IntroductionNatural killer (NK) cells are CD56 ϩ CD3 Ϫ large granular lymphocytes that comprise a key cellular compartment of the innate immune system. NK cells have been shown to exert antitumor activity against the malignant plasma cell clone in multiple myeloma (MM). 1-4 However, through several established mechanisms, the NK-cell versus MM effect is attenuated as the disease inexorably progresses. 5-9 MM is increasing in incidence and remains incurable despite the advent of potent novel therapies such as lenalidomide and bortezomib. 10 In fact, both lenalidomide and bortezomib have been shown to confer anti-MM activity, in part, through recovery or enhancement of the NK-cell versus MM effect. 11,12 The NK-cell versus MM effect is subject to modulation through intracellular signal transduction cascades initiated by activating and inhibitory receptors at the NK-cell surface interacting with ligands expressed on MM tumor cells. Programmed death 1 (PD-1), a member of the B7 family of cosignaling molecules, and its associated ligands PD-L1 and PD-L2 have been shown to play a key role in down-regulating the T-cell immune response. 13 The constitutive or inducible expression of PD-1 has been characterized on several immune cell subsets, including T, B, and dendritic cells; however, to date, comparatively little is known regarding PD-1 expression on NK cells and whether or not the PD-1/PD-L1 axis is involved in the NK-cell versus MM effect. 14 CT-011 (CureTech, LTD; previously CT-AcTibody or BAT) is a novel immunoglobulin G1 (IgG1) humanized monoclonal antibody (mAb) that modulates the immune response through interaction with PD-1, with previously demonstrated antitumor efficacy in experimental models of both solid and liquid tumors. [15][16][17] Several human malignancies, including MM, express cognate ligands for PD-1 (eg, PD-L1) and play a key role in tumor immunoevasion. 18,19 In a phase 1 clinical trial of patients with advanced hematologic malignancies including MM, CT-011 was demonstrated to be safe and well tolerated with evidence of single-agent clinical beneficial responses in 33% of the patients. 20 Given the results of this phase 1 study and the potential complementary mechanisms of action between CT-011 and lenalidomide, we hypothesized these agents in combination may represent a promising novel therapy for MM.Lenalidomide (Revlimid; Celgene) exerts efficacy in part through enhancement of the NK-cell versus MM effect, 11 an effect likely mediated through T-cell production of interleukin-2 (IL-2) in response to this agent. 21 The numbers of both T cells and NK cells are increased in patients receiving lenalidomide therapy 22 ; however, NK-cell killing is also enhanced, including antibodydependent cellular cytotoxicity and natural cytotoxicity. 23,24 Moreover, these events correlate with clinical responses to lenalidomide therapy in patients with MM. 22 In this report, we show that the PD-1/PD-L1 signaling axis mediates NK-cell activation and cytotoxicity against MM. We show that freshly isolated NK cells...

Multiple myeloma (MM) is an incurable hematological malignancy. Chimeric antigen receptor (CAR)-expressing T cells have been demonstrated successful in the clinic to treat B-lymphoid malignancies. However, the potential utility of antigen-specific CAR-engineered natural killer (NK) cells to treat MM has not been explored. In this study, we determined whether CS1, a surface protein that is highly expressed on MM cells, can be targeted by CAR NK cells to treat MM. We successfully generated a viral construct of a CS1-specific CAR and expressed it in human NK cells. In vitro, CS1-CAR NK cells displayed enhanced MM cytolysis and IFN-γ production, and showed a specific CS1-dependent recognition of MM cells. Ex vivo, CS1-CAR NK cells also showed similarly enhanced activities when responding to primary MM tumor cells. More importantly, in an aggressive orthotopic MM xenograft mouse model, adoptive transfer of NK-92 cells expressing CS1-CAR efficiently suppressed the growth of human IM9 MM cells and also significantly prolonged mouse survival. Thus, CS1 represents a viable target for CAR-expressing immune cells, and autologous or allogeneic transplantation of CS1-specific CAR NK cells may be a promising strategy to treat MM.

Human CD56bright natural killer (NK) cells possess little or no killer immunoglobulin-like receptors (KIRs), high interferon-γ (IFN-γ) production, but little cytotoxicity. CD56dim NK cells have high KIR expression, produce little IFN-γ, yet display high cytotoxicity. We hypothesized that, if human NK maturation progresses from a CD56bright to a CD56dim phenotype, an intermediary NK cell must exist, which demonstrates more functional overlap than these 2 subsets, and we used CD94 expression to test our hypothesis. CD94highCD56dim NK cells express CD62L, CD2, and KIR at levels between CD56bright and CD94lowCD56dim NK cells. CD94highCD56dim NK cells produce less monokine-induced IFN-γ than CD56bright NK cells but much more than CD94lowCD56dim NK cells because of differential interleukin-12–mediated STAT4 phosphorylation. CD94highCD56dim NK cells possess a higher level of granzyme B and perforin expression and CD94-mediated redirected killing than CD56bright NK cells but lower than CD94lowCD56dim NK cells. Collectively, our data suggest that the density of CD94 surface expression on CD56dim NK cells identifies a functional and likely developmental intermediary between CD56bright and CD94lowCD56dim NK cells. This supports the notion that, in vivo, human CD56bright NK cells progress through a continuum of differentiation that ends with a CD94lowCD56dim phenotype.

Glioblastoma (GB) remains the most aggressive primary brain malignancy. Adoptive transfer of chimeric antigen receptor (CAR)-modified immune cells has emerged as a promising anti-cancer approach, yet the potential utility of CAR-engineered natural killer (NK) cells to treat GB has not been explored. Tumors from approximately 50% of GB patients express wild-type EGFR (wtEGFR) and in fewer cases express both wtEGFR and the mutant form EGFRvIII; however, previously reported CAR T cell studies only focus on targeting EGFRvIII. Here we explore whether both wtEGFR and EGFRvIII can be effectively targeted by CAR-redirected NK cells to treat GB. We transduced human NK cell lines NK-92 and NKL, and primary NK cells with a lentiviral construct harboring a second generation CAR targeting both wtEGFR and EGFRvIII and evaluated the anti-GB efficacy of EGFR-CAR-modified NK cells. EGFR-CAR-engineered NK cells displayed enhanced cytolytic capability and IFN-γ production when co-cultured with GB cells or patient-derived GB stem cells in an EGFR-dependent manner. In two orthotopic GB xenograft mouse models, intracranial administration of NK-92-EGFR-CAR cells resulted in efficient suppression of tumor growth and significantly prolonged the tumor-bearing mice survival. These findings support intracranial administration of NK-92-EGFR-CAR cells represents a promising clinical strategy to treat GB.

Blockade of PD-L1 expression on tumor cells via anti-PD-L1 monoclonal antibody (mAb) has shown great promise for successful cancer treatment by overcoming T-cell exhaustion; however, the function of PD-L1 on natural killer (NK) cells and the effects of anti-PD-L1 mAb on PD-L1 + NK cells remain unknown. Moreover, patients with PD-L1 − tumors can respond favorably to anti-PD-L1 mAb therapy for unclear reasons. Here, we show that some tumors can induce PD-L1 on NK cells via AKT signaling, resulting in enhanced NK-cell function and preventing cell exhaustion. Anti-PD-L1 mAb directly acts on PD-L1 + NK cells against PD-L1 − tumors via a p38 pathway. Combination therapy with anti-PD-L1 mAb and NK cell-activating cytokines significantly improves the therapeutic efficacy of human NK cells against PD-L1 − human leukemia when compared with monotherapy. Our discovery of a PD-1-independent mechanism of antitumor efficacy via the activation of PD-L1 + NK cells with anti-PD-L1 mAb offers new insights into NK-cell activation and provides a potential explanation as to why some patients lacking PD-L1 expression on tumor cells still respond to anti-PD-L1 mAb therapy.

SUMMARY Little is known about the role of negative regulators in controlling natural killer (NK) cell development and effector functions. Foxo1 is a multifunctional transcription factor of the forkhead family. Using a mouse model of conditional deletion in NK cells, we found that Foxo1 negatively controlled NK cell differentiation and function. Immature NK cells expressed abundant Foxo1 and little Tbx21 relative to mature NK cells, but these two transcription factors reversed their expression as NK cells proceeded through development. Foxo1 promoted NK cell homing to lymph nodes through upregulating CD62L expression, and impaired late-stage maturation and effector functions by repressing Tbx21 expression. Loss of Foxo1 rescued the defect in late-stage NK cell maturation in heterozygous Tbx21+/− mice. Collectively, our data reveal a regulatory pathway by which the negative regulator Foxo1 and the positive regulator Tbx21 play opposing roles in controlling NK cell development and effector functions.

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...

Breast cancer brain metastases (BCBMs) are common in patients with metastatic breast cancer and indicate a poor prognosis. These tumors are especially resistant to currently available treatments due to multiple factors. However, the combination of chimeric antigen receptor (CAR)-modified immune cells and oncolytic herpes simplex virus (oHSV) has not yet been explored in this context. In this study, NK-92 cells and primary NK cells were engineered to express the second generation of EGFR-CAR. The efficacies of anti-BCBMs of EGFR-CAR NK cells, oHSV-1, and their combination were tested in vitro and in a breast cancer intracranial mouse model. In vitro, compared with mock-transduced NK-92 cells or primary NK cells, EGFR-CAR-engineered NK-92 cells and primary NK cells displayed enhanced cytotoxicity and IFN-γ production when co-cultured with breast cancer cell lines MDA-MB-231, MDA-MB-468, and MCF-7. oHSV-1 alone was also capable of lysing and destroying these cells. However, a higher cytolytic effect of EGFR-CAR NK-92 cells was observed when combined with oHSV-1 compared to the monotherapies. In the mice intracranially pre-inoculated with EGFR-expressing MDA-MB-231 cells, intratumoral administration of either EGFR-CAR-transduced NK-92 cells or oHSV-1 mitigated tumor growth. Notably, the combination of EGFR-CAR NK-92 cells with oHSV-1 resulted in more efficient killing of MDA-MB-231 tumor cells and significantly longer survival of tumor-bearing mice when compared to monotherapies. These results demonstrate that regional administration of EGFR-CAR NK-92 cells combined with oHSV-1 therapy is a potentially promising strategy to treat BCBMs.