Towards effective immunotherapy of myeloma: enhanced elimination of myeloma cells by combination of lenalidomide with the human CD38 monoclonal antibody daratumumab

Abstract

Background: In our efforts to develop novel effective treatment regimens for multiple myeloma we evaluated the potential benefits of combining the immunomodulatory drug lenalidomide with daratumumab. Daratumumab is a novel human CD38 monoclonal antibody which kills CD38+ multiple myeloma cells via antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity and apoptosis.

Design and methods: To explore the effect of lenalidomide combined with daratumumab, we first carried out standard antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity assays in which the CD38+ multiple myeloma cell line UM-9 and primary multiple myeloma cells isolated from patients were used as target cells. We also tested the effect of lenalidomide on daratumumab-dependent cell-mediated-cytotoxicity and complement-dependent cytotoxicity of multiple myeloma cells directly in the bone marrow mononuclear cells of multiple myeloma patients. Finally, we determined the daratumumab-dependent cell-mediated cytotoxicity using peripheral blood mononuclear cells of multiple myeloma patients receiving lenalidomide treatment.

Results: Daratumumab-dependent cell-mediated cytotoxicity of purified primary multiple myeloma cells, as well as of the UM-9 cell line, was significantly augmented by lenalidomide pre-treatment of the effector cells derived from peripheral blood mononuclear cells from healthy individuals. More importantly, we demonstrated a clear synergy between lenalidomide and daratumumab-induced antibody-dependent cell-mediated cytotoxicity directly in the bone marrow mononuclear cells of multiple myeloma patients, indicating that lenalidomide can also potentiate the daratumumab-dependent lysis of myeloma cells by activating the autologous effector cells within the natural environment of malignant cells. Finally, daratumumab-dependent cell-mediated cytotoxicity was significantly up-regulated in peripheral blood mononuclear cells derived from 3 multiple myeloma patients during lenalidomide treatment.

Conclusions: Our results indicate that powerful and complementary effects may be achieved by combining lenalidomide and daratumumab in the clinical management of multiple myeloma.

Figure 1.

Enhancement of DARA induced ADCC by preincubation of effector PBMC with LEN. PBMC from healthy individuals were incubated with LEN (3 μM) for 72 h prior to ADCC assays in which UM9 (A, B) and purified MM cells (C) were used after addition of DARA (0.1 μg/mL) or a control antibody against KLH (1 μg/mL). A representative DARA dose-response curve is shown in (B). Error bars represent the SEM of triplicate measurements. P values are calculated by the Tukey’s post hoc analysis of a repeated measures ANOVA.

Figure 2.

Absence of sensitization of MM cells by LEN for DARA-induced ADCC. Primary MM cells (N=7, left) or UM9 (n=2, right) where pre-treated for 24 h with LEN (3 mM). A standard ADCC was performed with DARA (0.1 mg/mL) and PBMC from healthy donors. Percentage lysis is calculated as indicated in the Design and Methods section. P values are calculated by a paired t-test.

Figure 3.

Improvement of DARA-induced ADCC by LEN in BM-MNC of MM patients. (A) BM-MNC of MM patients (n=14) were incubated for 48 h with LEN (3 μM) and DARA (0.1 μg/mL). Surviving MM cells were enumerated by FACS analysis of CD138⁺ cells. Percentage lysis of MM cells in LEN, DARA and LEN+ DARA treated patients were calculated using the MM survival in wells treated with the control anti-KLH antibody alone, which was indistinguishable from the medium control (thus 100% survival or no lysis). P values are calculated by the Tukey’s post hoc analysis of repeated measures ANOVA. (B) The observed effect (% lysis) of the combination treatment was compared to the expected proportional effect of the combined treatments. Mixed model analysis (see Design and Methods section) supports the conclusion that the combination treatment is synergistic.

Figure 4.

MM cell lysis in the BM of MM patients by the combination of DARA+LEN in the absence or presence of complement. BM-MNCs from MM patients (n=16) were subsequently incubated for 24 h with LEN and for 45 min with or without complement in the absence or presence of DARA. P values are calculated by the Tukey’s post hoc analysis of a one-way repeated measure ANOVA.

Figure 5.

Significant DARA-dependent ADCC in PBMC from patients treated with LEN. Frozen PBMC obtained from 3 patients (Pt 1–3) 0–7 days before (Pre), during (During) or one month after (Post) the first cycle of LEN treatment (25mg/daily) was tested in ADCC assays after addition of DARA and control antibody KLH. Control antibody against KLH did not induce any ADCC (data not shown) Error bars represent the SEM of the duplicate conditions. P values are calculated using a Student’s t-test.