P1/2, N=35, Recruiting, Washington University School of Medicine | Not yet recruiting --> Recruiting

P1/2, N=35, Not yet recruiting, Washington University School of Medicine | Trial completion date: Nov 2027 --> Feb 2028 | Initiation date: Jan 2024 --> Apr 2024 | Trial primary completion date: Nov 2025 --> Feb 2026

P1/2, N=30, Recruiting, M.D. Anderson Cancer Center | Not yet recruiting --> Recruiting

P1/2, N=35, Not yet recruiting, Washington University School of Medicine | Trial completion date: Aug 2027 --> Nov 2027 | Initiation date: Oct 2023 --> Jan 2024 | Trial primary completion date: Aug 2025 --> Nov 2025

P1/2, N=0, Withdrawn, M.D. Anderson Cancer Center | N=10 --> 0 | Trial completion date: May 2025 --> Nov 2023 | Initiation date: Feb 2024 --> Aug 2023 | Not yet recruiting --> Withdrawn | Trial primary completion date: May 2025 --> Nov 2023

P1/2, N=30, Not yet recruiting, M.D. Anderson Cancer Center

Importantly, mechanistic analysis indicated that the processes of the cell cycle, ribonucleoprotein complex biogenesis, and ribosome biogenesis were upregulated after combination treatment. This study implied the possibility of L-Norvaline as a modulator of the immune response in cancer and provided a new potential therapy combined with ADI-PEG 20.

Collectively, our data indicate that ADI-PEG20-inducible cytokines orchestrate argininosuccinate fuelling of ASS1-deficient mesothelioma by macrophages. This novel stromal-mediated resistance pathway may be leveraged to optimize arginine deprivation therapy for mesothelioma and related arginine-dependent cancers.

P1/2, N=108, Recruiting, Washington University School of Medicine | Not yet recruiting --> Recruiting

Gene expression patterns of MCF-12A and MCF-7 were very similar.In conclusion, metabolic fingerprinting reveals complex and differential regulation of L-arginine pathways in breast cancer cell lines. These analyses may help to understand differences in proliferation during L-arginine depletion, thus offering a perspective to further personalize treatment options within classical molecular breast cancer subtypes.

Therapeutic utilization of a metabolic defect in GBM along with cytostatic therapy provides a novel combination approach. Whether this SpyADI/MitA regimen will provide a safe alternative to combat GBM, will have to be addressed in subsequent (pre-)clinical trials.

Treatment of established tumors with the arginase inhibitor CB-1158 exhibited further increased CD8 T cell infiltration, beyond that seen with the macrophage-specific knockout, and sensitized the tumors to anti-PD1 immune checkpoint blockade. Our data demonstrate that Arg1 drives immune suppression in pancreatic cancer by depleting Arginine and inhibiting T cell activation.

Methods Cell lines were treated with ADI-PEG20 and A-1331852 (Bcl-xL inhibitor) and monitored for cell death using the Incucyte. Bcl-xL inhibition synergies with ADI-PEG20 to induce apoptosis. This study provides the preclinical rationale for combining ADI-PEG20 with next generation Bcl-xL inhibitors such as the Bcl-xL PROTAC in a phase I clinical trial.

P1, N=9, Completed, Memorial Sloan Kettering Cancer Center | Active, not recruiting --> Completed | Trial completion date: Apr 2024 --> Jan 2023 | Trial primary completion date: Apr 2024 --> Jan 2023

Alterations in arginine metabolism may sensitise cells to metabolic drugs and we find that BAP1-negative/ASS1-expressing MPM cell lines are more sensitive to ASS1 inhibition, although not to inhibition of purine synthesis by mizoribine. Importantly, BAP1w-/KO MeT5A become desensitised to arginine-deprivation by pegylated arginine deiminase (ADI-PEG20), phenocopying BAP1-negative/ASS1-expressing MPM cell lines. Implications: Our data reveal an inter-relationship between BAP1 and arginine metabolism, providing a potential means of identifying epithelioid MPM patients likely to benefit from ADI-PEG20.

P1, N=9, Active, not recruiting, Memorial Sloan Kettering Cancer Center | Trial completion date: Apr 2023 --> Apr 2024 | Trial primary completion date: Apr 2023 --> Apr 2024

The WWOX-rs13338697-GG genotype was associated with lower tissue WWOX and ASS1 levels and higher pretreatment plasma arginine levels, resembling an arginine auxotrophic phenotype requires excessive extracellular arginine supply. Silencing WWOX to mimic HCC with the WWOX-rs13338697-GG genotype further stimulated HCC cell response to hypoxia through increased HIF1A expression, leading to further reduction of ASS1 and thus increased cell susceptibility to ADI-PEG 20.

The fusion protein demonstrated selective cytotoxicity and reduced drug resistance in melanoma cells, thus would be a promising strategy for the improved efficacy in melanoma treatment. KEY POINTS: • Fusion of ADI with 30Kc19α enhances soluble expression and productivity of recombinant ADI in E. coli • 30Kc19α protects ADI from the proteolytic degradation by shielding effect, helping ADI to remain active • Intracellular delivery of ADI by 30Kc19α overcomes ADI resistance in melanoma cells by degrading intracellularly expressed arginine.

Mechanistically, ANXA2 signaled through the TLR2/MYD88 axis in neutrophils to induce ARG1 mRNA expression. The current study describes what we believe to be a novel mechanism by which ARG1 mRNA expression is regulated in neutrophils in cancer and highlights the central role that neutrophil lineage cells play in the suppression of tumor-infiltrating lymphocytes.

Small cell lung cancer (SCLC) displays marked arginine auxotrophy due to inactivation of the rate-limiting enzyme argininosuccinate synthetase 1 (ASS1), and as a consequence may be targeted with pegylated arginine deiminase or ADI-PEG20 (pegargiminase) and human recombinant pegylated arginases (rhArgPEG, BCT-100 and pegzilarginase). Moreover, recent work has identified an intriguing role for targeting arginine in combination with PD-1/PD-L1 immune checkpoint inhibitors and clinical trials are in progress. Thus, future studies of arginine-depleting agents with chemoimmunotherapy, the current standard of care for SCLC, may lead to enhanced disease control and much needed improvements in long-term survival for patients.

Imaging with proliferation biomarker 3'-deoxy-3'-[F] fluorothymidine (F-FLT) positron emission tomography (PET)-computed tomography (CT) was performed in a phase 1 study of pegargiminase with pemetrexed and cisplatin (ADIPemCis). Both early and late FLT PET-CT provide evidence of response to ADIPemCis therapy in MPM and NSCLC. We provide first-in-human FLT PET-CT data in MPM, indicating it is comparable with modified RECIST.

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.

The capacity of iNKT cells to restore antigen-specific T cell immunity was similarly demonstrated against myeloid-derived suppressor cells (MDSCs) in wild-type and Jα18 syngeneic lymphoma-bearing models in vivo. Thus, stimulation of iNKT cell activity has the potential as an immunotherapy against AML or as an adjunct to boost antigen-specific T cell immunotherapies in haematological or solid cancers.

These results demonstrated that LAP3 mediated IFN-γ-induced arginine depletion to malignant transformation of BMECs. Our findings provide a potential therapeutic target for breast cancer both in humans and dairy cows.

Pegylated arginase (BCT-100) is currently in phase I/II trials in relapsed pHGG. Our results suggest that therapeutic arginine depletion may also be useful in other tumour types and IHC analysis of patient tumour samples could help identify patients likely to benefit from this treatment.

This study aims at investigating the safety and tolerability of a triple combination consisting of ipilimumab and nivolumab immunotherapy and the metabolic therapy, ADI-PEG 20. Further, an IFNg-dependent inflammatory signature was observed in metastatic lesions in patients pre-treated with ICB compared with patients with no pretreatment. Multiplex immunohistochemistry demonstrated variable presence of tumor infiltrating CD8 lymphocytes and PD-L1 expression at the baseline in metastases.

ADIPemCis was well tolerated with modest disease stabilisation in metastatic UM. Further investigation of arginine deprivation is indicated in UM including combinations with immune checkpoint blockade and additional antimetabolite strategies.

ADI-PEG20 not only enhances the cellular sensitivity of Arginine succinate synthetase 1 positive GBM to ionising radiation by elevated production of nitric oxide (NO) and hence generation of cytotoxic peroxynitrites, but also promotes glioma-associated macrophages/microglia infiltration into tumors and turns their classical anti-inflammatory (pro-tumor) phenotype into a pro-inflammatory (anti-tumor) phenotype. Our results provide an effective, well-tolerated and simple strategy to improve GBM treatment which merits consideration for early evaluation in clinical trials.

Furthermore, the results also revealed a significant reduction in activities of ALT, AST, and ALP in the Peg-Arginase group compared to MNU untreated or native-Arginase groups, suggesting that Peg-Arginase may eliminate the hepatotoxic effect of the MNU. All in all, our results suggested that the arginine-depleting enzyme, Peg-Arginase, exerted an anticancer effect against both T-47D and MDA-MB-231 breast cancer cell lines through cell proliferation inhibition and apoptosis induction.

P1, N=9, Active, not recruiting, Memorial Sloan Kettering Cancer Center | Trial completion date: Apr 2022 --> Apr 2023 | Trial primary completion date: Apr 2022 --> Apr 2023

Concurrent IM injection of ADI-PEG 20 at 36 mg/m weekly and intravenous infusion of PLD at 20 mg/m biweekly had an acceptable safety profile in patients with advanced ASS1-deficient solid tumors. Further evaluation of this combination is under discussion.

Arginine deprivation with the therapeutic enzyme ADI-PEG20 (pegylated arginine deiminase) sensitises cancers deficient in the enzyme argininosuccinate synthase (ASS1) to the apoptosis initiating activity of TRAIL through tumour cell surface upregulation of death receptors DR4 and DR5...Conclusion Our study proposes a synergistic interaction between the arginine depleting enzyme ADI-PEG20 and anti-CD19 CAR-T for the treatment of ASS1 deficient B-ALL, whereby priming of death receptor signalling may underlie enhanced CAR-T cytotoxicity against CD19 + tumour cells. These data support an emerging framework for CAR-T optimisation based on targeting of the death receptor mediated extrinsic apoptosis pathway and can inform future refinements in the development of cellular immunotherapy.

The dose expansion of ADIPemCis confirmed the high clinical activity and good tolerability in ASS1-deficient poor-prognosis mesothelioma, underpinning an ongoing phase 3 study (ClinicalTrials.govNCT02709512). Notably, resistance to pegargiminase correlated with marked macrophage recruitment and-along with the tumor immune microenvironment-warrants further study to optimize arginine deprivation for the treatment of mesothelioma.

In the TRAP Phase 1 trial (NCT02029690) ADI-PEG 20 combined with 1st-line pemetrexed (PEM) and cisplatin (CDDP) chemotherapy revealed a 94% disease control rate (34/36) in MPM with a 33.3% partial response rate in biphasic and sarcomatoid subtypes...Patients who developed CDDP toxicity were switched to carboplatin... ATOMIC-meso is the largest first-line triplet chemotherapy study to assess the role of targeted arginine deprivation in aggressive subtypes of mesothelioma on a global scale (US, Europe, and Australasia). The overall blinded data is encouraging and an interim analysis for ORR is planned once the 176 patients have reached the first CT treatment response assessment scan. Pending a Go decision, a total of up to 386 patients will be randomized to complete ATOMIC-meso (NCT02709512).

Furthermore, the potent anti-leukemia activities of NEI-01 were observed in three different types of mouse models including human cell line-derived xenograft (CDX), mouse cell line-derived homografts in syngeneic mice and patient-derived xenograft (PDX). This preclinical data provide strong evidence to support the potential use of NEI-01 as a therapeutic approach in AML treatment.

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.

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.

ARG2 and ASS1 enzymes are extensively expressed in NSCLC stroma and cancer cells, respectively. Auxotrophic tumors have a poor prognosis, potentially by utilizing Arg, thus reducing Arg-dependent TIL anti-tumor activity. ASS1 expression in cancer cells would allow Arg fueling of TILs and enhance anti-tumor immunity. However, upregulation of ARG2 in CAFs may divert Arg from TILs, allowing immune escape. Identification of these three distinct phenotypes may be useful in the individualization of Arg-targeting therapies and immunotherapy.

ADI-PEG 20 treatment increased T cell infiltration in the low PD-L1 tumor microenvironment. The recommended phase 2 doses are 36 mg/m weekly for ADI-PEG 20 and 200 mg every 3 weeks for pembrolizumab.