NK Cell Effector Research Articles

Abstract PR15: A novel NK cell-targeted therapeutic strategy against pancreatic cancer

Abstract Recent years have witnessed an increased incidence of pancreatic adenocarcinoma (PDAC). Pancreatic tumors are poorly immunogenic; current immunotherapeutic strategies largely focus on boosting adaptive immunity while ignoring immunosuppressed innate immune players such as natural killer cells (NKs). NK cells respond to tumor insults by generating IFN-γ for T-cell activation. Multiple studies suggest that tumor cells evade NK cell-mediated killing by 1) developing “escape variants” and 2) regulating inhibitory and activating receptors on NK cells, imparting anergic/exhaustive phenotype inside the pancreatic tumor microenvironment. We posit that a therapeutic combination that not only overturns tumor-mediated NKs dysfunction but also boosts cytotoxic CD8 T cells will provide long-lasting therapeutic benefits in PDAC. Given this, we explored the efficacy of a unique therapeutic combination, tumor antigen-targeted IgE antibody (humanized anti-MUC1.IgE) in combination of anti-PD-L1 (for relieving T-cell exhaustion) and PolyICLC (for dendritic cell maturation) in a preclinical model of pancreatic cancer using mice transgenic for human MUC1 and FcϵRI (hMUC1/hFcϵRI). This therapeutic combination induced MUC1 specific rejection of two different human MUC1-expressing pancreatic tumor cell lines (Panc02.MUC1, KPC.MUC1) and prolonged the overall survival of mice challenged with subcutaneous and orthotopic tumors as compared to control counterparts. Additionally, this combination generated CD8 T-cell memory response as evidenced by MUC1 specific rejection/delays of tumors in mice rechallenged with MUC1-expressing tumors. Cytokine/chemokine profiling of anti-MUC1.IgE+anti-PD-L1+PolyICLC treated tumors further demonstrates a reduction in proinflammatory cytokines as compared to control counterparts. Most importantly, NK and CD8 T cells were involved in cell-mediated antitumor responses, as in vivo depletion of these subtypes abrogated the tumor-protective benefits in mice bearing orthotopic tumors. Anti-MUC1.IgE+anti-PD-L1+PolyICLC combination appears to increase circulating NKs and reverse NK cell exhaustion inside pancreatic tumor microenvironment. Additional data suggest that this therapeutic combination boosts tumor cell killing by NK cells in antibody-dependent cell cytotoxicity assays (ADCC). In sum, this is the first study to show that specific stimulation of IgE/FcϵRI axis in combination with PolyICLC and anti-PD-L1 can activate both CD8 T and NK cell effector pathways and provide long-lasting tumor-protective benefits against pancreatic cancer. This abstract is also being presented as Poster A48. Citation Format: Kamiya Mehla, Thomas C. Caffrey, Kelly A. O'Connel, Raghupathy Madiyalakan, Christopher F. Nicodemus, Michael A. Hollingsworth. A novel NK cell-targeted therapeutic strategy against pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr PR15.

Abstract IA09: Using high-dimensional machine-assisted analysis for biomarkers detection and guidance of immunotherapy to cancer

Abstract The emergence of immuno-oncology (IO) agents has led to unprecedented successes in the treatment of deadly cancers and sometimes even cures. These agents act by “releasing the brakes” and thereby unleashing the full potential of cells that fight tumors. Sadly, not every patient benefits from these new treatments. We need to know who will benefit and who may not. Here we approached the problem by designing a customized workflow by using high-dimensional single-cell mass cytometry (CyTOF) combined with machine-learning bioinformatics for the in-depth characterization of single immune cells in predicting and monitoring immune responses. The analysis is data driven, can be adapted to high-throughput approaches, and can model arbitrary trial designs such as batch effects and paired designs. We tested our workflow on two studies: a) Predict response to anti-PD-1 immunotherapy in melanoma. In our discovery cohort peripheral blood mononuclear cells (PBMCs) from 20 patients with stage IV melanoma before and after 12 weeks of anti-PD-1 therapy were analyzed. We observed a clear T-cell response on therapy. The most evident difference in responders before therapy was an enhanced frequency of CD14+ CD16+HLA-DRhi classical monocytes. We validated our results using conventional flow cytometry in an independent exploratory cohort of 31 patients before therapy. Finally, we correlated enhanced monocyte frequencies before therapy initiation with clinical response and could show association with lower hazard, extended progression-free and overall survival. b) Monitoring the immune response in anti-PD-1 immunotherapy-refractory non-small cell lung cancer to a novel combination immunotherapy of anti-PD-1 and an IL-15 superagonist. Twenty-one patients with non-small cell lung cancer who mostly were refractory to previous anti-PD-1 treatment received a novel combination therapy of IL-15 superagonist plus anti-PD-1. A response in the CD8+ T cell compartment was observed. Unexpectedly, our high-dimensional approach was able to detect and characterize a strong expansion of innate tumor-reactive effector NK cells starting around day 4 of therapy. Taken together, our unbiased artificial intelligence-driven immune workflow might support patient selection prior to therapy, select the right drug combination, and identify new druggable cell populations. Citation Format: Luis Cardenas, Silvia Guglietta, John Wrangle, Mark Rubinstein, Mark Robinson, Carsten Krieg. Using high-dimensional machine-assisted analysis for biomarkers detection and guidance of immunotherapy to cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr IA09.

Dysfunctional Natural Killer cells expanded from a liver with Hepatocellular Carcinoma show reduced killing against a unique HCC cell line

BackgroundThe liver has one of the largest reserves of NK cells in the body. NK cells in the liver are key defenses against viral hepatitis, liver fibrosis, and tumorigenesis. In the healthy liver, they exhibit higher cytotoxicity and secrete more cytokines compared to those isolated from peripheral blood. Hepatocellular carcinoma (HCC) is one of the most rapidly increasing cancers worldwide. Previously, NK cells have been shown to be dysfunctional in HCC. Additionally, NK cells are reduced in number intratumorally and peripherally and there is a decrease in the cytotoxic CD56dim subpopulation. Many studies have shown a downregulation of key signaling molecules involved in peripheral NK cell cytotoxic activity. We aimed to further investigate the level of dysfunction of NK cells isolated from HCC livers to document the nature of dysfunction of liver‐resident NK cells in eliminating HCC cell lines.MethodsBriefly, samples of tissue were collected from explanted livers and made into single cell suspensions. Next, lymphocytes were isolated using density‐based centrifugation. The resulting fraction was placed into RPMI‐1640 with IL‐2 and IL‐15. After two weeks, samples were taken to determine the number and purity of NK cells by flow cytometry. Target cell lines were labeled with chromium‐51 to test the killing capacities of NK cells isolated from healthy donor PBMC’s or HCC patient livers. NK cells were prepared in serial dilutions and in triplicate to generate a killing curve. After 4 hours of coincubation of effector NK cells with target cells, the released radioactive particles were measured and analyzed.ResultsTwo cell lines, Hep3B (ATCC) and HCO2 (generated in our lab), were used as target cells. Figures 1 and 2 depict the results of separate killing assays using the different target HCC cell lines. The effector cells in each assay consist of NK cells sourced from either healthy donor PBMC’s, cirrhotic patient PBMC’s, or HCC liver. Notably, the results show that even under the constant stimulation of IL‐2 and IL‐15, which are known interleukins that are involved in the proliferation and activation of NK cells and T‐cells, there is a large dysfunction in NK cells sourced from diseased livers. More strikingly, our results show that there is a specific inability of NK cells sourced from HCC livers to eliminate HCO2 cells which have been previously shown in our lab to be sorafenib‐resistant. Our hypothesis is that there is an innate dysfunction that is present in NK cells isolated from diseased livers that is not recoverable even when cultured in the presence of activating cytokines. Additionally, HCO2 cells possess a unique resistance to killing by NK cells isolated and expanded from an HCC liver.ConclusionHere we show that NK cells can be isolated and expanded, ex vivo, from human diseased livers. Additionally, we show that despite being stimulated for over 2 weeks with cytokines, expanded NK cells still show a marked reduction in killing capacity against a variety of HCC cell lines compared to NK cells expanded from PBMC’s from healthy and diseased donors. Our results confirm that there is an innate dysfunction that is present within NK cells isolated from diseased livers that is not recoverable even when cultured in the presence of activating cytokines.HCC cell lines (Hep3B, in circles, HCO2, in triangles) were labeled with Chromium‐51 for 2 hours. Target cells were rinsed with media and cocultured with NK cells at various effector‐to‐target ratios. NK cells were sourced from either PBMC’s from a cirrhotic patient (PBNK, in blue) or a liver with hepatocellular carcinoma (LNK, in green) and expanded ex vivo with IL‐2 and IL‐15.Figure 1

Simulated microgravity disarms human NK-cells and inhibits anti-tumor cytotoxicity in vitro

Long-duration spaceflight impairs natural killer (NK) cell function, which could compromise immune surveillance in exploration class mission crew. To determine if microgravity can impair NK-cell function, we established a rotary cell culture system to expose human peripheral blood mononuclear cells to simulated microgravity (SMG) in vitro. We found that 12 h of SMG suppressed NK-cell cytotoxic activity (NKCA) by ~50% against K562, U266 and 721.221 tumor target cells when returned to the 1G environment. Mass cytometry was used to identify 37 individual markers associated with NK-cell activation, maturation and cytotoxicity, revealing that SMG causes reductions in NK-cell degranulation and effector cytokine production. Extended flow cytometry confirmed that SMG lowered NK cell perforin and granzyme b expression by 25% and 17% respectively, but did not affect the surface expression of various activating (NKG2D, NKp30) and inhibitory (NKG2A, KLRG1) receptors or the ability of NK-cells to conjugate with target cells. Flow cytometry further revealed that SMG impaired NK-cell degranulation (reduced CD107a+ expression) and suppressed TNFα and IFNγ secretion in response to stimulation with K562 target cells. These findings indicate that SMG ‘disarms’ human NK-cells of cytolytic granules and impairs NKCA against a range of tumor target cells in vitro. Exposure to microgravity could be a factor that contributes to impaired NK-cell function during long duration space travel.

α2β1 Integrin Is Required for Optimal NK Cell Proliferation during Viral Infection but Not for Acquisition of Effector Functions or NK Cell-Mediated Virus Control.

NK cells play an important role in antiviral resistance. The integrin α2, which dimerizes with integrin β1, distinguishes NK cells from innate lymphoid cells 1 and other leukocytes. Despite its use as an NK cell marker, little is known about the role of α2β1 in NK cell biology. In this study, we show that in mice α2β1 deficiency does not alter the balance of NK cell/ innate lymphoid cell 1 generation and slightly decreases the number of NK cells in the bone marrow and spleen without affecting NK cell maturation. NK cells deficient in α2β1 had no impairment at entering or distributing within the draining lymph node of ectromelia virus (ECTV)-infected mice or at becoming effectors but proliferated poorly in response to ECTV and did not increase in numbers following infection with mouse CMV (MCMV). Still, α2β1-deficient NK cells efficiently protected from lethal mousepox and controlled MCMV titers in the spleen. Thus, α2β1 is required for optimal NK cell proliferation but is dispensable for protection against ECTV and MCMV, two well-established models of viral infection in which NK cells are known to be important.

Control of Metastases via Myeloid CD39 and NK Cell Effector Function.

Natural killer (NK) cell protection from tumor metastases is a critical feature of the host immune response to cancer, but various immunosuppression mechanisms limit NK cell effector function. The ectoenzyme, CD39, expressed on tumor-infiltrating myeloid cells, granulocytes, and lymphocytes, including NK cells, converts extracellular ATP (eATP) into AMP and, thus, potentially suppresses eATP-mediated proinflammatory responses. A CD39-targeting monoclonal antibody (mAb) that inhibits the mouse ectoenzyme CD39 suppressed experimental and spontaneous metastases in a number of different tumor models and displayed superior antimetastatic activity compared with the CD39 inhibitor POM1 and inhibitors and mAbs that block other members of the adenosinergic family (e.g., A2AR and CD73). The antimetastatic activity of anti-CD39 was NK cell and IFNγ dependent, and anti-CD39 enhanced the percentage and quantity of IFNγ produced and CD107a expression in lung-infiltrating NK cells following tumor challenge and anti-CD39 therapy. Using conditional Cd39 gene-targeted mouse strains and adoptive NK cell transfers, we showed that CD39 expressed on bone marrow-derived myeloid cells was essential for anti-CD39's antimetastatic activity, but NK cell expression of CD39 was not critical. The eATP receptor P2X7 and the NALP3 inflammasome, including downstream IL18, were critical in the mechanism of action of anti-CD39, and the frequency of P2X7 and CD39 coexpressing lung alveolar macrophages was specifically reduced 1 day after anti-CD39 therapy. The data provide a mechanism of action involving NK cells and myeloid cells, and anti-CD39 combined with anti-PD-1, NK cell-activating cytokines IL15 or IL2, or an inhibitor of A2AR to effectively suppress tumor metastases.

IL-15 and CD155 expression regulate LAT expression in murine DNAM1+ NK cells, enhancing their effectors functions.

NK cells are innate immune cells characterized by their ability to spontaneously lyse tumor and virally infected cells. We have recently demonstrated that IL-15-sufficient DC regulate NK cell effector functions in mice. Here, we established that among ITAM-proximal signaling molecules, the expression levels of the scaffold molecule Linker for Activation of T cells (LAT) and its transcription factor ELF-1 were reduced 4 days after in vivo depletion of DC. Addition of IL-15, a cytokine presented by DC to NK cells, regulates LAT expression in NK cells with a significant effect on the DNAM1+ subset compared to DNAM1- cells. We also found that LAT expression is regulated via interaction of the DNAM1 receptor with its ligand CD155 in both immature and mature NK cells, independently of NK cell education. Finally, we found that LAT expression within DNAM1+ NK cells might be responsible for enhanced calcium mobilization following the triggering of activating receptors on NK cells. Altogether, we found that LAT expression is tightly regulated in DNAM1+ NK cells, via interaction(s) with DC, which express CD155 and IL-15, resulting in rapid activation of the DNAM1+ subset during activating receptor triggering.

Abstract P3-01-02: B2 adrenergic receptor-mediated signaling regulates the immunosuppressive potential of myeloid-derived suppressor cells

Abstract Chronic stress is thought to suppress immunity; however, details about how chronic stress affects tumor growth and anti-tumor immune responses are still missing or poorly understood. Catecholamines (epinephrine, norepinephrine) released during sympathetic nerve stimulation can activate adrenergic receptors present on nearly every cell type including myeloid derived suppressor cells (MDSCs). MDSCs are increasingly recognized as critical players in tumor immunology because, in addition to their fundamental roles in suppressing effector NK cells, CD8+ T cells and CD4+ T cells, they also play a direct role in promoting tumor growth, differentiation and metastasis. Here, we demonstrate that the chronic adrenergic stress (as manipulated by a) housing temperature (which affects norepinephrine (NE) production, b) use of b-AR agonists or antagonists, or c) using β2-AR knockout mice regulates the accumulation and the pro-tumor phenotype of MDSCs. This same result was seen using two different murine models of breast cancer (4T1 and AT-3) and through the use of β2-AR knockout mice, we found that these effects are dependent on β2-adrenergic receptor (β2-AR) signaling. Using an agonist of β-AR signaling, we found that the activation of β2-AR signaling increases the expression of immunosuppressive molecules such as arginase-I and PDL-1 in MDSCs and enhances their immune-regulatory functions. β2-AR expression in MDSCs was induced by tumor promoting cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF). The survival of MDSCs is increased upon the activation of β2-AR on MDSCs in a Fas-FasL dependent manner. The regulation by β-AR signaling on MDSCs is dependent upon phosphorylation of STAT3 as a pivotal transcription factor in the function and expansion of MDSCs while MDSCs lacking β2-AR had poorer survival in both spleen and tumor. In murine tumor models, we found that the tumor promoting effects of β2-AR activation can be prevented by simple treatment of mice with β-blockers or by peripheral sympathectomy using 6-hydroxydopamine (6-OHDA). Furthermore, treatment with β-AR agonist increases the generation and immunosuppressive functions of MDSCs derived from human PBMCs in vitro. These data reveal a major, physiologically relevant role for nervous activity and adrenergic stress in MDSC-tumor biology and underscore the clinical potential for blockade of a previously unrecognized β2-AR-MDSC axis to improve immunotherapy. Keywords: β2 adrenergic receptor, MDSC, Chronic Stress, Tumor, STAT3 Citation Format: Hemn Mohammadpour, Cameron R. MacDonald, Guanxi Qiao, Minhui Chen, Bowen Dong, Bonnie L. Hylander, Philip L. McCarthy, Scott I. Abrams, Elizabeth A. Repasky. B2 adrenergic receptor-mediated signaling regulates the immunosuppressive potential of myeloid-derived suppressor cells [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-01-02.

Differentiation of c-Kit+ CD24+ natural killer cells into myeloid cells in a GATA-2-dependent manner.

Myeloid progenitor cells have generally been considered the predominant source of myeloid cells under steady-state conditions. Here we show that NK cells contributed to a myeloid cell lineage pool in naïve and tumor-bearing mice. Using fate tracing of NKp46+ cells, we found that myeloid cells could be derived from NK cells. Notably, among mature CD11b+ CD27+ NK cells, c-Kit+ CD24+ NK cells were capable of differentiating into a range of myeloid lineages in vitro and produced neutrophils and monocytes in vivo. The differentiation was completely inhibited by NK-stimulating cytokines. In addition to the potential for differentiation into myeloid cells, c-Kit+ CD24+ NK cells retained NK cell phenotypes and effector functions. Mechanistically, GATA-2 was necessary for the differentiation of c-Kit+ CD24+ NK cells. Therefore, we discovered that GATA-2-dependent differentiation of c-Kit+ CD24+ NK cells contributes to myeloid cell development and identified a novel pathway for myeloid lineage commitment under physiological conditions.

MAIT Cells Promote Tumor Initiation, Growth, and Metastases via Tumor MR1

Mucosal-associated invariant T (MAIT) cells are innate-like T cells that require MHC class I-related protein 1 (MR1) for their development. The role of MAIT cells in cancer is unclear, and to date no study has evaluated these cells in vivo in this context. Here, we demonstrated that tumor initiation, growth, and experimental lung metastasis were significantly reduced in Mr1 -/- mice, compared with wild-type mice. The antitumor activity observed in Mr1 -/- mice required natural killer (NK) and/or CD8+ T cells and IFNγ. Adoptive transfer of MAIT cells into Mr1 -/- mice reversed metastasis reduction. Similarly, MR1-blocking antibodies decreased lung metastases and suppressed tumor growth. Following MR1 ligand exposure, some, but not all, mouse and human tumor cell lines upregulated MR1. Pretreatment of tumor cells with the stimulatory ligand 5-OP-RU or inhibitory ligand Ac-6-FP increased or decreased lung metastases, respectively. MR1-deleted tumors resulted in fewer metastases compared with parental tumor cells. MAIT cell suppression of NK-cell effector function was tumor-MR1-dependent and partially required IL17A. Our studies indicate that MAIT cells display tumor-promoting function by suppressing T and/or NK cells and that blocking MR1 may represent a new therapeutic strategy for cancer immunotherapy. SIGNIFICANCE: Contradicting the perception that MAIT cells kill tumor cells, here MAIT cells promoted tumor initiation, growth, and metastasis. MR1-expressing tumor cells activated MAIT cells to reduce NK-cell effector function, partly in a host IL17A-dependent manner. MR1-blocking antibodies reduced tumor metastases and growth, and may represent a new class of cancer therapeutics.This article is highlighted in the In This Issue feature, p. 1.

Bruton tyrosine-kinase inhibitor on the rise: acalabrutinib in Waldenström macroglobulinemia

Bruton tyrosine-kinase inhibitor on the rise: acalabrutinib in Waldenström macroglobulinemia

NK Cell Metabolism and TGFβ – Implications for Immunotherapy

NK cells are innate lymphocytes which play an essential role in protection against cancer and viral infection. Their functions are dictated by many factors including the receptors they express, cytokines they respond to and changes in the external environment. These cell processes are regulated within NK cells at many levels including genetic, epigenetic and expression (RNA and protein) levels. The last decade has revealed cellular metabolism as another level of immune regulation. Specific immune cells adopt metabolic configurations that support their functions, and this is a dynamic process with cells undergoing metabolic reprogramming during the course of an immune response. Upon activation with pro-inflammatory cytokines, NK cells upregulate both glycolysis and oxphos metabolic pathways and this supports their anti-cancer functions. Perturbation of these pathways inhibits NK cell effector functions. Anti-inflammatory cytokines such as TGFβ can inhibit metabolic changes and reduce functional outputs. Although a lot remains to be learned, our knowledge of potential molecular mechanisms involved is growing quickly. This review will discuss our current knowledge on the role of TGFβ in regulating NK cell metabolism and will draw on a wider knowledge base regarding TGFβ regulation of cellular metabolic pathways, in order to highlight potential ways in which TGFβ might be targeted to contribute to the exciting progress that is being made in terms of adoptive NK cell therapies for cancer.

Concomitant deletion of SLAM-family receptors, NKG2D and DNAM-1 reveals gene redundancy of NK cell activating receptors in NK cell development and education.

NK cells recognize "unwanted" cells using a variety of germline-encoded activating receptors, such as the seven members of signaling lymphocyte activating molecule (SLAM)-family receptors (SFRs), natural killer cell group 2D (NKG2D), and DNAX accessory molecule-1(DNAM-1). Whether these receptors redundantly or synergistically regulate NK cell development and effector function remains poorly understood. By generating mice lacking SFRs, NKG2D, and DNAM-1, separately or in combination, we found that SLAMF6, one of the SFR members, was associated with NK cell differentiation, but its absence had no severe effect on NK cell differentiation and function, likely due to SFR redundancy. Moreover, we revealed that SFRs might work with other NK cell activating receptors in regulating NK cell development and function. We found that SFR deficiency caused an increase in immature NK cell subsets (CD27+ CD11b- ), and this effect was further augmented by the additional deficiency of NKG2D but not DNAM-1. However, SFR-deficient NK cells exhibited elevated responsiveness against "missing-self" hematopoietic targets, whereas the deletion of either NKG2D or DNAM-1 could partially abrogate the elevated effect of SFR deficiency on NK cell activation. Therefore, our results reveal the complexity of activating receptors in regulating NK cell differentiation and activation, extending our insights into the gene redundancy and compensatory effect of NK cell activating receptors.

NKTR-255, a Polymer-Conjugated IL-15 Enhances Antibody-Dependent Cellular Cytotoxicity Mediated By NK Cells in a B Cell Lymphoma Model

Background IL-15 is a cytokine that activates and provides survival benefit to T and NK cells and has great potential as an immunotherapeutic agent for the treatment of cancer. Exploiting the therapeutic value of native IL-15 has been challenging due to its unfavorable pharmacokinetic properties and poor tolerability. NKTR-255 is a polymer-conjugated IL-15 designed to retain binding affinity to the IL-15 receptor alpha chain and have decreased clearance to provide a sustained pharmacodynamics response. NKTR-255 has an enhanced immunotherapeutic effect when combined with certain targeted monoclonal antibodies that mediate tumor killing by antibody dependent cellular cytotoxicity (ADCC). Here we further characterized the effect of NKTR-255 on NK cell-mediated ADCC in a B cell lymphoma model. Methods For in vitro ADCC, NKTR-255 pre-treated human NK cells (effector cells) were co-cultured with RPMI-8226 (target cells) and with daratumumab for 3 hours. The ability of NK cells to lyse target cells was evaluated by detecting 7-AAD stained RPMI-8226. In the Daudi lymphoma model, SCID or SCID beige mice were inoculated IV with 1x107 Daudi cells on Day 0. NKTR-255 (0.3 mg/kg IV) was administered on Days 14, 21 and 28 and a single dose of antibody treatment was given on Day 14 (daratumumab 0.5 mg/kg IP). Survival rate was determined by onset of hind limb paralysis or moribundity as surrogate parameters. Concurrent vs staggered regimen assessment was conducted with NKTR-255 dosed on day 14 and daratumumab on Day 14, 17, or 21. For dose examination, high (0.5 mg/kg) and low (0.05 mg/kg) doses of daratumumab were combined with low (0.03 mg/kg) and high (0.3 mg/kg) doses of NKTR-255 in a concurrent manner. Daudi cells in the bone marrow were assessed 10 days after the NKTR-255 dose by flow cytometry. Results In vitro, NKTR-255 pre-treatment of human purified NK cells enhanced daratumumab-mediated ADCC against RPMI-8226. The ability of NKTR-255 to enhance in vitro ADCC was translated into an enhanced therapeutic efficacy of daratumumab in the Daudi lymphoma model with SCID mice. NKTR-255 combined with daratumumab synergistically provided long-term survival benefit in a NKTR-255 dose-dependent manner. To confirm the contribution of NK cells as effector cells for ADCC in vivo, the therapeutic effect of the combination treatment was assessed in the tumor model in SCID beige mice, which have a selective impairment of NK cell cytotoxic function. The long-term survival benefit by the combination treatment, which was observed in SCID mice (median survival of 27 days in control and 59.5 days in treatment), was attenuated in SCID beige mice (median survival of 23 days in control and 30 days in treatment). Concurrent vs staggered dose regimens and dose ranging for NKTR-255 and daratumumab were evaluated in the Daudi model with SCID mice to understand optimal therapeutic use of NKTR-255 in future clinical studies. Tumor depletion in bone marrow was most effective in concurrent treatment of NKTR-255 and daratumumab and three days-staggered treatment (NKTR-255 first and the antibody 3 days later). In contrast, a 7-day stagger showed no benefit of the combination in tumor depletion. Combination of low dose daratumumab and high dose NKTR-255 was equally effective in tumor depletion as a combination of high dose daratumumab and low dose NKTR-255. Conclusions NKTR-255 was shown to enhance in vivo therapeutic efficacy of daratumumab, a monoclonal antibody that mediates tumor killing by ADCC. The effect was mainly mediated by NK cells. The treatment scheduling experiments demonstrated that antibody should be present during NKTR-255-driven NK cell activation/expansion phase to efficiently deplete Daudi tumor cells in bone marrow. In addition, even low dose of daratumumab treatment showed effective tumor depletion when combined with high dose of NKTR-255. This suggests that NKTR-255 has a potential to expand the therapeutic window of existing tumor-targeted antibodies to patients with, for example, low antibody concentration in target tumor site or low expression of tumor antigen. Disclosures Miyazaki: Nektar Therapeutics: Employment, Equity Ownership. Kivimäe:Nektar Therapeutics: Employment, Equity Ownership. Hennessy:Nektar Therapeutics: Employment, Equity Ownership. Pena:Nektar Therapeutics: Employment, Equity Ownership. Quach:Nektar Therapeutics: Employment, Equity Ownership. Moffet:Nektar Therapeutics: Employment, Equity Ownership. Nieves:Nektar Therapeutics: Employment, Equity Ownership. Zhang:Nektar Therapeutics: Employment, Equity Ownership. Marcondes:Nektar Therapeutics: Employment, Equity Ownership. Madakamutil:Nektar Therapeutics: Employment, Equity Ownership. Zalevsky:Nektar Therapeutics: Employment, Equity Ownership.

Deletion of CISH in Human Pluripotent Stem Cell-Derived Natural Killer Cells Enhances Anti-Tumor Activity Via Metabolic Reprogramming

Cellular metabolism is now recognized to play a key role in regulating function and differentiation of immune cells. Immune cells undergo dynamic metabolic shifts to support their activity. Recent studies have shown the importance of metabolism to regulate NK cell function under different conditions. IL-15 can regulate NK cell metabolism in a dose- and time- dependent manner. However, whether improving metabolic fitness of NK cells can lead to improved anti-tumor activity is largely unknown. Here, we deleted CIS (encoded by the CISH gene in human induced pluripotent stem cells (iPSC)-derived NK cells (CISH-/- iPSC-NK) using CRISPR/Cas9 technology. CIS is known to function as a critical negative regulator of IL-15 signaling and a potent inhibitory checkpoint in NK cell-mediated anti-tumor activity. Surprisingly, more than 3000 differentially expressed genes were identified in CISH-/- iPSC-NK cells through RNA sequencing analysis. Gene ontology (GO) enrichment analysis showed that the top 2 up-regulated signaling pathways were associated with leukocyte differentiation and activation. Notably, more than 50 genes that involved in positive regulation of lymphocyte activation including genes associated with NK cell effector functions were significantly up-regulated in CISH-/- iPSC-NK cells compared with unmodified iPSC-NK cells. As expected, CISH-/- iPSC-NK cells exhibited increased sensitivity to IL-15 stimulation. Specifically, IL-15 stimulated JAK1 tyrosine phosphorylation, STAT3 and STAT5 phosphorylation were all dramatically increased in CISH-/- iPSC-NK cells, compared with unmodified iPSC-NK cells. In addition, when incubated with low concentrations of IL-15 (5 ng/ml), a concentration that does not support the growth and functions of unmodified iPSC-NK cells, CISH-/- iPSC-NK cells maintained significantly better expansion with >10 fold expansion for CISH-/- iPSC-NK cells in 3 weeks, whereas most of WT iPSC-NK cells died under these conditions (p<0.001). These CISH-/- iPSC-NK cells exhibited improved cytotoxic against acute myeloid leukemia (AML) cell lines K562 and Molm-13. More importantly, in an AML (MOLM-13) xenograft model, CISH-/- iPSC-NK cells displayed significantly increased in vivo persistence thus lead to significantly better control of tumor progression. To further understand the underling mechanisms contributing to the increased functionality in CISH-/- iPSC-NK cells, we then analyzed the metabolic profiles of these cells using Agilent Seahorse assays. After treatment with low concentration IL-15 for 7 days, CISH-/- iPSC-NK cells displayed both increased glucose metabolism and increased mitochondrial respiration profiles when compared with unmodified iPSC-NK cells. Increased glucose metabolism include a significant increase in basal glycolysis (p=0.0003, 84.5 ± 1.0 vs 61.1 ± 1.8 mpH/min, n=6), and glycolytic capacity (p=0.0049, 15.2±0.4 vs 11.7±0.5 mpH/min, n=6). And increased mitochondrial respiration profile is characterized by an increase in the spare respiratory capacity (p=0.0009, 16.3±1.9 vs 6.9±0.5 pmol/min, n=6), maximal respiration (p<0.0001, 31.6±1.9 vs 15.7±0.8 pmol/min, n=6) and ATP-linked respiration (p<0.0001, 11.5 ± 0.4 vs 7.0±0.3 pmol/min, n=6). Interestingly, limiting the increase of glucose metabolism and mitochondrial respiration via utilization of an mTOR inhibitor (rapamycin) attenuated the increased functionality in CISH-/- iPSC-NK cells. The findings presented here show that deletion of CISH in iPSC-NK cells enhance anti-tumor activity, at least in part via metabolic reprogramming. These findings suggest that improving NK cell functions for cancer immunotherapy through metabolic reprogramming provides a promising therapeutic strategy. Disclosures Guan: OncoImmune: Equity Ownership. Kaufman:FATE Therapeutics: Consultancy, Research Funding.

Ptcy + ATG Vs Ptcy Alone As Gvhd Prophylaxis for Peripheral Blood Stem Cells Haplo-Transplants: Comparison of NK and T Cell Effector Reconstitution

Backgound: A higher incidence of graft-versus-host disease (GVHD) has been observed after haplo-transplant of hematopoietic stem cells with post-transplant cyclophosphamide (PTCY) using peripheral blood stem cells (PBSC) instead of bone marrow as source of graft. Combining PTCY with anti-thymoglobulin (ATG) may help to reduce GVHD incidence. Here, early immune reconstitution, especially of NK and T cell compartments, was compared after both types of transplant (PTCY vs PTCY+ATG) investigating their influence on patients (pts) outcomes. Methods: This retrospective study included 58 adults who received a reduced-intensity conditioning (RIC) PBSC haplo-transplant with ciclosporine and mycophenolate mofetyl + PTCY (n=32, period: 08/2014-05-2017) or PTCY+ATG (N=26, period: 05/017-03/2019) as GVHD prophylaxis. The PTCY group received a Baltimore-based RIC regimen with fludarabine (n=10) or clofarabine (n=22). The PTCY+ATG group received a CloB2A1 regimen consisting of clofarabine 30 mg/m²/day (d), d-6 to -2, busulfan 3,4 mg/kg d-3 and d-2, ATG 2,5 mg/kg d-1. Both groups shared similar characteristics except for the median number of CD3+ T cells infused, significantly higher for CloB2A1 pts (27.5x107/kg vs 22.3, p=0.01). Samples from all pts were collected three times a week from d0 until d30, then at d60 and d90/100 in order to evaluate NK and T cells reconstitution by flow cytometry (FCM). All pts provided informed consent for data and samples collection before graft. Results: The median follow-up was 32 and 10,5 months for alive pts in PTCY and PTCY+ATG groups, respectively. Primary graft failure (PGF) was observed in 5 pts in the PTCY+ATG group (19%) vs none in the PTCY group (p=0.03). The PTCY and PTCY+ATG groups shared similar survivals at 1-year with PTCY OS of 71.5% vs PTCY+ATG 60,2% (p=0.73), DFS of 62,5% vs 58.6% (p=0,8) and GRFS of 46.8% vs 5.7% (p=0.47). Incidences of relapse and death were also comparable. Conversely, considering engrafted patients, the incidence of grade 2-4 acute GVHD was significantly lower in the CloB2A1 sub-group (23.8% vs 59%, p=0.02) despite higher numbers of CD3+ T cells infused in this group. The incidence of grade 3-4 acute GVHD was also lower in the CloB2A1 sub-group (4.7% vs 18.7%, p=0.29). Immune reconstitution of the PTCY group has been already reported (Willem, J Immunol 2019). Considering the PTCY+ATG group, T cell reconstitution did not influence the occurrence of acute post-transplant GVHD. Yet, in the group of pts with myeloid diseases (n=20), a higher frequency of CD56- T cells at d20 was associated with a higher relapse incidence (39% vs 18%, p=0.04). The NK cells frequency was lower at d+30 for patients with PGF (52.5% vs 19.5%, p=0.01). Pts who did not relapse were documented with higher d30 frequency of NKG2A+ NK cells (94.4% vs 72.1% of all lymphocytes, p=0.04). Specifically, these pts showed higher NKG2A+ KIR2DL2/3/S2- NK cell levels at d30 (74.1% vs 56.3%, p=0.05) and d60 (74.1% vs 55.5%, p=0.03). Inhibitory KIR/HLA incompatibilities, documented in 4 donor/recipient pairs, were associated with lower NK cell frequency at d60 (15% vs 69.2% of all lymphocytes, p= 0.01) and d100 (9% vs 61.5%, p=0.01) Comparing the PTCY vs the PTCY+ATG groups, the frequency of CD56- T cells was found to be significantly lower during the first month when using PTCy+ATG: d5 (34.3% vs 75.7% of all lymphocytes, p<0.0001), d20 (26.4% vs 54.8%, p<0.0001), d25 (35.9% vs 55.7%, p=0.01) and d30 (29.1% vs 48.1%, p=0.008). However, higher d5 (p=0.0001) and d30 (p=0.003) KIR2DL2/3/S2+ T cell levels were observed in this group. However, this proportion strongly decreased in comparison to the PTCY group at d100 (p>0.0001). NK cell levels were higher at d25 (34.9% vs 18.4% of all lymphocytes, p=0.03) and d30 (42.7% vs 27.9%, p=0.04) in the PTCY+ATG group. Specifically, faster reconstitutions at d20 of mature NKp46+ 2B4- (p=0.002), NKG2A+ (p=0.01) and NKG2A+ KIR2DL2/3/S2- (p=0.0004) cell subsets were found in this group. Conclusion: These results show that PTCy+ATG vs PTCy alone as GVHD prophylaxis limits acute grade 2-4 GVHD occurrence after RIC PBSC haplo-transplant, perhaps because of the combined effect of T and NK cell effector reconstitution. Indeed, the slower T cell reconstitution with PTCy+ATG may limit GvHD occurrence. In parallel, the quicker reconstitution of some NK cells sub-types may limit relapse occurrence. Larger prospective studies are needed to confirm these preliminary results. Disclosures Peterlin: Daiichi-Sankyo: Consultancy; Jazz Pharma: Consultancy; AbbVie Inc: Consultancy; Astellas: Consultancy. Chevallier:Jazz Pharmaceuticals: Honoraria; Daiichi Sankyo: Honoraria; Incyte: Consultancy, Honoraria.

1909P - Evaluation of antibody-dependent cell cytotoxicity (ADCC) in lung cancer cell lines treated with combined anti-EGFR and anti-PD-L1 therapy

BackgroundPrevious studies have demonstrated that IgG1 mAbs as cetuximab, stimulate Antibody-Dependent Cell Cytotoxicity (ADCC). Among immune checkpoint inhibitors, avelumab is the only fully human IgG1 anti-PD-L1 mAb with ADCC properties. Anti-PD-L1 and anti-EGFR mediated NK cytotoxicity is evaluated. MethodsLDH release was analyzed to study NK-mediated cytotoxicity by LDH Cytotoxicity Assay Kit and results was correlated to the level of PD-L1, EGFR and MHC-I cell surface expression analyzed by flow cytometry. NK-mediated cytotoxicity of the combination of anti-PD-L1 and anti-EGFR mAbs was studied in a panel of NSCLC cells lines encompassing different tumor types, using as effector NK cells isolated from healthy donors or NSCLC patients. ResultsPD-L1/EGFR/MHC-I expression levels correlated with enhanced ADCC lysis by the combination of avelumab and cetuximab as demonstrated by LDH assay, CD16 and CD107a mRNA. No significant difference in avelumab plus cetuximab-mediated ADCC between NK cells from healthy donors or from NSCLC patients was observed with a trend in favor of cancer patients, indicating that NK from cancer patients maintain lytic activity. ADCC capability of NK cells isolated from patients enrolled in the phase II study CAVE (Cetuximab-AVElumab)-lung, a single arm phase II clinical study of the combination of avelumab plus cetuximab in the second line treatment of metastatic non small cell lung cancer (NSCLC) patients (EUDRACT 2017- 004195-58) study resulted significantly enhanced after the experimental treatment compared to untreated baseline and healthy donors samples. ConclusionsThe combination of anti-EGFR and anti PD-L1 IgG1 antibodies is synergistic in terms of ADCC, where each antibody complements each other by promoting a more permissive immune reaction against the tumor, active also in otherwise immune-resistant cancers. Legal entity responsible for the studyUniversità degli studi della Campania Luigi Vanvitelli. FundingMerck KGaA. DisclosureF. Ciardiello: Advisory / Consultancy, Research grant / Funding (institution): Roche; Advisory / Consultancy, Research grant / Funding (institution): Bayer; Advisory / Consultancy, Research grant / Funding (institution): Amgen; Advisory / Consultancy: Merck; Advisory / Consultancy: Pfizer; Advisory / Consultancy: Sanofi; Advisory / Consultancy: Servier; Advisory / Consultancy: BMS; Advisory / Consultancy: Cellgene; Advisory / Consultancy: Lilly; Research grant / Funding (institution): AstraZeneca; Research grant / Funding (institution): Ipsen. F. Morgillo: Advisory / Consultancy: MSD; Advisory / Consultancy: Lilly; Research grant / Funding (institution): AstraZeneca. All other authors have declared no conflicts of interest.

Influenza Vaccination Primes Human Myeloid Cell Cytokine Secretion and NK Cell Function.

Cytokine-induced memory-like (CIML) NK cells generated in response to proinflammatory cytokines in vitro and in vivo can also be generated by vaccination, exhibiting heightened responses to cytokine stimulation months after their initial induction. Our previous study demonstrated that in vitro human NK cell responses to inactivated influenza virus were also indirectly augmented by very low doses of IL-15, which increased induction of myeloid cell-derived cytokine secretion. These findings led us to hypothesize that IL-15 stimulation could reveal a similar effect for active influenza vaccination and influence CIML NK cell effector functions. In this study, 51 healthy adults were vaccinated with seasonal influenza vaccine, and PBMC were collected before and up to 30 d after vaccination. Myeloid and lymphoid cell cytokine secretion was measured after in vitro PBMC restimulation with low-dose IL-15, alone or in combination with inactivated H3N2 virus; the associated NK cell response was assessed by flow cytometry. PBMC collected 30 d postvaccination showed heightened cytokine production in response to IL-15 compared with PBMC collected at baseline; these responses were further enhanced when IL-15 was combined with H3N2. NK cell activation in response to IL-15 alone (CD25) and H3N2 plus IL-15 (CD25 and IFN-γ) was enhanced postvaccination. We also observed proliferation of less-differentiated NK cells with downregulation of cytokine receptors as early as 3 d after vaccination, suggesting cytokine stimulation in vivo. We conclude that vaccination-induced "training" of accessory cells combines with the generation of CIML NK cells to enhance the overall NK cell response postvaccination.

Abstract 1538: Margetuximab mediates greater Fc-dependent anti-tumor activities than trastuzumab or pertuzumab in vitro

Abstract Margetuximab (M) is an investigational Fc-engineered anti-HER2 monoclonal antibody (mAb) with potential for greater immune-mediated anti-tumor activity than trastuzumab (T). M and T bind the same subdomain IV epitope, but M binds with higher affinity to activating Fc receptor, CD16A (FcγRIIIA), and lower affinity to inhibitory Fc receptor, CD32B (FcγRIIB). Pertuzumab (P) binds to a subdomain II epitope and has the same wild type Fc domain as T. We compared in vitro properties of M, T and P and combinations of M+P and T+P to evaluate Fc domain contributions to anti-HER2 mAb mediated activities. By surface plasmon resonance, M, T and P bound to the HER2 extracellular domain with similar high affinity. Binding affinities of M and T were unchanged if P was pre-bound, indicating lack of interaction. M, T and P exhibited comparable binding to HER2-expressing cell lines. Anti-proliferative activities of M and T toward HER23+ cells (N87, SKBR3 or BT474) in the absence or presence of ligands (EGF or HRG1β) were similar, whereas P exhibited weaker activity. In the absence of ligands, M+P or T+P were comparably as active as M or T alone. In presence of ligands, M+P and T+P were generally more active than M or T alone. Antibody-dependent cell-mediated cytotoxicity (ADCC) was evaluated with N87 and SKBR3 target cells (HER23+, expressing a reporter gene to measure viability) and NK effector cells from donors with differing CD16A158 genotypes. M was 7- to 84-fold more potent than T, as well as 69- to 744-fold more potent than P. Greater differences generally were seen with effector NK cells bearing the more common CD16A158 FF and VF genotypes than with the less common, high affinity CD16A158 VV genotype. While addition of P enhanced mean potency of T by 2- to 3-fold, no such effect was seen with M; however, M or M+P was 7- to 25-fold more potent than T+P. Results with JIMT-1 cells (HER22+) were similar, albeit with lower differential than with HER23+ target cells. NK cells were monitored by flow cytometry following incubation of peripheral blood mononuclear cells with HER2+ target cells and mAbs. M induced greater expression of markers of activation (CD137), cytolytic capability (granzyme B, perforin) and proliferation (Ki67) than T. An anti-HER2 mAb with an inactivated Fc domain was ineffective, indicating that induced changes in NK cells were Fc-dependent. In conclusion, M and T bind HER2 with similar affinity and exhibit comparable anti-proliferative activity that is enhanced by addition of P if ligands (EGF or HRG1β) are present. Relative to T, M mediates superior ADCC with effector cells of all CD16A genotypes and promotes greater NK cell activation and expansion. The M+P combination maintains superior ADCC compared to the T+P combination. Citation Format: Liqin Liu, Yinhua Yang, Robert Burns, Jonathan Li, Haiquan Li, Sergey Gorlatov, Paul Moore, Jeffrey Nordstrom. Margetuximab mediates greater Fc-dependent anti-tumor activities than trastuzumab or pertuzumab in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1538.

Abstract CT082: Phase (Ph) I/Ib study of NIZ985 with and without spartalizumab (PDR001) in patients (pts) with metastatic/unresectable solid tumors

Abstract Background: NIZ985 is a soluble IL-15/IL-15 receptor α heterodimer (hetIL-15) that binds IL-2/IL-15 receptor β/γ and promotes effector T-cell and NK cell expansion and activation. Spartalizumab is a humanized IgG4 mAb that blocks binding of programmed death 1 (PD-1) to programmed death ligand 1/2 (PD-L1/2). In preclinical studies, IL-15 and anti-PD-1/PD-L1 combinations demonstrated synergistic antitumor activity. Here we report dose escalation results from a Ph I/Ib study of NIZ985 as a single agent (SA) and in combination with spartalizumab (NCT02452268). Methods: Pts with metastatic/unresectable solid tumors received subcutaneous NIZ985 TIW (2 weeks on/2 weeks off); 0.25-4 μg/kg NIZ985 SA or 1 μg/kg NIZ985 with IV spartalizumab (400 mg, Q4W). Ph I endpoints: DLTs, safety, PK, pharmacodynamics (PD), preliminary efficacy, and recommended dose for expansion (RDE), statistically guided by a 3+3 study design. Results: As of Oct 9, 2018, 14 pts (median age 56.5 y [range 42.0-73.0]) received NIZ985 SA and 11 pts (median age 61 y [range 34.0-75.0]) received NIZ985 + spartalizumab. Treatment was discontinued in 13 (93%) and 10 (91%) pts receiving NIZ985 SA and NIZ985 + spartalizumab, respectively, mainly due to disease progression (57% and 82%); 1 pt in each group is ongoing. No DLTs were reported in the first cycle. AEs suspected to be drug related were reported in 100% of pts (36% grade 3/4) for NIZ985 SA and 100% of pts treated with the combination (27% grade 3/4). Common AEs [all grades; grade 3/4], defined as ≥50% of pts for any grade, were injection site reaction [100%; 7%], chills [64%; 0%], and fatigue [57%; 0%] in NIZ985 SA; and injection site reaction [100%; 9%], fatigue [82%; 0%], diarrhea [64%; 9%], and nausea [55%; 0%] in NIZ985 + spartalizumab. Median duration of NIZ985 exposure (range) across doses was 7.6 weeks (2.6-15.6) in NIZ985 SA and 21.6 weeks (5.6-53.6) in NIZ985 + spartalizumab. In 13 evaluable NIZ985 SA pts, confirmed best overall responses (BORs) were stable disease (SD; n=3, 23%), progressive disease (n=8, 62%), and unknown (n=2, 15%); SD is ongoing in 1 pt with spindle cell ocular melanoma. Confirmed BORs in 11 evaluable NIZ985 + spartalizumab pts were SD (n=5, 45%) and progressive disease (n=6, 55%); SD is ongoing in 1 pt with gastric adenocarcinoma. RDE was declared as 1 μg/kg NIZ985 (TIW; 2 weeks on/2 weeks off) for SA and with spartalizumab (400 mg, Q4W). PD analyses showed NIZ985 induces lymphocyte proliferation in peripheral blood. Preliminary antitumor activity, PK, and PD data from tumors and peripheral blood will be presented. Conclusions: NIZ985 SA and with spartalizumab was well tolerated in pts with metastatic/unresectable solid tumors, with no DLTs reported. RDEs were NIZ985 1 μg/kg (TIW, 2 weeks on/2 weeks off) for SA and with spartalizumab (400 mg, Q4W). PD data support proof of mechanism. Dose escalation with an alternate NIZ985 schedule is ongoing. Citation Format: Kevin Conlon, Rom Leidner, Douglas McNeel, Sumati Gupta, Andrea Wang-Gillam, Thomas A. Waldmann, George Pavlakis, Mirek Dostalek, Sema Kurtulus, Niladri Roy Chowdhury, Jessica O'Keeffe, Nehal S. Parikh, John A. Thompson. Phase (Ph) I/Ib study of NIZ985 with and without spartalizumab (PDR001) in patients (pts) with metastatic/unresectable solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT082.