Emerging evidence suggests that FcγR-mediated cross-linking of tumor-bound mAbs may induce signaling in tumor cells that contributes to their therapeutic activity. In this study, we show that daratumumab (DARA), a therapeutic human CD38 mAb with a broad-spectrum killing activity, is able to induce programmed cell death (PCD) of CD38+ multiple myeloma tumor cell lines when cross-linked in vitro by secondary Abs or via an FcγR. By comparing DARA efficacy in a syngeneic in vivo tumor model using FcRγ-chain knockout or NOTAM mice carrying a signaling-inactive FcRγ-chain, we found that the inhibitory FcγRIIb as well as activating FcγRs induce DARA cross-linking–mediated PCD. In conclusion, our in vitro and in vivo data show that FcγR-mediated cross-linking of DARA induces PCD of CD38-expressing multiple myeloma tumor cells, which potentially contributes to the depth of response observed in DARA-treated patients and the drug’s multifaceted mechanisms of action.

Daratumumab (DARA) is a human IgG1 therapeutic mAb that binds to CD38. Among other hematological malignancies, CD38 is expressed at high levels on multiple myeloma (MM) tumor cells (1). In 2015, the U.S. Food and Drug Administration has approved DARA for MM patients who have received at least three prior lines of therapy, including a proteasome inhibitor and an immunomodulatory agent, or patients double refractory to these agents. Approval was based on two phase II studies of DARA monotherapy (16 mg/kg) in heavily treated patients (2, 3). A pooled analysis of these studies revealed an overall response rate of 31%, including responses that deepened over time, and median overall survival of 19.9 mo (4). DARA is currently in multiple phase III clinical trials for the treatment of MM in relapsed and frontline settings. Additional studies are ongoing or planned to assess its potential in other malignant and premalignant diseases on which CD38 is expressed, such as smoldering myeloma and non–Hodgkin’s lymphoma.

Multiple mechanisms of action have been observed for DARA, including the Fc-dependent effector mechanisms, complement-dependent cytotoxicity (CDC), Ab-dependent cellular cytotoxicity (ADCC) (5), and Ab-dependent phagocytosis (ADCP) (6). Recent studies have revealed previously unknown immunomodulatory effects of DARA where CD38-expressing immunosuppressive regulatory T and B cells and myeloid-derived suppressor cells are highly sensitive to DARA treatment (7). It has also been shown that DARA can modulate the enzymatic activity of CD38 and potentially may lead to a reduction in immunosuppressive adenosine levels (8, 9). This shift away from an immunosuppressive environment may lead to the generation of protective immune responses. Indeed, a concomitant induction of helper and cytotoxic T cell increases in absolute cell counts and production of IFN-γ in response to viral peptides was observed. Additionally, an increase in T cell clonality in subjects who responded to DARA versus subjects who did not respond was observed, indicating an improved adaptive immune response (7).

ADCC and ADCP are induced by mAb binding to activating FcγRs on immune effector cells, for example, NK cells, macrophages, and polymorphonuclear cells (PMNs), to clustered IgG constant domains (Fc domains). Remarkably, it is becoming clear that Fc–FcγR interactions may also lead to signaling in the tumor cells, enhancing the agonistic activity of mAb or inducing programmed cell death (PCD). PCD includes all pathways leading to cell death mediated by activation of intracellular signaling. Agonistic therapeutic mAb targeting members of the death receptor family induce PCD via the extrinsic apoptosis pathway (10). PCD induced by agonistic mAb targeting these death receptors is enhanced by cross-linking, either by secondary Ab or, more physiologically, via binding to FcγRs (11, 12). This suggests that FcγRs on, for example, tumor-associated leukocytes could provide a cross-linking scaffold for antitumor mAb in vivo. Indeed, PCD induced by drozitumab, a human IgG1 mAb targeting death receptor 5, could be strongly enhanced by Fc cross-linking using activating FcγRs or the inhibitory FcγRIIb in vitro (13). Nevertheless, in vivo the antitumor activity of drozitumab was predominantly diminished in FcγRIIb−/− mice, whereas in the absence of activating FcγRs antitumor activity was not affected. This suggests that FcγRIIb-mediated cross-linking is sufficient for the antitumor effect of drozitumab in vivo, which corresponds to findings for other dead receptor targeting agonistic mAb (14, 15).

Ab-mediated cross-linking of Ags, not related to the death receptor family, may also induce PCD, but not via the classical apoptotic pathway (1619). This pathway is characterized by homotypic aggregation of cells involving cytoskeleton reorganization, lysosomal activation, and production of reactive oxygen species (20). This Ab-induced PCD pathway can be enhanced by Fc cross-linking secondary Ab (2123) or by FcγR-expressing cells (24).

In this study, we explored whether DARA induces PCD of CD38-expressing tumor cells via FcγR-mediated cross-linking. PCD was defined by morphological changes resulting in clustering of cells, phosphatidylserine translocation, loss of mitochondrial membrane potential, and loss of membrane integrity. To investigate the contribution of FcγRs in DARA-mediated PCD in vivo, we explored PCD induction in NOTAM (transgenic mice expressing physiological levels of signaling-abrogated activating FcγRs) and FcRγ-chain knockout mice. Our results show that both inhibitory and activating FcγRs can mediate DARA cross-linking, leading to phosphatidylserine translocation followed by cell death.

The MM cell lines UM-9, generated at the University Medical Center (Utrecht, the Netherlands) (25), and L363, obtained from the American Type Culture Collection transduced with GFP and luciferase marker genes (26), were transduced with human CD38 gene to increase CD38 expression levels. For this the amphotropic Phoenix packaging cell line (Phoenix Ampho) was transfected, using calcium phosphate precipitation, with the pQCXIN vector in which the gene encoding human CD38 was inserted. These cell lines are referred to as UM9-CD38 and L363-CD38 expressing CD38 in a range of 350,000–600,000 and 450,000–800,000 molecules/cell, respectively, as determined with QiFi analysis (QiFi kit, Dako, Glostrup, Denmark). IIA1.6 cells (mouse pre–B cell line, American Type Culture Collection) were transfected with human FcγRI (hFcγRI) and human FcRγ-chain as described previously (27); these cells are referred to as IIA1.6-hFcγRI. EL4 cells (mouse lymphoma cell line, American Type Culture Collection) and were retrovirally transduced with a GFP-IRES-luciferase construct as described previously (26). Subsequently, the pQCXIN vector containing the gene encoding human CD38 was inserted as described for the MM cell lines. Cells were subcloned by limiting dilution, resulting in a stable EL4-CD38 clone expressing 225,000–400,000 CD38 molecules/cell as determined with QuantiBrite analysis (BD Biosciences, Franklin Lakes, NJ). All cells were cultured in RPMI 1640 (Life Technologies, Carlsbad, CA), 10% heat-inactivated FCS (Bodinco, Alkmaar, the Netherlands), 50 U/ml penicillin (Life Technologies), and 50 μg/ml streptomycin (Life Technologies). Culture medium for the IIA1.6-hFcγRI cells was supplemented with 2.5 μg/ml methotrexate (Emthexate; Teva Pharmachemie, Haarlem, the Netherlands) and for the EL4-CD38 cells with 1 mg/ml geneticin (Life Technologies). UM9-CD38 and EL4-CD38 cells were labeled with CFSE or CellTrace Violet (Life Technologies) according to the manufacturer’s protocol.

Human IgG1 anti-CD38 mAb DARA was generated by immunization of HuMAb mice and was produced recombinantly as described previously (5). DARA-K322A, a Fc mutant lacking complement activation, was generated by mutating the lysine at position 322 to alanine as described previously (28, 29). F(ab′)2 fragments of rabbit anti-human IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) was used as Fc–cross-linking secondary Ab. F(ab′)2 fragments of the mouse FcγRIIb mAb K9.361 were generated according to the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA). PE-labeled annexin V and Via-Probe (7-aminoactinomycin D [7-AAD]) were purchased from BD Biosciences and used according to the manufacturer’s protocol. Briefly, cells were washed with binding buffer containing 10 mM HEPES (Merck, Darmstadt, Germany), 0.14 M NaCl (Merck), and 2.5 mM CaCl2 (Riedel-de Haen, Seelze, Germany) followed by annexin V and 7-AAD staining in binding buffer at room temperature for 15 min. Mitochondrial membrane potential depolarization was measured with the MitoProbe 1,1′,3,3,3′,3′-hexamethylindodicarbo–cyanine iodide [DilC1(5)] kit (Life Technologies), which was used according to the manufacturer’s protocol. Briefly, after washing in PBS (Pharmacy, University Medical Center, Utrecht, the Netherlands), cells were incubated for 20 min with 3 nM DilC1(5) at 37°C and 5% CO2. Cells were washed twice with RPMI 1640 medium supplemented with 10% FCS and penicillin/streptomycin to stop the DilC1(5) reaction. Numbers of cell surface CD38 molecules were determined with mouse anti-human CD38 Ab (BD Biosciences) and the QiFi kit (Dako) or QuantiBRITE tubes (BD Biosciences).

Labeled target cells were seeded at 1 × 105 cells per well in 24-well plates or 2 × 104 cells per well in 96-well plates and preincubated 30 min with varying concentrations of indicated mAb followed by incubation in the presence of either 5 μg/ml rabbit anti-human IgG F(ab′)2 fragments, IIA1.6-hFcγRI cells, IIA1.6 cells, or primary cells isolated from bone marrow and peritoneum of NOTAM transgenic mice at the indicated E:T ratio. Morphologic changes were visualized using the EVOS microscope (Advanced Microscopy Group, Life Technologies). PCD markers (annexin V, 7-AAD, and mitochondrial membrane potential depolarization) were analyzed for the labeled target cells after 4 or 24 h by flow cytometry using a FACSCanto II (BD Biosciences). Percentage of annexin V+, annexin V+/7-AAD+, and DilC5(1)low cells was calculated using FACSDiva software (BD Biosciences).

Experiments were performed with 8- to 19-wk-old FcRγ-chain knockout (FcRγ−/−) mice (30) and NOTAM mice (31) on a C57BL/6 background. Mice were bred at the specific pathogen-free facility of the Central Animal Laboratory of Utrecht University, and all experiments were approved by the local Animal Ethical Committee.

Mice were injected i.p. with 5 × 106 CFSE-labeled EL4-CD38 cells in 100 μl PBS and, directly after tumor cell inoculation, with 2 μg DARA-K322A or 100 μl PBS. To block FcγRIIb in vivo, 50 μg F(ab′)2 fragments of the anti-FcγRIIb mAb K9.361 was injected i.p. 30 min prior to tumor cell inoculation (blocking of FcγRIIb was confirmed on peritoneal effector cells by flow cytometry). Following 4 h incubation, the mice were euthanized and the peritoneum was washed with PBS containing 5 mM EDTA (Sigma-Aldrich, St. Louis, MO). Annexin V and 7-AAD staining of the CFSE+ EL4-CD38 cells was analyzed by flow cytometry as described above.

Data analysis was performed using GraphPad Prism version 5.0 (GraphPad Software, San Diego, CA). Data are reported as means ± SD. Differences between groups were analyzed using a Student unpaired t test or a Bonferroni multiple comparison test (a p value <0.05 was considered statistically significant).

We explored whether DARA causes PCD by cross-linking of CD38 on MM cell lines. As CD38 expression levels were shown to correlate with the level of DARA-induced ADCC and CDC on both MM cell lines and primary MM cells (32), we made use of MM cell lines L363-CD38 and UM9-CD38 transduced with human CD38 to increase CD38 expression to levels that are comparable to primary myeloma cells. Incubation of DARA-opsonized L363-CD38 and UM9-CD38 cells with Fc–cross-linking secondary Ab resulted in significant upregulation of phosphatidylserine translocation, as indicated by an increase in the number of annexin V+ cells (Fig. 1A). DARA-induced PCD was demonstrated by the subsequent significant loss of mitochondrial membrane potential for the UM9-CD38 cells (Fig. 1B) and a significant increase in the number of 7-AAD+ cells (Fig. 1C). An isotype control gave similar results as the medium control (data not shown). In the absence of secondary Abs, DARA did not increase any of the PCD markers studied (Supplemental Fig. 1).

FIGURE 1.

DARA induces PCD in MM cells after Ab-mediated cross-linking. PCD induction in L363-CD38 (left panels) or UM9-CD38 (right panels) cells after 24 h incubation with 0.1 μg/ml DARA or culture medium (no mAb) in the presence of Fc-cross-linking secondary Ab. Flow cytometric analysis of percentage annexin V+ cells (A), loss of mitochondrial membrane potential as measured by the decrease in DilC1(5) fluorescence (B), and percentage 7-AAD+ (dead) cells (C). Each bar shows mean ± SD of a representative experiment (n ≥ 2). **p < 0.01, ***p < 0.001, ****p < 0.0001 by unpaired t test.

FIGURE 1.

DARA induces PCD in MM cells after Ab-mediated cross-linking. PCD induction in L363-CD38 (left panels) or UM9-CD38 (right panels) cells after 24 h incubation with 0.1 μg/ml DARA or culture medium (no mAb) in the presence of Fc-cross-linking secondary Ab. Flow cytometric analysis of percentage annexin V+ cells (A), loss of mitochondrial membrane potential as measured by the decrease in DilC1(5) fluorescence (B), and percentage 7-AAD+ (dead) cells (C). Each bar shows mean ± SD of a representative experiment (n ≥ 2). **p < 0.01, ***p < 0.001, ****p < 0.0001 by unpaired t test.

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To study DARA-mediated PCD in a more physiological setting, we explored whether cells expressing FcγRs could mediate Fc–cross-linking of DARA-opsonized target cells, resulting in PCD. We used the hFcγRI-transduced murine B cell lymphoma cell line, IIA1.6-hFcγRI, which is unable to induce ADCC (data not shown). Coculture of IIA1.6-hFcγRI cells with either DARA-opsonized L363-CD38 or UM9-CD38 cells also resulted in significant upregulation of phosphatidylserine translocation (Fig. 2A). Phosphatidylserine translocation was subsequently followed, for a fraction of the annexin V+ cells, by a significant loss of mitochondrial membrane potential (Fig. 2B) and eventually a significant increase of cell death (Fig. 2C). Coculture of DARA-opsonized L363-CD38 and UM9-CD38 cells with hFcγRI IIA1.6 cells did not induce any of the PCD markers studied (data not shown), confirming the effect to be FcγR mediated. DARA-induced hFcγRI-mediated PCD was observed over a broad DARA concentration range.

FIGURE 2.

PCD induction of MM cell lines via FcγR-mediated cross-linking of DARA. GFP+ L363-CD38 (left panels) and CFSE-labeled UM9-CD39 (right panels) cells were cocultured with IIA1.6-hFcγRI cells (E:T ratio of 1:1) in the presence of indicated concentration mAb for 24 h. Flow cytometric analysis of percentage annexin V+ cells (A), loss of mitochondrial membrane potential (B), and percentage 7-AAD+ (dead) cells (C). Each bar shows mean ± SD of a representative experiment (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by Bonferroni multiple comparison test.

FIGURE 2.

PCD induction of MM cell lines via FcγR-mediated cross-linking of DARA. GFP+ L363-CD38 (left panels) and CFSE-labeled UM9-CD39 (right panels) cells were cocultured with IIA1.6-hFcγRI cells (E:T ratio of 1:1) in the presence of indicated concentration mAb for 24 h. Flow cytometric analysis of percentage annexin V+ cells (A), loss of mitochondrial membrane potential (B), and percentage 7-AAD+ (dead) cells (C). Each bar shows mean ± SD of a representative experiment (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by Bonferroni multiple comparison test.

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Incubation of DARA-opsonized L363-CD38 cells cross-linked either via Fc-cross-linking secondary Ab (Fig. 3, upper and middle panels) or with IIA1.6-hFcγRI cells (Fig. 3, bottom panel) induced clustering of the cells, which was demonstrated to correlate with PCD induction by CD20 mAb (16, 18, 33). FcγR-mediated clustering was effective over a broad concentration range (0.0001–10 μg/ml), whereas Fc–cross-linking mediated by a secondary Ab was restricted to a more narrow concentration range (0.01–10 μg/ml) (Fig. 3).

FIGURE 3.

DARA cross-linking–mediated clustering of L363-CD38 cells. Bright-field images of L363-CD38 cells after 24 h incubation with a range of DARA concentrations in the presence of 5 or 25 μg/ml Fc–cross-linking secondary Ab (upper and middle panels) or IIA1.6-hFcγRI cells (E:T ratio of 1:1) (bottom panel). Original magnification ×10.

FIGURE 3.

DARA cross-linking–mediated clustering of L363-CD38 cells. Bright-field images of L363-CD38 cells after 24 h incubation with a range of DARA concentrations in the presence of 5 or 25 μg/ml Fc–cross-linking secondary Ab (upper and middle panels) or IIA1.6-hFcγRI cells (E:T ratio of 1:1) (bottom panel). Original magnification ×10.

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The role of FcγR-mediated DARA-induced PCD in vivo was studied in NOTAM and FcRγ-chain knockout (FcRγ−/−) mice in a syngeneic peritoneal mouse model for which mouse EL4 lymphoma cells were transduced with human CD38. We first validated whether the EL4-CD38 cells were sensitive for PCD induction by DARA in vitro. DARA cross-linking with the secondary anti-Fc Ab or IIA1.6-hFcγRI cells indeed induced PCD in EL4-CD38 cells (Fig. 4A, 4B, Supplemental Fig. 2). FcγRI-mediated DARA cross-linking was, also on EL4-CD38 cells, effective over a broad concentration range. NOTAM mice have normal surface expression of all activating FcγRs, but without signaling capacity due to a signaling-deficient FcR-associated γ-chain (31) (Fig. 4C). Leukocytes in the NOTAM mice are therefore capable of FcγR-mediated Ab cross-linking, without inducing cytotoxicity via ADCC or ADCP (31). Leukocytes in FcRγ−/− mice lack expression of all activating FcγRs and solely express the inhibitory FcγRIIb. We explored ex vivo whether primary FcγR-expressing cells from NOTAM mice could mediate Fc–cross-linking of DARA-opsonized EL4-CD38 cells. Primary FcγR-expressing cells isolated from either bone marrow or peritoneum (Fig. 4D, 4E) can also efficiently mediate PCD induction after DARA cross-linking. No efficacy was observed in the presence of F(ab′)2 fragments of DARA (data not shown), confirming that the observed PCD was Fc mediated.

FIGURE 4.

PCD induction of EL4-CD38 cells via DARA cross-linking in vitro and ex vivo. CFSE-labeled EL4-CD38 cells were cocultured for 4 h with Fc–cross-linking secondary Ab, IIA1.6 cells, or IIA1.6-hFcγRI cells (E:T ratio of 1:1) in the presence of a serial dilution of DARA (A and B). (C) Cartoon depicting the available FcγRs in NOTAM and FcRγ−/− mice. The X indicates blocking of FcγRIIb receptors using K9.361 F(ab′)2 fragments. CellTrace Violet–labeled EL4-CD38 cells were cocultured for 24 h with primary cells isolated from bone marrow (BM; E:T ratio of 50:1) or peritoneum (PL; E:T ratio of 10:1) of NOTAM transgenic mice at a fixed concentration of 10 μg/ml DARA (D and E). Flow cytometric analysis of percentage annexin V+ cells (A and D) or 7-AAD+ cells (B and E). Each line shows mean ± SD of a representative experiment (n = 2).

FIGURE 4.

PCD induction of EL4-CD38 cells via DARA cross-linking in vitro and ex vivo. CFSE-labeled EL4-CD38 cells were cocultured for 4 h with Fc–cross-linking secondary Ab, IIA1.6 cells, or IIA1.6-hFcγRI cells (E:T ratio of 1:1) in the presence of a serial dilution of DARA (A and B). (C) Cartoon depicting the available FcγRs in NOTAM and FcRγ−/− mice. The X indicates blocking of FcγRIIb receptors using K9.361 F(ab′)2 fragments. CellTrace Violet–labeled EL4-CD38 cells were cocultured for 24 h with primary cells isolated from bone marrow (BM; E:T ratio of 50:1) or peritoneum (PL; E:T ratio of 10:1) of NOTAM transgenic mice at a fixed concentration of 10 μg/ml DARA (D and E). Flow cytometric analysis of percentage annexin V+ cells (A and D) or 7-AAD+ cells (B and E). Each line shows mean ± SD of a representative experiment (n = 2).

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To exclude a role for DARA-mediated CDC induction, we made use of a DARA-K322A mutant with reduced mouse C1q binding (29). The K322A mutation did not affect FcγR-mediated PCD induction (Supplemental Fig. 3). CFSE-labeled EL4-CD38 cells were inoculated i.p. directly followed by treatment with 2 μg/mouse DARA-K322A (∼0.1 mg/kg). After 4 h, the CFSE-labeled EL4-CD38 cells, harvested by peritoneal flush, were analyzed by flow cytometry for annexin V and 7-AAD staining (Fig. 5A). DARA-K322A induced an increase of annexin V+ cells and significantly increased the number of 7-AAD+ cells following 4 h incubation in the FcRγ−/− mice (Fig. 5B, 5C, top bars). This indicates that DARA-K322A–induced cross-linking via the inhibitory FcγRIIb is sufficient to induce PCD in vivo. Also in NOTAM mice, DARA-K322A treatment significantly increased the number of annexin V+ and 7-AAD+ cells (Fig. 5B, 5C, bottom bars). To explore which type of FcγR was important for PCD induction, mice were pretreated with FcγRIIb-specific blocking F(ab′)2 fragments of mAb K9.361 (Fig. 4C). As expected, in FcRγ−/− mice, FcγR-mediated PCD via DARA-K322A was abolished after blocking FcγRIIb (Fig. 5D, 5E, top bars). In NOTAM mice, significant PCD induction was still observed after blocking FcγRIIb, demonstrating that in addition to the inhibitory FcγRIIb, also activating FcγRs can mediate DARA cross-linking in vivo leading to PCD (Fig. 5D, 5E, bottom bars).

FIGURE 5.

In vivo PCD induction by Fc–cross-linking of DARA via activating and the inhibitory FcγR. (A) Scheme of the syngeneic peritoneal mouse model. NOTAM and FcRγ−/− mice were, as indicated, treated with FcγRIIb blocking F(ab′)2 fragments [K9.361 F(ab′)2] 30 min prior to tumor cell inoculation. Subsequently, 5 × 106 CFSE-labeled EL4-CD38 cells were inoculated i.p. followed by DARA-K322A (2 μg/mouse) or PBS treatment. After 4 h, tumor cells in the peritoneal wash were analyzed by flow cytometry. (B and D) Percentage annexin V+ cells. (C and E) Percentage 7-AAD+ cells (n = 4–6 mice/group). *p < 0.05, **p < 0.01, ***p < 0.001 by an unpaired t test (B and C) or a Bonferroni multiple comparison test (D and E).

FIGURE 5.

In vivo PCD induction by Fc–cross-linking of DARA via activating and the inhibitory FcγR. (A) Scheme of the syngeneic peritoneal mouse model. NOTAM and FcRγ−/− mice were, as indicated, treated with FcγRIIb blocking F(ab′)2 fragments [K9.361 F(ab′)2] 30 min prior to tumor cell inoculation. Subsequently, 5 × 106 CFSE-labeled EL4-CD38 cells were inoculated i.p. followed by DARA-K322A (2 μg/mouse) or PBS treatment. After 4 h, tumor cells in the peritoneal wash were analyzed by flow cytometry. (B and D) Percentage annexin V+ cells. (C and E) Percentage 7-AAD+ cells (n = 4–6 mice/group). *p < 0.05, **p < 0.01, ***p < 0.001 by an unpaired t test (B and C) or a Bonferroni multiple comparison test (D and E).

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In this study, we demonstrated that DARA can induce PCD in CD38-expressing MM cells after cross-linking by FcγR-expressing cells, both in vitro and ex vivo. Using the non–complement-binding DARA-K322A mutant in a syngeneic peritoneal mouse tumor model in NOTAM and FcRγ−/− mice, we showed that DARA-induced PCD also occurs in vivo and can be mediated by the inhibitory FcγRIIb as well as by activating FcγRs.

DARA-induced PCD was characterized by four phenotypic characteristics: morphological changes resulting in clustering of cells, phosphatidylserine translocation, loss of mitochondrial membrane potential, and loss of membrane integrity. Phosphatidylserine translocation is a marker for the initiation of PCD, which is a reversible process (34). In contrast, loss of the mitochondrial membrane potential and membrane integrity are irreversible and result in cell death (35). Cross-linking of DARA by both Fc–cross-linking secondary Ab or FcγRI-expressing cells induced PCD in the MM cell lines L363-CD38 and UM9-CD38. The number of annexin V+ cells was usually higher compared with the number of cells that lost membrane integrity (7-AAD+ cells), which may reflect the reversible nature of phosphatidylserine translocation. The low percentage of 7-AAD+ cells suggests that the contribution of PCD to the antitumor effect of DARA is limited. However, therapeutically, the relatively high number of annexin V+ cells may substantially enhance efficacy of other mechanisms of action of DARA. For instance, phosphatidylserine translocation is recognized by scavenger receptors on macrophages, resulting in phagocytosis of sensitized cells. We previously observed DARA to be highly potent in phagocytosis induction (6), and therefore phosphatidylserine translocation induced by FcγR-mediated cross-linking may potentially enhance phagocytosis. DARA-induced PCD possibly will represent an effective mechanism of action, especially under conditions when complement is depleted or when ADCC-mediating NK cells are exhausted during Ab treatment as previously described for anti-CD20 therapy (36, 37).

DARA-induced PCD was further characterized by morphologic changes resulting in clustering of the studied cells, so-called homotypic aggregation. Homotypic aggregation of cells was demonstrated to correlate with caspase-independent PCD induction for several therapeutic mAbs (16, 18, 20, 38, 39). We also did not detect caspase-3 cleavage upon DARA cross-linking with a secondary Ab in the L363-CD38 and UM9-CD38 MM cell lines (data not shown). However, on the Burkitt’s lymphoma cell line Ramos we did show caspase-3 activation upon cross-linking of DARA with secondary Ab (8). The homotypic aggregation–related caspase-independent and -dependent PCD was also shown for SAR650984, a chimeric CD38 Ab (40, 41), in the absence of secondary cross-linking. Nonetheless, we have previously shown that the direct effect of SAR650984 to induce PCD is comparable to DARA-induced PCD in the presence of a secondary Ab (8). As FcγRs are ubiquitously available in the tumor environment and because they also efficiently mediate DARA-induced PCD by cross-linking, PCD induction by DARA and SAR650984 may also be comparable in patients.

In a syngeneic peritoneal tumor model in FcRγ−/− mice, we showed that DARA-induced PCD was FcγR-dependent and feasible through binding to FcγRIIb in trans alone. This might be relevant at tumor sites where only FcγRIIb expressing cells, for example, B cells, reside. FcγRIIb distribution and expression levels were previously shown to be critical for efficacy of agonistic Abs targeting members of the tumor necrosis superfamily (TNFR) (42). In addition to trans binding of FcγRIIb to DARA, cis binding may also play a role, as demonstrated for a CD38-targeting mAb by Vaughan et al. (43). Primary mature MM cells have been shown to express FcγRIIb (44), suggesting that MM cells themselves might also mediate DARA cross-linking without a requirement for accessory cells, as was shown for CD40 mAb (13). PCD may therefore be an effective mechanism of action of DARA in bulky tumors to which access of effector cells may be limited (45) or under conditions of immune suppression, for example, owing to high MM tumor load or bone marrow–targeted therapy.

Using NOTAM mice in the syngeneic peritoneal tumor model, we showed that DARA-induced PCD can also be mediated by the murine activating FcγRs, as we observed significant PCD induction in the presence of FcγRIIb-specific blocking F(ab′)2 fragments. Accordingly, in the present study we show in vivo induction of PCD mediated via cross-linking by the activating FcγRs for a target that does not belong to the TNFR superfamily. Because NOTAM mice carry a signaling-inactive FcRγ-chain, the FcγR-mediated cross-linking of DARA resulting in PCD is FcγR signaling–independent, as was similarly observed for agonistic TNFR-targeting Abs (42). The long-term effect of DARA in this syngeneic i.p. model was studied in wild-type and NOTAM mice. Whereas DARA effectively delayed tumor outgrowth in wild-type mice, DARA no longer delayed tumor outgrowth in NOTAM mice (data not shown). We conclude that FcRγ-chain ITAM signaling is the main mechanism of action of DARA in this model and that, at least in this model, PCD induction by DARA cross-linking alone is not sufficient to prevent tumor outgrowth.

FcγR-mediated Ab cross-linking may be induced by various FcγR-expressing cell types such as PMNs, NK cells, monocytes, macrophages, dendritic cells, platelets, B cells, and endothelial cells, depending on the tumor niche. For MM cells, these might be NK cells, monocytes, or macrophages, as these are described to be in close proximity with MM cells in bone marrow tumor microenvironment (4648). In this study, we observed after 4 h F4/80+ macrophages as well as GR1+ PMNs in the peritoneal cavity (data not shown), suggesting a role for both effector cell types in DARA-induced PCD. However, we cannot exclude a role for FcγRIIb-mediated cross-linking by B cells present in the peritoneal cavity.

In summary, we have shown in vitro PCD induction by DARA through FcγR-mediated cross-linking on CD38-expressing MM cell lines. In vivo we observed that the inhibitory FcγRIIb as well as the activating FcγRs can mediate cross-linking of DARA on tumor cells, resulting in PCD induction. From these findings, we conclude that PCD induction by FcγR-mediated cross-linking may contribute to the antitumor activity of DARA. Based on these new findings, we hypothesize that DARA’s deep and durable responses in patients with MM are induced by the drug’s multifaceted mechanisms of action. The underlying mechanism of this PCD induction and the contribution to the overall antitumor effect of DARA require further investigation.

We thank Daniëlle Jacobs and Lukas Oomen for technical assistance and Michel de Weers for scientific input.

The online version of this article contains supplemental material.

Abbreviations used in this article:

7-AAD

7-aminoactinomycin D

ADCC

Ab-dependent cellular cytotoxicity

ADCP

Ab-dependent phagocytosis

CDC

complement-dependent cytotoxicity

DARA

daratumumab

DilC1(5)

1,1′,3,3,3′,3′-hexamethylindodicarbo–cyanine iodide

MM

multiple myeloma

PCD

programmed cell death

PMN

polymorphonuclear cell.

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M.B.O., J.J.L.v.B., and P.W.H.I.P. are Genmab BV employees and own warrant and/or stock. J.H.M.J., M.N., R.W.J.G., J.H.W.L., and P.B. received research funding from Genmab BV.

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