Abstract 4785: Preclinical characterization of the anti-tumor activity of the investigational anti-B7-H3 antibody-drug conjugate (ADC), vobramitamab duocarmazine (vobra duo), in pediatric sarcomas (pSC)

Background: pSC are a heterogeneous group of rare bone and soft tissue malignancies. Vobra duo (MGC018, MacroGenics, Inc., Rockville, MD, USA) is a duocarmycin-based ADC directed against B7-H3 that showed clinical anti-tumor activity in adults as well as preclinical activity in neuroblastoma. Materials and methods: A panel of human pSC cell lines expressing B7-H3, as assessed by flow cytometry, was used. Cytotoxicity, in response to vobra duo, was evaluated in 2D and 3D models. Proliferation was assessed by using the Livecyte instrument. Apoptosis was investigated by annexin V assay. Cell signaling focused on PI3K and its downstream effectors involved in cell proliferation, AKT and S6K, was investigated by phospho-specific western blot assays. The expression of ULK-1, ATG13, LC3A-I, LC3A-II, and LAMP-1, all genes involved in autophagy and lysosomal metabolism, was also studied. An in vivo experiment was carried out in athymic nude mice inoculated sub cutaneously with a rhabdomyosarcoma cell line. Results: In vitro, treatment with vobra duo induced a time-dependent reduction in cell viability in both monolayer and 3D cell culture models. Automated tracking of single-cell proliferation showed that vobra duo led to a significant increase in the doubling time of treated cells compared to untreated ones. A change in cell morphology, underlined by a significant increase of the cell area of vobra duo-treated cells, was observed. Following treatment with vobra duo, a down-modulation of phospho-AKT and phospho-S6K proteins was observed in both A204 (rhabdomyosarcoma) and U2OS (osteosarcoma) cells, the most treatment-sensitive and resistant lines, respectively, suggesting their potential involvement in the vobra duo-mediated anti-proliferative effect. In response to vobra duo, all cell lines underwent apoptotic cell death, although to a different extent and with different kinetics. A significant increase in executioner cleaved caspase-3 was observed, and a partial, although significant, reversion of apoptotic cell death was noted following pre-treatment with the pan-caspase inhibitor, Q-VD-OP-h. The expression of autophagic machinery’s proteins involved in induction, nucleation (ULK-1 and ATG13), elongation and maturation (LC3A-I and LC3A-II) phases, was significantly down-regulated after vobra duo treatment. Moreover, vobra duo significantly affected the expression of the glycosylated form of LAMP-1 protein, a marker of lysosomal membrane integrity and functionality. A single intravenous injection of 3 mg/Kg vobra duo induced a significant tumor growth delay in a mouse rhabdomyosarcoma model. Conclusions: Vobra duo shows anti-tumor activity toward in vitro and in vivo models of pSC. Further in depth pre-clinical assessment to support potential clinical application is warranted. Citation Format: Giovanna Bianchi, Gaia Rolandi, Fabio Pastorino, Daniela Segalerba, Nicoletta Pedemonte, Valeria Tomati, Eleonora Ciampi, Mirco Ponzoni, Chiara Brignole. Preclinical characterization of the anti-tumor activity of the investigational anti-B7-H3 antibody-drug conjugate (ADC), vobramitamab duocarmazine (vobra duo), in pediatric sarcomas (pSC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4785.