CD14+CD16+ Monocyte Binding to Myeloma Cells Is Required for Daratumumab Dependent Killing in Multiple Myeloma Patients
Authors: Storti Paola , Vescovini Rosanna , Marchica Valentina , Bolzoni Marina , Costa Federica , Vicario Emanuela , Toscani Denise , Dalla Palma Anna Benedetta , Accardi Fabrizio , Malavasi Fabio , Aversa Franco , Giuliani Nicola 
Published: October 2019

Abstract
Recently, the introduction of anti-CD38 monoclonal antibody, Daratumumab (DARA), in multiple myeloma (MM) therapy has improved the response rate of relapsed MM patients. However only a fraction of the DARA-treated patients respond, thus further studies on DARA mechanisms of action are needed. Because the antibody dependent cellular phagocytosis (ADCP) mediated by monocyte, is one of the mechanisms through DARA exerts its anti-MM activity, an ex-vivo approach was established in order to investigate which mechanisms or patient's immunological characteristics could influence DARA-mediated killing of MM cells.
Bone marrow mononuclear cells (BM-MNCs) obtained from 25 MM patients (12 newly diagnosed and 13 relapsed MM) were analyzed at time 0 (T0) by flow cytometry. We checked the % of plasma cells (PCs) (CD138+ cells), % of total monocytes (CD14+ cells) and their two subsets (CD14+CD16- or CD14+CD16+ cells), the ratio between % of CD14+ and % of CD138+ cells (CD14+:CD138+ ratio) and the median fluorescence intensity (MFI) of CD38 and of the immuno-check points CD47 on PCs and CD172a (SIRPα) on monocytes. Subsequently, BM-MNCs were treated with control IgG (10µg/ml), DARA (10µg/ml) and the F(Ab)2 portion of DARA (DARA F(Ab)2) (10µg/ml) for 48 hours and then the % variation of surviving 7AAD- CD138+ cell, the modification of the % of monocyte or subsets analyzed and the % of PCs that are attached to monocyte (identified as CD138+ cells also positive to CD14) were evaluated by flow cytometry.
Firstly, we found that DARA significantly exploited its anti-MM activity (median % variation of surviving CD138+ cells: 69.05%, p=0.0007) compared to IgG control while DARA F(Ab)2 did not have any killing effect (median % variation of surviving CD138+ cells: 97.33%) compared to the control.
Secondly, we detected that the % of total of monocytes and their subsets (CD14+CD16+ or CD14+CD16-) composition were not modulated by DARA or DARA F(Ab)2 treatment compared to IgG. Indeed, only in presence of DARA, we observed that a double positive population of CD138+CD14+ cell significantly increased compared to IgG control (mean %: 15.76 vs 3.14, p=<0.0001) and to DARA F(Ab)2 condition (mean %:15,76 vs 1,48, p=<0.004). Moreover, we found that monocytes involved in the double positive population CD138+CD14+ were almost completely CD14+CD16+ monocytes. Indeed, we have divided the patient' samples in high responder (HR) and low responder (LR) based on the median % of survival cell after DARA treatment (HR: <69.05% and LR:>69.05% of surviving PCs). Interestingly, in the HR group compared to LR group were significantly increased the % of double positive CD138+CD14+ (median % HR:21.59 vs LR: 7.41, p=0.035), the % of CD138+ cells bonded to CD14+CD16+ (median % HR:13.28 vs LR: 3.93, p=0.048) and the CD14+:CD138+ ratio measured at T0 (median HR:0.63 vs LR: 0.32, p=0.0158). Moreover, in 5 relapsed MM patient, we have correlated the ex-vivo response to DARA with the type of in vivo response after DARA single-agent treatment.
In addition in our cohort of patients, we observed that the % of surviving CD138+ cells after DARA treatment negatively correlate with the CD14+:CD138+ ratio at T0 (r:-0.628, p=0.0023), with the % of CD138+CD14+ population (r:-0.602, p=0.0039) and with the % of CD138+CD14+CD16+ population (r:- 0.657, p=0.0238) but did not correlate with the MFI of CD38 expression on PCs and the % of CD14+CD16+ at T0.
Finally, to go further inside the mechanism involved in DARA response, we have explored the role of the inhibitory axis CD47-CD172a in the ADCP DARA-induced in 5 ex-vivo samples from our cohort of MM patients. We found that the MFI of CD47 strongly positively correlated with the % of surviving CD138+ cells (r: 0.9897; p=0.010) after DARA treatment; on the other hand, we have not reported any correlation with the MFI of CD172a on monocytes. The results of these analyses are continuously updating increasing the number of samples tested.
In conclusion, these data highlight that monocyte binding to PCs, in particular those CD14+CD16+, plays a central role in DARA killing effect independently of % subset composition in the pre-treatment samples, suggesting that there are mechanisms that regulate the effectiveness of the monocyte-based killing effect on malignant PCs, as the immune-suppressive axis CD47-CD172a, giving the rationale design to identify new strategies to increase the efficiency of DARA-based therapeutic regimen.

Disclosures
Malavasi: Takeda Pharmaceutical Co: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen Pharmaceutica: Membership on an entity's Board of Directors or advisory committees, Research Funding. Aversa:Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas: Honoraria; Basilea: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Honoraria; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees. Giuliani:Takeda Pharmaceutical Co: Research Funding; Celgene Italy: Other: Avisory Board, Research Funding; Janssen Pharmaceutica: Other: Avisory Board, Research Funding.