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.

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.

533 Cross-species immunogenomic analysis identifies pathways of canine natural killer cell response to cytokine therapy, and reveals convergence of activated dog and human natural killer transcriptomes

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A569-A569
Author(s):  
Alicia Gingrich ◽  
Taylor Reiter ◽  
Sean Judge ◽  
Daniel York ◽  
Mio Yanagisawa ◽  
...  
Keyword(s):  
Gene Expression ◽  
Clinical Trial ◽  
Phase I ◽  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Ex Vivo ◽  
Phase I Clinical Trial ◽  
Gene Profiles

BackgroundNatural killer (NK) cells are key effectors of the innate immune system, but major differences between human and murine NK cells impede translation. Outbred dogs offer an important link for NK-based cancer immunotherapy studies. We compared gene expression profiles of dog NK signatures in vitro and from a phase I clinical trial of inhaled IL-15, and analyzed dog, mouse and human NK cells using a novel orthologous transcriptome.MethodsWe performed differential gene expression (DGE) using resting healthy donor CD5dim NK populations and following ex vivo activation using recombinant human (rh)IL-15 or co-culture with irradiated feeder cells. Eight dogs with naturally-occurring pulmonary metastases were enrolled on a Phase I clinical trial of inhaled rhIL-15 using a 3+3 cohort design with escalating doses of inhaled rhIL-15. Blood was collected from study dogs before, during, and after therapy. We compared DGE among healthy and cancer-bearing dogs and then across mouse, dog and human NK cells in resting and activated states using ~7000 1:1 orthologous genes.ResultsDGE revealed distinct transcriptional profiles between the ex vivo resting, IL-15 and co-cultured canine NK cells. Among treated patients, hierarchical clustering revealed that in vivo NK cell transcriptional signatures grouped by individual dog, and not amount of time exposed to treatment. PCA showed in vivo profiles of the clinical responders were distinctly separate from the non-responding patients (PC1 38%, PC2 12%). Patient in vivo NK cell transcription profiles most closely resembled those of ex vivo resting NK cells and not IL-15 treated or co-culture activated (PC1 43%, PC2 19%), likely reflecting key differences in activation. In cross-species analysis, PCA showed within-species spatial clustering of resting NK cells. After activation, variance between dog and human NK cells decreased, while variance between human and mouse NK cells increased (PC1 40%, PC2 28%).ConclusionsIn this first transcriptomic sequencing of dog NK cells, we demonstrate distinct gene profiles of ex vivo activated NK cells from healthy donors compared to circulating NK cells from dogs receiving inhaled rhIL-15 on a clinical trial. Baseline in vivo NK cell profiles appear to predict response to therapy more than changes over time. We also show distinct gene profiles of NK cells across the most commonly used mouse, dog, and human NK populations, with convergence of dog and human NK cells after activation. By defining the canine NK cell DGE signatures, these data fill a gap in translational NK studies.Ethics ApprovalThe canine clinical trial study was approved by IACUC and Clinical Trials Review Board (Inhaled IL-15 Immunotherapy for Treatment of Lung Metastases, Protocol #20179).

scholarly journals Autologous Natural Killer Cell-enrichment for Immune Cell Therapy: Preclinical Setting Phase, Shiraz Experience

2021 ◽  
Author(s):  
Somayeh Rezaeifard ◽  
Yuji Heike ◽  
Jun-Ichi Masuyama ◽  
Alireza Rezvani ◽  
Reza Vojdani ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Cell Therapy ◽  
Phase I ◽  
Nk Cells ◽  
Natural Killer ◽  
Peripheral Blood ◽  
Nk Cell ◽  
Healthy Individuals ◽  
Cell Product ◽  
Nk Cell Therapy

Natural killer (NK) cell therapy has proven to be a promising approach for the treatment of malignancies. Osaki method for ex-vivo autologous NK cell expansion has been recently introduced in Japan. To start clinical trial phase I at Shiraz University of Medical Sciences in collaboration with the Japanese group, this preclinical setting study aimed to evaluate the proliferative efficacy of the method, the activation status of expanded autologous NK cells, and the likely unwanted contamination of the final cell product. Peripheral blood mononuclear cells (PBMCs) were isolated from 5 healthy individuals' peripheral blood and transferred directly to the specified initial culture bag containing anti-CD52 and anti-CD3 and Interleukin (IL)-2. The cells were cultured for 14-17 days in an incubator, during which the cells received condition media, and underwent several passages into bigger culture bags. All the procedures were carried out in a cleanroom and associated facilities. Before and after activation PBMCs were analyzed for their phenotype and cytotoxic activity; using flow cytometry and cytokine release assay. Our results indicated that NK (CD3-CD16+/-CD56+) cells were expanded 510-fold on average (range 200-1100 fold), and the purity of NK cells per whole lymphocytes exceeded 68%. The expanded cells were highly lytic as indicated by in-vitro cytotoxic assay, with a strong expression of Natural killer group 2 member D (NKG2D) and CD16. The prepared final cell products were negative for HCV, HBV, HIV, mycoplasma, and endotoxin. In the preclinical phase, large numbers of activated and un-contaminated NK cells from healthy individuals' peripheral blood were successfully generated. The method seems to provide ample clean cell product with no contamination and has the potential to be used for NK cell therapy in future clinical trials, suitable to be infused back to the donors in phase I clinical trial.

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.

First Interim Results of a Phase I/II Study of Lenalidomide in Combination with Anti-PD-1 Monoclonal Antibody MDV9300 (CT-011) in Patients with Relapsed/Refractory Multiple Myeloma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1838-1838 ◽  
Author(s):  
Yvonne A. Efebera ◽  
Ashley E Rosko ◽  
Craig Hofmeister ◽  
Joe Benner ◽  
Courtney Bakan ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Monoclonal Antibody ◽  
T Cell ◽  
Partial Response ◽  
Phase I ◽  
Nk Cells ◽  
Disease Progression ◽  
Stable Disease ◽  
Nk Cell ◽  
Grade 3

Abstract Introduction: Multiple myeloma (MM) is associated with profound and widespread disarray of both the adaptive and innate arms of the immune system including loss of effector T cell function, humoral immune deficiency, and natural killer (NK) cell immunity. This immunosuppressive milieu is crucial to promoting disease progression. Standard treatment options (immunomodulators (IMIDs) and proteosome inhibitors, radiation, and high-dose corticosteroids) offer modest benefit, but also contribute to further immune suppression. Little is known regarding the mechanisms by which immune dysfunction and immunoevasion occur. Our group has characterized an important role for the programmed death receptor-1 (PD-1) / PD-L1 signaling axis in these processes. MDV9300 (formerly CT-011 / Pidilizumab) is a novel IgG1 humanized monoclonal antibody (mAb) that modulates the immune response through interaction with PD-1. Lenalidomide (Len) an IMID exerts efficacy in MM in part through enhancement of NK cell versus MM effect - an effect likely mediated through T cell production of interleukin (IL)-2. In our in-vitro study, pretreatment of NK cells with MDV9300 with or without Len enhanced immune complex formation between NK cells and MM tumor targets and also augmented NK cell activation and cytotoxicity against MM. We sought to determine the safety, tolerability and any early signs of efficacy in relapsed or refractory MM patients using MDV9300 in combination with Len. Methods: In the phase I portion, the primary endpoint is to determine the maximum tolerated dose (MTD) of the combination. Key eligibility criteria are relapsed or refractory disease but not progressed on Len 25 mg; ≥2 prior lines of therapy, absolute neutrophil count ≥ 1000/µL; Platelets ≥60,000/µL; and creatinine clearance of ≥ 40ml/min. Patients are treated with escalating doses of MDV9300 and Len utilizing a 3x3 escalation design (Table 1). If stable disease is the best response after 4 cycles, patients have the option of adding dexamethasone (20-40mg weekly). Len dose may be modified independently of MDV9300. Patients can receive a maximum of 12 cycles of therapy. Results: Twelve patients are evaluable to date. The median age was 68.5 (range 49-82) and the median time from diagnosis 4.98 years (range 1.54-12.62). At study entry, 67% had high risk cytogenetics (del 17p, complex karyotype, gain 1q) and the median number of prior treatment lines was 2 (range 2-11). 100% of patients had received prior Len, bortezomib and Dex, 50% alkylating agents (cyclophosphamide, oral melphalan, bendamustine), 75% autologous stem cell transplant, 25% pomalidomide and 33% carfilzomib. MDV9300 infusion has been well tolerated with only one grade 2 infusion related toxicity with sore throat. The patient received hydrocortisone with no further reaction observed. Grade 3/4 Anemia, neutropenia, and thrombocytopenia attributable to therapy have been seen in 25%, 23%, and 34% of patients, respectively. Other common grade 2-3 therapy related adverse events are fatigue (50%), anorexia (17%), and hypophosphatemia (17%). There has been no grade 3 or higher infection and no worsening of neuropathy from baseline. Len dose was reduced in 3 patients (25%) and increased in one. There has been no dose reduction in MDV9300. Dex 20 mg or less was added in 2 patients for muscle cramps and < PR after 3 cycles. To date 7 patients are off therapy; 1 due to grade 3 fatigue and 6 due to disease progression. Five patients continue on therapy at respective 12, 11, 9, 5 and 3 months. Responses to date have been 3 Very good partial response,1 partial response, 2 minimal response and 2 stable disease. Conclusion: The combination of steroid sparing MDV9300 and Len regimen has demonstrated an acceptable toxicity profile to date with evidence of anti-myeloma activity. This is the first reported combination anti-PD-1 based immune therapy for MM. Updated results will be presented at the meeting including the MTD dose for phase II. Table 1. MDV9300- mg/kg Intravenously given on day 3 every 28 days Lenalidomide- mg orally days 1-21 every 28 days DLT Evaluable DLTs Cohort 1 1.5 15 6 Grade 3 fatigue. Cohort extended to 6 Cohort 2 3 15 3 none Cohort 3 3 25 3 none Cohort 4 6 25 0 Acknowledgments: Drug has been provided by Medivation; The study is sponsored by the American Cancer Society Disclosures No relevant conflicts of interest to declare.

scholarly journals Autologous NK-cell-enrichment: preclinical setting phase, Shiraz experience

2020 ◽  
Author(s):  
Somayeh Rezaeifard ◽  
yuji Heike ◽  
Junichi Masuyama ◽  
Alireza rezvani ◽  
Reza vojdani ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Cell Therapy ◽  
Phase I ◽  
Nk Cells ◽  
Peripheral Blood ◽  
Nk Cell ◽  
Ex Vivo ◽  
University Hospital ◽  
Healthy Individuals ◽  
Therapy Regimen

Abstract Background: NK cell therapy has proven to be a promising approach for treatment of hematological malignancies and solid tumors. Masuyama et al. have recently introduced a new method for ex-vivo autologous NK cell expansion (Osaki method); resulting in the production of ample active NK cells for a promising cell therapy regimen. In order to start clinical trial phase I at Shiraz University of medical Sciences in collaboration with Masuyama clinic and St. Luck's International University Hospital, this preclinical setting study aimed to evaluate the proliferative efficacy of the method, the activation status of expanded autologous NK cells and the likely unwanted contamination of the final cell product.Methods: PBMCs were isolated from 30 ml of 5 healthy individuals' peripheral blood transferring directly to the specified initial culture bag containing antibodies for CD3, CD52 as well as IL-2 cytokine. The cells were cultured for 14-17 days in incubators; during which the cell received condition media, and underwent several passages into bigger culture bags. All the procedure was carried out in the clean room and associated facilities. Results: Our results indicated that NK cells were expanded 510-fold in average (range 200-1100 fold), and the purity of NK cells per whole lymphocytes exceeded 68%. The expanded cells were highly lytic as indicated by in-vitro cytotoxic assay; with strong expression of NKG2D and CD16. The prepared final cell products were negative for HCV, HBV, HIV, Mycoplasma and endotoxin. Conclusion: In the preclinical setting phase, large numbers of activated and un-contaminated NK cells from 30 ml of healthy individuals' peripheral blood were successfully generated. The method seems to provide ample clean cell product with no contamination; suitable to be infused back to the patients in phase I clinical trial.

A phase I clinical trial testing the safety of IL-21-expanded, off-the-shelf, natural killer cells for relapsed/refractory acute myeloid leukemia and myelodysplastic syndrome.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. TPS7562-TPS7562
Author(s):  
Sumithira Vasu ◽  
Nidhi Sharma ◽  
Lynn Odonnell ◽  
Kevin Bosse ◽  
Dean Anthony Lee
Keyword(s):  
Clinical Trial ◽  
Acute Myeloid Leukemia ◽  
Myelodysplastic Syndrome ◽  
Phase I ◽  
Nk Cells ◽  
Natural Killer ◽  
Myeloid Leukemia ◽  
Nk Cell ◽  
Third Party ◽  
Acute Myeloid

TPS7562 Background: Allogeneic transplantation (Allo-HCT) demonstrates the enduring and potent role of the immune system in the control and eradication of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). However, patients with relapsed, refractory (R/R) disease or comorbidities are not eligible for Allo-HCT. We sought to develop an allogeneic Natural killer (NK) cell-based immunotherapy approach to induce remission for these patients. The efficacy of haploidentical NK cells expanded ex vivo using a K562 feeder-cell line transfected with IL-21 and 41BBL has been established in R/R AML patients. However, haploidentical donor-derived NK cell manufacturing exceeds three weeks with the possibility of fulminant malignancy rendering patients ineligible for cellular therapy. To address this limitation we established a third-party NK cell bank derived from KIR and HLA-mismatched ‘ideal’ donors that allows scalable, affordable mass-production of large numbers of NK cells suitable for banking and immediate ‘off-the-shelf’ (OTS) administration to a broad population of recipients. Methods: This phase I study follows a 3+3 design to investigate the safety of mIL-21-expanded, third-party, OTS NK cells for treatment of R/R AML and MDS patients. Patients aged ≥18 or ≤80 years are enrolled into two cohorts: those <60 years and able to tolerate intensive chemo will receive Fludarabine 30mg/m2/day (days -6 to -2) and Cytarabine 2g/m2/day (days -6 to -2). Patients >60 years or <60 years and unable/unwilling to tolerate intensive chemo will receive Fludarabine 30mg/m2/day (days -5 to -2) and Decitabine 20mg/m2/day (days -6 to -2). All patients subsequently receive a total of 6 infusions of NK cells administered thrice weekly for two weeks (between days 0-21) and will be followed up to day 56 from first NK cell infusion. Three NK cell dose-levels: 1x107, 3x107 and 1x108 cells/kg/dose will be explored to determine maximum tolerated dose (MTD). 3-18 patients/cohort/dose may be enrolled for MTD determination plus an additional 10 patients/dose in an expansion phase (maximum 28/cohort = 56 total subjects). Primary objective is to determine safety and feasibility of NK cell infusions. Secondary objectives will explore rates of remission PFS, overall survival and measurable residual disease negativity, cell counts, infectious complications, and patients proceeding to transplant. Enrollment in dose level 1 has started. Clinical trial information: NCT04220684 .

scholarly journals Ex-Vivo Expanded NK Cell Therapy Combined with Elotuzumab for MRD after Autologous Stem Cell Transplantation: A Phase I/ II Clinical Trial in Progress

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3133-3133
Author(s):  
Shotaro Hagiwara ◽  
Yan-Hua Wang ◽  
Hirohito Kobayashi ◽  
Yutaka Kato ◽  
Norina Tanaka ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Multiple Myeloma ◽  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Phase I ◽  
Nk Cells ◽  
Cell Transplantation ◽  
Nk Cell ◽  
Residual Disease ◽  
Ex Vivo

Recent advances of monoclonal antibodies provide a potent anti-myeloma effect based on antibody-dependent cytotoxicity (ADCC). Elotuzumab is a monoclonal antibody which binds SLAMF7 on the surface of myeloma cells and induces ADCC by NK cells (Cancer Immunol Immunother 64:61-73,2015). Lenalidomide may enhance the killing activity ad IFN-gamma production in NK cells (Blood 126:50-60,2015). In Eloquent-2 trial, the combination therapy of Elotuzumab, Lenalidomide, and Dexamethasone (ELD) showed a significant relative reduction in the risk of disease progression or death (N Engl J Med 373:621-631,2015). However, the number of NK cells in patients with multiple myeloma is generally suppressed, and the activity is often exhausted (Cytometry 26:121-124,1996, Med Oncol 24:312-317,2007). It is unclear if they fully exhibit the ADCC against myeloma cells. Also, enough number of NK cells as effector cells is required to obtain effective ADCC. To enhance the ADCC activity of NK cells against multiple myeloma, cell therapy using expanded NK cells might be effective. Therefore, we conduct the clinical trial of ex-vivo expanded NK cell infusion therapy in combination with ELD for minimal residual disease (MRD) after autologous stem cell transplantation (ASCT). Clinical trial registry number: UMIN000033128 Study design: Non-randomized single-arm phase I/ II clinical trial. Study population: 20; Five for phase I part and 15 for phase II. Primary endpoint: Any grade of adverse events, and the response defined by IMWG criteria. Secondary endpoint: Overall survival, progression-free survival, and MRD. Study protocol: 1x106 - 5x107/kg of ex-vivo expanded NK cells are infused on day 2 of ELD regimen in patients who failed to achieve sCR or with detective MRD after ASCT. The NK cell infusion with ELD therapy will be repeated 4 cycles monthly. Major inclusion criteria: (1)Multiple myeloma diagnosed by IMWG diagnostic criteria, (2)Age: 20=<, 65>=, (3)ECOG Performance status0-2, (4)PR or better after induction therapy, (5) Transplant eligible patients without severe organ damages, (6)Patients failed to achieve sCR after stem cell transplantation or positive for minimal residual disease by 6-8 color Flow cytometry. Major exclusion criteria: (1) Patients who were previously transplanted, (2) Failed to obtain more than 2x106/kg of CD34+ cells, (3) Patients who are not eligible for harvest or transplant due to cardiac or pulmonary disorder, AST/ALT/total bilirubin>2.5x lower limit, serum Cr>=2.0mg/dl, (4) Patients who have severe allergy or uncontrollable active infection. Currently, two participants completed the protocol and are under the observation. Figure Disclosures Hagiwara: Bristol-Myers Squibb: Research Funding. Kanno:Agios Pharmaceuticals, Inc.: Honoraria. Tanaka:Bristol-Myers Squibb: Research Funding.

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 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.

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