Pegylated Arginine Deiminase Research Articles

Arginine Deiminase Induces Immunogenic Cell Death and Is Enhanced by N-acetylcysteine in Murine MC38 Colorectal Cancer Cells and MDA-MB-231 Human Breast Cancer Cells In Vitro.

The use of arginine deiminase (ADI) for arginine depletion therapy is an attractive anticancer approach. Combination strategies are needed to overcome the resistance of severe types of cancer cells to this monotherapy. In the current study, we report, for the first time, that the antioxidant N-acetylcysteine (NAC), which has been used in therapeutic practices for several decades, is a potent enhancer for targeted therapy that utilizes arginine deiminase. We demonstrated that pegylated arginine deiminase (ADI-PEG 20) induces apoptosis and G0/G1 phase arrest in murine MC38 colorectal cancer cells; ADI-PEG 20 induces Ca2+ overload and decreases the mitochondrial membrane potential in MC38 cells. ADI-PEG 20 induced the most important immunogenic cell death (ICD)-associated feature: cell surface exposure of calreticulin (CRT). The antioxidant NAC enhanced the antitumor activity of ADI-PEG 20 and strengthened its ICD-associated features including the secretion of high mobility group box 1 (HMGB1) and adenosine triphosphate (ATP). In addition, these regimens resulted in phagocytosis of treated MC38 cancer cells by bone marrow-derived dendritic cells (BMDCs). In conclusion, we describe, for the first time, that NAC in combination with ADI-PEG 20 not only possesses unique cytotoxic anticancer properties but also triggers the hallmarks of immunogenic cell death. Hence, ADI-PEG 20 in combination with NAC may represent a promising approach to treat ADI-sensitive tumors while preventing relapse and metastasis.

Pegylated arginine deiminase depletes plasma arginine but maintains tissue arginine availability in young pigs.

Pegylated arginine deiminase (ADI-PEG20) results in the depletion of arginine with the production of isomolar amounts of citrulline. This citrulline has the potential to be utilized by the citrulline recycling pathway regenerating arginine and sustaining tissue arginine availability. The goal of this research was to test the hypothesis that ADI-PEG20 depletes circulating arginine in pigs but maintains tissue arginine concentration and function, and to characterize the kinetics of citrulline and arginine. Two multitracer approaches (bolus dose and primed-continuous infusion) were used to investigate the metabolism of arginine and citrulline in Control (n = 7) and ADI-PEG20 treated (n = 8) pigs during the postprandial period. In addition, blood pressure was monitored by telemetry, and multiple tissues were collected to determine arginine concentration. Plasma arginine was depleted immediately after ADI-PEG20 administration, with an increase in plasma citrulline concentration (P < 0.01). The depletion of arginine did not affect (P > 0.10) blood pressure, whole body protein synthesis, or urea production. Despite the lack of circulating arginine in ADI-PEG20-treated pigs, most tissues were able to maintain concentrations similar (P > 0.10) to those in Control animals. The kinetics of citrulline and arginine indicated the high citrulline turnover and regeneration of arginine through the citrulline recycling pathway. ADI-PEG20 administration resulted in an absolute and almost instantaneous depletion of circulating arginine, thus reducing global availability without affecting cardiovascular parameters and protein metabolism. The citrulline produced from the deimination of arginine was in turn utilized by the citrulline recycling pathway restoring local tissue arginine availability.NEW & NOTEWORTHY Pegylated arginine deiminase depletes circulating arginine, but the citrulline generated is utilized by multiple tissues to regenerate arginine and sustain local arginine availability. Preempting the arginine depletion that occurs as result of sepsis and trauma with arginine deiminase offers the possibility of maintaining tissue arginine availability despite negligible plasma arginine concentrations.

Phase 1b study of pegylated arginine deiminase (ADI-PEG 20) plus Pembrolizumab in advanced solid cancers

ABSTRACT Background Pegylated arginine deiminase (ADI-PEG 20) is a metabolism-based strategy that depletes arginine, resulting in tumoral stress and cytotoxicity. Preclinically, ADI-PEG 20 modulates T-cell activity and enhances the therapeutic efficacy of programmed death-1 (PD-1) inhibition. Methods A phase 1b study, including a dose-escalation cohort and an expansion cohort, was undertaken to explore the effects of ADI-PEG 20 in combination with pembrolizumab, an anti-PD-1 antibody, for safety, pharmacodynamics, and response. CD3 levels and programmed death-ligand 1 (PD-L1) expression were assessed in paired biopsies collected prior to and after ADI-PEG 20 treatment but before pembrolizumab. Results Twenty-five patients, nine in the dose-escalation cohort and sixteen in the expansion cohort, were recruited. Treatment was feasible with adverse events consistent with those known for each agent, except for Grade 3/4 neutropenia which was higher than expected, occurring in 10/25 (40%) patients. Mean arginine levels were suppressed for 1–3 weeks, but increased gradually. CD3+ T cells increased in 10/12 (83.3%) subjects following ADI-PEG 20 treatment, including in three partial responders (p = .02). PD-L1 expression was low and increased in 3/10 (30%) of subjects. Partial responses occurred in 6/25 (24%) heavily pretreated patients, in both argininosuccinate synthetase 1 proficient and deficient subjects. Conclusions The immunometabolic combination was safe with the caveat that the incidence of neutropenia might be increased compared with either agent alone. ADI-PEG 20 treatment increased T cell infiltration in the low PD-L1 tumor microenvironment. The recommended phase 2 doses are 36 mg/m2 weekly for ADI-PEG 20 and 200 mg every 3 weeks for pembrolizumab.

MicroRNA-1291-5p Sensitizes Pancreatic Carcinoma Cells to Arginine Deprivation and Chemotherapy through the Regulation of Arginolysis and Glycolysis.

Cancer cells are dysregulated and addicted to continuous supply and metabolism of nutritional glucose and amino acids (e.g., arginine) to drive the synthesis of critical macromolecules for uncontrolled growth. Recent studies have revealed that genome-derived microRNA (miRNA or miR)-1291-5p (miR-1291-5p or miR-1291) may modulate the expression of argininosuccinate synthase (ASS1) and glucose transporter protein type 1 (GLUT1). We also developed a novel approach to produce recombinant miR-1291 agents for research, which are distinguished from conventional chemo-engineered miRNA mimics. Herein, we firstly demonstrated that bioengineered miR-1291 agent was selectively processed to high levels of target miR-1291-5p in human pancreatic cancer (PC) cells. After the suppression of ASS1 protein levels, miR-1291 perturbed arginine homeostasis and preferably sensitized ASS1-abundant L3.3 cells to arginine deprivation therapy. In addition, miR-1291 treatment reduced the protein levels of GLUT1 in both AsPC-1 and PANC-1 cells, leading to a lower glucose uptake (deceased > 40%) and glycolysis capacity (reduced approximately 50%). As a result, miR-1291 largely improved cisplatin efficacy in the inhibition of PC cell viability. Our results demonstrated that miR-1291 was effective to sensitize PC cells to arginine deprivation treatment and chemotherapy through targeting ASS1- and GLUT1-mediated arginolysis and glycolysis, respectively, which may provide insights into understanding miRNA signaling underlying cancer cell metabolism and development of new strategies for the treatment of lethal PC.SIGNIFICANCE STATEMENTMany anticancer drugs in clinical use and under investigation exert pharmacological effects or improve efficacy of coadministered medications by targeting cancer cell metabolism. Using new recombinant miR-1291 agent, we revealed that miR-1291 acts as a metabolism modulator in pancreatic carcinoma cells through the regulation of argininosuccinate synthase– and glucose transporter protein type 1–mediated arginolysis and glycolysis. Consequently, miR-1291 effectively enhanced the efficacy of arginine deprivation (pegylated arginine deiminase) and chemotherapy (cisplatin), offering new insights into development of rational combination therapies.

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

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. Due to increasing attention to it, we aimed to evaluate and compare 30 arginine deiminase proteins from different bacterial species through in silico analysis. 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. 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. 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.

Abstract 2217: Pegylated arginine deiminase sensitizes ASS1-negative and KRAS mutant non-small cell lung cancer to PD-1 blockade immunotherapy

Abstract Cancer cells display dysregulated amino acid synthesis underlying a key metabolic hallmark of tumorigenesis that may be exploited therapeutically via specific amino acid deprivation. In particular, we have identified that arginine deprivation therapy with pegylated arginine deiminase (ADI-PEG20), which hydrolyses exogenous arginine to citrulline and ammonia, is clinically active in argininosuccinate synthetase 1 (ASS1)-deficient non-small cell lung cancer (NSCLC) and enhances the activity of the antifolate pemetrexed. Moreover, prior studies have shown that ADI-PEG20 also induces tumoral PD-L1 expression and T cell infiltration and is additive with anti-PD-1/PD-L1 antibodies in melanoma and colorectal murine models. Here, we reverse translated ADI-PEG20 studies from patients with ASS1-deficient NSCLC, co-associating with KRAS mutations, to analyze the impact of arginine deprivation with PD1-based checkpoint immunotherapy using murine NSCLC cell line models. First, we showed the CMT64 cell line, which harbors the KRAS-G12V mutation and is resistant to anti-programmed death antibodies, was ASS1-negative and highly sensitive to ADI-PEG20 in vitro. Next, CMT64 tumor cells were implanted subcutaneously into the right flank of syngeneic immunocompetent C57BL/6 mice. Once tumors reached 80mm3 animals were randomly assigned to four groups and treated with vehicle control (PBS), ADI-PEG20 (12 mg/kg), anti-PD-1 antibodies (10 mg/kg) or the combination of ADI-PEG20 and PD-1 blockade. CMT64 tumors were refractory to PD-1 blockade while ADI-PEG20 monotherapy induced a modest tumor response, with a two-fold decrease in tumor growth compared to control (p=0.0058). In contrast, the combination of ADI-PEG20 and PD-1 blockade elicited robust anti-tumor activity with a five-fold reduction in CMT64 tumor volume compared to control (p=0.0003). Modulation of the tumor microenvironment was observed by fluorescence activated cell sorting. In particular, we noted that tumor-associated macrophages showed higher expression of MHCII upon ADI-PEG20 and anti-PD-1 therapy (p&amp;lt;0.05). Lastly, mice treated with ADI-PEG20 and anti-PD-1 therapy, survived longer than either ADI-PEG20 or anti-PD-1 therapy alone or PBS. In summary, arginine deprivation with ADI-PEG20 and PD-1 blockade is synthetically lethal in aggressive murine NSCLC and warrants further clinical investigation. A new clinical trial evaluating ADI-PEG20 with atezolizumab in combination with pemetrexed and platinum is opening in 2020 in patients with aggressive ASS1-deficient and PD-1/PD-L1 refractory lung adenocarcinoma (ClinicalTrials.gov Identifier: NCT03498222). Citation Format: Iuliia Pavlyk, Julie Foster, Katie Dexter, Jane Sobasowski, John Bomalarski, Chiara Berlato, Frances Balkwill, Peter W Szlosarek. Pegylated arginine deiminase sensitizes ASS1-negative and KRAS mutant non-small cell lung cancer to PD-1 blockade immunotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2217.

Abstract B14: Epigenetic remodeling of cancer metabolisms in cisplatin-resistant bladder cancer

Abstract Introduction and Objective: Alterations in DNA methylation are important epigenetic markers in bladder cancer (BC). These epigenome modifications drive the mechanisms of aggressive chemoresistant BC. Clinicopathologic biomarkers that indicate chemotherapeutic resistance are critical for better assessing treatment strategies for individual patients. Thus, in this study, we aimed to determine whether DNA methylation of certain metabolic enzymes is significantly altered in cisplatin-resistant BC cells. Methods: To characterize CpG methylation and nucleosome accessibility in cisplatin-resistant BC cells, the Illumina Infinium HM450 DNA methylation assay was performed. Perturbed gene expression was found to be associated with cisplatin resistance, and the biologic roles of spermidine/spermine N1-acetyltransferase (SAT1) and argininosuccinate synthase 1 (ASS1) were further studied using qRT-PCR analysis and various cell biology assays, including Western blot. Results: ASS1 and SAT1, genes for amino acid and polyamine metabolism catalysts, respectively, were found to be vastly hypermethylated, resulting in greatly downregulated expression. ASS1 expression is of particular interest because prior studies have demonstrated its potential association with BC stage and recurrence. In regard to chemoresistance, we found that aberrant expression or induced stimulation of SAT1 restored cisplatin sensitivity in the cell culture system. We also found that the addition of exogenous arginine deiminase through administration of ADI-PEG 20 (pegylated arginine deiminase) increased ASS1 expression and enhanced cisplatin's apoptotic effects. Conclusions: Our study demonstrates a novel mechanistic link between the epigenetic perturbation of SAT1 and ASS1 and cancer metabolism in cisplatin-resistant bladder cancer cells. These findings suggest potential utility of SAT1 and ASS1 as predictive biomarkers in resensitizing bladder cancer to chemotherapy and personalizing therapy. Citation Format: Austin Yeon, Sungyong You, Minhyung Kim, Amit Gupta, Myung Hee Park, Daniel Weisenberger, Gangning Liang, Jayoung Kim. Epigenetic remodeling of cancer metabolisms in cisplatin-resistant bladder cancer [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr B14.

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

BackgroundPre-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 MethodsPatients 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. ResultsBetween 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. ConclusionAlthough 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

BackgroundBased 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.MethodsThe 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.ResultsADI 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.ConclusionsMitochondrial 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.

Mono-PEGylation of a Thermostable Arginine-Depleting Enzyme for the Treatment of Lung Cancer.

L-arginine (L-Arg) depletion induced by randomly PEGylated arginine deiminase (ADI-PEG20) can treat arginosuccinate synthase (ASS)-negative cancers, and ADI-PEG20 is undergoing phase III clinical trials. Unfortunately, ASS-positive cancers are resistant to ADI-PEG20. Moreover, the yield of ADI production is low because of the formation of inclusion bodies. Here, we report a thermostable arginine-depleting enzyme, Bacillus caldovelox arginase mutant (BCA-M: Ser161->Cys161). An abundant amount of BCA-M was easily obtained via high cell-density fermentation and heat treatment purification. Subsequently, we prepared BCA-M-PEG20, by conjugating a single 20 kDa PEG monomer onto the Cys161 residue via thio-chemistry. Unlike ADI-PEG20, BCA-M-PEG20 significantly inhibited ASS-positive lung cancer cell growth. Pharmacodynamic studies showed that a single intraperitoneal injection (i.p). administration of 250 U/mouse of BCA-M-PEG20 induced low L-Arg level over 168 h. The mono-PEGylation of BCA-M prolonged its elimination half-life from 6.4 to 91.4 h (a 14-fold increase). In an A549 lung cancer xenograft model, a weekly administration of 250 U/mouse of BCA-M-PEG20 suppressed tumor growth significantly. We also observed that BCA-M-PEG20 did not cause any significant safety issue in mouse models. Overall, BCA-M-PEG20 showed excellent results in drug production, potency, and stability. Thereby, it has great potential to become a promising candidate for lung cancer therapy.

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.

ES03.04 Pegargiminase to Treat Mesothelioma

ES03.04 Pegargiminase to Treat Mesothelioma

MYC-Driven Small-Cell Lung Cancer is Metabolically Distinct and Vulnerable to Arginine Depletion.

Small-cell lung cancer (SCLC) has been treated clinically as a homogeneous disease, but recent discoveries suggest that SCLC is heterogeneous. Whether metabolic differences exist among SCLC subtypes is largely unexplored. In this study, we aimed to determine whether metabolic vulnerabilities exist between SCLC subtypes that can be therapeutically exploited. We performed steady state metabolomics on tumors isolated from distinct genetically engineered mouse models (GEMM) representing the MYC- and MYCL-driven subtypes of SCLC. Using genetic and pharmacologic approaches, we validated our findings in chemo-naïve and -resistant human SCLC cell lines, multiple GEMMs, four human cell line xenografts, and four newly derived PDX models. We discover that SCLC subtypes driven by different MYC family members have distinct metabolic profiles. MYC-driven SCLC preferentially depends on arginine-regulated pathways including polyamine biosynthesis and mTOR pathway activation. Chemo-resistant SCLC cells exhibit increased MYC expression and similar metabolic liabilities as chemo-naïve MYC-driven cells. Arginine depletion with pegylated arginine deiminase (ADI-PEG 20) dramatically suppresses tumor growth and promotes survival of mice specifically with MYC-driven tumors, including in GEMMs, human cell line xenografts, and a patient-derived xenograft from a relapsed patient. Finally, ADI-PEG 20 is significantly more effective than the standard-of-care chemotherapy. These data identify metabolic heterogeneity within SCLC and suggest arginine deprivation as a subtype-specific therapeutic vulnerability for MYC-driven SCLC.

Phase 1 dose-expansion study of pegylated arginine deiminase, cisplatin, and pemetrexed in patients with argininosuccinate synthetase 1 (ASS1)–deficient non-squamous non-small cell lung cancer.

9097 Background: Pegylated arginine deiminase (ADI-PEG20) targets ASS1-ve tumors, including non–small-cell lung cancer (NSCLC), by potentiating pemetrexed cytotoxicity via arginine depletion. In Beddowes et al (JCO 2017) we showed a 100% disease control rate in thoracic cancers treated with ADI-PEG20, cisplatin and pemetrexed (ADIPemCis). Thus, we tested ADIPemCis in a phase I dose-expansion cohort study of patients (pts) with non-squamous NSCLC. Methods: Good performance (ECOG 0-1) advanced non-squamous NSCLC pts were enrolled at the maximum tolerated dose (MTD) of ADIPemCis, using tumoral ASS1 loss as a selection biomarker. Pem (500mg/m2) and Cis (75mg/m2) were given every 3 weeks with weekly IM ADI-PEG20 (36mg/m2) for up to 4 cycles with maintenance ADI-PEG20 or Pem in responding pts. Primary endpoint was tumor response rate (RR by RECIST 1.1), with secondary endpoints including progression-free survival (PFS), overall survival (OS), and toxicity. We also measured plasma [arginine] and [citrulline], anti-ADI-PEG20 antibodies, and PD-L1 expression. Results: 21 of 70 screened pts (median age 60.1) were enrolled between April 15 and August 17. A confirmed partial response (PR) was observed in 55.6 % (n = 10/18 evaluable pts). Median PFS and OS were 4 months (95% CI 2.9-4.8) and 7.2 months (95% CI 5.1-18.4), respectively. 9% (n = 2/21) remain alive on subsequent therapies. 43% (n = 9/21) experienced grade 3/4 treatment-related toxicities, commonly non-febrile neutropenia. Plasma [arginine] declined rapidly and [citrulline] increased; both changes persisted at 16 weeks. 55% of pts’ tumors (n = 6/11 ) were PD-L1 &lt; 1% by immunohistochemistry. Conclusions: The ADIPemCis regimen is active and safe in ASS1-ve NSCLC pts almost doubling the expected RR. However, the short survival compared with ASS1-agnostic historical controls indicates that ASS1 (and frequent PD-L1) loss selects for a biologically more aggressive and immunorefractory NSCLC phenotype. The iTRAP study opening Q2 of 2019 will assess the safety and tolerability of ADIPemPlatinum(Carbo) with atezolizumab in pts with ASS1-deficient non-squamous NSCLC. Clinical trial information: NCT02029690.

A phase II study of ADI-PEG 20 in combination with gemcitabine and docetaxel for the treatment of soft tissue sarcoma.

TPS11079 Background: The combination of gemcitabine (G) and docetaxel (D) is a standard second line therapy for soft tissue sarcoma (STS) with a modest response rate. Recent studies have looked to add agents to enhance response. We have shown that argininosuccinate synthase 1 (ASS1) expression is silenced in 88% of all sarcomas (n = 708) (Bean et al., 2016, Cell Death and Disease), and that this loss is associated with a reliance on extracellular sources of the amino acid arginine. The arginine depleting enzyme PEGylated arginine deiminase (ADI-PEG 20) depletes extracellular arginine. Preclinical studies have demonstrated that arginine starvation and D administration induce c-Myc-driven hENT1 surface expression overcoming intrinsic cell surface G transporter related resistance. To test this hypothesis, we opened this multi-institutional randomized phase II trial examining the safety and efficacy of ADI-PEG 20 with G + D in STS (NCT03449901) in July of 2018. Methods: Eligible patients are adults with metastatic or unresectable histologically or cytologically confirmed FNCLCC grade 2 or 3 STS that would be standardly treated with G and/or D. Patients are treated with ADI-PEG 20 at a dose of 36 mg/m2 via intramuscular injection on Day -7 of Cycle 1 and then on Days 1, 8, and 15 of each subsequent cycle. G will be given intravenously at a dose of 750 mg/m2 over 90 minutes on Days 1 and 8 and D will be given intravenously at a dose of 75 mg/m2 over 60 minutes on Day 8 of each cycle. The median PFS of metastatic sarcoma patients receiving the standard G + D treatment was estimated to be 6.2 months in a randomized phase II study (Maki et. al., 2007, JCO). With the addition of ADI-PEG 20, we target to improve the median PFS to 9 months, a 45.2% (2.8 months or 12 weeks) improvement in patients treated on G + D + ADI-PEG 20 against the null hypothesis median PFS of 6.2 months to achieve 80% power to detect the improvement in PFS at a 5% alpha level. Tumor specimens (pre- and post-ADI-PEG 20 during week -1) and blood are collected for correlative studies including metabolomics, pharmacodynamics, immunogenicity and ASS1 biomarkers. Quality of life will be measured using FACT-G7. Clinical trial information: NCT03449901.

A phase II study of ADI-PEG 20 and FOLFOX6 in patients (pts) with advanced hepatocellular carcinoma (HCC).

TPS477 Background: Arginine depletion interferes with pyrimidine metabolism as well as DNA damage repair pathways, and preclinical data indicate that pairing pegylated arginine deiminase (ADI-PEG 20) with fluoropyrimidines or platinum enhances cytotoxicity in vitro and in vivo in HCC models. A prior phase 1 study of FOLFOX6 and ADI-PEG 20 established the safety and recommended phase 2 dose of the combination in pts with advanced gastrointestinal tumors (Harding et al. CCP 2018). For 23 treatment-refractory HCC pts who were treated at the recommended phase 2 dose on an expansion cohort of the phase 1, the objective response rate (ORR) was 21% (95% CI 7.5-43.7) and median progression-free survival (PFS) was 7.3 months. These data were favorable when compared to historic data for FOLFOX alone where the ORR was ~8% and PFS was 2.93 months and suggest greater clinical activity of the combination (Qin et al. JCO 2013). Prospective confirmation of these results is required. Methods: This is an international, multicenter, single-arm, open-label phase 2 trial of ADI-PEG 20 and FOLFOX6 for advanced HCC pts with Child-Pugh A liver function who progressed on ≥ 2 prior lines of prior systemic therapy (NCT02102022). The primary objective is to define the ORR by RECIST 1.1 as assessed by blinded independent central review. Secondary objectives include determination of safety, disease control rate (DCR), duration of response (DOR), PFS, overall survival (OS), serum arginine, citrulline and anti-ADI-PEG 20 levels over 24 weeks, and alpha-fetoprotein response. Eligible pts receive intravenous FOLFOX6 biweekly at standard doses and ADI-PEG 20 intramuscularly weekly at 36 mg/m.2 Cross-sectional imaging will be completed every 8 weeks until progression of disease. Based on a two-sided exact test of a one-sample proportion with an alpha of 0.05, under a presumed ORR of 22%, there is 80% power to yield 95% confidence interval of 15-26%, which will require 46 objective responses in 225 subjects. Futility will be assessed three times during the study based on having ORR data available for 56, 110, and 166 patients. This Phase 2 will be stopped for futility if the conditional power drops below 20% at any of these time points. Clinical trial information: NCT02102022.

Innate and adaptive resistance mechanisms to arginine deprivation therapies in sarcoma and other cancers

Many cancers lack functional expression of the enzyme argininosuccinate synthetase 1 (ASS1) that is necessary for synthesis of L-arginine. These cancers must import arginine for survival and growth, and this reliance can be targeted by arginine-degrading extracellular enzymatic drugs, most commonly PEGylated arginine deiminase. These enzymes can become targets of the immune system, reducing their effectiveness, but PEGylation improves the in vivo stability. Arginine deprivation causes cell death in some cancers, but others gain resistance by expressing ASS1 after a starvation response is induced. Other resistance mechanisms are possible and explored, but these have not been observed specifically in response to arginine deprivation. Future studies, especially focusing on the mechanisms of ASS1 upregulation and metabolic adaptations, may yield insights into preventing or taking advantage of resistance adaptations to make arginine deprivation therapy more effective.

Efficacy of arginine depletion by ADI-PEG20 in an intracranial model of GBM

Glioblastoma multiforme (GBM) remains a cancer with a poor prognosis and few effective therapeutic options. Successful medical management of GBM is limited by the restricted access of drugs to the central nervous system (CNS) caused by the blood brain barrier (BBB). We previously showed that a subset of GBM are arginine auxotrophic because of transcriptional silencing of ASS1 and/or ASL and are sensitive to pegylated arginine deiminase (ADI-PEG20). However, it is unknown whether depletion of arginine in peripheral blood in vivo has therapeutic activity against intracranial disease. In the present work, we describe the efficacy of ADI-PEG20 in an intracranial model of human GBM in which tumour growth and regression are assessed in real time by measurement of luciferase activity. Animals bearing intracranial human GBM tumours of varying ASS status were treated with ADI-PEG20 alone or in combination with temozolomide and monitored for tumour growth and regression. Monotherapy ADI-PEG20 significantly reduces the intracranial growth of ASS1 negative GBM and extends survival of mice carrying ASS1 negative GBM without obvious toxicity. The combination of ADI-PEG20 with temozolomide (TMZ) demonstrates enhanced effects in both ASS1 negative and ASS1 positive backgrounds.Our data provide proof of principle for a therapeutic strategy for GBM using peripheral blood arginine depletion that does not require BBB passage of drug and is well tolerated. The ability of ADI-PEG20 to cytoreduce GBM and enhance the effects of temozolomide argues strongly for its early clinical evaluation in the treatment of GBM.

Abstract B33: Expansion study of pegylated arginine deiminase (ADI-PEG20), pemetrexed, and cisplatin in patients with ASS1-deficient non-squamous non-small cell lung cancer (TRAP)

Abstract Background: Loss of the metabolic tumor suppressor argininosuccinate synthetase 1 (ASS1) sensitizes non-squamous non-small cell lung cancer (NSCLC) cells to arginine deprivation with pegylated arginine deiminase (ADI-PEG20), which also disrupts thymidine pools and potentiates pemetrexed (Pem) cytotoxicity. In the phase I dose-escalation TRAP study (NCT02029690) we showed that ADI-PEG20 with first-line PEM and cisplatin (Cis) chemotherapy (ADIPemCis) produced a 100% disease control rate (DCR) (n = 9) of ASS1-deficient thoracic cancers, with no additional toxicity (Beddowes et al., 2017). Here, we present the TRAP expansion cohort experience in non-squamous NSCLC. Methods: Good-performance (ECOG 0-1) NSCLC patients (pts) with nonresectable disease were enrolled in a phase I TRAP expansion cohort at the maximum tolerated dose (MTD) of ADIPemCis, using tumoral ASS1 loss as a selection biomarker. Pem (500mg/m2) and Cis (75mg/m2) were given every 3 weeks with weekly IM ADI-PEG20 (36mg/m2) for a maximum of 4 cycles with maintenance ADI-PEG20 or Pem in responding pts. Primary endpoint was tumor response rate (RECIST 1.1), with secondary endpoints including progression-free survival (PFS), overall survival (OS), and toxicity. We measured plasma arginine and citrulline concentrations, ADI-PEG20 antibodies, early molecular responses by 18-FLT-PET/CT, and biopsied pts on progression to explore resistance mechanisms. Results: 21 ASS1-deficient non-squamous NSCLC pts (median age 60) were enrolled out of 70 screened pts; 14% (3/21) had stable brain metastases at entry. Plasma arginine decreased with a reciprocal increase in plasma citrulline. The partial response rate was 48% (n=10/21) with a DCR of 85.7% (n=18/21). Median PFS was 2.6 months and median OS was 5.3 months. 23.8% (n=5/21) pts experienced grade 3/4 treatment-related toxicities, the most common being non-febrile neutropenia (14.3%). A partial metabolic response was detected in 57.1% (n=4/7) pts by 24 hrs of arginine deprivation using 18-FLT-PET/CT with EORTC response criteria. Upregulation of ASS1 expression was observed in 1/3 biopsies on progression. Conclusions: The ADIPemCis regimen is active in ASS1-deficient non-squamous NSCLC pts with a higher than expected response rate. However, the short survival (PFS and OS) compared with ASS1-agnostic historical controls indicates that ASS1 loss selects for a biologically more aggressive phenotype of NSCLC. Further studies are warranted exploring rationale combinations with ADIPemCis in patients with ASS1-deficient non-squamous NSCLC. Citation Format: Melissa Phillips, Teresa Szyszko, Peter Hall, Sukaina Rashid, Ramsay Khadeir, Louise Lim, Jeremy Steele, John Conibear, Paula Wells, Stephen Ellis, Xiaoxing Feng, Jim Thomson, Amanda Johnston, Bor-Wen Wu, John Bomalaski, Simon Pacey, Gary Cook, Peter Szlosarek. Expansion study of pegylated arginine deiminase (ADI-PEG20), pemetrexed, and cisplatin in patients with ASS1-deficient non-squamous non-small cell lung cancer (TRAP) [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr B33.

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.