WO2021257523A1

WO2021257523A1 - Use of arginase for treatment of influenza infections

Info

Publication number: WO2021257523A1
Application number: PCT/US2021/037362
Authority: WO
Inventors: Scott W. Rowlinson; Mark BADEAUX
Original assignee: Aeglea Biotherapeutics, Inc.
Priority date: 2020-06-15
Filing date: 2021-06-15
Publication date: 2021-12-23

Abstract

Described are methods for preventing and/or treating influenza infections with recombinant Arginase, such as PEGylated, cobalt-substituted recombinant human Arginase 1.

Description

USE OF ARGINASE FOR TREATMENT OF INFLUENZA INFECTIONS TECHNICAL FIELD [0001] The present disclosure generally relates to treatment of influenza infections. BACKGROUND [0002] Many antiviral compounds are designed to disrupt a single viral protein or process that is essential for viral replication. This approach has limited the overall therapeutic effectiveness and applicability of current antivirals due to restricted viral specificity, a propensity for development of drug resistance, and an inability to control deleterious host- mediated inflammation. Accordingly, there is a need for new therapies for the treatment of viral infections. SUMMARY [0003] One aspect of the present invention relates to a method of preventing or treating an influenza infection, the method comprising administering a pharmaceutical composition comprising a recombinant human arginase (rhARG) to a patient in need thereof. In one or more embodiments, the influenza disease is a respiratory disease. In one or more embodiments, the influenza virus is an influenza type A virus. In one or more embodiments, the influenza virus is an influenza type B virus. In one or more embodiments, the influenza virus is an influenza type C virus. In one or more embodiments, the influenza virus is an influenza type D virus. [0004] In one or more embodiments, the rhARG comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 1. In one or more embodiments, the rhARG is cobalt-substituted. In one or more embodiments, the rhARG comprises about 0.1 to about 2 µg Co per mg protein. In one or more embodiments, the rhARG is PEGylated. In one or more embodiments, the average number of PEG residues is about 8 to about 25 moles of PEG per mole of rhARG monomer. In one or more embodiments, the average number of PEG residues is about 8 to about 16 moles of PEG per mole of rhARG monomer. In one or more embodiments, each PEG residue has an average molecular weight of about 1,000 to about 10,000 Daltons. In one or more embodiments, each PEG residue has an average molecular weight of about 5,000 Daltons. [0005] In one or more embodiments, the pharmaceutical composition is administered intravenously. In one or more other embodiments, the pharmaceutical composition is administered subcutaneously. In one or more other embodiments, the pharmaceutical composition is administered intraperitoneally. [0006] In one or more embodiments, the pharmaceutical composition is administered at a dose of 0.05 to 2 mg/kg based on the weight of unPEGylated enzyme. In one or more embodiments, the pharmaceutical composition is administered at a dose of 0.1 to 0.5 mg/kg based on the weight of unPEGylated enzyme. In one or more embodiments, the pharmaceutical composition is administered at a dose of 0.27 mg/kg based on the weight of unPEGylated enzyme. [0007] In one or more embodiments, the pharmaceutical composition is administered once. In one or more other embodiments, the pharmaceutical composition is administered multiple times. [0008] In one or more embodiments, the pharmaceutical composition is administered once every day to once every two weeks. In one or more embodiments, the pharmaceutical composition is administered weekly. [0009] In one or more embodiments, the patient is co-administered a second therapy. In one or more embodiments, the second therapy is co-administered simultaneously with the rhARG. In one or more embodiments, wherein the second therapy is co-administered sequentially with the rhARG. [0010] In one or more embodiments, the second therapy comprises an antiviral therapy. In one or more embodiments, the second therapy comprises baloxavir. In one or more embodiments, the second therapy comprises an ion channel inhibitor. . In one or more embodiments, the second therapy comprises an inhibitor comprises a neuraminidase inhibitor. BRIEF DESCRIPTION OF THE DRAWINGS [0011] Further features of the present invention will become apparent from the following written description and the accompanying figures, in which: [0012] Figure 1 shows the amino acid and DNA sequence of Arginase 1, as well as the amino acid sequence of Arginase 2. Figure 1(a) shows the amino acid sequence of recombinant human Arginase 1 that was expressed in E. coli. (SEQ ID NO: 1); and Figure 1(b) shows the codon optimized DNA sequence (SEQ ID NO: 2) of recombinant human Arginase 1. The expressed monomer of Arginase 1 is missing the N-terminal methionine found in native human Arginase 1 monomer. Figure 1(c) shows the amino acid sequence of Arginase 2 that is missing the N-terminal methionine found in native human Arginase 2 monomer. [0013] Figure 2 is a schematic diagram of an exemplary process for the fermentation of E. coli and expression of Arginase 1. [0014] Figure 3 is a schematic diagram of an exemplary process for purification of recombinant human Arginase 1, cobalt-substituted recombinant human Arginase 1 and PEGylated cobalt-substituted recombinant human Arginase 1. Figure 3(a) shows an exemplary process including a cation exchange column (Column 1), an anion exchange column (Column 2) and a Capto multimodal column (Column 3), as well as a cobalt loading step. Figure 3(b) shows PEGylation of the Co-Arginase 1 intermediate, followed by final filtering and formulation to provide the drug substance. [0015] Figure 4 shows column chromatography purification of Arginase 1. Figure 4(a) shows loading the E. coli cell lysate onto a cation exchange column (Column 1), washing of the column, then elution with a high salt solution (to provide a First Protein Product). Protein loading and elution was assessed by measuring UV absorbance at 280 nm. Approximately 3 liters (L) of cell lysate was applied to the column, then the column washed with approx.1.5 L of buffer, then elution was performed with less than about 1 L. Figure 4(b) shows loading of the Column 1 eluted Arginase 1 (the First Protein Product) onto an anion exchange column (Column 2), the protein concentration was measured using absorbance at 280 nm. Arginase 1 was collected in the flow through from this Column 2 to provide a Second Protein Product. Figure 4(c) shows capture of Arginase 1 onto the Capto Multimodal Cation Exchange column (Column 3) and elution with a high salt solution to provide a Third Protein Product. [0016] Figure 5 shows the results of an analytical cation exchange HPLC method used to determine the charge heterogeneity profile of the Co-Arginase 1 intermediate sample eluted from Column 1 (also called First Protein Product). A 1 mg/ml sample of Arginase 1 was loaded onto a cation exchange column with a mobile phase of 20 mM MES, pH 6.0 buffer, with a flow rate of 1.0 mL/minute. A gradient of 0-500 mM NaCl was introduced over 40 minutes and the amount of protein eluted from this column was estimated by absorbance at 280 nm. Figure 5(a) shows a representative chromatogram of the charge heterogeneity species of Arginase 1. The Arginase 1 charge variants were eluted from this analytical HPLC column after 10-20 minutes. Figure 5(b) shows the same chromatogram as Figure 5(a) with greater magnification of the peaks. Figure 5(c) shows the assignment of Peak Numbers to the Arginase 1 cation exchange charge variants. Figure 5(d) shows a typical charge heterogeneity profile of drug substance resolved by an imaging capillary isoelectric focusing (iCIEF) method. [0017] Figure 6 shows the results of a LC/MS method to determine Arginase 1 gluconylation variants produced by expression of rhARG in E. coli. The LC/MS analysis identifies unmodified Arginase 1 (monomer), gluconylated Arginase1, phosphogluconylated Arginase 1, and 2 times (2X) gluconylated Arginase 1. The trace is from two separate production runs of drug intermediate. The overlay of mass spectra was summed over 33-35 minutes on a RP LCMS at 35°C; Spectra are normalized to the peak intensities of the signals due to unmodified Arginase 1. Peak intensities of variants are proportional to relative abundance. [0018] Figure 7 show the results of a gradient of 0.0 - 0.2 M NaCl applied to Column 1. Fractions were collected every 0.25 CV (Column Volume) through the gradient. The data represent two column 1 runs using two different lots of harvested cell slurry as feed material. The load factor used for the evaluations was 30 g/L. The gradient successfully separated different gluconoylated species while maintaining product recovery. [0019] Figure 8 shows the enzyme activity of Co-Arginase 1 intermediate and Co- rhARG1-PEG drug substance. Figure 8(a) shows a representative enzyme kinetic analysis of Co-Arginase 1 intermediate (conversion of arginine to ornithine with substrate concentrations over a range of 0 - 2 mM at 37°C). Figure 8(b) shows a representative enzyme kinetic analysis for Co-rhARG1-PEG drug substance. [0020] Figure 9 shows a pharmacokinetic analysis of Co-rhARG1-PEG drug substance. Figures 9(a) and (b) show mean (±SD) Arginase 1 concentration versus time profiles in patients following a single IV dose administration of Co-rhARG1-PEG: Part 1. Linear (a) and semi-log (b) plots are shown. Note that first mean BQL concentration is plotted at half of the LLOQ (0.125 µg/mL). Mean circulating drug concentrations in all patients increased with escalating doses of Co-rhARG1-PEG. Figures 9(c) - 9 (f) show mean (±SD) Co-rhARG1-PEG concentration versus time profiles in patients following QW (weekly) IV dose administration of Co-rhARG1-PEG: Part 2. Linear plots for week 1 (c) and week 8 (d); semi-log plots for week 1 (e) and week 8 (f). [0021] Figure 10 shows three representative integrated plots for pharmacokinetics (PK) and pharmacodynamics (PD)) in the Phase 1/2, open-label study to evaluate administration of Co-rhARG1-PEG to patients with Arginase 1 deficiency. Using the escalation stop criteria, the doses settled upon in Part 2 were 0.09 mg/kg for Patient 1, 0.12 mg/kg for Patient 3, and 0.04 mg/kg (for the period of Part 2 shown). By applying the dose escalation stopping criteria, other patients in the trial settled upon a variety of Part 2 dosing levels. These same criteria can be applied to adjust (increase or decrease) the dose of any patient that is already on Co-rhARG1- PEG in response to arginine levels that move outside preferred (healthy) ranges. [0022] Figure 11 shows a comparison of IV and subcutaneous administration of Co- rhARG1-PEG. The preferred plasma arginine concentrations for a patient are between 40 μM and 115 μM (dotted lines). Subcutaneous administration of Co-rhARG1-PEG results in arginine concentrations within this preferred range longer than via IV administration. Figure 11 (a) includes data from the first week subsequent to end of Part 2 and Figure 11 (b) excludes IV data from this week 1 extension. Plots are shown as an average of patient values and the data are drawn from the dose that the stopping criteria determined for each patient. [0023] Figure 12 shows plasma arginine and plasma guanidino compound levels after administration of Co-rhARG1-PEG. Figure 12(a) shows plasma arginine levels at baseline, after dose 1, after dose 8 and during the open label extension (OLE). Figure 12(b) shows plasma levels for guanidinoacetic acid (GAA), N-α-acetyl-L-arginine (NAA), α-keto-δ- guanidinovaleric acid (GVA) and argininic acid (ARGA) at baseline and during the OLE. [0024] Figures 13a and 13b show in vitro influenza A antiviral (13a) and cell viability (13b) results after incubation with Co-rhARG1-PEG. [0025] Figure 14 shows a study design for phase 1/2 trial 101A and a 102A open label extension. DETAILED DESCRIPTION [0026] Definitions [0027] “Influenza virus” is any virus of the family Orthomyxoviridae. Influenza viruses currently include four main subgroupings that are known to infect vertebrates: alpha (influenza A), beta (influenza B), gamma (influenza C) or delta (influenza D) type viruses. Influenza A includes several subtypes that are labeled according to an H number (for the type of hemagglutinin) and an N number (for the type of neuraminidase). Currently, there are 18 different HA subtypes and 11 different NA subtypes. In one or more embodiments, the influenza virus is of HA subtype H1, H2, H3, H5, H6, H7, H9 or H10. In one or more embodiments, the virus is of NA subtype N1, N2, N6, N7, N8 or N9. Exemplary influenza A viruses include those of subtype H1N1, H1N2, H2N2, H3N2, H5N1 and H7N9. [0028] “Influenza infection” is any infection caused by an influenza virus in a subject or patient. [0029] “Subject” and “patient” refer to either a human or a non-human, such as primates, mammals, and vertebrates. [0030] “Cytopathic effects” (CPEs) are distinct observable cell abnormalities due to viral infection. CPEs can include loss of adherence to the surface of the container, changes in cell shape from flat to round, shrinkage of the nucleus, vacuoles in the cytoplasm, fusion of cytoplasmic membranes and the formation of multinucleated syncytia, inclusion bodies in the nucleus or cytoplasm, and partial or complete cell lysis. [0031] “Multiplicity of infection” (MOI) means the number of virions that are added per cell during infection. For example, if ten million virions are added to ten million cells, the MOI is one. [0032] “Plaque assay” is an assay showing an area of clearing in a flat confluent growth of tissue or cells, such as that caused by the cytopathic effect of certain animal viruses in a sheet of cultured tissue cells. [0033] “Plaque forming unit” (PFU) is a measure of viable infectious entities (e.g. influenza virus particles or group of particles) in a sample or solution or inoculum, which is the smallest quantity that can produce a cytopathic effect in the host cell culture infected with the virus. The plaque being visible under the microscope and/or to the naked eye. The number of plaque forming units (PFUs) per unit volume is a conventional way to refer the titer of a virus in a specimen or inoculum. [0034] “Tissue culture infective/infectious dose 50” (TCID₅₀) is a potency unit defined as a minimal dose of infectious material at which preparation causes cytopathic effects (changes in the morphology and metabolism of tissue culture cells, indicating cell death, due to suspected infection) in the 50% of the tissue culture-containing flasks inoculated with that dilution of infectious material in the product potency assay or pathogen activity assay. [0035] “Viral titer” is an expression of the concentration of a virus, e.g. the concentration of influenza virus in a given volume. A titer is frequently determined by performing serial dilutions of the virus to obtain a quantitative and reproducible measure of the virus. Titer can be expressed as viral particles, or infectious particles per mL of fluid. Viral titer can alternatively be expressed as viral load, viral burden, and may correlate to the severity of an active viral infection. [0036] “PEGylated” refers to conjugation with polyethylene glycol (PEG), which has been widely used as a drug carrier, given its high degree of biocompatibility and ease of modification. (Harris et al., 2001). Attachment to various drugs, proteins, and liposomes has been shown to improve residence time and decrease toxicity. (Greenwald et al., 2000; Zalipsky et al., 1997). PEG can be coupled (e.g. covalently linked) to active agents through the hydroxyl groups at the ends of the chain and via other chemical methods; however, PEG itself is limited to at most two active agents per molecule. In a different approach, copolymers of PEG and amino acids have been explored as novel biomaterials which would retain the biocompatibility properties of PEG, but which would have the added advantage of numerous attachment points per molecule (providing greater drug loading), and which can be synthetically designed to suit a variety of applications (Nathan et al., 1992; Nathan et al., 1993). [0037] “Remdesivir” is a broad-spectrum antiviral medication currently developed by Gilead Sciences. [0038] As used herein, the term “rhARG1” refers to a recombinant human Arginase 1 enzyme, such as a recombinant enzyme having at least 98% sequence identity to SEQ ID NO: 1. [0039] “Co-rhARG1”, “Co-Arginase 1 intermediate” and the like refer to a rhARG1 that has at least some of the native manganese cofactor replaced with cobalt. In one or more embodiments, the Co-rhARG1 is an isolatable intermediate in a production and/or purification process for Co-rhARG1-PEG. [0040] “Co-rhARG1-PEG”, “PEGylated Co-Arginase 1” and the like refers to a Co- rhARG1 that has one or more PEG units covalently linked to the enzyme, such as at free amine(s) at the N-terminal amino acid and/or at one or more lysine residues. [0041] “Pegzilarginase” refers to a cobalt substituted, recombinant human arginase I enzyme that is covalently conjugated to monomethoxy polyethylene glycol (mPEG) that acts by catalyzing the same reaction as arginase 1, converting arginine into ornithine and urea. [0042] “High Salt Solution” refers to NaCl concentration of up to about 0.5 M. [0043] “Salt Gradient” refers to an increasing or decreasing salt concentration. Exemplary salt gradients include concentrations of NaCl ranging from about 0.01 to about 0.5M, such as about 0.1, about 0.02, about 0.03, about 0.04, about 0.05, 0.1, about 0.2, about 0.3, about 0.4 and about 0.5 M. [0044] “Room temperature” refers to at or about 15 ºC to about 25 ºC, such as about 20 to about 25 ºC. [0045] “Cation exchange” (CEX) chromatography column refers to a chromatography column uses a negatively charged ion exchange resin with an affinity for molecules having net positive surface charges. Cation exchange chromatography is used both for preparative and analytical purposes and can separate a large range of molecules from amino acids and nucleotides to large proteins. In one or more embodiments, Column 1 is a CEX column. [0046] “First Protein Product” refers to the protein product eluted from Column 1. [0047] “Anion exchange” (AEX) chromatography column refers to a chromatography column that separates substances based on their charges using an ion-exchange resin containing positively charged groups. In one or more embodiments, Column 2 is an AEX column. [0048] “Second Protein Product” refers to the protein product eluted from Column 2. [0049] “Capto Multimodal chromatography column” (MMC) utilizes a multimodal salt-tolerant “BioProcess” resin for capture and intermediate purification of proteins from large feed volumes by packed bed chromatography. In one or more embodiments, Column 3 is an MMC column. [0050] “Size exclusion chromatography” (SEC) column utilizes a chromatographic method where separation of different molecules or compounds occurs according to their size, and in some cases molecular weight. In one or more embodiments, Column 3 is an SEC column. [0051] “Third Protein Product” refers to the protein product eluted from Column 3. [0052] Methoxy PEG succinimidyl carboxymethyl ester is an amine reactive PEG product with a stable non-degradable linker between the PEG polymeric chain and the NHS ester. [0053] “High pressure homogenization” is a process that forces a stream of primarily liquid sample through a system which subjects it to any one of a number of forces which is intended to homogenize the sample and/or reduce the particle sizes of any components within it. [0054] “High-performance liquid chromatography” (HPLC) is a chromatographic method that is used to separate a mixture of compounds to identify, purify, or quantify the individual components of the mixture. [0055] “Imaging capillary isoelectric focusing” (iCIEF) is a capillary electrophoresis (CE) technique used to study physical properties of proteins. [0056] “Normalized water permeability test” (NWP) is a method for determining the cleanliness of a cassette after cleaning. This method involves measuring the passage of clean water through the membrane under standard pressure and temperature conditions. The rate of clean water flux through the membrane is measured as liters per membrane area per hour (L/m2-h). Water flux divided by the transmembrane pressure is the normalized water permeability or NWP (L/m2-h-bar). The NWP values are compared to initial (pre-process) levels and may be analyzed for trends over time. [0057] “Ultrafiltration/Diafiltration" (UF/DF) is a high yield, and robust separation process based on size exclusion. UF involves separation of components based on molecular weight or size. It is a pressure-driven process in which soluble macromolecules are retained while small molecular-weight particles and fluids pass through the membrane as waste. DF is used to exchange buffer solutions. Use of Arginase for Preventing or Treating Influenza Infections [0058] Viruses are reliant on host metabolism and macromolecular synthesis pathways for their replication. Many viruses, including influenza viruses, utilize the bioavailability of arginine, which is critical for many physiological and pathophysiological processes associated with either facilitating viral replication or progression of disease. [0059] Synthetic nucleoside analogues have proven a safe and effective means to inhibit HSV by targeting genomic replication. [0060] Arginine serves as a precursor for synthesis of protein, nitric oxide (NO), polyamines and nucleotides Arginine and its metabolites have critical functions in innate and adaptive immunity, inflammation, wound healing, and vascularization (Morris, 2006, 2007; Wu et al., 2009; Wu and Morris, 1998). However, these processes often contribute to an overactive immune response in addition to virus-induced disease. Therefore, arginine bioavailability may be linked to pathophysiological processes. [0061] As described herein, arginase is expected to effectively inhibit influenza virus replication, infectious virus production, cell-to-cell transmission, and virus-induced cytopathic effects. Limiting arginine-associated metabolic pathways is expected to be an effective antiviral treatment and when used in combination with nucleoside analogs its ability to control viral replication will be enhanced. [0062] The ability of recombinant arginase to modulate host arginine-associated metabolic pathways and control viral replication requires highly active enzyme and preferably a long half-life in vivo. [0063] Pegzilarginase has been assessed in vitro and in vivo with clinical trials to define its pharmacokinetic and pharmacodynamic characteristics. [0064] Influenza viruses have proteins that require incorporation of arginine for its infectivity and replication. Without wising to be bound by any particular theory, it is believed that the high activity of recombinant arginase (e.g. pegzilarginase) in vivo will lower the systemic concentration of arginine to a point where the supply of arginine becomes a rate limitation for influenza virus. Additionally, the lowering of arginine will also cause the production of NO to lower. Thus, alleviating an immune response molecule that likely contributes to generalize pulmonary damage in influenza virus patients. This slowing of viral replication and moderation of this deleterious immune-effector molecule will give the adaptive immune response more time to mount a focused suppression of the virus. Previous studies with pegzilarginase have demonstrated that systemic dosing does not adversely affect the immune response and in fact enhances adaptive immune responses. [0065] Accordingly, embodiments of the present invention pertain to administering recombinant arginase (e.g. pegzilarginase) for the treatment of influenza virus infections. In one or more embodiments, the recombinant human Arginase protein is recombinant human Arginase 1 (rhARG1) (SEQ ID NO: 1; shown Figure 1(a)). In other embodiments, the recombinant human Arginase protein is recombinant human Arginase 2 (rhARG2) (SEQ ID NO: 3; shown Figure 1(c)). Although specific reference is made herein to rhARG1, the methods, formulations and uses described herein can also be applied to rhARG2. [0066] Recombinant Human Arginase 1 [0067] Human Arginase 1, identified as hArg1, is a binuclear manganese metalloenzyme that catalyzes the hydrolysis of L-arginine (L-Arg) to yield L-ornithine and urea. Arginase 1 is a trimer of three non-covalently bound identical monomer units. Monomeric Arginase 1 is enzymatically active but less stable. The substitution of the native manganese (Mn²⁺) with cobalt (Co²⁺) in the active site of Arginase 1 enhances catalytic activity at physiological pH. The methods of producing cobalt-substituted Arginase 1 enzyme described herein provide an enzyme that is highly pure and highly active. The methods can also provide Co-Arginase 1 (Co-rhARG1) as an isolated intermediate in the manufacture of the drug substance. In one or more embodiments, the drug substance is PEGylated Co-Arginase 1 (Co-rhARG1-PEG). PEGylation of Co-Arginase 1 extends the circulating half-life significantly. Again, although specific reference is made herein to rhARG1, the methods, formulations and uses described herein can also be applied to rhARG2. [0068] The quantity of Co-rhARG1-PEG drug substance can be expressed as the mass amount of un-PEGylated enzyme. In one embodiment of the method, each mg (enzyme basis) of Co-rhARG1-PEG drug substance also contains approximately 1-2 mg of PEG, such as about 1.4 mg of PEG. [0069] Figure 1(a) shows the amino acid sequence that was expressed in E. coli. The hArg1 protein sequence was obtained from the NCBI database (UniProtKB: locus ARGI1_HUMAN, accession P05089). Overlapping oligonucleotides were used in a PCR reaction to generate Arginase 1 DNA that was codon optimized for expression in E. coli (Figure 1(b)). The 321 amino acid E. coli expressed monomer of Arginase 1 lacks the N- terminal methionine found in native human Arginase 1 monomer. The calculated molecular weight of Co-Arginase 1 is 34721.6 Daltons (Table 1). The calculated molecular weight of homotrimeric Co-Arginase 1 is 104164.8 Daltons. Arginase 1 does not have any disulfide bonds. [0070] Table 1: Structural Information of an Exemplary Co-Arginase 1 Intermediate

[0071] In one or more embodiments, the calculated molecular weight of monomeric Co-rhARG1-PEG is about 75-115 kDa. In one or more embodiments, the calculated molecular weight of homotrimeric Co-rhARG1-PEG is about 224-344 kDa. In one or more embodiments, the average number of PEG is between about 8 to about 25 moles of PEG/mole Co-Arginase 1 monomer, such as about 8 to about 16 moles of PEG/mole Co-Arginase 1 monomer. Exemplary amounts of PEG include about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15 and about 16 moles of PEG/mole Co-Arginase 1 monomer. In one or more embodiments, each PEG has an average molecular weight of about 1,000 to about 10,000 Daltons, such as about 1,000, about 2,000, about 3,000, about 4,000, about 5,000, about 6,000, about 7,000, about 8,000, about 9,000 or about 10,000 Daltons. In a particular embodiment, the average MW of the PEG is about 5,000 Daltons. [0072] In one or more embodiments, the Co-rhARG1-PEG comprises pegzilarginase. Pegzilarginase has the following two chemical names: a. poly(oxy-1,2-ethanediyl), α-(carboxymethyl)-ω-methoxy-, amide with arginase 1 [cobalt cofactor] (synthetic human) (1:10), trimer b. Des-Met¹-arginase-1 (liver-type arginase, EC 3.5.3.1) (Homo sapiens) from which manganese has been replaced with cobalt, an average of 10 primary amines (of N-terminal serines and N⁶-lysines) are amidified with [methoxypoly(ethyleneoxy)]acetyl, non-covalent homotrimer, produced in Escherichia coli [0073] Potential PEGylation sites of pegzilarginase are shown below:

[0074] The molecular formula for pegzilarginase is C₁₅₅₄H₂₄₉₂N₄₁₆O₄₅₃S₆ [C₃H₄O₂ (C₂H₄O)_n]_a monomer. The average molecular weight for pegzilarginase is 284 kDa for the trimer. The CAS registry number for pegzilarginase is 1659310-95-8. [0075] Human Arginase 1 catalyzes the fifth and final step in the urea cycle which is the conversion of L-arginine into L-ornithine and urea. The PEGylated drug substance, Co- rhARG1-PEG, catalyzes the same reaction. The assay to assess enzyme activity measures the conversion of L-arginine to L-ornithine during a fixed reaction time at pH 7.4 and 37°C. The amount of conversion of product is converted to a reaction rate and fit to the Michaelis-Menten equation to determine K_m and k_cat.

[0076] V_max is the maximum reaction rate achieved at saturating substrate concentration; K_m is the Michaelis-Menten binding constant to measure the substrate concentration yielding a velocity at the half of V_max. The enzymatic turnover number, k_cat is calculated by V_max/[E]. [0077] Specific activity is determined by dividing the reaction velocity at 2 mM arginine expressed in µmoles/minute by the enzyme concentration in mg. [0078] The values for Co-rhARG1-PEG drug substance for K_M and k_cat as measured in the enzyme activity assay typically range from 0.15-0.22 mM and approximately 200-300/sec respectively. Upon PEGylation of the Co-Arginase 1 intermediate to form the drug substance, compared to the unPEGylated intermediate, the enzyme activity is not significantly changed. However, PEGylation significantly increases the circulating half-life of the Co-rhARG1-PEG drug product compared to the Co-Arginase 1 intermediate. [0079] In one or more embodiments, the protein (e.g. Co-rhARG1 or Co-rhARG1- PEG) displays a k_cat/K_M greater than 200 mM^-1 s^-1 at pH 7.4. In a particular embodiment, the protein displays a k_cat/K_M between 200 mM^-1 s^-1 and 4,000 mM^-1 s^-1 at pH 7.4. In another embodiment, the protein displays a k_cat/K_M between 400 mM^-1 s^-1 and 2,500 mM^-1 s^-1 at pH 7.4 at 37° C. In a particular embodiment, the present invention contemplates a protein comprising an amino acid sequence of human Arginase 1 and a non-native metal cofactor, wherein said protein exhibits a k_cat/K_M greater than 400 mM^-1 s^-1 at 37° C., pH 7.4. Exemplary k_cat/K_M values include about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 800, about 900, about 1,000, about 1,100, about 1,200, about 1,500, about 2,000, about 2,500, about 3,000, about 3,500 and about 4,000 mM^-1 s^-1 at pH 7.4 at 37° C. [0080] In one or more embodiments, the rhARG1, Co-rhARG1 or Co-rhARG1-PEG can have at least 98%, 98.5%, 99% or 99.5% identity to SEQ ID NO: 1. In one or more embodiments, rhARG1, Co-rhARG1 or Co-rhARG1-PEG can have at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more deletions, substitutions and/or insertions to the amino acid sequence described by SEQ ID NO: 1. Various alignment algorithms and/or programs may be used to calculate the identity between two sequences, including FASTA, or BLAST which are available at the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov/). [0081] Using the methods described herein, it is possible to replace almost all the manganese cofactor in Arginase 1 with cobalt. The change to cobalt cofactor results in a change in the K_m for arginine from 2.8 mM to about 0.18 mM at pH 7.4. In one or more embodiments, the Co-rhARG1-PEG comprises about 0.1 to about 2 µg Co/mg protein. Exemplary cobalt loadings include about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9 and about 2 µg Co/mg protein. In various embodiments, the Co-rhARG1-PEG comprises less than about 1 µg Mn/mg protein, such as less than about 1, about 0.9, about 0.8, about 0.7, about 0.6, about 0.5, about 0.4, about 0.3, about 0.2, about 0.15, about 0.1, about 0.09, about 0.08, about 0.07, about 0.06, about 0.05, about 0.04, about 0.03, about 0.02 or about 0.01 µg Mn/mg protein. In a particular embodiment, Co-rhARG1-PEG drug substance contains about 2 µg Co/mg protein and about 0.05 µg Mn/mg protein. [0082] Production and Purification of rhARG1, Co-rhARG1 and PEGrhARG1 [0083] Shake Flask Expansion [0084] The purpose of the shake flask expansion/fermentation is to generate an inoculum to seed the production fermenter. Shake flask expansion creates cell mass for inoculation of the production reactor as well as extra for analytical purposes. A representative overview of the Arginase 1 fermentation processes can be seen in Figure 2. [0085] An aliquot of the inoculum medium is introduced into one 500 mL flask (a Primary flask) and six 3 L disposable flasks (Secondary flasks). The flasks are autoclaved, and post-sterile additions are transferred to each flask. Prior to inoculation, the primary medium is pre-warmed to the processing temperature of 37°C. Before secondary inoculation, the secondary flasks are pre-warmed to the processing temperature of 37°C. [0086] One vial of an Arginase 1-expressing E. coli working cell bank (WCB) is removed from cold storage and thawed. A target volume of thawed cells (approximately 1.1 mL) is added aseptically to the primary flask, and the flask is incubated at 37°C with agitation. Samples are removed from the flask hourly starting several hours post-inoculation to follow cell growth by optical density at 600 nm (OD600). Once the target OD600 of ≥ 1.0 is reached in the primary flask, a target volume (15 mL) of primary culture is aseptically transferred into each secondary flask. The secondary flasks are incubated at 37°C with agitation. Samples are removed from one secondary flask hourly starting at 4 h post-inoculation, increasing to every 30 min once the OD600 has reached ≥ 1.5. When the measurement has met the specified density of ≥ 2.0 OD600, the remaining secondary flasks are sampled. If the average OD600 of all secondary flasks meets a specified transfer criterion, the flasks are pooled, and the inoculum is transferred to the production fermenter. [0087] Production Fermentation [0088] The purpose of the Production Fermentation is to expand the shake flask culture and induce production of Arginase 1. Production fermentation can create large scale quantities of Arginase 1. Following a shake flask expansion phase to build cell mass, the fermentation process produces Arginase 1 (in E. coli) as a soluble protein. In one embodiment a 1500 L fermenter contains the initial batch medium including sterile additions before inoculation. After inoculation, inputs to the fermenter include nutrient feed, antifoam solution, addition of acid or base to maintain culture pH. A secondary vessel holds the nutrient feed medium. An automated control strategy maintains important parameters for consistent cell growth including dissolved oxygen, sparge rate, agitation rate, pH, pressure, and temperature. Arginase 1 expression is induced by addition of IPTG (isopropyl betta-D-1-thiogalactophranoside), with harvest occurring approximately 18 hours later. The performance of the fermenter is assessed at the end of production by monitoring cell density, percent solids, and the proportion of soluble Arginase 1. [0089] In a preferred embodiment, the fermentation medium is prepared directly in the production fermenter. Purified water is added to the fermentation medium to the required weight before in-place sterilization (SIP). Post-sterilization additions of kanamycin, glucose, and potassium phosphate are filter-sterilized into the production fermenter once the medium has cooled. If necessary, the sterile medium is brought to a designated pre-inoculation weight with purified water using a 0.2 µm sterile filter. The fermentation medium is titrated with base (ammonium hydroxide) to a controlled pH value. [0090] The production fermenter at 37ºC is inoculated aseptically using a pooled inoculum via a pressure-assisted transfer. Fermentation broth samples are collected at a regular frequency and measured for OD600 analysis from the time of inoculation until fermentation cool down. Glucose samples are taken at a regular interval beginning at 3 h post-inoculation and increasing in frequency after 9 h post-inoculation. Antifoam solution is added as needed during the fermentation process to avoid excessive foaming of the culture. Dissolved oxygen is controlled by an agitation cascade with oxygen sparge on demand. Culture pH is maintained using acid and base inputs. Growth medium is preferably maintained at 36-38 °C and at a pH of 7.0 – 7.4 with agitation and aeration. [0091] The nutrient feed consists of yeast extract, Martone B-1, L-cysteine HCl, and glycerol. The feed starts when the glucose concentration is less than 10 g/L (12 –14 h post- inoculation) and continues at a fixed rate until the end of production. Expression is triggered with addition of IPTG. Induction continues for 18 hours. Completion of the fermentation process is followed by a cool down in preparation for harvest operations. The production fermenter can generate titers of soluble Arginase 1 of approximately 6 g/L. An overview of the Production Fermentation can be seen in Figure 3. [0092] Harvest Operations [0093] Harvest operations capture cells containing soluble Arginase 1, break open the cells/lyse the cells, and clear the lysate of cell debris by using centrifugation and/or filtration. The recovered cell slurry can be frozen or kept at a low temperature for long-term storage. Harvest operations may collect the cells by centrifugation, lysed with two passes through a homogenizer or cell disruption under pressure (French press), centrifuged a second time, and membrane filtered prior to the first chromatography step. [0094] In a preferred embodiment, whole cells are separated from fermentation medium using a disc stack centrifuge. The resulting cell slurry resuspended in 25 mM HEPES, pH 7.6, followed by two passes through a homogenizer. The pH of the 25 mM HEPES can also be used in the range of pH 7.2-7.6. The lysed material is clarified using a centrifuge to remove cell debris, then membrane filtered through 0.2μm grade filters. In a preferred embodiment, harvest steps are performed at a target temperature of ≤ 15°C. [0095] In an alternative embodiment, cell disruption is performed using high pressure. Cell slurry is transferred to a homogenizer at a controlled rate and the homogenized outflow is passed through a heat exchanger to reduce the temperature increase seen during pressure homogenization. The chilled cells undergo two homogenization passes. The first pass lysis pool is transferred from the collection vessel back to the feed vessel. The hold duration between passes is minimized to reduce potential microbial growth. [0096] The post-lysis material is clarified by centrifugation to remove cellular debris from the soluble components of the lysate. The lysate is transferred at a controlled rate to a disk-stack, intermittent discharge, centrifuge. The clarified lysate is collected for further processing. [0097] The clarified lysate is filtered, such as with an about 0.2 µm filter. Process transition filters can also be used for microbial control during process operations. For this purpose, filters can be either 0.5 µm or 0.2 µm filters. This step also removes small particulates from clarified material that may not have separated during clarification operations. Prior to use, the filters are flushed extensively with purified water and equilibrated with 25 mM HEPES, pH 7.6 buffer. Each downstream process step can be preceded by a pre-filter to mitigate the potential for bioburden load. [0098] Purification of rhARG1, Co-rhARG1 and Co-rhARG1-PEG [0099] Regardless of the methods used to culture cells that express the rhARG1 (e.g. the fermentation processes described above), the purification methods described herein can be used to capture rhARG1 and further purify the enzyme. The purification methods can include optional steps such as loading with cobalt to produce Co-rhARG1 and/or reacting with a PEGylation reactant to provide Co-rhARG1-PEG. [00100] Various embodiments of the purification process relate to the use of a cation exchange (CEX) column to capture rhARG1. In one or more embodiments, the CEX column is the first column (“Column 1”) in system with multiple chromatography columns. The protein product eluted from this Column 1 is “First Protein Product”. [00101] In one or more embodiments, Column 1 uses cation exchange chromatography to bind rhARG1 at a pH in the range of about 7 to about 8, such as a pH of about 7.6. In one or more embodiments, the rhARG1 is bound in the absence of salt or at low salt concentrations. In one or more embodiments, the rhARG1 is eluted with a buffer having a high salt (e.g. NaCl) concentration, such as up to about 0.5 M NaCl. Exemplary salt concentrations include about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, 0.1, about 0.2, about 0.3, about 0.4 and about 0.5 M NaCl. [00102] In various embodiments, a salt gradient is used to separate different charge variants of rhARG1. Exemplary salt gradients are from about 0 to about 0.5 M NaCl, about 0 to about 0.4 M NaCl, about 0 to about 0.3 M NaCl, about 0 to about 0.2 M NaCl or about 0 to about 0.1 M NaCl. [00103] In one or more embodiments, the method further comprises loading the First Protein Product (optionally after cobalt substitution) onto an anion exchange (AEX) chromatography column (“Column 2”) and collecting the flow-through to provide a second protein product (“Second Protein Product”). In another aspect of the methods, the method further comprises loading the Second Protein Product onto third column which captures the Arginase 1 and is then eluted to provide a third protein product (“Third Protein Product”). In some embodiments, this third chromatography column (“Column 3”) may be a size exclusion chromatography (SEC) column or a multimodal chromatography (MMC) column. [00104] Various embodiments provide that the rhARG1 is loaded with Co to replace the Mn cofactor. In one or more embodiments, the Co loading is performed using a Co²⁺ salt such as CoCl₂. Incubation times are temperature dependent, such that lower cobalt substitution temperatures require longer incubation times and higher cobalt substitution temperatures do not require as long incubation times. The cobalt loading temperature may be as low as 1°C or greater than 50 °C, and corresponding incubation times can be as long as over 8 hours or less than 10 minutes. [00105] Various embodiments provide that the rhARG1 or Co-rhARG1 is reacted with a PEGylation reactant such as methoxy PEG succinimidyl carboxymethyl ester (MW 5000). The PEGylation reactant is typically provided in molar excess of 10-40 compared to the enzyme. Incubation times can be in the range of 0.5 to 4 hours. The pH during PEGylation can be about 8 to about 9, such as a pH of about 8.4. [00106] Administration of rhARG1, Co-rhARG1 and Co-rhARG1-PEG [00107] The rhARG1, Co-rhARG1 and Co-rhARG1-PEG as described herein (and compositions comprising them) can be administered via any appropriate route, including intravenously, intrathecally, subcutaneously, intramuscularly, intratumorally, and/or intraperitoneally. In one or more embodiments, the rhARG1, Co-rhARG1 and Co-rhARG1- PEG (or compositions comprising them) are administered intravenously (IV) or subcutaneously (SC). [00108] Compositions containing rhARG1, Co-rhARG1 and Co-rhARG1-PEG thereof can be provided in formulations together with physiologically tolerable liquid, gel or solid carriers, diluents, and excipients. Such compositions are typically prepared as liquid solutions or suspensions, as injectables. Suitable diluents and excipients are, for example, water, saline, dextrose, glycerol, or the like, and combinations thereof. In addition, if desired the compositions may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, stabilizing or pH buffering agents. [00109] Exemplary methods and instructions regarding the administration of rhARG1, Co-rhARG1 and Co-rhARG1-PEG (e.g. pegzilarginase) are provided below. Although the following description is specific to pegzilarginase, the methods and instructions are also applicable to other recombinant Arginase 1 and 2 enzymes. [00110] Blood Arginine Monitoring: [00111] After initiating treatment with pegzilarginase, plasma arginine monitoring may be performed to ensure low plasma arginine levels. [00112] Preparation and Administration Instructions [00113] Pegzilarginase is supplied as a frozen liquid formulation in 10 mL single-use glass vials that contain 5 mL of pegzilarginase at a concentration of either 1 mg/mL or 5 mg/mL. Each single-use glass vial of pegzilarginase is intended for use as a single intravenous injection or as a subcutaneous injection. Inspect pegzilarginase visually for particulate matter and discoloration prior to administration. Pegzilarginase is a colorless to slightly yellow or slightly pink solution. Discard if discolored, cloudy or if particulate matter is present in the vial. Remove the flip-top from the vial. Wipe the rubber stopper of the vial with alcohol swabs to disinfect. Use a sterile syringe with an 18G needle to remove the appropriate volume of drug from the vial. If more than one vial is required, please use a separate needle to draw the solution from each vial. Calculate the solution to be withdrawn from the vial for use in the syringe pump. Once the appropriate volume of drug has been drawn into the syringe, draw normal saline using a separate needle to achieve a total volume of 40 mL. Calculate the required amount of drug to be used as follows:

[00114] Administer pegzilarginase via intravenous infusion over 30 minutes using a syringe pump. [00115] Table 2: Weight-Based Dosing for Administration of 0.1 mg/kg Once Weekly

[00116] In one or more embodiments, the volume for a subcutaneous injection has a maximum volume, such as a maximum of 2 mL/injection for adult patients and/or a maximum volume of 1 mL/injection for pediatric patients. If the calculated volume for subcutaneous administration is greater than a maximum volume, then a higher vial concentration may be used (e.g.5 mg/mL instead of 1 mg/mL) and/or the volume may be split into multiple smaller injections (e.g. a 4 mL injection is split into 2 injections of 2 mL each). [00117] Dosage Forms and Strengths [00118] pegzilarginase injection is a colorless to slightly yellow or slightly pink solution available as follows in 10mL vials: a. Solution for Injection: 5 mL of 1.0 mg/mL b. Solution for Injection: 5 mL of 5.0 mg/mL [00119] Warnings and Precautions [00120] Hypersensitivity reactions may occur with administration of pegzilarginase. Monitor all patients for signs and symptoms of acute allergic reactions (e.g. urticaria, pruritus, erythema, hypotension, tachycardia) during and following pegzilarginase infusion. In case of severe hypersensitivity reactions, slow or discontinue the administration of pegzilarginase immediately and administer appropriate medical care. Consider premedication of patients with a non-sedating antihistamine prior to dosing. In the event that corticosteroids are required, they should be used with caution due to their potential to cause hyperammonemia. [00121] Pregnancy: Pregnancy Category B [00122] Reproduction studies have been performed in mice and rats at doses up to 100 mg/kg. There was no evidence of harm to the fetus due to pegzilarginase. There are, however, no adequate and well-controlled studies in pregnant women. Because animal reproduction studies are not always predictive of human response, pegzilarginase should be used during pregnancy only if clearly needed. [00123] Nursing Mothers [00124] It is not known if pegzilarginase is present in human milk. The developmental and health benefits of breastfeeding should be considered along with the mother’s clinical need for pegzilarginase and any potential adverse effects on the breastfed child from the drug. [00125] Description [00126] Pegzilarginase is a cobalt substituted, recombinant human arginase I enzyme that is covalently conjugated to monomethoxy polyethylene glycol (mPEG) that acts by catalyzing the same reaction as arginase 1, converting arginine into ornithine and urea. Human arginase 1 is a binuclear manganese metalloenzyme. To produce pegzilarginase, the manganese cofactor is replaced with cobalt to yield Co-Arginase I. The substitution of the native manganese (Mn+2) with cobalt (Co+2) in the active site of arginase I enhances the stability and catalytic activity at physiological pH. Pegylation extends the circulating half-life. The average molecular weight of pegzilarginase is approximately 284 kDa. Pegzilarginase has a specific activity ranging from approximately 320-600 units per mg of protein content. One activity unit is defined as the amount of enzyme required to convert 1 micromole of arginine to ornithine per minute at 37°C. [00127] Pegzilarginase is intended for intravenous or subcutaneous infusion and is supplied as a sterile, clear, colorless to slightly yellow or slightly pink solution formulated at a 1 mg/mL and at a 5 mg/mL concentration in a buffer containing 50 mM sodium chloride, 5 mM potassium phosphate, and 1.5% w/v glycerol, at a pH of 7.4. It is provided as a preservative-free, sterile solution in a clear, single-use, glass vial. Each vial of 1 mg/mL pegzilarginase drug product contains 5 mL of drug product (5 mg pegzilarginase per vial). Each vial of 5 mg/mL pegzilarginase drug product contains 5 mL of drug product (25 mg pegzilarginase per vial). Vials are stoppered with a coated rubber stopper and sealed with an aluminum flip off seal and are stored frozen at ≤ -60°C and thawed before use. [00128] Pharmacodynamics [00129] pegzilarginase treatment of adults and pediatric patients with Arginase 1 Deficiency resulted in the reduction of blood arginine concentrations from pre-treatment baseline values into the normal blood arginine range of 40 to 115 micromole/L. Maximum suppression of L-arginine was observed at approximately 8 hours post-dose, decreasing in a dose dependent manner with recovery to pre-dose levels occurring by 168 hours post dose. A strong correlation was observed between pegzilarginase and arginine, with an immediate suppressive effect on arginine following IV administration, and the maximum decrease of arginine concentration being reached within 24 hours post-dose. [00130] Pharmacokinetics [00131] Following IV administration to 14 subjects, pharmacokinetic samples were collected throughout the dosing interval from 0-168 hours to characterize the relationship between pegzilarginase pharmacokinetics and arginine. Across the dose range (0.015 mg/kg — 0.2 mg/kg), pegzilarginase exposure, as measured by C_max and AUC_0-168, increased approximately proportional to dose, with a 13-fold increase in dose resulting in a 14-fold increase in C_max and AUC_0-168. No accumulation of pegzilarginase was observed following a once weekly IV dosing regimen, with a T_1/2 of approximately 30 hours across the dose range, and low to moderate inter-subject variability (13 – 46 % CV) in the exposure metrics. [00132] Animal Toxicology and/or Pharmacology [00133] The pharmacologic effects of pegzilarginase on arginine levels were assessed in a neonatal transgenic mouse model of Arginase I and a tamoxifen-induced arginase deficiency model in adult mice. These models mimic the human disease in that a significant excess of circulating arginine and catabolites of arginine are present; however, unlike humans with Arginase I Deficiency, these animals develop severe and generally lethal hyperammonemia. Pharmacologic effects also were assessed in a rat arginine-induced model of hyperargininemia. Pegzilarginase reduced plasma arginine levels in a dose-dependent manner. [00134] The potential toxicity and TK of pegzilarginase were evaluated in postnatal day (PND) 21 (equivalent to a 2-year old human) juvenile rats administered once weekly IV bolus injections at 0.1, 0.3, and 1.0 mg/kg for 6 months followed by a 6-week recovery period. Pegzilarginase was well tolerated, with no test article-related mortality and no significant test article effects observed on: food consumption, coagulation, urinalysis, ophthalmoscopic examinations, sexual maturation, growth hormone analyses, bone marrow analyses, functional observation battery (FOB) evaluations and neurobehavioral testing (auditory startle habituation, motor activity, or Morris water swim maze). There were no pegzilarginase-related macroscopic findings at the end of the 6-month terminal and 6-week recovery intervals. Adverse microscopic changes were limited to the testes and epididymides and correlated with reduced weight of male reproductive organs and adverse sperm analyses findings at 0.3 and 1.0 mg/kg. At 1.0 mg/kg, an adverse effect was observed on sperm analyses with reduced sperm motility, lower caudal epididymal sperm counts, decreased sperm concentration, and increased percentage of abnormal sperm observed. These observations were considered a direct treatment-related effect and correlated with microscopic changes of subtle tubular degeneration in the testes at 0.3 mg/kg and 1.0 mg/kg. Following the 6-week recovery period for the control and 1.0 mg/kg groups, these changes were overall reversible with the exception of the increased percentage of abnormal sperm and sperm counts. The partial reversibility after 6 weeks was not unexpected because the normal sperm development cycle is approximately 9 weeks or longer than the 6-week recovery period. [00135] Importantly, there were no apparent PEGylation effects observed by histopathology. Toxicokinetic data indicated that pegzilarginase exposure was maintained throughout the study. In conclusion, the NOAEL in females was 1.0 mg/kg. In males, the NOAEL was 0.1 mg/kg based on microscopic changes in the testes at 0.3 mg/kg and 1.0 mg/kg. [00136] The potential toxicity and TK of pegzilarginase were evaluated following once weekly intravenous bolus injection to cynomolgus monkeys at doses of 0.1, 0.3, and 1.0 mg/kg for 13 weeks followed by a 4-week recovery period. Clinical signs observed at 1.0 mg/kg included decreased body weight, increased incidences of sparse hair (entire body), dry/discolored skin (entire body), tremors, inappetence, watery feces, decreased activity, ataxia, muscle wasting, and/or unkempt/hunched appearance. No treatment-related effects were noted in clinical pathology parameters (coagulation, growth hormone, and urinalysis), ECG and ophthalmic examinations, respiratory rate, and blood pressure assessments. [00137] How Supplied/Storage and Handling [00138] Pegzilarginase is supplied as a solution for injection. [00139] Pegzilarginase is supplied frozen (≤-60°C). Diluted pegzilarginase should be used immediately. If immediate use is not possible, diluted pegzilarginase may be stored for up to 8 hours at 2°C to 8°C (36°F to 46°F) during administration. [00140] EXAMPLES [00141] Before describing several exemplary embodiments of the disclosure, it is to be understood that the disclosure is not limited to the details of construction or process steps set forth in the following description. The disclosure is capable of other embodiments and of being practiced or being carried out in various ways. [00142] In the experimental disclosure which follows, the following abbreviations apply: eq (equivalents); M (Molar); μM (micromolar); mM (millimolar); N (Normal); mol (moles); mmol (millimoles); μmol (micromoles); nmol (nanomoles); g (grams); mg (milligrams); μg (micrograms); L (liters); ml (milliliters); μl (microliters); cm (centimeters); mm (millimeters); μm (micrometers); nm (nanometers); MW (molecular weight); PBS (phosphate buffered saline); min (minutes). [00143] Example 1: Cation Exchange Column Chromatography (Column 1) [00144] In a preferred embodiment, Arginase 1 is captured on a cation exchange column (CEX) to reduce product-related impurities and process-related impurities such as host cell proteins (HCP), DNA, and endotoxin (see Figure 3 for an overview of the purification process). In a particular embodiment, the first column (Column 1) chromatography step in the Arginase 1 purification process uses SP Sepharose FF resin and an inlet heat exchanger. Column 1 uses cation-exchange chromatography to bind Arginase 1 in the absence of salt at pH 7.6, and elute with a buffer of increased salt concentration (Figure 4(a)). In one embodiment the salt is NaCl and the elution from Column 1 is performed with 25 mM HEPES, 0.1M NaCl, pH 7.2-7.6 at room temperature. However, alternative embodiments are possible such as application of a NaCl gradient to Column 1. [00145] Figure 4(a) shows a representative purification of Arginase 1 on Column 1. Approximately three liters of clarified E. coli lysate were loaded on the cation exchange column. As can be seen from the high level of absorbance at 280 nm, a large amount of protein did not bind onto the column and is detected in the flow through. The column was then washed with approximately two liters of column wash solution. As detected by absorbance at 280 nm, a fraction enriched for Arginase 1 was then eluted with 0.1 M NaCl (final peak). [00146] Example 2: Cobalt Substitution [00147] In a preferred embodiment, the Arginase 1 native manganese co-enzyme is replaced by cobalt. During cobalt substitution (also called cobalt loading), one or both of the two manganese ions normally present in Arginase 1 are replaced with cobalt ions. A wide variety of temperatures can be used for the cobalt substitution step as well as a wide concentration of cobalt (See Table 2). Incubation times for cobalt substitution can be a short as 10 minutes and be performed at over 50 °C. Conversely, cobalt loading temperature can be as low as 1°C or 5°C and performed for over 8 hours. Also, the higher proportion of cobalt loaded into Arginase 1 leads to a higher specific activity. [00148] The Arginase 1 eluted from Column 1 (also called Column 1 Pool) can be held at room temperature for the cobalt substitution step. In one embodiment, Cobalt Chloride Stock Solution (0.5 M CoCl₂) is diluted 50-fold by adding it to Column 1 Pool at a defined rate, with final cobalt chloride concentration at 10 mM. The cobalt substitution is then mixed for two hours at 20°C. In another embodiment, Arginase 1 cobalt loading is performed in a solution of 10mM CoCl₂ for between 2 - 8 hours at room temperature. [00149] An overview of the cobalt loading step can be seen in Table 3. [00150] Table 3

[00151] Example 3: Ultrafiltration/Diafiltration 1 (UF/DF 1) [00152] UF/DF 1 removes free cobalt ions and exchanges the Co-Arginase 1 into a solution in preparation for anion exchange chromatography on. The UF/DF1 step uses membranes with a molecular weight cutoff of 30 kDa. One important function of this step is to reduce the levels of free cobalt and buffer exchange the Co-Arginase 1 Pool prior to anion exchange chromatography. Membranes are sanitized with cleaning solution (0.5 N NaOH) and rinsed with water. A normalized water permeability test (NWP) is performed followed by equilibration prior to use in production. Once the UF/DF system is equilibrated, the Co- Arginase 1 Pool is diafiltered against 25 mM HEPES, 0.1 M NaCl, pH 7.6, for three diavolumes, followed by four diavolumes of 50 mM Tris, pH 8.4. After diafiltration, the pool is recirculated and recovered from the system using two times the system’s hold-up volume with 50 mM Tris, pH 8.4. [00153] The UF/DF1 membranes are cleaned by performing a 2 M NaCl flush followed by a denaturing cleaning step using 0.5 N NaOH with a 30 minute recirculation. The system is flushed with purified water and the NWP tested to assess the effectiveness of the cleaning procedures. Membranes may be stored in 0.1 N NaOH. [00154] In an alternative embodiment, the first exchange of buffer is into 25mM HEPES, 0.1M NaCl, pH 7.2-7.6 and the second exchange is into 50mM Tris, pH 8.1-8.5. [00155] Example 4: Anion Column Chromatography (Column 2) [00156] A preferred embodiment of the Arginase 1 purification uses anther column which is an anion exchange column chromatography (“Column 2”). One embodiment of Column 2 is a Q Sepharose FF resin. One function of this Column 2 step is to reduce process- related impurities such as host-cell DNA and endotoxin from the UF/DF1 pool. Column 2 binds these impurities while Co-Arginase 1 flows though and is collected in the column effluent during the load and wash steps. In one embodiment, the anion exchange flow-thru chromatography for Column 2 is performed with Q Sepharose FF, and up to 40 g protein/L resin is loaded onto the column with buffer 50mM Tris, pH 8.1-8.5. [00157] In another embodiment of the methods, the First Protein Product is loaded onto an anion exchange column to capture impurities while Arginase 1 is retrieved in the flow through. Figure 4(b) is a representative chromatogram of Arginase 1 purification over an anion exchange column (Column 2). As can be seem from absorbance at 280 nm a large amount of protein is detected in the flow through. Impurities are captured on Column 2 and not eluted into the Column 2 Pool (also called Second Protein Product) which is further enriched for Arginase 1. [00158] Example 5: Capto Multimodal Column Chromatography (Column 3) [00159] In a preferred embodiment, the Arginase purification process uses a third column chromatography column (Column 3). In one embodiment, Column 3 is a Capto multimodal chromatography (MMC) column or alternatively a size exclusion column. Embodiments that use MMC capture Arginase 1 on the column while process-related impurities such as host cell proteins (HCP), DNA, and endotoxin are washed out in the flow through. In this embodiment, Co-Arginase 1 can be captured by the column in the absence of salt at pH 8.4 and then Co-Arginase 1 eluted with a buffer of increased salt concentration. A representative example of Capto Multimodal Cation exchange chromatography column is shown in Figure 4(c). [00160] In one embodiment, MMC chromatography (Column 3) uses approximately 15 column volumes to load up to 30 g protein/L resin, and the high salt step elution is performed with 50mM Tris, 250mM NaCl, pH 8.1-8.5. In several embodiments, the flow through from the anion exchange column (Column 2) is loaded onto a Capto MMC column at pH 8.4, washed, then the bound Co-Arginase 1 is eluted using 50 mM tromethamine, 250 mM sodium chloride. [00161] Example 6: Ultrafiltration/Diafiltration 2 (UF/DF 2) [00162] UF/DF 2 concentrates Arginase 1 and exchanges the protein into a pre- PEGylated intermediate. The UF/DF2 step uses membranes with a molecular weight cutoff of 30 kDa. An important function of this step is to buffer exchange the Column 3 Pool of the un- PEGylated Co-Arginase 1 intermediate prior PEGylation (or prior to additional filtration and storage). Membranes are sanitized with cleaning solution (0.5 N NaOH) and rinsed with water. Once the UF/DF system is equilibrated, Column 3 Pool (also called Third Protein Product) is diafiltered against 20 mM sodium phosphate, 50 mM sodium chloride, 1.5% (w/v) glycerol, pH 7.4, for five diavolumes. If the Column 3 pool concentration is < 8 g/l, the pool is further concentrated to 8 g/L. After diafiltration (and concentration, if necessary), the pool is recirculated and recovered from the system using two times the system’s holdup volume with 20 mM sodium phosphate, 50 mM sodium chloride, 1.5% (w/v) glycerol, pH 7.4. After recovery, a two-step dilution with diafiltration solution may be employed. The first dilution has a concentration target of 6 g/L and the second step has a concentration target of 5 g/L. Two steps may be used to reach the target. The second step may not be necessary if the concentration following the first dilution is within the targeted range. [00163] Example 7: Intermediate Filtration and UF/DF 3 [00164] Prior to the PEGylation reaction, the cobalt-containing Arginase 1 can be stored long-term, including frozen long-term. The intermediate Co-Arginase can be filtered through a 0.2 μm filter and can be frozen for long term storage. [00165] The UF/DF3 step uses membranes with a molecular weight cutoff of 30 kDa. One function of this step is to buffer exchange and concentrate the filtered UF/DF2 pool (fresh or thawed) to provide conditions optimal for PEGylation. If frozen Co-Arginase 1 intermediate is used as the starting material, thawing will be done at room temperature for up to 36 hours. The membranes are sanitized with cleaning solution (0.5 N NaOH) and rinsed with water. A normalized water permeability test (NWP) is performed followed by equilibration prior to use in production. Once the UF/DF system is equilibrated, the Co-Arginase 1 Intermediate is diafiltered against 0.1 M sodium phosphate, pH 8.4, for five diavolumes. After diafiltration, the pool is concentrated, recirculated, and recovered from the system using two times the system’s hold-up volume with 0.1 M sodium phosphate, pH 8.4. After recovery, a two-step dilution with diafiltration solution is employed. The first dilution has a concentration target of 11 g/L and the second step has a concentration target of 10 g/L. Two steps are utilized to facilitate the target is levels. The second step may not be necessary if the concentration following the first dilution is within the targeted range. [00166] Regarding the UF/DF 2 and UF/DF 3 steps, the first buffer exchange can be into 20mM Sodium Phosphate, 50mM NaCl, 1.5% Glycerol, pH 7.4, ≥5DV, and protein concentrated to approximately 5.0 mg/mL. The second buffer exchange can be made into 0.1M sodium phosphate, pH 8.1-8.5, and protein concentrated to approximately 10.0 mg/mL (in preparation for PEGylation of the drug substance). [00167] Example 8: PEGylation of Arginase 1 [00168] PEGylation covalently attaches PEG (polyethylene glycol) to the Co-Arginase 1 (drug substance) molecule (see Table 4 for a representative embodiment of the PEGylation step). In one embodiment, the PEGylation reaction covalently binds 5000 Da PEG molecules to Co-Arginase 1. In alternative embodiments, PEGylation can be performed prior to cobalt substitution of Arginase 1 or at other points in the production process. In one embodiment, the PEG conjugation reaction can use solid or liquid methoxy PEG succinimidyl carboxymethyl ester which reacts with sterically available lysines on Co-Arginase 1. The resulting PEGylated protein (Co-rhARG1-PEG) has a molecular weight of approximately 280 kDa. The PEGylated pool can be filtered and stored at 2-8°C until UF/DF4 operations. [00169] Table 4: PEGylation Process for Co-rhARG1 Drug Substance

[00170] In one embodiment, solid methoxy PEG succinimidyl carboxymethyl ester (MW 5000) can be added to the Arginase 1 containing solution at a 19.3x molar excess and incubation 0.5- 4.0 hours, pH 8.4. [00171] Following PEGylation, ultrafiltration/diafiltration removes unbound PEG, exchanges the Arginase 1 into a formulation buffer and concentrates the Arginase 1 for the formulation step. This UF/DF4 step uses membranes with a molecular weight cutoff of 100 kDa. One function of this step is to buffer exchange the PEG pool into the final formulation while removing free PEG. Membranes used for this purpose are sanitized with cleaning solution (0.5 N NaOH) and rinsed with water. Once the UF/DF system is equilibrated, the PEG Pool is diafiltered against 5 mM potassium phosphate, 50 mM sodium chloride, 1.5% (w/v) glycerol, pH 7.4 for ten diavolumes. After diafiltration, the pool is recovered from the system with pressure. The recovered UF/DF4 Pool is diluted to 5 g/L with 5 mM potassium phosphate, 50 mM sodium chloride, 1.5% (w/v) glycerol, pH 7.4, prior to the final filtration and fill steps. IN an alternative embodiment, Arginase 1 is exchanged into 20mM Sodium Phosphate, 50mM NaCl, 1.5% Glycerol, pH 7.4, and adjusted to a protein concentration of about 5.0 mg/mL. [00172] In some embodiments, the formulation buffer 5 mM potassium phosphate, 50 mM sodium chloride, 1.5% glycerol, pH 7.4 was found to enhance the stability upon storage of Arginase 1 compared other buffers such as sodium phosphate buffer. In one or more embodiments, the buffer 5 mM potassium phosphate comprises 1 mM K₂HPO₄ and 4 mM KH₂PO₄. [00173] Drug substance (Co-rhARG1-PEG) is a PEGylated cobalt-substituted human Arginase 1 made by conjugating activated PEG molecules with the e-amino group of lysines and the amine group of N-terminal amino acid. A dye-based fluorescent assay is used to determine the molar ratio of PEG molecules per protein using ortho phthaldialdehyde. Ortho- phthaldialdehyde reacts in the presence of thiols, specifically with primary amines, to form fluorescent derivatives. Measurement of the fluorescent signal allows for the quantitation of reactive free amines present in a protein molecule. Quantitation is based on a standard curve using N-acetyl lysine. The number of PEGylated amines per protein can be determined by subtracting the number of free amines as measured by the fluorescent assay of the PEGylated drug substance from the theoretical number of free amines present in the unconjugated Co- Arginase 1. The theoretical number of free amines from lysine residues plus the N-terminal amino acid is 25. Free unconjugated PEG in the drug substance is measured by SEC-HPLC with detection by refractive index. Results can be expressed as µg/mL of free PEG (see Table 5). [00174] Table 5: SEC-HPLC Method Parameters Free PEG Co-rhARG1-PEG Drug Substance

[00175] Example 9: CIEX-HPLC Characterization of Drug Intermediate [00176] During E. coli fermentation, various Arginase 1 charge variants may be produced. Charge variants can be analyzed by a cation exchange HPLC (CIEX-HPLC) method using a TSK gel cation exchange column. This type of analysis uses a mobile phase (A) of 20 mM MES, pH 6.0 and a mobile phase B of 20 mM MES, 500 mM NaCl pH 6.0; flow rate of 1.0 mL/minutes; run time of 40.0 minutes; column temperature of 22°C; and mobile phase gradient according to Table 6. [00177] Table 6: CIEX-HPLC Charge Variants Co-Arginase 1 Intermediate Gradient Program

[00178] Samples are diluted with formulation buffer prior to analysis. The results are described as percent charge variant distribution. A representative chromatogram is shown in Figure 5(a) where six predominant peaks are typically observed for Co Arginase 1 intermediate. [00179] Example 10: iCIEF Characterization of Drug Substance [00180] The drug intermediate is PEGylated to form the drug substance. PEGylation of the drug intermediate renders the use of the drug intermediate CIEX-HPLC method less suitable than other embodiments developed as part of this invention. An anion IEX-HPLC was evaluated but did not give an adequate separation. Alternatively, an imaging capillary isoelectric focusing (iCIEF) method was developed to analyze charge variants of the drug substance. [00181] Analytes in imaging capillary isoelectric focusing (iCIEF) migrate through a capillary by the counter-migration of hydronium ions (anolyte), and hydroxyl ions (catholyte) in the presence of an applied electric field. The sample is diluted in a matrix containing carrier ampholytes and pI markers. Separation of proteins occurs in two focusing steps. An initial prefocusing step establishes the pH gradient. Charge variants are more sharply focused and separated during a second higher voltage focusing step. An image of UV light absorption of the entire capillary is digitally captured every 30 seconds and after completion of the focusing steps. [00182] The results can be expressed as percent charge variant distribution. A representative electopherogram is shown in Figure 5(b) where nine predominant peaks are observed for drug substance. Peaks 3 and 4 are integrated together because the resolution between those peaks has been shown to be variable. The relative areas of these peaks are provided in Table 7: [00183] Table 7: iCIEF Characterization of Charge Variants of Co-rhARG1-PEG

[00184] Example 11: Enzyme Activity of Co-Arginase 1 Intermediate and Drug Substance [00185] The enzymatic assay used to measure activity and to establish identity of Co- Arginase 1 intermediate and Co-rhARG1-PEG drug substance monitors the conversion of arginine to ornithine. The reaction mixtures have one enzyme concentration tested at seven different arginine substrate concentrations over a range of 0 - 2 mM. The reactions are conducted for a fixed time at 37°C. The reaction time has been established to ensure that there is less than 10% consumption of substrate at any given substrate concentration. The reaction is quenched and the product, ornithine, is derivatized and quantified by reverse phase-UPLC. [00186] Examples of plots of reaction velocity vs substrate concentration are shown in Figure 8(a) (Co-Arginase 1 Intermediate) and Figure 8(b) (Co-rhARG1-PEG drug substance) along with representative K_cat, K_m, and K_cat/ K_m values. [00187] Example 12: Analysis of Cobalt and Manganese [00188] Cobalt, residual manganese, and free cobalt were measured using inductively coupled plasma mass spectrometry (ICP-MS). Samples were digested by microwaving and using 1% nitric acid and 6% hydrogen peroxide to release all the metals from the matrix. The resulting digestion was analyzed by ICP-MS. Cobalt and residual manganese samples were digested without any sample treatment. Free cobalt was measured on permeate samples that have been ultrafiltered to separate the enzyme from the permeate to measure cobalt that is not associated with the enzyme. Table 8 summarizes some of the characteristics of Co-Arginase 1 Intermediate. [00189] Table 8: Typical Co-Arginase 1 Intermediate Characteristics

[00190] Table 9: Typical Co-rhARG1-PEG Drug Substance Characteristics

[00191] Example 13: Co-Arginase 1 Intermediate Post-Translational Modifications [00192] Co-Arginase 1 intermediate post-translational modifications were detected using a variety of techniques such as peptide mapping, LC-MS intact mass spectrometry, and reverse phase LC/MS. The summary of all identified modifications is listed in Table 10. [00193] Table 10: Identified Modifications of Co-Arginase 1 Intermediate

[00194] Characterization determined that when Co-Arginase 1 intermediate modification was present, the predominant modification was N-terminal gluconoylation (confirmed by peptide mapping). Additional characterization of Arginase 1 modified species was performed by testing samples taken at three time points (fermentation, post-Column 1, and on drug intermediate from Column 3). The analytical methods typically require Arginase to be dissociated and analyzed as a monomer. Arginase 1 N-terminal gluconoylated (analyzed as a monomer) was typically 10.8 to 13.9%. Other modifications were N-terminal phosphogluconoylated monomer (4.3 to 6.5%), and di-gluconoylated monomer (0.7 to 1.2%), across the three timepoint samples. In a sample used from a standardized reference production run, the levels of un-modified Co-Arginase 1 (monomer) and Co-Arginase 1 intermediate were comparable at 80.6% to 83.6%, respectively. The standard condition used for the purification process (i.e. no salt gradient applied to Column 1) moderately altered the relative level of un- modified monomer carried through to Co-Arginase 1 intermediate (81.1 to 83.6%). [00195] Table 11: Co-Arginase 1 Characterization LC/MS Results

[00196] Example 14: Variation of Column 1 Conditions [00197] In alternative embodiments, a NaCl gradient can be applied to Column 1. Using a NaCl gradient over Column 1 enables separation of different Arginase 1 variants to select for preferred embodiments. Figure 7 shows a gradient from 0.0 - 0.2 M NaCl applied to Column 1. Individual fractions collected from the Column 1 elution were assayed by SE-HPLC, CEX- HPLC, and RP-HPLC. [00198] An analytical CEX-HPLC method was used that assigns Arginase 1 charge variants Peak Numbers of 1 through 6 (see Figure 5(c)). The Peak Numbers align with the various gluconoylation states as well as un-gluconoylated Arginase 1. This analysis showed six peaks in the Arginase 1 eluted from the NaCl gradient. Arginase 1 variants assigned Peak Numbers 1, 2, and 3 eluted early in the Column 1 elution peak. Peak 4 eluted through the highest concentration portion of the eluted Arginase 1 and peak 5 (unmodified Arginase 1) and peak 6 eluted later in the elution peak. Thus, the 0.0 – 0.2 M NaCl successfully separated different charge variants of Arginase 1. [00199] Alternative NaCl gradients can be used for Column 1 elution such as 0 – 0.5 M NaCl. The use of a NaCl gradients was found to reproducibly separate Arginase 1 into six distinct peaks enabling selection of specific Arginase 1 variants for further processing in the manufacture of drug substance or drug product. [00200] Further analysis of the first protein product (and the Arginase 1 variants) was also analyzed by LC/MS (see Figure 6). The LC/MS analysis identifies specific types of gluconylation generated by production of Arginase 1 in E. coli. The LC/MS analysis identifies unmodified Arginase 1, gluconylated Arginase1, phosphogluconylated Arginase 1, and 2 times (2X) gluconylated Arginase 1. [00201] Table 12 shows that the application of a 0-0.2 M NaCl gradient (and the corresponding fractionated CEX Peaks 1 - 6) produces fractions that have differing levels of gluconylation. Each of Peak Numbers 1-6 were analyzed by LC/MS. The data show that the dominant peak (Peak 5) has a high percentage of non-gluconylated Arginase 1 as well as high specific activity. Depending upon the desired characteristics different fractions (corresponding to Peaks 1-6) can be collected for further processing. Table 12: LC/MS analysis of drug intermediate Peaks 1 – 6.

[00202] In addition to varying NaCl concentrations on Column 1, different amounts of protein can be loaded on Column 1 to enhance purification of non-gluconylated Arginase 1 species. [00203] Varying the load factor of Column 1 and using a NaCl gradient over Column 1 can compensate for unexpected perturbations experienced during E. coli fermentation that produce gluconylated Arginase 1 species. [00204] Example 15: Variation in Fermentation Conditions [00205] Experiments were performed to determine the robustness of fermentation conditions for production of Arginase 1. Table 13 shows that fermentation of E. coli at the at sub-optimal pH of 7.6 produces more gluconylation that at the preferred pH 7.2 fermentation. Vessels B1, B8, and B12 used optimal conditions of fermentation: pH 7.2, Dissolved Oxygen 30%, feed rate of media 0.06 mL/min. Vessel B3 was used to ferment Arginase 1 expressing E. coli at pH 7.6 (a pH that is higher than optimal conditions). The increase in pH resulted in a higher proportion of phospho-gluconylated adducts (23% vs 10-12% in control runs) [00206] Table 13: Gluconylated Arginase 1 Observed in Fermentation Vessels

[00207] Example 16: Variation of Load Factor on Column 1 [00208] Different amounts of E. coli cell lysate were applied to Column 1 to determine the effect on purification of Arginase 1 charge variants, as well as yield and purity. Load factors of 15 - 60 g protein/L resin were used under various conditions showing a shift in the CIEX charge species profiles (Table 14). Higher load factors resulted in better separation of gluconylated variants (but depending on which fractions were collected a trade-off in yield may result). For example, with a load factor of 20 mg protein/mL resin peak 5 was 45.8% whereas with a load factor of 40 mg/mL this increased to 50.0%. [00209] Table 14: Effect of Column 1 Load Factor on Protein Product 1

[00210] Example 17: Phase 1/2 Clinical Investigation [00211] The drug product produced by the methods of this invention was used in a Phase 1/2, open-label study to evaluate administration of Co-rhARG1-PEG in Arginase 1 deficiency and hyperargininemia. The primary endpoint of this study was to evaluate the safety and tolerability of intravenous (IV) administration of Co-rhARG1-PEG in subjects with hyperargininemia/Arginase 1 deficiency. The secondary endpoints were: to determine the effects of study drug administered IV on plasma arginine concentrations; to determine the effects of study drug administered IV on plasma guanidino compounds (GCs); and to characterize the pharmacokinetic (PK) profile of study drug administered IV. Other endpoints include evaluation of clinical outcome assessments in capturing clinical benefit such as: 6- Minute Walk Test (6MWT), Gross Motor Function Measure (GMFM) Parts D and E, and Adaptive Behaviour Assessment System (ABAS). [00212] The Phase 1/2 data demonstrated that Co-rhARG1-PEG was highly effective in sustainably lowering plasma arginine. In addition, the control of plasma arginine levels in patients was accompanied by clinically meaningful responses in mobility and adaptive behavior. The treatment was generally well tolerated. Hypersensitivity reactions were infrequent and manageable with standard measures. [00213] The Co-rhARG1-PEG drug product supplied for the study was as a liquid formulation in 10 mL single-use glass vials containing 5 mL of formulated drug product at a concentration of 1 mg/mL. The drug was formulated in 50 mM NaCl, 1 mM KH₂PO₄, 4 mM KH₂PO₄, and 1.5% w/v glycerol. [00214] The Phase 1/2 study was conducted in two parts: Part 1 (Single Ascending Dose Escalation) and Part 2 (Repeated Dosing). The study design for this phase 1/2 trial 101A and the 102A open label extension is shown in Figure 14. [00215] Part 1 introduced the patient to the drug and was focused upon safety. Part 2 was designed to settle the patient on a consistent dose and look for markers of clinical effectiveness. Each part was preceded by a baseline assessment of arginine levels. All patients who participated in Part 1 could continue Arginase 1 dosing in Part 2 if they qualified for continued dosing. [00216] In the study, each patient received a starting dose that could escalate in Part 1 with a 2-week washout/observation period between each successive dose level. The possible doses for each patient in Part 1 were 0.015, 0.03, 0.06, 0.10, 0.15, 0.20, and 0.30 mg/kg, at 2- week intervals as needed to optimize plasma arginine. Any particular dose can be repeated, or a dose increased/decreased between the specified dose levels if emerging data from prior dose levels met certain criteria. For example, the escalation of dose may cease if one or more of the following dose escalation stopping criteria were met: the patient's plasma arginine level was < 40 μM for at least 40 (± 2) consecutive hours post-dosing for all samples collected during that time period or the patient's plasma arginine level averaged <115 μM for at least 112 (± 2) consecutive hours post-dosing for all samples collected during that time period. [00217] If none of these events occurred, the patient could be escalated to the next higher dose level of Arginase 1 every 2 weeks until any dose escalation stopping criterion was reached or the patient had received the highest dose under this protocol of 0.30 mg/kg. Ultimately, for treatment purposes, it is also possible that the dosing might increase over 0.30 mg/kg. [00218] Part 2 was a repeat-dosing period for patients who completed Part 1. Part 2 found a dose and regimen for each patient that safely optimized plasma arginine between 40 and 115 μM during repeat-dose administration, with emphasis on maintaining pre-dose levels below 150–200 μM. Several dose levels could have been used in Part 2 if the data indicated a potential to better investigate a dose-response outcome during repeat-dose administration. On- treatment arginine levels were also compared with arginine levels determined prior to treatment. [00219] Patients who completed Part 2 of 101A were eligible to participate in a long- term open-label extension (OLE) trial (NCT03378531). Treatment with 24 weekly IV doses with the option to switch to subcutaneous dosing for the remainder of the 3-year OLE period. [00220] Results [00221] Increases in mean C_max and mean AUC_0-168 appeared dose proportional in all patients. Mean (± SD) C_max was 0.428 ± 0.0915, 0.723 ± 0.247, 1.73 ± 0.538, 2.27 ± 0.238, and 6.13 (N=1) µg/mL for Co-rhARG1-PEG dose levels of 0.015, 0.03, 0.06, 0.1 and 0.2 mg/kg, respectively (Figure 9). [00222] Changes in the AUCs ( AUC_0-168, AUC_0-∞) appeared dose proportional across the studied dose range, noting that there no notable change between 0.06 and 0.1 mg/kg (with the data that was available). Mean clearance (CL) estimates ranged from 0.789 to 1.57 mL/hr/kg in all patients. The mean volume of distribution (Vss) estimates ranged from 35.3 to 52.1 mL/kg in all patients. [00223] Part 1 of the study helped select an optimal (individual) starting dose for each patient for Part 2 using the observed PD (arginine) response. In Week 1 of Part 2, there was a trend for increasing mean circulating drug concentrations in all patients with escalating doses of Co-rhARG1-PEG across the dose range evaluated. After the first dose of Co-rhARG1-PEG in Part 2, increases in mean C_max appeared dose proportional in all patients. Mean (± SD) C_max was 0.292 (N=1), 0.395 (N=1), 1.01 ± 0.221, 1.75 ± 0.391, 1.99 (N=1), 2.34 (N=1), and 2.87 ± 0.626 µg/mL for Co-rhARG1-PEG dose levels of 0.015, 0.03, 0.04, 0.06, 0.09, 0.1 and 0.12 mg/kg, respectively. [00224] In Part 2, Week 8, mean circulating drug concentrations in all patients generally increased with escalating doses of Co-rhARG1-PEG. There was no notable ADA impact in the available PK concentrations at Week 8. As a result of the data, achievement of steady state was assumed for most (13/14) patients at this time. After the 8th QW dose of Co-rhARG1-PEG, increases in mean C_max and AUC_0-168 appeared dose proportional in all patients. [00225] In addition to pharmacokinetic data, pharmacodynamic (arginine) data was collected (Figure 10). Patients with Arginase 1 deficiency were administered (weekly) QW IV doses of Co-rhARG1-PEG in Part 2 and the starting dose was selected in Part 1 based on the observed PD (arginine) response. After the first QW IV dose of Co-rhARG1-PEG, there were notable reductions in circulating arginine levels, particularly for Co-rhARG1-PEG doses at or above 0.04 mg/kg. In several instances, the individual arginine concentrations dropped below 40 µM. In addition, recoveries to starting arginine levels were incomplete in most patients at doses ≥ 0.04 mg/kg and just prior to administration of the second QW (weekly) Co-rhARG1- PEG dose. [00226] Overall, exposure to Co-rhARG1-PEG generally increased and there was increased arginine suppression with escalating dose. Individualized dose optimization was undertaken in Part 1 such that there were a range of doses with varying numbers of patients per dose level in Weeks 1 and 8 of Part 2. [00227] Example 18: Subcutaneous Administration [00228] After Part 2 of the Phase 1/2 study of Example 17 was complete, some patients were switched from IV administration of Co-rhARG1-PEG to subcutaneous administration. Surprisingly, subcutaneous administration of Co-rhARG1-PEG gave a pharmacodynamic profile that appeared superior to IV administration. Also unexpectedly, the same formulation as for IV administration was successfully used for subcutaneous administration of Co-rhARG1- PEG. [00229] The subcutaneous administration of Co-rhARG1-PEG maintained patient arginine levels within the preferred (healthy) target range for plasma arginine concentration longer than IV administration (Figure 11). The preferred optimized plasma arginine concentration in a patient is between 40 μM and 115 μM (during repeat-dose administration), with emphasis on maintaining levels below the pre-dose 150–200 μM. As can be seen in Figure 11, subcutaneous administration of Co-rhARG1-PEG results in arginine concentrations above the lower level of 40 μM and below the upper level of 115 μM. Surprisingly, subcutaneous administration gave arginine concentrations that are entirely in the preferred range. This means the patient will stay in the appropriate plasma range of arginine concentrations until another weekly Co-rhARG1-PEG dose is received. [00230] Example 19: Pharmacodynamic and Clinical Responses From Phase 1/2 Clinical Study and Open Label Extension [00231] 16 patients (11 paediatric and 5 adult) were enrolled into 101A Part 1 and 15 patients advanced into 101A Part 2. 2 patients withdrew from the trial for personal reasons (1 patient after Part 1 dose 3 and 1 patient after Part 2 dose 3). All 14 patients completing 101A Part 2 advanced into the OLE trial. [00232] Baseline characteristics for the patients are shown in Table 15. [00233] Table 15: Baseline Characteristics

[00234] An analysis of plasma arginine and guanidino compound levels found marked and sustained reductions in plasma arginine levels (Figure 12 (a) were demonstrated with a median reduction of 274 µM from baseline after 20 doses of pegzilarginase. Reductions in plasma arginine from baseline to dose 1, dose 8, and OLE were statistically significant (p<0.001). Plasma arginine reductions were accompanied by decreases in plasma guanidino compound (GC) levels. Figure 12(b) shows plasma levels for guanidinoacetic acid (GAA), N- α-acetyl-L-arginine (NAA), α-keto-δ-guanidinovaleric acid (GVA) and argininic acid (ARGA) at baseline and the reduction of plasma GC levels during the OLE. [00235] Data from all patients following 20 doses of pegzilarginase demonstrated marked and sustained reductions in plasma arginine. Pegzilarginase was well tolerated and the rates of treatment-related adverse events decreased over time. The improvements in arginine control and evidence of clinical benefit following pegzilarginase treatment provide further validation of the key endpoints and design elements of the pivotal Phase 3 PEACE trial (NCT03921541). [00236] Example 20: Pegzilarginase In Vitro Suppression of Influenza Virus [00237] This experiment was performed in order to understand the antiviral activity (as assessed by the amount of intracellular viral proteins produced) of pegzilarginase against a virulent strain of influenza A virus. In order to control for indirect antiviral effects related to reduced cell numbers and/or fitness of host cells, we also measured cell viability. In this experiment, A549 lung carcinoma cells were first seeded into 96 well plates and incubated for 24 hours at 37C. Influenza virus (A/Puerto Rico/8/1934; “PR8 strain”) was pre-diluted with either media, media containing the antiviral baloxavir (as a positive control), or media containing pegzilarginase, and added to the A549 cells. After incubation for one hour in order to permit viral entry, the media was replaced with fresh media containing the test compound of interest, then cells were incubated for an additional 48 hours at 37C. Cells were then permeabilized, treated with a blocking reagent to minimize non-specific antibody binding, then incubated with an antibody cocktail directed against influenza viral proteins. Cells were then washed and incubated with a horseradish peroxidase-conjugated secondary antibody. After a subsequent wash step, cells were incubated with substrate for horseradish peroxidase and absorbance at 490 nm was assessed using a spectrophotometer. In parallel, cell viability was measured in duplicate at 48 hours for each treatment and dose using an XTT-based system that utilizes the metabolic conversion of a tetrazolium salt as a surrogate for cell number and health. For the above experiments, baloxavir was tested in duplicate in a 5-fold dilution series ranging from 1000nM to 0.0128nM. Pegzilarginase were tested in duplicate in a 10-fold dilution series ranging from 10,000nm to 0.0001nM. [00238] The results of the viral infectivity assay are shown in Figure 13(a) and the results of the cell viability assay are shown in Figure 13(b). As can be seen from Figures 13(a) and (b), 1 nm pegzilarginase provided a marked decrease in PR8 infectivity, with less effect on the cell viability surrogate measurements than higher concentrations of pegzilarginase. [00239] Reference throughout this specification to “one embodiment,” “certain embodiments,” “various embodiments,” “one or more embodiments” or "an embodiment" means that a particular feature, structure, material, or characteristic described in connection with the embodiment is included in at least one embodiment of the disclosure. Thus, the appearances of the phrases such as “in one or more embodiments,” “in certain embodiments,” “in various embodiments,” “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily referring to the same embodiment of the disclosure. Furthermore, the particular features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments. [00240] Although the disclosure herein provided a description with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made to the present disclosure without departing from the spirit and scope thereof. Thus, it is intended that the present disclosure include modifications and variations that are within the scope of the appended claims and their equivalents.

Claims

What is claimed is: 1. A method of preventing or treating an influenza infection, the method comprising administering a pharmaceutical composition comprising a recombinant human Arginase (rhARG) to a patient in need thereof.

2. The method of claim 1, wherein the influenza infection comprises a respiratory infection.

3. The method of claim 1 or 2, wherein the influenza infection is caused by an influenza virus of type A or type B.

4. The method of claim 3, wherein the influenza virus is an influenza A virus.

5. The method of claim 3, wherein the influenza virus is an influenza B virus.

6. The method of any one of claims 1-5, wherein the rhARG comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 1.

7. The method of any one of claims 1-6, wherein the rhARG is cobalt-substituted.

8. The method of claim 7, wherein the rhARG comprises about 0.1 to about 2 µg Co per mg protein.

9. The method of any one of claims 1-8, wherein the rhARG is PEGylated.

10. The method of claim 9, wherein the average number of PEG residues is about 8 to about 25 moles of PEG per mole of rhARG monomer.

11. The method of claim 10, wherein the average number of PEG residues is about 8 to about 16 moles of PEG per mole of rhARG monomer.

12. The method of any one of claims 9-11, wherein each PEG residue has an average molecular weight of about 1,000 to about 10,000 Daltons.

13. The method of claim 12, wherein each PEG residue has an average molecular weight of about 5,000 Daltons.

14. The method of any one of claims 1-13, wherein the pharmaceutical composition is administered intravenously.

15. The method of any one of claims 1-13 wherein the pharmaceutical composition is administered subcutaneously.

16. The method of any one of claims 1-15, wherein the pharmaceutical composition is administered at a dose of 0.05 to 2 mg/kg based on the weight of unPEGylated enzyme.

17. The method of any one of claims 1-16, wherein the pharmaceutical composition is administered at a dose of 0.1 to 0.5 mg/kg based on the weight of unPEGylated enzyme.

18. The method of any one of claims 1-17, wherein the pharmaceutical composition is administered at a dose of 0.27 mg/kg based on the weight of unPEGylated enzyme.

19. The method of any one of claims 1-18, wherein the pharmaceutical composition is administered once every day to once every two weeks.

20. The method of any one of claims 1-19, wherein the pharmaceutical composition is administered weekly.

21. The method of any one of claims 1-20, wherein the patient is co-administered a second therapy.

22. The method of claim 21, wherein the second therapy is co-administered simultaneously with the rhARG.

23. The method of claim 21, wherein the second therapy is co-administered sequentially with the rhARG.

24. The method of any one of claims 21-23, wherein the second therapy comprises an antiviral therapy.

25. The method of any one of claims 21-24, wherein the second therapy comprises baloxavir.

26. The method of any one of claims 21-24, wherein the second therapy comprises an ion channel inhibitor.

27. The method of any one of claims 21-24, wherein the second therapy comprises an inhibitor comprises a neuraminidase inhibitor.