Elotuzumab is a monoclonal antibody in development for multiple myeloma (MM) that targets CS1, a cell surface glycoprotein expressed on MM cells. In preclinical models, elotuzumab exerts anti-MM efficacy via natural killer (NK)-cell-mediated antibody-dependent cellular cytotoxicity (ADCC). CS1 is also expressed at lower levels on NK cells where it acts as an activating receptor. We hypothesized that elotuzumab may have additional mechanisms of action via ligation of CS1 on NK cells that complement ADCC activity. Herein, we show that elotuzumab appears to induce activation of NK cells by binding to NK cell CS1 which promotes cytotoxicity against CS1(+) MM cells but not against autologous CS1(+) NK cells. Elotuzumab may also promote CS1–CS1 interactions between NK cells and CS1(+) target cells to enhance cytotoxicity in a manner independent of ADCC. NK cell activation appears dependent on differential expression of the signaling intermediary EAT-2 which is present in NK cells but absent in primary, human MM cells. Taken together, these data suggest elotuzumab may enhance NK cell function directly and confer anti-MM efficacy by means beyond ADCC alone.

IntroductionNatural killer (NK) cells exert cytotoxicity against multiple myeloma (MM), and some therapies for MM appear to recover or enhance NK-cell function against MM. [1][2][3][4][5] Lenalidomide in particular confers NK-cell expansion and activation associated with tumor cell apoptosis. 4,5 MM cells up-regulate the expression of ligands to NK cell-inhibitory killer cell immunoglobulin-like receptor (KIR) 6 and KIR-ligand mismatch in T cell-depleted, allogeneic stem cell transplantation may reduce the risk of relapse in MM patients, suggesting that this signaling axis may be particularly important. 7 IPH2101 is a human IgG4 mAb against common inhibitory KIR2DL-1, KIR2DL-2, and KIR2DL-3. 8 IPH2101 enhances NKcell function against malignant cells by preventing inhibitory KIR-ligand interaction and subsequent inhibitory signaling. 8 In the present study, we provide novel data characterizing mechanisms by which inhibitory KIR blockade augments NK-cell function against MM, sparing normal cells. In addition, we uncover novel immunomodulatory properties of lenalidomide that likely contribute to enhanced NK-cell activity. We demonstrate that a murine anti-inhibitory NK-cell receptor Ab and lenalidomide further augment NK-cell function against MM compared with either agent alone, leading to in vivo rejection of a lenalidomideresistant tumor. These data support the initiation of a steroidsparing, phase 2 trial of IPH2101 and lenalidomide in MM. Methods CellsCells were cultured in RPMI 1640 medium (Invitrogen) supplemented with 10% FBS (ICN Biomedicals) at 37°in 5% CO 2 . NK cells and PBMCs from healthy donors (American Red Cross, Columbus, OH, and Indiana Blood Center, Indianapolis, IN) and PBMCs and BM aspirates from patients with MM obtained per Institutional Review Board-approved protocols were prepared as described previously. 9 The MM cell lines U266 and K562 were from the American Type Culture Collection. We were unable to procure sufficient patient blood volume to enrich for NK cells from MM patient donors; therefore, experiments using patient-derived NK cells were conducted in PBMCs at effector:target (E:T) ratios based on the proportion of CD56 ϩ , CD3 Ϫ NK cells in patient PBMCs determined by flow cytometry. Abs and reagentsIPH2101 (and PE-labeled anti-IPH2101) were provided by Innate Pharma. Lenalidomide was from Toronto Research Chemicals and John C. Byrd (The Ohio State University, Columbus, OH). Flow Abs were from Beckman Coulter, BD Pharmingen, eBioscience, R&D Systems, and Miltenyi Biotec. NKG2D-blocking Ab was from BioLegend. Abs against TRAIL, DNAM-1, and HLA class I (and isotypes) were from BD Biosciences, and 7-aminoactinomycin D was from Sigma-Aldrich. Antigen expression assaysU266 cells were stained with 7-amino-actinomycin D and PE-Ab, incubated at 4°C for 15 minutes, and washed with MACS buffer. Ten thousand cells and QuantiBRITE PE beads (BD Biosciences) were collected with a FACSCalibur flow cytometer (BD Biosciences) and analyzed using FlowJo Version 7.6.1 software (TreeStar). The median PE...

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1966 Introduction: A potent natural killer (NK) cell versus multiple myeloma (MM) effect has been reported in clinical observations and characterized mechanistically in translational studies. However, the NK cell versus MM effect is lost as the disease methodically progresses due, in part, to upregulation of MHC Class 1 antigens on MM tumor cells which serve as ligands to inhibitory KIR on NK cells. Our group has shown that IPH-2101, a novel IgG4 monoclonal blocking antibody against commonly expressed inhibitory KIR (KIR2DL-1, -2 and -3), may augment NK cell function against MM tumor cells. A single-agent phase 1 open-label, dose escalation study of IPH-2101 is nearing completion to determine the safety, tolerability and biologic effects of inhibitory KIR blockade as a novel form of therapy for MM. Methods: A single-agent, open-label, Phase 1 dose escalation study is being performed with 7 cohorts in standard 3+3 design. The protocol was amended to include 7 additional patients with less heavily pretreated disease in the final cohort. Doses from 0.0003 mg/kg to 3mg/kg are being evaluated. Patients enrolled on the 7 cohorts had heavily pre-treated, relapsed/refractory MM with at least one prior therapy, whereas 7 additional patients accrued to the final cohort are limited to one prior therapy only. IPH-2101 is administered intravenously once every 28 days, with 4 cycles of therapy possible as determined by safety, tolerability and disease characteristics. Extensive pharmacodynamic (PD), pharmacokinetic (PK), KIR occupancy, and correlative biologic data are being collected. Results: Currently, 32 patients have been enrolled, and 5 patients remain on study in the final cohort. IPH-2101 has been well tolerated without determination of a dose limiting toxicity (DLT). 1 patient at dose level 1 was replaced and 3 additional patients were enrolled at dose level 4 due to an SAE (acute renal failure) deemed possibly related to drug. PK and PD data closely approximate those predicted by preclinical modeling. Full KIR occupancy across the dosing interval appears to be achieved in most cases with the 1mg/kg dose. No evidence of autoimmunity and no deleterious effect on NK cell maturation has been observed to date. Ongoing correlative studies will characterize NK cell phenotype, cytotoxicity, and effects of IPH-2101 on immunomodulation. To date, no objective responses have been observed; however, preliminary analyses suggest that several patients may have achieved disease stabilization. Conclusions: IPH-2101 improves autologous NK cell-mediated cytotoxicity against MM tumor cells and appears to be well tolerated and safe with achievement of biologic endpoints independent of DLT or maximally tolerated dose. Preliminary data suggest that several patients may have disease stabilization on therapy. Updated correlative, biologic data will be presented at the meeting. Disclosures: No relevant conflicts of interest to declare.