Background: Malignant cells rely on buffering oxidative stress through mitochondrial PRX3. Oxidative stress is selectively lethal to cancer cells and may be leveraged therapeutically. RSO-021 is a naturally-occurring, sulfur-rich, cyclic oligopeptide of the thiopeptide class, which covalently inactivates PRX3, leading to catastrophic oxidative stress and cell death. We conducted a phase 1 study to explore safety and identify the maximum tolerated dose (MTD) of intra-pleural RSO-021 in patients with mesothelioma, or other predominantly pleural cancer, with pleural effusion. Methods: MITOPE was a multicenter trial of weekly intrapleural RSO-021 (90, 120, and 180 mg) with a 3 + 3 dose-escalation design. The primary objective was to assess RSO-021 safety/tolerability. Secondary objectives included establishing local and systemic pharmacokinetic profiles and early anti-tumor activity. We defined dose-limiting toxicity (DLT) as grade (G) ≥3 non-hematological AEs with exceptions for some manageable G3 AEs that resolved within 72 h and complicated G4 hematological AEs or those lasting >7 days. We also studied inflammatory and pharmacodynamic markers in local/systemic samples. Efficacy was assessed using mRECIST 1.1 (mesothelioma) and RECIST 1.1 (other tumors). Results: Of 15 patients treated (90 mg/wk, n=7; 120 mg/wk, n=6; 180 mg/wk, n=2), 12 (80%) had ≥3 prior anti-cancer regimens; 12 (80%) had mesothelioma. See table for AE and efficacy data. The commonest all-grade treatment-related AEs (TRAE) were fatigue (33%), pyrexia (20%), and elevated creatinine (20%). Three patients (20%) had DLTs: G3 dyspnea and G3 acute inflammatory response at 120 mg/wk; G3 dyspnea at 180 mg/wk. MTD was 90 mg/wk. Pro-inflammatory cytokines in pleural fluid/serum correlated with AEs suggestive of local inflammation. Prophylaxis mitigated local inflammatory TRAEs. PRX3 target binding in cell pellets from pleural fluid confirmed on-target activity. Systemic exposure was minimal. Disease control was 70% in 10 evaluable patients. The partial response at 90 mg/wk was a 59% reduction in mesothelioma target lesions. Median PFS at MTD was 5.7 months (95% CI, 1.4-NR). Two patients had disease control (DC) >30 wks. Conclusions: Weekly intrapleural RSO-021 was safe with signs of anti-tumor activity in patients with pleural mesothelioma. Phase 2 exploration of this novel agent is ongoing at 2 doses, as a single agent and in combination. Clinical trial information: NCT05278975.