Pluripotent Stem Cell-Derived Human Natural Killer Cells with Potent Anti-Multiple Myeloma Activity,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4034-4034
Author(s):  
David A. Knorr ◽  
Zhenya Ni ◽  
Allison Bock ◽  
Vijay G. Ramakrishnan ◽  
Shaji Kumar ◽  
...  
Keyword(s):  
Stem Cells ◽  
Multiple Myeloma ◽  
Stem Cell ◽  
Nk Cells ◽  
Natural Killer ◽  
Tumor Cells ◽  
Adoptive Immunotherapy ◽  
Target Cell ◽  
Peripheral Blood ◽  
Nk Cell

Abstract Abstract 4034 Natural Killer (NK) cells are lymphocytes of the innate immune system with anti-viral and anti-cancer activity. Over the past decade, they have gained interest as a promising cellular source for use in adoptive immunotherapy for the treatment of cancer. Most notably, NK cells play an important role in the graft-vs-tumor effect seen in allogeneic hematopoietic stem cell transplantation (allo-HSCT), and a better understanding of NK cell biology has translated into improved transplant outcomes in acute myelogenous leukemia (AML). Small studies have demonstrated a role for NK cell activity in multiple myeloma (MM) patients receiving allo-HSCT. Investigators have also utilized haplo-identical killer immunoglobulin-like receptor (KIR) mismatched NK cells for adoptive immunotherapy in patients with multiple myeloma (MM). Our group has focused on the development of NK cells from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) as a novel starting source of lymphocytes for immunotherapy. We have previously demonstrated potent anti-tumor activity of hESC-derived NK cells in vitro and in vivo against a variety of different targets. We have also shown that iPSC-derived NK cells from a variety of different somatic cell starting sources posses potent anti-tumor and anti-viral activity. Here, we demonstrate hESC- and iPSC-derived NK cell development in a completely defined, feeder-free system that is amenable to clinical scale-up. These cultures contain a pure population of mature NK cells devoid of any T or B cell contamination, which are common adverse bystanders of cellular products isolated and enriched from peripheral blood. Our cultures are homogenous for their expression of CD56 and express high levels of effector molecules known to be important in anti-MM activity, including KIR, CD16, NKG2D, NKp46, NKp44, FasL and TRAIL. We have now tested the activity of hESC- and iPSC-derived NK cells against MM tumor cells in order to provide a universal source of lymphocytes for adoptive immunotherapy in patients with treatment refractory disease. We find that similar to peripheral blood NK cells (PB-NK), hESC- and iPSC-derived NK cells are cytotoxic against 3 distinct MM cell lines in a standard chromium release cytotoxicity assay. Specifically, activated PB-NK cells killed 48.5% of targets at 10 to 1 effector to target ratios, whereas hESC (46.3%) and iPSC (42.4%) derived NK cells also demonstrated significant anti-MM activity. Also, hESC- and iPSC-derived NK cells secrete cytokines (IFNγ and TNFα) and degranulate as demonstrated by CD107a surface expression in response to MM target cell stimulation. When tested against freshly isolated samples from MM patients, hESC- and IPSC-derived NK cells respond at a similar level as activated PB-NK cells, the current source of NK cells used in adoptive immunotherapy trials. These MM targets (both cell lines and primary tumor cells) are known to express defined ligands (MICA/B, DR4/5, ULBP-1, BAT3) for receptors expressed on NK cells as well as a number of undefined ligands for natural cytotoxicity receptors (NCRs) and KIR. As these receptor-ligand interactions drive the anti-MM activity of NK cells, we are currently evaluating expression of each of these molecules on the surface of both the effector and target cell populations. Not only do hESC- and iPSC-derived NK cells provide a unique, homogenous cell population to study these interactions, they also provide a genetically tractable source of lymphocytes for improvement of the graft-vs-myeloma effect and could be tailored on a patient specific basis using banks of hESC-or iPSC-derived NK cells with defined KIR genotypes for use as allogeneic or autologous effector cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The PD-1/PD-L1 axis modulates the natural killer cell versus multiple myeloma effect: a therapeutic target for CT-011, a novel monoclonal anti–PD-1 antibody

Blood ◽  
2010 ◽  
Vol 116 (13) ◽  
pp. 2286-2294 ◽  
Author(s):  
Don M. Benson ◽  
Courtney E. Bakan ◽  
Anjali Mishra ◽  
Craig C. Hofmeister ◽  
Yvonne Efebera ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Immune Response ◽  
Nk Cells ◽  
Natural Killer ◽  
Tumor Cells ◽  
Cell Function ◽  
Nk Cell ◽  
Killer Cell ◽  
Cell Immune Response ◽  
Cell Versus

Abstract T-cell expression of programmed death receptor-1 (PD-1) down-regulates the immune response against malignancy by interacting with cognate ligands (eg, PD-L1) on tumor cells; however, little is known regarding PD-1 and natural killer (NK) cells. NK cells exert cytotoxicity against multiple myeloma (MM), an effect enhanced through novel therapies. We show that NK cells from MM patients express PD-1 whereas normal NK cells do not and confirm PD-L1 on primary MM cells. Engagement of PD-1 with PD-L1 should down-modulate the NK-cell versus MM effect. We demonstrate that CT-011, a novel anti–PD-1 antibody, enhances human NK-cell function against autologous, primary MM cells, seemingly through effects on NK-cell trafficking, immune complex formation with MM cells, and cytotoxicity specifically toward PD-L1+ MM tumor cells but not normal cells. We show that lenalidomide down-regulates PD-L1 on primary MM cells and may augment CT-011's enhancement of NK-cell function against MM. We demonstrate a role for the PD-1/PD-L1 signaling axis in the NK-cell immune response against MM and a role for CT-011 in enhancing the NK-cell versus MM effect. A phase 2 clinical trial of CT-011 in combination with lenalidomide for patients with MM should be considered.

scholarly journals Chemiluminescence response of human natural killer cells. I. The relationship between target cell binding, chemiluminescence, and cytolysis.

1982 ◽  
Vol 156 (2) ◽  
pp. 492-505 ◽  
Author(s):  
S L Helfand ◽  
J Werkmeister ◽  
J C Roder
Keyword(s):  
Tumor Cell ◽  
Nk Cells ◽  
Natural Killer ◽  
Tumor Cells ◽  
Cell Lines ◽  
Target Cell ◽  
Nk Cell ◽  
Target Antigen ◽  
Target Cells ◽  
Tumor Cell Lines

The binding of tumor cells or fetal fibroblasts to human natural killer (NK) cells led to a rapid chemiluminescence response within seconds of target-effector interaction. The degree of chemiluminescence was dependent on the concentration of NK-enriched lymphocytes or target cells, and plasma membrane vesicles from K562 also induced a chemiluminescence response. Mild glutaraldehyde treatment of effector cells abrogated their ability to generate chemiluminescence, whereas K562 target cells treated in the same way were almost fully able to induce a chemiluminescence response to NK-enriched lymphocytes. These results show a directionality of response with NK as the responders and tumor cells as the stimulators. A survey of eight different tumor cell lines and fetal fibroblast lines revealed a striking correlation (r greater than 0.93, P less than 0.001) between the ability of a given line to bind to NK-enriched lymphocytes, induce chemiluminescence, and to be lysed. Three differentiated sublines of K562 grown in butyrate and cloned induced little chemiluminescence compared with the K562 parent, and they were selectively resistant to NK-mediated binding and cytolysis. In addition, treatment of K562 cells with higher concentrations of glutaraldehyde for longer periods led to varying degrees of target antigen preservation, as measured in cold target competition assays and in conjugate formation. The degree of NK target antigen preservation correlated directly with the ability of the cells to induce chemiluminescence (r greater than 0.95). The degree of NK activation was also important because interferon-pretreated effectors generated more chemiluminescence upon stimulation with K562 or MeWo targets. Monocytes or granulocytes did not contribute to the chemiluminescence induced by NK-sensitive targets. Some NK-resistant tumor cell lines were sensitive to monocyte-mediated cytolysis and also induced chemiluminescence in monocytes but not NK cells. These results show that the target structures recognized by the NK cell may play a role in NK activation because the degree of chemiluminescence was directly proportional to the ability of a given target cell line to bind to the NK cell and to be lysed.

scholarly journals Autologous Activated and Expanded Natural Killer Cells Are Safe and Clinically Actives in Multiple Myeloma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1856-1856
Author(s):  
Alejandra Leivas ◽  
Antonio Pérez-Martínez ◽  
María Jesús Blanchard ◽  
Estela Martín Clavero ◽  
Dario Campana ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Multiple Myeloma ◽  
Phase I ◽  
Nk Cells ◽  
Disease Progression ◽  
Natural Killer ◽  
Peripheral Blood ◽  
Nk Cell ◽  
Phase I Clinical Trial ◽  
End Products

Abstract Multiple myeloma (MM) remains an incurable disease, despite that it has had a huge increase in survival in part due to new drugs as proteasome inhibitors and immunomodulatory drugs; however new therapeutic venues are required. Immune-based therapies are having an important relevance to control cancer, and are a new therapeutic armamentarium. Natural killer (NK) cells have an important role as natural control of tumor cells; based on that, NK cell infusions could be a novel treatment strategy to treat MM. By co-culture with the genetically modified cell line K562-mb15-41BBL it is possible to expand ex vivo large numbers of activated NK (NKAE) cells from MM patients. NK cell therapy has some challenges to be answered in real clinical practice: Could they be used out of transplantation setting? Could they be used with other anti-myeloma drugs? Could they be infused and expanded several times? To answer these questions we have designed a phase I clinical trial to make multiple infusions of autologous NKAE cells together with anti-myeloma drugs bortezomib or lenalidomide in MM (NCT02481934). Five MM patients on 2nd or later relapse have been enrolled in this phase I clinical trial to date. To activate and expand NK cell, peripheral blood mononuclear cell (PBMCs) were co-cultured with K562-mb15-41BBL cells and 100 IU/ml IL-2. We collected 200 ml of peripheral blood (PB) from patients every cycle (n=4) to produce autologous NKAEs under GMP conditions and cells were harvested on day 14 and 21 for infusions. Four cycles of pharmacological treatment with 2 infusions of 7.5x106 autologous NKAEs/kg on day 1 and 8 of each cycle were performed. NKAEs purity and T regulatory cells (Treg) were analyzed by flow cytometry. NK cells presence in PB was also assessed by PB smear examination before and after each infusion. Serum cytokines concentration was determined by cytometric bead assay. Safety of NKAE end products was verified by real time-PCR of c-MYC and telomerase on NKAE from the 2th and 3rd week of expansion. BCR-ABL PCR studies were performed on NKAE cultures and on PB samples from the patients after treatment. Three patients received lenalidomide-based treatment and 2 bortezomib-based treatment. Patients received a total of 35 NKAEs infusions. We have not observed any serious toxicity attributable to NKAE infusion. Two patients had grade II neutropenia, which did not require dose adjustment. The 5 MM patients enrolled had 23% (±11%) NK cells of PBMCs. We collected a mean of 21x106 NK cells from PB. After 1 week NKAEs number increased x13 with 71% of NKAEs, at 2nd week the fold of NKAE cells expansion was x30 with a purity of 92%. We collected 550x106 (±50x106) NKAEs from culture for the first infusion. At 3rd week NKAEs number increased 45 times (fig.1.A). NKAEs infusion was completely safe; expression of c-Myc and telomerase was not altered in NKAE end products. The expression of BCR-ABL disappeared from cultures after the first week, and was undetectable in PB after NKAE therapy. Contamination of autologous T cells on NKAE end products was not significant; less than 4%. NKAE cells were detectable on PB after infusions; percentage of PB NK cells increased a mean of 5% and expression of activatory receptors NKp30 and NKG2D and apoptosis ligands TRAIL and FasL increased on PBMCs after infusion. PB smear showed an increase fold of activated circulating lymphocytes change of x3.8 (p<0.05). There was no variation on Treg CD4+CD25+CD127- during therapy. Serum levels of IFN-γ increased progressively until the 7th day of cycle and IL-10 levels showed an increase at the end of cycle. Patient 01 achieved a partial response and maintained it for 13 months after NKAEs infusion. Patient 02 started NKAEs infusion while in relapse and, achieved stable disease, which was maintained for 9 months before disease progression. Of note, bone marrow infiltration by MM plasma cells decreased at least 50% at the end of NKAE treatment in these two patients. Patient 03 had disease progression 2 months after stopping treatment due to unrelated toxicity. Patients 04 and 05 recently finished NKAEs treatment and achieved disease stabilization 4 months after the first NKAE infusion (fig.1.B). Clinical-grade NKAEs can be obtained from MM patients undergoing treatment, and multiple infusions of NKAEs are feasible without toxicity. NKAEs showed clinical anti-myeloma activity. These results warrant further development of NKAEs infusion as a treatment modality for MM. Disclosures Lahuerta: Janssen Cilag, Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees.

scholarly journals Repopulation of Lymphocytes and Natural Killer Cells on Day 15 Following First Autologous Stem Cell Transplantation in Myeloma Patients Correlates with the Number of Reinfused Lymphocytes and Natural Killer T Cells in the Autologous Graft

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5820-5820
Author(s):  
Matevz Skerget ◽  
Barbara Skopec ◽  
Samo Zver
Keyword(s):  
Body Weight ◽  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Nk Cells ◽  
Natural Killer ◽  
Peripheral Blood ◽  
Nk Cell ◽  
Induction Treatment ◽  
Cd34 Cells ◽  
Lymphocyte Number

Abstract Introduction Autologous hematopoietic stem cell transplantation (aHSCT) is still considered standard of care for eligible myeloma patients following induction treatment. Beside the effect of a high dose melphalan, several studies proposed a beneficial effect of immune graft versus myeloma effect following aHSCT. The higher number of lymphocytes and natural killer (NK) cells on day 15 after aHSCT correlated with a better longterm outcome. We have previously published the data on the influence of different mobilization regimens on the number of collected lymphocytes and NK cells and their number on day 15 post aHSCT. In this abstract we present the data on the correlation between the dose of reinfused lymphocytes, NK cells and CD34+ cells and the number of lymphocytes and NK cells on day 15 after aHSCT. Methods We prospectively enrolled 48 newly diagnosed myeloma patients following induction treatment with bortezomib and dexamethasone. They were mobilized with either filgrastim (10 mcg/kg body weight daily), pegfilgrastim (12 mg one-time injection) or cyclophosphamide (4 g/m2) followed by filgrastim (10 mcg/kg body weight daily). The lymphocyte and NK cell (CD16+/56+) counts in the stem cell concentrate and on day 15 post aHSCT in peripheral blood were determined with the flow cytometer. Pearson r correlation was used to associate the dose of infused cells and their number on day 15 after aHSCT in peripheral blood. Scatter plots and linear regression were used for the data presentation. SPSS Statistics v21 (IBM, USA) was used for the analysis. The study complied with the Declaration of Helsinki and was approved by the local ethical committee. All patients signed a written informed consent. Results We established a correlation between the dose of infused lymphocytes in the autologous graft and their number in the peripheral blood on day 15 after aHSCT (Figure 1; Pearson r = 0.27, p = 0.04). There was also a correlation between the dose of infused NK cells and their number in the peripheral blood on day 15 post aHSCT (Figure 2; Pearson r = 0.6, p < 0.01). However, there was no correlation between the number of reinfused CD34+ cells and the number of lymphocytes on day 15 after aHSCT (Figure 3). Conclusion The early immune repopulation is important for better overall survival after the aHSCT in multiple myeloma and lymphoma patients. The data from our study showed that the higher numbers of reinfused lymphocytes and NK cells, but not the total number of CD 34+ cells, significantly impact earlier lymphocyte and NK cell engraftment. We concluded that with the aim to achieve the best possible outcomes of aHSCT for our patients, we must make sure to reinfuse not only sufficient number of CD 34+ cells, but also of lymphocytes and NK cells. Figure 1 Correlation between a dose of the infused lymphocytes and their number in the peripheral blood on day 15 after aHSCT (Pearson r = 0.27, p = 0.04). Figure 1. Correlation between a dose of the infused lymphocytes and their number in the peripheral blood on day 15 after aHSCT (Pearson r = 0.27, p = 0.04). Figure 2 Correlation between the dose of the infused NK cells and their number in the peripheral blood on day 15 post aHSCT (Pearson r = 0.6, p < 0.01). Figure 2. Correlation between the dose of the infused NK cells and their number in the peripheral blood on day 15 post aHSCT (Pearson r = 0.6, p < 0.01). Figure 3 Correlation between the dose of infused CD34+ cells and the total lymphocyte number in the peripheral blood on day 15 post aHSCT. Figure 3. Correlation between the dose of infused CD34+ cells and the total lymphocyte number in the peripheral blood on day 15 post aHSCT. Disclosures Skopec: amgen: Consultancy, Honoraria; novartis: Honoraria.

Autologous Activated and Expanded Natural Killer Cells Kill Clonogenic Myeloma Cells: A New Therapeutic Option for Multiple Myeloma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3467-3467
Author(s):  
Alejandra Leivas ◽  
Ruth M Risueño ◽  
Antonio Pérez-Martínez ◽  
María Jesús Blanchard ◽  
Dario Campana ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Multiple Myeloma ◽  
Phase I ◽  
Nk Cells ◽  
Natural Killer ◽  
Cell Lines ◽  
Peripheral Blood ◽  
Nk Cell ◽  
Maximum Effect ◽  
Healthy Donors

Abstract Introduction Multiple myeloma (MM)remains an incurable disease because most of the available drugs do not destroy clonogenic tumor cell (CTC). Natural Killer (NK) cells exert cytotoxicity against MM cells; improving NK cell cytotoxicity might be part of the mechanism of action of effective anti-myeloma drugs such as lenalidomide or bortezomib. By coculture with the genetically-modified K562-mb15-41BBL cell line it is possible to expand ex vivo large numbers of activated NK cells from MM patients. We are conducting a phase I clinical trial to evaluate feasibility, safety and tolerability of these NK cells (termed “NKAEs”) infused in MM patients an autologous setting (EudraCT 2012-000514-11). Because the activity of NKAEs against MM CTCs is unknown, we addressed this issue and analyzed NK cell ligands and receptors pathways mediating CTCs destruction. Methods and Patients Peripheral blood (PB) was collected from MM patients (n=36) or healthy donors (n=14).To activate and expand NK cell from MM patients, peripheral blood mononuclear cell were co-cultured with K562-mb15-41BBL cells and 100 IU/ml IL-2. We used time-resolved fluorescence to detect activity of NK cells on bulk MM cells and methylcellulose clonogenic assays to determine NK cell specific activity on MM CTCs. We analyzed NK and MM receptor expression profile by flow cytometry and Real Time PCR, and identified the “side population” (SP) by DyeCycle Violet efflux. Three MM patients on 2nd or later relapse have been enrolled in the phase I clinical trial to date. We collected 200 ml of PB from patients to produce autologous NKAEs under GMP conditions and cells were harvested on day 14 and 21 for infusions. Four cycles of pharmacological treatment with 2 infusions of 7.5 x 106 autologous NKAEs/kg on day 1 and 8 of each cycle were performed. Results NK cells from patients (n=20) produced 26.6±12.7% lysis of bulk MM cells, similar to NK cells from healthy donors (17±7.8%), while cytotoxicity by NKAEs from MM patients was 68± 0.7% (n=3) at 8:1 ratio. In methylcellulose assays, MM cell killing was higher on CTCs (47±16.8% in MM patients and 57±8% in healthy donors) than on corresponding bulk MM cells (p<0.01), with a maximum effect at 32:1 (58±22% and 87.5±6.5%, respectively). In contrast, killing of CTCs with patient NKAEs (n=6) was 81±13% (8:1) (figure 1), with a strong dose-dependent relationship (maximum effect 95.1±6% at 32:1). NKAEs (n=5) showed over-expression of NKG2D and NKp30 receptors compared to NK cells (n=18). Blocking NKAEs NKp30 or NKG2D prior to methylcellulose assay (n=5) caused a significant increase in colony growth. Flow cytometric analysis of MM cells demonstrated that the SP cells have same expression profile of NKG2D ligands when compared to non-SP cells in 7 MM cell lines. Nevertheless, they showed down-regulation of apoptosis receptors and expression of DNAM-1 ligands. The NKp30 ligand B7H6 was downregulated in both MM cell lines and bone marrow MM cells. Two MM patients undergoing lenalidomide treatment and 1 MM patient who received bortezomib and bendamustine treatment, all with persistent or progressing disease, have been enrolled in the clinical trial and received a total of 20 NKAEs infusions. We observed grade II and III neutropenia, which did not require dose adjustment. MM patients had 15% (±5%) NK cells of PB mononuclear cells. We collected 30 x 106 (±17 x 106) NK cells from patients PB. After 1 week NKAEs number increased x9 fold, at 2nd week fold was x29. We collected 550 x 106 (±50x 106) NKAEs from culture for the first infusion. At 3rd week NKAEs number reached 1077 x 106 and 87.5% (±11.5%) purity of NKAEs (figure 2). All patients are still alive after one 1 year of starting the treatment. One patient achieved a partial response and maintained it for 13 months after NKAEs infusions. Another patient, who started NKAEs infusion while in relapse, achieved stable disease and maintained it for 11 months, after which disease progressed. The third patient progressed two months after stopping the treatment for unrelated toxicity. Conclusion NKAEs from MM patients have enhanced cytotoxicity against MM CTCs, which is mediated through NKG2D receptor and their cognate ligands. Clinical grade NKAEs can be obtained from MM patients even during treatment, and multiple infusions of NKAEs are feasible without notable toxicities. These results and clinical observations warrant further development of NKAEs infusion as a treatment modality for refractory MM. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

542 Expansion of cytotoxic NK Cells from PBMCs using individualized cytokine combination

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A578-A578
Author(s):  
Andreia Maia ◽  
Joana Lerias ◽  
Markus Maeurer ◽  
Mireia Castillo-Martin
Keyword(s):  
Flow Cytometry ◽  
Nk Cells ◽  
Natural Killer ◽  
Peripheral Blood ◽  
Blood Donors ◽  
Nk Cell ◽  
Tumour Cells ◽  
List Type ◽  
Mhc I ◽  
Cytokine Combination

BackgroundAdoptive immunotherapy relies on the use of T-cells to target tumour cells, through Major Histocompatibility Complex (MHC) Class I recognition(1). However, many tumours display alterations in the MHC-I pathway, a well-described immune evasion mechanism(2). Natural Killer (NK) cells recognize transformed cells independently from the presence of MHC-I and may be a reliable therapeutic option for patients with altered tumour MHC-I expression. The source of NK cells may be autologous or allogeneic and NK cells are also clinically relevant recipients of transgenic receptors (TCRs or antibodies) targeting tumour cells. NK cells have been categorized according to their CD56 and CD16 surface expression into different subpopulations: cytotoxic (CD56+CD16+) and regulatory (CD56brightCD16-)(3). Expanding cytotoxic NK cells is challenging, since the frequency of NK cells is low in peripheral blood(4) and there is also – at this point – not an optimal expansion protocol available.The goal of this project is to determine the best cytokine combination that facilitates expansion of cytotoxic NK cells that either target tumor cells directly or serve as recipients for transgenic receptors.MethodsPeripheral Blood Mononuclear Cells (PBMCs) were extracted using Ficoll methodology from blood donors and cultured in T25 flasks with Cell Genix Medium supplemented with 10% human serum and antibiotics. NK cells were expanded supplemented with feeder cells (ratio 1:1) and different cytokine combinations (1000 U/mL of IL-2, 10 U/ml of IL-12, 180 U/mL of IL-15 and/or 1 U/mL of IL-21) during 20 days. The immunophenotype of expanded NK cells was analyzed at days 0, 5, 10, 15 and 20 by flow cytometry. The cytotoxicity of NK cells was measured by a CD107a Assay or by a Total Cytotoxicity and Apoptosis Assay at days 10 and 20. Thirteen different cytokine combinations were tested.Results4/13 cytokine combinations produced a statistically significant increase of the absolute number of NK cells with a higher percentage of cytotoxic NK cells (figure 1). However, induction of cytotoxicity was not associated with a strong NK cell expansion. The regulatory NK cells subset (CD56brightCD16-) showed the highest percentage of CD107a-expressing cells, more than the CD56+CD16+, the most cytotoxic subpopulation of NK cells.Abstract 542 Figure 1Representative percentage of NK cells in total lymphocytes (A), CD56+CD16+ subpopulation in total NK cells (B), and CD56brightCD16- subpopulation amongst total NK cells (C) at different time points (5, 10, 15 and 20 days) expanded from PBMCs* p-value < 0.05ConclusionsThis work shows that we are able to grow and efficiently expand NK cells from PBMCs with different cytokine combinations leading to clinically relevant NK cell numbers as well as cytotoxic functions. This enables to produce NK cell products for therapy and as recipients for transgenic tumor antigen-specific receptors.AcknowledgementsThe authors would like to thank the Champalimaud Foundation Biobank, the Vivarium Facility and the Flow Cytometry Platform of the Champalimaud Centre for the Unknown.Ethics ApprovalThis study was approved by the Champalimaud Foundation Ethics Committee and by the Ethics Research Committee of NOVA Medical School of NOVA University of Lisbon.ConsentWritten informed consent was obtained from the blood donors to use their samples for research purposes.ReferencesRosenberg SA, Restifo NP, Yang JC, Morgan RA, Mark E. Adoptive cell transfer: a clinical path to effective cancer immunotherapy. Nat Rev Cancer 2008;8(4):299–308.Aptsiauri N, Ruiz-Cabello F, Garrido F. The transition from HLA-I positive to HLA-I negative primary tumors: the road to escape from T-cell responses. Curr Opin Immunol 2018;51:123–32.Di Vito C, Mikulak J, Mavilio D. On the way to become a natural killer cell. Front Immunol. 2019;10(August):1–15.Zotto G Del, Antonini F, Pesce S, Moretta F, Moretta L. Comprehensive phenotyping of human PB NK Cells by Flow Cytometry. 2020;1–9.

scholarly journals Role and Modulation of NK Cells in Multiple Myeloma

Hemato ◽  
2021 ◽  
Vol 2 (2) ◽  
pp. 167-181
Author(s):  
Marie Thérèse Rubio ◽  
Adèle Dhuyser ◽  
Stéphanie Nguyen
Keyword(s):  
Multiple Myeloma ◽  
Monoclonal Antibodies ◽  
Nk Cells ◽  
Tumor Cells ◽  
Nk Cell ◽  
Ex Vivo ◽  
Killer Cell ◽  
Proteasome Inhibitors ◽  
Disease Evolution ◽  
Cell Functions

Myeloma tumor cells are particularly dependent on their microenvironment and sensitive to cellular antitumor immune response, including natural killer (NK) cells. These later are essential innate lymphocytes implicated in the control of viral infections and cancers. Their cytotoxic activity is regulated by a balance between activating and inhibitory signals resulting from the complex interaction of surface receptors and their respective ligands. Myeloma disease evolution is associated with a progressive alteration of NK cell number, phenotype and cytotoxic functions. We review here the different therapeutic approaches that could restore or enhance NK cell functions in multiple myeloma. First, conventional treatments (immunomodulatory drugs-IMids and proteasome inhibitors) can enhance NK killing of tumor cells by modulating the expression of NK receptors and their corresponding ligands on NK and myeloma cells, respectively. Because of their ability to kill by antibody-dependent cell cytotoxicity, NK cells are important effectors involved in the efficacy of anti-myeloma monoclonal antibodies targeting the tumor antigens CD38, CS1 or BCMA. These complementary mechanisms support the more recent therapeutic combination of IMids or proteasome inhibitors to monoclonal antibodies. We finally discuss the ongoing development of new NK cell-based immunotherapies, such as ex vivo expanded killer cell immunoglobulin-like receptors (KIR)-mismatched NK cells, chimeric antigen receptors (CAR)-NK cells, check point and KIR inhibitors.

scholarly journals 150 GAIA-102: a new class off-the-shelf allogeneic NK-like cells that can eliminate solid tumors

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A163-A163
Author(s):  
Yui Harada ◽  
Yoshikazu Yonemitsu
Keyword(s):  
Nk Cells ◽  
Solid Tumors ◽  
Tumor Cells ◽  
Adoptive Immunotherapy ◽  
Nk Cell ◽  
Peritoneal Dissemination ◽  
Checkpoint Inhibitors ◽  
Antitumor Effects ◽  
Car T

BackgroundCancer immunotherapy has been established as a new therapeutic category since the recent success of immune checkpoint inhibitors and a type of adoptive immunotherapy, namely chimeric antigen receptor-modified T cells (CAR-T). Although CAR-T demonstrated impressive clinical results, serious adverse effects (cytokine storm and on-target off-tumor toxicity) and undefined efficacy on solid tumors are important issues to be solved. We’ve developed a cutting-edge, simple, and feeder-free method to generate highly activated and expanded human NK cells from peripheral blood (US9404083, PCT/JP2019/012744, PCT/JP2020/012386), and have been conducting further investigation why our new type of NK cells, named as GAIA-102, are so effective to kill malignant cells.MethodsCryopreserved PBMCs purchased from vendors were mixed and processed by using LOVO and CliniMACS® Prodigy (automated/closed systems). CD3+ and CD34+ cells were depleted, and the cells were cultured with high concentration of hIL-2 and 5% UltraGRO® for 14 days in our original closed system. Then, we confirmed the expression of surface markers, CD107a mobilization and cell-mediated cytotoxicity against various tumor cells and normal cells with or without monoclonal antibody drugs in vitro and antitumor effects against peritoneal dissemination model using SKOV3 in vivo.ResultsImportantly, we’ve found that our GAIA-102 exhibited CD3-/CD56bright/CD57- immature phenotype that could kill various tumor cells efficiently from various origins, including Raji cells that was highly resistant to NK cell killing. More importantly, massive accumulation, retention, infiltration and sphere destruction by GAIA-102 were affected neither by myeloid-derived suppressor cells nor regulatory T-lymphocytes. GAIA-102 was also effective in vivo to murine model of peritoneal dissemination of human ovarian cancer; thus, these findings indicate that GAIA-102 has a potential to be an ‘upward compatible’ modality over CAR-T strategy, and would be a new and promising candidate for adoptive immunotherapy against solid tumors.ConclusionsWe now just started GMP/GCTP production of this new and powerful NK cells and first-in-human clinical trials in use of GAIA-102 will be initiated on 2021.Ethics ApprovalThe animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee of Kyushu University (approval nos. A30-234-0 and A30-359-0).

scholarly journals Production of colony-stimulating activity by human natural killer cells: analysis of the conditions that influence the release and detection of colony-stimulating activity

Blood ◽  
1989 ◽  
Vol 74 (1) ◽  
pp. 156-164
Author(s):  
V Pistoia ◽  
S Zupo ◽  
A Corcione ◽  
S Roncella ◽  
L Matera ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Nk Cells ◽  
Natural Killer ◽  
Peripheral Blood ◽  
Nk Cell ◽  
K562 Cells ◽  
Cell Suspensions ◽  
Normal Peripheral Blood ◽  
Colony Stimulating Activity ◽  
Culture Supernatants

Highly purified natural killer (NK) cell suspensions were tested for their capacity to release colony-stimulating activity (CSA) in vitro. NK cell suspensions comprised primarily CD16+ cells and were devoid of CD3+ T cells, CD15+ monocytes, and of B cells. CSA was detected in the NK cell supernatants and sustained the growth of myeloid colonies from both normal peripheral blood and bone marrow. CSA could be in part inhibited by pretreating NK cell culture supernatants with a specific goat anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) antiserum. The inhibition, however, was never complete, a finding that suggests that additional factors were responsible for CSA. Incubation of NK cells with K562 cells (an NK-sensitive target) or with normal bone marrow cells resulted in the appearance of a strong colony- inhibiting activity (CIA) in the culture supernatants. Such CIA was demonstrable in an experimental system where bone marrow or peripheral blood progenitors were induced to form myeloid colonies in the presence of conditioned medium by CSA-producing giant cell tumor (GCT) cells. Stimulation of NK cells with NK-insensitive targets failed to induce CIA production. Neutralizing antitumor necrosis factor (TNF) monoclonal antibodies (MoAbs) were found capable of inhibiting CIA present in the supernatants of NK cells stimulated with K562 cells. Following treatment with anti-TNF antibodies, CSA was again detectable in the same supernatants. This finding indicates that induction of TNF production did not concomitantly switch off CSA production by NK cells. Pretreatment of NK cells with recombinant interleukin-2 (rIL-2) or gamma interferon (r gamma IFN) did not change the amount of CSA released. However, treatment with rIL-2 caused the appearance of a factor in the NK cell supernatants capable of sustaining the formation of colonies of a larger size.

scholarly journals 799 Neoantigen-cytokine-chemokine multifunctional natural killer cell engager for immunotherapy of solid tumors

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A834-A834
Author(s):  
Xue Yao ◽  
Sandro Matosevic
Keyword(s):  
Tumor Microenvironment ◽  
Cell Therapy ◽  
Nk Cells ◽  
Natural Killer ◽  
Solid Tumors ◽  
Tumor Cells ◽  
Nk Cell ◽  
Killer Cell ◽  
Nk Cell Therapy

BackgroundThe effectiveness of natural killer (NK) cell-based immunotherapy against solid tumors is limited by the lack of specific antigens and the immunosuppressive tumor microenvironment (TME). Glioblastoma multiforme (GBM) is one such heavily immunosuppressive tumor that has been particularly hard to target and remains without a viable treatment. The development of novel approaches to enhance the efficacy of NK cells against GBM is urgently needed. NK cell engagers (NKCE) have been developed to enhance the efficacy of NK cell therapy.MethodsTo improve the clinical efficacy of NK cell therapy, we are developing a new generation of multi-specific killer engagers, which consists of a neoantigen-targeting moiety, together with cytokine and chemokine-producing domains. Neoantigens are new antigens formed specifically in tumor cells due to genome mutations, making them highly specific tools to target tumor cells. Our engager has been designed to target Wilms' tumor-1 (WT-1), a highly specific antigen overexpressed in GBM among other solid tumors. This is done through the generation of an scFv specific targeting the complex of WT-1126-134/HLA-A*02:01 on the surface of GBM. On the NK cell side, the engager is designed to target the activating receptor NKp46. Incorporation of the cytokine IL-15 within the engager supports the maturation, persistence, and expansion of NK cells in vivo while favoring their proliferation and survival in the tumor microenvironment. Additionally, our data indicated that the chemokine CXCL10 plays an important role in the infiltration of NK cells into GBM, however, GBM tumors produce low levels of this chemokine. Incorporation of a CXCL10-producing function into our engager supports intratumoral NK cell trafficking by promoting, through their synthetic production, increased levels of CXCL10 locally in the tumor microenvironment.ResultsCollectively, this has resulted in a novel multifunctional NK cell engager, combining neoantigen-cytokine-chemokine elements fused to an activating domain-specific to NK cells, and we have investigated its ability to support and enhance NK cell-mediated cytotoxicity against solid tumors in vitro and in vivo against patient-derived GBM models. The multi-specific engager shows both high tumor specificity, as well as the ability to overcome NK cell dysfunction encountered in the GBM TME.ConclusionsWe hypothesize that taking advantage of our multi-functional engager, NK cells will exhibit superior ex vivo expansion, infiltration, and antitumor activity in the treatment of GBM and other solid tumors.

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