Method and composition for treating arginase 1 deficiency

Abstract

To provide a method to treat a subject with arginase 1 (ARG1) deficiency (ARG1-D).SOLUTION: A method comprises administering an arginase to a subject in an amount sufficient to reduce a plasma level of arginine in the subject to below 200 μmol / L within about 2 to about 4 days after initial administration of the arginase.SELECTED DRAWING: None

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims 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.

[0002] Sequence Listing Reference The sequence listing associated with this application has been submitted in electronic format via EFS-Web and is hereby incorporated by reference in its entirety. The name of the text file containing the sequence listing is 218107-0011-00-WO_ST25. The size of the text file is 6,247 bytes and was created on December 4, 2018. [Technical field]

[0003] background The present invention discloses methods of treating Arginase 1 Deficiency in a patient and compositions for treating Arginase 1 Deficiency (ARG1-D) in a patient. [Background technology]

[0004] Arginase 1 deficiency (also known as hyperargininemia or argininemia) is caused by a deficiency or loss of activity of the arginase 1 protein, for example 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 and progressive disease. It is estimated to occur in 1 in 300,000 to 1 million individuals. ARG1-D is an autosomal recessive urea cycle disorder that results in toxic accumulation of arginine and other guanidino compounds (GCs). Clinical features of ARG1-D typically manifest during early infancy, although some patients may manifest shortly after birth. Symptoms of the disease include spasticity, growth retardation, stroke, protein avoidance, episodic hyperammonemia, nausea, and vomiting. In adolescence, when the disease progresses, patients may lose mobility, be unable to speak or understand, and suffer from malnutrition and vitamin D deficiency.Further progression of the disease and its associated symptoms includes liver damage due to elevated transaminases and hepatic fibrosis, severe spasticity and muscle contractures, mental retardation, and limited life span.Unfortunately, dietary restrictions and nitrogen scavengers are insufficient to prevent disease progression due to chronic and significant increase in arginine and other guanidino compounds and episodic hyperammonemia.

[0005] When ARG1-D patients are provided with a low protein diet, arginine levels can be reduced to approximately 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," Amino Acids 47:1751-1762 (2015)). De novo arginine synthesis, influenced by dietary protein intake, accounts for approximately 5-15% of plasma arginine synthesis, and the major source of arginine is turnover of body tissues (Wu, G. et al., "Arginine metabolism: nitric oxide and beyond," Biochem J 336(part 1):1-17 (1998)). Thus, dietary protein restriction has limited effect on plasma arginine levels, and circulating levels of arginine in subjects remain high due to the inability to degrade excess arginine.

[0006] Non-clinical studies of most treatment methods using ARG1-D animal models have not shown that lowering plasma arginine levels has a substantial effect on disease outcome. Considering that most of these approaches do not address the root cause of the disease, namely correcting the cytosolic deficiency of ARG1 in the liver, the US Food and Drug Administration (FDA) has questioned whether increasing plasma arginase levels can have the same overall effect as correcting intracellular ARG1 activity, including reducing arginine-derived guanidino compounds that may contribute to disease pathogenesis. Although there is literature evidence that dietary arginine restriction can reduce plasma arginine levels, the beneficial impact of dietary approaches on disease expression is limited due to chronically significant elevation of plasma arginine levels, which are well above both physiological normal ranges and medical guidelines. This is supported by a group in which progression of spasticity was reported in 4 of 11 patients despite dietary therapy and reduction in plasma arginine (Prasad et al., "Argininemia: a treatable genetic cause of progressive spastic diplegia simulating cerebral palsy-case reports and literature review," J. Child Neurol, Vol. 12, pp. 301-309 (1997)).In line with this rationale, some investigators have suggested that high levels of arginine metabolites, such as arginate, guanidinoacetate, β-guanidinopropionic acid, β-guanidinobutryic acid, and N-α-acetylarginine, may be important in causing the neurological sequelae in ARG1-deficient patients (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., "A long-term survival case of arginase deficiency with severe multicystic white matter and compound mutations," Brain Dev. 33:45-48 (2011); Wyse et al., "In vitro stimulation of oxidative stress in cerebral cortex of rats by the guanidino compounds accumulating in (Lambert et al., "Hyperargininemia: intellectual and motor improvement related to changes in biochemical data," J. Pediatr., (1991) vol. 118, no. 3, pp. 420-4). Lambert et al. were able to show that a low-arginine diet can reduce blood arginine levels within approximately one month after strict dietary control, but that guanidinoacetic acid and α-keto-δ-guanidinovaleric acid (GVA) levels were not changed, meaning that diet is unlikely to rapidly control GC levels as a therapeutic intervention.Given the disorder and its underlying complex mechanisms, new therapies are needed to rapidly lower and control high levels of arginine in patients to address this unmet medical need. Summary of the Invention

[0007] Newly disclosed herein are methods, compounds, and compositions for treating ARG1-D patients, preferably obtaining a rapid response in the patient, where the patient is a human, and may be an adult, child, or infant.

[0008] Methods of treating arginase 1 (ARG1) deficiency (ARG1-D) in a subject are provided, 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 a first administration of arginase. The subject's plasma level of arginine can be reduced to within a range of 40 μmol / L to 115 μmol / L after a first administration of arginase. The subject can be a human adult, human child, or human infant (e.g., less than 12 months of age) with ARG1-D.

[0009] The arginase may be wild-type arginase I (e.g., SEQ ID NO: 2) or arginase II (e.g., SEQ ID NO: 1). The arginase may be PEGylated arginase 1 and may have a cobalt metal cofactor instead of a manganese metal cofactor. The PEGylated arginase 1 may be a pegzilarginase.

[0010] The disclosed method of treatment can further involve administration of at least one of N-α-acetylarginine (NAArg), arginic acid (ArgA), GVA, guanidinoacetic acid (GAA), and arginine to a normal level at least once in the subject, less than 7 days, less than 3 days, less than 2 days, and / or less than 1 day after the first administration. The method of treatment with arginase can result in the plasma level of GAA reaching a normal level after administration. The method can result in an amount sufficient to reduce the plasma level of arginine in the subject to improve one or more characteristics, such as resting spasticity, spasticity-related leg spasms, adaptive behavior, and Patient-Reported Outcomes Measurement Information System (PROMIS) physical function score.

[0011] This method of treatment contemplates administering intravenously from about 0.005 to about 1.00 mg / kg / patient body weight. Other ranges or uses for intravenous administration can include 0.01-0.5 mg / kg, 0.01-0.2 mg / kg, 0.015-0.25 mg / kg, and 0.015-0.075 mg / kg, with increments of 0.005 between the recited ranges also being contemplated.

[0012] This method of treatment contemplates administering subcutaneously from about 0.01 to about 1.50 mg / kg / patient body weight. Other ranges or uses for intravenous administration can include 0.015-0.75 mg / kg, 0.015-0.30 mg / kg, 0.015-0.25 mg / kg, and 0.015-0.075 mg / kg, with increments of 0.005 between the recited ranges also being contemplated.

[0013] Another method contemplates administering arginase to a 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 two-fold, where the plasma level is assayed about 24-48 hours after administration. Another method contemplates that the administered dose reduces the plasma level of NAArg by at least three-fold. Another method contemplates that the administered dose reduces the plasma level of GAA by at least two-fold. The arginase of the disclosed therapeutic methods can be administered to a patient intravenously or subcutaneously, or a combination of both. The arginase can be administered to a subject daily, weekly, bimonthly, or monthly.

[0014] A nitrogen scavenger may also be administered to the subject. The disclosed compositions having arginase may include a nitrogen scavenger. The disclosed methods of treatment with arginase may be administered to a subject with a nitrogen scavenger, and in some cases, the subject may further be placed on a low-arginine diet. Another method of treating a subject with arginase includes administering a nucleic acid operably linked to an adenoviral vector for delivery to the subject, which produces arginase in the subject when administered to the subject. The amount of arginase produced by adenoviral vector delivery may be therapeutically effective 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 two-fold, where the plasma level is assayed about 24-48 hours after administration of arginase.

[0015] Another method contemplates improving neuromotor function after the first administration of arginase. Neuromotor function can be, but is not limited to, one or more of steps, walking, spasticity, and / or awakening. Another method contemplates that the subject shows at least one of the following after the first administration of arginase: reduced resting spasticity, reduced spasticity-related lower limb cramps, adaptive behavior, and improved PROMIS-T score, compared to at least one of spasticity, behavior, and PROMIS-T score before administration of arginase. This method contemplates an acute phase response to treatment with arginase, such that one or more toxic metabolites (e.g., GAA) are reduced to normal levels or eliminated.

[0016] The method contemplates administering to the subject at least one repeated dose of arginase in an amount that reduces 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 8 of the at least one repeated dose, the subject can show at least one improvement in (a) the mobility or (b) adaptive behavior, compared to the baseline of the mobility or adaptive behavior of the subject before treatment. 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 contemplates that the subject has a minimal clinically important difference (MCID) of more than 1 after 9 days of treatment. The plasma level of arginine is correlated with the MCID.

[0017] Also contemplated is a composition comprising PEGylarginase and a pharma- ceutically acceptable buffer. The composition can comprise about 10% glycerol, and the pharma-ceutically acceptable buffer can be phosphate buffered saline in addition to a desired amount of PEGylarginase or other arginase. Provided is the use of a composition comprising arginase (e.g., PEGylarginase) or a nucleic acid encoding arginase in the manufacture of a medicament for treating ARG1-D.

[0018] Also contemplated are arginase, for example PEGylarginase, formulated into red blood cell ghosts.

[0019] A method of rapidly reducing plasma levels of at least one compound selected from the group consisting of arginine, N-α-acetylarginine (NAArg), arginic acid (ArgA), α-keto-δ-guanidinovaleric acid (GVA), and guanidinoacetic acid (GAA) to normal levels in a subject with arginase 1 (ARG1) deficiency (ARG1-D), comprising administering to the subject a therapeutically effective amount of a composition comprising PEGylated arginase, wherein the PEGylated arginase is administered intravenously to the subject at an initial dose of 0.005 mg / kg to 1.00 mg / kg, and weekly thereafter either subcutaneously or intravenously. Another method contemplates administering the PEGylated arginase intravenously at an initial dose of 0.005 mg / kg to 0.50 mg / kg. Another method contemplates administering the PEGylated arginase intravenously at an initial dose of 0.005 mg / kg to 0.20 mg / kg. Another method contemplates that the PEGylated arginase is PEGylarginase. Another method contemplates that the plasma level of at least one compound selected from the group consisting of ArgA, NAArg, GVA, GAA, or arginine is reduced to normal levels in the subject less than 3 days, less than 2 days, and / or less than 1 day after the first administration of the PEGylated arginase. Another method contemplates that 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, where the plasma level is assayed about 24-48 hours after administration of the PEGylated arginase.

[0020] A method of rapidly reducing plasma levels of at least one compound selected from the group consisting of arginine, N-α-acetylarginine (NAArg), arginic acid (ArgA), α-keto-δ-guanidinovaleric acid (GVA), and guanidinoacetic acid (GAA) to normal levels in a subject with arginase 1 (ARG1) deficiency (ARG1-D), comprising administering to the subject a therapeutically effective amount of a composition comprising PEGylated arginase, wherein the PEGylated arginase is administered to the subject subcutaneously at an initial dose of 0.01 mg / kg to 1.50 mg / kg, and then weekly, either subcutaneously or intravenously. Another method contemplates administering the PEGylated arginase subcutaneously at an initial dose of 0.015 mg / kg to 0.75 mg / kg. Another method contemplates administering the PEGylated arginase subcutaneously at an initial dose of 0.015 mg / kg to 0.30 mg / kg. Another method contemplates that the plasma level of at least one compound selected from the group consisting of ArgA, NAArg, GVA, GAA, or arginine decreases to normal levels in the subject less than 3 days, less than 2 days, and / or less than 1 day after the first administration of PEGylated arginase.Another method contemplates that the dose of 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, where the plasma level is assayed about 24-48 hours after administration of the PEGylated arginase. [Brief description of the drawings]

[0021] [Figure 1A-B] Figure 1A shows the plasma concentration of arginine. Figure 1B shows the plasma concentration of arginine acid (ArgA). [Figure 1C-D]Figure 1C shows the plasma concentration of GVA. Figure 1D shows the plasma concentration of N-α-acetylarginine (NAArg). Arginine levels are the average of values ​​obtained from two ARG1-D patients treated with 0.03 mg / kg PEG-dilarginase using a validated (good laboratory practice, or GLP) assay. ArgA, GVA, and NAArg levels were analyzed by non-GLP assays using pooled samples from the same two ARG1-D patients treated with 0.03 mg / kg PEG-dilarginase. The normal range for humans is indicated by "*". The normal range for arginine in human serum is 40 μmol / L to 115 μmol / L. See, e.g., Lueneburg, N. et al., "Reference intervals for plasma L-arginine and the L-arginine: asymmetric dimethylarginine ratio in the Framingham Offspring Cohort," J. Nutr., Vol. 141, No. 12, pp. 2186-2190 (2011). The normal range for ArgA is 0.025 μmol / L to 0.100 μmol / L, the normal range for GVA is less than 0.050, and the normal range for NAArg is less than 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., Vol. 27, No. 3, pp. 297-303 (1990). [Diagram 2]Figure 2A shows the plasma concentration of arginine. Figure 2B shows homoarginine (HArg). Arginine levels are the average of values ​​obtained from two ARG1-D patients treated with 0.03 mg / kg PEG-dilarginase using a validated (GLP) assay. HArg values ​​were obtained by a non-GLP assay using pooled samples from the same two ARG1-D patients treated with 0.03 mg / kg PEG-dilarginase. "*" refers to the normal range in human serum as described in Luneburg, N. et al. (2011) for arginine or Marescau et al. (1990) for guanidino compounds. The normal range for HArg is less than 0.500 μmol / L to 2.80 μmol / L. [Figure 3A-B] Figure 3A shows the plasma concentration of arginine. Figure 3B shows the plasma concentration of arginine acid (ArgA). [Figure 3C-D] Figure 3C is the plasma concentration of GVA. Figure 3D is the plasma concentration of N-α-acetylarginine (NAArg). Arginine levels are the average of values ​​obtained from two ARG1-D patients treated with 0.015 mg / kg PEG-dilarginase ("●") and 0.03 mg / kg PEG-dilarginase ("□") using a validated (GLP) assay. ArgA, GVA, and NAArg levels were obtained by non-GLP assays using pooled samples from the same two ARG1-D patients treated with 0.015 mg / kg and 0.03 mg / kg PEG-dilarginase. "*" refers to the normal range in human serum as described in Luneburg, N. et al. (2011) for arginine or Marescau et al. (1990) for guanidino compounds. [Figure 4A] FIG. 4A shows GAA data in two patients, patient no. 120-101 (FIG. 4A) and patient no. 120-102 (FIG. 4B), before (eg, at screening) and after treatment with pegylarginase. [Figure 4B]FIG. 4B shows data on GAA in two patients, Patient 120-101 (FIG. 4A) and Patient 120-102 (FIG. 4B), before (e.g., at screening) and after treatment with pegylarginase. In each case, data on GAA levels at screening and during treatment in parts 1 and 2 are shown. "FUP" stands for follow-up. The normal range for GAA is 0.400 μmol / L to 3.00 μmol / L. See, e.g., Marescau et al. (1990). [Figure 5A] The effect of arginase treatment on plasma levels of arginine and GCs was examined in part 1 of a clinical trial using a single dose (NAA refers to NAArg). [Figure 5B] In FIG. 5B, the effect of arginase treatment on arginine and GC plasma levels was examined in the second part of the clinical trial, which included repeated dosing. Data shown in part 2 include patients who received all eight doses administered according to the clinical protocol. For both FIG. 5A and FIG. 5B, "a" indicates baseline arginine levels in patients, "b" indicates post-dose nadir, "c" indicates levels 7 days after dosing, "d" indicates arginine levels before dosing, "e" indicates all post-dose values, "f" indicates the normal range of arginine plasma levels in healthy patients, between 40 μmol / L and 115 μmol / L, as described in Luneburg, N. et al. (2011), and "n" represents the number of patients. The post-dose nadir is the lowest value after dosing in part 1, and "all post-dose values" in part 2 include all post-dose values, except for the value immediately before the next dose. [Figure 6] Results of the 6-minute walk test for three patients, Patient No. 1 ("a"), Patient No. 2 ("b"), and Patient No. 5 ("c"), are shown at baseline, dose 8, dose 20, and dose 32 (no data available for Patient No. 5 at doses 20 and 32). Doses 8, 20, and 32 are administered from the start of the repeat dosing. [Figure 7] An overview of the phase 1 / 2 study and the open-label extension study is provided. [Figure 8] Time-dependent improvement in plasma arginine with repeated administration of pegylarginase in patients in part 2 and open-label extension. "BL" is baseline, "F / U" is follow-up, "OLE" is open-label extension, and "n" is the number of patients at each time point. It is desirable to maintain arginine in patient plasma below 200 μmol / L. 100% (5 / 5) of patients who completed part 2 achieved consistent arginine reduction levels below the recommended guidelines. Arginine reduction was accompanied by sustained reductions in plasma GC (GVA, ArgA, NAA, GAA). [Figure 9] Treatment with PEGylarginase improved clinical outcomes from baseline after only eight repeated doses. Each point in Figure 9 represents one rating for one patient. Favorable ratings are indicated by filled circles (●) and neutral ratings are indicated by open circles (○). [Figure 10] FIG. 10 shows arginine concentrations over 9 weeks for patients who responded to PEGylarginase treatment (responders) and patients who did not respond to PEGylarginase treatment (non-responders) based on the patient's MCID improvement. The range of plasma arginine above medical guidelines is 200 μmol / L or higher, and the normal range for arginine in humans is 40 μmol / L to 115 μmol / L, as described in Luneburg, N. et al. (2011). The MCID is determined as described below. A "responder" can be an individual with an MCID greater than 1, and a non-responder can be an individual with an MCID less than 1. Non-responder data points are indicated with black diamonds and responder data points are indicated with white diamonds. The mean of the non-responder data points is indicated by line "a" and the mean of the responder data points is indicated by line "b." DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] At the time of seeking approval of the study disclosed in the Examples below, the US Food and Drug Administration (FDA) believed that the scientific literature did not support a clear and consistent relationship between the reduction of plasma arginine levels by dietary arginine restriction and objective improvement in disease progression in ARG1-D patients. Initially, the FDA argued that no direct clinical benefit was expected from administering PEGylarginase to ARG1-D patients. The FDA expressed concern about whether PEGylarginase and such drugs could address the underlying enzyme deficiency in ARG1-D patients. The FDA questioned whether peripheral circulation of arginase-containing medications would have a realistic effect on the metabolism of intrahepatic arginine levels and the production of related arginine metabolites. As a result, the FDA initially postponed testing in pediatric patients until treatment of adult patients was possible, but then allowed testing in pediatric patients to continue.

[0023] What has surprisingly been discovered is a method of treating ARG1-deficient patients that rapidly reduces arginine and at least one of the guanidino compounds (NAArg) levels to within normal levels within three days (Figure 1).

[0024] definition As used herein, the terms "treating," "to treat," or "treatment" include inhibiting, slowing, arresting, reducing, ameliorating, or reversing the progression or severity of an existing symptom, disorder, condition, or disease associated with ARG1-D. Treatment may be applied prophylactically or therapeutically.

[0025] The term "effective amount" as used herein refers to an amount of PEGylated arginase, such as wild-type arginase or PEGylarginase, that when administered has a desired effect, such as reducing plasma levels of one or more of arginine, arginic acid (ArgA), GVA, N-α-acetylarginine (NAArg), GAA, and homoarginine (HArg). Effective amounts may vary depending on factors such as the weight of the patient. For example, an effective amount may range from 0.005 to 1.00 mg / kg / patient body weight for intravenous administration of PEGylated arginase, including increments of 0.005 between these ranges. Effective amounts for subcutaneous (sc) administration of PEGylated arginase to ARG1-D patients include 0.01 to 1.50 mg / kg / patient body weight, including increments of 0.01 between these ranges. One example is administering the compound to achieve arginine, ArgA, GVA, GAA, and / or NAArg in a range equivalent to the normal level of each compound in a normal human not suffering from ARG1 deficiency. Effective doses can also improve muscle strength, the patient's walking ability (i.e., ability to run, walk, bike, climb stairs unsupported), and improve cognitive ability (e.g., improvement in Wechsler Intelligence Scale for Children (WISC) test) and / or adaptive behavior (e.g., improvement in Adaptive Behavior Assessment Scale (ABAS) or Vineland Adaptive Behavior Scale (VABS) test) (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 guanidino compounds are shown in the table below. Normal levels of arginine are described in Luneburg, N. et al. (2011).

[0026] The plasma levels of arginine and / or guanidino compounds may be reduced to the normal range or normal levels, which may mean that at some point following administration of the initial dose and / or repeated doses of arginase, the plasma levels of arginine and / or one or more guanidino compounds have values ​​within the ranges shown in the table below or 40 μmol / L to 115 μmol / L for arginine as described in Luneburg, N. et al. (2011). For example, the patient's plasma levels of arginine and / or one or more guanidino compounds may oscillate within and outside the normal range during treatment with arginase as disclosed herein. The patient is considered to have plasma levels of the analyzed arginine and / or guanidino compounds reduced to the normal range or normal levels. As another example, a patient may have an average amount of plasma levels of arginine and / or one or more guanidino compounds that are within the normal range (e.g., 40-115 μmol / L for arginine as described in the table below or in Luneburg, N. et al. (2011)) after receiving one or more doses of arginase disclosed herein. Thus, the compositions and methods disclosed herein may reduce a subject's plasma levels of arginine and / or guanidino compounds to normal levels or within the normal range at least once after receiving an initial dose and / or repeated doses of arginase. It has been observed that in some patients, plasma levels of arginine and / or guanidino compounds may be maintained, on average, within the normal level or range following the methods disclosed herein.

[0027] [Table 1] Normal ranges of guanidino compounds in human serum, urine, and cerebrospinal fluid (CSF) TIFF2024028760000001.tif75170

[0028] The wild-type arginase may be based on human Arginase I or Arginase II. Wild-type human Arginase II has the following sequence (Uniprot / P78540): MSLRGSLSRLLQTRVHSILKKSVHSVAVIGAPFSQGQKRKGVEHGPAAIREAGLMKRLSSLGCHLKDFGDLSFTPVPKDDLYNNLIVNPRSVGLANQELAEVVSRAVSDGYSCVTLGGDHSLAIGTISGHARHCPDLCVVWVDAHADINTPLTTSSGNLHGQPVSFLLRELQDKVPQLPGFSWIKPCISSASIVYIGLRDVDPPEHFILKNYDIQYFSMRDIDRLGIQKVMERTFDLLIGKRQRPIHLSFDIDAFDPTLAPATGTPVVGGLTYREGMYIAEEIHNTGLLSALDLVEVNPQLATSEEEAKTTANLAVDVIASSFGQTREGGHIVYDQLPTPSSPDESENQARVRI (SEQ ID NO: 1).

[0029] Wild-type human Arginase I has the following sequence (Uniprot / P05089): MSAKSRTIGIIGAPFSKGQPRGGVEEGPTVLRKAGLLEKLKEQECDVKDYGDLPFADIPNDSPFQIVKNPRSVGKASEQLAGKVAEVKKNGRISLVLGGDHSLAIGSISGHARVHPDLGVIWVDAHTDINTPLTTTSGNLHGQPVSFLLKELKGKIPDVPGFSWVTPCISAKDIVYIGLRDVDPGEHYILKTLGIKYFSMTEVDRLGIGKVMEETLSYLLGRKKRPIHLSFDVDGLDPSFTPATGTPVVGGLTYREGLYITEEIYKTGLLSGLDIMEVNPSLGKTPEEVTRTVNTAVAITLACFGLAREGNHKPIDYLNPPK (SEQ ID NO: 2).

[0030] The PEGylarginase disclosed herein has the sequence of Arginase I in SEQ ID NO:2, but has a cobalt metal cofactor instead of a manganese metal cofactor. PEGylarginase is also PEGylated as described in U.S. Patent No. 8,440,184.

[0031] "Administering" refers to injection of a therapeutically effective amount of a compound and compositions containing the disclosed compounds. For example, and without limitation, administration may be intravascular (iv) or subcutaneous (sc). Compositions of the invention may also be administered intramuscularly (im).

[0032] The term "about" is understood by those of skill in the art and will vary to some extent depending on the context in which it is used. As used herein, "about" is meant to encompass ±10%, ±5%, or ±1%.

[0033] The term "pegylated" refers to conjugation with polyethylene glycol (PEG), which is widely used as a drug carrier given its high degree of biocompatibility and ease of modification (see, e.g., Harris et al., Clin. Pharmacokinet., Vol. 40, No. 7, pp. 539-51 (2001)). PEG can be coupled (e.g., covalently attached) to active agents via hydroxyl groups at the ends of the chains 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 explored as novel biomaterials that retain the biocompatible properties of PEG but have the added advantage of multiple attachment points per molecule (increasing drug loading).

[0034] The PEGylated arginase variant can be formulated according to known methods to prepare a medicamentously useful composition. ARG1-D patients can be administered wild-type arginase protein (either arginase I or arginase II) that naturally contains manganese metal cofactor, or wild-type arginase protein that is PEGylated and contains manganese metal cofactor. In another example, ARG1-D patients can be administered arginase with cobalt metal cofactor instead of the natural manganese metal cofactor. The cobalt metal cofactor containing arginase can be further PEGylated, with Co-ArgI-PEG (also referred to herein as AEB1102, PEGylarginase, or Co-hArgI), as described in U.S. Patent No. 8,440,184, which is incorporated herein by reference, being an exemplary form. An exemplary form, such as PEGylarginase, has approximately 12 5K (5000 dalton) PEG units per monomer attached to one or more lysines present in the protein sequence of PEGylarginase. The desired formulation is a stable lyophilized formulation that is reconstituted with an appropriate diluent or high-purity aqueous solution, along with any pharma- ceutically 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 to be delivered into red blood cell ghosts (also called artificial red blood cells). Another approach would be to administer arginine deiminase (ADI)-PEG20 (Polaris Pharma) to ARG1-D patients via im, sc, or iv, or using red blood cell ghosts as described.

[0035] As used herein, the term "portion" when in reference to a protein (as in "a portion of a given protein") refers to an arginase fragment, where the fragment has arginase activity in the urea cycle.

[0036] As used herein, the terms "protein" and "polypeptide" refer to compounds that contain amino acids joined through peptide bonds and are used interchangeably.

[0037] As used herein, the term "fusion protein" refers to a chimeric protein that contains a protein of interest (i.e., human arginase or a variant thereof) bound (or operably linked) to an exogenous protein fragment (a fusion partner consisting of a non-arginase protein). The fusion partner may enhance serum half-life, solubility, or both. It may also provide an affinity tag (e.g., His tag) that allows purification of the recombinant fusion protein from the host cell or culture supernatant, or both.

[0038] The terms "in operable combination," "in operable order," and "operably linked" refer to the linking of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and / or the synthesis of a desired protein molecule is produced. The terms also refer to the linking of amino acid sequences in such a manner that a functional protein is produced.

[0039] As used herein, the term "K m " refers to the Michaelis-Menton constant of an enzyme, which is defined as the concentration of a specific substrate at which a given enzyme produces half its maximum velocity in an enzyme-catalyzed reaction.

[0040] As used herein, the term "k cat " refers to the number of turnovers or the number of substrate molecules that each enzyme site converts to product per unit time when the enzyme is operating at maximum efficiency.

[0041] As used herein, the term "K cat / K m " is the specificity constant, which is a measure of how efficiently an enzyme converts a substrate into a product.

[0042] The term "Mn-hArgI" refers to human Arginase I with a Mn(II) metal cofactor. The term "Co-hArgI" refers to human Arginase I (mutated or native) with a Co(II) metal cofactor.

[0043] The term “IC 50 " is the half maximal (50%) inhibitory concentration (IC) and is therefore a measure of efficacy.

[0044] The term "gene" refers to a DNA sequence that includes control and coding sequences necessary for the production of a polypeptide, such as arginase or a precursor thereof. The polypeptide may be encoded by a full length coding sequence or any portion of the coding sequence, so long as the desired enzymatic activity of arginase, which reduces arginine to ornithine, is retained.

[0045] The term "subject" refers to animals, such as mammals, including humans.

[0046] 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 naturally occurring source. A wild type gene is that which is most frequently observed in a population and is therefore arbitrarily designated 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) when compared to the wild type gene or gene product. It should be noted that naturally occurring mutants can be isolated. These are identified by the fact that they have altered characteristics when compared to the wild type gene or gene product.

[0047] The following abbreviations are used herein: Abbreviation ADA Anti-drug antibodies ARG or ARG Arginine ARG1-D Arginase 1 deficiency Arg1- / - mice, arginase-deficient mice ArgA arginine acid AUC Area under the plasma concentration-time curve BQL Below quantification limit Co-ArgI-PEG Cobalt-substituted and PEGylated Arginase 1 EOI End of injection F / U Follow-up GC Guanidino Compounds Good Laboratory Practice (GLP) GVA α-keto-δ-guanidinovaleric acid GAA Guanidinoacetic acid HArg Homoarginine iv intramuscularly or intramuscularly (IV) K2EDTA Plasma Dipotassium Ethylenediaminetetraacetate Tube for Anticoagulation LC-MS / MS Liquid Chromatography Tandem Mass Spectrometry NAArg N-acetyl-arginine PEG Polyethylene glycol sc subcutaneous, subcutaneous, or SC SOI implantation completed wt wild type

[0048] We investigated whether PEGylarginase, a recombinant human arginase enzyme produced in Escherichia coli, could reduce arginine levels to the normal range in neonatal and adult mouse models of ARG1 deficiency. PEGylated for stability and half-life, and has a cobalt metal cofactor instead of a manganese metal cofactor. The cobalt metal cofactor enhances catalytic activity and improves arginase stability. Treatment with PEGylarginase reduced plasma arginine and total brain arginine, but administration of the drug failed to treat hyperammonemia in mice. The lack of reduction in arginine levels in liver tissue was theorized to be possibly due to the failure of the administered enzyme PEGylarginase to enter the liver. In an adult mouse model of ARG1-D, administration of PEGylarginase failed to improve animal survival. In neonatal arginase I-deficient mice (Arg1), the closest simulation of human disease in a mouse model, we performed a randomized controlled trial of PEGylarginase I-deficient mice (Arg1-D), which is a mouse model of human disease. - / - Peglyarginase was tested in mice (Burrage et al., "Human recombinant arginase enzyme reduces plasma arginine in mouse models of arginase deficiency," Hum. Mol. Genetics, vol. 24, no. 22, pp. 6417-6417 (2015)). Multiple dose studies reduced plasma and brain arginine levels to the normal range. However, liver arginine levels were not beneficially affected, and the levels of untreated Arg1 were significantly reduced. - / - There was no improvement in survival compared to mice, which was unexpected.

[0049] Unlike the human clinical manifestations of arginase I deficiency, Arg1 - / -Mice have severe hyperammonemia that is believed to be the cause of premature death (e.g., Carvalho, DR, et al., "Clinical features and neurologic progression of hyperargininemia," Pediatr. Neurol, Vol. 46, No. 6, pp. 369-74 (2012)). Hyperammonemia is a milder complication in human patients with the disorder. Given that elevated plasma arginine rather than hyperammonemia is the primary treatment challenge in human patients with arginase I deficiency, it may be speculated that Co-ArgI-PEG may have therapeutic utility, but the degree of utility and response cannot be reasonably predicted, and no trials or therapeutic models have been conducted or developed, even assuming as far back as 1995. See, e.g., Uchino, T., et al., "Molecular basis of phenotypic variation in patients with argininemia," Hum. Genet., Vol. 96, No. 3, pp. 255-60 (1995).

[0050] I. Arginase Wild-type arginase is a manganese-containing enzyme. It is the last enzyme in the urea cycle. Arginase is the fifth and final step in the urea cycle, a series of biophysical reactions in mammals by which the body processes harmful ammonia. Specifically, arginase converts L-arginine to L-ornithine and urea.

[0051] L-arginine is the nitrogen donor substrate for nitric oxide synthase (NOS), which produces L-citrulline and nitric oxide (NO). M Arginase may also play a role in regulating NOS activity, although its affinity for L-arginine (2-5 mM) has been reported to be much higher than that of NOS (2-20 μM). Under certain conditions, arginase I is Cys-S-nitrosylated, resulting in a higher affinity for L-arginine and a reduced availability of the substrate for NOS.

[0052] Arginase is a homotrimeric enzyme with an α / β fold of a parallel eight-stranded β sheet surrounded by several helices. The enzyme contains a binuclear metal cluster that is essential for generating hydroxide for the nucleophilic attack on the guanidinium carbon of L-arginine. The natural metal cofactor for arginase is Mn 2+ These Mn 2+ The ions coordinate water, orienting and stabilizing the molecule, allowing water to act as a nucleophile to attack L-arginine, hydrolyzing it to ornithine and urea.

[0053] Mammals have two arginase isozymes (EC 3.5.3.1) that catalyze the hydrolysis of L-arginine to urea and L-ornithine. The arginase I gene is located on chromosome 6 (6q23), is highly expressed in the cytosol of hepatocytes, and functions in nitrogen removal as the final step of the urea cycle. The arginase II gene is present on chromosome 14 (14q24.1). Arginase II is localized in mitochondria in tissues such as kidney, brain, and skeletal muscle, and is thought to provide L-ornithine for proline and polyamine biosynthesis (Lopez et al., FEBS J., vol. 272, pp. 4540-48 (2005)).

[0054] Arginase has been studied for nearly 50 years as a way to degrade 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., Vol. 8, No. 7, pp. 248-253 (2002)). Although native arginase is cleared from the circulation within minutes (Savoca et al., Cancer Biochem. Biophys, Vol. 7, pp. 261-268 (1984)), a single injection of PEG-arginase MW 5,000 into rats was sufficient to achieve near-total arginine depletion for approximately 3 days (Cheng et al., Cancer Res, Vol. 67, pp. 309-17 (2007)).

[0055] ADI, a bacterial arginine hydrolase that shows good kinetics and stability, was tested in vitro. Unfortunately, ADI is a bacterial enzyme and therefore induces strong immune responses and side effects in most patients, making it unsuitable for long-term administration in ARG1-D patients, who require regular dosing.

[0056] For clinical use in ARG1-D patients, it is essential that arginase be designed to last for a long time (e.g., several days) in the circulation. In the absence of any modifications, human arginase has a half-life in the circulation of only a few minutes, mainly because its size is not large enough to avoid filtration by the kidney. Unmodified human arginase is highly susceptible to inactivation in serum and degraded with a half-life of only 4 hours.

[0057] II. PEGylation of Arginase Variants In one aspect of the present invention, methods and compositions related to PEGylated arginase are disclosed. Specifically, PEGylation of arginase at engineered cysteine ​​residues (e.g., substituting the third residue at the N-terminus) can be used to produce homogeneous PEGylated arginase compositions. A method for isolating PEGylated arginase based on the temporary interruption of polymerization is also disclosed.

[0058] PEGylation is the process of covalently attaching PEG polymer chains to another molecule, usually a drug or therapeutic protein. "PEGylation" can be achieved by incubating a reactive derivative of PEG with a target macromolecule. It increases the hydrodynamic size (size in solution) of the drug or therapeutic protein, prolonging circulation time by decreasing renal clearance. PEGylation can also confer water solubility to hydrophobic drugs and proteins.

[0059] The first step in PEGylation can be suitable functionalization of the PEG polymer at one or both terminal domains of the protein or internally to an amino acid such as lysine. PEG activated at each end with the same reactive moiety is known as "homobifunctional", whereas when the functional groups present are different, the PEG derivative is termed "heterobifunctional" or "heterofunctional". Chemically active or activated derivatives of PEG polymers are prepared in order to attach PEG to the desired molecule.

[0060] The selection of suitable functional group for PEG derivative is based on the type of available reactive group on the molecule that will be coupled to PEG.For protein, typical reactive amino acids include lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, and tyrosine.PEG can also be attached to polypeptide using N-terminal amino group and C-terminal carboxylic acid.

[0061] Techniques used to form PEG derivatives include reacting PEG polymers with groups that react with hydroxyl groups, typically anhydrides, acid chlorides, chloroformates, and carbonates. PEGylation chemistries can also use functional groups such as aldehydes, esters, and amides available for conjugation. Heterobifunctional PEGs are very useful for linking two entities where a hydrophilic, flexible, and biocompatible spacer is required. Preferred terminal groups for heterobifunctional PEGs are maleimide, vinyl sulfone, pyridyl disulfide, amine, carboxylic acid, and H-hydroxysuccinimide (NHS) esters.

[0062] The most common modifiers or linkers are based on methoxy PEG (mPEG) molecules. Their activity relies on the addition of protein-modifying groups at the alcohol termini. Polyethylene glycol (PEG diol) can be used as a precursor molecule. Both ends of this diol can then be modified to form heterodimeric or homodimeric PEG-linked molecules (as shown for example with PEG bis-vinylsulfone).

[0063] Proteins are generally 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 vinylsulfone. All four show strong cysteinyl specificity under mild conditions and neutral to slightly alkaline pH, but each has some drawbacks. The amides formed with maleimides can be somewhat unstable under alkaline conditions, so formulation options with this linker can be somewhat limited. The amide bonds formed with iodo-PEG are more stable, but free iodine can modify tyrosine residues under some conditions. PEG thiols form disulfide bonds with protein thiols, but this bond can also be unstable under alkaline conditions. PEG-vinylsulfone reactivity is relatively slow compared to maleimide and iodo-PEG. However, the thioether bond formed is quite stable. The slow reaction rate also makes the PEG-vinylsulfone reaction easier to control.

[0064] Site-specific PEGylation at natural cysteinyl residues is rarely performed because these residues are usually in the form of disulfide bonds or are required for biological activity. Alternatively, site-directed mutagenesis can be used to incorporate cysteinyl PEGylation sites in thiol-specific linkers. The cysteine ​​mutation must be designed so that it is accessible to PEGylation reagents and still biologically active after PEGylation.

[0065] Amine-specific modifying agents include PEG-NHS ester, PEG tresylate, PEG aldehyde, PEG isothiocyanate, and several others. These amine-specific agents generally react under mild conditions and are highly specific for amino groups.

[0066] Site-specific PEGylation can be difficult because most proteins have multiple lysine residues. Fortunately, because these reagents react with unprotonated amino groups, it is possible to PEGylate lower pK amino groups by performing the reaction at a lower pH. In general, the pK of α-amino groups is 1-2 pH units lower than the ε-amino groups of lysine residues. High selectivity for the N-terminus can often be achieved by PEGylating molecules at pH 7 or lower. However, this is only possible if the N-terminal portion of the protein is not required for bioactivity. Nevertheless, the pharmacokinetic benefits of PEGylation often outweigh any significant loss of in vitro bioactivity, resulting in products with much greater in vivo bioactivity, regardless of the chemistry of PEGylation.

[0067] III. Proteins and Peptides In certain embodiments, the present invention relates to compositions comprising at least one protein or peptide, such as a stabilized arginase multimer. These peptides may be included in a fusion protein or may be conjugated to an agent.

[0068] A. Proteins and Peptides As used herein, a protein or peptide generally refers to, but is not limited to, a protein of more than about 200 amino acids up to the full length sequence translated from a gene, a polypeptide of about 100 or more amino acids, and / or a peptide of about 3 to about 100 amino acids. For convenience, the terms "protein," "polypeptide," and "peptide" are used interchangeably herein.

[0069] 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 contiguous, without any non-amino acids interrupting the sequence of amino acid residues. Other exemplary sequences may include one or more non-amino acid moieties. For example, the sequence of residues of a protein or peptide may be interrupted by one or more non-amino acid moieties.

[0070] Thus, the term "protein or peptide" encompasses amino acid sequences that contain at least one of the 20 common amino acids found in naturally occurring proteins, and may contain at least one modified or unusual amino acid, including, but not limited to, those set forth below.

[0071] TIFF2024028760000002.tif187170

[0072] IV. Nucleic Acids and Vectors The nucleic acid sequence encoding the arginase polypeptide of interest can be a stabilized multimeric arginase. 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 that are rarely utilized in E. coli, which can interfere with expression. Therefore, each gene or its variant can be codon-optimized for E. coli expression, for example, as described in U.S. Pat. No. 8,440,184. A variety of vectors, such as fusion multimeric arginase or cysteine-substituted arginase, can also be used to express the protein of interest. Exemplary vectors include, but are not limited to, plasmid vectors, viral vectors, transposons, ghost red blood cells, or liposome-based vectors.

[0073] V. Host cells Host cells, preferably eukaryotic cells, can be used and transformed to allow expression and secretion of arginase and its fusion multimers. The host cells can be bacteria, mammalian cells, yeast, or filamentous fungi. Various bacteria include the genera Escherichia and Bacillus. Yeasts belonging to Saccharomyces, Kluyveromyces, Hansenula, or Pichia can also 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.

[0074] Examples of bacterial host organisms that can be used include, for example, Escherichia coli MC1061, derivatives of Bacillus subtilis BRB1, Staphylococcus aureus SAI123, or Streptococcus lividans. Exemplary yeasts that can be used as host cells include, for example, Saccharomyces cerevisiae AH22 and Schizosaccharomyces pombe, and exemplary filamentous fungi include, for example, Aspergillus nidulans, Aspergillus awamori, and Trichoderma reesei.

[0075] Examples of commonly available mammalian host cells include Chinese hamster ovary cells (CHO-K1, American Type Culture Collection (ATCC) No. CCL61), rat pituitary cells (GH1, ATCC, No. CCL82), HeLaS3 cells (ATCC, No. CCL2.2), rat hepatoma cells (H-4-II-E, ATCC No. CRL1548), SV40 transformed monkey kidney cells (COS-1, ATCC, No. CRL1650), and mouse embryo cells (NIH-3T3, ATCC, No. CRL1658). The above are illustrative, but not limiting, of the many possible host organisms known in the art.

[0076] Mammalian host cells expressing arginase and / or their fusion multimers can be under conditions typically used to culture the parent cell line. Generally, cells are cultured in standard mammalian cell culture media containing physiological salts and nutrients, such as standard Roswell Park Memorial Institute medium (RPMI), Minimum Essential Medium (MEM), Improved Minimum Essential Medium (IMEM), or Dulbecco's Minimum Essential Medium (DMEM), typically supplemented with 5-10% serum, such as fetal bovine serum (FBS). Culture conditions are also standard, e.g., cultures are incubated at 37°C in stationary or roller cultures until the desired level of protein is achieved.

[0077] VI. Protein purification The protein or polypeptide of interest can be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity), unless otherwise indicated. Analytical methods particularly suited to the preparation of a pure peptide are ion exchange chromatography, gel exclusion chromatography, polyacrylamide gel electrophoresis, affinity chromatography, immunoaffinity chromatography, and isoelectric focusing. A particularly efficient method of purifying peptides is fast performance liquid chromatography (FPLC) or even high performance liquid chromatography (HPLC).

[0078] A purified protein or peptide is intended to refer to a composition that is isolatable from other components, where the protein or peptide is purified to any extent relative to its naturally available state. Thus, an isolated or purified protein or peptide also refers to a protein or peptide that is free from the environment in which it may naturally occur. In general, "purified" refers to a protein or peptide composition that has been subjected to fractionation to remove various other components and that substantially retains its expressed biological activity. When the term "substantially purified" is used, this designation refers to a composition in which the protein or peptide forms a major component of the composition, e.g., a composition in which the protein constitutes about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the composition.

[0079] Exemplary protein purification techniques include the use of ammonium sulfate, PEG, antibodies, and the like, or heat denaturation, followed by chromatographic steps such as centrifugation, ion exchange, gel filtration, reverse phase, hydroxylapatite, and affinity chromatography, isoelectric focusing, gel electrophoresis, and combinations of these and other techniques.

[0080] VII. Pharmaceutical Compositions The arginase described herein can be administered systemically or locally.Arginase and compositions containing them can be administered intravenously, intrathecally, subcutaneously, intramuscularly, intratumorally, and / or intraperitoneally, or combinations thereof.The compounds described herein and compositions containing them can be administered alone or in combination with arginine scavengers and / or arginine-reduced diets.

[0081] The composition containing arginase or a part thereof can be provided as a formulation with physiologically acceptable liquid, gel or solid carriers, diluents, and excipients. Such compositions are typically prepared as injections, as liquid solutions or suspensions. Suitable diluents and excipients are, for example, water, saline, dextrose, or glycerol, and the like, and combinations thereof. In addition, if desired, the composition may contain small amounts of auxiliary substances, such as wetting or emulsifying agents, stabilizers, or pH buffers. When clinical applications are contemplated, it may be necessary to prepare pharmaceutical compositions (expression vectors, virus stocks, proteins, antibodies, and drugs) in a form appropriate for the intended application. In general, the pharmaceutical compositions of the present invention comprise an effective amount of one or more arginase variants or additional agents dissolved or dispersed in a pharma- ceutically acceptable carrier.

[0082] The phrase "pharmaceutical or pharmacologically acceptable" refers to molecules and compositions that do not produce adverse, allergic, or other undesirable reactions, as appropriate, when administered to a subject, such as a human. The preparation of pharmaceutical compositions containing at least one arginase variant, such as a stabilized multimeric arginase or a PEGylated arginase, isolated by the methods disclosed herein, or additional active ingredients, is known to those of skill in the art in light of the present disclosure, as exemplified by Remington's, Pharmaceutical Sciences, 18th Edition (1990). Additionally, it is understood that for administration to animals (e.g., humans), the formulations should meet the standards of sterility, pyrogenicity, general safety, and purity required by the FDA Office of Biological Standards.

[0083] As used herein, "pharmaceutical acceptable carriers" include any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial, antifungal), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, dyes, materials such as, and combinations thereof, known to those skilled in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th Edition (1990)). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the pharmaceutical compositions is contemplated.

[0084] A pharmaceutical composition containing arginase can include different types of carriers depending on whether it is to be administered in solid, liquid, or aerosol form and whether it needs to be sterile for the route of administration, such as injection. The present invention can be administered intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intramuscularly, subcutaneously, intratumorally, topically, by injection, infusion, continuous infusion, via catheter, in lipid compositions (e.g., liposomes), or by other methods known to those skilled in the art (e.g., Remington's Pharmaceutical Sciences), or any combination of the foregoing.

[0085] The arginase variants can be formulated and composed in free base, neutral, or salt form. Pharmaceutically acceptable salts include acid addition salts, such as those formed with free amino groups of the proteinaceous composition, or with inorganic acids, such as hydrochloric acid or phosphoric acid, or with 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, potassium, ammonium, calcium, or ferric hydroxides, or organic bases, such as isopropylamine, trimethylamine, histidine, or procaine. Once formulated, the solution can be administered in a therapeutically effective amount in a manner compatible with the administration formulation. The formulations are easily administered in a variety of dosage forms, such as formulations for parenteral administration, such as injections, or aerosols for delivery to the lungs, or drug-release capsules for administration in the digestive tract.

[0086] The composition for administration can be provided in a pharma- ceutically acceptable carrier with or without an inert diluent. The carrier must be absorbent and includes liquid, semi-solid, i.e., paste, or solid carriers. Except where any common vehicle, agent, diluent, or carrier is harmful to the recipient or to the therapeutic effectiveness of the composition contained therein, its use in an administrable composition for use in carrying out the method of the present invention is appropriate. Examples of carriers or diluents include fats, oils, water, saline, lipids, liposomes, resins, binders, bulking agents, and the like, or combinations thereof. The composition can also include various antioxidants to retard oxidation of one or more components. In addition, the use of various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparaben, propylparaben), chlorobutanol, phenol, sorbic acid, thimerosal, or combinations thereof, can improve the shelf life of the pharmaceutical composition.

[0087] The composition may be brought into association with the carrier in any convenient and practical manner, i.e., by dissolving, suspending, emulsifying, mixing, encapsulating, absorbing, etc. Such procedures are routine for those skilled in the art.

[0088] A pharmaceutical lipid vehicle can be used in a composition comprising an arginase variant. The lipid vehicle composition can include one or more lipids and an aqueous solvent. As used herein, the term "lipid" is defined to include any of a wide range of substances that are characteristically insoluble in water and extractable with an organic solvent. Examples include compounds that contain long-chain aliphatic hydrocarbons and their derivatives. Lipids can be natural or synthetic (i.e., engineered or produced by humans). However, lipids are usually biological materials. Biological lipids include, for example, neutral lipids, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulfatides, lipids with ether- and ester-linked fatty acids, polymerizable lipids, and combinations thereof. Of course, compounds other than those specifically described herein that are understood by those skilled in the art as lipids are also included in the compositions and methods of the present invention.

[0089] The actual dosage of the composition comprising arginase described herein to a patient can be determined by physical and physiological factors such as the patient's weight, the severity of the condition, the type of disease being treated, previous or current therapeutic interventions, idiopathic disease, and the route of administration. Depending on the dosage and route of administration, the preferred dose and / or the number of times of administration of the effective amount can vary from subject to subject. The dosage is based on the amount required for the patient to achieve normal levels of at least one of arginine, HArg, ArgA, GVA, GAA, and NAArg. The ARG1-D patient level of one or more of these five compounds is generally evaluated until the normal range is obtained in the patient's plasma. Tissue levels of the five compounds can also be evaluated, but are not necessarily required or can be performed less frequently than plasma level tests.

[0090] For ARG1-D patients, the initial administration can be intravenous administration at a dose of 0.005-1.00 mg / kg of arginase per kg of patient, and any 0.005 amount in the range of 0.005-1.00 mg / kg / patient body weight, such as 0.02 mg / kg or 0.035 mg / kg. Exemplary intravenous doses or arginase can be administered daily, weekly, bimonthly, or monthly. Alternatively, the arginase composition can be administered initially, or subcutaneously only, or any combination of intravenous or subcutaneous administration. Subcutaneous or intramuscular administration can be at a dose of 0.01-1.50 mg / kg / patient body weight, and any 0.01 amount in the range of 0.01-1.50 mg / kg / patient body weight, such as 0.08 mg / kg. Subcutaneous or intramuscular administration can be daily, weekly, bimonthly, or monthly.

[0091] A pharmaceutical composition may, for example, contain at least about 0.1% of active compound. In other embodiments, the active compound may, for example, comprise from about 2% to about 75% of the weight of the unit, or from about 25% to about 60%, and any range derivable therein. Of course, the amount of active compound in each therapeutically useful composition may be prepared to obtain a suitable dosage in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, shelf life of the product, and other pharmacological reasons will be contemplated by those skilled in the art of preparing such pharmaceutical formulations, and as such, various dosages and treatment regimens may be desirable. An exemplary formulation of PEGylarginase is formulated in a buffer containing 5 mM potassium phosphate, 50 mM sodium chloride, and 1.5% glycerol (w / v / ), at pH 7.4.

[0092] VIII. Treatment method In addition to administering arginase protein to ARG1-D patients, other treatment methods are also contemplated.For example, patients can be treated with gene therapy vectors, such as adenovirus-based gene delivery vectors that contain wild-type arginase.Wild-type protein can target specific organs, such as the liver.For example, as described in U.S. Patent No. 8,398,968, various viral packaging systems have been created to reintroduce protein into patients by gene therapy.

[0093] Another method to treat ARG1-D patients is to use the CRISPR (clustered regularly interspaced short palindromic repeats) system, where the defective gene on the chromosome is engineered to edit the error so that the patient can produce normal amounts of wild-type arginase.As mentioned before, there are two arginase genes located on different chromosomes that need to be edited.CRISPR genome editing is described in, for example, US Patent Application Publication No. 20170240922, US Patent Application Publication No. 20170283830, US Patent Application Publication No. 20170224843, and US Patent Application Publication No. 20170191078.

[0094] Another method of delivering arginase is to encapsulate the arginase protein in resealed red blood cells (RBCs), which can be administered to a patient. Encapsulation of arginase can be generally performed as described in U.S. Patent Application Publication Nos. 20160095884 and 20140154797. The lysis / resealing process for preparing red blood cells containing arginase comprises suspending the spheroid concentrate in an isotonic solution with a hematocrit level of 65% or more and cooling at 1°C to 8°C, measuring the osmotic fragility based on a sample of red blood cells from the same spheroid concentrate, preferably a sample of the suspension, and a lysis and internalization procedure of the active ingredient (i.e., arginase) at a temperature maintained at about 1°C to 8°C in the same chamber, including circulating in a dialysis cartridge a red blood cell suspension with a hematocrit level of 65% or more and a hypotonic lysis solution refrigerated at 1°C to 8°C, the lysis parameters being adjusted according to the previously measured osmotic fragility, and resealing in a second chamber at a temperature of 30°C to 40°C using a hypertonic solution. EXAMPLES

[0095] The open-label Phase 1 / 2 study enrolled 12 adult and pediatric patients (7 children and 5 adults) diagnosed with ARG1-D. In part 1, patients received escalating doses of PEGylarginase at 2-week intervals. In part 2, patients received PEGylarginase (AEB1102) intravenously (IV) at weekly intervals for 8 weeks.

[0096] 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 Measure 66 item (GMFM-66), and PROMIS) were assessed. GMFM-66 is a tool to assess various aspects of gross motor function from part A to part E. Guanidino compounds (GCs) were analyzed both before and after PEG-dilarginase administration to evaluate the therapeutic effect of PEG-dilarginase in patients. GCs selected for analysis from patients included α-keto-δ-guanidinovalerate (GVA), arginine acid (ArgA), homoarginine, N-α-acetylarginine (NAArg), and GAA in plasma.

[0097] Methods. A biochemical analytical method was developed to quantify GCs (GAA, GVA, ArgA, NAArg) from patient-derived K2EDTA plasma. Patient-derived plasma proteins were precipitated with 10% trichloroacetic acid solution and separated on a Shimadzu Nexera® using an Imtakt Intrada Amino Acid 50x2mm (product no. WAA22) column. Analytes were detected by LC-MS / MS using an Applied Biosystems MDS Sciex API 5500™. Isotopically labeled standards of each GC were used to allow quantification of each plasma sample. A detailed method protocol is provided at the end of this example.

[0098] Results of study in two adult female patients Two adult female patients, ages 24 and 25, were treated with PEGylarginase IV once weekly for 8 weeks. Both patients have moderate to severe neurocognitive and neuromotor impairments, including spasticity. Both patients continued pre-PEGylarginase treatment standard during the PEGylarginase administration period. Weekly intravenous (IV) administration of 0.04 mg / kg PEGylarginase was well tolerated. In addition to the expected decrease in plasma arginine (Figure 1), a decrease over time in the concentrations of the analyzed GCs (e.g., ArgA, NAArg, GVA) was observed following PEGylarginase treatment. The decrease in ArgA, NAArg, and GVA plasma levels occurred within 24 hours of the first infusion and was maintained below baseline levels throughout the 8 weeks of administration each patient received.

[0099] Plasma samples from both patients initially used for arginine / ornithine sample analysis in the single ascending dose portion of the Phase 1 study (PEG-101A) were pooled to help establish the assay range during GC method development. These pooled samples were then used to quantify GC after the method development phase was completed. Both patients received a single dose of PEG-101A at 0.015 mg / kg and 0.03 mg / kg / patient body weight and were observed for 2 weeks during the dosing period.

[0100] Using these pooled patient samples in our previously reported non-GLP assay (detailed methods at the end of this example), we observed a time-dependent decrease in ArgA (approximately 3-fold), GVA (approximately 4-fold), and NAArg (approximately 2-fold) concentrations from ARG1-D patients treated with 0.03 mg / kg PEGylarginase. The decrease in GC levels paralleled the decrease in plasma arginine measured using the GLP assay (Figure 1A-D).

[0101] As reflected in Figures 3A-3D, two patients (i.e., Nos. 120-101 and 120-102) were administered PEGylarginase at 0.015 mg / kg / patient body weight or 0.03 mg / kg / patient body weight, and plasma arginine and guanidino compound levels were measured as follows.

[0102] [Table 2] GC levels in two patients at a dose of 0.015 mg / kg / patient. TIFF2024028760000003.tif87170*Average of 2 subjects, **Pooled samples from 2 subjects. If a value is not listed in Tables 2 and 3, the patient was not tested at the time because the tests are expensive and blood levels are generally not thought to change rapidly.

[0103] [Table 3] GC levels at a dose of 0.03 mg / kg / patient body weight in two patients. TIFF2024028760000004.tif91170*Average of 2 subjects, **Pooled samples from 2 subjects.

[0104] [Table 4] The doses and concentrations tested were as follows: TIFF2024028760000005.tif255170TIFF2024028760000006.tif255170TIFF20240287600 00007.tif255170TIFF2024028760000008.tif255170TIFF2024028760000009.tif255170

[0105] [Table 5] PK concentrations (concentration data available for the dose escalation portion of the study). PK data for two patients are as follows: TIFF2024028760000010.tif255170TIFF2024028760000011.tif255170TIFF2024028760000012.tif255170

[0106] [Table 6] Individual and mean PK parameters of PEGylarginase in hyperargininemic patients after a single IV infusion (data for dose escalation only). TIFF2024028760000013.tif255170TIFF2024028760000014.tif255170

[0107] Data. The observed relationships between plasma arginine and ArgA, GVA, and NAArg concentrations are generally consistent with data published by Marescau et al. (1990), in which patients placed on a protein-restricted diet were analyzed. In the Marescau et al. (1990) study, GC levels decreased concomitantly with the decrease in arginine levels.

[0108] For the data presented herein, homoarginine (HArg) levels were also shown, as well as ArgA, GVA, and NAArg levels analyzed. HArg levels were examined because HArg was significantly elevated in the serum of ARG1-D patients in the study by Marescau et al. (1990). In ARG1-D patients treated with PEGylarginase, HArg levels did not appear to be regulated (Figure 2). This result is consistent with the data reported by Marescau et al. (1990) that serum HArg levels do not decrease in patients maintained on a protein-restricted diet. Further conditions that can be evaluated by treating patients are improving muscle strength, the patient's walking ability (i.e., ability to run, walk, cycle, and climb stairs unsupported), improving cognitive ability (e.g., improvement in WISC tests), or improving adaptive behavior (e.g., improvement in ABAS or VABS tests).

[0109] GC data were also generated from pooled plasma samples from two adult patients treated with a low dose of PEGylarginase, 0.015 mg / kg / patient body weight. A decrease in plasma arginine concentration was observed with the low dose of PEGylarginase. However, this decrease was not as pronounced as the decrease in plasma arginine observed with the 0.03 mg / kg dose.

[0110] Considering the findings of these two patients above, we conclude that a dose of 0.04 mg / kg of pegdilarginase was well tolerated in adult patients with ARG1-D when administered intravenously to patients once a week with patience. Patients showed a significant reduction in GCs that paralleled the reduction in arginine levels. The effect on arginine and GC levels is based on these two adult patients and is considered to be chemically more significant than the effect historically achieved by strict dietary arginine restriction. One ARG1-D patient in the study successfully administered 0.2 mg / kg. Maximum tolerated dose (MTD) in solid tumors was established at doses from 0.33 mg / kg up to a maximum of 0.48 mg / kg.

[0111] Of course, patients with elevated arginine levels (e.g., 600-800 μM arginine) may be treated with PEGylarginase at doses higher than 0.50 mg / kg. Patients with elevated arginine levels may require arginase administration in the range of 0.005-1.00 mg / kg of subject body weight. Dosage ranges of 0.005 mg / kg to 0.50 mg / kg of subject body weight and / or 0.005 mg / kg to 0.20 mg / kg of subject body weight are also contemplated.

[0112] The bioavailability of a drug can be measured by measuring the amount of drug in the blood over a specified period of time from IV and / or subcutaneous injection. For calculation purposes, it is assumed that 100% of the drug administered intravenously enters the bloodstream, but not all of the drug administered subcutaneously enters the blood from the subcutaneous space. Bioavailability can be determined by dividing the total subcutaneous volume by the total IV volume. The bioavailability of subcutaneously administered PEGylarginase was determined to be approximately 60% of the IV dose based on a comparison of intravenous and subcutaneous administration in cynomolgus monkeys. Thus, patients with high levels of arginine can be successfully treated with as much as 1.5 mg / kg of arginase. Based on bioavailability data, the dosage range for subcutaneous administration of PEGylarginase may be, for example, 0.01 to about 1.5 mg / kg subject body weight, with 0.015 mg / kg to 0.75 mg / kg subject body weight and / or 0.015 mg / kg to about 0.30 mg / kg subject body weight being used.

[0113] Figures 4A and 4B show data on GAA in two patients, Patient No. 120-101 (Figure 4A) and Patient No. 120-102 (Figure 4B), before (e.g., at screening) and after treatment with PEGylarginase. In each case, data on GAA levels at screening and during administration of Parts 1 and 2 are shown. In both patients, GAA was reduced after administration of 0.015 mg / kg / subject body weight, with greater effects observed at higher doses. Notably, a rapid reduction in GAA was observed immediately after administration of PEGylarginase. Furthermore, with early treatment, both patients were in the normal range for plasma levels of GAA within 24 hours after administration. For example, in Figure 4B, the patient's GAA level on day 14 was 4,350 nM, which decreased to a low level of 1,350 nM within 24 hours after administration of arginase. Thus, treatment with arginase can rapidly remove toxic metabolites such as GAA or GVA and is useful as an acute treatment. PEGylarginase removes the indicated toxic metabolites at a much faster rate than an arginine-reduced diet alone.

[0114] Patient characteristics and safety Baseline assessments were performed on patients in the Phase 1 / 2 study. Patients in this study had significant disease burden at baseline assessments shown in the table below. Patients were on a protein restricted diet.

[0115] For the Berg balance test, deficit balance is defined as moderate or high risk of falling (score ≦40). For the 6MWT, deficit balance is defined as below the age-adjusted range for healthy subjects as described in Geiger et al., "Six-minute walk test in children and adolescents," J. Pediatr., April 2007, Vol. 150, No. 4, pp. 395-399, and Enright et al., "Reference equations for the six-minute walk in healthy adults," Am. J. Respir. Crit. Care Med., November 1998, Vol. 158, No. 5, Part 1, pp. 1384-1387. For GMFM Part E, deficits are defined as <68 based on the minimal clinically meaningful difference (MCID) as described by Oeffinger et al., "Outcome tools used for ambulatory children with cerebral palsy: responsiveness and minimum clinically important differences," Dev. Med. Child Neurol, 2008, Vol. 50, No. 12, pp. 918-925. If multiple baseline clinical laboratory assessments are available, the assessment immediately prior to dosing is reported. For PROMIS, deficit baseline is defined as a T-score <40.

[0116] [Table 7] Baseline assessments were performed for patients in the Phase 1 / 2 study. TIFF2024028760000015.tif129170

[0117] The majority of treatment-related adverse events (AEs) were mild, as shown in the table below. Treatment-related AEs observed in two or more patients included hypersensitivity (n=3, all moderate), pruritus (n=3, all mild), and dry skin (n=2, both mild). In total, more than 130 infusions were performed in all patients in the study. Four types of moderate hypersensitivity reactions were observed in three patients (two considered serious adverse events (SAEs)). These AEs were managed with infusion rate adjustments and administration of antihistamines and, in some cases, corticosteroids. One SAE of hyperammonemia was assessed as not related to treatment with pegylarginase.

[0118] [Table 8] Adverse events TIFF2024028760000016.tif27170

[0119] The PEGylarginase ADA effect was tested. A bridging assay using Meso Scale Discovery electrochemiluminescence was validated to detect antibodies against AEB1102 (Co-ArgI-PEG) in rat, monkey, and human serum. 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 antibodies.

[0120] 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 wells of streptavidin-coated plates. After incubation and washing, 150 μL of 2X Read Buffer T (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. Note that the confirmation assay was pre-incubated with 150 μg / mL AEB1102 (drug). High, medium, and low concentrations of positive and negative controls were included in each test. Cut points, screening, and confirmation were assigned statistically, resulting in false positive rates of approximately 5% and 1%, respectively. The positive control was an anti-Co-ArgI-PEG affinity purified polyclonal antibody, and the negative control (NC) was aliquoted pooled normal rat, cynomolgus monkey, or human serum. Positive and non-specific binding (NSB) controls were used to monitor assay performance.

[0121] A direct binding assay was validated to detect antibodies against PEG, allowing the detection of anti-PEG antibodies that may be pre-existing or develop upon treatment following administration of AEB1102.

[0122] Wells of Starwell C8 Maxisorp (96-well format plate) were coated with 100 μL of 2 μg / mL monoPEGylated bovine serum albumin (BSA) (BSA-mPEG) 5 kDa (Life Diagnostics), 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 minimum required dilution (MRD) of 50 in dilution buffer were added to the plate in duplicate (100 μL / well). 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. The reaction was then stopped approximately 10-20 minutes later by adding 100 μL per well of 3,3',5,5'-tetramethylbenzidine (TMB) substrate, followed by 100 μL per well of stop solution. Plates were read on a Synergy2 plate reader at 450 (detection) and 620 (background). Cut points, screening, and confirmation, were statistically assigned, resulting in false positive rates of approximately 5 and 1%, respectively. This method was used for screening, titration, and confirmation. Samples to be titrated were subjected to at least seven two-fold serial dilutions in negative pooled human serum.

[0123] Transient low titer anti-PEG ADA (anti-drug antibodies) were detected in Part 1 of the patient study (see table below). At the start of repeat dosing, 6 / 7 patients had no detectable ADA. Tolerance to PEGylarginase was unexpectedly rapid, as shown in Table 9 below.

[0124] [Table 9] ADA evaluation for PEGylarginase and PEG. TIFF2024028760000017.tif30170

[0125] *One patient with pretreatment anti-PEG ADA had a decline in titers during Part 1 and had 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.

[0126] Figures 5A and 5B show the effect of arginine and GC. GAA was elevated in ARG1-D patients. GAA levels in these patients were reduced by treatment with PEGylarginase. The repeat dose graph shown in Figure 5B includes patients who received all eight doses in Part 2. The upper limit of normal (ULN) for GC is based on a study of GC in healthy adults. PEGylarginase is highly effective in reducing arginine levels to the normal range in single and repeat doses (Figure 5B). GAA, which is elevated in ARG1-D patients, is also reduced by treatment with PEGylarginase. Reductions in GVA, ArgA, GAA, and NAA concentrations over time were observed after IV QW (weekly) treatment with PEGylarginase. Reductions in GVA, ArgA, GAA, and NAA levels occurred within 24 hours of the first infusion, and reductions from baseline levels were maintained through 8 weeks of treatment. Serum pegylarginase levels were measured at multiple time points around dose 1 and dose 8.

[0127] FIG. 6 presents the results of the 6 minute walk test for three patients (no data available for patient 5 ("c") at dose 20 and dose 32). Neuromotor results are shown in the table below. Doses 8, 20, and 32 are measured from the start of the repeat dose (2nd dose). PROMIS, specifically the PROMIS physical function domain. The 6MWT (6 minute walk test), BBS (Berg Balance Scale), GMFM part E, and PROMIS data shown in the table below are presented as raw data at baseline and as change from baseline at other time points. For the 6MWT, the MCID is 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. 2017-04-26, vol. 12, no. 1, p. 78; for the BBS, the MCID is defined from the analysis of Downs et al., "The Berg Balance Scale", J. Physiother., 2015-01, vol. 61, no. 1, p. 46; for the GMFM, the MCID is defined from the analysis of Oeffinger et al. (2008); and for the PROMIS, the MCID is based on physical function with 0.5 standard deviations or T-score of 5 points. The following table and Figure 6 show the improvement of neuromotor function in patients after administration of PEGylarginase. Neuromotor function may 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 steps, walking, spasticity, and awakening. For example, an affected patient may walk on tiptoes instead of the normal gait (heel to toe). Improved neuromotor function may mean that such a patient has an improved walking ability, or walks normally instead of tiptoe. Improved neuromotor function may be increased mobility, such as no longer needing to use a walking aid (e.g., a "walker" or cane), or using a walking aid less.Improved neuromotor function may mean improved posture and / or improved communication / socialization.

[0128] [Table 10] Neuromotor function evaluation TIFF2024028760000018.tif131170

[0129] GAA is associated with stroke 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, Vol. 111, No. 1, pp. 16-25). Results show that treatment with PEGylarginase reduces GAA levels in patients and reduces plasma arginine and associated GCs. Levels of one or more guanidino compounds such as GAA (see, e.g., FIG. 5B) are reduced or eliminated within 24-48 hours after administration of arginase. Thus, arginase is useful as an acute treatment to reduce or eliminate toxic metabolites in patients. Clinical improvement was observed with repeated administration of PEGylarginase after 8 weeks. PEGylarginase was generally well tolerated by patients. Most associated AEs were mild and manageable with standard measures.

[0130] Treatment with PEGylarginase unexpectedly rapidly reduced symptoms of hyperargininemia in ARG1-D patients. These improvements were evident at the biochemical level. Plasma levels of one or more arginine and guanidino compounds can be reduced to normal levels based on initial and / or repeated administration of arginase. Furthermore, administration of arginase improved adaptive behavior and / or neuromotor function in ARG1-D patients. All patients who participated in the study were receiving standard or conventional treatment for hyperargininemia during the study period. Such standard or conventional treatments include dietary restrictions that limit protein intake, which can increase arginine levels, and the use of nitrogen scavenger drugs. Despite these standard treatments, all patients still exhibited high arginine levels. Treatment with arginase rapidly and persistently reduces plasma levels of one or more arginine and other guanidino compounds. The effects of arginase treatment (e.g., PEGylarginase) were unexpectedly rapid, occurring within 24-48 hours after administration. In contrast, standard or conventional treatment for hyperargininemia may not result in any improvement for up to 2 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, Vol. 41, No. 9, pp. 1021-1024). Furthermore, patients treated with PEGylarginase were able to eat more liberally due to the lowering of arginine, and therefore obtain more protein.

[0131] Phase 2 clinical data demonstrate improvement in ARG1-D-related disease symptoms after plasma arginine lowering with arginine-depleting agent In parts 1 and 2, the open-label phase 1 / 2 study described above was continued, and an open-label extension study was continued. As shown in Figure 7, in phase 1 / 2, in part 1, 16 patients received a single ascending dose of pegdilarginase intravenously every other week for weeks 4-10, as previously described. In part 2, repeated doses of pegdilarginase (8 weekly doses) were administered intravenously for 10 weeks, as previously described. Nine patients started part 2, and six patients completed the repeated doses. In the open-label extension study, three patients were started on intravenous pegdilarginase.

[0132] The following table shows the patient characteristics, patient disease burden in the study, and open-label extension. 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 plasma arginine levels at baseline. Seven of 10 patients showed deficits in both mobility and adaptive behavior. Median plasma arginine was based on the mean of all plasma arginine values ​​for each patient before the first dose. For other biochemical parameters, assessments immediately prior to dosing were used. For clinical laboratory assessments, abnormal values ​​were defined as being outside the reference range. For height percentiles, deficits were defined as 10% or less of normal values ​​provided by the CDC. For the 6MWT, Berg balance, GMFM part E, and PROMIS were applied as previously described. Deficits were defined as a standard score of less than 85 for practical, social, conceptual, or combined in the Adaptive Behavior Assessment System, Third Edition (ABAS). For the 6MWT, the MCID (see Figure 9) was defined as a 9% change from baseline, for GMFM Part E, a change of 1.8 to 4.0 points depending on the individual patient's Gross Motor Ability Classification System level, for the BBS, a change of 7 points, for the ABAS, a change in General Adaptive Composite (GAC) standard score of 7.5 points, and for PROMIS, a change of 5 points.

[0133] [Table 11] Baseline assessments were performed for patients in the Phase 1 / 2 study. TIFF2024028760000019.tif158170

[0134] FIG. 8 shows the time-dependent improvement of plasma arginine with repeated dosing for patients in the second part 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 is shown for each time point. According to the treatment guidelines given by Haeberle et al., "Suggested guidelines for the diagnosis and management of urea cycle disorders," Orphanet. J. Rare Dis., 2012, vol. 7, p. 32, plasma arginine levels should be maintained below 200 μmol / L. FIG. 8 shows that 100% (6 / 6) of patients who completed the second part achieved consistent arginine lowering levels below the recommended guidelines. The reduction in arginine lowering plasma levels was accompanied by a significant and sustained reduction in plasma levels of GCs (GVA, ArgA, NAA, GAA).

[0135] Treatment with pegdilarginase also improved clinical outcomes from baseline after eight repeated doses, as illustrated in Figure 9. Each point in Figure 9 represents one assessment for one patient. Favorable assessments are indicated by filled circles, and neutral assessments are indicated by open circles. 67% (4 / 6) of patients showed improvement equal to or greater than the MCID in tests of mobility and / or adaptive behavior after only 8 weeks of repeated pegdilarginase administration. The criteria for these tests have been previously described for the 6MWT, Berg Balance Scale, GMFM Part E, PROMIS, and ABAS. Thirty-three percent of assessments exceeded the MCID favorably, while no unfavorable assessments exceeded the MCID. One-third of ABAS assessments exceeded the MCID favorably, and two-thirds trended toward improvement at week 8.

[0136] [Table 12] Summarizes the observations of some patients in the study by the assessors. TIFF2024028760000020.tif118170

[0137] Pegdilarginase administration generally resulted in mild AEs, if any. All patients received 180 infusions of pegdilarginase. Low titer treatment-emergent ADAs were detected in 6 / 16 patients in Part 1. All patients in Part 2 had undetectable ADA levels by the fifth infusion. Two or more patients with treatment-related AEs of at least moderate severity included hypersensitivity (four events in three patients, three considered serious adverse events (SAEs) by the investigators), which were managed with medications such as infusion rate adjustment and administration of antihistamines, and in some cases corticosteroids.

[0138] Figure 10 shows an analysis of mean arginine levels by clinical response performed on patients who participated in Part 2. The mean arginine levels of patients were found to be lower in patients who showed a clinical response of >1 MCID improvement on neuromotor or adaptive behavioral assessment (n=4) than in patients who showed an MCID improvement of <1 on neuromotor or adaptive behavioral assessment (n=2).

[0139] In conclusion, the progressive nature of this disease highlights a significant unmet medical need for pharmacological treatment methods that offer the potential to lower arginine levels beyond levels achievable with current standard disease management, and thus slow or halt the progression of neuromotor, neurocognitive, and / or adaptive behavioral deterioration observed in ARG1-D patients, despite standardized disease management approaches utilizing severe dietary protein restriction and the use of ammonia scavengers to address disease symptoms alone. PEGylarginase, an engineered human arginase 1, has provided direct evidence of a significant and sustained reduction in plasma arginine and related GC levels in ARG1-D patients, accompanied by improvements in neuromotor function and / or adaptive behavior in some patients.

[0140] The improvements observed in patients with ARG1-D with PEGylarginase in the phase 1 / 2 study of the aforementioned trial were sustained in the open-label extension study. PEGylarginase was highly effective in sustainably lowering elevated plasma arginine, which is believed to underlie the pathology of ARG1-D symptoms. The reduction in plasma arginine was accompanied by improvements in mobility and adaptive behavior after only 8 weeks of repeated dosing. PEGylarginase was well tolerated by patients. Most treatment-related AEs were mild. Hypersensitivity reactions could be managed with standard methods, and all patients continued on study treatment. Comprehensive baseline profiling of patients with ARG1-D showed quantifiable deficits in mobility and / or adaptive behavior in 94% (15 / 16) of patients.

[0141] Analysis of α-K-δ-GVA, (R,S)-ArgA, homoarginine HCl, and Nα-acetyl-L-arginine in K2EDTA human plasma by LC-MS / MS. TIFF2024028760000021.tif172170TIFF2024028760000022.tif94170

[0142] The commercially available reagents used are as follows: TIFF2024028760000023.tif96170

[0143] Treated human plasma [THP]. To 20.0 mL of K2EDTA human plasma, add 0.059 mL of AEB1102. Place in a polypropylene vial (PPV) and store at approximately -70°C. Breath is from the plasma used.

[0144] Treated human lipemic plasma [TLP]. To 20.0 mL of K2EDTA lipemic human plasma, add 0.059 mL of PEGylarginase. Place in PPV and store at approximately -70°C. Breath is from used plasma.

[0145] Treatment Human Whole Blood [THB]. 20.0 mL of K2EDTA human whole blood is added with 0.059 mL of PEGylarginase. Place in PPV and store at approximately 4°C. Breath is from used plasma.

[0146] 1mg / mL nor-NOHA [NOHA] Dissolve the entire contents of a 5 mg vial of nor-NOHA in 5.00 mL of MQ. Place in PPV and store at approximately -70°C for up to one month.

[0147] 10% mannitol solution (w / v) [MT1] Weigh out approximately 1.00 g of mannitol and dissolve in 10 mL of MQ. Stir until dissolved. Store at room temperature for up to 1 month.

[0148] Non-oxidizing matrix [NM1] Centrifuge 20 mL of K2EDTA human plasma at 3500 rcf 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 0.180 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 just before using the plasma). Mix well. Place in PPV and store at approximately -70°C up to the expiration date of the plasma component.

[0149] Oxide Matrix [AM1] 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 just before using the plasma). Mix well. Place in PPV and store at approximately -70°C up to the expiration date of the plasma component.

[0150] 10% (w / v) TCA [BAC-359]. Weigh approximately 50 g of TCA into a solvent bottle. Add 500 mL of MQ using a graduated cylinder. Stir until dissolved. The solution can be stored at room temperature for up to one month. This solution will be used as the protein precipitation solution. Chill this solution on ice before using it for sample extraction.

[0151] 0.1% FA and 0.05% PFHx in MQ [BAC-360]. Measure 1000mL of MQ into a graduated cylinder and add to the solvent storage bottle. Add 1mL of FA and 0.5mL of PFHx using a pipette. Mix thoroughly. The solution can be stored at room temperature for up to 1 month. This solution will be used as Mobile Phase A (MPA). This solution should be made in a 1L Teflon vessel.

[0152] 0.1% FA and 0.05% PFHx in ACN [BAC-361]. Measure 1000 mL of ACN into a graduated cylinder and add to the solvent storage bottle. Add 1 mL of FA and 0.5 mL of PFHx using a pipette. Mix thoroughly. This solution can be stored at room temperature for up to 1 month. This solution will be used as Mobile Phase B (MPB). This solution should be made in a 1 L Teflon vessel.

[0153] 80:20:0.3(v / v / v) of MeOH:MQ:FA[BAC-409] Combine 800 mL of MeOH (methanol) and 200 mL of MQ in a solvent storage bottle using a graduated cylinder. Add 3.00 mL of FA using a pipette. Mix thoroughly. Store at room temperature for up to 1 month. This solution can be used as Mobile Phase A (MPA).

[0154] 100 mM Ammonium Formate in MQ [BAC-409] Measure out 1000 mL of MQ using a graduated cylinder. Place approximately 6.306 g of ammonium formate into a tare boat. Transfer the ammonium formate to the solvent storage bottle containing the MQ rinse. Transfer the remaining MQ to the bottle. Stir until dissolved. Store at room temperature for up to 1 month. This solution can be used to prepare Mobile Phase B (MPB).

[0155] 70:30 (v / v) 100 mM ammonium formate:MeOH [BAC-410] Combine 700 mL of BAC-408 and 300 mL of MeOH in a solvent storage bottle using a graduated cylinder. Mix thoroughly. Store at room temperature up to the expiration date of the BAC-408. This solution can be used as Mobile Phase B (MPB).

[0156] MQ:FA[BAC-001] at 1000:1 (v / v). Add 1000 mL of MQ to the solvent bottle using a graduated cylinder. Add 1 mL of FA using a pipette. Mix thoroughly. This solution can be stored at room temperature for up to 1 month. This solution can be used for the R0 solution.

[0157] IPA:Acetone:CAN:FA [BAC-083] in a ratio of 40:10:50:0.05 (v / v / v / v) Combine 400 mL IPA, 100 mL acetone, and 500 mL ACN using a graduated cylinder and transfer into an appropriately sized solvent storage bottle. Stir to mix. Using a pipette, transfer 0.500 mL FA to the bottle. Mix thoroughly. Store at room temperature for up to 1 month. Alternatively, a purchased solution may be used in place of the prepared solution. If using a purchased solution, store the solution at room temperature following standard procedures. This solution can be used for R3 solution.

[0158] 50:25:25 (v / v / v) IPA:ACN:MeOH [BAC-011]. Combine 500 mL IPA, 250 mL ACN, and 250 mL MeOH in a solvent storage bottle using a graduated cylinder. Mix thoroughly. The solution can be stored at room temperature for up to one month. This solution can be used for heavy-duty cleaning of needles.

[0159] Preparation of stock solutions. Stock solutions should be prepared in duplicate and compared prior to use. The volumes prepared may be varied provided proportionality and final concentrations are maintained and recorded.

[0160] GVA Stock Solution (10,000 μM) [S01]. Weigh out the equivalent of 3 mg of GVA (MW 173.17) after applying the correction factor into an amber glass vial. Dissolve and dilute to 10,000 μM in MQ. Mix thoroughly. Divide the solution into 0.075 mL aliquots and store the solution in PPV at approximately -70°C. Thaw the stock on wet ice prior to use.

[0161] ArgA Stock Solution (10,000 μM) [S02]. Weigh out the equivalent of 3 mg of ArgA (MW 175.19) after applying the correction factor into an amber glass vial. Dissolve in MQ and dilute to 10,000 μM. 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 on wet ice prior to use.

[0162] HArg Stock Solution (40,000 μM) [S03]. Weigh out the equivalent of 15 mg of HArg (MW 224.69) after applying the correction factor into an amber glass vial. Dissolve and dilute to 40,000 μM in MQ. Mix thoroughly. Divide the solution into 0.100 mL aliquots and store the solution in PPV at approximately -70°C. Thaw the stock on wet ice prior to use.

[0163] NAArg Stock Solution (20,000 μM) [S04]. Weigh out the equivalent of 6 mg of NAArg (MW 216.24) after applying the correction factor into an amber glass vial. Dissolve and dilute to 20,000 μM in MQ. Mix thoroughly. Divide the solution into 0.075 mL aliquots and store the solution in PPV at approximately -70°C. Thaw the stock on wet ice prior to use.

[0164] GAA stock solution (40,000 μM) [S05]. Weigh out the equivalent of 14 mg GAA (MW 117.11) after applying the correction factor into an amber glass vial. Dissolve in [BAC-009] and dilute to 40,000 μM. Mix thoroughly. Divide the solution into 0.150 mL aliquots and store the solution protected from light in PPV at approximately -70 °C. Thaw the stock on wet ice prior to use.

[0165] Preparation of Internal Standard (IS) Solution. The volume prepared may vary provided that proportionality and final concentration are maintained and recorded.

[0166] GVA- 13 C6 IS stock solution (4,150 μM) [I01]. After applying the correction factor, 3 mg equivalent of GVA- 13 Weigh out C6 (MW 215.65). Dissolve in MQ and dilute to 4150 μM. Mix thoroughly. Divide solution into 0.050 mL aliquots and store solution in PPV at approximately -70° C. Thaw stock on wet ice prior to use.

[0167] ArgA- 13 C6 IS stock solution (5,000 μM) [I02]. After applying the correction factor, 3 mg equivalent of ArgA- 13 Weigh out C6 (MW 181.14). Dissolve in MQ and dilute to 5,000 μM. Mix thoroughly. Divide solution into 0.050 mL aliquots and store solution in PPV at approximately -70° C. Thaw stock on wet ice prior to use.

[0168] HArg-d4 IS stock solution (5,000 μM) [I03]. Weigh out the equivalent of 3 mg of HArg-d4 (MW 265.17) after applying the correction factor into an amber glass vial. Dissolve and dilute to 5000 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 on wet ice prior to use.

[0169] NAArg- 13 C6 IS stock solution (5,000 μM) [I04]. After applying the correction factor, 3 mg equivalent of NAArg- 13 Weigh out C6 (MW 222.19). Dissolve in MQ and dilute to 5,000 μM. Mix thoroughly. Divide solution into 0.050 mL aliquots and store solution in PPV at approximately -70° C. Thaw stock on wet ice prior to use.

[0170] GAA-13C2 IS stock solution (5,000 μM) [I05] After applying the correction factor, 3 mg equivalent of GAA- 13 Weigh out C2 (MW 119.09). Dissolve in [BAC-009] and dilute to 5,000 μM. Mix thoroughly. Divide solution into 0.050 mL aliquots and store solution PFL in PPV at approximately -70°C. Thaw stock on wet ice prior to use.

[0171] Heavy IS working solution (2.075 μM I01, 2.50 μM I02, I04) [HI06] Combine 0.020 mL each of [I01], [I02], and [I04] with 39.940 mL of [1N HCl] in PPV using a pipette. Mix thoroughly. Prepare solutions on wet ice. Dispose of solutions after use.

[0172] IS Working Solution (5,000 nM) [I05]. Combine 0.020 mL each of [I01], [I02], [I04], and [I05] with 39.920 mL of [1 N HCl] in PPV using a pipette. Mix thoroughly. Prepare the solution on wet ice. Dispose of the solution after use.

[0173] Preparation of stock comparison solutions Prepare comparator solutions in PPV on wet ice using a pipette as shown in the table below. Mix thoroughly. Store refrigerated. Stock comparator solutions used to establish stability should be evaluated within the established process sample stability window. Volumes prepared may be varied as long as proportionality and final concentrations are maintained and recorded. TIFF2024028760000024.tif255170

[0174] SC6 can be referred to as SST for short-term storage comparisons and SLT for long-term storage comparisons. SC6 can be used to compare I05 stock solution preparations. (*) If only one concentration is listed, GVA- 13 The C6 concentration is 0.83 times the value shown.

[0175] Sample extraction. 1. Transfer 0.050 mL of each calibration, quality control (QC), blank, and experimental sample to a 96-well plate on wet ice. Diluted samples should be diluted with 1x PBS at the appropriate dilution factor prior to sample processing. 2. Add 0.100 mL of [1N HCl] to each matrix blank sample. 3. Add 0.100 mL of [I05] to each calibration sample, QC sample, blank sample with IS, and experimental sample. Do not add [I06] to GQC (GAA LLOQ (Lower Limit of Quantitation) (QC) samples, if present. 4. Vortex mix at 1600 rpm for 5 minutes. 5. Add 0.400 mL of ice cold [BAC-359] to each sample. 6. Vortex mix at 1000 rpm for 5 minutes. 7. Centrifuge at 3500rcf for 5 minutes. 8. Transfer 0.300 mL of the supernatant to a clean 96-well plate using a TomTec or multi-channel pipette. 9. Centrifuge at 3500rcf for 5 minutes. 10. Until analysis is complete, store the processed samples in the sample compartment of the LC instrument or in a refrigerator at approximately 4°C.

[0176] Ultra-high performance liquid chromatography (UHPLC) setup. TIFF2024028760000025.tif103170

[0177] Gradient Program: TIFF2024028760000026.tif55170

[0178] Mass spectrometer parameters. Parameters other than mass analyzer, interface, and scan mode can be modified for optimal performance. The exact mass transitions may vary slightly from instrument to instrument due to the unit resolution of the quadrupole mass analyzer. TIFF2024028760000027.tif68170

[0179] TIFF2024028760000028.tif92170

[0180] Direct binding ELISA for detection of antibodies against PEG in human serum A. Reagents / Buffers Dilution Buffer / Assay Buffer (4% Bovine Gamma-Globulin (BGG) in 1x PBS) Assay diluent was prepared by adding 4 g of BGG to 100 mL of 1x PBS. The solution was 0.22 μM filtered and stored at 2-8°C for up to 2 weeks. Volumes were measured as needed.

[0181] Competition buffer (assay buffer + 100 μg / mL PEG) Competition buffer was prepared by adding 100 μg PEG per mL of assay buffer. Competition buffer was made fresh on the day of the assay. Volumes were measured as needed.

[0182] Stop liquid (1M H3PO4) 21.4 mL of 85% (11.7 M) H3PO4 was added to 228.6 mL of dI (deionized) H2O, mixed well and stored in an acid cabinet for up to one year. Volume was measured as needed.

[0183] Additional Reagents (see table below) TIFF2024028760000029.tif253170

[0184] Reagent Source CAT Number Batch / Lot Number Carbonate Coating Buffer BioWorld 40320016-1 L16020109JC Dilution Buffer / Assay Buffer (4% BioAgilytix N / A RP08Jul16JHJ04, BGG, 1x in PBS) RP13Jul16MB01, RP11Jul16JHJ02, RP18Jul16JHJ01 Bovine gamma-globulin Millipore 82-041 1269 Wash Buffer (1x PBS) BioAgilytix N / A RP11Jul16JHJ01, RP12Jul16MBM01, RP13Jul16JHJ01, RP18Jul16JHJ02, RP08Jul16JHJ03 Polypropylene plate Costar 3365 09516000 Nunc Immuno Starwell C8 Thermo 441653 125663 Maxisorp Plate TMB Microwell Proxidase Kit KPL 50-76-00 10158819 Stop solution (1M H3PO4)BioAgilytix N / A RP08Jul16MBM01, RP26May16kml03, RP18Jul16MBM01 Human IgG Jackson 009-000-003 126258 Immunoresearch Human IgM Jackson 009-000-012 124517 Immunoresearch

[0185] B. Screening Assay Procedure Wells of 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 (these controls were omitted from post-cut point testing) in carbonate coating buffer according to the plate map. Plates were sealed, briefly shaken at approximately 450 rpm to distribute across wells, and incubated at 37° C. for approximately 1 hour. After incubation, wells were washed 3 times with 1× PBS. A wash program included an overflow with suction setting and 10 seconds shaking after each dispense (Program 29_Wash_Shake) to ensure thorough washing of the Starwell plate. This program was used for all wash steps in this procedure.

[0186] After washing, the plate was inverted and tapped dry on absorbent paper. 300 μL of dilution buffer was then added to all wells of the plate. The dilution buffer contains 4% BGG, and is therefore also considered "inhibiting buffer." The plate was covered and incubated at room temperature with shaking (approximately 450 rpm) for at least 1 hour, but not more than 3 hours.

[0187] After the blocking incubation period, the plates were washed, inverted, and tapped dry on absorbent paper. Controls and samples diluted to a minimum dilution factor (MRD) of 50 in dilution buffer were added (100 μL / well) to the plates in duplicate according to the plate map. The plates were then sealed and incubated at room temperature with shaking (approximately 450 rpm) for approximately 1 hour.

[0188] The plate was washed, inverted, tapped dry on absorbent paper, and 100 μL of detection antibody was added to the appropriate wells according to the plate map. Mouse anti-PEG antibodies were detected using goat anti-mouse IgG-Fc-HRP diluted 1:5,000, and anti-human antibodies were detected using rabbit anti-human IgG / A / M diluted 1:30,000 in dilution buffer and added to the appropriate wells. The plate was sealed and incubated for approximately 1 hour at room temperature with shaking (approximately 450 rpm). The plate was washed, inverted, tapped dry on absorbent paper, and 100 μL of TMB substrate per well was added. The plate was covered and incubated for approximately 10-20 minutes at room temperature with shaking (approximately 450 rpm). The reaction was stopped by adding 100 μL of stop solution per well. To ensure complete reaction, the plate was briefly shaken.

[0189] C. Confirmatory Assay Procedures Wells of a Starwell C8 Maxisorp were coated with 100 μL of 2 μg / mL BSA mPEG 5K, or 500 ng / mL human IgG, or 500 ng / mL (these controls were omitted from post-cut point testing) in carbonate coating buffer according to the plate map below. The plate was sealed, shaken briefly at approximately 450 rpm to distribute across the wells, and incubated at 37° C. for approximately 1 hour.

[0190] After incubation, the wells were washed 3 times with (1x PBS) using program 29_Wash_Shake to ensure thorough washing of the star-well plate. This program was used for all washing steps in this procedure. After washing, the plate was inverted and tapped dry on absorbent paper. Then, 300 μL of dilution buffer was added to all wells of the plate as a blocking step. The plate was covered and incubated at room temperature with shaking (approximately 450 rpm) for at least 1 hour but not more than 3 hours.

[0191] While the star well plate was blocked, samples and controls were diluted to 50x MRD in dilution buffer and competition buffer containing PEG at 100 μg / mL in a polypropylene plate. The diluted samples and controls were incubated at room temperature with shaking (approximately 450 rpm) for approximately 1 hour.

[0192] After the blocking incubation period, the star well plate was washed, inverted and tapped dry on absorbent paper. Controls and samples pre-incubated with and without PEG were added to the plate in duplicate (100 μL / well) according to the plate map. The plate was then sealed and incubated at room temperature with shaking (approximately 450 rpm) for approximately 1 hour.

[0193] After washing, the plate was inverted, tapped dry on absorbent paper, and 100 μL of detection antibody was added to the appropriate wells according to the plate map. Mouse anti-PEG antibody was detected using (goat anti-mouse IgG-Fc-HRP) diluted 1:5,000, and anti-human antibody was detected using (rabbit anti-human IgG / A / M) diluted 1:30,000 in dilution buffer and added to the appropriate wells. The plate was sealed and incubated for approximately 1 hour at room temperature with shaking (approximately 450 rpm). The plate was washed, inverted, tapped dry on absorbent paper, and 100 μL of TMB substrate per well was added. The plate was covered and incubated for approximately 10-20 minutes at room temperature with shaking (approximately 450 rpm). The reaction was stopped by adding 100 μL of stop solution per well. The plate was shaken briefly to ensure proper mixing and then read on a Synergy2 plate reader at 450 (detection) 620 (background).

[0194] D. Titration Method Procedure The titer assay followed the same procedure as the screening assay. Samples to be titrated were subjected to at least seven two-fold serial dilutions in negative pool human serum. A pool of normal humans was screened during quality check and the one with the lowest background was selected to be used as the negative pool in check. These titer dilutions were diluted to a minimum dilution factor of 50 in dilution buffer and added to the plate in duplicate (100 μL / well) according to the plate map.

[0195] E. Method Confirmation Method validation includes specificity, assay sensitivity, selectivity / matrix interference, drug resistance, prozone (adhesion) effect, titration assay linearity, intra- and interassay precision, short-term and freeze-thaw stability, and establishment of screening, validation, and titration cut points.

[0196] Two sets of controls were used for assay validation. One set was 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. The second set was prepared using human samples that showed high anti-PEG response in the screening assay and high inhibition in the confirmation assay. This sample would match the expected existing anti-PEG sample. A human anti-PEG low concentration control was prepared by diluting an identified high concentration human sample into the negative control serum to generate a sample that would result in a low (LPC-h) signal in the assay. The undiluted high concentration human sample was used as the HPC-h. Both sets of controls were included in each study where applicable. The controls should show a signal proportional to the level, i.e. high concentrations are greater than low concentrations, and low concentrations are greater than the cut point. Immunoglobulin controls (human IgG and human IgM coated on the plate) were also included in the cut point study to verify the performance of the detection antibody cocktail.

[0197] F. Cut-point Analysis Mouse anti-PEG screening cut point. For data evaluation using mouse anti-PEG SPC, a screening cut point was set using two times the standard deviation of the mean of all negative control samples generated during validation (n=55 plates). This calculation yielded a correction factor of 0.01467 that, when added to the mean of the NCs on each plate, produced a cut point specific to the mouse anti-PEG SPC samples.

[0198] Human Screening Assay Cut Points. Screening assay cut points were established using 98 normal human serum samples. Six measurements were performed for each sample in a minimum of seven replicates by three analysts. Data were evaluated using SAS JMP® software (version 12 or later) to remove statistical outliers, measure variance, and establish cut-off points. For statistical outlier determination, responses were normalized by dividing the mean signal of the sample by the mean of the anti-human NC samples on each plate.

[0199] G. Cut Point Determination Human anti-PEG screening cut points. Parametric and non-parametric screening drift cut point factors were determined using normalized values. First, a parametric method with Tukey's biweight estimation was used to calculate robust estimates of the mean and standard deviation (SD) of the ratios after removing outliers. Parametric drift cut point factors at a 5% false positive error rate were then determined by multiplying the SD value by the 95th quantile of the t-distribution (with degrees of freedom equal to the number of ratio values ​​minus 1) and adding the product to the mean. Non-parametric 5% error rate cut point factors were determined by calculating the empirical 95th quantile value of the ratio values.

[0200] Human confirmatory cut points. Confirmatory cut point values ​​were established with a 1% false positive error rate by the procedure recommended by Shankar G et al. Cut point values ​​were determined by assigning a lower limit of specific inhibition with the 14th sample excluded from the screen cut point assignment as a biological outlier. These samples had consistently higher % inhibition values ​​than samples screened as negative. The percentage of change from the unspiked sample was calculated for each sample using the following formula: TIFF2024028760000030.tif14170

[0201] Parametric confirmatory cut points were determined by first calculating Tukey's biweight estimates of the mean and SD of all % inhibition values ​​retained in the analysis. Cut point values ​​were then calculated by multiplying each SD value by a coefficient equal to the first quantile of the t-distribution (with degrees of freedom equal to the number of inhibition values ​​minus 1) and subtracting this product to the mean. Nonparametric cut point values ​​were also determined based on the empirical first quantile.

[0202] G. Specificity The addition of high levels of human IgG did not affect the signal generated in the assay compared to unspiked samples. H.PEG durability

[0203] Assay interference was assessed by performing two-fold serial dilutions of PEG starting at a concentration of 100 μg / mL in the presence of concentrations of anti-PEG surrogate positive controls corresponding to HPC-m and LPC-m, as well as concentrations of human anti-PEG HPC-h and LPC-h. Zero-spiked controls consisting of each control without PEG were also performed. This was performed in at least two duplicates by at least two analysts. Human-specific screening cut points were used to determine tolerability in running these assays. HPC-m, HPC-h, and LPC-h tolerated all concentrations of PEG tested up to 100 μg / mL.

[0204] I. Drug resistance of PEGylarginase Assay interference was evaluated by performing two-fold serial dilutions of PEGylarginase starting at a concentration of 150 μg / mL in the presence of concentrations of anti-PEG surrogate positive controls corresponding to HPC-m and LPC-m, and concentrations of human anti-PEG HPC-h and LPC-h. Zero-spiked controls consisting of each control without PEG were also performed. Drug resistance was evaluated using anti-human specific cut points. HPC-m was tolerated up to 9.38 μg / mL in one evaluation and up to 37.5 μg / mL in the second evaluation, with a mean drug resistance of 23.4 μg / mL. LPC-m was not resistant to Co-Arg1PEG in one evaluation and up to 2.34 μg / mL in the second evaluation. HPC-h was tolerated up to 9.38 μg / mL in one evaluation and up to 18.8 μg / mL in the second evaluation, with a mean drug resistance of 14.1 μg / mL. At one evaluation, LPC-h was tolerated down to 4.69 μg / mL, and at a second evaluation, it was tolerated down to 9.38 μg / mL, with a mean drug tolerance of 7.04 μg / mL.

[0205] Neuromotor and Neurocognitive Testing A physical examination will be completed with assessment of neurological and neuromotor function, including but not limited to the 6MWT, GMFM, Berg Balance Scale, Ashworth Modified, and Purdue Pegboard Test. Assessments may be performed over one or more days. Assessments may be recorded at baseline and at subsequent time points.

[0206] The 6MWT is widely used in clinical trials to globally evaluate the cardiovascular, pulmonary, and neuromuscular systems in numerous disease states. It measures the distance a patient can walk on level ground in 6 minutes. The American Thoracic Society (ATS) has published a standardized method for performing the test (ATS Statement Guidelines for the Six-Minute Walk, Am. J. Respir. Crit. Care Med., vol. 166, pp. 111-117 (2002)), which is well tolerated and easy to administer. This procedure was followed for the 6MWT. Modifications to the test method, approved by the sponsor, may be made to account for the patient's condition and logistical considerations at the study site. Non-ambulatory patients were not administered the 6MWT. Results of the 6MWT are recorded as distance completed in meters and as percentage change from baseline.

[0207] The Berg Balance Scale measures balance in patients with balance disorders by assessing their performance in specific functional tasks. There are 14 tasks that assess specific aspects of balance, such as sit-to-stand transitions, transfers, turns, and one-legged standing. Furthermore, most tasks require the subject to maintain that position for a specific period of time.

[0208] The GMFM is a clinical scale designed to assess changes in gross motor skills by observing a range of activities, including lying, rolling, walking, running, and jumping. Each item on the GMFM uses a four-point scoring system that measures the extent to which a person can initiate and complete a movement across five dimensions (A-E): A: lying and rolling, B: sitting, C: crawling and kneeling, D: standing, and E: walking, running, and jumping.

[0209] The modified Ashworth scale was developed to assess spasticity in patients with central nervous system (CNS) lesions and is used to measure the resistance to passive movement around a joint due to spasticity. The scale is well tolerated and easy to administer. The scale cannot distinguish between spasticity and soft tissue stiffness. The scale uses a scoring scale with six scoring options ranging from 0 (no spasticity) to 4 (complete stiffness), as shown in the table below.

[0210] TIFF2024028760000031.tif61170

[0211] The Purdue Pegboard Test is a test that measures manipulative dexterity and bimanual coordination speed in fine motor tasks using both sides of the body (Tiffin et al., 1948). The test involves two distinct abilities: (1) gross movements of the arm, hand, and fingers, and (2) fine motor limbs, also known as "fingerprint" dexterity. Poor performance on the Pegboard Test is indicative of impairments in complex visually guided or coordinated movements that are thought to be mediated by circuits involving the basal ganglia.

[0212] The table below lists further neurocognitive, developmental and quality of life (QOL) assessments that can be performed on patients undergoing treatment to assess improvement over time. TIFF2024028760000032.tif238170TIFF2024028760000033.tif188170 aPROMIS (patients who started a phase 1 / 2 trial with a PROMIS assessment) or PedsQL measurement model for the Pediatric Quality of Life Inventory (patients who were not in a phase 1 / 2 trial with a PROMIS assessment).

[0213] List of references ATS Statement Guidelines for the Six-Minute Walk, Am. J. Respir. Crit. Care Med., vol. 166, pp. 111-117 (2002) Burrage et al., "Human recombinant arginase enzyme reduces plasma arginine in mouse models of arginase deficiency," Hum. Mol. Genetics, Vol. 24, No. 22, pp. 6417-27 (2015) Carvalho, D.R. et al., "Clinical features and neurologic progression of hyperargininemia," Pediatr. Neurol, Vol. 46, No. 6, pp. 369-74 (2012) Cheng et al., Cancer Res, 67, 309-17 (2007) Deignan et al., "Increased plasma and tissue guanidine compounds in a mouse model of hyperargininemia," Mol. Genet. Metab., 93, 172-178 (2008) Dillon et al., "Biochemical characterization of the arginine degrading enzymes arginase and arginine deiminase and their effect on nitric oxide production," Med. Sci. Monit., Vol. 8, No. 7, pp. BR248-253 (2002) Downs et al., “The Berg Balance Scale,” J.Physiother.”, 2015, Volume 61, No. 1, Page 46 Enright et al., "Reference equations for the six-minute walk in healthy adults," Am. J. Respir. Crit. Care Med., 1998, Vol. 158, No. 5, Part 1, pp. 1384-1387 Geiger et al., "Six-minute walk test in children and adolescents," J. Pediatr., 2007, Vol. 150, No. 4, pp. 395-399 Haberle et al., "Suggested guidelines for the diagnosis and management of urea cycle disorders," Orphanet. J. Rare Dis., 2012, Vol. 7, p. 32 Harris et al., Clin. Pharmacokinet., Vol. 40, No. 7, pp. 539-51 (2001) Lambert et al., “Hyperargininemia: intellectual and motor improvement related to changes in biochemical data,” J. Pediatr, 1991, vol. 118, no. 3, pp. 420-4. Lopez et al., FEBS J., 272, 4540-48 (2005) Luneburg, N. et al., “Reference intervals for plasma L-arginine and the L-arginine:asymmetric dimethylarginine ratio in the Framingham Offspring Cohort.” J. Nutr., Vol. 141, No. 12, pp. 2186-2190 (2011). Marescau et al., “Guanidino compound analysis as a complementary diagnostic parameter for “hyperargininemia: Follow-up of guanidino compound levels during therapy”, Pediatric.Res. , Vol. 27, No. 3, pp. 297-303 (1990) Marescau et al., “The pathobiochemistry of uremia and hyperargininemia further demonstrates a "metabolic relationship between urea and guanidinosuccinic acid", 1992, Vol. 41, No. 9, pp. 1021-1024 Oeffinger et al., “Outcome tools used for ambulatory children with cerebral palsy: "Responsiveness and minimum clinically important differences," Dev. Med. Chile Neurol., 2008, Vol. 50, No. 12, pp. 918-925 Prasad et al., "Argininemia: a treatable genetic cause of progressive spastic diplegia simulating cerebral palsy-case reports and literature review," J. Child Neurol., vol. 12, pp. 301-309 (1997). Remington, The Science and Practice of Pharmacy, 19th ed., edited by Gennaro, Mack Publishing Co., Easton, PA (1995). Savoca et al., Cancer Biochem. Biophys, 7, 261-268 (1984) 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, Vol. 12, No. 1, p. 78 Schlune et al., "Hyperargininemia due to arginase 1 deficiency: the original patients and their natural history, and a review of the literature," Amino Acids, Vol. 47, pp. 1751-1762 (2015). Segawa et al., "A long-term survival case of arginase deficiency with severe multicystic white matter and compound mutations," Brain Dev., Vol. 33, pp. 45-48 (2011) Stockler-Ipsiroglu et al., "Guanidinoacetate methyltransferase (GAMT) deficiency: outcomes in 48 individuals and recommendations for diagnosis, treatment and monitoring," Mol. Genet. Metab., Vol. 111, No. 1, pp. 16-25 (2014) Uchino, T. et al., "Molecular basis of phenotypic variation in patients with argininemia," Hum. Genet., Vol. 96, No. 3, pp. 255-60 (1995) Wu, G. et al., "Arginine metabolism: nitric oxide and beyond," Biochem. J., Vol. 336, Part 1, pp. 1-17 (1998) Wyse et al., "In vitro stimulation of oxidative stress in cerebral cortex of rats by the guanidino compounds accumulating in hyperargininemia," Brain Res., 2001, Vol. 923, No. 1-2, pp. 50-7 and U.S. Patent Publication No.: U.S. Patent Application Publication No. 20170240922, U.S. Patent Application Publication No.: 20170283830, U.S. Patent Application Publication No.: 20170224843, U.S. Patent Application Publication No.: 20170191078, U.S. Patent Application Publication No.: 20160095884, U.S. Patent No.: ...

Claims

1. A pharmaceutical product comprising PEGylated arginase for reducing the plasma level of at least one compound selected from the group consisting of arginine, N-α-acetylarginine (NAArg), arginic acid (ArgA), α-keto-δ-guanidinovaleric acid (GVA), and guanidinoacetic acid (GAA) to a normal level in a subject having arginase 1 (ARG1) deficiency (ARG1-D), PEGylated arginase is a medication administered intravenously at an initial dose of 0.005 mg / kg to 1.00 mg / kg of patient body weight, followed by weekly subcutaneous or intravenous administration.

2. The pharmaceutical product according to claim 1, wherein the PEGylated arginase is pegdiarginase.

3. The pharmaceutical product according to claim 1 or 2, wherein the initial intravenous dose is 0.005 mg / kg to 0.20 mg / kg of patient body weight.

4. The pharmaceutical product according to claim 3, wherein the initial intravenous administration is a dose of approximately 0.10 mg / kg patient body weight.

5. The pharmaceutical product according to any one of claims 1 to 4, wherein the subsequent weekly dose is 0.005 mg / kg to 0.20 mg / kg of patient body weight.

6. The pharmaceutical product according to claim 5, wherein the subsequent weekly administration is a dose of 0.10 mg / kg patient body weight.

7. The pharmaceutical product according to any one of claims 1 to 6, wherein subsequent weekly administration is subcutaneous.

8. The pharmaceutical product according to any one of claims 1 to 6, wherein subsequent weekly administration is intravenous.

9. The pharmaceutical product according to any one of claims 1 to 8, wherein, after repeated weekly intravenous administrations, the repeated weekly intravenous administrations are changed to subcutaneous administrations of substantially the same dose.

10. The pharmaceutical product according to any one of claims 1 to 9, wherein the weekly dose is adjusted based on the patient's measured plasma arginine levels.

11. The pharmacopoeia according to claim 10, wherein if the measured plasma arginine level is lower than 50 μM, the weekly dose administered is reduced by 0.05 mg / kg patient body weight.

12. The pharmacopoeia according to claim 10 or 11, wherein if the measured plasma arginine level is higher than 150 μM, the weekly dose is increased by 0.05 mg / kg patient body weight.

13. The pharmaceutical product according to any one of claims 10 to 12, wherein the weekly dose administered does not exceed 0.20 mg / kg patient body weight.

14. The pharmaceutical product according to any one of claims 1 to 13, wherein PEGylated arginase is administered in an amount sufficient to reduce the patient's plasma arginine level to less than 200 μmol / L within 2 to 4 days after the initial administration.

15. The pharmaceutical product according to claim 14, wherein the plasma arginine level decreases within the range of 40 μmol / L to 115 μmol / L.

16. The pharmaceutical product according to any one of claims 1 to 15, wherein the plasma level of at least one compound selected from the group consisting of ArgA, NAArg, GVA, GAA, and arginine decreases to a normal level within 7 days after the first administration.

17. The pharmaceutical product according to claim 16, wherein the plasma level decreases to a normal level in less than three days.

18. A pharmacopoeia according to any one of claims 1 to 17, wherein repeated weekly administration maintains plasma arginine levels below 200 μmol / L for at least 30 weeks.

19. The pharmaceutical product according to claim 18, wherein the plasma arginine level is maintained at less than 200 μmol / L for at least 40 weeks.

20. The pharmacopoeia according to any one of claims 1 to 19, wherein, after repeated weekly administration, the patient exhibits improved mobility and / or adaptive behavior compared to baseline.

21. The pharmaceutical product according to any one of claims 1 to 20, wherein the PEGylated arginase comprises human arginase 1 having a cobalt metal cofactor instead of a manganese metal cofactor.

22. The pharmaceutical product according to any one of claims 1 to 21, wherein the PEGylated arginase comprises the amino acid sequence of SEQ ID NO: 2.

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