Background: The treatment of metastatic or unresectable osteosarcoma after standard chemotherapy remains a significant clinical challenge. Connexin 43 (Cx43) hemichannel has been suggested to be a key regulator of bone homeostasis and represents a new target for bone and breast cancer. ALMB-0168, a first-in-class therapeutic antibody agonist for Cx43 hemichannel, has been shown to suppress the growth and migration of osteosarcoma and breast cancer bone metastases in preclinical studies. Methods: Patients ≥16 years with histologically confirmed osteosarcoma who progressed after standard chemotherapy were eligible. This study consists of accelerated titration followed by a 3+3 design with 7 planned ALMB-0168 dose levels (1, 3, 6, 12, 18, 24, and 30 mg/kg) administered intravenously once every 3 weeks and then dose expansion at the potential recommended phase 2 dose (RP2D). Primary endpoints are safety and tolerability. Adverse events are rated according to the NCI CTCAE v5.0. Key secondary endpoints are overall response rate (ORR) and disease control rate (DCR) assessed using RECIST v1.1. Results: As of August 21, 2022, 14 patients (10 males, 4 females) with median age 27.5 years (range 16–38 years) were enrolled; ECOG PS was 0 in 7 patients (50.0%), 1 in 6 patients (42.9%) and 2 in 1 patient (7.1%). 5 patients received ≥2 prior lines of therapy. Six dose levels (1-24 mg/kg) have been completed in this ongoing study with no dose-limiting toxicities reported. Treatment related adverse events (TRAEs) of any grade occurred in 10 (71.4%) patients and were Grade 3 in 1 patient (infectious pneumonia); no events were Grade 4 or 5. Common TRAEs ( > 10%) were proteinuria (21.4%), anemia (21.4%), hematuria (14.3%), and increased aspartate aminotransferase (14.3%). No Cx43-related cardiac events or severe hepatic events were observed. A total of 13 patients were evaluable for response. ORR was 15.4% (2/13, 95% CI: 1.9–45.5%), including 2 partial responses (PR), 1 patient each at 6 mg/kg and 18 mg/kg. The patient at 6 mg/kg, who had ≥3 prior lines of therapy and lung metastases, achieved durable disease control with stable disease (SD) for 33 weeks followed by PR for 8+ weeks (at the time of analysis). The DCR was 53.8% (7/13, 95% CI: 25.1–80.8%) with 2 PRs and 5 SDs. Conclusions: ALMB-0168 demonstrated encouraging efficacy and tolerable safety in patients with metastatic or unresectable osteosarcoma after standard chemotherapy in a phase 1 dose-escalation trial. Dose escalation is ongoing and dose expansion will start at the potential RP2D levels. Clinical trial information: NCT04886765.