Expression Profile of CD38 and Related Ectoenzymes in Myeloma Bone Niche: A Rational Basis for the Use of Daratumumab to Inhibit Osteoclast Formation and Activity
Authors: Toscani Denise , Bolzoni Marina , Costa Federica , Quarona Valeria , Marchica Valentina , Mancini Cristina , Martella Eugenia , Bonomini Sabrina , Storti Paola , Chillemi Antonella , Accardi Fabrizio , Dalla Palma Benedetta , Craviotto Luisa , Malavasi Fabio , Aversa Franco , Giuliani Nicola 
Published: October 2019

Abstract
The relationship between multiple myeloma (MM) cells and osteoblast (OB)s and osteoclast (OCL)s into the bone marrow (BM) niche has a critical role in the pathophysiology of MM and in the development of bone disease in MM patients.
Recently, Daratumumab (DARA), a human anti-CD38 monoclonal antibody has been developed with broad-spectrum killing activity including complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and, at least in part, modulation of CD38 enzymatic activity. In pre-clinical studies, DARA has been shown to effectively kill MM cells and clinical trials are ongoing. However, the effects of DARA in the context of the MM bone niche and on MM-induced bone remodeling alterations are unknown.
In this study, we have firstly evaluated the expression profile of CD38 and its related ectoenzymes by bone niche cells and, thereafter, we investigated the effect of DARA on OCL and OB formation and bone remodeling. The study design included immunohistochemistry for CD38, CD73, CD39, CD203a (PC-1), CD157 and CD31 on bone biopsies in a cohort of 38 patients with newly diagnosed MM and 14 patients with monoclonal gammopathy of uncertain significance (MGUS). Moreover, the expression profile of these antigens was performed by flow cytometry on primary BM CD138+ cells (sample number=16), mesenchymal stromal cells, OBs, monocytes and OCLs and on the related human cell lines.
As expected, we found that CD38 was highly expressed by MM cells that were also positive for CD203a and for CD39 and CD31 at variable level but not for CD157 and CD73. CD38 was also expressed by BM monocytes but not by OBs, OCLs and BM stromal cells. Interestingly, we found that OBs expressed CD73 and CD203a. Any significant difference was not observed in the expression of CD38 and related ectoenzymes between MM and MGUS patients. In line with CD38 expression profile by MM cells and the niche, we further demonstrated that DARA binds both MM cells and monocytes, but not OBs and OCLs. Consistently, we lack to find any significant effect of DARA on OB formation from BM stromal cells or OBs proliferation and survival. Thus, we investigated the effect of DARA (1-25 ug/ml) as compared to human IgG isotype control on OCL formation and activity in the presence of RANKL and M-CSF, using either CD138- cell fraction or purified CD14+ cells from MM BM samples. OCLs were evaluated by both TRAP staining and a fluorimetric osteolysis assay. We found that DARA, between 10 and 25 ug/ml, with a dose dependent effect, significantly inhibited OCL formation and activity from BM mononuclear cells and from the CD138- cell fraction, but not from purified CD14+ cells. The inhibitory effect on OCL formation by DARA was observed when the antibody was present for all the culture period (21-28 days). On the other hand DARA did not show any effect on late OCL progenitors and mature OCLs. Accordingly CD38 expression by monocytes cultured in a pro-osteoclastogenic medium disappeared after 7 days.
In conclusion, our data indicate that DARA inhibit osteoclastogenesis, likely mediated by ADCC, targeting monocytes and early OCL progenitors. This evidence supports the use of an anti-CD38 based approach as a treatment for MM bone disease.

Disclosures
Malavasi: Janssen: Honoraria, Research Funding. Giuliani:Celgene Italy: Other: Research Grant; Janssen Pharamceutical: Other: Research Grant.