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

Citation: Blood Cancer Journal (2011) 1, e41; doi:10.1038/bcj.2011.42 & 2011 Macmillan Publishers Limited All rights reserved 2044-5385/11

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LETTER TO THE EDITORThe therapeutic human CD38 antibody daratumumab improves the anti-myeloma effect

of newly emerging multi-drug therapies

Blood Cancer Journal (2011) 1, e41; doi:http://dx.doi.org/10.1038/bcj.2011.42

Web End =10.1038/bcj.2011.42 ; published online 28 October 2011

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 signicantly improved the overall survival of MM patients.1,2 All treatment strategies, including multidrug regimens, however, are eventually hampered by the development of drug resistance.3 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 udarabine, cyclophosphamide or LEN.4,5

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.6 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 signicantly improved by LEN, mainly because of the potent capacity of LEN to activate the effector cells of ADCC.7 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 benet of combining targeted DARA therapy with newly emerging multi-drug chemotherapy regimens. To this end, we used a recently developed ex vivo ow cytometry-based assay platform,7 which enables us to enumerate and subsequently deduce the drug/antibody-mediated lysis of primary CD138 MM cells directly in bone marrow samples from the MM patients. The assays are performed with bone marrow mono-nuclear cells (BM-MNC), thus without the need for separating malignant cells from autologous effector cells and tumor-supporting accessory cells, such as stromal cells. With this ex vivo assay system, we rst addressed the benets of combining DARA with both LEN and BORT, since not only LEN but also BORT may enhance the therapeutic efcacy of DARA by sensitizing tumor cells for antibody-mediated lysis. In a series of experiments, we incubated BM-MNC from 16 MM patients, containing 220% malignant plasma cells, either with medium alone or with combinations of LEN, BORT and DARA at

carefully selected individual concentrations inducing half-maximal 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 CD138 MM cells using single-platform ow 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 signicantly increased the MM cell lysis by more than twofold in all combinations (P 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 signicant improvement of MM cell lysis by DARA was observed in the LEN/BORT low-responder subset particularly (Figure 1c) and the effect was synergistic. This group included cells from ve 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 ve LEN/BORT-resistant patients (Figure 1d, as illustrated by the fact that observed levels of MM cell lysis with DARA/LEN/BORT treatment were signicantly 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 anti-tumorigenic properties of LEN and BORT, despite the development of drug resistance. Taken together, these results indicate the potential clinical benets 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 benets of combining DARA with LEN and BORT, our further investigation focused on two recently introduced and so far the most successful rst-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 signicantly increased the treatment efcacy by almost doubling the lysis levels

Letter to the Editor

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Figure 1 Addition of DARA to BORT-LEN signicantly increases the MM cell lysis in BM-MNC of MM patients, particularly in LEN/BORT low responder or refractory patients. (a) BM-MNC from MM patients (n 16), containing 220% CD138 MM cells as detected by ow cytometry

were incubated with LEN (3 mM), BORT (3 nM) and DARA (10 mg/ml) alone or in combination for 48 h in RPMI 10% fetal bovine serum in 96-well

u bottom plates in fully humidied incubators at 37 1C, 5% CO2-air mixture. Surviving MM cells were enumerated by the single-platform uorescence-activated cell sorting analysis of CD138 cells in the presence of Tru-Count beads (Beckman Coulter, Miami, FL, USA). Percentage lysis of MM cells in LEN, DARA and LEN DARA-treated conditions were calculated using the MM survival of wells treated with the control KLH

antibody alone. Differences between indicated groups were tested for signicance in repeated measures analysis of variance (ANOVA), using Bonferonnis post-hoc multiple comparison tests with two-tailed 95% condence intervals. In bd, data are analyzed for low LEN/BORT responders, high LEN/BORT responders and LEN/BORT refractory patients, respectively. aP-values were calculated using a repeated measures ANOVA. bP-values were calculated by a paired t-test. cExpected values were calculated to test the null hypothesis that there is no synergism between DARA and LEN/BORT using the following formula: % expected lysis 100 %survival after DARA %survival after LEN/BORT.

especially at lower doses of the cocktails. These results illustrate that targeted immunotherapy of MM by DARA holds a signicant potential to improve the clinical outcome of currently available novel combination therapies.

Recent studies have indicated that combination of multiple drugs are superior over single- or double-agent combinations.2 Addition of new drugs to the available regimens can mediate their clinical benet because of the induction of a higher rate of initial complete responses, which in turn improves the relapse free and overall survival.8 Contingent on the premise that the combined agents have non-overlapping and synergistic mechanisms of actions, immediate and effective targeting of tumors with multiple agents appears a successful strategy to improve the clinical outcome of MM therapy. Indeed, such a strategy is in full agreement with the emerging concept that the genetic signature of MM, and consequently,

the individual patients susceptibility to a specic agent will be highly heterogeneous and this eventually may lead to drug resistance. Nevertheless, the complete response rate of the best chemotherapeutic combinations is currently o50%, and all current combination therapies eventually induce drug resistance.2,9 In this respect, DARA, with its immediate and effective cell-mediated cytotoxic effects against MM cells, and the observed remarkable synergy with LEN/BORT even in LEN/BORT refractory patients, may potentially improve the achievement of rst CR in MM when combined with these agents either alone or in multidrug chemotherapy regimens. In conclusion, our study, in which we demonstrate the potential benets of combining DARA-mediated targeted therapy with newly emerging chemotherapy options, warrants the evaluation of this approach in MM in clinical phase I/II trials.

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References

1 Brenner H, Gondos A, Pulte D. Recent major improvement in long-term survival of younger patients with multiple myeloma. Blood 2008; 111: 25212526.

2 Cavo M, Rajkumar SV, Palumbo A, Moreau P, Orlowski R, Blade J et al. International Myeloma Working Group consensus approach to the treatment of multiple myeloma patients who are candidates for autologous stem cell transplantation. Blood 2011; 117: 60636073.

3 Orlowski RZ, Nagler A, Sonneveld P, Blade J, Hajek R, Spencer A et al. Randomized phase III study of pegylated liposomal doxorubicin plus bortezomib compared with bortezomib alone in relapsed or refractory multiple myeloma: combination therapy improves time to progression. J Clin Oncol 2007; 25: 38923901.

4 Hallek M, Pug N. State of the art treatment of chronic lymphocytic leukaemia. Blood Rev 2011; 25: 19.

5 Ahmadi T, Chong EA, Gordon A, Leinbach L, Aqui NA, Nasda SD et al. Phase II trial of lenalidomideFdexamethasoneFrituximab in relapsed or refractory indolent B-cell or mantle cell lymphomas resistant to rituximab. Blood (ASH Annual Meeting Abstracts) 2010; 116: 3962.

6 de Weers M, Tai YT, van der Veer M, Bakker JM, Vink T, Jacobs DC et al. Daratumumab, a novel therapeutic human CD38 monoclonal antibody, induces killing of multiple myeloma and other hematological tumors. J Immunol 2011; 186: 18401848.

7 van der Veer M, de Weers M, van Kessel B, Bakker JM, Wittebol S,

Parren PW et al. Towards effective immunotherapy of myeloma: enhanced elimination of myeloma cells by combination of lenalidomide with the human CD38 monoclonal antibody daratumumab. Haematologica 2011; 96: 284890.8 Chanan-Khan AA, Giralt S. Importance of achieving a complete response in multiple myeloma, and the impact of novel agents. J Clin Oncol 2010; 28: 26122624.9 Ludwig H, Beksac M, Blade J, Cavenagh J, Cavo M, Delforge M et al. Multiple myeloma treatment strategies with novel agents in 2011: a European perspective. Oncologist 2011; 16: 388403.

This work is licensed under the Creative Commons Attribution-NonCommercial-No Derivative Works3.0 Unported License. To view a copy of this license, visit http:// creativecommons.org/licenses/by-nc-nd/3.0/

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Figure 2 DARA increases response against MM in triple chemotherapy treatments. BM-MNC form patients (n 7) were incubated

with increasing dilutions of a cocktail of (a) LEN, BORT and DEX (1 dilution contains: 1 m

M LEN, 1 nM BORT and 1 mM DEX) or with a cocktail of (b) MEL, PRED, BORT (1 dilution contains 1 m

M MEL,

1 mM PRED and 1 nM BORT) in the presence or absence of DARA (10 mg/ml) for 48 h. Surviving MM cells were enumerated by uorescence-activated cell sorting analysis of CD138 cells.

P-values were calculated by a paired t-test. nPo0.05, nnnPo0.001.

Letter to the Editor

Conict of interest

This work was supported by research grants from the UMC

Utrecht and Genmab BV provided to TM and HML.

MS van der Veer1, M de Weers2, B van Kessel1, JM Bakker2, S Wittebol3, PWHI Parren2, HM Lokhorst4 and T Mutis1

1Department of Clinical Chemistry and Hematology, University Medical Center Utrecht, Utrecht, Netherlands;

2Genmab, Utrecht, Netherlands;

3Department of Internal Medicine, Meander Medical Center, Amersfoort, Netherlands and

4Department of Hematology, University Medical Center Utrecht, Utrecht, Netherlands E-mail: mailto:[email protected]

Web End [email protected]

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