The Mechanism of Action of the Anti-CD38 Monoclonal Antibody

Published OnlineFirst January 28, 2019; DOI: 10.1158/1078-0432.CCR-18-1597

Translational Cancer Mechanisms and Therapy Clinical Cancer Research The Mechanism of Action of the Anti-CD38 Monoclonal Antibody Isatuximab in Multiple Myeloma Laura Moreno1, Cristina Perez1, Aintzane Zabaleta1, Irene Manrique1, Diego Alignani1, Daniel Ajona1,2,3, Laura Blanco1, Marta Lasa1, Patricia Maiso1, Idoia Rodriguez1, Sonia Garate1, Tomas Jelinek1, Victor Segura1, Cristina Moreno1, Juana Merino1, Paula Rodriguez-Otero1, Carlos Panizo1, Felipe Prosper1, Jesus F. San-Miguel1, and Bruno Paiva1

Abstract

Purpose: Knowledge about the mechanism of action (MoA) triggered by CD38lo and CD38hi tumor plasma cells (PC), of monoclonal antibodies (mAb) is required to understand (iv) antibody-dependent cellular phagocytosis (ADCP) was which patients with multiple myeloma (MM) beneﬁt the most triggered only by CD38hi MM cells, whereas (v) complement- from a given mAb, alone or in combination therapy. Although dependent cytotoxicity could be triggered in less than half of there is considerable research about daratumumab, knowl- the patient samples (those with elevated levels of CD38). edge about other anti-CD38 mAbs remains scarce. Furthermore, we showed that isatuximab depletes CD38hi Experimental Design: We performed a comprehensive B-lymphocyte precursors and natural killer (NK) lymphocytes analysis of the MoA of isatuximab. ex vivo—the latter through activation followed by exhaustion Results: Isatuximab induces internalization of CD38 but and eventually phagocytosis. not its signiﬁcant release from MM cell surface. In addition, we Conclusions: This study provides a framework to under- uncovered an association between levels of CD38 expression stand response determinants in patients treated with isatux- and different MoA: (i) Isatuximab was unable to induce direct imab based on the number of MoA triggered by CD38 levels apoptosis on MM cells with CD38 levels closer to those in of expression, and for the design of effective combinations patients with MM, (ii) isatuximab sensitized CD38hi MM cells aimed at capitalizing disrupted tumor–stroma cell protection, to bortezomib plus dexamethasone in the presence of stroma, augmenting NK lymphocyte–mediated ADCC, or facilitating (iii) antibody-dependent cellular cytotoxicity (ADCC) was ADCP in CD38lo MM patients.

Introduction cancer immunotherapy, monoclonal antibodies (mAb) are at Growing knowledge about tumor and immune cell biology the forefront of recent clinical development with two new drugs led to continuous development of immunotherapies against approved in 2015 for the treatment of relapsed/refractory each of the four major nodes of vulnerability in the cancer– disease (2). immune relationship: direct targeting of surface antigens; Characteristics that make antigens attractive as targets for mAb- boosting of numbers and functioning of immune effectors; based therapy include, amongst others, the density of expression activating tumor antigen-speciﬁc immunity, and; overcoming of the target molecule by malignant and benign cells (3). Other inhibitory immune suppression (1). Multiple myeloma (MM) desirable characteristics of target tumor antigens vary depending is no exception to this; however, among the four major nodes of on the mAb construct and the mechanism of action (MoA) triggered by such constructs (3). Some mAbs that target antigens on the surface of malignant cells can induce apoptosis by direct 1Clinica Universidad de Navarra, Centro de Investigacion Medica Aplicada transmembrane signaling (4). There is also evidence that mAbs (CIMA), Instituto de Investigacion Sanitaria de Navarra (IDISNA), CIBER-ONC kill target cells by complement-dependent cytotoxicity (CDC; number CB16/12/00369 and CB16/12/00489, Pamplona, Spain. 2Solid Tumors ref. 5), or by inducing antibody-dependent cellular cytotoxicity Program, Centro de Investigacion Medica Aplicada (CIMA), Instituto de Investigacion Sanitaria de Navarra (IDISNA), CIBER-ONC number CB16/12/ (ADCC; ref. 6) and phagocytosis (ADCP; ref. 7). Thus, exquisite 00443, Pamplona, Spain. 3Department of Biochemistry and Genetics, School understanding about the MoA of novel mAbs is warranted, of Sciences, University of Navarra, Pamplona, Spain. because precise identiﬁcation of the (one or more) effector Note: Supplementary data for this article are available at Clinical Cancer mechanisms triggered by these can have considerable impact on Research Online (http://clincancerres.aacrjournals.org/). optimal selection of patients' candidates to receive a given mAb based on tumor phenotypes, the design of effective treatment L. Moreno, C. Perez, and A. Zabaleta contributed equally to this work. combinations, and correct immune monitoring to predict treat- Corresponding Author: Bruno Paiva, CIMA, Avda. Pio XII 55, 31008 Pamplona, ment failure (i.e., precision medicine). Navarra. Phone: 349-4919-4700, ext. 1038; E-mail: [email protected] CD38 is a type II transmembrane glycoprotein without an doi: 10.1158/1078-0432.CCR-18-1597 internal signaling domain that, although at variable levels (8), 2019 American Association for Cancer Research is expressed on malignant plasma cells (PC) from all patients with

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informed consent was given by each patient, according to the Translational Relevance local ethics committee and the Helsinki Declaration. We used the Greater knowledge about the immune effector mechanisms RPMI-8226, MM1S, OPM2, and H929 MM cell lines for studying of monoclonal antibodies is a prerequisite for better predic- internalization or release of CD38 after isatuximab binding. The tion of patients' response and optimal monitoring of treat- effect of isatuximab on proliferation and apoptosis was measured ment effects. Here, we performed a comprehensive analysis using diphenyltetrazolium bromide (MTT) and flow cytometry about the mechanism of action of isatuximab in multiple assays, whereas the ability of isatuximab to trigger CDC, ADCP myeloma. Our results underline similarities and differences and ADCC was determined using various flow cytometry meth- between isatuximab and other anti-CD38 monoclonal anti- ods. In parallel, we evaluated the immune modulatory effects of bodies and unveiled a direct association between the levels of isatuximab in the series of patients described above, and purified CD38 expression and the mechanisms triggered by isatuxi- NK lymphocytes from healthy individuals to understand the mab; accordingly, antibody-dependent cellular cytotoxicity mechanisms behind their activation and depletion after treatment emerges as the most prevalent effector mechanism by which with isatuximab, using fluorescence-activated cell sorting (FACS) isatuximab eliminates tumor cells. They also provide informa- and gene expression profiling (GEP). We also evaluated isatux- / tion that could explain, at least in part, why patients with lower imab MoA in NOD/scid/gc (NSG) xenotransplant models, expression of CD38 may respond poorly to these drugs than which were inoculated with firefly luciferase-expressing MM1S patients with higher CD38 expression. We also provide new cells in the presence or absence of immune effector cells [i.e., insight about the paradoxical depletion of natural killer (NK) peripheral blood mononucleated cells (PBMC) obtained from lymphocytes following treatment with anti-CD38 monoclonal healthy donors]. We examined the potential of an anti-CD137 antibodies; further development of therapeutic strategies to mAb, lenalidomide and bortezomib to enhance isatuximab- overcome this phenomenon should become a priority. mediated activation of NK lymphocytes and prevent their exhaustion, using flow cytometry methods. The Wilcoxon signed rank test was used to evaluate the statistical significance of the differences observed between CD16 mean MM (9). CD38 mAbs currently available or being developed for the fluorescence intensity (MFI) levels of NK lymphocytes untreated treatment of MM include daratumumab, isatuximab, MOR202, versus treated with isatuximab, whereas the Mann–Whitney U and Ab19. Except the latter, all other three mAbs are under clinical and the Kruskal–Wallis tests were used to estimate the statistical development but, so far, only daratumumab has been approved significance of differences observed between two or more groups, for the treatment of patients with MM. Coincidently, the MoA of respectively. Correlation studies were performed using the Pear- daratumumab has been considerably investigated and several son test. Survival curves were plotted according to the Kaplan– effector mechanisms were identified: tumor cell apoptosis upon Meier method and compared using the log-rank test. The SPSS FcgR cross-linking (10), CDC (11), ADCP (12), and ADCC (13). software (version 20.0; SPSS Inc.) was used for all statistical tests. More recently, Krejcik and colleagues (14, 15) have shown that þ daratumumab depletes CD38 ve immune regulatory cells, which promoted the expansion of TCR-related T lymphocytes. Although Results these findings are surprising because they suggest that daratumu- Isatuximab induces internalization of CD38 but not its mab is capable of depleting cells with considerable lower levels of significant release from MM cell surface CD38, they also indicate that drugs able to enhance the immune Recent evidence indicates that daratumumab induces rapid system may be preferred partners to combine with anti-CD38 (i.e., less than 4 hours) redistribution of CD38 molecules and mAbs (16). Accordingly, both preclinical (11, 13, 17, 18) and formation of polar aggregates leading to the release of CD38 in clinical data have confirmed the efficacy of daratumumab in microvesicles (25). Thus, we sought to investigate whether isa- combination with lenalidomide (19, 20) or pomalidomide (21). tuximab has a similar effect on CD38 density in the surface of MM Isatuximab has also demonstrated single-agent and combination cells. First, we started by selecting the H929, MM1S, OPM2, and activity in heavily pretreated patients with relapsed/refractory RPMI-8226 cell lines to investigate MM cells with a well-defined MM (22); however, preclinical data to support the clinical devel- range of specific isatuximab antibody-binding capacity (SABC) opment of isatuximab are scarce (23, 24). and CD38 antigen density. Thus, when compared with MM Here, we aimed to gain an understanding of isatuximab MoA. patients (n ¼ 13; median SABC of 120.486), anti-CD38 SABC Our results allowed us to identify thresholds of CD38 expression found in H929, MM1S and OPM2 cell lines was considerably required for triggering different MoA, which can be used for lower (7.079, 10.421, and 21.886, respectively) and equivalent to optimal patient selection and to design effective treatment com- anti-CD38 SABC values found in only one patient (Fig. 1A). By binations. In addition, we unveil potential mechanisms by which contrast, RPMI-8226 MM cells displayed SABC levels comparable natural killer (NK) lymphocytes decrease after treatment with to the median value of patients with MM (137.759 vs. 120.486, anti-CD38 mAbs. respectively). Therefore, H929, MM1S, and OPM2 cell lines are representative of patients whose tumor PCs have low CD38 expression (CD38lo), whereas RPMI-8226 cells are representative Materials and Methods of patients with high CD38 levels (CD38hi). To measure CD38 A complete description of methods and techniques used in this occupancy and changes in CD38 surface expression upon treat- study is available in the Supplementary Methods ment with isatuximab, we used monoclonal (targeting a single Briefly, ex vivo studies were performed in bone marrow samples epitope that competes with isatuximab) and polyclonal (targeting from 13 patients with MM (six newly diagnosed and seven with multiple epitopes) anti-CD38–FITC-conjugated antibodies, to relapse/refractory disease). All samples were collected after label the four MM cell lines after treatment with isatuximab for

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Figure 1. Isatuximab induces internalization of CD38 but not its significant release from MM cell surface. A, SABC and CD38 antigen density in RPMI-8226, OPM2, MM1S, and H929 MM cell lines, as well as on primary tumor PCs (n ¼ 13 patients). B and C, MM cell lines were cultured in triplicates and in the presence of isatuximab (10 mg/mL) for 24, 48, 72, and 96 hours (h). After 96 h, cells were collected for flow cytometry immunophenotyping; half were labeled with a mAb, whereas the other half were labeled with a polyclonal antibody to measure the MFI levels of the two CD38-FITC–conjugated antibodies. Baseline values of each cell line were normalized, and the variability of CD38 MFI levels is shown every 24 h. D, MM1S and RPMI-8226 MM cell lines were treated with A647-labeled isatuximab for 0.5, 1, 2, 4, 24, and 48 hours (h). After treatment, cells were washed with PBS, and flow cytometry analysis showed that, over time, there was an increase in MFI of the CD38/isatuximab-A647 signal (measured intracellularly and in the surface membrane) with respect to the control sample. E, Surface membrane CD38- isatuximab complexes were measured with a secondary antibody FITC–anti-human IgG Fab after treatment with unlabeled isatuximab for 0.5, 1, 2, 4, 24, and 48 hours (h). No significant differences on CD38-isatuximab surface levels were noted with respect to the Control-T0 [MM cells incubated with A647-labeled isatuximab or unlabeled isatuximab (10 mg/mL) at 4C for 15 minutes]. The MFI levels measured in each time point were normalized with respect to the Control- T0. All experiments were performed in triplicate.

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24 up to 96 hours. Although the MFI of CD38 measured with the cells was not reduced by treatment with isatuximab (Supplemen- monoclonal antibody was significantly lower after the first 24 tary Fig. S2F and S2G). Thus, our results suggest that isatuximab hours compared with baseline and remained low thereafter has no direct effect on MM cell lines with CD38lo and CD38hi (median decrease of 73% in all cell lines, P ¼ 0.04; Fig. 1B), there levels but may sensitize CD38hi MM cells to combined bortezo- was no significant variation in the MFI of CD38 when using the mib and dexamethasone through mechanisms other than dis- polyclonal antibody (median variation of 12% in all cell lines; P ¼ rupting tumor–stromal cell adhesion. 0.36; Fig. 1C). Similarly, RPMI-8226 cells treated during 24 hours and cultured in RPMI medium with 10% normal human serum Isatuximab activates complement in MM cells with elevated (NHS) or heat-inactivated NHS (HI-NHS), in presence or absence levels of CD38 of PBMCs and stained with the polyclonal anti-CD38–FITC- In the absence of a direct effect on MM cells with CD38 levels conjugated antibody, displayed continuous intracellular and sur- comparable to that of most MM patients, we then explored face expression of CD38 despite the formation of CD38 polar which immune effector mechanisms mediate the anti-myeloma aggregates (Supplementary Fig. S1). activity of isatuximab. To determine whether isatuximab trig- To further investigate whether CD38 persisted in MM cells after gered activation of complement, we measured the deposition of binding to isatuximab, RPMI-8226 and MM1S MM cells were C3-related fragments in the four MM cells lines as well as in cultured up to 48 hours at 4Cor37C in the presence of primary tumor PCs (n ¼ 9 patients). Although no deposition of unlabeled versus A647-labeled isatuximab. Flow cytometry anal- C3-related fragments was noted in CD38lo and CD38hi MM cell ysis of MM cells cultured at 37C in the presence of isatuximab- lines (Fig. 2A), there was a non-significant increment of A647 revealed a significant increase in MFI levels after 24 and 48 C3-related fragments deposition in primary tumor PCs (mean hours (Fig. 1D). By contrast, MFI levels remained unaltered when C3 MFI levels of 1.8 and 7.0 in the absence versus presence of MM cells were cultured with isatuximab-A647at 4C (Supple- isatuximab; P ¼ 0.13). More detailed analyses showed a sig- mentary Fig. S1D). To confirm the presence of surface CD38- nificant correlation between anti-CD38 SABC and deposition isatuximab complexes over time, we cultured RPMI-8226 and of C3-related fragments in primary tumor PCs (r ¼ 0.86; P ¼ MM1S MM cells with unlabeled isatuximab and stained them 0.003; Fig. 2B), as well as a trend for a correlation between with a secondary FITC goat anti-human IgG Fab antibody to tumor PC depletion and deposition of C3-related fragments measure the amount of isatuximab bound to CD38 in the surface (r ¼ 0.61; P ¼ 0.08; Fig. 2C). Thus, our results suggest that of MM cells. No significant differences were observed in the MFI although isatuximab may induce CDC in patients with elevated measured in the FITC channel during 48 hours (Fig. 1E). Thus, our levels of CD38, immune effector mechanisms other than CDC results indicate that isatuximab effectively binds CD38 and may must mediate the activity of isatuximab against MM cells with induce internalization but not significant release of CD38 from lower levels of CD38 expression. MM cells. Isatuximab induces ADCC in MM cells with a broad range of Isatuximab sensitizes CD38hi MM cells to bortezomib plus CD38 expression and selective ADCP in CD38hi MM cells dexamethasone but does not induce apoptosis by direct To determine whether isatuximab triggered ADCC, we started transmembrane signaling on MM cells with CD38 levels closer by incubating CD38lo and CD38hi MM cells in absence or pres- to those in MM patients ence of the drug, with or without donor NK lymphocytes. When After demonstrating that there was no significant release of MM cells were incubated only with isatuximab, survival was CD38 after binding to isatuximab, we then investigated whether nearly 100%, thus reproducing our previous findings that indi- the drug had a direct antiproliferative or pro-apoptotic effect on cated lack of direct apoptosis. By contrast, significant cell death MM cells. Upon treating CD38lo and CD38hi MM cells with was observed when both isatuximab and NK lymphocytes were increasing concentrations of isatuximab, we have not observed cultured with MM1S, OPM2 and RPMI-8226 but not with H929 a significant (P > 0.05) effect on viability, proliferation or survival MM cells (Fig. 3A), which had the lowest isatuximab anti-CD38 of MM cells (Supplementary Fig. S2A–S2C). To understand SABC (Fig. 1A). We also evaluated the potential of isatuximab whether, in the absence of direct apoptosis, isatuximab was to trigger ADCC against primary tumor PCs (n ¼ 13 patients) modulating MM cells at the molecular level, we analyzed the and observed significant tumor PC depletion (median 51%; transcriptome of CD38hi RPMI-8226 MM cells after treatment P ¼ 0.003); importantly, we noted a significant correlation with isatuximab. No significant gene deregulation (using a B between depletion of tumor PCs and their anti-CD38 SABC statistic cutoff B > 0 and log FC > 1) induced by treatment with (r ¼ 0.58; P ¼ 0.04; Fig. 3B). isatuximab was observed when compared with untreated RPMI- Because, in addition to NK lymphocytes, part of T lympho- 8226 MM cells (Supplementary Fig. S2D). cytes also express FcRIIIA (CD16A), we sought to determine the Because CD38 may synergize with the CXCR4 pathway and relative contribution of each subset to the ADCC triggered by cooperate in CXCL12-mediated homing (26), we investigated isatuximab. Accordingly, we used FACS to culture CD38hi whether CD38 blocking by isatuximab could increase the efficacy RPMI-8226 MM cells either with donor PBMCs, or with PBMCs of anti-myeloma drugs known to have reduced effect in the without NK lymphocytes (W/o NK), or with PBMCs without þ presence of stroma (e.g., bortezomib and dexamethasone; ref. 27). CD16 ve T lymphocytes (W/o T), in the presence or absence of Interestingly, we observed that in the presence of stroma, isatux- isatuximab. Our results show that the percentage of tumor PCs imab significantly increased the percentage of dying CD38hi remained stable in the absence of NK lymphocytes, whereas RPMI-8226 cells after treatment with bortezomib plus dexameth- significant tumor depletion was noted in the absence of þ asone (mean increment, 12%; P ¼ 0.006; Supplementary Fig. CD16 ve T lymphocytes (Fig. 3C), thus indicating that NK S2E). However, adhesion of RPMI-8226 and MM1S MM cells to lymphocytes are the critical mediators of ADCC triggered by the HS5 stromal cell line or patient-derived bone marrow stromal isatuximab.

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Figure 2. Isatuximab may induce complement activation in MM cells with elevated levels of CD38. A, Deposition of C3-derived fragments in CD38lo (H929, MM1S, and OPM2) and CD38hi (RPMI-8226) MM cells after complement activation in the presence of 10 mg/mL of isatuximab for 24 hours (treated) or saline (untreated). B, Deposition of C3-derived fragments in tumor PCs from primary bone marrow samples (n ¼ 9 patients) untreated and treated with 10 mg/mL of isatuximab for 24 hours was measured, and correlated with the levels of SABC and CD38 antigen density per tumor PC. C, Correlation between C3-fragment deposition and depletion of tumor PCs from primary bone marrow samples (n ¼ 9 patients) untreated and treated with 10 mg/mL of isatuximab for 24 hours. Data are expressed as the ratio of the MFI between cells incubated with 10% serum and cells incubated with its respective complement inactivated serum. Bar graphs represent the median the 95% conﬁdence intervals (from three independent experiments). The statistical signiﬁcance was evaluated using the Mann–Whitney U test.

Afterwards, we investigated if isatuximab also triggered ADCP cells, indicating that ADCP and ADCC were triggered in vivo by by labeling MM cells with violet proliferation dye (VPD) prior isatuximab (Fig. 3E and F). culturing them with unlabeled M2-like macrophages. Thus, the þ intensity of VPD measured in CD11b ve macrophages, after Effect of isatuximab on other immune cells þ culture with VPD ve MM cells and in the presence vs absence of Immune modulation triggered by daratumumab due to deple- þ isatuximab, would be a surrogate for ADCP. Our results show tion of CD38 ve immune regulatory cells has been recently non-significant ADCP against CD38lo MM cells, but significant described (14, 15). Thus, we investigated whether isatuximab ADCP against CD38hi RPMI-8226 MM cells (median of 43%, also depleted specific immune cell subsets by treating primary P ¼ 0.005; Fig. 3D). bone marrow samples from 13 patients with MM with isatux- To evaluate isatuximab MoA in vivo, we inoculated NOD/scid/ imab. As a control for the effect of isatuximab, we previously gc / (NSG) mice (which are deficient in NK cells but retain demonstrated that the drug significantly depleted tumor PCs phagocytic cells) with firefly luciferase-expressing MM1S cells (median of 51%; P ¼ 0.003; Fig. 3B). Interestingly, we observed and treated with isatuximab in the presence or absence of immune significant depletion of CD38hi B-lymphocyte precursors (median effector cells (i.e., PBMCs from healthy donors). Our results of 54%; P ¼ 0.009), basophils (median of 26.5%; P ¼ 0.006) and show that treatment with isatuximab significantly prolonged NK lymphocytes (median of 18%; P ¼ 0.002) after treatment with survival of mice in the absence and presence of immune effector isatuximab. By contrast, no differences were noted in the relative

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Figure 3. Isatuximab induces ADCC in MM cells with a broad range of CD38 levels and selective ADCP in CD38hi MM cells. A, ADCC was measured according to the percentage of Annexin V–positive CD38lo (H929, MM1S, and OPM2) and CD38hi (RPMI-8226) MM cells cocultured with donor NK lymphocytes, and after treatment with 10 mg/mL of isatuximab for 4 hours. B, Tumor PC depletion was determined in bone marrow samples from 13 patients with MM after treatment with 10 mg/mL of isatuximab for 24 hours, and correlated with the SABC and CD38 antigen density per tumor PC. C, To determine the relative contribution of NK and CD16þve T lymphocytes to the ADCC triggered by isatuximab against CD38hi RPMI-8226 MM cells, we used FACS to coculture CD38hi RPMI-8226 MM cells either with donor PBMCs, with PBMCs W/o NK, or with PBMCs without CD16þve W/o T, in the presence versus absence of 10 mg/mL of isatuximab for 24 hours. D, CD38lo (H929, MM1S, and OPM2) and CD38hi (RPMI-8226) MM cells were labeled with VPD and were preincubated with 10 mg/mL of isatuximab before adding monocyte-derived M2-like macrophages obtained from healthy donors in a 1:4 :MM cell ratio. VPD was measured on CD11bþve macrophages after 2 hours of coculture. All experiments were performed in triplicate. In A, C,andD, bars represent median values and vertical lines the upper bound of the 95% conﬁdence intervals; the statistical signiﬁcance was evaluated using the Kruskal–Wallis (A and C) and the Mann–Whitney U (D) tests. E, Mice (n ¼ 7 per group) were inoculated with 5 x 106 MM1S-GFP-Luc cells on day 0, and either were left untreated or were treated with isatuximab (20 mg/kg) on days 7 and 14. F, In another two groups of mice (n ¼ 7 per group), 10 x 106 PBMCs were administered on day 8. Survival was monitored twice weekly.

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Figure 4. Activation of NK lymphocytes upon treatment with isatuximab. A, Heat map of genes with significantly different expression (using a B statistic cutoff B > 0 and log FC > 1) from NK lymphocytes cocultured with CD38hi RPMI-8226 MM cells, left untreated versus treated with 10 mg/mL of isatuximab for 24 hours. B, CD16 MFI levels were measured on NK lymphocytes from bone marrow samples of 13 patients with MM treated with 10 mg/mL of isatuximab for 24 hours. C and D, The percentage of CD69 (C) and CD137 (D) expression on NK lymphocytes cocultured with RPMI-8226 MM cells, left untreated or treated with increasing concentrations of an Fc blocker (0.5, 1, and 2.5 mg/mL), in the absence versus presence of 10 mg/mL of isatuximab. Experiments in A, C, and D were performed in triplicate. In C and D, bars represent median values and vertical lines the upper bound of the 95% confidence intervals; the statistical significance was evaluated using the Wilcoxon (B) and Kruskal–Wallis (C and D) tests. percentage of regulatory T cells (Treg), and T lymphocytes were not whose upregulation was also observed at the protein (antigen) immune modulated after exposure to isatuximab (Supplementary level (Fig. 4D). These results suggest that isatuximab induces NK- þ Fig. S3). Other hematopoietic cells (including CD34 veCD19 ve lymphocyte activation beyond tumor–NK cell cross-talk. Because hematopoietic progenitors, neutrophils or monocytes) were not in previous experiments with primary patient samples (n ¼ 13), depleted by isatuximab (Supplementary Fig. S3E and S3F). we observed significant downregulation of CD16 MFI levels on NK lymphocytes after treatment with isatuximab (62% down- Activation and depletion of NK lymphocytes upon treatment regulation, P < 0.001; Fig. 4B), we thus investigated whether with isatuximab isatuximab was activating NK lymphocytes through Fc binding, Because NK-mediated ADCC was the only MoA developed by or if activation was also being triggered after binding to CD38 on isatuximab to kill CD38lo and CD38hi MM cells, but at the same CD38hi NK lymphocytes. We observed that in the presence of time isatuximab was depleting (CD38hi) NK lymphocytes, we increasing concentrations of an Fc blocker containing specialized sought to gain further insight into the mechanism behind the human IgG, NK lymphocytes cultured with RPMI-8226 showed direct effect of isatuximab on NK-lymphocyte depletion. Thus, we increased expression of the activation markers CD69 and CD137 compared the transcriptome of NK lymphocytes FACSorted after a in a dose-dependent manner. Of note, the extent of activation 24-hour culture with CD38hi RPMI-8226 MM cells, in the pres- induced by the Fc blocker was consistently lower than the extent of ence or absence of isatuximab. Interestingly, NK lymphocytes activation observed in the presence of isatuximab alone. Thus, the showed deregulated expression of 70 genes in the presence of combination of Fc blocker with isatuximab induced a significant isatuximab (Fig. 4A), for which Ingenuity analysis attributed increment in the expression of the activation markers CD69 and biological processes such as cell chemotaxis, cytolysis and defense CD137 compared with NK lymphocytes treated with Fc blocker in response. One such gene was TNFRSF9 (i.e., 4-1BB or CD137), the absence of isatuximab (Fig. 4C and D). Altogether, these

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Figure 5. Depletion of NK lymphocytes upon treatment with isatuximab. A, Activation of VPD-labeled NK lymphocytes (NK-VPD) was measured according to the percentage of CD69 expression, in the presence or absence of VPD-unlabeled NK lymphocytes (NK) from the same subject, and after treatment with 10 mg/mL of isatuximab for 24 hours. Cell death was determined according to the percentage of Annexin V–positive cells in the same experimental conditions. B, Activation of NK lymphocytes was measured according to the percentage of CD69 expression, in the presence of isatuximab (10 mg/mL) and anti-CD137 (mouse IgG1, clone 4B4-1, k isotype, Biolegend) alone or in combination. Cell survival was determined according to the percentage of Annexin V–negative cells in the same experimental conditions. C, NK lymphocytes were labeled with VPD and were incubated with 10 mg/mL of isatuximab for 24 hours before coculturing them with monocyte-derived M2-like macrophages obtained from healthy donors in 1:1, 1:2, and 1:4 :NK cell ratios. VPD was measured on CD11bþve macrophages after 2 hours of coculture. All experiments were performed in triplicate. Bars represent median values and vertical lines the upper bound of the 95% conﬁdence intervals; the statistical signiﬁcance was evaluated using the Mann–Whitney U tests.

results suggest that in the presence of target cells, isatuximab compared with isatuximab alone (Fig. 5B); accordingly, no dif- activates NK lymphocytes by Fc binding and CD38 transmem- ferences in tumor cell depletion were noted by adding the anti- brane signaling. CD137 mAb (Supplementary Fig. S4A). Equivalent results were Afterwards, we investigated whether the depletion of CD38hi found using similar experimental conditions in primary patient NK lymphocytes occurred due to coating and activation by bone marrow samples (Supplementary Fig. S4B). We also inves- isatuximab (i.e., fratricide). Accordingly, VPD-labeled NK lym- tigated whether lenalidomide could prolong isatuximab-mediat- phocytes (VPD-NK) preincubated with isatuximab were cultured ed activation of NK lymphocytes and prevent their exhaustion, by with VPD-unlabeled NK lymphocytes (NK) from the same sub- pretreating PBMCs with increasing doses of lenalidomide before ject. Our results show that in the absence of isatuximab, there was culture with RPMI-8226, MM1S and OPM2 MM cells, in the no NK-lymphocyte activation or depletion; however, exposure to presence or absence of isatuximab. However, no significant dif- isatuximab induced significant activation (mean 17% increment ferences were noted (Supplementary Fig. S5A and S5B). As in CD69 expression, P ¼ 0.001) and death of VPD-labeled NK expected, immune suppression of NK lymphocytes induced by lymphocytes (mean 27% increment, P ¼ 0.027), without any proteasome inhibition after pretreatment with bortezomib differences in activation and death rates when VPD-NK lympho- resulted in lower NK cell viability (Supplementary Fig. S5C). cytes were cultured with VPD-unlabeled NK lymphocytes Because recent findings suggesting that SLAMF7 could be (Fig. 5A). These results suggest that rather than fratricide, activa- critical for phagocytosis of hematopoietic cells (7) and, coinci- tion followed by exhaustion would be responsible, at least in part, dently, some NK lymphocytes co-express SLAMF7 and CD38 with for depletion of NK lymphocytes after treatment with isatuximab. levels above that of RPMI-8226 MM cells (Fig. 6), we investigated Driven by the upregulation of CD137, we subsequently evaluated whether phagocytosis could also contribute to the depletion of if an anti-CD137 agonist mAb could prolong isatuximab-medi- some NK lymphocytes. Accordingly, we labeled increasing num- ated activation of NK lymphocytes and prevent their exhaustion. bers of NK lymphocytes with VPD and cultured them with M2-like However, both viability and activation of NK lymphocytes was macrophages, in the presence or absence of isatuximab; the þ identical after treatment with isatuximab plus anti-CD137 as percentage of VPD ve macrophages would thus indicate the extent

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Figure 6. Schematic representation of the different mechanisms of action possibly triggered by isatuximab according to the levels of CD38 expression in normal and tumor cells. We measured, in normal bone marrow samples from healthy individuals (n ¼ 3), the levels of CD38 expression in T-, NK- and B-lymphocyte subsets, normal PCs, distinct myeloid cells and nucleated red blood cells. Using the Infinicyt software (Cytognos; Salamanca, Spain), we merged the FCS files from normal bone marrow samples with those from H929, MM1S, OPM2, and RPMI-8226 cell lines, as well as from bone marrow samples from 13 patients with MM. The levels of CD38 expression (measured with the clone HB7 conjugated with APCH7; BD Biosciences) are represented by population-band histograms and ordered from the lowest to highest expression detected among normal bone marrow cells, MM cell lines, and primary tumor PCs. Of note, patient MM-01 was studied at relapse after being treated with daratumumab. Levels of CD38 expression required to trigger different MoA induced by isatuximab are represented by dashed lines as a schematic representation in accordance with the results reported in this study. of NK-lymphocyte phagocytosis. Interestingly, our results showed An antigen that can be effectively targeted by a cancer-specific a non-significant increment of NK-lymphocyte phagocytosis mAb needs to be found on the surface of tumor cells in numbers with isatuximab (P ¼ 0.16; Fig. 5C), suggesting that some NK high enough to trigger one or more effector mechanisms. Here, we lymphocytes (possibly those with co-expression of SLAMF7 and showed that continuous exposure to effective concentrations of CD38hi) could be phagocyted by M2-like macrophages in the isatuximab does not result in a decrease of CD38 numbers on the presence of isatuximab. surface of MM cells, which contrasts with daratumumab-induced polar aggregates and release of CD38 in microvesicles almost immediately after antigen–antibody binding in vitro (25). These Discussion findings suggest that sequential analyses of antigen saturation and Several anti-CD38 mAbs have been developed for the treatment turnover could be valuable to design cost-effective anti-CD38 of MM (28). Thus, in-depth understanding of their MoA is of treatment schedules. utmost importance to select patients who, according to their Some mAbs can induce tumor cell death in the absence of tumor and immune phenotypes, are potential candidates to immune effector mechanisms; the strength of this effect varies benefit the most from a given mAb, alone or within rational considerably depending on the mAb, the target antigen and the treatment combinations. Although there is considerable target cell (32, 33). Direct killing by daratumumab (upon cross- research about the MoA of daratumumab (10, 11, 12–15, 17, linking) and isatuximab (without cross-linking) has been previ- 25, 29, 30, 31), preclinical data about other anti-CD38 mAbs ously reported, which is in contrast with the data shown here. in clinical development remain scarce (23, 24). Here, we per- However, such results were obtained in Burkitt's lymphoma or formed a comprehensive analysis on the MoA of isatuximab in CD38-overexpressing MM cell lines (i.e., following lentiviral MM, and uncovered a direct association between the levels of transduction; refs. 10, 23, 24), and it should be noted that, as CD38 expression and the mechanisms triggered by isatuximab; compared with normal PCs, CD38 density is downregulated in accordingly, ADCC emerges as the most prevalent effector mech- clonal PCs from nearly half of patients with MM (8), to levels that anism by which isatuximab eliminates tumor PCs. are similar to that of parental (non-transduced) MM cell lines

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(Fig. 6). Here, we showed that isatuximab had no effect on viability, primary tumor PCs (median of 51%) was similar to that reported proliferation and survival of four different MM cell lines with for daratumumab in other preclinical studies (11, 13, 17, 30), variable levels of CD38 expression. It was also surprising to see suggesting that in the event of different efficacy between both anti- that isatuximab does not induce gene expression changes in MM CD38 mAbs, such difference would be related to mechanisms cells with normal CD38 expression. Functions ascribed to CD38 other than ADCC. þ include receptor-mediated adhesion, signaling events, or bifunc- It has been reported that daratumumab depletes CD38 ve tional ectoenzymatic activities that contribute to intracellular cal- immune regulatory cells which have substantially lower MFI cium mobilization (34), although none of these functions have levels of CD38 as compared with normal and tumor PCs as well been actually demonstrated on MM cells. Furthermore, given the as MM cell lines (Fig. 6; ref. 14). These observations are surprising end-stage long-lived and quiescent phenotype of bone marrow because daratumumab (refs. 40, 41; or isatuximab; ref. 22) has PCs, it could be hypothesized that rather than signaling, CD38 acts not shown significant hematologic toxicity, suggesting that most as a marker of adhesion to the bone marrow stroma (35, 36). mechanisms triggered by these drugs require high CD38 density Interestingly, we demonstrated that tumor cells became more and spare all other cells with considerable lower MFI levels sensitive to the combined administration of bortezomib and of CD38 (14). Here, we demonstrated that, although isatuxi- dexamethasone when CD38 was blocked by isatuximab; however, mab significantly killed primary tumor PCs, it also depleted those findings appear to be unrelated to a disruption of tumor- B-lymphocyte precursors and basophils. The density of CD38 in stroma cell adhesion. Thus, other functions ascribed to CD38 such B-lymphocyte precursors and basophils are the second and third as its ectoenzymatic activity should be considered. CD38 together highest (after PCs) among all bone marrow cells (Fig. 6), and over- with CD203a (PC-1) and CD73 may flank the CD39/CD73 canon- laps with that found in immature/transitional B lymphocytes ical pathway in the production of adenosine (ADO), which could (42, 43), which are also depleted by daratumumab (14). favor tumor survival (37, 38). Accordingly, Morandi and collea- Altogether, these data suggest that anti-CD38 mAbs can target gues (39) unveiled that MM PCs express high levels of ectoenzymes B lymphocytes with elevated levels of CD38, but although this contributing to higher catabolism of ADO, and production of ADO may be valuable if these cells are immune suppressive (14), it has been demonstrated in cultures of MM and stromal cell could also delay the regeneration of mature B lymphocytes lines (25). Notwithstanding, our results suggest that direct apo- (including normal PCs and normal immunoglobulin production; ptosis triggered by isatuximab may only occur in selected patients ref. 44). By contrast, and as expected on the basis of the observa- (i.e., those in which CD38 is expressed at elevated levels). tion that isatuximab had no effect on H929 MM cells (Fig. 6), The ability of a given mAb to fix complement and to induce other hematopoietic cells with CD38 MFI levels below the former CDC is partly dependent on antigen concentration, the orienta- were not depleted by the drug. Namely, Tregs were not depleted by tion of the antigen in the membrane, and if the antigen is present isatuximab in our experimental conditions, which supports the þ in the surface as a monomer or polymer. CDC can also depend on findings of Krejcik and colleagues (14) that only rare CD38 ve and the mAb isotype and the characteristics of the target cell, including not CD38 /lo Tregs were eliminated during treatment with dar- whether the malignant cell expresses complement-regulatory atumumab. Similarly, there were no significant differences in the proteins (3). Here, we showed that isatuximab triggered the percentage of neutrophils and monocytes after exposure to isatux- deposition of C3-related fragments (i.e., a pre-requisite for CDC) imab, consistent with the low levels of CD38 expression found in in less than half of the MM patient samples included in this these cell types (Fig. 6). Because daratumumab induces release of þ study. Although CDC has been previously ascribed as a killing CD38 from the surface of MM and other CD38 ve hematopoietic þ mechanism to daratumumab and isatuximab, this has been cells (15, 25, 29), it is uncertain if the drug depletes CD38 ve demonstrated in Burkitt's lymphoma cell lines (11, 23) and in immune regulatory cells, or if these cells become undetectable after primary patient samples without depletion of NK lymphocytes release of CD38 from their surface (in this case, monitoring of (23, 29, 30); thus, the contribution of ADCC to the killing of MM CD38 with another mAb such as HuMax-003 would render false- cells in the later experiments cannot be ruled out, as deposition negative results). Accordingly, the EuroFlow uses a multiepitope of C3 was not measured. Furthermore, surface expression of anti-CD38 antibody and simultaneous surface plus intracellular complement inhibitory proteins such as CD46, CD55, and CD59 staining to measure CD38 expression during MRD assessment (9). was not associated with clinical response to daratumumab in the That notwithstanding, it cannot be excluded that other mechan- GEN501 and SIRIUS studies (29). Although the results obtained isms beyond cell depletion induced by anti-CD38 mAbs can with MM cell lines should be interpreted with caution, our lead to decreased levels of immune suppressive cells as well as findings suggest that CDC may occur only in selected patients enhanced adaptive immunity (e.g., immune modulation after (i.e., those in which CD38 is expressed at elevated levels). robust lymphocyte activation). Altogether, these findings urge for mAbs can induce cytotoxicity by binding to FcRs, which are in-depth and longitudinal monitoring of patients with MM (i.e., expressed by a variety of immune effector cells, including from baseline to MRD and eventually disease progression) to fully NK lymphocytes as well as macrophages (i.e., ADCC and ADCP, understand both tumor and immune cell mechanisms behind the respectively; ref. 3). ADCP triggered by daratumumab and isatux- response and resistance to anti-CD38 mAbs. imab has also only been demonstrated against Burkitt's lympho- Another interesting finding of this study was that, whereas NK- ma cell lines (12, 23) and in primary patient samples without lymphocytes emerged as the most relevant immune subset for depletion of NK lymphocytes (12). Here, we showed that isatux- the efficacy of isatuximab, anti-CD38 mAbs may induce their imab only induced significant ADCP in CD38hi MM cells. By depletion through exhaustion and, eventually, CD38/SLAMF7- contrast, the extent of ADCC was significant in all but H929 cells mediated phagocytosis (7). These results, if applied more broadly, (the one with the lowest anti-CD38 SABC), and detectable against could help to explain the rapid decrease in absolute counts of tumor PCs in most patient samples (11 out of 13, Fig. 4B). NK lymphocytes after the first infusions of daratumumab in Interestingly, the extent of ADCC triggered by isatuximab against patients enrolled into the GEN501, GEN503 and SIRIUS

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studies (14, 15). Accordingly, drugs able to enhance or prolong Authors' Contributions the functionality of NK lymphocytes (e.g., lenalidomide; refs. 20, Conception and design: C. Perez, A. Zabaleta, J.F. San-Miguel, B. Paiva 22; or pomalidomide; ref. 19) emerge as attractive partners to Development of methodology: L. Moreno, C. Perez, A. Zabaleta, I. Manrique, combine with anti-CD38 mAbs. On the basis of the upregulation M. Lasa, P. Maiso, S. Garate, B. Paiva Acquisition of data (provided animals, acquired and managed patients, of CD137 expression on NK lymphocytes following CD16 liga- provided facilities, etc.): C. Perez, A. Zabaleta, I. Manrique, D. Alignani, tion to isatuximab, we explored whether the combination of anti- D. Ajona, L. Blanco, M. Lasa, I. Rodriguez, S. Garate, P. Rodriguez-Otero, CD38 plus anti-CD137 mAbs could increase activation and F. Prosper, B. Paiva prolong the lifespan of NK lymphocytes; however, our results Analysis and interpretation of data (e.g., statistical analysis, biostatistics, were not comparable to those reported in lymphoma with computational analysis): L. Moreno, C. Perez, A. Zabaleta, I. Manrique, CD137 stimulation and anti-CD20 mAbs (45). Similarly, we D. Ajona, M. Lasa, S. Garate, V. Segura, C. Moreno, J. Merino, J.F. San-Miguel, B. Paiva found that pretreatment with lenalidomide did not increase Writing, review, and/or revision of the manuscript: L. Moreno, C. Perez, isatuximab-mediated activation of NK lymphocytes or prevent- A. Zabaleta, I. Manrique, D. Ajona, M. Lasa, T. Jelinek, P. Rodriguez-Otero, ed their exhaustion and death. Thus, further investigations are C. Panizo, F. Prosper, J.F. San-Miguel, B. Paiva warranted to assess if immunotherapies other than immunomodu- Administrative, technical, or material support (i.e., reporting or organizing latory drugs can potentiate ADCC, as well as to define if there is a data, constructing databases): D. Alignani, B. Paiva correlation between the absolute counts of NK lymphocytes and Study supervision: B. Paiva patients' response to anti-CD38 mAb therapy. Acknowledgments Greater knowledge about the immune effector mechanisms of This study was supported by the Centro de Investigacion Biomedica en Red— anti-CD38 mAbs is a prerequisite for better prediction of patients' Area de Oncología—del Instituto de Salud Carlos III (CIBERONC; CB16/12/ response and optimal monitoring of treatment effects. Here, we 00369 CB16/12/00489 and CB16/12/00443), formerly named as Cooperative performed a comprehensive analysis about the MoA of isatux- Research Thematic Network (grant numbers RD12/0036/0058 and RD12/ imab in MM; our results underline similarities and differences 0036/0068) of the Red de Cancer (Cancer Network of Excellence); Instituto between isatuximab and other anti-CD38 mAbs. They also pro- de Salud Carlos III/Subdireccion General de Investigacion Sanitaria (FIS num- ~ vide information that could explain, at least in part, why patients ber PI17/01243 and FIS number PI17/00411); Asociacion Espanola Contra el Cancer (GCB120981SAN and Accelerator); and Fundacion Areces. The authors with lower expression of CD38 may respond poorly to these also acknowledge the Navarra Blood and Tissue Bank, Navarrabiomed Biobank, drugs compared with patients with higher CD38 expression. Navarra Health Department for the samples from healthy donors. This study We also provide new insight about the paradoxical depletion of was supported internationally by the Black Swan Research Initiative of the NK lymphocytes following treatment with anti-CD38 mAbs; International Myeloma Foundation, the European Research Council (ERC) further development of therapeutic strategies to overcome this 2015 Starting Grant (MYELOMANEXT), the Multiple Myeloma Research Foun- phenomenon should become a priority. dation (MMRF) Immunotherapy Networks of Excellence, and the 2017 Euro- pean Hematology Association (EHA) non-clinical advanced research grant fi fl (3680644). This work was also supported by an independent grant from Sano , Disclosure of Potential Con icts of Interest and the authors would like to acknowledge the intellectual input from Francisco P. Rodriguez-Otero reports receiving speakers bureau honoraria from Cel- Aldrian and Zhili Song. gene, Janssen, and Bristol-Myers Squibb, and is a consultant/advisory board member for Celgene, Janssen, and Takeda. C. Panizo reports receiving speakers bureau honoraria from Roche Pharma, Janssen, and Bristol-Myers Squibb, and The costs of publication of this article were defrayed in part by the is a consultant/advisory board member for Takeda and Janssen. J.F. San-Miguel payment of page charges. This article must therefore be hereby marked is a consultant/advisory board member for Takeda, Janssen, Sanofi, Bristol- advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate Myers Squibb, Amgen, Novartis, and Celgene. B. Paiva reports receiving speakers this fact. bureau honoraria from Celgene, is a consultant/advisory board member for Amgen, Celgene, Janssen, Karyopharm, Takeda, and Sanofi, and reports receiving commercial research support from Sanofi, Celgene, and Takeda. No poten- Received May 22, 2018; revised November 26, 2018; accepted January 14, tial conflicts of interest were disclosed by the other authors. 2019; published first January 28, 2019.

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The Mechanism of Action of the Anti-CD38 Monoclonal Antibody Isatuximab in Multiple Myeloma

Laura Moreno, Cristina Perez, Aintzane Zabaleta, et al.

Clin Cancer Res Published OnlineFirst January 28, 2019.

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