SummaryCheckpoint inhibitors have revolutionized cancer treatment. However, only a minority of patients respond to these immunotherapies. Here, we report that blocking the inhibitory NKG2A receptor enhances tumor immunity by promoting both natural killer (NK) and CD8+ T cell effector functions in mice and humans. Monalizumab, a humanized anti-NKG2A antibody, enhanced NK cell activity against various tumor cells and rescued CD8+ T cell function in combination with PD-x axis blockade. Monalizumab also stimulated NK cell activity against antibody-coated target cells. Interim results of a phase II trial of monalizumab plus cetuximab in previously treated squamous cell carcinoma of the head and neck showed a 31% objective response rate. Most common adverse events were fatigue (17%), pyrexia (13%), and headache (10%). NKG2A targeting with monalizumab is thus a novel checkpoint inhibitory mechanism promoting anti-tumor immunity by enhancing the activity of both T and NK cells, which may complement first-generation immunotherapies against cancer.

Key Points• Blockade of inhibitory KIRs with MHC class I antigens on lymphoma cells by anti-KIR antibodies augments NK-cell spontaneous cytotoxicity.• In combination with anti-CD20 mAbs, anti-KIR induces enhanced NK cell-mediated, rituximab-dependent cytotoxicity against lymphoma.

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...

In human blood, 1% to 5% of lymphocytes are ␥␦ T cells; they mostly express the ␥␦ T-cell receptor ( IntroductionThe success of therapeutic monoclonal antibodies (mAbs) in the treatment of cancer can be attributed to their multiple bioactivities. Their mechanism of action combines antibody-dependent cellular cytotoxicity (ADCC), complement-mediated cytotoxicity, antibodydependent phagocytosis, direct cytotoxic activity, and inhibition of receptor signaling. 1 ADCC occurs when cytolytic effector cells expressing a receptor for the Fc region of the IgG class of antibodies (Fc␥ receptors) bind to antibodies on the surface of target cells. In humans, Fc␥ receptors comprise CD16 (Fc␥RIIIA-B), CD32 (Fc␥RIIA-C), and CD64 (Fc␥RI), which all bind the same region on IgG Fc but with low-to-medium (CD16, CD32) or high (CD64) affinities. 2 Several lines of evidence suggest that enhancing ADCC induced by therapeutic mAbs may directly improve their clinical efficacy. First, in mice bearing xenografted tumors, the efficacy of the therapeutic mAbs rituximab (RTX) and trastuzumab (TTZ) relies upon cell-surface expression of Fc␥R. 3 Second, ADCC is essential for the clinical efficacy of RTX in B-cell lymphoma patients and depends on the affinity of Fc␥RIIIA for the IgG. 4,5 Third, optimizing the affinity of RTX, TTZ, and alemtuzumab (ALZ) for Fc␥RIIIA increases their ADCC and their efficacy in preclinical and clinical studies. [6][7][8] Finally, recruitment and activation of additional cell effectors for ADCC might also enhance the cytolytic activity of anticancer mAbs. 9,10 The cytolytic effector cells involved in ADCC are CD16 ϩ (ie, Fc␥RIIIA)-positive natural killer (NK) cells and other CD8 ϩ cytolytic T lymphocytes, which release perforin through immunologic synapses to kill target cells. In addition, human CD4 Ϫ CD8 Ϫ ␥␦ T cells from peripheral blood might provide an important reservoir of cytolytic effector cells for ADCC. In most humans and nonhuman primate species, the majority of circulating ␥␦ T lymphocytes expresses the V␥9 T-cell receptor, with CD4 Ϫ CD8 Ϫ TCRV␥9 ϩ cells representing 1% to 3% of mononuclear cells. All these cells respond to stimulation with nonpeptide phosphoantigens (PAgs), which are small, phosphorylated metabolites produced by the cholesterol pathway in microbial pathogens and tumor cells. In addition to natural PAgs, the synthetic analog BrHPP 11 selectively stimulates TCRV␥9 ϩ ␥␦ T lymphocytes. PAg-stimulated ␥␦ T cells proliferate, secrete pro-inflammatory cytokines and chemokines, and, most importantly, kill leukemia, lymphoma, and carcinoma cells. 12,13 Several studies involving macaque monkeys 14,15 and clinical studies in cancer patients [16][17][18][19][20][21][22] have demonstrated in vivo the potential of PAg-activated TCRV␥9 ϩ ␥␦ T lymphocytes for cancer immunotherapy.The mechanism by which PAgs stimulate ␥␦ T cell-mediated cancer cell killing is unclear. The number of circulating ␥␦ T lymphocytes increases 50-to 100-fold in humans treated with BrHPP and IL2 (our unpublished observations...

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