1726PA peptidase-potentiated alkylating agent melflufen is effective anti-neoplastic agent in osteosarcoma

Abstract Background High-grade osteosarcoma (HGOS) is the most common primary malignant bone tumor. In metastatic patients, survival rates are poor and remained virtually unchanged over the past 30 years. Furthermore, recent randomized trials failed to improve the efficacy of current chemotherapy. Here we tested the anti-neoplastic activity of a novel peptide-conjugated alkylator melflufen in preclinical osteosarcoma setting. Due to its high lipophilicity melflufen rapidly enters tumor cells where it is immediately cleaved by peptidases leading to entrapment and enrichment of alkylating payload. Overexpression of peptidases is often seen in tumor cells and can thus lead to increased melfufen sensitivity. In osteosarcoma, over-expression of aminopeptidase N (ANPEP/CD13) has been associated with poor survival and metastasis suggesting that melflufen may represent a valid new therapeutic option. Methods Gene expression of ANPEP/CD13 in patient samples from GEO datasets was analyzed using standard tools. Anti-neoplastic activity of melflufen and was evaluated in a panel of 11 osteosarcoma cell lines including early passage patient-derived primary lines. In vitro anti-neoplastic activity was examined using survival and apoptosis assays. In vivo activity of melflufen was tested in a chicken embryo xenograft model. Results Melflufen has demonstrated superior anti-neoplastic activity in comparison to other alkylating agents including melphalan, cyclophosphamide, ifosfamide, busulfan, and bendamustine. Melflufen was active in all osteosarcoma cell lines including the cells resistant to methotrexate, etoposide, and talazoparib. Anti-neoplastic activity of melflufen could be hindered by ANPEP inhibitor bestatin. Furthermore, in vivo administration of melflufen reduced tumor growth and metastasis of highly aggressive 143B osteosarcoma cells in a xenograft model. Conclusions Melflufen has demonstrated promising results in preclinical osteosarcoma model showing anti-neoplastic activity superior to other alkylating agents and PARP inhibitor talazoparib. Melflufen’s favorable toxicity profile suggests that it might represent a novel valid therapeutic approach for osteosarcoma patients with poor response to chemotherapy and metastasis. Legal entity responsible for the study The authors. Funding Oncopeptides AB. Disclosure A. Slipicevic: Full / Part-time employment: Oncopeptides AB. K. Byrgazov: Research grant / Funding (self): Oncopeptides AB. F. Lehmann: Full / Part-time employment: Oncopeptides AB; Leadership role: EpiEndo Pharmaceuticals. T. Lion: Honoraria (self), Advisory / Consultancy, Research grant / Funding (self): Incyte. All other authors have declared no conflicts of interest.