Daratumumab, a Novel Potent Human Anti-CD38 Monoclonal Antibody, Induces Significant Killing of Human Multiple Myeloma Cells: Therapeutic Implication.
Authors: Tai Yu-Tzu , de Weers Michel , Li Xian-Feng , Song Weihua , Nahar Sabikun , Bakker Joost M , Vink Tom , Jacobs Daniëlle , Oomen Lukas , Bleeker Wim K , Munshi Nikhil C. , van de Winkel Jan G. J. , Parren Paul W. H. I. , Richardson Paul , Anderson Kenneth C. 
Published: October 2019

Abstract
Abstract 608
CD38 is a type II transmembrane glycoprotein highly expressed in many hematological malignancies including multiple myeloma (MM), representing a promising target for monoclonal antibody (mAb)-based targeted immunotherapy. We here investigated the mechanisms of anti-MM activity of daratumumab, a novel anti-CD38 mAb currently in clinical development, to treat human MM. Daratumumab was generated in human Ig transgenic HuMab-mice® and was selected for its excellent binding characteristics. It strongly reacts against a panel of CD38-expressing MM cell lines (>94%, 17/18) that are sensitive or resistant to current treatments for MM: median fluorescence intensity (MFI) observed was 378-20764 and 15-140 for daratuzumab and isotype control Ab, respectively. U266 cells, which lack CD38 mRNA, did not bind daratuzumab. Significantly, daratuzumab induced NK-mediated antibody-dependent cellular cytotoxicity (ADCC) against CD38-expressing MM cells but not CD38-negative U266 cells, confirming its specific targeting. Daratuzumab-mediated MM cell lysis was dose-dependent, with maximum lysis of the CD38-expressing MM cell lines (n=17) at concentrations of 0.01-0.1μg/ml. We also determined potential effects of daratumumab on NK and peripheral blood mononuclear cells (PBMC) effector cells, since these cells express CD38, albeit at lower levels than MM cells. Overnight pretreatment of PBMCs or purified NK cells from 2 normal donors with daratumumab (2 μg/ml) did not significantly alter daratumumab-induced ADCC against MM1S target cells. The bone marrow (BM) microenvironment has been shown to protect MM cells against apoptosis and stimulate their growth; we therefore next asked whether daratumumab effectively induced ADCC against MM cells in the presence of BM stromal cells (BMSCs). Daratumumab induced a similar, dose-dependent, ADCC of dexamethasone-sensitive MM1S as well as dexamethasone-resistant MM1R cells both in the presence and absence of BMSCs, suggesting that daratumumab could indeed effectively induce ADCC in the BM microenvironment. Significantly, our findings translated to patient cells, since daratumumab induced ADCC of both allogeneic (n=2) and autologous (n=9) patient MM cells. We next turned our attention to complement-mediated cytotoxicity (CDC). In calcein AM-labeling assays in the presence of normal human serum, daratumumab induced dose-dependent CDC of LP-1 and XG-1 MM cells expressing low levels of CD59 and CD55 both in the absence or presence of BMSCs. Effective daratumumab-induced CDC of CD138-purified MM cells was seen for 6 of 9 patients. Daratumumab-induced CDC was not affected by the expression of complement inhibitor proteins in this cohort of patients. Finally, daratumumab induced apoptosis in Ramos and Daudi cells when crosslinked by anti-IgG antibody, and it induced cell death in CD138-purified patient MM cells, as assessed by annexin V/PI staining. Taken together, our results indicate that daratumumab is able to effectively kill patient MM tumor cells via several cytotoxic mechanisms, supporting clinical development of daratumumab to improve patient outcome in MM.

Disclosures:
de Weers: Genmab: Employment. Bakker:Genmab: Employment. Vink:Genmab: Employment. Jacobs:Genmab: Employment. Oomen:Genmab: Employment. Bleeker:Genmab: Employment. Munshi:Millennium Pharmaceuticals: Honoraria, Speakers Bureau. van de Winkel:Genmab: Employment. Parren:Genmab: Employment. Richardson:Keryx Biopharmaceuticals: Honoraria. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.