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Elotuzumab is a humanized therapeutic monoclonal antibody directed to the surface glycoprotein SLAMF7 (CS1, CRACC, CD319), which is highly expressed on multiple myeloma (MM) tumor cells. Improved clinical outcomes have been observed following treatment of MM patients with elotuzumab in combination with lenalidomide or bortezomib. Previous work showed that elotuzumab stimulates NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC), via Fc-domain engagement with FcγRIIIa (CD16). SLAMF7 is also expressed on NK cells, where it can transmit stimulatory signals. We tested whether elotuzumab can directly activate NK cells via ligation with SLAMF7 on NK cells in addition to targeting ADCC through CD16. We show that elotuzumab strongly promoted degranulation and activation of NK cells in a CD16-dependent manner, and a non-fucosylated form of elotuzumab with higher affinity to CD16 exhibited enhanced potency. Using F(ab')2 or Fc-mutant forms of the antibody, the direct binding of elotuzumab to SLAMF7 alone could not stimulate measurable CD69 expression or degranulation of NK cells. However, the addition of soluble elotuzumab could costimulate calcium signaling responses triggered by multimeric engagement of NKp46 and NKG2D in a CD16-independent manner. Thus, while elotuzumab primarily stimulates NK cells through CD16, it can also transduce effective "trans"-costimulatory signals upon direct engagement with SLAMF7, since these responses did not require direct co-engagement with the activating receptors. Trans-costimulation by elotuzumab has potential to reduce activation thresholds of other NK cell receptors engaging with their ligands on myeloma target cell surfaces, thereby potentially further increasing NK cell responsiveness in patients.

Stable surface expression of human inhibitory killer cell Ig-like receptors (KIRs) is critical for controlling NK cell function and maintaining NK cell tolerance toward normal MHC class I+ cells. Our recent experiments, however, have found that Ab-bound KIR3DL1 (3DL1) readily leaves the cell surface and undergoes endocytosis to early/recycling endosomes and subsequently to late endosomes. We found that 3DL1 internalization is at least partially mediated by an interaction between the μ2 subunit of the AP-2 clathrin adaptor complex and ITIM tyrosine residues in the cytoplasmic domain of 3DL1. Disruption of the 3DL1/μ2 interaction, either by mutation of the ITIM tyrosines in 3DL1 or mutation of μ2, significantly diminished endocytosis and increased surface expression of 3DL1 in human primary NK cells and cell lines. Furthermore, we found that the 3DL1/AP-2 interaction is diminished upon Ab engagement with the receptor, as compared with untreated cells. Thus, we have identified AP-2–mediated endocytosis as a mechanism regulating the surface levels of inhibitory KIRs through their ITIM domains. Based on our results, we propose a model in which nonengaged KIRs are internalized by this mechanism, whereas engagement with MHC class I ligand would diminish AP-2 binding, thereby prolonging stable receptor surface expression and promoting inhibitory function. Furthermore, this ITIM-mediated mechanism may similarly regulate the surface expression of other inhibitory immune receptors.

Natural killer (NK) cells participate in host defense by surveying for and ultimately killing virally infected or malignant target cells. NK cell cytotoxicity is a tightly regulated process that proceeds stepwise from adhesion and activation to the secretion of preformed lytic granule contents onto a diseased or stressed cell. We previously characterized rapid dynein-dependent lytic granule convergence to the microtubule-organizing center (MTOC) as an early, prerequisite step in NK cell cytotoxicity. Although multiple activating receptors can trigger granule convergence, the specific signal or signals responsible remained unknown. Using live cell confocal microscopy, NK cell lytic granule movement after NK cell activation was captured and measured. Using inhibitors of common early signaling mediators, we show that Src kinases are required for lytic granule convergence, but downstream signals that promote actin rearrangement, MTOC polarization, and calcium mobilization are not. Exposure to interleukin 2 was also sufficient to induce lytic granule convergence, which required noncanonical Src-dependent signaling. Thus, NK cell lytic granule convergence, prompted by specific receptor-mediated and soluble cytokine signals, depends on a directly downstream early Src kinase-dependent signal and emphasizes the importance of this step in readying NK cells for cytotoxicity.

By exploiting NK cell LROs (known as lytic granules) as a model, a new role is defined for Arl8b in regulating motility and exocytosis of lytic granules of NK cells. Not only lytic granules but also the MTOC is unable to polarize toward the immune synapse formed between the NK cell and its target in Arl8b-depleted NK cells., Natural killer (NK) lymphocytes contain lysosome-related organelles (LROs), known as lytic granules, which upon formation of immune synapse with the target cell, polarize toward the immune synapse to deliver their contents to the target cell membrane. Here, we identify a small GTP-binding protein, ADP-ribosylation factor-like 8b (Arl8b), as a critical factor required for NK cell–mediated cytotoxicity. Our findings indicate that Arl8b drives the polarization of lytic granules and microtubule-organizing centers (MTOCs) toward the immune synapse between effector NK lymphocytes and target cells. Using a glutathione S-transferase pull-down approach, we identify kinesin family member 5B (KIF5B; the heavy chain of kinesin-1) as an interaction partner of Arl8b from NK cell lysates. Previous studies showed that interaction between kinesin-1 and Arl8b is mediated by SifA and kinesin-interacting protein (SKIP) and the tripartite complex drives the anterograde movement of lysosomes. Silencing of both KIF5B and SKIP in NK cells, similar to Arl8b, led to failure of MTOC-lytic granule polarization to the immune synapse, suggesting that Arl8b and kinesin-1 together control this critical step in NK cell cytotoxicity.

Natural killer (NK) cell-mediated cytotoxicity is governed by the formation of a lytic immune synapse in discrete regulated steps, which give rise to an extensive array of cellular checkpoints in accessing NK cell-mediated cytolytic defense. Appropriate progression through these cell biological steps is critical for the directed secretion of specialized secretory lysosomes and subsequent target cell death. Here we highlight recent discoveries in the formation of the NK cell cytolytic synapse as well as the molecular steps and cell biological checkpoints required for this essential host defense process.

Multiple myeloma (MM) is a malignancy of plasma cells characterized by a monoclonal expansion of tumor cells in bone marrow and accumulation of a monoclonal protein in blood and urine, which ultimately leads to end-stage organ damage. Despite advances in therapy, MM remains an incurable disease. Recent investigations have revealed a new therapeutic target for MM, SLAMF7 (CS1), which is expressed at high levels on MM tumor cells. SLAMF7 is also found on NK, CD8+ T, CD4+ T and activated B cells. Elotuzumab (HuLuc63) is a humanized IgG1 anti-SLAMF7 antibody, which can facilitate killing of primary MM cells by NK cells via antibody-dependent cellular cytotoxicity (ADCC) and has shown promise in treating relapsed MM. A key question in understanding elotuzumab’s mechanism of action is whether it can directly activate NK cells via SLAMF7 binding, in addition to Fc-mediated activation via binding of CD16 (FcγRIIIA). We have examined the in vitro effect of elotuzumab on NK cells in fresh human peripheral blood from healthy donors and MM patients incubated with or without MM cell lines that have varied levels of SLAMF7 surface expression. Cytotoxic degranulation of NK cells was quantified by staining for CD107a (marker of degranulation) after 2 hours, and direct effects of elotuzumab on NK cells were assessed by measuring upregulation of the CD69 activation marker after 24 hours of antibody exposure. Degranulation of fresh primary NK cells toward SLAMF7-bearing MM target cell lines was stimulated by addition of elotuzumab. Intensity of the degranulation response was dependent upon the concentration of elotuzumab and the expression level of SLAMF7 on the MM target cells. While elotuzumab alone could stimulate low-level degranulation of NK cells in fresh PBL from healthy donors, the degranulation response was stronger if SLAMF7-expressing MM target cells were added in combination with elotuzumab. Addition of F(ab’)2 elotuzumab or an Fc mutant form of elotuzumab that cannot bind CD16 did not stimulate degranulation, implying that binding of the antibody to SLAMF7 on NK cells and MM cells, without concomitant engagement of NK cell Fc receptors, is insufficient to trigger degranulation. Soluble intact elotuzumab stimulated CD69 expression and degranulation by fresh primary NK cells in the absence of MM target cells, which peaked at 1 µg/ml and only modestly increased up to 100 µg/ml elotuzumab. In the same assays, we only observed modest stimulation of CD69 expression using soluble F(ab’)2 or Fc mutant elotuzumab. In contrast, plate-bound F(ab’)2 elotuzumab stimulated robust CD69 expression, indicating that the antibody can induce direct stimulation of NK cells through SLAMF7, but strong stimulation requires aggregation of the antibody. In soluble form, a non-fucosylated version of elotuzumab with greater affinity for CD16 stimulated significantly stronger degranulation and CD69 expression than intact elotuzumab, both in the presence or absence of MM target cells, and with both high and low SLAMF7 expression on target cells. Importantly, elotuzumab was found to induce similar degranulation by NK cells from freshly isolated peripheral blood of MM patients as compared to healthy donors. Taken together, our data support the hypothesis that binding of soluble elotuzumab to SLAMF7 can weakly activate NK cells, but strong activation and degranulation require antibody aggregation or engagement of the Fc domain with CD16. Elotuzumab variants with enhanced CD16 binding, such as a non-fucosylated version, can augment ADCC against myeloma cell lines, and warrant further study as a potential strategy to improve clinical efficacy in MM patients. Disclosures Cohen: Celgene: Member, Independent Response Adjudication Committee Other; Janssen: Advisory Board, Advisory Board Other; Bristol-Myers Squibb: Advisory Board, Advisory Board Other, Research Funding; Onyx: Advisory Board, Advisory Board Other. Campbell:Bristol-Myers Squibb: Advisory Board Other, Research Funding; Janssen: Advisory Board, Advisory Board Other; Conkwest: Consultancy, Patented NK-92 cell lines, Patented NK-92 cell lines Patents & Royalties, Research Funding.