scholarly journals Membrane bound IL-21 based NK cell feeder cells drive robust expansion and metabolic activation of NK cells

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Evelyn O. Ojo ◽  
Ashish Arunkumar Sharma ◽  
Ruifu Liu ◽  
Stephen Moreton ◽  
Mary-Ann Checkley-Luttge ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cell Line ◽  
Large Scale ◽  
Nk Cell ◽  
Aerobic Glycolysis ◽  
Third Party ◽  
Feeder Cell ◽  
Adoptive Therapy ◽  
Highly Active ◽  
Membrane Bound

Abstract NK cell adoptive therapy is a promising cancer therapeutic approach, but there are significant challenges that limiting its feasibility and clinical efficacy. One difficulty is the paucity of clinical grade manufacturing platforms to support the large scale expansion of highly active NK cells. We created an NK cell feeder cell line termed ‘NKF’ through overexpressing membrane bound IL-21 that is capable of inducing robust and sustained proliferation (>10,000-fold expansion at 5 weeks) of highly cytotoxic NK cells. The expanded NK cells exhibit increased cytotoxic function against a panel of blood cancer and solid tumor cells as compared to IL-2-activated non-expanded NK cells. The NKF-expanded NK cells also demonstrate efficacy in mouse models of human sarcoma and T cell leukemia. Mechanistic studies revealed that membrane-bound IL-21 leads to an activation of a STAT3/c-Myc pathway and increased NK cell metabolism with a shift towards aerobic glycolysis. The NKF feeder cell line is a promising new platform that enables the large scale proliferation of highly active NK cells in support of large scale third party NK cell clinical studies that have been recently intiatied. These results also provide mechanistic insights into how membrane-bound IL-21 regulates NK cell expansion.

scholarly journals 188 Development of WU-NK-101, a feeder cell-free expanded allogeneic memory NK cell product with potent anti-tumor activity

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A200-A200
Author(s):  
Ryan Sullivan ◽  
Mary Mathyer ◽  
Jennifer Govero ◽  
John Dean ◽  
Andrew Martens ◽  
...  
Keyword(s):  
Nk Cells ◽  
Large Scale ◽  
Nk Cell ◽  
Feeder Cell ◽  
Free Expansion ◽  
Anti Tumor Activity ◽  
Memory Nk Cells

BackgroundAllogeneic Natural Killer (NK) cells are emerging as a safe and effective modality for the treatment of cancer, overcoming several limitations associated with adoptive T cell therapies. Cytokine induced memory-NK cells offer several advantages over conventional NK cells, including enhanced functional persistence, efficacy, and metabolic fitness. Additionally, unlike iPSC and cord blood derived NK cells, they do not require engineering to enable functionality. Here we describe the use of WU-PRIME, a GMP-grade fusion protein complex to generate memory NK cells, and WU-EXPAND, a feeder cell free expansion system to expand memory-NK cells and create WU-NK-101. Further cryopreservation enables the large-scale, off-the-shelf manufacture of memory NK for cancer immunotherapy, with high anti-tumor activity.MethodsNK cells derived from healthy donor leukopheresate were either activated with WU-PRIME and then expanded with WU-EXPAND to form WU-NK-101 or immediately expanded with WU- EXPAND as controls and then cryopreserved. We compared NK cell expansion as well as post- thaw NK cell functionality as assessed by cytokine secretion and short-term and long-term anti- tumor functionality, long-term persistence in NSG mice, as well as anti-tumor activity in vivo.ResultsNK cells activated with WU-PRIME followed by WU-EXPAND (WU-NK-101), expand robustly in large-scale reactions, over 250-fold in 14 days. The cells maintain durable expression of CD25 after expansion, as well as several other hallmarks of the memory-NK phenotype as assessed by mass cytometry. As compared to cells expanded with WU-EXPAND only, WU-NK-101 cells have improved in vitro activity against K562 cells, as well as AML cell lines (TF-1, THP-1, and HL-60). Notably, this functionality is maintained long-term upon repeated challenge. In vivo, WU-NK-101 cells, compared to expanded NK cells have improved in vivo persistence (figure 1; 50,290 v. 9,623, p<0.0001). In vivo anti-tumor activity was also assessed in leukemia models, where Memory NK cells demonstrate superior anti-tumor activity compared to expanded NK cells.Abstract 188 Figure 1NK cell persistence in tumor-bearing mice. 10e6 cryopreserved NK cells were injected into K562 tumor-bearing mice, and supported with 50,000IU human IL-2 every other day. After 9 days, blood was harvested by cheek bleed and assessed for NK cells (hCD45+, CD56+, CD3) in the blood by flow cytometry.ConclusionsThe data demonstrate that WU-NK-101 generated using a feeder cell-free expansion system has a memory phenotype and improved in vitro and in vivo anti-tumor activity compared to conventional NK cells. This activation and expansion platform will enable the development and clinical translation of multiple allogeneic NK cell therapies.

scholarly journals IMMU-47. PROTEIN PHOSPHATASE 2A INHIBITION IN NK CELL THERAPY FOR TREATMENT OF MEDULLOBLASTOMA

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii115-ii115
Author(s):  
Rongze Olivia Lu ◽  
Winson Ho ◽  
Brandon Chiou
Keyword(s):  
Nk Cells ◽  
Cell Line ◽  
Protein Phosphatase ◽  
Nk Cell ◽  
Protein Phosphatase 2A ◽  
T Cell Response ◽  
Intrinsic Activity ◽  
Cd107a Expression ◽  
Phosphatase 2A

Abstract Checkpoint immunotherapy (ICB) thus far has shown limited efficacy against brain tumors, such as medulloblastoma (MB). Its low mutational burden is thought to result in a paucity of neoantigen to trigger an effective T-cell response. Natural killer (NK) cells, can recognize tumor cells independently of neoantigens, making them appealing against MBs. Modulation of NK cells to enhance cytotoxicity against MBs could be a novel treatment strategy. Protein Phosphatase 2A (PP2A), a ubiquitous serine/threonine phosphatase, has been shown to inhibit IFNg and Granzyme B production by NK cells. We hypothesize that NK92, a transformed human NK cell line, has intrinsic activity against human MB cells and that inhibiting PP2A pharmacologically can enhance cytotoxicity of NK92 cells. We performed NK cytotoxicity assay and granulation assay against human MB cell line D425. We also used a small molecular inhibitor, LB100, to modulate PP2A activity in NK92. NK92 cells were co-cultured with D425, in increasing E:T (Effector:Target) ratio for 4 hours. D425 cells were pre-labeled with CellTrace Violet dye. The percentage of D425 (Violet+) cells in apoptosis (Cas3/7+) or necrosis (AAD+) were compared with different ET ratios to quantify NK mediated cell cytotoxicity. We also measured CD107a expression in NK92 to assess granulation with LB100 treatment. D425 cells were sensitive to NK92 killing. Percentage of D425 cells either apoptotic or necrotic increased with increasing ET ratio, suggesting that there was NK92 mediated cytotoxicity. Percentage of killed D425 cells ranged from 18% at baseline (without NK92) to 80% at ET ratio of 20. Inhibition of PP2A using LB100, enhanced NK92 degranulation. CD107a+ NK92 cells increased from 19% to 28% with 8uM of LB100. NK92 cells are cytotoxic against MB cells in vitro and inhibition of PP2A in NK cells can enhance their activity against MB cells.

scholarly journals Aggressive natural killer cell leukemia in an adult with establishment of an NK cell line

Blood ◽  
1986 ◽  
Vol 67 (4) ◽  
pp. 925-930 ◽  
Author(s):  
LA Fernandez ◽  
B Pope ◽  
C Lee ◽  
E Zayed
Keyword(s):  
Platelet Count ◽  
Nk Cells ◽  
Natural Killer ◽  
Cell Line ◽  
Peripheral Blood ◽  
Nk Cell ◽  
Killer Cell ◽  
Chromosome 1 ◽  
Cell Leukemia ◽  
Wbc Count

Abstract There have been many reports of cases in which chronic increases in the numbers of natural killer (NK) cells have been reported. Whether this is reactive or neoplastic in nature has been debated. We report the first case of an aggressive NK cell leukemia in an adult with establishment of an NK cell line. A 70-year-old man had two spontaneous episodes of jejunal perforation and one month later developed a severe febrile illness with moderate splenomegaly. Hemoglobin was 13.1 g/L, and WBC count was 1.8 X 10(9)/L with 2% large granular lymphocytes (LGLs). Platelet count was 143 X 10(9)/L; prothrombin time (PT) and partial thromboplastin time (PTT) were normal. Bone marrow was infiltrated with 25% to 30% LGLs; serum lysozyme was normal. Serum LDH was initially 1,191 U/L and rose to 6,408 (normal 240 to 525 U/L). Ten days later, the WBC count increased to 99.9 X 10(9)/L with 70% LGL cells; the PT and PTT increased, and the platelet count dropped. No bacterial or viral cause of fever was identified. The cells from peripheral blood were LGLs that stained positively for acid phosphatase. All of the LGLs reacted with a monoclonal antibody reactive with NK cells (LEU-11b). Functionally, the patient's peripheral blood mononuclear cells (PBMs) demonstrated 100 times more lytic activity against K562 tumor cell lines than did normal PBMs. The patient's PBMs were propagated in vitro. The cultured cells showed the morphological, cytochemical, immunological, and functional characteristics of NK cells. In addition, partial trisomy involving chromosome 1 q with duplication in regions of q21 through q31 was observed in all metaphases analyzed. The extra chromosome 1q with duplication in regions q21 through q31 was translocated to the p- terminal of chromosome 5. One percent to 5% of normal PBMs comprise NK cells; in most cases, leukemias arise from normal phenotypic counterparts. This case demonstrated that aggressive NK cell leukemia may occur in adults. In addition, the chromosomal abnormalities suggest that this is not a reactive process but a malignancy.

Soluble and Membrane Bound MICA in Myeloma Do Not Adversely Effect Regulation of Natural Killer Cell Function Via NKG2D..

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2462-2462
Author(s):  
Jumei Shi ◽  
Susann Szmania ◽  
Fenghuang Zhan ◽  
John Shaughnessy ◽  
Bart Barlogie ◽  
...  
Keyword(s):  
Cell Surface ◽  
Nk Cells ◽  
Cell Function ◽  
Nk Cell ◽  
Elevated Serum ◽  
Serum Levels ◽  
Down Regulation ◽  
Nkg2d Expression ◽  
Mica Gene ◽  
Membrane Bound

Abstract The human MHC class I -related chain gene A (MICA), a highly polymorphic MHC-encoded cell-surface glycoprotein, is a stress-induced and transformation related molecule absent from most normal tissues. Expression of MICA has been reported on a variety of mostly epithelial tumors. MICA can activate Natural Killer (NK) cells via interacting with the immunoreceptor NKG2D. Soluble MICA (sMICA), when shedded from tumor by the action of matrix-metalloproteinases (MMPs), can be detected at high levels in the sera of cancer patients. High levels of sMICA can down-regulate NKG2D expression and lead to functional impairment of NK cells thus providing for a mechanism of tumor escape. Gene array expression profiling of bone marrow biopsies revealed high levels of MMP2 and MMP9 in the bone marrow microenvironment, but not in the purified myeloma cells of the same patients. Since we are developing allogeneic NK cell therapy as a novel treatment for high-risk myeloma we decided to study whether the high activity of MMP2 and MMP9 resulted in 1) elevated sMICA levels in myeloma, and 2) affected NKG2D expression by NK cells. We found by ELISA assays that 28% (13/46) of patients with MM at diagnosis contained elevated serum levels of sMICA (median: 265.2 pg/ml; range: 125.9 – 806.5 pg/ml). sMICA levels were low in 16 control healthy donors tested (median: 0.8 pg/ml; range: 0 – 91 pg/ml). Next, we correlated sMICA levels with indicators of tumor burden and prognosticators of outcome in MM. We observed that sMICA levels in the serum of MM patients was not associated with Ras associated oncogene (RAN) expression, presence of abnormal cytogenetics, elevated CRP, elevated b2-microglobulin or elevated serum M-protein levels. There was also no correlation between MICA gene expression in purified MM cells and increased sMICA levels in the serum. We therefore examined several myeloma cell lines and found that high MICA gene expression does not always correlate with MICA expression at the cell surface as detected by flow cytometry. We hypothesize that the lack of correlation between MICA RNA expression and sMICA may be due to variation in translation of the MICA mRNA or failure to transport the MICA protein to the cell surface. Further, we tested the expression of NKG2D on CD3−/CD56+ cells of 5 MM patients with elevated sMICA serum levels and detected no down-regulation of NKG2D on NK cells compared to controls comprising MM patients with normal sMICA levels (n=3) or normal donors (n=5). Recent studies have suggested that tumor-membrane bound MICA may also play a role in the down-regulation of NKG2D on NK cells. We next incubated normal donor NK cells with a) the high membrane bound MICA expressing MM cell line U266 or b) serum from MM patients containing high MICA levels neither of which resulted in down-regulation of NKG2D. We conclude that despite the high expression of MMP2 and MMP9 both soluble or membrane bound MICA do not down-regulate NKG2D on NK cells and hence do not adversely affect NK cell function in MM. One explanation may be that the sMICA levels we found in myeloma patients were in the order of pg/ml, whilst the levels reported in epithelial tumors are 2–3 logs higher.

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

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 5012-5012
Author(s):  
Elena Gitelson ◽  
Alexander W. MacFarlane ◽  
Kerry S Campbell ◽  
R. Katherine Alpaugh ◽  
Tahseen I. Al-Saleem ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cell Line ◽  
Peripheral Blood ◽  
Partial Remission ◽  
Nk Cell ◽  
Identical Twin ◽  
Effector Memory ◽  
Target Cells ◽  
Tumor Target ◽  
Memory Phenotype

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

NK Cell Mediated Cytotoxicity Against Malignant Promyelocytes Enhanced By Arsenic Trioxide: Potential Clinical Relevance

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1455-1455
Author(s):  
Ansu Abu Alex ◽  
Hamenth Kumar P ◽  
Saravanan Ganesan ◽  
Nithya Balasundaram ◽  
Kavitha M Lakshmi ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cell Line ◽  
Cell Lines ◽  
Nk Cell ◽  
Single Agent ◽  
Adaptive Immune System ◽  
Cytolytic Activity ◽  
Leukemic Cells ◽  
Ligand Expression ◽  
The Impact

Abstract NK cells are primary effectors of the innate immune response against cells that have undergone malignant transformation. Several lines of evidence indicate that the expression level of NK ligands on leukemic cells affects the sensitivity of the leukemic cells to cytolytic activity by NK cells. Various agents have been evaluated for their ability to induce these ligands on leukemic cells to augment the NK cell mediated anti-leukemia effect. There is substantial evidence that has established the importance of the adaptive immune system in the treatment of acute promyelocytic leukemia (APL) (Rose Ann Padua et al. Nat Med 2003). While there is significant data which address the mechanisms of arsenic trioxide (ATO) on malignant promyelocytes, limited data is available of its effect on the innate and adaptive immune system. We undertook a series of experiments to address the impact of ATO on NK cell receptor and malignant promyelocyte ligand expression and its effect on NK cell mediated cytotoxicity. We also evaluated NK cell reconstitution in patients treated with ATO and the impact of KIR genotypes on relapse. We first evaluated the cytotoxic activity of NK92MI (NK cell line) against 5 different myeloid (K562, U937, HL60, UF1, NB4) and 2 lymphoid cell lines (Jurkat E6.1, SUP-B15) by CFSE/ 7AAD cytotoxicity assay. Target (T) cells (1x 105/100 µL/well) pre-treated with CFSE were co-cultured with effector NK cells (E) at a E:T ratio of 1:1, 2:1 and 5:1 for 5 hours at 37°C in 96 well plates. The percentage cytolytic activity of the NK cells was then calculated after adding 7AAD and acquired in FACS Calibur (Becton Dickinson, San Jose, CA, USA). Significant cytolytic activity was noted against K562 and NB4 cell lines. At the highest E:T ratio there was a median 22% cytolytic activity against NB4 (N=5). We observed that NB4 when treated overnight with 1µM ATO (>99% viability retained after this exposure) significantly increased the cytotoxic effect of NK92MI cell line at all the E:T ratios as shown in figure 1A (n=5; P=0.0023). No other cell line showed a similar increase in cytotoxic effect following exposure to ATO at these concentrations (data not shown). We next evaluated the effect of exposure of NB4 cells to ATO at 1µM for 6 hours on NK ligand expression by flowcytometry. As shown in figure 1B there was a significant increase in activating ligand MICA/B in NB4 cell lines (n=3; P=0.016) which was not seen in any of the other cell lines. Similar significant increased expression of Nectin-2 (DNAM-1 ligand) and HLA Class I was seen. Exposure of NK92MI to ATO for 6 hours at 1uM (non cytotoxic dose:IC50-3.8uM) resulted in increased expression of activating receptors NKG2D, NKP30 and KIR2DS4 (figure 1C) and inhibitory receptor NKG2A and decrease in inhibitory receptors KIR3DL1/DL2. There were no changes in the expression of NKP46, KIR2DL1, KIR2DL2 and DNAM1 receptors. We undertook a prospective study to evaluate the pattern of NK (CD56+CD3-) reconstitution in patients with newly diagnosed APL treated at our center with a single agent ATO regimen (Mathews et al. JCO 2011). The mean NK cell counts in patients were below the 2SD deviation level of the normal range even after completion of therapy (approximately a year)(figure 1D). All other subsets evaluated (CD4, CD8, CD3, CD19, CD56+CD3+, CD4CD45RO) had returned to levels within the normal range by the end of consolidation therapy (approximately 3 months from diagnosis). KIR genotyping was done on 55 patients with APL who received treatment with single agent ATO based regimen. The median follow up of this cohort was 20 months and 14 cases relapsed following initial therapy. The presence or absence of 17 KIR genes was done by PCR-SSP method (KIR Typing kit, Miltenyi Biotech Inc, CA). There was no association with any specific genotype or haplotype with risk of relapse. In summary we have noted that there is up regulation of receptors on NK cells and ligands on malignant promyelocytes following exposure to ATO that favors NK cell mediated cytotoxicity. In-vitro we have demonstrated a significant increase in NK cell mediated cytolytic activity against malignant promyelocytes exposed to ATO even at relatively low E:T ratios. This could be an important mechanism by which ATO induces durable remissions in patients with APL. The delayed NK cell recovery following treatment with ATO raises the possibility of using NK cell therapy to augment the effect of ATO in the treatment of patients.Figure 1Figure 1. Disclosures: No relevant conflicts of interest to declare.

Decitabine Or 5-Azacitidine Pre-Treatment Does Not Compromise The Novel Fc-Engineered Antibody Mab 33.1-Mediated ADCC Against Primary AML Blasts - A Rationale For Combination Therapy

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3959-3959
Author(s):  
Shun He ◽  
Carolyn Cheney ◽  
Susan P. Whitman ◽  
Jianhua Yu ◽  
Sumithira Vasu ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cell Line ◽  
Cell Lines ◽  
Nk Cell ◽  
Hl60 Cell ◽  
Adcc Activity ◽  
Term Survival ◽  
Effector Cells ◽  
Blast Cells ◽  
Pre Treatment

Abstract Introduction Acute Myeloid leukemia (AML) in patients older than 60 years is a devastating diagnosis with long-term survival rates of 10%. Elderly patients have poor survival both due to chemoresistance and presence of concomitant comorbidities rendering them ineligible for induction chemotherapy. Hence novel treatment options are warranted in this patient population. Promising activity of monoclonal antibodies such as alemtuzumab and rituximab for chronic lymphocytic leukemia (CLL) and rituximab for lymphomas has raised the potential use of antibody therapies in AML. CD33 is expressed on greater than 90% of AML blast cells while absent from all non-hematopoietic tissues. Hence CD33 is a viable target for antibody-based therapeutics in AML. Here, we tested the ex vivo efficacy of the mAb 33.1, a fully human anti-CD33 antibody Fc-engineered for increased binding to Fcγ receptors on AML cell lines and primary AML blasts. The goals of this study are to evaluate 1) the efficacy of mAb33.1 on purified allogeneic and autologous natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC) against primary AML Blasts; 2) to evaluate efficacy of mAb 33.1 in combination with azanucleosides (i.e. decitabine, 5-azacitidine) that are currently used in AML therapy on NK cell-mediated ADCC against primary AML blasts; and 3) to correlate the levels of surface expression of CD33 on AML blasts to the mAb 33.1 mediated ADCC. Methods mAb 33.1 mediated NK cell activation was determined by NK degranulation as determined by CD107a induction, and ADCC was determined by standard 4-hour 51Cr-release assay. An AML cell line HL60 and a total of 15 AML blast samples were used as targets in this study. NK cells enriched from normal donor PBMC (for allogeneic assays) or sorted from AML blast samples (for autologous assays) were used as effector cells. Results The mAb 33.1 induced potent ADCC activity (>40%) compared to control non-Fc engineered antibody at the concentration of 10 μg/ml in the HL60 cell line. For the AML blasts, mAb 33.1 mediated significantly higher ADCC activity when compared to the control antibody (p<0.05). The relative cytotoxicity mediated by mAb 33.1 varied among different patients, ranging from 4.4% to 65.8%. Subsequent quantification of CD33 showed that there is a positive correlation between ADCC activity and the number of surface CD33 molecules on the AML blasts. Induction of CD107a expression was also observed in both allogeneic and autologous NK cells when the blasts were labeled with mAb 33.1. Pre-treatment of the NK cells and/or target blasts with decitabine or 5-azacitidine for 48hrs, did not alter the mAb 33.1 mediated ADCC activity or CD107 induction. Conclusion mAb33.1 mediated potent ADCC activity and NK activation against AML cell lines and primary AML blasts. Both autologous and allogeneic NK cell-mediated ADCC against primary blast cells from AML patients was observed. The level of NK cell-mediated ADCC was positively associated with the levels of the surface CD33 expression on target AML blasts. Pre-treatment of either AML blasts and/or NK effector cells with Decitabine or 5-azacitidine did not compromise mAb 33.1-mediated ADCC. These pre-clinical studies support further clinical development of mAb 33.1 in combination with relevant anti-AML therapies such as decitabine or 5-azacitidine in patients with CD33 expression. Disclosures: Heider: boehringer-ingelheim: Employment.

scholarly journals Effects of IL-2 on MMP Expression in Freshly Isolated Human NK Cells and the IL-2-independent NK Cell Line YT

2010 ◽  
Vol 33 (5) ◽  
pp. 475-481 ◽  
Author(s):  
Karin Edsparr ◽  
Frank M. Speetjens ◽  
Adri Mulder-Stapel ◽  
Ronald H. Goldfarb ◽  
Per H. Basse ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cell Line ◽  
Nk Cell

scholarly journals Proliferation of Highly Cytotoxic Human Natural Killer Cells by OX40L Armed NK-92 With Secretory Neoleukin-2/15 for Cancer Immunotherapy

2021 ◽  
Vol 11 ◽  
Author(s):  
Meng Guo ◽  
Chen Sun ◽  
Yuping Qian ◽  
Liye Zhu ◽  
Na Ta ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Cell Expansion ◽  
Large Scale ◽  
Cell Function ◽  
Treatment Effectiveness ◽  
Nk Cell ◽  
Expansion Mechanism ◽  
Human Natural Killer Cells

Adoptive natural killer (NK) cell transfer has been demonstrated to be a promising immunotherapy approach against malignancies, but requires the administration of sufficient activated cells for treatment effectiveness. However, the paucity of clinical-grade to support the for large-scale cell expansion limits its feasibility. Here we developed a feeder-based NK cell expansion approach that utilizes OX40L armed NK-92 cell with secreting neoleukin-2/15 (Neo-2/15), a hyper-stable mimetic with a high affinity to IL-2Rβγ. The novel feeder cells (NK92-Neo2/15-OX40L) induced the expansion of NK cells with a 2180-fold expansion (median; 5 donors; range, 1767 to 2719) after 21 days of co-culture without added cytokines. These cells were highly cytotoxic against Raji cells and against several solid tumors in vivo. Mechanistically, NK92-Neo2/15-OX40L induced OX40 and OX40L expression on expanded NK cells and promoted the OX40-OX40L positive feedback loop, thus boosting NK cell function. Our data provided a novel NK cell expansion mechanism and insights into OX40-OX40L axis regulation of NK cell expansion.

scholarly journals CD38low Natural Killer Cells Transiently Expressing CD16F158V m-RNA Potentiates the Therapeutic Activity of Daratumumab Against Multiple Myeloma with Minimal Effector NK Cell Fratricide

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3199-3199 ◽  
Author(s):  
Subhashis Sarkar ◽  
Sachin Chauhan ◽  
Arwen Stikvoort ◽  
Alessandro Natoni ◽  
John Daly ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cell Line ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Nk Cell ◽  
Ex Vivo ◽  
Advisory Committees ◽  
Cd38 Expression ◽  
Rpmi 8226

Abstract Introduction: Multiple Myeloma (MM) is a clonal plasma cell malignancy typically associated with the high and uniform expression of CD38 transmembrane glycoprotein. Daratumumab is a humanized IgG1κ CD38 monoclonal antibody (moAb) which has demonstrated impressive single agent activity even in relapsed refractory MM patients as well as strong synergy with other anti-MM drugs. Natural Killer (NK) cells are cytotoxic immune effector cells mediating tumour immunosurveillance in vivo. NK cells also play an important role during moAb therapy by inducing antibody dependent cellular cytotoxicity (ADCC) via their Fcγ RIII (CD16) receptor. Furthermore, 15% of the population express a naturally occurring high affinity variant of CD16 harbouring a single point polymorphism (F158V), and this variant has been linked to improved ADCC. However, the contribution of NK cells to the efficacy of Daratumumab remains debatable as clinical data clearly indicate rapid depletion of CD38high peripheral blood NK cells in patients upon Daratumumab administration. Therefore, we hypothesize that transiently expressing the CD16F158V receptor using a "safe" mRNA electroporation-based approach, on CD38low NK cells could significantly enhance therapeutic efficacy of Daratumumab in MM patients. In the present study, we investigate the optimal NK cell platform for generating CD38low CD16F158V NK cells which can be administered as an "off-the-shelf"cell therapy product to target both CD38high and CD38low expressing MM patients in combination with Daratumumab. Methods: MM cell lines (n=5) (MM.1S, RPMI-8226, JJN3, H929, and U266) and NK cells (n=3) (primary expanded, NK-92, and KHYG1) were immunophenotyped for CD38 expression. CD16F158V coding m-RNA transcripts were synthesized using in-vitro transcription (IVT). CD16F158V expression was determined by flow cytometry over a period of 120 hours (n=5). 24-hours post electroporation, CD16F158V expressing KHYG1 cells were co-cultured with MM cell lines (n=4; RPMI-8226, JJN3, H929, and U266) either alone or in combination with Daratumumab in a 14-hour assay. Daratumumab induced NK cell fratricide and cytokine production (IFN-γ and TNF-α) were investigated at an E:T ratio of 1:1 in a 14-hour assay (n=3). CD38+CD138+ primary MM cells from newly diagnosed or relapsed-refractory MM patients were isolated by positive selection (n=5), and co-cultured with mock electroporated or CD16F158V m-RNA electroporated KHYG1 cells. CD16F158V KHYG1 were also co-cultured with primary MM cells from Daratumumab relapsed-refractory (RR) patients. Results: MM cell lines were classified as CD38hi (RPMI-8226, H929), and CD38lo (JJN3, U266) based on immunophenotyping (n=4). KHYG1 NK cell line had significantly lower CD38 expression as compared to primary expanded NK cells and NK-92 cell line (Figure 1a). KHYG1 electroporated with CD16F158V m-RNA expressed CD16 over a period of 120-hours post-transfection (n=5) (Figure 1b). CD16F158V KHYG1 in-combination with Daratumumab were significantly more cytotoxic towards both CD38hi and CD38lo MM cell lines as compared to CD16F158V KHYG1 alone at multiple E:T ratios (n=4) (Figure 1c, 1d). More importantly, Daratumumab had no significant effect on the viability of CD38low CD16F158V KHYG1. Moreover, CD16F158V KHYG1 in combination with Daratumumab produced significantly higher levels of IFN-γ (p=0.01) upon co-culture with CD38hi H929 cell line as compared to co-culture with mock KHYG1 and Daratumumab. The combination of CD16F158V KHYG1 with Daratumumab was also significantly more cytotoxic to primary MM cell ex vivo as compared to mock KHYG1 with Daratumumab at E:T ratio of 0.5:1 (p=0.01), 1:1 (p=0.005), 2.5:1 (p=0.003) and 5:1 (p=0.004) (Figure 1e). Preliminary data (n=2) also suggests that CD16F158V expressing KHYG1 can eliminate 15-17% of primary MM cells from Daratumumab RR patients ex vivo. Analysis of more Daratumumab RR samples are currently ongoing. Conclusions: Our study provides the proof-of-concept for combination therapy of Daratumumab with "off-the-shelf" CD38low NK cells transiently expressing CD16F158V for treatment of MM. Notably, this approach was effective against MM cell lines even with low CD38 expression (JJN3) and primary MM cells cultured ex vivo. Moreover, the enhanced cytokine production by CD16F158V KHYG1 cells has the potential to improve immunosurveillance and stimulate adaptive immune responses in vivo. Disclosures Sarkar: Onkimmune: Research Funding. Chauhan:Onkimmune: Research Funding. Stikvoort:Onkimmune: Research Funding. Mutis:Genmab: Research Funding; OnkImmune: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Research Funding; Celgene: Research Funding; Novartis: Research Funding. O'Dwyer:Abbvie: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; BMS: Research Funding; Glycomimetics: Research Funding; Onkimmune: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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