Unlocking the Potential of Arginine Deprivation Therapy: Recent Breakthroughs and Promising Future for Cancer Treatment.

Arginine is a semi-essential amino acid that supports protein synthesis to maintain cellular functions. Recent studies suggest that arginine also promotes wound healing, cell division, ammonia metabolism, immune system regulation, and hormone biosynthesis-all of which are critical for tumor growth. These discoveries, coupled with the understanding of cancer cell metabolic reprogramming, have led to renewed interest in arginine deprivation as a new anticancer therapy. Several arginine deprivation strategies have been developed and entered clinical trials. The main principle behind these therapies is that arginine auxotrophic tumors rely on external arginine sources for growth because they carry reduced key arginine-synthesizing enzymes such as argininosuccinate synthase 1 (ASS1) in the intracellular arginine cycle. To obtain anticancer effects, modified arginine-degrading enzymes, such as PEGylated recombinant human arginase 1 (rhArg1-PEG) and arginine deiminase (ADI-PEG 20), have been developed and shown to be safe and effective in clinical trials. They have been tried as a monotherapy or in combination with other existing therapies. This review discusses recent advances in arginine deprivation therapy, including the molecular basis of extracellular arginine degradation leading to tumor cell death, and how this approach could be a valuable addition to the current anticancer arsenal.

Arginine deiminase produced by lactic acid bacteria as a potent anti-cancer drug.

Bacterial-based cancer immunotherapy has recently gained widespread attention due to its exceptional mechanism of rich pathogen-associated molecular patterns in anti-cancer immune responses. Contrary to conventional cancer therapies such as surgery, chemotherapy, radiation and phototherapy, bacteria-based cancer immunotherapy has the unique ability to suppress cancer by selectively accumulating and growing in tumours. In the view of this, several bacterial strains are being used for the treatment of cancer. Of which, lactic acid bacteria are a powerful, albeit still inadequately understood bacteria that possess a wide source of bioactive chemicals. Lactic acid bacteria metabolites, such as bacteriocins, short-chain fatty acids, exopolysaccharides show antitumour property. Amino acid pathways, which have lately been focussed as a new strategy to cancer therapy, are key element of the adaptability and dysregulation of metabolic pathways identified in proliferation of tumour cells. Arginine metabolism, in particular, has been shown to be critical for cancer therapy. As a result, better understanding of arginine metabolism in LAB and cancer cells could lead to new cancer therapeutic targets. This review will outline current advances in the interaction of arginine metabolism with cancer therapy and propose an arginine deiminase expression system to combat cancer more effectively.

The serine hydrolase ABHD6 controls survival and thermally induced seizures in a mouse model of Dravet syndrome.

Evidence suggests that inhibition of α/ß hydrolase-domain containing 6 (ABHD6) reduces seizures; however, the molecular mechanism of this therapeutic response remains unknown. We discovered that heterozygous expression of Abhd6 (Abhd6+/-) significantly reduced the premature lethality of Scn1a+/- mouse pups, a genetic mouse model of Dravet Syndrome (DS). Both Abhd6+/- mutation and pharmacological inhibition of ABHD6 reduced the duration and incidence of thermally induced seizures in Scn1a+/- pups. Mechanistically, the in vivo anti-seizure response resulting from ABHD6 inhibition is mediated by potentiation of gamma-aminobutyric acid receptors Type-A (GABAAR). Brain slice electrophysiology showed that blocking ABHD6 potentiates extrasynaptic (tonic) GABAAR currents that reduce dentate granule cell excitatory output without affecting synaptic (phasic) GABAAR currents. Our results unravel an unexpected mechanistic link between ABHD6 activity and extrasynaptic GABAAR currents that controls hippocampal hyperexcitability in a genetic mouse model of DS. BRIEF SUMMARY: This study provides the first evidence for a mechanistic link between ABHD6 activity and the control of extrasynaptic GABAAR currents that controls hippocampal hyperexcitability in a genetic mouse model of Dravet Syndrome and can be targeted to dampened seizures.

Mono-PEGylated thermostable Bacillus caldovelox arginase mutant (BCA-M-PEG20) induces apoptosis, autophagy, cell cycle arrest and growth inhibition in gastric cancer cells.

Gastric cancer is one of the most common malignant solid tumors in the world, especially in Asia with high mortality due to a lack of effective treatment. The potential usage of the newly constructed arginine-depleting enzyme-mono-PEGylated Bacillus caldovelox arginase mutant (BCA-M-PEG20), an effective drug against multiple cancer cell lines such as cervical and lung cancers, for the treatment of gastric cancer was demonstrated. Our results indicated that BCA-M-PEG20 significantly inhibited argininosuccinate synthetase (ASS)-positive gastric cancer cells, MKN-45 and BGC-823, while another arginine-depleting enzyme, arginine deiminase (ADI, currently under Phase III clinical trial), failed to suppress the growth of gastric cancer cells. In vitro studies demonstrated that BCA-M-PEG20 inhibited MKN-45 cells by inducing autophagy and cell cycle arrest at the S phase under 0.58 U/mL (IC50 values). Significant caspase-dependent apoptosis was induced in MKN-45 after the treatment with 2.32 U/mL of BCA-M-PEG20. In vivo studies showed that administrations of BCA-M-PEG20 at 250 U/mouse twice per week significantly suppressed about 50% of tumor growth in the MKN-45 gastric cancer xenograft model. Taken together, BCA-M-PEG20 demonstrated a superior potential to be an anti-gastric cancer drug.

Assessment of pegylated arginine deiminase and modified FOLFOX6 in patients with advanced hepatocellular carcinoma: Results of an international, single-arm, phase 2 study.

BACKGROUND: Arginine starvation depletes the micronutrients required for DNA synthesis and interferes with both thymidylate synthetase activity and DNA repair pathways in preclinical models of hepatocellular carcinoma (HCC). Pegylated arginine deiminase (ADI-PEG 20), an arginine degrader, potentiates the cytotoxic activity of platinum and pyrimidine antimetabolites in HCC cellular and murine models. METHODS: This was a global, multicenter, open-label, single-arm, phase 2 trial of ADI-PEG 20 and modified 5-fluorouracil, leucovorin, and oxaliplatin (mFOLFOX6) in patients who had HCC with Child-Pugh A cirrhosis and disease progression on ≥2 prior lines of treatment. The primary objective was the objective response rate assessed according to Response Evaluation Criteria in Solid Tumors, version 1.1. Secondary objectives were to estimate progression-free survival, overall survival, safety, and tolerability. Eligible patients were treated with mFOLFOX6 intravenously biweekly at standard doses and ADI-PEG-20 intramuscularly weekly at 36 mg/m2 . RESULTS: In total, 140 patients with advanced HCC were enrolled. The median patient age was 62 years (range, 30-85 years), 83% of patients were male, 76% were of Asian race, 56% had hepatitis B viremia, 10% had hepatitis C viremia, 100% had received ≥2 prior lines of systemic therapy, and 39% had received ≥3 prior lines of systemic therapy. The objective response rate was 9.3% (95% confidence interval [CI], 5.0%-15.4%), with a median response duration of 10.2 months (95% CI, 5.8 months to not reached). The median progression-free survival was 3.8 months (95% CI, 1.8-6.3 months), and the median overall survival was 14.5 months (95% CI, 13.6-20.9 months). The most common grade ≥3 treatment-related events were neutropenia (32.9%), white blood cell count decrease (20%), platelet count decrease (19.3%), and anemia (9.3%). CONCLUSIONS: Concurrent mFOLFOX6 plus ADI-PEG 20 exhibited limited antitumor activity in patients with treatment-refractory HCC. The study was terminated early, and no further evaluation of the combination will be pursued. LAY SUMMARY: Arginine is an important nutrient for hepatocellular carcinoma (HCC). The depletion of arginine with pegylated arginine deiminase (ADI-PEG 20), an arginine degrader, appeared to make chemotherapy (FOLFOX) work better in animal models of HCC and in patients with HCC on an early phase clinical trial. To formally test this hypothesis in the clinical setting, a large, global, phase 2 clinical trial was conducted of ADI-PEG 20 and FOLFOX in the treatment of patients with refractory HCC. The study showed limited activity of ADI-PEG 20 and FOLFOX in advanced HCC and was stopped early.

Phase 1, pharmacogenomic, dose-expansion study of pegargiminase plus pemetrexed and cisplatin in patients with ASS1-deficient non-squamous non-small cell lung cancer.

INTRODUCTION: We evaluated the arginine-depleting enzyme pegargiminase (ADI-PEG20; ADI) with pemetrexed (Pem) and cisplatin (Cis) (ADIPemCis) in ASS1-deficient non-squamous non-small cell lung cancer (NSCLC) via a phase 1 dose-expansion trial with exploratory biomarker analysis. METHODS: Sixty-seven chemonaïve patients with advanced non-squamous NSCLC were screened, enrolling 21 ASS1-deficient subjects from March 2015 to July 2017 onto weekly pegargiminase (36 mg/m2 ) with Pem (500 mg/m2 ) and Cis (75 mg/m2 ), every 3 weeks (four cycles maximum), with maintenance Pem or pegargiminase. Safety, pharmacodynamics, immunogenicity, and efficacy were determined; molecular biomarkers were annotated by next-generation sequencing and PD-L1 immunohistochemistry. RESULTS: ADIPemCis was well-tolerated. Plasma arginine and citrulline were differentially modulated; pegargiminase antibodies plateaued by week 10. The disease control rate was 85.7% (n = 18/21; 95% CI 63.7%-97%), with a partial response rate of 47.6% (n = 10/21; 95% CI 25.7%-70.2%). The median progression-free and overall survivals were 4.2 (95% CI 2.9-4.8) and 7.2 (95% CI 5.1-18.4) months, respectively. Two PD-L1-expressing (≥1%) patients are alive following subsequent pembrolizumab immunotherapy (9.5%). Tumoral ASS1 deficiency enriched for p53 (64.7%) mutations, and numerically worse median overall survival as compared to ASS1-proficient disease (10.2 months; n = 29). There was no apparent increase in KRAS mutations (35.3%) and PD-L1 (<1%) expression (55.6%). Re-expression of tumoral ASS1 was detected in one patient at progression (n = 1/3). CONCLUSIONS: ADIPemCis was safe and highly active in patients with ASS1-deficient non-squamous NSCLC, however, survival was poor overall. ASS1 loss was co-associated with p53 mutations. Therapies incorporating pegargiminase merit further evaluation in ASS1-deficient and treatment-refractory NSCLC.

Arginine deprivation as a strategy for cancer therapy: An insight into drug design and drug combination.

Extensive studies have shown that cancer cells have specific nutrient auxotrophy and thus have much a higher demand for certain nutrients than normal cells. Amino acid deprivation has attracted much attention in cancer therapy with positive outcomes from clinical trials. Arginine, as one of the conditionally essential amino acids, plays a pivotal role in cellular division and metabolism. Since many types of cancer cells exhibit decreased expression of argininosuccinate synthetase and/or ornithine transcarbamylase, they are auxotrophic for arginine, which makes arginine deprivation an accessible choice for cancer treatment. Arginine deiminase (ADI) and human arginase (hArg) are the two major protein drugs used for arginine deprivation and are undergoing many clinical trials. However, the clinical application of ADI and hArg is facing some common problems, including their short half-lives, immunogenicity and inconsistent production, which underlines the importance of improving these drugs using protein engineering techniques. Thus, we systematically review the latest studies of protein engineering and anti-cancer studies based on in vitro, in vivo and clinical models of ADI and hArg, and we include the latest studies on drug combinations consisting of ADI/hArg with chemotherapeutic drugs.

Biomimetic microbioreactor-supramolecular nanovesicles improve enzyme therapy of hepatic cancer.

A novel biomimetic nanovesicle-loaded supramolecular enzyme-based therapeutics has been developed. Here, using a biomimetic lipid-D-α-tocopherol polyethylene glycol succinate (TPGS) hybrid semi-permeable membrane, cyclodextrin supramolecular docking, metal-ion-aided coordination complexing, we combined multiple functional motifs into a single biomimetic microbioreactor-supramolecular nanovesicle (MiSuNv) that allowed effective transport of arginine deiminase (ADI) to hepatic tumor cells to enhance arginine depletion. We compared two intercalated enzyme-carrying supermolecular motifs mainly comprising of 2-hydroxypropyl-ß-cyclodextrin and sulfobutyl-ether-ß-cyclodextrin, the only two cyclodextrin derivatives approved for injection by the United States Food and Drug Administration. The ADI-specific antitumor effects were enhanced by TPGS (one constituent of MiSuNv, having synergistic antitumor effects), as ADI was separated from adverse external environment by a semi-permeable membrane and sequestered in a favorable internal microenvironment with an optimal pH and metal-ion combination. ADI@MiSuNv contributed to cell cycle arrest, apoptosis and autophagy through the enhanced efficacy of enzyme treatment against Hep3B xenograft tumors in rats.

Phase II Study of Arginine Deprivation Therapy With Pegargiminase in Patients With Relapsed Sensitive or Refractory Small-cell Lung Cancer.

BACKGROUND: Pre-clinical studies indicated that arginine-deprivation therapy using pegylated arginine deiminase (pegargiminase, ADI-PEG 20) may be effective in patients with argininosuccinate synthetase 1 (ASS1)-deficient small-cell lung cancer (SCLC). PATIENTS AND METHODS: Patients were enrolled into either a 'sensitive' disease cohort (≥ 90 days response to first-line chemotherapy) or a 'refractory' disease cohort (progression while on chemotherapy or < 90 days afterwards or ≥ third-line treatment). Patients received weekly intramuscular pegargiminase, 320 IU/m2 (36.8 mg/m2), until unacceptable toxicity or disease progression. The primary endpoint was tumor response assessed by Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 with secondary endpoints including tolerability, pharmacodynamics, and immunogenicity. RESULTS: Between January 2011 and January 2014, 22 patients were enrolled: 9 in the sensitive disease cohort and 13 in the refractory disease cohort. At a pre-planned interim analysis, the best overall response observed was stable disease in 2 patients in each cohort (18.2%). Owing to the lack of response and slow accrual in the sensitive disease cohort, the study was terminated early. Pegargiminase treatment was well-tolerated with no unexpected adverse events or discontinuations. CONCLUSION: Although pegargiminase monotherapy in SCLC failed to meet its primary endpoint of RECIST-confirmed responses, more recent molecular stratification, including MYC status, may provide new opportunities moving forward.

Intracellular expression of arginine deiminase activates the mitochondrial apoptosis pathway by inhibiting cytosolic ferritin and inducing chromatin autophagy.

BACKGROUND: Based on its low toxicity, arginine starvation therapy has the potential to cure malignant tumors that cannot be treated surgically. The Arginine deiminase (ADI) gene has been identified to be an ideal cancer-suppressor gene. ADI expressed in the cytosol displays higher oncolytic efficiency than ADI-PEG20 (Pegylated Arginine Deiminase by PEG 20,000). However, it is still unknown whether cytosolic ADI has the same mechanism of action as ADI-PEG20 or other underlying cellular mechanisms. METHODS: The interactions of ADI with other protein factors were screened by yeast hybrids, and verified by co-immunoprecipitation and immunofluorescent staining. The effect of ADI inhibiting the ferritin light-chain domain (FTL) in mitochondrial damage was evaluated by site-directed mutation and flow cytometry. Control of the mitochondrial apoptosis pathway was analyzed by Western Blotting and real-time PCR experiments. The effect of p53 expression on cancer cells death was assessed by siTP53 transfection. Chromatin autophagy was explored by immunofluorescent staining and Western Blotting. RESULTS: ADI expressed in the cytosol inhibited the activity of cytosolic ferritin by interacting with FTL. The inactive mutant of ADI still induced apoptosis in certain cell lines of ASS- through mitochondrial damage. Arginine starvation also generated an increase in the expression of p53 and p53AIP1, which aggravated the cellular mitochondrial damage. Chromatin autophagy appeared at a later stage of arginine starvation. DNA damage occurred along with the entire arginine starvation process. Histone 3 (H3) was found in autophagosomes, which implies that cancer cells attempted to utilize the arginine present in histones to survive during arginine starvation. CONCLUSIONS: Mitochondrial damage is the major mechanism of cell death induced by cytosolic ADI. The process of chromatophagy does not only stimulate cancer cells to utilize histone arginine but also speeds up cancer cell death at a later stage of arginine starvation.

Enzyme-based autophagy in anti-neoplastic management: From molecular mechanisms to clinical therapeutics.

Autophagy is an evolutionarily conserved self-cannibalization process commonly found in all eukaryotic cells. Through autophagy, long-lived or damaged organelles, superfluous proteins, and pathogens are sequestered and encapsulated into the double-membrane autophagosomes prior to fusion with lysosomes for ultimate degradation and recycling. Given that autophagy is deemed both protective and detrimental in malignancies, the clinical therapeutic utilization of autophagy modulators in cancer has attracted immense attentions over the past decades. Dependence of tumor cells on autophagy during amino acid insufficiency or deprivation has prompted us to explore the underlying autophagy regulatory mechanisms to inject amino acid degrading enzymes and enzyme-based strategies into therapeutic maneuvers of autophagy in cancer.

Amino Acids Sequence-based Analysis of Arginine Deiminase from Different Prokaryotic Organisms: An In Silico Approach.

BACKGROUND: Arginine deiminase is a bacterial enzyme, which degrades L-arginine. Some human cancers such as hepatocellular carcinoma (HCC) and melanoma are auxotrophic for arginine. Therefore, PEGylated arginine deiminase (ADI-PEG20) is a good anticancer candidate with antitumor effects. It causes local depletion of L-arginine and growth inhibition in arginineauxotrophic tumor cells. The FDA and EMA have granted orphan status to this drug. Some recently published patents have dealt with this enzyme or its PEGylated form. OBJECTIVE: Due to increasing attention to it, we aimed to evaluate and compare 30 arginine deiminase proteins from different bacterial species through in silico analysis. METHODS: The exploited analyses included the investigation of physicochemical properties, multiple sequence alignment (MSA), motif, superfamily, phylogenetic and 3D comparative analyses of arginine deiminase proteins thorough various bioinformatics tools. RESULTS: The most abundant amino acid in the arginine deiminase proteins is leucine (10.13%) while the least amino acid ratio is cysteine (0.98%). Multiple sequence alignment showed 47 conserved patterns between 30 arginine deiminase amino acid sequences. The results of sequence homology among 30 different groups of arginine deiminase enzymes revealed that all the studied sequences located in amidinotransferase superfamily. Based on the phylogenetic analysis, two major clusters were identified. Considering the results of various in silico studies; we selected the five best candidates for further investigations. The 3D structures of the best five arginine deiminase proteins were generated by the I-TASSER server and PyMOL. The RAMPAGE analysis revealed that 81.4%-91.4%, of the selected sequences, were located in the favored region of arginine deiminase proteins. CONCLUSION: The results of this study shed light on the basic physicochemical properties of thirty major arginine deiminase sequences. The obtained data could be employed for further in vivo and clinical studies and also for developing the related therapeutic enzymes.

Robust LC-MS/MS methods for analysis of heparan sulfate levels in CSF and brain for application in studies of MPS IIIA.

Aim: Accumulation of heparan sulfate (HS) is associated with the neurodegenerative disorder Mucopolysaccharidosis type IIIA (MPS IIIA). Here, we compare HS levels in brain and cerebrospinal fluid (CSF) of MPS IIIA mice after treatment with a chemically modified sulfamidase (CM-rhSulfamidase). Materials & methods: Two LC-MS/MS methods were adapted from literature methodology, one to measure HS metabolites (HSmet), the other to measure digests of HS after heparinase treatment (HSdig). Results: The HSmet and HSdig methods showed similar relative reduction of HS in brain after CM-rhSulfamidase administration to MPS IIIA mice and the reduction was reflected also in CSF. Conclusion: The results of the two methods correlated and therefore the HSdig method can be used in clinical studies to determine HS levels in CSF from patients with MPS IIIA.

Exploiting Arginine Auxotrophy with Pegylated Arginine Deiminase (ADI-PEG20) to Sensitize Pancreatic Cancer to Radiotherapy via Metabolic Dysregulation.

Distinct metabolic vulnerabilities of cancer cells compared with normal cells can potentially be exploited for therapeutic targeting. Deficiency of argininosuccinate synthetase-1 (ASS1) in pancreatic cancers creates auxotrophy for the semiessential amino acid arginine. We explored the therapeutic potential of depleting exogenous arginine via pegylated arginine deiminase (ADI-PEG20) treatment as an adjunct to radiotherapy. We evaluated the efficacy of treatment of human pancreatic cancer cell lines and xenografts with ADI-PEG20 and radiation via clonogenic assays and tumor growth delay experiments. We also investigated potential mechanisms of action using reverse-phase protein array, Western blotting, and IHC and immunofluorescence staining. ADI-PEG20 potently radiosensitized ASS1-deficient pancreatic cancer cells (MiaPaCa-2, Panc-1, AsPc-1, HPAC, and CaPan-1), but not ASS1-expressing cell lines (Bxpc3, L3.6pl, and SW1990). Reverse phase protein array studies confirmed increased expression of proteins related to endoplasmic reticulum (ER) stress and apoptosis, which were confirmed by Western blot analysis. Inhibition of ER stress signaling with 4-phenylbutyrate abrogated the expression of ER stress proteins and reversed radiosensitization by ADI-PEG20. Independent in vivo studies in two xenograft models confirmed significant tumor growth delays, which were associated with enhanced expression of ER stress proteins and apoptosis markers and reduced expression of proliferation and angiogenesis markers. ADI-PEG20 augmented the effects of radiation by triggering the ER stress pathway, leading to apoptosis in pancreatic tumor cells.

Arginine metabolism and deprivation in cancer therapy.

Certain cancer cells with nutrient auxotrophy and have a much higher nutrient demand compared with normal human cells. Arginine as a versatile amino acid, has multiple biological functions in metabolic and signaling pathways. Depletion of this amino acid by arginine depletor is generally well tolerated and has become a targeted therapy for arginine auxotrophic cancers. However, the modulatory eff ;ect of arginine on cancer cells is very complicated and still controversial. Therefore, this article focuses on arginine metabolism and depletion therapy in cancer treatment to provide systemical review on this issue.

Arginine Deiminase: Current Understanding and Applications.

BACKGROUND: Arginine deiminase (ADI), an arginine catabolizing enzyme, is considered as an anti-tumor agent for the treatment of arginine auxotrophic cancers. However, some obstacles limit its clinical applications. OBJECTIVE: This review will summarize the clinical applications of ADI, from a brief history to its limitations, and will discuss the different ways to deal with the clinical limitations. METHOD: The structure analysis, cloning, expression, protein engineering and applications of arginine deiminase enzyme have been explained in this review. CONCLUSION: Recent patents on ADI are related to ADI engineering to increase its efficacy for clinical application. The intracellular delivery of ADI and combination therapy seem to be the future strategies in the treatment of arginine auxotrophic cancers. Applying ADIs with optimum features from different sources and or ADI engineering, are promising strategies to improve the clinical application of ADI.

Arginine-Depleting Enzymes - An Increasingly Recognized Treatment Strategy for Therapy-Refractory Malignancies.

Arginine auxotrophy occurs in certain tumor types and is usually caused by the silencing of argininosuccinate synthetase 1 or arginine lyase genes. Such tumors are often associated with an intrinsic chemoresistance and thus a poor prognosis. Arginine auxotrophy however renders these tumors vulnerable to treatment with arginine-degrading enzymes. Among the most frequently applied arginine-degrading agents are bacterial arginine deiminases (ADI). The anti-cancerous effects of ADI derived from different bacteria were extensively studied in numerous preclinical cell culture and xenograft models. Mycoplasma-derived ADI-PEG20 is most commonly used and is currently under clinical investigation as a single agent therapeutic as well as in combination with different antineoplastic compounds. Mechanistically, ADI is capable of reducing metabolic activity in tumor cells, contributing to autophagy, senescence and apoptosis in arginine auxotrophic cells. Although clinical trials are promising, the resistance development upon initial treatment response is an increasing challenge. Furthermore, interference of ADI with the tumor microenvironment is poorly understood. In the present review, we outline recent experimental ADI-based treatment approaches and their translation into the clinic. Furthermore, we summarize new insights into the molecular mechanisms underlying the anti-cancer effects of ADI that might facilitate the refinement of ADI-based combination therapy approaches.

Sensitivity of Colorectal Cancer to Arginine Deprivation Therapy is Shaped by Differential Expression of Urea Cycle Enzymes.

Tumors deficient in the urea cycle enzymes argininosuccinate synthase-1 (ASS1) and ornithine transcarbamylase (OTC) are unable to synthesize arginine and can be targeted using arginine-deprivation therapy. Here, we show that colorectal cancers (CRCs) display negligible expression of OTC and, in subset of cases, ASS1 proteins. CRC cells fail to grow in arginine-free medium and dietary arginine deprivation slows growth of cancer cells implanted into immunocompromised mice. Moreover, we report that clinically-formulated arginine-degrading enzymes are effective anticancer drugs in CRC. Pegylated arginine deiminase (ADI-PEG20), which degrades arginine to citrulline and ammonia, affects growth of ASS1-negative cells, whereas recombinant human arginase-1 (rhArg1peg5000), which degrades arginine into urea and ornithine, is effective against a broad spectrum of OTC-negative CRC cell lines. This reflects the inability of CRC cells to recycle citrulline and ornithine into the urea cycle. Finally, we show that arginase antagonizes chemotherapeutic drugs oxaliplatin and 5-fluorouracil (5-FU), whereas ADI-PEG20 synergizes with oxaliplatin in ASS1-negative cell lines and appears to interact with 5-fluorouracil independently of ASS1 status. Overall, we conclude that CRC is amenable to arginine-deprivation therapy, but we warrant caution when combining arginine deprivation with standard chemotherapy.

Reversal of indoleamine 2,3-dioxygenase-mediated cancer immune suppression by systemic kynurenine depletion with a therapeutic enzyme.

Increased tryptophan (Trp) catabolism in the tumor microenvironment (TME) can mediate immune suppression by upregulation of interferon (IFN)-γ-inducible indoleamine 2,3-dioxygenase (IDO1) and/or ectopic expression of the predominantly liver-restricted enzyme tryptophan 2,3-dioxygenase (TDO). Whether these effects are due to Trp depletion in the TME or mediated by the accumulation of the IDO1 and/or TDO (hereafter referred to as IDO1/TDO) product kynurenine (Kyn) remains controversial. Here we show that administration of a pharmacologically optimized enzyme (PEGylated kynureninase; hereafter referred to as PEG-KYNase) that degrades Kyn into immunologically inert, nontoxic and readily cleared metabolites inhibits tumor growth. Enzyme treatment was associated with a marked increase in the tumor infiltration and proliferation of polyfunctional CD8+ lymphocytes. We show that PEG-KYNase administration had substantial therapeutic effects when combined with approved checkpoint inhibitors or with a cancer vaccine for the treatment of large B16-F10 melanoma, 4T1 breast carcinoma or CT26 colon carcinoma tumors. PEG-KYNase mediated prolonged depletion of Kyn in the TME and reversed the modulatory effects of IDO1/TDO upregulation in the TME.