The Anti-Leukemia Effect of Natural Killer-Derived Exosomes

Circulating NK cells are the effector arm of the innate immune system. As such, they recognize transformed cells as "non-self" and kill them. Numerous ex vivo studies have demonstrated the ability of NK cells to kill allogeneic leukemia cells. Based on these in vitro studies, several past and ongoing clinical trials have shown allogenic NK cells have a strong anti-leukemia effect. However, the use of NK cells as an "off the shelf" product is limited since they, like most cellular products, induce an allo-reactive immunogenic response which limits efficacy and increases toxicity. Exosomes are nano scaled extracellular vesicles that are released by various types of cells including NK cells. Since the exosomal cargo reflects, in part, the molecular makeup of its cell of origin, we hypothesized that NK-derived exosomes maintain the anti-leukemia effect of their cell of origin. To test this hypothesis, we exposed leukemia cells to NK-derived exosomes and tested their ability to eliminate them. As a source for NK-derived exosomes we used the NK-92MI cell line. This is a genetically altered cell line which constitutively expresses IL-2 that augments the cytotoxic activity of the cells and is routinely used in clinical trials. We cultured these cells in exosome free medium for 48 hours and extracted the exosomes by ultracentrifugation. Nanoparticle analyzing system showed that the pellet was enriched with the typical ~100nm sized vesicles and these particles were visualized by electron microscopy. Western immunoblotting confirmed that these particles express CD63, a known exosomal biomarker. Subsequently, we subjected CML K562, ALL Jurkat and AML HL-60 to NK-92MI-Exo stained with PKH-26 and showed by flow cytometry that these cells uptake the exosomes in a dose- and time- dependent manner. As controls, we used exosomes derived from the human embryonic kidney 293 (HEK-293) cell line. LDH release assay showed a marked increase in LDH activity in NK-92MI-Exo exposed leukemia cells but not in HEK-293-Exo exposed leukemia cells across all cell lines tested. Similarly, flow cytometry of leukemia cells double stained for annexin/PI showed that the rate of apoptosis was markedly increased in NK-92MI-Exo exposed leukemia cells but not in HEK-293-Exo exposed leukemia cells. Together, these assays indicate that NK-92MI-Exo are cytotoxic to various leukemia cell lines and that this effect is specific to NK-derived exosomes. Encouraged by our preliminary results, we harvested leukemia cells from 4 patients with acute myeloid leukemia (AML), 4 patients with acute lymphoblastic leukemia (ALL), 4 patients with chronic lymphocytic leukemia (CLL) and normal B-cells from healthy volunteers and exposed them to NK-92MI-Exo. Similar to the cell line results, we found a marked cytotoxic effect to NK-92MI-Exo on these leukemia cells but not on normal B-cells from healthy individuals whereas HEK-293-Exo had only a minimal or no effect on primary leukemia cells. Clonal assay on peripheral blood sample from 2 CML patients showed that the number of colony forming unit -granulocyte, erythrocyte, megakaryocyte monocyte (CFU-GEMM) is significantly reduced following exposure to NK-92MI-Exo, suggesting that these exosomes target leukemia progenitor cells. Proteomics analysis of NK-92MI-Exo revealed that similar to NK cytotoxic granules, the exosomal cargo include granozymes known to induce apoptosis of target cells but unlike NK cytotoxic granules the exosomal cargo does not include perphorines, known to perforate target cell membrane. Taken together our data suggests that NK-derived exosomes have a potent cytotoxic effect across a wide range of leukemia cells. This effect is specific to NK-exosomes. Furthermore, NK-derived exosomes preferentially kill leukemia cells but not normal cells. We also show that these exosomes are toxic to leukemia progenitor cells. Proteomics analysis suggested that NK-cytotoxic granules and exosomes use different strategies to enter target-cells. Whether NK-derived exosomes may become a-cellular therapeutic strategy to combat leukemia remains to be determined. Figure Disclosures No relevant conflicts of interest to declare.

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Influence of Btk Inhibitors on Antitumor Activity of Natural Killer Cells

Blood ◽

10.1182/blood.v124.21.2742.2742 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2742-2742

Author(s):

Marta Siernicka ◽

Kamil Bojarczuk ◽

Malgorzata Bobrowicz ◽

Michal Dwojak ◽

Beata Pyrzynska ◽

...

Keyword(s):

Flow Cytometry ◽

Clinical Trials ◽

Antitumor Activity ◽

Nk Cells ◽

Natural Killer ◽

Cytokine Secretion ◽

Target Cells ◽

Btk Inhibitor ◽

Hodgkin's Lymphomas ◽

Btk Inhibitors

Abstract Small molecule Bruton's tyrosine kinase (Btk) inhibitors are extensively studied in preclinical investigations and clinical trials in the treatment of hematological malignancies derived from B-cells. Ibrutinib, due to its safety and efficiency, has been recently approved for the treatment of patients with Chronic Lymphocytic Leukemia (CLL) who received at least one prior therapy. Moreover, another Btk inhibitor AVL-292 is currently tested in clinical trials in patients with B Cell Non-Hodgkin's Lymphomas, CLL and Waldenstrom Macroglobulinemia. Despite a huge therapeutic potential of Btk inhibitors we have recently demonstrated that both natural killer (NK) cells cytotoxicity and degranulation are significantly impaired upon ibrutinib treatment (Bojarczuk et al., Leukemia 2014). Since NK cells are effectors of innate immune system capable to kill tumor cells directly and in the mechanism of antibody dependent cell-mediated cytotoxicity (ADCC), which constitutes one of the major mechanism of monoclonal antibodies widely used in hematooncology, in our ongoing studies we are focused to determine in details the influence of various Btk inhibitors on NK cells cytotoxicity, degranulation, cytokine secretion and expression of activatory/inhibitory receptors. All experiments were performed fully in vitro using human primary NK cells isolated from PBMC of healthy donors as well as NK92 and NK92.CD16 cell lines. To determine cytolytic activity of NK cells CFSE/PI flow cytometry assay was used. Cytokine secretion and NK cells degranulation, evaluated as the expression of CD107a on the surface of NK cells, were determined with flow cytometry upon incubation with target cells for 4 h. Phenotype of NK cells pre-incubated with tested compounds was determined with flow cytometry using antibodies conjugated with fluorochromes. The initial results of our studies show that various Btk inhibitors differentially regulate NK cells antitumor activity. Ibrutinib and AVL-292 which covalently bind to cysteines at the position 481 significantly inhibit NK cells cytotoxicity. Interestingly, we have observed that pre-incubation of NK cells with ibrutinib as well as AVL-292 results in down-regulation of CD25 and CD69. This effect was not observed upon treatment of NK cells with CGI-1746, a reversible Btk inhibitor which blocks phosphorylation of BTK in Y551 and Y223. Expression of NK cells activatory receptors such as NKp30, NK44, NKp46, NKG2D, DNAM-1 and CD16 remained unchanged. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Identifying Mechanisms of Enhanced NK Cell Cytotoxicity Using Permanent NK Cell Lines.

Blood ◽

10.1182/blood.v104.11.3842.3842 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3842-3842

Author(s):

Garnet Suck ◽

Donald R. Branch ◽

Joanna Vergidis ◽

Soad Fahim ◽

Armand Keating

Keyword(s):

Flow Cytometry ◽

Nk Cells ◽

Cell Line ◽

Cell Lines ◽

Nk Cell ◽

Active Form ◽

The Other ◽

Alternative Activation ◽

Target Cells ◽

Nk Cytotoxicity

Abstract Although NK cells are promising candidates for adoptive immunotherapy and at least one permanent cell line is in clinical trials, further studies evaluating efficacy and mechanisms of action are warranted. As a first step towards identifying the most potent effector cells, we investigated the molecular mechanisms of cytotoxicity of the three natural killer lines, KHYG-1, NK-92 and YT, and the NK-T cell line, SNT-8, under standardized culture conditions with human serum as the only serum source. We confirmed the previously established differential killing potential of the 4 cell lines against target K562 cells using a new method based on detecting Annexin V (+) target cells by flow cytometry. By labeling the NK cells with specific antibodies, the assay is designed to screen any target cell for NK cytotoxicity. In contrast to previous reports, we found KHYG-1 the most cytotoxic, followed by NK-92, SNT-8 and YT. Genotypic and transcriptional phenotypic analysis of the cell lines for killer cell Ig-like receptors (KIRs) by SSP-PCR showed that inhibitory KIRs outnumbered activating KIRs in all cases but did not explain the differential cytotoxicity. A correlation with cytotoxicity was found with expression of the activating type II C lectin-like receptor, NKG2D: KHYG-1, 99%+; NK-92, 91%+; SNT-8, 6%+ and YT, 2%+. Moreover, the ITAM-bearing adaptor molecule DAP12, involved in the alternative activation signaling pathway via NKG2C-CD94 and activating KIRs, was detected only for KHYG-1 by immunoblotting, These data suggest that the superior cytotoxicity of KHYG-1 may be due, in part, to the additional activation of this alternative pathway that is not triggered in the other lines. The downstream signaling molecules involved in NK cytotoxicity, including the tyrosine phosphatases SHP-1, SHP-2 and SHIP-1 (inhibitory), as well as SHIP-2, the tyrosine kinases ZAP-70, Syk, PI3K and the MAP kinase phospho-ERK-2 (activating) were compared among the lines by immunoblotting followed by densitometry normalized to b-actin or ERK-2 for phospho-ERK-2. We found that the activating kinase Syk was expressed only in NK-92 and KHYG-1 at even higher levels. Also, phospho-ERK-2, was hyperphosphorylated only in KHYG-1. Perforin, granzyme A and granzyme B, present in cytotoxic granules, were compared by RT-PCR and intracellular flow cytometry and/or immunoblotting. Perforin was found to be almost exclusively fully processed to the active 60 kD form only in KHYG-1, in contrast to the other lines, which displayed approximately half the levels of the active form. These data provide a further explanation for the superior cytotoxicity of KHYG-1 and demonstrate the value of comparing cell lines with diverse cytotoxic potential as a means of elucidating cell killing mechanisms. It is conceivable that targeted modifications to the signaling pathways for cytotoxicity in this model will lead to the generation of activated NK cells with even greater efficacy.

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Natural killer-mediated lysis of normal and malignant target cells, and its regulation by monocytes.

Journal of Experimental Medicine ◽

10.1084/jem.162.2.472 ◽

1985 ◽

Vol 162 (2) ◽

pp. 472-486 ◽

Cited By ~ 27

Author(s):

K Oshimi ◽

Y Oshimi ◽

M Satake ◽

H Mizoguchi

Keyword(s):

Nk Cells ◽

Natural Killer ◽

B Lymphocytes ◽

Leukemia Cells ◽

Target Cells ◽

Large Granular Lymphocytes ◽

Percoll Density Gradient ◽

Granular Lymphocytes ◽

Density Gradient Sedimentation

After depletion of monocytes, natural killer (NK) cells were partially purified from peripheral blood by Percoll density gradient sedimentation. The NK cells were then cultured for 1 d and assayed for their cytotoxicity against various types of normal and malignant target cells. All types of target cells tested were found to be susceptible to NK cells. The susceptible targets were autologous T and B lymphocytes, mitogen-induced T and B blasts, monocytes, large granular lymphocytes, autologous or allogeneic lymphoma and leukemia cells isolated from patients, and cultured cell lines, including those resistant to interferon-activated lymphocytes. Such a broad spectrum of cytotoxicity was demonstrated in 1 d of culture, and freshly prepared NK cells were not cytotoxic, or, if anything, were less cytotoxic. Monocytes and their supernatants, added throughout the course of culture, markedly inhibited the development of their cytotoxicity. These results may suggest that, although NK cells having ability to lyse autologous normal and malignant target cells are present in vivo, their lytic activity is regulated by coexisting monocytes.

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Immune Function in Follicular Lymphoma (FL): Comparison of Patient with Her Healthy Identical Twin Reveals Enhanced NK Cell Responses in the Patient During Spontaneous Remission [KC1].

Blood ◽

10.1182/blood.v114.22.5012.5012 ◽

2009 ◽

Vol 114 (22) ◽

pp. 5012-5012

Author(s):

Elena Gitelson ◽

Alexander W. MacFarlane ◽

Kerry S Campbell ◽

R. Katherine Alpaugh ◽

Tahseen I. Al-Saleem ◽

...

Keyword(s):

Nk Cells ◽

Cell Line ◽

Peripheral Blood ◽

Partial Remission ◽

Nk Cell ◽

Identical Twin ◽

Effector Memory ◽

Target Cells ◽

Tumor Target ◽

Memory Phenotype

Abstract Abstract 5012 Identical twins are an excellent model in which to study tumor-specific immune responses (Gitelson et al Br J Haem 2002) as it can be postulated that the immune systems are identical. Spontaneous remissions in FL occur but the immunologic mechanisms remain elusive. We investigated adaptive and innate immunologic phenotypes and responses in a 41-year old patient with untreated grade 1 FL, and her healthy identical twin sister. Patients and methods The patient has had a waxing and waning course of FL over 7 years and currently is in spontaneous partial remission (> 50% reduction of generalized lymphadenopathy) and her peripheral blood was negative for t(14:18) by PCR at the time of this analysis. Immunologic responses of her twin, as well as another set of healthy identical twin sisters, were investigated as controls. We studied peripheral blood using 8-color multiparametric flow cytometry for frequencies and phenotypes of NK and regulatory T cells (Tregs). FOXP3+ cells were further analyzed for naïve, central memory and effector memory phenotype. Activation of NK cells and degranulation in response to tumor target cells as measured by Lysosomal-Associated Membrane Protein 1 (LAMP-1) surface expression were investigated. An erythroblastoid cell line (K562) and an EBV transformed MHC-I deficient lymphoblastoid cell line (721.221) were used as targets. Results NK studies revealed that activated CD69+ NK cells were increased in percentage in FL patient (4.43%) compared to her healthy twin (1.82%), and had increased mean fluorescence intensity (MFI) of 1057 vs 357, respectively. In contrast, almost identical frequencies and MFI of activated NK cells were found in the set of healthy twins (0.55% vs 0.54% and 153 vs 153, respectively). NK cells of FL patient exhibited elevated degranulation compared to the healthy twin in response to stimulation by either target cell: LAMP-1 staining MFI 1907 vs 1395, respectively for 721.221 target cells and 1394 vs 1122, respectively for K562 cells, whereas no difference was detected in non-stimulated NK cells (MFI 536 vs 506, respectively). In addition, Killer Cell Immunoglobulin-like Receptor (KIR) analysis revealed a deficit in the percentage of NK cells staining with an antibody recognizing KIR2DL1/S1 in the patient, but not in her healthy twin (7.0% vs 15.1% of NK cells, respectively), whereas no other differences in KIR receptor expression profiles were found for other KIR or in comparing the set of healthy twins. Analysis of frequencies of circulating Tregs revealed no difference between FL patient and her healthy twin: CD4+CD25+FOXP3+ cells represented 4.92% vs 5.78%, respectively of total CD4 cells and CD8+CD25+FOXP3+ cells represented 1.25% vs 1.37%, respectively of total CD8 cells. Analysis of T cell subsets revealed that Tregs in FL patient and in her twin were mainly of effector memory phenotype: CCR7-/CD45RA-/CD45RO+ (82.6% vs 74.4%, respectively) and central memory phenotype: CCR7+/CD45RA-/CD45RO+ (10.7 vs 14.3%, respectively). Conclusions NK cell studies of FL patient revealed increased numbers of activated CD69+ NK cells which correlated with increased degranulation response to tumor target cells and a deficit in the NK cell repertoire, as noted by the deficiency in NK cells expressing KIR2DL1/S1 in the patient compared to the healthy twin, while no differences in frequencies of circulating Tregs or Treg phenotypes were identified. Although the current results are based upon a single sampling, due to limited availability, the observed potentiation of NK cell activity in the patient with FL suggest a potentially important role of NK cells in tumor control during spontaneous partial remission. Follow-up studies of persisting remission, temporary flare-up or at disease progression will be of interest. Disclosures No relevant conflicts of interest to declare.

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Fc-Engineered NKG2D-IgG1 Fusion Proteins Target Leukemia Cells for Antibody-Dependent Cellular Cytotoxicity (ADCC) of NK Cells

Blood ◽

10.1182/blood.v118.21.1537.1537 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1537-1537 ◽

Cited By ~ 1

Author(s):

Julia Hilpert ◽

Katrin Baltz-Ghahremanpour ◽

Benjamin J Schmiedel ◽

Lothar Kanz ◽

Gundram Jung ◽

...

Keyword(s):

Nk Cells ◽

Fusion Proteins ◽

Nk Cell ◽

Conflicts Of Interest ◽

Leukemia Cell ◽

Leukemic Cells ◽

Leukemia Cells ◽

Target Cells ◽

Cellular Cytotoxicity ◽

Antibody Dependent Cellular Cytotoxicity

Abstract Abstract 1537 The capability of anti-tumor antibodies to recruit Fc-receptor (FcR) bearing effector cells like NK cells, a feature considered critical for therapeutic success, can be markedly improved by modifications of the human IgG1 part. At present, Fc-engineered antibodies targeting leukemia cells are yet not available. The various ligands of the NK cell-activating immunoreceptor NKG2D (NKG2DL) are generally absent on healthy cells but upregulated on malignant cells of various origins including leukemia. We aimed to take advantage of the tumor-restricted expression of NKG2DL by using them as target-antigens for Fc-optimized NKG2D-IgG1 fusion proteins targeting leukemia cells for antibody-dependent cellular cytotoxicity (ADCC) and IFN-g production of NK cells. NKG2D-IgG1 fusion proteins with distinct modifications in their Fc portion were generated as previously described (Lazar 2006; Armour 1999). Compared to wildtype NKG2D-Fc (NKG2D-Fc-WT), the mutants (S239D/I332E and E233P/L234V/L235A/DG236/A327G/A330S) displayed highly enhanced (NKG2D-Fc-ADCC) and abrogated (NKG2D-Fc-KO) affinity to the NK cell FcgRIIIa receptor but comparable binding to NKG2DL-expressing target cells. Functional analyses with allogenic NK cells and leukemia cell lines as well as primary leukemic cells of AML and CLL patients revealed that NKG2D-Fc-KO significantly (p<0.05, Mann-Whitney U test) reduced NK cytotoxicity and IFN-g production (about 20% and 30% reduction, respectively), which can be attributed to blockade of NKG2DL-mediated activating signals. Treatment with NKG2D-Fc-WT significantly (p<0.05, Mann-Whitney U test) enhanced NK reactivity (about 20% and 100% increase in cytotoxicity and cytokine production, respectively). The effects observed upon treatment with NKG2D-Fc-ADCC by far exceeded that of NKG2D-Fc-WT resulting in at least doubled NK ADCC and IFN-g production compared to NKG2D-Fc-WT. When applied in combination with Rituximab in analyses with CLL cells, a clear additive effect resulting in a more than four-fold increase of ADCC and FcgRIIIa-induced IFN-g production was observed. The NKG2D-Fc fusion proteins did not induce NK reactivity against healthy blood cells, which is in line with the tumor-restricted expression of NKG2DL. Of note, treatment with NKG2D-Fc-ADCC also significantly (p<0.05, Mann-Whitney U test) enhanced reactivity (up to 70% increase) of NK cells against NKG2DL-positive AML and CLL cells among patient PBMC in an autologous setting. Together, our results demonstrate that Fc-engineered NKG2D-Fc-ADCC fusion proteins can effectively target NKG2DL-expressing leukemia cells for NK anti-tumor reactivity. In line with the hierarchically organized potential of the various activating receptors governing NK reactivity and due to their highly increased affinity to the FcgRIIIa receptor, NKG2D-Fc-ADCC potently enhances NK anti-leukemia reactivity despite the inevitable reduction of activating signals upon binding to NKG2DL. Due to the tumor-restricted expression of NKG2DL, Fc-modified NKG2D-Ig may thus constitute an attractive means for immunotherapy of leukemia. Disclosures: No relevant conflicts of interest to declare.

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Expression and Immunomodulatory Function of RANKL in Leukemia

Blood ◽

10.1182/blood.v118.21.245.245 ◽

2011 ◽

Vol 118 (21) ◽

pp. 245-245

Author(s):

Benjamin J Schmiedel ◽

Tina Baessler ◽

Miyuki Azuma ◽

Lothar Kanz ◽

Helmut R. Salih

Keyword(s):

Nk Cells ◽

Cell Function ◽

Nk Cell ◽

Conflicts Of Interest ◽

Immune Surveillance ◽

Leukemia Cells ◽

Target Cells ◽

Cytotoxic Lymphocytes ◽

Hematopoietic Malignancies ◽

Immunomodulatory Function

Abstract Abstract 245 The TNF family member RANKL and its receptors RANK and osteoprotegerin (OPG) are key regulators of bone remodelling, but have also been shown to influence progression of malignancies like breast cancer (Tan et al., Nature 2011), myeloma (Sordillo et al., Cancer 2003) and CLL (Secchiero et al. J Cell Physiol. 2006). NK cells are cytotoxic lymphocytes that play an important role in tumor immune surveillance especially of hematopoietic malignancies. Their reactivity is influenced by a variety of activating and inhibitory molecules expressed by their target cells including several members of the TNF family. Recently, we reported that RANK, upon interaction with RANKL which can be expressed by malignant hematopoietic cells, mediates signals that impair NK reactivity (Schmiedel et al., Blood 2010 116,21:893–893). Here we extended these analyses and comprehensively studied the expression and immunomodulatory function of RANKL in leukemia. Analysis of primary leukemia cells revealed substantial RANKL surface expression in a high proportion of the investigated cases (AML, 47 of 65 (72%); ALL, 16 of 21 (76%); CML, 6 of 10 (60%); CLL, all 54 (100%)). Signaling via surface-expressed RANKL into the malignant cells mediated the release of cytokines like TNF, IL-6, IL-8 and IL-10 which have been shown to act as autocrine and paracrine growth and survival factors in leukemia. Moreover, the factors released upon RANKL signaling upregulated RANK expression on NK cells. In line, NK cells from leukemia patients (n=75) displayed significantly (p<0.001, Mann-Whitney U-test) higher RANK expression compared to healthy controls (n=30) confirming our notion that RANK-RANKL interaction may contribute to leukemia pathophysiology. We further found that RANK-RANKL interaction, beyond directly inhibiting NK cell function via RANK, may contribute to evasion of leukemia cells from NK immunosurveillance by creating an NK inhibitory cytokine milieu. This was revealed by impaired cytotoxicity and degranulation in response to leukemia targets following exposure of the NK cells to the factors released upon RANKL signaling by leukemia cells. Notably, the RANKL-mediated cytokine release of leukemia cells could be disrupted by the clinically approved RANKL antibody Denosumab/AMG162. Thus, RANKL signaling may trigger a “vicious cycle” comprising of release of immunosuppressive cytokines and also upregulation of RANK on NK cells. The latter both directly inhibits NK reactivity and may result in augmented RANKL signaling into leukemia cells. Our data suggest that therapeutic modulation of the RANK/RANKL system e.g. with Denosumab/AMG162, which is approved for treatment of osteolysis, may be a promising strategy to reinforce NK reactivity against hematopoietic malignancies. 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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Panobinostat Induces Upregulation of CD38 and Augments the Anti-Myeloma Efficacy of Daratumumab in Pre-Clinical Models

Blood ◽

10.1182/blood.v128.22.4481.4481 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4481-4481 ◽

Cited By ~ 1

Author(s):

Estefania Garcia-Guerrero ◽

Tea Gogishvili ◽

Sophia Danhof ◽

Martin Schreder ◽

Celine Pallaud ◽

...

Keyword(s):

Multiple Myeloma ◽

Flow Cytometry ◽

Cell Line ◽

Myeloma Cell ◽

Target Cells ◽

Myeloma Cell Line ◽

Myeloma Cells ◽

Cd38 Expression ◽

Clinical Models ◽

Pre Treatment

Abstract Background: Immunotherapy with monoclonal antibodies (mAbs) has recently entered the clinical arena in multiple myeloma, including Daratumumab that targets CD38 on malignant plasma cells. The efficacy of mAbs depends on antigen density and expression of accessory ligands on target cells to initiate cell- and complement-dependent effector mechanisms. Here, we investigate the use of the histone deacetylase inhibitor (HDACi) Panobinostat to modulate target antigen expression and ligand profile on myeloma in favor of potent mAb-mediated recognition and destruction. We show that Panobinostat augments CD38 expression specifically on myeloma cells and demonstrate powerful synergy with anti-CD38 mAb Daratumumab in pre-clinical models. Methods: The myeloma cell line MM1.S and primary myeloma cells were treated with titrated doses of Panobinostat (0, 10, 25 nM) and expression of CD38 and a panel of additional target molecules including B-cell maturation antigen (BCMA) and SLAMF7, as well as accessory ligands analyzed by flow cytometry at 24, 48 and 72 hours. Antibody-dependent cellular cytotoxicity (ADCC) against Panobinostat treated and untreated myeloma cells was analyzed at 4 and 20 hours after addition of PBMC at an effector to target ratio of 25:1 in the presence of Daratumumab (1, 10, 50 ug/mL) or an isotype control antibody. Results: We first treated the myeloma cell line MM1.S with Panobinostat and analyzed its direct cytotoxic anti-myeloma effect. Consistent with previous work, the percentage of live MM1.S myeloma cells had decreased to 85% and 50% after 48 hours of exposure to 10 and 25 nM respectively. We analyzed expression of CD38 on residual live, i.e. 7-AAD negative MM1.S cells by flow cytometry and observed a 1.5 (10 nM) and 2-fold (25 nM) increase of CD38 expression by mean fluorescence intensity (MFI) compared to baseline levels and untreated control cells. The increase in CD38 expression was already detectable after 24 hours and plateaued between 48 and 72 hours. We confirmed our observation in primary myeloma cells from multiple donors (n=4) and detected an even stronger increase to 2 (10 nM) and 4-fold (25 nM) higher CD38 expression compared to untreated cells at 48 hours. Interestingly, expression of BCMA and SLAMF7 was not increased after Panobinostat treatment at all tested concentrations and time points in both MM1.S and primary myeloma. We confirmed that Panobinostat-induced upregulation of CD38 specifically occurred in myeloma, and neither observed this phenomenon in a panel of leukemia and lymphoma cell lines including Raji (Burkitt) and JeKo-1 (mantle cell), nor on resting/activated primary CD8+ and CD4+ T cells that we isolated from peripheral blood of several donors (n=3). Next, we were interested in determining whether the increase in CD38 expression enabled superior anti-myeloma activity of the anti-CD38 mAb Daratumumab. Panobinostat pre-treatment was done for 48 hours at 10 nM as this is a clinically achievable serum level with currently approved regimens. Indeed, significantly higher ADCC was mediated by Daratumumab at all tested concentrations (1, 10 and 50 ug/mL) against MM1.S that we had exposed to Panobinostat. At 4 hours, ADCC was 45% and 25% in Panobinostat-treated and untreated MM1.S respectively, and at 20 hours, near-complete, >90% ADCC of Panobinostat-pre-treated MM1.S had occurred, whereas only 65% of MM1.S were eliminated by Daratumumab without Panobinostat pre-treatment. These data were confirmed in multiple experiments with MM1.S and PBMC from different donors, and with primary myeloma cells. Experiments to evaluate synergy of Panobinostat and Daratumumab therapy in a xenograft model (NSG/MM1.S) are ongoing. Conclusions: Our data demonstrate that the HDACi Panobinostat induces upregulation of CD38 on myeloma and a subsequent dramatic increase of Daratumumab-mediated ADCC in pre-clinical models. These data suggest that Panobinostat could be used synergistically with Daratumumab in a clinical setting to increase response rates and extend duration of responses to Daratumumab. Panobinostat has a known ability to modulate the transcriptional profile of myeloma cells and our data demonstrate for the first time that this ability can be utilized to augment the therapeutic index of antibody-based immunotherapy in multiple myeloma. Disclosures Pallaud: Novartis: Employment. Lehmann:Novartis: Employment. Hudecek:Novartis: Research Funding.

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The Addition of Interleukin (IL)-15 to ANTI-CD3, IL-2 and FLT-3 Results in Significant Increase in Expansion, Activation, Proliferation and Survival of Cord Blood (CB) NK Cells and Cytotoxic T-Lymphocyte (CTL) Subsets: Potential for Adoptive Cellular Immunotherapy (ACI).

Blood ◽

10.1182/blood.v104.11.4952.4952 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4952-4952

Author(s):

Prakash Satwani ◽

Allison F. O’Neill ◽

Janet Ayello ◽

Lynn Simpson ◽

Dustin Cairo ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Proliferation ◽

Nk Cells ◽

Mononuclear Cells ◽

Nk Cell ◽

Colorimetric Assay ◽

Annexin V ◽

Cellular Immunotherapy ◽

Target Cells ◽

Color Flow

Abstract A limitation of CB as a source for ACI is the limited number of immunoeffector T and NK cell subsets. IL-15 is an important lymphokine regulating NK activity, T-cell proliferation and CTL activities. We have previously demonstrated reduced IL-15 mRNA expression and protein production from CB which may in part contribute to the immaturity of CB cellular immunity (Qian/Cairo et al, Blood 1997). NK cells are characterized by NK receptors which exist in both activatory and inhibitory forms (Moretta et al, Ann Rev Immun 2001). We have previously demonstrated the ex vivo expansion, maturation and activation of fresh CB derived T lymphocytes with anti-CD3, IL-2, IL-12 and IL-7 (Robinson/Cairo et al, Exp Hem 2001). For this study, we compared the expansion, proliferation, activation and survival of CB mononuclear cells (MNC) with IL-2, anti-CD3, FLT-3 ± IL-15. CB MNC were isolated by Ficoll density centrifugation and incubated overnight in AIM-V media. The non-adherent MNC fraction was cultured in AIM-V media alone and with IL-2 (5ng/ml), anti-CD3 (50ng/ml), FLT-3 (50ng/ml) and IL-15 (10 &100ng/ml) for 48 hrs. Cell proliferation was determined by the colorimetric assay system using WST-1. Expression of T & NK cell subsets were analyzed by three color flow cytometry using CD3, CD8, CD4, CD25, CD45RO, CD16, CD56, CD158a, CD158b, NKB1, CD94 & NKG2A monoclonal antibodies. Apoptosis markers were determined by measuring Annexin-V & PI by flow cytometry. Cytotoxicity was determined by europium release assay using K562 & Daudi target cells at 20:1 E:T ratio. Expansion of lymphocyte subsets of CD4+/25+, CD8+/25+, CD3+/45RO+ & CD94/NKG2A was significantly increased with IL-2, anti-CD3, FLT-3 + IL-15(100ng/ml) compared to IL-2, anti-CD3, FLT-3 and compared to media (CD4+/25+: 67.2±1.2 vs 39.19±5.3 vs 3.54±0.3%, p<0.01; CD8+/25+: 57.8±8.6 vs 16.94±0.3 vs 0.99±0.4%, p<0.01; CD3+/45RO+: 77.13±1.0 vs 29.96±1.9 vs 6.69±0.3%, p<0.001; CD94/NKG2A: 3.4±0.68 vs 0.29±0.092 vs 0.23±0.09%, p<0.01, respectively). We also noted a significant increase in CD3−/16+/56+ with IL-2, anti-CD3, FLT-3 + IL-15(100ng/ml) compared to media alone (p<0.01). There was no significant increase in expansion of CD3+/16+/56+, CD3−/56+/158a, CD3−/56+/158b or CD3−/56+/NKB1 in various culture conditions described above. IL-2, anti-CD3, FLT-3 + IL-15(100ng/ml) resulted in significant improvement in cell survival compared to media alone (Annexin-V staining) (3.3±0.26 vs 15.51±3.3%, p<0.05). We also noted increase in cell proliferation with IL-2, anti-CD3, FLT-3 + IL-15(100ng/ml) as compared to IL-2, anti-CD3, FLT-3 vs. media alone (1.11±0.09 vs 0.82±0.01 vs 0.69±0.03 OD, p<0.05). We also noted significant increase in cytotoxicity with IL-2, anti-CD3, FLT-3 + IL-15(100ng/ml) as compared to IL-2, anti-CD3, FLT-3 vs media alone (K562–57.81±8.1 vs 17.5±1.4 vs 2.68±0.6%, p<0.01; Daudi-79.8±1.7 vs 23.69±1.5 vs 6.38±0.6, p<0.001) The combination of IL-2 + anti-CD3 + FLT-3 + IL-15 appears to enhance expansion and cytotoxicity of CB CTL and NK subsets in short term culture in part secondary to inhibition of apoptosis. Future studies are warranted to determine if the combination of anti-CD3 + IL-2 + FLT-3 + IL-15 can enhance CB CTL and NK cells for ACI to induce GVHM effects following UCBT.

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Natural Killer Cells with NKDG2D Cytotoxicity but NKp30-Dull Phenotype Predominate in Myelodysplastic Syndrome (MDS).

Blood ◽

10.1182/blood.v106.11.3436.3436 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3436-3436

Author(s):

Fanqi Bai ◽

Jeffrey S. Painter ◽

Cantor Alan ◽

Zou JianXiang ◽

Sheng Wei ◽

...

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Cell Line ◽

Cell Lines ◽

Nk Cell ◽

B Cell Lymphoma ◽

Target Cells ◽

Nkg2d Ligands ◽

Human Stress ◽

Normal Controls

Abstract Natural Killer (NK) function in patients with MDS as measured by non-MHC-restricted cytotoxicity and activation-dependent cell cytotoxicity (ADCC) are reduced in patients with MDS, however, the mechanisms of the functional impairment are not known. Tumor cytolysis occurs through orchestrated control by inhibitory NK receptors (NKRs) and activating NKRs, which control signaling events that lead to polarized movement of perforin-containing granules toward the NK-tumor contact area. We found that NK cells from 23 out of 35 patients with MDS (66%) displayed reduced lysis of K562 tumor cells compared to age-matched normal controls (p<0.01). To better characterize this defect, we evaluated patient NK function against differential tumor targets including the MDS1 cell line established from an MDS patients. We found that MDS1 incited non-MHC-restricted lysis. Unactivated PBMCs, unactivated NK cells, NK cell lines (NK92 and NKL) but not purified unactivated T cells from normal donors killed MDS1 in 4-hr 51Cr-release assays. Normal NK cells and NK cell lines were also found to rapidly redistrubute perforin granules after exposure to MDS1suggesting that a perforin-dependent lytic pathway was activated. We then performed simultaneous cytolytic assays with K562, MDS1, and the 721.221 B cell lymphoma cell line as target cells. We found that NK cells from MDS patients had greater lytic activity against MDS1 (average 24% vs. average 8% at 50:1 Effector:Target ratio, respectively, p<0.01) Antibody-blocking experiments demonstrated that the NKL cell line and PBMCs from 8 out of 10 MDS patients predominantly used the NKG2D activating receptor to kill MDS1. Consistent with this finding, we showed that MDS1 cells express the major human stress-inducible endogenous proteins MICA and MICB, which are NKG2D ligands. In contast, lysis by NK92 cells and normal PBMCs was not appreciably reduced by NKG2D blocking antibodies suggesting that other unidentified NKR(s) also mediate lysis. To identify the NKRs expressed in MDS patients, we performed immunophenotyping for both the activating NKRs and inhibitory NKRs compared to age-matched normal controls. We found that two activating receptors, NKp30 and CD244 (2B4), were significantly reduced on NK cells from all MDS patients regardless of their ability to lyse NK targets. Inhibitory NKR expression and function were normal. Interestingly, NKG2D expression correlated with reduced cytolytic function. Similar to studies on normal NK cells with low NKp30 and NKp46 (NCRdull) phenotypes, these results suggest that low NKp30 expression leads to predominant NKG2D utilization for tumor cell lysis, which is reduced in MDS patients with defective NK function. Our findings provide critical information about potential importance for immunosurviellance through NKG2D-NKG2D ligands.

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