This was a phase 1/2, open-label, dose-escalation study of arginine deiminase linked via succinimidyl succinate to polyethylene glycol of 20,000 molecular weight (ADI-PEG 20) in subjects with advanced melanoma. ADI-PEG 20 was administered intramuscularly (IM) at escalating doses weekly for 9 weeks (cycle 1) or 8 weeks (subsequent cycles). The primary objectives were to the establish the safety, tolerability, and clinical efficacy of ADI-PEG 20. Secondary objectives included evaluation of the metabolic activity by [18F]-fluorodeoxyglucose positron emission tomography (FDG PET), pharmacodynamics, correlation of immunogenicity with clinical response, and correlation of argininosuccinate synthetase (ASS) tumor expression with clinical response.

A 3+3 design was implemented during phase 1, in which 3 to 6 subjects were enrolled sequentially into the following escalating dose cohorts:

• Cohort 1 (40 IU/m^2)
• Cohort 2 (80 IU/m^2)
• Cohort 3 (160 IU/m^2)

Subjects were monitored for dose-limiting toxicity (DLT) during the first 2 weeks of cycle 1, with DLT defined as any grade 3 or higher toxicity. The maximum tolerated dose (MTD) was defined as the cohort in which < 33% of subjects (ie, 0/3 or 1/6 subjects in a cohort) experienced DLT. In phase 2, the MTD cohort was expanded in up to 25 patients.

Subjects who completed treatment in cycle 1 without DLT were eligible to initiate cycle 2 at week 10 provided that a computed tomography (CT) scan showed either enlargement of existing disease without accompanying symptoms OR stable disease or improvement with no unacceptable toxicity. The same radiologic criteria applied for initiation of subsequent cycles. Subjects could continue to receive study treatment until disease progression.

Inclusion Criteria:

1. Histologically confirmed malignant melanoma, American Joint Committee on Cancer (AJCC) stage III (unresectable) or IV. Subjects with uveal and mucosal melanomas were eligible.

2. Measurable disease using the Response Evaluation Criteria in Solid Tumors (RECIST).

3. Pathology slides reviewed by the Memorial Hospital Department of Pathology or New York University (NYU) Department of Pathology for confirmation of melanoma diagnosis.

4. Karnofsky performance status of 80% or more.

5. Adequate organ and marrow function, as defined below:

   • white blood cell count ≥ 3000/µL
   • absolute neutrophil count ≥ 1500/µL
   • platelet count ≥ 100,000/µL
   • total bilirubin ≤ 2.5 x institutional upper limit of normal (ULN)
   • lactate dehydrogenase ≤ 1.5 x institutional ULN
   • albumin ≥ 3.0 mg/dL
   • creatinine ≤ 2.0 mg/dL

6. Expected survival of at least 3 months.

7. Age ≥ 18 years.

8. Able and willing to give valid written informed consent.

Exclusion Criteria:

1. Receipt of chemotherapy, immunotherapy, or radiotherapy within 3 weeks prior to first dosing of study agent or lack of recovery from adverse events (AEs) due to agents administered more than 3 weeks earlier. For nitrosoureas, at least 6 weeks must have elapsed.
2. Any other malignancy that required concomitant therapy.
3. Any medical condition that could have made it difficult for the subject to complete the full course of treatments, at the discretion of the Principal Investigator or co-Principal Investigators.
4. Metastatic disease to the central nervous system, unless treated and stable.
5. Known human immunodeficiency virus (HIV) positivity.
6. Mental impairment that may have compromised the ability to give informed consent and comply with the requirements of the study.
7. Lack of availability for clinical follow-up assessments.
8. Participation in any other clinical trial involving another investigational agent within 3 weeks prior to enrollment.
9. Pregnant women or women who were nursing. Women of child-bearing potential and sexually active men must have used appropriate contraception during the course of this study. Women of child-bearing potential must not have been pregnant (negative β human chorionic gonadotropin within 2 weeks of treatment) or nursing during treatment.
10. History of seizure disorder.

Safety Analysis Set (all subjects who received at least 1 dose of ADI-PEG 20)

All adverse events (AEs) occurring between the signing of informed consent and the off-study date were documented, regardless of the causal relationship to study drug. AEs occurring after the first dose of study drug were considered treatment emergent (i.e., TEAEs).

AE documentation included onset/resolution dates, severity using NCI CTCAE (v4.0), seriousness, study drug action taken, treatment, and outcome. In summaries, treatment-related AEs included those with a "possible", "probable", or "definite" relationship to study drug; preferred terms were counted only once per subject at the maximum reported grade