CD38-expressing Multiple Myeloma Cell Research Articles

Nicotinamide-Expanded Allogeneic Natural Killer Cells with CD38 Deletion, Expressing an Enhanced CD38 Chimeric Antigen Receptor, Target Multiple Myeloma Cells.

Natural killer (NK) cells are a vital component of cancer immune surveillance. They provide a rapid and potent immune response, including direct cytotoxicity and mobilization of the immune system, without the need for antigen processing and presentation. NK cells may also be better tolerated than T cell therapy approaches and are susceptible to various gene manipulations. Therefore, NK cells have become the focus of extensive translational research. Gamida Cell's nicotinamide (NAM) platform for cultured NK cells provides an opportunity to enhance the therapeutic potential of NK cells. CD38 is an ectoenzyme ubiquitously expressed on the surface of various hematologic cells, including multiple myeloma (MM). It has been selected as a lead target for numerous monoclonal therapeutic antibodies against MM. Monoclonal antibodies target CD38, resulting in the lysis of MM plasma cells through various antibody-mediated mechanisms such as antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity, and antibody-dependent cellular phagocytosis, significantly improving the outcomes of patients with relapsed or refractory MM. However, this therapeutic strategy has inherent limitations, such as the anti-CD38-induced depletion of CD38-expressing NK cells, thus hindering ADCC. We have developed genetically engineered NK cells tailored to treat MM, in which CD38 was knocked-out using CRISPR-Cas9 technology and an enhanced chimeric antigen receptor (CAR) targeting CD38 was introduced using mRNA electroporation. This combined genetic approach allows for an improved cytotoxic activity directed against CD38-expressing MM cells without self-inflicted NK-cell-mediated fratricide. Preliminary results show near-complete abolition of fratricide with a 24-fold reduction in self-lysis from 19% in mock-transfected and untreated NK cells to 0.8% of self-lysis in CD38 knock-out CAR NK cells. Furthermore, we have observed significant enhancements in CD38-mediated activity in vitro, resulting in increased lysis of MM target cell lines. CD38 knock-out CAR NK cells also demonstrated significantly higher levels of NK activation markers in co-cultures with both untreated and αCD38-treated MM cell lines. These NAM-cultured NK cells with the combined genetic approach of CD38 knockout and addition of CD38 CAR represent a promising immunotherapeutic tool to target MM.

Half-Life Extended Nanobody-Based CD38-Specific Bispecific Killercell Engagers Induce Killing of Multiple Myeloma Cells.

CD38 is a target for immunotherapy of multiple myeloma. Llama-derived CD38-specific nanobodies allow easy reformatting into mono-, bi- and multispecific proteins. To evaluate the utility of nanobodies for constructing CD38-specific nanobody-based killer cell engagers (nano-BiKEs), we generated half-life extended nano-BiKEs (HLE-nano-BiKEs) by fusing a CD38-specific nanobody to a CD16-specific nanobody for binding to the Fc-receptor on NK cells and further to an albumin-specific nanobody to extend the half-life in vivo. HLE-nano-BiKEs targeting three different epitopes (E1, E2, E3) of CD38 were expressed in transiently transfected HEK-6E cells. We verified specific and simultaneous binding to CD38 on myeloma cells, CD16 on NK cells, and to albumin. We tested the capacity of these HLE-nano-BiKEs to mediate cytotoxicity against CD38-expressing multiple myeloma cell lines and primary myeloma cells from human bone marrow biopsies in bioluminescence and flowcytometry assays with NK92 cells as effector cells. The results revealed specific time- and dose-dependent cytolysis of CD38+ myeloma cell lines and effective depletion of CD38-expressing multiple myeloma cells from primary human bone marrow samples. Our results demonstrate the efficacy of CD38-specific HLE-nano-BiKEs in vitro and ex vivo, warranting further preclinical evaluation in vivo of their therapeutic potential for the treatment of multiple myeloma.

P-005: Novel strategy of elotuzumab and zoledronic acid with Th1-like γδT cells against myeloma

<h3>Background</h3> We are now in the new era of immunotherapies against multiple myeloma (MM) such as anti-CD38 and anti-SLAMF7 therapeutic monoclonal antibodies (mAbs); however, MM progression falls into attenuation and exhaustion of effector cells via immune checkpoint axes such as PD-1/PD-L1 and TIGIT/CD155, leading to hamper the clinical efficacy of these therapeutic mAbs. <h3>Methods</h3> To overcome this issue, we have been studying the utilization of Vδ2 γδT cells in peripheral blood mononuclear cells, which can be expanded and induced as Th1-like phenotype ex vivo using aminobisphosphonates in combination with IL-2, as effectors for MM treatment. The present study was aimed to develop the novel therapeutic strategies of γδT cells in combination with anti-MM mAbs. <h3>Results</h3> The next generation IMiDs, cereblon E3 ligase modulators (CELMoDs) of iberdomide and CC-92480 have been developed. CELMoDs was able to expand and activate γδT cells at much lower dose compared to IMiDs of lenalidomide and pomalidomide, in combination with zoledronic acid (ZA) ex vivo. Since the expanded Th1-like γδT cells highly expressed CD16, FcγRIIIa without expression of checkpoint molecules such as PD-1, TIGIT, CTLA-4, and LAG-3, we next examined whether anti-MM mAbs, elotuzumab (ELO), daratumumab (DARA), and isatuximab (ISA), induced ADCC in the presence of the γδT cells. DARA and ISA did not induce cytotoxicity against CD38-expressing MM cells in the presence of the γδT cells; however, addition of ELO further increased cytotoxicity of the γδT cells against SLAMF7-expressing MM.1S and OPM-2 cells but not RPMI 8226 cells with marginal SLAMF7 expression, suggesting induction of ELO's ADCC with the γδT cells. We next examined the cytotoxic effect of the combinatory treatment on osteoclasts (OCs) because MM cells resides in the bone marrow and the interaction between MM cells and OCs forms vicious cycle of MM cell growth and bone destruction. Of note, OCs, which can be induced from monocytes using M-CSF and RANK ligand, highly expressed SLAMF7 along with elevation of NFATc1 transcription factor. Although the expanded γδT cells can target OCs, addition of ELO augmented the cytotoxic effect of γδT cells on OCs, suggesting the emergence of ELO's ADCC with Th1-like γδT cells towards OCs. As γδT cells recognize not only neoantigens but also phosphoantigens (pAgs), we treated MM cells and OCs with ZA to enhance the expression of pAgs, and γδT cells then incubated with the ZA-treated cells. ZA-stimulation augmented the cytotoxicity of γδT cells towards MM cells and OCs even in the presence of ELO. <h3>Conclusions</h3> Taken together, our findings demonstrated the effective usage of ELO and Th1-like γδT cells with ZA against MM cells and ambient cells in MM bone marrow. Further study is warranted on the effective revitalization and expansion of Th1-like γδT cells using CELMoDs.

Adaptability of Antibody-Coupled T Cell Receptor (ACTR) Engineered Autologous T Cells in Combination with Daratumumab over CAR-Based Approaches

Chimeric antigen receptor (CAR)-T cells have demonstrated profound clinical responses against CD19+ B cell malignancies, with ongoing trials in multiple myeloma (MM). Although BCMA CAR-T cells show promise in MM, development of CAR-T against other MM targets has been problematic due to target expression on T cells themselves. Specifically, efforts to develop CAR-T cell therapies targeting CD38 have been hampered by the upregulation of CD38 on activated T cells and consequent induction of CAR-T mediated autolysis that occurs during manufacturing. To overcome this challenge during CAR-T production, investigators have taken several approaches including multiple rounds of T cell expansion, addition of CD38 blocking antibody, and CD38 gene knockout in T cells prior to CAR transduction (1,2,3).The Antibody-Coupled T Cell Receptor (ACTR) technology is a universal, engineered T cell therapy that combines the Fc receptor, CD16, with endogenous T cell signaling and costimulatory domains (4). ACTR T cells function by engaging the Fc domain of therapeutic antibodies opsonized on target cells, resulting in T cell activation and tumor cell killing. In contrast to CAR-T cell constructs that express the antigen targeting single chain variable fragment (scFv), ACTR T cell activity is regulated through antibody administration and is therefore not susceptible to target-mediated autolysis during T cell production.The recent approval of the CD38-targeting antibody, daratumumab, has improved the treatment of multiple myeloma through its tumor targeting and immunomodulating capabilities. Here, we leveraged the ability of the ACTR T cell platform to decouple the targeting antibody from the engineered T cell signaling moieties, and evaluated the anti-myeloma activity of ACTR T cells in combination with daratumumab. We demonstrated that in contrast to CD38-targeting CAR-T cells, ACTR T cells exhibited enhanced viability and expansion of the chimeric construct throughout the manufacturing process. We then evaluated the anti-myeloma activity of ACTR T cells in combination with daratumumab. ACTR T cells mediated robust cytotoxicity, cytokine production, and proliferation in response to a range of daratumumab-opsonized target cell lines. Notably, the target specific killing and cytokine production mediated by ACTR T cells in combination with daratumumab was greater than the activity mediated by daratumumab alone. Recognizing the augmented activity of ACTR T cells in combination with daratumumab, we systematically investigated the potential for on-target off-tumor toxicity. In a peripheral blood mononuclear cell coculture assay, ACTR T cells in combination with daratumumab selectively targeted CD38-expressing multiple myeloma cells over CD38-expressing normal B cells, T cells, and natural killer cells. Furthermore, despite low levels of CD38 expression reported on erythrocytes, ACTR T cells in combination with daratumumab did not mediate hemolysis. Taken together, our findings demonstrate the adaptability of the ACTR platform to function in combination with daratumumab, and bypass the challenges of scFv-based CAR-T cell production to safely and effectively target CD38-positive multiple myeloma.

Daratumumab and Nanobody-Based Heavy Chain Antibodies Inhibit the ADPR Cyclase but not the NAD+ Hydrolase Activity of CD38-Expressing Multiple Myeloma Cells.

Simple SummaryMultiple myeloma is a hematological malignancy of antibody-producing plasma cells in the bone marrow. Nucleotides released from cells in the tumor microenvironment act as inflammatory danger signals. CD38 and other enzymes on the surface of cancer cells hydrolyze these nucleotides to immunosuppressive mediators, thereby hampering anti-tumor immune responses. Daratumumab and other CD38-specific antibodies mediate killing of tumor cells by natural killer cells, macrophages, and the complement system. Here, we investigated whether CD38-specific antibodies also inhibit the enzyme activity of CD38-expressing tumor cells, thereby providing a potential second mode of action. Our results showed that daratumumab and nanobody-based heavy chain antibodies inhibit the ADPR cyclase but not the NAD+ hydrolase activity of CD38. Thus, there remains a need for better CD38-inhibitory antibodies.The nucleotides ATP and NAD+ are released from stressed cells as endogenous danger signals. Ecto-enzymes in the tumor microenvironment hydrolyze these inflammatory nucleotides to immunosuppressive adenosine, thereby, hampering anti-tumor immune responses. The NAD+ hydrolase CD38 is expressed at high levels on the cell surface of multiple myeloma (MM) cells. Daratumumab, a CD38-specific monoclonal antibody promotes cytotoxicity against MM cells. With long CDR3 loops, nanobodies and nanobody-based heavy chain antibodies (hcAbs) might bind to cavities on CD38 and thereby inhibit its enzyme activity more potently than conventional antibodies. The goal of our study was to establish assays for monitoring the enzymatic activities of CD38 on the cell surface of tumor cells and to assess the effects of CD38-specific antibodies on these activities. We monitored the enzymatic activity of CD38-expressing MM and other tumor cell lines, using fluorometric and HPLC assays. Our results showed that daratumumab and hcAb MU1067 inhibit the ADPR cyclase but not the NAD+ hydrolase activity of CD38-expressing MM cells. We conclude that neither clinically approved daratumumab nor recently developed nanobody-derived hcAbs provide a second mode of action against MM cells. Thus, there remains a quest for “double action” CD38-inhibitory antibodies.

Isatuximab Acts Through Fc-Dependent, Independent, and Direct Pathways to Kill Multiple Myeloma Cells.

Isatuximab is a monoclonal antibody targeting the transmembrane receptor and ectoenzyme CD38, a protein highly expressed on hematological malignant cells, including those in multiple myeloma (MM). Upon binding to CD38-expressing MM cells, isatuximab is thought to induce tumor cell killing via fragment crystallizable (Fc)-dependent mechanisms, including antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC), as well as via direct Fc-independent mechanisms. Here, these mechanisms of action were investigated in MM and diffuse large B-cell lymphoma (DLBCL) cell lines, as well as in peripheral blood mononuclear cells derived from healthy donors, and in MM patient-derived samples. Our findings show that isatuximab-mediated cytotoxicity occurred primarily via ADCC and ADCP in MM cell lines and via ADCC and apoptosis in DLBCL cell lines expressing high levels of CD38. We identified the programmed cell death-1/programmed cell death-ligand 1 (PD-1/PD-L1) pathway and MM cell-secreted transforming growth factor-beta (TGF-β) as tumor cell-related features that could suppress CD38-mediated ADCC. Furthermore, we established that isatuximab can directly activate natural killer (NK) cells and promote NK cell-mediated cytotoxicity via crosslinking of CD38 and CD16. Finally, isatuximab-induced CDC was observed in cell lines with high CD38 receptor density (>250,000 molecules/cell) and limited expression of inhibitory complement regulatory proteins (CD46, CD55, and CD59; <50,000 molecules/cell). Taken together, our findings highlight mechanistic insights for isatuximab and provide support for a range of combination therapy approaches that could be tested for isatuximab in the future.

Targeting CD38-Expressing Multiple Myeloma and Burkitt Lymphoma Cells In Vitro with Nanobody-Based Chimeric Antigen Receptors (Nb-CARs).

The NAD-hydrolyzing ecto-enzyme CD38 is overexpressed by multiple myeloma and other hematological malignancies. We recently generated CD38-specific nanobodies, single immunoglobulin variable domains derived from heavy-chain antibodies naturally occurring in llamas. Nanobodies exhibit high solubility and stability, allowing easy reformatting into recombinant fusion proteins. Here we explore the utility of CD38-specific nanobodies as ligands for nanobody-based chimeric antigen receptors (Nb-CARs). We cloned retroviral expression vectors for CD38-specific Nb-CARs. The human natural killer cell line NK-92 was transduced to stably express these Nb-CARs. As target cells we used CD38-expressing as well as CRISPR/Cas9-generated CD38-deficient tumor cell lines (CA-46, LP-1, and Daudi) transduced with firefly luciferase. With these effector and target cells we established luminescence and flow-cytometry CAR-dependent cellular cytotoxicity assays (CARDCCs). Finally, the cytotoxic efficacy of Nb-CAR NK-92 cells was tested on primary patient-derived CD38-expressing multiple myeloma cells. NK-92 cells expressing CD38-specific Nb-CARs specifically lysed CD38-expressing but not CD38-deficient tumor cell lines. Moreover, the Nb-CAR-NK cells effectively depleted CD38-expressing multiple myeloma cells in primary human bone marrow samples. Our results demonstrate efficacy of Nb-CARs in vitro. The potential clinical efficacy of Nb-CARs in vivo remains to be evaluated.

CC-92480, a Novel Cereblon E3 Ligase Modulator, Is Synergistic with Dexamethasone, Bortezomib, and Daratumumab in Multiple Myeloma

Introduction: CC-92480 is a novel cereblon E3 ligase modulator (CELMoD) with enhanced autonomous cell-killing and immunomodulatory activity against multiple myeloma (MM) cells. CC-92480 is currently in phase 1 development in a late-line myeloma patient population (NCT03374085). Here, we sought to characterize the antitumor activity of CC-92480 in combination with dexamethasone (DEX), bortezomib (BORT), or daratumumab (DARA) in MM cell lines in vitro and xenograft mouse models in vivo. Methods: CC-92480 activity in combination with DEX was evaluated in MM cell lines. Apoptosis was measured by quantification of caspase-3 activation. The effect of BORT on CC-92480-induced Ikaros and Aiolos degradation was determined by concurrent treatment of MM cells with BORT and CC-92480. β5-site proteasome activity was also determined in the same experiment. The in vitro activity of CC-92480 in combination with BORT was characterized using washout experiments to more faithfully model the short in vivo exposure but more prolonged, gradually diminishing proteasome inhibitory activity of BORT. Apoptosis and cell viability of CC-92480 with BORT were analyzed by flow cytometry. The effect of CC-92480 on CD38 expression was also evaluated across a panel of MM cell lines. The effect of CC-92480 in combination with DARA was characterized with antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) assays. CC-92480 in combination with DEX or BORT was tested in a lenalidomide-resistant (H929-1051) xenograft mouse model. Female SCID mice were inoculated with H929-1051 cells in the right hind leg. For the DEX combination, groups of tumor-bearing mice (n = 9-10) were dosed with vehicle, DEX, or CC-92480 once daily (QD), or CC-92480 in combination with DEX throughout the study, starting when the tumor volumes reached approximately 115 mm3. For combination with BORT, mice (n = 9-10/group) were dosed with vehicle, CC-92480, or BORT, or the CC-92480 and BORT combination starting when the tumor volumes reached approximately 500 mm3. CC-92480 was administered orally QD for 3 days and BORT as a single intravenous dose. Tumor volumes were measured twice a week for the duration of the studies. Results: CC-92480 synergized with DEX in reducing cell viability and potentiated DEX-induced apoptosis in a concentration-dependent manner in MM cell lines. Of note, the combination showed activity at concentrations of both DEX and CC-92480 that had minimal activity as single agents. In the xenograft model with H929-1051 cells, the combination of CC-92480 and DEX significantly inhibited tumor growth (−84%) when compared with either agent alone (−34% and −20% for CC-92480 and DEX, respectively) and was classified as a synergistic effect using the fractional product method. Although proteasome activity is required for CC-92480-induced degradation of Ikaros and Aiolos, CC-92480 nevertheless maintained its ability to efficiently degrade Ikaros and Aiolos in the presence of doses of BORT that cause clinically relevant levels of proteasome inhibition. The in vitro combination of CC-92480 with BORT resulted in greater cytotoxic activity on MM cells than either single agent alone. The in vivo efficacy of CC-92480 and BORT, administered concurrently, showed a strongly synergistic effect with a near complete or complete tumor regression in every animal, and 6 of 9 animals remained tumor-free through an observation period extending 157 days after the control group was terminated. Anti-CD38 therapies, including DARA and isatuxumab, target CD38-expressing MM cells for killing by immune cells through cytotoxic and phagocytic mechanisms. In a panel of MM cell lines, CC-92480 treatment caused increased cell surface expression of CD38 (2-3 times that of control). Pretreatment of MM cells with CC-92480 resulted in increased DARA-mediated ADCC and ADCP compared with DMSO-treated controls. Conclusions: The strong preclinical synergy in MM cell killing exhibited by CC-92480 in combination with DEX, BORT, and with an anti-CD38 antibody (DARA), highlights its potential to bring clinical benefit to patients with MM in combination with these agents and supports the rationale for testing these combinations in clinical studies. Disclosures Wong: Celgene Corporation: Employment, Equity Ownership. Narla:Celgene Corporation: Employment, Equity Ownership. Leisten:Celgene Corporation: Employment. Bauer:Celgene Corporation: Employment, Equity Ownership. Groza:Celgene Corporation: Employment, Equity Ownership. Gaffney:Celgene: Employment. Havens:Celgene: Equity Ownership; Pfizer: Employment, Equity Ownership. Choi:AnaptysBio Inc: Employment, Equity Ownership; Celgene Corporation: Equity Ownership, Other: Formerly Employed. Lopez-Girona:Celgene Corporation: Employment. Hansen:Celgene Corporation: Employment. Cathers:Celgene Corporation: Equity Ownership; Global Blood Therapeutics (GBT): Employment. Carmichael:Celgene plc: Employment, Equity Ownership. Pierce:Celgene Corporation: Employment, Equity Ownership.

Synergistic in Vitro Activity of MOR202, a Human CD38 Antibody, in Combination with Pomalidomide

Background: MOR202 is a fully human anti-CD38 antibody currently being tested in a Phase I/IIa clinical trial in multiple myeloma (MM). It mediates antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) in MM cells with high potency (EC50 ~200 pM) representing a possible promising new therapy for MM patients. In this in vitro study, we evaluated the synergistic potential of MOR202 and pomalidomide (POM), a newly approved IMiD® immunomodulatory agent in MM therapy.Methods: Using flow cytometry analysis, POM was evaluated in relation to its effects on several parameters anticipated to be relevant for anti-tumor activity when combined with MOR202. This included the induction of direct cytotoxicity and CD38 upregulation in several MM cell lines, as well as the activation of human immune effector cells derived from peripheral blood mononuclear cells of healthy donors. On a functional level the interaction of MOR202 and POM was assessed using FACS-based ADCC assays. Different incubation schemes prior to the ADCC assays were evaluated in order to distinguish the influence of POM on ADCC activity when pre-incubated for 72 hours either on target or effector cells or on both in parallel. The observed combination effects were analyzed for synergistic potential. Experiments were carried out in triplicate and mean values (±SEM) were calculated.Results: POM as a single agent showed cytotoxic effects on MM cell lines with high potency (EC50 ~150 nM) and additionally induced an up to 2.7-fold upregulation of CD38 (EC50 ~20 nM) on CD38-expressing MM cell lines. Both effects were maximal at the last tested time point of 72 hours and strongest on cell lines with comparably lower CD38 expression levels. In combination with the observed activation of effector cells these POM-mediated mechanisms lead to a synergistically enhanced cytotoxic activity of MOR202. This synergistic benefit ranged between 1.2-fold and 3.1-fold above theoretical additivity depending on the cell line used and was most pronounced in the case of strong CD38 upregulation. [Display omitted] Conclusions: The cytotoxic activity of MOR202 on MM cells was enhanced and synergized when combined with the immunomodulator agent POM via multiple mechanisms, namely direct cytotoxicity, CD38 upregulation and activation of effector cells. These results provide a mechanistic rationale for combining MOR202 and POM and warrant further evaluation in the clinical setting. DisclosuresEndell:MorphoSys AG: Employment, Patents & Royalties. Boxhammer:Morphosys AG: Employment, Patents & Royalties. Steidl:MorphoSys AG: Employment, Patents & Royalties.

SAR650984 (SAR) Directly Promotes Homotypic Adhesion-Related Multiple Myeloma (MM) Cell Death and SAR-Induced Anti-MM Activities Are Enhanced By Pomalidomide, More Potently Than Lenalidomide

SAR650984 (SAR) is a naked humanized IgG1 monoclonal antibody (mAb) selectively targeting the membrane protein CD38 in early clinical development to treat multiple myeloma (MM) and other CD38+ hematological malignancies. SAR has demonstrated encouraging single agent activity in relapsed/refractory (R/R) MM patients (ASCO abstract #8532) and even better efficacy when combined with Dexamethasone and Lenalidomide (Len), without reaching a maximum tolerated dose in patients with heavily pretreated MM (ASCO Abstract #8512). It functions through multiple mechanisms including antibody dependent cytotoxicity (ADCC), complement dependent cytotoxicity (CDC), and direct killing against CD38-positive tumor cells including MM. Although SAR induces lysis of all CD38-expressing MM cell lines via ADCC, it only significantly induces direct killing of MOLP8 cells that express the highest CD38 surface density (~580,000/cell) among > 17 MM cell lines. We first sought to determine whether direct cell death induced by SAR depends on CD38 levels on MM cell membrane by generating RPMI8226 cells overexpressing CD38 (R-CD38) (Abstract #67338). R-CD38 cells express > 6-fold higher CD38 mRNA and surface protein levels than parental RPMI8226 cells (577,304/cell vs. 128,713/cell). Direct MM cell killing by SAR was determined using caspase 3/7 activity and CellTiter-Glo luminescent cell viability assays without goat anti-human IgG crosslinking, in the presence or absence of IL-6 or bone marrow stromal cells (BMSCs). Following overnight incubation, SAR significantly induced homotypic aggregation (HA) of R-CD38, but not control RPMI8226 cells, associated with dose-dependent activation of pro-apoptotic caspase 3/7 in R-CD38, but not control cells. Importantly, SAR decreased the viability of R-CD38, but not control cells, regardless of the presence of IL-6 or BMSCs. Direct cell death induced by SAR depends on SAR-induced HA in MM cells since SAR only blocked survival of R-CD38 and MOLP8 MM cells that show significant HA. Thus, direct apoptosis induced by SAR depends on the level of CD38 surface expression, which may contribute to clinical responses in R/R MM expressing higher CD38 levels. Next, we evaluated the combination effect of Len or Pomalidomide (Pom) with SAR on MM cells. BM mononuclear cells from MM patients were incubated with SAR (10 mg/ml) with or without 10 mM of Len or Pom overnight, followed by flow cytometric analysis to determine % Annexin V/PI staining of CD138+/BCMA+ MM cells. As expected, Pom alone induced slightly higher % of Annexin V+/PI+ MM cells than Len (41 + 1.8 % vs 49 + 1.5 %). Either combination further increased the % of double positive MM patient cells when compared with individual agent alone (from 40 + 2.1% to 70 + 3.1% combined with Len; from 40 + 2.1% to 86 + 3.4% combined with Pom). In addition, PBMC effectors from normal donors (n=4) were pretreated with Len or Pom (5 mM) for 3-7 days and used for SAR-mediated ADCC assays against MM cells (MM1S, MM1R, RPMI8226, R-38, MOLP8), with or without HS-5 or BMSCs from patients. Pom, more potently than Len, further increased SAR-induced MM cell lysis regardless of the presence of BMSCs. Moreover, additional pretreatment of MM cells with Pom overnight further enhanced SAR-induced ADCC by Pom-pretreated PBMC effectors. Both MOLP8 and R-CD38 are relative resistant to direct cytotoxicities induced by Len or Pom. Significantly, Pom, also more potently than Len, augmented direct toxicities induced by SAR in MOLP8 and R-CD38 MM cells. Taken together, we here demonstrate that SAR directly induces apoptosis of MM cells with higher CD38 levels; and that Pom, more effectively than Len, increases SAR-induced MM cell killing via apoptosis and ADCC. These data strongly support SAR as a monotherapy or in combination treatment to improve the outcome of MM patients. DisclosuresCai:Sanofi: Employment. Song:Sanofi: Employment. Yang:Sanofi: Employment. Adrian:Sanofi: Employment. Munshi:Celgene: Consultancy; Onyx: Consultancy; Janssen: Consultancy; Sanofi-Aventis: Consultancy; Ocopep: Consultancy, Equity Ownership, Patents & Royalties. Anderson:Celgene: Consultancy; Onyx: Consultancy; Gilead Sciences: Consultancy; Sanofi-Aventis US: Consultancy; Acetylon: Scientific Founder Other; Oncoprep: Scientific Founder Other.

Daratumumab, a Novel Potent Human Anti-CD38 Monoclonal Antibody, Induces Significant Killing of Human Multiple Myeloma Cells: Therapeutic Implication.

Abstract 608CD38 is a type II transmembrane glycoprotein highly expressed in many hematological malignancies including multiple myeloma (MM), representing a promising target for monoclonal antibody (mAb)-based targeted immunotherapy. We here investigated the mechanisms of anti-MM activity of daratumumab, a novel anti-CD38 mAb currently in clinical development, to treat human MM. Daratumumab was generated in human Ig transgenic HuMab-mice® and was selected for its excellent binding characteristics. It strongly reacts against a panel of CD38-expressing MM cell lines (>94%, 17/18) that are sensitive or resistant to current treatments for MM: median fluorescence intensity (MFI) observed was 378-20764 and 15-140 for daratuzumab and isotype control Ab, respectively. U266 cells, which lack CD38 mRNA, did not bind daratuzumab. Significantly, daratuzumab induced NK-mediated antibody-dependent cellular cytotoxicity (ADCC) against CD38-expressing MM cells but not CD38-negative U266 cells, confirming its specific targeting. Daratuzumab-mediated MM cell lysis was dose-dependent, with maximum lysis of the CD38-expressing MM cell lines (n=17) at concentrations of 0.01-0.1μg/ml. We also determined potential effects of daratumumab on NK and peripheral blood mononuclear cells (PBMC) effector cells, since these cells express CD38, albeit at lower levels than MM cells. Overnight pretreatment of PBMCs or purified NK cells from 2 normal donors with daratumumab (2 μg/ml) did not significantly alter daratumumab-induced ADCC against MM1S target cells. The bone marrow (BM) microenvironment has been shown to protect MM cells against apoptosis and stimulate their growth; we therefore next asked whether daratumumab effectively induced ADCC against MM cells in the presence of BM stromal cells (BMSCs). Daratumumab induced a similar, dose-dependent, ADCC of dexamethasone-sensitive MM1S as well as dexamethasone-resistant MM1R cells both in the presence and absence of BMSCs, suggesting that daratumumab could indeed effectively induce ADCC in the BM microenvironment. Significantly, our findings translated to patient cells, since daratumumab induced ADCC of both allogeneic (n=2) and autologous (n=9) patient MM cells. We next turned our attention to complement-mediated cytotoxicity (CDC). In calcein AM-labeling assays in the presence of normal human serum, daratumumab induced dose-dependent CDC of LP-1 and XG-1 MM cells expressing low levels of CD59 and CD55 both in the absence or presence of BMSCs. Effective daratumumab-induced CDC of CD138-purified MM cells was seen for 6 of 9 patients. Daratumumab-induced CDC was not affected by the expression of complement inhibitor proteins in this cohort of patients. Finally, daratumumab induced apoptosis in Ramos and Daudi cells when crosslinked by anti-IgG antibody, and it induced cell death in CD138-purified patient MM cells, as assessed by annexin V/PI staining. Taken together, our results indicate that daratumumab is able to effectively kill patient MM tumor cells via several cytotoxic mechanisms, supporting clinical development of daratumumab to improve patient outcome in MM. Disclosures:de Weers:Genmab: Employment. Bakker:Genmab: Employment. Vink:Genmab: Employment. Jacobs:Genmab: Employment. Oomen:Genmab: Employment. Bleeker:Genmab: Employment. Munshi:Millennium Pharmaceuticals: Honoraria, Speakers Bureau. van de Winkel:Genmab: Employment. Parren:Genmab: Employment. Richardson:Keryx Biopharmaceuticals: Honoraria. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.