The therapeutic human CD38 antibody daratumumab improves the anti-myeloma effect of newly emerging multi-drug therapies

Multiple myeloma (MM) is an incurable malignancy of antibody-producing clonal plasma cells. The mean life expectancy of patients has remained at o3 years during the past few decades. The introduction of a new generation drug regimens including an immunomodulatory agent such as lenalidomide (LEN) or a proteasome inhibitor such as bortezomib (BORT) used alone or in combination with classical anti-MM drugs melphalan (MEL), dexamethasone (DEX) or prednisone (PRED) has significantly improved the overall survival of MM patients. All treatment strategies, including multidrug regimens, however, are eventually hampered by the development of drug resistance. Targeted immunotherapy, based on human antibodies against relevant tumor antigens has shown to be a feasible and highly promising approach in hematological malignancies that can be effectively combined with chemotherapy to further increase the potency of anti-tumor effects. For instance in several B-cell malignancies, clinically approved human antibodies against CD20 are now being successfully combined with fludarabine, cyclophosphamide or LEN. Building on this knowledge and to achieve a similar goal in the MM setting, we recently generated daratumumab (DARA), a human CD38 antibody with broad-spectrum killing activity. We have shown that DARA mediates strong lysis of MM cells via CDC (complement-dependent cytotoxicity) as well as ADCC (antibody-dependent cellular cytotoxicity), although the potency of autologous ADCC was donor-dependent. In our initial work to combine DARA with novel chemotherapeutics, we have demonstrated that DARA-mediated cellular lysis of MM cells is significantly improved by LEN, mainly because of the potent capacity of LEN to activate the effector cells of ADCC. Current clinical practice, however, shows that the future of successful MM treatment lies in the use of drug combination regimens. It appears essential to identify regimens in which individual components synergize to obtain the greatest achievable effects. Therefore, we now explored the potential clinical benefit of combining targeted DARA therapy with newly emerging multi-drug chemotherapy regimens. To this end, we used a recently developed ex vivo flow cytometry-based assay platform, which enables us to enumerate and subsequently deduce the drug/antibody-mediated lysis of primary CD138þ MM cells directly in bone marrow samples from the MM patients. The assays are performed with bone marrow mononuclear cells (BM-MNC), thus without the need for separating malignant cells from autologous effector cells and tumorsupporting accessory cells, such as stromal cells. With this ex vivo assay system, we first addressed the benefits of combining DARA with both LEN and BORT, since not only LEN but also BORT may enhance the therapeutic efficacy of DARA by sensitizing tumor cells for antibody-mediated lysis. In a series of experiments, we incubated BM-MNC from 16 MM patients, containing 2–20% malignant plasma cells, either with medium alone or with combinations of LEN, BORT and DARA at carefully selected individual concentrations inducing halfmaximal lysis of MM cells. An antibody against an irrelevant antigen (Keyhole Limpet Hemocyanin (KLH)) was used as an isotype control. After 48 h, we harvested the cells, labeled them with a monoclonal CD138 antibody and enumerated the surviving CD138þ MM cells using single-platform flow cytometry, to assess the percentage of MM cell lysis in each sample (Figure 1a) relative to that obtained with the control antibody KLH, which induced negligible MM cell lysis (data not shown). LEN and BORT alone or in combination caused low to moderate lysis of MM cells (mean lysis 10%, 18% and 25%, respectively). Addition of DARA significantly increased the MM cell lysis by more than twofold in all combinations (P1⁄4 0.001). The highest MM cell lysis was observed with the triple LEN-BORT-DARA combination. Notably, combination with DARA seemed to improve MM cell lysis especially in the samples that poorly responded to LEN and BORT (MM cell lysis o30%; Figure 1a). To evaluate this, we analyzed the data of LEN/BORT high and low responders separately (Figures 1b and c). A significant improvement of MM cell lysis by DARA was observed in the LEN/BORT lowresponder subset particularly (Figure 1c) and the effect was synergistic. This group included cells from five patients who had been treated in the clinic with LEN and/or BORT without success (Figure 1d). Interestingly, the synergy between DARA and LEN/BORT treatment was also apparent for cells from the five LEN/BORTresistant patients (Figure 1d, as illustrated by the fact that observed levels of MM cell lysis with DARA/LEN/BORT treatment were significantly higher than the expected levels of MM cell lysis, calculated on the assumption that there was no treatment interaction). Although we have only been able to evaluate a small number of samples from resistant patients to date, this remarkable synergy suggests the maintenance of antitumorigenic properties of LEN and BORT, despite the development of drug resistance. Taken together, these results indicate the potential clinical benefits of combining DARA with these two novel anti-MM agents and warrant further investigation even in patients who are low responders or have become resistant to the latter drugs. After showing the potential benefits of combining DARA with LEN and BORT, our further investigation focused on two recently introduced and so far the most successful first-line combination therapies based on these two novel agents, namely the triple combinations of LEN, BORT, DEX, abbreviated as RVD, and of MEL, PRED, BORT, abbreviated as MPV. To assess the impact of combining DARA with these combination chemotherapies, we prepared cocktails of these agents, by mixing them at concentrations causing ±30% of the maximal lytic effect on various MM cell lines (data not shown). We then incubated BM-MNC of MM patients with serial dilutions of these cocktails alone or in the presence of DARA and assessed MM cell lysis. As expected, cocktails of RDV (Figure 2a) as well as MPV (Figure 2b) induced dose-dependent lysis of MM cells. Addition of DARA to both RDV and MPV significantly increased the treatment efficacy by almost doubling the lysis levels Citation: Blood Cancer Journal (2011) 1, e41; doi:10.1038/bcj.2011.42 & 2011 Macmillan Publishers Limited All rights reserved 2044-5385/11