METHODS FOR PRODUCTION OF HUMAN RECOMBINANT ARGINASE 1 AND USES THEREOF
Described are methods for producing recombinant Arginase, such as PEGylated, cobalt-substituted recombinant human Arginase 1. Also described are pharmaceutical compositions comprising such recombinant Arginase, as well as methods of treatment and uses of such recombinant Arginase.
1 .- 45 . (canceled)
46 . A composition comprising a recombinant human Arginase protein, wherein the protein comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 1, wherein the protein is in a complex with a non-native metal cofactor, wherein the non-native metal cofactor is cobalt, and wherein the protein is covalently linked to a polyethylene glycol at one or more of the amino acid residues: K16, K32, K38, K40, K47, K67, K74, K82, L87, K88, K152, K154, K171, K222, K223, K312 and K321.
47 . The composition of claim 46 , wherein the protein comprises an amino acid substitution at a position selected from the group consisting of H100, D123, H125, D127, D231, D233, W121, D180, S229, C302, and E255.
48 . The composition of claim 46 , wherein the protein comprises at least one amino acid substitution selected from the group consisting of D180S, S229C, S229G, C302F, C302I, E255Q, D180E, and S229A.
49 . The composition of claim 47 , wherein the amino acid substitution is at C302.
50 . The composition of claim 46 , wherein the protein comprises at least two amino acid substitutions.
51 . The composition of claim 46 , wherein the protein is a truncated arginase I protein.
52 . The composition of claim 46 , wherein the protein further comprises an exogenous protein fragment.
53 . The composition of claim 52 , wherein the exogenous protein fragment comprises the Fc region of an immunoglobulin or a portion of the Fc region of an immunoglobulin.
54 . The composition of claim 46 , wherein the specific activity of the protein is in the range of about 400 U/mg to about 700 U/mg.
55 . The composition of claim 46 , wherein the protein displays a k cat /K m for the hydrolysis of arginine in the range of about 200 mM −1 s −1 to about 4,000 mM −1 s −1 at pH 7.4 when assayed in vitro.
56 . The composition of claim 46 , wherein the protein displays a k cat /K m for the hydrolysis of arginine in the range of about 400 mM −1 s −1 to about 2,500 mM −1 s −1 at pH 7.4 when assayed in vitro.
57 . The composition of claim 46 , wherein the molar ratio of PEG to the protein is in the range of about 7 moles/mole to about 15 moles/mole.
58 . The composition of claim 46 , wherein the total cobalt content of the composition is in the range of about 9 pg/mL to about 15 pg/mL.
59 . The composition of claim 46 , wherein the composition produces at least 9 peaks when loaded on imaging capillary isoelectric focusing (iCIEF), wherein peak 1 is less than 20%, peak 2 is less than 30%, peak 3+4 is in the range of 10-30%, peak 5 is in the range of 15-30%, peak 6 is in the range of 10-25%, peak 7 is less than 25%, peak 8 is less than 15%, and peak 9 is less than 8%.
60 . The composition of claim 46 , wherein the composition produces at least 9 peaks when loaded on iCIEF, wherein peak 1 is in the range of 5-7%, peak 2 is in the range of 8-11%, peak 3+4 is in the range of 16-20%, peak 5 is in the range of 21-24%, peak 6 is in the range of 21-22%, peak 7 is in the range of 14-15%, peak 8 is in the range of 5-8%, and peak 9 is in the range of 2-3%.
61 . A pharmaceutical composition comprising the composition of claim 46 and a pharmaceutically acceptable carrier.
62 . The pharmaceutical composition of claim 61 , wherein the composition comprises potassium phosphate, sodium chloride and glycerol.
63 . The pharmaceutical composition of claim 61 , wherein the composition comprises about 50 M NaCl, about 1 mM K 2 HPO 4 , about 4 mM KH 2 PO 4 , and about 1.5% w/v glycerol.
64 . A method of treating Arginase 1 deficiency, the method comprising administering the pharmaceutical composition of claim 61 to a patient.
65 . The method of claim 64 , wherein the pharmaceutical composition is administered intravenously or subcutaneously.
66 . The method of claim 64 , wherein the pharmaceutical composition is administered at a dose of 0.1 mg/kg based on the weight of unPEGylated enzyme.
67 . The method of claim 64 , further comprising monitoring the patient's plasma arginine levels.
68 . The method of claim 64 , wherein the dose is adjusted according to the following algorithm:
(a) if the plasma arginine level is >150 pM, a single 168-hour sample will be used to increase the dose by 2 dose levels in the table below (not to exceed 0.20 mg/kg) if the 2 doses prior to this sample were a) the same dose level in mg/kg, and b) consecutive (with no missed doses);
(b) If the plasma arginine levels from 2 sequential 168-hour samples (regardless of missed doses) are both <50 mM, the dose is decreased by 1 dose level in the table below, not to decrease below 0.05 mg/kg.
Dose Level (a)
Dose
1 (Minimum Possible Dose)
0.05 mg/kg
2 (Starting Dose)
0.10 mg/kg
3
0.15 mg/kg
4 (Maximum Possible Dose)
0.20 mg/kg
(a) Pegzilarginase dosing starts at level 2. 0.10 mg/kg. Dose increases when required, are by 2 dose levels. Dose decreases are by 1 dose level.