Human arginase I: a potential broad-spectrum anti-cancer agent

Abstract

With high rates of morbidity and mortality, cancer continues to pose a serious threat to public health on a global scale. Considering the discrepancies in metabolism between cancer and normal cells, metabolism-based anti-cancer biopharmaceuticals are gaining importance. Normal cells can synthesize arginine, but they can also take up extracellular arginine, making it a semi-essential amino acid. Arginine auxotrophy occurs when a cancer cell has abnormalities in the enzymes involved in arginine metabolism and relies primarily on extracellular arginine to support its biological functions. Taking advantage of arginine auxotrophy in cancer cells, arginine deprivation, which can be induced by introducing recombinant human arginase I (rhArg I), is being developed as a broad-spectrum anti-cancer therapy. This has led to the development of various rhArg I variants, which have shown remarkable anti-cancer activity. This article discusses the importance of arginine auxotrophy in cancer and different arginine-hydrolyzing enzymes that are in various stages of clinical development and reviews the need for a novel rhArg I that mitigates the limitations of the existing therapies. Further, we have also analyzed the necessity as well as the significance of using rhArg I to treat various arginine-auxotrophic cancers while considering the importance of their genetic profiles, particularly urea cycle enzymes.

Introduction

In spite of recent advances in the therapeutic field, cancer remains a severe hazard that challenges the progress and development made thus far. According to the World Health Organization (WHO), cancer is the second leading cause of death globally, with an estimated 10 million deaths reported (Cancer-WHO 2021). In India, around 2.25 million people are living with different types of cancer, and every year approximately 1.2 million new cases are being registered (Cancer Statistics-India Against Cancer 2020). Cancer incidence and fatalities are estimated to grow to 29.5 and 16.3 million, respectively, by 2040 (Kulothungan et al. 2022). Cancer refers to a vast range of disorders that can affect any region of the body, defined by uncontrolled proliferation, invasion, and metastasis as a result of a multistep process that results in the buildup of multiple genetic abnormalities (Gupta and Massagué 2006). There are several methods and medications available to treat cancer, with many more under investigation. Some therapies are “local,” such as radiation therapy and surgery, and are used to treat a specific tumour or body part. Because drug therapies such as chemotherapy, targeted therapy, and immunotherapy may influence the entire body, they are commonly referred to as “systemic” treatments. For certain cancers, a single form of treatment may be appropriate, whereas for others optimum combination therapy may be required (Hellman and Vokes 1996; Society AC 2022). These treatments, while useful, have their own set of drawbacks. Chemotherapy, for example, has little selectivity between normal and cancer cells and causes severe toxicity (Padma 2015). Targeted treatment using a monoclonal antibody or a small molecule inhibitor to target one or more receptors in cancer cells is also less effective in cancers that are weakly vascularized or located in inaccessible areas (Zhou et al. 2014). Thus, these problems have driven the quest for a safe and broad-spectrum agent(s) to effectively combat the global cancer threat. Arginine depletion by administering arginine-hydrolyzing enzymes has been shown to inhibit the growth and progression of various arginine-auxotrophic cancers with lower expression of urea cycle enzymes.

Thus, this article discusses the importance of arginine auxotrophy in cancer and different arginine-hydrolyzing enzymes that are in various stages of their clinical development and reviews the need for a novel rhArg I that mitigates the limitations of the existing therapies. Further, we have also analyzed the necessity as well as the significance of using rhArg I to treat various arginine-auxotrophic cancers, while considering the importance of their genetic profiles, particularly urea cycle enzymes.

Auxotrophy in cancer

In addition to being involved in the synthesis of peptides and proteins, amino acids are known to regulate crucial cellular processes in both normal and cancerous cells (Andersen et al. 2014). To ensure that cellular activities are not disrupted, the physiological level of amino acids must be maintained in the body (Patil et al. 2016). Cancer cells require additional nutrients than healthy cells due to their rapid growth and multiplication. Furthermore, some cancer cells become incapable of synthesizing one or more amino acids (Fernandes et al. 2017). To continue their growth and metabolism, cancer cells, therefore, become dependent on exogenous amino acid sources, or ‘auxotrophic’ for such amino acids. Since such auxotrophic cancer cells are destroyed when the availability of amino acids is limited, studies are now being conducted to develop amino acid deprivation therapy (Wang et al. 2021). Also, normal cells are unaffected in such conditions since they are less demanding and can synthesize these amino acids (Fernandes et al. 2017). For example, children with acute lymphoblastic leukaemia (ALL) have been effectively treated with the l-asparaginase enzyme, which serves as the rationale for amino acid depletion for cancer therapy (Pieters et al. 2011; Phillips et al. 2013; Lomelino et al. 2017). 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 phases of their development (Table 1) (Jeon et al. 2016; Wang et al. 2021).

Table 1.

Amino acid targets in amino acid deprivation therapy

Targeted amino acid	Enzyme used for amino acid depletion	References
Arginine	Arginase 1, arginine deiminase	Dillon et al. (2004), Bowles et al. (2008)
Glutamine	Glutaminase	Jiang et al. (2019)
Methionine	Methioninase	Hu and Cheung (2009)
Threonine	Threonine deaminase	Greenfield and Wellner (1977)
Asparagine	Asparaginase	Egler et al. (2016)
Lysine	Lysine-α-oxidase	Lukasheva et al. (2021)

Arginine auxotrophy

Arginine is a cationic, semi-essential amino acid that is required for the synthesis of amino acids such as proline and glutamate, as well as polyamines, urea, and agmatine (Wu and Morris 1998; Anakha et al. 2022). It has immunomodulatory properties like T cell receptor expression, formation of immunological memory, required for the production of nitric oxide (NO) through inducible nitric oxide synthase (NOS), which results in vasodilation, promotes the production of growth hormones, ammonia detoxification, and aids in wound healing (Alba-Roth et al. 1988; Bronte and Zanovello 2005; Stechmiller et al. 2005; Luiking et al. 2012). The enzymes argininosuccinate synthetase (ASS1) and argininosuccinate lyase (ASL) are responsible for converting citrulline to arginine. ASS1 converts citrulline and aspartic acid to argininosuccinate, which is then converted to arginine and fumaric acid by ASL. Arginase I then hydrolyses arginine into ornithine and urea and ornithine transcarbamylase (OTC) acts on ornithine and converts it to citrulline (Fig. 1) (Feun et al. 2008). ASS1 and/or ASL and/or OTC gene deficiencies or low levels of expression result in arginine auxotrophy, which makes such cancer cells entirely reliant on extracellular arginine for survival and maintenance (Tsai et al. 2009; Delage et al. 2010). The transcriptional silencing (hypermethylation) of OTC and ASS1 genes are thought to be the cause of their downregulation in cancer cells (Delage et al. 2012). Normal cells enter the G₀/G₁ phase of the cell cycle, become quiescent and survive for several weeks when arginine levels fall, whereas cancer cells continue to go through the cell cycle despite the lack of arginine, resulting in a significant imbalance and cell death (Fung and Chan 2017; Chen et al. 2021). Thus, arginine auxotrophy renders these cancers amenable to arginine-degrading enzyme therapy (Fig. 1).

Fig. 1.

The expression of ASS1 and OTC in normal cells ensures a steady supply of arginine through the catalysis of citrulline and ornithine, respectively. However, the loss of ASS1 and OTC expression causes arginine metabolism to be dysfunctional in arginine-auxotrophic cancer. ADI-PEG 20 and rhArg I deplete extracellular arginine to citrulline and ornithine, respectively, which cause the apoptosis and autophagy of the cancer cells (the figure was created using free medical images available from Servier Medical Art at: smart.servier.com)

Enzymes such as human arginase I (hArg I), NOS, arginine deiminase (ADI) and arginine decarboxylase were explored for arginine-auxotrophic cancer therapy and hArg I and ADI are found to be potential drug candidates (Fultang et al. 2016; Patil et al. 2016). For a variety of reasons, other enzymes were found to be not suitable. Arginine decarboxylase, for example, can efficiently metabolize arginine, but is hazardous to human cells as it generates agmatine and CO₂ (Stasyk et al. 2015). ADI metabolizes arginine to ammonia, and citrulline and is a promising anti-cancer agent (Kozai et al. 2009) (Fig. 1). ADI-PEG 20 (pegargiminase), is a US FDA-approved engineered arginine deiminase molecule, developed by Polaris pharmaceuticals, USA, with reduced immunogenicity and enhanced plasma stability (compared to the native enzyme) and is being used for the treatment of hepatocellular carcinoma (HCC) as well as malignant melanomas (Tsai et al. 2009; Dhankhar et al. 2018; Drugbank online-Accession Number-DB06592). ADI-PEG 20 is currently being developed for a range of arginine-auxotrophic cancers including pancreatic cancer, AML, non-small cell lung cancer, mesothelioma, melanoma, etc., and to date, certain phase I and phase II studies have had promising results! However, there are several major limits to using ADI-PEG 20, which includes the generation of a high level of ammonia that has been linked to hyperammonaemia, which causes neurological damage as well as neutropenia in many patients on ADI-PEG 20 treatment (Yoon et al. 2013). Strong neutralizing antibody responses resulting in anaphylactic events in recipients have also been observed due to the microbial origin of the ADI enzyme (Fung and Chan 2017). In light of these limitations of ADI-PEG 20, recombinant hArg I (rhArg I) has emerged as a potential candidate for the development of a broad-spectrum anti-cancer drug for arginine-auxotrophic cancers.

Human arginase I (hArg I)

hArg I (EC 3.5.3.1) is a homotrimeric, manganese (Mn²⁺) metalloenzyme (Mol. wt. ~105 kDa) that catalyzes the hydrolysis of arginine to form urea and ornithine (Stone et al. 2010). Mn²⁺, which acts as a cofactor, and contributes to catalysis by generating a metal-bound hydroxide ion (OH⁻) from a water molecule that serves as a nucleophile attacking the guanidium carbon of arginine (Romero et al. 2012). Humans have two isoforms of the arginase enzyme: arginase I (Arg I), which is mostly found in hepatocytes, and arginase II (Arg II) present in non-hepatic organs (Srivastava and Ratha 2013). Both isoforms have ~60% amino acid sequence similarity, although they differ in gene location and functionality. The Arg I gene is found on chromosome 6 (6q.23) in humans, and its main physiological role is in the urea cycle. The Arg II gene, on the other hand, is found on chromosome 14 (14q.24.1) and is involved in cellular development and proliferation through proline and polyamine synthesis (Ash 2004; Romero et al. 2012). It has been demonstrated that Arg I knock-out animals exhibit lethal phenotypes, whereas Arg II knock-out animals show minimal physiological repercussions (Fultang et al. 2016). Furthermore, hyperargininaemia is a rare arginase I deficiency disorder in humans, caused by the formation of shortened protein due to a point mutation (R291X) in its gene, which leads to stunted development and hyperammonaemia (Lavulo et al. 2001). As discussed above, arginine auxotrophy is identified as a major characteristic of human malignancies, which has been discovered in a wide range of advanced solid tumours, including prostate carcinoma, HCC, malignant melanoma, pancreatic carcinoma, breast cancer, and many more (Dillon et al. 2004; Bowles et al. 2008; Kim et al. 2009a, b). This metabolic anomaly is also seen in acute myeloid leukaemia (AML), as well as lymphomas including Hodgkin’s lymphoma and non-Hodgkin’s lymphoma (Delage et al. 2012; Miraki-Moud et al. 2015). Arginine depletion by administering hArg I enzyme has been shown to inhibit the growth and progression of such arginine-auxotrophic cancers with lower expression of OTC and/or ASS1 enzymes (Cheng et al. 2007; Lam et al. 2010). However, at physiological pH, the native form of hArg I is less efficient since it has an optimum pH of 9.6. Moreover, it has a K_m of 10.5 mM for arginine, indicating that a large quantity of the enzyme is required to obtain the desired outcomes. Also, the circulatory half-life of hArg1 is ~30 min, which contributes to its poor pharmacokinetic and pharmacodynamic characteristics. This limits the usefulness of this enzyme in its native form, necessitating the quick development of modifications to overcome these constraints (Patil et al. 2016; Anakha et al. 2022). To address this issue, PEGylated- and fusion-rhArg I molecules are being developed by pharmaceutical firms and are under various stages of their clinical development.

BCT-100, developed by Bio-cancer Treatment International Limited, Hong Kong, is a PEGylated (PEG5000) recombinant human arginase I (rhArg I) with improved pharmacokinetic properties (in vivo half-life of ~3 days) and enhanced arginine-hydrolyzing activity (Table 2) (Cheng et al. 2007). Weekly administration of BCT-100 (1600 U/kg) depletes arginine and has a favourable toxicity profile, according to a clinical study (Yau et al. 2015). BCT-100 is now being tested in phase II/III clinical studies for AML as well as liver cancer (Bio Cancer 2022). Nevertheless, there have been reports showing that BCT-100 has reduced catalytic activity and stability in serum at physiological pH (Georgiou and Stone 2013, 2014).

Table 2.

Recombinant human arginase I (rhArg I) under clinical development

Therapeutic	Developed by	Modifying modality	Development stage	Condition or disease	References
BCT-100	Bio-cancer Treatment International Limited, Hong Kong	PEGylation (multiple and random)	Phase III	Hepatocellular carcinoma, leukaemia, lymphoma, melanoma, prostate cancer, paediatric AML	ClinicalTrials.gov Identifier: NCT00988195, NCT01551628, NCT02089633, NCT02285101, NCT02089763, NCT03455140
Pegzilarginase (AEB1102)	Aeglea BioTherapeutics, USA	PEGylation (multiple and random)	Phase II	Arginase I deficiency or hyperargininaemia, small-cell lung cancer	ClinicalTrials.gov Identifier: NCT03378531, NCT03921541, NCT05676853, NCT03371979
Pegtomarginase (PT01)	Athenex, Inc. USA	PEGylation (single and specific)	Phase I	Advanced solid malignancies	ClinicalTrials.gov Identifier: NCT04136834
N-ABD094-rhArg	Hong Kong Polytechnic University	Fusion protein (ABD as HLEP)	Pre-clinical	Obese-related metabolic disorders	Leung and Shum (2020)
LBURGINAZE (EHA-3)	National Institute of Pharmaceutical Education and Research (NIPER), Mohali, India	Fusion protein (HSA fragment as HLEP)	Pre-clinical	Liver### cancer	Pande et al. (2022)

ABD albumin-binding domain; AML acute myeloid leukaemia; HLEP half-life extension partner; HSA human serum albumin; PEG polyethylene glycol; EHA-3 engineered human arginase-3

Pegzilarginase (AEB1102) is another PEGylated rhArg I developed by Aeglea BioTherapeutics, USA, with improved plasma stability and arginine-hydrolyzing activity due to the replacement of Mn²⁺ present in the active site of the native form with Co²⁺ (Table 2) (Glazer et al.2011). This molecule, which is also known as HuArgI (Co)-PEG5000, has proved to be beneficial in decreasing plasma arginine levels in mouse models of arginase deficiency and shows considerable inhibition of HCC and pancreatic cancer xenografts in animal studies (Mauldin et al. 2012; Burrage et al. 2015). It was approved by the US FDA in 2019 and granted the designation of breakthrough therapy for the treatment of arginase I deficiency or hyperarginaemia. Aeglea BioTherapeutics and Immedica recently signed a marketing partnership for pegzilarginase for hyperarginaemia in Europe and the Middle East (Aeglea 2022).

Pegtomarginase (PT01), developed by Athenex, Inc. USA, is a linear PEGylated form of rhArg I with two cysteines changed to serines at positions 168 and 303, leaving a single cysteine at position 45 and allowing site-specific PEGylation (PEG 20000) via maleimide–thiol conjugation (Table 2) (Yu et al. 2020). In mice xenograft models of prostate cancer and pancreatic cancer, preclinical studies with pegtomarginase revealed predictable pharmacodynamic behaviour (Athenex 2022). It is now being tested in phase I clinical study for the treatment of advanced solid tumours (ClinicalTrials.gov Identifier: NCT04136834).

Despite the fact that PEGylated protein therapies have shown to be effective and have been in clinical use for a long time, it is now recognized that they too have a number of drawbacks. As PEG is a non-biodegradable polymer, it causes vacuolization in the hepatic, renal as well splenic regions of the body (Gaberc-Porekar et al. 2008). Another severe concern of PEGylated protein therapeutics are its unexpected characteristics, such as lower activity, heterogeneity and increased aggregation in some cases. It is also reported that the efficacy of PEGylated therapeutics has been reduced due to the formation of antibodies against them, which causes them to be removed quickly (Langenheim and Chen 2009; Fee and Alstine 2011; Li et al. 2013; Haeckel et al. 2016). Because of these drawbacks, 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 domain of another protein, has emerged as a potent method for improving therapeutic protein pharmacokinetic features (Kontermann 2011). Protein clearance processes like receptor-mediated endocytosis, proteolysis and hepatic and renal clearance are being used to develop fusion proteins (Kintzing et al. 2016). 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 (Zaman et al.2019). Human transferrin (Strohl 2015), human IgG Fc domain (Sockolosky et al. 2012) and human serum albumin (HSA)-full length or domain (Dennis et al. 2002) 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 (Chen et al. 2013). N-ABD094-rhArg is a rhArg I fusion protein with an albumin-binding domain developed by Hong Kong Polytechnic University for the treatment of obese-related disorders, which has shown to be effective in xenograft models with increased circulatory life (~7 days) and pharmacokinetic properties (Table 2) (Leung and Shum 2020). We have also developed hArg I variants using the fusion technique to treat arginine-auxotrophic cancers, in which the hArg I enzyme is linked via a peptide linker to a variety of HLEPs (Pande et al. 2022). The ‘lead’ engineered human arginase I (EHA-3 or LBURGINAZE), which has the third domain of HSA serves as the HLEP, in conjunction with 5-fluorouracil shows enhanced in vivo circulatory half-life and has considerable anti-cancer effectiveness in HCC xenograft model, indicating its potential utility as an anti-cancer agent (Table 2) (Pande et al. 2022).

ASS1 and/or OTC expression in arginine-auxotrophic cancer

Arginine depletion by enzymatic approaches is now being used/explored to treat a variety of malignancies. However, drug resistance induction was found to be a significant barrier to employing arginine deprivation-inducing therapeutics (Haines et al. 2011). As discussed above, cells meet their arginine requirements in physiological conditions by taking it directly from the circulation or through its biosynthesis (catalyzed by urea cycle enzymes). Arg I enzyme catalyze the conversion of arginine to urea and ornithine, which is then converted to citrulline by OTC. The two-step conversion of citrulline to arginosuccinate and then to arginine is mediated by ASS1 and ASL (Feun et al. 2008) (Fig. 1). As a result, the efficiency of arginine deprivation therapy using any arginine-depleting enzyme is reliant on these recycling enzymes. It is reported that ASS1 acts as a tumour suppressor gene in addition to its enzymatic activity. ASS1 deficiency was found in approximately half of all nasopharyngeal cancer patients; it was found to be highly associated with advanced tumour status and is a potential biomarker of poor disease-free survival (Lan et al. 2014; Huang et al. 2021). Patients with bladder cancer as well as myxofibrosarcoma also show similar features. ASS1 epigenetic silencing has also been shown to increase tumour cell proliferation and migration in bladder cancer, demonstrating that ASS1 can act as a tumour suppressor gene (Huang et al. 2013; Allen et al. 2014). Similarly, reduced or deficient expression of OTC enzyme in HCC, prostate cancer and melanoma have been shown to increase their sensitivity toward rhArg I-induced growth inhibition (Cheng et al. 2007; Lam et al. 2010; Hsueh et al. 2012). These findings suggest that any arginine-degrading enzyme can be used in clinical settings depending on the patient’s genetic profiles, particularly, ASS1 and/or OTC expression. In this context, many in vitro studies have been conducted and it has been observed that cancerous cells with ASL and/or ASS1 deficits are more susceptible to ADI-PEG20 treatment, whereas those with ASS1 and/or OTC impairments are more responsive to rhArg I therapeutics (Hsueh et al. 2012; Lam et al. 2010; Allen et al. 2014; Qiu et al. 2015). Interestingly, more than 25 arginine-auxotrophic tumours have been reported so far; however, around 9 of them only had more than 60% ASS1 and/or OTC deficiency in immunohistochemical (IHC) studies (Table 3).

Table 3.

ASS1 and/or OTC deficiency in different cancer using immunohistochemical studies

Arginine-auxotrophic cancer	Ratio of patients with minimal/deficit expression of ASS1 and/or OTC enzymes with total enrolled patients	Percentage of ASS1 and/or OTC deficiency	References
Hepatocellular carcinoma	51/51-ASS1 30/42-OTC	100%-ASS1 71%-OTC	Dillon et al. (2004) He et al. (2019)
Prostate cancer	13/13-ASS1	100%	Dillon et al. (2004)
Hodgkin’s lymphoma	173/179; 50/50-ASS1	98%	Delage et al. (2012)
Non-Hodgkin’s lymphoma	288/303-ASS1	95%	Delage et al. (2012)
Malignant melanoma	119/119; 75/102-ASS1	88%	Dillon et al. (2004); Khadeir et al. (2015)
Pancreatic cancer	41/47; 7/14-ASS1	69%	Bowles et al. (2008); Liu et al. (2014)
Breast cancer	95/149-ASS1	63.8%	Qiu et al. 2014
Malignant pleural mesothelioma	52/82-ASS1	63%	Szlosarek et al. (2006)
Osteosarcoma	39/62-ASS1	63%	Kobayashi et al. (2010)
Nasopharyngeal carcinoma	64/124-ASS1	51.6%	Lan et al. (2014)
Myxofibrosarcoma	44/90-ASS1	48.8%	Huang et al. (2013)
Small cell lung carcinoma	7/16-ASS1	43.7%	Kelly et al. (2011)
Ovarian cancer	23/54-ASS1	42.5%	Nicholson et al. (2009)
Bladder cancer	190/478-ASS1	39.7%	Allen et al. (2014)
Glioblastoma	8/22-ASS1	36%	Syed et al. (2013)
Renal cell carcinoma	6/21-ASS1	28.5%	Yoon et al. (2007)
Esophageal carcinoma	6/32-ASS1	18.7%	Lagarde et al. (2008)
Seminoma	2/12-ASS1	16.6%	Dillon et al. (2004)

The nine arginine-auxotrophic cancers with more than 60% ASS1 and/or OTC impairments in IHC studies are discussed here, along with the potential therapeutic role of arginine-hydrolyzing enzymes in their treatment.

Hepatocellular carcinoma

Hepatocellular carcinoma (HCC), also called malignant hepatoma, is a major type of primary liver cancer that accounts for around 75% of all liver cancer cases (Petrick and McGlynn 2019; Dasgupta et al. 2020). With an estimated 905,677 new cases and 830,180 deaths in 2020, HCC is the sixth most often diagnosed cancer and the second major cause of cancer mortality globally (Globocan World 2020). In India, HCC is the fourth leading cause of cancer deaths with approximately 66,279 deaths in 2020 (Fig. 2) (Globocan India 2020). The optimal therapy for HCC is a multidisciplinary strategy that takes into account the tumour stage, patient performance status, and liver function reserve. The short-term survival of HCC has improved in recent years due to major breakthroughs in locoregional as well as surgical treatments, but the recurring disease remains a serious concern, and this has fuelled the search for novel target agents (Fig. 3) (Raza and Sood 2014). Growth inhibition of the human hepatoma cell line (HLE) by the administration of 5 ng/ml mycoplasma-derived ADI revealed the possibility of arginine-deprivation therapy for HCC. It is also reported that the urea cycle is suppressed in most HCC cell lines, and free arginine levels are elevated 5- to 20-fold in animals with HCC (Moyer and Pitot 1974; Miyazaki et al. 1990). In addition, an IHC study of HCC biopsy samples obtained from 51 patients to determine ASS1 expression exhibited 100% ASS1 deficiency (Table 3) (Dillon et al. 2004). Interestingly, Feun et al. conducted a study on various HCC cell lines and found that the arginine auxotrophy of these cell lines were caused by a deficiency of ASS1 enzyme, which made them sensitive to mycoplasma-derived ADI (Feun and Savaraj 2006). As a result, ADI-PEG 20, or pegargiminase was developed and got certified as an ‘orphan drug’ by US-FDA and the European agency for HCC treatment (Polaris pharmaceuticals 2022). It is currently being studied in phase III clinical trials for advanced HCC (ClinicalTrials.gov Identifier: NCT01287585). Recently, IHC studies show a minimal or absent expression of OTC in 71% (30 out of 42) of HCC cell lines collected from patients suffering from advanced HCC, which indicates its functional role in HCC progression (Table 3) (He et al. 2019).

Fig. 2.

Arginine-auxotrophic cancer statistics, 2020 (the figure was created using Prism—GraphPad software)

Fig. 3.

Adverse effects of different treatments of cancer (the figure was created using free medical images available from Servier Medical Art at: smart.servier.com)

Based on these findings, arginine-hydrolyzing enzymes are also being studied alone as well as with combinations, for the treatment of HCC. BCT-100 completed phase II clinical trials and shows effective results in patients with advanced HCC, when it is administered alone (1600 U/kg/week in 16 patients) as well as in dose-escalated PACOX administration, which is a combination regimen of BCT-100 with capecitabine and oxaliplatin drugs (ClinicalTrials.gov Identifier: NCT01092091, NCT02089633). Furthermore, in vivo study using EHA-3 in combination with 5-fluorouracil in a mouse xenograft model of HCC (10 mg/kg/week) showed a significant reduction in the growth rate of the tumour, suggesting its tumour-suppressing effect (Pande et al. 2022). Hence, these findings imply that arginine depletion utilizing rhArg I variants could be a feasible strategy for the management of HCC.

Malignant melanoma

Malignant melanoma, a neoplasm of melanocytes, occurs due to genetic abnormalities in melanocytes present in the skin, genitals, mouth, ear, anal area, and so on (Tolleson 2005). It is considered the deadliest form of skin cancer, which makes up less than 5% of all skin cancer cases (Lee et al. 2012). Although it was long thought to be rare, the annual incidence has risen considerably in recent decades with an estimated 57,043 deaths and 324,635 new cases in 2020 (Globocan World 2020). When compared to the global statistics, India had a low incidence of melanoma, with 2296 fatalities and 3916 new cases, a 0.7% rise over the previous Indian statistic report (Fig. 2) (Globocan India 2018, 2020). The US-FDA has authorized many drugs for its treatment such as dacarbazine, interleukin-2, vemurafenib, etc. (Domingues et al. 2018). Apart from these, surgical excision, targeted therapy, radiation treatment, immunotherapy and/or photodynamic therapy may be used depending on the characteristics of the tumour (Batus et al. 2013; Miller et al. 2016; Van Zeijl et al. 2017). Despite the fact that these surgical techniques and treatments have made remarkable progress, there is still a pressing need to reduce the rising burden caused by their limitations such as lack of selectivity, reduced efficacy, and gastrointestinal and cutaneous toxicity (Fig. 3) (Widakowich et al. 2007; Li et al. 2010; Austin et al. 2017). Real-time quantitative PCR (qPCR) and IHC studies conducted in melanoma cell lines show ASS1 deficiency, and hence its susceptibility toward arginine-degrading enzymes (Table 3) (Dillon et al. 2004; Khadeir et al. 2015; Sugimura et al. 1992). Furthermore, a dose-escalation (40, 80, or 160 IU/m²) clinical trial with ADI-PEG 20 reveals 100% plasma arginine depletion in 30 out of 31 advanced melanoma patients in 8 days (ClinicalTrials.gov Identifier: NCT00520299). In this context, a phase I study employing ADI-PEG20 (36 mg/m²) in combination with ipilimumab (1 mg/kg) and nivolumab (240 mg/kg) is now being conducted in nine advanced uveal melanoma patients (ClinicalTrials.gov Identifier: NCT03922880). Apart from this, BCT-100 (2.7 mg/kg) is also in its phase I clinical trial using 22 patients with advanced melanoma (ClinicalTrials.gov Identifier: NCT02285101). Thus, the development of a safe and long-acting variant of rhArg I will be a milestone in the treatment of melanoma.

Prostate cancer

Prostate cancer, the most common cancer in males after the age of 50 years, is a condition in which nonlocalized tumours are potentially very dangerous and difficult to treat (Kim et al. 2009a, b). It is the second most prevalent cancer in males and the third most common cancer globally with an estimated 1,414,259 new cases and 375,304 fatalities (Globocan World 2020). Despite the fact that prostate cancer rates in India are lower than in Western nations, it is the fifth most common cancer in men, with an estimated 16,783 deaths and 34,540 new cases (Fig. 2) (Globocan India 2020; Mathur et al. 2020). Prostatectomy, chemotherapy, biphosphonate therapy, targeted therapy, external beam radiation therapy, brachytherapy, and immunotherapy are the major treatments available for prostate cancer (Prostate Cancer Treatment (PDQ®)–Patient Version 2022). However, these therapies have a number of drawbacks, including bladder incontinence, rectal ulcers and bleeding, erectile dysfunction, proctitis, and many more (Fig. 3) (Pearlstein et al. 2019). IHC analysis of 13 prostate tumour biopsy specimens revealed total ASS1 impairment, and paved the possibility for therapy with an arginine-degrading enzyme (Table 3) (Dillon et al. 2004). Moreover, the Polaris group completed a phase 1 clinical trial and concluded that weekly administration of ADI-PEG 20 to castration-resistant prostate cancer patients results in increased tumour cell apoptosis with manageable toxicity (ClinicalTrials.gov Identifier: NCT01497925). Later studies revealed that ADI therapy, or any arginine deprivation therapy, effectively kills ASS1-deficient prostate tumour cells by inducing the formation of mitochondrial reactive oxygen species (mitoROS), morphological changes in mitochondria and decreased synthesis of mitochondrial metabolites, all of which led to mitochondrial dysfunction (Changou et al. 2014; Cheng et al. 2018). Henceforth, these results suggested that arginine depletion employing an enzyme (rhArg I) that hydrolyses arginine is a promising treatment strategy for prostate cancer.

Hodgkin’s lymphoma and non-Hodgkin’s lymphoma

Lymphoma is a malignant haematologic cancer that originates in the lymph system, comes in almost 70 distinct varieties and is broadly classified into Hodgkin’s lymphoma (HL) and non-Hodgkin’s lymphoma (NHL) (Ng et al. 2011). HL most commonly affects young adults (20s–30s) having a high rate of cure using available treatments such as radiotherapy, steroid therapy, chemotherapy, and combined modality therapy (Shanbhag and Ambinder 2018). However, people who had these treatments for HL have a higher chance of getting other diseases or ailments later in life, according to the reports. The development of a second form of cancer such as breast cancer, AML, NHL, and lung cancer later in life is an unusual, but a highly significant adverse effect of HL therapy (Rathore and Kadin 2010; Shanbhag and Ambinder 2018). Infertility has also been reported in teens and adults treated with the ABVD (doxorubicin, bleomycin, vinblastine and dacarbazine) and BEACOPP (bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine and prednisone) chemotherapies, which are often used to treat HL (Amin et al. 2021). In addition to this, the prevalence of HL has been rising in recent years, with an estimated 23,376 annual deaths and representing around 10% of newly diagnosed lymphoma instances globally (Fig. 2) (Globocan World 2020).

NHL, on the other hand, is the ninth most common cancer in India and the twelfth most prevalent cancer globally in terms of new incidence and mortality (Fig. 2) (Globocan India 2020; Globocan World 2020). These lymphoproliferative tumours are found to be less predictable than HL, with a higher proclivity for spreading to extranodal sites. Approximately, a quarter of NHL cases occur in non-nodal sites, with the majority of these including both nodal and extranodal regions. NHL accounts for around 5% of head and neck cancers and has a wide variety of symptoms that are similar to HL (Armitage et al. 2017; Singh et al. 2020). Despite the availability of several therapies for NHL, such as chemotherapy, radiation and bone marrow transplant, it has been noted that similar to HL treatments, there is a greater chance of acquiring late effects (Armitage 1993). This includes left ventricular dysfunction in NHL patients treated with doxorubicin (dosage >200 mg/m²) (Haddy et al. 1998; Moser et al. 2006), high risk of acquiring secondary malignancies such as HCC, bladder cancer, glioblastoma, AML, melanoma, etc. (Travis et al. 1993; Hemminki et al. 2008), a higher chance of being permanently sterile in patients who had cyclophosphamide and radiation treatment in the pelvic area (Mudie et al. 2006) and the risk of developing osteoporosis (Fig. 3) (Westin et al. 2013).

Burkitt lymphoma, a type of NHL that requires arginine for proliferation, paved the way for arginine-degrading enzyme treatment for lymphomas (Osunkoya et al. 1970). The majority of lymphoma cell lines lack ASS1 and are vulnerable to mycoplasma-derived ADI (Delage et al. 2012). Also, in vitro rhArg I-treatment studies (0.02–2 IU/ml) in NHL cell lines revealed considerable cell growth inhibition due to G₁ phase arrest, as well as ASS1 and OTC deficiency as determined by western blot analysis (Zeng et al. 2013). The Polaris Group has completed a phase II clinical study with ADI-PEG 20 in 18 NHL patients who had previously failed systemic therapy (ClinicalTrials.gov Identifier: NCT01910025). Thus, these data suggest that arginine depletion using rhArg I could inhibit lymphoma proliferation.

Pancreatic cancer

The prevalence of pancreatic cancer (PC) has grown in recent years and is one of the leading causes of cancer-related death with a poor prognosis. With 1,414,259 new cases and 375,304 deaths worldwide, it is now the third most common cancer (Fig. 2) (Globocan World 2020). The majority of patients have no symptoms while their illness develops into advanced pancreatic metastasis, which explains the low 5-year survival rate of patients (2–9%) (Gillen et al. 2010; McGuigan et al. 2018). Available treatments include pancreatectomy, radiation therapy such as proton beam therapy and SBRT (Stereotactic body radiation therapy), immunotherapy and chemotherapy, with most regimens comprising capecitabine, nab-paclitaxel, oxaliplatin, 5-fluorouracil, leucovorin and others (McGuigan et al. 2018; Robatel and Schenk 2022). Most of these therapies are less effective and have a number of drawbacks, including peripheral neuropathy, a high risk of diabetes, hand–foot syndrome, and other complications (Fig. 3) (Kubota 2011; Robatel and Schenk 2022). As a result, novel therapies targeting the metabolic activity of tumour are becoming more appealing and are expected to aid pancreatic cancer patients in the future. IHC studies conducted on pancreatic biopsy specimens show an ASS1 deficiency rate of 69% (41/47), indicating the feasibility of arginine-depletion treatment employing enzymes (Table 3) (Bowles et al. 2008). Additionally, in a clinical trial conducted with 18 advanced pancreatic adenocarcinoma, patients were given ADI-PEG 20 (36 mg/m²) weekly for 3 weeks in combination with nab-paclitaxel (125 mg/m²) and gemcitabine (1000 mg/m²). When compared to the toxicity of the FDA-approved first-line therapy for stage 3 pancreatic adenocarcinoma, gemcitabine and nab-paclitaxel regimen, this phase 1/1B clinical trial shows moderate toxicity, providing the chance for a more effective pancreatic cancer treatment (ClinicalTrials.gov Identifier: NCT02101580; Lowery et al. 2017) (Fig. 3). Thus, the successful development of rhArg I variants will provide a potentially ground-breaking treatment regimen for pancreatic cancer, which will help the suffering patients.

Breast cancer

Breast cancer is the most frequent cancer worldwide and in India ranks first in terms of incidence and mortality rate (Fig. 2) (Globocan World 2020; Globocan India 2020). Based on the presence or absence of molecular markers human epidermal growth factor receptor 2 (HER2), progesterone receptors and oestrogen receptors, breast cancer is divided into three groups. Around 15% of all breast cancer patients have triple negative cancer, which lacks these three molecular markers, whereas ~70% of patients have HER2-negative or hormone receptor-positive cancer and the remaining ~15% have HER2-positive cancer (Waks and Winer 2018; Bhattacharyya et al. 2020). Mastectomy or lumpectomy, radiation therapy such as brachytherapy, intra-operative or external beam radiation, hormone therapy and chemotherapy regimen (doxorubicin, gemcitabine, cisplatin, capecitabine, cyclophosphamide, etc.) are some of the therapies available (Richie and Swanson 2003; Sharma et al. 2010). Despite the fact that 70–80% of people with breast cancer may be treated in the early stages with currently available medicines, advanced cancer with metastasis is considered incurable (Harbeck et al. 2019). Apart from that, a major drawback of most of these treatments is the substantial threat of recurrence (Bhattacharyya et al. 2020). Interestingly, ASS1 deficiency in cytoplasmic ASS1 staining was seen in roughly 64% of samples from breast cancer patients (Table 3) (Qiu et al. 2014). Moreover, ADI-PEG 20 (36 mg/m²) in combination with doxorubicin (240 mg/m²) was given weekly for around 4 months to nine patients with HER2-negative metastatic breast cancer and this phase I clinical trial revealed a satisfactory safety profile, as well as lower arginine levels in the blood (ClinicalTrials.gov Identifier: NCT01948843; Yao et al. 2022). Therefore, utilizing rhArg I to manage breast cancer will be a promising therapeutic approach.

Malignant pleural mesothelioma

Malignant pleural mesothelioma (MPM) is an incurable, deadly form of mesothelioma that is primarily caused by frequent exposure to asbestos. In spite of extensive asbestos manufacturing and supply constraints, MPM prevalence keeps rising, with 30,870 new cases globally (Fig. 2) (Globocan World 2020; Asciak et al. 2021). According to statistics, MPM patients have a short median survival time of 9–13 months, with fewer biomarkers and no treatment options. Because it has such a broad range of symptoms, diagnosing it can be difficult, making treatments such as oncoviral therapies, angiogenesis inhibitors, radiation therapy, and microRNA replacement ineffective (Nicolini et al. 2020). IHC studies demonstrated a 63% minimal or loss of ASS1 expression in biopsies collected from 82 MPM patients (Table 3) (Szlosarek et al. 2006). Furthermore, ~94% disease management rate was achieved in patients with non-epithelioid ASS1-deficient MPM subtypes by the administration of ADI-PEG 20 (18, 27 and 36 mg/m²) in combination with cisplatin (75 mg/m²) and pemetrexed (500 mg/m²) every 3 weeks (ClinicalTrials.gov Identifier: NCT02029690). ADI-PEG20 or placebo (36 mg/m²) coupled with the conventional dosage of cisplatin and pemetrexed for a period of 18 weeks is now being tested on 386 MPM patients in phase II/III study (ClinicalTrials.gov Identifier: NCT02709512; Szlosarek et al. 2017). These findings indicate that the treatment of MPM via arginine depletion using an enzyme is very effective.

Osteosarcoma

Osteosarcoma (OS) is defined by the formation of a premature osteoid matrix by mesenchymal spindle cells (Campanacci 2013). In adolescents and young adults, it is the most prevalent primary bone cancer with a global incidence rate of 3.25 new cases per million every year, despite its rarity (Misaghi et al. 2018; Osteosarcoma-American Cancer Society 2022). Generally, adjuvant and neoadjuvant chemotherapy, as well as amputation or limb salvage are used to treat OS. Cisplatin, ifosfamide, doxorubicin and methotrexate combined with the antidote leucovorin are the common treatment regimens used in chemotherapy (O’Kane et al. 2015; Misaghi et al. 2018). Despite the fact that these therapies have been proven to be beneficial, it has been observed that considerable OS patients may acquire fatal secondary malignancies or severe treatment-related difficulties, suggesting the necessity of novel therapies (Longhi et al. 2006; Durfee et al. 2016; Schwartz et al. 2016). IHC examination of samples from 62 osteosarcoma patients revealed a 63% ASS1 deficit, suggesting that arginine-degrading enzyme therapy could be used to treat OS (Table 3) (Kobayashi et al. 2010). A phase II clinical trial is presently underway employing ADI-PEG 20 (36 mg/m²) in conjunction with docetaxel (60 mg/m²) and gemcitabine (600 mg/m²), which are the standard chemotherapy regimens for soft tissue sarcoma treatments (ClinicalTrials.gov Identifier: NCT03449901). These results imply that arginine deprivation using arginine-hydrolyzing enzymes (rhArg I) could prevent OS proliferation.

Conclusion

Cancer, a common and enduring disease that is neither an epidemic nor a pandemic, took more than 10 million lives globally in 2020. Depending on the type and stage of cancer, numerous types of therapy options are available, which include chemotherapy, radiation therapy, surgery, targeted therapy, photodynamic therapy, and many more. The quest for a safe and broad-spectrum agent that can be effective against a wide range of cancers has been spurred by the severity of side effects linked to some of these treatments and/or their specificity towards a particular cancer (Society AC 2022). Therefore, it has been shown that focusing on the metabolism of cancer cells may be a more effective strategy with fewer side effects (Vettore et al. 2020; Lieu et al. 2020). In this context, numerous malignancies have been identified that are arginine auxotrophic and, thus, treatments based on the enzymes that break down arginine appear to be promising (Patil et al. 2016). As a result, arginine deprivation, which can be induced by rhArg I, has been explored as a novel approach to treating cancer, since it makes use of the characteristic arginine auxotrophy in cancers (Burrage et al. 2015). However, the native enzyme has poor pharmacokinetic properties, which constrains its development for clinical use. PEGylated-rhArg I molecules, such as BCT-100, pegzilarginase (AEB1102) and pegtomarginase (PT01), are being developed by the pharmaceutical companies to address this issue, which are at different phases of their clinical development. However, because of the limitations associated with the clinical use of PEGylated proteins, the development of recombinant variants employing fusion technology, in which the therapeutic protein is genetically fused to another protein/domain, has emerged as an effective approach to enhance its pharmacokinetic features. Aberrant expression of ASS1 and/or OTC is essential for the effectiveness of arginine deprivation therapy and only nine out of the more than 25 arginine-auxotrophic cancers have been reported thus far exhibited 60% or more ASS1 and/or OTC deficiency. Therefore, taking into account the significance of genetic profiles, particularly these urea cycle enzymes, the successful development of fusion variants of rhArg I with increased circulatory half-life will be a step toward generating a safer and longer-acting Arg I molecule for the treatment of the nine arginine-auxotrophic cancers discussed in this literature.

Abbreviations

ADI

Arginine deiminase

ALL

Acute lymphoblastic leukaemia

AML

Acute myeloid leukaemia

ASL

Argininosuccinate

ASS1

Argininosuccinate synthetase

BCT

Bio-cancer treatment

CO₂

Carbon dioxide

Co

Cobalt

EHA

Engineered human arginase

HCC

Hepatocellular carcinoma

HER

Human epidermal growth factor

HL

Hodgkin’s lymphoma

HLEPs

Half-life extension partners

HSA

Human serum albumin

IHC

Immunohistochemistry

mitoROS

Mitochondrial reactive oxygen species

MPM

Malignant pleural mesothelioma

Mn

Manganese

NO

Nitric oxide

NHL

Non-Hodgkin’s lymphoma

OTC

Ornithine transcarbamylase

OS

Osteosarcoma

PC

Pancreatic cancer

PEG

Polyethylene glycol

rhArg I

Recombinant human arginase I

SBRT

Stereotactic body radiation therapy

WHO

World Health Organization

Author contributions

AJ and YRP contributed equally. AJ, YRP and NS were involved in conceptualization, data curation and writing the manuscript. AHP was involved in conceptualization, visualization, investigation, supervision, reviewing and editing. All authors read and approved the final manuscript.

Funding

The authors would like to thank the Department of Biotechnology (New Delhi, Government of India; grant # BT/PR23283/MED/30/1953/2018) and the National Institute of Pharmaceutical Education and Research, S.A.S. Nagar (NPLC-AHP), for providing financial support.

Data availability

Not Applicable.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.