USE OF ARGINASE FOR TREATMENT OF CORONAVIRUS INFECTION TECHNICAL FIELD [0001] The present disclosure generally relates to treatment of coronavirus infections such as COVID-19. 5 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- 10 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 treating a coronavirus 15 disease, 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 coronavirus disease is a respiratory disease. In one or more embodiments, the coronavirus disease is COVID-19. In one or more embodiments, the coronavirus is SARS- CoV-2. In one or more embodiments, the RNA sequence of the coronavirus is at least 80, 85, 20 90, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, or 99.9% identical to SEQ ID NO: 4. [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 25 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 5 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 10 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 15 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 20 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 remdesivir. In one or more 25 embodiments, the second therapy comprises an immune checkpoint inhibitor. In one or more embodiments, the immune checkpoint inhibitor comprises one or more of a PD-1 inhibitor or a PD-L1 inhibitor. In one or more embodiments, the immune checkpoint inhibitor comprises one or more of pembrolizumab, ipilimumab, atezolizumab or nivolumab. In one or more embodiments, the immune checkpoint inhibitor comprises pembrolizumab. 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 5 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 10 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 15 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. 20 [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 25 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 30 (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, 5 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 10 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 15 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^; Spectra are normalized to the peak intensities of the signals due to unmodified Arginase 1. Peak intensities of variants are proportional to relative 20 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 25 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^). Figure 8(b) shows a representative enzyme kinetic analysis 30 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 5 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). 10 [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 15 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 ^M 20 and 115 ^M (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. 25 [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-^-acetyl-L-arginine (NAA), ^-keto-^- guanidinovaleric acid (GVA) and argininic acid (ARGA) at baseline and during the OLE. 30 [0024] Figure 13 shows the RNA sequence of SARS-CoV-2 (SEQ ID NO: 4) as reported by Wu et al. (GenBank # MN908947). [0025] Figure 14 shows the results of Co-rhARG1-PEG and remdesivir on in vitro SARS-CoV-2 infection. Figure 14(a) shows IC50 (infection: blue squares) and CC50 (toxicity: green circles) for Co-rhARG1-PEG on SARS-CoV-2 infected Calu3 cells. Figure 14(b) shows IC50 (infection: blue squares) and CC50 (toxicity: green circles) for remdesivir on SARS- 5 CoV-2 infected Calu3 cells. [0026] Figure 15 shows an exemplary clinical trial protocol for evaluating the effectiveness of pegzilarginase therapy for the treatment of COVID-19. DETAILED DESCRIPTION [0027] Definitions 10 [0028] “Coronavirus” is any virus of the family Coronaviridae. Coronaviruses currently include four main subgroupings: alpha, beta, gamma or delta type coronaviruses. Exemplary alpha coronaviruses (^-CoV) include: human coronavirus 229E (HCoV-229E); human coronavirus NL63 (HCoV-NL63). Exemplary beta coronaviruses (^-CoV) include: human coronavirus OC43 (HCoV-OC43); human coronavirus HKU1 (HCoV-HKU1); MERS-CoV 15 (the beta coronavirus that causes Middle East Respiratory Syndrome, or MERS); SARS-CoV (the beta coronavirus that causes severe acute respiratory syndrome, or SARS); and SARS- CoV-2 (the beta coronavirus that causes COVID-19). [0029] “Coronavirus infection” is any infection caused by a coronavirus. Exemplary coronavirus infections include, but are not limited to, SARS, COVID-19 and MERS. 20 [0030] “COVID-19” is coronavirus disease 2019, a potentially severe respiratory disease characterized by fever, coughing and shortness of breath. It is caused by the coronavirus SARS-CoV-2. [0031] “Subject” and “patient” refer to either a human or a non-human, such as primates, mammals, and vertebrates. 25 [0032] “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. [0033] “50% cytotoxic concentration” (CC50) is the drug concentration that reduces the cell viability by 50% when compared to untreated controls. The CC50 can be viewed as an approximate measure of drug toxicity in vitro. Alternatively, CC50 may include cytostatic effects in addition to, or independent of, cytotoxic effects. 5 [0034] “50% inhibitory concentration” (IC50) is the drug concentration that inhibits 50% of viral replication when compared to the virus control. The IC50 can be viewed as an approximate measure of inhibitory potential of a drug to inhibit virus. [0035] “Minimal inhibitory concentration” (MCC) is the minimum (or lowest) concentration of drug that results in an observable suppression of viral load. 10 [0036] “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. [0037] “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 15 in a sheet of cultured tissue cells. [0038] “Plaque forming unit” (PFU) is a measure of viable infectious entities (e.g. coronavirus 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 20 plaque forming units (PFUs) per unit volume is a conventional way to refer the titer of a virus in a specimen or inoculum. [0039] “Selectivity index” (SI) of a drug is the ratio of CC50 over IC50. [0040] “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 25 (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. [0041] “Viral titer” is an expression of the concentration of a virus, e.g. the concentration of coronavirus in a given volume. A titer is frequently determined by performing 30 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. [0042] “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 5 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 10 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). [0043] “Remdesivir” is a broad-spectrum antiviral medication currently developed by 15 Gilead Sciences. [0044] An “immune checkpoint inhibitor” is a type of drug that blocks proteins called checkpoints that are made by some types of immune system cells, such as T cells, and some cancer cells. An immune checkpoint inhibitor can comprise one or more of a PD-1 inhibitor or a PD-L1 inhibitor. Immune checkpoint inhibitors can comprise pembrolizumab, ipilimumab, 20 atezolizumab, nivolumab, or pembrolizumab. [0045] 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. [0046] “Co-rhARG1”, “Co-Arginase 1 intermediate” and the like refer to a cobalt 25 substituted rhARG1, i.e. a rhARG1 that has at least some of the native manganese cofactor replaced with cobalt. Suitably, a Co-rhARG1 has at least 50% of the native manganese cofactor replaced with cobalt, more suitably at least 60%, 70%, 80%, 90%, 95% or 98%. Most suitably, 100% or substantially 100% of the native manganese cofactor is replaced with cobalt. In one or more embodiments, the Co-rhARG1 is an isolatable intermediate in a production 30 and/or purification process for Co-rhARG1-PEG. [0047] “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. [0048] “Pegzilarginase” refers to a cobalt substituted, recombinant human arginase I 5 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. [0049] “High Salt Solution” refers to NaCl concentration of up to about 0.5 M. [0050] “Salt Gradient” refers to an increasing or decreasing salt concentration. Exemplary salt gradients include concentrations of NaCl ranging from about 0.01 to about 10 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. [0051] “Room temperature” refers to at or about 15 ^o C to about 25 ^o C, such as about 20 to about 25 ^o C. [0052] “Cation exchange” (CEX) chromatography column refers to a chromatography 15 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. [0053] “First Protein Product” refers to the protein product eluted from Column 1. 20 [0054] “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. [0055] “Second Protein Product” refers to the protein product eluted from Column 2. 25 [0056] “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. [0057] “Size exclusion chromatography” (SEC) column utilizes a chromatographic 30 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. [0058] “Third Protein Product” refers to the protein product eluted from Column 3. [0059] Methoxy PEG succinimidyl carboxymethyl ester is an amine reactive PEG 5 product with a stable non-degradable linker between the PEG polymeric chain and the NHS ester. [0060] “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 10 it. [0061] “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. [0062] “Imaging capillary isoelectric focusing” (iCIEF) is a capillary electrophoresis 15 (CE) technique used to study physical properties of proteins. [0063] “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 20 (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. [0064] “Ultrafiltration/Diafiltration" (UF/DF) is a high yield, and robust separation process based on size exclusion. UF involves separation of components based on molecular 25 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 Treating Coronavirus Infections 30 [0065] Viruses are reliant on host metabolism and macromolecular synthesis pathways for their replication. Many viruses, including coronaviruses, utilize the bioavailability of arginine, which is critical for many physiological and pathophysiological processes associated with either facilitating viral replication or progression of disease. [0066] Synthetic nucleoside analogues have proven a safe and effective means to inhibit HSV by targeting genomic replication. 5 [0067] 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 10 bioavailability may be linked to pathophysiological processes. [0068] As described herein, arginase is expected to effectively inhibit coronavirus 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 15 viral replication will be enhanced. [0069] 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. [0070] Pegzilarginase has been assessed in vitro and in vivo with clinical trials to 20 define its pharmacokinetic and pharmacodynamic characteristics. [0071] Coronaviruses 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 25 limitation for coronavirus. 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 coronavirus 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 30 pegzilarginase have demonstrated that systemic dosing does not adversely affect the immune response and in fact enhances adaptive immune responses. [0072] Accordingly, embodiments of the present invention pertain to administering recombinant arginase (e.g. pegzilarginase) for the treatment of coronavirus diseases such as coronavirus disease 2019 (COVID-19). In one or more embodiments, the recombinant human Arginase protein is recombinant human Arginase 1 (rhARG1) (SEQ ID NO: 1; shown Figure 5 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. [0073] Patients with severe COVID-19 tend to have a high viral load and a long virus- 10 shedding period (Liu et al.). This finding suggests that the viral load of COVID-19 might be a useful marker for assessing disease severity/prognosis and provide a mechanism to measure the efficacy of a therapeutic in suppression of viral load leading to clinical benefit of arginase. [0074] Patients with COVID-19 can be classified as severe (based upon increasing severity): (1) respiratory distress (^30 breaths per min); (2) oxygen saturation at rest ^93%; (3) 15 ratio of partial pressure of arterial oxygen to fractional concentration of oxygen inspired air ^300 mm Hg; or (4) severe disease complications (e.g., respiratory failure, requirement of mechanical ventilation, septic shock, or non-respiratory organ failure). If COVID-19 patients do not present with symptoms 1-4 they are considered to have mild clinical symptoms. One early study found that 46 (61%) individuals were classified as mild cases and 30 (39%) were 20 classified as severe cases (Liu et al.). [0075] The mean viral load of severe cases was around 60 times higher than that of mild cases, suggesting that higher viral loads might be associated with severe clinical outcomes. Mild cases also showed significantly lower viral loads for the first 12 days after onset of disease symptoms compared to severe cases. 25 [0076] Mild cases were found to have an early viral clearance, with 90% of these patients repeatedly testing negative on RT-PCR by day 10 post-onset. By contrast, all severe cases still tested positive at or beyond day 10 post-onset. [0077] Thus, there are several indicators of successful treatment with arginase that can be measured. One success parameter will show a greater proportion of patients clearing the 30 virus by day 10 post-onset. An alternative measure of success will show a viral load approaching 60 times lower than that which is considered severe. A range of successful indications could be a viral load that is 5 to 60 times lower than a viral load that is considered severe, suitably 20 to 60 times lower or 40 to 60 times lower. Examples of successful indications include a viral load which is 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 times lower than a viral load that is considered severe. 5 [0078] Another alternative indicator of successful treatment will be a lower proportion of COVID-19 patients in any one of clinical classifications (1) through (4) above. Another marker of success of arginase treatment is a lower proportion of COVID-19 patient death or a longer period of survival compared to untreated patients. [0079] Recombinant Human Arginase 1 10 [0080] 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 (Mn ²⁺ ) with cobalt (Co ²⁺ ) in the active site of Arginase 1 enhances catalytic 15 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 20 significantly. Again, although specific reference is made herein to rhARG1, the methods, formulations and uses described herein can also be applied to rhARG2. [0081] 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 25 1.4 mg of PEG. [0082] 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. coli30 (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. [0083] Table 1: Structural Information of an Exemplary Co-Arginase 1 Intermediate ^^^ 5 [0084] 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 amount of PEG is from about 8 to about 25 moles of PEG/mole Co-Arginase 1 10 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, or 3,000 to 7,000 Daltons, such as about 1,000, about 2,000, about 3,000, about 4,000, 15 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. [0085] In one or more embodiments, the Co-rhARG1-PEG comprises pegzilarginase. Pegzilarginase has the following two chemical names: a. poly(oxy-1,2-ethanediyl), ^-(carboxymethyl)-^-methoxy-, amide with arginase 20 1 [cobalt cofactor] (synthetic human) (1:10), trimer b. Des-Met ¹ -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 N ⁶ -lysines) are amidified with [methoxypoly(ethyleneoxy)]acetyl, non-covalent homotrimer, produced in 25 Escherichia coli [0086] Potential PEGylation sites of pegzilarginase are shown below:

[0087] The molecular formula for pegzilarginase is C1554H2492N416O453S6 [C3H4O2 (C ₂ H ₄ O) _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. 5 [0088] 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 10 equation to determine Km and kcat. [0089] Vmax 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 Vmax. The enzymatic turnover number, kcat is 15 calculated by V _max /[E]. [0090] Specific activity is determined by dividing the reaction velocity at 2 mM arginine expressed in µmoles/minute by the enzyme concentration in mg. [0091] The values for Co-rhARG1-PEG drug substance for KM and kcat as measured in the enzyme activity assay typically range from 0.15-0.22 mM and approximately 200-300/sec 20 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. [0092] In one or more embodiments, the protein (e.g. Co-rhARG1 or Co-rhARG1- PEG) displays a kcat/KM greater than 200 mM ^-1 s ^-1 at pH 7.4. In a particular embodiment, the 5 protein displays a kcat/KM from 200 mM ^-1 s ^-1 to 4,000 mM ^-1 s ^-1 at pH 7.4. In another embodiment, the protein displays a kcat/KM from 400 mM ^-1 s ^-1 to 2,500 mM ^-1 s ^-1 or 800 mM ^-1 s- 1 to 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 kcat/KM greater than 400 mM ^-1 s ^-1 at 37° 10 C., pH 7.4. Exemplary kcat/KM 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. [0093] In one or more embodiments, the rhARG1, Co-rhARG1 or Co-rhARG1-PEG 15 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 20 available at the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov/). [0094] 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 Km for arginine from 2.8 mM to about 0.18 mM at pH 7.4. In one or more 25 embodiments, the Co-rhARG1-PEG comprises about 0.1 to about 2 µg, such as about 0.5 to 2 µg or about 1-2 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 30 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. Typical ranges for Mn include, for example, 0 to 1 µg, 0.001 to 0.5 µg, 0.001 to 0.2 µg or 0.001 to 0.1 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. 5 [0095] Production and Purification of rhARG1, Co-rhARG1 and PEGrhARG1 [0096] Shake Flask Expansion [0097] 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 10 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. [0098] 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 15 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. [0099] 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. 20 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 25 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. 30 [00100] Production Fermentation [00101] 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 5 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 10 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. [00102] In a preferred embodiment, the fermentation medium is prepared directly in the 15 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 20 (ammonium hydroxide) to a controlled pH value. [00103] 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 25 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 ^ and at a pH of 7.0 – 7.4 with agitation and aeration. 30 [00104] 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 5 Production Fermentation can be seen in Figure 3. [00105] Harvest Operations [00106] 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. 10 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. [00107] In a preferred embodiment, whole cells are separated from fermentation 15 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 0.2^m grade filters. In a preferred embodiment, harvest steps are performed at a target temperature of ^ 15°C. 20 [00108] 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 25 between passes is minimized to reduce potential microbial growth. [00109] 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. 30 [00110] 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 5 potential for bioburden load. [00111] Purification of rhARG1, Co-rhARG1 and Co-rhARG1-PEG [00112] 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 10 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. [00113] 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 15 the first column (“Column 1”) in system with multiple chromatography columns. The protein product eluted from this Column 1 is “First Protein Product”. [00114] 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. 20 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. [00115] In various embodiments, a salt gradient is used to separate different charge 25 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. [00116] In one or more embodiments, the method further comprises loading the First Protein Product (optionally after cobalt substitution) onto an anion exchange (AEX) 30 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. 5 [00117] 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 Co ²⁺ 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^ or 10 greater than 50 ^, and corresponding incubation times can be as long as over 8 hours or less than 10 minutes. [00118] 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. 15 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. [00119] Administration of rhARG1, Co-rhARG1 and Co-rhARG1-PEG [00120] The rhARG1, Co-rhARG1 and Co-rhARG1-PEG as described herein (and compositions comprising them) can be administered via any appropriate route, including 20 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) by injection or infusion. [00121] Compositions containing rhARG1, Co-rhARG1 and Co-rhARG1-PEG thereof 25 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 30 emulsifying agents, stabilizing or pH buffering agents. [00122] 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. 5 [00123] Blood Arginine Monitoring: [00124] After initiating treatment with pegzilarginase, plasma arginine monitoring may be performed to ensure low plasma arginine levels. [00125] Preparation and Administration Instructions [00126] Pegzilarginase is supplied as a frozen liquid formulation in 10 mL single-use 10 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 15 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 20 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: Amount of 1.0 mg/mL pegzilarginase = Patient Weight (kg) x dose level (mg/kg) Amount of 5.0 mg/mL pegzilarginase = Patient Weight (kg) x dose level (mg/kg) 25 5 [00127] Administer pegzilarginase via intravenous infusion over 30 minutes using a syringe pump. [00128] Table 2: Weight-Based Dosing for Administration of 0.1 mg/kg Once 30 Weekly

[00129] 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 5 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). [00130] Dosage Forms and Strengths [00131] pegzilarginase injection is a colorless to slightly yellow or slightly pink solution available as follows in 10mL vials: 10 a. Solution for Injection: 5 mL of 1.0 mg/mL b. Solution for Injection: 5 mL of 5.0 mg/mL [00132] Warnings and Precautions [00133] 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 5 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. [00134] Pregnancy: Pregnancy Category B 10 [00135] 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. 15 [00136] Nursing Mothers [00137] 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. [00138] Description 20 [00139] 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 25 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 30 ornithine per minute at 37°C. [00140] 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 5 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. 10 [00141] Pharmacodynamics [00142] 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 15 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. [00143] Pharmacokinetics 20 [00144] 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 Cmax and AUC0-168, increased approximately proportional to dose, with a 13-fold increase in dose resulting in a 14-fold 25 increase in Cmax and AUC0-168. 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. [00145] Animal Toxicology and/or Pharmacology [00146] The pharmacologic effects of pegzilarginase on arginine levels were assessed in 30 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. 5 [00147] The potential toxicity and toxicokinetics (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, 10 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 15 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 20 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. 25 [00148] 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. 30 [00149] 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 5 noted in clinical pathology parameters (coagulation, growth hormone, and urinalysis), ECG and ophthalmic examinations, respiratory rate, and blood pressure assessments. [00150] How Supplied/Storage and Handling [00151] Pegzilarginase is supplied as a solution for injection. [00152] Pegzilarginase is supplied frozen (^-60°C). Diluted pegzilarginase should be 10 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. [00153] EXAMPLES [00154] 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 15 forth in the following description. The disclosure is capable of other embodiments and of being practiced or being carried out in various ways. [00155] In the experimental disclosure which follows, the following abbreviations apply: eq (equivalents); M (Molar); ^M (micromolar); mM (millimolar); N (Normal); mol (moles); mmol (millimoles); ^mol (micromoles); nmol (nanomoles); g (grams); mg 20 (milligrams); ^g (micrograms); L (liters); ml (milliliters); ^l (microliters); cm (centimeters); mm (millimeters); ^m (micrometers); nm (nanometers); MW (molecular weight); PBS (phosphate buffered saline); min (minutes). [00156] Example 1: Cation Exchange Column Chromatography (Column 1) 25 [00157] 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. 30 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. [00158] Figure 4(a) shows a representative purification of Arginase 1 on Column 1. 5 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). 10 [00159] Example 2: Cobalt Substitution [00160] 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 15 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 ^. Conversely, cobalt loading temperature can be as low as 1^ or 5^ and performed for over 8 hours. Also, the higher proportion of cobalt loaded into Arginase 1 leads to a higher specific activity. 20 [00161] 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^. In another embodiment, Arginase 1 cobalt loading is performed in a solution of 25 10mM CoCl2 for between 2 - 8 hours at room temperature. [00162] An overview of the cobalt loading step can be seen in Table 3. Table 3 [00163] Example 3: Ultrafiltration/Diafiltration 1 (UF/DF 1) [00164] UF/DF 1 removes free cobalt ions and exchanges the Co-Arginase 1 into a 5 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 by10 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. 15 [00165] 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. [00166] In an alternative embodiment, the first exchange of buffer is into 25mM 20 HEPES, 0.1M NaCl, pH 7.2-7.6 and the second exchange is into 50mM Tris, pH 8.1-8.5. [00167] Example 4: Anion Column Chromatography (Column 2) [00168] A preferred embodiment of the Arginase 1 purification uses anther column which is an anion exchange column chromatography (“Column 2”). One embodiment of 5 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 10 resin is loaded onto the column with buffer 50mM Tris, pH 8.1-8.5. [00169] 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 15 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. [00170] Example 5: Capto Multimodal Column Chromatography (Column 3) 20 [00171] 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 25 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). [00172] In one embodiment, MMC chromatography (Column 3) uses approximately 15 30 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. 5 [00173] Example 6: Ultrafiltration/Diafiltration 2 (UF/DF 2) [00174] 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 10 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 15 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 20 concentration following the first dilution is within the targeted range. [00175] Example 7: Intermediate Filtration and UF/DF 3 [00176] 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 25 0.2 ^m filter and can be frozen for long term storage. [00177] 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. 30 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 5 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. [00178] Regarding the UF/DF 2 and UF/DF 3 steps, the first buffer exchange can be into 10 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). 15 [00179] Example 8: PEGylation of Arginase 1 [00180] 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 20 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^ until UF/DF4 operations. 25 [00181] Table 4: PEGylation Process for Co-rhARG1 Drug Substance

Step Process Operation Add solid methoxy PEG succinimidyl carboxymethyl ester (MW 5000) and incubate for greater than 15 min pH 8.4 ll [00182] 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. 5 [00183] 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 10 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. 15 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. [00184] 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 KH ₂ PO ₄ . [00185] Drug substance (Co-rhARG1-PEG) is a PEGylated cobalt-substituted human Arginase 1 made by conjugating activated PEG molecules with the ε-amino group of lysines 5 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 10 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 15 with detection by refractive index. Results can be expressed as µg/mL of free PEG (see Table 5). [00186] Table 5: SEC-HPLC Method Parameters Free PEG Co-rhARG1-PEG Drug Substance 20 [00187] Example 9: CIEX-HPLC Characterization of Drug Intermediate [00188] 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. [00189] Table 6: CIEX-HPLC Charge Variants Co-Arginase 1 Intermediate 5 Gradient Program [00190] 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 10 intermediate. [00191] Example 10: iCIEF Characterization of Drug Substance [00192] 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 15 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. [00193] Analytes in imaging capillary isoelectric focusing (iCIEF) migrate through a 20 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. [00194] The results can be expressed as percent charge variant distribution. A representative electopherogram is shown in Figure 5(d) where nine predominant peaks are 5 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: [00195] Table 7: iCIEF Characterization of Charge Variants of Co-rhARG1-PEG a 10 [00196] Example 11: Enzyme Activity of Co-Arginase 1 Intermediate and Drug Substance [00197] 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 15 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^. 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. 20 [00198] 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. [00199] Example 12: Analysis of Cobalt and Manganese [00200] 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 5 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. 10 [00201] Table 8: Typical Co-Arginase 1 Intermediate Characteristics ^^^ B^ B^ $B^ $B^ ^B^ B^ ^^ ^^ ^^ ^ &^ [00202] Table 9: Typical Co-rhARG1-PEG Drug Substance Characteristics $B^

[00203] Example 13: Co-Arginase 1 Intermediate Post-Translational Modifications [00204] Co-Arginase 1 intermediate post-translational modifications were detected using a variety of techniques such as peptide mapping, LC-MS intact mass spectrometry, and 5 reverse phase LC/MS. The summary of all identified modifications is listed in Table 10. [00205] Table 10: Identified Modifications of Co-Arginase 1 Intermediate [00206] 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 10 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%), 15 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%). 5 [00207] Table 11: Co-Arginase 1 Characterization LC/MS Results d mer [00208] Example 14: Variation of Column 1 Conditions [00209] In alternative embodiments, a NaCl gradient can be applied to Column 1. Using 10 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. [00210] An analytical CEX-HPLC method was used that assigns Arginase 1 charge 15 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 20 peak 6 eluted later in the elution peak. Thus, the 0.0 – 0.2 M NaCl successfully separated different charge variants of Arginase 1. [00211] 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 25 manufacture of drug substance or drug product. [00212] 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 5 (2X) gluconylated Arginase 1. [00213] 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 10 specific activity. Depending upon the desired characteristics different fractions (corresponding to Peaks 1-6) can be collected for further processing. [00214] Table 12: LC/MS analysis of drug intermediate Peaks 1 – 6. ^ ^+^ 15 ^ [00215] 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. [00216] Varying the load factor of Column 1 and using a NaCl gradient over Column 1 20 can compensate for unexpected perturbations experienced during E. coli fermentation that produce gluconylated Arginase 1 species. [00217] Example 15: Variation in Fermentation Conditions [00218] Experiments were performed to determine the robustness of fermentation 25 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 5 higher proportion of phospho-gluconylated adducts (23% vs 10-12% in control runs) [00219] Table 13: Gluconylated Arginase 1 Observed in Fermentation Vessels ^^^ ^^ ^ 10 [00220] Example 16: Variation of Load Factor on Column 1 [00221] 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 15 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%. [00222] Table 14: Effect of Column 1 Load Factor on Protein Product 1

[00223] Example 17: Phase 1/2 Clinical Investigation [00224] 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 5 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 10 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). [00225] 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 5 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. [00226] 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 10 concentration of 1 mg/mL. The drug was formulated in 50 mM NaCl, 1 mM K2HPO4, 4 mM KH2PO4, and 1.5% w/v glycerol. [00227] 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 the graphic below: 15 [00228] 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 20 continued dosing. [00229] 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 ^M for at least 40 (± 2) consecutive hours post-dosing for all samples collected during that 5 time period or the patient's plasma arginine level averaged <115 ^M for at least 112 (± 2) consecutive hours post-dosing for all samples collected during that time period. [00230] 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. 10 Ultimately, for treatment purposes, it is also possible that the dosing might increase over 0.30 mg/kg. [00231] 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 ^M during repeat-dose administration, with emphasis on maintaining pre-dose levels 15 below 150–200 ^M. 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. [00232] Patients who completed Part 2 of 101A were eligible to participate in a long- 20 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. [00233] Results [00234] Increases in mean Cmax and mean AUC0-168 appeared dose proportional in all patients. Mean (± SD) Cmax was 0.428 ± 0.0915, 0.723 ± 0.247, 1.73 ± 0.538, 2.27 ± 0.238, and 25 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). [00235] 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 30 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. [00236] 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 5 in Part 2, increases in mean Cmax appeared dose proportional in all patients. Mean (± SD) Cmax 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. [00237] In Part 2, Week 8, mean circulating drug concentrations in all patients generally 10 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 Cmax and AUC0-168 appeared dose proportional in all patients. [00238] In addition to pharmacokinetic data, pharmacodynamic (arginine) data was 15 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 20 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. [00239] Overall, exposure to Co-rhARG1-PEG generally increased and there was increased arginine suppression with escalating dose. Individualized dose optimization was 25 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. [00240] Example 18: Subcutaneous Administration [00241] After Part 2 of the Phase 1/2 study of Example 17 was complete, some patients 30 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. [00242] The subcutaneous administration of Co-rhARG1-PEG maintained patient 5 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 ^M and 115 ^M (during repeat-dose administration), with emphasis on maintaining levels below the pre-dose 150–200 ^M. As can be seen in Figure 11, subcutaneous administration of Co-rhARG1-PEG results in arginine concentrations 10 above the lower level of 40 ^M and below the upper level of 115 ^M. 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. 15 [00243] Example 19: Pharmacodynamic and Clinical Responses From Phase 1/2 Clinical Study and Open Label Extension [00244] 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 20 Part 2 advanced into the OLE trial. [00245] Baseline characteristics for the patients are shown in Table 15. [00246] Table 15: Baseline Characteristics [00247] 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 5 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- ^-acetyl-L-arginine (NAA), ^-keto-^-guanidinovaleric acid (GVA) and argininic acid (ARGA) at baseline and the reduction of plasma GC levels during the OLE. 10 [00248] 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 15 (NCT03921541). Example 20: Use of Pegzilarginase for the Treatment of Coronavirus Disease Such As COVID-19 [00249] Pegzilarginase is an engineered wild-type human arginase enzyme, altered both 20 by replacing its manganese cofactor with cobalt and by pegylating the molecule. This enzyme possesses high catalytic activity, serum stability, and a PK profile that entails once-per-week dosing; pegzilarginase is capable of rapid and sustained depletion of plasma arginine. In addition, the safety profile for pegzilarginase has been well-established in a variety of therapeutic contexts. Importantly, monotherapy dose escalation safety trials in the oncology 25 space, wherein patients’ baseline arginine levels are similar to those of healthy patients, have established that doses of 0.2 -0.27 mg/kg delivered intravenously once weekly are well- tolerated, safe, and able to provide sustained arginine depletion. [00250] Additionally, a combination study with pembrolizumab (#NCT03371979) showed an increase in disease control relative to measures of single-agent pembrolizumab activity in SCLC: The combination of pegzilarginase with pembrolizumab in an unselected small-cell lung cancer (SCLC) patient population shows a disease control rate (DCR) of 46% 5 (NCT03371979, study ongoing). In comparison, single-agent pembrolizumab evinced a DCR of 30% in an unselected SCLC patient cohort (KEYNOTE-158) and a DCR of 37.5% in a trial of PD-L1+ SCLC (KEYNOTE-028). This suggests that pegzilarginase does not impair the adaptive arm of the immune response responsible for tumor control in immune checkpoint blockade therapy, e.g. pembrolizumab. 10 [00251] While not wishing to be bound by any particular theory, based on the established effects of arginine deprivation on cellular metabolic pathways and on existing knowledge concerning viral replication cycles, it is believed that pegzilarginase may potentially exert both direct and indirect antiviral activity. [00252] Direct antiviral activity may occur through inhibition of protein translation, as 15 viruses are obligated to use host translational machinery to synthesize proteins, and the amino acid arginine is an essential constituent of all of the putative proteins in the SARS-C0V-2 genome (GenBank accession number MN908947). [00253] Arginine depletion alters several key cellular metabolic programs, including the pyrimidine nucleotide synthesis pathway, as cells that lack expression of the enzyme 20 argininosuccinate synthase 1 (ASS1), e.g. epithelial cells of the lower respiratory tract (Human Protein Atlas data), are forced to express this protein in order to synthesize arginine in the absence of an extracellular arginine reservoir from which to draw. Aspartate that would otherwise be used to generate pyrimidines is then funneled into arginine synthesis (Rabinovich et al.). In support of the concept of arginase-mediated direct antiviral activity, arginase been 25 shown to limit viral load in vitro in herpes simplex virus (Dulfary Sanchez et al.), where it was found to have greater antiviral activity than acyclovir. Additionally, in vivo, arginase reduced viral load in patients with comorbid hepatocellular carcinoma and hepatitis C (Izzo et al.). However, arginine depletion has yet to be tested in the context of SARS-CoV-2 infection. [00254] Also, because of the potential for pegzilarginase to limit synthesis of 30 nucleotides necessary for replication of the viral genome, this mechanism of action may synergize with other antiviral therapies. Remdesivir, a purine nucleotide analog that acts to inhibit RNA polymerase in the related coronavirus that causes Middle East Respiratory Syndrome (MERS) (Gordon et al.), essentially “poisons” the purine nucleotide pool. Thus, its efficacy may be enhanced by co-depletion of the pyrimidine nucleotide pool through degradation of arginine. 5 [00255] Moreover, indirect arginase antiviral activity may occur through immunomodulatory mechanisms. Nitric oxide (NO) is a key inflammatory mediator, and production of nitric oxide by pro-inflammatory type M1 macrophages (via inducible nitric oxide synthase; iNOS) and endothelial cells (endothelial nitic oxide synthase; eNOS) may contribute to the inflammatory phenotype. In COVID-19, the lung microenvironment is often 10 characterized by a hyperinflammatory state that is associated with acute tissue injury and poor patient outcome (Fu et al.). L-arginine is the substrate for the NOS-mediated production of nitric oxide, thus, depletion of circulating arginine may curb the inflammatory response in the lung by limiting NO production. [00256] Depletion of extracellular arginine causes mammalian cells to initiate autophagy 15 (Kim et al.), the process of “self eating” that may be used to recycle macromolecules. Autophagy has been demonstrated to increase the display and presentation of antigen to immune cells of the adaptive immune system, thereby enhancing the antiviral immune response (Romao et al., Paludan et al.). [00257] Accordingly, the methods described herein are expected to be useful for the 20 treatment of coronavirus diseases such as COVID-19. [00258] An exemplary protocol for a trial to investigate the efficacy of pegzilarginase for the treatment of COVID-19 is shown in Figure 15. In this exemplary trial protocol, the key inclusion criteria include confirmation of SARS-CoV-2 infection via PCR, hospitalization with fever, SpO2>94% on air and pulmonary infiltrates. The key exclusion criteria include the 25 subject being unlikely to survive more than 48 hours, SpO2<94% on air, known hypersensitivity to polyethylene glycol (PEG), or the subject is unable to provide consent. The subjects will be administered IV pegzilarginase at a dose of 0.27 mg/kg or placebo. The primary outcome includes clinical status at day 14 (6 pt ordinal). The secondary outcomes includes time to hospital discharge, normalization of fever, or mechanical ventilation/death; 6 30 pt ordinal over time; viral load reduction and/or % of patients with viral clearance; and safety and tolerability. [00259] Example 21: Pegzilarginase In Vitro Suppression of Coronavirus using Pegzilarginase Monotherapy [00260] Experimental Rationale 5 [00261] Suppression of coronavirus growth can be detected in cell lines that are susceptible to infection. Viral expansion can be assessed by several different techniques such as Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) which detects the viral genome; Western blot which detects viral protein using, for example, the anti-SARS-CoV-2 nucleocapsid antibody; immunofluorescence (IF) to detect viral protein using anti-SARS-CoV- 10 2 nucleocapsid antibody; and viral titer by applying supernatant in a limiting dilution series to fresh cultures of permissive cells and measuring plaques, i.e., areas of local cell death. [00262] Infection after low-multiplicity inoculation with severe acute respiratory syndrome coronavirus (SARS-CoV) has been shown in African Green monkey kidney epithelial cell lines (Vero, Vero E6), human colon adenocarcinoma cell line (CaCo-2), Human 15 liver carcinoma cell line (HepG2), and Human lung epithelial cancer cell line (Calu-3). [00263] The cellular entry point for SARS-CoV and SARS-CoV-2 (which causes COVID-19 disease) is the same (human Angiotensin-converting enzyme 2; hACE2). Given the genetic similarity between SARS-CoV-2 and SARS-CoV, upon low-multiplicity inoculation with SARS-CoV-2, infection is expected to similarly progress in the above cell 20 lines. [00264] In addition to using highly pathogenic strains of coronavirus for experiments, other less pathogenic strains of coronavirus can be used to predict the effect of Pegzilarginase on SARS-CoV-2. Other members of the family Coronaviridae include human coronavirus OC43 (HCoV-OC43), which is a beta coronavirus (^-CoV); human coronavirus HKU1 25 (HCoV-HKU1), ^-CoV; human coronavirus 229E (HCoV-229E), which is an alpha coronavirus (^-CoV); human coronavirus NL63 (HCoV-NL63), ^-CoV. These strains, which cause mild symptoms, offer the advantage of handling in a lower bio-security lab facility while still modelling the effectiveness of arginase treatment of SARS-CoV-2. [00265] To confirm the extent of viral expansion in various cell lines, cells are initially 30 grown in appropriate media to approximately 60%, 70%, 80%, 90% confluence then coronavirus, for example SARS-CoV-2, is introduced to the cells. The cell monolayers are infected with coronavirus at low multiplicity of infection (MOI) of, for example, 0.001-0.005; 0.005-0.01; 0.01-0.1; 0.1-0.5; 0.5-2; 2-10. A typical low MOI is 0.1 infectious viral units/cell. Cells can be examined periodically (e.g. daily) for cytopathic effect (CPE). When CPE reaches approximately 75% to 90%, the cell culture medium is harvested for virus titration by using 5 50% tissue culture infective/infectious dose (TCID50) assay or plaque assay (for example on Vero E6 cells). If no CPE is observed, culture supernatant is harvested, typically six days post- infection (dpi) but possibly sooner or later than three to six dpi. [00266] The same monitoring of viral expansion can be performed under conditions limiting viral access to the amino acid arginine. By comparing the viral expansion in the 10 presence versus the absence of arginine, or by comparing viral expansion with an abundance of arginine or a shortage of arginine, or using a negative control where enzymatically deactivated Pegzilarginase (mock enzyme) is used, the effect of arginase-1 treatment on viral replication can be assessed. The concentration in vitro of arginine under limiting conditions predicts the effectiveness of arginase-1 treatment in vivo. Arginase-1 can be introduced to the cultured cells 15 anytime between zero to eight hours prior to the introduction of coronavirus or SARS-CoV-2. Alternatively, Arginase 1 can be used to treat the infected cell line after introduction of coronavirus and then the suppression on an already expanding population of virus can be assessed. A single dose of arginase or multiple doses are used to maintain a culture environment of active arginase. Multiple doses between 1ng/mL and 10ug/mL of 20 pegzilarginase can be introduced into cell culture once, twice, etc. over a one, two, three etc. hour period. Alternatively, a single dose of between 1ng/mL and 10ug/mL of pegzilarginase can be introduced once into cell culture to cover the entire duration of the in vitro experiment. [00267] Cells treated with arginase are monitored for signs of cytotoxicity or loss of cell viability due to arginase treatment in the absence of virus. This provides one control to 25 differentiate between virus-associated damage to cells verses any potential damage caused by treatment. Lack of treatment-associated cellular damage is also an initial indicator of in vivo safety. Previous studies have indicated that there is no cellular damage caused by Pegzilarginase. [00268] Experimental Design 30 [00269] The experimental design for measuring the effect of suppression of coronavirus using pegzilarginase monotherapy is as follows. [00270] Successful treatment (e.g. suppression of viral replication) will be assessed by using any of the following endpoints: qRT-PCR (to detect viral genome), Western blot (using anti-coronavirus antibody such as anti-SARS-CoV-2 nucleocapsid antibody), immunofluorescence (IF, using anti-coronavirus antibody such as anti-SARS-CoV-2 5 nucleocapsid antibody), viral titer (by applying supernatant in a limiting dilution series to fresh cultures of permissive cells and measuring plaques, i.e., areas of local cell death. Coronaviruses other than SARS-CoV-2 may also be used in addition to or instead of SARS- CoV-2, including alpha or beta coronaviruses such as HCoV-229E, HCoV-NL63, HCoV- OC43 and/or HCoV-HKU1, as well as appropriate antibodies for Western blot and IF. 10 [00271] Cell line models to be used include Vero/VeroE6 (Green Monkey kidney epithelial cell lines), HepG2 (Human liver carcinoma cell line), CaCo-2 (Human colon adenocarcinoma cell line) and/or Calu-3 (Human lung epithelial cancer cell line). [00272] Cells will be cultured at 37^ in appropriate media until cells reach approximately 70% confluence. Between 8 and 0 hours prior to viral infection of the cell 15 culture, pegzilarginase will be added to the culture media at a range of doses including one or more of 1ng/mL, 10 ng/mL, 100 ng/mL, 1 ug/mL and 10ug/mL. After addition of Pegzilarginase, the cells will be infected with coronavirus (e.g. SARS-CoV-2 virus) at a multiplicity of infection (MOI) of 0.1 infectious viral units/cell. [00273] For IF, fix and stain cells at 24 hours after infection; for Western blot (WB), 20 lyse cells and perform WB at 24 hours after infection; for viral titering, collect supernatant after observing virally induced cytopathic affect, add the collected supernatant to permissive cultured cells in a limiting dilution series, count plaques at a predetermined cytopathic effect time point later; for qRT-PCR, collect cells or cell media supernatant and extract RNA, perform cDNA synthesis, then detect/quantify viral genome using qPCR 25 [00274] Example 22: In Vitro Suppression of Coronavirus using Pegzilarginase Combination Treatment with Remdesivir [00275] The experimental design for a study measuring the effect in vitro suppression of coronavirus using pegzilarginase combination treatment with remdesivir is as follows. 30 [00276] Successful suppression of viral replication will be assessed by using any of the following endpoints: qRT-PCR (to detect viral genome), Western blot (using anti-coronavirus antibody such as anti-SARS-CoV-2 nucleocapsid antibody), immunofluorescence (IF, using anti-coronavirus such as antibody anti-SARS-CoV-2 nucleocapsid antibody), viral titer (by applying supernatant in a limiting dilution series to fresh cultures of permissive cells and measuring plaques, i.e., areas of local cell death. Coronaviruses other than SARS-CoV-2 may 5 also be used in addition to or instead of SARS-CoV-2, including alpha or beta coronaviruses such as HCoV-229E, HCoV-NL63, HCoV-OC43 and/or HCoV-HKU1, as well as appropriate antibodies for Western blot and IF. [00277] Cell line models to be used include Vero/VeroE6 (Green Monkey kidney epithelial cell lines), HepG2 (Human liver carcinoma cell line), CaCo-2 (Human colon 10 [00278] Cells will be cultured at 37^ in appropriate media until cells reach approximately 70% confluence. Anytime between 8 and 0 hours before viral infection, will treat cell culture with a range of doses (multiple doses between 1ng/mL and 10ug/mL) of pegzilarginase, with remdesivir (at 5µM or the equivalent of a 1.25 mg/kg in vivo dose), or the combination of pegzilarginase (covering the dose range as used above for single-agent dosing)15 plus remdesivir (5µM or 1.25 mg/kg). Then cells will be infected with coronavirus (e.g. SARS- CoV-2 virus) at a MOI of 0.1 infectious viral units/cell. [00279] For IF, fix and stain cells at 24 hours after infection; for Western blot, lyse cells and perform WB at 24 hours after infection; for viral titering, collect supernatant after observing virally induced cytopathic effects, add to permissive cultured cells in a limiting 20 dilution series, count plaques 24-72 hours later. [00280] Example 23 Coronavirus Infection in Mouse Models [00281] Study Rationale [00282] Human angiotensin-converting enzyme 2 (ACE2) is a type I transmembrane 25 metallocarboxypeptidase with homology to Angiotensin-converting enzyme (ACE). ACE2 is attached to the outer surface of cells in a variety of organs including in the lungs, arteries, heart, kidney, and intestines. ACE2 has been confirmed as the entry point for coronaviruses including HCoV-NL63, SARS-CoV and SARS-CoV-2. [00283] Cells resistant to coronavirus infection become permissive to infection upon30 incorporation of human ACE2 into the cell membrane. Incorporation of the human ACE2- coding DNA sequence into the genome of wild-type mice similarly create hACE2 transgenic mouse strains that model coronavirus infection e.g. with SARS-CoV-2. This model of lethal infection with SARS-CoV will similarly be useful for SARS-CoV-2 studies of pathogenesis and for the assessment of antiviral therapy with arginase. [00284] Mouse Models of Coronavirus Infection 5 [00285] An example of a coronavirus animal model is the transgenic Keratin-18 human ACE2 transgenic mouse model McCray et al. In this model, ACE2 expression is regulated by the human cytokeratin 18 (K18) promoter in epithelial cells. When infected with SARS-CoV, these animals show a prominent infection in the airway epithelia with subsequent alveolar involvement and extrapulmonary virus spreading to the brain. Infection results in macrophage 10 and lymphocyte infiltration in the lungs and upregulation of proinflammatory cytokines and chemokines in both the lung and the brain. These characteristics are similar to COVID-19 presentation in humans and the presence of ACE2 on the surface of cells make these animals susceptible to SARS-CoV-2. [00286] Another example of a coronavirus-susceptible animal model is the Human 15 ACE2 transgenic knock-in mouse model (Yang et al). The hACE2 gene is expressed in a range of tissues and organs including in lung, heart, kidney, and intestine. These transgenic mice are susceptible to SARS-CoV infection which is associated with severe pathologic changes that resembled human SARS infection. The presence of ACE2 on the surface of cells make these animals susceptible to SARS-CoV-2. 20 [00287] A typical experimental design for a transgenic mouse model of coronavirus infection measures pathological markers of disease which are assessed in addition to a survival study. Typically, eight-week-old transgenic mice are inoculated intranasally with, for example, 210,000 plaque-forming units (PFU) of SARS-CoV-2 (or alternatively a less pathogenic strain of coronavirus). The ideal number of PFUs to infect a transgenic mouse can be determined by 25 titrating the amount of virus introduced into the mouse for each route of administration. For example, a range of PFU of one to one million PFU will inform the level of pathogenesis in the infected mouse as well as the rate of viral expansion or survival time. For example, the number of PFUs in an intranasal inoculum could be 1, 10, 100, 100,000, or 1,000,00 or any combination in between these PFU amounts. A control group of mice can be similarly be 30 inoculated with mock or inactive virus or carrier liquid that does not contain any virus. Infected mice then receive active or mock treatment. [00288] Treated mice will have arginase introduced by any route that is effective including intravenously, subcutaneously, and intraperitoneally. The arginase treated group of animals inoculated with SARS-CoV-2 virus will be treated with a dose of Pegzilarginase equivalent to, for example, 3mg/kg pegzilarginase 6 hours prior to the time of inoculation. An 5 alternative dosing strategy is a dose of pegzilarginase equivalent to a single dose of, for example, 0.1, 1.0, 2.0, 3.0, 4.0, or 5.0 mg/kg. The timing of treatment is flexible and can be, for example, 6, 5, 4, 3, 2, 1, or 0 hours prior to inoculation. Alternatively, multiple doses of pegzilarginase can be given both prior, and after, inoculation with coronavirus. [00289] Animal health is monitored, and body weight measured daily, morbidity is 10 observed, and animals sacrificed if moribund. Animal tissue is collected for assessment of viral load and assessment of pathological effects and immunological profile. Several different methods are used to assess pathology including fixing tissues for histological examination. Studies are also performed to determine the increased survival of animals treated with arginase. [00290] In a typical experiment, between days 3 and 5 post-infection nasopharyngeal 15 fluid is collected and the level of viral load (for example the virus titer) is quantified by adding the collected fluid to freshly cultured Vero cells in a limiting dilution assay. Blood is also collected for immunophenotyping (discrimination of immune cell populations by antibody- binding profile of cell surface markers), cytokine analysis, and measurement of nitric oxide (NO). Upon euthanization, animal organs are preserved for a range of measurements including 20 lung immunophenotyping, qRT-PCR measurement of viral load, organ immunophenotyping, and measurement of tissue NO levels. [00291] Study Design [00292] The experimental design for a study measuring the effect of treatment on survival is as follows. Groups of eight-week-old transgenic mice will be intranasally inoculated 25 with 30µL of DMEM with or without virus. The group with virus will receive 30µL of 7x10 ⁶ plaque-forming units (PFU) per mL of SARS-CoV-2 virus (and/or a less pathogenic strain such as HCoV-229E, HCoV-NL63, HCoV-OC43 and/or HCoV-HKU1). Half of the animals inoculated with SARS-CoV-2 virus will be treated with a single dose of 3mg/kg pegzilarginase six hours prior to the time of inoculation. The other half of animals will be treated with a mock 30 treatment or inactivated enzyme or with a carrier such as buffer. Animal health will be monitored, body weight recorded daily, and moribund mice will be sacrificed as appropriate noting the day of sacrifice post-inoculation. At the time of sacrifice lung tissue will be collected, fixed, and examined histologically. [00293] The experimental design for a study specifically assessing pathology of coronavirus infection is as follows. Inoculate 8-week old transgenic mice intranasally with 5 30µL of DMEM with or without 7x10 ⁶ PFU/mL of SARS-CoV-2 virus. Half of the SARS- CoV-2 virus-inoculated animals will be treated with a single dose of 3mg/kg pegzilarginase six hours prior to the time of inoculation. [00294] On days 3 and 5 post-infection, nasopharyngeal fluid will be collected and the viral titer determined by adding the fluid to freshly cultured Vero cells in a limiting dilution 10 assay. Blood will be collected and immunophenotyped, cytokines analyzed, and nitric oxide (NO) measured. Organs (for example, lung, kidney, brain) will be collected from euthanized animals and these organs will be used for immunophenotyping, measuring viral load via qRT- PCR, and measurement of tissue nitric oxide (NO) levels. 15 [00295] Example 24 Spontaneous Infection in Ferrets [00296] Study Rationale [00297] Ferrets (for example Mustela putorius furo) provide an appropriate model of SARS-CoV-2 infection and transmission that recapitulates aspects of the human disease. Infection can be induced via intranasal inoculation with a small volume of liquid containing 20 SARS-CoV-2 (typical volumes are in the range of 0.2 mL and 0.5 mL). Upon infection with SARS-CoV-2 ferrets exhibit elevated body temperature and virus replication. Infected ferrets shed virus in nasal washes, saliva, urine, and feces up to 8 days post-infection. By two days post-contact, SARS-CoV-2 is detected in all naive direct contact uninoculated littermates. Also, indirect contact ferrets are positive for viral RNA suggesting this model recapitulates 25 airborne transmission observed in human disease. Viral antigens are detected in nasal turbinate, trachea, lungs, and intestine with acute bronchiolitis present in infected lungs. Thus, ferrets represent an infection and transmission animal model of COVID-19 that confirms the anti-viral effect of Pegzilarginase. [00298] Ferret SARS-CoV-2 infection also recapitulates the human COVID-19 disease 30 by showing gastrointestinal involvement and viral load in this compartment. Therefore, viral load in fecal and urine specimens from infected ferrets are also a useful measure of disease. Further recapitulation of human disease is shown by ferrets experiencing lung histopathology upon infection. At day four post-infection ferrets show increased immune infiltration and cell debris in the alveolar wall, bronchial epithelium, and bronchial lumen, evidencing an acute bronchiolitis caused by coronavirus infection. SARS-CoV-2-infected ferrets show high virus 5 titers in upper respiratory tracts (nasal washes) and consequently transmitted to naive ferrets by direct contact at high efficiency, suggesting that SARS-CoV-2 ferret model recapitulates aspects of human infection and transmission. [00299] Treatment of infected ferrets can be by single or multiple doses of arginase, particularly Pegzilarginase, given by the intravenous, subcutaneous, or ntraperitonealroute. 10 [00300] A variety of in vivo measurements can be made in animal models to indicate successful treatment, also called endpoints. Endpoints that can be used to assess the anti-viral effects of Pegzilarginase include animal survival, suppression of loss of body weight which animals typically experience upon exposure to a pathogens, titres of virus in various tissues and fluids in the animal (viral titer in these samples can be assessed using Vero or VeroE6 15 cells), an example of an animal fluid that can be assessed for viral titer is nasopharyngeal fluid. Other endpoint measurements include determining viral load in lung tissue via, for example, qRT-PCR; immunophenotyping of myeloid and lymphoid immune cells in peripheral blood and lung tissues; inflammatory cytokine measurements in peripheral blood and lung tissue; nitric oxide (NO; actually downstream nitrate and nitrite metabolites) measurement in samples, 20 or example in peripheral blood and lung tissue; lung histology can also be examined to differentiate successful treatment compared to typical pathology (stains such as hematoxylin and eosin staining can be done on formalin-fixed, paraffin-embedded tissues). Additional clinical symptoms for animal models include sneezing, coughing, nasal discharge, lethargy, fever, viral shedding and transmission to liter mates. 25 [00301] Study Design [00302] SARS-CoV-2 is recapitulated in the ferret model of disease. SARS-CoV-2- infected ferrets exhibit elevated body temperatures and virus replication. SARS-CoV-2- infected ferrets shed virus in nasal washes (as well as saliva, urine, and feces) and the disease can be transmitted to litter mates as well as likely transmitted to ferrets in adjacent housing that 30 is merely separated via an air permeable partition. [00303] Ferrets will be inoculated via the intranasal (IN) route with 10 ^5.5 TCID50 of SARS-CoV-2 (NMC-nCoV02) and/or coronaviruses other than SARS-CoV-2 may also be used in addition to or instead of SARS-CoV-2, including alpha or beta coronaviruses such as HCoV-229E, HCoV-NL63, HCoV-OC43 and/or HCoV-HKU1. One group of infected ferrets 5 will receive a single intravenous dose of 3mg/kg pegzilarginase six hours prior to intranasal inoculation whereas one group will receive PBS. Another group will receive a combination of 3mg/kg pegzilarginase and an initial intravenous or peritoneal dose of 2.5 mg/kg of remdesivir at the same time pegzilarginase is first administered. Subsequent doses of remdesivir will be given at 1.25 mg/kg at the same time as pegzilarginase or daily for up to nine days post- 10 infection. [00304] To investigate SARS-CoV-2 replication and shedding in each group of ferrets, a variety of samples will be collected, including blood, nasal washes, saliva, urine, and fecal specimens for up to 12 days post-inoculation (and including samples taken prior to infection). Samples can be collected every day or every other day post-infection. Collected ferret 15 secretions will be resuspended in cold phosphate-buffered saline (PBS) containing antibiotics (5% penicillin/streptomycin). [00305] To further assess viral replication in infected ferret organs, additional ferrets (both treated and PBS treated) will be sacrificed at 4, 8, and 12 days post-infection. Nasal turbinate, trachea, lung, kidney, and intestine tissues will be collected from each animal. 20 [00306] For titration of virus using RNA, total RNA will be extracted from samples, cDNA will be synthesized, and viral RNA copy number will be quantitated using quantitative real-time RT-PCR (qRT-PCR) targeted towards the spike protein and open reading frame (ORF1a). The number of viral RNA copies will be calculated by comparison to the number of copies in a standard control. 25 [00307] To evaluate the infectious virus titer in each specimen, collected nasal washes and saliva specimens will be assessed by plaque assay using Vero cells. [00308] The temperature of the ferrets will be measured beginning at day 0 until day 9 post-inoculation. Elevation of body temperatures, such as from 38.1°C to 40.3°C, is indicative of infection. The temperatures of the infected animals will be compared to uninfected animals. 30 [00309] To further assess the effects of infection and treatment in ferrets, immunohistochemistry (IHC) and histopathological examinations will be conducted. Tissue samples will be collected from sacrificed ferrets and incubated in 10% neutral-buffered formalin (which also achieves virus inactivation) for tissue fixation before they are embedded in paraffin. The embedded tissues will be sectioned and dried for 3 days at room temperature. [00310] To detect the viral antigens by IHC, mouse polyclonal antibody raised against 5 inactivated SARS-CoV-2 virions will be used as a primary antibody. IHC analyses shows number of cells in the nasal turbinate, trachea, lung, and intestine sections of infected ferrets positive for SARS-CoV-2 antigen (successful treatment of SARS-CoV-2 infection is confirmed by a reduction in the number of positive cells upon treatment). [00311] Lung histopathology progresses without intervention such that by day 4 post- 10 infection there is increased immune infiltration and cell debris in the alveolar wall, bronchial epithelium, and bronchial lumen (acute bronchiolitis). Successful treatment will be confirmed by reduction in these clinical markers of disease. References 15 Chu et al., The SARS-CoV ferret model in an infection–challenge study, https://www.sciencedirect.com/science/article/pii/S004268220 7008458. Dulfary Sanchez M et al. Antiviral Research 2016; 132: 13-25. Fu et al. Virologica Sinica 2020; e-pub ahead of print. Gordon et al. Journal of Biological Chemistry 2020; e-pub ahead of print. 20 The Human Protein Atlas; www.proteinatlas.org. Izzo F et al. Journal of Gastroenterology and Hepatology 2007; 22(1): 86-91. Kim et al. Autophagy 2009 May; 5(4): 567-568. Kim et al., 2020, Cell Host & Microbe 27, 1–6, https://doi.org/10.1016/j.chom.2020.03.023. Liu et al. Lancet Infect Dis 2020; Viral dynamics in mild and severe cases of COVID-19. 25 McCray et al. Paludan C et al. Science 2005; 307: 593-596. Rabinovich et al. Nature 2015; 527(7578): 379-383. Romao S et al. Semin Cancer Biol 2013; 23: 391-396. Yang et al 30 [00312] Example 25 Comparative Study between Pegzilarginase and Remdesivir for In Vitro Suppression of Coronavirus in Calu-3 Cells [00313] Study Rationale [00314] A variety of cell types can be used in vitro to study SARS-CoV-2. Calu-3 cells 5 are human lung adenocarcinoma cells derived from metastatic pleural effusion. These epithelial cells can be used for growth of SARS-CoV-2 and measurement of inhibitory properties of potential therapeutics. The cells are adherent in cell culture which facilitates immunohistochemical staining techniques to visualize viral infection and quantitate the level of viral suppression. 10 [00315] Calu-3 cells can be grown in Eagle’s Minimum Essential Medium optionally supplemented with 10% fetal bovine serum. Cell monolayers can be detached from culture flasks with 0.25% (w/v) Trypsin – 0.53 mM EDTA solution for approximately 5-15 minutes until the cell layer is dispersed. These cells are susceptible to infection by a variety of coronavirus types including USA-WA1/2020. 15 [00316] SARS-CoV-2 Isolate: (USA-WA1/2020) is an enveloped, positive-sense single- stranded RNA virus from the Coronaviridae family and the Betacoronaviridae genus. USA- WA1/2020 is a US reference strain used in research, drug discovery, and vaccine testing. When cells are infected with coronavirus, the strain typically shows cytopathic effect observable on the second day of infection of the cell culture. This strain can also be used for 20 plaque assays. [00317] Experimental Design [00318] Calu-3 cells were plated in 384 well plates. After culturing for approximately 18 hours, test compounds were added to sequential wells in descending concentration. Remdesivir was resuspended in DMSO, starting at no more than 50µM final concentration. Negative 25 controls (DMSO or pegzilarginase formulation buffer) and positive controls (remdesivir) were included in the plates. Calu3 (ATCC, HTB-55) cells were pretreated with test compounds for 2 hours prior to infection with SARS-CoV-2 (isolate USA WA1/2020) at a MOI=0.5. Forty- eight hours post-infection cells were fixed and processed for automated microscopy for quantification of infection (dsRNA+ cells/total cell number) and cell number. For the 30 automated microscopy, the fixed cells were incubated with a fluorophore-tagged RNA probe specific to the SARS-CoV-2 RNA genome, then washed, counterstained, and finally imaged by automated microscopy. Quantification of infection was based upon staining of viral genome using RNA and calculated as a percentage using the total cell number. Sample well data was normalized to aggregated DMSO control wells. [00319] In a particular embodiment, Calu-3 cells were treated with various 5 concentrations of pegzilarginase or control. For negative control, the cells were treated with a low concentration of DMSO. The DMSO is expected to have negligible effect on cell growth. The cells were cultured for 48 hours. After 48 hours, viability and % infection were calculated for both pegzilarginase treated cells and DMSO treated cells. The viability (average Percentage of Control (POC)) is the number of viable cells (average of triplicate wells) after 48 hours of 10 culture as a percentage of total observable cells in the culture. Th “% Infection” is an average of the percentage of cells after culture for 48 hours that are infected with SARS-CoV-2 virus compared to the control. The viability and the % infection data of the pegzilarginase treated cells were normalized to the viability and the % infection data of the DMSO treated cells, respectively. Table 16 shows normalized data for viability and % infection. 15 Table 16: Normalized Data ^ Viability is the number of viable cells ^values represent average Percentage of Control (POC)=(sample well measurement/aggregated 20 DMSOavg)*100 for n=3 replicates. [00320] The normalized data was plotted against drug concentration. The plots were used to calculate an IC50 (for infection) and CC50 (toxicity) and the Selective Index (SI). Figure 14(a) shows IC50 (infection: blue squares) and CC50 (toxicity: green circles) for pegzilarginase. Figure 14(b) shows IC50 (infection: blue squares) and CC50 (toxicity: green circles) for remdesivir. [00321] An ideal drug candidate will suppress viral growth at a concentration that has minimal or no effect on cell viability. It is also acceptable that the drug has a cytostatic effect 5 but that any cytotoxic effect is slow to develop. The reduced availability of arginine in vitro may cause cells to slow their growth or stop expanding (i.e. cytostatic effect) but not necessarily cause cell death. Accordingly, this data shows that pegzilarginase has a positive effect in suppressing viral growth at concentrations that have little to no effect on cell viability. 10 [00322] Example 26 Coronavirus Infection in Syrian Golden Hamsters [00323] Study Rationale [00324] Treatment of human SARS-CoV-2 infection with Pegzilarginase will be predicted using Syrian Golden Hamsters. The Syrian Golden Hamster is a powerful model of human disease caused by infection with SARS-CoV-2 virus. Infected animals develop high 15 levels of SARS-Cov-2 in lungs, intestines, and other tissues that display the protein receptor viral entry point, angiotensin-converting enzyme 2 (ACE2). Viral RNA is detected in lung from day 1 post infection and reaches a peak level at day 4 post infection. Furthermore, viral antigens are found in nasal mucus, bronchial epithelial cells, and in lung consolidation 2-5 days post-inoculation with virus. SARS-CoV-2 is also transmitted efficiently between hamsters via 20 aerosols or direct contact. [00325] The intranasal route of administration with SARS-CoV-2 reproducibly leads to broncho-interstitial pneumonia with high viral lung loads and virus shedding. The Syrian hamster is a highly susceptible model with potentially as few as five infectious particles leading to disease (infectious dose 50 (ID50) = 5). 25 [00326] Study Design [00327] Infection and Pathology [00328] Syrian hamsters (Mesocricetus auratus), 6-8 weeks of age are inoculated via intranasal instillation with 50 ul of varying concentrations of inoculum (from ~1 to 1 x 10 ⁵ tissue culture dose 50 (TCID ₅₀ ) into each naris. Hamsters are weighed and monitored for 30 symptoms of disease. Swabs are taken throughout the duration of infection. [00329] Virus titration can be performed in Vero E6 cells. Viral genome load can be determined using RNA extracted from blood, tissues, and swab sample with methods described above including qRT-PCR. [00330] For histopathology and immunohistochemistry tissues are fixed in 10% neutral 5 buffered formalin. [00331] Pegzilarginase Maximum Tolerated Dose (MTD) [00332] The dose range of Pegzilarginase that will not cause harmful side effects will be determined. The ultimate dose will be one that suppresses viral pathology without causing harm to the host. A single high or lose dose (and vehicle control) will be administered to 10 groups of hamsters. Blood (and plasma) will be collected at pre-dose, and 24, 48, 72, and 96 hours in relation to Pegzilarginase administration. Body weights will be concomitantly measured. An MTD would be expected to reduce body weight by less than 20%. [00333] Table 16: Study Design 15 t *single dose [00334] Pegzilarginase Treatment of SARS-CoV-2 Infection [00335] Pegzilarginase (iv) and SARS-CoV-2 (intranasal) will be administered simultaneously at Day 0. The dose range maximum will be no more than the MTD and no less 20 than 0 mg/kg. The high dose can be up to the MTD and the low dose can be anything above 0 mg/kg. Blood samples, body weight, and clinically relevant parameters will be measured until termination of the study at Day 4. [00336] The study endpoints will be viral load (titer) and lung histopathology. Direct viral tittering (TCID50) will be assessed from the lower respiratory tract (lung tissue 25 homogenate as measured using a virally permissible cell line). Viral load will also be assessed using RNA quantification in lung tissue. Lung histopathology will be assessed with H&E staining. [00337] Table 17: Study Design 5 of **single dose [00338] Reference throughout this specification to “one embodiment,” “certain 10 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 15 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. [00339] Although the disclosure herein provided a description with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of 20 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.