The Therapeutic CD38 Monoclonal Antibody Daratumumab Induces

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The Therapeutic CD38 Monoclonal Antibody Daratumumab Induces Programmed Cell Death via Fcγ Receptor−Mediated Cross-Linking This information is current as of October 2, 2021. Marije B. Overdijk, J. H. Marco Jansen, Maaike Nederend, Jeroen J. Lammerts van Bueren, Richard W. J. Groen, Paul W. H. I. Parren, Jeanette H. W. Leusen and Peter Boross J Immunol published online 17 June 2016

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published June 17, 2016, doi:10.4049/jimmunol.1501351 The Journal of Immunology

The Therapeutic CD38 Monoclonal Antibody Daratumumab Induces Programmed Cell Death via Fcg Receptor–Mediated Cross-Linking

Marije B. Overdijk ,*,1 J. H. Marco Jansen ,†,1 Maaike Nederend,† Jeroen J. Lammerts van Bueren ,* Richard W. J. Groen ,‡ Paul W. H. I. Parren,*,x,{ Jeanette H. W. Leusen ,†2 and Peter Boross †,2

Emerging evidence suggests that FcgR-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 FcgR. By comparing DARA efﬁcacy in a syngeneic in vivo tumor model Downloaded from using FcRg-chain knockout or NOTAM mice carrying a signaling-inactive FcRg-chain, we found that the inhibitory FcgRIIb as well as activating FcgRs induce DARA cross-linking–mediated PCD. In conclusion, our in vitro and in vivo data show that FcgR- 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. The Journal of Immunology, 2016, 197: 000–000. http://www.jimmunol.org/ aratumumab (DARA) is a human IgG1 therapeutic mAb lignant and premalignant diseases on which CD38 is expressed, that binds to CD38. Among other hematological ma- such as smoldering myeloma and non–Hodgkin’s lymphoma. D lignancies, CD38 is expressed at high levels on multiple Multiple mechanisms of action have been observed for DARA, myeloma (MM) tumor cells (1). In 2015, the U.S. Food and Drug including the Fc-dependent effector mechanisms, complement- Administration has approved DARA for MM patients who have dependent cytotoxicity (CDC), Ab-dependent cellular cytotoxic- received at least three prior lines of therapy, including a protea- ity (ADCC) (5), and Ab-dependent phagocytosis (ADCP) (6). some inhibitor and an immunomodulatory agent, or patients Recent studies have revealed previously unknown immunomod- double refractory to these agents. Approval was based on two ulatory effects of DARA where CD38-expressing immunosup-

phase II studies of DARA monotherapy (16 mg/kg) in heavily pressive regulatory T and B cells and myeloid-derived suppressor by guest on October 2, 2021 treated patients (2, 3). A pooled analysis of these studies revealed cells are highly sensitive to DARA treatment (7). It has also been an overall response rate of 31%, including responses that deep- shown that DARA can modulate the enzymatic activity of CD38 ened over time, and median overall survival of 19.9 mo (4). and potentially may lead to a reduction in immunosuppressive DARA is currently in multiple phase III clinical trials for the adenosine levels (8, 9). This shift away from an immunosup- treatment of MM in relapsed and frontline settings. Additional pressive environment may lead to the generation of protective studies are ongoing or planned to assess its potential in other ma- immune responses. Indeed, a concomitant induction of helper and cytotoxic T cell increases in absolute cell counts and production of IFN-g in response to viral peptides was observed. Additionally, an † *Genmab, 3584 CM Utrecht, the Netherlands; Immunotherapy Laboratory, Labora- increase in T cell clonality in subjects who responded to DARA tory for Translational Immunology, University Medical Center, 3584 CX Utrecht, the Netherlands; ‡Department of Cell Biology, University Medical Center, 3584 CX versus subjects who did not respond was observed, indicating an Utrecht, the Netherlands; xDepartment of Cancer and Inﬂammation Research, Insti- improved adaptive immune response (7). tute of Molecular Medicine, University of Southern Denmark, 5000 Odense, Den- mark; and {Department of Immunohematology and Blood Transfusion, Leiden ADCC and ADCP are induced by mAb binding to activating University Medical Center, 2300 RC Leiden, the Netherlands FcgRs on immune effector cells, for example, NK cells, macro- 1M.B.O. and J.H.M.J. contributed equally to this work. phages, and polymorphonuclear cells (PMNs), to clustered IgG 2J.H.W.L. and P.B. contributed equally to this work as cosenior authors. constant domains (Fc domains). Remarkably, it is becoming clear ORCID: 0000-0002-4365-3859 (P.W.H.I.P.). that Fc–FcgR interactions may also lead to signaling in the tumor Received for publication June 15, 2015. Accepted for publication May 25, 2016. cells, enhancing the agonistic activity of mAb or inducing programmed cell death (PCD). PCD includes all pathways leading Address correspondence and reprint requests to Dr. Jeanette H.W. Leusen, Immuno- therapy Laboratory, F03.821, Laboratory for Translational Immunology, University to cell death mediated by activation of intracellular signaling. Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, the Netherlands. Agonistic therapeutic mAb targeting members of the death re- E-mail address: [email protected] ceptor family induce PCD via the extrinsic apoptosis pathway The online version of this article contains supplemental material. (10). PCD induced by agonistic mAb targeting these death re- Abbreviations used in this article: 7-AAD, 7-aminoactinomycin D; ADCC, Ab-dependent ceptors is enhanced by cross-linking, either by secondary Ab or, cellular cytotoxicity; ADCP, Ab-dependent phagocytosis; CDC, complement-dependent cytotoxicity; DARA, daratumumab; DilC1(5), 1,19,3,3,39,39-hexamethylindodicarbo– more physiologically, via binding to FcgRs (11, 12). This suggests cyanine iodide; MM, multiple myeloma; PCD, programmed cell death; PMN, that FcgRs on, for example, tumor-associated leukocytes could polymorphonuclear cell. provide a cross-linking scaffold for antitumor mAb in vivo. In- Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 deed, PCD induced by drozitumab, a human IgG1 mAb targeting

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1501351 2 DARATUMUMAB INDUCES PROGRAMMED CELL DEATH death receptor 5, could be strongly enhanced by Fc cross-linking aminoactinomycin D [7-AAD]) were purchased from BD Biosciences using activating FcgRs or the inhibitory FcgRIIb in vitro (13). and used according to the manufacturer’s protocol. Briefly, cells were Nevertheless, in vivo the antitumor activity of drozitumab was washed with binding buffer containing 10 mM HEPES (Merck, Darm- 2/2 stadt, Germany), 0.14 M NaCl (Merck), and 2.5 mM CaCl2 (Riedel-de predominantly diminished in FcgRIIb mice, whereas in the Haen, Seelze, Germany) followed by annexin V and 7-AAD staining in absence of activating FcgRs antitumor activity was not affected. binding buffer at room temperature for 15 min. Mitochondrial membrane This suggests that FcgRIIb-mediated cross-linking is sufficient for potential depolarization was measured with the MitoProbe 1,19,3,3,39,39- the antitumor effect of drozitumab in vivo, which corresponds to hexamethylindodicarbo–cyanine iodide [DilC1(5)] kit (Life Technologies), which was used according to the manufacturer’s protocol. Briefly, after findings for other dead receptor targeting agonistic mAb (14, 15). washing in PBS (Pharmacy, University Medical Center, Utrecht, the Ab-mediated cross-linking of Ags, not related to the death receptor Netherlands), cells were incubated for 20 min with 3 nM DilC1(5) at 37˚C family, may also induce PCD, but not via the classical apoptotic and 5% CO2. Cells were washed twice with RPMI 1640 medium supple- pathway (16–19). This pathway is characterized by homotypic ag- mented with 10% FCS and penicillin/streptomycin to stop the DilC1(5) gregation of cells involving cytoskeleton reorganization, lysosomal reaction. Numbers of cell surface CD38 molecules were determined with mouse anti-human CD38 Ab (BD Biosciences) and the QiFi kit (Dako) or activation, and production of reactive oxygen species (20). This Ab- QuantiBRITE tubes (BD Biosciences). induced PCD pathway can be enhanced by Fc cross-linking secondary Ab (21–23) or by FcgR-expressing cells (24). CD38 cross-linking in vitro In this study, we explored whether DARA induces PCD of Labeled target cells were seeded at 1 3 105 cells per well in 24-well plates or CD38-expressing tumor cells via FcgR-mediated cross-linking. 2 3 104 cells per well in 96-well plates and preincubated 30 min with varying PCD was defined by morphological changes resulting in cluster- concentrations of indicated mAb followed by incubation in the presence of either 5 mg/ml rabbit anti-human IgG F(ab9) fragments, IIA1.6-hFcgRI cells, ing of cells, phosphatidylserine translocation, loss of mitochon- 2

IIA1.6 cells, or primary cells isolated from bone marrow and peritoneum of Downloaded from drial membrane potential, and loss of membrane integrity. To NOTAM transgenic mice at the indicated E:T ratio. Morphologic changes were investigate the contribution of FcgRs in DARA-mediated PCD visualized using the EVOS microscope (Advanced Microscopy Group, Life in vivo, we explored PCD induction in NOTAM (transgenic mice Technologies). PCD markers (annexin V, 7-AAD, and mitochondrial mem- expressing physiological levels of signaling-abrogated activating brane potential depolarization) were analyzed for the labeled target cells after 4 or 24 h by ﬂow cytometry using a FACSCanto II (BD Biosciences). Percentage FcgRs) and FcRg-chain knockout mice. Our results show that of annexin V+,annexinV+/7-AAD+, and DilC5(1)low cells was calculated both inhibitory and activating FcgRs can mediate DARA cross- using FACSDiva software (BD Biosciences). linking, leading to phosphatidylserine translocation followed by http://www.jimmunol.org/ Mice cell death. Experiments were performed with 8- to 19-wk-old FcRg-chain knockout (FcRg2/2) mice (30) and NOTAM mice (31) on a C57BL/6 background. Materials and Methods Mice were bred at the speciﬁc pathogen-free facility of the Central Animal Cell culture Laboratory of Utrecht University, and all experiments were approved by the local Animal Ethical Committee. The MM cell lines UM-9, generated at the University Medical Center (Utrecht, the Netherlands) (25), and L363, obtained from the American Syngeneic peritoneal mouse model Type Culture Collection transduced with GFP and luciferase marker genes Mice were injected i.p. with 5 3 106 CFSE-labeled EL4-CD38 cells in 100

(26), were transduced with human CD38 gene to increase CD38 expression by guest on October 2, 2021 levels. For this the amphotropic Phoenix packaging cell line (Phoenix ml PBS and, directly after tumor cell inoculation, with 2 mg DARA-K322A Ampho) was transfected, using calcium phosphate precipitation, with the or 100 ml PBS. To block FcgRIIb in vivo, 50 mg F(ab9)2 fragments of the pQCXIN vector in which the gene encoding human CD38 was inserted. anti-FcgRIIb mAb K9.361 was injected i.p. 30 min prior to tumor cell These cell lines are referred to as UM9-CD38 and L363-CD38 expressing inoculation (blocking of FcgRIIb was confirmed on peritoneal effector CD38 in a range of 350,000–600,000 and 450,000–800,000 molecules/cell, cells by flow cytometry). Following 4 h incubation, the mice were eu- respectively, as determined with QiFi analysis (QiFi kit, Dako, Glostrup, thanized and the peritoneum was washed with PBS containing 5 mM EDTA (Sigma-Aldrich, St. Louis, MO). Annexin V and 7-AAD staining of Denmark). IIA1.6 cells (mouse pre–B cell line, American Type Culture + Collection) were transfected with human FcgRI (hFcgRI) and human the CFSE EL4-CD38 cells was analyzed by flow cytometry as described FcRg-chain as described previously (27); these cells are referred to as above. IIA1.6-hFcgRI. EL4 cells (mouse lymphoma cell line, American Type Statistical analysis Culture Collection) and were retrovirally transduced with a GFP-IRES- luciferase construct as described previously (26). Subsequently, the Data analysis was performed using GraphPad Prism version 5.0 (GraphPad pQCXIN vector containing the gene encoding human CD38 was inserted Software, San Diego, CA). Data are reported as means 6 SD. Differences as described for the MM cell lines. Cells were subcloned by limiting di- between groups were analyzed using a Student unpaired t test or a Bon- lution, resulting in a stable EL4-CD38 clone expressing 225,000–400,000 ferroni multiple comparison test (a p value ,0.05 was considered statis- CD38 molecules/cell as determined with QuantiBrite analysis (BD Bio- tically significant). sciences, 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 Results mg/ml streptomycin (Life Technologies). Culture medium for the IIA1.6- DARA-mediated cross-linking induces PCD in MM cell lines hFcgRI cells was supplemented with 2.5 mg/ml methotrexate (Emthexate; Teva Pharmachemie, Haarlem, the Netherlands) and for the EL4-CD38 We explored whether DARA causes PCD by cross-linking of CD38 cells with 1 mg/ml geneticin (Life Technologies). UM9-CD38 and EL4- on MM cell lines. As CD38 expression levels were shown to CD38 cells were labeled with CFSE or CellTrace Violet (Life Technolo- correlate with the level of DARA-induced ADCC and CDC on both gies) according to the manufacturer’s protocol. MM cell lines and primary MM cells (32), we made use of MM Abs and reagents cell lines L363-CD38 and UM9-CD38 transduced with human CD38 to increase CD38 expression to levels that are comparable Human IgG1 anti-CD38 mAb DARA was generated by immunization of HuMAb mice and was produced recombinantly as described previously (5). to primary myeloma cells. Incubation of DARA-opsonized L363- DARA-K322A, a Fc mutant lacking complement activation, was generated CD38 and UM9-CD38 cells with Fc–cross-linking secondary Ab by mutating the lysine at position 322 to alanine as described previously resulted in significant upregulation of phosphatidylserine translo- (28, 29). F(ab9)2 fragments of rabbit anti-human IgG (Jackson Immuno- cation, as indicated by an increase in the number of annexin V+ Research Laboratories, West Grove, PA) was used as Fc–cross-linking cells (Fig. 1A). DARA-induced PCD was demonstrated by the secondary Ab. F(ab9)2 fragments of the mouse FcgRIIb mAb K9.361 were generated according to the manufacturer’s instructions (Thermo subsequent significant loss of mitochondrial membrane potential Fisher Scientific, Waltham, MA). PE-labeled annexin V and Via-Probe (7- for the UM9-CD38 cells (Fig. 1B) and a significant increase in the The Journal of Immunology 3

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-hFcgRI cells (Fig. 3, bottom panel) induced clustering of the cells, which was demonstrated to correlate with PCD induction by CD20 mAb (16, 18, 33). FcgR- mediated clustering was effective over a broad concentration range (0.0001–10 mg/ml), whereas Fc–cross-linking mediated by a secondary Ab was restricted to a more narrow concentration range (0.01–10 mg/ml) (Fig. 3). EL4-CD38 cells are sensitive for PCD induction after DARA cross-linking in vitro and ex vivo The role of FcgR-mediated DARA-induced PCD in vivo was studied in NOTAM and FcRg-chain knockout (FcRg2/2) 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-hFcgRI cells indeed induced PCD in EL4-CD38 cells (Fig. 4A, 4B, Supplemental Fig. 2). FcgRI-mediated Downloaded from DARA cross-linking was, also on EL4-CD38 cells, effective over a broad concentration range. NOTAM mice have normal surface expression of all activating FcgRs, but without signaling capacity due to a signaling-deficient FcR-associated g-chain (31) (Fig. 4C). Leukocytes in the NOTAM mice are therefore capable of FcgR-mediated Ab cross-linking, without inducing cytotoxicity http://www.jimmunol.org/ via ADCC or ADCP (31). Leukocytes in FcRg2/2 mice lack expression of all activating FcgRs and solely express the inhibitory FcgRIIb. We explored ex vivo whether primary FcgR-expressing cells from NOTAM mice could mediate Fc–cross-linking of DARA- FIGURE 1. DARA induces PCD in MM cells after Ab-mediated cross- opsonized EL4-CD38 cells. Primary FcgR-expressing cells iso- linking. PCD induction in L363-CD38 (left panels) or UM9-CD38 (right lated from either bone marrow or peritoneum (Fig. 4D, 4E) can also panels) cells after 24 h incubation with 0.1 mg/ml DARA or culture me- efficiently mediate PCD induction after DARA cross-linking. No dium (no mAb) in the presence of Fc-cross-linking secondary Ab. Flow efficacy was observed in the presence of F(ab9)2 fragments of by guest on October 2, 2021 cytometric analysis of percentage annexin V+ cells (A), loss of mito- DARA (data not shown), confirming that the observed PCD was chondrial membrane potential as measured by the decrease in DilC1(5) Fc mediated. fluorescence (B), and percentage 7-AAD+ (dead) cells (C). Each bar shows mean 6 SD of a representative experiment (n $ 2). **p , 0.01, ***p , Both inhibitory and activating FcgRs can induce 0.001, ****p , 0.0001 by unpaired t test. DARA-mediated PCD in vivo To exclude a role for DARA-mediated CDC induction, we made number of 7-AAD+ cells (Fig. 1C). An isotype control gave use of a DARA-K322A mutant with reduced mouse C1q binding similar results as the medium control (data not shown). In the (29). The K322A mutation did not affect FcgR-mediated PCD absence of secondary Abs, DARA did not increase any of the PCD induction (Supplemental Fig. 3). CFSE-labeled EL4-CD38 cells markers studied (Supplemental Fig. 1). were inoculated i.p. directly followed by treatment with 2 mg/ mouse DARA-K322A (∼0.1 mg/kg). After 4 h, the CFSE- FcgRI-mediated cross-linking of DARA induces PCD in MM labeled EL4-CD38 cells, harvested by peritoneal flush, were cell lines analyzed by flow cytometry for annexin V and 7-AAD staining To study DARA-mediated PCD in a more physiological setting, we (Fig. 5A). DARA-K322A induced an increase of annexin V+ explored whether cells expressing FcgRs could mediate Fc–cross- cells and significantly increased the number of 7-AAD+ cells linking of DARA-opsonized target cells, resulting in PCD. We following4hincubationintheFcRg2/2 mice (Fig. 5B, 5C, top used the hFcgRI-transduced murine B cell lymphoma cell line, bars). This indicates that DARA-K322A–induced cross-linking IIA1.6-hFcgRI, which is unable to induce ADCC (data not via the inhibitory FcgRIIb is sufficient to induce PCD in vivo. shown). Coculture of IIA1.6-hFcgRI cells with either DARA- Also in NOTAM mice, DARA-K322A treatment significantly opsonized L363-CD38 or UM9-CD38 cells also resulted in increased the number of annexin V+ and 7-AAD+ cells (Fig. 5B, significant upregulation of phosphatidylserine translocation 5C, bottom bars). To explore which type of FcgR was important (Fig. 2A). Phosphatidylserine translocation was subsequently for PCD induction, mice were pretreated with FcgRIIb-specific + followed, for a fraction of the annexin V cells, by a significant blocking F(ab9)2 fragments of mAb K9.361 (Fig. 4C). As ex- loss of mitochondrial membrane potential (Fig. 2B) and eventually pected, in FcRg2/2 mice, FcgR-mediated PCD via DARA- a significant increase of cell death (Fig. 2C). Coculture of DARA- K322A was abolished after blocking FcgRIIb (Fig. 5D, 5E, top opsonized L363-CD38 and UM9-CD38 cells with hFcgRI2 bars). In NOTAM mice, significant PCD induction was still IIA1.6 cells did not induce any of the PCD markers studied (data observed after blocking FcgRIIb, demonstrating that in addition not shown), confirming the effect to be FcgR mediated. DARA- to the inhibitory FcgRIIb, also activating FcgRs can mediate induced hFcgRI-mediated PCD was observed over a broad DARA DARA cross-linking in vivo leading to PCD (Fig. 5D, 5E, concentration range. bottom bars). 4 DARATUMUMAB INDUCES PROGRAMMED CELL DEATH

FIGURE 2. PCD induction of MM cell lines via FcgR-mediated cross-linking of DARA. GFP+ L363- CD38 (left panels) and CFSE-labeled UM9-CD39 (right panels) cells were cocultured with IIA1.6- hFcgRI 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 + C 6

7-AAD (dead) cells ( ). Each bar shows mean SD Downloaded from of a representative experiment (n =3).*p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001 by Bonferroni multiple comparison test. http://www.jimmunol.org/ by guest on October 2, 2021 Discussion potential and membrane integrity are irreversible and result in cell In this study, we demonstrated that DARA can induce PCD in death (35). Cross-linking of DARA by both Fc–cross-linking CD38-expressing MM cells after cross-linking by FcgR-expressing secondary Ab or FcgRI-expressing cells induced PCD in the cells, both in vitro and ex vivo. Using the non–complement- MM cell lines L363-CD38 and UM9-CD38. The number of binding DARA-K322A mutant in a syngeneic peritoneal mouse annexin V+ cells was usually higher compared with the number tumor model in NOTAM and FcRg2/2 mice, we showed that of cells that lost membrane integrity (7-AAD+ cells), which may DARA-induced PCD also occurs in vivo and can be mediated by reﬂect the reversible nature of phosphatidylserine translocation. the inhibitory FcgRIIb as well as by activating FcgRs. The low percentage of 7-AAD+ cells suggests that the contribution DARA-induced PCD was characterized by four phenotypic of PCD to the antitumor effect of DARA is limited. However, characteristics: morphological changes resulting in clustering therapeutically, the relatively high number of annexin V+ cells of cells, phosphatidylserine translocation, loss of mitochondrial mem- may substantially enhance efﬁcacy of other mechanisms of action brane potential, and loss of membrane integrity. Phosphatidylserine of DARA. For instance, phosphatidylserine translocation is rec- translocation is a marker for the initiation of PCD, which is a re- ognized by scavenger receptors on macrophages, resulting in versible process (34). In contrast, loss of the mitochondrial membrane phagocytosis of sensitized cells. We previously observed DARA

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 5or25mg/ml Fc–cross-linking secondary Ab (upper and middle panels)or IIA1.6-hFcgRI cells (E:T ratio of 1:1) (bottom panel). Original magniﬁcation 310. The Journal of Immunology 5

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-hFcgRI cells (E:T ratio of 1:1) in the presence of a serial di- lution of DARA (A and B). (C) Cartoon depicting the available FcgRs in NOTAM and FcRg2/2 mice. The X indicates blocking of

FcgRIIb receptors using K9.361 F Downloaded from

(ab9)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 http://www.jimmunol.org/ concentration of 10 mg/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 6 SD of a representative experiment (n = 2). by guest on October 2, 2021

to be highly potent in phagocytosis induction (6), and there- cross-linking, PCD induction by DARA and SAR650984 may also fore phosphatidylserine translocation induced by FcgR-mediated be comparable in patients. cross-linking may potentially enhance phagocytosis. DARA- In a syngeneic peritoneal tumor model in FcRg2/2 mice, we induced PCD possibly will represent an effective mechanism of showed that DARA-induced PCD was FcgR-dependent and feasible action, especially under conditions when complement is depleted through binding to FcgRIIb in trans alone. This might be relevant at or when ADCC-mediating NK cells are exhausted during Ab tumor sites where only FcgRIIb expressing cells, for example, treatment as previously described for anti-CD20 therapy (36, 37). B cells, reside. FcgRIIb distribution and expression levels were DARA-induced PCD was further characterized by morphologic previously shown to be critical for efficacy of agonistic Abs tar- changes resulting in clustering of the studied cells, so-called geting members of the tumor necrosis superfamily (TNFR) (42). In homotypic aggregation. Homotypic aggregation of cells was dem- addition to trans binding of FcgRIIb to DARA, cis binding may onstrated to correlate with caspase-independent PCD induction for also play a role, as demonstrated for a CD38-targeting mAb by several therapeutic mAbs (16, 18, 20, 38, 39). We also did not Vaughan et al. (43). Primary mature MM cells have been shown to detect caspase-3 cleavage upon DARA cross-linking with a sec- express FcgRIIb (44), suggesting that MM cells themselves might ondary Ab in the L363-CD38 and UM9-CD38 MM cell lines (data also mediate DARA cross-linking without a requirement for ac- not shown). However, on the Burkitt’s lymphoma cell line Ramos cessory cells, as was shown for CD40 mAb (13). PCD may there- we did show caspase-3 activation upon cross-linking of DARA fore be an effective mechanism of action of DARA in bulky tumors with secondary Ab (8). The homotypic aggregation–related to which access of effector cells may be limited (45) or under caspase-independent and -dependent PCD was also shown for conditions of immune suppression, for example, owing to high MM SAR650984, a chimeric CD38 Ab (40, 41), in the absence of tumor load or bone marrow–targeted therapy. secondary cross-linking. Nonetheless, we have previously shown Using NOTAM mice in the syngeneic peritoneal tumor model, that the direct effect of SAR650984 to induce PCD is comparable we showed that DARA-induced PCD can also be mediated by to DARA-induced PCD in the presence of a secondary Ab (8). As the murine activating FcgRs, as we observed significant PCD FcgRs are ubiquitously available in the tumor environment and induction in the presence of FcgRIIb-specific blocking F(ab9)2 because they also efficiently mediate DARA-induced PCD by fragments. Accordingly, in the present study we show in vivo 6 DARATUMUMAB INDUCES PROGRAMMED CELL DEATH Downloaded from http://www.jimmunol.org/

FIGURE 5. In vivo PCD induction by Fc–cross-linking of DARA via activating and the inhibitory FcgR. (A) Scheme of the syngeneic peritoneal mouse 2/2 model. NOTAM and FcRg mice were, as indicated, treated with FcgRIIb blocking F(ab9)2 fragments [K9.361 F(ab9)2] 30 min prior to tumor cell inoculation. Subsequently, 5 3 106 CFSE-labeled EL4-CD38 cells were inoculated i.p. followed by DARA-K322A (2 mg/mouse) or PBS treatment. After 4 by guest on October 2, 2021 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). induction of PCD mediated via cross-linking by the activating lines. In vivo we observed that the inhibitory FcgRIIb as well as the FcgRs for a target that does not belong to the TNFR superfamily. activating FcgRs can mediate cross-linking of DARA on tumor cells, Because NOTAM mice carry a signaling-inactive FcRg-chain, the resulting in PCD induction. From these findings, we conclude that FcgR-mediated cross-linking of DARA resulting in PCD is FcgR PCDinductionbyFcgR-mediated cross-linking may contribute to the signaling–independent, as was similarly observed for agonistic antitumor activity of DARA. Based on these new findings, we hy- TNFR-targeting Abs (42). The long-term effect of DARA in this pothesize that DARA’s deep and durable responses in patients with syngeneic i.p. model was studied in wild-type and NOTAM mice. MM are induced by the drug’s multifaceted mechanisms of action. Whereas DARA effectively delayed tumor outgrowth in wild-type The underlying mechanism of this PCD induction and the contribution mice, DARA no longer delayed tumor outgrowth in NOTAM mice to the overall antitumor effect of DARA require further investigation. (data not shown). We conclude that FcRg-chain ITAM signaling is the main mechanism of action of DARA in this model and that, at Acknowledgments least in this model, PCD induction by DARA cross-linking alone We thank Danie¨lle Jacobs and Lukas Oomen for technical assistance and is not sufficient to prevent tumor outgrowth. Michel de Weers for scientific input. FcgR-mediated Ab cross-linking may be induced by various FcgR-expressing cell types such as PMNs, NK cells, monocytes, Disclosures macrophages, dendritic cells, platelets, B cells, and endothelial M.B.O., J.J.L.v.B., and P.W.H.I.P. are Genmab BVemployees and own war- cells, depending on the tumor niche. For MM cells, these might be rant and/or stock. J.H.M.J., M.N., R.W.J.G., J.H.W.L., and P.B. received NK cells, monocytes, or macrophages, as these are described to be research funding from Genmab BV. in close proximity with MM cells in bone marrow tumor microenvironment (46–48). In this study, we observed after 4 h F4/80+ macrophages as well as GR1+ PMNs in the peritoneal cavity (data References not shown), suggesting a role for both effector cell types in DARA- 1. Lin, P., R. Owens, G. Tricot, and C. S. Wilson. 2004. Flow cytometric immu- nophenotypic analysis of 306 cases of multiple myeloma. Am. J. Clin. Pathol. induced PCD. However, we cannot exclude a role for FcgRIIb- 121: 482–488. mediated cross-linking by B cells present in the peritoneal cavity. 2. Lokhorst, H. M., T. Plesner, J. P. Laubach, H. Nahi, P. Gimsing, M. Hansson, M. C. Minnema, U. Lassen, J. Krejcik, A. Palumbo, et al. 2015. Targeting CD38 In summary, we have shown in vitro PCD induction by DARA with daratumumab monotherapy in multiple myeloma. N. Engl. J. Med. 373: through FcgR-mediated cross-linking on CD38-expressing MM cell 1207–1219. The Journal of Immunology 7

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