately 24 to 48 hours after administration of the arginase.

[0018] Another method anticipates improving neuromotor function after the initial administration of arginase. Neuromotor function can be, but is not limited to, one or more of stepping, walking, spasticity, and / or alertness. Another method anticipates that after the initial administration of arginase, the subject will exhibit at least one of: reduced resting spasticity, fewer leg cramps associated with spasticity, adaptive behavior, and improved PROMIS T score, compared to at least one of spasticity, behavior, and PROMIS T score before administration of arginase. One method anticipates that an acute response to arginase treatment will result in a reduction in or clearance of one or more toxic metabolites (e.g., GAA).

[0019] The method anticipates administering at least one repeated dose of arginase to the subject to reduce the plasma level of arginine to less than 200 μmol / L. The plasma level of arginine can be reduced to a level less than 200 μmol / L for at least 30 weeks and / or at least 40 weeks. After receiving at least one repeated dose eight times, the subject can show at least one of the following improvements relative to the baseline of the subject's mobility or adaptive behavior before treatment: (a) mobility or (b) adaptive behavior. The plasma level of at least one of NAArg, ArgA, GVA, GAA or arginine can be reduced compared to the baseline plasma level in the subject. Another method anticipates that after 9 days of treatment, the subject's minimum clinically important difference (MCID) is greater than 1. The plasma level of arginine is related to the MCID.

[0020] Also contemplated is a composition comprising a polyethylene glycol arginase and a pharmaceutically acceptable buffer. The composition may comprise about 10% glycerol, and the pharmaceutically acceptable buffer may be phosphate-buffered saline in addition to the desired amount of polyethylene glycol arginase or other arginase. Provided is the use of a composition comprising an arginase (e.g., polyethylene glycol arginase) or a nucleic acid encoding an arginase in the preparation of a medicament for treating ARG1-D.

[0021] It is also contemplated to formulate arginase (such as pegylated arginase) in red blood cell ghosts.

[0022] A method for rapidly reducing the plasma level of at least one compound selected from the group consisting of arginine, N-α-acetylarginine (NAArg), argininic acid (ArgA), α-keto-δ-guanidinovaleric acid (GVA), guanidinoacetic acid (GAA) in a subject with arginase 1 (ARG1) deficiency (ARG1-D) to normal levels, the method comprising administering to the subject a composition comprising a therapeutically effective amount of a PEGylated arginase, wherein the PEGylated arginase is initially administered intravenously at 0.005 mg / kg to 1.00 mg / kg and then administered subcutaneously or intravenously to the subject weekly. Another method contemplates administering the PEGylated arginase initially at 0.005 mg / kg to 0.50 mg / kg intravenously. Another method contemplates administering the PEGylated arginase initially at 0.005 mg / kg to 0.20 mg / kg intravenously. Another method contemplates administering the PEGylated arginase as a PEGylated arginase. Another method contemplates reducing the plasma level of at least one compound selected from the group consisting of ArgA, NAArg, GVA, GAA, and arginine in a subject to normal levels in less than 3 days, 2 days, and / or 1 day after the initial administration of the PEGylated arginase. Another method contemplates administering to the subject a dose of the PEGylated arginase sufficient to reduce the plasma level of at least one compound selected from the group consisting of ArgA, NAArg, GVA, GAA, and arginine by at least 2-fold, wherein the plasma level is determined about 24 to 48 hours after administration of the PEGylated arginase.

[0023] A method for rapidly reducing the plasma level of at least one compound selected from the group consisting of arginine, N-α-acetylarginine (NAArg), argininic acid (ArgA), α-keto-δ-guanidinovaleric acid (GVA), guanidinoacetic acid (GAA) in a subject with arginase 1 (ARG1) deficiency (ARG1-D) to normal levels, the method comprising administering to the subject a composition comprising a therapeutically effective amount of PEGylated arginase, wherein the PEGylated arginase is initially administered at 0.01 mg / kg to 1.50 mg / kg subcutaneously and then administered to the subject weekly subcutaneously or intravenously. Another method contemplates administering the PEGylated arginase initially at 0.015 mg / kg to 0.75 mg / kg subcutaneously. Another method contemplates administering the PEGylated arginase initially at 0.015 mg / kg to 0.30 mg / kg subcutaneously. Another method contemplates reducing the plasma level of at least one compound selected from the group consisting of ArgA, NAArg, GVA, GAA, and arginine in a subject to normal levels in less than 3 days, 2 days, and / or 1 day after the initial administration of the PEGylated arginase. Another method contemplates administering to the subject a dose of the PEGylated arginase sufficient to reduce the plasma level of at least one compound selected from the group consisting of ArgA, NAArg, GVA, GAA, and arginine by at least 2-fold, wherein the plasma level is determined about 24 to 48 hours after administration of the PEGylated arginase. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1. Figure 1A Arginine, Figure 1B argininic acid (ArgA), Figure 1C GVA and Figure 1DPlasma concentrations of N-α-acetylarginine (NAArg). Arginine levels are the mean values ​​obtained using a validated (Good Laboratory Practice, or GLP) assay from two ARG1-D patients treated with 0.03 mg / kg of pegylated arginase. ArgA, GVA, and NAArg levels were determined using a non-GLP assay using pooled samples from the same two ARG1-D patients treated with 0.03 mg / kg of pegylated arginase as described above. Normal ranges in humans are indicated by an "*". The normal range of arginine in human serum is 40 μmol / L to 115 μmol / L. See, e.g., Lüneburg, N. et al., "Reference intervals for plasma L-arginine and the L-arginine:asymmetricdimethylarginine ratio in the Framingham Offspring Cohort." J.Nutr. 141(12):2186-2190(2011). The normal range for ArgA is <0.025 μmol / L to 0.100 μmol / L; the normal range for GVA is <0.050; and the normal range for NAArg is <0.025 μmol / L to 0.255 μmol / L. See, e.g., Marescau et al., “Guanidino compound analysis as a complementary diagnostic parameter for hyperargininemia: Follow-up of guanidino compound levels during therapy,” Pediatric. Res. 27(3):297-303(1990).

[0025] Figure 2 shows Figure 2A Arginine and Figure 2BPlasma concentration of homoarginine (HArg). Arginine levels are the mean values ​​obtained using a validated (GLP) assay from two ARG1-D patients treated with 0.03 mg / kg pegylated arginase. HArg values ​​were obtained using a non-GLP assay using pooled samples from the same two ARG1-D patients treated with 0.03 mg / kg pegylated arginase as described above. "*" indicates the normal range in human serum as described in Lüneburg, N. et al. (2011) for arginine or Marescau et al. (1990) for guanidino compounds. The normal range for HArg is <0.500 μmol / L to 2.80 μmol / L.

[0026] Figure 3 shows Figure 3A Arginine, Figure 3B argininic acid (ArgA), Figure 3C GVA and Figure 3D Plasma concentrations of N-α-acetylarginine (NAArg). Arginine levels are the mean of those obtained using a validated (GLP) assay from two ARG1-D patients treated with 0.015 mg / kg ("●") and 0.03 mg / kg PEGylated arginase ("□"). ArgA, GVA, and NAArg levels were obtained using a non-GLP assay using pooled samples from the same two ARG1-D patients treated with 0.015 mg / kg and 0.03 mg / kg PEGylated arginase as described above. "*" indicates the normal range in human serum as described in Lüneburg, N. et al., (2011) for arginine or in Marescau et al., (1990) for guanidino compounds.

[0027] Figure 4A and Figure 4B Two patients, Patient 120-101 ( Figure 4A ) and patient 120-102( Figure 4B ). In both cases, data for GAA levels at screening and during dosing in Parts 1 and 2 are shown. "FUP" indicates follow-up. The normal range for GAA is 0.400 μmol / L to 3.00 μmol / L. See, e.g., Marescau et al., (1990).

[0028] Figure 5A The effects of arginase treatment on plasma levels of arginine and GC were examined in part 1 of the clinical study, which used a single dose (NAA refers to NAArg). Figure 5B The effects of arginase treatment on plasma levels of arginine and GC were examined in Part 2 of the clinical study, which employed repeated doses. The data presented for Part 2 include patients who received all eight (8) doses according to the clinical protocol. Figure 5A and 5B In both cases, "a" represents the baseline arginine level in the patient, "b" represents the lowest point after dosing, "c" represents the level 7 days after dosing, "d" represents the arginine level before dosing, "e" represents the level after all doses, and "f" represents the normal range of arginine plasma levels in healthy patients, which is 40 μmol / L to 115 μmol / L as described in Lüneburg, N. et al. (2011), and "n" represents the number of patients. The lowest point after dosing is the lowest value after dosing in Part 1; "after all doses" in Part 2 includes all values ​​after dosing, except the value just before the next dose.

[0029] Figure 6 Results of the 6-minute walk test are shown for three patients (i.e., patients 1 ("a"), 2 ("b"), and 5 ("c") at baseline, dose 8, dose 20, and dose 32 (data were not available for patient 5 ("c") at doses 20 and 32). Doses 8, 20, and 32 were initiated with repeated doses.

[0030] Figure 7 An overview of the Phase 1 / 2 study and the open-label extension study is provided.

[0031] Figure 8 Repeated dosing of PEGylated arginase resulted in time-dependent improvements in plasma arginine in patients in Part 2 and the open-label extension study. "BL" is baseline; "F / U" is follow-up; "OLE" is the open-label extension study, and "n" is the number of patients at each time point. The goal was to maintain plasma arginine at ≤200 μmol / L. 100% (5 / 5) of patients who completed Part 2 achieved consistent post-decline arginine levels that were below the recommended targets. The decrease in arginine was accompanied by a sustained reduction in plasma GCs (GVA, ArgA, NAA, and GAA).

[0032] Figure 9 .After only 8 repeated doses, pegarginase treatment improved clinical outcomes from baseline. Figure 9 Each point in the graph represents an evaluation of a patient. A favorable evaluation is represented by a solid circle "●", while a neutral evaluation is represented by an open circle "○".

[0033] Figure 10Arginine concentrations over a nine-week period are shown for patients who responded to pegylated arginase treatment (responders) and patients who did not respond to pegylated arginase treatment (non-responders), based on the patient's MCID improvement. The range of plasma arginine above the medical threshold is ≥200 μmol / L; as described in Lüneburg, N. et al. (2011), the normal range of arginine in humans is 40 μmol / L to 115 μmol / L. The MCID was determined as follows. A "responder" can be an individual with an MCID > 1; a non-responder can be an individual with an MCID < 1. Non-responder data points are represented by closed diamonds; responder data points are represented by open diamonds. The average of the non-responder data points is represented by line "a," and the average of the responder data points is represented by line "b." DETAILED DESCRIPTION

[0034] In seeking approval for the studies disclosed in the examples below, the U.S. Food and Drug Administration (FDA) concluded that the scientific literature did not support a clear and consistent relationship between lowering plasma arginine levels through dietary arginine restriction and objective improvement in disease progression in patients with ARG1-D. Initially, the FDA stated that they did not see the prospect of direct clinical benefit from administering pegylated arginase to patients with ARG1-D. The FDA raised concerns about the ability of pegylated arginase and similar drugs to address the underlying enzyme defect in patients with ARG1-D. The FDA questioned whether peripheral circulation of arginase-containing drugs would have any realistic effect on the metabolism of arginine levels in the liver and the production of related arginine metabolites. As a result, the FDA initially delayed testing in pediatric patients until treatment could be conducted in adult patients, at which time the FDA allowed testing to continue in pediatric patients.

[0035] Surprisingly, a method for treating patients with ARG1 deficiency was discovered that rapidly reduced the levels of arginine and at least one guanidinyl compound (NAArg) to within normal levels within 3 days (Figure 1).

[0036] definition

[0037] As used herein, the terms "treating," "to treat," or "treatment" include inhibiting, slowing, halting, alleviating, ameliorating, or reversing the progression or severity of an existing symptom, disorder, condition, or disease associated with ARG1-D. Treatment may be performed prophylactically or therapeutically.

[0038] The term "effective amount" as used herein refers to the amount of wild-type arginase or pegylated arginase (such as pegylated arginase) administered that will have the desired effect, such as reducing the plasma level of one or more of the following compounds: arginine, argininic acid (ArgA), GVA, N-α-acetylarginine (NAArg), GAA, and homoarginine (HArg). The effective amount may vary depending on factors such as the patient's weight. For example, for intravenous administration of pegylated arginase, the effective amount may be in the range of 0.005 to 1.00 mg / kg of patient body weight (including every 0.005 value between those ranges). An effective amount of pegylated arginase for subcutaneous (sc) administration to an ARG1-D patient includes 0.01 to 1.50 mg / kg of patient body weight (including every 0.01 value between those ranges). One example is to administer a compound to achieve a range of arginine, ArgA, GVA, GAA, and / or NAArg levels that is equivalent to the normal level of each compound in a normal person who does not have ARG1 deficiency. The effective amount can also improve muscle strength, the patient's walking ability (i.e., the ability to run, walk, ride a bicycle, climb stairs without support), and improve cognitive ability (e.g., Wechsler Intelligence Scale for Children (WISC) test improvement) and / or adaptive behavior (e.g., Adaptive Behavior Assessment Scale (ABAS) or Vineland Adaptive Behavior Scale (VABS) test improvement) (Lopata et al., "Comparison of Adaptive Behavior Measures for Children with HFASDs," Autism Research and Treatment, Vol. 2013, pp. 1-10, (2013)). Normal levels of guanidine compounds are provided in the table below. Normal levels of arginine are described by Lüneburg, N. et al. (2011).

[0039] The plasma level of arginine and / or guanidino compound can be reduced to a normal range or normal level, which can mean that at some point after administration of an initial dose and / or repeated doses of arginase, the plasma level of arginine and / or one or more guanidino compounds is within the range provided in the table below or within the range of 40 μmol / L to 115 μmol / L of arginine as described in Lüneburg, N. et al. (2011). For example, during treatment with an arginase as disclosed herein, a patient's plasma level of arginine and / or one or more guanidino compounds may fluctuate within or exceed the normal range. It is considered that the patient's measured plasma level of arginine and / or guanidino compound has been reduced to a normal level or within a normal range. As another example, after receiving one or more doses of an arginase as disclosed herein, the patient's average plasma level of arginine and / or one or more guanidino compounds can be within a normal range (e.g., as provided in the table below or within the range of 40-115 μmol / L of arginine as described in Lüneburg, N. et al. (2011)). Thus, the compositions and methods disclosed herein can reduce the plasma levels of arginine and / or guanidinyl compounds in a subject to normal levels or a normal range at least once after the patient receives an initial dose and / or repeated doses of arginase. In some patients, it has been observed that according to the methods disclosed herein, the plasma levels of arginine and / or guanidinyl compounds can be maintained at average normal levels or within a normal range.

[0040] Table 1: Normal ranges of guanidine compounds in human serum, urine, and cerebrospinal fluid (CSF)

[0041]

[0042] * = detection limit. Quantity discussed in Marescau et al., (1990).

[0043] Wild-type arginase can be based on human arginase I or arginase II. Wild-type human arginase II has the following sequence (Uniprot P78540):

[0044] MSLRGSLSRLLQTRVHSILKKSVHSVAVIGAPFSQGQKRKGVEHGPA

[0045] AIREAGLMKRLSSLGCHLKDFGDLSFTPVPKDDLYNNLIVNPRSVGL

[0046] ANQELAEVVSRAVSDGYSCVTLGGDHSLAIGTISGHARHCPDLCVV

[0047] WVDAHADINTPLTTTSSGNLHGQPVSFLLRELQDKVPQLPGFSWIKPC

[0048] ISSASIVYIGLRDVDPPEHFILKNYDIQYFSMRDIDRLGIQKVMERTFD

[0049] LLIGKRQRPIHLSFDIDAFDPTLAPATGTPVVGGLTYREGMYIAEEIH

[0050] NTGLLSALLDLVEVNPQLATSEEEAKTTANLAVDVIASSFGQTREGGHIVYDQLPTPSSPDESENQARVRI (SEQ ID NO: 1).

[0051] The wild-type human arginase I sequence has the following sequence (Uniprot / P05089):

[0052] MSAKSRTIGIIGAPFSKGQPRGGVEEGPTVLRKAGLLEKLKEQECDV

[0053] KDYGDLPFADIPNDSPFQIVKNPRSVGKASEQLAGKVAEVKKNGRIS

[0054] LVLGGDHSLAIGSISGHARVHPDLGVIWVDAHTDINTPLTTTSGNLH

[0055] GQPVSFLLKELKGKIPDVPGFSWVTPCISAKDIVYIGLRDVDPGEHYI

[0056] LKTLGIKYFSMTEVDRLGIGKVMEETLSYLLGRKKRPIHLSFDVDGL

[0057] DPSFTPATGTPVVGGLTYREGLYITEEIYKTGLLSGLDIMEVNPSLGKTPEEVTRTVNTAVAITLACFGLAREGNHKPIDYLNPPK(SEQ ID NO:

[0058] 2).

[0059] The PEGylated arginase disclosed herein has the sequence of arginase I, SEQ ID NO: 2, with a cobalt metal cofactor in place of a manganese metal cofactor. PEGylated arginase is also PEGylated as described in US Patent No. 8,440,184.

[0060] "Administering" refers to injecting a therapeutically effective amount of a disclosed compound or composition containing the compound. For example, but not limited to, administration can be intravenous (iv) or subcutaneous (sc). The compositions of the present invention can also be administered intramuscularly (im).

[0061] The term "about" is understood by persons of ordinary skill in the art and will vary to some extent depending on the context in which it is used. As used herein, "about" is intended to encompass variations of ±10%, ±5%, or ±1%.

[0062] The term "PEGylation" refers to conjugation with polyethylene glycol (PEG), which is widely used as a drug carrier due to its high biocompatibility and ease of modification (see, for example, Harris et al., Clin. Pharmacokinet. 40(7):539-51, 2001). PEG can be coupled (e.g., covalently linked) to active agents via the hydroxyl groups at the chain termini and by other chemical methods; however, PEG itself is limited to a maximum of two active agents per molecule. In a different approach, copolymers of PEG and amino acids have been investigated as novel biomaterials that would retain the biocompatibility properties of PEG but would have the additional advantage of having many attachment points per molecule (thus providing a greater drug loading capacity).

[0063] PEGylated arginase variants can be formulated according to known methods to prepare pharmaceutically useful compositions. Wild-type arginase proteins (arginase I or arginase II) that naturally contain a manganese metal cofactor can be administered to ARG1-D patients, or wild-type arginase proteins that have been PEGylated and contain a manganese metal cofactor can be administered. In another example, arginase with a cobalt metal cofactor instead of the natural manganese metal cofactor can be administered to ARG1-D patients. Arginase containing a cobalt metal cofactor can be further PEGylated, an exemplary form being Co-ArgI-PEG (also referred to herein as AEB1102, PEGylated arginase, or Co-hArgI), such as described in U.S. Patent No. 8,440,184, which is incorporated herein by reference. One exemplary form, such as PEGylated arginase, has approximately twelve 5K (5000 Dalton) PEG units per monomer attached to one or more lysines present in the protein sequence of PEGylated arginase. The desired formulation is a stable lyophilizate that can be reconstituted with an appropriate diluent or a high-purity aqueous solution with optional pharmaceutically acceptable carriers, preservatives, excipients, or stabilizers (see Remington, The Science and Practice of Pharmacy, 19th ed., Gennaro, ed., Mack Publishing Co., Easton, PA 1995). The drug can be formulated for delivery in the form of erythrocyte ghosts (also known as engineered erythrocytes). Another approach is to administer arginine deiminase (ADI)-PEG 20 (Polaris Pharma) to ARG1-D patients intramuscularly, subcutaneously, or intravenously, or using the erythrocyte ghosts.

[0064] As used herein, the term "portion" when referring to a protein (as in "a portion of a given protein") refers to a fragment of arginase, wherein the fragment has arginase activity in the urea cycle.

[0065] As used herein, the terms "protein / albumin" and "polypeptide" refer to compounds comprising amino acids linked by peptide bonds and are used interchangeably.

[0066] As used herein, the term "fusion protein" refers to a chimeric protein comprising a protein of interest (i.e., human arginase or a variant thereof) linked (or operably linked) to an exogenous protein fragment (a fusion partner composed of a non-arginase protein). The fusion partner can enhance serum half-life, solubility, or both. It can also provide an affinity tag (e.g., a His tag) to allow purification of the recombinant fusion protein from host cells or culture supernatant, or both.

[0067] The terms "in operable combination," "in an operable order," and "operably linked" refer to the linkage of nucleic acid sequences in such a manner as to produce a nucleic acid molecule capable of directing the transcription of a given gene and / or the synthesis of a desired protein molecule. The term also refers to the linkage of amino acid sequences in such a manner as to produce a functional protein.

[0068] As used herein, the term "K m ” refers to the Michaelis-Menten constant of an enzyme and is defined as the concentration of a specific substrate at which a given enzyme produces half of its maximum rate in an enzyme-catalyzed reaction.

[0069] As used herein, the term "k cat ” refers to the turnover number or number of substrate molecules converted into product per unit time per enzyme site, where the enzyme is working at its highest efficiency.

[0070] As used herein, the term "K cat / K m ” refers to the specificity constant, which is a unit of measurement of the efficiency of an enzyme in converting a substrate into a product.

[0071] The term "Mn-hArgI" refers to human arginase I with a Mn(II) metal cofactor. The term "Co-hArgI" refers to human arginase I (mutant or native) with a Co(II) metal cofactor.

[0072] The term "IC 50 ” is the half-maximal (50%) inhibitory concentration (IC) and is therefore a measure of effectiveness.

[0073] The term "gene" refers to a DNA sequence that contains the control and coding sequences necessary to produce a polypeptide such as arginase or its precursor. The polypeptide is encoded by the full-length coding sequence or any portion of the coding sequence, as long as the required enzymatic activity of arginase to reduce arginine to ornithine is retained.

[0074] The term "subject" refers to animals, such as mammals, including humans.

[0075] The term "wild-type" refers to a gene or gene product that has the characteristics of that gene or gene product when isolated from a natural source. A wild-type gene is the gene most commonly observed in the human population and is therefore arbitrarily designated as the "normal" or "wild-type" form of the gene. In contrast, the term "modified" or "variant" or "mutant" refers to a gene or gene product that exhibits modifications in sequence and / or functional properties (i.e., altered characteristics) compared to a wild-type gene or gene product. It should be noted that naturally occurring mutants can be isolated; they are identified by the fact that they have altered properties compared to a wild-type gene or gene product.

[0076] This article uses the following abbreviations:

[0077] Abbreviations

[0078] ADA anti-drug antibodies

[0079] ARG or Arg Arginine

[0080] ARG1-D Arginase 1 Deficiency

[0081] Arg1- / - mice with arginase deficiency

[0082] ArgA argininic acid

[0083] AUC Area under the plasma concentration-time curve

[0084] BQL Below the limit of quantitation

[0085] Co-ArgI-PEG Cobalt-substituted and PEGylated arginase 1

[0086] EOI End of Infusion

[0087] F / U follow-up

[0088] GC Guanidine compounds

[0089] GLP Good Laboratory Practice

[0090] GVA α-keto-δ-guanidinovaleric acid

[0091] GAA Guanidine acetic acid

[0092] HArg homoarginine

[0093] iv intravascular or intravascular (IV)

[0094] K2EDTA plasma tubes treated with dipotassium EDTA to prevent clotting

[0095] LC-MS / MS liquid chromatography-tandem mass spectrometry

[0096] NAArg N-acetylarginine

[0097] PEG polyethylene glycol

[0098] sc subcutaneous, subcutaneous or SC

[0099] SOI Infusion Start

[0100] wt wild type

[0101] Pegylated arginase, a recombinant human arginase produced in Escherichia coli (E. Coli), was studied to see if it could reduce arginine levels to normal in neonatal mice in an adult mouse model of ARG1 deficiency. Pegylated arginase was pegylated for stability and half-life and had a cobalt metal cofactor instead of a manganese metal cofactor. The cobalt metal cofactor increased catalytic activity and improved the stability of arginase. Although pegylated arginase treatment did reduce plasma arginine and total brain arginine, administration of the drug failed to treat hyperammonemia in mice. Theoretically, the absence of a decrease in arginine levels in liver tissue could be because the administered enzyme, pegylated arginase, did not enter the liver. In the adult mouse model of ARG1-D, administration of pegylated arginase failed to improve animal survival. Pegylated arginase was tested in newborn mice with arginase I deficiency (Arg1- / - mice), the closest model of human disease in mice (Burrage et al., “Human recombinant arginase enzyme reduces plasma arginine in mouse models of arginase deficiency,” Hum. Mol. Genetics 24(22):6417-27 (2015)). Multiple-dose studies reduced plasma and brain arginine levels to normal ranges; however, compared with untreated Arg1 mice, the - / - Unexpectedly, liver arginine levels were not beneficially affected and survival was not improved compared with controls.

[0102] Unlike the clinical manifestations of human arginase I deficiency, Arg1 - / -Mice develop severe hyperammonemia, which appears to be the cause of their premature death (see, e.g., Carvalho, DR et al., “Clinical features and neurologic progression of hyperargininemia,” Pediatr. Neurol., 46(6):369-74 (2012)). Hyperammonemia is a complication that is less severe in human patients with this condition. Given that the primary therapeutic challenge in human patients with arginase I deficiency is elevated plasma arginine rather than hyperammonemia, it has been suggested that Co-ArgI-PEG may have therapeutic utility, but the extent of its utility and response cannot be reasonably predicted, and even though the hypothesis was proposed in 1995, no tests or treatment models have been conducted or developed. See, e.g., Uchino, T. et al., “Molecular basis of phenotypic variation in patients with argininemia,” Hum. Genet. 96(3):255-60 (1995).

[0103] I. Arginase

[0104] Wild-type arginase is a manganese-containing enzyme. It is the final enzyme in the urea cycle. Arginase is the fifth and final step in the urea cycle, a series of biophysical reactions in mammals that release harmful ammonia. Specifically, arginase converts L-arginine into L-ornithine and urea.

[0105] L-arginine is a nitrogen-supplying substrate for nitric oxide synthase (NOS), which can produce L-citrulline and nitric oxide (NO). M (2-5mM) than L-arginine NOS K M Arginase I is much higher than that of NOS (2-20 μM), but it may also play a role in regulating NOS activity. Under certain conditions, arginase I is Cys-S-nitrosylated, resulting in a higher affinity for L-arginine and reduced utilization of NOS substrates.

[0106] Arginase is a homotrimeric enzyme with an α / β-sheet consisting of eight parallel β-sheets surrounded by several helices. The enzyme contains a dinuclear metal cluster that is essential for the generation of the hydroxide required for the nucleophilic attack on the guanidine carbon of L-arginine. The natural metal cofactor of arginase is Mn 2+ These Mn 2+ The ions coordinate to the water, orienting and stabilizing the molecule and enabling the water to act as a nucleophile and attack the L-arginine, hydrolyzing it to ornithine and urea.

[0107] Mammals have two arginase isoenzymes (EC 3.5.3.1) that catalyze the hydrolysis of L-arginine into urea and L-ornithine. The arginase I gene is located on chromosome 6 (6q23) and is highly expressed in the cytoplasm of hepatocytes, where it functions to denitrify the final step of the urea cycle. The arginase II gene is found on chromosome 14 (14q24.1). Arginase II is mitochondrially localized in tissues such as the kidney, brain, and skeletal muscle, and is thought to provide a supply of L-ornithine for the biosynthesis of proline and polyamines (Lopez et al., FEBS J. 272:4540-48, 2005).

[0108] Arginase has been studied for nearly 50 years as a method for degrading extracellular L-arginine (Dillon et al., "Biochemical characterization of the arginine degrading enzymes arginase and arginine deiminase and their effect on nitric oxide production," Med. Sci. Monit., 8(7): BR248-253 (2002). Although native arginase is cleared from the circulation within minutes (Savoca et al., Cancer Biochem. Biophys. 7: 261-268, 1984), a single injection of PEG-arginase MW 5,000 in rats is sufficient to achieve near-complete arginine depletion within about 3 days (Cheng et al., Cancer Res. 67: 309-17, 2007).

[0109] The bacterial arginine hydrolase ADI has been tested in vitro and has shown promising kinetics and stability. Unfortunately, ADI is a bacterial enzyme and, therefore, induces strong immune responses and adverse reactions in most patients, making it unsuitable for long-term administration in patients with ARG1-D, who require regular dosing.

[0110] For clinical use in patients with ARG1-D, arginase must be modified to allow it to persist in the circulation for extended periods (e.g., several days). Without any modification, human arginase has a half-life of only a few minutes in the circulation, primarily because it is not large enough to avoid filtration by the kidneys. Unmodified human arginase is readily inactivated in serum and has a degradation half-life of only 4 hours.

[0111] II. Pegylation of Arginase Variants

[0112] In certain aspects of the present invention, methods and compositions related to PEGylated arginase are disclosed. Specifically, PEGylation of arginase at an engineered cysteine ​​residue (e.g., replacing the third residue from the N-terminus) can be used to produce a homogeneous PEGylated arginase composition. Also disclosed are methods for isolating PEGylated arginase based on temporary disruption of polymerization.

[0113] PEGylation is the process of covalently attaching a PEG polymer chain to another molecule, typically a drug or therapeutic protein. PEGylation can be achieved by incubating a reactive derivative of PEG with the target macromolecule. This increases the hydrodynamic size (size in solution) of the drug or therapeutic protein, extending its circulation time by reducing renal clearance. PEGylation can also impart water solubility to hydrophobic drugs and proteins.

[0114] The first step in PEGylation can be the appropriate functionalization of a PEG polymer at one or both terminal domains of a protein or within an opposing amino acid such as lysine. PEGs that are activated at each terminus with the same reactive moiety are referred to as "homobifunctional," while PEG derivatives are referred to as "heterobifunctional" or "heterofunctional" if the functional groups present are different. Chemically active or activated derivatives of PEG polymers are prepared to attach PEG to the desired molecule.

[0115] The selection of suitable functional groups for PEG derivatives is based on the type of available reactive groups on the molecule to be coupled to PEG. For proteins, typical reactive amino acids include lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, and tyrosine. The N-terminal amino group and the C-terminal carboxylic acid can also be used to connect PEG to polypeptides.

[0116] The technology for forming PEG derivatives includes reacting the PEG polymer with a group that can react with a hydroxyl group (usually anhydrides, acid chlorides, chloroformates and carbonates). Pegylation chemistry can also use functional groups that can be used for conjugation, such as aldehydes, esters, amides, etc. Heterobifunctional PEGs are very suitable for connecting two entities where hydrophilicity, flexibility and biocompatibility spacers are required. Preferred end groups for heterobifunctional PEGs are maleimides, vinyl sulfones, disulfide pyridines, amines, carboxylic acids and H-hydroxysuccinimide (NHS) esters.

[0117] The most common modifier or linker is based on the methoxy PEG (mPEG) molecule. Its activity depends on the addition of a protein-modifying group to the alcohol terminus. Polyethylene glycol (PEG diol) can be used as a precursor molecule; the diol is then modified at both ends to produce heterodimeric or homodimeric PEG-linked molecules (as shown in the example of PEG bisvinyl sulfone).

[0118] Proteins are typically PEGylated at nucleophilic sites, such as unprotonated thiols (cysteinyl residues) or amino groups. Examples of cysteinyl-specific modification reagents include PEG maleimide, PEG iodoacetate, PEG thiol, and PEG vinyl sulfone. All four have strong cysteinyl specificity under mild conditions and neutral to slightly alkaline pH, but each has some defects. The amide formed by maleimide can be slightly unstable under alkaline conditions, so there may be some limitations in the formulation selection using this linker. The amide bond formed by iodine PEG is more stable, but free iodine can modify tyrosine residues under some conditions. PEG thiol forms a disulfide bond with protein thiols, but this bond can also be unstable under alkaline conditions. The reactivity of PEG-vinyl sulfone is relatively slow compared to maleimide and iodine PEG; however, the thioether bond formed is extremely stable. Its slower reaction rate can also make the PEG-vinyl sulfone reaction easier to control.

[0119] Site-specific PEGylation at native cysteinyl residues is rarely performed because these residues are often in the form of disulfide bonds or are required for biological activity. In another aspect, site-directed mutagenesis can be used to incorporate cysteinyl PEGylation sites for thiol-specific linkers. Cysteine ​​mutations must be designed to make them accessible to PEGylation reagents and still have biological activity after PEGylation.

[0120] Amine-specific modifiers include PEG NHS esters, PEG triflates, PEG aldehydes, PEG isothiocyanates, etc. These amine-specific reagents usually react under mild conditions and are very specific for amino groups.

[0121] Site-specific PEGylation is challenging due to the multiple lysine residues on most proteins. Fortunately, because these reagents react with unprotonated amino groups, it is possible to direct PEGylation to lower pK amino groups by reacting at a lower pH. Generally speaking, the pK of α-amino groups is 1-2 pH units smaller than the pK of the ε-amino group of lysine residues. By PEGylating the molecule at pH 7 or lower, high selectivity for the N-terminus can usually be achieved. However, this is only feasible if the N-terminal portion of the protein is not required for biological activity. In addition, the pharmacokinetic benefits of PEGylation are generally stronger than the significant loss of in vitro biological activity, resulting in products with greater in vivo biological activity, regardless of PEGylation chemistry.

[0122] III. Proteins and Peptides

[0123] In certain embodiments, the present invention relates to compositions comprising at least one protein or peptide, such as a stabilized arginase multimer. These peptides can be included in a fusion protein or conjugated to a reagent.

[0124] A. Proteins and peptides

[0125] As used herein, protein or peptide generally refers to, but is not limited to, proteins of greater than about 200 amino acids up to the full-length sequence translated from a gene; polypeptides of greater than about 100 amino acids; and / or peptides of about 3 to about 100 amino acids. For convenience, the terms "protein," "polypeptide," and "peptide" are used interchangeably herein.

[0126] As used herein, "amino acid residue" refers to any naturally occurring amino acid, any amino acid derivative, or any amino acid mimetic known in the art. Exemplary residues of a protein or peptide are continuous, without any non-amino acids interrupting the sequence of amino acid residues. Other exemplary sequences may contain one or more non-amino acid moieties. For example, a sequence of residues of a protein or peptide may be interrupted by one or more non-amino acid moieties.

[0127] Thus, the term "protein or peptide" encompasses proteins comprising at least one of the 20 common amino acids found in naturally occurring proteins, and may comprise at least one modified or unnatural amino acid, including but not limited to those shown below:

[0128] Full acronym

[0129] Aad 2-aminoadipic acid

[0130] Baad 3-aminoadipic acid

[0131] Bala β-alanine, β-alanine

[0132] Abu 2-aminobutyric acid

[0133] 4Abu 4-aminobutyric acid, pipecolic acid

[0134] Acp 6-aminohexanoic acid

[0135] Ahe 2-aminoheptanoic acid

[0136] Aib 2-aminoisobutyric acid

[0137] Baib 3-aminoisobutyric acid

[0138] Apm 2-aminopimelate

[0139] Dbu 2,4-diaminobutyric acid

[0140] Des desmosin

[0141] Dpm 2,2'-diaminopimelanediol

[0142] Dpr 2,3-diaminopropionic acid

[0143] EtGly N-ethylglycine

[0144] EtAsn N-ethylasparagine

[0145] Hyl Hydroxylysine

[0146] AHyl Isohydroxylysine

[0147] 3Hyp 3-hydroxyproline

[0148] 4Hyp 4-hydroxyproline

[0149] Ide isodesmosin

[0150] Alloisoleucine

[0151] MeGly N-methylglycine, sarcosine

[0152] MeIle N-methylisoleucine

[0153] MeLys 6-N-methyllysine

[0154] MeVal N-methylvaline

[0155] Nva norvaline

[0156] Nle norleucine

[0157] Orn Ornithine

[0158] IV. Nucleic Acids and Vectors

[0159] The nucleic acid sequence encoding the target arginase polypeptide can be a stable polyarginase. Depending on the expression system used, the nucleic acid sequence can be selected based on conventional methods. For example, human arginase I and II contain multiple codons, which are rarely used in Escherichia coli (E. coli) and may interfere with expression; therefore, the respective genes or variants thereof can be codon optimized for expression in E. coli (E. coli), as described in, for example, U.S. Patent No. 8,440,184. A variety of vectors can also be used to express the target protein, such as fusion polyarginase or cysteine-substituted arginase. Exemplary vectors include, but are not limited to, plasmid vectors, viral vectors, transposons, blood ghost red blood cells, or liposome-based vectors.

[0160] V. Host cells

[0161] Host cells, preferably eukaryotic cells, can be used for transformation to allow expression and secretion of arginase and its fusion polymers. The host cell can be bacteria, mammalian cells, yeast or filamentous fungi. Various bacteria include Escherichia and Bacillus. Yeast belonging to the genus Saccharomyces, Kluyveromyces, Hansenula or Pichia can be used as host cells. Various species of filamentous fungi can be used as expression hosts, including the following genera: Aspergillus, Trichoderma, Neurospora, Penicillium, Cephalosporium, Achlya, Podospora, Endothia, Mucor, Cochliobolus, and Pyricularia.

[0162] Examples of useful bacterial host organisms include, for example, Escherichia coli MC1061, Bacillus subtilis BRB1 derivatives, Staphylococcus aureus SAI123, or Streptococcus lividans. Exemplary yeasts that can be used as host cells include, for example, Saccharomyces cerevisiae AH22 and Schizosaccharomyces pombe; exemplary filamentous fungi, such as Aspergillus nidulans, Aspergillus awamori, and Trichoderma reesei.

[0163] Examples of publicly available mammalian host cells include Chinese hamster ovary cells (CHO-K1; American Type Culture Collection (ATCC) No. CCL61), rat pituitary cells (GH1; ATCC CCL82), HeLa S3 cells (ATCC No. CCL2.2), rat hepatoma cells (H-4-II-E; ATCC CRL 1548), SV40-transformed monkey kidney cells (COS-1; ATCC CRL 1650), and murine embryonic cells (NIH-3T3; ATCC CRL 1658). The foregoing is illustrative and non-limiting of the many possible host organisms known in the art.

[0164] Mammalian host cells expressing arginase and / or its fusion multimers are under conditions commonly used to culture the parent cell line. Typically, cells are cultured in standard mammalian cell culture media containing physiological salts and nutrients, such as standard Roswell Park Memorial Institute medium (RPMI), minimal essential medium (MEM), modified minimal essential medium (IMEM) or Dulbecco's minimal essential medium (DMEM), typically supplemented with 5-10% serum, such as fetal bovine serum (FBS). Culture conditions are also standard, for example, cultures are statically cultured or rotated at 37°C until the desired protein level is achieved.

[0165] VI. Protein Purification

[0166] Unless otherwise specified, the protein or polypeptide of interest can be further purified using chromatography and electrophoresis techniques to achieve partial or complete purification (or purification to homogeneity). Particularly suitable analytical methods for preparing pure peptides are ion exchange chromatography, gel exclusion chromatography, polyacrylamide gel electrophoresis, affinity chromatography, immunoaffinity chromatography, and isoelectric focusing. Particularly effective methods for purifying peptides are fast high performance liquid chromatography (FPLC) or even high performance liquid chromatography (HPLC).

[0167] Purified protein or peptide means a composition that is separable from other components, wherein the protein or peptide is purified to any degree relative to its naturally obtained state. Therefore, an isolated or purified protein or peptide also refers to a protein or peptide that is separated from the environment in which it may naturally occur. Generally speaking, "purified" will refer to a protein or peptide composition that has been fractionated to remove various other components, and the composition substantially retains its expressed biological activity. Where the term "substantially purified" is used, this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the protein in the composition.

[0168] Exemplary protein purification techniques include precipitation using ammonium sulfate, PEG, antibodies, etc., or by heat denaturation followed by centrifugation; chromatographic steps such as ion exchange, gel filtration, reverse phase, hydroxyapatite, and affinity chromatography; isoelectric focusing; gel electrophoresis; and combinations of these and other techniques.

[0169] VII. Pharmaceutical Compositions

[0170] Arginases described herein can be administered systemically or topically. Arginases and compositions comprising them can be administered intravenously, intrathecally, subcutaneously, intramuscularly, intratumorally, and / or intraperitoneally, or a combination thereof. The compounds described herein and compositions comprising them can be administered alone or in combination with an arginine scavenger and / or an arginine-reduced diet.

[0171] Compositions containing arginase or a part thereof can be provided in a formulation together with physiologically tolerable liquids, gels or solid carriers, diluents and excipients. Such compositions are typically prepared into liquid solutions or suspensions as injections. Suitable diluents and excipients are, for example, water, saline, glucose, glycerol, etc. and combinations thereof. In addition, if necessary, the composition may contain a small amount of auxiliary substances, such as wetting agents or emulsifiers, stabilizers or pH buffers. In the case of expected clinical applications, it may be necessary to prepare pharmaceutical compositions - expression vectors, viral stock solutions, proteins, antibodies and drugs in a form suitable for the expected application. Typically, the pharmaceutical composition of the present invention comprises an effective amount of one or more arginase variants or other reagents dissolved or dispersed in a pharmaceutically acceptable carrier.

[0172] The term "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that are generally nontoxic to recipients at the dosages and concentrations employed, i.e., do not produce adverse, allergic or other untoward reactions when administered to a subject (such as a human), as appropriate. The preparation of pharmaceutical compositions comprising at least one arginase variant, such as a stabilized polyarginase or PEGylated arginase isolated by the methods disclosed herein, or additional active ingredients in light of the present disclosure are known to those skilled in the art, as exemplified by Remington's Pharmaceutical Sciences, 18th edition, 1990. In addition, for animal (e.g., human) administration, it will be understood that the formulations should meet the sterility, pyrogenicity, general safety and purity standards required by the FDA Office of Biological Standards.

[0173] As used herein and as known to those of ordinary skill in the art, "pharmaceutically acceptable carriers" include any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, dyes, such similar materials, and combinations thereof (see, e.g., Remington's Pharmaceutical Sciences, 18th ed., 1990). Unless any conventional carrier is incompatible with the active ingredient, its use in pharmaceutical compositions is contemplated.

[0174] The pharmaceutical composition comprising arginase may comprise different types of carriers, depending on whether it is to be administered in solid, liquid or aerosol form, and whether sterility is required for routes of administration such as injection. The present invention may be administered intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intramuscularly, subcutaneously, intratumorally, topically, by injection, infusion, continuous infusion, by catheter, in lipid compositions (e.g., liposomes), or by any other method or combination thereof known to those of ordinary skill in the art (see, e.g., Remington's Pharmaceutical Sciences).

[0175] The modified arginase variants can be formulated into compositions in free base, neutral or salt form. Pharmaceutically acceptable salts include acid addition salts, such as those formed by the free amino groups of the protein composition, or those formed by inorganic acids (e.g., hydrochloric acid or phosphoric acid) or organic acids (such as acetic acid, oxalic acid, tartaric acid or mandelic acid). Salts formed with free carboxyl groups can also be derived from inorganic bases, such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or ferric hydroxide; or organic bases, such as isopropylamine, trimethylamine, histidine or procaine. When formulated, the solution can be administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The preparation is easily administered in a variety of dosage forms, such as formulated for parenteral administration, such as injection solutions, or for delivery to the lungs in aerosols, or formulated for dietary administration, such as drug release capsules.

[0176] The composition for use can be provided in a pharmaceutically acceptable carrier with or without an inert diluent. The carrier should be able to absorb and include liquid, semisolid, i.e., pasty or solid carriers. Unless any conventional medium, reagent, diluent or carrier is harmful to the recipient or to the therapeutic effectiveness of the composition contained therein, otherwise its purposes in the composition for practicing the method of the present invention are suitable. The example of carrier or diluent includes fat, oil, water, saline solution, lipid, liposome, resin, binding agent, filler etc., or its combination. The composition also can include various antioxidants to delay the oxidation of one or more components. In addition, preservatives such as various antibacterials and antifungals are used, including but not limited to parahydroxybenzoates (for example, methylparaben, propylparaben), chlorobutanol, phenol, sorbic acid, thimerosal or its combination can improve the life span of the pharmaceutical composition.

[0177] The composition is combined with the carrier in any convenient and practical manner, i.e., by solution, suspension, emulsification, admixture, encapsulation, absorption, etc. Such procedures are routine to those skilled in the art.

[0178] Pharmaceutical lipid carriers can be used for compositions comprising arginase variants. The lipid carrier composition can comprise one or more lipids and an aqueous solvent. As used herein, the term "lipid" will be defined as including any one of a wide range of substances characterized by being insoluble in water and extractable using an organic solvent. Examples include compounds containing long-chain aliphatic hydrocarbons and derivatives thereof. Lipids can be naturally occurring or synthetic (i.e., artificially designed or produced). However, lipids are typically biological substances. Biological lipids include, for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, sphingolipids, glycolipids, thioesters, lipids and polymerizable lipids with ether and ester-linked fatty acids, and combinations thereof. Of course, the compositions and methods of the present invention also encompass compounds that those skilled in the art will understand as lipids that are different from those specifically described herein.

[0179] The actual dosage of a composition comprising an arginase as described herein for a patient can be determined by physical and physiological factors such as body weight, severity of the condition, type of disease being treated, prior or concurrent therapeutic interventions, the patient's spontaneous onset, and the route of administration. Depending on the dosage and route of administration, the preferred dosage and / or number of administrations of an effective amount can vary depending on the subject. The dosage will depend on the amount required for the patient to achieve normal levels of at least one or more of the following compounds: arginine, HArg, ArgA, GVA, GAA, and NAArg. Patient levels of ARG1-D are typically assessed for one or more of five (5) compounds until a normal range is achieved in the patient's plasma. Tissue levels of the five (5) compounds can also be assessed, but this may not be necessary or may be performed less frequently than plasma level testing.

[0180] For patients with ARG1-D, the initial administration can be a dose of 0.005 to 1.00 mg / kg of arginase per kilogram of patient intravenously, and any 0.005 dose between 0.005 and 1.00 mg / kg of patient body weight, such as 0.02 mg / kg or 0.035 mg / kg. Exemplary intravenous doses or arginase can be administered daily, weekly, twice monthly, or monthly. Alternatively, the arginase composition can be initially or only administered subcutaneously or in any combination of intravenous or subcutaneous administration. Subcutaneous or intramuscular administration can be a dose of 0.01 to 1.50 mg / kg of arginase per kilogram of patient body weight, and 0.01 amounts within the range of 0.01 to 1.50 mg / kg of patient body weight, such as 0.08 mg / kg. Subcutaneous or intramuscular administration can be daily, weekly, twice monthly, or once monthly.

[0181] The pharmaceutical composition can include, for example, at least about 0.1% of the active compound. In other embodiments, the active compound can include, for example, between about 2% to about 75% of the unit weight, or between about 25% to about 60%, and any scope deducible therefrom. Of course, the amount of the active compound in each treatment available composition can be prepared in this manner for the appropriate dosage obtained in any given unit dose of the compound. Those skilled in the art of preparing this type of pharmaceutical formulation will expect factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life and other pharmaceutical considerations, so multiple doses and treatment regimens can be desired. An exemplary formulation of polyethylene glycol arginase is prepared in a buffer solution comprising 5mM potassium phosphorus, 50mM sodium chloride and 1.5% glycerol (w / v / ), at a pH of 7.4.

[0182] VIII. Treatment Methods

[0183] In addition to administering arginase protein to ARG1-D patients, other therapeutic approaches are also contemplated. For example, patients can be treated with gene therapy vectors, such as adenovirus-based gene delivery vectors containing wild-type arginase. The wild-type protein can be targeted to specific organs, such as the liver. Various viral packaging systems have been established for reintroducing proteins into patients via gene therapy, for example, as described in U.S. Patent No. 8,398,968.

[0184] Another approach to treating ARG1-D patients is through the use of a CRISPR (clustered regularly interspaced short palindromic repeats) system, in which a defective gene on a chromosome is manipulated to edit the error so that the patient can produce wild-type arginase under normal circumstances. As described above, it may be necessary to edit two arginase genes located on different chromosomes. CRISPR genome engineering is described in, for example, U.S. Patent Publication Nos. 20170240922; 20170283830; 20170224843; and 20170191078.

[0185] Another method of delivering arginase is to encapsulate the arginase protein in resealed red blood cells (RBCs) that can be re-administered to the patient. Encapsulated arginase can generally be as described in U.S. Patent Publication Nos. 20160095884 and 20140154797. A lysis / resealing method for preparing red blood cells containing arginase comprises placing a pellet concentrate in an isotonic solution of a suspension having a hematocrit level equal to or greater than 65% and refrigerating at 1°C to 8°C; measuring osmotic fragility based on a sample of red blood cells from the same pellet concentrate, preferably based on a sample of the suspension; performing a lysis and internalization step of the active ingredient (i.e., arginase) in the same chamber at a temperature maintained at about 1°C to 8°C, comprising circulating a suspension of red blood cells having a hematocrit level equal to or greater than 65% and a hypotonic lysis solution refrigerated at a temperature of 1°C to 8°C in a dialysis cassette; adjusting lysis parameters based on the previously measured osmotic fragility; and then resealing in a second chamber at a temperature of 30°C to 40°C with a hypertonic solution.

[0186] Example

[0187] In an open-label, phase 1 / 2 study, 12 adult and pediatric patients (7 children and 5 adults) diagnosed with ARG1-D were enrolled. In part 1, patients received escalating doses of pegylated arginase every 2 weeks. In part 2, patients received intravenous (IV) pegylated arginase (AEB1102) every other week for 8 weeks.

[0188] Safety, pharmacokinetics, pharmacodynamics (plasma arginine and GCs (e.g., GVA, ArgA, NAA, GAA)) and standardized clinical measures (e.g., 6-minute walk test (6MWT), Berg balance scale (BBS), gross motor function test scale (GMFM-66) and PROMIS) were assessed. The GMFM-66 is a tool that assesses various aspects of gross motor function from parts A to E. Guanidine compounds (GCs) were analyzed before and after the administration of PEG arginase to assess the therapeutic effect of PEG arginase on patients. GCs selected for analysis in patients included α-keto-δ-guanidinovalerate (GVA), argininic acid (ArgA), homoarginine, N-α-acetylarginine (NAArg) and GAA in plasma.

[0189] Methods. A bioanalytical procedure was developed for the determination of GC (GAA, GVA, ArgA, and NAAg) from K2EDTA plasma of patients. Plasma proteins from patients were precipitated with 10% trichloroacetic acid solution and analyzed using Imtakt Intrada amino acid 50 x 2 mm (PN: WAA22) columns at Shimadzu Separation was performed using an Applied Biosystems / MDS Sciex API 5500 TM Analytes were detected by LC-MS / MS. Isotopically labeled standards for each GC were used to achieve quantification for each plasma sample. A detailed method protocol is provided at the end of this example.

[0190] Results of a study of two adult female patients

[0191] Two adult female patients, aged 24 and 25 years, received weekly intravenous infusions of PEG arginase for 8 weeks. Both patients had moderate to severe neurocognitive and neuromotor deficits, including spasticity. Both patients continued to receive PEG arginase pre-treatment standards throughout the entire process of PEG arginase administration. Weekly intravenous (IV) doses of 0.04 mg / kg PEG arginase were well tolerated. In addition to the expected decrease in plasma arginine, a time-dependent decrease in the concentration of measured GCs (e.g., ArgA, NAArg, and GVA) was also observed after treatment with PEG arginase (Figure 1). The decrease in ArgA, NAArg, and GVA plasma levels occurred within 24 hours after the first infusion and continued to remain below baseline levels within 8 weeks of dosing for each patient.

[0192] In the single ascending dose portion of the Phase I study (PEG-arginase-101A), plasma samples from two patients initially analyzed for arginine / ornithine samples were pooled to help set the assay range during GC method development. Following completion of the method development phase, these pooled samples were used for quantitative GC. Both patients received single doses of 0.015 mg / kg and 0.03 mg / kg of patient body weight of PEG-arginase, with a 2-week observation period between doses.

[0193] Using these pooled patient samples in a previously described non-GLP assay (detailed methods are provided at the end of the example), time-dependent decreases in the concentrations of ArgA (approximately 3-fold), GVA (approximately 4-fold), and NAArg (approximately 2-fold) were observed in patients with ARG1-D who received 0.03 mg / kg of PEGylated arginase. The decrease in GC levels paralleled a decrease in plasma arginine, as determined using the GLP assay ( Figure 1A -D).

[0194] like Figure 3A As reflected in Table 1, two patients (i.e., 120-101 and 120-102) were administered pegylated arginine at either 0.015 mg / kg or 0.03 mg / kg of patient body weight, and plasma arginine and guanidino compound levels were determined as follows:

[0195] Table 2: GC levels in two patients at a dose of 0.015 mg / kg.

[0196]

[0197] *Average of two subjects; **Pooled samples from two subjects. If no value is listed in Tables 2 and 3, the patient was not tested at that time point because these assays are very expensive and blood levels are generally not thought to change rapidly.

[0198] Table 3: GC levels in two patients at a dose of 0.03 mg / kg patient body weight.

[0199]

[0200]

[0201] *Average of two subjects, **Combined samples from two subjects.

[0202] Table 4: Dosage and concentration tests were performed as follows:

[0203]

[0204]

[0205]

[0206]

[0207]

[0208]

[0209] Note that due to patient scheduling, the sample scheduled for 120 hours was actually drawn within 144 hours.

[0210] Table 5: PK concentrations (concentration data available for the dose escalation portion of the trial); PK data for two patients are as follows:

[0211]

[0212]

[0213]

[0214]

[0215]

[0216] Note that due to patient scheduling, the sample planned for 120 hours was actually drawn at 144 hours. Additionally, the PK and pharmacodynamic (PD) data were provided by different vendors, so the format of the two datasets will differ.

[0217] Table 6: Individual and mean PK parameters of pegylated arginase in patients with hyperarginineemia following a single IV infusion (data available for dose escalation only).

[0218]

[0219]

[0220] CV: coefficient of variation; NR: not reported (N<3).

[0221] The data. The observed relationship between plasma arginine and ArgA, GVA, and NAAg concentrations is generally consistent with the data published by Marescau et al. (1990) in their analysis of patients maintained on a protein-restricted diet. In the study by Marescau et al. (1990), GC levels decreased concomitantly with decreasing arginine levels.

[0222] For the data provided herein, not only the levels of ArgA, GVA and NAArg were measured, but also the levels of homoarginine (HArg). Since Harg in the serum of ARG1-D patients was significantly elevated in the study by Marescau et al. (1990), HArg levels were examined. In ARG1-D patients treated with PEGylated arginase, HArg levels did not appear to be regulated (Figure 2). This result is consistent with the data reported by Marescau et al. (1990), as serum HArg levels in patients who maintained a protein-restricted diet did not decrease. Other conditions that can be assessed by patient treatment are: improved muscle strength, the patient's ability to walk (i.e., the ability to run, walk, ride a bicycle, climb stairs without support), improved cognitive abilities (e.g., WISC test improvements), or adaptive behavioral changes (e.g., ABAS or VABS tests).

[0223] GC data were also generated from pooled plasma samples obtained from two (2) adult patients who received a lower dose of PEGylated arginase, 0.015 mg / kg patient body weight. A decrease in plasma arginine concentration was observed at the lower PEGylated arginase dose; however, this decrease was not as profound as the decrease in plasma arginine observed at the 0.03 mg / kg dose.

[0224] Considering the findings from the two patients presented above, it was concluded that adult patients with ARG1-D tolerated pegylated arginase well at a dose of 0.04 mg / kg administered intravenously once weekly. The patients had significantly reduced GCs, which paralleled the reduction in arginine levels. Based on these two adult patients, the effects on arginine and GC levels appear to be scientifically more significant than those historically achieved with strict dietary arginine restriction. One ARG1-D patient in this study was successfully dosed at 0.2 mg / kg. The maximum tolerated dose (MTD) for solid tumors was determined to be 0.33 mg / kg, and doses as high as 0.48 mg / kg have been reported.

[0225] Of course, patients with high arginine levels (e.g., 600-800 μM arginine) can be treated with higher doses of polyethylene glycol arginase than 0.50 mg / kg. Patients with high arginine levels may require administration of arginase in the range of 0.005 to 1.00 mg / kg of subject body weight. Dosage ranges of 0.005 mg / kg to 0.50 mg / kg and / or 0.005-0.20 mg / kg of subject body weight are also contemplated.

[0226] The bioavailability of a drug can be determined by measuring the amount of drug in the blood from an intravenous and / or subcutaneous injection over a specific time period. For calculation purposes, it is assumed that 100% of the intravenously administered drug enters the bloodstream, while not all of the subcutaneously administered drug enters the blood from the subcutaneous space. Bioavailability can be determined by dividing the total subcutaneous amount by the total intravenous amount. Based on a comparison of intravenous and subcutaneous administration in cynomolgus monkeys, the bioavailability of subcutaneously administered polyethylene glycol arginase was determined to be approximately 60% of that administered intravenously. Therefore, patients with high arginine levels can successfully receive up to 1.5 mg / kg of arginase treatment. Based on bioavailability data, the dosage range for subcutaneously administered polyethylene glycol arginase can be, for example, 0.01 to about 1.5 mg / kg of subject body weight; 0.015 mg / kg to 0.75 mg / kg; and / or 0.015 mg / kg to about 0.30 mg / kg of subject body weight.

[0227] Figure 4A and Figure 4B Two patients, Patient 120-101 ( Figure 4A ) and patient 120-102( Figure 4B ). In both cases, data for GAA levels at screening and during dosing in Parts 1 and 2 are shown. In both patients, GAA decreased after administration of 0.015 mg / kg subject body weight, with a greater effect observed at the higher dose. Notably, a rapid decrease in GAA was observed shortly after administration of PEGylated arginase. Furthermore, initial treatment resulted in normal GAA plasma levels in both patients within 24 hours of administration. For example, in Figure 4B In a study conducted on patients with GAA, day 14, the patient's GAA level was 4,350 nM, which decreased to 1,350 nM within 24 hours of arginase administration. Therefore, treatment with arginase can rapidly clear toxic metabolites, such as GAA or GVA, and can be used as an acute treatment. Pegylated arginase cleared the designated toxic metabolites much more rapidly than an arginine-reduced diet alone.

[0228] Patient characteristics and safety

[0229] Baseline assessments were performed on patients in the Phase 1 / 2 study. The table below provides baseline assessments that demonstrate the significant disease burden in patients in this study. Patients were on a protein-restricted diet.

[0230] For Berg balance, deficit balance is defined as moderate or high risk of falling (i.e., score ≤ 40). For 6MWT, deficit is defined as below the age-adjusted range for normal individuals in Geiger et al., "Six-minute walk test in children and adolescents," J. Pediatr., 2007 Apr; 150(4): 395-399, and Enright et al., "Reference equations for the six-minute walk in healthy adults," Am. J. Respir. Crit. Care Med., 1998 Nov.; 158(5 Pt 1): 1384-1387. For GMFM part E, deficit is defined as < 68 based on the minimum clinically important difference (MCID) of Oeffinger et al., "Outcome tools used for ambulatory children with cerebral palsy: responsiveness and minimum clinically important differences," Dev. Med. Child Neurol., 2008 50(12): 918-925. If multiple baseline laboratory assessments are available, they should be reported immediately before dosing.For PROMIS, baseline impairment is defined as a T score <40.

[0231] Table 7: Baseline assessments of patients in the Phase 1 / 2 study.

[0232]

[0233]

[0234] As shown in the table below, the most treatment-related adverse events ("AEs") were mild. Treatment-related AEs occurring in ≥2 patients were hypersensitivity (n=3, all moderate), pruritus (n=3, all mild), and dry skin (n=2, both mild). In total, more than 130 infusions were administered to all patients in the study. Four moderate hypersensitivity reactions were observed in three patients (two of which were considered serious adverse events (SAEs)). These adverse events were managed with modification of the infusion rate and administration of antihistamines (and in some cases, corticosteroids). There was one SAE of hyperammonemia that was assessed as unrelated to pegarginine treatment.

[0235] Table 8: Adverse events.

[0236] Patients with Part 1 (n=12) Part 2 (n=4) Any related AE 6(50％) 3(75％) Any SAE 3(25％) 0 Any relevant SAEs 2(17％) 0

[0237] The effect of PEGylated arginase ADA was investigated. A bridging assay using the Meso Scale Discovery electrochemiluminescence method was validated to detect antibodies to AEB1102 (Co-Arg1-PEG) in rat, monkey, and human sera. The method used biotinylated AEB1102 (B-AEB1102 or B-Co-ArgI-PEG) to capture ADA and ruthenium-labeled AEB1102 (Ru-AEB1102 or Ru-Co-ArgI-PEG) to detect the antibodies.

[0238] During validation, a master mix (Mm) of B-Co-ArgI-PEG and Ru-Co-ArgI-PEG was prepared in assay buffer to a final concentration of 1.0 μg / mL B-AEB1102 and 1.0 μg / mL Ru-AEB1102. Diluted samples and controls were added to the master mix in the wells of a streptavidin-coated plate. After incubation and washing, 150 μL of 2X Reading Buffer T (from Meso Scale Discovery) was added to each well. Samples were read on a Sector Imager 6000. This method was used for screening, titration, and confirmation, with the confirmatory assay preincubated with 150 μg / mL AEB1102 (drug). Positive and negative controls at high, medium, and low concentrations were included in each run. Statistical distributions for cutpoints, screening, and confirmation were performed to provide approximately 5% and 1% false positive rates, respectively. The positive control was an anti-Co-ArgI-PEG affinity purified polyclonal antibody, and the negative control (NC) was aliquoted from pooled normal rat, cynomolgus monkey, or human serum. Positive and nonspecific binding (NSB) controls were used to monitor assay performance.

[0239] A direct binding assay was validated to detect antibodies against PEG, thereby allowing the detection of possible pre-existing anti-PEG antibodies or treatment-emergent events following administration of AEB1102.

[0240] Wells of a Starwell C8 Maxisorp (96-well format) were coated with 100 μL of 2 μg / mL mono-PEGylated bovine serum albumin (BSA) (BSA-mPEG) 5kDa (LifeDiagnostics) or 500 ng / mL human IgG (Jackson ImmunoResearch Laboratories) or 500 ng / mL human IgM (Jackson ImmunoResearch Laboratories) in carbonate coating buffer (BioWorld). Controls and samples diluted to a 50-fold minimum required dilution (MRD) in dilution buffer were added to the plate in duplicate (100 μL / well). The dilution buffer contained 4% bovine gamma globulin in 1× phosphate-buffered saline (PBS). 100 μL of detection antibody was added to the appropriate wells. Mouse anti-PEG antibodies (Jackson ImmunoResearch Laboratories) were detected using goat anti-mouse IgG-Fc-HRP (Jackson ImmunoResearch Laboratories) diluted 1:5,000, and anti-human antibodies were detected using rabbit anti-human IgG / A / M (Jackson ImmunoResearch Laboratories) diluted 1:30,000 in dilution buffer and added to the appropriate wells. 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB) substrate was then added to each well, and after approximately 10 to 20 minutes, the reaction was terminated by adding 100 μL of stop solution to each well. Plates (multiple) were read on a Synergy 2 plate reader at 450 (detection) and 620 (background). Statistical distributions were made for the cutoff points, screening, and confirmation to provide approximately 5% and 1% false positive rates, respectively. This method was used for screening, titer, and confirmation. The sample to be titrated was serially diluted a minimum of seven times, 2-fold, in negative pooled human serum.

[0241] Transient, low-titer anti-PEG ADA (anti-drug antibodies) were detected in the first part of the patient study (see table below). At the start of repeated dosing, no detectable ADA was detected in 6 of 7 patients. As shown in Table 9 below, tolerance to PEGylated arginase developed unexpectedly rapidly.

[0242] Table 9: ADA assessment of pegylated arginase and PEG.

[0243]

[0244] *One patient who was using anti-PEG ADA prior to treatment had declining titers during Part 1, with undetectable ADA at the final Part 1 dose. However, this patient did not continue into Part 2 of the study for reasons unrelated to the study; this patient is not included in n=7.

[0245] Figure 5A and 5B The results showed the role of arginine and GC. GAA was elevated in patients with ARG1-D. GAA levels in these patients were reduced by treatment with PEGylated arginine. Figure 5B The repeated dose figures shown in the Table include patients who received all 8 doses in Part 2. The upper limit of normal (ULN) for GC is based on studies of GC in healthy adults. Pegylated arginase was highly effective in reducing arginine levels to within the normal range with single and repeated doses ( Figure 5B ). GAG (elevated in patients with ARG1-D) can also be reduced by pegylated arginase treatment. Following IV Qw (once a week) treatment with pegylated arginase, a time-dependent decrease in GVA, ArgA, GAA, and NAA concentrations was observed. The decrease in GVA, ArgA, GAA, and NAA levels occurred within 24 hours of the first infusion and remained reduced from baseline levels throughout the 8-week dosing process. Serum pegylated arginase levels were measured at multiple time points around doses 1 and 8.

[0246] Figure 6Results of the 6-minute walk test for three patients are provided (data were not available for patient 5 ("c") at doses 20 and 32. The table below provides neuromotor results. Doses 8, 20, and 32 were measured starting with the repeat dose (second dose). PROMIS, specifically the PROMIS physical function domain. The data in the table below for the 6MWT (6-minute walk test), BBS (Berg Balance Scale), GMFM Part E, and PROMIS are shown as raw values ​​at baseline and changes from baseline at other time points. The MCID for the 6MWT was defined from the analysis of Schrover et al., “Minimal clinically important difference for the 6-min walk test: literature review and application to Morquio A syndrome,” Orphanet. J. Rare Dis., 2017 Apr 26;12(1):78; for the BBS, the MCID was defined from the analysis of Downs et al., “The Berg Balance Scale,” J. Physiother., 2015 Jan;61(1):46; for the GMFM, the MCID was defined from the analysis of Oeffinger et al., 2008, and for PROMIS, the MCID was based on physical function with 0.5 standard deviation, or a T score of 5. The following table and Figure 6 It has been demonstrated that the patient's neuromotor function is improved after administering polyethylene glycol arginase. Neuromotor function can refer to muscle or nerve function, and can be clinically assessed in patients using, for example, PROMIS, 6MWT, BBS and GMFM. Examples of neuromotor function include, but are not limited to, climbing stairs, walking, spasms and alertness. For example, a patient with the disease may walk on tiptoe rather than normally (heel to toe). Improved neuromotor function may refer to that such a patient has the ability to walk better or walk normally, rather than walking on tiptoe. Improved neuromotor function may refer to enhanced mobility, such as no longer needing to use a walker (for example, a "walker" or crutch) or needing to use a walker less. Improved neuromotor function may refer to improved posture and / or improved communication / social interaction.

[0247] Table 10: Neuromotor function assessment.

[0248]

[0249]

[0250] GAA is associated with seizures in patients with GAMT (guanidinoacetate methyltransferase) deficiency (Stockler-Ipsiroglu et al., "Guanidinoacetate methyltransferase (GAMT) deficiency: outcomes in 48 individuals and recommendations for diagnosis, treatment and monitoring," Mol. Genet. Metab., 2014; 111(1): 16-25). The results showed that treatment with PEGylated arginine reduced GAA levels in patients, as well as plasma arginine and related GCs. The levels of one or more guanidino compounds such as GAA (see, e.g., Figure 5B ) is reduced or cleared within 24 to 48 hours after administration of arginase. Therefore, arginase can be used as an acute therapy to reduce or clear toxic metabolites in patients. Clinical improvement was observed after repeated doses of pegylated arginase after 8 weeks. Patients generally tolerated pegylated arginase well. Most associated adverse events were mild and manageable with standard measures.

[0251] Unexpectedly, treatment with polyethylene glycol arginase rapidly alleviated the symptoms of hyperargininemia in patients with ARG1-D. These improvements were manifested at the biochemical level. Based on the initial and / or repeated administration of arginase, the plasma levels of one or more arginine and guanidine compounds can be reduced to normal levels. In addition, the administration of arginase improved the adaptive behavior and / or neuromotor function of patients with ARG1-D. All patients in this study had received standard treatment for hyperarginemia or received conventional treatment during the study. This standard or conventional treatment involves dietary restriction to limit the intake of proteins that can increase arginine levels, and the use of nitrogen scavenging drugs. Despite these standard treatments, all patients still had high arginine levels. Treatment with arginase can quickly and continuously reduce the plasma levels of one or more of arginine and other guanidine compounds. The therapeutic effect of arginase (e.g., polyethylene glycol arginase) is unexpectedly rapid, occurring within 24-48 hours after administration. In contrast, standard or conventional treatments for hyperargininemia may not show any improvement within two years (Marescau et al., 1990; Marescau et al., "The pathobiochemistry of uremia and hyperargininemia further demonstrates a metabolic relationship between urea and guanidinosuccinic acid," 1992 41(9):1021-1024). In addition, patients receiving pegylated arginase therapy may have a more liberal diet due to the reduction in arginine and may therefore consume more protein.

[0252] Phase 2 clinical data show that lowering plasma arginine with an arginine-depleting agent improves ARG1-D-related disease manifestations

[0253] The open-label extension study will continue with Parts 1 and 2 from the open-label Phase 1 / 2 study described above. Figure 7 As shown in the , 16 patients in the first part of the phase 1 / 2 study received single ascending doses of pegylated arginase intravenously every other week for 4 to 10 weeks, as described above. In part 2, repeated doses (8 doses per week) of pegylated arginase were administered intravenously for 10 weeks, as described above. Nine patients started part 2 treatment, and six completed the repeated doses. In the open-label extension study, three patients began intravenous pegylated arginase.

[0254] The following table shows the patient characteristics and indicates the disease burden of the patients in the study and open-label extension study. The median plasma arginine was based on calculating the mean of all plasma arginine values ​​for each patient before the first dose and determining the median of these values. The table shows that all 16 patients had elevated baseline plasma arginine. Seven of the ten patients had deficits in both mobility and adaptive behavior. The median plasma arginine was based on the mean of all plasma arginine values ​​for each patient before the first dose. For other biochemical parameters, the assessment immediately before dosing was used. For laboratory assessments, abnormalities were defined as being outside the reference range. For height percentiles, deficits were defined as ≤10% of the CDC-provided normal value. For the 6MWT, the Berg balance, GMFM part E, and PROMIS were applied as described above. For the Adaptive Behavior Assessment System (ABAS), third edition, deficits were defined as a standard score <85 at the practical, social, conceptual, or composite level. For the MCID of the 6MWT (see Figure 9 ) was defined as a 9% change from baseline; for GMFM part E, as a 1.8 to 4.0-point change, depending on each patient's Gross Motor Function Classification System level; for BBS, as a 7-point change; for ABAS, as a 7.5-point change in the General Adaptive Composite (GAC) standard score; and for PROMIS, as a 5-point change.

[0255] Table 11: Baseline assessments of patients in the Phase 1 / 2 study.

[0256]

[0257] Figure 8 Time-dependent improvement in plasma arginine with repeated dosing for patients in Part 2 and the open-label extension study. BL is baseline; F / U is follow-up; and n is the number of patients at each time point. The median arginine level for all patients at each time point is indicated. et al., “Suggested guidelines for the diagnosis and management of urea cycle disorders,” Orphanet. J. Rare Dis., 2012 7:32, describe treatment targets that aim to maintain plasma arginine levels at ≤200 μmol / L. Figure 8 100% (6 / 6) of patients who completed Part 2 achieved consistent levels of reduced arginine below the recommended guidelines. The reduction in plasma levels of arginine was accompanied by a significant and sustained decrease in plasma levels of GC (GVA, ArgA, NAA, GAA).

[0258] like Figure 9As shown, treatment with pegylated arginase also improved clinical outcomes from baseline after 8 repeated doses. Figure 9 Each point in the table represents an assessment for one patient. Favorable assessments are indicated by a solid circle "●", while neutral assessments are indicated by an open circle "○". 67% (4 / 6) of patients showed improvements exceeding the MCID on tests of mobility and / or adaptive behavior after only 8 weeks of repeated administration of pegarginase. The criteria for these tests have been previously described for the 6MWT, Berg Balance Scale, GMFM Part E, PROMIS, and ABAS. 33% of the assessments exceeded the MCID favorably, while none exceeded the MCID unfavorably. 1 / 3 of the ABAS assessments exceeded the MCID favorably, and 2 / 3 of the assessments trended towards improvement at 8 weeks.

[0259] Table 12: Summary of assessors' observations for some patients in the study.

[0260]

[0261]

[0262] Administration of pegylated arginase generally resulted in mild AEs, if any. All patients received 180 doses of pegylated arginase. In Part 1, low titers of treatment-emergent ADA were detected in 6 / 16 patients. All patients in Part 2 had undetectable ADA levels at the fifth dose. Patients with treatment-related AEs ≥ 2 of at least moderate severity included hypersensitivity reactions (4 events in 3 patients; 3 were considered serious adverse events (SAEs) by the investigator), which can be managed with infusion rate adjustments and medications such as antihistamines and, in some cases, corticosteroids.

[0263] Figure 10 Shown are mean arginine values ​​analyzed by clinical response for patients who participated in Part 2. Patients with a clinical response >1 MCID improvement in neuromotor or adaptive behavior assessments (n=4) were found to have lower arginine levels than those with <1 MCID improvement in neuromotor or adaptive behavior assessments (n=2).

[0264] In summary, despite standard disease management approaches that address only disease symptoms with strict dietary protein restriction and the use of ammonia scavengers, the progressive nature of the disease highlights a significant unmet medical need for pharmacological treatments that would lower arginine levels to levels not currently achievable with standard disease management, thereby offering the potential to slow or halt the progression of neuromotor, neurocognitive, and / or adaptive behavioral deterioration observed in patients with ARG1-D. The engineered human arginase 1, pegylated arginase, has directly demonstrated a significant and sustained reduction in plasma arginine and related GC levels in patients with ARG1-D, accompanied by improvements in neuromotor function and / or adaptive behavior in some patients.

[0265] Improvements observed in patients with ARG1-D treated with pegylated arginase in the Phase 1 / 2 arm of the previously described study persisted in the open-label extension study. Pegylated arginase was highly effective in consistently reducing elevated plasma arginine, thought to underlie the pathological basis of ARG1-D symptoms. After just eight weeks of repeated dosing, the reduction in plasma arginine was accompanied by improvements in mobility and adaptive behavior. Pegylated arginase was well tolerated by patients. Most treatment-related adverse events were mild; hypersensitivity reactions were manageable with standard measures, and all patients continued on study treatment. A comprehensive baseline analysis of patients with ARG1-D demonstrated quantifiable deficits in mobility and / or adaptive behavior in 94% (15 / 16) of patients.

[0266] Method for the analysis of α-K-δ-GVA, (R,S)-ArgA, Homoarginine HCL, and Nα-acetyl-L-arginine in K2EDTA human plasma by LC-MS / MS.

[0267]

[0268]

[0269]

[0270] The commercial reagents used are as follows:

[0271]

[0272]

[0273] Therapeutic human plasma [THP]. Add 0.059 mL of AEB1102 to 20.0 mL of K2EDTA human plasma. Store in polypropylene vials (PPV) at approximately -70°C. The expiration date is the expiration date of the plasma used.

[0274] Therapeutic human lipemic plasma [TLP]. Add 0.059 mL of polyethylene glycol arginase to 20.0 mL of K2EDTA lipemic human plasma. Store in PPV at approximately -70°C. The expiration date is the expiration date of the plasma used.

[0275] Treated human whole blood (THB). Add 0.059 mL of polyethylene glycol arginase to 20.0 mL of K2EDTA human whole blood. Store in PPV at approximately 4°C. The expiration date is the expiration date of the whole blood used.

[0276] 1mg / mL nor-NOHA[NOHA]

[0277] Dissolve the entire contents of a 5 mg nor-NOHA vial in 5.00 mL of MQ and store in PPV at approximately -70°C for up to 1 month.

[0278] 10% mannitol solution (w / v) [MT1]

[0279] Weigh approximately 1.00 g of mannitol and dissolve it in 10 mL of MQ. Stir to dissolve. Store at room temperature for up to 1 month.

[0280] Non-acidified matrix [NM1]

[0281] Centrifuge 20 mL of K2EDTA human plasma at 3500 rcf for 5 minutes. Use a pipette to add 0.059 mL of AEB1102 to the plasma. Incubate the plasma at 37°C for approximately 3 hours. Add 0.180 mL of Nor-NOHA to the plasma vial. Add 0.200 mL of MT1 to the treated plasma to achieve 0.1% (v / v) mannitol (this step can be performed before using the plasma). Mix well. Store in PPV at approximately -70°C until the expiration date of the plasma component.

[0282] Acidified matrix [AM1]

[0283] Centrifuge 20 mL of K2EDTA human plasma at 3500 rcf (relative centrifugal force) for 5 minutes. Add 0.059 mL of AEB1102 to the plasma using a pipette. Incubate the plasma at 37°C for approximately 3 hours. Add 0.400 mL of GLA to the plasma vial. Add 0.180 mL of Nor-NOHA to the plasma vial. Add 0.200 mL of MT1 to the treated plasma to achieve 0.1% (v / v) mannitol (this step can be performed before using the plasma). Mix well. Store in PPV at approximately -70°C until the expiration date of the plasma component.

[0284] 10% (w / v) TCA [BAC-359]. Weigh approximately 50 g of TCA into a solvent bottle. Using a graduated cylinder, add 500 mL of MQ. Stir to dissolve. The solution can be stored at room temperature for up to 1 month. This solution is used as a protein precipitation solution. Chill the solution on ice before using it for sample extraction.

[0285] 0.1% FA and 0.05% PFHx in MQ [BAC-360]. Use a graduated cylinder to measure 1000 mL of MQ and add it to the solvent storage bottle. Use a pipette to add 1 mL of FA and 0.5 mL of PFHx. Mix thoroughly. The solution can be stored at room temperature for up to 1 month. This solution serves as mobile phase A (MPA). This solution must be prepared in a 1-L Teflon container.

[0286] 0.1% FA and 0.05% PFHx in ACN [BAC-361]. Use a graduated cylinder to measure 1000 mL of ACN and add it to the solvent storage bottle. Use a pipette to add 1 mL of FA and 0.5 mL of PFHx. Mix thoroughly. This solution can be stored at room temperature for up to 1 month. This solution serves as mobile phase B (MPB). This solution must be prepared in a 1-L Teflon container.

[0287] 80:20:0.3(v / v / v)MeOH:MQ:FA[BAC-409]

[0288] Use a graduated cylinder to mix 800 mL of MeOH (methanol) and 200 mL of MQ in a solvent storage bottle. Use a pipette to add 3.00 mL of FA. Mix thoroughly. Store at room temperature for up to 1 month. This solution can be used as mobile phase A (MPA).

[0289] 100 mM ammonium formate in MQ [BAC-409]

[0290] Use a graduated cylinder to measure 1000 mL of MQ. Weigh approximately 6.306 g of ammonium formate into a tared weighing boat. Rinse with MQ to transfer the ammonium formate to a solvent storage bottle. Transfer the remaining MQ to a flask. Stir to dissolve. Store at room temperature for up to one month. This solution can be used to prepare mobile phase B (MPB).

[0291] 70:30 (v / v) 100 mM ammonium formate:methanol [BAC-410]

[0292] Using a graduated cylinder, combine 700 mL of BAC-408 and 300 mL of MeOH in a solvent storage bottle. Mix thoroughly. Store at room temperature until the expiration date of BAC-408. This solution can be used as mobile phase B (MPB).

[0293] 1000:1(v / v)MQ:FA[BAC-001].

[0294] Use a graduated cylinder to add 1000 mL of MQ to the solvent bottle. Use a pipette to add 1 mL of FA. Mix thoroughly. This solution can be stored at room temperature for up to 1 month. This solution can be used for the R0 solution.

[0295] 40:10:50:0.05 (v / v / v / v) IPA: Acetone: CAN: FA [BAC-083]

[0296] Using a graduated cylinder, combine 400 mL of IPA, 100 mL of acetone, and 500 mL of ACN and transfer to an appropriately sized solvent storage bottle. Stir to mix. Using a pipette, transfer 0.500 mL of FA to the bottle. Mix thoroughly. Store at room temperature for up to 1 month. Alternatively, a purchased solution can be used instead of the prepared solution. If using a purchased solution, store the solution at room temperature according to standard procedures. This solution can be used for R3.

[0297] 50:25:25 (v / v / v) IPA:ACN:MeOH [BAC-011]. Combine 500 mL of IPA, 250 mL of ACN, and 250 mL of MeOH in a solvent bottle using a graduated cylinder. Mix thoroughly. The solution can be stored at room temperature for up to 1 month. This solution is used as a powerful needle rinse.

[0298] Preparation of Stock Solutions Prepare stock solutions in duplicate and compare before use. The quantity prepared can be changed, provided the ratios and final concentrations are maintained and recorded.

[0299] GVA stock solution (10,000 μM) [S01]. After applying the correction factor to an amber glass vial, weigh an amount of GVA (MW 173.17) equivalent to 3 mg. Dissolve and dilute to 10,000 μM with MQ. Mix thoroughly. Divide the solution into 0.075 mL aliquots and store the solution in PPV at approximately -70°C. Thaw the stock solution on wet ice before use.

[0300] ArgA stock solution (10,000 μM) [S02]. After applying the correction factor to an amber glass vial, weigh an amount of ArgA (MW 175.19) equivalent to 3 mg. Dissolve and dilute to 10,000 μM with MQ. Mix thoroughly. Sonicate the stock solution for approximately 10 minutes. Divide the solution into 0.075 mL aliquots and store the solution in PPV at approximately -70°C. Thaw the stock solution on wet ice before use.

[0301] HArg stock solution (40,000 μM) [S03]. After applying the correction factor to an amber glass vial, weigh an amount of HArg (MW 224.69) equivalent to 15 mg. Dissolve and dilute to 40,000 μM with MQ. Mix thoroughly. Divide the solution into 0.100 mL aliquots and store the solution in PPV at approximately -70°C. Thaw the stock solution on wet ice before use.

[0302] NAArg stock solution (20,000 μM) [S04]. After applying the correction factor to an amber glass vial, weigh an amount of NAArg (MW 216.24) equivalent to 6 mg. Dissolve and dilute to 20,000 μM with MQ. Mix thoroughly. Divide the solution into 0.075 mL aliquots and store the solution in PPV at approximately -70°C. Thaw the stock solution on wet ice before use.

[0303] GAA stock solution (40,000 μM) [S05].

[0304] After applying the correction factor to an amber glass vial, weigh an amount of GAA (MW 117.11) equivalent to 14 mg. Dissolve and dilute to 40,000 μM with [BAC-009]. Mix thoroughly. Divide the solution into 0.150 mL aliquots and store the solution protected from light (PFL) in PPV at approximately -70°C. Thaw the stock solution on wet ice before use.

[0305] Preparation of Internal Standard (IS) Solution. The quantity prepared can be varied, provided the ratios and final concentrations are maintained and recorded.

[0306] GVA- 13 C6 IS stock solution (4,150 μM) [I01]. After applying the correction factor to the amber glass vial, weigh an amount of GVA- 13 C6 (MW 215.65). Dissolve in MQ and dilute to 4150 μM. Mix thoroughly. Divide the solution into 0.050 mL aliquots and store the solution in PPV at approximately -70°C. Thaw the stock solution on wet ice before use.

[0307] ArgA- 13 C6 IS stock solution (5,000 μM) [I02]. After applying the correction factor to the amber glass vial, weigh an amount equivalent to 3 mg of ArgA- 13 C6 (MW 181.14). Dissolve and dilute to 5,000 μM in MQ. Mix thoroughly. Divide the solution into 0.050 mL aliquots and store the solution in PPV at approximately -70°C. Thaw the stock solution on wet ice before use.

[0308] HArg-d4 IS stock solution (5,000 μM) [I03]. After applying the correction factor to an amber glass vial, weigh an amount of HArg-d4 (MW 265.17) equivalent to 3 mg. Dissolve and dilute to 5000 μM with MQ. Mix thoroughly. Divide the solution into 0.050 mL aliquots and store the solution in PPV at approximately -70°C. Thaw the stock solution on wet ice before use.

[0309] NAArg- 13 C6 IS stock solution (5,000 μM) [I04]. After applying the correction factor to the amber glass vial, weigh an amount equivalent to 3 mg of NAAg- 13 C6 (MW 222.19). Dissolve in MQ and dilute to 5,000 μM. Mix thoroughly. Divide the solution into 0.050 mL aliquots and store the solution in PPV at approximately -70°C. Thaw the stock solution on wet ice before use.

[0310] GAA-13C2 IS stock solution (5,000 μM) [I05].

[0311] After applying the correction factor to the amber glass vial, weigh an amount equivalent to 3 mg of GAA- 13 C2 (MW 119.09). Dissolve and dilute to 5,000 μM with [BAC-009]. Mix thoroughly. Divide the solution into 0.050 mL aliquots and store the solution PFL in PPV at approximately -70°C. Thaw the stock solution on wet ice before use.

[0312] Heavy IS working solution (2.075 μM I01; 2.50 μM I02, I04) [HI06]

[0313] Combine 0.020 mL of each of [I01], [I02], and [I04] with 39.940 mL of [1N HCl] in PPV using a pipette. Mix thoroughly. Prepare the solution on wet ice. Discard the solution after use.

[0314] IS working solution (5,000 μM) [I05]. Combine 0.020 mL of each of [I01], [I02], [I04], and [I05] with 39.920 mL of [1N HCl] in PPV using a pipette. Mix thoroughly. Prepare the solution on wet ice. Discard the solution after use.

[0315] Preparation of stock comparison solutions.

[0316] Prepare the comparison solution in PPV on wet ice using a pipette as shown in the table below. Mix thoroughly. Store frozen. The storage comparison solution used to establish stability should be evaluated within the established stability window of the processed samples. The prepared quantity may vary, provided the ratio and final concentration are maintained and recorded.

[0317]

[0318] For short-term stock comparisons, SC6 can be referred to as SST, and for long-term stock comparisons, SC6 can be referred to as SLT. SC6 can be used to compare I05 stock solution preparations. (*) When only one concentration is listed, GVA- 13 The C6 concentration was 0.83 times the indicated value.

[0319] Sample extraction.

[0320] 1. On wet ice, transfer 0.050 mL of each calibrant, quality control (Qc), blank, and experimental sample to a 96-well plate. Prior to sample processing, diluted samples must be diluted to the appropriate dilution factor with 1X PBS.

[0321] 2. Add 0.100 mL of [1N HCl] to each matrix blank sample.

[0322] 3. Add 0.100 mL of [I05] to each calibration, QC, blank with IS, and experimental sample. Do not add [I06] to GQC (GAA LLOQ (Lower Limit of Quantitation) (QC) samples, if present.

[0323] 4. Vortex mix at 1600 rpm for 5 minutes.

[0324] 5. Add 0.400 mL of ice-cold [BAC-359] to each sample.

[0325] 6. Vortex mix at 1000 rpm for 5 minutes.

[0326] 7. Centrifuge at 3500 rcf for 5 minutes.

[0327] 8. Use a TomTec or multichannel pipette to transfer 0.300 mL of supernatant to a clean 96-well plate.

[0328] 9. Centrifuge at 3500 rcf for 5 minutes.

[0329] 10. Store the processed samples at approximately 4°C in the sample compartment of the LC instrument or in a refrigerator until analysis.

[0330] Ultra-high performance liquid chromatography (UHPLC) setup.

[0331]

[0332] Gradient program:

[0333]

[0334] Mass spectrometer parameters. Parameters other than the mass spectrometer, interface, and scan mode can be modified to achieve optimal performance. Due to the unit resolution of a quadrupole mass spectrometer, the exact mass transition may vary slightly between instruments.

[0335]

[0336]

[0337]

[0338] Direct Binding ELISA for Detection of Anti-PEG Antibodies in Human Serum

[0339] A. Reagents / Buffers

[0340] Dilution buffer / assay buffer (4% bovine gamma globulin (BGG) in 1X PBS)

[0341] Prepare the assay diluent by adding 4 g of BGG to 100 mL of 1X PBS. Filter the solution through a 0.22 μM filter and store at 2-8°C for up to 2 weeks. Scale the volume as needed.

[0342] Competition buffer (assay buffer + 100 μg / mL PEG)

[0343] Prepare competition buffer by adding 100 μg of PEG per 1 mL of assay buffer. Prepare competition buffer fresh on the day of the assay. Scale volumes as needed.

[0344] Stop solution (1M H3PO4)

[0345] Add 21.4 mL of 85% (11.7 M) H3PO4 to 228.6 mL of dI (deionized) H2O, mix thoroughly and store in an acid cabinet for up to 1 year. Scale volumes as needed.

[0346] Other reagents (see table below)

[0347]

[0348]

[0349]

[0350] B. Screening Assay Procedure

[0351] According to the plate map, the wells of a Starwell C8 Maxisorp (96-well format plate) were coated with 100 μL of 2 μg / mL BSA mPEG 5K (5,000 Daltons) or 500 ng / mL human IgG or 500 ng / mL human IgM in carbonate coating buffer (these controls were excluded from the post-cut point run). The plate(s) were sealed, shaken briefly at approximately 450 rpm to ensure distribution throughout the wells, and incubated at 37°C for approximately 1 hour. After incubation, the wells were washed 3 times with 1X PBS. The washer program contained an overflow device with an aspiration setting, and each dispense was followed by shaking for 10 seconds (Program 29_Wash_Shake) to ensure complete cleaning of the star-shaped well plate. This program was used for all wash steps in this program.

[0352] After washing, the plate(s) were inverted and patted dry on absorbent paper. 300 μL of dilution buffer was then added to all wells of the plate(s). The dilution buffer contained 4% BGG and was therefore also referred to as "blocking buffer." The plate(s) were covered and incubated at room temperature with shaking (approximately 450 rpm) for a minimum of 1 hour, but no more than 3 hours.

[0353] After the blocking incubation period, the plate (multiple) was washed, inverted and patted dry on absorbent paper. According to the plate map, the control and sample diluted to 50 times the minimum required dilution (MRD) in dilution buffer were added to the plate in duplicate (100 μL / well). The plate (multiple) was then sealed and incubated at room temperature with shaking (about 450 rpm) for about 1 hour.

[0354] The plate(s) are washed, inverted and patted dry on absorbent paper, and 100 μL of detection antibody is added to the appropriate wells according to the plate map. Mouse anti-PEG antibodies are detected using goat anti-mouse IgG-Fc-HRP diluted 1:5,000, and anti-human antibodies are detected using rabbit anti-human IgG / A / M diluted 1:30,000 in dilution buffer and added to the appropriate wells. The plate(s) are sealed and incubated at room temperature with shaking (approximately 450 rpm) for approximately 1 hour. The plate(s) are washed, inverted and patted dry on absorbent paper, and 100 μL of TMB substrate is added to each well. The plate(s) are covered and incubated at room temperature with shaking (approximately 450 rpm) for approximately 10-20 minutes. The reaction is stopped by adding 100 μL of stop solution to each well. Briefly shake the plate(s) to ensure

[0355] C. Confirmatory Assay Procedures

[0356] According to the following plate map, 100 μL of 2 μg / mL BSA-mPEG 5K, or 500 ng / mL human IgG, or 500 ng / mL human IgM in carbonate coating buffer (these controls were excluded from the post-cleavage run) were coated onto the wells of a Starwell C8 Maxisorp plate. The plate(s) were sealed, shaken briefly at approximately 450 rpm to ensure distribution throughout the wells, and incubated at 37°C for approximately 1 hour.

[0357] After incubation, the wells were washed 3 times with 1X PBS using program 29_Wash_Shake to ensure complete cleaning of the Starwell plate. This program was used for all washing steps in this program. After washing, the plate (multiple) was flipped over and patted dry on absorbent paper. 300 μL of dilution buffer was then added to all wells of the plate (multiple) as a blocking step. The plate (multiple) was covered and incubated at room temperature with shaking (approximately 450 rpm) for at least 1 hour, but no more than 3 hours.

[0358] While the Starwell plates (multiple) are blocked, samples and controls are diluted to 50-fold MRD in dilution buffer and competition buffer containing 100 μg / mL PEG in polypropylene plates. The diluted samples and controls are incubated at room temperature for approximately 1 hour with shaking (approximately 450 rpm).

[0359] After the blocking incubation period, the star-shaped well plate (multiple) was washed, inverted, and patted dry on absorbent paper. According to the plate map, controls and samples with and without PEG pre-incubation were added to the plate in duplicate (100 μL / well). The plate (multiple) was then sealed and incubated at room temperature with shaking (approximately 450 rpm) for approximately 1 hour.

[0360] 4,647,851 cells / well.After washing, plate (multiple) is inverted and patted dry on absorbent paper, and 100 μ L of detection antibody is added to appropriate wells according to plate diagram. Use goat anti-mouse IgG-Fc-HRP dilution 1:5,000 to detect mouse anti-PEG antibody, and use rabbit anti-human IgG / A / M dilution 1:30,000 in dilution buffer to detect anti-human antibody, and add to appropriate wells. Plate (multiple) is sealed and incubated at room temperature for about 1 hour with shaking (about 450rpm). Plate (multiple) is washed, inverted and patted dry on absorbent paper, and then every hole TMB substrate is added 100 μ L. Cover plate (multiple) and incubated at room temperature for about 10-20 minutes with shaking (about 450rpm). Stop reaction by adding 100 μ L stop solution in every well. Briefly shake plate (multiple) to ensure appropriate mixing, then read with 450 (detection) 620 (background) readings on Synergy 2 plate reader.

[0361] D. Titration Procedure

[0362] Titer determination follows the same procedure as the screening assay. The sample to be titrated is diluted at least seven times in a two-fold serial dilution in negative pooled human serum. The normal human pool is screened during the identification process, and the human pool with the lowest background is selected as the negative pool in the validation. These titer dilutions are diluted to a minimum required dilution of 50 times using dilution buffer and added to the plate in duplicate (100 μL / well) according to the plate map.

[0363] E. Method Validation

[0364] Validation of the method included assessment of specificity, assay sensitivity, selectivity / matrix interference, drug tolerance, prozone (hook) effect, titration assay linearity, intra- and interassay precision, short-term and freeze-thaw stability, and establishment of screening, confirmation, and titration cut points.

[0365] Two sets of controls are used for assay validation. One set is prepared using a mouse anti-PEG surrogate positive control spiked into pooled normal human serum (NC) to produce high (HPC-m) and low (LPC-m) signals in the assay. A second set is prepared using a human sample that has a high anti-PEG response in the screening assay and a high inhibitory effect in the confirmatory assay. This sample will be consistent with the expected pre-existing anti-PEG sample. A human anti-PEG low control is prepared by diluting the identified high human sample into the negative control serum to produce a sample that results in a low (LPC-h) signal in the assay. Undiluted high-level human samples are used as HPC-h. Both sets of controls are included in each run (if applicable). The controls should show signals proportional to their levels, i.e., high is greater than low, and low is greater than the cut point. Immunoglobulin controls (human IgG and human IgM coated on the plate) are also included in the cut point run to verify the performance of the detection antibody mixture.

[0366] F. Cut-point analysis

[0367] Mouse Anti-PEG Screening Cut Points To evaluate the data using mouse anti-PEG SPC, the screening cut point was set using the standard deviation of the mean of all negative control samples generated during the 2-fold validation process (n=55 plates). This calculation yielded a correction factor of 0.01467, which was added to the mean of the NCs on each plate to generate a cut point specific for mouse anti-PEG SPC samples.

[0368] Human screening assay cut point. Screening assay cut point was determined using 98 individual normal human serum samples. Each sample was assayed 6 times in a minimum of 7 runs by 3 analysts. The software (version 12 or higher) evaluates the data, eliminates statistical outliers, identifies differences and establishes cut points. To identify statistical outliers, responses were normalized by dividing the mean signal of the sample by the mean of the anti-human NC samples per plate.

[0369] G. Determination of tangent point

[0370] Human anti-PEG screening cut points. Parametric and nonparametric screening floating cut factors were determined using normalized values. First, after excluding outliers, a parametric method using Tukey's two-weight procedure was used to calculate robust estimates of the mean and standard deviation (SD) of the ratios. Then, a parametric floating cut factor with a false positive error rate of 5% was determined by multiplying the SD value by the 95th percentile of the t-distribution (degrees of freedom equal to the number of ratio values ​​minus 1) and adding the product to the mean. A nonparametric 5% error rate cut factor was determined by calculating the empirical 95th percentile of the ratio values.

[0371] Confirmatory cut points were determined using the procedure recommended by Shankar G et al. with a 1% false-positive error rate. Cut points were determined by assigning a lower limit of specific inhibition using 14 samples excluded from the screening cut point assignment as biological outliers. These samples consistently had higher % inhibition values ​​compared to samples that screened negative. The percent change from the unspiked sample was calculated for each sample using the following formula:

[0372]

[0373] Parametric confirmatory cut points were determined by first calculating the Tukey biweighted estimates of the mean and SD of all inhibition percentages retained in the analysis. Cut points were then calculated by multiplying the SD values ​​by a factor equal to the first percentile of the t distribution (with degrees of freedom equal to the number of inhibition values ​​minus 1) and subtracting the mean from the product. Nonparametric cut points were also determined based on the empirical first percentile.

[0374] G. Specificity

[0375] The addition of high levels of human IgG did not affect the signal generated in the assay compared to unspiked samples.

[0376] H.PEG resistance

[0377] Assay interference was assessed by performing a two-fold serial dilution of PEG starting at a concentration of 100 μg / mL in the presence of an anti-PEG surrogate positive control equal to that of HPC-m and LPC-m, as well as human anti-PEG HPC-h and LPC-h. A zero spike control consisting of each control without PEG was also run. This was performed in duplicate by at least two analysts over a minimum of two runs. Human-specific screening cut points were used to determine tolerance in these assay runs. HPC-m, HPC-h, and LPC-h tolerated all concentrations of PEG tested, up to 100 μg / mL.

[0378] I. Drug Tolerance of Pegylated Arginase

[0379] Assay interference was assessed by performing two-fold serial dilutions of PEGylated arginase starting at a concentration of 150 μg / mL in the presence of anti-PEG surrogate positive controls equal to those of HPC-m and LPC-m, as well as human anti-PEG HPC-h and LPC-h. A zero-spiked control consisting of each control without PEG was also run. Drug tolerance was assessed using anti-human-specific cut points. HPC-m tolerated 9.38 μg / mL in one run and up to 37.5 μg / mL in a second run, for an average drug tolerance of 23.4 μg / mL. LPC-m was intolerant to Co-Arg1 PEG in one run and up to 2.34 μg / mL in a second run. HPC-h tolerated 9.38 μg / mL in one run and up to 18.8 μg / mL in a second run, for an average drug tolerance of 14.1 μg / mL. In one run, LPC-h was tolerated to 4.69 μg / mL, and in the second run, 9.38 μg / mL was tolerated, for an average drug tolerance of 7.04 μg / mL.

[0380] Neuromotor and neurocognitive testing methods

[0381] Physical examinations will be completed with neurological and neuromotor function assessments, including but not limited to the 6MWT, GMFM, Berg balance scale, modified Ashworth scale, and Purdue pegboard test. Assessments may be continued for more than one day. Assessments may be recorded at baseline and at subsequent time points thereafter.

[0382] The 6MWT has been widely used in clinical research to comprehensively assess the cardiovascular, pulmonary, and neuromuscular systems in a variety of disease situations. It measures the distance a patient can walk on a flat surface in 6 minutes. The American Thoracic Society (ATS) has published a standardized method (see, ATS Statement Guidelines for the Six-Minute Walk; Am. J. Respir. Crit. Care Med., 166: 111-117, 2002) for conducting the test, which is well tolerated and easy to administer. The 6MWT follows this procedure. With the approval of the sponsor, modifications to the method may be allowed to take into account the patient's disease condition and logistical considerations of the test site. Ambulatory patients did not receive the 6MWT. The results of the 6MWT are recorded as the distance completed (in meters) and the percentage change relative to baseline.

[0383] The Berg Balance Scale measures balance in patients with impaired balance by assessing performance on certain functional tasks. Fourteen tasks assess aspects of balance, such as sitting to standing, transferring, turning, and standing on one leg. Additionally, most tasks require the subject to maintain a posture for a specific amount of time.

[0384] The GMFM is a clinical measure designed to assess changes in gross motor function through a range of activities, such as lying, rolling, walking, running, and jumping. Each item on the GMFM uses a 4-point scoring system to determine how well a person can initiate and complete a movement, covering five dimensions (A through E). A: lying and rolling, B: sitting, C: crawling and kneeling, D: standing, and E: walking, running, and jumping.

[0385] The modified Ashworth scale is used to assess spasticity in patients with central nervous system (CNS) pathology and measures resistance to passive motion around joints due to spasticity. The scale is well tolerated and easy to administer. It cannot differentiate between spasticity and soft tissue stiffness. The scale uses a 0 (no spasticity) to 4 (total stiffness) scale with six scoring options, as shown in the table below:

[0386]

[0387] The Purdue Pegboard Test is a test of manipulative dexterity and bimanual coordination that measures the speed of performance while using both sides of the body to perform fine motor movements (Tiffin et al., 1948). The test involves two distinct abilities: (1) gross motor movements of the arms, hands, and fingers, and (2) fine motor limbs, also known as "fingerprint" dexterity. Poor performance on the Pegboard Test is a sign of deficiencies in complex, visually guided, or coordinated movements, most likely mediated by circuits involving the basal ganglia.

[0388] The table below shows additional neurocognitive, developmental, and quality of life (QOL) measures that can be administered to treated patients to assess improvement over time.

[0389]

[0390]

[0391]

[0392] a PROMIS (for patients who started Phase 1 / 2 studies using PROMIS) or the Pediatric Quality of Life Inventory (PedsQL) measurement model (for patients who started Phase 1 / 2 studies not using PROMIS).

[0393] References

[0394] ATS Statement Guidelines for the Six-Minute Walk,Am.J.Respir.Crit.Care Med.,166:111-117,2002

[0395] Burrage et al., “Human recombinant arginase enzyme reduces plasmaarginine in mouse models of arginase deficiency,” Hum.Mol.Genetics 24(22):6417-27(2015)

[0396] Carvalho, DR, et al., “Clinical features and neurologic progression of hyperargininemia,” Pediatr. Neurol., 46(6):369-74(2012)

[0397] Cheng et al., Cancer Res. 67:309-17, 2007

[0398] Deignan et al., "Increased plasma and tissue guanidine compounds in amouse model of hyperargininemia," Mol.Genet.Metab.93:172-178, 2008

[0399] Dillon et al.,“Biochemical characterization of the argininedegradingenzymes arginase and arginine deiminase and their effect on nitricoxideproduction,”Med.Sci.Monit.,8(7):BR248-253(2002)

[0400] Downs et al.,“The Berg Balance Scale,”J.Physiother.,”2015 61(1):46

[0401] Enright et al.,“Reference equations for the six-minute walk inhealthyadults,”Am.J.Respir.Crit.Care Med.,1998 158(5 Pt 1):1384-1387

[0402] Geiger et al.,“Six-minute walk test in children and adolescents,”J.Pediatr.,2007,150(4):395-399

[0403] et al.,“Suggested guidelines for the diagnosis and managementofurea cycle disorders,”Orphanet.J.Rare Dis.,2012 7:32

[0404] Harris et al.,Clin.Pharmacokinet.40(7):539-51,2001

[0405] Lambert et al.,“Hyperargininemia:intellectual and motorimprovementrelated to changes inbiochemical data,”J.Pediatr.,1991,118(3):420-4

[0406] Lopez et al.,FEBS J.272:4540-48,2005

[0407] Lüneburg,N.et al.,“Reference intervals for plasma L-arginine and theL-arginine:asymmetric dimethylarginine ratio in the FraminghamOffspringCohort.” J.Nutr. 141(12):2186-2190(2011)。

[0408] Marescau et al.“Guanidino compound analysis as acomplementarydiagnostic parameter for

[0409] hyperargininemia:Follow-up of guanidino compound levelsduringtherapy,”Pediatric.Res.27(3):297-303(1990)

[0410] Marescau et al.,“The pathobiochemistry of uremia andhyperargininemiafurther demonstrates a

[0411] metabolic relationship between urea and guanidinosuccinic acid,”199241(9):1021-1024

[0412] Oeffinger et al.,“Outcome tools used for ambulatory children withcerebralpalsy:responsiveness and minimum clinically important differences,”Dev.Med.Chile Neurol.,2008;50(12):918-925

[0413] Prasad et al.,“Argininemia:a treatable genetic cause of progressivespasticdiplegia simulating cerebral palsy-case reports and literature review”J.ChildNeurol.12:301-309,1997

[0414] Remington,The Science and Practice of Pharmacy,19th ed.,Gennaro,ed.,Mack Publishing Co.,Easton,PA 1995

[0415] Savoca et al.,Cancer Biochem.Biophys.7:261-268,1984

[0416] Schrover et al.,“Minimal clinically important difference for the 6-min walktest:literature review and application to Morquio A syndrome,”Orphanet.J.RareDis.,2017 12(1):78

[0417] Schlune et al.,“Hyperargininemia due to arginase 1 deficiency:theoriginalpatients and their natural history,and a review of the literature,”Amino Acids 47:1751-1762,2015

[0418] Segawa et al.,“A long-term survival case of arginase deficiency withseveremulticystic white matter and compound mutations,”Brain Dev.33:45-48,2011

[0419] Stockler-Ipsiroglu et al., “Guanidinoacetate methyltransferase (GAMT) deficiency: outcomes in 48 individuals and recommendations for diagnosis, treatment and monitoring,” Mol. Genet. Metab., 111(1):16-25, 2014

[0420] Uchino, T., et al., “Molecular basis of phenotypic variation in patients with argininemia,” Hum. Genet. 96(3):255-60(1995)

[0421] Wu, G. et al., “Arginine metabolism: nitric oxide and beyond,” Biochem. J., 336(Pt 1), 1-17, 1998

[0422] Wyse et al., “In vitro stimulation of oxidative stress in cerebral cortex of rats by the guanidino compounds accumulating in hyperargininemia,” Brain Res. 2001, 923(1-2):50-7 as well as U.S. Patent Publications: U.S.Pub. 20170240922; U.S.Pub. 20170283830; U.S.Pub. 20170224843; U.S.Pub. 20170191078; U.S.Pub. 20160095884; U.S.Pat. 8,398,968; U.S.Pat. 8,440,184; U.S.Pub. 20160095884; and U.S.Pub. 20140154797.

Claims

1. A method of treating arginase 1 (ARG1) deficiency (ARG1-D) in a subject, comprising administering to the subject an amount of arginase sufficient to reduce the subject's plasma level of arginine to less than 200 μmol / L within about 2 to about 4 days after the initial administration of the arginase. 2 . The method of claim 1 , wherein following initial administration of the arginase, the subject's plasma level of arginine decreases to within the range of 40 μmol / L to 115 μmol / L. The method according to claim 1 , wherein the arginase is PEGylated arginase 1.

4. The method according to any one of claims 1 to 3, wherein the arginase has a cobalt metal cofactor instead of a manganese metal cofactor.

5. The method according to any one of claims 1 to 4, wherein the arginase is pegzilarginase.

6. The method of any one of claims 1 to 5, wherein the plasma level of at least one compound selected from the group consisting of N-α-acetylarginine (NAArg), argininic acid (ArgA), α-keto-δ-guanidinovaleric acid (GVA), guanidinoacetic acid (GAA), and arginine is reduced to normal levels in the subject at least once less than 7 days after the initial administration of the arginase.

7. The method of claim 6, wherein the plasma level of at least one compound selected from the group consisting of ArgA, NAArg, GVA, GAA, and arginine is reduced to normal levels in the subject at least once less than 3 days after the initial administration of the arginase.

8. The method of claim 6, wherein the plasma level of at least one compound selected from the group consisting of ArgA, NAArg, GVA, GAA, and arginine is reduced to normal levels in the subject at least once less than 2 days after the initial administration of the arginase.

9. The method of claim 6, wherein the plasma level of at least one compound selected from the group consisting of ArgA, NAArg, GVA, GAA, and arginine is reduced to normal levels in the subject at least once less than 1 day after the initial administration of the arginase.

10. The method of any one of claims 1-5, wherein the amount sufficient to reduce the subject's plasma level of arginine improves a characteristic selected from the group consisting of resting spasticity, leg cramps associated with spasticity, adaptive behavior, and a Patient-Reported Outcomes Measurement Information System (PROMIS) physical function score.

11. The method of any one of claims 1-5, wherein guanidinoacetic acid (GAA) plasma levels in the subject are brought to normal levels.

12. The method of any one of claims 1 to 11, wherein the arginase is administered to the subject intravenously or subcutaneously.

13. The method of any one of claims 1 to 12, wherein the arginase is administered intravenously to the subject at a dose of about 0.005 to about 1.00 mg / kg of the subject's body weight.

14. The method of claim 13, wherein the arginase is administered intravenously to the subject at a dose of about 0.005 to about 0.50 mg / kg of the subject's body weight.

15. The method of claim 14, wherein the arginase is administered intravenously to the subject at a dose of about 0.005 to about 0.20 mg / kg of the subject's body weight.

16. The method of any one of claims 1 to 11, wherein the arginase is administered subcutaneously to the subject at a dose of about 0.01 to about 1.50 mg / kg of the subject's body weight.

17. The method of claim 16, wherein the arginase is administered subcutaneously to the subject at a dose of about 0.015 to about 0.75 mg / kg of the subject's body weight.

18. The method of claim 17, wherein the arginase is administered subcutaneously to the subject at a dose of about 0.015 to about 0.30 mg / kg of the subject's body weight.

19. The method of any one of claims 1 to 11, wherein the arginase is administered to the subject at a dose sufficient to reduce the plasma level of at least one compound selected from the group consisting of ArgA, NAArg, GVA, GAA, and arginine in the subject by at least 2-fold, wherein the plasma level is determined about 24 to 48 hours after administration.

20. The method of claim 19, wherein the administered dose reduces the plasma level of NAAg by at least 3-fold.

21. The method of claim 19, wherein the administered dose reduces the plasma level of GAA by at least 2-fold.

22. The method of any one of claims 1 to 21, wherein the arginase is administered to the subject daily, weekly, twice monthly, or monthly.

23. The method of any one of claims 1 to 22, further comprising administering a nitrogen scavenger to the subject.

24. The method of claim 1, wherein the arginase is administered in the form of a nucleic acid operably linked to an adenoviral vector for delivery to the subject and for production of arginase in the subject following administration to the subject.

25. A method for rapidly reducing the plasma level of at least one compound selected from the group consisting of arginine, N-α-acetylarginine (NAArg), argininic acid (ArgA), α-keto-δ-guanidinovaleric acid (GVA), guanidinoacetic acid (GAA) in a subject with arginase 1 (ARG1) deficiency (ARG1-D) to normal levels, the method comprising administering to the subject a composition comprising a therapeutically effective amount of PEGylated arginase, wherein the PEGylated arginase is initially administered intravenously at a dose of 0.005 mg / kg to 1.00 mg / kg and then administered subcutaneously or intravenously weekly thereafter.

26. The method of claim 25, wherein the PEGylated arginase is initially administered intravenously at a dose of 0.005 mg / kg to 0.50 mg / kg and thereafter administered subcutaneously or intravenously weekly.

27. The method of claim 26, wherein the PEGylated arginase is initially administered intravenously at a dose of 0.005 mg / kg to 0.20 mg / kg and thereafter administered subcutaneously or intravenously weekly.

28. The method of any one of claims 25-27, wherein the PEGylated arginase is pegzilarginase.

29. The method of any one of claims 25-28, wherein the plasma levels of at least one compound selected from the group consisting of ArgA, NAArg, GVA, GAA, and arginine are reduced to normal levels in the subject in less than 3 days after the initial administration of the PEGylated arginase.

30. The method of any one of claims 25-28, wherein the plasma levels of at least one compound selected from the group consisting of ArgA, NAArg, GVA, GAA, and arginine are reduced to normal levels in the subject in less than 2 days after the initial administration of the PEGylated arginase.

31. The method of any one of claims 25-28, wherein the plasma level of at least one of ArgA, NAArg, GVA, GAA, and arginine in the subject is reduced to normal levels less than 1 day after initial administration of the PEGylated arginase.

32. The method of any one of claims 25-28, wherein the dose of the PEGylated arginase administered to the subject is sufficient to reduce the plasma level of at least one compound selected from the group consisting of ArgA, NAArg, GVA, GAA, and arginine by at least 2-fold, wherein the plasma level is determined about 24 to 48 hours after administration of the PEGylated arginase.

33. A method for rapidly reducing the plasma level of at least one compound selected from the group consisting of arginine, N-α-acetylarginine (NAArg), argininic acid (ArgA), α-keto-δ-guanidinovaleric acid (GVA), guanidinoacetic acid (GAA) in a subject with arginase 1 (ARG1) deficiency (ARG1-D) to normal levels, the method comprising administering to the subject a composition comprising a therapeutically effective amount of PEGylated arginase, wherein the PEGylated arginase is initially administered subcutaneously at 0.01 mg / kg to 1.50 mg / kg and then administered subcutaneously or intravenously weekly thereafter.

34. The method of claim 33, wherein the PEGylated arginase is initially administered subcutaneously at 0.015 mg / kg to 0.75 mg / kg and then administered subcutaneously or intravenously weekly thereafter.

35. The method of claim 34, wherein the PEGylated arginase is initially administered subcutaneously at 0.015 mg / kg to 0.30 mg / kg and then administered subcutaneously or intravenously weekly thereafter.

36. The method of any one of claims 33-35, wherein the PEGylated arginase is pegzilarginase.

37. The method of claims 33-36, wherein the plasma levels of at least one compound selected from the group consisting of ArgA, NAArg, GVA, GAA, and arginine are reduced to normal levels in the subject in less than 3 days after the initial administration of the PEGylated arginase.

38. The method of any one of claims 33-36, wherein the plasma levels of at least one compound selected from the group consisting of ArgA, NAArg, GVA, GAA, and arginine are reduced to normal levels in the subject in less than 2 days after the initial administration of the PEGylated arginase.

39. The method of any one of claims 33-36, wherein the plasma levels of at least one compound selected from the group consisting of ArgA, NAArg, GVA, GAA, and arginine are reduced to normal levels in the subject less than 1 day after the initial administration of the PEGylated arginase.

40. The method of any one of claims 33-36, wherein the dose of the PEGylated arginase administered to the subject is sufficient to reduce the plasma level of at least one compound selected from the group consisting of ArgA, NAArg, GVA, GAA, and arginine by at least 2-fold, wherein the plasma level is determined about 24 to 48 hours after administration of the PEGylated arginase.

41. The method of claim 22, wherein the subject is administered at least one repeated dose of the arginase in an amount that reduces the plasma level of arginine to less than 200 μmol / L.

42. The method of claim 41, wherein the plasma level of arginine is reduced to below 200 μmol / L for at least 30 weeks.

43. The method of claim 42, wherein the plasma level of arginine is reduced to below 200 μmol / L for at least 40 weeks.

44. The method of claim 43, wherein after receiving eight repeated doses of at least one, the subject exhibits at least one improvement in: (a) mobility or (b) adaptive behavior relative to a baseline of the subject's mobility or adaptive behavior before treatment.

45. The method of claim 22, wherein the plasma level of at least one compound selected from the group consisting of NAArg, ArgA, GVA, GAA, or arginine is reduced compared to baseline plasma levels in the subject.

46. ​​The method of any one of claims 25 to 41, wherein the subject's minimal clinically important difference (MCID) is greater than 1 after 9 days of treatment.

47. The method of claim 46, wherein the plasma level of arginine correlates with the MCID.

48. A composition comprising polyethylene glycol arginase and a pharmaceutically acceptable buffer.

49. The composition of claim 49, further comprising about 10% glycerol, wherein the pharmaceutically acceptable buffer is phosphate buffered saline.

50. The composition of claim 49, wherein the PEGylated arginase is formulated in red blood cell ghosts.

Images