Elsevier

Journal of Controlled Release

Volume 158, Issue 1, 28 February 2012, Pages 171-179
Journal of Controlled Release

Strategies for optimizing the serum persistence of engineered human arginase I for cancer therapy

https://doi.org/10.1016/j.jconrel.2011.09.097Get rights and content

Abstract

Systemic l-arginine depletion following intravenous administration of l-arginine hydrolyzing enzymes has been shown to selectively impact tumors displaying urea cycle defects including a large fraction of hepatocellular carcinomas, metastatic melanomas and small cell lung carcinomas. However, the human arginases display poor serum stability (t1/2 = 4.8 h) whereas a bacterial arginine deiminase evaluated in phase II clinical trials was reported to be immunogenic, eliciting strong neutralizing antibody responses. Recently, we showed that substitution of the Mn2+ metal center in human Arginase I with Co2+ (Co-hArgI) results in an enzyme that displays 10-fold higher catalytic efficiency for L-Arg hydrolysis, 12–15 fold reduction in the IC50 towards a variety of malignant cell lines and, importantly a t1/2 = 22 h in serum. To investigate the utility of Co-hArgI for L-Arg depletion therapy in cancer we systematically investigated three strategies for enhancing the persistence of the enzyme in circulation: (i) site specific conjugation of Co-hArgI engineered with an accessible N-terminal Cys residue to 20 kDa PEG-maleimide (Co-hArgI-CPEG-20K); (ii) engineering of the homotrimeric Co-hArgI into a linked, monomeric 110 kDa polypeptide (Co-hArgI x3) and (iii) lysyl conjugation of 5 kDa PEG-N-hydroxysuccinimide (NHS) ester (Co-hArgI-KPEG-5K). Surprisingly, even though all three formulations resulted in proteins with a predicted hydrodynamic radius larger than the cut-off for renal filtration, only Co-hArgI amine conjugated to 5 kDa PEG remained in circulation for sufficiently long durations. Using Co-hArgI-KPEG-5K labeled with an end-terminal fluorescein for easy detection, we demonstrated that following intraperitoneal administration at 6 mg/kg weight, a well tolerated dose, the circulation t1/2 of the protein in Balb/c mice is 63 ± 10 h. Very low levels of serum L-Arg (< 5 μM) could be sustained for over 75 h after injection, representing a 9-fold increase in pharmacodynamic efficacy relative to similarly prepared Mn2+-containing hArgI conjugated to 5 kDa PEG-NHS ester (Mn-hArgI-KPEG-5K). The favorable pharmacokinetic and pharmacodynamic properties of Co-hArgI-KPEG-5K reported here, coupled with its human origin which should reduce the likelihood of adverse immune responses, make it a promising candidate for cancer therapy.

Introduction

Many tumors exhibit metabolic deficiencies in one or more amino acid biosynthesis or salvage pathways and as a result, are forced to rely upon uptake of these amino acids from the serum for growth. Systemic depletion of tumor-essential amino acid results in apoptosis of the malignant cells with minimal side effects to normal cells. Enzyme-mediated depletion of extracellular amino acids has the added benefit that it can target sensitive tumors even when those that are poorly vascularized or are sequestered in inaccessible locations such as CNS or testes. Thus unlike antibody drugs, the function of enzyme therapeutics is not dependent on tumor penetration.

A large number of hepatocellular carcinomas, melanomas, renal cell and prostate carcinomas [1], [2], [3] do not express the urea cycle enzyme, argininosuccinate synthase (ASS) and thus are sensitive to l-arginine (L-Arg) depletion. Recently, Cheng et al. [4] demonstrated that many HCC cells are deficient in ornithine transcarbamylase expression and thus also rely on external sources of L-Arg. Whereas non-malignant cells enter into quiescence (G0) when depleted of L-Arg and remain viable for several weeks, hepatocellular carcinomas experience cell cycle defects that lead to the re-initiation of DNA synthesis even though protein synthesis is inhibited, in turn resulting in major imbalances and rapid cell death [5], [6]. The selective toxicity of L-Arg depletion for HCC, melanoma and other urea cycle deficient cancer cells has been extensively demonstrated in vitro, in xenograft animal models and in clinical trials [1], [5], [7], [8].

Arginine deiminase (ADI) (EC 3.5.3.6) an enzyme found in prokaryotes and archea but not in mammals, catalyzes the efficient hydrolysis of L-Arg to l-Citrulline (L-Cit) plus ammonia [9]. The bacterial Mycoplasma arginini enzyme, a homodimer of two 48 kDa polypeptide chains, has been selected for clinical evaluation due to its high catalytic rate under physiological conditions [5]. The anti-tumor activity of ADI is likely related to the induction of apoptosis under conditions of amino acid stress, although additional mechanisms such as localized ammonia toxicity and inhibition of neovascularization may play a role [10]. Since ADI is cleared rapidly from circulation, conjugation to 20 kDa MW polyethylene glycol (PEG) was employed to generate a molecule suitable for clinical applications. However, meta-analysis of phase II clinical data indicated that ADI is immunogenic, leading to the generation of neutralizing antibodies that coincided with an increase in serum L-Arg to pre-treatment levels [11], [12]. This is not surprising since adverse antibody responses to heterologous enzymes have been reported in numerous instances [13], [14], [15].

Since human proteins are much less likely to be immunogenic than their heterologous counterparts, arginine hydrolyzing enzymes of human origin have also been considered for L-Arg depletion therapy [16]. Humans produce two Mn2+-dependent l-Arginase isozymes (EC 3.5.3.1) that catalyze the hydrolysis of L-Arg to urea and l-ornithine (L-Orn). The Arginase I (hArgI) gene, located on chromosome 6 (6q.23), is highly expressed in the cytosol of hepatocytes and functions in nitrogen removal in the final step of the urea cycle. The Arginase II gene, located on chromosome 14 (14q.24.1), is localized to the mitochondria in tissues such as kidney, brain, and skeletal muscle where it is thought to provide a supply of L-Orn for proline and polyamine biosynthesis [17]. In early clinical studies, administration of Mn2+-human arginase I (Mn-hArgI) by transhepatic arterial embolisation led to partial remission of HCC in several patients [18]. Unfortunately, under physiological conditions (pH 7.4 and serum L-Arg ~ 120 μM) Mn-hArgI has minimal activity due to a high pH optimum (~ 9.5), high KM (2.3 mM) and low stability (t1/2 in serum = 4.8 h) [19].

We recently reported that substitution of the Mn2+ cofactor with Co2+ in hArgI (Co-hArgI) markedly improves its pharmacological properties, namely: (i) lowers the pH optimum to 7.4; (ii) lowers the KM from 2.3 mM to 120 μM without an appreciable effect on kcat and (iii) increases the t1/2 in serum to ~ 22 h. The enhanced pharmacological properties of Co-hArgI translate into a 12–15 fold improvement in the killing of melanomas and hepatocellular carcinomas in vitro[19].

hArgI is a homotrimer with a subunit MW of ~35 kDa. Even though the M.W. of the trimer is 105 kDa and therefore larger than the limit for glomerular filtration, in mice, the enzyme is cleared from circulation within 30 min[20]. This may be due to the filtration of monomeric hArgI in equilibrium with trimer. Although sedimentation experiments did not reveal the formation of appreciable amounts of monomer at equilibrium, the continuous removal of the monomeric form in the kidneys could result in the gradual loss of the protein [21]. Cheng and coworkers reported that conjugation of multiple lysyl residues in hArgI to PEG 5 kDa, extended its circulation half-life (PD) in mice to 12 h[22]. More recently the same group reported that Mn-hArgI conjugated to PEG 5 kDa maintained low concentration of L-Arg for approximately 3 days in a single patient [23], [24].

Here we evaluated three strategies for conferring a long circulation half-life to the serum stable Co-hArgI form of arginase: (i) Site specific conjugation of Co-hArgI engineered with an accessible N-terminal Cys residue to 20 kDa PEG-maleimide, a process that necessitated dissociation of the trimer into monomers after reaction with 20 kDa PEG-maleimide by low pH size exclusion chromatography followed by reassembly of the PEGylated trimer. (ii) Engineering of a linked, monomeric 110 kDa Co-hArgI polypeptide which could be expressed at preparative levels in bacteria following genetic optimization (Co-hArgI x3). (iii) Amine conjugation with 5 or 10 kDa PEG-N-hydroxysuccinimide (NHS) ester (Co-hArgI-KPEG-5K or Co-hArgI-KPEG-10K). Even though the first two approaches resulted in near homogenous mono-PEGylated and homotrimeric Co-hArgI respectively, surprisingly, they failed to confer long circulation persistence in Balb/c mice. In contrast, Co-hArgI-KPEG-5K resulted in a circulation t1/2 of 63 ± 10 h. In Balb/c mice we determined that the optimal dose for sustained L-Arg depletion is 6–8 mg/kg. IP injection of Co-hArgI-KPEG-5K at 6 mg/kg weight was shown to deplete serum L-Arg to below the detection limits for 78 ± 10 h. The improved pharmacodynamics of this molecule correspond to a 9-fold longer serum persistence than an equivalent dose of the authentic, Mn2+-hArgI conjugated to 5 K PEG in an identical fashion [19]. Thus, Co-hArgI-KPEG-5K exhibits PK and PD characteristics suitable for clinical development.

Section snippets

Materials

DNA modifying enzymes and reagents were from NEB (Ipswich, MA), l-Arginine and various buffers and chemicals were obtained from Sigma (St. Louis, MO), various PEG materials were obtained from JenKem Technology USA (Allen, TX) and Nanoc Inc (New York, NY).

Construction of an hArgI-C gene for site-specific Cys-PEGylation

We constructed a gene (named hArgI-C) containing the previously described E. coli codon optimized gene of human Arginase I (hArgI) containing a codon for Cys on the third residue (N terminal sequence Met-Gly-Cys), followed by an N-terminal 6 x

Construction of high M.W. Co-hArgI variants

Earlier studies have revealed that bovine arginase is cleared from circulation in mice with a t1/2 of < 1 h and that increasing its hydrodynamic radius via amine conjugation to PEG resulted in markedly increased persistence in circulation (12 h) [20]. However, in contrast to reports by Cheng and coworkers [24] we had reported earlier that Mn-hArgI is rapidly deactivated in serum with a t1/2 of only 4.8 h and that this phenomenon is due to the chelation of the Mn2+ metal cofactor of the enzyme by

Discussion

Previously, non-PEGylated bovine arginase was reported to have a very short circulation half-life of < 1 h in the mouse [20]. Similarly, in a more recent report it was also found that even large doses of recombinant human hArgI could not fully deplete L-Arg in a rat model [38]. Thus, to capitalize on the high catalytic activity and serum stability of Co-hArgI it was necessary to formulate the enzyme in a manner that confers much longer circulation half-life and results in prolonged depletion of

Conclusion

Arginase is a promising chemotherapeutic agent for the treatment of L-Arg auxotrophic tumors. In this work we compared three different approaches for improving the pharmacokinetic and pharmacodynamic behavior of Co2+ substituted hArgI. Modification with PEG-5K NHS esters was shown to increase the retention of the enzyme in circulation by about 2 orders of magnitude. Moreover, in the mouse model Co-hArgI-KPEG-5K resulted in a 9-fold better pharmacodynamics, i.e. complete depletion of L-Arg in

Acknowledgements

The authors would like to acknowledge the graphical assistance of Dr. Jack Borrok. This project was supported by grants NIHRO1 CA 139059 and by the Cancer Prevention and Research Initiative of Texas (CPRIT) L.C. was supported by a fellowship from the Arnold & Mabel Beckman Foundation.

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