Human Cytokine-Induced Memory-like (CIML) NK Cells Are Active Against Myeloid Leukemia in Vitro and in Vivo

Abstract NK cells are innate lymphoid cells that mediate anti-leukemia responses. The ability of MHC-haploidentical NK cells to recognize and eliminate AML blasts have been established in the setting of stem cell transplantation and early phase adoptive NK cell immunotherapy trials. However, the optimal approach to prepare human NK cells for maximal anti-leukemia capacity is unclear. As one form of innate NK cell memory, cytokine-induced memory-like (CIML) NK cells are induced by a brief (16 hour) pre-activation of human NK cells with the combination of IL-12, IL-15, and IL-18, while control NK cells from the same donor are activated by IL-15 only. In published work, this combined IL-12, IL-15, and IL-18 pre-activation results in enhanced proliferation and augmented IFN-gamma responses to cytokine or activating receptor-based re-stimulation following a rest period of 1 – 6 weeks. We hypothesized that CIML NK cells exhibit improved anti-leukemia properties compared to control NK cells from the same individual. Purified primary human CIML NK cells [both CD56bright and CD56dim subsets] produce more IFN-gamma, compared to control NK cells, upon re-stimulation with K562 cells or primary AML blasts after 7 days of rest (p<0.05 and p<0.001, N=5). CIML NK cells also exhibit higher granzyme B protein expression (p<0.01; N=8), and increased cytotoxicity against K562 leukemia targets in vitro (p<0.001, 2.5:1 and 5:1 E:T ratios). We next established a NOD-SCID-gamma-c-/- (NSG) xenograft model to investigate primary human CIML NK cell responses in vivo, with survival supported by low dose IL-2 administered every other day. Seven days following injection of 4 million NK cells / mouse, human CIML NK cells traffic to the bone marrow, spleen, liver and blood, and exhibited better in vivo expansion and persistence, compared to control NK cells (p=0.05 in the blood and bone marrow). Further, the characteristic enhanced functionality of CIML compared to control NK cells when restimulated with K562 targets was retained when assessed ex vivo 7 days post-transfer (p<0.05). Next, we investigated the ability of CIML versus control NK cells from the same donor to clear K562 AML cells in vivo. First, luciferase expressing K562 cells (1 million / mouse) were engrafted into sub-lethally irradiated (250 cGy) NSG mice. On day 3 after K562 challenge, primary human CIML or control NK cells from the same donor (4 million / mouse) were injected, which were supported in vivo using low dose IL-2. CIML NK cells exhibited significantly improved in vivo leukemia clearance as evidenced by whole mouse bioluminescence imaging (see Figure, P=0.03, N=7 mice per group). Thus, human CIML NK cells exhibit enhanced in vitro and in vivo anti-leukemia effects, compared to control NK cells. Based on these findings, a first-in-human phase 1 study of CIML NK cells in relapsed/refractory AML is currently underway. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Expanded Natural Killer (NK) Cells for Immunotherapy: Fresh and Made to Order

Blood ◽

10.1182/blood.v120.21.1912.1912 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1912-1912

Author(s):

Susann Szmania ◽

Natalia Lapteva ◽

Tarun K. Garg ◽

Joshuah D Lingo ◽

Amy D Greenway ◽

...

Keyword(s):

High Risk ◽

Nk Cells ◽

Mononuclear Cells ◽

Nk Cell ◽

Conflicts Of Interest ◽

K562 Cells ◽

Clinical Grade ◽

Additional Advantage

Abstract Abstract 1912 Introduction Remarkable increases in the dose and activity of NK cells can be achieved by co-culture with the HLA class I deficient cell line K562 that has been genetically modified to express membrane-bound IL15 and the co-stimulatory molecule 41BB-ligand (K562-mb15-41BBL; Fujisaki et al. Cancer Res. 2009;69:4010–4017). We are conducting a clinical trial utilizing these ex-vivo expanded NK cells (ENK) which are produced at the Center for Cell and Gene Therapy (CAGT) at Baylor and then shipped to the University of Arkansas for Medical Sciences (UAMS) for infusion to high-risk relapsed multiple myeloma (MM) patients using the NHLBI-PACT mechanism. Here we report on the characteristics of the ENK cell products sent fresh versus frozen. Methods Apheresis products were collected from MM patients or healthy donors (HD), cryopreserved, and then shipped to CAGT for GMP grade production, as described (Lapteva et al. Cytotherapy 2012; in press). Briefly, mononuclear cells from thawed and ficolled apheresis products were cultured in Stem Cell Growth Medium (CellGenix) supplemented with 10% fetal bovine serum and 10 U/mL IL2 with stimulator cells at a ratio of 1 NK cell to 10 irradiated K562-mb15-41BBL cells (developed at St. Jude Children's Research Hospital, Memphis, TN). Cells were harvested on day 8–9; products from HD were CD3-depleted. Clinical-grade products were shipped to UAMS overnight either cryopreserved in a dry shipper (n=7) or fresh in 5% human serum albumin on cold packs at 1–11°C (n=4). Cell purity, expression of activating molecules, and viability by 7AAD exclusion was assessed by flow cytometry. Standard 4h chromium-release assays were used to assess potency against K562 cells at a 20:1 ENK: K562 ratio. Student's t-Test was used to determine significance. Results From 0.9–1.5×107 starting NK cells, the total number of ENK cells produced was 5.4×109 (range 1.8–24×109). The fold NK-cell expansion was significantly lower for MM patients (n=5, median 22, 12–70 fold) than for HD (n=6, median 95, 31–160 fold; p<0.05). At harvest, median CD3+/CD56+ NK cell purity was 70% (52–88); CD3 depletion of HD products increased CD3+/CD56+ purity to 93% (86–95) resulting in a median CD3+/CD56- T cell content of 0.02% (0.04–1.02). Overall, median viability was 93% (67–98) and potency (defined as lysis of K562 cells at a 20:1 E:T ratio) was 74% (26–92). One product derived from a patient with 21% CD138+ MM cells in the apheresis collection had low expansion (12-fold), viability (66.7%) and potency (26%). For cryopreserved products, viability immediately after thawing was acceptable (median 94%, 75–99) but recovery of viable cells varied from 61% to 100% and thawed ENK failed to lyse K562 cells unless rested overnight. Further, recovery was extremely poor after overnight incubation (median 16%, 10–21). We therefore validated shipment of fresh ENK products. In contrast with frozen NK cells, the median recovery for fresh clinical products post-shipping was 101% (87–151). We confirmed that NK purity, viability, potency and expression of the key activating molecules NKG2D, NKp30, NKp44 and CD226 were retained up to 48h after transfer. ENK further increased by 34% after 72h in vitro incubation in the presence of IL2. Significant in vivo expansion of ENK was observed after infusion of fresh ENK cell products (n=3) but not after infusion of thawed products (n=3, see separate abstract). An additional advantage was that the fresh cells arrived ready to infuse and changes in release criteria relying on rapid and in process testing significantly reduced the time from apheresis collection to ENK infusion, an important consideration when treating high-risk MM patients who can experience rapid disease progression. Conclusion We conclude that large numbers of clinical grade ENK cells can be generated from both MM patient and HD derived apheresis products by co-culture with IL2 and K562-mb15-41BBL although less vigorous expansion was observed with patient-derived cells. Upon thawing, cryopreserved ENK cells exhibited inferior recovery and potency, and survived poorly during further in vitro culture. In contrast, freshly formulated and shipped ENK cells have excellent recovery and retain cytolytic ability. Robust in vivo expansion was only seen after infusion of fresh ENK cells. Production assistance by CAGT allowed for the rapid implementation of a novel therapy utilizing fresh ENK cells for poor prognosis MM patients. Disclosures: No relevant conflicts of interest to declare.

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A Novel Method to Study the in Vivo Trafficking and Homing of Adoptively Transferred NK Cells in Rhesus Macaques and Humans

Blood ◽

10.1182/blood.v124.21.659.659 ◽

2014 ◽

Vol 124 (21) ◽

pp. 659-659 ◽

Cited By ~ 3

Author(s):

Jan Davidson-Moncada ◽

Noriko Sato ◽

Robert F Hoyt ◽

Robert N Reger ◽

Marvin Thomas ◽

...

Keyword(s):

Nk Cells ◽

Adoptive Transfer ◽

Nk Cell ◽

Conflicts Of Interest ◽

Raji Cell ◽

K562 Cells ◽

Ct Imaging ◽

Pet Ct ◽

Hematological Cancers

Abstract Adoptive transfer of allogeneic or autologous natural killer (NK) cells is now being developed for therapy of both hematological and solid malignancies. The efficacy of NK immunotherapy to mediate anti-tumor effects will ultimately be dependent on their ability to traffic and home to the tumor microenvironment. Recent data suggest expanded NK cells are ineffective at homing to the bone marrow (BM) and lymph nodes (LN) where hematological malignancies reside. A variety of techniques to maintain and/or enforce expression of homing receptors in NK cells are now being explored in preclinical models to improve their localization to the BM and LN. Historically, xenogeneic human into mouse or mouse into mouse models have been utilized for preclinical development of adoptive NK transfer. These experiments often use fluorescent dye-labeled NK cells and require repeated invasive biopsies, which can be confounded by sampling error, or the requirement for post mortem analysis. Here we present a method to track in real time and in vivo adoptively infused zirconium-89 (89Zr) labelled NK cells by PET imaging. A rhesus macaque (RM) model was used for these preclinical experiments as RM and human NK cells have similar expansion kinetics, and have greater similarity than mice in their phenotype, function, and homing receptors and ligands. PBMCs collected from the PB of 13 RMs were enriched for NK cells by CD3+ T-cell depletion and were then expanded for 14 days by culturing with irradiated human EBV-LCL cells in X-VIVO 20 media containing 10% human AB serum and 500 IU/μl of human IL-2. RM NK cells expanded a mean 145±41 fold and contained >99% pure CD3- and CD56+ cells. The phenotype and tumor cytotoxicity of RM NK cells were similar to NK cells expanded from humans (n=3) using similar expansion cultures; at a 10:1 E:T ratio, 67% and 73% of K562 cells were lysed by RM and human NK cell respectively. To label NK cells, 89Zr was conjugated to oxine, which readily permeabilized the cellular membrane and was retained in the cells. Expanded NK cells from both humans and RM showed no changes in CD16 or CD56 expression for up to 6 days following radiolabeling. Human and RM NK cell viability 0 to 24 hours following radiolabelling was 60-100% then declined to 20-30% after 6 days. 89Zr retention by both human and RM NK cells was 75-80% in the first 24 hours of culture but gradually declined with time, decreasing to 20-30% after 7 days of culture. Culturing radiolabeled human NK cells for 24-36 hours with different cellular populations including Ramos and Raji cell lines and normal human PBMCs revealed no significant transfer of radioactivity (max 2% above baseline), establishing that 89Zr was not transferred from labeled to unlabeled cells. Oxine labeling did not alter the cytotoxicity of human or RM NK cells vs K562 cells compared to unlabeled controls. 89Zr-oxine labeling of expanded RM NK cells is currently being used to quantify NK cell trafficking and survival following adoptive transfer in autologous macaques. In these experiments, RM recipients of adoptively infused 89Zr labeled NK cells receive concurrent deferoxamine to chelate and then enhance renal excretion of any free 89Zr that is released from dead cells. In the experiments shown below, 13 x 107 autologous ex vivo expanded 89Zr-labeled RM NK cells were injected IV into a 5.7 kg RM and tracked by sequential PET/CT imaging for 7 days. Up to 1-hour post infusion, most NK cell activity was restricted to the lungs. By 4 hours, NK cells began to traffic from the lungs to the liver and spleen. By 2 days, NK cells were no longer detectable in the lungs and resided largely in the liver and spleen, where they remained for the remainder of the 7 day imaging period. During the entire observation period, little to no NK cell radioactivity was detected in the LN or BM. In conclusion, 89Zr oxine labelling of NK cells followed by PET/CT imaging represents a powerful tool to track the in vivo fate of adoptively transferred NK cells. The RM model presented here provides a method to evaluate and optimize various strategies aimed at altering the phenotype of NK cells, with the goal of improving their homing to the BM and LN where hematological cancers reside. These preclinical in vitro and in vivo data suggest this technology could be safely extended to humans and could be applied to other cellular populations besides NK cells. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Stem Cell Factor Enhances Interleukin-2–Mediated Expansion of Murine Natural Killer Cells In Vivo

Blood ◽

10.1182/blood.v90.9.3647 ◽

1997 ◽

Vol 90 (9) ◽

pp. 3647-3653 ◽

Cited By ~ 26

Author(s):

Todd A. Fehniger ◽

William E. Carson ◽

Ewa Mrózek ◽

Michael A. Caligiuri

Keyword(s):

Nk Cells ◽

Cell Expansion ◽

Stem Cell Factor ◽

Low Dose ◽

Nk Cell ◽

Interleukin 2 ◽

Innate Immune ◽

Ifn Γ

Abstract The administration of low dose interleukin-2 (IL-2) results in a selective expansion of natural killer (NK) cells in vivo, and promotes the differentiation of NK cells from hematopoietic precursor cells in vitro. We have previously shown that stem cell factor (SCF ), the ligand to the c-kit tyrosine kinase receptor, enhances IL-2–induced NK cell proliferation and differentiation in vitro. Here, we investigated the effects of SCF plus IL-2 delivered to mice in vivo. Eight-week-old C57BL/6 mice were treated with a continuous subcutaneous infusion of IL-2 (1 × 104 IU/d) plus a daily intraperitoneal dose of SCF (100 μg/kg/d), IL-2 alone, SCF alone, or vehicle alone for 8 weeks. The in vivo serum concentration of IL-2 ranged between 352 ± 12.0 pg/mL and 606 ± 9.0 pg/mL, achieving selective saturation of the high affinity IL-2 receptor, while the peak SCF serum concentration was 296 ± 13.09 ng/mL. Alone, the daily administration of SCF had no effect on the expansion of NK cells. The continuous infusion of IL-2 alone did result in a significant expansion of NK1.1+CD3− cells compared to mice treated with placebo or SCF. However, mice treated with both SCF and IL-2 showed an increase in the absolute number of NK cells that was more than twofold that seen with IL-2 alone, in the spleen (P ≤ .005), bone marrow (P ≤ .025), and blood (P < .05). NK cytotoxic activity against YAC-1 target cells was significantly higher for mice treated with SCF plus IL-2, compared to mice treated with IL-2 alone (P ≤ .0005). Interferon-γ (IFN-γ) production in cytokine-activated splenocytes was also greater for the SCF plus IL-2 group, over IL-2 treatment alone (P ≤ .01). The effect of SCF plus IL-2 on NK cell expansion was likely mediated via NK cell precursors, rather than mature NK cells. In summary, we provide the first evidence that SCF can significantly enhance expansion of functional NK cells induced by the prolonged administration of low dose IL-2 in vivo. Since the NK cell is a cytotoxic innate immune effector and a potent source of IFN-γ, this therapeutic strategy for NK cell expansion may serve to further enhance innate immune surveillance against malignant transformation and infection in the setting of cancer and/or immunodeficiency.

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Interleukin-2-activated natural killer cells can support hematopoiesis in vitro and promote marrow engraftment in vivo

Blood ◽

10.1182/blood.v80.3.670.670 ◽

1992 ◽

Vol 80 (3) ◽

pp. 670-677 ◽

Cited By ~ 45

Author(s):

WJ Murphy ◽

JR Keller ◽

CL Harrison ◽

HA Young ◽

DL Longo

Keyword(s):

Growth Factors ◽

Nk Cells ◽

Nk Cell ◽

Interleukin 2 ◽

Hematopoietic Growth Factors ◽

Ifn Gamma ◽

Gm Csf ◽

Growth Promoting

Abstract Purified natural killer (NK) cells were obtained from mice with severe combined immune deficiency (SCID) to ascertain their effect on hematopoiesis. When activated and propagated with recombinant human interleukin-2 (rhIL-2) in vitro, SCID spleen cells maintained a phenotypic and lytic spectrum consistent with a pure population of activated NK cells. When added with syngeneic bone marrow cells (BMC) in soft agar, the activated NK cells could support hematopoietic growth in vitro without the addition of exogenous hematopoietic growth factors. However, when syngeneic BMC were added along with cytokines to produce optimal growth conditions, the addition of NK cells was then inhibitory for hematopoietic colony formation. Antibodies to interferon- gamma (IFN-gamma) partially reversed the inhibitory effects. Supernatants from the NK-cell cultures could also exert these effects on hematopoiesis, although to a lesser extent. Analysis of the NK cell RNA demonstrated that activated NK cells express genes for hematopoietic growth factors such as granulocyte-macrophage colony- stimulating factor (GM-CSF), granulocyte CSF (G-CSF), and IL-1 beta. The NK cells were also found to express IFN-gamma, transforming growth factor-beta 1 (TGF-beta 1), and tumor necrosis factor-alpha (TNF-alpha) mRNA. Analysis of the NK-cell supernatants using factor-dependent myeloid progenitor cell lines showed that the NK cells were producing G- CSF and growth-promoting activity that could not be attributed to IL-1, IL-3, IL-4, IL-5, IL-6, GM-CSF, G-CSF, macrophage CSF (M-CSF), or stem cell factor. The transfer of activated NK cells with BMC into lethally irradiated syngeneic mice resulted in greater BMC engraftment in the recipients. Thus, these results using a pure population of activated NK cells indicate that when activated, these cells can produce a variety of growth factors for hematopoiesis and exert significant hematopoietic growth-promoting effects in vivo.

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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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The IL-15 Super-Agonist ALT-803 Promotes Superior Activation and Cytotoxicity of Ex Vivo Expanded NK Cells Against AML

Blood ◽

10.1182/blood.v126.23.3090.3090 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3090-3090 ◽

Cited By ~ 1

Author(s):

Folashade Otegbeye ◽

Nathan Mackowski ◽

Evelyn Ojo ◽

Marcos De Lima ◽

David N. Wald

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Nk Cells ◽

Myeloid Leukemia ◽

Nk Cell ◽

Ex Vivo ◽

Leukemia Cells ◽

Activating Receptor

Abstract Introduction: A crucial component of the innate immune response system, natural killer (NK) cells are uniquely competent to mediate anti-myeloid leukemia responses. NKG2D is an activating receptor on the surface of NK cells that engages stress ligands MICA and MICB, typically upregulated on myeloid leukemia cells. Adoptive transfer of NK cells is a promising treatment strategy for AML. Strategies to optimize the anti-leukemia effect of NK cell adoptive transfer are an area of active research. These include attempts to enhance NK cell activity and to maintain the activation status and proliferation of the NK cells in vivo. Traditionally, IL-2 has been used to maintain the in vivo proliferation of adoptively transferred NK cells, but it leads to unwanted proliferation of regulatory T cells and suboptimal NK cell proliferation. IL-15 may be superior to IL-2, without the effects on T regulatory cells. The IL-15 superagonist, ALT-803 exhibits >25 fold enhancement in biological activity as compared to IL-15. ALT-803 is a fusion protein of an IL-15 mutant and the IL-15Rα/Fc complex that has recently entered clinical trials as a direct immunomodulatory agent in cancer clinical trials We hypothesized ALT-803 would augment the activity and/or proliferation of adoptively transferred NK cells in vitro and in a mouse model system.. Methods: Human NK cells were isolated from healthy donor peripheral blood and were expanded over a 21-day period in co-culture with irradiated K562 cells genetically modified to express membrane-bound IL-21. (Somanchi et al. 2011 JoVE 48. doi: 10.3791/2540) The NK cells were expanded with IL-2 (50mU/mL) and/or ALT-803 (200ng/mL). On Day 21, NK cells were examined for cytotoxicity against AML cells as well as by flow cytometry for expression of known activating receptors. An NSG murine xenograft model of human AML was developed to test the in vivo function of NK cells expanded above. Briefly, NSG mice (n=5 per group) were non-lethally irradiated and each injected IV with 5 x106 OCI-AML3 leukemic cells. Two days later, each mouse received weekly NK cell infusions for 2 weeks. Mice that received NK cells expanded with IL2 got cytokine support with IL-2 (75kU IP three times a week). Mice infused with ALT-803 expanded cells (alone or in combination with IL2) received ALT-803 (0.2mg/kg IV weekly). One control group received OCI cells but were infused weekly only with 2% FBS vehicle, no NK cells. Leukemic burden in each mouse was assessed by flow cytometry of bone marrow aspirates on day 28 following start of NK cell infusions). This time point was chosen as the control mice appeared moribund. Results: ALT-803 did not have any differential effect on the proliferation of the NK cells ex vivo as compared to IL-2. However, the presence of ALT-803 either alone or in combination with IL-2 resulted in a significant increase (30% increase, p<0.0001) in the cytotoxic activity of the NK cells against leukemia cells as compared with IL-2 alone in vitro (figure 1). In addition, the percentages of NK cells that express the activating receptor NKG2D as well as CD16 were significantly higher (p<0.001 for both) after ALT-803 exposure (figure 1). Finally, in the murine xenograft AML model, ALT-803 expanded NK cells, which were also supported in vivo with ALT-803, resulted in an 8-fold reduction in disease burden in the bone marrow (p<0.0001). Importantly the efficacy of NK cells in the ALT-803 injected mice was significantly higher (3-fold, p= 0.0447) than IL-2 treated mice (figure 2). Discussion: Our results suggest that the presence of ALT-803 during ex-vivo expansion of NK cells results in increased activation and cytotoxicity against AML cells. In addition our results using a murine model of human AML show that the use of ALT-803 in combination with adoptively transferred NK cells provides a significant anti-leukemic benefit as compared to IL-2. Future studies to test larger panels of leukemia cells as well as other cancer cell lines are currently in progress. It is hoped that this work will lead to an improvement in the efficacy of adoptively transferred NK cells for AML patients due to an improvement in survival and activity of the NK cells. Disclosures Wald: Invenio Therapeutics: Equity Ownership.

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Endothelial JAK3 Expression Enhances Pro-Hematopoietic Angiocrine Function of Sinusoidal Endothelial Cells

Blood ◽

10.1182/blood-2019-122449 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2488-2488 ◽

Cited By ~ 1

Author(s):

José Gabriel Barcia Durán

Keyword(s):

Bone Marrow ◽

Endothelial Cells ◽

Nk Cell ◽

Conflicts Of Interest ◽

Fluorescent Microscopy ◽

Hematopoietic Stem ◽

Interleukin Receptors ◽

Sinusoidal Endothelium

Unlike Jak1, Jak2, and Tyk2, Jak3 is the only member of the Jak family of secondary messengers that signals exclusively by binding the common gamma chain of interleukin receptors IL2, IL4, IL7, IL9, IL15, and IL21. Jak3-null mice display defective T and NK cell development, which results in a mild SCID phenotype. Still, functional Jak3 expression outside the hematopoietic system remains unreported. Our data show that Jak3 is expressed in endothelial cells across hematopoietic and non-hematopoietic organs, with heightened expression in the bone marrow and spleen. Increased arterial zonation in the bone marrow of Jak3-null mice further suggests that Jak3 is a marker of sinusoidal endothelium, which is confirmed by fluorescent microscopy staining and single-cell RNA-sequencing. We also show that the Jak3-null niche is deleterious for the maintenance of long-term repopulating hematopoietic stem and progenitor cells (LT-HSCs) and that Jak3-overexpressing endothelial cells have increased potential to expand LT-HSCs in vitro. In addition, we identify the soluble factors downstream of Jak3 that provide endothelial cells with this functional advantage and show their localization to the bone marrow sinusoids in vivo. Our work serves to identify a novel function for a non-promiscuous tyrosine kinase in the bone marrow vascular niche and further characterize the hematopoietic stem cell niche of sinusoidal endothelium. Disclosures No relevant conflicts of interest to declare.

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Interleukin-2-activated natural killer cells can support hematopoiesis in vitro and promote marrow engraftment in vivo

Blood ◽

10.1182/blood.v80.3.670.bloodjournal803670 ◽

1992 ◽

Vol 80 (3) ◽

pp. 670-677 ◽

Cited By ~ 1

Author(s):

WJ Murphy ◽

JR Keller ◽

CL Harrison ◽

HA Young ◽

DL Longo

Keyword(s):

Growth Factors ◽

Nk Cells ◽

Nk Cell ◽

Interleukin 2 ◽

Hematopoietic Growth Factors ◽

Ifn Gamma ◽

Gm Csf ◽

Growth Promoting

Purified natural killer (NK) cells were obtained from mice with severe combined immune deficiency (SCID) to ascertain their effect on hematopoiesis. When activated and propagated with recombinant human interleukin-2 (rhIL-2) in vitro, SCID spleen cells maintained a phenotypic and lytic spectrum consistent with a pure population of activated NK cells. When added with syngeneic bone marrow cells (BMC) in soft agar, the activated NK cells could support hematopoietic growth in vitro without the addition of exogenous hematopoietic growth factors. However, when syngeneic BMC were added along with cytokines to produce optimal growth conditions, the addition of NK cells was then inhibitory for hematopoietic colony formation. Antibodies to interferon- gamma (IFN-gamma) partially reversed the inhibitory effects. Supernatants from the NK-cell cultures could also exert these effects on hematopoiesis, although to a lesser extent. Analysis of the NK cell RNA demonstrated that activated NK cells express genes for hematopoietic growth factors such as granulocyte-macrophage colony- stimulating factor (GM-CSF), granulocyte CSF (G-CSF), and IL-1 beta. The NK cells were also found to express IFN-gamma, transforming growth factor-beta 1 (TGF-beta 1), and tumor necrosis factor-alpha (TNF-alpha) mRNA. Analysis of the NK-cell supernatants using factor-dependent myeloid progenitor cell lines showed that the NK cells were producing G- CSF and growth-promoting activity that could not be attributed to IL-1, IL-3, IL-4, IL-5, IL-6, GM-CSF, G-CSF, macrophage CSF (M-CSF), or stem cell factor. The transfer of activated NK cells with BMC into lethally irradiated syngeneic mice resulted in greater BMC engraftment in the recipients. Thus, these results using a pure population of activated NK cells indicate that when activated, these cells can produce a variety of growth factors for hematopoiesis and exert significant hematopoietic growth-promoting effects in vivo.

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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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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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