Selinexor Enhances NK Cell Activation Against Lymphoma Cells Via Downregulation of HLA-E

Abstract Introduction: Natural killer (NK) cells are powerful immune effectors which induce direct cytotoxicity, promote adaptive immune responses and mediate antibody dependent cellular cytotoxicity (ADCC). Enhancement of NK cell activity against cancer is currently the focus of intense research efforts and strategies include CAR-NK, stimulatory antibodies, cytokines and checkpoint inhibitors. Upregulation of exportin-1 (XPO1) is common in human cancers and high expression is negatively associated with survival in various cancers including diffuse large B cell lymphoma (DLBCL). Targeted inhibition of XPO1 by the selective inhibitor selinexor leads to cancer cell death via accumulation of tumour suppressor proteins in the nucleus, dysregulation of growth regulatory proteins and blockade of oncogene protein translation. The therapeutic efficacy of XPO1 inhibition has led to FDA approval of the oral XPO1 inhibitor selinexor for the treatment of multiple myeloma and DLBCL. The effect of selinexor on NK cell activity has not previously been investigated and was therefore addressed in this study. Methods: The B lymphoma cell lines JeKo-1, SU-DHL-4 and Ramos were incubated with selinexor (50-2000nM) for 18 hours before analysis. Flow cytometry was used to assess cell surface expression of activating and inhibitory ligands for NK cells. For NK based assays, peripheral blood derived NK cells were isolated from healthy donors and incubated with IL-15 (1ng/ml) overnight prior to co-culture with target lymphoma cells for a further 4 hours. Cytotoxicity was assessed using propidium iodide staining of target cells and degranulation of NK cells was assessed by measurement of CD107a. Whole blood samples from colorectal cancer patients (n=11) at pre-treatment and 3 weeks post selinexor monotherapy were assessed by flow cytometry for CD45+CD3-CD19-CD56+ NK cells. Results: Selinexor pre-treatment of target lymphoma cells significantly increased NK cell mediated cytotoxicity against SU-DHL-4 (2.2 Fold increase, p<0.01), JeKo-1 (2 Fold increase, p<0.01) and Ramos (1.7 Fold increase, p<0.01) cells. In accordance with this, selinexor pre-treatment of target cells also increased the activation of NK cells against SU-DHL-4, JeKo-1 and Ramos cells as measured by CD107a expression in both CD56 bright and CD56 dim NK cell sub-groups. To identify the mechanism behind this, we measured expression of activating and inhibitory ligands for NK cells on SU-DHL-4 cells after incubation with selinexor. No significant changes in expression of activating ligands (MICA/B, ULBP-2/5/6, ULBP-1, Vimentin, B7H6, CD54) were evident. In contrast, selinexor significantly (p<0.001) reduced the surface expression of HLA-E on SU-DHL-4 cells by 50%. Selinexor mediated downregulation of HLA-E was also evident in Ramos (60% reduction, p<0.001) and JeKo-1 cells (20% reduction, p<0.01). HLA-E binds the ITIM containing receptor NKG2A, a key inhibitory receptor for NK cells and subsets of T cells. In accordance with this, selinexor pre-treatment of SU-DHL-4 cells selectively increased NKG2A+ NK cell activation (p<0.01) following co-culture. To examine the effect of selinexor on NK cells in patients, we assessed the proportion of NK cells in the peripheral blood of 11 colorectal cancer patients at pre-treatment and three weeks post selinexor monotherapy. % NK cells of CD45+ peripheral blood lymphocytes following treatment with selinexor was increased 2-fold (Median 5% pre-treatment vs 10% post selinexor). In addition, increased abundance of the less mature and less cytotoxic CD56 bright subset of NK cells was associated with poor response to therapy (Median 4% responders (n=3) vs 20% non-responders (n=8)). Larger patient datasets are required to confirm these effects and this analysis is currently ongoing. The effect of selinexor on NK cells in patients with lymphoma is also currently under investigation. Conclusions: The NKG2A:HLA-E axis is a novel immune checkpoint target and our data identifies that selinexor sensitises lymphoma cells to NK cell mediated killing via disruption of this interaction. In addition, we provide initial evidence that NK cells may be associated with clinical response to selinexor. This data indicates that NK cells may contribute to the therapeutic efficacy of selinexor and that selinexor may synergise with NK cell targeted therapies for the treatment of lymphoma. Disclosures Walker: Karyopharm Therapeutics: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Johnson: Morphosys: Honoraria; Kymera: Honoraria; Kite Pharma: Honoraria; Incyte: Honoraria; Genmab: Honoraria; Celgene: Honoraria; Bristol-Myers: Honoraria; Epizyme: Consultancy, Research Funding; Boehringer Ingelheim: Consultancy; Novartis: Honoraria; Takeda: Honoraria; Oncimmune: Consultancy; Janssen: Consultancy. Cragg: BioInvent International: Consultancy, Research Funding; GSK: Research Funding; UCB: Research Funding; iTeos: Research Funding; Roche: Research Funding. Forconi: Novartis: Honoraria; Roche: Honoraria; Janssen: Consultancy, Honoraria, Speakers Bureau; AbbVie: Consultancy, Honoraria, Speakers Bureau; Gilead: Research Funding. Landesman: Karyopharm Therapeutics: Current Employment, Current equity holder in publicly-traded company. Blunt: Karyopharm Therapeutics: Research Funding.

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NK-Cell Reconstitution after HLA-Identical Blood and Marrow Transplantations for CML: The Recovery of NKG2D Is Inversely Correlated with NKG2A and It Promotes the GVL Effect.

Blood ◽

10.1182/blood.v110.11.4879.4879 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4879-4879

Author(s):

Juan Tong ◽

Huilan Liu ◽

Liangquan Geng ◽

Zimin Sun ◽

Baolin Tang ◽

...

Keyword(s):

Nk Cells ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Nk Cell ◽

Linear Regression Analysis ◽

Cell Activity ◽

Target Cells ◽

Peripheral Blood Stem Cells ◽

Peripheral Blood Mononuclear ◽

Blood Stem Cells

Abstract Natural killer (NK) cell alloreactivity is reported to mediate strong graft versus leukemia (GVL) effect in patients after allogeneic stem-cell transplantation. NKG2D receptors recognize human MHC class Ichain related A and B (MICA/B) and UL16-binding protein 1∼4(ULBP 1∼4) on target cells, thereby regulating NK cell activity. To examine the recovery of NKG2D, NKG2A and other receptors expression by NK cells, we used flow cytometry to evaluate samples from 11 chronic myeloid leukemia patients and their donors in the year following unmanipulated HLA completely matched peripheral blood stem cells plus bone marrow transplantation. Peripheral blood mononuclear cells from patients and their donors were tested in standard 51Cr release assays against cultured K562 targets to determine the cytotoxicity of the NK cells in the same intervals. There is no mismatched immunoglobulin-like receptor (KIR) ligand in both GVH and HVG direction. The reconstitution of KIR2DL1 (CD158a) after this transplantation protocol was very slow and these receptors didn’t reach normal value in the year and KIR2DL2 (CD158b) was much better. The NKG2D increased and the NKG2A decreased quickly at the same time after engraftment, and used linear regression analysis we demonstrated that NKG2A recovery was inversely correlated with NKG2D recovery in the year following transplantation. The ratio of NKG2D/NKG2A was directly associated with the capacity of NK-cell cytotoxicity. Thus, the reconstitution of NKG2D makes contribution to the recovery of the NK cytotoxicity. These results reveals that the NK cells generated after HLA matched blood plus bone morrow transplantation of CML patients are promoted at an immature state characterized by specific phenotypic features and enhanced functioning, having potential impact for immune responsiveness and transplantation outcome.

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CSL362: A Monoclonal Antibody to Human Interleukin-3 Receptor (CD123), Optimized for NK Cell-Mediated Cytotoxicity of AML Stem Cells

Blood ◽

10.1182/blood.v120.21.3598.3598 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3598-3598 ◽

Cited By ~ 4

Author(s):

Samantha J. Busfield ◽

Mark Biondo ◽

Mae Wong ◽

Hayley S. Ramshaw ◽

Erwin M Lee ◽

...

Keyword(s):

Stem Cells ◽

Monoclonal Antibody ◽

Mouse Model ◽

Nk Cells ◽

Peripheral Blood ◽

Research Funding ◽

Nk Cell ◽

Target Cells ◽

Cell Leukemia ◽

Interleukin 3

Abstract Abstract 3598 The interleukin-3 receptor alpha chain (IL-3Rα/CD123) is expressed in a variety of hematological malignancies including AML, MDS, B-ALL, Hodgkin's lymphoma, hairy cell leukemia, systemic mastocytosis, plasmacytoid dendritic cell leukemia and CML. In AML, the majority of AML blasts express CD123 and this receptor is selectively over expressed on CD34+CD38− leukemic stem cells (LSC) compared to normal hematopoietic stem cells. This difference may provide a biological advantage to the leukemic cells given the survival and proliferation-promoting activities of IL-3, whilst at the same time providing an opportunity to target these malignant cells selectively. We have shown previously that 7G3, a mouse monoclonal antibody (mAb) which blocks IL-3 binding to CD123, is capable of eliminating human LSC in a mouse model of human AML by a combination of mechanisms, including engagement of the innate immune system via Fc-dependent mechanisms (Jin et al., 2009 Cell Stem Cell, 5:31). We have subsequently humanised and affinity-matured this antibody and, in addition, have engineered the Fc-domain to optimise potential cytotoxicity against AML cells. The resultant antibody, CSL362, retains the ability to neutralise IL-3 and has enhanced affinity for the FcγRIIIa (CD16) on NK cells. In vitro studies have demonstrated that the increased affinity for CD16 correlates with greater antibody-dependent cell-mediated cytotoxicity (ADCC) against CD123 expressing cell lines compared to CSL360, a non Fc-engineered anti-CD123 mAb. The improved activity was evident as both an increased maximal level of target cell lysis and as a shift in the EC50 of the antibody to lower concentrations. Importantly, both primary AML blasts and CD34+CD38−CD123+LSC were susceptible to CSL362-induced ADCC and this was seen even in samples that were resistant to ADCC by a non Fc-engineered anti-CD123 mAb. In an AML xenograft mouse model, where treatment with the antibody was initiated 4 weeks after engraftment of leukemia cells, CSL362 was more effective in reducing leukemic growth than the non Fc-engineered anti-CD123 mAb. The evaluation of neutrophils, monocytes, macrophages and NK cells in ADCC assays has revealed that the major effector cell responsible for CSL362-mediated cytotoxicity in human peripheral blood is the NK cell. In clinical samples we have been able to demonstrate autologous depletion ex vivo of target AML blasts (collected at diagnosis and cryopreserved) following incubation with CSL362 and peripheral blood mononuclear cells (taken from the same patient at first remission), indicating that NK cell number and function is sufficiently preserved in such patients for CSL362-directed killing of leukemic target cells. The pre-clinical data generated thus far strongly support the clinical development of CSL362 for the treatment of AML in patients with adequate NK cell function. A Phase 1 study of CSL362 in patients with CD123 positive AML in remission is underway (Clinical Trials.gov identifier: NCT01632852). Disclosures: Busfield: CSL Limited: Employment. Biondo:CSL Limited: Employment. Wong:CSL Limited: Employment. Ramshaw:CSL Limited: Research Funding. Lee:CSL Limited: Research Funding. Martin:CSL Limited: Employment. Ghosh:CSL Limited: Employment. Braley:CSL Limited: Employment. Tomasetig:CSL Limited: Employment. Panousis:CSL Limited: Employment. Vairo:CSL Limited: Employment. Roberts:CSL Limited: Research Funding. DeWitte:CSL Behring: Employment. Lock:CSL Limited: Consultancy, Research Funding. Lopez:CSL Limited: Consultancy, Research Funding. Nash:CSL Limited: Employment.

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Engineered Interleukin-15 Autocrine Signaling Invigorates Anti-CD123 CAR-NK Cells

Blood ◽

10.1182/blood-2021-146609 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2806-2806

Author(s):

Ilias Christodoulou ◽

Michael Koldobskiy ◽

Won Jin Ho ◽

Andrew Marple ◽

Wesley J. Ravich ◽

...

Keyword(s):

Nk Cells ◽

Peripheral Blood ◽

Research Funding ◽

Nk Cell ◽

Xenograft Model ◽

Blood Analysis ◽

Target Cells ◽

Interleukin 15 ◽

Peripheral Blood Analysis

Abstract Introduction : Acute Myeloid Leukemia (AML) is an aggressive neoplastic disorder with poor outcomes in children and adults. NK cell adoptive transfer is an anti-cancer immunotherapy that has promise for AML treatment. We aimed to improve NK cell anti-tumor efficacy with expression of a Chimeric Antigen Receptor (CAR) on the cell surface. Our CAR consists of an extracellular single-chain variable fragment targeting the AML-associated antigen CD123 (IL3Rα) and intracellular domains derived from 2B4 and TCRζ. We sought to improve the persistence and long-term functionality of our CAR-NKs by introducing transgenic interleukin-15 (IL15). Methods: CD3-depleted PBMCs were first activated with lethally irradiated feeder cells, then transduced with transiently produced replication incompetent γ-retrovirus (αCD123.2B4.ζ, αCD123.2B4.ζ-IRES-sIL15, sIL15-IRES-mOrange) on day 4 of culture. CAR expression was measured on day 8 using FACS. Secretion of IL15 was verified with ELISA. Cytotoxicity was measured using ffLuc expressing target cells and bioluminescence (BL) measurement. In serial stimulation assays, target cells were repleted daily to maintain a 1:1 effector:target ratio. Immunophenotype and cell counts were assessed by FACS. Transcriptomic analysis (RNAseq) was performed on RNA derived from NK cells purified on D10. Xenograft modeling was performed using NSG mice engrafted with MV-4-11.ffLuc or MOLM-13.ffLuc AML cell lines. Mice were treated with NK cells on D4 or D4-7-10. Untreated mice served as controls. Tumor growth was serially tracked in vivo using BL imaging. NK cell persistence and expansion were measured in peripheral blood. Results: The 2B4.ζ CAR was well expressed on the surface of transduced NK cells (median transduction efficiency 95%, range 85-97%, n=3). 2B4.ζ CAR-NK treatment prolonged survival of AML engrafted mice when compared to treatment with unmodified NKs (median survival: 63 vs 55 days; n=8 mice; p=0.014). Serial peripheral blood analysis revealed a steady decline in circulating NK cells, which were undetectable in all cohorts within 21 days. NK cells were then engineered for constitutive secretion of IL15, with and without CAR expression. 2B4.ζ/sIL15 CAR-NKs had the most potent 24h-cytotoxicity against CD123+ targets (Fig. 1). After a 10-day chronic stimulation with MV-4-11, 2B4.ζ/sIL15- and sIL15-NKs expanded (x1.2 and x5.9 respectively), while NK cells without sIL15 decreased in number. In this assay, only 2B4.ζ/sIL15 CAR-NKs exhibited sustained tumor killing. Transcriptomic analysis after 10 days of serial stimulation showed sample clustering dependent on IL15 secretion. Differential gene expression analysis (DESeq2) identified upregulation of genes associated with cell cycle progression, apoptosis regulation, chemokine signaling, and NK cell mediated cytotoxicity in NK cells secreting IL15 compared to those without. In multiparameter flow cytometric analysis, 2B4.ζ/sIL15 CAR-NKs had a higher percentage of NK cells populating clusters defined by higher surface expression of NK cell activating receptors (NKp30, NKG2D, LFA-1) compared to 2B4.ζ and unmodified NK cells. In our MV-4-11 xenograft model, NKs armed with secreted IL15 expanded in vivo and had improved persistence. A single dose (D4) of 2B4.ζ/sIL15 CAR-NKs demonstrated an initial antitumor response, equivalent to that seen following 3 doses (D4-7-10) of 2B4.ζ CAR-NKs. However, mice treated with IL15-secreting NKs had short survival (Fig. 2). Compared to control mice, peripheral blood analysis showed increasing systemic hIL15 and higher levels of hTNFα. In our more aggressive MOLM-13 xenograft model, both single dose 2B4.ζ/sIL15 CAR-NK and multiple dose 2B4.ζ CAR-NK treatment prolonged survival compared to treatment with unmodified NKs. (27 and 26 vs 20 days; n=5 mice; p<0.01; Fig. 2). Conclusion: 2B4.ζ CAR-NKs have limited antitumor efficacy and short persistence in vivo. NK cells armored with secreted IL15 have enhanced anti-AML cytotoxicity and in vitro persistence. Introduction of IL15 secretion confers a distinctly activated phenotype that is maintained during chronic antigen stimulation. Constitutive local IL15 secretion improves in vivo NK cell persistence but may cause lethal toxicity when employed against AML. These results warrant further study and should impact the development of CAR-NK clinical products for patients with AML. Figure 1 Figure 1. Disclosures Ho: Rodeo Therapeutics/Amgen: Patents & Royalties; Exelixis: Consultancy; Sanofi: Research Funding. Bonifant: Kiadis Pharma: Research Funding; BMS: Research Funding; Merck, Sharpe, Dohme: Research Funding.

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Expansion of Dysfunctional CD56-Negative NK Cells Is a Hallmark of NK Cell Activation in Ph+ Leukemia Patients Treated with Dasatinib

Blood ◽

10.1182/blood.v128.22.1904.1904 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1904-1904

Author(s):

Ken-ichi Ishiyama ◽

Toshio Kitawaki ◽

Naoshi Sugimoto ◽

Akifumi Takaori-Kondo ◽

Norimitsu Kadowaki

Keyword(s):

Nk Cells ◽

Peripheral Blood ◽

Research Funding ◽

Cell Activation ◽

Nk Cell ◽

Pharmaceutical Research ◽

Chronic Viral Infection ◽

Cell Counts ◽

Cmv Reactivation ◽

Dasatinib Treatment

Abstract [Background] Tyrosine kinase inhibitors (TKIs), imatinib, nilotinib and dasatinib, are key drugs for the treatment of Philadelphia chromosome-positive (Ph+) leukemia. Dasatinib treatment markedly increases the number of large granular lymphocytes (LGLs) including NK cells in peripheral blood, which associates with a better prognosis. We previously showed that dasatinib expands CMV-associated highly differentiated NK cells in Ph+ leukemia patients through reactivation of CMV (Ishiyama, et al., Leukemia, 2016). NK cells consist of conventional CD56bright NK cells and CD56dim NK cells, and also rare CD56-negative (CD56neg) NK cells, which have been reported to increase in chronic viral infection such as HIV and HCV. Here, we show that CD56neg NK cells increase in CMV seropositive (CMV+) patients treated with dasatinib (DA), but not in those treated with other TKIs (OT). However, characteristics and clinical implications of CD56neg NK cells in CMV+DA patients remain unknown. Therefore, we sought to examine the phenotypic and functional properties of CD56neg NK cells in these patients. [Methods] We assessed NK cell subsets in 36 DA and 26 OT patients. NK cells were defined as lin-CD16+ or lin-CD56+ cells in peripheral blood. NK-cell marker expression and cytotoxic activity were compared between CD56neg NK cells and CD56dim NK cells in CMV+ DA patients with 5% or more of CD56neg NK cells. In vivo phosphorylation of intracellular signaling molecules in NK cells were evaluated by flow cytometry combined with phospho-specific antibodies (BD PhosflowTM). [Results] CD56neg NK cells exclusively increased in CMV+ DA group, compared to CMV- DA and CMV+ OT groups (median count: 20.9/μl, 1.1/μl, 1.5μl; p < 0.001. Median percentage: 4.5%, 0.8%, 0.4% of total NK cells; p < 0.001). CD56neg NK cell counts strongly correlated with total NK cell counts in CMV+ DA (r = 0.720, p < 0.001). CD56neg NK cells gradually increased over a period of one year after starting dasatinib. When we compared CD56neg NK cells and CD56dim NK cells in CMV+ DA, CD56neg NK cells showed decreased expression of activating NK cell markers including NKG2C, CD57, NKG2D, NKp30 and NKp46. In contrast, expression of PD-1 increased. CD56neg NK cells also showed lower rate of degranulation and IFN-γ production in functional assays. CMV+ DA patients with 4% or more of CD56neg NK cells significantly achieved deep molecular responses at 1 year after starting dasatinib than those with lower CD56neg NK cells (10/14, 3/14; p < 0.05). Phosflow analysis showed enhanced phosphorylation of ZAP70 in NK cells from CMV+ DA compared to CMV+ healthy controls (MFI/Isotype = 24.6 vs. 7.4, p < 0.01). [Discussion] CD56neg NK cells exclusively increased in CMV+ DA, and their increase persisted during dasatinib therapy. This suggests the similarity with the previous findings that CMV-associated differentiation in NK cells is enhanced during dasatinib therapy in CMV+ DA, which reflects the NK cell activation in response to CMV reactivation. In addition, CD56neg NK cells exhibited downregulation of activating receptors, upregulation of PD-1, and lower cytotoxic activity, indicating that these cells are the anergic and exhausted population as seen in chronic viral infection. These findings suggest that CD56neg NK cells accumulate owing to chronic stimulation by reactivated CMV during dasatinib therapy. ZAP70 is an immediate downstream tyrosine kinase of activating NK cell receptors, and is regulated by Src-family kinases, which is potentially inhibited by the kinase inhibitor property of dasatinib. Intriguingly, phosphorylation of ZAP70 in CD56dim NK cells was enhanced in CMV+ DA, although they had taken dasatinib a few hours before collecting blood samples. This finding suggests that strong activation of NK cells by CMV reactivation in CMV+ DA may overcome the direct suppressive effect of dasatinib on NK cell activation. [Conclusion] The accumulation of CD56neg NK cells is a consequence of NK cell activation specifically observed in dasatinib-treated patients. The NK cell activation is likely to be a response to the CMV reactivation induced by dasatinib treatment. Assessing CD56neg NK cells in peripheral blood could be a practical clinical indicator for the immunomodulatory effect of dasatinib, which significantly affects the prognosis. Disclosures Takaori-Kondo: Chugai Pharmaceutical: Research Funding; Astellas Pharma: Research Funding; Merck Sharp and Dohme: Speakers Bureau; Pfizer: Research Funding; Toyama Chemical: Research Funding; Takeda Pharmaceutical: Research Funding; Kyowa Kirin: Research Funding; Eisai: Research Funding; Cognano: Research Funding; Janssen Pharmaceuticals: Speakers Bureau; Shionogi: Research Funding; Mochida Pharmaceutical: Research Funding; Alexion Pharmaceuticals: Research Funding; Bristol-Myers Squibb: Speakers Bureau.

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The human cytomegalovirus protein UL147A downregulates the most prevalent MICA allele: MICA*008, to evade NK cell-mediated killing

10.1101/2020.07.17.208462 ◽

2020 ◽

Author(s):

Einat Seidel ◽

Liat Dassa ◽

Esther Oiknine-Djian ◽

Dana G. Wolf ◽

Vu Thuy Khanh Le-Trilling ◽

...

Keyword(s):

Nk Cells ◽

Human Cytomegalovirus ◽

Immune Evasion ◽

Cell Activation ◽

Nk Cell ◽

Cell Activity ◽

Asymptomatic Infection ◽

Target Cells ◽

Hcmv Disease ◽

Infected Cells

AbstractNatural killer (NK) cells are innate immune lymphocytes capable of killing target cells without prior sensitization. NK cell activity is regulated by signals received from activating and inhibitory receptors. One pivotal activating NK receptor is NKG2D, which binds a family of eight ligands, including the major histocompatibility complex (MHC) class I-related chain A (MICA). Human cytomegalovirus (HCMV) is a ubiquitous betaherpesvirus causing morbidity and mortality in immunosuppressed patients and congenitally infected infants. HCMV encodes multiple antagonists of NK cell activation, including many mechanisms targeting MICA. However, only one of these mechanisms counters the most prevalent MICA allele, MICA*008. Here, we discover that a hitherto uncharacterized HCMV protein, UL147A, specifically targets MICA*008 to proteasomal degradation, thus hindering the elimination of HCMV-infected cells by NK cells. Mechanistic analyses disclose that the non-canonical GPI anchoring pathway of immature MICA*008 constitutes the determinant of UL147A specificity for this MICA allele. These findings advance our understanding of the complex and rapidly evolving HCMV immune evasion mechanisms, which may facilitate the development of antiviral drugs and vaccines.Author SummaryHuman cytomegalovirus (HCMV) is a common pathogen that usually causes asymptomatic infection in the immunocompetent population, but the immunosuppressed and fetuses infected in utero suffer mortality and disability due to HCMV disease. Current HCMV treatments are limited and no vaccine has been approved, despite significant efforts. HCMV encodes many genes of unknown function, and virus-host interactions are only partially understood. Here, we discovered that a hitherto uncharacterized HCMV protein, UL147A, downregulates the expression of an activating immune ligand allele named MICA*008, thus hindering the elimination of HCMV-infected cells. Elucidating HCMV immune evasion mechanisms could aid in the development of novel HCMV treatments and vaccines. Furthermore, MICA*008 is a highly prevalent allele implicated in cancer immune evasion, autoimmunity and graft rejection. In this work we have shown that UL147A interferes with MICA*008’s poorly understood, nonstandard maturation pathway. Study of UL147A may enable manipulation of its expression as a therapeutic measure against HCMV.

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PD-1 Is Expressed at Low Levels on All Peripheral Blood Natural Killer Cells but Is a Significant Suppressor of NK Function Against PD-1 Ligand Expressing Tumor Targets

Blood ◽

10.1182/blood-2019-127261 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 621-621

Author(s):

Zachary Davis ◽

Martin Felices ◽

Todd R Lenvik ◽

Sujan Badal ◽

Peter Hinderlie ◽

...

Keyword(s):

T Cells ◽

Nk Cells ◽

Board Of Directors ◽

Peripheral Blood ◽

Research Funding ◽

Cytokine Production ◽

Nk Cell ◽

Fold Increase ◽

Advisory Committees ◽

Fund Research

Checkpoint blockade has become a promising immunotherapy for the treatment of a variety of malignancies. In particular, the receptor programmed death-1 (PD-1) has become a focus of intense study due to its expression on and negative regulation of T-cell function. The ligand for PD-1, PD-L1, is upregulated on many tumors and, as a result, can suppress antigen-specific T-cells thereby limiting their anti-tumor response. Pharmacological PD-1/PD-L1 axis disruption can occur with either Pembrolizumab and Nivolumab (PD-1 antagonists) and Avelumab and Atezolizumab (PD-L1 antagonists). These antibodies (mAbs) are being used to treat melanoma, non-small cell lung cancer, kidney, bladder and head and neck cancer with varying degrees of success. Like T-cells, natural killer cells (NK) also have potent antitumor cytolytic properties. The expression and functional effects of PD-1 on NK cells remain unclear due to difficulties in receptor detection and efficacy of receptor blockade by available commercial reagents. While some studies have been unable to detect PD-1 on resting NK cells, others have identified PD-1 expression only on specific NK populations under certain conditions (e.g. Cytokine stimulation or virus infection). Here, we identify PD-1 expression on peripheral blood NK cells. Using commercial reagents (Figure 1A) and a FITC-labeled clinical mAb (Pembrolizumab, Pembro), we detect low yet consistent PD-1 expression on all circulating, resting NK cells. Since FITC-Pembro mean fluorescent intensity was low and a high proportion of FITC labeled NK cells overlapped with the isotype control (Figure 1B), we designed a short-chain variable fragment (scFv) of the mAb to determine whether the smaller scFv molecule has better binding and functional activity than the intact mAb. The Pembro scFv bound to resting NK cells with a distinct fluorescent peak compared to the native Prembro from which the scFv was derived (Figure 1B). Compared to intact Prembro, use of the Pembro scFv as a PD-1 antagonist resulted in a 2-fold increase of NK cell cytolytic activity and a 3-4 fold increase in cytokine production against the PD-L1 expressing CML target, K562 (Figure 1C-D) and the AML target, THP-1 (Figure 1E-F). While PD-1 blockade enhanced NK cell degranulation and target cell killing, a greater functional enhancement was seen for interferon-γ production. PD-1 signaling inhibits PI3K induced pAkt and NK function. PD-1/PD-1 ligand blockade by the Pembro scFv resulted in increased NK cell pAKT in the presence of PD-L1 and NK activating NKG2D-ligand-expressing THP-1 cells. In addition to natural cytotoxicity, NK-mediated ADCC was also enhanced with PD-1 blockade. CD33 mAb immunoconjugates have been used to treat AML. Combined anti-CD33 mAb and PD-1 blockade against THP-1 cells resulted in a small but significant increase in NK cell degranulation and a 4-fold increase in cytokine production compared to anti-CD33 mAb without PD-1 blockade (Figure 1G-H). Since stimulation with IL-15, a cytokine that effectively lowers the NK activation threshold, abrogated the benefits of Pembro scFv in diminishing PD-1 inhibitory effects on NK cells, PD-1 control of NK function appears limited to be mostly relevant to resting NK cells. To understand the physiologic expression of PD-1 in vivo, we studied samples taken from AML patients receiving matched sibling donor transplantation at the University of Minnesota. Increased PD-1 on reconstituting NK cells in BMT recipients up to day 100 post-transplant was shown by both flow-cytometric (Figure 2A) and mass-cytometric (CyTOF) analyses (Figure 2B). Blockade of PD-1 on these cells significantly enhanced both NK degranulation (Figure 2C) and cytokine production (Figure 2D) against K562 targets. A similar increase in NK function was observed with PD-1 blockade in AML patients receiving umbilical cord transplants (not shown). These data indicate that PD-1 is present on human NK cells and PD-1 ligation negatively regulates NK function against PD-L1 expressing tumor targets. The observation that functional PD-1 is expressed on NK cells under resting conditions strongly suggests that the use of a PD-1 antagonist, in combination with NK cell therapy, should be clinically effective for treatment of cancer. Disclosures Felices: GT Biopharma.: Other: consulting funds, Research Funding. Blazar:Kamon Pharmaceuticals, Inc: Membership on an entity's Board of Directors or advisory committees; Tmunity: Other: Co-Founder; BlueRock Therapeutics: Membership on an entity's Board of Directors or advisory committees; Regeneron Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Five Prime Therapeutics Inc: Co-Founder, Membership on an entity's Board of Directors or advisory committees; KidsFirst Fund: Research Funding; Childrens' Cancer Research Fund: Research Funding; Leukemia and Lymphoma Society: Research Funding; Abbvie Inc: Research Funding; Alpine Immune Sciences, Inc.: Research Funding; RXi Pharmaceuticals: Research Funding; Fate Therapeutics, Inc.: Research Funding; Magenta Therapeutics and BlueRock Therapeuetics: Membership on an entity's Board of Directors or advisory committees. Vallera:GT Biopharma, Inc.: Consultancy, Research Funding. Miller:Fate Therapeutics, Inc: Consultancy, Research Funding; GT BioPharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; CytoSen: Membership on an entity's Board of Directors or advisory committees; OnKImmune: Membership on an entity's Board of Directors or advisory committees; Dr. Reddys Laboratory: Membership on an entity's Board of Directors or advisory committees; Moderna: Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: Keytruda. PD-1 blockade on NK cells for tumor immunotherapy

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Optimizing the Procedure to Manufacture Clinical-Grade NK Cells for Adoptive Immunotherapy

Cancers ◽

10.3390/cancers13030577 ◽

2021 ◽

Vol 13 (3) ◽

pp. 577

Author(s):

Adrián Fernández ◽

Alfonso Navarro-Zapata ◽

Adela Escudero ◽

Nerea Matamala ◽

Beatriz Ruz-Caracuel ◽

...

Keyword(s):

Nk Cells ◽

Cell Activation ◽

Mononuclear Cells ◽

Nk Cell ◽

Fold Increase ◽

Clinical Grade ◽

Transcriptome Profile ◽

Expansion Process ◽

Peripheral Blood Mononuclear ◽

Activation Status

Natural killer (NK) cells represent promising tools for cancer immunotherapy. We report the optimization of an NK cell activation–expansion process and its validation on clinical-scale. Methods: RPMI-1640, stem cell growth medium (SCGM), NK MACS and TexMACS were used as culture mediums. Activated and expanded NK cells (NKAE) were obtained by coculturing total peripheral blood mononuclear cells (PBMC) or CD45RA+ cells with irradiated K562mbIL15-41BBL or K562mbIL21-41BBL. Fold increase, NK cell purity, activation status, cytotoxicity and transcriptome profile were analyzed. Clinical-grade NKAE cells were manufactured in CliniMACS Prodigy. Results: NK MACS and TexMACs achieved the highest NK cell purity and lowest T cell contamination. Obtaining NKAE cells from CD45RA+ cells was feasible although PBMC yielded higher total cell numbers and NK cell purity than CD45RA+ cells. The highest fold expansion and NK purity were achieved by using PBMC and K562mbIL21-41BBL cells. However, no differences in activation and cytotoxicity were found when using either NK cell source or activating cell line. Transcriptome profile showed to be different between basal NK cells and NKAE cells expanded with K562mbIL21-41BBL or K562mbIL15-41BBL. Clinical-grade manufactured NKAE cells complied with the specifications from the Spanish Regulatory Agency. Conclusions: GMP-grade NK cells for clinical use can be obtained by using different starting cells and aAPC.

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miR-1224 contributes to ischemic stroke-mediated natural killer cell dysfunction by targeting Sp1 signaling

Journal of Neuroinflammation ◽

10.1186/s12974-021-02181-4 ◽

2021 ◽

Vol 18 (1) ◽

Author(s):

Yan Feng ◽

Yan Li ◽

Ying Zhang ◽

Bo-Hao Zhang ◽

Hui Zhao ◽

...

Keyword(s):

Ischemic Stroke ◽

Nk Cells ◽

Natural Killer ◽

Brain Ischemia ◽

Cell Activation ◽

Nk Cell ◽

Cell Activity ◽

Passive Transfer ◽

Immune Defense ◽

Nk Cell Activity

Abstract Background Brain ischemia compromises natural killer (NK) cell-mediated immune defenses by acting on neurogenic and intracellular pathways. Less is known about the posttranscriptional mechanisms that regulate NK cell activation and cytotoxicity after ischemic stroke. Methods Using a NanoString nCounter® miRNA array panel, we explored the microRNA (miRNA) profile of splenic NK cells in mice subjected to middle cerebral artery occlusion. Differential gene expression and function/pathway analysis were applied to investigate the main functions of predicted miRNA target genes. miR-1224 inhibitor/mimics transfection and passive transfer of NK cells were performed to confirm the impact of miR-1224 in NK cells after brain ischemia. Results We observed striking dysregulation of several miRNAs in response to ischemia. Among those miRNAs, miR-1224 markedly increased 3 days after ischemic stroke. Transfection of miR-1224 mimics into NK cells resulted in suppression of NK cell activity, while an miR-1224 inhibitor enhanced NK cell activity and cytotoxicity, especially in the periphery. Passive transfer of NK cells treated with an miR-1224 inhibitor prevented the accumulation of a bacterial burden in the lungs after ischemic stroke, suggesting an enhanced immune defense of NK cells. The transcription factor Sp1, which controls cytokine/chemokine release by NK cells at the transcriptional level, is a predicted target of miR-1224. The inhibitory effect of miR-1224 on NK cell activity was blocked in Sp1 knockout mice. Conclusions These findings indicate that miR-1224 may serve as a negative regulator of NK cell activation in an Sp1-dependent manner; this mechanism may be a novel target to prevent poststroke infection specifically in the periphery and preserve immune defense in the brain.

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151 Potentiating the Large-Scale Expansion and Engineering of Peripheral Blood-Derived CAR NK Cells for Off-the-Shelf Application

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.151 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A159-A159

Author(s):

Michael Whang ◽

Ming-Hong Xie ◽

Kate Jamboretz ◽

Hadia Lemar ◽

Chao Guo ◽

...

Keyword(s):

T Cells ◽

Cell Therapy ◽

Nk Cells ◽

Peripheral Blood ◽

Nk Cell ◽

Adoptive Cell Therapy ◽

Therapeutic Window ◽

Target Cells ◽

Healthy Donors ◽

Custom Made

BackgroundPeripheral blood natural killer (NK) cells are mature cytotoxic innate lymphocytes possessing an inherent capacity for tumor cell killing, thus making them attractive candidates for adoptive cell therapy. These NK cells are also amenable to CRISPR and chimeric antigen receptor (CAR) genomic engineering for enhanced functions. Moreover, NK cells possess an inherent capacity for off-the-shelf therapy since they are not known to cause graft-versus-host disease, unlike T cells. Presently, approved CAR cell therapy is custom-made from each patient‘s own T cells, a process that can limit patient pool, narrow therapeutic window, and contribute to product variability. In this study, we investigate whether peripheral blood NK cells from a selected donor can be edited, engineered, and expanded sufficiently for off-the-shelf use in a wide patient population.MethodsUsing the CRISPR/Cas9 system, we knocked out CISH expression in isolated peripheral blood NK cells from 3 healthy donors. Subsequently, we expanded edited NK cells by using IL-2 and sequential stimulations using NKSTIM, a modified K562 stimulatory cell line expressing membrane-bound form of IL-15 (mbIL-15) and 4-1BBL. IL-12 and IL-18 were added twice during expansion to drive memory-like NK cell differentiation. We transduced the expanded NK cells to express engineered CD19-targeted CAR and mbIL-15 during an interval between the first and second NKSTIM pulses. We assessed NK cell cytotoxicity against Nalm6 target cells by IncuCyte.ResultsIsolated peripheral blood NK cells from 3 healthy donors were successfully edited using CRISPR/Cas9, engineered to express high levels of CAR, extensively expanded using a series of NKSTIM pulses in the presence of IL-2, and differentiated into memory-like NK cells using IL-12 and IL-18. Interestingly, NK cells from the 3 donors exhibited distinct outcomes. NK cells from one donor reached a peak expansion limit of approximately 7-million-fold before undergoing contraction whereas NK cells from two donors continued to expand over the length of the study surpassing 100-million-fold expansion, without appearing to have reached a terminal expansion limit. At the end of the study, NK cells from one donor exceeded 1-billion-fold expansion and maintained 88% cytolytic activity compared to Nkarta’s standard process control in a 72-hour IncuCyte assay.ConclusionsIn this study, we demonstrate that healthy donor-derived peripheral blood NK cells are capable of expanding over billion-fold while maintaining potency. These results provide a rationale for the development of off-the-shelf CAR NK cell therapies using NK cells from donors selected to provide optimal product characteristics.Ethics ApprovalHuman samples were collected with written informed consent by an approved vendor.

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Adrenal tumors provide insight into the role of cortisol in NK cell activity

Endocrine Related Cancer ◽

10.1530/erc-21-0048 ◽

2021 ◽

Author(s):

Andrew E. Greenstein ◽

Mouhammed Amir Habra ◽

Subhagya A. Wadekar ◽

Andreas Grauer

Keyword(s):

Immune Response ◽

Nk Cells ◽

Cell Activation ◽

Nk Cell ◽

Phase 1 ◽

Cell Activity ◽

The Cancer Genome Atlas ◽

Adrenal Tumors ◽

Steroid Synthesis

Elevated glucocorticoid (GC) activity may limit tumor immune response and immune checkpoint inhibitor (ICI) efficacy. Adrenocortical carcinoma (ACC) provides a unique test case to assess correlates of GC activity, as approximately half of ACC patients exhibit excess GC production (GC+). ACC multi-omics were analyzed to identify molecular consequences of GC+ and assess the rationale for combining the glucocorticoid receptor (GR) antagonist relacorilant with an ICI. GC status, mRNA expression, and DNA mutation and methylation data from 71 adrenal tumors were accessed via The Cancer Genome Atlas. Expression of 858 genes differed significantly between GC- and GC+ ACC cases. KEGG pathway analysis showed higher gene expression of 3 pathways involved in steroid synthesis and secretion in GC+ cases. Fifteen pathways, most related to NK cells and other immune activity, showed lower expression. Hypomethylation was primarily observed in the steroid synthesis pathways. Tumor-infiltrating CD4+ memory (P=.003), CD8+ memory (P=.001), and NKT-cells (P=.014) were depleted in GC+ cases; tumor-associated neutrophils were enriched (P=.001). Given the pronounced differences between GC+ and GC- ACC, the effects of cortisol on NK cells were assessed in vitro (NK cells from human PBMCs stimulated with IL-2 or IL-12/15). Cortisol suppressed, and relacorilant restored, NK cell activation, proliferation, and direct tumor cell killing. Thus, GR antagonism may increase the abundance and function of NK and other immune cells in the tumor microenvironment, promoting immune response in GC+ ACC and other malignancies with GC+. This hypothesis will be tested in a phase 1 trial of relacorilant + ICI.

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