Arginine deiminase, a potential anti-tumor drug

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Cancer Letters 261 (2008) 1–11 www.elsevier.com/locate/canlet

Mini-review

Arginine deiminase, a potential anti-tumor drug Ye Ni a, Ulrich Schwaneberg b, Zhi-Hao Sun a

a,*

Laboratory of Biocatalysis, School of Biotechnology, Jiangnan University, The Key Laboratory of Industrial Biotechnology, Ministry of Education, 1800 Lihu Road, Wuxi 214122, PR China b School of Engineering and Science, Jacobs University Bremen, Campus Ring 8, 28759 Bremen, Germany Received 31 July 2007; received in revised form 21 November 2007; accepted 23 November 2007

Abstract Arginine deiminase (ADI; EC 3.5.3.6), an arginine-degrading enzyme, has been studied as a potential anti-tumor drug for the treatment of arginine-auxotrophic tumors, such as hepatocellular carcinomas (HCCs) and melanomas. Studies with human lymphatic leukemia cell lines further suggest that ADI is a potential anti-angiogenic agent and is effective in the treatment of leukemia. For instance ADI-PEG-20, patented by Pheonix Pharmacologic Inc., is currently in clinical trials for the treatment of HCC (Phase II/III) and melanoma (Phase I/II). This review summarizes results on recombinant expression, structural analysis, PEG (polyethylene glycerol) modification, in vivo anti-cancer activities, and clinical studies of ADI. Discussions on heterogeneous expression of ADI, directed evolution for improving enzymatic properties, and HSA-fusion for increased in vivo activity conclude this review.  2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Arginine deiminase; Arginine; Anti-tumor; Cancer; Drug

1. Introduction Arginine deiminase is a potential cancer therapy agent for the treatment of arginine-auxotrophic tumors, such as hepatocellular carcinomas and melanomas. The hepatocellular carcinoma (HCC) accounts annually, as one of the most common cancers worldwide, for approximately 1 million new cases. Between 1992 and 2002, the incidence rate (per 100,000 persons) of HCC in US is 8.6, and the mortality rate is as high as 6.5 due to the low responses of hepatoma to chemotherapeutic treat-

*

Corresponding author. Tel./fax: +86 510 85918252. E-mail address: [email protected] (Z.-H. Sun).

ments. Besides, HCC is often diagnosed at an advanced stage, when potentially curative surgical or local ablative therapies are not feasible. Consequently, the development of an efficacious approach to the therapy of HCC is required urgently [1,2]. Studies also indicate that ADI might be more potent for the therapy of acute lymphoblastic leukemia than L-asparaginase [3]. Its anti-angiogenic activity suggests that ADI could become a novel anti-cancer drug targeting the neovascularization-related tumors [4,5]. Current studies have been focused on its in vivo inhibitory effect towards HCCs, leukemia, melanomas, and human umbilical vein endothelial cells (HUVEC) [3,5,6], clinical trials for HCC (Phase III) and melanoma (Phase I/II) [2,6], and the PEG formulation of ADI to improve its efficacy

0304-3835/$ - see front matter  2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2007.11.038

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as a clinical drug, including serum half-life and antigenicity [1,7]. Recent reviews have been concentrated on the relationships between arginine catabolic enzymes and cancer therapy [8], intermediary metabolism of tumor cells and their sensitivity towards arginine deprivation [9], and evaluation of ADI-PEG-20 as a potential anti-cancer drug [2]. Here, we review recent progress in ADI’s anti-cancer activities, clinical studies, and reports on cloning, expression, structural analysis and chemical modification. This review ends with a discussion section focused on challenges in future studies on ADI, including heterogeneous expression, protein engineering to overcome insufficient properties, and formulation strategies for improved in vivo application of ADI. 2. ADI and its anti-tumor activity 2.1. ADIs: properties and origins Arginine deiminase catalyzes the first step of the arginine deiminase (ADI) pathway by hydrolyzing arginine to citrulline and ammonium. The ADI pathway comprises three enzymes, arginine deiminase (ADI; converts arginine to citrulline and ammonia), ornithine transcarbamylase (OTC; citrulline to ornithine and carbamoyl phosphate), and carbamate kinase (CK; carbamoyl phosphate to ammonia and CO2) (Fig. 1). One mole of ATP is yielded by phosphorylation of ADP for each mole of arginine degraded in the ADI pathway; arginine is therefore a major energy source in many microorganisms such as Mycoplasma, Pseudomonas, BacilArginin H2O ADI NH4+ Citrulline Pi OTC Ornithine

Carbamoyl phosphate ADP CK ATP NH3+CO2

Fig. 1. Arginine deiminase pathway.

lus, etc. [10]. Since F. Horn’s first report on ADI in Bacillus pyocyaneus in 1933 [11], several ADI genes have been identified, purified, and characterized from bacteria, archaea and some eukaryotes excluding mammalian cells. Prominent examples are from Pseudomonas [12–16], Mycoplasma [17– 20], Halobacterium [21], Lactobacillus [22], Lactococcus [23], and a few eukaryotes [24–26]. Table 1 shows the specific activities (from 5.4 to 140.3 IU/ mg) and pH optima (from 5.6 to 7.6) of various ADIs. In addition subunit structures of various ADIs were solved (such as ADIs from Mycoplasma arginini and Pseudomonas aeruginosa). 2.2. ADI: anti-tumor activity Amino acid-degrading enzymes such as L-asparaginase (EC 3.5.1.1) and arginase (EC 3.5.3.1) are known to inhibit the growth of some tumor cell lines by depleting extracellular supply of respective amino acids. Arginine is a nonessential amino acid for humans since it could be generated from citrulline via urea cycle enzymes argininosuccinate synthetase (ASS) and argininosuccinate lyase (AL) in normal cells and tissues. And an adequate supply of arginine is vital for normal cell growth. However, some human tumor cells do not express ASS and are therefore auxotrophic for arginine. As an argininedegrading enzyme, ADI has been demonstrated to inhibit the growth of arginine-auxotrophic tumors including melanomas and HCCs [1,2], and to induce cell cycle arrest and apoptosis of human leukemia cells. Additionally anti-angiogenic effect on endothelial cells have been reported due to ADI activity [4,5]. Anti-proliferative and anti-angiogenic activities make ADI a highly potential agent for cancer therapy. ADI isolated from Pseudomonas putida was first applied by Jones on murine leukemia lymphoblast as an anti-tumor agent in vitro, but its activity was not detectable in vivo [27]. Later, Takaku and coworkers successfully used ADI purified from Mycoplasma to inhibit growth of several murine and human tumor cell lines in vitro and in vivo [19,20,28,29]. Their results show that its Kmvalue for L-arginine (0.3 mM) is about 30 times lower than arginase. It is remarkable that an effective dose (5 ng/ml) of ADI is three magnitudes lower than that of arginase [20]. Gong and co-workers suspected first that ADI extracted from M. arginini might have anti-angiogenic effect beyond its anti-tumor activity. Their

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Table 1 Comparison of ADIs from different microorganisms Sources

Subunits

Mw (kDa)

Specific activity (IU/mg)

Optimum pH

Reference

Pseudomonas putida Halobacterium saliniarium Mycoplasma arginini Mycoplasma hominis Giardia intestinals Pseudomonas aeruginosa Lactococcus lactis ssp. lactis Pseudomonas plecoglossicida

Dimeric Homodimeric Homodimeric Homodimeric Homodimeric Tetrameric Homotrimeric N.A.

120 105 90 96 124 184 140 N.A.

58.8 5.4 (90 nkat/mg) 44.5 35 41 N.A. 140.3 0.476a

6.0 7.6 6.5 6.0 N.A. 5.6 7.2 6.5

[12,13] [21] [19] [20] [24] [14,15] [23] [16]

a

The PpADI was not purified to homogeneity, and activity was calculated using 0.476 IU/mg DCW (dry cell weight).

results showed that ADI inhibits growth of tumor and vascular endothelial cells by arresting cell cycle and inducing apoptosis [30]. Beloussow et al. were attracted by the anti-angiogenic effect of ADI and first demonstrated the in vitro inhibition effect of recombinant ADI on the migration and tube formation of HUVEC [4]. Wheatley et al. showed that the anti-proliferative activity of ADI was not its sole role as an anti-tumor agent. Their neovascularization studies with endothelial cells suggested that ADI significantly inhibits tube formation and the inhibitory effect is likely associated with NO suppression due to the depletion of arginine, which represents a precursor of NO [5]. However, no experimentally proven hypothesis has yet been given on the mechanism of ADI’s inhibition effect on tumor angiogenic. ADI is a potentially better therapeutic agent for the treatment of leukemia than L-asparaginase, which has been used for the treatment of acute leukemia for over 20 years. Ensor et al’s. work showed that ADI is highly specific for arginine without converting other amino acids [1]. L-Asparaginase degrades asparagine and glutamine, and some deleterious side effects were reported to be caused by the degradation of glutamine [31]. Gong et al’s. study with human lymphatic leukemia cell lines indicated that ADI is much more potent compared with L-asparaginase. ADI inhibited the growth of cultured leukemia cells at concentrations of 5–10 ng/ml, which were about 20–100 times lower than those of L-asparaginase. Arginine deprivation caused by ADI-catalyzed deimination of arginine in human serum, is speculated to be one mechanism of ADI’s inhibitory effect on human lymphatic leukemia [3]. Furthermore, an in vivo study showed that ADI treatment does not have serious side-effects such as anaphylactic shock, coagulopathies, and liver toxicity, which have been reported for L-asparaginase [3,32].

Arginine is further involved in several pathways for regulation and maintenance of cellular function, such as protein synthesis, NO production, polyamine synthesis, urea cycle. Its complex metabolic pathways require metabolic analysis when it comes to the arginine deprivation effect of ADI. In Wheatly’s recent review discussing the intermediary metabolism, it was concluded that intermediates of urea cycle would be a clear indicator for the sensitivity of cells to arginine deprivation. The latter is critical for deciding on an optimal treatment strategy for specific tumor [9]. Shen et al. also investigated recently the anti-tumor mechanism of recombinant ADI (rADI), in which arginine metabolic pathways was modulated by ASS activity or rADI. A main outcome of this study was that the compartmentalization of ADI is affecting arginine’s utilization in different pathways [33]. In conclusion, ADI is effective in the eradication or control of arginine-auxotrophic tumors such as melanomas and HCCs, in which an active citrulline to arginine recycling pathway is absent. Furthermore, it has been reported that ADI has inhibitory effects on the proliferation of human lymphatic leukemia cell lines. Although the anti-tumor mechanism of ADI has not yet been understood completely, studies show that the arginine-degradation action of ADI is closely related to the cell apoptotic death and several biological pathways such as urea cycle, NO generation, polyamine synthesis, which have significant influence on tumor cell growth and angiogenesis. 2.3. ADI in clinical studies In vivo studies proved that ADI injection was effective in inhibiting melanomas and HCCs in experimental mice [19]. However, the use of ADI as a routine anti-cancer agent is limited by its low

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efficiency which is caused by its strong antigenicity in mammals as a microbial-active enzyme and mainly by its short serum half-life (around 4 h in mice). Therefore large daily doses of 0.2 mg/mouse (i.e. 9 IU/mouse) for 14 days are required for native ADI to be efficacious in implanted mice [19]. By formulating ADI with PEG, Clark et al. reduced the dose to a weekly injection of 5 IU/mouse. The ADI-PEGs were prepared using PEGs with various size, structure, and linkers. Their studies (pharmacodynamics, pharmacokinetics, and immunogenicity) demonstrated that the conjugation of ADI with PEG of 20,000 Da resulted in an optimal formulation method for its application as a therapy agent. ADI-PEG-20 (ADI-ssPEG20,000) has the same effect on melanomas and HCCs in vitro as native ADI, whereas it is much more effective in vivo [1]. The half-life of PEGylated ADI increases from 4 h to 7 days in mice at a weekly dose of 5 IU, which will keep the serum arginine under the detectable level [7]. In March 1999 and April 1999, ADI-PEG-20, PEGylated recombinant mycoplasmal ADI was designated the US orphan drug status by FDA for the treatment of HCCs and invasive malignant melanomas. European Agency for the Evaluation of Medicinal Products (EMEA) also granted ADIPEG-20 orphan drug status for the treatment of HCC in July 2005. At present, ADI-PEG-20 is being developed by Pheonix Pharmacologic Inc. for the potential treatment of HCC and melanoma (with their respective trade name Hepacid and Melanocid). Clinical trials for the treatment of HCC (Phase III) and melanoma (Phase II) are ongoing in Italy, and for HCC (Phase II) and melanoma (Phase I) in the United States [2]. In dose-escalation Phase I/II studies with ADIPEG-20 (20–320 U/m2) for the treatment of patients with advanced or unresectable HCC and metastatic melanoma, promising results were observed. Four of 19 HCC patients are still alive after over 680 days

and over 50% of 28 melanoma patients receiving >160 U/m2 doses experienced partial regression of lesions. The efficacy of ADI-PEG-20 in Phase I/II trials show that it could potentially be used as an efficient anti-tumor agent [2,6]. The following side effects and inconsistency in clinical case studies remain however to be elucidated to validate ADI-PEG-20 as an anti-tumor drug. Side effects include: effect of elevated ammonia level, an immunogenicity problem of the microbial protein ADI, and an unstable OBD (optimal biological dose) level in different clinical trials [2]. 3. ADI’s cloning, expression, structure analysis, and chemical modifications 3.1. Cloning and expression of ADI Various ADI genes have been cloned and overexpressed in Escherichia coli strains to further understand their functions in arginine metabolism, cell growth, and biological activities including anti-tumor activity (Table 2). Burne et al. isolated ADI pathway genes from Streptococcus sanguis chromosomal DNA by generating a gene library with bacteriophage lambda and subsequent expression in E. coli. Mapping studies employing subcloned genes demonstrated that the ADI pathway genes, including ADI, OTC, and CK are tightly clustered on the S. sanguis chromosome [34]. The complete nucleotide sequence of ADI gene in M. arginini was first reported by Kondo et al. who used a phage library for gene isolation. The partial amino acid sequences of the purified ADI protein were determined and used for the isolation of ADI encoding gene [35]. Later in the same year, Ohno et al. also cloned and sequenced the ADI encoding gene in M. arginini. However, these two sequences had major differences at the C termini [18].

Table 2 Cloning and expression of ADIs from different microorganisms Sources

Specific activity (IU/mg protein)

Expression vector

Expression host

Reference

Streptococcus sanguis Mycoplasma arginini Giardia intestinals Mycoplasma arginini Pseudomonas aeruginosa Lactococcus lactis ssp. lactis

0.115 33.6 36.5 32.7 N.A. 140.3

pUC19 pMK2 pQE-30 pTTQ118 pET30.b pGEM-T Easy

E. E. E. E. E. E.

[34] [36] [37] [4] [38] [23]

coli coli coli coli coli BL21(DE3)pLysS coli BL21

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Misawa et al. established a high-level expression system for Mycoplasma ADI[36]. The ADI gene was cloned from M. arginini and five tryptophan codons (TGA, which codes for the stop codon in E. coli) were mutated before cloning into the expression vector. The recombinant M. arginini (MaADI) was expressed cytoplasmically as inclusion bodies in E. coli and refolded in active form by guanidine treatment. Renatured MaADI showed comparable anti-tumor activity in vitro and in vivo against two mouse tumor cell lines [36]. For the first time, Knodler et al. identified, cloned and over-expressed the ADI from a eukaryotic organism, Giardia intestinalis. The ADI coding gene was amplified from Giardia genomic DNA using degenerate oligonucleotides based on sequences derived from tryptic peptide sequences of the purified enzyme. The G. intestinalis ADI (GiADI) was successfully expressed in E. coli and the 6·His-tagged rADI was purified in a single step using chelation column chromatography [37]. Beloussow et al. reported the cloning of an ADI encoding gene from M. arginini genomic cDNA and its high level expression in E. coli. The arginine– sepharose purified recombinant M. arginini ADI (MaADI) has a molecular weight of 48 kDa and a specific activity of 37 U/mg. Further in vitro studies showed that rADI had anti-angiogenic activity on HUVEC [4]. Oudjama et al. cloned an ADI encoding gene arcA from P. aeruginosa and expressed it in E. coli. The crystal structure of the purified recombinant P. aeruginosa (PaADI) was further solved [38]. ADI encoding gene arcA from Lactococcus lactis ssp. lactis ATCC 7962 (LADI) has recently been cloned and expressed in E. coli BL21. The recombinant ADI was purified through anion exchange and gel filtration chromatography. The molecular weight of native LADI is around 140 kDa. The deduced amino acid sequence of LADI shows only 35% homology with MaADI. Despite low identity, the sequence alignment reveals that LADI shares a conserved substrate (arginine) binding site and water molecule interacting site with MaADI [23]. 3.2. Structure analysis In 1970s, Kakimoto et al. diffracted the first ADI crystals from P. putida through multiple-step chromatography followed by crystallization. The ADI has a calculated molecular weight of around

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120,000 Da and maximum absorbance at 278 nm [12,13]. Almost three decades later, the X-ray crystal structure of ADI from P. aeruginosa (PaADI) (PDB ID: 1RXX, 2ABR, 2ACI, 2A9G, 2AAF) [14,15,38,39] and M. arginini (MaADI) (PDB ID: 1S9R, 1LXY) [40] were determined and structure models have been established to understand the relationship between protein structure and its catalytic activity. Oudjama et al. over-expressed the ADI from P. aeruginosa in E. coli and the selenomethionyl (SeMet)-containing rADI was produced to help in structure solution. The purified SeMet enzyme was concentrated by ultra-filtration and crystallized by a sitting-drop vapour-diffusion method. The PaADI crystals exhibit tetragonal symmetry structure (P41212 or P43212) and its complete MAD (multiwavelength anomalous diffraction) data set was col˚ resolution with beamline BM30 at lected to 3.2 A ESRF [38]. Two years later, Galkin et al. determined the crystal structure of ADI from P. aeruginosa ˚ and (PaADI) by MAD to a resolution of 2.45 A developed a first molecular model using QUANTA software. The electron density map shows that SeMet-containing rADI consists of four protein molecules in the asymmetrical unit (A, B, C, D) with 1630 amino acid residues in total. The PaADI crystal has tetrameric structures which are comprised of molecules A and B, and molecules C and D, respectively (Fig. 2A). The overall folding of ADI exhibits a 5-fold bbab subunit in symmetrical barrel arrangement and an additional a-helix inserted between two bbab subunits. The typical catalytic triad Cys-His-Glu/Asp conserved in arginine modifying enzymes is preserved in PaADI, which comprises Cys406, His278, and Glu224 (Fig. 2A). The proposed substrate-binding model shows that the catalytic center contains abundant charged residues (e.g. Asp280, His405, Glu13, and Arg165). The ionpairing interactions between these residues contribute to the integrity and activity of ADI. In addition, Asp116, Asp227, Arg185, Arg401, His278, Glu118, and Glu224 are part of an extended charged network. The proposed catalytic mechanism suggests that the active site of PaADI is blocked by Arg401, and therefore a conformational transition is taken place upon the substrate binding. Based on the proposed reaction mechanism, Cys406 is as a key residue responsible for the nucleophilic attack on the guanidinium carbon atom of arginine in the

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Fig. 2. Structural comparison of ADIs from P. aeruginosa and M. arginini. The structures were visualized with the graphics program VMD [41]. (A) PaADI (PDB ID: 1RXX) and stereo view of its catalytic triad (C406-H278-E224). (B) MaADI (PDB ID: 1S9R) and stereo view of its catalytic triad (C398-H269-E213) with substrate L-arginine.

first step of the catalytic reaction, and His278 serves as a general base in the proton transfer chain [14]. Evidence for this substrate-mediated proton transfer mechanism is recently presented by Wang et al. through molecular dynamics and density functional studies [42]. Further kinetic structural studies of PaADI mutants were reported by Lu et al. The contributions of the conserved catalytic core in the guanidino-modifying enzyme superfamily (GMSF): Cys406, His278 and Asp166, neighbor residue Asp280, active site gating residue Arg401, arginine-binding residues Arg185, Arg234, Asn160, as well as hydrogen bond partner Glu224, were evaluated by site-directed mutagenesis for substrate

analogues, including Nx-methyl-Arg, Nx-aminoArg, and agmatine. Dramatic reductions in the PaADI turnover were observed when substratebinding residues were replaced, indicating that electrostatic interactions are important for high catalytic activity [15]. In 2004, the crystal structure of M. arginini ADI (MaADI), which shares 27% sequence identity with ˚ resolution PaADI, was determined at 1.6 and 2.0 A by Das et al. using multiple isomorphous replacement (MIR) method with two reaction intermediates. The MaADI crystal features a characteristic 5-fold pseudosymmetrical structure with typical bbab arrangement in each element. The overall structure of MaADI resembles a ‘‘clip-on-fan’’ with

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five blades, each corresponding to a repeated bbab element. The structure model indicates that the substrate-binding pocket is located at the center of the molecular, in which a solvent channel leads to the surface of MaADI. However, substantial conformational changes in MaADI may be required upon the reaction since shape and size of the channel would not allow the import/export of the substrate arginine and product citrulline. Similar to PaADI, MaADI harbours a catalytic triad of identical amino acids (Cys398-His269-Glu213) (Fig. 2B). A six-step reaction mechanism representing the chemical steps of MaADI has also been proposed based upon the crystal structure of its covalent complex with two key reaction intermediates. The mechanism involves the binding of L-arginine to the enzyme through polar interactions, followed by the removal of a proton from Cys398 to result in a tetrahedral intermediate via nucleophilic attack on guanidinium group Cf atom of arginine, the expulsion of ammonia, the protonation of His269/ Glu218 and the subsequent formation of a covalent link with Cf atom. The product L-citrulline is finally released upon the breakage of the covalent bond to Cys398 [40]. In summary, the crystal structures of PaADI and MaADI share the same catalytic triad (Cys-HisGlu) and both crystal structures form a characteristic 5-fold pseudosymmetrical barrel with bbab subunits. The proposed reaction mechanism of both ADIs suggests that Cys406 and Cys398 residues in the catalytic triad are responsible for the nucleophilic attack of their respective thiol group on the guanidino carbon (Cf). Simultaneously, His278 and His269 serve as proton acceptors during the formation of a tetrahedral carbon intermediate (Table 3). Table 3 Summary of crystal structures of PaADI and MaADI

Resolution ˚) (A Catalytic triad Nucleophilic residue Proton acceptor Overall structure Reference

PaADI (PDB ID: 1RXX)

MaADI (PDB ID: 1S9R)

2.45

1.6 and 2.0

Cys406-His278-Glu224 Cys406

Cys398His269-Glu213 Cys398

His278

His269

5-Fold pseudosymmetrical barrel with bbab subunit [14]

[40]

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3.3. Chemical modifications of ADI The in vivo application of ADI as an anti-cancer agent encounters two major challenges: strong antigenicity and short circulating half-life (around 4 h) in plasma. PEG is a well-known chemical modifier, which could be covalently linked to the amino groups of ADI. It has a number of advantages such as low antigenicity, low toxicity and extended circulating half-life and has been successfully used for the modification of three FDA approved PEGylated proteins: PEG-asparaginase, PEG-adenosine deaminase and PEG-interferon-a-2b. Formulation of ADI with PEG has been reported to enhance its potency as anti-tumor drug [1,2,6,7,29]. Takaku et al. modified ADI purified from M. arginini with PEG and studied its clearance time in mice plasma. The average molecular weight of the ADI-PEG was estimated to be around 400 kDa. The activity of the PEG modified ADI was 25.5 U/mg protein, about half of that of the native ADI. The degree of modification of the amino groups per molecule was determined to be 51%. The in vivo study showed that the plasma L-arginine disappeared within 5 min after 5 U of ADI-PEG was injected into mice, and the L-arginine level remained undetectable for over 8 days. Ten daily injections of 5 U of native enzyme were required to attain the same effects as for one PEGylated dose. These results indicate that PEGylated ADI has a significantly longer serum half-life and could therefore be a more promising anti-tumor drug than native ADI [29]. Other important properties such as toxicology and immunology of the PEGylated ADI were not evaluated in this study. Clark’s group systematically studied the properties and clinical effect of ADI-PEGylation. First, they studied the effect of different PEG formulation on the pharmacological properties of recombinant Mycoplasma hominis ADI. The result showed that the enzyme activity was not affected by the molecular weight of PEG when 8–12 moles of PEG were attached to one mole of ADI protein. Size, structure, and the linker of PEG affect the in vitro activity of ADI-PEGs to a minor extend (8–12 IU/mg) at similar degrees of PEGylation. However, the pharmacodynamics (pK) and pharmacokinetics (pD) of ADI-PEG were correspondingly increased when PEG size increased from 5 to 20 kDa. No further increase was observed for PEG sizes over 20 kDa. The data suggests that formulation of ADI with PEG of 20 kDa is an optimal PEGylation size for

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cancer therapy [7]. Later in the same year, the same group reported the inhibition effect of ADI-PEG-20 on human melanomas and HCCs in vitro and in vivo. Their result was consistent with what has been reported by Takaku et al. [29]. One dose of ADI-PEG-20 (5 U/mouse) reduced the plasma arginine below detectable levels for around 7 days. While the same dose of native ADI resulted in 50– 60% reduction in arginine level after 24 h of injection. After another 24 h the arginine concentration bounced back normal levels. In vivo studies with melanomas and HCC implanted mice showed that ADI-PEG-20 significantly increased their survival rate, in which over 50% of ADI-PEG-treated mice survived for 24 weeks and all ADI-treated mice died within 7 weeks [1]. Clark et al. have applied ADIPEG-20 in the Phase I/II studies on patients with unresectable HCC. The results showed that optimal biologic dose (160 U/m2) was sufficient to reduce the plasma arginine from 130 lmol/L to undetectable level (<2 lmol/L) for over 7 days. The therapeutic dose was well tolerated among all 19 patients enrolled. Ten of the patients had complete or partial response and the average survival period of 19 patients was 410 days [6]. 4. Challenges for ADI applications 4.1. Non-E. coli hosts for ADI expression So far, ADIs from different microorganisms have only been expressed in E. coli strains. Studies using expression hosts such as Bacillus subtilis (capable of secreting proteins), yeasts capable of post-translational modification, specifically methyltrophic Pichia pastoris and GRAS organism Saccharomyces cerevisiae, would be of interests to understand the influence of different host strains on enzymatic properties of rADI and response in treatments. Compared with the most-frequent used Gramnegative bacterium E. coli, Bacillus subtilis is considered as GRAS and free of endotoxin (LPS), which is pyrogenic in human and other mammals. Most importantly, the naturally highly secretory capacity of B. subtilis enables simplified downstream protein purification procedures. Over recent years, a number of protease deficient B. subtilis mutant strains (e.g. WB800), and promoter systems (e.g. vegI), have been developed to overcome its major problems as heterologous protein expression host: high level production of proteases and lack of genetic engineering tools [43,44]. Yeasts such as Pi. pastoris

and Saccharomyces cerevisiae are attractive expression hosts due to their powerful genetic engineering tools, high cell density cultivation protocols, and ability of post-translational modification. A variety of vectors and host strains with different genotypes are commercially available for the expression of different heterologous proteins in S. cerevisiae (e.g. strains INVSc1, YPH, and vectors pYES2, pYC2, pESC) and Pi. pastoris (e.g. strains SMD1168, GS115, KM71, and vectors pPIC9K, pHIL-D2, pPICZ). 4.2. Protein engineering of ADI for improved enzymatic properties In the past decade, directed evolution has been successfully used as powerful algorithm to design proteins with desired properties. A directed evolution experiment comprises iterative cycles of diversity generation and screening for improved protein variants [45]. Numerous enzymes, such as subtilisin E [46], b-lactamase [47], b-galactosidase [48], dioxygenase [49], and peroxidase [50] have been improved in organic solvents resistance, activity towards novel substrates, and thermal stability. Pseudomonas plecoglossicida CGMCC 2039 (16S rRNA GenBank Accession No. EF645247), a strain exhibits high ADI activity, was isolated from local soil samples, and ADI encoding gene from P. plecoglossicida CGMCC 2039 (GenBank Accession No. EU030267) was cloned and expressed in E. coli BL21(DE3). The Preliminary in vitro studies of PpADI (ADI from P. plecoglossicida) showed over 98% inhibition rate towards the HepG2 cell line without obvious side effect on normal cells [16]. However, PpADI has an acidic optimal pH of 6.5 and is reduced in activity under physiological pH conditions (50% residual activity at pH 7; less than 15% residual activity at pH 7.5), which represents an obstacle in ADI’s in vivo application. Similar acidic pH optimum were observed for ADIs from M. hominis [20] (50% residual activity at pH 7; 10% residual activity at pH 7.5), M. arginini [19,20], P. aeruginosa [14,15], P. putida (no detectable activity in vivo) [27] (Table 1). The acidic pH optimum of ADI is likely associated with the proposed reaction mechanism, in which protons are required for the deimination of L-arginine in the proton transfer chain [14,40]. Directed evolution approach has been proven to be effective in evolving pH stability. One such successful example is D-pantonohydrolase from Fusarium moniliforme, in which the enzyme activity

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under acidic pH 5.5 was successfully improved for 10.5-fold by combining two rounds of DNA shuffling with three rounds of error-prone PCR [51]. Similar directed evolution strategies could be applied for improving ADIs activity under physiological conditions. The structural models of MaADI and PaADI [14,15,38–40] provide further opportunities to increase turnover frequencies of arginine by site-directed mutagenesis of amino acids involved in arginine binding and/or orientation. 4.3. Modifications of PpADI through HSA fusion and PEGylation PEGylation, has been one of the most successful strategies for the chemical modification of pharmaceutical proteins and was first reported in the modification of albumin and catalase in 1970s [52,53]. Clinical Phase I/II studies with ADI-PEG-20 showed that PEGylated ADI have significantly prolonged serum half-life and are effective for patients with unresectable HCC and metastatic melanoma. However, limitations still exists in the use of PEG. First, the polydisperse property of the synthetic polymers PEG inevitably yields a population of drug conjugates, which might lead to different pharmacological properties, such as immunogenicity and body-residence time. Another possible side-effect is the accumulation of PEGs in liver at higher molecular weights (over 30 kDa), which might cause macromolecular syndrome [54]. Human serum albumin (HSA) is the most prevalent naturally occurring blood protein (at 40 g/L) in the circulation and has a molecular weight of 66.5 kDa. HSA has a long half-life in human body of over two weeks. Therefore it is an attractive fusion partner for desired pharmaceutical proteins and an alternative for PEGylation. The Albumin Fusion Technology is patented by Human Genome Sciences, Inc. (HGS) [55]. It has been successfully applied in the development of several pharmaceutical products (Albuferon-Alpha, Albugon, Albuleukin, Albutropin, and Albuferon-Beta) which have shown promising activity profiles in Phase I/II clinical trials and an elongated half-life. Clinical studies of albumin-fusions indicated advantages of HSAfusion proteins in terms of safety, efficacy, and convenience [56]. Consequently, the construction of HSA-ADI fusion protein will potentially provide a promising strategy for improving the in vivo antitumor activity of ADI with fewer undesirable side effects. A HSA fusion to ADI has to our best knowl-

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edge not yet been reported. Applying these two strategies in modifying ADI will likely accelerate the development of ADI into a therapeutic protein for cancer treatment. 5. Conclusions The use of amino acid-depriving enzymes is an innovative strategy in therapy of specific auxotrophic tumors. L-Asparaginase is probably the bestknown example for its successful application in the treatment of acute leukemia. ADI, an argininedegrading enzyme, is in the clinical trials for the therapy of HCCs and melanomas, for which there is currently no efficacious treatment available. Progress in ADI research raises hopes to use of ADI as anti-leukemic agent. Besides, ADI’s anti-angiogenic activity suggests that it could become effective for the treatment of angiogenesis-dependent diseases, such as breast cancer. Compared with traditional chemotherapy, ADI has several advantages including high specificity for targeting malignant cells and low toxicity for patients. The progress in the ADI research will undoubtedly facilitate the pursuit of effective treatments to combat cancers and have profound influence on human health. Acknowledgements We thank Dr. Danilo Roccatano (Jacobs University Bremen) for generating the protein structural images in this review. The authors are grateful to the financial support for this research from the State Key Basic Research and Development Plan of China (No. 2003CB716008), and Program for Changjiang Scholars and Innovative Research Team in University (IRT0532). References [1] C.M. Ensor, F.W. Holtsberg, J.S. Bomalaski, M.A. Clark, Pegylated arginine deiminase (ADI-ssPEG20,000 mw) inhibits human melanomas and hepatocellular carcinomas in vitro and in vivo, Cancer Res. 62 (2002) 5443–5450. [2] L.J. Shen, W.C. Shen, Drug evaluation: ADI-PEG-20 – a PEGylated arginine deiminase for arginine-auxotrophic cancers, Curr. Opin. Mol. Ther. 8 (2006) 240–248. [3] H. Gong, F. Zo¨lzer, G. von Recklinghausen, W. Havers, L. Schweigerer, Arginine deiminase inhibits proliferation of human leukaemia cells more potently than asparginase by inducing cell cycle arrest and apoptosis, Leukemia 14 (2000) 826–829. [4] K. Beloussow, L. Wang, J. Wu, D. Ann, W.C. Shen, Recombinant arginine deiminase as a potential anti-angiogenic agent, Cancer Lett. 183 (2002) 55–162.

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