In VivoActivation of Human NK Cells by Treatment with an Interleukin-15 Superagonist Potently Inhibits AcuteIn VivoHIV-1 Infection in Humanized Mice

ABSTRACTNatural killer (NK) cells with anti-HIV-1 activity may inhibit HIV-1 replication and dissemination during acute HIV-1 infection. We hypothesized that the capacity of NK cells to suppress acutein vivoHIV-1 infection would be augmented by activating them via treatment with an interleukin-15 (IL-15) superagonist, IL-15 bound to soluble IL-15Rα, an approach that potentiates human NK cell-mediated killing of tumor cells.In vitrostimulation of human NK cells with a recombinant IL-15 superagonist significantly induced their expression of the cytotoxic effector molecules granzyme B and perforin; their degranulation upon exposure to K562 cells, as indicated by cell surface expression of CD107a; and their capacity to lyse K562 cells and HIV-1-infected T cells. The impact of IL-15 superagonist-induced activation of human NK cells on acutein vivoHIV-1 infection was investigated by using hu-spl-PBMC-NSG mice, NOD-SCID-IL2rγ−/−(NSG) mice intrasplenically injected with human peripheral blood mononuclear cells (PBMCs) which develop productivein vivoinfection after intrasplenic inoculation with HIV-1. IL-15 superagonist treatment potently inhibited acute HIV-1 infection in hu-spl-PBMC-NSG mice even when delayed until 3 days after intrasplenic HIV-1 inoculation. Removal of NK cells from human PBMCs prior to intrasplenic injection into NSG mice completely abrogated IL-15 superagonist-mediated suppression ofin vivoHIV-1 infection. Thus, thein vivoactivation of NK cells, integral mediators of the innate immune response, by treatment with an IL-15 superagonist increases their anti-HIV activity and enables them to potently suppress acutein vivoHIV-1 infection. These results indicate thatin vivoactivation of NK cells may represent a new immunotherapeutic approach to suppress acute HIV-1 infection.IMPORTANCEEpidemiological studies have indicated that NK cells contribute to the control of HIV-1 infection, andin vitrostudies have demonstrated that NK cells can selectively kill HIV-1-infected cells. We demonstrated thatin vivoactivation of NK cells by treatment with an IL-15 superagonist that potently stimulates the antitumor activity of NK cells markedly inhibited acute HIV-1 infection in humanized mice, even when activation of NK cells by IL-15 superagonist treatment is delayed until 3 days after HIV-1 inoculation. NK cell depletion from PBMCs prior to their intrasplenic injection abrogated the suppression ofin vivoHIV-1 infection observed in humanized mice treated with the IL-15 superagonist, demonstrating that activated human NK cells were mediating IL-15 superagonist-induced inhibition of acute HIV-1 infection. Thus,in vivoimmunostimulation of NK cells, a promising therapeutic approach for cancer therapy, may represent a new treatment modality for HIV-1-infected individuals, particularly in the earliest stages of infection.

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Optimizing Lentiviral Transduction of Human Natural Killer Cells

Blood ◽

10.1182/blood.v118.21.4714.4714 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4714-4714 ◽

Cited By ~ 2

Author(s):

Su Su ◽

Dawn M Betters ◽

Muthalagu Ramanathan ◽

Keyvan Keyvanfar ◽

Aleah Smith ◽

...

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Cell Line ◽

Nk Cell ◽

K562 Cells ◽

Cell Phenotype ◽

Human Renal Cell Carcinoma ◽

Hiv 1

Abstract Abstract 4714 The development of an efficient method to genetically modify natural killer (NK) cells could be used to characterize NK cell differentiation, acquisition of self-tolerance, tumor trafficking in vivo, as well as to manipulate NK cells to enhance their activity against infectious diseases and tumors. Although HIV-1 based lentiviral vectors (LVs) have been used to efficiently transfer genes into human T-cells, little data exists on LV transduction of either fresh or in vitro expanded human NK cells or its effects on NK cell phenotype and cytolytic function. In this study, we used an HIV-based LV expressing enhanced green fluorescence protein (EGFP) driven by a murine stem cell virus long terminal repeat (MSCV-LTR) promoter to transduce CD3− and CD56+ and/or CD16+ human NK cells that were either resting, IL-2 activated, or expanded in vitro using an irradiated EBV-LCL feeder cell line. We observed that resting NK cells were difficult to transduce with LVs, even at high multiplicities of infection (MOI), with transduction efficiencies (TE) in the range of only 3–14%. The efficiency of LV transduction improved when the NK cells were pre-stimulated in vitro with IL-2: TE improved to 21±0.2% in NK cells cultured for 24 hours in media containing IL-2 (200 U/mL) and 28.7±12.9% in NK cells that underwent in vitro expansion over 9 days prior to transduction using irradiated EBV-LCL feeder cells and media containing IL-2 (200U/mL). Subsequently, we evaluated incremental MOIs (3-200) to optimize LV transduction of expanded NK cells; optimal transduction was achieved using a spinoculation protocol at a MOI of 25 which resulted in the highest transduction efficiencies with the least amount of cell death. Increasing the MOI above this level resulted in a small increase in transduction, but was offset by an increase in NK cell apoptosis/death. Using a one-round, non-spinoculation protocol and an MOI of 30, we obtained a median transduction efficiency of 29% (range 16–41) with excellent retention of NK cell viability. This optimized protocol was used to transduce expanded NK cells with a LV vector encoding an shRNA targeting a region of the NK cell inhibitory receptor transcript NKG2A. Following transduction, surface expression of NKG2A decreased significantly on expanded NK cells compared to non-transduced expanded NK cells and “scramble transduced” LV controls; at a MOI of 10, the MFI of NKG2A on expanded human NK cells decreased 35% compared to non-transduced and LV transduced scramble controls (median MFI 428, 673, 659 in shRNA, non-transduced and scramble LV control transduced NK cells respectively). A comparison of transduction efficiencies using LVs expressing EGFP driven by MSCV-LTR, EF1a, and Ubi promoters showed MSCV-LTR mediated the highest level of gene expression in expanded NK cells. Transduced NK cells maintained stable EGFP transgene expression in vitro, which peaked 5 days following LV transduction and remained stable for an additional 9 days. The phenotype of lentiviral transduced NK cells was similar to non-transduced NK cells. Specifically, expression of CD56, CD16, granzyme A and B, perforin, the inhibitory receptors NKG2A, KIR3DL1, KIR3DL2, and KIR2DL1/DL2, and the activating receptors NKG2D, NCRs NKp46, and NKp30 were not altered in either fresh or expanded NK cells following LV transduction, although we did observe a significant reduction in NKp44 expression in LV transduced cells (22% compared to 50% on untransduced NK cells; 0.02). Furthermore, NK cell function, as assessed by cytokine production and cytotoxicity vs tumor targets was not altered in LV transduced NK cells. A 51Cr release cytotoxicity assay showed GFP+ NK cells, flow sorted following LV transduction of expanded NK cells, had similar cytotoxicity against K562 cells and human renal cell carcinoma cells (RCC) compared to non-transduced expanded NK cell controls (figures). In conclusion, we show that an HIV-1 based lentiviral vector driven by a MSCV-LTR, mediated efficient and stable gene transfer in IL-2 activated and in vitro expanded human NK cells. This study provides valuable insights for methods to optimize the long-term expression of LV transduced genes in human NK cells which could be used to improve their anti-tumor function in vivo. Target: K562 cells Target: RCC cell line Disclosures: No relevant conflicts of interest to declare.

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Assessment of antitumor function of NK cells expanded with exosomes from K562.mb21 cells.

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.7_suppl.132 ◽

2017 ◽

Vol 35 (7_suppl) ◽

pp. 132-132 ◽

Cited By ~ 1

Author(s):

Jeremiah Oyer ◽

Sarah B. Gitto ◽

Sara Khederzadeh ◽

Kari Shaver ◽

Dean A. Lee ◽

...

Keyword(s):

Nk Cells ◽

Cell Expansion ◽

Nk Cell ◽

Ex Vivo ◽

K562 Cells ◽

In Vitro Cytotoxicity ◽

Feeder Cell ◽

Nsg Mice

132 Background: NK cells can kill malignant cells to provide innate immunity against tumors. Due to their low abundance in blood, a focus is to expand NK cells ex vivo having enhanced anti-tumor cytotoxicity to be used as a treatment. Our group has pioneered a cell-free method using plasma membrane (PM) particles derived from K562 cells expressing 41BBL and membrane-bound IL-21 (K562.mb21) which were developed for NK cell expansion. Compared to feeder cell based methods for NK cell expansion, PM21-particles improve safety and allow for potential wide-spread dissemination, and also allows direct in vivo use. Exosomes, vesicles naturally secreted by cells, may yet be another novel feeder cell free way for NK cell expansion and may have further advantageous therapeutic dimensions. Methods: EX21-exosomes and PM21-particles were prepared from K562.mb21 cells and characterized by Nanosight and Western blot analysis. CD3-depleted PBMCs were cultured with EX21 for 14 days, NK cell amounts were monitored and media changed every 2-3 days. In vitro cytotoxicity against K562 cells were comparatively assessed for EX21-NK cells and PM21-NK cells. In vivo anti-tumor efficacy of EX21- and PM21-NK cells was assessed in NSG mice implanted ip with SKOV3_luc ovarian tumor cells (1 x 106 cells seeded for 4 days). SKOV3-bearing mice were treated with vehicle, or two doses of EX21-NK cells or PM21-NK cells (1 x 107, in 5 day intervals), and with or without in vivo administration of EX21 (10 µg, 3x/week) or PM21-particles (600 µg, 3x/week). All groups were injected ip with IL-2 (10 KU, 3x/week). Survival analysis was performed with a Log-rank (Mantel-Cox) test. Results: NK cells cultured with EX21 expanded 530 fold (344-710) over 14 days compared to 735 fold (667-802) in presence of PM21-particles. Treatment of SKOV3 engrafted NSG mice with NK cells, expanded with either EX21 or with PM21, allowed significant ( < 0.0001) increase in survival compared to untreated animals (41-44 vs 29 days post treatment). Ip delivery of EX21 to SKOV3 bearing mice had no effect on survival in either untreated control or EX21-NK cell treated groups. Conclusions: EX21 efficiently expands NK cells and EX21-NK cells have equal anti-tumor effect as PM21-NK cells, both in vitro and in vivo.

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In Vitro Preactivation of PBMCs with PM-mb21-41BBL Particles Stimulates in Vivo Expansion of NK Cells Under Low IL-2 in NSG Mice

Blood ◽

10.1182/blood.v124.21.3842.3842 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3842-3842

Author(s):

Jeremiah L Oyer ◽

Veethika Pandey ◽

Robert Y Igarashi ◽

Dominic A Colosimo ◽

Melhem M. Solh ◽

...

Keyword(s):

Cancer Treatment ◽

Nk Cells ◽

Peripheral Blood ◽

Cell Expansion ◽

Nk Cell ◽

K562 Cells ◽

High Dose ◽

Nsg Mice

Abstract NK cell immunotherapy as a cancer treatment shows promise, but methods for consistent expansion of NK cells from a small fraction (~5%) of peripheral blood mononuclear cells (PBMCs) to therapeutically effective amounts are not widely accessible. Strategies that promote in vivo expansion rely on high-dose IL-2 in combination with lymphodepletion and suffer from severe treatment related toxicities as well as limited expansion. On the other hand, in vitro methods are available for robust NK cell expansion, but rely on prolonged in vitro co-culture with feeder cells that is costly and complex and requires high dose IL-2 (25,000-35,000 U) for persistence after injection into mice. An optimal NK cell expansion method would rely on off the shelf reagents, would not require long-term complex cultures, and would induce a rapid, sustained in vivo expansion of NK cells from unsorted PBMCs under low concentration of IL-2. We have developed a method to prepare particles from K562 cells engineered to express specific cytokines, and such particles stimulate NK cell expansion. Particles prepared using K562 cells expressing IL-21 and 41BBL (PM21 particles) cause highly specific expansion of cytotoxic NK cells from unsorted PBMCs that achieves 95% NK cells in ~14 days and ~10,000 fold expansion of NK in about 21 days. NK cell expansion is consistent using different preparations of PM-particles or leukocyte sources, and the PM-particles retain expansion efficacy with storage. The PM-mb21-41BBL particles were also used to stimulate in vivo NK cell expansion in NSG mice under ultralow IL-2 (1,000 units per injection, 3 injections per week, per mouse). Unsorted human PBMCs, either shortly pre-activated in culture for two days or not pre-activated, were injected (2 x 106 PBMCs per mice by i.p.) and human CD56+CD3- NK cells and other relevant hCD45+ lymphocytes were monitored in the peripheral blood over time and in tissues and fluids at the time of euthanization. In vivo NK cell expansion was observed in mice injected with PBMCs that were pre-activated with PM21 particles. The extent of NK cell expansion observed in the peripheral blood was in levels that would be relevant for clinical cancer treatment (>50,000 NK cells/mL of mouse blood 14 days post injection). In vivo NK cell expansion was further confirmed with analogous experiments using PBMCs that were stained with CellTrace Violet to monitor proliferation. Notably, NK cells were also found in spleen, bone marrow, lung, liver and brain. These results taken together provide proof of principle that PM-particle technology is effective for direct in vivo NK cell expansion from unsorted PBMCs under low dose IL-2. Disclosures No relevant conflicts of interest to declare.

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Genetically Reengineered K562 Cells Significantly Expand Cord Blood (CB) Natural Killer (NK) Cells Ex-Vivo Expanded with Increased NK Cell Activation and Cytolytic Activity Both In-Vitro and In-Vivo: Potential Use In Adoptive Cellular Immunotherapy (ACI)

Blood ◽

10.1182/blood.v116.21.3928.3928 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3928-3928

Author(s):

Michele Levin ◽

Janet Ayello ◽

Frances Zhao ◽

Andrew Stier ◽

Lauren Tiffen ◽

...

Keyword(s):

Nk Cells ◽

Nk Cell ◽

Ex Vivo ◽

Granzyme B ◽

K562 Cells ◽

Cytolytic Activity ◽

Scid Mice ◽

Activation Marker

Abstract Abstract 3928 Background: NK cells play a role in reducing relapse in hematological malignancy following AlloSCT (Dunbar et al, Haematologica, 2008). NK cell limitations include lack of tumor recognition and/or limited numbers of viable and functional NK cells (Shereck/Cairo et al, Ped Bld Can, 2007). NK ACI provide safe and effective therapy against tumor relapse; yet NK cells are limited to specific cancer types and not all patients demonstrate optimal response (Ruggieri et al. Science, 2002; Ljunggren et al. Nat Rev Immuno, 2007). To circumvent these limitations, methods to expand and activate PBMNCs with genetically engineered K562 cells expressing membrane bound IL-15 and 41BB ligand (K562-mbIL15-41BBL [modK562]; Imai/Campana et al, Blood, 2005) have shown to significantly increase NK cells in number and maintain heterogeneous KIR expression (Fusaki/Campana et al BJH, 2009). We have shown that CB NK cells can be activated/expanded and exhibit enhanced cytolytic activity when cultured in a cytokines/antibody cocktail (Ayello/Cairo et al, BBMT, 2006; Exp Heme, 2009). Objective: To evaluate CBNK expansion, activation, cytolytic mechanism and function against Burkitt lymphoma (BL) tumor target and its influence on NK cell mediated in-vitro and in-vivo cytotoxicity in NOD-SCID mice following stimulation with modK562 cells (generously supplied by D.Campana, St Jude's Children's Hospital, Memphis, Tx). Methods: Following 100GY irradiation, modK562cells were incubated 1:1 with CBMNCs in RPMI+IL-2 (10IU/ml) for 7 days in 5%CO2, 37°C. NK activation marker (LAMP-1), perforin and granzyme B were determined by flow cytometry. Cytotoxicty was determined via europium assay at 20:1 E:T ratio with Ramos (BL) tumor targets (ATCC). The mammalian expression construct (ffLucZeo-pcDNA (generously supplied by L.Cooper, MD, PhD) was transfected to BL cells using lipofectin and selected by zeocin for stable transfection. Six week old NOD-SCID mice received 5×106 BL cells subcutaneously. Upon engraftment, xenografted NOD-SCID mice were divided in 5 groups: injected with PBS (control), BL only, 5×106 wildtype (WT) K562 expanded (E) CBNK cells, modK562 expanded (E) CB NK cells (5×106) and modK562 expanded (E) CBNK cells (5×107). Ex-vivo ECBNK cells were injected weekly for 5 weeks and xenografted NOD-SCID mice were monitored by volumetric measurement of tumor size (Tomayko/Reynolds, Can Chemother Pharmac, 1989), bioluminescent imaging (Inoue et al Exp Heme, 2007) and survival. The survival distribution for each group was estimated using the Fisher exact test. Results: On Day 0, NK cells (CD56+/3-) population was 3.9±1.3%. After 7 days, modK562 expanded CBNK cells was significantly increased compared to WTK562 and media alone (72±3.9 vs 43±5.9 vs 9±2.4%, p<0.01). This represented a 35-fold or 3374±385% increase of the input NK cell number. This was significantly increased compared to WTK562 (1771±300%, p<0.05). ModK562 ECBNK cells demonstrated increased perforin and granzyme B expression compared to WTK562 (42±1.5 vs 15±0.5%,p<0.001; 22±0.5 vs 11±0.3%,p<0.001, respectively). Cytotoxicity was against BL tumor targets was significantly increased (42±3 vs 18±2%,p<0.01), along with NK activation marker expression, CD107a (p<0.05). At 5 weeks, in-vivo studies demonstrated increased survival of NOD-SCID mice receiving both 5×106 and 5×107 modK562 ECBNK cells when compared to those with no treatment (p=0.05, p=0.0007, respectively). There was no difference in survival when comparing mice that received 5×106 vs 5×107 modK562 ECBNK cells (p=0.0894) at 5 weeks. Tumor volume of mice receiving either dose of modK562 ECBNK cells was significantly less than those receiving WTK562 ECBNK cells (1.92±0.57 and 0.37±0.05 vs 3.41±0.25, p=0.0096 and p=0.0001, respectively). Conclusions: CBMNCs stimulated and expanded with modK562 cells results in significant expansion of CBNK cells with enhanced in-vitro cytotoxicity, significant receptor expression of NK activation marker (LAMP-1), and perforin and granzyme B. Furthermore, modK562 ECBNK cells leads to increased survival and lower tumor burden of NOD-SCID mice xenografted with BL. Future directions include modK562 ECBNK cells to be genetically modified to express chimeric antigen receptor CD20 (MSCV-antiCD20-41BB-CD3 ζ) against CD20+ hematologic malignancies for future studies to evaluate whether targeting enhances in-vitro and in-vivo cytotoxicity. Disclosures: No relevant conflicts of interest to declare.

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Human Cytokine-Induced Memory-like NK Cells Exhibit in Vivo Anti-Leukemia Activity in Xenografted NSG Mice and in Patients with Acute Myeloid Leukemia (AML)

Blood ◽

10.1182/blood.v126.23.101.101 ◽

2015 ◽

Vol 126 (23) ◽

pp. 101-101

Author(s):

Rizwan Romee ◽

Rosario Maximillian ◽

Melissa M Berrien-Elliott ◽

Julia A Wagner ◽

Brea A Jewell ◽

...

Keyword(s):

Nk Cells ◽

Myeloid Leukemia ◽

Low Dose ◽

Nk Cell ◽

Leukemia Cells ◽

Cell Frequency ◽

Nsg Mice ◽

Post Infusion

Abstract Natural killer (NK) cells mediate anti-AML responses and previously published clinical trials of adoptive allogeneic NK cell therapy provide proof-of-principle that NK cells may eliminate leukemia cells in patients. However, complete remissions occur in 30-50% of patients with active AML and are typically of limited duration. Thus, improvements are needed for this promising cellular immunotherapy strategy. Following paradigm-shifting studies in mice, it was established that human NK cells exhibit an innate 'memory-like' responses following a brief, combined pre-activation with IL-12, -15, and -18 (Romee R et. al., Blood, 2012). These long-lived memory-like NK cells have an enhanced ability to produce IFN-g in response to restimulation with cytokines or activating receptor ligation, even following extensive proliferation. We hypothesized that memory-like NK cells exhibit enhanced responses to myeloid leukemia. Compared to control NK cells from the same donor, IL-12/15/18-induced memory-like NK cells produced significantly increased IFN-g upon co-culture with primary AML blasts in vitro (P<0.001), following 7 days of rest in low dose IL-15 vitro. In addition, memory-like NK cells had increased granzyme B expression (P<0.01), and enhanced killing of K562 leukemia targets in vitro (P<0.05). Utilizing an in vivo xenograft model of human NK cells in NSG mice (Leong J et. al., BBMT, 2014), IL-12/15/18-induced memory-like NK cells that differentiated in NSG mice for 7 days exhibited increased IFN-g responses after ex vivo re-stimulation with K562 leukemia, confirming their memory-like functionality (P<0.05). To test in vivo responses to human leukemia in this model, luciferase-expressing K562 cells were engrafted into NSG mice (1x106/mouse, IV), and on day 3, groups of mice were injected with IL-12/15/18-pre-activated or control NK cells from the same donor (4x106/mouse). Mice treated with a single dose of memory-like NK cells exhibited significantly improved in vivo leukemia control measured by whole mouse bioluminescent imaging (P=0.03), as well as overall survival (P<0.05), compared to mice treated with control or no NK cells. Based on these pre-clinical findings, we initiated a first-in-human clinical trial of HLA-haploidentical IL-12/15/18-induced memory-like NK cells in patients with AML (NCT01898793). Relapsed/refractory (rel/ref) AML patients receive lymphodepleting non-myeloablative flu/cy conditioning, infusion of a single dose of CD56+CD3- memory-like donor NK cells, followed by two weeks of low dose rhIL-2. Three patients were treated at dose level 1 (0.5x106 cells/kg) and two patients treated at dose level 2 (1.0x106/kg) with no DLTs observed, and accrual continues. Correlative analyses utilizing donor-specific HLA mAbs allow tracking of donor memory-like NK cell frequency and function following adoptive transfer. Donor memory-like NK cells were detectable in the PB and BM of all tested patients with informative HLA (4/5), peak in frequency at 7-8 days post-infusion, and contract after 14-21 days as expected following recipient T cell recovery (Figure). Memory-like NK cells exhibit significantly increased Ki67%+ as a marker of proliferation at day 7 [97.8+1.0% (donor) vs. 21.6+5.5% (recipient), mean+SEM, P<0.001]. Moreover, functional analyses of NK cells at days 7-8 post-infusion reveal increased numbers of donor IFN-g+ NK cells following restimulation with K562 leukemia cells in the same blood [1009+590 (donor) vs. 8+3 (recipient) IFN-g+ NK cells] or BM [686+423 (donor) vs. 4+2 (recipient) IFN-g+ NK cells] samples. Two of four evaluable patients treated with memory-like NK cells had leukemia free BM and PB at days 14 post-therapy, which correlated with BM NK cell frequency and IFN-g production (Figure). CIML007 had rel/ref AML with 48% BM blasts pre-therapy, and had no evidence of leukemia on day 14, 28, and 100 BM biopsies, and has an ongoing complete remission more than 100 days after this therapy. CIML009 had 80% BM blasts pre-therapy, and had no evidence of leukemia on day 14 BM biopsy post-infusion. Thus, human IL-12/15/18-induced memory-like NK cells expand and have enhanced anti-AML function following adoptive transfer in patients, thereby constituting a promising translational innovation for immunotherapy of AML. Figure 1. Figure 1. Disclosures Fehniger: Celgene: Research Funding.

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Treatment of Ex Vivo Expanded NK Cells with Daratumumab F(ab')2 Fragments Protects Adoptively Transferred NK Cells from Daratumumab-Mediated Killing and Augments Daratumumab-Induced Antibody Dependent Cellular Toxicity (ADCC) of Myeloma

Blood ◽

10.1182/blood.v126.23.4244.4244 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4244-4244 ◽

Cited By ~ 6

Author(s):

Elena Cherkasova ◽

Luis Espinoza ◽

Ritesh Kotecha ◽

Robert N. Reger ◽

Maria Berg ◽

...

Keyword(s):

Nk Cells ◽

Adoptive Transfer ◽

Nk Cell ◽

Ex Vivo ◽

Cell Killing ◽

Cellular Toxicity ◽

Myeloma Cells ◽

Nsg Mice

Abstract Daratumumab is a fully humanized monoclonal antibody (IgG1) that targets CD38 expressed on myeloma cells. Daratumumab kills myeloma cells through antibody dependent cellular toxicity (ADCC), compliment dependent cytotoxicity (CDC), and antibody dependent phagocytosis (ADCP). In early clinical trials, daratumumab has showed significant anti-myeloma activity in patients with treatment refractory disease. In vivo, daratumumab has been found to induce NK cell lymphopenia of unclear etiology. We found that NK cells isolated from the peripheral blood of healthy and cancer patients expressed variable surface levels of CD38 (Fig. 1A). Further, surface expression of CD38 increased substantially when NK cells underwent ex vivo cytokine activation by culturing cells overnight in IL-2 containing media or ex vivo expansion using irradiated EBV-LCL feeder cells (Fig. 1B). Remarkably, daratumumab induced apoptosis of expanded NK cells in a dose dependent manner, with substantial NK cell apoptosis occurring within 2 hours following in vitro exposure to daratumumab at a concentration of 1 and 10 ug/ml (Fig. 1C). Further, adoptive transfer of ex vivo expanded human NK cells into NSG mice that had been pre-treated with daratumumab showed daratumumab induced NK cell killing in vivo: the numbers of NK cells isolated from the lungs, blood, spleen and bone marrow of NSG mice 24 hours after infusion of expanded human NK cells was reduced by 90% in mice that were pretreated with 1 mg/kg of daratumumab i.p. compared to controls that had not received the antibody (Fig. 1D). In vitro experiments showed NK cell killing by daratumumab occurred as a consequence of ADCC and was dependent on NK cell CD16 expression; when CD56+ NK cells were sorted by FACS into CD16 positive and negative populations, only NK cells expressing CD16 were killed by daratumumab, with no effect on NK cell viability occurring in the CD16- NK cell. Further, we observed that NK cells obtained from donors who have high affinity FCgR3 as a consequence of a single nucleotide polymorphism in the FCGR3A gene resulting in an amino acid substitution at position 158 (F158V) in CD16 were more sensitive to daratumumab killing compared to NK cells isolated from donors carrying the low affinity CD16 polymorphism. Although NK cell counts and NK reduction in peripheral blood and bone marrow were not associated with daratumumab clinical response in myeloma studies, NK cells play an important role in mediating antitumor responses through ADCC following mAb therapy. In this regard, combining mAb therapy with adoptive transfer of ex vivo expanded NK cells could be utilized as a strategy to potentiate the antitumor effects of mAbs. To overcome daratumumab-mediated killing of adoptively transferred NK cells in daratumumab-treated patients, we blocked CD38 on the surface of NK cells by pretreating them with daratumumab F(ab')2 fragments. The F(ab')2 fragments that were generated using pepsin cleavage of daratumumab were confirmed to bind and block the CD38 epitope expressed on NK cells. Importantly, these F(ab')2 fragments remained bound to the surface of NK cells for at least 96 hours, did not induce NK cell apoptosis, protected NK cells from daratumumab-mediated NK cell killing, and bolstered their tumor cytotoxicity against daratumumab-treated myeloma targets. In vitro experiments showed NK cell tumor cytotoxicity vs myeloma cells in daratumumab-containing media was significantly higher by NK cells that had CD38 blocked with F(ab')2 fragments compared to unblocked controls (Fig. 1E). Importantly, pretreatment with daratumumab F(ab')2 fragments also protected human NK cells from daratumumab-mediated killing in vivo; expanded NK cells pretreated with F(ab')2 fragments prior to adoptive transfer into NSG mice that had been treated with daratumumab were detectable at significantly higher numbers in the blood compared to untreated NK cell controls (Fig. 1F). Conclusion: Expression of CD38 on activated NK cells makes them susceptible to killing by daratumumab, which could compromise the ability of adoptively transferred NK cells to bolster ADCC following treatment with this mAb. Pretreatment of ex vivo expanded NK cells with daratumumab F(ab')2 fragments protects cells from daratumumab-mediated killing, potentially offering a strategy to augment the anti-tumor effects of adoptively transferred NK cells in myeloma patients that have received daratumumab treatment. Disclosures No relevant conflicts of interest to declare.

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127 Preclinical evaluation of NKX019, a CD19-targeting CAR NK Cell

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0127 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A140-A140

Author(s):

Nadege Morisot ◽

Sarah Wadsworth ◽

Tina Davis ◽

Nicole Dailey ◽

Kyle Hansen ◽

...

Keyword(s):

T Cells ◽

B Cell ◽

Nk Cells ◽

Half Life ◽

Nk Cell ◽

Cytolytic Activity ◽

Nsg Mice ◽

Animal Care

BackgroundNatural killer (NK) cells are highly effective and fast-acting cytolytic cells capable of eradicating target cells with limited adverse effects such as cytokine release syndrome (CRS) or graft-versus-host disease. Chimeric antigen receptors (CARs)-engineered NK cells have been recently used against leukemia with encouraging clinical outcomes.1 The surface antigen CD19, expressed by B-lymphoblasts, represents an ideal CAR target against B cell acute lymphoblastic leukemia (B-ALL). We developed a highly potent CD19 -directed CAR NK cell therapy, NKX019, with an extended in vivo half-life aimed at killing CD19-expressing target.MethodsNK cells isolated from healthy PBMCs were expanded in the presence of NKSTIM cells, IL-2, IL-12, IL-18 and transduced with both a CD19-targeted CAR construct and a membrane-bound form of IL-15 (mbIL-15). Control (non-engineered) NK cells were produced in parallel. Cytotoxic activity of NKX019 against CD19+ B-ALL cell line (REH), pre-B ALL cell line (Nalm-6), allogeneic PBMCs was assessed using Incucyte® or flow cytometry. NSG mice bearing either Nalm-6.fluc (Nalm6) or REH.fluc (REH) tumor received different concentrations of NKX019 or control NK cells. In-life analysis of tumor-bearing and naïve NSG mice include: 1) bioluminescence imaging, 2) clinical observations, 3) serum cytokines and 4) CAR+ NK cell persistency.ResultsNKX019 showed enhanced cytolytic activity against REH and Nalm-6 tumor cells compared to control NK cells and CAR19+ T cells. The superiority of NKX019 over CAR19+ T cells was more pronounced at the earlier time point (24 hours) with near identical calculated EC50 observed at 72 hours for both cell types. Increased cytolytic activity of NKX019 was limited to CD19+ cells in bulk PBMCs. Consistent with our in vitro observations, NKX019 controlled Nalm-6 and REH tumor growth in doses as low as 2 × 106 cells/kg for up to 30 days with no apparent increase in cytokines commonly associated with CRS. Increased Nalm-6 tumor growth coincided with an apparent decrease in measurable NKX019 in the periphery. In tumor-naïve NSG mice, NKX019 was detectable in the blood for up to 9 weeks post-infusion consistent with its extended half-life.ConclusionsNKX019 expresses mbIL-15 and is produced in the presence of IL-12 and IL-18, resulting in enhanced in vitro expansion and longer in vivo half-life than non-engineered NK cells. NKX019 also exhibited advantages compared to CAR19+ T cells including faster cytotoxic kinetics and limited production of cytokines associated with CRS. A first-in-human trial of NKX019 in B cell malignancies is planned for 2021.Ethics ApprovalThe animal procedures described in this abstract were conducted in accordance with Explora BioLabs Animal Care and Use Protocol approved by Explora BioLabs Institutional Animal Care and Use Committee.ReferenceLiu, et al. 2020 NEJM

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Human NK cell development in hIL-7 and hIL-15 knockin NOD/SCID/IL2rgKO mice

Life Science Alliance ◽

10.26508/lsa.201800195 ◽

2019 ◽

Vol 2 (2) ◽

pp. e201800195 ◽

Cited By ~ 6

Author(s):

Masashi Matsuda ◽

Rintaro Ono ◽

Tomonori Iyoda ◽

Takaho Endo ◽

Makoto Iwasaki ◽

...

Keyword(s):

Nk Cells ◽

Immune Cells ◽

Nk Cell ◽

Cell Development ◽

Innate Immune ◽

Humanized Mice ◽

In Vivo Model ◽

Hematopoietic Stem ◽

Nsg Mice

The immune system encompasses acquired and innate immunity that matures through interaction with microenvironmental components. Cytokines serve as environmental factors that foster functional maturation of immune cells. Although NOD/SCID/IL2rgKO (NSG) humanized mice support investigation of human immunity in vivo, a species barrier between human immune cells and the mouse microenvironment limits human acquired as well as innate immune function. To study the roles of human cytokines in human acquired and innate immune cell development, we created NSG mice expressing hIL-7 and hIL-15. Although hIL-7 alone was not sufficient for supporting human NK cell development in vivo, increased frequencies of human NK cells were confirmed in multiple organs of hIL-7 and hIL-15 double knockin (hIL-7xhIL-15 KI) NSG mice engrafted with human hematopoietic stem cells. hIL-7xhIL-15 KI NSG humanized mice provide a valuable in vivo model to investigate development and function of human NK cells.

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Eomes and T-Bet Expression Are Required By Mature Primary Human NK Cells for Anti-Leukemia Responses In Vivo

Blood ◽

10.1182/blood-2021-149693 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 194-194

Author(s):

Pamela Wong ◽

Carly C. Neal ◽

Lily Chang ◽

Julia A Wagner ◽

Melissa M. Berrien-Elliott ◽

...

Keyword(s):

Nk Cells ◽

Research Funding ◽

Nk Cell ◽

Ex Vivo ◽

T Box ◽

Nsg Mice ◽

Healthy Donors ◽

And Function

Abstract Natural Killer (NK) cells are innate lymphoid cells that respond to hematologic cancers via cytotoxicity (perforin/granzyme and death receptors) and cytokine/chemokine production, yet the molecular determinants underlying their proliferation, function, and persistence are poorly understood. There are promising reports of pre-clinical and clinical NK cell responses to leukemia and lymphoma, which represent a nascent cellular therapy for these blood cancers. The T-box transcription factors (TFs) Eomes and T-bet are expressed by NK cells throughout their lifespan, and are required for development as evidenced by NK cell loss in Eomes and T-bet deficient mice. However, the roles of these TFs in mature human NK cell molecular programs and functions remain unclear. We hypothesized Eomes and T-bet, which are the only T-box TFs expressed in NK cells, are critical regulators of NK cell homeostasis and functionality, and are necessary for proper mature NK cell responses. To address this, we utilized the CRISPR-Cas9 system to genetically delete both Eomes and T-bet in primary human NK cells isolated from healthy donors, and investigated their role beyond guiding NK cell development, specifically in the anti-leukemia response. Gene-editing of primary human NK cells has been technically challenging, thus most reports that modified NK cells were performed with cell lines, in vitro-differentiated, or highly expanded NK cells that likely do not reflect primary human NK cell biology. Here, we introduced Cas9 mRNA and sgRNA targeting T-bet and Eomes by electroporation into unexpanded primary human NK cells isolated from healthy donors using the MaxCyte GT system. We observed highly efficient reductions of Eomes and T-bet protein expression, quantified by flow cytometry (p < 0.0001, Fig A-B) without viability differences between control (sgRNA targeting TRAC, an unexpressed locus in NK cells), and Eomes/T-bet double CRISPR-edited (DKO) cells after one week in vitro. To study Eomes and T-bet in NK cell anti-leukemia response, control or DKO primary human NK cells were engrafted into NSG mice, supported with human IL-15, and challenged with K562 leukemia cells. Utilizing bioluminescent imaging to visualize leukemia burden, we observed that NK cells lacking both TFs were unable to suppress leukemia growth in vivo. To understand the mechanism responsible for impaired leukemia control, we investigated in vivo persistence and proliferation, cytotoxic effector molecule expression, as well as ex vivo degranulation and cytokine production of DKO NK cells compared to control NK cells. DKO or control human NK cells were transferred into NSG mice and supported with human IL-15. After 2-3 weeks, significantly fewer (<30%) DKO NK cells persisted compared to control NK cells: spleen (5-fold decrease, control 240e3±65e3 vs DKO 47e3±15e3 NK cells, p<0.01, Figure C), blood (6-fold decrease, p<0.01), and liver (4-fold decrease, p<0.05). Using intracellular flow cytometry, double T-bet/Eomes CRISPR-edited NK cells that lacked both Eomes and T-bet protein after in vivo transfer were identified. A proliferative defect was evident in flow-gated DKO (62±6% undivided), compared to unedited (WT) NK cells (4±2% undivided) assessed by CellTrace Violet dilution (Figure D). In addition, there were marked reductions in granzyme B and perforin protein (p<0.001) in flow-gated DKO NK cells compared to controls. To assess DKO NK cell functional capacity, we performed an ex vivo functional assay on NK cells from spleens of the NSG mice as effectors, and K562 targets or IL-12/15/18 stimulation for 6 hours. Degranulation to K562 targets was impaired (p<0.05), and IFN-γ production was reduced (p<0.0001) after cytokine stimulation in flow-gated DKO NK cells (Figure E). Thus, CRISPR-editing of unexpanded, primary human NK cells revealed that Eomes and T-bet are required by mature human NK cells for their function and homeostasis, distinct from their role in development. This is translationally relevant, as defects in proliferation and function of human DKO NK cells manifested markedly reduced response against human leukemia cells in vivo in xenografts. These findings expand our understanding of key molecular regulators of mature NK cell homeostasis and function, with the potential to provide new avenues to enhance NK cell therapy. Figure 1 Figure 1. Disclosures Berrien-Elliott: Wugen: Consultancy, Patents & Royalties: 017001-PRO1, Research Funding. Foltz-Stringfellow: Kiadis: Patents & Royalties: TGFbeta expanded NK cells; EMD Millipore: Other: canine antibody licensing fees. Fehniger: HCW Biologics: Research Funding; Compass Therapeutics: Research Funding; Affimed: Research Funding; ImmunityBio: Research Funding; Wugen: Consultancy, Current equity holder in publicly-traded company, Patents & Royalties: related to memory like NK cells, Research Funding; Kiadis: Other; OrcaBio: Other; Indapta: Other.

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Abstract 49: Over-activation Of Natural Killer Cells Due To Dysfunction Of Cd16-mediated Signaling In Adamts13-deficient Ttp Patients With A History Of Relapse

Circulation Research ◽

10.1161/res.115.suppl_1.49 ◽

2014 ◽

Vol 115 (suppl_1) ◽

Author(s):

FANG ZHOU ◽

Darise Farris

Keyword(s):

Nk Cells ◽

Cell Activation ◽

Nk Cell ◽

K562 Cells ◽

Antibody Treatment ◽

History Of ◽

Relapse Group

Thrombotic thrombocytopenic purpura (TTP) is a rare life-threaten vascular autoimmune disease. There is no effective method to treat it in clinical trials since pathogenesis of TTP has not been fully elucidated. Here we investigate the role of NK cells in relapse development of TTP. Our results showed that the frequencies of CD3 − CD56 dim CD16 − and CD3 − CD56 bri CD16 − NK cells are increased in TTP patients with a history of relapse. Expression of CD107a, granzyme A and IFN-γ by CD3 − CD56 dim NK cells following in vitro stimulation with PMA/ionomycin / monensin is improved in the relapse group, compared with those on NK cells derived from TTP patients without relapse development. NK cells isolated from TTP patients with a history of relapse indicated stronger cytotoxicity to target K562 cells than those of NK cells derived from TTP patients without relapse development, suggesting prior activation of NK cells in vivo . Treatment with anti-human CD16 antibody up-regulates cytotoxicity of NK cells derived from TTP patients without relapse development. However, Anti-human CD16 antibody treatment does not affect cytotoxicity of NK cells isolated from TTP patients with a history of relapse, suggesting inability of CD16-mediated signaling in NK cells derived from TTP patients with relapse development. These data provide evidence of altered NK cell activation and/or licensing in TTP patients with a history of relapse modulated by CD16-mediated signaling and a new avenue of investigation into mechanisms of TTP immunopathogenesis.

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