ALBERT

Description: Cell membrane protein CS1 is highly expressed by tumor cells from the majority of multiple myeloma (MM) patients (〉95%) regardless of cytogenetic abnormalities and response to current treatments. Furthermore, CS1 is detected in MM patient sera and correlates with active MM. However, its role in MM pathophysiology is undefined. In the present study, we first generated CS1 null OPM2 MM cells using lentiviral CS1 short interfering RNA. Specific CS1 knockdown was confirmed by depletion of CS1 mRNA and membrane protein, whereas CS1 was expressed in parental OPM2 and OPM2 cells infected with control lentiviral vector (cntOPM2). Immunoblotting of phopho-site of multiple kinase screen analysis showed decreased phosphorylation of ERK1/2, AKT, and STAT3 in CS1null OPM2 cells vs. cntOPM2 cells. Serum deprivation markedly blocked survival at earlier time points in CS1null OPM2 cells vs. cntOPM2 cells. Earlier apoptosis in CS1null OPM2 cells correlated with earlier activation of caspases, PARP cleavage, and increased proapoptotic proteins BNIP3, BIK. CS1 knockdown further delayed development of OPM2 tumor and prolonged survival in mice. CS1null OPM2 cells failed to grow tumors in the majority of mice (n=8) at 5 weeks after cell inoculation, whereas cntOPM2 cells formed tumors within 1.5 weeks in all animals (n=8). Interestingly, CS1 was expressed in tumors that developed late in mice injected with CS1null OPM2 cells. Concomitantly, we overexpressed CS1 in CS1-low expressing U266 cells by transfecting an expressing plasmid pflagCS1 or control vector. Enforced CS1 expression enhanced U266 cell growth and survival. In contrast to the majority of U266 cells (〉95%) that grow in suspension in standard tissue culture flasks, all U266CS1 cells exhibited adherent growth and homotypic adhesion. Importantly, overexpressed CS1 increased adhesion of U266 and MM1S cells to BMSCs. Furthermore, U266CS1 cells formed more and larger colonies in methylcellulose than U266 cells. Interestingly, tumors that developed in mice injected with U266 cells expressed significantly higher levels of CS1 than injected U266 cells; moreover, exercised tumors grew in an adherent manner in vitro. Overlapping differentially expressed genes in U266CS1 vs. U266 and CS1null OPM2 vs. cntOPM2 was next analyzed by gene expression profiling. Importantly, c-maf pathway was significantly upregulated in U266CS1 vs. U266 cells and downregulated in CS1null OPM2 vs. cntOPM2 cells, as evidenced by differentially expressed c-maf and its target genes, i.e., cyclin D2, integrin αE/β7 at both mRNA and protein levels. Myeloma cell adhesion-induced VEGF secretion by BMSCs was greater with U266CS1 than U266 cells. Finally, immunoblotting showed upregulation of c-maf and cyclin D2 in U266 tumors overexpressing CS1. These studies provide direct evidence of the role of CS1 in myeloma pathogenesis, define molecular mechanisms regulating its effects, and further support novel therapies targeting CS1 in MM.

Description: Abstract 609 HM1.24/CD317 or BST2, a cell surface protein highly expressed on malignant plasma cells, represents a potential target of immunotherapy for multiple myeloma (MM). Here we characterized XmAb®5592, a novel Fc-engineered and humanized anti-HM1.24 antibody (Ab), and studied mechanisms of its anti-MM activity. XmAb®5592, with double amino acid substitution in Fc region of the wild type IgG1, has approximately 40-fold and 10-fold increases in affinity for Fc gamma receptor III (FcγRIIIa) and (FcγRIIa), respectively, expressed on effector cells including NK cells. The Fv region of XmAb®5592 was humanized and engineered to achieve high affinity and specificity of binding to HM1.24-expressing target cells. XmAb®5592 reacts against a panel of MM cell lines (n=19) which are both sensitive or resistant to current anti-MM conventional and novel therapies. Importantly, it triggers 10-100-fold higher antibody-dependent cell-mediated cytotoxicity (ADCC) against these MM cell lines than a native/non Fc-engineered version (anti-HM1.24 IgG1) of the Ab. Specifically, the maximum specific lysis of MM1S, MM1R, and RPMI8226 target cells induced by XmAb®5592 is at a concentration of 0.001-0.01 μg/ml, whereas the IgG1 analog did not induce maximum cell lysis until 0.1 μg/ml. The maximum 100% specific lysis of INA-6 target cells occurred at 0.1 μg/ml of XmAb5592, in contrast to 60% maximum lysis induced by10 μg/ml of the IgG1 analog. Since the bone marrow (BM) microenvironment induces resistance in MM cells to conventional therapies, we next asked whether XmAb®5592 induced ADCC against MM cells even in the presence of BM stromal cells (BMSCs). Importantly, XmAb®5592 triggered significant ADCC against MM1S, MM1R, and INA-6 MM cells in the context of BMSCs. XmAb5592 also reacts against patient MM cells, and triggers robust ADCC against CD138-purified patient MM cells in assays using NK effector cells from normal donors. Furthermore, cross-linked XmAb5592 inhibited RMPI 8226 cell growth in the absence of effector cells. The in vivo efficacy of XmAb®5592 was next evaluated in murine subcutaneous (sc) xenograft murine models using RPMI 8226 cells. Administration of XmAb5592 (9mg/kg, ip, 2x/week for 4 weeks) led to a significant reduction in growth of established tumors in vivo compared to a non-engineered IgG1 anti-HM1.24 analog. At termination of the study. 7/15 mice were tumor free in the XmAb- treated group versus only 1/15 tumor free mice in the IgG1 analog treated group. An anti-HM1.24 antibody with Fc region engineered to completely ablate binding to FcγRs (knock-out) behaved equivalent to the PBS vehicle control in these studies, again underlining the significance of interaction with FcγR for anti-tumor efficacy. These results therefore suggest that XmAb5592, an anti-HM1.24 antibody engineered for improved effector function and antitumor potency in vitro and in vivo, is a promising next-generation immunotherapeutic for MM. Disclosures: Muchhal: Xencor: Employment. Horton:Xencor: Employment. Nguyen:Xencor: Employment. Karki:Xencor: Employment. Desjarlais:Xencor: Employment. Munshi:Millennium Pharmaceuticals: Honoraria, Speakers Bureau. Richardson:Keryx Biopharmaceuticals: Honoraria. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Description: Abstract 4064 HM1.24, an immunological target highly expressed on majority of multiple myeloma (MM) cells, has not been effectively targeted with therapeutic monoclonal antibodies (mAbs). Recently, XmAb5592, a novel Fc-domain engineered humanized anti-HM1.24 mAb with specific Fc-domain modification, was shown to induce 〉10-fold antibody-dependent cellular cytotoxicity (ADCC) and antibody dependent cellular phagocytosis against MM cells, when compared with the humanized normal anti-HM1.24 IgG1 (XmAb5627) from which it is derived (ASH Abstract#609, 2009). Here we investigated whether XmAb5592, when combined with other anti-MM drugs, further enhanced ADCC against MM cell lines and primary patient MM cells using Calcein-AM release ADCC assays and flow cytometric analysis for cell membrane CD107a to specifically quantitate NK cell (CD56+CD3-) activation. Addition of lenalidomide (10 μ M) in standard ADCC assays increased XmAb5592 (0.1 μ g/ml)-induced cell lysis against MM1S, MM1R, and RPMI8226 cells in the presence of peripheral blood mononuclear cells (PBMCs) from normal donors (n=2). Specifically, XmAb5592 (0.01, and 0.1 μ g/ml) combined with lenalidomide (2.5, 5, 10 μ M), in the presence or absence of IL-2 (100 units/ml), synergistically induced NK-mediated RPMI8226 MM cell lysis, as evidenced by combination index (CI) 〈 1 (0.08-0.89). Pre-incubation of PBMCs with additional IL-2 (100 units/ml) enhanced even stronger XmAb5592-induced cytotoxicity against MM cells than pretreatment with lenalidomide alone. Lenalidomide also enhanced PBMC effectors to kill primary patient MM cells. Cell surface CD107a, as a functional marker for NK cell activation dependent on granzyme B secretion, was further determined following target myeloma cell incubation, with or without NK cells and in the presence of mAbs. NK cells were activated by as low as 0.001 μ g/ml of XmAb5592 only in the presence of MM1S tumor cells. In contrast, neither XmAb6166, an Fc-domain knockout of XmAb5592 without NK binding, nor XmAb4614, a similar Fc-engineered mAb targeting respiratory syncytial virus (RSV) antigen, induced any cell surface CD107a on NK cells incubated with MM cells. Specific XmAb5592-induced CD107a-dependent NK cell degranulation further correlated with IFNγ secretion (r=0.7, p=0.03). XmAb5592 induced 〉 10-fold more potent NK degranulation than XmAb5627, which significantly correlated with MM cell lysis by ADCC, even in the presence of bone marrow stromal cells (BMSCs). Moreover, no significant HM1.24 was expressed on NK cells stimulated with or without IFN-α, suggesting minimal NK toxicity. XmAb5592 more potently (〉10-fold) than XmAb5627 induces homotypic aggregation and adhesion of NK cells, which was further enhanced by lenalidomide. Finally, minimal HM1.24 expression was confirmed on different PBMC subsets including CD15+ (PMN), CD19+ (B), CD14+ (MC), and CD3+ (T) cells. These results indicate that lenalidomide further potentiates XmAb5592-induced myeloma cell killing via NK-mediated ADCC, providing a rationale to combine both novel drugs to improve patient outcome in MM. Disclosures: Muchhal: Xencor Inc: Employment. Desjarlais:Xencor Inc: Employment. Richardson:Gentium: Membership on an entity's Board of Directors or advisory committees, Research Funding. Munshi:Millennium Pharmaceuticals: Honoraria, Speakers Bureau. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Description: Abstract 3013 Daratumumab is a novel fully human therapeutic CD38-specific monoclonal antibody (mAb) that is currently in phase I/II safety and dose finding clinical studies in MM. We recently demonstrated that daratumumab induces antibody dependent cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) against multiple myeloma (MM) cells (ASH Abstract #608, 2009). Significantly, daratumumab induces ADCC-mediated autologous lysis against MM patient cells. In addition, when cross-linked, daratumumab directly induces Ramos lymphoma cell death. We here studied whether daratumumab directly kills MM cells and whether daratumumab could be combined with other anti-MM drugs to further enhance its direct cytotoxicity. Direct daratumumab-induced MM cell death was determined using CellTiter-Glo luminescent cell viability assay and Annexin V/PI flow cytometry analysis, with or without goat anti-human IgG crosslinking. Following 48h incubation, daratumumab (0.1-10 μg/ml), when cross-linked, directly induced cytoxicity against dexamethasone (dex)-sensitive MM1S and dex-resistant MM1R cells, as evidenced by decreased cell viability in a dose-dependent manner. Importantly, cross-linked daratumumab increased caspase 3/7 activities in a dose-dependent fashion, as assessed by the Caspase-Glo® 3/7 luminescence assay. Furthermore, daratumumab upregulated Annexin V+ and Annexin V+/PI+ cells in freshly isolated CD138+ MM patient cells, from 7.7% to 20.6% and 10.9% to 15.4 %. Therefore, cross-linked daratumumab can directly trigger apoptosis of patient myeloma cells. Cell viability assay was further performed on MM1S cells when daratumumab (0.1, 1, 10 μg/ml) was combined with dex (0.5 and 1 μM) or bortezomib (2.5, 5, and 10 nM). Following 48–72h incubation with daratumumab, both dex and bortezomib synergistically inhibited MM cell viability, as determined by combination index (CI) 〈 0.5 at given combined concentrations of these drugs. Enhanced caspase 3/7 activation was also seen when daratumumab was combined with dex. To evaluate combination cytotoxicity induced by lenalidomide with daratumumab, peripheral blood mononuclear effector cells (PBMCs) from normal donors (n=2) were pretreated with lenalidomide (2 μM) for 3 days followed by daratumumab-mediated ADCC assays against MM1S cells. Using calcein-AM release measurements, lenalidomide-pretreated PBMCs further augmented daratumumab-induced MM1S cell lysis, whereas daratumumab-pretreated PBMCs did not alter ADCC. Taken together, our studies show that daratumumab directly induces MM cell death via activation of caspase 3/7 and daratumumab induced synergistic cytotoxicity with dex or bortezomib. Moreover, lenalidomide augments daratumumab-induced ADCC against MM cells. These results further support combination clinical trials of conventional and novel anti-MM drugs with daratumumab in MM. Disclosures: Weers: Genmab: Employment. Parren:Genmab: Employment. Richardson:Keryx Biopharmaceuticals: Honoraria. Munshi:Millennium Pharmaceuticals: Honoraria, Speakers Bureau. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Description: Abstract 608 CD38 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.

Description: Abstract 3848 Poster Board III-784 The mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway plays a crucial role in the pathogenesis of human multiple myeloma (MM) by promoting interactions of MM cells with bone marrow stromal cells (BMSCs) that secrete cytokines and growth factors for MM cell growth, survival, and resistance to chemotherapeutic drugs. Accumulating studies have supported targeting this signaling pathway in MM. Here we investigate cytotoxicity of AS703026, a novel selective MEK1/2 inhibitor with highly oral bioavailability, in MM cell lines and patient MM cells and define its mechanisms of action. AS703026, more potently (∼9-10 fold) than AZD6244, inhibits growth and survival of MM cells and cytokine-induced osteoclast differentiation. It specifically blocks baseline and adhesion-induced pERK1/2, but not pSTAT3. Selective MEK1/2 inhibition by AS703026 led to a cessation of cell proliferation accompanied by G0-G1 cell cycle arrest, as shown by increased subG0 cells, and concurrently abolished S phase cells. AS703026 also reduced expression of c-maf oncogene in a time-dependent manner, suggesting a MEK1/2-dependent regulation of c-maf that may contribute MM cell growth inhibition. AS703026 further induced apoptosis in MM cells, as manifested by caspase 3 and PARP cleavages in a time-dependent manner. It blocked osteoclastogenesis in vitro, as measured by number of TRAP-positive multinuclear cells following culturing PBMCs with RANKL and M-CSF. Importantly, AS703026 sensitized drug-resistant MM cells to a broad spectrum of conventional (dexamethasone, melphalan), as well as novel or emerging (lenalidomide, perifosine, bortezomib, rapamycin) anti-MM therapies. Synergistic or additive cytotoxicity (combination index 〈 1) induced by these combinations was further validated by annexin-V/PI staining and flow cytometric analysis. Combining these agents led to a significantly increased apoptosis and cell death than AS703026 alone, confirming enhanced cytotoxicity against MM cells. In vivo studies demonstrate that treatment of MM cell line H929-bearing mice with AS703026 (n=4 at 30 mg/kg; n=6 at 15 mg/kg), but not vehicle alone (n=6), blocked MM tumor growth in a dose-dependent manner (p

Description: CS1 is highly expressed on tumor cells from the majority of multiple myeloma (MM) patients regardless of cytogenetic abnormalities or response to current treatments. Furthermore, CS1 is detected in MM patient sera and correlates with active disease. However, its contribution to MM pathophysiology is undefined. We here show that CS1 knockdown using lentiviral short-interfering RNA decreased phosphorylation of ERK1/2, AKT, and STAT3, suggesting that CS1 induces central growth and survival signaling pathways in MM cells. Serum deprivation markedly blocked survival at earlier time points in CS1 knockdown compared with control MM cells, associated with earlier activation of caspases, poly(ADP-ribose) polymerase, and proapoptotic proteins BNIP3 and BIK. CS1 knockdown further delayed development of MM tumor and prolonged survival in mice. Conversely, CS1 overexpression promoted myeloma cell growth and survival by significantly increasing myeloma adhesion to bone marrow stromal cells (BMSCs) and enhancing myeloma colony formation in semisolid culture. Moreover, CS1 increased c-maf–targeted cyclin D2-dependent proliferation, -integrin β7/αE-mediated myeloma adhesion to BMSCs, and -vascular endothelial growth factor-induced bone marrow angiogenesis in vivo. These studies provide direct evidence of the role of CS1 in myeloma pathogenesis, define molecular mechanisms regulating its effects, and further support novel therapies targeting CS1 in MM.

Description: The bone marrow (BM) microenvironment consists of extracellular-matrix and the cellular compartment including immune cells. Multiple myeloma (MM) cell and BM accessory cell interaction promotes MM survival via both cell-cell contact and cytokines. Immunomodulatory agents (IMiDs) target not only MM cells, but also MM cell-immune cell interactions and cytokine signaling. Here we examined the in vitro effects of IMiDs on cytokine signaling triggered by interaction of effector cells with MM cells and BM stroma cells. IMiDs diminished interleukin-2, interferonγ, and IL-6 regulator suppressor of cytokine signaling (SOCS)1 expression in immune (CD4T, CD8T, natural-killer T, natural-killer) cells from both BM and PB of MM patients. In addition, coculture of MM cells with healthy PBMCs induced SOCS1 expression in effector cells; conversely, treatment with IMiDs down-regulated the SOCS1 expression. SOCS1 negatively regulates IL-6 signaling and is silenced by hypermethylation in MM cells. To define the mechanism of inhibitory-cytokine signaling in effector cells and MM cells, we next analyzed the interaction of immune cells with MM cells that were epigenetically modified to re-express SOCS1; IMiDs induced more potent CTL responses against SOCS1 re-expressing–MM cells than unmodified MM cells. These data therefore demonstrate that modulation of SOCS1 may enhance immune response and efficacy of IMiDs in MM.

Description: Aurora-A is a mitotic kinase that regulates mitotic spindle formation and segregation. In multiple myeloma (MM), high Aurora-A gene expression has been correlated with centrosome amplification and proliferation; thus, inhibition of Aurora-A in MM may prove to be therapeutically beneficial. Here we assess the in vitro and in vivo anti-MM activity of MLN8237, a small-molecule Aurora-A kinase inhibitor. Treatment of cultured MM cells with MLN8237 results in mitotic spindle abnormalities, mitotic accumulation, as well as inhibition of cell proliferation through apoptosis and senescence. In addition, MLN8237 up-regulates p53 and tumor suppressor genes p21 and p27. Combining MLN8237 with dexamethasone, doxorubicin, or bortezomib induces synergistic/additive anti-MM activity in vitro. In vivo anti-MM activity of MLN8237 was confirmed using a xenograft-murine model of human-MM. Tumor burden was significantly reduced (P = .007) and overall survival was significantly increased (P 〈 .005) in animals treated with 30 mg/kg MLN8237 for 21 days. Induction of apoptosis and cell death by MLN8237 were confirmed in tumor cells excised from treated animals by TdT-mediated dUTP nick end labeling assay. MLN8237 is currently in phase 1 and phase 2 clinical trials in patients with advanced malignancies, and our preclinical results suggest that MLN8237 may be a promising novel targeted therapy in MM.