Development and characterization of fused human arginase I for cancer therapy

Recombinant human arginase I (rhArg I) have emerged as a potential candidate for the treatment of varied pathophysiological conditions ranging from arginine-auxotrophic cancer, inflammatory conditions and microbial infection. However, rhArg I have a low circulatory half-life, leading to poor pharmacokinetic and pharmacodynamic properties, which necessitating the rapid development of modifications to circumvent these limitations. To address this, polyethylene glycol (PEG)ylated-rhArg I variants are being developed by pharmaceutical companies. However, because of the limitations associated with the clinical use of PEGylated proteins, there is a dire need in the art to develop rhArg I variant(s) which is safe (devoid of limitations of PEGylated counterpart) and possess increased circulatory half-life. In this study, we described the generation and characterization of a fused human arginase I variant (FHA-3) having improved circulatory half-life. FHA-3 protein was engineered by fusing rhArg I with a half-life extension partner (domain of human serum albumin) via a peptide linker and was produced using P. pastoris expression system. This purified biopharmaceutical (FHA-3) exhibits (i) increased arginine-hydrolyzing activity in buffer, (ii) cofactor - independency, (iii) increased circulatory half-life (t1/2) and (iv) potent anti-cancer activity against human cancer cell lines under in vitro and in vivo conditions.


Introduction
L-arginine is a semi-essential amino acid which not only serves as a building block of peptides and proteins but also serves as a precursor for the synthesis of a variety of important metabolites, intermediate and precursors in the body.It is thus involved in many processes such as ammonia detoxification, hormone secretion, and immunomodulation [1].The urea cycle enzymes argininosuccinate synthetase 1 (ASS1) and argininosuccinate lyase (ASL) are responsible for the synthesis of arginine from citrulline in the

ASL
Argininosuccinate lyase ASS1 Argininosuccinate synthetase 1 BMGY Buffered complex glycerol medium BMMY Buffered methanol-complex medium DMEM Dulbecco's Modified Eagle's medium DMSO Dimethyl sulfoxide FHA Fused human arginase I HCC Hepatocellular carcinoma HLEPs Half-life extension partners HSA Human serum albumin Abhay H. Pande apande@niper.ac.in; abbupande@yahoo.co.in hepatocytes.On the other hand, arginase I (Arg I), ornithine transcarbamylase (OTC) and ASS1 catabolize arginine to ornithine and urea, citrulline, and argininosuccinate, respectively [2,3].Arginine auxotrophy arises due to deficiency or minimal expression of the genes for ASS1 and/or OTC and such cells are therefore completely reliant on extracellular arginine for survival and maintenance [4].The transcriptional silencing (hypermethylation) of OTC and ASS1 genes is thought to be the cause of their downregulation in cancer cells [5].Several amino acids are now being targeted for the treatment of various cancers and many of the enzymes that are employed to deplete these amino acids are in the advanced stages of their development [6,7].Human arginase I (hArg I) is a homotrimeric, manganese (Mn 2+ )-dependent metalloenzyme (of ~ 105 kDa) that carries out the hydrolysis of L-arginine to form urea and L-ornithine [8].Arginine depletion by administering the hArg I enzyme has been shown to inhibit the growth and progression of various arginineauxotrophic cancers with lower expression of OTC and/ or ASS1 enzymes [9,10].However, at physiological pH, the native form of hArg I is less efficient with a high K m value (10.5 mM) for the arginine as well as poor circulatory half-life (̴ 30 min), which constrains the usefulness of arginase I in its native form [2,11].To address these issues, PEGylated-recombinant hArg I (PEG-rhArg I) molecules are being developed by pharmaceutical firms and are in various stages of their clinical development [2,9,12,13].However, clinical use of PEGylated-proteins is known to have several severe safety and other concerns, viz., PEG toxicity, immunogenicity, hypersensitivity to PEG, problems due to heterogeneity of PEGylated proteins and increased aggregation in some cases [14][15][16][17][18].Because of these drawbacks of PEGylated proteins, there is a dire need in the field to produce rhArg I variants that are both safe and have a longer circulatory half-life.
In this regard, the development of fusion proteins, in which a therapeutic protein is genetically linked to the full length or domain of another protein, has emerged as a potent method for improving therapeutic protein pharmacokinetic features [18,19].The development of half-life extension technology, in which therapeutic proteins are fused with half-life extension partners (HLEPs), has been shown to significantly improve the circulatory half-lives of these proteins and hence their overall pharmacokinetic properties [20].Human transferrin [21], human IgG Fc domain [22], and human serum albumin (HSA)-full length or domain [23] are some of the effective HLEPs which can be used to enhance the half-life of rhArg I by fusing it with any of these HLEPs using appropriate linkers [18,24].
In the present study, we have developed and characterized FHA-3, which is a rhArg I fusion protein with an albumin binding domain (as HLEP).Purified FHA-3 exhibits increased circulatory half-life with potent anti-cancer activity against human cancer cell lines under in vitro and in vivo conditions.

Expression and purification of FHA-3
The recombinant plasmid encoding FHA-3 was amplified (in E. coli DH5α cells) and was subjected to restriction digestion with Bgl II, by using a standard protocol.The digested reaction was subjected to agarose gel electrophoresis (0.8%) and the linearized pPIC9K-FHA-3 fragment was purified using GeneJet gel extraction kit.Competent Pichia pastoris cells (SMD1168) were transformed with linearized pPIC9K-FHA-3 fragment and were plated on minimal dextrose (MD) plates (1.34% (w/v) yeast nitrogen base without amino acids and without ammonium sulphate, 2% (w/v) dextrose, 1.5% (w/v) agar) and grown at 30 o C for 72-96 h.Colonies obtained on the MD plates were plated on YPD (1% (w/v) yeast extract, 2% (w/v) peptone and 2% (w/v) dextrose, 1.5% (w/v) agar) plates containing an increasing concentration of Geneticin sulphate (G418) antibiotic and grown at 30 o C for 4-5 days to screen for multicopy transformants.Colonies present in the plate containing the highest concentration of antibiotic were screened for the expression of recombinant protein by inoculating in 10 ml of BMGY (Buffered complex glycerol medium containing 1% (w/v) yeast extract, 2% (w/v) peptone, 1% (w/v) glycerol, 1.34% (w/v) yeast nitrogen base without amino acids and without ammonium sulphate, 0.1 M phosphate buffer pH 6.0) media (containing 50 μg/ml kanamycin and 100 μg/ml G418) and the cultures were grown for 24 h at 30 o C, 250 rpm.Subsequently, 1% culture was transferred to the 500 ml of BMGY media and grown further at 30 o C for 48 h.The cultures were then centrifuged to remove BMGY media and the cell mass was washed with BMMY (Buffered methanol-complex medium containing 1% (w/v) yeast extract, 2% (w/v) peptone, 1.34% (w/v) yeast nitrogen base without amino acids and without ammonium sulphate, 0.1 M phosphate buffer pH 6.0 and 1% (v/v) methanol) and resuspended in fresh BMMY media.The cultures were grown further at 30 o C and induced with 1% methanol every 24 h for 5 days, which was then centrifuged to collect the media without cell mass.The presence of recombinant protein in cultivating media was determined by western blot analysis and the clones showing (maximum) expression of target protein were then selected for production of FHA-3 protein.
A single colony of the selected clone was used and the cultivation of microbe and expression of recombinant protein was done by following the procedure essentially described above.After 5 days of induction of the culture with methanol, the cultivating media was collected and subjected to affinity chromatography using (50 ml) of Ni-NTA resin.Elution of the bounded protein was done in 20 mM Tris-HCl buffer (pH 8) containing 350 mM imidazole and 250 mM NaCl and the eluted fractions were collected and analyzed for both protein content (by Bradford assay) as well as arginine-hydrolyzing activity (using AutoZyme urea detection kit form Accurex Biomedical Pvt Ltd, Mumbai, India).Qualitative analysis of these fractions was done by performing SDS-PAGE and western blot analysis.Fractions containing enzymatically active protein were pooled and dialyzed against 10 mM Tris HCl buffer containing 150 mM NaCl, pH 8.0.Dialyzed sample was then subjected to endotoxin removal step by using High-Capacity Endotoxin Removal Spin Columns and concentrated using Amicon® Ultra-15 centrifugal filter units.After adding 10% glycerol (final concentration), the concentrated protein sample was then kept at -80°C in aliquots for further use.

Enzymatic activity assay
A coupled spectrophotometric assay was used to determine the arginine-hydrolyzing activity of FHA-3 as described in Stone et al. with modifications [8].Briefly, the enzyme was activated by mixing (80 μl) of the enzyme with 20 μl of either MnSO 4 or CoCl 2 (final concentration 10 mM) and incubating the mixture at 50 o C for 20 min.Then the activated enzyme was brought to room temperature and used in the activity assay.The arginine-hydrolyzing activity of FHA-3 was determined by incubating activated FHA-3 (20 μl) with 40 μl of arginine substrate (final concentration 50 mM) in 100 mM HEPES buffer of pH 8.5 or pH 7.5 (at 30 o C) and the urea generated in the reaction was then determined using AutoZyme urea detection kit.The specific activity of FHA-3 (U/mg) defines the amount of FHA-3 required to convert 1 μmole of L-arginine to 1 μmole of L-ornithine and 1 μmole of urea per min at pH 8.5 or 7.5 at 30°C per mg of protein.

In vitro plasma stability of FHA-3
In vitro plasma stability of FHA-3 was determined by following the procedure described previously [25].Briefly, blood samples were collected from healthy human volunteers in an anti-coagulant (3.8% tri-sodium citrate) containing tube, and plasma was separated by centrifugation.The protocol was approved by the Institutional Ethics Committee (IEC No: IEC/52/2021).FHA-3 protein (10 μM final concentrations) was added to the plasma and the mixtures were incubated at 25°C.Aliquots were withdrawn from the incubated mixture at designated time points and the stability of the exogenously added FHA-3 enzyme was checked

Cell viability assay
The in vitro anticancer activity of FHA-3 was determined by measuring cytotoxicity against selected cancer cell lines, by following the procedure described previously [27,28], with slight modifications.Briefly, cancer cells (3 × 10 5 per well) were seeded in each well of flat-bottom 24-well plates containing DMEM supplemented with 10% FBS at 37°C (in 95% humidity and 5% CO 2 ) and allowed to grow overnight After attaining 80% confluence, FHA-3 was added in designated concentration (0-50 U/ml final concentration) to the cells and the plates were further incubated for 72 h at 37 o C and viability of the cells was determined by MTT assay [29].For this, MTT solution (final concentration of 0.5 mg/mL) was added to the well and the plates were further incubated for 4 h at 37°C to induce reaction.After removing the supernatant, formazan crystal was dissolved in DMSO (500 μL) and the absorbance was measured at a dual-wavelength of 550 and 630 nm using a multi-mode automated microplate reader (Flex station III, Molecular Devices, Sunnyvale, CA, USA).The results were expressed as percentage cell viability, assuming the viability of untreated control cells as 100%.The concentration of enzymes required for 50% inhibition of the cells in culture was defined as the IC 50 , and IC 50 values were determined by using GraphPad Prism 6 (GraphPad Software, Inc.).Two independent experiments were performed for each study and all measurements were performed in triplicates.Data were represented as mean ± S.D.

Xenograft nude mice tumor model
BALB/c nude mice [CrTac: NCr-Foxn1 nu (NCRNU-F)] and RMS(P) PL feed were purchased from the Vivo Bio Tech Ltd., Hyderabad, India, and transferred to a pathogenfree environment of National Toxicology Center (NTC) in NIPER, SAS Nagar.The animals were housed in sterile and clean cages with HEPA filters with free access to diet and water under controlled conditions of temperature: 25°C, humidity: 50%; and a 12 h light/dark cycle.All the animals were acclimatized for one week before the start of experiments and were properly maintained by taking all necessary precautions and care.
In vivo anticancer activity of FHA-3 was determined in the hepatocellular carcinoma (HCC) xenograft model by following the procedures as described previously [27,28], with slight modifications.Briefly, 2 × 10 6 viable HepG2 cells were dispersed in 1:1 (v/v) Phosphate buffered saline (PBS) and matrigel in a final volume of 100 μl and injected subcutaneously into the right flanks of each mouse (3-4 weeks old BALB/c nude mice) and the tumors were allowed to grow in the animals.The sizes of tumors were measured by monitoring the arginine-hydrolyzing activity by using 50 mM arginine substrate.The activity of the control plasma samples (in which FHA-3 enzyme was not added) was taken as 100%.

In vivo pharmacokinetic analysis
Male adult Sprague-Dawley (SD) rats (230-250 g; 4-6 weeks old) were procured from the Central Animal Facility of our Institute (NIPER, SAS Nagar) and were maintained under standard environmental conditions with controlled temperature (22 ± 2°C), humidity (50 ± 10%), and a 12 h light/dark cycle.Animals were provided feed and water ad libitum.All animals were in good condition without any macroscopic changes in the skin and tail.Institutional Animal Ethics Committee approval was obtained in advance (IAEC No. IAEC/21/50) for the execution of research in accordance with CPCSEA rules, which are outlined in the Institute of Laboratory Animal Resources' (ILAR, USA) standards.
In vivo pharmacokinetic properties of FHA-3 were determined in SD rats by following the procedures described in the literature [16,26], with slight modifications.Briefly, 3 mg/kg of FHA-3 protein was administered in SD rats (n = 6) as a single intravenous dosage (in the tail vein) and ~ 0.3 ml blood samples were collected from the animals (from the retro-orbital route under anesthesia) at the designated time-points (pre-dose, 1 h, 4 h, 8 h, 24 h, 48 h, 72 h, 120 h) in a heparinized tube.Collected blood samples were immediately centrifuged and clear plasma was separated, aliquoted, and stored at -80 o C until further use.The amount of FHA-3 protein present in plasma was determined by using the His-tag ELISA detection kit by following the procedure described by the manufacturer.Results obtained after ELISA were fitted into the standard curve and the amount of protein present in plasma was calculated.Noncompartment model for pharmacokinetic analysis was used to calculate pharmacokinetic parameters, Mean Residence Time (MRT) and half-life (t 1/2 ) by using PK solver software.

Cell culture
Hepatocellular carcinoma -HepG2, prostate cancer -DU145, and lung cancer -A549 cell lines were maintained in DMEM medium with (10%) fetal bovine serum, (1%) antibiotic solution (streptomycin (100 mg/ml) and penicillin (10,000 U/ml)) in an incubator with 5% CO 2 , 95% humidity at 37°C.Cells were allowed to grow up to approximately 80% confluency before experimentation.microscopic images were captured with the OLYMPUS DP 72 camera attached to the microscope.

Statistical analysis
The data obtained in the results are expressed as mean ± S.D. The statistical analysis was performed using GraphPad Prism 8.The t-test was done for statistical comparison between two groups, whereas the one-way analysis of variance (ANOVA) followed by Tukey's test was performed for the comparison between more than two different groups.A p-value of less than 0.05 was considered to be significant.

Expression and purification of FHA-3
Using the fusion-protein engineering approach, amino acid sequences of the third domain of human serum albumin (HSA), linker peptide and rhArg I were used to design FHA-3 (Fig. 1A).We used P. pastoris expression system for the production of FHA-3 protein as it is one of the widely used systems for the production of heterologous proteins.The native-like folding of target proteins is one of the most significant benefits of the yeast expression system over the bacterial expression system [30,31].Plasmids with the individually with a Vernier Caliper twice a week.After attaining the desired tumor volume (> 80 mm 3 ), tumor-bearing animals were randomly divided into 4 groups (n = 7) and treated with indicated samples (0.9% saline solution, 5-FU (10 mg/kg once a week), FHA-3 (300 U/mouse twice a week) and a combination of FHA-3 + 5-FU) by injecting the sample intraperitoneally for three weeks.Tumor dimensions were measured in situ during the treatment by Caliper measurement and tumor volume (0.5 x L x W 2 ) was calculated and plotted.After the completion of the treatment period, the animals were sacrificed (by CO 2 inhalation) and the tumors were excised and weighed.

Histological examination of tumor tissue
Histological examination of extracted tumor tissue was done by following the procedure described previously [27,28].Briefly, after the completion of the treatment schedule, animals were sacrificed and tumors were isolated from each animal, sliced, and fixed in 10% v/v formal saline.Thereafter, the tumors were subsequently embedded in paraffin; sections of 5 μm were prepared and mounted on pre-coated slides.Sections were stained with hematoxylin and eosin to observe the structural changes.A cover slip was mounted on the stained sections using DPX and observed at 40x magnification using the OLYMPUS BX51 microscope.The The addition of FHA-3 protein increased the arginase activity of the plasma and there was no significant decrease in the arginase activity of the plasma even after 24 h of incubation.This indicated that the exogenously added FHA-3 protein was active and stable in human plasma for at least 24 h (Fig. 2A).

In vivo pharmacokinetic analysis
In vivo pharmacokinetic properties of FHA-3 were determined in SD rats.Single intravenous dose of FHA-3 (3 mg/ kg ) was given to the animals in their tail veins.The FHA-3 protein present in the plasma was determined using a His-Tag ELISA detection kit and blood samples drawn at pre -determined intervals.The quantity of His-tagged protein present in the sample (plasma) was determined from the standard curve generated by plotting His-tagged protein standards of known concentration (kit reagent) and the corresponding absorbance values (Fig. 2B inset).The pharmacokinetic parameters were then calculated using the average data from all six animals using a non-compartmental model(Fig.2B) [8,16,26,34].Analysis of kinetic parameters using PK solver shows that half-life (t 1/2 ), and MRT of FHA-3 molecule in SD rat were 29.6 h, and 41.5 h, respectively.The reported half-life of different arginase variants (unfused-, PEGylated-and fusion-rhArg I) in the literature ranges from 4 h to 4 days [8,16,26,32,36].

Cell viability assay
The in vitro anticancer activity of FHA-3 was determined by measuring cytotoxicity against selected human cancer cell lines: HepG2 -liver cancer, DU145 -prostate cancer, and A549 -lung cancer.To standardize the assay, we first used recombinant human liver rhArg I (OriGene Technologies, Inc. Cat # TP304649) and HepG2 cells.Cells were grown and treated with an increasing concentration of rhArg I and the cytotoxicity were determined by measuring the cell viability using the MTT assay.It was observed that rhArg I exhibited a dose-dependent decrease in the cell viability (data not shown) validating our assay.Then, under a similar experimental set-up, we determined the in vitro anticancer activity of FHA-3 against three cancer cell lines (Fig. 3).As evident, FHA-3 exhibited considerable cytotoxicity towards these cancer cell lines in a dose-dependent manner.The IC 50 obtained for HepG2, DU145 and A549 cells were 0.13 ± 0.05, 0.27 ± 0.05 and 0.35 ± 0.15 U/ml, respectively.The IC 50 value of different arginase variants towards HCC cell lines reported in the literature ranges from 0.08 to 0.24 U/ml [9,16]; against DU145 ~ 0.02 U/ml [37] and A549 from 2 to 100 U/ml [9,38].Our results suggest that, as expected, depletion of arginine in the medium by treating inducible gene expressing FHA-3 (pPIC9K-FHA-3) were amplified in E. coli DH5α cells and subjected to restriction digestion with Bgl II, by using a standard protocol.Linearized pPIC9K-FHA-3 fragment was purified using GeneJet gel extraction kit and was used for the transfection of competent P. pastoris SMD1168 cells.The cells were first plated on the MD plate and the colonies obtained on the MD plate were then collected and plated on the YPD plates containing an increasing concentration of G418 (0.25-4.0 mg/ml).Western blotting was used to monitor the concentration of the target recombinant protein in the growth medium in order to screen colonies found on G418 plates with the highest concentration of G418 for the expression of the target recombinant protein.The clones showing (maximum) expression of target protein were then selected for production of FHA-3 and the recombinant protein was expressed as described in materials and methods.Cultivating media was collected by centrifugation and subjected to affinity chromatography on Ni-NTA column (Fig. 1B).Fractions containing active protein were pooled and subjected to dialysis as well as endotoxin removal steps.The sample was then concentrated and stored using 10% glycerol (final concentration) at -80 o C, in aliquots.Molecular weight of engineered protein was confirmed by SDS PAGE and western blot analysis (Fig. 1C-D), which matches with the theoretical molecular weight of designed protein (~ 60 kDa; 548 amino acid residues).As can be seen, a good amount of active and substantially pure protein was obtained.Routinely, using above-mentioned protocol we were able to produce 2.0 ± 0.5 mg of FHA-3 protein per liter culture of this particular clone (yield value is an average of 5 independent batches).

Enzymatic activity assay
The specific activity of purified FHA-3 was found to be 916 ± 276 U/mg when Mn 2+ was used as a cofactor and the reaction was done in the buffer of pH 8.5.Similarly, a specific activity of 946 ± 296 U/mg was observed when Co 2+ was used as a cofactor and the reaction was done in the buffer of pH 7.5.It is important to note here that the specific activity (arginine-hydrolyzing) of 389-518 has been reported for unfused wild-type hArg 1 in the literature [31][32][33][34].Similarly, for different PEGylated and fusion variants of hArg 1, the specific activity reported in the literature ranges from 205 to 1219 U/mg [13,34,35].Another crucial finding is that when either Co 2+ or Mn 2+ was utilized as a cofactor, the activity of the FHA-3 variant was nearly similar.

In vitro plasma stability of FHA-3
In vitro plasma stability of FHA-3 protein in pooled human blood plasma was determined as described previously [25].with HepG2 cells, the tumors were allowed to develop, and then the specified samples were administered intraperitoneally for three weeks: 0.9% saline solution, 5-FU (10 mg/kg once a week), FHA-3 (300 U/mouse twice a week), and a combination of FHA-3 + 5-FU.After the completion of the treatment period, the animals were sacrificed and the tumors were excised and weighed.Treatment of animals with either FHA-3 or 5-FU has resulted in suppression of the rate of tumor growth.However, administration of a combination of the cell culture with FHA-3 resulted in a concentrationdependent killing of the cells.The results also indicate that among the three cancer cell lines tested, HepG2 cells are most sensitive to FHA-3 treatment.

Tumor xenograft
In vivo anticancer activity of FHA-3 was determined in the HCC xenograft mice model.Animals were implanted Fig. 3 In vitro anticancer activity of FHA-3 against cancer cell line.Cells were grown in 24-wells plates for 24 h and then challenged with increasing concentration of FHA-3 (0-50 U/ml) and incubated further for an additional 72 h.The viability of the cells was then determined using MTT assay and the data was used to calculate IC 50 values by the Hill slope method.Assays were performed in triplicate and repeated two times and the data are expressed as a mean ± SD Fig. 2 (A) In vitro plasma stability of FHA-3 protein.FHA-3 protein was mixed with human plasma and the mixture was incubated at 25 o C and the stability of exogenously added FHA-3 was studied by determining arginase activity (using 50 mM arginine as substrate).Intrinsic arginase activity of the pooled plasma in which FHA-3 enzyme was not added was taken as 100% (Bar A).Bar B and C denote plasma arginase activity after 0 min and 24 h after addition of FHA-3, respectively.Each data points represent mean ± SD of two independent experiments.(B) In vivo pharmacokinetic properties of FHA-3.SD rats (n = 6) were injected (i.v.) with 3 mg/kg FHA-3 per animal.
Blood samples were withdrawn at designated time-points and used to determine the amount of FHA-3 protein present in the plasma by using His-Tag ELISA detection kit.His-tagged protein standards of known concentration (kit reagent) and the corresponding absorbance values are used to plot a standard curve (inset) and the amount of Histagged protein present in the sample (plasma) was calculated.Average data of all animals was then used to determine the pharmacokinetic parameters by using a non-compartmental model approach.Error bar represents Mean ± SD Moreover, the FHA-3 + 5-FU combination treatment group exhibited a significant reduction in tumor volume relative to the control group as well as the individually drug-treated group.This reduction in tumor growth rate was observed after only one week of the treatment and a decrease in the tumor volume (compared to the control group) was sustained throughout the study.Similarly, at the termination of the study where tumors from each treatment group were excised and weighed, a considerable reduction in the final tumor weights of treated animals was observed (Fig. 4C).Specifically, the average tumor weight of the combination FHA-3 and 5-FU resulted in a significant inhibition in the tumor growth.A representative image of tumor-bearing animals at the end of the experiment is given in Fig. 4A.During the treatment period, the volume of the growing tumor was checked.In the control group (where only 0.9% saline solution was given) progressive tumor growth was observed throughout the experimental study (Fig. 4B).When the animals were treated with 5-FU (10 mg/kg once a week) or FHA-3 (300 U twice a week), substantial inhibitions in the growth rate of the tumor was observed.Interestingly, FHA-3 was more potent in suppressing tumor growth than 5-FU.both the compounds (FHA-3 + 5-FU).These results are in agreement with a previous study where arginase treatment has been shown to cause a high grade of necrosis in hepatocellular carcinoma (HCC) [12].However, compared to 5-FU or FHA-3 treatment alone, the FHA-3 + 5-FU combination treatment group displayed extensive necrosis (Fig. 5).

Discussion
Numerous cancerous cells were reports as arginine-auxotrophic, i.e., they either lack or produce low levels of an enzyme(s) involved in the synthesis of arginine and, thus depend on the body pool of arginine for their growth and maintenance.Thus, enzyme mediated depletion of arginine using rhArg I have been identified as a potential strategy for treating such auxotrophic cancers [2].
In this study, we have designed fused human arginase I (FHA-3) with improved PK properties, using fusion protein treatment groups was significantly lower than the control group.Further, the effect of treatments on the survival of HCC tumor-bearing mice was evaluated using the Kaplan-Meier method (Fig. 4D), as described previously [27,28].Compared to 5-FU or FHA-3 treatment alone, substantial protection from the tumor-related deaths was observed in the group treated with a combination of 5-FU with FHA-3.

Effect of FHA-3 treatment on tumor histoarchitecture
Animals were sacrificed and the excised tumor tissues were used for histopathology to determine their structural/morphological changes with different treatments.Widespread cellular proliferation was evident in the tumor tissue of control group animals.However, noticeable changes were observed in the tumor tissue of the treated group animals.Conspicuous (interstitial) spaces were noted in between the tumor cells of the treated group, signifying cytotoxicity of fragment is known to increase not only the overall hydrodynamic radius of the fused molecule (thereby decreasing its clearance from the kidney), but also increases the plasma recirculation time of the fused molecule by utilizing the FcRn-mediated recycling mechanism [18].Many HSA-(or its domain-) fused protein therapeutics are developed, and many more are in various developmental stages, that exhibit improved pharmacokinetic properties, compared to their unfused counterparts [18,[40][41][42].
Furthermore, administration of rhArg I has been shown to inhibit the growth of many arginine-auxotrophic cancers.Anti-cancer activity of FHA-3 was determined in both in vitro (using different cancer cell lines) and in vivo conditions (using the HCC xenograft mice model).In vitro anticancer activity determination of FHA-3 suggests that depletion of arginine in the medium by treating the cell culture with increasing concentration of FHA-3 results in a concentration-dependent killing of the cancer cells.Like other variants of rhArg I reported in the literature (unfused protein, PEGylated protein, or fusion protein), FHA-3 was found to be more effective towards HepG2 cells [9].In vivo anticancer efficacy determination of FHA-3 in HepG2-induced HCC xenograft mice model suggest that treatment of tumor-bearing animals with FHA-3 (300U; twice a week) reduced the growth of the tumor and protected the animals from tumor-related death more effectively than 5-FU treatment.Treatment of animals with a combination of 5-FU + FHA-3 was found to be more potent than either 5-FU or FHA-3 alone in reducing the growth and increasing the survival of tumorbearing animals.Moreover, histological data clearly indicate that highest extent of cytotoxic damage was observed in tumor tissue of animal treated with FHA-3 + 5-FU combination.Also, the results of the histology were in corroboration with the tumor morphometric parameters (tumor size, tumor weight and tumor volume), suggesting that arginase could act as a potential anticancer agent by synergizing the activity of other chemotherapeutic agents such as 5-fluorouracil.5-FU is a pyrimidine analog used as an anti-cancer drug to treat multiple solid tumors [43].This was expected and the results are in agreement with the literature reports where a combination of rhArg 1 with different chemotherapeutic agents has been shown to augment tumor regression rates [9,10,16].Literature suggests that the arginine depletion approach can be combined with other types of chemotherapeutic drugs in the treatment of a variety of arginine-auxotrophic cancers and some combinations have also undergone (and undergoing) clinical trials [40].Thus, it seems FHA-3 can be used in combination with other molecular targeting or cytotoxic anti-cancer agents (viz.chemotherapeutic, radiation therapy, monoclonal antibody, autophagy technology and a lab-scale (industrially feasible) process was developed to produce FHA-3 protein using P. pastoris expression system.P. pastoris expression system is widely used for the production of therapeutic proteins as the mechanism of protein expression in this system is very close to the ones used in mammalian cells.Moreover, compared with the prokaryotic expression system, this system offers significant advantages which include post-translational modifications of recombinant proteins, secretory expression of recombinant proteins, and generation of stable cell lines by insertion of a linearized foreign DNA in a chromosome of yeast via recombination [30].It is also proposed that P. pastoris can produce high yields of recombinant proteins with the similarity of glycosylation to the mammalian cells [31].
Enzymatic characterization of FHA-3 revealed two important observations: (i) the specific activity of FHA-3 variant is considerably higher than the specific activity reported for wild-type hArg I and, (ii) there are no major differences in the arginase activity of FHA variants when Mn 2+ (which is a native cofactor of the enzyme) is substituted with Co 2+ .In vitro plasma stability studies indicate that FHA-3 retained almost full (arginase) activity when incubated in human plasma for up to 24 h.We were not expecting these patterns as it is reported in the literature that the substitution of two Mn 2+ ions with Co 2+ leads to a significant increase in the affinity of the enzyme towards arginine substrate and also shifts the optimal pKa of the enzyme from 8.5 to 7.5 [8,12,36,38].It was claimed that the combination of these effects leads to ~ 10-fold increase in the catalytic activity of the Co 2+ -containing enzyme at physiological pH, as compared to its Mn 2+ -containing counterpart [12].Also, it is reported that, like FHA-3 used in this study, native human Arg I contains two Mn 2+ ions as cofactor and incubation of rhArg I in human serum results in a loss of these Mn 2+ ions leading to enzyme inactivation and shorter serum halflife [8,12,38,39].We do not know the reasons for the 'cofactor-ambiguity' shown by FHA-3 protein; however, we speculate that the fusion of rhArg I with HLEP (HSA fragment) might have modified the architecture (chemical environment) of the active site of the enzyme in a subtle way such that cofactor substitution has no major effect on the arginine-hydrolyzing activity of the fused enzyme.Also, such subtle modification of the active site in FHA-3 might have facilitated the retention of the Mn 2 + cofactor in the active site and prevented the enzyme from inactivation in plasma.We are in process of further investigating these interesting results.
Pharmacokinetic analysis of FHA-3 in rats suggests that it has an in vivo circulatory half-life of 29.6 h which is ~ 7-folds greater than that of Mn-rhArg I [8].FHA-3 protein contains the third domain of HSA as a half-life extension partner.Fusion of protein therapeutic with either HSA or its suffering and pain.Our approved protocol is following the guidelines of the Committee for the Purpose of Control and Supervision of Experiment on Animals (CPCSEA), Ministry of Social Justice and Environment, Government of India.Rules of CPCSEA are laid down as per ILAR (Institute of Laboratory Animal Resources, USA) guidelines and prior permission was sought from the Institutional Animal Ethics Committee (IAEC No. IAEC/21/50) for conducting the study.

Conclusions
Depletion of circulating arginine by administering argininehydrolyzing enzyme has emerged as a powerful approach to treat various diseases/conditions in which lowering of arginine concentration has been shown to provide therapeutic effects.Towards this, rhArg I is being developed as a potential 'broad-spectrum' therapeutic agent.However, the poor in vivo circulatory half-life of rhArg I is a major obstacle in the development of rhArg I for therapeutic use.In the present study, we have used the 'fusion' approach to improve the pharmacokinetic properties of the enzyme.rhArg I is genetically fused to HLEP (via peptide linker) to generate FHA variants.A lab-scale process is developed to produce 'lead' FHA variant and biological characterization of lead variant (FHA-3) revealed that this variant not only possesses improved pharmacokinetic properties but also exhibits anti-cancer activities.At this juncture, we propose that FHA-3 is a potential candidate (either alone or in combination with other agents) for the development of treatment of not only arginine-auxotrophic cancers but also for the other conditions in which lowering of arginine has been shown to provide therapeutic effects.

Fig. 1
Fig. 1 Panel A is cartoon of fused human arginase 1 used in this study.Panel B is a chromatogram showing the resolution of protein on and Ni-Sepharose 6 column.(Blue circled) and (Red circled) denote protein content (measured by Bradford's assay) and arginine-hydrolyzing activity (measured by using kit) of the eluted fractions, respectively.

Fig. 4
Fig. 4 In vivo anticancer activity of FHA-3 in HCC xenograft mice model.HepG2 cells (2 × 10 6 ) were injected subcutaneously into 3-4 weeks old BALB/c nude mice and the tumors were allowed to grow.When the size of tumors reached desired size, the animals were randomly divided into 4 groups (n = 7) and treated with 0.9% saline solution, 5-FU (10 mg/kg per week), FHA-3 (300 U/mouse twice a week) and a combination of FHA-3 + 5-FU, for three weeks.A representative image of animals taken at the end of experiment is given in Panel A. In Panel A, (1) 0.9% saline solution, (2) 10 mg/ml of 5-FU, (3) 300 U of FHA-3, and (4) combination of 300 U of FHA-3 + 10 mg/ ml of 5-FU.Tumor volume was measured every week during the

Fig. 5
Fig. 5 Effect of FHA-3 treatment on tumor histoarchitecture.Representative images (taken at 40X) of hematoxylin and eosin-stained tumor tissue excised from animals from different groups after a treatment period of 3 weeks