Background: ADI-PEG-20 (pegylated arginine deiminase), an arginine degrading enzyme, and FOLFOX each exhibit clinical activity in a HCC pt subset. Preclinical data indicate that arginine depletion interferes with folate and pyrimidine metabolism as well as DNA damage repair pathways, suggesting a role for pairing AGI-PEG-20 with cytotoxic chemotherapy. Methods: This is a single center phase 1 trial of mFOLFOX-6 and ADI-PEG-20 in treatment-refractory advanced GI tumors to assess safety and tolerability, and to identify the recommended phase 2 dose (R2PD). mFOLFOX6 was administered intravenously biweekly at standard fixed doses and ADI-PEG-20 intramuscularly weekly at 18 or 36 mg/m2. Toxicity was assessed by CTCAE v4.03 and efficacy by RECIST v1.1. A HCC RP2D expansion was completed to explore efficacy. Serum arginine, citrulline, and anti-ADI-PEG-20 antibodies were monitored every other week. Results: 17 pts were treated—7 pts with GI tumors in dose escalation and 10 HCC pts in RP2D dose expansion. Histologies included HCC (13), pancreatic (3) and fibrolamellar carcinoma (1). All HCC pts had Child-Pugh A liver function and had progressed on sorafenib. Treatment-related grade ≥ 3 laboratory adverse events (AEs) occurred in 47% of pts, including neutropenia (4), thrombocytopenia (3), anemia (2), lymphocytopenia (2) and hyponatremia (1). Treatment-related grade ≥ 3 clinical AEs occurred in 2 pts, notably grade 3 fatigue and a grade 3 cardiac event not otherwise specified. No dose limiting toxicities, treatment-related deaths, or cases of hepatic failure were observed. Efficacy data for the 13 HCC pts revealed 3 (23%) PR, 6 (46%) SD, 3 PD and 1 NE. Arginine levels were depleted with therapy (mean ± SEM: baseline 85.3 ± 13.0 uM, 8 wks 4 ± 3.0 uM, 16 wk 1.1 ± 0.8uM). Anti-ADI-PEG-20 antibodies were not detected at 8 weeks (mean ± SEM: baseline 0.7 ± 0.5, 8 wks 1.5 ± 1). Conclusions: Concurrent mFOLFOX6 plus ADI-PEG-20 intramuscularly at 36 mg/m2 weekly shows an acceptable safety profile with rapid and durable reduction in serum arginine. Further evaluation of this combination is warranted in advanced HCC pts. Clinical trial information: NCT02102022