Purpose: The CD38 cell surface antigen is expressed in diverse hematologic malignancies including multiple myeloma, B-cell non-Hodgkin lymphoma (NHL), B-cell chronic lymphocytic leukemia, B-cell acute lymphoblastic leukemia (ALL), and T-cell ALL. Here, we assessed the antitumor activity of the anti-CD38 antibody SAR650984.

Experimental Design: Activity of SAR650984 was examined on lymphoma, leukemia and multiple myeloma cell lines, primary multiple myeloma samples, and multiple myeloma xenograft models in immunodeficient mice.

Results: We identified a humanized anti-CD38 antibody with strong proapoptotic activity independent of cross-linking agents, and potent effector functions including complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, and antibody-dependent cellular phagocytosis (ADCP), equivalent in vitro to rituximab in CD20+ and CD38+ models. This unique antibody, termed SAR650984, inhibited the ADP-ribosyl cyclase activity of CD38, likely through an allosteric antagonism as suggested by 3D structure analysis of the complex. In vivo, SAR650984 was active in diverse NHL, ALL, and multiple myeloma CD38+ tumor xenograft models. SAR650984 demonstrated single-agent activity comparable with rituximab or cyclophosphamide in Daudi or SU-DHL-8 lymphoma xenograft models with induction of the proapoptotic marker cleaved capase-7. In addition, SAR650984 had more potent antitumor activity than bortezomib in NCI-H929 and Molp-8 multiple myeloma xenograft studies. Consistent with its mode of action, SAR650984 demonstrated potent proapoptotic activity against CD38+ human primary multiple myeloma cells.

Conclusion: These results validate CD38 as a therapeutic target and support the current evaluation of this unique CD38-targeting functional antibody in phase I clinical trials in patients with CD38+ B-cell malignancies. Clin Cancer Res; 20(17); 4574–83. ©2014 AACR.

Translational Relevance

The CD38 antigen is a promising target for an antibody-based therapeutic as it is expressed in a number of hematologic malignancies including multiple myeloma, B-cell non-Hodgkin lymphoma, B-cell chronic lymphocytic leukemia, B-cell acute lymphoblastic leukemia (ALL), and T-cell ALL. On the basis of the success of rituximab in treating CD20+ lymphoma, targeting CD38 with a functional antibody is a promising therapeutic approach for these diverse indications. SAR650984, a novel anti-CD38 antibody described here, induces a unique combination of effects on cancer cells including complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, antibody-dependent cellular phagocytosis, and apoptosis, and demonstrates potent in vitro and in vivo activity against cells derived from these malignancies. Consequently, clinical trials with this antibody in patients with CD38+ hematologic malignancies are on-going (NCT01084252, NCT01749969), with initial signs of promising efficacy.

Rituximab, a chimeric anti-CD20 antibody, dramatically improved the treatment outcome of both indolent and aggressive non-Hodgkin lymphoma (NHL) through its use as a single agent or in combination with chemotherapy (1, 2). Although rituximab ultimately depletes B cells through binding to CD20, its mechanism of action is attributed to a combination of direct apoptosis induction and activation of immune effector mechanisms, including antibody-dependent cell-mediated cytotoxicity (ADCC) and phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC; ref. 3). Despite the success of rituximab, incomplete treatment responses and emergence of resistance represent important limitations (1). In an attempt to overcome these limitations, a plethora of second-generation anti-CD20 antibodies has been introduced into clinical development, most notably ofatumumab and obinutuzumab which have gained regulatory approval in B-cell chronic lymphocytic leukemia (CLL; ref. 4). CD20 expression is confined to a B-cell subset, restricting the utility of these agents to patients with CLL and B-cell NHL. Therefore, identification of new targets with a broader expression spectrum and potential for antibody mechanisms similar to rituximab could yield novel antibody therapeutics for a wider range of hematologic malignancies.

The cell surface molecule CD38 represents such an opportunity because it is expressed in multiple hematologic malignancies derived from both lymphoid and myeloid lineages, including multiple myeloma, a CD20 negative B-cell malignancy (5). CD38 is a 45 kDa type II transmembrane glycoprotein that has been described as both a receptor and a multifunctional enzyme involved in the production of nucleotide-metabolites (6–8). It has been proposed to associate with cell surface proteins in lipid rafts, regulate cytoplasmic Ca2+ flux, and mediate signal transduction in lymphoid and myeloid cells (9–12). During hematopoiesis, CD38 is expressed on CD34+ stem cells and lineage-committed progenitors of lymphoid, erythroid, and myeloid cells, while pluripotent early stem cells are defined as CD34+CD38. CD38 expression is absent during the intermediate stages of lymphocyte maturation, then reappears during the final stages of maturation, and continues through the plasma cell stage (13–16).

CD38 expression is an important prognostic factor in B-CLL that identifies patients with aggressive disease and poor survival (17–19). It has been proposed that CD38 plays a role in the proliferation and survival of CLL cells (20). High CD38 expression has been reported on plasma cells as well as their malignant counterparts, multiple myeloma cells, leading to its widespread use as a surface marker, combined with CD138, to enumerate malignant myeloma cells in patient bone marrow (5, 21). Overall, CD38 expression in hematologic malignancies and its correlation with disease progression make CD38 an attractive target for antibody-based therapeutics. Several anti-human CD38 antibodies differing in their functional properties against various malignant cells and cell lines have been described in the literature (10, 22–27). A recently developed antibody, daratumumab, is currently undergoing clinical trials in patients with multiple myeloma and reported to have a manageable safety profile and early signs of efficacy (28–30). Another anti-CD38 antibody, MOR202, is being evaluated in a phase I/IIa trial in patients with relapsed/refractory multiple myeloma (31, 32).

Here, we describe the generation of a new humanized anti-CD38 antibody, SAR650984, selected for its multiple mechanisms of action. SAR650984 demonstrated potent proapoptotic activity in the absence of cross-linking agents, elicited immune effector mechanisms such as CDC, ADCC, and ADCP against CD38+ malignant cells, and in addition strongly inhibited CD38 enzymatic activity. This unique combination of activities translated into strong in vivo efficacy of SAR650984 against diverse CD38+ tumor xenograft models.

Cell culture

Ramos, Raji, Daudi, and Namalwa lymphoma cells were obtained from the American Type Culture Collection (ATCC). All other cell lines were obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ). Cells were cultured in RPMI-1640 medium supplemented with 10% FBS, 2 mmol/L glutamine, and 1% penicillin-streptomycin (Life Technologies) at 37°C in a humidified 5% CO2 incubator.

Antibody generation

CD38-binding murine monoclonal antibodies were generated by standard hybridoma technology following immunizations with murine 300-19 cells transfected to express the full-length human CD38 antigen. Antibodies were chimerized (ch) as huIgG1-κ and humanized (hu) by variable domain resurfacing (33). Chimeric antibodies, chHB7 and chOKT10, were produced on the basis of sequences derived from their respective murine hybridomas (ATCC HB-136, CRL-8022). Ab005 (a surrogate antibody for daratumumab) and huAT13/5 were produced on the basis of published sequences (Genbank: ADS96869 and ADS96865, corresponding to sequences 17 and 12 from US7829673 for Ab005, CAA03366 and CAA03368 for huAT13/5). IB4 and SUN-4B7 were a gift from F. Malavasi, University of Turin, Italy. Rituximab, cyclophosphamide (CPA), and bortezomib were obtained from commercial sources.

Apoptosis induction

Cells were incubated at 2 × 105 cells/mL in complete medium (RPMI-1640, 10% FBS, 2 mmol/L l-glutamine) with 1.5 μg/mL (10 nmol/L) of indicated antibodies for 20 hours at 37°C with 5% CO2. Cells were stained with Annexin V-FITC in accordance with the manufacturer's instructions (Life Technologies). Samples were analyzed by flow cytometry on a FACSCalibur flow cytometer with CellQuest Pro (v5.2) for acquisition control and data analysis (both BD Biosciences).

Binding assay

Antibodies were incubated with 5 × 104 cells per assay in RPMI-1640 with 2% normal goat serum and 0.1% sodium azide (both Sigma-Aldrich) for 3 hours on ice followed by incubation with FITC-conjugated goat-anti-mouse or anti-human IgG antibody (Jackson ImmunoResearch) for 1 hour on ice. Samples were analyzed on a FACSCalibur flow cytometer as above. Sigmoidal dose–response curves were generated and EC50 values calculated using GraphPad Prism (GraphPad Software).

Expression analysis

Cells were incubated in PBS with 0.1% sodium azide and 0.5% BSA (Sigma-Aldrich) at 2 × 105 cells per assay with 5 μg/mL anti-CD38-PE or 16 μg/mL anti-CD20-PE (BD Biosciences) for 2 hours on ice. Samples were washed, fixed, and acquired in conjunction with QuantiBRITE beads (BD Biosciences) by flow cytometry according to the manufacturer's instructions. Values for antibodies bound per cell (ABC) were extrapolated from the standard curve.

ADCC, ADCP, and CDC assays

ADCC activity with isolated natural killer (NK) cells and 5 × 104 target cells at an effector:target (E:T) ratio of 3:1 was measured by lactate dehydrogenase release (Cytotoxicity Detection Kit, Roche) after 4-hour incubation using standard protocols (34). The degree of specific lysis was calculated as follows: %lysis = (experimental release − spontaneous release)/(maximum release - spontaneous release) × 100.

Monocyte-derived macrophages were prepared by isolating monocytes from peripheral blood using CD14+ beads and culturing them in the presence of 50 ng/mL hGM-CSF (both Miltenyi Biotec) for 7 days. ADCP was evaluated using 2 × 105 Ramos target cells labeled with PKH26 (Sigma-Aldrich), then incubated with antibodies and isolated human monocyte-derived macrophages in round-bottom 96-well plates at 37°C for 3 hours at an E:T ratio of 3:1. Subsequently, samples were stained with anti-CD11b-Alexa Fluor-488 (AF; Life Technologies) to identify macrophages, washed and fixed with 4% paraformaldehyde. Cell-associated fluorescence of PKH26 (FL2, Target cells) and AF (FL1, Macrophages) was measured by flow cytometry using a FC500 cytometer. Dual-labeled cells were considered as phagocytic events, and the degree of phagocytosis was determined as: %phagocytosis = 100 × [# of FL2+FL1+ events/(# of FL2+FL1+ events + # of FL2+FL1- events)].

In CDC assays, antibodies were incubated with 5 × 104 target cells per well in the presence of 5% human complement (Sigma-Aldrich) for 2 hours as previously described (35). Viability of remaining cells was determined by AlamarBlue assays (Life Technologies).

Primary multiple myeloma samples

Bone marrow aspirates from patients with multiple myeloma were obtained from Hospital Edouard Herriot (Lyon, France) after written informed consent under protocols approved by an Institutional Review Board. Mononuclear cells were prepared by standard Ficoll–Paque centrifugation. Cells were incubated at 106 cells per sample with 100 μg/mL SAR650984 for 18 hours at 37°C in culture media containing 50% autologous serum (all patient samples contained > 18 mg/mL of IgG; see Supplementary Table S1). Samples were stained with anti-CD38-APC (HB7, BD Biosciences) and anti-CD138-PC5 (BA38, Beckman Coulter) antibodies to identify multiple myeloma cells and the degree of apoptosis were measured by Annexin-V-FITC staining. Samples were analyzed by flow cytometry on a FACSCalibur or a FACS Canto II (both BD Biosciences).

Enzymatic activity assay

A recombinant soluble human CD38-Fc fusion protein was produced, and enzymatic activity assays carried out as described in the Supplementary Data section (36).

3D structure analysis

Crystals of the non-glycosylated soluble human CD38 antigen (extracellular domain R45-I300) and the SAR650984 Fab fragment were obtained and analyzed as described in the Supplementary Data section. High-resolution data for crystals up to 1.53 Å were collected at beamline ID-29 at the European Synchrotron Radiation Facility (ESRF).

In vivo xenograft studies

Female CB.17 SCID mice (Charles River Laboratories) were inoculated subcutaneously with 1 × 107 cells. Treatment was initiated as indicated and tumor volumes were measured twice weekly for the duration of the study. %T/C was calculated as the median tumor volume of each treated (T) group divided by the median tumor volume of the vehicle control (C) group. Activity was assessed according to National Cancer Institute (NCI, Bethesda, MD) standards (%T/C ≤42% = active, %T/C value ≤12% = highly active).

For survival studies, SCID mice were inoculated intravenously with 5 × 106 cells per mouse through lateral tail vein injection. Animals were sacrificed when hind leg paralysis was present or body weight decreased by >20% of pretreatment weight. Kaplan–Meier analysis was performed for survival studies and %increase of lifespan (%ILS) was calculated as %ILS = 100 × (median survival of treated group − median survival of control group)/median survival of control group. In accordance with NCI standards, %ILS > 25% was considered active. Induction of cleaved caspase-7 in tumors was evaluated by immunoblot analysis as described in the Supplementary Data section.

Antibody generation and selection

We generated a large panel of murine monoclonal antibodies that specifically bound to human CD38 (huCD38). To identify antibodies with intrinsic anticancer cell activity, antibodies were screened for their ability to induce apoptosis in lymphoma cells in the absence of any cross-linking agents. One of the antibodies identified had notably strong proapoptotic activity, inducing 35% Annexin V-positive Daudi cells compared with 6% Annexin V-positive cells in the absence of antibody (Fig. 1A). In contrast, other anti-CD38 antibodies generated in our screen, such as clones 7 and 23, did not induce a significant increase in Annexin V staining of cells. Previously generated anti-CD38 antibodies huAT13/5, chOKT10, IB4, and SUN-4B7 also resulted at best in a minor (14% or less) increase in the percentage of Annexin V-positive Daudi cells. This murine lead clone was therefore chosen for further analysis based on its unique proapoptotic activity and humanized by variable domain resurfacing. The chimeric and humanized versions of the lead clone retained specific, high-affinity binding to human CD38 with an apparent Kd of 0.2 nmol/L (Fig. 1B). Its humanized IgG1 version was designated as SAR650984.

Functional activity of the humanized anti-CD38 antibody SAR650984

Superior proapoptotic activity by SAR650984 over rituximab in the absence of cross-linking agents was seen in Raji, Daudi, and Ramos lymphoma cells, which express CD20 and CD38 at similar levels (Fig. 2A and B). In addition, SAR650984 induced apoptosis in CD38+CD20 SU-DHL-8 diffuse large B-cell lymphoma (DLBCL) cells, Molp-8 multiple myeloma cells, and DND-41 T-ALL cells (Fig. 2A).

Immune effector functions mediated by rituximab were reported to contribute to its clinical activity. We therefore compared immune effector activities of SAR650984 in CD38+CD20+ cell lines with those of rituximab. SAR650984 induced strong CDC in the presence of human serum as well as strong ADCC via human NK cells in Raji and Daudi cells, with similar activity as rituximab (Fig. 2C and D). In addition, SAR650984 mediated ADCP with human macrophages against Ramos cells to a similar extent as rituximab at 10 μg/mL (Fig. 2E).

SAR650984 activity against a panel of diverse cell lines

CD38 is expressed on a wider range of malignant cell types than CD20, providing the potential for an expanded utility of a therapeutic anti-CD38 antibody (5). We therefore characterized a larger panel of cell lines for their CD38 expression level and SAR650984 in vitro activity.

CD38 levels were determined by quantitative flow cytometry and ranged from 790,000 to 59,000 ABC in the 15 cell lines tested representing B-cell NHL, multiple myeloma, B-CLL, T-cell acute lymphoblastic leukemia (ALL), and B-cell ALL (Fig. 3A). Most notably, the three multiple myeloma cell lines evaluated ranked among the six highest expressors with 790,000 to 233,000 ABC. SAR650984 induced apoptosis in eight of 15 evaluated cell lines with up to 90% Annexin V-positive cells and EC50 values from 2 to 16 ng/mL (Fig. 3B). Strong CDC activity was seen in seven of 15 cell lines evaluated with up to 90% maximum lysis and EC50 values varying widely from 8 to 230 ng/mL (Fig. 3C). SAR650984-mediated potent ADCC against all 15 cell lines evaluated with maximum lysis of 30% to 90% of target cells and EC50 values of the dose–response ranged from approximately 0.2 to 8 ng/mL (Fig. 3D). SAR650984 mediated ADCP with 60% phagocytosed Ramos cells, compared with 25% in untreated samples, with an EC50 value of 5 ng/mL (Fig. 3E). Although ADCC activity was seen against all CD38+ cell lines tested regardless of CD38 expression level, apoptosis induction and CDC activity were most often seen against cell lines with the highest CD38 levels (Fig. 3A).

Binding of SAR650984 to normal human peripheral blood cells was assessed by flow cytometry. SAR650984 specifically bound to monocytes and T cells (Supplementary Fig. S1A and S1B) with the same affinity seen on tumor cells. We observed low levels of CD38 expression in normal human blood cells with 3,000 to 14,000 ABC, much lower than levels of CD20 expression on B cells (Supplementary Fig. S1C). SAR650984 treatment did not deplete peripheral blood cells cultured in vitro (Supplementary Fig. S1D), whereas rituximab resulted in B-cell depletion and alemtuzumab in both B- and T-cell depletion.

Apoptosis induction in primary multiple myeloma patient cells

The ability of SAR650984 to induce apoptosis without the addition of external cross-linking agent was also tested in primary cells isolated from bone marrow aspirates of 7 patients with multiple myeloma. SAR650984 noticeably increased the percentages of Annexin V-positive cells over background levels in all multiple myeloma samples tested with a mean increase of 25% Annexin V-positive cells (Fig. 3F).

Enzymatic activity

Inhibition of the ADP ribosyl-cyclase enzymatic activity of human CD38 by SAR650984 and other anti-CD38 antibodies was evaluated in vitro. SAR650984 inhibited the enzymatic function of 5 nmol/L recombinant CD38 almost completely at concentrations of 200 and 20 nmol/L with partial inhibition at 2 nmol/L concentration (Fig. 4A). A human IgG1 isotype control antibody had no measurable inhibitory effect in these experiments. Other anti-CD38 antibodies tested, Ab005 (a surrogate antibody for daratumumab), chHB7, huAT13/5, and chOKT10, resulted in much lower or no inhibitory effect (Fig. 4B).

3D structure of the huCD38/SAR650984-Fab complex

Mapping of the epitope and identification of the paratope were achieved by determining the crystal structure of soluble human CD38 (huCD38) in complex with the Fab fragment from SAR650984 at 1.53 Å resolution (PDB ID code: 4 CMH; Fig. 4C). Residues that are part of the huCD38 epitope bound by SAR650984-Fab are highlighted in Fig. 4D and notably include a continuous segment from Met110 to Cys119. Mutations introduced to silence the four glycosylation sites of CD38 are not part of the huCD38/SAR650984-Fab interface.

A close-up view of the epitope shows that the interface between huCD38 and SAR650984-Fab involves only part of the residues in the CDR loops (Fig. 4E). Heavy chain CDR Loop H3 protrudes out of the structure of SAR650984-Fab and is particularly critical for complex stabilization, whereas Loop H1, H2, L1, L2, and L3 are located at the periphery of the interface. Four salt-bridges form the interactions between H1 and H2 with Arg194 and Lys111 of huCD38 (Supplementary Fig. S2). Binding of SAR650984-Fab induces local conformational changes in the huCD38 structure. The most significant conformational change affects the segment Gln115-Thr116-Val117 of huCD38 that is shifted by approximately 2.5 Å toward the light chain, creating enough space to accommodate the H3 loop of SAR650984-Fab (Fig. 4E). However, the configuration of key residues involved in the ADP-ribosyl cyclase activity of CD38 is maintained and the catalytic site remains accessible (Fig. 4F), suggesting that SAR650984 is likely an allosteric antagonist.

In vivo activity against lymphoma and leukemia models

We evaluated the in vivo efficacy of SAR650984 in comparison with established lymphoma treatments such as rituximab and chemotherapy. SAR650984 showed similar antitumor activity as rituximab in a disseminated xenograft model established from CD20+ CD38+ Daudi lymphoma cells (Fig. 5A). Treatment with 40 mg/kg SAR650984 or rituximab twice weekly for 3 weeks markedly increased survival with a median increase in lifespan (%ILS) of 68% for SAR650984 and 52% for rituximab as compared with untreated control animals. SAR650984 was also highly efficacious in a disseminated NALM-6 B-ALL xenograft model, with a median%ILS of >200% at doses of ≥ 2.5 mg/kg (Fig. 5B). In a subcutaneous xenograft model derived from SU-DHL-8, a diffuse large B-cell lymphoma cell line, SAR650984 was active at all doses tested with T/C values of 8%, 14%, 14%, or 26% at the 40, 20, 10, or 5 mg/kg dose, respectively (Fig. 5C). No significant weight loss was observed as a result of SAR650984 treatment in any of these studies. CPA, an established lymphoma treatment component, was inactive at the 56 mg/kg dose level, active at 145 and 90 mg/kg (T/C values of 3% or 17%, respectively), and was not tolerated at a higher dose of 234 mg/kg. SU-DHL-8 tumors excised after a single SAR650984 treatment were analyzed for the levels of cleaved caspase-7, a marker for apoptotic pathway activation, in comparison with untreated tumors. SAR650984 induced a 64% or 169% increase in cleaved caspase-7 levels after 14 or 24 hours, respectively (Fig. 5D).

In vivo activity against multiple myeloma models

Next, we examined the utility of SAR650984 in subcutaneous xenograft models derived from CD20CD38+ multiple myeloma cell lines, Molp-8 and NCI-H929, in comparison with bortezomib, an established multiple myeloma treatment. In a study with palpable Molp-8 tumors, SAR650984 was well tolerated and active at 40, 25, and 15 mg/kg administered twice weekly for 3 weeks with T/C values of 8%, 10%, and 12%, respectively (Fig. 6A). Bortezomib treatment at 0.7 or 0.4 mg/kg administered twice weekly for 2 weeks was inactive, whereas higher doses of 1.2 or 1.9 mg/kg were toxic.

Similarly, in a study with established NCI-H929 tumors, SAR650984 treatment was well tolerated and highly active at 40, 20, and 10 mg/kg ×6 with 10 of 10 tumor-free survivors and at 5 mg/kg ×6 with 9 of 10 tumor-free survivors at study end (Fig. 6B). Bortezomib administered twice weekly for 2 weeks was highly active at 0.6 mg/kg (T/C 7.8%; 4/10 tumor-free survivors) and at 1 mg/kg (10/10 tumor-free survivors). A higher bortezomib dose of 1.6 mg/kg was toxic, whereas a dose of 0.4 mg/kg was inactive.

The introduction of therapeutic monoclonal anti-CD20 antibodies, such as rituximab, ofatumumab, and very recently obinutuzumab, is improving outcomes for patients with B-cell malignancies, although unmet medical need remains. It would be desirable to develop antibody therapeutics for other hematologic diseases, in particular multiple myeloma, that could also combine high efficacy with low systemic toxicity. Our goal was to build upon the success story of CD20 antibody therapy by developing a novel multifunctional antibody directed against a different target with expanded utility in a range of hematologic malignancies. Here, we describe the generation and characterization of SAR650984, a CD38-targeting antibody with a unique combination of antitumor activities.

The ability of SAR650984 to induce apoptosis in vitro was superior to rituximab in B-cell lymphoma cell lines with similar CD38 and CD20 expression, with potent activity even in the absence of cross-linking agents. This type of enhanced proapoptotic activity has been described for type II anti-CD20 antibodies such as tositumumab or obinutuzumab (37). In addition, SAR650984 induced apoptosis in multiple myeloma and T-ALL cell lines, which are typically CD20, as well as a CD20 DLBCL cell line. Similarly to rituximab, SAR650984 mediated ADCC and ADCP effector activities with potent ADCC activity seen against cell lines derived from diverse CD38+ malignancies. Unlike type II anti-CD20 antibodies, SAR650984 also induced CDC in lymphoma cell lines with activity comparable with rituximab. SAR650984 demonstrated potent in vivo antitumor activities similar to rituximab in both disseminated and subcutaneous CD20+CD38+ lymphoma models. Against DLBCL SU-DHL-8 tumors SAR650984 activity compared favorably with CPA, a component of the standard treatment regimen CHOP, whose in vivo activity was limited by toxicity. In this model, SAR650984 also induced cleaved caspase-7, a marker of apoptotic cell death. Notably, SAR650984 was active against Molp-8 and NCI-H929 multiple myeloma xenograft tumors, whereas bortezomib, an agent commonly used in multiple myeloma treatment, was only active against NCI-H929 tumors.

Several anti-CD38 antibodies with various functional properties have been identified previously. Although some of these can mediate in vitro killing of CD38+ cell lines via ADCC and/or CDC effector function, their direct impact on cell proliferation or apoptosis remains unclear (10, 22–24). For example, the agonistic antibody IB4 was reported to prevent apoptosis or induce proliferation in some contexts (10, 38), and induce apoptosis in another (9). The more recently developed antibody daratumumab has been shown to mediate ADCC as well as ADCP but to induce apoptosis only in the presence of stromal cells or cross-linking agents (28, 39). It was selected on the basis of its CDC activity, which may be linked to its unique epitope. Another recent addition, MOR202 primarily possesses ADCC activity with CDC and ADCP also being observed with no reported intrinsic proapoptotic activity (31, 32).

Analysis of the 3D structure of SAR650984-Fab with human CD38 revealed that SAR650984 binds to a discontinuous epitope which includes amino acids located opposite to the catalytic site of CD38. Although significant conformational changes are observed in CD38 upon binding by SAR650984, the overall configuration of key residues involved in the CD38 enzymatic activity and access to the catalytic site appear conserved (40–42). Nevertheless, SAR650984 strongly inhibits CD38 enzymatic activity suggesting that SAR650984 is an allosteric rather than an orthosteric antagonist of CD38. This is different from HB7, an antibody that does not compete with SAR650984 (Supplementary Fig. S3), binds at a different location in the C-terminal portion of the CD38 extracellular domain (Supplementary Fig. S4), and does not inhibit CD38 enzymatic activity (Fig. 4B). The SAR650984 binding mode appears unique as other anti-CD38 antibodies tested here or described elsewhere show no or much less potent inhibition of the enzymatic activity.

CD38 enzymatic activities produce nucleotide-metabolites including cADPR, which induce Ca2+ mobilization and signaling (7, 8, 43). However, the physiologic role of CD38 is still unknown, as functional studies have mainly used cell lines ectopically overexpressing CD38 and anti-CD38 antibodies, which are nonphysiologic ligands (44, 45). Therefore, the exact interplay between enzymatic activity, receptor function and consequences on signaling pathways and cellular response remains to be further documented. As a consequence, the physiologic impact of SAR650984 inhibition of CD38 enzymatic activity remains to be deciphered and how the functional differences observed between various anti-CD38 antibodies affect their therapeutic utility requires further investigation. The contribution of distinct mechanisms of action of SAR650984 to clinical efficacy may also differ depending on the type of hematologic malignancy, tumor biology, and tumor microenvironment.

Use of chemotherapy and autologous stem cell transplantation and the availability of new therapeutic agents including proteasome, kinase, and histone deacetylase inhibitors, as well as immune-modulatory drugs have improved survival in patients with myeloma (46, 47). However, the majority of patients will eventually relapse underscoring the need for additional therapeutic options. Moreover, many of these small-molecule agents have significant toxicities, such as neuropathy, that limit their dosing and their potential for use in combination therapy. Drawing on the example of rituximab, with its favorable safety profile and success in combination therapy for lymphoma, a functional CD38 antibody could offer the prospect of a highly effective therapy in multiple myeloma that might also be used readily in combination with other agents to improve clinical outcomes. On the basis of the results described here, SAR650984 has shown potent in vitro and in vivo activity against diverse CD38+ lymphoma, leukemia as well as multiple myeloma-derived cell line models and can induce apoptosis in primary multiple myeloma cells. Therefore, it presents an exciting opportunity for a novel antibody therapeutic in CD38+ hematologic malignancies and is currently in clinical testing in patients with multiple myeloma with early signs of efficacy (48).

T. Chittenden holds ownership interest (including patents) in ImmunoGen. No potential conflicts of interest were disclosed by the other authors.

Conception and design: M.-C. Wetzel, V.S. Goldmacher, C. Lahoute, C. Dumontet, P. Lejeune, P.U. Park, V. Blanc

Development of methodology: M.-C. Wetzel, Q. Zhou-Liu, P. Ferrari, P.U. Park

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): J. Deckert, M.-C. Wetzel, L.M. Bartle, A. Skaletskaya, F. Vallée, S. Pouzieux, C. Dumontet, A. Plesa, M. Chiron, P.U. Park

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): J. Deckert, L.M. Bartle, F. Vallée, C. Dumontet, A. Plesa, M. Chiron, P. Lejeune, P.U. Park, V. Blanc

Writing, review, and/or revision of the manuscript: J. Deckert, M.-C. Wetzel, F. Vallée, Q. Zhou-Liu, C. Dumontet, M. Chiron, P. Lejeune, T. Chittenden, P.U. Park, V. Blanc

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): Q. Zhou-Liu, P.U. Park

Study supervision: M.-C. Wetzel, P. Lejeune, T. Chittenden, P.U. Park

The authors thank Daniel Tavares, Michele Mayo, Hélène Bastien, Josiane Le Parc, François Michoux, Anne Pommeret, Dominique Quarteronet, Florence Attenot, and Catherine Geslin for technical support, Ti Cai for critical reading of the manuscript, and Walter Blättler, Thierry Hercend, Michel Streuli, and John Lambert for guidance and helpful discussions.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Bello
C
,
Sotomayor
EM
. 
Monoclonal antibodies for B-cell lymphomas: rituximab and beyond
.
Hematology Am Soc Hematol Educ Program
2007
:
233
42
.
2.
Dotan
E
,
Aggarwal
C
,
Smith
MR
. 
Impact of rituximab (rituxan) on the treatment of B-cell non-Hodgkin's mymphoma
.
P T
2010
;
35
:
148
57
.
3.
Alduaij
W
,
Illidge
TM
. 
The future of anti-CD20 monoclonal antibodies: are we making progress
?
Blood
2011
;
117
:
2993
3001
.
4.
Cang
S
,
Mukhi
N
,
Wang
K
,
Liu
D
. 
Novel CD20 monoclonal antibodies for lymphoma therapy
.
J Hematol Oncol
2012
;
5
:
64
.
5.
Konopleva
M
,
Rissling
I
,
Andreeff
M
. 
CD38 in hematopoietic malignancies
.
Chem Immunol
2000
;
75
:
189
206
.
6.
Quarona
V
,
Zaccarello
G
,
Chillemi
A
,
Brunetti
E
,
Singh
VK
,
Ferrero
E
, et al
CD38 and CD157: a long journey from activation markers to multifunctional molecules
.
Cytometry B Clin Cytom
2013
;
84
:
207
17
.
7.
Lee
HC
,
Munshi
C
,
Graeff
R
. 
Structures and activities of cyclic ADP-ribose, NAADP and their metabolic enzymes
.
Mol Cell Biochem
1999
;
193
:
89
98
.
8.
Berthelier
V
,
Tixier
JM
,
Muller-Steffner
H
,
Schuber
F
,
Deterre
P
. 
Human CD38 is an authentic NAD(P)+ glycohydrolase
.
Biochem J
1998
;
330
:
1383
90
.
9.
Morra
M
,
Zubiaur
M
,
Terhorst
C
,
Sancho
J
,
Malavasi
F
. 
CD38 is functionally dependent on the TCR/CD3 complex in human T cells
.
FASEB J
1998
;
12
:
581
92
.
10.
Konopleva
M
,
Estrov
Z
,
Zhao
S
,
Andreeff
M
,
Mehta
K
. 
Ligation of cell surface CD38 protein with agonistic monoclonal antibody induces a cell growth signal in myeloid leukemia cells
.
J Immunol
1998
;
161
:
4702
8
.
11.
Zilber
MT
,
Gregory
S
,
Mallone
R
,
Deaglio
S
,
Malavasi
F
,
Charron
D
, et al
CD38 expressed on human monocytes: a coaccessory molecule in the superantigen-induced proliferation
.
Proc Natl Acad Sci U S A
2000
;
97
:
2840
5
.
12.
Deaglio
S
,
Vaisitti
T
,
Billington
R
,
Bergui
L
,
Omede
P
,
Genazzani
AA
, et al
CD38/CD19: a lipid raft-dependent signaling complex in human B cells
.
Blood
2007
;
109
:
5390
8
.
13.
Reinherz
EL
,
Kung
PC
,
Goldstein
G
,
Levey
RH
,
Schlossman
SF
. 
Discrete stages of human intrathymic differentiation: analysis of normal thymocytes and leukemic lymphoblasts of T-cell lineage
.
Proc Natl Acad Sci U S A
1980
;
77
:
1588
92
.
14.
Tedder
TF
,
Clement
LT
,
Cooper
MD
. 
Discontinuous expression of a membrane antigen (HB-7) during B lymphocyte differentiation
.
Tissue Antigens
1984
;
24
:
140
9
.
15.
Terstappen
LW
,
Huang
S
,
Safford
M
,
Lansdorp
PM
,
Loken
MR
. 
Sequential generations of hematopoietic colonies derived from single nonlineage-committed CD34+CD38- progenitor cells
.
Blood
1991
;
77
:
1218
27
.
16.
Ferrero
E
,
Malavasi
F
. 
The metamorphosis of a molecule: from soluble enzyme to the leukocyte receptor CD38
.
J Leukoc Biol
1999
;
65
:
151
61
.
17.
Damle
RN
,
Wasil
T
,
Fais
F
,
Ghiotto
F
,
Valetto
A
,
Allen
SL
, et al
Ig V gene mutation status and CD38 expression as novel prognostic indicators in chronic lymphocytic leukemia
.
Blood
1999
;
94
:
1840
7
.
18.
Ibrahim
S
,
Keating
M
,
Do
KA
,
O'Brien
S
,
Huh
YO
,
Jilani
I
, et al
CD38 expression as an important prognostic factor in B-cell chronic lymphocytic leukemia
.
Blood
2001
;
98
:
181
6
.
19.
Hamblin
TJ
,
Orchard
JA
,
Ibbotson
RE
,
Davis
Z
,
Thomas
PW
,
Stevenson
FK
, et al
CD38 expression and immunoglobulin variable region mutations are independent prognostic variables in chronic lymphocytic leukemia, but CD38 expression may vary during the course of the disease
.
Blood
2002
;
99
:
1023
9
.
20.
Deaglio
S
,
Vaisitti
T
,
Aydin
S
,
Ferrero
E
,
Malavasi
F
. 
In-tandem insight from basic science combined with clinical research: CD38 as both marker and key component of the pathogenetic network underlying chronic lymphocytic leukemia
.
Blood
2006
;
108
:
1135
44
.
21.
Lin
P
,
Owens
R
,
Tricot
G
,
Wilson
CS
. 
Flow cytometric immunophenotypic analysis of 306 cases of multiple myeloma
.
Am J Clin Pathol
2004
;
121
:
482
8
.
22.
Stevenson
FK
,
Bell
AJ
,
Cusack
R
,
Hamblin
TJ
,
Slade
CJ
,
Spellerberg
MB
, et al
Preliminary studies for an immunotherapeutic approach to the treatment of human myeloma using chimeric anti-CD38 antibody
.
Blood
1991
;
77
:
1071
9
.
23.
Ausiello
CM
,
Urbani
F
,
Lande
R
,
la Sala
A
,
Di Carlo
B
,
Baj
G
, et al
Functional topography of discrete domains of human CD38
.
Tissue Antigens
2000
;
56
:
539
47
.
24.
Goldmacher
VS
,
Bourret
LA
,
Levine
BA
,
Rasmussen
RA
,
Pourshadi
M
,
Lambert
JM
, et al
Anti-CD38-blocked ricin: an immunotoxin for the treatment of multiple myeloma
.
Blood
1994
;
84
:
3017
25
.
25.
Ellis
JH
,
Barber
KA
,
Tutt
A
,
Hale
C
,
Lewis
AP
,
Glennie
MJ
, et al
Engineered anti-CD38 monoclonal antibodies for immunotherapy of multiple myeloma
.
J Immunol
1995
;
155
:
925
37
.
26.
Maloney
DG
,
Donovan
K
,
Hamblin
TJ
. 
Antibody therapy for treatment of multiple myeloma
.
Semin Hematol
1999
;
36
:
30
3
.
27.
Stevenson
GT
,
Bell
AJ
,
Evans
TR
,
French
RR
,
Glennie
MJ
,
Hamblin
TJ
, et al
Mechanisms in removal of tumor by antibody
.
Cell Biophys
1994
;
24
25
:
45–50
.
28.
de Weers
M
,
Tai
YT
,
van der Veer
MS
,
Bakker
JM
,
Vink
T
,
Jacobs
DC
, et al
Daratumumab, a novel therapeutic human CD38 monoclonal antibody, induces killing of multiple myeloma and other hematological tumors
.
J Immunol
2011
;
186
:
1840
8
.
29.
Gimsing
P
,
Plesner
T
,
Nahi
H
,
Lokhorst
H
,
Valentin
M-L
,
Lisby
S
, et al
A Phase I/II, Dose-Escalation Study of Daratumumab, A CD38 Mab in Patients with Multiple Myeloma - Preliminary Safety Data
.
ASH Annual Meeting Abstracts
2011
;
118
:
1873
.
30.
Plesner
T
,
Lokhorst
H
,
Gimsing
P
,
Nahi
H
,
Lisby
S
,
Richardson
PG
. 
Daratumumab, a CD38 Monoclonal Antibody in Patients with Multiple Myeloma - Data From a Dose-Escalation Phase I/II Study
.
ASH Annual Meeting Abstracts
2012
;
120
:
73
.
31.
Tesar
M
,
Springer
M
,
Swoboda
B
,
Brocks
B
,
Popp
A
,
Broders
O
, et al
Fully human antibody MOR202 against CD38 for the treatment of multiple myeloma and other blood-borne malignancies.
J Clin Oncol
2007
;
25
:
8106
.
32.
Endell
J
,
Boxhammer
R
,
Wurzenberger
C
,
Ness
D
,
Steidl
S
. 
The Activity of MOR202, a Fully Human Anti-CD38 Antibody, Is Complemented by ADCP and Is Synergistically Enhanced by Lenalidomide in Vitro and in Vivo
.
ASH Annual Meeting Abstracts
2012
;
120
:
4018
.
33.
Roguska
MA
,
Pedersen
JT
,
Keddy
CA
,
Henry
AH
,
Searle
SJ
,
Lambert
JM
, et al
Humanization of murine monoclonal antibodies through variable domain resurfacing
.
Proc Natl Acad Sci U S A
1994
;
91
:
969
73
.
34.
Shields
RL
,
Namenuk
AK
,
Hong
K
,
Meng
YG
,
Rae
J
,
Briggs
J
, et al
High resolution mapping of the binding site on human IgG1 for Fc gamma RI, Fc gamma RII, Fc gamma RIII, and FcRn and design of IgG1 variants with improved binding to the Fc gamma R
.
J Biol Chem
2001
;
276
:
6591
604
.
35.
Gazzano-Santoro
H
,
Ralph
P
,
Ryskamp
TC
,
Chen
AB
,
Mukku
VR
. 
A non-radioactive complement-dependent cytotoxicity assay for anti-CD20 monoclonal antibody
.
J Immunol Methods
1997
;
202
:
163
71
.
36.
Graeff
RM
,
Walseth
TF
,
Fryxell
K
,
Branton
WD
,
Lee
HC
. 
Enzymatic synthesis and characterizations of cyclic GDP-ribose. A procedure for distinguishing enzymes with ADP-ribosyl cyclase activity
.
J Biol Chem
1994
;
269
:
30260
7
.
37.
Mossner
E
,
Brunker
P
,
Moser
S
,
Puntener
U
,
Schmidt
C
,
Herter
S
, et al
Increasing the efficacy of CD20 antibody therapy through the engineering of a new type II anti-CD20 antibody with enhanced direct and immune effector cell-mediated B-cell cytotoxicity
.
Blood
2010
;
115
:
4393
402
.
38.
Zupo
S
,
Rugari
E
,
Dono
M
,
Taborelli
G
,
Malavasi
F
,
Ferrarini
M
. 
CD38 signaling by agonistic monoclonal antibody prevents apoptosis of human germinal center B cells
.
Eur J Immunol
1994
;
24
:
1218
22
.
39.
Overdijk
MB
,
Verploegen
S
,
Marijn
B
,
van Egmond
M
,
Groen
RWJ
,
Martens
ACM
, et al
Phagocytosis Is A Mechanism of Action for Daratumumab
.
ASH Annual Meeting Abstracts
2012
;
120
:
4504
.
40.
Liu
Q
,
Kriksunov
IA
,
Graeff
R
,
Munshi
C
,
Lee
HC
,
Hao
Q
. 
Crystal structure of human CD38 extracellular domain
.
Structure
2005
;
13
:
1331
9
.
41.
Liu
Q
,
Kriksunov
IA
,
Graeff
R
,
Munshi
C
,
Lee
HC
,
Hao
Q
. 
Structural basis for the mechanistic understanding of human CD38-controlled multiple catalysis
.
J Biol Chem
2006
;
281
:
32861
9
.
42.
Liu
Q
,
Graeff
R
,
Kriksunov
IA
,
Lam
CM
,
Lee
HC
,
Hao
Q
. 
Conformational Closure of the Catalytic Site of Human CD38 Induced by Calcium
.
Biochemistry
2008
;
47
:
13966
73
.
43.
Lee
HC
,
Graeff
RM
,
Walseth
TF
. 
ADP-ribosyl cyclase and CD38. Multi-functional enzymes in Ca+2 signaling
.
Adv Exp Med Biol
1997
;
419
:
411
9
.
44.
Kitanaka
A
,
Suzuki
T
,
Ito
C
,
Nishigaki
H
,
Coustan-Smith
E
,
Tanaka
T
, et al
CD38-mediated signaling events in murine pro-B cells expressing human CD38 with or without its cytoplasmic domain
.
J Immunol
1999
;
162
:
1952
8
.
45.
Hoshino
S
,
Kukimoto
I
,
Kontani
K
,
Inoue
S
,
Kanda
Y
,
Malavasi
F
, et al
Mapping of the catalytic and epitopic sites of human CD38/NAD+ glycohydrolase to a functional domain in the carboxyl terminus
.
J Immunol
1997
;
158
:
741
7
.
46.
Munshi
NC
,
Anderson
KC
. 
New strategies in the treatment of multiple myeloma
.
Clin Cancer Res
2013
;
19
:
3337
44
.
47.
Gentile
M
,
Recchia
AG
,
Mazzone
C
,
Lucia
E
,
Vigna
E
,
Morabito
F
. 
Perspectives in the treatment of multiple myeloma
.
Expert Opin Biol Ther
2013
;
13
:
S1
22
.
48.
Martin
TG
,
Strickland
SA
,
Glenn
M
,
Zheng
W
,
Daskalakis
N
,
Mikhael
JR
. 
SAR650984, a CD38 Monoclonal Antibody In Patients With Selected CD38 +Hematological Malignancies- Data From a Dose-Escalation Phase I Study
.
ASH Annual Meeting Abstracts
2013
;
122
:
284
.