Background Melflufen (melphalan flufenamide) is a first-in-class peptide-drug conjugate (PDC) that targets aminopeptidases and rapidly releases alkylating agents into tumor cells. Osteolytic lesions are a hallmark of multiple myeloma and occur in almost all MM patients, causing severe bone pain and pathological fractures. Previous reports indicate that melphalan enhances osteoclastogenesis. On other other hand, melflufen is able to suppress proliferation of malignant myeloid cells including osteoclast precursors, monocytes. In the present study we therefore evaluated the direct effects of melflufen and melphalan on human osteoclastogenesis using cultured peripheral blood CD14+ monocytes. Methods CD14+ osteoclast precursors were isolated from peripheral blood. Osteoclastogenesis was stimulated by adding RANKL and M-CSF to CD14+ osteoclast precursors and assessed by tartrate-resistant acid phosphatase (TRAP) staining using para-nitrophenyl phosphate (PNPP). Osteoclastic activity was assessed by two assays. First, TRAP activity was measured by quantification of resorption pits on osteo assay synthetic surface made of crystalline calcium phosphate coating mimicking living bone material. Second, collagen degradation was quantified by release of C-terminal telopeptide of type I collagen (CTx) from mineralized bone slices. The proliferation of mature osteoclasts and their precursors in the presence of melflufen and melphalan was assessed using cytotoxicity assays. RNA isolated from osteoclast precursors and mature osteoclasts was subject to gene expression analysis by microarray. Results Melflufen inhibited RANKL-induced osteoclast formation at 1 µM concentration, while 25 µM melphalan was needed to initiate osteoclastogenesis inhibition. However, only extremely high concentrations of melflufen (10 µM) and melphalan (100 µM) affected mature osteoclasts and their TRAP activity. Cytotoxicity assays have demonstrated that melflufen, but not melphalan, can inhibit proliferation of monocytes, osteoclast progenitor cells, at clinically relevant concentrations. When added during osteoclastogenesis, melflufen has significantly inhibited TRAP phosphatase activity on crystalline calcium phosphate-coated surface at 1 µM, whereas 25 µM of melphalan was required to achieve the same effect. As for collagen degradation, the addition of 1 µM melflufen completely suppressed the CTx fragment release. In a parallel assay, 10 µM of melphalan was necessary to produce the same effect. Noteworthy, we did not observe any positive effect of melphalan on osteoclastogenesis or proliferation of osteoclasts. Gene expression analysis showed that mature osteoclasts express higher levels of bone lytic genes ACP5/TRAP, CTSK, MMP2, MMP7, and MMP9. In addition, CD276 and PDCD1LG2 mRNAs encoding B7 family immune checkpoint molecules B7-H3 and PD-L2 are also strongly up-regulated upon osteoclastogenesis. Discussion Myeloma patients often suffer from severe bone pain associated with osteolytic bone lesions. These lesions are the result of excessive osteoclastogenesis induced by multiple myeloma in the bone marrow microenvironment. Moreover, this process creates an immunosuppressive microenvironment since osteoclasts express immune checkpoint molecules known to negatively control activity of T lymphocytes (PD-L2, B7-H3) and NK cells (B7-H3). In this study, we have shown that a novel anti-myeloma PDC melflufen efficiently inhibits osteoclastogenesis and associated collagen degradation and bone resorption when used at clinically relevant concentrations. Melphalan could also induce a reduction of bone resorption and collagen release, however high and clinically irrelevant concentrations were necessary to obtain these effects. Given these results, it is plausible that melflufen-treated MM patients might achieve a reduction in bone pain, and this should be assessed in more detail in the follow-up studies. Also, elimination of immunosuppressive osteoclasts by melflufen may provide a synergistic effect between melflufen and immunotherapy while treating MM patients. This hypothesis is currently being tested in clinical trials. Disclosures Byrgazov: Oncopeptides AB: Current Employment. Gullbo:Theradex: Current Employment; Oncopeptides AB: Consultancy, Current Employment, Current equity holder in publicly-traded company. Slipicevic:Oncopeptides AB: Current Employment. Lehmann:Oncopeptides AB: Current Employment, Current equity holder in publicly-traded company. Larsson:Oncopeptides AB: Current equity holder in publicly-traded company. Fryknäs:Oncopeptides AB: Research Funding.