Composition and application of arginine depletion agents for cancer, obesity, metabolic disorders, and related complications and comorbidities.

Abstract

To provide arginine depleting proteins with improved pharmacokinetics.SOLUTION: The present disclosure relates to arginase albumin binding domain (ABD) fusion proteins and methods of preparation and use thereof. Also provided are methods involving arginine depletion for the treatment of obesity, metabolic disorders, and related complications and comorbidities.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 678,300, filed on 31 May 2018, and this reference constitutes an integral part of this specification with respect to the entirety of said Provisional Patent Application.

[0002] This disclosure relates to arginase albumin-binding domain (ABD) fusion proteins, and methods for preparing and using the same. Also provided are methods for performing arginine depletion to treat obesity, metabolic disorders, and / or related complications and / or comorbidities. [Background technology]

[0003] Arginase is a hydrolytic enzyme that catalyzes the catabolism of arginine into ornithine and urea, and is present in most organisms. In humans, there are two isoforms of arginase, namely arginase type I and type II, which differ in tissue distribution and molecular characteristics.

[0004] Arginase I is a 35kD protein that is mainly found in the cytoplasm of the liver and exists as a trimer. Arginase II, a protein of approximately 38.5kD, is usually found in the mitochondria of cells and is thought to be involved in regulating intracellular arginine / ornithine concentrations.

[0005] In some types of tumors, arginine nutrition is required because there is a deficiency or low production level of arginine nosuccinate synthetase (ASS) and / or ornithine transcarbamoylase (OTC), which are necessary for arginine synthesis. By depriving these tumor cells of arginine, the significant vulnerability of these tumor cells is exploited, leading to rapid tumor cell death. Therefore, enzyme-mediated arginine depletion has been investigated as a potential strategy for selectively destroying tumor cells.

[0006] The main obstacle to enzyme-mediated arginine depletion in cancer treatment is the short circulating half-life (several minutes to several hours) of arginase due to its low molecular weight (<50 kDa), which leads to rapid excretion by renal clearance. Maintaining effective therapeutic concentrations in systemic circulation may require frequent dosing schedules, which can lead to patient compliance issues.

[0007] Considering this problem, various strategies have been developed to improve the pharmacokinetic properties of therapeutic proteins. It has been shown that PEGylation (i.e., PEGylation) of various therapeutic proteins increases the hydrodynamic radius of the protein, delaying its excretion by renal clearance. Fusion protein strategies based on the recycling mechanism of the neonatal Fc receptor (FcRn) have also been investigated, and it has been shown that fusion of immunoglobulin, albumin, or albumin-binding peptide fragment crystallizable (Fc) regions to the protein increases the circulating half-life of the protein. However, there remains a need for the development of arginine-depleted proteins with improved pharmacokinetics.

[0008] Obesity, metabolic disorders, and associated complications and comorbidities are considered some of the most serious global public health problems of the 21st century and represent a significant socioeconomic burden. Furthermore, the metabolic consequences of obesity can lead to other disorders, such as type 2 diabetes, impaired glucose tolerance, insulin resistance, hypercholesterolemia, dyslipidemia, hypertension, cardiovascular disease, and cancer. Therefore, there is a great need for safe and effective methods for the prevention and / or treatment of obesity, metabolic disorders, and associated complications and comorbidities. Obesity is a result of an imbalance between energy intake and expenditure. Lifestyle interventions alone may not be sufficient to help individuals lose weight sufficiently or sustain weight loss and improve their health. Anti-obesity drugs such as orlistat (Xenical), lorcaserin (Velvic), and phentermine and topiramate combination (Xumia) can reduce appetite and fat absorption, leading to weight loss. However, these drugs have various side effects, including decreased absorption of fat-soluble vitamins, headaches, fatigue, dizziness, diarrhea, and fecal incontinence. Bariatric surgery, such as gastric banding, can treat severe obesity and achieve long-term weight loss, and liposuction, which removes unwanted localized fat deposits, is popular for cosmetic reasons. However, these types of invasive procedures also carry considerable risks. Therefore, there is a need to develop improved methods for treating obesity, metabolic disorders, and associated complications and comorbidities. [Overview of the Initiative]

[0009] To address one or more of the above-mentioned needs, an arginase ABD fusion protein with improved in vivo half-life and arginine catabolic activity is provided. The fusion protein described herein is useful in the treatment of diseases or conditions in which arginine depletion has a therapeutic effect, such as cancer, viral infections, multiple sclerosis, rheumatoid arthritis, autoimmune diseases, congenital hyperargininemia, graft-versus-host disease (GvHD), inflammation, obesity and metabolic disorders, and associated complications and comorbidities. The fusion protein can be rapidly generated and purified from crude protein. The fusion protein can be used alone or in combination with at least one other agent to provide a synergistic effect in the treatment or prevention of disease.

[0010] Furthermore, methods are provided for treating obesity, metabolic disorders, and / or related complications and / or comorbidities by depleting the target arginine concentration (for example, by administering an arginine depleting agent to the subject).

[0011] In the first aspect, a fusion protein comprising an albumin-binding domain (ABD) polypeptide and an arginase polypeptide is provided.

[0012] Provided in the first embodiment of the first aspect is a fusion protein of the first aspect, wherein the ABD polypeptide comprises a polypeptide sequence having at least 93% sequence homology with SEQ ID NO: 66, SEQ ID NO: 67, or SEQ ID NO: 68.

[0013] A second embodiment of the first aspect is provided which a fusion protein of the first aspect is provided in which the arginase polypeptide comprises a polypeptide sequence having at least 95% sequence homology with SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, or SEQ ID NO: 72.

[0014] A third embodiment of the first aspect is provided which a fusion protein of the first aspect is provided in which the ABD polypeptide comprises a polypeptide sequence having at least 93% sequence homology with SEQ ID NO: 66, SEQ ID NO: 67, or SEQ ID NO: 68, and the arginase polypeptide comprises a polypeptide sequence having at least 95% homology with SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, or SEQ ID NO: 72.

[0015] Provided in the fourth embodiment of the first aspect is a fusion protein of the third embodiment of the first aspect, wherein the ABD polypeptide comprises a polypeptide sequence having at least 93% sequence homology with SEQ ID NO: 66, and the arginase polypeptide comprises a polypeptide sequence having at least 95% sequence homology with SEQ ID NO: 69.

[0016] Provided in the fifth embodiment of the first aspect is a fusion protein of the fourth embodiment of the first aspect, further comprising a peptide linker connecting the C-terminus of the ABD polypeptide and the N-terminus of the arginase polypeptide, wherein the peptide linker is a linear polypeptide having 1 to 20 amino acids.

[0017] Provided in the sixth embodiment of the first aspect is a fusion protein of the fifth embodiment of the first aspect, wherein the peptide linker comprises a polypeptide sequence having at least 90% sequence homology with SEQ ID NO: 73 or SEQ ID NO: 74.

[0018] Provided in the seventh embodiment of the first aspect is a fusion protein of the sixth embodiment of the first aspect, wherein the ABD polypeptide comprises a polypeptide sequence having at least 93% sequence homology with SEQ ID NO: 67, and the arginase polypeptide comprises a polypeptide sequence having at least 95% homology with SEQ ID NO: 72.

[0019] Provided in the eighth embodiment of the first aspect is a fusion protein of the seventh embodiment of the first aspect, wherein the peptide linker is a polyhistidine linker having 4 to 8 histidine amino acids.

[0020] A ninth embodiment of the first aspect is provided which is a fusion protein of the first aspect, comprising a polypeptide sequence having at least 98% sequence homology with SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 75, or SEQ ID NO: 76.

[0021] A tenth embodiment of the first aspect is provided which a fusion protein of the first aspect comprises a polypeptide sequence having at least 98% sequence homology with SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 75, and SEQ ID NO: 76.

[0022] Provided in the eleventh embodiment of the first aspect is a fusion protein of the first aspect, comprising a polypeptide selected from the group consisting of SEQ ID NOs: 49, SEQ ID NOs: 50, SEQ ID NOs: 75, and SEQ ID NOs: 76.

[0023] In the second aspect, a pharmaceutical composition is provided comprising the fusion protein of the first aspect and a pharmaceutically acceptable carrier, excipient, or combination thereof.

[0024] Provided in the third aspect is a method for treating cancer in a subject requiring cancer treatment, the method comprising the step of administering a therapeutically effective amount of the fusion protein of the first aspect to the subject.

[0025] Provided in the fourth aspect is a method for treating a condition in a subject requiring treatment of at least one condition selected from the group consisting of obesity, metabolic disorders and related complications, the method comprising the step of administering a therapeutically effective amount of an arginine depletion agent to the subject.

[0026] Provided in the first embodiment of the fourth aspect is a method in which the metabolic disorder is selected from the group consisting of obesity, impaired glucose tolerance, hyperglycemia and diabetes mellitus, and the associated complication is one or more pathological conditions selected from the group consisting of diabetic nephropathy, diabetic retinopathy, diabetic vascular disorder, diabetic neuropathy, hypercholesterolemia, dyslipidemia, hypertriglyceridemia, hyperleptinemia, steatosis, steatohepatitis, fibrosis, cirrhosis, chronic low-grade inflammation, hypertension, cardiovascular disease and whitening of brown fat.

[0027] A second embodiment of the fourth aspect provides a method in which the metabolic disorder is insulin resistance.

[0028] A third embodiment of the fourth aspect provides a method for treating obesity that includes at least one of preventing an increase in fat mass and reducing fat mass.

[0029] A fourth embodiment of the fourth aspect provides a method for selecting a metabolic disorder from the group consisting of fatty liver, renal steatosis, pancreatic steatosis, and cardiac steatosis.

[0030] A fifth embodiment of the fourth aspect provides a method for maintaining the arginine concentration in the serum of a subject at less than 50 μM.

[0031] A sixth embodiment of the fourth aspect is provided in which the arginine depletion agent is an arginine catabolic enzyme.

[0032] Provided in the seventh embodiment of the fourth aspect is the method of the sixth embodiment of the fourth aspect, wherein the arginine catabolic enzyme is an arginase protein, an arginine deiminase protein, or an arginine decarboxylase protein.

[0033] Provided in the eighth embodiment of the fourth aspect is the method of the seventh embodiment of the fourth aspect, wherein the arginase protein, arginine deiminase protein, or arginine decarboxylase protein further comprises one or more polyethylene glycol (PEG) groups.

[0034] Provided in the ninth embodiment of the fourth aspect is the method of the eighth embodiment of the fourth aspect, wherein the arginase protein comprises a polypeptide having SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, or SEQ ID NO: 104.

[0035] Provided in the tenth embodiment of the fourth aspect is the method of the seventh embodiment of the fourth aspect, wherein the arginase protein, arginine deiminase protein, or arginine decarboxylase protein further comprises an albumin-binding domain, human serum albumin, or human IgGFc domain.

[0036] Provided in the eleventh embodiment of the fourth aspect is the method of the tenth embodiment of the fourth aspect, wherein the arginine catabolic enzyme is a fusion protein comprising an ABD polypeptide and an arginase polypeptide, an ABD polypeptide and an arginine deiminase polypeptide, or an ABD polypeptide and an arginine decarboxylase polypeptide.

[0037] Provided in the twelfth embodiment of the fourth aspect is the method of the eleventh embodiment of the fourth aspect, wherein the arginine catabolic enzyme is the fusion protein of the first aspect.

[0038] Provided in the thirteenth embodiment of the fourth aspect is the method of the twelfth embodiment of the fourth aspect, wherein the arginine catabolic enzyme comprises a polypeptide having at least 98% sequence homology with SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 75, or SEQ ID NO: 76.

[0039] Provided in the fifth aspect is a method for treating a condition in a subject requiring treatment of at least one condition selected from the group consisting of viral infections, multiple sclerosis, rheumatoid arthritis, autoimmune diseases, congenital hyperargininemia, graft-versus-host disease (GvHD), and inflammation, the method comprising the step of administering a therapeutically effective amount of the fusion protein of the first aspect to the subject.

[0040] The above-mentioned and other purposes and features of this disclosure will become apparent from the following description of the invention in conjunction with the accompanying drawings. [Brief explanation of the drawing]

[0041] [Figure 1]This shows the purification of the albumin-binding domain (ABD) recombinant human arginase (rhArg) fusion protein N-ABD094-rhArg (SEQ ID NO: 50) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Lane 1: SDS-PAGE low-pass marker (Bio-rad), Lane 2: N-ABD094-rhArg (SEQ ID NO: 50) 20-fold dilution, Lane 3: N-ABD094-rhArg (SEQ ID NO: 50) 10-fold dilution. [Figure 2A] An exemplary N-ABD094-rhArg gene (SEQ ID NO: 38) is shown, illustrating the primer binding sites. [Figure 2B] The protein sequence of the N-ABD094-rhArg (SEQ ID NO: 50) fusion protein with a theoretical pI / Mw of 6.07 / 42555.38 is shown. [Figure 3] This shows the protein sequence of the genetically modified Bacillus caldovelox arginase BCA(S161C)-His (SEQ ID NO: 89), which has a single-site mutation from serine to cysteine ​​and a C-terminal histidine tag. The cysteine ​​residue at position 161 and the 6×His tag are highlighted. [Figure 4] This shows the protein sequence alignment of BCA(S161C)-His (SEQ ID NO: 89), BCA-6×His-ABD fusion protein BHA (SEQ ID NO: 75), and BCA-ABD-6×His fusion protein BAH (SEQ ID NO: 76) by CLUSTALW. Alignment scores: BCA vs. BHA: 99.6721, BCA vs. BAH: 98.0328, BHA vs. BAH: 97.5138. [Figure 5A] This shows the elution profile of BHA (SEQ ID NO: 75) purified from E. coli grown in a 500 mL shaking flask culture by nickel-packed 5 mL HiTrap chelating HP column chromatography (single step). mAU: milliabsorption units, 100% B = 0.5 M imidazole. [Figure 5B]This shows the SDS-PAGE analysis of the column fraction from nickel affinity chromatography for BHA (SEQ ID NO: 75). M: SDS-PAGE molecular weight marker, low range (Bio-Rad), FT: flow-through, F2-F10: elution fractions from the column. [Figure 5C] This shows the elution profile of BAH (SEQ ID NO: 76) purified from E. coli cells grown in a 500 mL shaking flask culture by nickel-packed 5 mL HiTrap chelating HP column chromatography (single step). mAU: milliabsorption units, 100% B = 0.5 M imidazole. [Figure 5D] This shows the SDS-PAGE analysis of the column fraction from nickel affinity chromatography for BAH (SEQ ID NO: 76). M: SDS-PAGE molecular weight marker, low frequency (Bio-Rad), FT: flow-through, F2-F14: elution fractions from the column. [Figure 6A] The chromatogram shows the large-scale purification of genetically modified BCA(S161C)-His (SEQ ID NO: 89) using an XK50 nickel affinity column with a total volume of 196 mL. Similar to the small-scale purification, a 4-segment elution gradient was employed, and the elution profile was monitored using absorbance at 280 nm. [Figure 6B] This shows SDS-PAGE analysis of selected fractions from large-scale purification of genetically modified BCA(S161C)-His (SEQ ID NO: 89). Lane 1 is a low molecular weight marker. Lane 2 is total protein from cell lysates. Lane 3 represents soluble protein collected after heat treatment. Lane 4 shows protein flow-through from an XK50 nickel affinity column. Lane 5 is nonspecific binding protein (pooled fraction from A6-C3). Lanes 6-10 represent fractions E2, E3, F7, G3, and A'5, respectively. [Figure 7]This shows the binding of N-ABD-rhArg fusion protein (SEQ ID NO: 49) to HSA in undenatured PAGE. 30 picomoles of HSA were mixed with 7.5, 15, 30, 60, and 120 picomoles of N-ABD-rhArg (SEQ ID NO: 49), as shown in lanes 5-9, respectively. The mobility of 30 picomoles of HSA and N-ABD-rhArg (SEQ ID NO: 49) in undenatured PAGE is shown in lanes 2 and 3, respectively. Lane 1 is empty or blank. Lane 10 shows 120 picomoles of N-ABD-rhArg (SEQ ID NO: 49). [Figure 8] This shows the binding of BHA fusion protein (SEQ ID NO: 75) to HSA in undenatured PAGE. 60 picomoles of HSA were mixed with 6, 12, 60, 300, and 600 picomoles of BHA fusion protein (SEQ ID NO: 75), as shown in lanes 1-5, respectively. The mobility of 60 picomoles of HSA and BHA fusion protein (SEQ ID NO: 75) in undenatured PAGE is shown in lanes 6 and 7, respectively. The band labeled "BCA-ABD" represents BHA (SEQ ID NO: 75). [Figure 9] This study demonstrates the pharmacodynamics of BCA(S161C)-His (SEQ ID NO: 89) and BHA fusion protein (SEQ ID NO: 75) in relation to mouse plasma arginine. Each BALB / c mouse was administered 250 U of either BCA(S161C)-His (SEQ ID NO: 89) or BHA fusion protein (SEQ ID NO: 75) by intravenous injection (iv) or intraperitoneal injection (ip). Plasma samples were collected at the time indicated in the figure after injection. Time 0 refers to the plasma sample collected before injection. The amount of arginine in each sample was determined using a Biochrom30 amino acid analyzer. In these plots, arginine concentrations below the detection limit (3 μM) are considered as 0 μM. Each point represents the mean ± SD of three mice. [Figure 10] This shows the plasma arginine concentrations of BALB / c mice measured with a Biochrom30 amino acid analyzer after intraperitoneal injection of single doses of N-ABD-rhArg (SEQ ID NO: 49) ((A) 125U, (B) 250U, and (C) 500U). N-ABD-rhArg (SEQ ID NO: 49) was injected on day 0. [Figure 11]This shows the SDS-PAGE analysis of the formation of PEGylated His-rhArg (SEQ ID NO: 101). Lanes 1 and 8: SDS-PAGE low molecular weight marker (Bio-rad), Lanes 2 and 3: His-rhArg (SEQ ID NO: 101), Lanes 4 and 5: 60% elution, Lanes 6 and 7: 30% elution, Lanes 9 and 10: PEGylated His-rhArg (SEQ ID NO: 101). The molecular weight of PEG is 5000. The PEG used was methoxypolyethylene glycol succinimidylpropionate (mPEG-SPA 5000). The protein surface lysine residue of His-rhArg (SEQ ID NO: 101) is covalently bonded to PEG (5000amu) via a propionamidyl (SPA) linker (as described in U.S. Patent No. 8,679,810, which hereby constitutes part of this specification). [Figure 12] The results of the preparation of N-ABD094-rhArg (SEQ ID NO: 50) are shown. Lane 1: SDS-PAGE molecular weight marker, Lane 2: Cell soluble product, Lane 3: After heat treatment, Lane 4: Flow-through, Lane 5: 30% elution, Lane 6: 60% elution, Lane 7: After tangential flow filtration (TFF). The major protein band in Lane 6 or Lane 7 is purified N-ABD094-rhArg (SEQ ID NO: 50). [Figure 13] This shows the plasma arginine concentrations of C57BL / 6J male mice fed solid feed. These concentrations were measured at various time points after intraperitoneal injection of a single dose of 500U of N-ABD094-rhArg (SEQ ID NO: 50) in saline solution using an Agilent 6460 liquid chromatography / electrospray ionization triple quadrupole mass spectrometer (detection limit: 0.3 μM arginine). The results indicate that plasma arginine concentrations can be depleted to less than 1 μM over 10 days. Each time point represents the mean ± SEM of 5 mice. [Figure 14]This shows the plasma concentrations of N-ABD094-rhArg (SEQ ID NO: 50) at various time points after injection of a single dose of 500U into C57BL / 6J male mice. Human arginase was detected by ELISA (n=5). The circulating half-life of this drug is approximately 4 days. Each time point represents the mean ± SEM of 5 mice. [Figure 15] This study shows a significant reduction in fat mass in normal, lean C57BL / 6J male mice [solid feed (rhArg) group] fed solid feed and treated with N-ABD094-rhArg (SEQ ID NO: 50). (A) Significant reduction in visceral and subcutaneous fat mass after treatment with 500U of N-ABD094-rhArg (SEQ ID NO: 50) at 10-day intervals for 4 weeks, compared to a vehicle (saline) treated control [solid feed (vehicle) group]. *P<0.05, independent t-test. Data are shown ±SEM, n=5 for each group. (B) Hematoxylin and eosin (H&E) stained paraffin sections of visceral white adipose tissue (WAT) from mice treated with N-ABD094-rhArg (SEQ ID NO: 50) showed a marked reduction in adipocyte size. [Figure 16] This shows the expression levels of several key lipid synthesis-related genes in various tissues, measured using real-time qRT-PCR. Significant downregulation of lipid synthesis-related genes in (A) visceral white adipose tissue (WAT) and (B) liver after treatment with 500U of N-ABD094-rhArg (SEQ ID NO: 50) at 10-day intervals for 4 weeks [solid feed (rhArg) group] compared to vehicle-treated controls [solid feed (vehicle) group]. Gene expression levels are expressed in comparison to vehicle-treated mice [solid feed (vehicle) group] (set to 1). *P<0.05, independent t-test. Data are expressed mean ± SEM, n=5 for each group. [Figure 17]This study demonstrates that treatment with N-ABD094-rhArg (SEQ ID NO: 50) significantly improves insulin sensitivity. (A) Insulin loading tests showed that mice injected with 500 U of N-ABD094-rhArg (SEQ ID NO: 50) at 10-day intervals [solid feed (rhArg) group] showed a significant increase in sensitivity to the hypoglycemic effect of insulin immediately after 2 weeks compared to vehicle-treated controls [solid feed (vehicle) group]. *P<0.05 vs. solid feed (rhArg) group, independent t-test. Data are expressed as mean ± SEM, n=5 for each group. (B) MTT assay showed that treatment of primary mouse hepatocytes with 5 U / mL of N-ABD094-rhArg (SEQ ID NO: 50) for 48 hours did not affect cell viability compared to untreated controls (con). (C) Western blot analysis of phosphorylated Akt (pAkt) (marker of insulin signaling) protein concentration in primary mouse hepatocytes depleted of arginine for 24 hours using 5 U / mL N-ABD094-rhArg (SEQ ID NO: 50) before exposure to 100 nM insulin for 20 minutes. Hepatocytes treated with N-ABD094-rhArg (SEQ ID NO: 50) showed higher levels of Akt phosphorylation upon insulin stimulation, suggesting enhanced insulin signaling. Lane 1: con, Lane 2: 5 U / mL N-ABD094-rhArg (SEQ ID NO: 50), Lane 3: 100 nM insulin, Lane 4: 5 U / mL N-ABD094-rhArg (SEQ ID NO: 50) + 100 nM insulin. [Figure 18]In vehicle-treated C57BL / 6J male mice (8 weeks to 12 weeks old) fed a high-fat diet (HFD) containing 60 kcal% fat [HFD (vehicle) group], (A) body weight and (B) size increased significantly. However, simultaneous treatment with 300 U of N-ABD094-rhArg (SEQ ID NO: 50) (once a week) in HFD-fed male mice (8 weeks to 12 weeks old) [HFD (rhArg) group] effectively prevented weight gain. Their body weight remained comparable to that of lean vehicle-treated control mice fed a corresponding low-fat diet (LFD) containing 10 kcal% fat [LFD (vehicle) group]. Data are expressed as mean ± SEM, with n=5 for each group. [Figure 19] This study demonstrates that simultaneous treatment with 300 U of N-ABD094-rhArg (SEQ ID NO: 50) [HFD(rhArg) group] (once a week for 12 weeks) in HFD-fed male mice effectively prevented HFD-induced white adipose tissue (WAT) enlargement. (A) Weight of major visceral (perigonadal, perirenal, and mesenteric) and subcutaneous (inguinal) fat bodies. (B) H&E stained sections of visceral WAT showed that simultaneous treatment with N-ABD094-rhArg prevented HFD-induced white adipocyte hypertrophy. (C) Expression levels of several important lipid-producing genes in WAT compared to lean control [LFD(vehicle) group] (set to 1). These genes were dramatically downregulated in HFD-fed mice treated with N-ABD094-rhArg. *P<0.05, Fischer's LSD followed by one-way ANOVA. The data is expressed as mean ± SEM, and n=5 for each group. [Figure 20] This study demonstrates the suppression of HFD-induced upregulation of lipid synthesis transcription factors (Pparg and Srebp1c) and the simultaneous downregulation of key lipid-producing enzymes (Acc1 and Scd1) in skeletal muscle of mice treated with 300U of N-ABD094-rhArg (SEQ ID NO: 50) [HFD(rhArg) group] (once a week for 12 weeks). *P<0.05, one-way ANOVA followed by Fisher's LSD. Data are expressed as mean ± SEM, with n=5 in each group. [Figure 21]This study demonstrates that simultaneous treatment with 300U of N-ABD094-rhArg (SEQ ID NO: 50) [HFD(rhArg) group] (once a week) effectively prevented HFD-induced obesity in C57BL / 6J female mice. (A) Body weight of mice fed HFD and treated with N-ABD094-rhArg or vehicle for 12 weeks from 8 weeks of age. Vehicle-treated female mice fed LFD were used as lean controls [LFD(vehicle) group]. (B) Weight of major visceral (perigonadal, perirenal, and mesenteric) and subcutaneous (inguinal) fat pads. *P<0.05, one-way ANOVA followed by Fisher's LSD. Data are expressed as mean ± SEM, with n=5 in each group. [Figure 22] This study demonstrates that simultaneous treatment with 300U of N-ABD094-rhArg (SEQ ID NO: 50) [HFD(rhArg) group] (once a week) effectively prevented HFD-induced insulin resistance and impaired glucose tolerance in C57BL / 6J female mice. (A) Insulin tolerance test (ITT) performed at 7 weeks of HFD feeding. (B) Glucose tolerance test (GTT) performed at 11 weeks of HFD feeding. The results for ITT and GTT are expressed as area under the curve (AUC). *P<0.05, Fisher's LSD was performed after one-way ANOVA. Data are expressed as mean ± SEM, with n=5 in each group. [Figure 23]This study demonstrates that arginine depletion by adding N-ABD094-rhArg (SEQ ID NO: 50) (rhArg), bovine arginase, or natural arginine deiminase (ADI) to the culture medium, or by using an arginine-free medium, inhibited the differentiation of mouse 3T3-L1 preadipocytes into adipocytes. Adding arginase metabolites such as L-ornithine or urea, or the ADI metabolite L-citrulline, to the culture medium did not inhibit adipogenesis or lipid synthesis. (*The medium contained 0.4 mM L-arginine, which is converted to 0.4 mM L-ornithine and 0.4 mM urea by arginase, and to 0.4 mM L-citrulline by ADI). Cells grown in the medium without the addition of arginine depletion agents or arginase / ADI metabolites were used as a control. Lipids in cells were stained with Oil Red O six days after exposure to a specific differentiation-inducing factor added to the culture medium. [Figure 24] This study demonstrates that N-ABD094-rhArg (SEQ ID NO: 50) can suppress adipogenesis and lipidogenesis genes in 3T3-L1 preadipocytes. Mouse 3T3-L1 preadipocytes were cultured for 6 days in a medium supplemented with specific factors to induce differentiation into adipocytes. Cells grown in a medium without these factors remained undifferentiated. 2 U / mL of N-ABD094-rhArg (SEQ ID NO: 50) (rhArg) was added to the medium. Cells grown in the medium without N-ABD094-rhArg were used as a control. mRNA expression levels of several important (A) transcription factors of adipogenesis and (B) enzymes in the lipidogenesis pathway were determined by real-time qRT-PCR and compared with the undifferentiated (control) group (set to 1). *P<0.05, one-way ANOVA followed by Fisher's LSD. Data are expressed as mean ± SEM, with n=3 for each group. [Figure 25]This study shows the response of 3T3-L1 preadipocytes to various concentrations of arginine. Mouse 3T3-L1 preadipocytes were cultured for 6 days in arginine-free medium with or without the addition of various concentrations of L-arginine. Cells cultured in standard arginine-containing medium supplemented with differentiation-inducing factors were used as positive (+ve) controls for differentiation, and cells cultured in standard arginine-containing medium without differentiation-inducing factors were used as negative (-ve) controls. One group of cells was cultured in standard arginine-containing medium supplemented with differentiation-inducing factors, and differentiation was inhibited by treatment with 5 U / mL of N-ABD094-rhArg (SEQ ID NO: 50) (rhArg). (A) Cell lipids were stained with Oil Red O, and the staining intensity was quantified by measuring the optical density (expressed as a percentage relative to the +ve control group). (B) mRNA levels of several important lipid synthesis-related genes are expressed in comparison to cells cultured in arginine-free medium (set to 1). The data is expressed as mean ± SEM, and n=3 for each group. [Figure 26] This study demonstrates that simultaneous treatment with 300 U of N-ABD094-rhArg (SEQ ID NO: 50) [HFD(rhArg) group] (once a week) in HFD-fed male mice effectively prevented HFD-induced insulin resistance. Insulin loading tests (ITT) were performed at (A) week 6 and (B) week 11 of HFD feeding, and the results are expressed as area under the curve (AUC). *P<0.05, one-way ANOVA followed by Fisher's LSD. Data are expressed as mean ± SEM, with n=5 in each group. [Figure 27] This study demonstrates that simultaneous treatment with 300 U of N-ABD094-rhArg (SEQ ID NO: 50) [HFD(rhArg) group] (once a week) in HFD-fed male mice effectively prevented HFD-induced impaired glucose tolerance. Glucose tolerance tests (GTTs) were performed at (A) week 5 and (B) week 10 of HFD feeding, and the results are expressed as area under the curve (AUC). *P<0.05, one-way ANOVA followed by Fisher's LSD. Data are expressed as mean ± SEM, with n=5 in each group. [Figure 28]This study demonstrates that simultaneous treatment with 300 U of N-ABD094-rhArg (SEQ ID NO: 50) [HFD(rhArg) group] (once a week) in HFD-fed male mice effectively prevented HFD-induced increases in (A) fasting blood glucose (measured using a blood glucose meter) and (B) plasma insulin concentration (measured using ELISA). (C) HOMA-IR score (calculated using a standard formula as a measure of insulin resistance) indicates that simultaneous treatment with N-ABD094-rhArg prevented the development of HFD-induced insulin resistance. *P<0.05, Fisher's LSD was performed following one-way ANOVA. Data are expressed as mean ± SEM, with n=5 in each group. [Figure 29] This study demonstrates that simultaneous treatment with 300 U of N-ABD094-rhArg (SEQ ID NO: 50) [HFD(rhArg) group] (once a week) in HFD-fed male mice prevented / suppressed HFD-induced increases in fasting plasma (A) leptin concentration, (B) total cholesterol concentration, (C) triglyceride concentration, and (D) free fatty acid concentration. *P<0.05, one-way ANOVA followed by Fisher's LSD. Data are expressed as mean ± SEM, with n=5 in each group. [Figure 30] This study demonstrates that HFD-induced hepatomegaly was prevented by simultaneous treatment with 300 U of N-ABD094-rhArg (SEQ ID NO: 50) [HFD(rhArg) group] (once a week for 12 weeks) in HFD-fed male mice. (A) Representative image of the entire fresh liver. (B) Liver weight. *P<0.05, one-way ANOVA followed by Fisher's LSD. Data are expressed as mean ± SEM, n=5 for each group. [Figure 31]This study demonstrates that simultaneous treatment of HFD-fed male mice with 300 U of N-ABD094-rhArg (SEQ ID NO: 50) [HFD(rhArg) group] (once a week for 12 weeks) effectively prevented HFD-induced fatty liver disease. (A) Staining of liver sections with Oil Red O showed widespread lipid accumulation in HFD-fed vehicle-treated mice [HFD(vehicle) group], which was absent in mice treated with N-ABD094-rhArg. (B) Treatment with N-ABD094-rhArg prevented an increase in triglyceride concentration in the liver of HFD-fed mice. (C) N-ABD094-rhArg suppressed HFD-induced upregulation of lipid synthesis-related genes in the liver. *P<0.05, one-way ANOVA followed by Fisher's LSD. Data are expressed as mean ± SEM, with n=5 in each group. [Figure 32] This study demonstrates that simultaneous treatment with 300 U of N-ABD094-rhArg (SEQ ID NO: 50) [HFD(rhArg) group] (once a week for 12 weeks) in HFD-fed male mice prevented the HFD-induced increase in serum alanine aminotransferase (ALT) concentration (typically measured as a biomarker of liver injury). *P<0.05, one-way ANOVA followed by Fisher's LSD. Data are expressed as mean ± SEM, with n=5 in each group. [Figure 33] This study demonstrates that simultaneous treatment with 300 U of N-ABD094-rhArg (SEQ ID NO: 50) in HFD-fed male mice [HFD(rhArg) group] (once a week for 12 weeks) prevented HFD-induced weight gain in (A) the kidney, (B) the pancreas, and (C) the heart. *P<0.05, one-way ANOVA followed by Fisher's LSD. Data are expressed as mean ± SEM, with n=5 in each group. [Figure 34]This study shows that simultaneous treatment of HFD-fed male mice with 300 U of N-ABD094-rhArg (SEQ ID NO: 50) [HFD(rhArg) group] (once a week for 12 weeks) effectively suppressed (A) HFD-induced upregulation of pro-inflammatory adipokin mRNA levels in visceral WAT (expressed in comparison to lean control mice [LFD(vehicle) group] (set to 1)) and (B) suppressed the increase in HFD-induced serum concentrations of inflammatory cytokines. *P<0.05, one-way ANOVA followed by Fisher's LSD. Data are expressed as mean ± SEM, with n=5 in each group. [Figure 35] This study demonstrates that simultaneous treatment of male mice with 300 U of N-ABD094-rhArg (SEQ ID NO: 50) [HFD(rhArg) group] (once a week for 12 weeks) effectively prevented HFD-induced whitening of brown adipose tissue (BAT). (A) Representative image of the entire fresh interscapular BAT. (B) Weight of the interscapular BAT. (C) H&E stained paraffin sections revealed that cells with large single pores (lipid globules), a typical histological feature of white adipocytes, were abundant in the BAT of vehicle-treated HFD-fed mice [HFD(vehicle) group], while they were almost absent in the BAT of N-ABD094-rhArg-treated HFD-fed mice. *P<0.05, one-way ANOVA followed by Fisher's LSD. Data are expressed mean ± SEM, n=5 for each group. [Figure 36] This study shows that simultaneous treatment of male mice with 300 U of N-ABD094-rhArg (SEQ ID NO: 50) [HFD(rhArg) group] (once a week for 12 weeks) significantly upregulated mRNA levels of major thermogenerative factors (Ucp1 and Pgc1a) in BAT, and suppressed HFD-induced downregulation of the fatty acid oxidation gene Acox1 in BAT. *P<0.05, one-way ANOVA followed by Fisher's LSD. Data are expressed as mean ± SEM, with n=5 in each group. [Figure 37]This study shows a significant decrease in body weight after treatment with N-ABD094-rhArg (SEQ ID NO: 50) in C57BL / 6J male mice with pre-existing obesity induced by HFD feeding (from 5 weeks to 12 weeks of age). (A) Body weight of mice treated with 600U of N-ABD094-rhArg [HFD(rhArg) group] (once a week for 12 weeks). (B) Representative images of mice after 12 weeks of treatment. Data are expressed as mean ± SEM, with n=5 in each group. [Figure 38] This study demonstrates that administering 600 U of N-ABD094-rhArg (SEQ ID NO: 50) once a week for 12 weeks to male mice with pre-existing HFD-induced obesity [HFD(rhArg) group] effectively reduced white adipose tissue. (A) The weight of the major visceral (perigonadal, perirenal, and mesenteric) and subcutaneous (inguinal) fat bodies in obese mice was nearly equivalent to that of lean control mice [LFD(rhArg) group] after treatment with N-ABD094-rhArg. *P<0.05, one-way ANOVA followed by Fisher's LSD. Data are expressed as mean ± SEM, n=5 in each group. (B) H&E stained sections of visceral WAT showed that treatment with N-ABD094-rhArg significantly reduced the size of adipocytes in obese mice. [Figure 39] This study demonstrates that insulin resistance could be effectively reversed in male mice with pre-existing HFD-induced obesity by administering 600 U of N-ABD094-rhArg (SEQ ID NO: 50) once a week [HFD(rhArg) group]. Insulin loading tests (ITTs) were performed before N-ABD094-rhArg treatment and at 4, 8, and 12 weeks after treatment. ITT results are expressed as area under the curve (AUC). *P<0.05, one-way ANOVA followed by Fisher's LSD. Data are expressed mean ± SEM, with n=5 in each group. [Figure 40]This study demonstrates that administering 600 U of N-ABD094-rhArg (SEQ ID NO: 50) once weekly to male mice with pre-existing HFD-induced obesity [HFD(rhArg) group] effectively reversed impaired glucose tolerance. Glucose tolerance tests (GTTs) were performed before N-ABD094-rhArg treatment and at 3, 7, and 11 weeks after treatment. GTT results are expressed as area under the curve (AUC). *P<0.05, one-way ANOVA followed by Fisher's LSD. Data are expressed mean ± SEM, with n=5 in each group. [Figure 41] This study demonstrates that administering 600 U of N-ABD094-rhArg (SEQ ID NO: 50) once weekly to male mice with pre-existing HFD-induced obesity [HFD(rhArg) group] effectively reversed hyperglycemia, hyperinsulinemia, and insulin resistance. (A) Fasting blood glucose, (B) Fasting plasma insulin, and (C) HOMA-IR scores in obese mice were corrected to near-normal levels after treatment with N-ABD094-rhArg. *P<0.05, one-way ANOVA followed by Fisher's LSD. Data are expressed mean ± SEM, n=5 in each group. [Figure 42] This study demonstrates that administering 600 U of N-ABD094-rhArg (SEQ ID NO: 50) once weekly to male mice with pre-existing HFD-induced obesity [HFD(rhArg) group] effectively reversed hyperleptinemia and hypercholesterolemia. (A) Fasting plasma leptin and (B) Fasting plasma total cholesterol in obese mice were corrected to near-normal levels after treatment with N-ABD094-rhArg. *P<0.05, one-way ANOVA followed by Fisher's LSD. Data are expressed mean ± SEM, n=5 in each group. [Figure 43]This study demonstrates that hepatomegaly could be effectively reversed in male mice with pre-existing HFD-induced obesity by administering 600 U of N-ABD094-rhArg (SEQ ID NO: 50) once a week for 12 weeks [HFD(rhArg) group]. (A) Representative images of the fresh whole liver show that the livers of obese mice returned to normal size after N-ABD094-rhArg treatment. (B) The liver weight of obese mice decreased dramatically after N-ABD094-rhArg treatment and was comparable to that of lean control mice [LFD(rhArg) group]. *P<0.05, one-way ANOVA followed by Fisher's LSD. Data are expressed mean ± SEM, n=5 in each group. [Figure 44] This study demonstrates that fatty liver disease could be effectively reversed in male mice with pre-existing HFD-induced obesity by administering 600 U of N-ABD094-rhArg (SEQ ID NO: 50) once a week for 12 weeks [HFD(rhArg) group]. In the livers of obese mice treated with N-ABD094-rhArg, (A) dramatic lipid clearance was shown by oil red O staining of liver sections, (B) triglyceride concentrations decreased to levels comparable to lean control mice [LFD(vehicle)], and (C) mRNA levels of several important lipid synthesis-related genes were significantly downregulated. Expression levels are expressed in comparison to lean control (set to 1). *P<0.05, one-way ANOVA followed by Fisher's LSD. Data are expressed mean ± SEM, n=5 in each group. [Figure 45] This study demonstrates that administering 600 U of N-ABD094-rhArg (SEQ ID NO: 50) once weekly for 12 weeks to male mice with pre-existing HFD-induced obesity [HFD(rhArg) group] significantly reduced serum concentrations of alanine transaminase (ALT) and aspartate transaminase (AST), common biomarkers of liver injury. *P<0.05, one-way ANOVA followed by Fisher's LSD. Data are expressed mean ± SEM, n=5 for each group. [Figure 46]This study demonstrates that renal steatosis could be reversed in male mice with pre-existing HFD-induced obesity by administering 600 U of N-ABD094-rhArg (SEQ ID NO: 50) once a week for 12 weeks [HFD(rhArg) group]. Obese mice treated with N-ABD094-rhArg showed significant reductions in (A) kidney weight, (B) triglyceride concentration in the kidneys, (C) vacuolated structures in the renal tubules of H&E-stained kidney sections, and (D) ectopic accumulation of lipid droplets in the glomeruli of oil red O-stained kidney sections. *P<0.05, one-way ANOVA followed by Fisher's LSD. Data are expressed mean ± SEM, n=5 in each group. [Figure 47] This study demonstrates that pancreatic steatosis could be reversed in male mice with pre-existing HFD-induced obesity by administering 600 U of N-ABD094-rhArg (SEQ ID NO: 50) once a week for 12 weeks [HFD(rhArg) group]. Obese mice treated with N-ABD094-rhArg showed a significant decrease in (A) pancreatic weight, (B) excessive interlobular and intralobular accumulation of white adipose tissue observed in H&E-stained sections of the pancreas, and (C) triglyceride concentration in the pancreas. *P<0.05, one-way ANOVA followed by Fisher's LSD. Data are expressed mean ± SEM, and n=5 in each group. [Figure 48] This study demonstrates that cardiac steatosis could be reversed in male mice with pre-existing HFD-induced obesity by administering 600 U of N-ABD094-rhArg (SEQ ID NO: 50) once a week for 12 weeks [HFD(rhArg) group]. Obese mice treated with N-ABD094-rhArg showed a significant decrease in (A) cardiac weight, (B) excessive accumulation of lipid droplets in the heart, and (C) triglyceride concentration in the heart. *P<0.05, one-way ANOVA followed by Fisher's LSD. Data are expressed as mean ± SEM, with n=5 in each group. [Figure 49]This study demonstrates that administering 600 U of N-ABD094-rhArg (SEQ ID NO: 50) once a week for 12 weeks to male mice with pre-existing HFD-induced obesity [HFD(rhArg) group] significantly reduced triglyceride concentrations in skeletal muscle. *P<0.05, one-way ANOVA followed by Fisher's LSD. Data are expressed as mean ± SEM, with n=5 in each group. [Figure 50] This study demonstrates that administering 600 U of N-ABD094-rhArg (SEQ ID NO: 50) once weekly for 12 weeks to male mice with pre-existing HFD-induced obesity [HFD(rhArg) group] significantly downregulated (A) mRNA levels of the pro-inflammatory adipokin Mcp1 in visceral WAT (expressed compared to lean control mice [LFD(vehicle) group] (set to 1)) and (B) significantly reduced serum concentrations of the pro-inflammatory cytokine Mcp1. *P<0.05, one-way ANOVA followed by Fisher's LSD. Data are expressed mean ± SEM, with n=5 in each group. [Figure 51] This study demonstrates that administering 600 U of N-ABD094-rhArg (SEQ ID NO: 50) once a week for 12 weeks to male mice with pre-existing HFD-induced obesity [HFD(rhArg) group] suppressed inflammation and fibrosis in the liver. In the livers of obese mice treated with N-ABD094-rhArg, significant downregulation of (A) pro-inflammatory genes and (B) pro-fibrotic genes was observed, while (C) upregulation of anti-inflammatory genes was observed. mRNA levels are expressed in comparison to lean controls [LFD(vehicle)] (set to 1). *P<0.05, one-way ANOVA followed by Fisher's LSD. Data are expressed mean ± SEM, with n=5 in each group. [Figure 52]This study demonstrates that administering 600 U of N-ABD094-rhArg (SEQ ID NO: 50) once a week for 12 weeks to male mice with pre-existing HFD-induced obesity [HFD(rhArg)] suppressed inflammation and fibrosis in the kidneys. In the kidneys of obese mice treated with N-ABD094-rhArg, (A) significant downregulation of pro-inflammatory genes and (B) pro-fibrosis genes was observed, and (C) significant suppression of hyperdeposition of collagen fibers in the renal tubules and around the glomeruli was observed. mRNA levels are expressed in comparison to lean controls [LFD(vehicle) group] (set to 1). *P<0.05, one-way ANOVA followed by Fisher's LSD. Data are expressed as mean ± SEM, with n=5 in each group. [Figure 53] This study demonstrates that administering 600 U of N-ABD094-rhArg (SEQ ID NO: 50) once a week for 12 weeks to male mice with pre-existing HFD-induced obesity [HFD(rhArg) group] suppressed inflammation and fibrosis in the pancreas. Significant downregulation of (A) pro-inflammatory genes and (B) pro-fibrotic genes was observed in the pancreases of obese mice treated with N-ABD094-rhArg. mRNA levels are expressed in comparison to lean controls [LFD(vehicle) group]. *P<0.05, one-way ANOVA followed by Fisher's LSD. Data are expressed mean ± SEM, with n=5 in each group. [Figure 54] This study demonstrates that administering 600 U of N-ABD094-rhArg (SEQ ID NO: 50) once weekly for 12 weeks to male mice with pre-existing HFD-induced obesity [HFD(rhArg) group] effectively reversed the whitening of brown adipose tissue (BAT). In obese mice treated with N-ABD094-rhArg, (A) the weight of interscapular BAT, (B) the morphology and size of interscapular BAT, and (C) the histological features shown in H&E stained sections of BAT were similar to those of lean control mice. *P<0.05, one-way ANOVA followed by Fisher's LSD. Data are expressed mean ± SEM, n=5 in each group. [Figure 55]This study demonstrates that administering 600 U of N-ABD094-rhArg (SEQ ID NO: 50) once weekly to male mice with pre-existing HFD-induced obesity [HFD(rhArg) group] significantly reduced (A) systolic and diastolic blood pressure and (B) heart rate in obese mice to levels comparable to lean control mice [LFD(vehicle) group] within 5 weeks after drug treatment. *P<0.05, one-way ANOVA followed by Fisher's LSD. Data are expressed as mean ± SEM, with n=10 in each group. [Figure 56] This study shows that non-neutralizing anti-drug antibodies were generated in C57BL / 6J male mice [LFD(rhArg) group] (weekly, starting at 5 weeks of age and continued for 8 months) that were fed LFD and intraperitoneally injected with 500 U of N-ABD094-rhArg (SEQ ID NO: 50). (A) Using an ELISA assay, anti-rhArg antibodies with an average antibody titer of 108 were detected in the plasma of LFD-fed mice treated with N-ABD094-rhArg. (B) After incubating the plasma with 1000 U / mL of N-ABD094-rhArg (rhArg*) for 1 hour, arginase activity was measured. No decrease in arginase activity was observed, which indicates that the antibodies against the drug generated in the mice were not neutralizing antibodies. Data are expressed as mean ± SEM, with n=3 in each group. [Figure 57] This study shows that administering 500 U of N-ABD094-rhArg (SEQ ID NO: 50) to LFD-fed C57BL / 6J male mice [LFD(rhArg) group] (once a week, starting at 5 weeks of age for 8 months) did not induce adverse effects on the liver or kidneys. LFD-fed mice treated with N-ABD094-rhArg showed no significant differences compared to LFD-fed mice treated with vehicles [LFD(vehicle) group] in (A) serum concentrations of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) (usually used as biomarkers to assess liver function or liver disease), and (B) serum creatinine concentration and urinary albumin / creatinine (usually used as biomarkers to assess renal function or renal disease). Data are expressed as mean ± SEM, with n=3 in each group. [Figure 58] This study shows that administering 500 U of N-ABD094-rhArg (SEQ ID NO: 50) to LFD-fed C57BL / 6J male mice [LFD(rhArg) group] (once a week, starting at 5 weeks of age and continuing for 8 months) did not induce any adverse vascular effects. LFD-fed mice treated with N-ABD094-rhArg showed no significant differences compared to LFD-fed mice treated with vehicles [LFD(vehicle) group] in (A) phenylephrine-induced vascular smooth muscle contraction, (B) acetylcholine-induced endothelium-dependent relaxation, and (C) endothelium-independent relaxation in response to the nitric oxide donor sodium nitroprusside. Data are expressed as mean ± SEM, with n=3 in each group. [Figure 59] This study demonstrates that administering 300 U of PEGylated His-rhArg (SEQ ID NO: 101) once a week for four weeks to C57BL / 6J male mice with pre-existing HFD-induced obesity [HFD (PEG-rhArg) group] effectively reduced the mice's body weight to a level comparable to lean control mice fed solid feed on vehicles [solid feed (vehicle) group]. Data are expressed as mean ± SEM, with n=6 in each group. [Figure 60] This study demonstrates that administering 300 U of PEGylated His-rhArg (SEQ ID NO: 101) once a week for 4 weeks to male mice with pre-existing HFD-induced obesity [HFD(PEG-rhArg) group] effectively reduced fat mass in major visceral (perigonadal, perirenal, and mesenteric) and subcutaneous (inguinal) storage, and reduced the weight of the liver, kidneys, pancreas, and heart. *P<0.05, one-way ANOVA followed by Fisher's LSD. Data are expressed as mean ± SEM, n=4 in each group. [Figure 61]This study demonstrates that administering 300 U of PEGylated His-rhArg (SEQ ID NO: 101) once weekly to male mice with pre-existing HFD-induced obesity [HFD(PEG-rhArg) group] resulted in (A) a significant decrease in fasting blood glucose and (B) a significant increase in insulin sensitivity in an insulin tolerance test (ITT) conducted two weeks after drug treatment. ITT results are expressed as area under the curve (AUC). *P<0.05, Fisher's LSD was performed following one-way ANOVA. Data are expressed as mean ± SEM, with n=6 in each group. [Figure 62] This study demonstrates that administering 25-100 U of N-ABD094-rhArg-Co2+ [SEQ ID NO: 50 (cobalt substitution)] once a week for 5 weeks to male C57BL / 6J mice with pre-existing HFD-induced obesity effectively reduced the mice's body weight. Data are expressed as mean ± SEM, n=3. [Figure 63] This study demonstrates the anticancer effect of a BHA fusion protein (SEQ ID NO: 75) against the MKN45 gastric cancer cell line. MKN45 proliferation was inhibited in a dose-dependent manner. Survival rates were determined by a colorimetric assay of the MTT endpoint. Data are expressed as mean ± SD, and n=3. [Figure 64] This study shows the effect of N-ABD-rhArg (SEQ ID NO: 49) on tumor volume in nude mice receiving xenografts of 4T1 breast cancer. The "Experimental Group" curve represents the experimental group, which received 500U of N-ABD-rhArg (SEQ ID NO: 49) once a week, while the "Control Group" curve represents the control group, which received PBS once a week. *P<0.05, **P<0.01 vs. experimental group, t-test, n=4-6. [Figure 65] This study shows the effect of N-ABD-rhArg (SEQ ID NO: 49) on relative tumor volume in nude mice receiving xenografts of 4T1 breast cancer. The "Experimental Group" curve represents the experimental group, which received 500U of N-ABD-rhArg (SEQ ID NO: 49) once a week. The "Control Group" curve represents the control group, which received PBS once a week. *P<0.05 vs. experimental group, t-test, n=4-6. [Figure 66]This study shows a significant difference in tumor weight between the experimental and control groups. The experimental group received 500 U of N-ABD-rhArg (SEQ ID NO: 49) once a week by injection. The control group received PBS once a week. *P<0.05 vs. experimental group, t-test, n=4-6. [Figure 67] The images show tumors collected from nude mice in the control group and the N-ABD-rhArg (SEQ ID NO: 49) treatment group. [Figure 68] This study shows the effect of N-ABD-rhArg (SEQ ID NO: 49) on relative tumor volume in 4T1 breast cancer allogeneic grafts. The curve for "500U ABD-rhArg" represents the experimental group that received 500U of N-ABD-rhArg (SEQ ID NO: 49) once a week. The curve for "250U ABD-rhArg" represents the experimental group that received 250U of N-ABD-rhArg (SEQ ID NO: 49) once a week. The curve for "500U PEG-rhArg" represents the experimental group that received 500U of PEGylated His-rhArg (SEQ ID NO: 101) once a week. The curve for "Control Group" represents the control group that received PBS once a week. **P<0.01, ***P<0.001 vs experimental group, t-test, n=6~8. [Figure 69] The data shows a significant difference in tumor weight between the experimental group and the control group. The "500U ABD-rhArg" data represents the group that received 500U of N-ABD-rhArg (SEQ ID NO: 49) once a week. The "250U ABD-rhArg" data represents the group that received 250U of N-ABD-rhArg (SEQ ID NO: 49) once a week. The "500U PEG-rhArg" data represents the group that received 500U of PEGylated His-rhArg (SEQ ID NO: 101) once a week. The "Control Group" data represents the control group that received PBS once a week. *P<0.05, **P<0.01 vs experimental group, t-test, n=6~8. [Figure 70]Figure 70A shows the mean number of metastatic nodules from the lungs of 4T1 allografts. (A) The data for "500U ABD-rhArg" represents the group that received 500U of N-ABD-rhArg (SEQ ID NO: 49) once a week. The data for "250U ABD-rhArg" represents the group that received 250U of N-ABD-rhArg (SEQ ID NO: 49) once a week. The data for "500U PEG-rhArg" represents the group that received 500U of PEGylated His-rhArg (SEQ ID NO: 101) once a week. The data for the "control group" represents the control group that received PBS once a week. *P<0.05, **P<0.01 vs experimental group, t-test, n=6~8. (B) Scatter plot of the number of metastatic nodules from the lungs of 4T1 allografts shown in Figure 70A. [Modes for carrying out the invention]

[0042] This disclosure generally relates to a fusion protein comprising an arginase polypeptide and an ABD polypeptide, a method of using the same, and a method of preparing the same. The fusion protein described herein is a highly effective arginine depletor, and its fusion with the ABD polypeptide significantly extends its half-life compared to known arginases and arginase derivatives. The disclosed fusion protein is useful in the treatment of diseases and conditions in which arginine depletion is therapeutically effective, such as cancer, viral infections, multiple sclerosis, rheumatoid arthritis, autoimmune diseases, congenital hyperargininemia, graft-versus-host disease (GvHD), inflammation, obesity, metabolic disorders, and associated complications and comorbidities.

[0043] Furthermore, this disclosure generally relates to methods of treating subjects requiring treatment for obesity, metabolic disorders, and associated complications and comorbidities by depleting the arginine levels of the subjects. Arginine depletion in subjects results in a reduction of body weight and fat mass, improved glucose tolerance and insulin responsiveness, normalization of blood glucose levels, endocrine and metabolic profiles, and reduction of inflammation, steatosis, and fibrosis. Any agent capable of depleting the arginine levels of a subject can be used in the methods of treating obesity, metabolic disorders, and associated complications and comorbidities described herein.

[0044] Definition of Terms The definitions of terms used herein are intended to incorporate the current level of understanding of each term in the field of biotechnology. Examples are provided where applicable. Unless otherwise specified in individual cases, these definitions apply to each term used herein, or to those terms as subclasses.

[0045] As used herein, the terms “half-life” or “half-life” refer to the time it takes for the concentration of a drug, such as the fusion protein or arginine depletor described herein, to decrease by half in vitro or in vivo, for example, after injection into a mammal. In certain cases, the plasma arginine concentration after injection is used herein as a surrogate indicator of the drug’s half-life. In such cases, the term “treatment period” is used to refer to the length of time that a particular dose of the arginine depletor can maintain the plasma arginine concentration below a specific threshold concentration at which the desired therapeutic effect is observed. In certain embodiments, the threshold concentration of plasma arginine is less than 50 μM, less than 40 μM, less than 30 μM, less than 20 μM, less than 10 μM, less than 5 μM, less than 3 μM, or below the detection limit of conventional analytical instruments. For example, if the arginine catabolic enzyme described herein is injected and the plasma arginine concentration is depleted to below the detection limit of the Biochrom30 amino acid analyzer (detection limit is 3 μM) within 7 days, the treatment period is 7 days, and the half-life is, for example, about 7 days.

[0046] As used herein, the terms “to bond” or “bonded” mean linking or integrating two or more compounds by a bonding or non-bonding interaction to keep them together, and this term includes direct or indirect bonding, such as bonding a first polypeptide to a second polypeptide or other molecule, as well as embodiments in which one or more intermediate compounds, such as polypeptides (e.g., linkers), are positioned between the first polypeptide and the second polypeptide or other molecule.

[0047] As used herein, the terms “protein” or “polypeptide” refer to an organic polymer composed of two or more amino acid monomers and / or analogs thereof. The term “polypeptide” encompasses amino acid polymers of any length, including full-length proteins and peptides, and their analogs and fragments. Polypeptides of three or more amino acids are also called oligopeptides. As used herein, the terms “amino acid,” “amino acid monomer,” or “amino acid residue” refer to any of the 20 naturally occurring amino acids (including synthetic amino acids with non-natural side chains, and including both D and L optical isomers). The term “amino acid analog” refers to an amino acid that is otherwise identical to its natural amino acid analog, except that one or more individual atoms are substituted with a different atom, isotope, or functional group.

[0048] As used herein, the term “non-natural amino acid” means any amino acid other than one of the 20 common natural amino acids, selenocysteine, or pyrrolidine, modified amino acids, and / or amino acid analogs.

[0049] As used herein, the term “fusion protein” means a chimeric protein comprising proteins or functional protein fragments of different origins (e.g., arginase or its variants) covalently linked by amide, ester, urea, carbamate, ether and / or disulfide bonds.

[0050] As used herein, the term “mutant” means a polynucleotide or nucleic acid that is different from the reference nucleic acid or polypeptide but retains its essential properties. Generally, mutants are very similar overall to the reference nucleic acid or polypeptide and are identical in many regions.

[0051] The mutant may, for example, include an amino acid sequence of the parent polypeptide sequence in which at least one conserved amino acid is substituted. Alternatively, the mutant may include an amino acid sequence of the parent polypeptide sequence in which at least one non-conserved amino acid is substituted. In this case, it is preferable that the non-conserved amino acid substitution does not interfere with or inhibit the biological activity of the functional mutant. The non-conserved amino acid substitution can enhance the biological activity of the mutant, thereby increasing its biological activity compared to the parent polypeptide.

[0052] When used in relation to polypeptides, the term “functional fragment” means any portion of the polypeptide in question that retains the biological activity of the polypeptide from which it comprises (the parent polypeptide). A functional fragment can be any fragment containing adjacent amino acids of the polypeptide from which it comprises, provided that its biological activity is still at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of that of the parent polypeptide, or substantially equivalent to or higher than that. With respect to the parent polypeptide, a functional fragment may contain, for example, about 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or more of that of the parent polypeptide.

[0053] The functional fragment may contain additional amino acids at its amino-terminus, carboxy-terminus, or both, such as amino acids not found in the amino acid sequence of the parent polypeptide.

[0054] The amino acid substitutions in the described polypeptides may be conservative amino acid substitutions. Conservative amino acid substitutions are known in the art and include, for example, amino acid substitutions in which one amino acid having a specific physical and / or chemical property is replaced with another amino acid having the same or similar chemical or physical property. For example, conservative amino acid substitutions can include substituting an acidic / negatively charged polar amino acid with another acidic / negatively charged polar amino acid (e.g., Asp or Glu), substituting an amino acid with a nonpolar side chain with another amino acid with a nonpolar side chain (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.), substituting a basic / positively charged polar amino acid with another basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.), substituting an uncharged amino acid with a polar side chain with another uncharged amino acid with a polar side chain (e.g., Asn, Gln, Ser, Thr, Tyr, etc.), substituting an amino acid with a β-branched side chain with another amino acid with a β-branched side chain (e.g., Ile, Thr, Val), or substituting an amino acid with an aromatic side chain with another amino acid with an aromatic side chain (e.g., His, Phe, Trp, Tyr).

[0055] The terms “homology (%)” and “sequence identity (%)” are used interchangeably in this specification to refer to comparisons between polynucleotides and polypeptides when used in relation to polypeptide or polynucleotide sequences, and are determined by comparing two optimally aligned sequences on a comparison window, in which case the portion of the polynucleotide or polypeptide sequence within the comparison window may include additions or deletions (i.e., gaps) relative to the reference sequence (which does not include additions or deletions) to optimize the alignment of the two sequences. The percentage is calculated by determining the number of positions where identical nucleic acid bases or amino acid residues exist in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Homology is evaluated using any of the various sequence comparison algorithms and programs known in the art. Examples of such algorithms and programs include, but are by no means limited to, TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW [Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85(8):2444-2448; Altschul et al., 1990, J. Mol. Biol. 215(3):403-410; Thompson et al., 1994, Nucleic Acids Res. 22(2):4673-4680; Higgins et al. 1996, Methods Enzymol. 266:383-402; Altschul et al., 1990, J. Mol. Biol. 215(3):403-410; Altschul et al., 1993, Nature Genetics 3:266-272].In certain embodiments, the homology of protein and nucleic acid sequences is evaluated using a basic local alignment search tool ("BLAST") that is well known in the art (see, for example, Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2267-2268; Altschul et al., 1990, J. Mol. Biol. 215:403-410; Altschul et al., 1993, Nature Genetics 3:266-272; Altschul et al., 1997, Nuc. Acids Res. 25:3389-3402).

[0056] As used herein, terms such as “to treat,” “treating,” and “curing” mean reducing or improving a disorder / disease and / or its associated symptoms. It will be understood that treating a disorder or condition does not necessarily mean completely eliminating the disorder, condition or its associated symptoms, although this is not impossible. In certain embodiments, treatment includes prevention of the disorder or condition and / or its associated symptoms. As used herein, the terms “prevention” or “preventing” mean any measure that inhibits the onset of a disorder, condition or its associated symptoms, or at least delays their onset. Prevention can be described as primary, secondary, and tertiary levels, where a) primary prevention avoids the onset of the disease, b) secondary prevention activities aim to treat the disease early, thus increasing opportunities for intervention to prevent disease progression and the appearance of symptoms, and c) tertiary prevention mitigates the adverse effects of an already established disease by restoring function and reducing disease-related complications.

[0057] As used herein, the terms “catabolism” or “catabolism” refer to a chemical reaction in which one molecule becomes another, for example, a smaller molecule. For example, arginine catabolic enzyme refers to any enzyme that can react with arginine to convert arginine into other molecules such as ornithine, citrulline, and agmatine.

[0058] As used herein, the term “subject” means any animal (e.g., mammal) (e.g., humans, non-human primates, dogs, cats and rodents, etc.).

[0059] As used herein, the term "BMI" refers to the ratio obtained by dividing body weight (kg) by the square of height (meters).

[0060] As used herein, the term “overweight” refers to a BMI of 25–30 in adults. For individuals under 20 years of age, “overweight” is defined as a BMI of 85–95 percent compared to that of a person of the same age.

[0061] As used herein, the term “obesity” refers to a BMI of 30–40 in adults. For individuals under 20 years of age, “obesity” is defined as a BMI greater than 95 percent compared to others of the same age. As used herein, this term may encompass both obesity and morbid obesity.

[0062] As used herein, the term “morbid obesity” refers to a BMI of 40 or greater in adults.

[0063] As used herein, the term “lipidemia” refers to a condition in which fat accumulates in tissues such as liver tissue, kidney tissue, pancreatic tissue, myocardial tissue, or other muscle tissue.

[0064] As used herein, the term "fibrosis" means a condition in which there is an excessive deposition of fibrous connective tissue, including collagen, in an organ or tissue such as liver tissue, kidney tissue, pancreatic tissue, or heart tissue.

[0065] As used herein, the term “hypercholesterolemia” refers to a condition in which blood cholesterol levels are elevated compared to the normal mean blood cholesterol levels in each reference group of the same ethnic background, age, and sex. In humans, this term specifically refers to blood total cholesterol levels above approximately 200 mg / dL, and especially above approximately 240 mg / dL.

[0066] As used herein, the terms “dyslipidemia” and “hyperlipidemia” refer to a condition in which the levels of lipids / fats such as cholesterol, cholesterol esters, phospholipids, triglycerides and / or lipoproteins in the bloodstream are abnormal and elevated compared to the normal mean values ​​of blood lipids / fats in a reference group of the same ethnic background, age, and sex.

[0067] The fusion proteins described herein include arginase polypeptides. Arginase polypeptides may be derived from arginase proteins expressed by any organism that expresses arginase. Examples of arginases include those produced by bacteria such as bacilli, Agrobacterium, cyanobacteria, and mycobacteria, and mammals such as cattle, pigs, sheep, goats, rodents, and humans. When the arginase polypeptide is derived from human arginase, it may be arginase type 1 (ARG1) or arginase type 2 (ARG2).

[0068] The arginase polypeptide may include the full-length arginase polypeptide or its functional fragments and / or variants.

[0069] Arginase is a manganese-containing enzyme. As shown in Example 32, one or more manganese ions present in the fusion protein described herein are converted into one or more Co 2+ or Ni 2+ Substitution with divalent cationic metals such as Co can enhance the catalytic activity of the fusion protein. Therefore, in certain embodiments, the fusion protein described herein is Co 2+ or Ni 2+It contains one or more divalent metals other than manganese such as. In certain embodiments, the fusion protein is Co 2+ and Ni 2+ and contains one or more metals selected from. In certain embodiments, the fusion protein is two Co 2+ ions or two Ni 2+ ions. In other embodiments, the fusion protein contains two Mn 2+ ions.

[0070] In certain embodiments, the arginase polypeptide is wild-type human ARG1. In certain embodiments, the arginase polypeptide comprises a sequence having at least 95% sequence homology with SEQ ID NO: 69. For example, the arginase polypeptide can comprise a polypeptide sequence having at least 96%, 97%, 98%, 99%, 99.1%, 99.4% or 99.7% homology with SEQ ID NO: 69. In certain embodiments, the sequence of the arginase polypeptide may differ from SEQ ID NO: 69 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or 30 amino acid modifications (e.g., insertions, substitutions, deletions, etc.). In certain embodiments, the arginase polypeptide comprises a polypeptide having conservative amino acid substitutions, non-conservative amino acid substitutions, or combinations thereof.

[0071] In certain embodiments, the arginase polypeptide is Bacillus caldovelox arginase (BCA). In certain embodiments, the arginase polypeptide comprises a sequence having at least 95% sequence homology with SEQ ID NO: 70. For example, the arginase polypeptide can comprise a polypeptide sequence having at least 96%, 97%, 98%, 99%, 99.3% or 99.7% homology with SEQ ID NO: 70. In certain embodiments, the sequence of the arginase polypeptide may differ from SEQ ID NO: 70 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or 30 amino acid modifications (e.g., insertions, substitutions, deletions, etc.). In certain embodiments, the arginase polypeptide comprises a polypeptide having conservative amino acid substitutions, non-conservative amino acid substitutions, or combinations thereof.

[0072] In certain embodiments, the arginase polypeptide is BCA, and serine 161 is substituted with cysteine, as shown in SEQ ID NOs. 71 and 72. Substituting serine with cysteine ​​allows for site-specific incorporation of a chemical moiety, which can further improve the properties of the fusion protein. For example, the side chain of cysteine ​​161 can be reacted with a appropriately activated PEG moiety to form a PEGylated arginase that can be incorporated into the resulting fusion protein. PEGylated fusion protein further protects the fusion protein described herein from various active degradation mechanisms in tissues or cells, thereby increasing the retention time of the fusion protein and consequently improving its therapeutic potential. Therefore, in certain embodiments, the arginase polypeptide includes a sequence having at least 95% sequence homology to SEQ ID NOs. 71 or 72. For example, the arginase polypeptide may include polypeptide sequences having at least 96%, 97%, 98%, 99%, 99.3%, or 99.7% homology to SEQ ID NOs. 71 or 72. In certain embodiments, the sequence of the arginase polypeptide may differ from SEQ ID NO: 71 or SEQ ID NO: 72 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 amino acid modifications (e.g., insertions, substitutions, deletions, etc.). In certain embodiments, the arginase polypeptide comprises a polypeptide having conserved amino acid substitutions, non-conserved amino acid substitutions, or combinations thereof.

[0073] When the fusion proteins described herein are PEGylated, the molecular weight of the PEG group can be about 5,000 to about 20,000 amu, about 5,000 to about 15,000 amu, about 5,000 to about 12,000 amu, about 7,000 to about 12,000 amu, or about 7,000 to about 10,000 amu. In certain embodiments, the molecular weight of the PEG group is about 4,000 to 10,000 amu. In certain embodiments, the PEG group is PEG4000 or PEG7000.

[0074] The PEG group can be covalently bonded to the fusion protein either directly or via a linker. Alternatively, the PEG group can be covalently bonded to the fusion protein by a reaction between the cysteine ​​or lysine side chain present in the fusion protein and the PEGylation reagent. In addition, the PEG group can be covalently bonded to the N-terminal amine of the protein.

[0075] In certain embodiments, the fusion protein is covalently bonded to PEG via a propionic acid linker. In other embodiments, the fusion protein is C2-C 10 The PEG group is covalently bonded to the PEG via a C2-C9, C2-C8, C2-C7, C2-C6, C2-C5, or C2-C4 linear or branched carboxylic acid linker. In certain embodiments, the PEG group is bonded to an arginase polypeptide.

[0076] The fusion proteins described herein also include albumin-binding domain (ABD) polypeptides. Numerous studies have shown the potential of albumin binding to extend the half-life of therapeutic proteins. However, designing fusion proteins containing ABD polypeptides can be challenging because fusion of ABD polypeptides into protein therapies can affect the efficacy of the protein therapy, the binding affinity of the ABD polypeptide, and the solubility of the fusion protein. Therefore, the selection of the ABD polypeptide, its binding site, and the construction of the necessary linker are not simple processes and often require time-consuming trial and error to obtain fusion properties with the desired characteristics. For example, Figures 9, 10, and 13 show that the therapeutic duration (and half-life) of N-ABD094-rhArg (SEQ ID NO: 50) is unexpectedly much longer than that of BHA and BAH. While BHA, BAH, and N-ABD094-rhArg all exhibit surprisingly long therapeutic durations (and half-lives), it was not predicted that N-ABD094-rhArg would have such a long therapeutic duration (and half-life).

[0077] Generally, a long-acting drug is desirable. The type, length, and mobility of the linker, and the fusion of the bioactive peptide or protein to the C or N terminus of the half-life extension module, can significantly affect the activity of the fusion protein. In this disclosure, various arginases are fused to ABD molecules via appropriate linkers to preserve both the albumin-binding ability and arginase enzyme activity of the ABD. Good stability and solubility are also essential. Achieving this is extremely difficult and challenging. Unlike common HSA or Fc fusions, little is known about ABD fusions. This disclosure provides examples of linker designs that can be used to generate functional arginase-ABD fusions. The activity of genetically engineered arginase fusion proteins (e.g., N-ABD094-rhArg, N-ABD-rhArg) is optimized in this disclosure by either or both linker engineering or the position of the bioactive protein (arginase) relative to the half-life extension module (ABD).

[0078] In many cases, protein engineering can be used to overcome the loss of activity. For example, linker engineering was used to restore activity lost when IFN-α2b was fused to HSA [Prot Exp Purif. 2008; 61:73-7]. Direct fusion of IFN-α2b to HSA resulted in an unstable protein with little biological activity. The effects of various linkers on IFN-α2b activity were tested in the fusion model. Peptide linkers are known to affect the expression, activity, and pharmacokinetics of fusion proteins [Adv Drug Deliv Rev. 2013; 65:1357-69]. The two main types of peptide linkers are: (i) mobile linkers (e.g., (G4S)) n (n=1~4, Sequence ID 107). (ii) α-Helical linker [A(EAAAK) n A] x (n=2~4 and x=1 or 2, Sequence ID 108), and XP nRigid linkers such as (X is A, K, or E, n=1-10). In the case of mobile linkers, one advantage is that mobility may be necessary to obtain the proper orientation of the bioactive part of the molecule with respect to the congener receptor. However, with mobile linkers, a large space cannot be obtained between the fusion partner and the bioactive protein. On the other hand, with rigid linkers, a larger space can be obtained but mobility is lacking. In the case of the IFN-α2b-HSA fusion protein, the mobile linker resulted in approximately 39% of the activity compared to natural IFN-α2b, while the rigid XP linker and α-helical linker resulted in 68% and 115% of the activity of natural IFN-α2b, respectively [Prot Exp Purif. 2008;61:73-7].

[0079] Certain linkers can negatively affect the properties of fusion proteins. For example, using a short leucine-glutamic acid (LE) linker fused to transferrin (Tf) with granulocyte colony-stimulating factor (G-CSF) resulted in only about 10% of the activity of natural G-CSF. (G4S)3 (SEQ ID NO: 109) or α-helical linker [A(EAAAK)] n A] m The insertion of (n=2-4, m=1 or 2, SEQ ID NO: 108) significantly increased the activity of the fusion protein compared to G-CSF-LE-Tf. In the case of a fusion protein constructed with the linker (A(EAAAK)4ALEA-(EAAAK)4A) (SEQ ID NO: 105), biological activity close to that of natural G-CSF was obtained [Pharm Res. 2006;23:2116-21]. In other examples, the C-terminus of the Fc moiety can be directly linked to the N-terminus of the IFN-β moiety via a peptide bond, but Gillies et al [US7,670,595B2] further link the Fc moiety and the IFN-β moiety via a linker peptide. The linker peptide is located between the C-terminus of the Fc moiety and the N-terminus of the mature IFN-β moiety. In this case, the linker peptide is preferably composed of serine and glycine residues such as the amino acid sequence G4SG4SG3SG (SEQ ID NO: 106). All of these findings indicate that testing linker technology is crucial for the success of fusion protein research and development programs.

[0080] The importance of fusion site for activity has been demonstrated using a different approach [Curr Pharmaceut Biotechnol. 2014;15:856-63]. Brain natriuretic peptide (BNP) was fused to the N-terminus or C-terminus of HSA in various forms. The results showed that BNP-HSA, BNP2-HSA (two copies of BNP), and BNP4-HSA (all fused to the N-terminus of HSA) were inactive. However, HSA-BNP2 fused to the C-terminus of HSA exhibited activity comparable to that of natural BNP and was persistent.

[0081] These examples demonstrate the importance of optimizing activity by making considerable efforts to optimize the reads of fusion proteins, either through linker engineering, the position of the bioactive protein or peptide relative to the half-life extension module, or both. Importantly, the present invention uses a novel ABD fusion approach to conjugate ABD and arginase to each other while maintaining arginase activity. We successfully generated a stable, soluble arginase-ABD fusion molecule that can bind to FcRn in a pH-dependent manner, enabling efficient endosome recycling. For example, surprisingly, in this disclosure, we found that in the case of rhArg fused to ABD, the terminal half-life in protein circulation was dramatically extended from a few minutes to four days in mice. In summary, various different approaches are available to fine-tune the pharmacokinetic properties of arginases and ensure appropriate residence times in circulation for various pathological conditions. Another important challenge in the development of therapeutic proteins is minimizing immunogenicity and avoiding adverse effects. Therefore, it is important to note that low immunogenicity of human arginase was observed, and that deimmunization of ABD (e.g., ABD094), which was also used in this disclosure as a gene fusion partner to extend the in vivo half-life, was successful.

[0082] Albumin-binding proteins are three helical protein domains found in various surface proteins expressed by Gram-positive bacteria. Albumin-binding proteins derived from Streptococcus protein G have 214 amino acids and contain three albumin-binding domains (ABD1-3) used to bind to human serum albumin and evade the host immune system. ABD3 corresponds to a 46-amino acid sequence and has been demonstrated to bind to human serum albumin. It has been the subject of numerous studies and affinity maturation studies on human serum albumin in developing ABD polypeptides with various properties (e.g., binding affinity and binding selectivity). Such studies have resulted in the generation of a considerable number of ABD polypeptides with diverse characteristics.

[0083] Albumin-binding proteins are found in other bacteria. For example, naturally occurring albumin-binding proteins include certain surface proteins from Gram-positive bacteria, such as Streptococcus M proteins (e.g., M1 / Emm1, M3 Emm3, M12 / Emml2, EmmL55 / Emm55, Emm49 / EmmL49, and protein H), Streptococcus proteins G, MAG, and ZAG, and PPL and PAB from certain strains of Finegoldia magna.

[0084] In certain embodiments, the fusion protein described herein comprises an ABD polypeptide derived from the streptococcal protein G albumin-binding domain. In certain embodiments, the ABD polypeptide is the complete streptococcal protein G albumin-binding domain 3 or a functional fragment and / or variant thereof.

[0085] In certain embodiments, the fusion protein comprises an ABD polypeptide having a polypeptide sequence having at least 93% sequence homology to SEQ ID NO: 66. For example, the ABD polypeptide may contain a polypeptide sequence having at least 94%, 96%, or 98% homology to SEQ ID NO: 66. In certain embodiments, the sequence of the ABD polypeptide may differ from SEQ ID NO: 66 by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., insertions, substitutions, deletions, etc.). In certain embodiments, the ABD polypeptide may contain a polypeptide having conserved amino acid substitutions, non-conserved amino acid substitutions, or a combination thereof.

[0086] In certain embodiments, the fusion protein comprises an ABD polypeptide having a polypeptide sequence having at least 93% sequence homology to SEQ ID NO: 67. For example, the ABD polypeptide may contain a polypeptide sequence having at least 94%, 96%, or 98% homology to SEQ ID NO: 67. In certain embodiments, the sequence of the ABD polypeptide may differ from SEQ ID NO: 67 by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., insertions, substitutions, deletions, etc.). In certain embodiments, the ABD polypeptide may contain a polypeptide having conserved amino acid substitutions, non-conserved amino acid substitutions, or a combination thereof.

[0087] In certain embodiments, the fusion protein comprises an ABD polypeptide having a polypeptide sequence having at least 93% sequence homology to SEQ ID NO: 68. For example, the ABD polypeptide may contain a polypeptide sequence having at least 93%, 95%, or 97% homology to SEQ ID NO: 68. In certain embodiments, the sequence of the ABD polypeptide may differ from SEQ ID NO: 68 by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., insertions, substitutions, deletions, etc.). In certain embodiments, the ABD polypeptide may contain a polypeptide having conserved amino acid substitutions, non-conserved amino acid substitutions, or a combination thereof.

[0088] The relative positions of the ABD polypeptide and the arginase polypeptide can vary. For example, the ABD polypeptide can precede the arginase polypeptide (e.g., the arginase polypeptide can bind directly or indirectly from the C-terminus of the ABD polypeptide), or the arginase polypeptide can precede the ABD polypeptide (e.g., the ABD polypeptide can bind directly or indirectly from the C-terminus of the arginase polypeptide).

[0089] In certain embodiments, the fusion protein may contain one or more arginase polypeptides and / or one or more ABD polypeptides. For example, the fusion protein may have the general structures ABD-rhArg-ABD, ABD094-rhArg-ABD094, ABD-BCA-ABD, ABD094-BCA-ABD094, rhArg-ABD-rhArg, rhArg-ABD094-rhArg, BCA-ABD-BCA, or BCA-ABD094-BCA.

[0090] ABD polypeptides and arginase polypeptides can be bound directly by covalent bonds, or indirectly via peptide linkers.

[0091] A peptide linker, or linker, is a polypeptide typically having an amino acid length in the range of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more, and is designed to facilitate the functional linkage that forms a linkage fusion protein between an ABD polypeptide and an arginase polypeptide. The term "functional linkage" refers to a linkage that facilitates the proper folding of polypeptides into a three-dimensional structure, which allows the linkage fusion protein to exhibit some or all of the functional aspects or biological activities of the proteins from which the polypeptide components originate.

[0092] The polypeptide linker can be positioned between the N-terminus of the ABD polypeptide and the C-terminus of the arginase polypeptide, or between the N-terminus of the arginase polypeptide and the C-terminus of the ABD polypeptide.

[0093] Peptide linkers can include naturally occurring amino acids, unnatural amino acids, and combinations thereof.

[0094] In certain embodiments, the peptide linker may include glycine, serine, asparagine, or a combination thereof. Examples of peptide linkers include polyglycine, (GS) n (Sequence ID 110) and (GGS) n Linkers including (Sequence ID 111) (n is 1-30) are mentioned. Further examples of peptide linkers include mobile linkers (e.g., (G4S) n (n=1~4, Sequence ID 107), or rigid linker (e.g., α-helical linker [A(EAAAK) n A] x (n=2~4 and x=1 or 2 (Sequence ID 108), and XP n Examples include (where X is A, K, or E, and n=1 to 10). In certain embodiments, the peptide linker is (A(EAAAK)4ALEA-(EAAAK)4A) (SEQ ID NO: 105), G4SG4SG3SG (SEQ ID NO: 106), GS(N) n GSG(n=1~10)(Sequence ID 112), and GS(Q) n Examples include GSG (n=1~10) (SEQ ID NO: 113). In certain embodiments, the peptide linker includes a polypeptide sequence having at least 90% sequence homology to SEQ ID NO: 73. For example, the peptide linker may include a polypeptide having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology to SEQ ID NO: 73, or being identical to SEQ ID NO: 73.

[0095] In certain embodiments, the peptide linker may include glycine, serine, asparagine, or a combination thereof. In certain embodiments, the peptide linker may include a polypeptide sequence having at least 90% sequence homology to SEQ ID NO: 74. For example, the peptide linker may include a polypeptide having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology to SEQ ID NO: 74, or being identical to SEQ ID NO: 74.

[0096] Purification tags can be used to improve the ease of purifying fusion proteins by affinity chromatography or other methods. A well-known purification tag is the hexahistidine (6×His) tag, which is a sequence of six histidine residues. Therefore, in certain embodiments, the fusion protein further comprises a polyhistidine containing 4 to 8 histidine amino acids, such as a 6×His tag. The polyhistidine can be located at the C-terminus, N-terminus, or between the ABD polypeptide and the arginase polypeptide.

[0097] When polyhistidine is positioned between the ABD polypeptide and the arginase polypeptide, it can act as a peptide linker or be included in addition to the peptide linker. For example, the fusion protein of SEQ ID NO: 75 contains a polypeptide linker consisting of six histidines at positions 300-305, which helps to link the ABD polypeptide and the arginase polypeptide and can be advantageously used to purify the fusion protein by affinity chromatography.

[0098] The polyhistidine tag can be removed as needed after purification is complete using techniques commonly known in the art. For example, the N-terminal polyhistidine tag can be removed using an exopeptidase (e.g., Qiagen TAGZyme), and a suitable amino acid sequence can be placed before the C-terminal polyhistidine tag to facilitate removal of the polyhistidine tag using an endopeptidase. Thus, fusion proteins without the N-terminal and / or C-terminal polyhistidine tags are included within the scope of this disclosure.

[0099] In certain embodiments, the ABD polypeptide includes a polypeptide sequence having at least 93% sequence homology to SEQ ID NO: 66, SEQ ID NO: 67, or SEQ ID NO: 68, and the arginase polypeptide includes a polypeptide sequence having at least 95% homology to SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, or SEQ ID NO: 72. For example, the ABD polypeptide includes a polypeptide sequence having at least 94%, 95%, 96%, 97%, 98%, or 99% sequence homology to SEQ ID NO: 66, SEQ ID NO: 67, or SEQ ID NO: 68, or is identical to SEQ ID NO: 66, SEQ ID NO: 67, or SEQ ID NO: 68, and the arginase polypeptide includes a polypeptide sequence having at least 96%, 97%, 98%, or 99% sequence homology to SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, or SEQ ID NO: 72, or is identical to SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, or SEQ ID NO: 72.

[0100] In certain embodiments, the ABD polypeptide comprises a polypeptide sequence having at least 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology with SEQ ID NO: 66, or being identical to SEQ ID NO: 66, and the arginase polypeptide comprises a polypeptide sequence having at least 95%, 96%, 97%, 98%, or 99% sequence homology with SEQ ID NO: 69, or being identical to SEQ ID NO: 69.

[0101] In certain embodiments, the fusion protein further comprises a peptide linker containing a polypeptide sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology with SEQ ID NO: 73 or SEQ ID NO: 74, or is identical to SEQ ID NO: 73 or SEQ ID NO: 74.

[0102] Examples of fusion proteins include fusion proteins that have at least 95%, 96%, 97%, 98%, or 99% homology with SEQ ID NOs. 49, 50, 51, 52, 53, 54, 55, 56, 75, and 76, or that are identical to these SEQ ID NOs.

[0103] The duration of treatment for the effect of the fusion protein on plasma arginine concentration depends on the amount of fusion protein administered and can be approximately 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, or 15 days or longer. In certain embodiments, the duration of fusion protein treatment is approximately 5 to 20 days, 5 to 19 days, 5 to 18 days, 5 to 17 days, 5 to 16 days, 5 to 15 days, 6 to 15 days, 7 to 15 days, 7 to 14 days, 7 to 13 days, 7 to 12 days, 7 to 11 days, or 8 to 11 days.

[0104] In certain embodiments, the half-life of the fusion protein is approximately 1 to 10 days, 1 to 9 days, 1 to 8 days, 1 to 7 days, 1 to 6 days, 1 to 5 days, 1 to 4 days, 1 to 3 days, or 1 to 2 days. In other embodiments, the half-life of the fusion protein is approximately 6 to 30 hours.

[0105] The arginase activity of the fusion proteins described herein may be substantially the same as, lower than, or higher than that of the arginase polypeptide from which they are derived. One unit of arginase activity is defined as the amount of fusion protein [e.g., BHA (SEQ ID NO: 75), BAH (SEQ ID NO: 76), N-ABD-rhArg (SEQ ID NO: 49), or N-ABD094-rhArg (SEQ ID NO: 50)] or arginase [e.g., BCA (SEQ ID NO: 70)] that catalyzes the production of 1 μmol of urea per minute under standard assay conditions. The specific activity of the enzyme is expressed in units of activity per mg of protein. Under standard diacetylmonoxime (DAMO) assay conditions (37°C, pH 7.4), the specific activity of the fusion protein may be about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, or about 40% lower or higher than that of the corresponding arginase polypeptide into which it is incorporated. In certain embodiments, the fusion protein may have a specific activity that is about 5% to about 40%, about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, about 20% to about 30%, about 20% to about 35%, about 15% to about 30%, about 15% to about 25%, or about 10% to about 20% lower or higher than the corresponding arginase polypeptide incorporated therein.

[0106] In certain embodiments, the arginase activity of the fusion protein is substantially unaffected by the presence of HSA. This is advantageous because the binding of the ABD fusion protein to HSA can have detrimental effects on the activity of the fusion protein.

[0107] Polynucleotide sequences encoding the fusion proteins described herein are also provided, either as isolated polynucleotides, as part of an expression vector, or as part of a linear DNA sequence (e.g., a linear DNA sequence used for in vitro transcription / translation, a vector suitable for prokaryotic or eukaryotic expression, secretion, and / or compositional indication). While certain exemplary polynucleotides are disclosed herein, other polynucleotides encoding the fusion proteins described herein are also within the scope of this disclosure, given codon selection or degeneracy of the genetic code in a given expression system.

[0108] The polynucleotides described herein can be produced by chemical synthesis, such as solid-phase polynucleotide synthesis in an automated polynucleotide synthesizer, and constructed into complete single-stranded or double-stranded molecules. Alternatively, the polynucleotides of the present invention can be produced by other techniques, such as PCR and subsequent conventional cloning. Techniques for producing or obtaining polynucleotides of a given known sequence are well known in the art.

[0109] The polynucleotides described herein may include at least one non-coding sequence, such as a promoter or enhancer sequence, an intron, or a polyadenylation signal. The polynucleotide sequence may also include additional sequences encoding additional amino acids, such as a marker or tag sequence (e.g., a polyhistidine (6×His) or HA tag to facilitate protein purification or detection), a signal sequence, or a fusion protein partner (e.g., a cDNA encoding a bioactive agent).

[0110] Provided in other embodiments are vectors comprising at least one of the polynucleotides described herein. Such vectors may be plasmid vectors, viral vectors, baculovirus expression vectors, transposon-based vectors, or any other vector suitable for introducing the polynucleotides of the present invention into a given organism or genetic background by any means. Such vectors may be expression vectors comprising nucleic acid sequence elements that can control, regulate, induce, or enable the expression of the polypeptide encoded by the vector. Such elements may include transcription enhancer binding sites, RNA polymerase initiation sites, ribosome binding sites, and other sites that promote the expression of the polypeptide encoded in a given expression system. Such expression systems may be cell-based or cell-free systems as are well known in the Art.

[0111] In many bacterial expression systems, the start codon typically encodes methionine, resulting in the production of proteins that begin with N-terminal methionine in such systems. However, it is well known that certain bacterial enzymes, such as methionine aminopeptidase (MetAP), can catalyze the hydrolytic cleavage of N-terminal methionine from newly synthesized polypeptides. This is commonly observed when the neighboring amino acid is, for example, Gly, Ala, Ser, or Thr [In vivo processing of N-terminal methionine in E. coli, FEBS Lett. 1990 Jun 18;266(1-2):1-3]. Therefore, certain embodiments of the fusion proteins described herein include variants in which the N-terminal methionine of the protein is absent.

[0112] The fusion proteins described herein can be isolated using separation procedures well known in the art for capture, immobilization, distribution, or sedimentation, and can be purified to the extent required for commercial use.

[0113] For therapeutic use, the fusion proteins described herein may be prepared as pharmaceutical compositions comprising a therapeutically effective amount of the fusion protein described herein as an active ingredient in a pharmaceutically acceptable carrier. The term “carrier” means a diluent, adjuvant, excipient, or vehicle used for administering the active compound. Examples of such vehicles include liquids, such as water, and oils of petroleum, animal, plant, or synthetic origin (e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc.). For example, 0.9% saline and 0.3% glycine can be used. Such solutions are sterile and generally free of particulate matter. Such solutions can be sterilized by conventionally known sterilization techniques (e.g., filtration). The composition may include pharmaceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as pH adjusters and buffers, stabilizers, thickeners, lubricants, and colorants. The concentration of fusion proteins in such pharmaceutical formulations can vary considerably, for example, from less than approximately 0.5% by weight (usually at least approximately 1% by weight) to as high as 15 or 20% by weight, and is selected primarily based on the required dose, volume, viscosity, etc., depending on the specific administration method chosen. Suitable vehicles and formulations are described, for example, in Remington: The Science and Practice of Pharmacy, 21st Edition, Troy, DB ed., Lipincott Williams and Wilkins, Philadelphia, Pa. 2006, Part 5, Pharmaceutical Manufacturing pp. 691-1092 (see pp. 958-989 in particular).

[0114] As is well known in the art, the method of administration for the therapeutic use of the fusion protein described herein can be any suitable route for delivering the drug to the host, for example, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous, pulmonary), mucosal administration (oral, intranasal, vaginal, rectal), use of formulations of tablets, capsules, solutions, suspensions, powders, gels, particles, inclusion in syringes, implantable devices, osmotic pumps, cartridges, micropumps, or other means recognized by those skilled in the art. Site-specific administration can be performed, for example, by intra-articular delivery, intra-bronchial delivery, intraperitoneal delivery, intracapsular delivery, intra-cartilage delivery, intracavitary delivery, intracerebral delivery, intracolonic delivery, intracervical delivery, intragastric delivery, intrahepatic delivery, intracardiac delivery, intraosseous delivery, intrapelvic delivery, intraperitoneal delivery, intrapleural delivery, intraprostatic delivery, intrapulmonary delivery, intrarectal delivery, intrarenal delivery, intraretinal delivery, intraspinal delivery, synovial delivery, intrathoracic delivery, intrauterine delivery, intravascular delivery, intrabladder delivery, intrafocal delivery, vaginal delivery, rectal delivery, buccal delivery, sublingual delivery, intranasal delivery, or percutaneous delivery.

[0115] The fusion proteins described herein can be used as arginine depletion agents. Such agents are useful in treating diseases or conditions in which arginine depletion has a therapeutic effect, such as cancer, certain viral infections, multiple sclerosis, rheumatoid arthritis, autoimmune diseases, congenital hyperargininemia, graft-versus-host disease (GvHD), and, as detailed herein, obesity, metabolic disorders, and associated complications and comorbidities.

[0116] Arginine-dependent tumors are dependent on extracellular arginine due to the downregulation of ASS or OTC, and depend on extracellular arginine sources for survival. Arginine depletion agents have been shown to be cytotoxic to arginine-dependent tumors, and their use in cancer treatment is currently being investigated in clinical trials.

[0117] Accordingly, the fusion proteins described herein can be used to treat cancers such as pancreatic cancer, leukemia, melanoma, head and neck cancer, colorectal cancer, lung cancer, breast cancer, prostate cancer, cervical cancer, liver cancer, nasopharyngeal cancer, esophageal cancer, sarcoma, gastric cancer, mesothelioma, lymphoma, bladder cancer, bone cancer, endometrial cancer, ovarian cancer, kidney cancer, eye cancer, neuroblastoma, glioblastoma, malignant peripheral nerve sheath tumor (MPNST), and brain cancer. The cancers may be arginine-dependent cancers, such as cancers characterized by downregulation of at least one of ASS and OTC.

[0118] The fusion proteins described herein can also be used to treat viral infections, such as human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), herpes simplex virus (HSV), human papillomavirus (HPV), Epstein-Barr virus (EBV), herpesviruses, varicella-zoster virus, and influenza.

[0119] As detailed herein, the fusion proteins described herein may also be used to treat at least one condition selected from the group consisting of obesity, metabolic disorders, and associated complications and / or comorbidities.

[0120] As shown in the following experiments, arginine depletion can dramatically suppress steatosis. This specification refers to N-ABD094-rhArg (SEQ ID NO: 50), PEGylated His-rhArg (SEQ ID NO: 101), and N-ABD094-rhArg-Co 2+The therapeutic potential of ABD arginase fusion proteins or PEGylated arginases, such as [SEQ ID NO: 50 (cobalt substitution)], for the treatment of obesity and related metabolic disorders was investigated. Data presented herein show, for example, that arginine depletion by N-ABD094-rhArg (SEQ ID NO: 50) significantly reduces fat mass, suppresses lipid synthesis, and enhances insulin sensitivity in normal lean mice and obese mice. For instance, arginine depletion by N-ABD094-rhArg (SEQ ID NO: 50) was found to prevent and mitigate obesity, metabolic disorders, and related complications and comorbidities (e.g., insulin resistance).

[0121] The effects of arginine depletion described herein are surprising and unexpected, because many studies have shown that increasing dietary intake of several amino acids (AAs), including arginine, significantly affects glucose and lipid metabolism, and that AA supplementation can lead to weight loss. Furthermore, since arginine is a semi-essential amino acid, its depletion does not have adverse effects on the subjects.

[0122] A mouse model of high-fat diet-induced obesity (DIO) was used. Lean mice initiated on a high-fat diet were simultaneously treated with N-ABD094-rhArg (SEQ ID NO: 50) to test its effectiveness in preventing the development of DIO and associated metabolic disorders. Mice with pre-existing DIO were treated with N-ABD094-rhArg (SEQ ID NO: 50) to test its effectiveness in reducing obesity, metabolic disorders, and associated complications and comorbidities.

[0123] As shown in the following examples, treatment with N-ABD094-rhArg (SEQ ID NO: 50) reduced body weight and fat mass, improved glucose tolerance and insulin responsiveness, normalized endocrine and metabolic profiles, alleviated inflammation, steatosis and fibrosis, and prevented or reversed the whitening of brown fat.

[0124] Provided herein is a method for treating obesity and / or metabolic disorders in subjects requiring treatment, the method comprising the step of administering a therapeutically effective dose of an arginine depletion agent to the subject.

[0125] The arginine depletor can be any arginine depletor known in the art that is capable of reducing the plasma and / or cellular arginine concentration in the subject. The arginine depletor can be a small molecule or a protein.

[0126] The protein may be a fusion protein and / or a chemically modified protein (e.g., a PEGylated protein). Examples of proteins include those capable of catalyzing the catabolism of arginine to other products, such as proteins having arginase activity, arginine deiminase activity, arginine decarboxylase activity, or arginine 2 monooxygenase activity.

[0127] The arginase may be any arginase known in the art, such as those produced by bacteria, fungi, fish, humans, cattle, pigs, rabbits, rodents, primates, sheep, and goats. For example, Bacillus caldovelox arginase, Thermus thermophilus arginase, Capra hircus arginase I, Heterocephalus glaber arginase I, Bos taurus arginase I, Sus scrofa arginase I, Plecoglossus altivelis arginase I, Salmo salar arginase I, Oncorhynchus mykiss arginase I, Osmerus mordax arginase I, Hyriopsis cumingii arginase I, Rattus norvegicus arginase I, Mus musculus arginase I, Homo sapiens (human) arginase I, Pan troglodytes arginase I, Oryctolagus cuniculus arginase I, Rattus norvegicus arginase II, Mus musculus arginase II, Homo Examples include human sapiens arginase II, Bostaurus arginase II, Heterocephalus glaber arginase II, Pan troglodytes arginase II, Oryctolagus cuniculus arginase II, Delftia arginase, Bacillus coagulans arginase, Hoeflea phototrophica arginase, and Roseiflexus castenholzii arginase.Other examples include arginase derived from Bacillus methanolicus, Bacillus sp. NRRL B-14911, Planococcus donghaensis, Paenibacillus dendritiformis, Desmospora sp., Methylobacter tundripaludum, Stenotrophomonas sp., Microbacterium laevaniformans, Porphyromonas uenonis, Agrobacterium sp., Octadecabacter arcticus, Agrobacterium tumefaciens, Anoxybacillus flavithermus, Bacillus pumilus, Geobacillus thermoglucosidasius, Geobacillus thermoglucosidans, Brevibacillus laterosporus, Desulfotomaculum ruminis, Geobacillus kaustophilus, Geobacillus thermoleovorans, Geobacillus thermodenitrificans, Staphylococcus aureus, Halophilic archaeon DL31, Halopigerxanaduensis, Natrialba magadii, Plasmodium falciparum, Helicobacter pylori, etc.

[0128] The arginine deiminase may be any arginine deiminase known in the art, for example, one produced from Mycoplasma, Lactococcus, Pseudomonas, Streptococcus, Escherichia, Mycobacterium, or Bacillus microorganisms. Examples of arginine deiminases include, but are not limited to, those produced by Mycoplasma hominis, Mycoplasma arginini, Mycoplasma arthritidis, Clostridium perfringens, Bacillus licheniformis, Borrelia burgdorferi, Borrelia afzellii, Enterococcus faecalis, Lactococcus lactis, Bacillus cereus, Streptococcus pyogenes, Steptococcus pneumoniae, Lactobacillus sake, Giardia intestinalis, Mycobacterium tuberculosis, Pseudomonas plecoglossicida, Pseudomonas putida, and Pseudomonas aeruginosa.

[0129] Arginine decarboxylase is any arginine decarboxylase known in the art, e.g., Escherichia coli., Salmonella typhimurium, Chlamydophila pneumoniae, Methanocaldococcus jannaschii, Paramecium bursaria Chlorella virus 1, Vibrio vulnificus YJ016, Campylobacter jejuni subsp., Trypanosoma cruzi, Sulfolobus solfataricus, Bacillus licheniformis, Bacillus cereus, Carica papaya, Nicotianatobacum, Glycine max, Lotus coniculata, Vibrio vulnificus, Vibrio cholerae, Mus musculus, Thermoga, Rattus norvegicus, Homo sapiens, Bos taurus, Susscrofa, Thermus thermophiles, Thermus This can be attributed to the production of plants such as parvatiensis, Thermus aquaticus, Thermus thermophilus, Thermus islandicus, Arabidopsis thaliana, and Avena sativa.

[0130] Arginine 2-monooxygenase can be produced from any arginine 2-monooxygenase known in the art, such as Arthrobacter globiformis IFO12137, Arthrobacter simplex IFO12069, Brevibacterium helvolum IFO12073, Helicobacter cinaedi CCUG18818, Streptomyces griseus, etc.

[0131] Arginine decarboxylase, arginine deiminase, arginine 2-monooxygenase, and arginase can be complete proteins or functional fragments and / or variants thereof. Arginine decarboxylase, arginine deiminase, arginine 2-monooxygenase, and arginase can be modified, for example, by fusion of the protein or its functional fragment and / or variant with human serum albumin, albumin-binding domains, immunoglobulin Fc regions, PEG groups, or combinations thereof to improve their pharmacokinetic properties.

[0132] The arginine catabolic enzymes described herein can be genetically engineered to include specific sites on the enzyme to which PEG can selectively bind. The selected PEGylation site is preferably located at a site removed from the enzyme's active site and is typically exposed to a solvent to enable reaction with the PEGylation reagent.

[0133] For example, Cys 45 - Human arginase I (HAI) and Cys 161 -Bacillus caldovelox arginase (BCA) can be generated to react with thiol-specific PEG molecules. A covalent bond can be formed between the PEG compound and the free cysteine ​​of the modified arginase by the binding of a single free cysteine ​​residue of the modified arginase to a maleimide group (MAL) bound to the PEG compound. Sequence IDs 102 and 104 are Cys 45 It contains a variant (C168S / C303S) designed for site-specific PEGylation, so it can be PEGylated as needed. Sequence ID 89 is also Cys 161 Because it contains a variant (S161C) designed for site-specific PEGylation, it can be PEGylated as needed.

[0134] In certain embodiments, the arginase may include SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, or SEQ ID NO: 104, wherein SEQ ID NOs: 102 and 104 may optionally include polyethylene glycol groups (PEG).

[0135] Any PEGylating reagent known in the art can be used to covalently bond PEG to the arginine catabolic enzyme described herein. Examples of PEGylating reagents include, but are not limited to, mPEG-ALD (methoxypolyethylene glycol-propionaldehyde), mPEG-MAL (methoxypolyethylene glycol-maleimide), mPEG-NHS (methoxypolyethylene glycol-N-hydroxy-succinimide), mPEG-SPA (methoxypolyethylene glycol-succinimidylpropionate), and mPEG-CN (methoxypolyethylene glycol-cyanuryl chloride).

[0136] The molecular weight of the PEG group can be approximately 5,000 to 20,000 amu, approximately 5,000 to 15,000 amu, approximately 5,000 to 12,000 amu, approximately 7,000 to 12,000 amu, or approximately 7,000 to 10,000 amu. In certain embodiments, the molecular weight of the PEG group is approximately 2,000 amu to 10,000 amu. In certain embodiments, the PEG group is PEG4000, PEG5000, PEG6000, or PEG7000.

[0137] The PEG group can be covalently bonded to the arginase directly or via a linker. In certain embodiments, the arginase is covalently bonded to PEG via a propionic acid linker. In other embodiments, the arginase is C2-C 10 It is covalently bonded to PEG via C2-C9, C2-C8, C2-C7, C2-C6, C2-C5, or C2-C4 linear or branched carboxylic acid linkers.

[0138] In another embodiment, a method for treating a condition in a subject requiring treatment of at least one condition selected from the group consisting of obesity, metabolic disorders, and associated complications and / or comorbidities includes administering a low-arginine diet or a substantially arginine-free diet to the subject.

[0139] A method for treating at least one condition selected from the group consisting of obesity, metabolic disorders, and associated complications and / or comorbidities may also include the prevention of at least one condition selected from the group consisting of obesity, metabolic disorders, and associated complications and / or comorbidities.

[0140] Metabolic disorders include obesity, hypercholesterolemia, dyslipidemia, steatosis, insulin resistance, impaired glucose tolerance, hyperglycemia, and diabetes mellitus or combinations thereof. Associated complications and / or comorbidities include diabetic retinopathy, diabetic nephropathy, diabetic vascular disease, diabetic neuropathy, fatty liver disease, fibrosis, cirrhosis, inflammation, hypertension, cardiovascular disease, and whitening of brown fat or combinations thereof.

[0141] In certain embodiments, metabolic disorders include, for example, the treatment and / or prevention of insulin resistance in subjects with type 1 or especially type 2 diabetes. As shown in the following examples, arginine depletion results in increased insulin sensitivity in subjects.

[0142] Methods for treating obesity may include reducing or preventing an increase in fat mass in a subject. Treatment of a subject with an arginine depletion agent can reduce the mRNA expression levels of several adipogenic transcription factors and lipid-producing enzymes (e.g., Pparg, Srebp1c, Acc, and Scd1) in visceral white adipose tissue (WAT), liver, and skeletal muscle compared to an untreated subject on a high-fat diet. In certain embodiments, the reduction in mRNA expression levels of lipid-producing regulators is, for example, at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% compared to the corresponding mRNA expression levels in an untreated subject on a high-fat diet. In certain embodiments, treatment of a subject with an arginine depletion agent suppresses the mRNA expression of one or more genes selected from the group consisting of Pparg, Srebp1c, Acc, and Scd1 in the subject's WAT, liver, and / or skeletal muscle.

[0143] Obesity is a result of, and / or may be related to, metabolic disorders (e.g., hyperglycemia, hyperinsulinemia) and / or other factors such as overeating and lack of exercise.

[0144] Metabolic disorders may also include the treatment and / or prevention of steatosis in the subject, such as cardiac steatosis, fatty liver, renal steatosis, pancreatic steatosis, and muscular steatosis.

[0145] Metabolic disorders may also include the treatment and / or prevention of fibrosis in the subject, such as hepatic fibrosis, renal fibrosis, pancreatic fibrosis, and cardiac fibrosis.

[0146] Metabolic disorders may include, for example, the treatment of hypercholesterolemia and dyslipidemia by lowering the concentrations of total cholesterol, LDL cholesterol, apolipoprotein B, and / or triglycerides in plasma.

[0147] Treatment of a subject with an arginine depletion agent can reduce plasma leptin concentration by, for example, at least about 10%, 20%, 30%, 40%, 50%, 60%, or 70% compared to the corresponding plasma leptin concentration in an untreated subject with HFD. In certain embodiments, plasma leptin concentration can be reduced by about 10% to 70%, 10% to 60%, 10% to 50%, 10% to 40%, or 10% to 30% compared to the corresponding plasma leptin concentration in an untreated subject with HFD.

[0148] Treatment of a subject with an arginine depletion agent can reduce plasma triglyceride concentrations by, for example, at least about 20%, about 30%, or about 40% compared to the corresponding plasma triglyceride concentrations in untreated subjects with HFD. In certain embodiments, plasma triglyceride concentrations can be reduced by about 5% to about 40%, about 5% to about 30%, or about 5% to about 20% compared to the corresponding plasma triglyceride concentrations in untreated subjects with HFD.

[0149] Treatment of a subject with an arginine depletion agent can reduce the plasma total cholesterol concentration by, for example, at least about 5%, about 10%, or about 15% compared to the corresponding plasma total cholesterol concentration of an untreated subject with HFD. In certain embodiments, the plasma total cholesterol concentration can be reduced by about 5% to about 15%, about 5% to about 10%, or about 10% to about 15% compared to the corresponding plasma total cholesterol concentration of an untreated subject with HFD.

[0150] Treatment of a target with an arginine depletion agent can reduce the plasma free fatty acid concentration by, for example, at least about 5%, about 10%, about 15%, or about 20% compared to the corresponding plasma free fatty acid concentration in an untreated HFD target. In certain embodiments, the plasma free fatty acid concentration can be reduced by about 5% to about 20%, about 5% to about 15%, or about 5% to about 10% compared to the corresponding plasma free fatty acid concentration in an untreated HFD target.

[0151] The plasma arginine concentration of the subject whose therapeutic effect needs to be observed can vary based on many factors, including the subject's condition, the type and severity of the disease, and / or the patient's condition and / or dietary composition. The selection of the target plasma arginine concentration is well within the skill of those skilled in the art. In certain embodiments, the plasma arginine concentration is less than about 100 μM, less than about 90 μM, less than about 80 μM, less than about 70 μM, less than about 60 μM, less than about 50 μM, less than about 40 μM, less than about 30 μM, less than about 20 μM, less than about 10 μM, or less than about 5 μM. In certain embodiments, the plasma arginine concentration is approximately 0.1 μM to 100 μM, approximately 0.1 μM to 90 μM, approximately 0.1 μM to 80 μM, approximately 0.1 μM to 70 μM, approximately 0.1 μM to 60 μM, approximately 0.1 μM to 50 μM, approximately 0.1 μM to 40 μM, approximately 0.1 μM to 30 μM, approximately 0.1 μM to 20 μM, or approximately 0.1 μM to 10 μM. In certain embodiments, the arginine concentration is below the detection limit of a Biochrom 30 amino acid analyzer (e.g., less than approximately 3 μM) and / or below the detection limit of an Agilent 6460 liquid chromatography / electrospray ionization triple quadrupole mass spectrometer (e.g., less than approximately 0.3 μM).

[0152] Determining the duration of treatment (for example, the period during which plasma arginine concentration is maintained in a depleted state in the subject) is well within the skill of those skilled in the art. In certain embodiments, the duration of treatment is approximately 1 week, 2 weeks, 3 weeks, 4 weeks, 8 weeks, 12 weeks, 16 weeks, 20 weeks, 24 weeks, 28 weeks, 32 weeks, 36 weeks, 40 weeks, 44 weeks, 48 ​​weeks, 52 weeks, 56 weeks or longer.

[0153] Arginine depletion has been shown to be an effective treatment for many conditions, including viral infections (U.S. Patent No. 8,507,245, which is included in this specification), multiple sclerosis, rheumatoid arthritis, autoimmune diseases (U.S. Patent No. 9,789,169, which is included in this specification), congenital hyperargininemia (ClinicalTrials.gov Identifier: NCT02488044), graft-versus-host disease (GvHD) (Hallet W., et al., Exploiting arginase to prevent GVHD, Blood 2010 116:5440-5441), and inflammation (Sahin E., et al., Macrophage PTEN regulates expression and secretion of arginase I modulating innate and adaptive immune responses, J Immunol. 2014 Aug). Examples include, but are not limited to, 15;193(4):1717-27) and retinal neurovascular degeneration (Fouda AY. et al., Arginase 1 promotes retinal neurovascular protection from ischemia through suppression of macrophage inflammatory responses Cell Death Dis. 2018 Sep 25;9(10):1001).

[0154] Accordingly, further provided herein are methods for treating a condition in a subject requiring treatment of at least one condition selected from the group consisting of viral infections, multiple sclerosis, rheumatoid arthritis, autoimmune diseases, congenital hyperargininemia, graft-versus-host disease (GvHD), and inflammation, the methods comprising the step of administering a therapeutically effective amount of the fusion protein described herein.

[0155] Further objectives, advantages, and novel features of this disclosure will become apparent to those skilled in the art by considering the following embodiments, which are not intended to be limiting. [Examples]

[0156] Construction of ABD and ABD094 fusion genes The N-ABD gene for N-terminal fusion was constructed by mixing primers 01-08 (SEQ ID NOs: 1-8) listed in Table 1 (each primer at a concentration up to 20 nM) with iProof DNA polymerase (Bio-rad) 1× reaction buffer, 0.2 mM dNTPs, and 0.5 units of iProof DNA polymerase (Bio-rad) in a 50 μL reaction volume and performing a PCR reaction. The PCR program was set as follows: (1) 98°C for 10 seconds, (2) 50°C for 20 seconds, (3) 72°C for 15 seconds, (4) repeating (1)-(3) 35 times. Next, 1 μL of the PCR reaction product was subjected to a new PCR reaction in a 50 μL reaction volume with iProof DNA polymerase (Bio-rad) 1× reaction buffer, 0.2 mM dNTPs, 0.2 μM primers (N-ABDHind-F): 5'-GATCTTAAGCATATGCATCATCACCATC-3' (SEQ ID NO: 9), primers (N-ABDHind-R): 5'-ACAGCTAAAAGCTTATCAGCCTAGGATCCGCCGCTACCATTG-3' (SEQ ID NO: 93), and 0.5 units of iProof DNA polymerase. The PCR program was set as follows: (1) 98°C for 10 seconds, (2) 50°C for 20 seconds, (3) 72°C for 15 seconds, (4) repeat (1) to (3) 35 times, (5) 72°C for 5 minutes. The construction of the N-ABD094 gene was the same as the procedure for constructing the N-ABD gene, except that primers 09-16 (sequence numbers 9-16) listed in Table 1 were used instead of primers 01-08 (sequence numbers 1-8) listed in Table 1.

[0157] The C-ABD gene for C-terminal fusion was constructed by mixing primers 17-24 (each primer at concentrations up to 20 nM) listed in Table 1 with iProof DNA polymerase 1× reaction buffer, 0.2 mM dNTPs, and 0.5 units of iProof DNA polymerase in a 50 μL reaction volume and performing a PCR reaction. The PCR program was set as follows: (1) 98°C for 10 seconds, (2) 50°C for 20 seconds, (3) 72°C for 15 seconds, (4) repeating (1)-(3) 35 times. Next, 1 μL of the PCR reaction product was subjected to a new PCR reaction in a 50 μL reaction volume with 1× reaction buffer for iProof DNA polymerase, 0.2 mM dNTPs, 0.2 μM primer (C-ABDNde-F): 5'-TAGCTGATCATATGTTATGCGATGGATCCAGTAACAAC-3' (SEQ ID NO: 91), 0.2 μM primer (C-ABDHind-R): 5'-GACCCTAAAAGCTTAATGGTGATGGTGATGATG-3' (SEQ ID NO: 92), and 0.5 units of iProof DNA polymerase. The PCR program was set as follows: (1) 98°C for 10 seconds, (2) 50°C for 20 seconds, (3) 72°C for 15 seconds, (4) repeat (1) to (3) 35 times, (5) 72°C for 5 minutes. The construction of the C-ABD094 gene was the same as the procedure for constructing the C-ABD gene, except that primers 25-32 (sequence numbers 25-32) listed in Table 1 were used instead of primers 17-24 (sequence numbers 17-24) listed in Table 1.

[0158] PCR fragments N-ABD, N-ABD094, C-ABD, or C-ABD094 were digested with restriction enzymes NdeI and HindIII, ligated to the vector pRSET-lac which had been pre-digested with the same enzymes, and transformed into E. coli Top10 to generate plasmids pN-ABD, pN-ABD094, pC-ABD, and pC-ABD094, respectively. Plasmid pRSET-lac was modified from pRSET-A (Invitrogen) by replacing the T7 promoter with the lac promoter of pGEMT-Easy Vector (Promega).

[0159] The construction of N-ABD or N-ABD094 fusions of the human arginase I gene was performed by mixing 1× reaction buffer for iProof DNA polymerase, 0.2 mM dNTPs, 100 ng of human arginase IcDNA, and 0.5 units of iProof DNA polymerase in a reaction volume of 50 μL, along with 0.2 μM primers HARGBam-F (5'-TTAGCTGGGGATCCGCCAAGTCCAGAACCATAGG-3') (SEQ ID NO: 93) and 0.2 μM primers HARGHind-R (5'-GAGATCAAAGCTTACTTAGGTGGGTTAAGGTAGTC-3') (SEQ ID NO: 94), along with 0.2 mM dNTPs, 100 ng of human arginase IcDNA, and 0.5 units of iProof DNA polymerase. The PCR program was set as follows: (1) 98°C for 10 seconds, (2) 50°C for 20 seconds, (3) 72°C for 30 seconds, (4) repeat (1) to (3) 35 times, (5) 72°C for 5 minutes. PCR fragments of the human arginase I gene were digested with restriction enzymes BamHI and HindIII, and ligated to vectors pN-ABD or pN-ABD094 that had been pre-digested with the same enzymes. These ligations transformed Escherichia coli ER2566 (NEB) to produce plasmids pN-ABD-rhArg and pN-ABD094-rhArg, respectively.

[0160] The construction of C-ABD or C-ABD094 fusions of the human arginase I gene was performed by mixing 0.2 μM primer HARGNde-F(5'-TAGGCTGCATATGAGCGCCAAGTCCAGAACCATAG-3') (SEQ ID NO: 95) and 0.2 μM primer HARGBam-R(5'-ATCAGCTAGGATCCCTTAGGTGGGTTAAGGTAGTCAATAGG-3') (SEQ ID NO: 96) with 1× reaction buffer for iProof DNA polymerase (Bio-rad), 0.2 mM dNTPs, 100 ng human arginase IcDNA, and 0.5 units of iProof DNA polymerase (Bio-rad) in a reaction volume of 50 μL. The PCR program was set as follows. (1) 98°C for 10 seconds, (2) 50°C for 20 seconds, (3) 72°C for 30 seconds, (4) repeat (1) to (3) 35 times, (5) 72°C for 5 minutes. PCR fragments of the human arginase I gene were digested with restriction enzymes NdeI and BamHI, and ligated to vectors pC-ABD or pC-ABD094 that had been previously digested with the same enzymes. These were then used to transform E. coli ER2566 (NEB) to produce plasmids pC-ABD-rhArg and pC-ABD094-rhArg, respectively.

[0161] The construction of N-ABD or N-ABD094 fusions of the Bacillus caldovelox arginase (BCA) gene was performed by mixing 1× reaction buffer for iProof DNA polymerase, 0.2 mM dNTPs, 100 ng of Bacillus caldovelox genomic DNA, and 0.5 units of iProof DNA polymerase in a reaction volume of 50 μL, along with 0.2 μM primers BCAbam-F (5'-ATGCTAGTGGATCCATGAAGCCAATTTCAATTATCG-3') (SEQ ID NO: 97) and 0.2 μM primer BCAHind-R (5'-TCAGCCTAAAGCTTACATGAGTTTTTCACCAAACAACG-3') (SEQ ID NO: 3), along with 0.2 mM dNTPs and 0.2 μM iProof DNA polymerase. The PCR program was set up as follows: (1) 98°C for 10 seconds, (2) 50°C for 20 seconds, (3) 72°C for 30 seconds, (4) repeat (1) to (3) 35 times, (5) 72°C for 5 minutes. The PCR fragment Bacillus caldovelox arginase gene was digested with restriction enzymes BamHI and HindIII, and ligated to vectors pN-ABD or pN-ABD094 that had been previously digested with the same enzymes. These ligations transformed Escherichia coli ER2566 (NEB) to produce plasmids pN-ABD-BCA and pN-ABD094-BCA, respectively.

[0162] The construction of C-ABD or C-ABD094 fusions of the Bacillus caldovelox arginase (BCA) gene was performed by mixing 1× reaction buffer for iProof DNA polymerase, 0.2 mM dNTPs, 100 ng of Bacillus caldovelox genomic DNA, and 0.5 units of iProof DNA polymerase in a reaction volume of 50 μL, along with 0.2 μM primers BCANde-F (5'-ATGCTAGCCATATGAAGCCAATTTCAATTATCGGG-3') (SEQ ID NO: 99) and 0.2 μM primer BCABam-R (5'-TCAGCTAAGGATCCCATGAGTTTTTCACCAAACAACG-3') (SEQ ID NO: 100), along with 0.2 mM dNTPs and 0.2 μM Bacillus caldovelox genomic DNA. The PCR program was set as follows: (1) 98°C for 10 seconds, (2) 50°C for 20 seconds, (3) 72°C for 30 seconds, (4) repeat (1) to (3) 35 times, (5) 72°C for 5 minutes. The PCR fragment Bacillus caldovelox arginase gene was digested with restriction enzymes NdeI and BamHI, and ligated to vectors pC-ABD or pC-ABD094 that had been previously digested with the same enzymes. These were then used to transform E. coli ER2566 (NEB) to produce plasmids pC-ABD-BCA and pC-ABD094-BCA, respectively.

[0163] [Table 1-1]

[0164] [Table 1-2]

[0165] [Table 1-3] [Examples]

[0166] Expression and purification of ABD-rhArg or ABD094-rhArg fusion proteins by fermentation To generate the N-ABD-rhArg (SEQ ID NO: 49) fusion protein, E. coli ER2566 transformed with pN-ABD-rhArg was grown in 50 mL of seed medium (1.5 g yeast extract, 0.25 g NaCl, and 5 mg ampicillin) at 30°C in an orbital shaker at 250 rpm for 16 hours. Next, the seed culture was transferred to 1 L of fermentation medium (10 g yeast extract, 16 g tryptone, 6.7 g Na2HPO4.7H2O, 3.41 g KH2PO4, 2.41 g NH4Cl, 0.67 g (NH4)2SO4, 10 g glycerol, 1 g glucose, MgSO4.7H2O, 1 mM CaCl2, and 0.1 g ampicillin), and grown at 28°C at a minimum speed of 500 rpm with an air supply of 1 L / min, maintaining the pH at 7.4. The stirring speed was automatically increased to maintain a minimum dissolved oxygen level of at least 20% pO2. When the absorbance of the culture at a wavelength of 600 nm reached an optical density (OD) of 15, 500 mL of culture medium (10 g yeast extract, 16 g tryptone, 2.41 g NH4Cl, 0.67 g (NH4)2SO4, 20 g glycerol) was continuously supplied at a rate of 31.25 mL / hour for 16 hours. Cells were collected by centrifugation, and proteins were purified using a Ni-IDA Sepharose column. Cultures with purified proteins exceeding 1 g / L were typically obtained. Next, endotoxins were removed from the purified proteins by the TritonX-114 method [Aida Y. and Pabst MJ (1990) Journal of Immunological Methods. 132: 191-195, this reference constitutes part of this specification]. The procedure for generating the fusion proteins N-ABD094-rhArg (SEQ ID NO: 50), C-ABD-rhArg (SEQ ID NO: 51), C-ABD094-rhArg (SEQ ID NO: 52), N-ABD-BCA (SEQ ID NO: 53), N-ABD094-BCA (SEQ ID NO: 54), C-ABD-BCA (SEQ ID NO: 55), and C-ABD094-BCA (SEQ ID NO: 56) was the same as the procedure for generating N-ABD-rhArg (SEQ ID NO: 49), except that the cells were transformed with the respective plasmids accordingly.Figure 1 and Table 2 show an example of protein purification of the aforementioned albumin-binding domain recombinant human arginase (rhArg), namely N-ABD094-rhArg (SEQ ID NO: 50).

[0167] [Table 2]

[0168] Figure 12 shows the preparation results. 709 mg of N-ABD094-rhArg (SEQ ID NO: 50) was obtained, with a specific activity of 205 U / mg and a protein concentration of 10.5 mg / mL. [Examples]

[0169] Construction and cloning of the N-ABD094-rhArg gene (SEQ ID NO: 38) The N-ABD094-rhArg (SEQ ID NO: 38) gene was synthesized by duplication extension PCR based on the N-ABD-rhArg gene (SEQ ID NO: 37). Two PCR reactions were performed. First, a pRSET / lac plasmid containing the N-ABD094-rhArg gene (SEQ ID NO: 38) was used. The rhArg region was isolated and amplified in the first PCR reaction using ABD094-0F (SEQ ID NO: 57) and HuArgHinBam-R (SEQ ID NO: 64). Isolation and purification of the PCR product were performed using a 0.7% agarose gel (Biorad) (30 minutes at 100V) and a gel band purification kit (GE Healthcare Life Science). Next, ABD094 was added to the PCR product by a second PCR reaction. A 20-fold diluted primer mixture containing ABD094-0F, ABD094-1F, ABD094-2F, ABD094-3F, ABD094-4F, and ABD094-5F (SEQ ID NOs. 57-62) was first prepared in sterile Milli-Q water. The primer mixture and abdNde-F overlap with each other. The second PCR reaction was performed with abdNde-F (SEQ ID NO. 63), HuArgHinBam-R (SEQ ID NO. 64), and the 20-fold diluted primer mixture. The PCR products were isolated and purified using the same method as described above. The primer sequences are shown in Table 3. The primer binding sites are shown in Figure 2A. The DNA sequence and protein sequence of the obtained N-ABD094-rhArg fusion gene (SEQ ID NO. 38) are shown in Figure 2B.

[0170] [Table 3]

[0171] PCR products and pRSET / lac vectors were double digested with BamHI-HF and NdeI (New England BioLabs) at 37°C for 2 hours. Isolation and purification of both inserts and vectors were performed using a 0.7% agarose gel (Biorad) (100V for 30 minutes) and a gel band purification kit (GE Healthcare Life Science). Next, the inserts and vectors were ligated with T4 ligase (New England BioLabs) at room temperature for 20 minutes. Competent cells were transformed with recombinant DNA and spread on LB plates containing 50 μg / mL kanamycin. The LB plates were incubated overnight at 37°C. Eight colonies were selected, and the presence of N-ABD094-rhArg was confirmed by PCR using abdNde-F and HuArgHinBam-R. Colonies that tested positive by PCR were further grown overnight at 30°C in 5 mL of 2×TY containing 50 μg / mL kanamycin. Bacterial broth was centrifuged in a 1.7 mL microcentrifuge tube at 16100 × g for 1 minute. The bacterial pellet was resuspended in a solubilization buffer (50 mM Tris-HCl, 0.1 M NaCl, 1 mM MnCl2, pH 7.4) and destroyed by repeated freezing and thawing in liquid nitrogen. The cell lysates were centrifuged to obtain a protein solution. The presence of arginase was tested by an arginase activity assay using DAMO. [Examples]

[0172] Construction of a BCA expression vector The gene encoding BCA (Pubmed registration number: U48226) was obtained from GenScript (where it was newly synthesized and inserted into the pUC57 cloning vector). A single cysteine ​​residue was introduced by site-directed mutagenesis, and a 6-histidine tag was added to the C-terminus to facilitate the protein purification process. The genetically modified BCA gene was subcloned into the pET-3a vector and transformed into BL21(DE3) competent cells for BCA expression. The crystal structure of BCA (PDB: 2CEV) can be searched in the Protein Data Bank. The protein sequence of the genetically modified BCA(S161C)-His (SEQ ID NO: 89) is shown in Figure 3. [Examples]

[0173] Construction of a BCA-ABD expression vector A pair of primers encoding the albumin-binding domain (ABD) were constructed and amplified by duplicate PCR. Specifically, 200 ng of primers were used as templates and amplified with one unit of iProof high-fidelity DNA polymerase (Bio-Rad). The primers were incubated at 98°C for 30 seconds, followed by 15 amplification cycles including thermal denaturation at 98°C for 10 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 15 seconds.

[0174] 5'-Sense-ABD GCGCAGCATGATGAAGCCGTGGATGCGAACAGCTTAGCTGAAGCTAAAGTCTTAGCTAACAGAGAACTTGACAAATATGGAGTAAGTGACTATTACAAGAACC (SEQ ID NO: 77) 5'-Antisense-ABD TTAAGGTAATGCAGCTAAAATTTCATCTATCAGTGCTTTTACACCTTCAACAGTTTTGGCATTGTTGATTAGGTTCTTGTAATAGTCACTTACTCCAT (SEQ ID NO: 78)

[0175] The ABD coding gene constructed using this pair of primers was amplified using two different primer sets to construct BCA-6×His-ABD (also known as BHA) (SEQ ID NO: 75) and BCA-ABD-6×His (also known as BAH) (SEQ ID NO: 76). To construct BHA, ABD was first amplified using the following pair of primers. The forward primer was designed for duplicate PCR with BCA via a 6-histidine tag, and the BamHI restriction site and stop codon were introduced for ligation with the pET-3a vector using the reverse primer.

[0176] ABD cloning forward primers: 5'-GCATCACCATCACCATCACGCGCAGCATGATGAAG-3'(Sequence ID 79) Reverse primers for ABD cloning: 5'-cgGGATCCTTAAGGTAATGCAGCTAAAATTTCATCTAT-3'(Sequence ID 80)

[0177] The PCR products obtained by ABD amplification were subjected to 1.5% agarose gel electrophoresis and purified. Meanwhile, genetically modified BCA containing the extra mutation (V20P) was cloned using the following pair of primers with the stop codon removed.

[0178] Forward primers for BCA cloning: 5'-ggaattccCATATGAAGCCAATTTCAATTATCG-3'(Sequence ID 81) Reverse primer for BCA cloning: 5'-GTGATGGTGATGGTGATGCA-3'(Sequence ID 82)

[0179] The BCA amplified with the above-mentioned primers was subjected to 1% agarose gel electrophoresis, and the PCR product was purified. The PCR products of BCA and ABD described above were ligated by duplicate PCR. Similarly, 200 ng of BCA and ABD were used as templates and amplified with one unit of iProof high-fidelity DNA polymerase. BCA and ABD were incubated at 98°C for 30 seconds, followed by 15 amplification cycles including thermal denaturation at 98°C for 10 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds. The product obtained by duplicate PCR was used as a template, and further amplification was performed using the following pair of primers.

[0180] Forward primers for BHA cloning: 5'-ggaattccCATATGAAGCCAATTTCAATTATCG-3'(Sequence ID 83) Reverse primer for BHA cloning: 5'-cgGGATCCTTAAGGTAATGCAGCTAAAATTTCATCTAT-3'(Sequence ID 84)

[0181] The PCR product was subjected to 1% agarose gel electrophoresis, and the band corresponding to BHA was excised from the gel and purified.

[0182] To construct BAH, ABD was first amplified with the following pair of primers. The forward primer was designed for direct overlap PCR with BCA, and the BamHI restriction site, 6-histidine tag, and stop codon were introduced via the reverse primer.

[0183] ABD cloning forward primers: 5'-CGTTGTTTGGTGAAAAACTCATGGCGCAGCATGATGAAG-3'(Sequence ID 85) Reverse primers for ABD cloning: 5'-cgGGATCCTTAGTGATGGTGATGGTGATGAGGTAATGCAGCTAAAATTTCATCTAT-3'(Sequence ID 86)

[0184] PCR products obtained by ABD amplification were subjected to 1.5% agarose gel electrophoresis to purify the PCR products. Genetically modified BCAs containing the extra mutation (V20P) were cloned using the following pair of primers with the stop codon and 6-histidine tag removed.

[0185] Forward primers for BCA cloning: 5'-ggaattccCATATGAAGCCAATTTCAATTATCG-3'(Sequence ID 81) Reverse primer for BCA cloning: 5'-GTGATGGTGATGGTGATGCA-3'(Sequence ID 82)

[0186] The BCA amplified with the above-mentioned primers was subjected to 1% agarose gel electrophoresis, and the PCR product was purified. The PCR products of BCA and ABD described above were ligated by duplicate PCR. Similarly, 200 ng of BCA and ABD were used as templates and amplified with one unit of iProof high-fidelity DNA polymerase. BCA and ABD were incubated at 98°C for 30 seconds, followed by 15 amplification cycles including thermal denaturation at 98°C for 10 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds. The product obtained by duplicate PCR was used as a template, and further amplification was performed using the following pair of primers.

[0187] Forward primer for BAH cloning: 5'-ggaattccCATATGAAGCCAATTTCAATTATCG-3'(Sequence ID 87) Reverse primer for BAH cloning: 5'-cgGGATCCTTAGTGATGGTGATGGTGATGAGGTAATGCAGCTAAAATTTCATCTAT-3'(Sequence ID 88)

[0188] PCR products were subjected to 1% agarose gel electrophoresis, and the band corresponding to BHA was excised from the gel and purified. The PCR products encoding BHA and BAH and the pET-3a vector were double digested with NdeI and BamHI. BHA and BAH were ligated into the pET-3a vector and used to transform TOP10 competent E. coli cells. The nucleotide sequences of BHA and BAH were confirmed by DNA sequencing. The pET-3a vector containing the BHA or BAH insert was transformed into a BL21(DE3) vector for protein expression. [Examples]

[0189] Protein constructs of BHA and BAH The BCA protein sequence used in this study is the same as that used by Bewley et al. (Structure 7, 435-448, 1999) (PDB ID: 2CEV, this reference constitutes part of this specification), except for the inclusion of a 6×His tag at the C-terminus and the S161C mutation. Both BCA-6×His-ABD (BHA) and BCA-ABD-6×His (BAH) constructs were designed, and the E. coli cell stock can express both BHA and BAH. In both BHA and BAH, the BCA portion is a mutant of BCA (V20P) in which the valine residue at position 20 is mutated to proline, and the ABD portion is the wild type of ABD with the linker sequence (AQHDEAVDANS) (Jonsson et al., 2008, Protein Eng. Des. Sel. 21, 515-527). The ABD portion is either located at the very C-terminus of the fusion protein (in the case of BHA) or fused between BCA and the 6× histidine tag (in the case of BAH). Figure 4 shows the alignment of the protein sequences of BCA, BHA, and BAH used in this study.

[0190] BCA (wild-type) amino acid sequence (PDB ID: 2CEV): MKPISIIGVPMDLGQTRRGVDMGPSAMRYAGVIERLERLHYDIEDLGDIPIGKAERLHEQGDSRLRNLKAVAEANEKLAAAVDQVVQRGRFPLVLGGDHSIAIGTLAGVAKHYERLGVIWYDAHGDVNTAETSPSGNIHGMPLAASLGFGHPA LTQIGGYSPKIKPEHVVLIGVRSLDEGEKKFIREKGIKIYTMHEVDRLGMTRVMEETIAYLKERTDGVHLSLDLDGLDPSDAPGVGTPVIGGLTYRESHLAMEMLAEAQIITSAEFVEVNPILDERNKTASVAVALMGSLFGEKLM (SEQ ID NO: 70)

[0191] BCA(S161C)-His amino acid sequence: MKPISIIGVPMDLGQTRRGVDMGPSAMRYAGVIERLERLHYDIEDLGDIPIGKAERLHEQGDSRLRNLKAVAEANEKLAAAVDQVVQRGRFPLVLGGDHSIAIGTLAGVAKHYERLGVIWYDAHGDVNTAETSPSGNIHGMPLAASLGFGHPALTQ IGGYCPKIKPEHVVLIGVRSLDEGEKKFIREKGIKIYTMHEVDRLGMTRVMEETIAYLKERTDGVHLSLDLDGLDPSDAPGVGTPVIGGLTYRESHLAMEMLAEAQIITSAEFVEVNPILDERNKTASVAVALMGSLFGEKLMHHHHHH (SEQ ID NO: 71)

[0192] BHA amino acid sequence: MKPISIIGVPMDLGQTRRGPDMGPSAMRYAGVIERLERLHYDIEDLGDIPIGKAERLHEQGDSRLRNLKAVAEANEKLAAAVDQVVQRGRFPLVLGGDHSIAIGTLAGVAKHYERLGVIWYDAHGDVNTAETSPSGNIHGMPLAASLGFGHPALTQIGGYCPKIKPEHVVLIGVRSLDEGEKKFIREKGIKIYTMHEVDRLGMTRVMEETIAYLKERTDGVHLSLDLDGLDPSDAPGVGTPVIGGLTYRESHLAMEMLAEAQIITSAEFVEVNPILDERNKTASVAVALMGSLFGEKLMHHHHHHAQHDEAVDANSLAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAALP(SEQ ID NO: 75)

[0193] BAH amino acid sequence: MKPISIIGVPMDLGQTRRGPDMGPSAMRYAGVIERLERLHYDIEDLGDIPIGKAERLHEQGDSRLRNLKAVAEANEKLAAAVDQVVQRGRFPLVLGGDHSIAIGTLAGVAKHYERLGVIWYDAHGDVNTAETSPSGNIHGMPLAASLGFGHPALTQIGGYCPKIKPEHVVLIGVRSLDEGEKKFIREKGIKIYTMHEVDRLGMTRVMEETIAYLKERTDGVHLSLDLDGLDPSDAPGVGTPVIGGLTYRESHLAMEMLAEAQIITSAEFVEVNPILDERNKTASVAVALMGSLFGEKLMAQHDEAVDANSLAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAALPHHHHHH(SEQ ID NO: 76)

Example

[0194] Generation of BHA and BAH E. coli cell stocks expressing BHA and BAH were used. To prepare seed cultures, a small amount of E. coli BL21(DE3) glycerol stock was seeded into 10 mL of LB medium supplemented with 100 μg / mL ampicillin. The seed cultures were grown overnight (approximately 16 hours) at 37°C with shaking at 700 rpm. Next, 2.5 mL of the overnight seed culture was used and seeded into 250 mL of LB medium containing 100 μg / mL ampicillin. The inoculated cells were grown at 37°C with shaking at 280 rpm for 2-4 hours, and the optical density (OD) at a wavelength of 600 nm was measured. 600 This was continued until the ratio was 0.6-0.8. Next, IPTG (0.2 mM) was added to the culture medium to induce overexpression of the target protein. The cells were grown for a further 4 hours and then harvested by centrifugation at 4500 rpm.

[0195] Protein purification by nickel affinity chromatography The E. coli cell pellet was resuspended in a buffer containing 50 mM Tris-HCl and 100 mM NaCl (pH 7.4), and then disrupted by sonication. Next, the cells were removed by centrifugation at 10,000 rpm for 40 minutes. 2+The supernatant was added to a 5 mL HiTrap chelating column (GE Healthcare) that had been packed with the appropriate solution and equilibrated with Buffer A (20 mM sodium phosphate, 0.5 M NaCl, pH 7.4) using an AKTA purifier. Proteins were eluted using an imidazole gradient of 0 to 0.5 M, with various ratios of Buffer A and Buffer B being used (0.5 M imidazole in Buffer A). The fractions were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using a 12% polyacrylamide gel, and those containing the target protein were pooled. The pooled fractions were concentrated, and the buffer of the concentrated and pooled fractions was replaced with 20 mM sodium phosphate (pH 7.4) using a centrifugal filter (Amicon) or a tangential flow filter (TFF) (Millipore). For BHA and BAH, the membrane cutoff value was 30,000 MW. Next, the remaining solution was diluted to the desired concentration, sterile filtered through a 0.2 μm syringe filter, and stored at 4°C.

[0196] Chromatograms of the purified BHA show three peaks during the elution process as the imidazole concentration is increased (Figure 5A). The first two peaks fuse, followed by a third peak that occurs when the imidazole concentration reaches approximately 0.13 M (Figure 5A). SDS-PAGE analysis reveals that the third peak is mainly composed of a target protein of approximately 40 kDa (Figure 5B, lanes F4-F8). Since the molecular weight of the BHA monomer calculated based on the amino acid sequence is 39.44 kDa, this target protein is highly likely to be BHA. Therefore, it is suggested that BHA expression and purification were successful. The fractions containing relatively pure BHA were pooled together for further processing.

[0197] Chromatograms of purified BAH show three peaks during the elution process as the imidazole concentration is increased (Figure 5C). The second and third peaks fuse, occurring when the imidazole concentration reaches approximately 0.18 M (Figure 5C). Analysis by SDS-PAGE reveals that this fused peak is primarily composed of a target protein of approximately 40 kDa (Figure 5D, lanes F6-F14), which is likely BAH. Compared to BHA, which elutes at approximately 0.13 M imidazole, BAH appears to bind more strongly to nickel affinity columns. This is likely because inserting the ABD domain between BCA and the 6× histidine tag causes the 6× histidine tag to be more exposed, allowing BAH to interact with nickel ions in the column more effectively than BHA. Fractions containing relatively pure BAH were pooled together, and buffers were exchanged using a tangential flow filtration (TFF) system (Millipore) instead of centrifugal filtration.

[0198] One unit of enzyme activity is defined as the amount of enzyme (BCA, BHA, or BAH) that catalyzes the production of 1 μmol of urea per minute under standard assay conditions. The specific activity of an enzyme is expressed in units of activity per mg of protein. Under standard DAMO assay conditions (37°C, pH 7.4), the specific activity of freshly purified BHA (SEQ ID NO: 75) is 220.6 ± 15.8 U / mg, which is close to the value of BCA(S161C)-His (SEQ ID NO: 89) (243.30 U / mg).

[0199] In the case of fusion proteins containing BCA polypeptide and ABD polypeptide, a very high protein yield of approximately 290 mg of protein per liter of shaking flask culture was obtained for BHA (SEQ ID NO: 75). The yield of ABD fusion protein depends heavily on the expression system used. The yield of ABD fused with sCR1 has been reported to be in the range of 0.4 to 1.1 μg per liter of Chinese hamster ovary cell culture every 24 hours (Makrides et al., 1996, J. Pharmacol. Exp. Ther. 277, 534-542). Approximately 2 to 15 mg of single-stranded bispecific antibody-ABD fusion protein can be obtained from any liter of HEK293 cell culture (Hopp et al., 2010, Protein Eng. Des. Sel. 23, 827-834; Stork et al., 2007, Protein Eng. Des. Sel. 20, 569-576). The yield of ABD-TolA fusion protein expressed in E. coli BL21 host was 20 mg / L (LB broth medium) (Ahmad et al., 2012, Proteins 80, 774-789). In comparison, the yield of BHA was significantly higher. Our results indicate that BHA (SEQ ID NO: 75) is easy to produce and cost-effective. [Examples]

[0200] Expression and purification of BCA(S161C)-His (SEQ ID NO: 89) Overnight cultures of Escherichia coli strain BL21(DE3)pLysS, transformed with plasmid pET-3a-BCA(S161C)-His, were prepared in 5 mL of LB medium containing 100 μg / mL ampicillin and grown in a shaking flask at 37°C for 16 hours with shaking at 280 rpm. For 250 mL of LB medium containing 100 μg / mL ampicillin, 2.5 mL of the overnight culture was added, and the optical density (OD) was measured. 600The cells were cultured at 37°C with shaking at 280 rpm until the γ-saturation reached 0.8-0.9 at 600 nm. Isopropyl β-D-thio-galactopyranoside (IPTG) was added to a final concentration of 0.2 mM. After 4 hours of induction, the cells were collected by centrifugation at 12000 × g for 20 minutes at 4°C. The cell pellet could be lysed by sonication and purified, or stored at -20°C until needed. The cell pellet was suspended in 20 mL of solubilization buffer (50 mM Tris-HCl, 100 mM NaCl, pH 7.4) containing 10 mM MnCl2. First, the cells were lysed by sonication on ice for 10 minutes with pulses generated every 30 seconds at 40% amplitude. Cell fragments were removed by centrifugation at 12000 × g for 20 minutes at 4°C. The supernatant was incubated at 70°C for 15 minutes, and the precipitate formed during the heat treatment process was removed by centrifugation. The soluble fraction was filtered through a 0.22 μm filter and loaded onto a 5 mL HisTrap chelating HP column. The flow-through fraction was collected during sample loading, and unbound proteins were washed away with 5 column volumes (CV) of start buffer or until the baseline stabilized. The target protein bound to the column was eluted by competitive gradient elution using imidazole (0.5 M imidazole in the start buffer). Using an Amicon Ultra-15 centrifuge unit with a cutoff value of 10 kDaMW, the target fractions were identified, combined, and pooled to exchange buffer with 20 mM sodium phosphate at pH 7.4 according to the manufacturer's instructions. The samples were filtered through a 0.22 μm filter under sterile conditions and stored at 4°C until needed. [Examples]

[0201] Purification of BCA(S161C)-His (SEQ ID NO: 89) from fed-batch fermentation Fermentation of BCA(S161C)-His-expressing E. coli cells in a 5L bioreactor resulted in a wet cell weight of 248.86g. The cell pellet was resuspended in a solubilization buffer containing deoxyribonuclease and ribonuclease to degrade the large amount of DNA and RNA released by homogenization using these enzymes, thereby reducing the viscosity of the cell soluble. After passing the cell soluble through a homogenizer three times, the viscosity was further reduced by incubation at 37°C for 15 minutes, followed by centrifugation to obtain the soluble fraction. In the first step of purification, heat treatment was performed at 70°C for 15 minutes to remove non-target impurities and precipitate heat-unstable proteins, and the heat-stable BCA(S161C)-His (SEQ ID NO: 89) was recovered by centrifugation.

[0202] The soluble fraction containing the target protein was further purified using a 196 mL Ni affinity column packed with an XK50 column. Specifically, BCA(S161C)-His (SEQ ID NO: 89) was eluted from the Ni affinity column using a 4-segment elution program. The first elution gradient of 0–30% elution buffer was intended to remove proteins nonspecifically bound to the column. The second segment was maintained at 30%, thereby completely eluting nonspecific bound proteins. The third segment eluted the target protein by increasing the elution buffer concentration from 30% to 70%. The fourth segment, with 70–100% elution buffer, eluted any remaining proteins still bound to the column. The fractions with absorbance at a UV wavelength of 280 nm were analyzed by SDS-PAGE, and 24 fractions (50 mL each) containing the target protein, E1–A'5, were pooled together (Figures 6A and 6B). Based on an understanding of the hexameric structure of BCA (wild-type) (SEQ ID NO: 70), a pooled fraction containing a high concentration of imidazole was desalted using a tangential flow filtration (TFF) system, with a 30 kDa NMWC membrane used for TFF. The purified BCA(S161C)-His (SEQ ID NO: 89) was retained in PBS buffer, filtered through a 0.2 μm filter under sterile conditions, and stored at 4°C until use. Approximately 1096 mg of BCA(S161C)-His (SEQ ID NO: 89) protein was obtained through a two-step pilot-scale purification, with a specific activity of 264 U / mg. [Examples]

[0203] Confirmation of arginase enzyme activity The activity of BCA(S161C)-His (SEQ ID NO: 89) was assayed by a direct colorimetric method using diacetyl monooxime (DAMO) to determine the amount of urea, the product of the enzymatic reaction. Specifically, 0.3 mL of BCA solution, 0.15 mL of water, and a step dilution of 720 mM arginine substrate (pH 7.4) were pre-incubated separately at 37°C for 10 minutes. 0.3 mL of arginine substrate was added to the pre-incubated enzyme solution to carry out the enzymatic reaction. The reaction mixture was incubated at 37°C for exactly 5 minutes, and the reaction was stopped by adding the reaction stop reagent [50% trichloroacetic acid (TCA)]. A sample blank was prepared in the same manner, but the same amount of enzyme solution was added after the TCA solution.

[0204] The urea formed by the above reaction was quantified using the DAMO direct colorimetric method described by the World Health Organization (WHO). This protocol has been adopted by the WHO as a standard operating procedure (SOP) for measuring blood urea concentration. Several reagents were prepared according to the SOP. The mixed acid reagent was prepared by slowly adding 100 mL of concentrated sulfuric acid to 400 mL of distilled water, and then adding 0.3 mL of the acid reagent stock solution (0.5 g of ferric chloride hexahydrate dissolved in 15 mL of distilled water, diluted to 25 mL with concentrated phosphoric acid). The mixed color reagent was prepared by mixing 35 mL of color reagent stock solution A (1 g of DAMO dissolved in 50 mL of distilled water) and 35 mL of color reagent stock solution B (0.25 g of thiosemicarbazide dissolved in 50 mL of distilled water), and then diluting with distilled water to 500 mL. The working urea standard solution was prepared by adding 5 mL of stock urea solution (0.5 g of urea dissolved in 50 mL of benzoic acid) to 45 mL of benzoic acid (1 g / dL). The DAMO assay was performed by adding 100 μL of a 20-fold diluted reaction mixture to a glass test tube, and then adding 3 mL of a newly prepared chromogenic reagent, which was prepared by mixing distilled water, mixed chromogenic reagent, and mixed acid reagent in a 1:1:1 ratio. The test tube was incubated at 100°C for 15 minutes and then cooled to room temperature for 5 minutes. The absorbance at 540 nm was measured. One unit of arginase is defined as the amount of enzyme that converts 1 micromolar of arginine to ornithine and urea per minute at 37°C and pH 7.4. The enzyme activity was calculated using the following formula and the absorbance value of the sample was subtracted from the blank.

[0205] ΔA 540nm Sample = A 540nm Sample-A 540nm Sample blank The amount of urea produced is calculated from the standard curve. Units / mL enzyme = (μmol of free urea) (df) / (5) (0.3) df = Dilution factor of enzyme 0.3 = Volume of enzyme used (mL) 5 = Assay time per unit (min) [Examples]

[0206] Measurement of Protein Concentration Protein concentration was determined by the Bradford assay. The protein assay dye reagent concentrate (Bio-rad) was diluted 5-fold before use. 20 μL of the sample was mixed with 1 mL of the diluted dye reagent and incubated at room temperature for 10 minutes. To determine the protein concentration, a Quick Start bovine serum albumin standard set (Bio-rad) was used as the standard in the range of 0 - 1 mg / mL, and the mixture was measured at 595 nm.

Example

[0207] Enzymatic Activity of the BHA (SEQ ID NO: 75) Fusion Protein To find out whether the enzymatic activity is affected by the presence of HSA, the arginine catabolic activity of BHA (SEQ ID NO: 75) was assayed. From the results, it was found that the activity of BHA (SEQ ID NO: 75) was not affected by each amount of HSA added to the activity assay. The molar ratios of BHA:HSA tested in the binding study were 1:10, 1:5, 1:1, 5:1 and 10:1. Under all these conditions, the measured specific activities of BHA (SEQ ID NO: 75) alone and the BHA-HSA complex were very similar and were approximately 200 U / mg.

Example

[0208] Binding of N-ABD-rhArg (SEQ ID NO: 49) or BHA (SEQ ID NO: 75) to HSA Revealed by Native PAGE Analysis When fusing the ABD to rhArg or BCA, it is important that the fusion protein retains both albumin-binding ability and enzymatic activity. To demonstrate the binding ability of the ABD in the fusion protein, a certain amount of human serum albumin (HSA) was incubated with N-ABD-rhArg (SEQ ID NO: 49) or BHA (SEQ ID NO: 75) at various molar ratios. Analysis of the reaction mixture was performed in the native state by native PAGE that separates proteins by size and conformation.

[0209] In Figure 7, only the mobilities of HSA and N-ABD-rhArg (SEQ ID NO: 49) are shown in lanes 2 and 3 of the undenatured PAGE, respectively (Figure 7). A fixed amount of HSA (30 picomoles) was titrated against N-ABD-rhArg (SEQ ID NO: 49) at various molar ratios, and the results are shown in lanes 5 to 9. Increasing the amount of N-ABD-rhArg (SEQ ID NO: 49) in the binding reaction proportionally decreased the amount of free HSA, indicating the specific binding of N-ABD-rhArg (SEQ ID NO: 49) to HSA. In Figure 8, only the mobilities of HSA and BHA (SEQ ID NO: 75) are shown in lanes 6 and 7 of the undenatured PAGE, respectively (Figure 8). A fixed amount of HSA (60 picomoles) was titrated against BHA (SEQ ID NO: 75) at various molar ratios, and the results are shown in lanes 1 to 5. Increasing the amount of BHA (SEQ ID NO: 75) in the binding reaction proportionally decreased the amount of free HSA, indicating the specific binding of BHA (SEQ ID NO: 75) to HSA. [Examples]

[0210] In vivo study of BHA (SEQ ID NO: 75) Six 8-week-old BALB / c mice were used in an in vivo study of BHA (SEQ ID NO: 75). Blood samples were collected from each mouse before BHA (SEQ ID NO: 75) injection and used as controls (0 hours). The mice were randomly divided into two groups: one for intraperitoneal (ip) injection and the other for intravenous (iv) injection. BHA (SEQ ID NO: 75) (250U) was injected into each mouse, and blood samples were collected 6, 24, 72, and 120 hours after injection. Plasma (16 μL) from each blood sample was analyzed using an amino acid analyzer (Biochrom) to obtain the arginine and ornithine content.

[0211] As shown in Figure 9, the in vivo arginine depletion effect of BHA (BHA SEQ ID NO: 75) was studied in BALB / c mice and compared with that of BCA(S161C)-His (SEQ ID NO: 89). The results showed that plasma arginine concentration rapidly returned to normal within 24 hours after injection of BCA(S161C)-His (SEQ ID NO: 89) (Figure 9). In contrast, plasma arginine concentration remained undetectable for at least 24 hours after injection of BHA (SEQ ID NO: 75) (Figure 9). This indicates that ABD fusion extended the circulating half-life of BCA(S161C)-His (SEQ ID NO: 89) in vivo. This suggests that ABD fusion is a feasible and promising strategy for improving the pharmacodynamic properties of arginine depletion enzymes. The route of administration does not appear to affect the in vivo arginine depletion effect of BHA (SEQ ID NO: 75) (Figure 9). Furthermore, no apparent side effects were observed, and all mice showed good tolerance to BHA (SEQ ID NO: 75). In summary, the fusion proteins described herein, such as BHA (SEQ ID NO: 75), demonstrated the feasibility of the ABD fusion strategy by reducing plasma arginine to undetectable levels for a much longer period (at least 24 hours) compared to using natural BCA(S161C)-His (SEQ ID NO: 89) without ABD fusion (8 hours). [Examples]

[0212] In vivo pharmacodynamic studies of N-ABD-rhArg (SEQ ID NO: 49) The anticancer effects of arginase have been studied for quite some time (Greenberg and Sassenrath, 1953, Cancer Res. 13, 709-715). Arginase was found to have very good anticancer effects in vitro. However, due to its short plasma half-life, little effect was observed in vivo. Plasma arginine levels decreased to their lowest levels 3 hours after injection of native arginase into C3H mice. In the same model, arginine levels rose to normal baseline levels 5 hours after injection (Greenberg and Sassenrath, 1953, Cancer Res. 13, 709-715). Therefore, arginase was considered an ineffective anticancer agent due to its short plasma half-life. This suggests that plasma half-life can affect the efficacy of enzyme drugs in vivo. N-ABD-rhArg (SEQ ID NO: 49) and N-ABD094-rhArg (SEQ ID NO: 50) showed similar specific activity. We found that fusion with ABD (SEQ ID NO: 103) is an effective method for extending the plasma half-life of rhArg. To reduce the manufacturing cost of rhArg (SEQ ID NO: 103) with an extended plasma half-life, we developed N-ABD-rhArg (SEQ ID NO: 49) and N-ABD094-rhArg (SEQ ID NO: 50).

[0213] This study used 15 female BALB / c mice (10 weeks old). They were kept in groups of 5 mice per cage. Single doses of N-ABD-rhArg (SEQ ID NO: 49) (500U, 250U, and 125U, respectively) were administered to the mice by intraperitoneal injection. Blood samples were collected from the saphenous vein using heparinized capillaries and centrifuged at 800×g to obtain plasma. L-arginine concentration was measured in 16 microliters of plasma samples using a Biochrom30 amino acid analyzer. The results are shown in Figure 10. When plasma arginine concentrations were measured, it was found that in BALB / c mice, 500U of N-ABD-rhArg (SEQ ID NO: 49) could deplete plasma arginine to below the detection limit (3 μM) of the Biochrom30 amino acid analyzer for at least 9 days, 250U of N-ABD-rhArg (SEQ ID NO: 49) could deplete serum arginine to below the detection limit of the Biochrom30 amino acid analyzer for at least 7 days, and 125U of N-ABD-rhArg (SEQ ID NO: 49) could deplete serum arginine to below the detection limit of the Biochrom30 amino acid analyzer for at least 3 days. These results clearly suggest that fusion with ABD can extend the plasma half-life of rhArg. Compared to BHA (SEQ ID NO: 75) (Figure 9), N-ABD-rhArg (SEQ ID NO: 49) can deplete arginine to very low levels in mice for a considerably longer period, highlighting the importance of the rational design requirements for fusion proteins. [Examples]

[0214] In vivo pharmacodynamic and pharmacokinetic studies of N-ABD094-rhArg (SEQ ID NO: 50) This study used 50 C57BL / 6J male mice (10-12 weeks old). 500 U of N-ABD094-rhArg (SEQ ID NO: 50) was administered to the mice by single-dose intraperitoneal injection. Blood samples were collected from the saphenous vein using heparinized capillaries and centrifuged at 800 × g to obtain plasma. Using an Agilent 6460 liquid chromatography / electrospray ionization triple quadrupole mass spectrometer (detection limit: 0.3 μM L-arginine), 5 microliters of plasma samples were subjected to L-arginine concentration measurement by liquid chromatography-tandem mass spectrometry. The N-ABD094-rhArg (SEQ ID NO: 50) concentration was measured using a human arginase I ELISA (enzyme-linked immunosorbent assay) kit (Abcam) that specifically detects human arginase and does not react with mouse arginase.

[0215] After injection, serum samples were collected at 2, 4, 8, 16, and 24 hours (Day 1), at 24-hour intervals from Day 2 to Day 12, and at 72-hour intervals from Day 12 to Day 30. To balance the number of mice used and minimize the impact on pharmacokinetic and pharmacodynamic studies resulting from the removal of N-ABD094-rhArg (SEQ ID NO: 50) from mice as a result of blood collection, the mice were divided into 10 groups (n=5 in each group) and subjected to blood collection at the time points indicated in parentheses below. Group 1 (hour 2), Group 2 (hour 4), Group 3 (hour 8), Group 4 (hour 16), Group 5 (hour 24), Group 6 (days 2, 7, and 12), Group 7 (days 3, 8, and 18), Group 8 (days 4, 9, and 21), Group 9 (days 5, 10, 15, and 24), and Group 10 (days 0, 6, 11, 27, and 30). The pharmacodynamic and pharmacokinetic results are shown in Figures 13 and 14, respectively.

[0216] Measurements of plasma arginine concentrations revealed that intraperitoneal injection of 500 U of N-ABD094-rhArg (SEQ ID NO: 50) depleted plasma arginine levels to 1 μM within 2 hours of injection (Figure 13). Plasma arginine levels remained below 1 μM for 10 days, then gradually increased over several days (Table 4). Plasma arginine levels returned to half of the baseline level between days 18 and 21. These results suggest that N-ABD094-rhArg (SEQ ID NO: 50) can maintain very low levels of plasma arginine (below 1 μM) in mice for a considerably long period.

[0217] [Table 4]

[0218] Pharmacokinetic parameters are calculated using a two-compartment model.

[0219] The emission rate constant ke is calculated using the following formula.

number

[0220] Plasma half-life t 1 / 2 It is calculated using the following formula.

number

[0221] The area under the curve (AUC) is calculated using the trapezoidal rule with the following formula.

number

[0222] For comparison, we use the half-life of natural rhArg reported in the literature. The efflux half-life of the fusion protein N-ABD094-rhArg (SEQ ID NO: 50) is 3.9 ± 0.3 days (Figure 14), which means it is eliminated much more slowly than the non-fusion protein rhArg, which has a very short half-life. Specifically, human arginase has a circulating half-life of only a few minutes (Georgiou et al., US8440, 184B2), indicating that binding dramatically extends the circulating time. These results clearly suggest that the plasma half-life of rhArg can be significantly extended by fusion with ABD. The circulating half-life of natural ADI (several hours) was extended by PEGylation (Ensor et al., Cancer Res 2002, 62:5443-5450). The circulating half-life of PEGylated ADI is 2-3 days, and only PEGylated ADI (not natural ADI) was effective in inhibiting tumor growth in vivo. When natural rhArg and PEGylated rhArg were administered to rats by intraperitoneal injection, 1500 U of PEGylated rhArg per rat (6 U / g rat) was able to deplete circulating arginine to below the detection limit of the amino acid analyzer (3 μM) for 6 days, whereas even at high doses of natural rhArg (7500 U / rat), circulating arginine only decreased to about 20 μM within 3 hours of injection, and arginine returned to basal levels about 2 days after injection (Tsui et al., Cancer Cell Int. 2009 Apr 17;9:9). Natural arginase is eliminated from circulation within minutes (Savoca et al., Cancer Biochem. Biophy's. 7:261-268, 1984). For clinical use, it is essential to design arginase to persist in circulation for days or even weeks. Without any modification, the half-life of human arginase is only a few minutes in circulation because the size of the arginase is not large enough to evade filtration by the kidney (Georgiou et al., US8440,184B2). Therefore, in this invention, we have developed a completely new and highly improved arginase form with dramatically improved circulatory persistence for therapeutic use. [Examples]

[0223] Treatment with N-ABD094-rhArg (SEQ ID NO: 50) reduces fat mass, suppresses lipid synthesis, and improves insulin sensitivity in normal, lean mice.

[0224] Our data from a mouse model showed that single intraperitoneal injection of 500U of N-ABD094-rhArg (SEQ ID NO: 50) into 8-week-old normal lean C57BL / 6J male mice maintained plasma arginine concentrations below 1 μM for at least 10 days, indicating a long circulating half-life of N-ABD094-rhArg (SEQ ID NO: 50) (Figure 13). Therefore, normal lean C57BL / 6J male mice fed a standard solid diet were treated with intraperitoneal injections of 500U of N-ABD094-rhArg (SEQ ID NO: 50) at 10-day intervals for 4 weeks. After 4 weeks of treatment, visceral (perigonadal, perirenal, and mesenteric fat pads) and subcutaneous (inguinal fat pad) fat mass decreased by more than 25% compared to control mice injected with a vehicle (physiological saline) (Figure 15A). Histological examination of visceral white adipose tissue (WAT) revealed a significant decrease in adipocyte size (Figure 15B). Further analysis of lipid synthesis showed that in mice treated with N-ABD094-rhArg (SEQ ID NO: 50), mRNA levels of several important lipid-producing enzymes, including acetyl-CoA carboxylase 1 (Acc1), fatty acid synthase (Fasn), and stearoyl-CoA desaturase 1 (Scd1), were dramatically suppressed in visceral WAT (Figure 16A) and liver (Figure 16B).

[0225] Obesity is strongly associated with insulin resistance. Therefore, we conducted studies both in vivo and in vitro to determine whether N-ABD094-rhArg (SEQ ID NO: 50) treatment affects insulin sensitivity. Insulin loading tests (ITT) showed that mice treated with N-ABD094-rhArg (SEQ ID NO: 50) had significantly increased insulin sensitivity after 2 weeks of drug treatment (Figure 17A). Next, we specifically examined hepatic insulin sensitivity by treating primary hepatocytes prepared from C57BL / 6J male mice with a medium containing 5 U / mL of N-ABD094-rhArg (SEQ ID NO: 50), but this concentration did not affect cell viability (Figure 17B). Primary hepatocytes were cultured in N-ABD094-rhArg (SEQ ID NO: 50) or control (without N-ABD094-rhArg) medium for 24 hours, and then stimulated with 100 nM insulin for 20 minutes. Next, cells were collected and subjected to Western blot analysis to detect phosphorylated protein kinase B (pAkt), which is commonly used as an insulin signaling marker. The results showed that Akt phosphorylation was significantly increased in the N-ABD094-rhArg (SEQ ID NO: 50) treated group compared to the control group, indicating enhanced hepatic insulin signaling (Figure 17C).

[0226] Insights from normal, lean mice revealed that N-ABD094-rhArg (SEQ ID NO: 50) has a long circulating half-life, reduces fat mass, suppresses lipid synthesis, and improves insulin sensitivity both in vivo and in vitro. These results support the potential use of ABD-rhArg fusion proteins for the prevention and treatment of obesity and related metabolic disorders (e.g., insulin resistance and hepatic steatosis (fatty liver)). [Examples]

[0227] Treatment with N-ABD094-rhArg (SEQ ID NO: 50) effectively prevents HFD-induced obesity. Treatment with recombinant human arginase [N-ABD094-rhArg (SEQ ID NO: 50)] can significantly reduce fat mass, suppress lipid synthesis, and improve insulin sensitivity in normal lean mice.

[0228] The effects of N-ABD094-rhArg (SEQ ID NO: 50) on steatosis and various metabolic parameters in obese animals were investigated. A high-fat diet-induced obesity (DIO) mouse model [Wang and Liao, 2012, Methods Mol. Biol. 821:421-433] (this model shares many characteristics with human obesity and is widely used in the testing of future anti-obesity agents [Vickers et al., 2011, Br. J. Pharmacol. 164(4):1248-1262]) was used. Specifically, the C57BL / 6J mouse strain, the most commonly used mouse strain for polygenic obesity models, was used in the study. C57BL / 6J mice fed a high-fat diet frequently exhibit significant weight gain, become obese, and develop hyperinsulinemia, hyperglycemia, impaired glucose tolerance, and insulin resistance [Gallou-Kabani et al., 2007, Obesity 15(8):1996-2005].

[0229] To confirm the efficacy of N-ABD094-rhArg (SEQ ID NO: 50) in preventing diet-induced obesity and associated metabolic disorders in mice, male C57BL / 6J mice were fed a high-fat diet containing 60 kcal% fat (HFD, Research Diets, D12492) from 8 weeks of age to 12 weeks, and simultaneously injected intraperitoneally with 200-300 U of N-ABD094-rhArg (SEQ ID NO: 50) or physiological saline as a vehicle control at 7-day intervals. C57BL / 6J mice fed a corresponding low-fat diet containing 10 kcal% fat (LFD, Research Diets, D12450J) and injected with physiological saline were used as lean controls for various measurements.

[0230] The study was conducted separately in two cohorts (n=5 in each group). Mice were housed in groups of 5. Similar results were obtained in the two independent cohorts. Data from one of the mouse cohorts are summarized in Examples 18-22.

[0231] Compared to lean controls fed LFD and treated with a vehicle [referred to as the LFD (vehicle) group], vehicle-treated male mice fed HFD [referred to as the HFD (vehicle) group] showed a significant increase in body weight (Figure 18A) and size (Figure 18B), and the amount of white adipose tissue (WAT) increased significantly in major visceral (perigonadal, perirenal, and mesentery) storage and subcutaneous (inguinal) storage (Figure 19A). However, mice fed HFD but simultaneously treated with N-ABD094-rhArg (SEQ ID NO: 50) [referred to as the HFD (rhArg) group] were able to effectively prevent the weight gain and WAT hypertrophy caused by HFD intake. Histological examination of WAT revealed that N-ABD094-rhArg (SEQ ID NO: 50) treatment significantly suppressed adipocyte hypertrophy (Figure 19B). Analysis of mRNA expression levels in gWAT using real-time qRT-PCR further demonstrated dramatic downregulation of several key adipogenic transcription factors, including peroxisome proliferator-activated receptor γ (Pparg), sterol regulatory element-binding protein 1c (Srebp1c), and lipid-producing enzymes (Acc1 and Scd1) (Figure 19C). These genes were also significantly downregulated in the skeletal muscle of HFD-fed mice treated with N-ABD094-rhArg (SEQ ID NO: 50) (Figure 20), suggesting that the anti-lipidogenic effect of N-ABD094-rhArg (SEQ ID NO: 50) is not limited to WAT.

[0232] After confirming the anti-obesity effect of N-ABD094-rhArg (SEQ ID NO: 50) in male mice, the effect of N-ABD094-rhArg (SEQ ID NO: 50) on female mice (n=5 in each group) was studied to determine if there were any sex differences. The results showed that simultaneous treatment with N-ABD094-rhArg (SEQ ID NO: 50) was equally effective in promoting weight gain (Figure 21A) and protecting against excessive accumulation of WAT (Figure 21B) in major visceral (perigonadal, perirenal, and mesenteric) and subcutaneous (inguinal) storage in female mice fed HFD (starting at 8 weeks of age for 12 weeks), and improved insulin sensitivity (Figure 22A) and glucose tolerance (Figure 22B).

[0233] To test the effects of N-ABD094-rhArg (SEQ ID NO: 50) on adipocytes, we used mouse 3T3-L1 preadipocytes [Green and Meuth, 1974, Cell 3:127-133], a cell-based model commonly used to study adipogenesis. N-ABD094-rhArg (SEQ ID NO: 50) effectively inhibited adipogenesis and lipidogenesis in 3T3-L1 cells (Figure 23), and significantly suppressed mRNA levels of several adipogenic transcription factors (Figure 24A) and lipid-producing enzymes (Figure 24B). Similar inhibitory effects were observed when 3T3-L1 preadipocytes were cultured in bovine arginase, other arginine-depleting enzymes—arginine deiminase (ADI)—or in arginine-free media (Figure 23). Arginase converts L-arginine to L-ornithine and urea, while ADI converts L-arginine to L-citrulline. However, L-ornithine, urea, or L-citrulline were unable to inhibit adipogenesis and lipid synthesis in 3T3-L1 cells (Figure 23).

[0234] Furthermore, supplementing arginine-free culture media with L-arginine at various concentrations demonstrated the dose-dependent effects of arginine on adipogenesis and lipid synthesis (Figure 25A), and the arginine threshold concentration for activating important adipogenic transcription factors such as Cebpa, Pparg, and the lipid-producing enzyme Scod1 was 10–40 μM (Figure 25B).

[0235] In other words, these findings indicate that the anti-lipidogenesis and anti-lipidogenesis effects of N-ABD094-rhArg (SEQ ID NO: 50) are mediated through arginine deficiency.

[0236] In summary, these findings support the possibility of using arginine depletion via ABD-rhArg fusion protein or other arginase forms (e.g., rhArg-PEG, BCA-PEG, or BCA-ABD) or arginine depletion enzymes (e.g., ADI-PEG, ADI-ABD, ADC-PEG, or ADC-ABD) as a prophylactic anti-obesity therapy. [Examples]

[0237] Treatment with N-ABD094-rhArg (SEQ ID NO: 50) prevents HFD-induced insulin resistance, impaired glucose tolerance, and metabolic disorders. Obesity is generally associated with insulin resistance, impaired glucose tolerance, and metabolic disorders. Indeed, compared to lean control mice fed LFD (low-fructose dextrose) and vehicle-treated mice fed HFD (high-fructose dextrose) and vehicle-treated mice showed a significant decrease in sensitivity to the blood glucose-lowering effect of injected insulin in an insulin tolerance test (ITT) conducted 6 weeks after HFD feeding (Figure 26A), and this worsened further at 11 weeks (Figure 26B). Along with decreased insulin sensitivity, these mice showed a significant decrease in the efficiency of returning blood glucose levels to baseline after D-glucose injection in a glucose tolerance test (GTT) within 5 weeks after HFD feeding (Figure 27A), indicating the development of impaired glucose tolerance, which worsened further 10 weeks after HFD consumption (Figure 27B). In fact, fasting (5-hour) blood glucose (Figure 28A) and fasting plasma insulin concentration (Figure 28B) were significantly elevated, and the HOMA-IR score (Figure 28C), a measure of insulin resistance, was higher in vehicle-treated HFD-fed mice compared to lean control mice. Fasting plasma leptin concentration (Figure 29A), total cholesterol concentration (Figure 29B), triglyceride concentration (Figure 29C), and free fatty acid concentration (Figure 29D) were significantly elevated when measured 12 weeks after HFD feeding. Notably, simultaneous treatment with N-ABD094-rhArg (SEQ ID NO: 50) completely prevented the development of HFD-induced insulin resistance, impaired glucose tolerance, hyperglycemia, hyperinsulinemia, hyperleptinemia, hypercholesterolemia, hypertriglyceridemia, and hyperfree fatty acidemia.

[0238] In other words, these results indicate that N-ABD094-rhArg (SEQ ID NO: 50) can effectively prevent insulin resistance and impaired glucose tolerance, which are precursors to the development of type 2 diabetes. It can also prevent other metabolic disorders such as leptin resistance and dyslipidemia associated with obesity.

[0239] In summary, these results support the possibility of using arginine depletion by ABD-rhArg fusion proteins or other arginase forms (e.g., rhArg-PEG, BCA-PEG, or BCA-ABD) or arginine depletion enzymes (e.g., ADI-PEG, ADI-ABD, ADC-PEG, or ADC-ABD) as a prophylactic antidiabetic, anti-dyslipidemia, anti-atherosclerosis, and anti-metabolic disorder therapy. [Examples]

[0240] Treatment with N-ABD094-rhArg (SEQ ID NO: 50) prevents HFD-induced stiliosis. Steroid disease refers to the abnormal accumulation of lipids within cells. Non-alcoholic fatty liver disease (NALF) is a common comorbidity associated with obesity. In the early stages, it may be asymptomatic, but if the liver becomes inflamed, it can progress to steatohepatitis, then to fibrosis, and in more advanced stages, to cirrhosis, significantly increasing the risk of hepatocellular carcinoma. In fact, in vehicle-treated mice fed HFD, the liver was significantly enlarged and lighter in color (Figure 30A), and its weight was almost double that of lean control mice (Figure 30B). Staining liver sections with Oil Red O revealed widespread accumulation of large lipid droplets (Figure 31A), and accordingly, triglyceride concentrations increased tenfold (Figure 31B). As if in conjunction with these findings, serum concentrations of alanine aminotransferase (ALT), which is commonly measured as a biomarker of liver damage, were significantly elevated (Figure 32). Notably, simultaneous treatment with N-ABD094-rhArg (SEQ ID NO: 50) effectively prevented excessive lipid accumulation in the liver of HFD-fed mice, with liver weight, triglyceride content, and serum ALT concentration being comparable to that of lean controls. Consistent with these findings, HFD induced significant upregulation of several key adipogenic transcription factors and lipid-producing enzymes in the liver, while treatment with N-ABD094-rhArg (SEQ ID NO: 50) significantly suppressed the upregulation of these genes (Figure 31C). In other words, these results indicate that N-ABD094-rhArg (SEQ ID NO: 50) is highly effective in preventing fatty liver disease.

[0241] Obesity is associated with the excessive accumulation of lipids not only in the liver but also in the kidneys (renal steatosis), pancreas (pancreatic steatosis), and heart (cardiac steatosis), leading to lipotoxicity and dysfunction of these organs. In fact, vehicle-treated mice fed HFD diets showed a significant increase in the weight of the kidneys (Figure 33A), pancreas (Figure 33B), and heart (Figure 33C). Simultaneous treatment with N-ABD094-rhArg (SEQ ID NO: 50) effectively prevented the weight gain of these organs, suggesting that N-ABD094-rhArg (SEQ ID NO: 50) can prevent excessive lipid accumulation and lipotoxicity in these organs.

[0242] In summary, these results support the idea that ABD-rhArg fusion proteins can effectively prevent steatosis and that arginine depletion by N-ABD094-rhArg (SEQ ID NO: 50) or other arginase forms (e.g., rhArg-PEG, BCA-PEG, or BCA-ABD) or arginine depletion enzymes (e.g., ADI-PEG, ADI-ABD, ADC-PEG, or ADC-ABD) can be used as a prophylactic anti-steatosis therapy. [Examples]

[0243] Treatment with N-ABD094-rhArg (SEQ ID NO: 50) prevents HFD-induced chronic inflammation. Excessive hypertrophy of the perigonal thoracic thromboemboli (WAT) increases the production of many adipokines, leading to chronic low-level inflammation that contributes to the development of insulin resistance. Monocyte chemotactic protein-1 (Mcp1) is one of the major pro-inflammatory adipokines secreted from the WAT and controls macrophage infiltration into the WAT, insulin resistance in obesity, and fatty liver. In fact, we found that McP1 mRNA levels were significantly upregulated in the perigonal WAT of vehicle-treated mice fed HFD (Figure 34A). Concurrently, serum concentrations of circulating McP1, as measured by enzyme-linked immunosorbent assay (ELISA), were significantly elevated compared to lean controls (Figure 34B). Other pro-inflammatory adipokines such as interleukin-1β (Il1b) and tumor necrosis factor-α (Tnfa) also showed high mRNA expression in the perigonal WAT (Figure 34A). Notably, simultaneous treatment with N-ABD094-rhArg (SEQ ID NO: 50) suppressed the upregulation of these adipokines in the perigonate WAT, thereby inhibiting the increase in serum McP1 concentration.

[0244] These results demonstrate that N-ABD094-rhArg (SEQ ID NO: 50) can prevent chronic low-grade inflammation associated with obesity, supporting its potential as a prophylactic anti-inflammatory therapy for arginine depletion by ABD-rhArg fusion proteins or other arginase forms (e.g., rhArg-PEG, BCA-PEG, or BCA-ABD) or arginine depletion enzymes (e.g., ADI-PEG, ADI-ABD, ADC-PEG, or ADC-ABD). [Examples]

[0245] Treatment with N-ABD094-rhArg (SEQ ID NO: 50) prevents HFD-induced whitening and deregulation of fatty acid oxidation in brown adipose tissue, and enhances unshivering thermogenesis. Brown adipose tissue (BAT) plays a role in dissipating oxidative fuels into heat (non-shivering thermogenesis). Recent studies have shown that this heat production has a significant capacity in energy expenditure to combat obesity. Conversely, whitening of BAT is associated with obesity and impairs BAT function. In fact, in vehicle-treated mice fed with high-fiber dextrinsic (HFD), the interscapular BAT was significantly enlarged (Figure 35A), and its weight was twice that of lean control mice (Figure 35B). Histological examination revealed widespread accumulation of large monolobular cells scattered among multilobed brown adipose cells (Figure 35C), suggesting the conversion of brown adipose cells to white-like cells. However, in HFD-fed mice simultaneously treated with N-ABD094-rhArg (SEQ ID NO: 50), the weight, size, and histological appearance of BAT were similar to those of lean control mice. Treatment with N-ABD094-rhArg (SEQ ID NO: 50) significantly upregulated BAT-specific gene uncoupling protein 1 (Ucp1), which is important for thermogenesis, and also upregulated peroxisome proliferator-activated receptor γ coactivator 1-α (Pgc1α), an important transcriptional regulator of Ucp1 in brown adipocytes. Furthermore, it was found that HFD-induced downregulation of acyl-CoA oxidase 1 (Acox1), a major gene involved in fatty acid oxidation, was suppressed (Figure 36). The fact that N-ABD094-rhArg (SEQ ID NO: 50) treatment was able to prevent Acox1 downregulation suggests that N-ABD094-rhArg (SEQ ID NO: 50) may normalize fatty acid oxidation.

[0246] In other words, these results indicate that treatment with N-ABD094-rhArg (SEQ ID NO: 50) can effectively prevent the whitening of brown adipose tissue and the deregulation of fatty acid oxidation in BAT associated with obesity, thereby increasing non-shivering thermogenesis to combat the steatotic effects of HFD.

[0247] These findings support the potential of ABD-rhArg fusion proteins or other arginase forms (e.g., ABD094-rhArg, BCA-PEG, or BCA-ABD) or arginine-depleting enzymes (e.g., ADI-PEG, ADI-ABD, ADC-PEG, or ADC-ABD) as prophylactic anti-obesity therapies for arginine depletion. [Examples]

[0248] Treatment with N-ABD094-rhArg (SEQ ID NO: 50) effectively suppresses HFD-induced obesity. After confirming that simultaneous treatment with N-ABD094-rhArg (SEQ ID NO: 50) effectively prevents HFD-induced obesity and associated complications and comorbidities (e.g., insulin resistance, impaired glucose tolerance, hyperglycemia, dyslipidemia, fatty liver, chronic inflammation, and whitening of BAT), we investigated the effectiveness of N-ABD094-rhArg (SEQ ID NO: 50) in suppressing obesity and reversing these disorders in mice with pre-existing diet-induced obesity (DIO). Male C57BL / 6J mice were fed HFD (from 5 weeks of age to 12 weeks) to induce obesity and associated complications and comorbidities. Next, the mice were divided into two groups according to body weight and treated with 600-700 U of N-ABD094-rhArg (SEQ ID NO: 50) or physiological saline as a vehicle control at 7-day intervals for 12 weeks while continuing to be fed HFD. One group of mice was fed a corresponding low-fat diet (LFD) and injected with saline throughout the study to serve as a lean control for various measurements. The study was conducted separately in two cohorts (n=5 in each group). Mice were housed in groups of five. Similar results were obtained in the two independent cohorts. Data from one of the mouse cohorts are summarized in Examples 23-28.

[0249] Twelve weeks of HFD feeding increased the body weight of male mice to over 50g, which was significantly heavier than lean control male mice fed LFD (Figures 37A and 37B). After treatment with N-ABD094-rhArg (SEQ ID NO: 50), the body weight of HFD-induced obese mice gradually decreased. By five weeks, body weight had decreased by more than 30%, similar to lean control mice. Body weight was maintained at this level until the completion of the 12-week treatment. In DIO mice treated with N-ABD094-rhArg (SEQ ID NO: 50), the weight of the fat body was significantly reduced in major visceral storage (perigonadal, perirenal, and mesenteric WAT) and subcutaneous storage (inguinal WAT), which was quite similar to lean control mice (Figure 38A). Histological examination of WAT revealed a significant reduction in adipocytes in DIO mice treated with N-ABD094-rhArg (SEQ ID NO: 50) (Figure 38B).

[0250] Together with the results of Example 18, these findings support the possibility that ABD-rhArg fusion proteins or other arginase forms (e.g., rhArg-PEG, BCA-PEG, or BCA-ABD) or arginine-depleting enzymes (e.g., ADI-PEG, ADI-ABD, ADC-PEG, or ADC-ABD) can be used for the therapeutic treatment of obesity. [Examples]

[0251] Treatment with N-ABD094-rhArg (SEQ ID NO: 50) reverses HFD-induced insulin resistance and impaired glucose tolerance, normalizing metabolic disorders. Mice fed HFD for 12 weeks developed peripheral insulin resistance (Figure 39) and impaired glucose tolerance (Figure 40), characteristics of prediabetes, compared to lean control mice before treatment. However, within 4 weeks of treatment with N-ABD094-rhArg (SEQ ID NO: 50), DIO mice showed a significant increase in insulin sensitivity, reaching a level comparable to lean control mice. The normalization of insulin sensitivity in N-ABD094-rhArg (SEQ ID NO: 50)-treated mice could be maintained until the end of the 12-week drug treatment while continuing to feed HFD. Furthermore, in mice treated with N-ABD094-rhArg (SEQ ID NO: 50), glucose tolerance significantly improved after 3 weeks of treatment and fully recovered to a level similar to lean control mice after 7 weeks of treatment. Consistent with these improvements, fasting blood glucose (Figure 41A) and fasting plasma insulin (Figure 41B) in DIO mice treated with N-ABD094-rhArg (SEQ ID NO: 50) recovered to levels similar to those of lean control mice by 4 weeks of rhArg treatment and remained within the normal range until the end of treatment. This resulted in a dramatic decrease in the HOMA-IR score from 35.1 in mice before treatment to 1.2 in mice 4 weeks after treatment (Figure 41C), suggesting a recovery of insulin resistance. Fasting plasma leptin concentration (Figure 42A) and fasting total cholesterol concentration (Figure 42B) also decreased significantly to normal levels.

[0252] In summary, our findings demonstrate the high efficacy of N-ABD094-rhArg (SEQ ID NO: 50) in reversing insulin resistance, impaired glucose tolerance, hyperglycemia, hyperinsulinemia, hyperleptinemia, and hypercholesterolemia within four weeks of treatment.

[0253] In other words, these findings support the idea that arginine depletion by ABD-rhArg fusion proteins or other arginase forms (e.g., rhArg-PEG, BCA-PEG, or BCA-ABD) or arginine depletion enzymes (e.g., ADI-PEG, ADI-ABD, ADC-PEG, or ADC-ABD) can be used as insulin sensitivity enhancers and for the therapeutic treatment of prediabetes and type 2 diabetes characterized by insulin resistance, impaired glucose tolerance, and hyperglycemia, as well as other disorders associated with insulin resistance. It can also be used for the therapeutic treatment of hyperleptinemia and hypercholesterolemia. [Examples]

[0254] Treatment with N-ABD094-rhArg (SEQ ID NO: 50) reverses HFD-induced stiliosis. Fatty liver was induced in male mice by 12 weeks of HFD feeding (Figures 30 and 31). After extending HFD feeding for another 12 weeks, the liver became even more enlarged and appeared paler in color (Figure 43A). Liver weight was more than twice that of lean control mice (Figure 43B). Large amounts of lipid accumulation spread throughout the liver (Figure 44A), and triglyceride content increased accordingly (Figure 44B). The expression levels of the important adipogenic transcription factors Pparg and Srebp1c were downregulated to levels comparable to lean controls, the expression level of the lipid-producing enzyme Acc1 was further reduced, and Scd1 was significantly suppressed (Figure 44C). Compared to mice fed HFD for 12 weeks (Example 20, Figure 32), mice fed HFD for 24 weeks showed a further increase in serum alanine aminotransferase (ALT) concentration and a significant increase in serum aspartate aminotransferase (AST) concentration (Figure 45), indicating further progression of liver damage. However, in DIO mice treated with N-ABD094-rhArg (SEQ ID NO: 50), liver size was significantly reduced, and its weight became comparable to that of lean controls (Figure 43B). Furthermore, the liver recovered to a reddish color (Figure 43A), which correlated well with a dramatic clearance of lipid droplets (Figure 44A), and triglyceride concentrations decreased to levels comparable to those of lean controls (Figure 44B). Notably, plasma ALT and AST concentrations were comparable to those of lean controls, suggesting that treatment with N-ABD094-rhArg (SEQ ID NO: 50) can prevent further progression and normalize existing liver damage. In summary, our findings demonstrate the remarkable effect of N-ABD094-rhArg (SEQ ID NO: 50) on the retrograde progression of fatty liver disease.

[0255] Obesity is associated with excessive lipid accumulation not only in the liver but also in the kidneys (renal steatosis), pancreas (pancreatic steatosis), and heart (cardiac steatosis), leading to lipotoxicity and dysfunction of these vital organs. Indeed, vehicle-treated DIO mice showed a significant increase in the weight of the kidneys (Figure 46A), pancreas (Figure 47A), and heart (Figure 48A). Histological examination revealed the accumulation of vacuoles resembling lipid droplets in the renal tubules (Figure 46C). Oil red O staining of kidney sections revealed ectopic lipid accumulation in the glomeruli (Figure 46D). Extensive intralobular and interlobular accumulation of WAT patches was observed in the pancreas (Figure 47B), and islands of white adipocytes were seen between myocardial fibers (Figure 48B). Triglyceride concentrations were significantly elevated in the kidneys (Figure 46B), pancreas (Figure 47C), and heart (Figure 48C). Furthermore, a significant increase in triglyceride concentration was also observed in skeletal muscle (Figure 49). These findings indicate the development of steatosis in these organs in vehicle-treated DIO mice. However, treatment with N-ABD094-rhArg (SEQ ID NO: 50) significantly reduced the weight of the kidneys (Figure 46A), pancreas (Figure 47A), and heart (Figure 48A) of DIO mice to levels comparable to those of lean controls. Histological examination revealed a significant decrease in renal tubular vacuoles (Figure 46C) and renal glomerular lipids identified by oil red O staining (Figure 46D). A significant decrease in WAT was observed in the pancreas (Figure 47B) and heart (Figure 48B). Triglyceride levels in the kidneys (Figure 46B), pancreas (Figure 47C), heart (Figure 48C), and skeletal muscle (Figure 49) of DIO mice treated with N-ABD094-rhArg (SEQ ID NO: 50) recovered to levels comparable to those of lean controls. These findings suggest that N-ABD-rhArg (SEQ ID NO: 50) can effectively reverse steatosis in the kidneys, pancreas, heart, and possibly other organs.

[0256] In summary, these findings support the possibility that ABD-rhArg fusion proteins or other arginase forms (e.g., rhArg-PEG, BCA-PEG, or BCA-ABD) or arginine-depleting enzymes (e.g., ADI-PEG, ADI-ABD, ADC-PEG, or ADC-ABD) could be used for the therapeutic treatment of steatosis. [Examples]

[0257] Treatment with N-ABD094-rhArg (SEQ ID NO: 50) reduces HFD-induced chronic inflammation and fibrosis. As shown in Example 21, HFD feeding induces chronic inflammation. In vehicle-treated DIO mice, the expression of the pro-inflammatory adipokine McP1 in the perigonate WAT was upregulated (Figure 50A), and serum McP1 concentration was significantly increased (Figure 50B). On the other hand, in DIO mice treated with N-ABD094-rhArg (SEQ ID NO: 50), McP1 expression in the perigonate WAT was significantly downregulated, and serum McP1 concentration was normalized to a level comparable to that of lean control mice (Figure 50B), suggesting that N-ABD094-rhArg treatment can reduce HFD-induced chronic inflammation in the WAT.

[0258] In the livers of vehicle-treated DIO mice, there was significant upregulation of several pro-inflammatory genes, including interleukin 1α and 1β (Il1a and Il1b) (Figure 51A). Chronic inflammation is associated with fibrosis. Indeed, there was significant upregulation of transforming growth factor β (Tgfb), an important pro-fibrosis gene (Figure 51B). Treatment with N-ABD094-rhArg (SEQ ID NO: 50) significantly downregulated these pro-inflammatory genes and significantly upregulated several anti-inflammatory genes, including interleukin 10 (Il10), interleukin 22 (Il22), and interferon γ (Infg) (Figure 51C). Expression of the pro-fibrosis gene Tgfb was restored to levels comparable to those of lean controls. Similar to the liver, the kidneys of vehicle-treated DIO mice showed significant upregulation of several pro-inflammatory genes, including McP1, tumor necrosis factor α (Tnfa), and Il1b (Figure 52A). The pro-fibrosis gene collagen type 1 α1 chain (Col1a1) was also significantly upregulated, which is consistent with the findings of excessive collagen fiber deposition identified by Sirius Red staining in the kidneys (Figure 52C). Notably, treatment of DIO mice with N-ABD094-rhArg (SEQ ID NO: 50) was able to downregulate the expression of these pro-inflammatory genes to levels comparable to those of lean controls. The pro-fibrosis gene Col1a1 was greatly suppressed, and the amount of collagen fiber was significantly reduced. Similarly, in the pancreas, treatment with N-ABD094-rhArg (SEQ ID NO: 50) significantly downregulated pro-inflammatory genes such as McP1 (Figure 52A) and the important pro-fibrosis gene Tgfb (Figure 53B).

[0259] In summary, these findings suggest that N-ABD094-rhArg treatment can effectively alleviate chronic inflammation and fibrosis in the liver, kidneys, pancreas, and possibly other organs.

[0260] Fatty liver (hepatic steatosis) can be asymptomatic in its early stages. When the liver becomes inflamed, it progresses to steatohepatitis, then to fibrosis, and in more advanced stages, to cirrhosis, significantly increasing the risk of hepatocellular carcinoma. Our findings suggest that N-ABD094-rhArg treatment can halt the progression of liver disease and reverse liver damage.

[0261] In other words, these results support the possibility that arginine depletion by ABD-rhArg fusion proteins or other arginase forms (e.g., rhArg-PEG, BCA-PEG, BCA-ABD) or arginine depletion enzymes (e.g., ADI-PEG, ABD-ADI, ADC-PEG, or ADC-ABD) can be used for the therapeutic treatment of chronic inflammation and fibrosis. [Examples]

[0262] Treatment with N-ABD094-rhArg (SEQ ID NO: 50) reverses the whitening of brown fat. After prolonged HFD feeding, vehicle-treated DIO mice showed further increases in interscapular BAT size (Figure 54A) and weight (Figure 54B) compared to lean control mice, which correlated with the presence of a large number of hypertrophied white-like unilocular cells (Figure 54C). In contrast, treatment of DIO mice with N-ABD094-rhArg (SEQ ID NO: 50) dramatically restored interscapular BAT to a size and weight comparable to lean control mice and almost completely eliminated the white-like unilocular cells. In summary, these findings demonstrate the superior ability of N-ABD094-rhArg (SEQ ID NO: 50) to reverse the whitening of brown adipose tissue.

[0263] In summary, these results support the possibility that ABD-rhArg fusion proteins or other arginase forms (e.g., rhArg-PEG, BCA-PEG, or BCA-ABD) or arginine-depleting enzymes (e.g., ADI-PEG, ADI-ABD, ADC-PEG, or ADC-ABD) can be used for the therapeutic treatment of obesity via the reversal of brown adipose tissue whitening. [Examples]

[0264] Treatment with N-ABD094-rhArg (SEQ ID NO: 50) lowers blood pressure. Obesity is associated with cardiovascular disease and hypertension. DIO mice treated with 600U of N-ABD094-rhArg (SEQ ID NO: 50) at 7-day intervals for 5 weeks were subjected to blood pressure measurement using the tail cuff method with the CODA non-invasive blood pressure system (Kents Scientific Corporation). The systolic and diastolic blood pressure (Figure 55A) and heart rate (Figure 55B) of DIO mice treated with N-ABD094-rhArg (SEQ ID NO: 50) were significantly lower than those of DIO mice treated with vehicle, and were quite similar to those of lean control mice. From Example 25, it can be seen that N-ABD094-rhArg (SEQ ID NO: 50) can effectively reverse renal and cardiac steatohepatia, which may have a beneficial effect on lowering blood pressure.

[0265] In summary, these findings support the possibility that ABD-rhArg fusion proteins or other arginase forms (e.g., rhArg-PEG, BCA-PEG, or BCA-ABD) or arginine-depleting enzymes (e.g., ADI-PEG, ADI-ABD, ADC-PEG, or ADC-ABD) could be used for the therapeutic treatment of hypertension. [Examples]

[0266] N-ABD0094-rhArg (SEQ ID NO: 50) does not induce neutralizing antibodies and maintains its effectiveness even with long-term use. The generation of neutralizing antibodies can affect the pharmacodynamics, pharmacokinetics, stability, and efficacy of a drug. To confirm the immunogenic response, 8-week-old C57BL / 6J male mice fed LFD were intraperitoneally injected with 500 U of N-ABD094-rhArg (SEQ ID NO: 50) or physiological saline as a vehicle control at 7-day intervals for 8 months. Despite the presence of anti-drug antibodies against N-ABD094-rhArg (SEQ ID NO: 50) (Figure 56A), these antibodies did not have a neutralizing effect on arginase activity (Figure 56B), which is consistent with the finding that plasma arginine concentrations were maintained below the detection limit (3 μM) of the Biochrom30 amino acid analyzer throughout the study period.

[0267] To evaluate the safety of long-term use, serum and urine samples were collected from LFD-fed mice treated with N-ABD094-rhArg (SEQ ID NO: 50) for 8 months. ALT and AST, commonly used biomarkers for liver injury, were analyzed, as well as serum creatinine and urinary albumin / creatinine ratio to assess renal function. Drug-treated LFD-fed mice showed no significant differences in any of these parameters compared to vehicle-treated LFD-fed mice (Figures 57A and 57B). Furthermore, when the functional response and vascular reactivity of isolated arteries were examined using wire myography, there were no significant differences between LFD-fed mice treated with N-ABD094-rhArg (SEQ ID NO: 50) and LFD-fed mice treated with vehicle in terms of phenylephrine-induced vascular smooth muscle contraction (Figure 58A), acetylcholine-induced endothelium-dependent relaxation (Figure 58B), and endothelium-independent relaxation in response to the nitric oxide donor sodium nitroprusside (Figure 58C).

[0268] In another study, diet-induced obese C57BL / 6J male mice were treated with 600 U of N-ABD094-rhArg (SEQ ID NO: 50) for 20 weeks while continuing to be fed HFD. By 5 weeks, their body weight had decreased to a level comparable to that of lean control mice and remained at this level without rebound. Regular monitoring of plasma arginine levels showed that arginine remained below the detection limit (3 μM) of the Biochrom30 amino acid analyzer. These results support the continued effectiveness of N-ABD094-rhArg (SEQ ID NO: 50) throughout the treatment period. In other words, these findings support the safety and effectiveness of N-ABD094-rhArg (SEQ ID NO: 50) for long-term use. [Examples]

[0269] PEGylation protocol for recombinant human arginase (rhArg) Recombinant human arginase (His-rhArg SEQ ID NO: 101) was obtained by purification using affinity chromatography. Before PEGylation, purified His-rhArg (SEQ ID NO: 101) was diafiltration with 20 mM sodium phosphate (pH 8.0) to adjust the protein concentration to 2 mg / mL. The PEGylation reagent was prepared with 20 mM sodium phosphate (pH 8.0). The molar ratio of PEG reagent to rhArg was 20:1. After adding the PEG reagent to His-rhArg (SEQ ID NO: 101), the mixture was incubated at room temperature for 4 hours with stirring. Next, the reaction mixture was diafiltration with 20 mM sodium phosphate (pH 7.4) by tangential flow filtration (TFF). Excess PEG was removed by flow-through affinity chromatography, and the protein was eluted with 0.5 M imidazole to recover the PEGylated His-rhArg (SEQ ID NO: 101). Non-derivativeized His-rhArg (SEQ ID NO: 101) was separated by a separate affinity chromatography. The reaction mixture was then analyzed by SDS-PAGE using a 12% polyacrylamide gel. The gel was stained with Coomassie blue for molecular weight determination or with a 0.1 M iodine solution for free PEG detection.

[0270] For example, 150 mg of PEGylated His-rhArg (SEQ ID NO: 101) with a specific activity of 245 U / mg and a concentration of 7.5 mg / mL was obtained from the preparation. Figure 11 and Table 5 show examples of PEG-rhArg production and analysis.

[0271] [Table 5] [Examples]

[0272] Treatment with PEGylated His-rhArg [SEQ ID NO: 101] suppresses obesity and improves insulin sensitivity. The production of PEGylated His-rhArg (SEQ ID NO: 101) is described in Example 30. C57BL / 6J male mice with pre-existing diet-induced obesity (DIO) were intraperitoneally injected with 300 U of PEGylated His-rhArg [SEQ ID NO: 101] or physiological saline as a vehicle control at 7-day intervals for 5 weeks, while continuing to be fed HFD. The body weight of mice treated with PEGylated His-rhArg (SEQ ID NO: 101) gradually decreased to 30 g, which was comparable to that of lean control mice treated with a vehicle and fed solid feed (Figure 59). Fat body weight significantly decreased in major visceral storage (perigonadal, perirenal, and mesenteric WAT) and subcutaneous storage (inguinal WAT), and was comparable to that of lean control mice at the end of the study (Figure 60). Liver, kidney, pancreas, and heart weights were significantly lower than those of DIO mice treated with a vehicle and comparable to those of lean control mice (Figure 60). Regular monitoring of plasma arginine levels confirmed that arginine remained below the detection limit (3 μM) of the Biochrom30 amino acid analyzer throughout the study.

[0273] Two weeks after treatment with PEGylated His-rhArg [SEQ ID NO: 101], DIO mice already had lower fasting blood glucose levels (Figure 61A) and significantly higher insulin sensitivity (Figure 61B) compared to DIO mice treated with the vehicle.

[0274] In other words, these findings support the idea that PEGylated His-rhArg [SEQ ID NO: 101] has similar anti-lipidemia and insulin resistance-improving effects as N-ABD094-rhArg [SEQ ID NO: 50]. Arginine deficiency induced by PEGylated His-rhArg (SEQ ID NO: 101) has therapeutic effects in treating obesity and diseases associated with insulin resistance. [Examples]

[0275] Metal ion substitution Wild-type arginase is usually Mn 2+ It contains metal ions (D'Antonio & Christianson, 2011, Biochemistry, 50:8018-27). Metal ion substitution is a stronger form of ABD094-rhArg (low K). m Value and high k cat / K m It is known that this is a method for preparing the value. Purified N-ABD094-rhArg (SEQ ID NO: 50) dissolved in 20 mM Tris-HCl buffer (pH 7.4) is mixed with 20 mM of different divalent metal ions (Mn 2+ Ni 2+ or ki 2+ After mixing with ) and incubating at 52-55°C for 15-60 minutes, the buffer was replaced with PBS buffer (pH 7.4).

[0276] The sample was subjected to studies on enzyme activity and enzyme dynamics, and specific activity and catalytic efficiency (k cat / K m The metal content was determined by applying inductively coupled plasma atomic emission spectrometry (ICP-OES, Agilent 700 series ICP emission spectrometer) to the sample.

[0277] Each protein sample (100-200 μL) was added to 10 mL of 1% w / v HNO3, and various elements and wavelengths were selected for ICP-OES analysis. The metal-to-protein ratio was determined by dividing the metal concentration by the protein concentration. The Pierce® BCA protein assay was used, and protein concentrations were evaluated according to the manufacturer's instructions (Thermo Fisher Scientific).

[0278] Co 2+ or Ni 2+ When metal ions are added to N-ABD094-rhArg (SEQ ID NO: 50), Mn 2+ Removal and Co 2+ or Ni 2+ The data was successfully incorporated.

[0279] Cobalt substitution enzyme (N-ABD094-rhArg-Co 2+ ) is much smaller in Km (0.27~0.35mM), k cat High (250~316s) -1 ), catalyst efficiency k cat / K m High (905~917s) -1 mM -1 It was found that the original enzyme (N-ABD094-rhArg-Mn 2+ ) In the case of K m 1.37~1.89mM, k cat 82~98s -1 , k cat / K m is 44~72s -1 mM -1 Therefore, Mn 2+ Co 2+ It can be substituted with saturated Co 2+ The protein-to-protein ratio was approximately 2-4:1. These data suggest that a more potent form of N-ABD094-rhArg (SEQ ID NO: 50) or other fusion proteins described herein may be more potent than other divalent metal ions (e.g., Ni). 2+ or ki 2+ This suggests that it can be manufactured by substitution with ). [Examples]

[0280] N-ABD094-rhArg-Co 2+ Treatment with [SEQ ID NO: 50 (Cobalt Substitution)] suppresses obesity. N-ABD094-rhArg-Co 2+ The production of [SEQ ID NO: 50 (Cobalt Substitution)] is described in Example 32. In C57BL / 6J male mice with pre-existing diet-induced obesity (DIO), 25-100U of N-ABD094-rhArg-Co was introduced while continuing to feed them HFD. 2+ [SEQ ID NO: 50 (cobalt substitution)] was administered intraperitoneally for 5 weeks. The body weight of the mice gradually decreased from 55g to 30g within 5 weeks of treatment, similar to the effect of 600-700U of N-ABD094-rhArg (Figure 37) (Figure 62). This result suggests that Mn 2+ Co 2+ Substitution with this protein significantly enhances the efficacy of the ABD-rhArg fusion protein in reducing weight. [Examples]

[0281] Mammalian cell culture and proliferation assays Cancer cell lines (e.g., MKN45, AGS, BGC823, HCT-15, and HCT-116) were maintained in RPMI medium containing 10% FBS and 100 units / mL of penicillin / streptomycin according to a standard protocol.

[0282] In the cell proliferation assay, cancer cells (5 × 10) in 100 μL of growth medium were measured. 3 The cells were seeded into each well of a 96-well plate and incubated for 24 hours. The culture medium was replenished with arginine-free medium (AFM) supplemented with various concentrations of arginine, and with fresh medium containing various concentrations of arginine-depleting enzymes, which were used in combination with citrulline. The plates were further incubated at 37°C for 1 to 3 days in a constant temperature incubator with a humid atmosphere of 95% air and 5% CO2.

[0283] Quantitative cell proliferation assays were performed using the MTT (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide) assay, as widely described elsewhere. The amount of arginine-depleting enzyme required to kill 50% of the cells in the culture was measured using IC50. 50 This was defined as follows. [Examples]

[0284] Inhibition of proliferation of various human cancer cell lines by various arginine-depleting enzymes (ADI, BCA, and ADC) The cytotoxicity of arginine deiminase (ADI), bacterial arginase (BCA), and arginine decarboxylase (ADC) was confirmed in comparative studies using five tumor cell lines (A375, HeLa, BxPC-3, PANC-1, and HCT116).

[0285] Since both ADI and BCA catalyze reactions in the urea cycle, the antitumor effects of both ADI and BCA are related to the concentration of ASS in cells. In contrast, since the catalytic products of ADC are not intermediates in the urea cycle, the antitumor effect of ADC may be less affected by the presence of ASS. Our results show that ADI, BCA, and ADC all exert inhibitory effects in the cell lines studied. ADI is effective against A375, HCT116, BxPC-3, and PANC-1, but not against HeLa cells, while both BCA and ADC are effective against all five of these cancer cell lines tested (Table 6). Although ADC is the least potent arginine-depleting enzyme, it is more advantageous than ADI and BCA in terms of efficacy, especially in ASS-positive cancer cells (Table 6).

[0286] The sensitivity of tumor cells to ADI shows a clear relationship with intracellular ASS concentration (Table 6). In summary, ADCs and BCAs can inhibit the proliferation of human cancer cell lines in an ASS-independent manner compared to ADIs.

[0287] Table 6. IC scores for ADI, BCA, and ADC against five human cancer cell lines. 50And maximum cytotoxicity. Cells were treated for 72 hours before MTT analysis. Data are the average of three experiments performed three times. IC 50 The value is defined as the amount of enzyme required to inhibit cell viability by 50%. Maximum cytotoxicity is expressed as the maximum percentage of non-viable cells obtained by each enzyme. ADI-resistant cells are defined as cells in which more than 50% survive at the maximum dose of the ADI tested. [Table 6] [Examples]

[0288] Cytotoxicity of BHA (SEQ ID NO: 75) against gastric cancer cells The in vitro efficacy of albumin-binding arginase BHA (SEQ ID NO: 75) was tested by incubating MKN45 gastric cancer cell lines with various concentrations of BHA (SEQ ID NO: 75) for 72 hours and confirming the viability of MKN45 cells by MTT assay. As shown in Figure 63, the proliferation of MKN45 cancer cells was inhibited in a dose-dependent manner by BHA (SEQ ID NO: 75). This further confirmed the cytotoxicity of the arginine-depleted fusion protein under culture conditions containing albumin and other different proteins, by adding fetal bovine serum (FBS) to the culture medium. [Examples]

[0289] Evaluation of apoptosis by Annexin V-FITC and PI double staining. To evaluate the induction of apoptosis by BCA arginase (SEQ ID NO: 71), 3 × 10⁶ units were used one day before various treatment conditions. 5Cells were seeded in a 6-well plate. The medium was removed and replaced with complete medium supplemented with BCA (SEQ ID NO: 71) at various concentrations. After exposure to BCA for 24, 48, and 72 hours, both suspension and adherent cells were collected. Specifically, suspension cells in the medium were collected by centrifugation at 100 × g for 3 minutes at room temperature, while adherent cells were washed with PBS and removed by incubation with 0.25% trypsin solution at 37°C for 3 minutes. After adding complete medium, cells were collected by centrifugation. The cells were washed with PBS and resuspended in 0.1 mL of binding buffer (0.01 M HEPES, pH 7.4, 0.14 M NaCl, 2.5 mM CaCl2) containing propidium iodide (PI) and Annexin V-FITC. The cells were stained in the dark at room temperature for 15 minutes, and after adding 0.4 mL of binding buffer to each tube, flow cytometry analysis was performed using FACSAria (BD biosciences). For each sample, the hemocytometer was configured to acquire data from 10,000 cells, and the data was analyzed using FACSAria software.

[0290] Flow cytometry results showed that apoptosis was induced in HCT116 human colon cancer cells in a time-dependent manner upon treatment with BCA (SEQ ID NO: 71) at a dose of 50 μg / mL. Approximately 16%, 21%, and 25% of HCT116 cancer cells underwent apoptosis at 24, 48, and 72 hours after BCA treatment, respectively. [Examples]

[0291] Cell viability assay and the efficacy of concomitant drugs These studies used breast cancer cell lines (e.g., human MCF-7 cells and MDA-MB-231 cells). Cancer cells (5 × 10⁻⁶) 3Cells were seeded into a 96-well plate (TPP) containing 100 μL of complete medium. The plate was incubated overnight in an incubator to allow the cells to adhere to the plate. Next, His-rhArg arginase (SEQ ID NO: 101) (2 U / mL to 0.0032 U / mL) and the autophagy inhibitor chloroquine (CQ, 100 μM to 0.16 μM) were added to the 96-well plate. For the drug combination group, His-rhArg (SEQ ID NO: 101) (2 U / mL to 0.0032 U / mL) was added to the wells along with 20 μM CQ (non-constant ratio method). Each drug dose was administered three times in three wells to obtain the mean change in cell viability. After 68 hours of incubation, 10 μL of 5 mg / mL MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide] solution was added. The plate was returned to the incubator and incubated for a further 4 hours. To dissolve formazan, an insoluble compound formed by MTT, 100 μL of a 0.01 M HCl solution of 10% SDS was added. The plate was incubated overnight at 37°C. The color change was measured by a spectrophotometer at 570 nm. Cell viability was calculated using the control setup (no drug) as a base, and dose-response curves were plotted using this data with the software Prism 6.0 (GraphPad). IC 50 A value was found. The interaction or combined effect of His-rhArg (sequence number 101) and CQ can be calculated using the combination index (CI). The CI value was calculated using CalcuSyn software (Biosoft).

[0292] MCF-7 cancer cells were sensitive to arginine starvation by arginase for 72 hours (Table 7). CQ also showed a cytotoxic effect on MCF-7 (Table 8). In the combination drug group, there was a synergistic effect (CI<1) between His-rhArg (SEQ ID NO: 101) and CQ (Table 9). Table 10 shows the IC50 for His-rhArg (SEQ ID NO: 101), CQ, and rhArg+CQ on MCF-7 cancer cells. 50 This indicates.

[0293] [Table 7]

[0294] [Table 8]

[0295] [Table 9]

[0296] [Table 10]

[0297] Experimental data revealed that MDA-MB-231 breast cancer cells are sensitive to arginine starvation induced by His-rhArg (SEQ ID NO: 101) and CQ (Tables 11 and 12). The combination of His-rhArg (SEQ ID NO: 101) and CQ also showed synergistic cytotoxicity against MDA-MB-231 cells. The CI value was less than 1 (Table 13). Table 14 shows the IC50 values ​​of His-rhArg (SEQ ID NO: 101), CQ, and His-rhArg (SEQ ID NO: 101) + CQ against MDA-MB-231 breast cancer cells. 50 This indicates.

[0298] [Table 11]

[0299] [Table 12]

[0300] [Table 13]

[0301] [Table 14]

[0302] The combination therapy of His-rhArg (SEQ ID NO: 101) (0.016, 0.08, or 0.4 U / mL) and CQ (20 μM) was also tested for the human hepatocellular carcinoma cell line "RedFluHepG2". A synergistic effect (CI value less than 1) was observed. [Examples]

[0303] Transwell migration assay and infiltration assay These studies used breast cancer cell lines (human MDA-MB-231 cells and mouse 4T1 cells). Both migration and invasion assays were performed using a 24-well Costar (Corning) Transwell system (polycarbonate membrane insert, pore size 8.0 μm). Before performing the assays, cells were cultured in appropriate serum-free medium for at least 24 hours. One day before seeding cells into the upper chamber, 100 μL of diluted Matrigel was added to the upper chamber of the insert to prepare for the invasion assay. Serum-free medium (100 μL) was added to the upper chamber used for the migration assay. The 24-well plate was incubated overnight in an incubator. The following day, cells were trypsinized and counted. In the studies, 3 × 10⁶ cells were counted. 4 4 T1 cells or 5 × 10 4 Individual MDA-MB-231 cells were seeded in the upper chamber with 200 μL of appropriate serum-free medium. Appropriate complete medium (750 μL) was added to the lower chamber. In the drug-treated group, 1 U / mL of His-rhArg (SEQ ID NO: 101) was added to both chambers. 24-well plates were incubated at 37°C in a 5% CO2 humidified atmosphere for 20 hours (4T1 cells) or 24 hours (MDA-MB-231 cells).

[0304] After incubation, the medium in the upper chamber was removed. The Transwell insert was washed twice with 1×PBS. Cold 100% methanol was added to both the upper and lower chambers for 10 minutes to fix the cells. The insert was washed twice with 1×PBS. Then, a 1×PBS solution of 1% crystal violet was added to both chambers for 15 minutes to stain the cells. The insert was washed twice with 1×PBS. The cells on the surface of the upper chamber were removed with a cotton swab. The insert was air-dried overnight. Photographs were taken with a confocal microscope. From the results, it was found that when the cells were treated with 1 U / mL of His-rhArg (SEQ ID NO: 101), the number of cancer cells that could migrate and infiltrate through the Transwell insert decreased, indicating that arginine depletion by arginase can inhibit the migration and infiltration of cancer cells.

Example

[0305] N-ABD-rhArg (SEQ ID NO: 49) inhibits the growth and metastasis of highly malignant breast cancer cells in vivo In a study on a xenograft model of highly malignant 4T1 breast cancer cells, 10 female nude mice (11 - 12 weeks old) were used. The mice were divided into groups of 5 per cage and given food and water ad libitum. 4T1 cancer cells were harvested by trypsin treatment, washed twice with 1×PBS, and resuspended in 1×PBS in preparation for inoculation. Cancer cells (1×10 6 individual 4T1 cells) were subcutaneously injected into the left flank of the mice. When the average length of the tumors reached 6 mm 3 days after inoculation, the mice were randomly divided into 2 groups. Either 500 U (3.2 mg, 230 μL) of N-ABD-rhArg (SEQ ID NO: 49) or 1×PBS was injected into the mice once a week. Blood samples were collected from the supine vein. The tumor size and body weight of the mice were monitored regularly. The tumor volume was estimated by the following formula. Tumor volume (mm 3 ) = length × width 2 / 2

[0306] In the control group treated with PBS injection, the tumors necrotized, so the mice were sacrificed 16 days after cancer cell inoculation (i.e., 13 days after the start of the experiment). After sacrifice, the tumors were removed from the mice and their weight was measured. The lungs were also removed and washed with 1×PBS. The lungs were fixed overnight with Bouin's fixative and then washed with 1×PBS. The number of lung metastatic nodules was measured.

[0307] Importantly, 500U of albumin-binding arginase N-ABD-rhArg (SEQ ID NO: 49) depleted arginine in BALB / c mice to levels below the detection limit (3 μM) of the Biochrom30 amino acid analyzer for at least 10 days (Figure 10). Therefore, the efficacy of 500U of N-ABD-rhArg (SEQ ID NO: 49) was studied by administering it once a week by intraperitoneal injection to nude mice with 4T1 breast cancer xenografts. Table 15 shows that 500U of N-ABD-rhArg (SEQ ID NO: 49) depleted arginine to undetectable levels during the experiment.

[0308] [Table 15]

[0309] Figures 64-67 show that 500U of N-ABD-rhArg (SEQ ID NO: 49) can inhibit the growth of 4T1 breast cancer xenografts in nude mice. There was a significant difference in estimated tumor volume between the control and experimental groups 12 and 13 days after the start of the experiment (Figures 64 and 65). There was also a significant difference in tumor size between the two groups (Figures 66 and 67).

[0310] These results suggest that arginine depletion by weekly injections of 500 U of N-ABD-rhArg (SEQ ID NO: 49) significantly inhibits the growth of xenografts of 4T1 high-grade breast cancer in nude mice. In vivo studies demonstrate that N-ABD-rhArg (SEQ ID NO: 49) can safely and effectively inhibit cancer growth in nude mice.

[0311] Furthermore, we found that arginine starvation could inhibit the metastasis of 4T1 breast cancer cells, which was consistent with in vitro results. After the mice in the treatment group were sacrificed, their lungs were collected and carefully examined for signs of metastasis. Three metastatic nodules were observed in three different lungs of the control group, but no metastatic nodules were observed in the N-ABD-rhArg (SEQ ID NO: 49) treated group (Table 16). These results convincingly demonstrate that arginine starvation by N-ABD-rhArg (SEQ ID NO: 49) can inhibit tumor metastasis. This suggests that arginine may play an important role in cell migration and tumor metastasis. Arginine depletion by N-ABD-rhArg (SEQ ID NO: 49) may inhibit several pathways important for cancer cell metastasis (Knott et al., 2018, Nature, 554:378-381).

[0312] [Table 16] [Examples]

[0313] Cytotoxicity of human colon cancer cell lines by N-ABD094-rhArg (SEQ ID NO: 50) fusion enzyme The in vitro efficacy of an ABD-rhArg fusion protein was tested in human colon cancer cell lines. Human colon cancer cell lines (HCT116, LOVO, and HT29) were used in 5 × 10⁶ samples. 3 Seeds were seeded at a density of cells / well in 96-well plates. One day after incubation, the culture medium was replaced with medium containing N-ABD094-rhArg (SEQ ID NO: 50) at various concentrations (2, 0.4, 0.08, 0.016, and 0.0032 U / mL). On day 3 [3 days after N-ABD094-rhArg (SEQ ID NO: 50) treatment], an MTT assay was performed to obtain IC50. 50The value was determined. The medium was replaced with MTT solution (1 mg / mL) (Invitrogen), and incubated at 37 °C for 4 hours. After incubation, the MTT solution was replaced with dimethyl sulfoxide (DMSO), and the absorbance at 570 nm with reference to 650 nm was measured. The cell viability was determined by dividing the absorbance of N-ABD094-rhArg-treated cells by the average absorbance of untreated cells. The IC 50 value (95% confidence interval) of N-ABD094-rhArg (SEQ ID NO: 50) against cancer cell lines was analyzed using software Prism 6.0. Three independent experimental sets (n = 3) were performed for each cell line. The results are shown in Table 17.

[0314]

Table 17

[0315] All tested colorectal cancer cell lines were highly sensitive to N-ABD094-rhArg (SEQ ID NO: 50) fusion enzyme in vitro, and the IC 50 value was 0.22 - 0.58 U / mL. HCT116 was the most sensitive tumor with an IC 50 of 0.22 U / mL. The IC 50 values of LOVO and HT29 were 0.38 U / mL and 0.58 U / mL, respectively.

Example

[0316] The combination of N-ABD094-rhArg (SEQ ID NO: 50) and chloroquine exhibits a synergistic cytotoxic effect on human colorectal cancer cell lines The IC 50 value of autophagy inhibitor chloroquine (CQ) was determined as follows. Colorectal cancer cell lines HCT116, LOVO and HT29 were seeded separately, and after 1 day of incubation, the medium was replaced with medium containing each concentration of CQ (100, 20, 4, 0.8, 0.16 μM). The MTT assay was performed on the third day. Three independent experimental sets (n = 3) were performed for each cell line. The results are shown in Table 18.

[0317] [Table 18]

[0318] CQ IC for HCT116, LOVO, and HT29 50 The values ​​were >100 μM, 15 μM, and 71 μM, respectively. Non-constant ratio analysis was performed in the drug combination assay (Hastak et al., 2010, Cancer Res. 70(20):7970-7980). Colon cancer cells were seeded, and one day after incubation, the culture medium was treated with N-ABD094-rhArg (SEQ ID NO: 50) at various concentrations (2, 0.4, 0.08, 0.016, 0.0032 U / mL) and its IC50. 50 The culture medium was replaced with a fixed amount of CQ combination according to the specified formula. The MTT assay was performed on day 3. The combination index (CI) of N-ABD094-rhArg (SEQ ID NO: 50) and CQ combination treatment was analyzed using the software CalcuSyn version 2.1. Three independent experimental sets (n=3) were performed for each cell line. The results are shown in Table 19.

[0319] [Table 19]

[0320] The results show that when 0.08 U / mL or 0.4 U / mL of N-ABD094-rhArg (SEQ ID NO: 50) was used in combination with 20 μM of CQ, the CI value was <1.0 in all three colon cancer cell lines. This suggests that N-ABD094-rhArg (SEQ ID NO: 50) is effective on its own and synergistically effective when used in combination with CQ. In summary, the autophagy inhibitor chloroquine (CQ) enhances the cell death-inducing effect of the N-ABD094-rhArg (SEQ ID NO: 50) fusion protein in human colon cancer cells. [Examples]

[0321] N-ABD-rhArg (SEQ ID NO: 49) and PEGylated His-rhArg (SEQ ID NO: 101) both inhibit the proliferation and metastasis of high-grade breast cancer cells in vivo. Other 4T1 breast cancer allograft studies used female nude mice (5-6 weeks old). The mice were divided into groups of 5 per cage and given free access to food and water. 4T1 breast cancer cells (1 × 10⁶) 5 The mice were subcutaneously injected with PEG-rhArg (500U) into the right flank. Eleven days after inoculation, when the average tumor length reached 8 mm, the mice were randomly divided into four groups: one control group (n=8) and three experimental groups: 500U PEGylated His-rhArg (SEQ ID NO: 101) (n=8), 500U N-ABD-rhArg (SEQ ID NO: 49) (n=6), and 250U N-ABD-rhArg (SEQ ID NO: 49) (n=8). The control group mice were injected with 1×PBS weekly. In the 500U ABD-rhArg group and the 250U ABD-rhArg group, mice were injected with 500U and 250U ABD-rhArg, respectively, once a week, and one group was injected with 500U PEG-rhArg once a week. In the PBS control group, the tumors necrotized, so the mice were sacrificed 24 days after inoculation (i.e., 14 days after the start of the experiment). After sacrifice, the mice were dissected. The tumors were collected from the mice and weighed. The lungs were also collected and washed with 1×PBS. The lungs were fixed overnight with Bouin's fixative and then washed with 1×PBS. The number of lung metastatic nodules was counted.

[0322] Regarding the results, Figure 68 shows that both 500U of ABD-rhArg and 500U of PEG-rhArg significantly inhibited the growth of high-grade 4T1 solid tumors. However, the low dose of 250U of ABD-rhArg did not inhibit tumor growth. This result was consistent with the changes in tumor weight in the different groups (Figure 69). Therefore, ABD-rhArg inhibited tumor growth in a dose-dependent manner. Importantly, the data clearly showed that arginine starvation (by either ABD-rhArg or PEG-rhArg) inhibited or suppressed tumor metastasis to the lungs (Figures 70A and 70B). Metastatic nodules originating from the lungs were significantly reduced in both the 500U PEG-rhArg and 500U ABD-rhArg groups compared to the control group. Histological sections of lungs stained with hematoxylin and eosin (H&E) revealed that the nodules were formed by a series of dense breast cancer cells. These results suggested that the small nodules were metastatic lesions. In summary, arginine depletion by long-acting arginase inhibits metastasis of high-grade cancer cells in a mouse model. Since cancer metastasis is a leading cause of death in patients (Knott et al., 2018, Nature, 554:378-381), this finding may offer new possibilities for more effective cancer therapies. [Sequence Listing Free Text]

[0323] Sequence ID 1: ABD primer 1, synthesized in the laboratory. Sequence ID 2: ABD primer 2, synthesized in the laboratory. Sequence ID 3: ABD primer 3, synthesized in the laboratory. Sequence ID 4: ABD primer 4, synthesized in the laboratory. Sequence ID 5: ABD primer 5, synthesized in the laboratory. Sequence ID 6: ABD primer 6, synthesized in the laboratory. Sequence ID 7: ABD primer 7, synthesized in the laboratory. Sequence ID 8: ABD primer 8, synthesized in the laboratory. Sequence ID 9: ABD primer 9, synthesized in the laboratory. Sequence ID No. 10: ABD primer 10, synthesized in the laboratory. Sequence ID 11: ABD primer 11, synthesized in the laboratory. Sequence ID No. 12: ABD primer 12, synthesized in the laboratory. Sequence ID 13: ABD primer 13, synthesized in the laboratory. Sequence ID 14: ABD primer 14, synthesized in the laboratory. Sequence ID 15: ABD primer 15, synthesized in the laboratory. Sequence ID 16: ABD primer 16, synthesized in the laboratory. Sequence ID 17: ABD primer 17, synthesized in the laboratory. Sequence ID 18: ABD primer 18, synthesized in the laboratory. Sequence ID 19: ABD primer 19, synthesized in the laboratory. Sequence ID 20: ABD primer 20, synthesized in the laboratory. Sequence ID 21: ABD primer 21, synthesized in the laboratory. Sequence ID 22: ABD primer 22, synthesized in the laboratory. Sequence ID 23: ABD primer 23, synthesized in the laboratory. Sequence ID No. 24: ABD primer 24, synthesized in the laboratory. Sequence ID 25: ABD primer 25, synthesized in the laboratory. Sequence ID 26: ABD primer 26, synthesized in the laboratory. Sequence ID 27: ABD primer 27, synthesized in the laboratory. Sequence ID 28: ABD primer 28, synthesized in the laboratory. Sequence ID 29: ABD primer 29, synthesized in the laboratory. Sequence ID 30: ABD primer 30, synthesized in the laboratory. Sequence ID 31: ABD primer 31, synthesized in the laboratory. Sequence ID 32: ABD primer 32, synthesized in the laboratory. Sequence ID 33: His-ABD-linker, synthesized in the laboratory. Sequence ID 34: His-ABD094-linker, synthesized in the laboratory. Sequence ID 35: Linker-ABD-His, synthesized in the laboratory. Sequence ID 36: Linker-ABD094-His, synthesized in the laboratory. Sequence ID 37: N-ABD-rhArg gene, synthesized in the laboratory. Sequence ID 38: N-ABD094-rhArg gene, synthesized in the laboratory. Sequence ID 39: C-ABD-rhArg gene, synthesized in the laboratory. Sequence ID 40: C-ABD094-rhArg gene, synthesized in the laboratory. Sequence ID 41: N-ABD-BCA gene, synthesized in the laboratory. Sequence ID 42: N-ABD094-BCA gene, synthesized in the laboratory. Sequence ID 43: C-ABD-BCA gene, synthesized in the laboratory. Sequence ID 44: C-ABD094-BCA gene, synthesized in the laboratory. Sequence ID 45: His-ABD-linker, synthesized in the laboratory. Sequence ID 46: His-ABD094-linker, synthesized in the laboratory. Sequence ID 47: Linker-ABD-His, synthesized in the laboratory. Sequence ID 48: Linker-ABD094-His, synthesized in the laboratory. Sequence ID 49: N-ABD-rhArg, synthesized in the laboratory. Sequence ID 50: N-ABD094-rhArg, synthesized in the laboratory. Sequence ID 51: C-ABD-rhArg, synthesized in the laboratory. Sequence ID 52: C-ABD094-rhArg, synthesized in the laboratory. Sequence ID 53: N-ABD-BCA, synthesized in the laboratory. Sequence ID 54: N-ABD094-BCA, synthesized in the laboratory. Sequence ID 55: C-ABD-BCA, synthesized in the laboratory. Sequence ID 56: C-ABD094-BCA, synthesized in the laboratory. Sequence ID 57: ABD094-0F primer, synthesized in the laboratory. Sequence ID 58: ABD094-1F primer, synthesized in the laboratory. Sequence ID 59: ABD094-2F primer, synthesized in the laboratory. Sequence ID 60: ABD094-3F primer, synthesized in the laboratory. Sequence ID 61: ABD094-4F primer, synthesized in the laboratory. Sequence ID 62: ABD094-5F primer, synthesized in the laboratory. Sequence ID 63: abdNde-F primer, synthesized in the laboratory. Sequence ID 64: HuArgHinBam-R primer, synthesized in the laboratory. Sequence ID 65: N-ABD094-rhArg gene, synthesized in the laboratory. Sequence ID 66: ABD based on Streptococcus sp. G148, synthesized in the laboratory. Sequence ID 67: ABD based on Streptococcus sp. G148, synthesized in the laboratory. Sequence ID 68: Linker-less ABD094, synthesized in the laboratory. Sequence ID 69: Xaa at position 1 is either methionine or absent. Sequence ID 71: BCA (S161C) arginase, synthesized in the laboratory. Sequence ID 72: BCA (V20P, S161C) arginase, synthesized in the laboratory. Sequence ID 73: Linker sequence 1, synthesized in the laboratory. Sequence ID 74: Linker sequence 2, synthesized in the laboratory. Sequence ID 75: BHA fusion protein, synthesized in the laboratory Sequence ID 76: BAH fusion protein, synthesized in the laboratory. Sequence ID 77: 5'-Sense-ABD primer, synthesized in the laboratory. Sequence ID 78: 5'-Antisense-ABD primer, synthesized in the laboratory. Sequence ID 79: Forward primer for ABD cloning, synthesized in the laboratory. Sequence ID 80: Reverse primer for ABD cloning, synthesized in the laboratory. Sequence ID 81: Forward primer for BCA cloning, synthesized in the laboratory. Sequence ID 82: Reverse primer for BCA cloning, synthesized in the laboratory. Sequence ID 83: Forward primer for BHA cloning, synthesized in the laboratory. Sequence ID 84: Reverse primer for BHA cloning, synthesized in the laboratory. Sequence ID 85: Forward primer for ABD cloning, synthesized in the laboratory. Sequence ID 86: Reverse primer for ABD cloning, synthesized in the laboratory. Sequence ID 87: Forward primer for cloning BAH, synthesized in the laboratory. Sequence ID 88: Reverse primer for cloning BAH, synthesized in the laboratory. Sequence ID 89: BCA (S161C)-His, synthesized in the laboratory. Sequence ID 90: N-ABDHind-R primer, synthesized in the laboratory. Sequence ID 91: C-ABDNde-F primer, synthesized in the laboratory. Sequence ID 92: C-ABDHind-R, synthesized in the laboratory. Sequence ID 93: HARGBam-F primer, synthesized in the laboratory. Sequence ID 94: HARGHind-R primer, synthesized in the laboratory. Sequence ID 95: HARGNde-F, synthesized in the laboratory. Sequence ID 96: HARGBam-R primer, synthesized in the laboratory. Sequence ID 97: BCABam-F primer, synthesized in the laboratory. Sequence ID 98: BCAHind-R primer, synthesized in the laboratory. Sequence ID 99: BCANde-F primer, synthesized in the laboratory. Sequence ID 100: BCABam-R primer, synthesized in the laboratory. Sequence ID 101: His-rhArg, synthesized in the laboratory. Sequence ID 102: His-rhArg-mono-Cys, synthesized in the laboratory. Sequence ID 104: rhArg-mono-Cys, synthesized in the laboratory. Sequence ID 105: A(EAAAK)4ALEA-(EAAAK)4A peptide linker, synthesized in the laboratory. Sequence ID 106: G4SG4SG3SG peptide linker, synthesized in the laboratory. SEQ ID NO: 107: (G4S)n peptide linker, synthesized in the laboratory. Amino acids ranked 5th to 20th can be omitted. Sequence ID 108: [A(EAAAK)nA]x peptide linker, synthesized in the laboratory. The amino acids in positions 12-21 are not necessary. The amino acids in positions 23-44 are not necessary. Sequence ID 109: (G4S)3 peptide linker, synthesized in the laboratory. Sequence ID 110: (GS)n peptide linker, synthesized in the laboratory. Amino acids ranked 3-60 are not necessary. Sequence ID 111: (GGS)n peptide linker, synthesized in the laboratory. Amino acids ranked 4-90 are not necessary. Sequence ID 112: GS(N)nGSG peptide linker, synthesized in the laboratory. The amino acids in positions 4-12 are not necessary. Sequence ID 113: GS(Q)nGSG peptide linker, synthesized in the laboratory. The amino acids in positions 4-12 are not necessary.

Claims

1. A fusion protein for improving the circulating half-life of arginase, comprising an albumin-binding domain (ABD) polypeptide, an arginase polypeptide, and a peptide linker connecting the C-terminus of the ABD polypeptide to the N-terminus of the arginase polypeptide, wherein the fusion protein comprises (a) or (b) below. (a) The ABD polypeptide selected from the group consisting of Sequence ID No. 66, Sequence ID No. 67, and Sequence ID No. 68, The arginase polypeptide selected from the group consisting of SEQ ID NOs: 69, 70, 71, 72, and 103, The peptide linker selected from the group consisting of SEQ ID NOs: 73, 74, 105, 106, 107, 108, 109, 110, 111, 112, and 113, or (b) A polypeptide sequence having at least 95% sequence identity with SEQ ID NO: 49 and SEQ ID NO:

50.

2. The fusion protein according to claim 1, wherein the fusion protein contains two Mn²⁺ ions.

3. The fusion protein according to claim 1, wherein the fusion protein contains one or more divalent metals selected from the group consisting of Co²⁺ and Ni²⁺.

4. A pharmaceutical composition comprising the fusion protein described in claim 1 and a pharmaceutically acceptable carrier, excipient, or combination thereof.

5. The use of the fusion protein according to claim 1 in the manufacture of a pharmaceutical product for treating cancer in a person requiring cancer treatment.

6. The use of the fusion protein according to claim 1 in the manufacture of a pharmaceutical product for treating a condition in a subject who requires treatment for at least one condition selected from the group consisting of viral infections, multiple sclerosis, rheumatoid arthritis, autoimmune diseases, congenital hyperargininemia, graft-versus-host disease (GvHD), and inflammation.

7. Use of the fusion protein according to Claim 1 in the manufacture of a pharmaceutical product for treating a condition in a subject who requires treatment of at least one condition selected from the group consisting of obesity, impaired glucose tolerance, hyperglycemia, diabetes mellitus, diabetic nephropathy, diabetic retinopathy, diabetic vascular disorder, diabetic neuropathy, hypercholesterolemia, dyslipidemia, hypertriglyceridemia, hyperleptinemia, hyperinsulinemia, steatosis, steatohepatitis, fibrosis, cirrhosis, chronic low-grade inflammation, hypertension, cardiovascular disease, whitening of brown fat, insulin resistance, preventing an increase in fat mass and reducing fat mass, fatty liver, renal steatosis, pancreatic steatosis and cardiac steatosis.

Images