Background: ADI-PEG 20 exploits the different metabolic ability for synthesizing arginine (Arg) between normal and neoplastic cells to reduce tumor cell growth. Preclinical and clinical studies have shown that cancers which are either Arg auxotrophic, with silencing of argininosuccinate synthetase (ASS1), or Arg non-auxotrophic, respond to ADI-PEG 20 monotherapy or ADI-PEG 20 combined with various chemotherapies, respectively. ADI-PEG 20 has shown efficacy as monotherapy in ASS1 negative mouse glioblastoma (GBM) models and the combination of ADI-PEG 20 with temozolomide (TMZ) and with radiation (RT) in both ASS1 negative and ASS1 positive mouse GBM models. Based on these rationales, ADI-PEG 20 was added to standard RT + TMZ in patients with newly diagnosed GBM. This is the first clinical trial combining ADI-PEG 20 with RT. Methods: This phase IB, open-label, single-arm, standard 3+3 dose escalation with a recommended phase 2 dose (RP2D) expansion study (NCT04587830) was initiated in June 2020. Weekly ADI-PEG 20 is added to concurrent RT + TMZ and to 6 cycles of adjuvant TMZ (Stupp protocol). ADI-PEG 20 could be continued for up to 2 years. RANO criteria are used to determine response by evaluating MRI at 1, 3 and 6 months after RT, and every 3 months thereafter. Major eligibility criteria are age 20-75 years with newly diagnosed, histologically confirmed GBM with Karnofsky performance status ≥ 60. Endpoints include safety, pharmacodynamics, immunogenicity, progression free survival (PFS) and overall survival (OS). Results: Cohorts 1 (18 mg/m²) and 2 (36 mg/m²) were completed without dose limiting toxicity (DLT). Enrollment to cohort 3 (RP2D phase, 36 mg/m²) is ongoing with 23/26 patients. The major adverse events (AEs) were fatigue (52%), constipation (39%) and neutrophil decrease (39%). Dermatologic or allergic reactions occurred in 12/23 (52%), and all were grade 1-2 except for anaphylactic shock in 1 and vasculitis/rash in 1. 22/23 are alive, with median PFS = 9.5 months. The first 6 study patients are all alive for at least 11 months, with the longest at 1.5 years. 10 are off treatment due to progressive disease in 6, severe AE in 2, consent withdrawal in 1, and medical decision in 1. Mean peripheral blood Arg levels were suppressed ( < 10uM) for 4-6 weeks in most subjects, with a reciprocal elevation of citrulline levels. Anti-ADI-PEG 20 antibodies tended to increase as peripheral Arg levels increased. Conclusions: The addition of ADI-PEG 20 to RT + TMX was safe, and no DLT was observed. The RP2D of ADI-PEG 20 was determined to be 36mg/m². AEs were those typically seen with RT + TMZ, with perhaps an increase in rash reported with the addition of ADI-PEG 20. Anaphylaxis and vasculitis were seen (1 subject with each), and have been observed previously with ADI-PEG 20. The preliminary OS data are encouraging. A registration phase 2/3 trial of this triplet is being considered. Clinical trial information: NCT04587830.