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.

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.

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 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.

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.

Patients’ Peripheral Blood as a Possible Source of Donor-Derived NK Cells for Ex Vivo Expansion and Genetic Modification for Post-Transplant Cellular Immunotherapy In Cord Blood Recipients

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2094-2094
Author(s):  
Chihaya Imai ◽  
Sakiko Yoshida ◽  
Takayuki Takachi ◽  
Masaru Imamura ◽  
Ryosuke Hosokai ◽  
...  
Keyword(s):  
Cord Blood ◽  
Nk Cells ◽  
Genetic Modification ◽  
Peripheral Blood ◽  
Nk Cell ◽  
Ex Vivo ◽  
K562 Cells ◽  
Cellular Immunotherapy ◽  
Healthy Individuals ◽  
Expansion Range

Abstract Abstract 2094 Haploidentical natural killer (NK) cells can induce and consolidate remission in patients with high-risk acute myeloid leukemia (AML) (Rubnitz et al. J Clin Onc 24: 371, 2010). Recently, significantly reduced relapse rates were observed in AML patients who received killer immunoglobulin-like receptor ligand-mismatched cord blood, suggesting effective alloreactivity of cord blood-derived NK cells (Willemze et al. Leukemia 23: 492, 2009). Cord blood transplantation (CBT) is an effective alternative source for allogeneic hematopoietic cell transplantation in both children and adults. However, its therapeutic efficacy for malignant diseases is limited by the lack of available donor effector cells, such as cytotoxic T lymphocytes, lymphokine-activated killer cells, NK-like T cells and NK cells, for treatment of hematological relapse and posttransplant lymphoproliferative disorder and/or for scheduled posttransplant cellular immunotherapy against refractory diseases. We previously reported a method that induces NK cells to proliferate and reliably allows their genetic modification in healthy individuals and leukemia patients in remission receiving maintenance chemotherapy (Imai et al. Blood 106: 376, 2005). To explore the possibility of using patients’ peripheral blood as a source for posttransplant NK cell therapy, we used our method to expand donor-derived NK cells from peripheral blood of CBT recipients early after engraftment. We also examined whether NK cells can be rendered cytotoxic against original leukemia blasts by transferring an antigen-specific artificial immunoreceptor gene. This study was approved by an institutional ethical committee. Patients received CBT for consolidation of hematological malignancy (n=7), neuroblastoma (n=1) or resolution of refractory EBV-associated hemophagocytic syndrome (n=1) with myeloablative (n=7) or reduced intensity conditioning (RIC) regimens (n=2). The patients were enrolled in the study after engraftment and peripheral blood was obtained after appropriate written consent was obtained. A chimerism study using short tandem repeat assays showed complete donor chimerism in all patients except one who received RIC-CBT. The peripheral blood was obtained at a median of 92 days post-CBT (range: 46–303 days) and subjected to ex vivo activation and expansion using a previously described protocol with slight modifications. Briefly, peripheral blood was coincubated with modified K562 cells expressing membrane-bound IL-15 and 4-1BB ligand (K562-mb15-41BBL) in the presence of low-dose IL-2 (10 U/mL). Most patients were on maintenance immunosuppressive therapy with calcineurin inhibitors with (n=3) or without (n=6) systemic corticosteroids. After 7 days of culture, a median 11.0-fold expansion (range: 5.3–28.9-fold) was observed in all but one patient who had been administered chemotherapy with Mylotarg for relapsed AML a few days before the blood sampling. The expansion rate in the first week was less efficient in CBT recipients than in healthy individuals (>20-fold), probably because of the immunosuppressants administered. However, an additional 2-week culture in the presence of high-dose IL-2 (1000 U/mL) yielded a median 206-fold expansion (range: 101–1381-fold in 21 days). The expanded NK cells exhibited upregulation of activating receptors including NKG2D, NCRp30 and NCRp44, and vigorous cytotoxicity against K562 cells (86.8–97.7% at an E/T ratio of 1:1). The NK cells were susceptible to retroviral genetic modification with the MSCV-IRES-GFP vector (median GFP-positive cells, 52.7%, n=10). Finally, peripheral NK cells from patients with acute lymphoblastic leukemia were expanded and transduced with the chimeric immunoreceptor gene anti-CD19-BB-ζ. The donor-derived NK cells expressed large amounts of anti-CD19 chimeric receptors on their surface and killed original leukemia blasts that were highly resistant to NK cell lysis (e.g. anti-CD19 vs. non-signaling receptor: 69% vs. 0% at an E/T ratio of 1:1). These results suggest that, in CBT recipients, ex vivo expansion and genetic modification of donor-derived NK cells from the patients’ peripheral blood is feasible. Because peripheral blood can be easily and repeatedly obtained, the method described here will allow multiple scheduled infusions. This preliminary study may lead to a novel strategy for posttransplant donor-NK cell therapy in CBT recipients. Disclosures: No relevant conflicts of interest to declare.

scholarly journals 151 Potentiating the Large-Scale Expansion and Engineering of Peripheral Blood-Derived CAR NK Cells for Off-the-Shelf Application

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A159-A159
Author(s):  
Michael Whang ◽  
Ming-Hong Xie ◽  
Kate Jamboretz ◽  
Hadia Lemar ◽  
Chao Guo ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Therapy ◽  
Nk Cells ◽  
Peripheral Blood ◽  
Nk Cell ◽  
Adoptive Cell Therapy ◽  
Therapeutic Window ◽  
Target Cells ◽  
Healthy Donors ◽  
Custom Made

BackgroundPeripheral blood natural killer (NK) cells are mature cytotoxic innate lymphocytes possessing an inherent capacity for tumor cell killing, thus making them attractive candidates for adoptive cell therapy. These NK cells are also amenable to CRISPR and chimeric antigen receptor (CAR) genomic engineering for enhanced functions. Moreover, NK cells possess an inherent capacity for off-the-shelf therapy since they are not known to cause graft-versus-host disease, unlike T cells. Presently, approved CAR cell therapy is custom-made from each patient‘s own T cells, a process that can limit patient pool, narrow therapeutic window, and contribute to product variability. In this study, we investigate whether peripheral blood NK cells from a selected donor can be edited, engineered, and expanded sufficiently for off-the-shelf use in a wide patient population.MethodsUsing the CRISPR/Cas9 system, we knocked out CISH expression in isolated peripheral blood NK cells from 3 healthy donors. Subsequently, we expanded edited NK cells by using IL-2 and sequential stimulations using NKSTIM, a modified K562 stimulatory cell line expressing membrane-bound form of IL-15 (mbIL-15) and 4-1BBL. IL-12 and IL-18 were added twice during expansion to drive memory-like NK cell differentiation. We transduced the expanded NK cells to express engineered CD19-targeted CAR and mbIL-15 during an interval between the first and second NKSTIM pulses. We assessed NK cell cytotoxicity against Nalm6 target cells by IncuCyte.ResultsIsolated peripheral blood NK cells from 3 healthy donors were successfully edited using CRISPR/Cas9, engineered to express high levels of CAR, extensively expanded using a series of NKSTIM pulses in the presence of IL-2, and differentiated into memory-like NK cells using IL-12 and IL-18. Interestingly, NK cells from the 3 donors exhibited distinct outcomes. NK cells from one donor reached a peak expansion limit of approximately 7-million-fold before undergoing contraction whereas NK cells from two donors continued to expand over the length of the study surpassing 100-million-fold expansion, without appearing to have reached a terminal expansion limit. At the end of the study, NK cells from one donor exceeded 1-billion-fold expansion and maintained 88% cytolytic activity compared to Nkarta’s standard process control in a 72-hour IncuCyte assay.ConclusionsIn this study, we demonstrate that healthy donor-derived peripheral blood NK cells are capable of expanding over billion-fold while maintaining potency. These results provide a rationale for the development of off-the-shelf CAR NK cell therapies using NK cells from donors selected to provide optimal product characteristics.Ethics ApprovalHuman samples were collected with written informed consent by an approved vendor.

scholarly journals Small Molecule Screening Identifies Rho-Associate Protein Kinase (ROCK) As a Regulator of NK Cell Cytotoxicity Against Cancer

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3607-3607
Author(s):  
Grace Lee ◽  
Sheela Karunanithi ◽  
Zachary Jackson ◽  
David Wald
Keyword(s):  
Cell Therapy ◽  
Cytotoxic Activity ◽  
Nk Cells ◽  
Nk Cell ◽  
Ex Vivo ◽  
Akt Activation ◽  
Nk Cell Cytotoxicity ◽  
Rock Inhibition ◽  
Nk Cell Therapy

NK cells are a subset of lymphocytes that directly recognize and lyse tumor cells without the limitation of antigen specific receptor recognition. In addition to behaving as cytotoxic effector cells, NK cells unlike T cells are not thought to elicit graft versus host disease. The combination of these characteristics makes NK cells a powerful tool for adoptive cell therapy. Despite the promise of NK cell therapy, key hurdles in achieving significant clinical efficacy include both generating sufficient numbers of highly tumoricidal NK cells and maintaining the cytotoxic activity of these cells in vivo despite the immunosuppressive tumor microenvironment. Our lab and others have developed several feeder cell line-based expansion modules that robustly stimulate the ex vivo proliferation of NK cells. However, strategies to enhance and sustain the activity of NK cells once administered in vivo are still limited. In order to identify strategies to enhance the cytotoxic activity of NK cells, we developed a high-throughput small molecule screen (Figure 1A) that involved a calcein-based cytotoxicity assay of ex vivo expanded and treated NK cells against ovarian cancer cells (OVCAR-3). 20,000 compounds were screened and the screen was found to be highly robust (Z'&gt;0.59). We identified 29 hits that led to at least a 25% increase in cytotoxicity as compared to DMSO control-treated NK cells. One of the most promising hits was the pan-ROCK inhibitor, Y-27632 that led to an 30% increase in NK killing of the OVCAR-3 cells. We validated that ROCK inhibition leads to enhanced NK cell cytotoxic activity using Y-27632 (Figure 1B) as well as other well-established ROCK inhibitors such as Fasudil using a flow cytometry based killing assay. Y-27632 increased NK cell cytotoxicity in a dose- and time- dependent manner. ROCK inhibition consistently led to ~10-25% increase in NK cell cytotoxic activity directed against a variety of ovarian (Figure 1C) and other solid tumor cell lines (Figure 1D). Interestingly, we found that the NK hyperactivation persists for up to 48hrs after washing off the drug that may enable ex vivo stimulation before NK cell infusion. Our preliminary results showed that ROCK inhibition activates PI3K-dependent Akt activation (Figure 1E). We hypothesize that ROCK inhibition restores Akt activation which may be critical for NK cell activating receptor pathways and our current investigations will test these hypotheses. ROCK inhibitors, such as Y-27632 and Fasudil have been utilized in both preclinical and clinical studies for a variety of diseases such as atherosclerosis, neurodegenerative disorders, and ocular diseases. However, the consequences of ROCK inhibition in NK cells has not been thoroughly investigated. Our work shows a promising novel strategy to significantly enhance NK cell therapy against cancer that has high translational potential. Disclosures No relevant conflicts of interest to declare.

scholarly journals Directed Differentiation of Mobilized Hematopoietic Stem and Progenitor Cells into Functional NK Cells with Enhanced Antitumor Activity

Cells ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 811
Author(s):  
Pranav Oberoi ◽  
Kathrina Kamenjarin ◽  
Jose Francisco Villena Ossa ◽  
Barbara Uherek ◽  
Halvard Bönig ◽  
...  
Keyword(s):  
Nk Cells ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Cell Expansion ◽  
Nk Cell ◽  
Ex Vivo ◽  
Feeder Cells ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells

Obtaining sufficient numbers of functional natural killer (NK) cells is crucial for the success of NK-cell-based adoptive immunotherapies. While expansion from peripheral blood (PB) is the current method of choice, ex vivo generation of NK cells from hematopoietic stem and progenitor cells (HSCs) may constitute an attractive alternative. Thereby, HSCs mobilized into peripheral blood (PB-CD34+) represent a valuable starting material, but the rather poor and donor-dependent differentiation of isolated PB-CD34+ cells into NK cells observed in earlier studies still represents a major hurdle. Here, we report a refined approach based on ex vivo culture of PB-CD34+ cells with optimized cytokine cocktails that reliably generates functionally mature NK cells, as assessed by analyzing NK-cell-associated surface markers and cytotoxicity. To further enhance NK cell expansion, we generated K562 feeder cells co-expressing 4-1BB ligand and membrane-anchored IL-15 and IL-21. Co-culture of PB-derived NK cells and NK cells that were ex-vivo-differentiated from HSCs with these feeder cells dramatically improved NK cell expansion, and fully compensated for donor-to-donor variability observed during only cytokine-based propagation. Our findings suggest mobilized PB-CD34+ cells expanded and differentiated according to this two-step protocol as a promising source for the generation of allogeneic NK cells for adoptive cancer immunotherapy.

Ex Vivo Activated Natural Killer (NK) Cells from Myeloma Patients Kill Autologous Myeloma and Killing Is Enhanced by Elotuzumab

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3666-3666
Author(s):  
Tarun K. Garg ◽  
Susann Szmania ◽  
Jumei Shi ◽  
Katie Stone ◽  
Amberly Moreno-Bost ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Therapy ◽  
Nk Cells ◽  
Nk Cell ◽  
Ex Vivo ◽  
K562 Cells ◽  
Scid Mice ◽  
Target Cells ◽  
Recent Trial ◽  
Nk Cell Therapy

Abstract Immune-based therapies may improve outcome for multiple myeloma (MM) by eradicating chemo-resistant disease. Our recent trial utilizing IL2 activated, killer immunoglobulin-like receptor-ligand mismatched NK cell transfusions from haplo-identical donors yielded (n) CR in 50% of patients. Unfortunately, after NK cell therapy, 2/10 patients had progressive disease, and the median duration of response for the other 8/10 patients was only 105 days (range 58–593). This may have been due to an insufficient dose of alloreactive NK cells and early rejection. Furthermore, appropriate donors were identified for only 30% of otherwise eligible patients. We therefore investigated whether NK cells from MM patients could be expanded and activated to kill autologous MM. We then examined whether pre-treatment of MM cell targets with elotuzumab, a humanized antibody to the MM tumor antigen CS1, could further enhance NK cell-mediated lysis. PBMC from 5 MM patients were co-cultured for 14 days with irradiated K562 cells transfected with 4-1BBL and membrane bound IL15 in the presence of IL2 (300U/ml) as previously described (Imai et al, Blood2005;106:376–383). The degree of NK cell expansion, NK immunophenotype, and ability to kill MM (4 hour 51Cr release assays) were assessed. To determine the ability of ex vivo expanded NK cells to traffic to bone marrow, activated NK cells were injected into the tail vein of NK cell depleted NOD-SCID mice, which were then sacrificed after 48 hours. Flow cytometry for human CD45, CD3, and CD56 was performed on cells from blood, marrow and spleen. There was an average 64-fold expansion of NK cells (range: 8–200) after 2 weeks of co-culture with K562 transfectants. Expansion of T cells was not observed. The NK cell activating receptor NKG2D, and natural cytotoxicity receptors NKp30, NKp44, and NKp46 were up-regulated following the expansion. Expanded NK cells were able to kill autologous MM (E:T ratio 10:1, average 31%, range 22–41%), whereas resting NK cells did not. Pretreatment of autologous MM cells with elotuzumab increased the activated NK cell-mediated killing by 1.7-fold over target cells pretreated with an isotype control antibody. This level of killing was similar to that of the highly NK kill-sensitive cell line K562 (Figure). Autologous PHA blasts and CD34+ stem cells were not killed. Activated human NK cells were detectable in the bone marrow of NOD-SCID mice 48 hours after injection. Ex vivo activation of NK cells from MM patients with K562 transfectants can induce killing of autologous MM and produce large numbers of NK cells for potential therapy. The addition of elotuzumab to activated NK cell therapy enhances anti-MM effects by ADCC thus invoking an additional NK cell-mediated mechanism of MM killing. Importantly, ex vivo activated NK cells traffic to the bone marrow in mice. Autologous NK cell therapy eliminates the issues related to allo-donor availability and early NK cell rejection, and could provide an option for patients refractory to chemotherapy agents. Figure Figure

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