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 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.

Re-Targeting of an NK Cell Line (NK92) with Specificity for CD19 Efficiently Kills Human B-Precursor Leukemia Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2747-2747 ◽  
Author(s):  
Annette Romanski ◽  
Christoph Uherek ◽  
Gesine Bug ◽  
Tina Muller ◽  
Claudia Rossig ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Nk Cell ◽  
Antibody Fragment ◽  
Surface Expression ◽  
Cytolytic Activity ◽  
Leukemia Cells ◽  
Cell Cytotoxicity ◽  
Nk Cell Cytotoxicity ◽  
B Lineage

Abstract The continuously growing Natural killer (NK) cell line NK-92 is highly cytotoxic against malignant cells of various origin without affecting normal human cells. It is the only NK cell line that has entered clinical trials to date. Acute lymphoblastic leukemias (ALL) show variable sensitivity towards NK cells including NK-92-lysis. All T-ALL cell lines tested (MOLT-3, MOLT-4 and CEM-T) display moderate sensitivity to NK cytotoxicity with approximately 22±2 – 24±2% killing at an effector to target (E:T) ratio of 10:1, respectively. Comparable results were obtained with primary patient derived T-ALLs (n=4), displaying specific killing rates of 17±5 – 20±2%. In contrast, B lineage ALL cell lines (NALM-6, SEM, REH, JKB, BV173, SupB15, TOM-1, TMD5) and cells derived from patients (n=11) were resistant to NK-92-mediated lysis, with specific killing rates ranging from 2±1% to 13±3% at an E:T ratio of 10:1. Approaches to overcome resistance of B-precursor ALL by re-targeting may shed light on the specific reasonable mechanism conferring resistance and lead to novel therapeutic strategies. Here we have generated genetically modified NK-92 cells expressing a chimeric antigen receptor specific for the pan B cell antigen CD19 which is universally expressed by B lineage leukemia cells. This receptor fragment kindly provided by H. Zola (Child Health Research Institute North Adelaide, Australia) consists of the CD19 specific scFv(CD19) antibody fragment, a flexible hinge region, the CD3 ζ chain and a Myc-tag. Transduced cells were selected with G418, and surface expression of the chimeric scFv(CD19)-ζ construct was verified by FACS analysis using the Myc-tag-specific mAb 9E10. No difference in cytotoxic activity of NK-92 and transduced NK-92-scFv(CD19)-ζ cells towards CD19 negative targets (K562, MOLT-4) was found. In contrast, NK-92-scFv(CD19)-ζ cells specifically and efficiently lysed CD19 expressing B precursor leukemia cells (NALM-6, SEM, B173, SupB15, TOM-1, TMD5) including cells that were completely resistant to cytolytic activity of parental NK-92 cells. Killing of SupB15 and BV173 cells was 10,0±0,8% and 12,0±8,5% with parental NK92 vs. 58,2±14,8% and 52,3±4,1% lysis with NK92 cells expressing the CD19 specific scFv(CD19) antibody fragment. These results demonstrate that efficient retargeting of NK-cell cytotoxicity can be achieved, and might allow the generation of potent cell-based therapeutics. Since the widespread resistance of B precursor ALL blasts to NK cell cytotoxicity is not caused by mechanisms commonly relevant in tumors, this model may allow dissection of possible mechanisms of resistance.

Lenalidomide Effects on NK Function in Patients with Myelodysplastic Syndrome (MDS).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3437-3437 ◽  
Author(s):  
P.K. Epling-Burnette ◽  
Fanqi Bai ◽  
Jeffrey S. Painter ◽  
Julie Y. Djeu ◽  
Alan F. List
Keyword(s):  
Nk Cells ◽  
Cell Line ◽  
Cell Lines ◽  
Cellular Response ◽  
Nk Cell ◽  
Leukemia Cell ◽  
T Test ◽  
Leukemia Cell Line ◽  
Immunomodulatory Effects

Abstract Lenalidomide, which is a 4-amino-glutarimide analogue of thalidomide, has significant erythropoietic activity in patients with lower-risk MDS (List et al, NEJM, 351:26,2004). Although its precise target of action in MDS is not known, lenalidomide modulates cellular response to varied stimuli including inhibition of angiogenic response and endotoxin induction of inflammatory cytokines and enhancement of antigen-induced immunologic response and erythropoietin receptor signaling. Clinical investigations in multiple myeloma indicate that the immunomodulatory effects of thalidomide and lenalidomide extends to the expansion of natural killer (NK) cells. We recently found that MDS patients have defective NK function, araising in part to reduced expression of activating NK receptors (NKRs) NKp30, CD244 (2B4), and NKG2D. To determine if lenalidomide may restore NK function in MDS, we investigated the effects of in vitro treatment with lenalidomide on NK function and phenotype. Lytic function was studied using peripheral blood mononuclear cells (PBMCs) as effector cells and the leukemia cell line, K562, as a target in standard 4-hr 51Cr-release assays at 12:1 and 25:1 effector:target (E:T) ratios. Among eight MDS patient’s specimens evaluated, five patients had significant increase in tumor lysis after treatment with 1 μM lenalidomide for 72 hours (p ≤ 0.01, T-test). In similar experiments using PBMCs from normal donors, we found that NK lysis of K562 was 42% ± 15 (25:1 E:T ratio) pre-treatment which increased to 71% ± 17 (25:1 E:T ratio) after treatment, which was statistically significant (p ≤ 0.01, T-test). We also examined the in vitro effects of lenalidomide on lytic activity by the NK cell lines, NK92 and NKL, which was significantly increased after drug treatment. To discern the mechanisms of lenalidomide action in NK cell lines and normal NK cells, we evaluated NKR display by flow cytometry, and NKR function by antibody redirected cytotoxicity using the FcγR+ murine mastocytoma (P815) target cell line. Using NK92 and NKL cells, treatment with lenalidomide 1 μM for 72 hrs increased lysis by anti-NKG2D and anti-CD244 activating antibodies. We also found that NKG2D surface expression was increased on normal NK cells after lenalidomide treatment in vitro. These results suggest that some MDS patients may have improved NK function through the immunomodulatory effects of lenalidomide. The relationship between in vitro NK responsiveness to lenalidomide and in vivo hematological response warrants investigation in patients with MDS.

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

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3436-3436
Author(s):  
Fanqi Bai ◽  
Jeffrey S. Painter ◽  
Cantor Alan ◽  
Zou JianXiang ◽  
Sheng Wei ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Cell Line ◽  
Cell Lines ◽  
Nk Cell ◽  
B Cell Lymphoma ◽  
Target Cells ◽  
Nkg2d Ligands ◽  
Human Stress ◽  
Normal Controls

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

Autologous Expanded Natural Killer Cells As a New Therapeutic Option for High-Risk Myeloma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2918-2918
Author(s):  
Tarun K. Garg ◽  
Junaid Khan ◽  
Susann Szmania ◽  
Amy D Greenway ◽  
Joshuah D Lingo ◽  
...  
Keyword(s):  
High Risk ◽  
Natural Killer Cells ◽  
Nk Cells ◽  
Natural Killer ◽  
Cell Line ◽  
Nk Cell ◽  
Tumor Burden ◽  
Cytolytic Activity ◽  
Killer Cells

Abstract Abstract 2918 Natural killer cells (NK) have the unique ability to kill target cells without priming. While their therapeutic potential against various malignancies is becoming more apparent, it has been restricted to the allogeneic setting; NK cells are inhibited by autologous targets by engaging killer immunoglobulin-like receptors with their ligands. Another major challenge to the clinical utility of NK cells is obtaining a sufficient number of NK cells for infusion. Co-culture of blood mononuclear cells (PBMNC) with the leukemic cell line K562, genetically modified to express membrane-bound IL15 and the co-stimulatory molecule 41BBL (K562mbIL15-41BBL) in the presence of IL2 results in robust expansion and activation of NK cells. To determine if NK cells derived from myeloma (MM) patients can be used therapeutically in the autologous setting, we explored the expansion of NK cells from MM patients, their gene expression profiles (GEP), and their ability to kill autologous and allogeneic MM cells from high-risk patients in vitro and in vivo, and compared these to NK cells from healthy donors (HD). PBMNC from MM patients (N=30) co-cultured with irradiated K562mbIL15-41BBL cells expanded a median of 351 fold (range20–10, 430), comparable to the expansion of HD-derived NK cells (N=15, median 803, range 127–1, 727; p=0.5). GEP of MM non-exp-NK differed from HD non-exp-NK in the expression of only one gene (PRKCi), underexpessed in MM (false discovery rate (FDR) <0.05, p-value <3×10−10). GEP of exp-NK cells from both MM patients and HD was very different from non-exp-NK cells (8 pairs each, 10, 639 differentially overexpressed and 26, 057 underexpressed probe sets, FDR <0.05). Genes associated with proliferation, cytolytic activity, activation, adhesion, migration and cell cycle regulation were highly up-regulated in exp-NK cells. Standard chromium release assays demonstrated that MM exp-NK cells killed both allogeneic and autologous primary MM cells more efficiently compared to non-exp-NK cells, via a perforin mediated mechanism. Blocking studies revealed that the natural cytotoxicity receptors, activating receptors, and DNAX accessory molecule (DNAM-1) played a central role in target cell lysis. The killing ability of MM patient and HD derived exp-NK cells was very similar against allogeneic targets, while primary MM targets were more resistant to killing by autologous exp-NK. The anti-MM activity of allogeneic and autologous exp-NK cells was further examined in vivo. NOD/SCID/IL2R γ-null mice were implanted subcutaneously with a human fetal bone, and primary MM cells or luciferase-transfected OPM2 MM cell line were engrafted into the bone. The tumor burden was determined by ELISA for human Ig and/or bio-imaging. The mice were randomized to control and exp-NK treatment groups. A total of 160 ×106 exp-NK cells, in 4 doses 48 hrs apart, were injected in the exp-NK treatment group via tail vein injection. The mice were administered 1000U of IL2 subcu daily to support the NK cells. The mice were bled on days 7, 14, 21 & 28 for the assessment of human Ig by ELISA and enumerating circulating NK cells by flow cytometry. Exp-NK treated mice had a significantly reduced MM burden by ELISA (p<0.04) on day 21, and exp-NK could be detected in the murine blood up to day 28 post-administration in both primary MM and OPM2 tumor bearing mice. The mice were sacrificed and the tumors were harvested after 4 weeks. A noticeable reduction in tumor burden in the exp-NK cell treated mice was confirmed by histology. NK cells were detected by immunohistochemistry (CD57 or CD16) in the hu-bone implants harvested 28 days after infusion. In conclusion, MM patient-derived NK cells have a similar expansion potential, and MM exp-NK cells have cytolytic activity against allogeneic targets similar to those of HD exp-NK cells, and somewhat reduced activity against autologous targets. These exp-NK cells have significant activity against the aggressive cell line OPM2 and high-risk autologous primary MM cells in vivo. Exp-NK cells trafficked to MM tumors and persisted in the myelomatous hu bone microenvironment for 4 weeks. The anti-MM activity of autologous exp-NK cells is exciting and avails a new therapeutic avenue for patients with GEP-defined high-risk disease. A phase II clinical trial of allogeneic and autologous exp-NK cell therapy for relapsed/refractory high-risk MM is in progress at our institution. Disclosures: No relevant conflicts of interest to declare.

NKG2D ligand expression in AML increases in response to HDAC inhibitor valproic acid and contributes to allorecognition by NK-cell lines with single KIR-HLA class I specificities

Blood ◽  
2008 ◽  
Vol 111 (3) ◽  
pp. 1428-1436 ◽  
Author(s):  
Stefan Diermayr ◽  
Heike Himmelreich ◽  
Bojana Durovic ◽  
Arina Mathys-Schneeberger ◽  
Uwe Siegler ◽  
...  
Keyword(s):  
Valproic Acid ◽  
Cell Surface ◽  
Nk Cells ◽  
Cell Lines ◽  
Nk Cell ◽  
Human Leukocyte ◽  
Hla Class I ◽  
Inhibitory Effect ◽  
Class I ◽  
The Impact

Abstract This study exploited alloreactivity of natural killer (NK) cells for augmenting the recognition of human acute myeloid leukemia (AML). To circumvent the inhibitory effect of killer immunoglobulin receptor (KIR) signaling, we generated NK-cell lines with single KIR specificities for major human leukocyte antigen (HLA) class I allotypes. We demonstrated efficient cytolysis of KIR-HLA class I–mismatched primary AML blasts even at low effector-to-target ratios. To define the impact of tumor-associated activating NKG2D-ligands (NKG2D-L), 66 AML patients at diagnosis were analyzed. NKG2D-L were selectively expressed on monoblastic cells in AML M4 and M5 yet absent or weakly expressed on myeloblastic cells in all AML subtypes. Paucity of cell-surface NKG2D-L was not the result of shedding because levels of soluble ULBP1 ligand measured in AML plasma were in the normal range. Notably, purified NKG2D-L+ monoblastic cells were more susceptible to NK-mediated killing than NKG2D-L− myeloblastic cells. Accordingly, induction of cell-surface NKG2D-L by treatment with the histone deacetylase inhibitor, valproic acid, rendered cells more sensitive to NK cytolysis. These data suggest that adoptive transfer of selected populations of alloreactive HLA class I–mismatched NK cells in combination with pharmacologic induction of NKG2D-L merits clinical evaluation as novel approaches to immunotherapy of human AML.

scholarly journals CD19 Target Activated Natural Killer (CD19.TaNK) Cellular Therapy: A Novel immunotherapeutic Approach to the Treatment of Non-Hodgkin Lymphoma (NHL)

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4174-4174
Author(s):  
Ravi Dashnamoorthy ◽  
Afshin Beheshti ◽  
Saheli Sarkar ◽  
Pooja Sabhachandani ◽  
Frank C. Passero ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cell Lines ◽  
Nk Cell ◽  
Resistant Cell ◽  
Cytolytic Activity ◽  
Lymphoma Cells ◽  
Cd20 Antibody ◽  
Anti Cd20 ◽  
Targeted Immunotherapy ◽  
Cd20 Antibodies

Abstract Background: Continued improvement in the treatment of NHL is desired, especially via the incorporation of 'targeted' immunotherapy agents. This is especially important in B-cell NHL (bNHL) as resistance to rituximab anti-CD20 antibody, and now second-generation antibodies (e.g., obinutuzumab), may occur. Activated NK-92 (aNK-92) is a continuously growing cell line consisting of "pure" (100%) activated NK cells. These cells were subsequently bioengineered to express human anti-CD19 chimeric antigen receptor (CAR) recognizing CD19+ B cells. The goal of this project was to investigate the specificity and the efficacy of a novel 'off the shelf' targeted immunotherapy, CD19.TaNK, in a multitude of B-cell NHL cell lines, including anti-CD20 antibody resistant cell lines. Methods: Using gene expression profiling, Gene Set Enrichment Analysis and Ingenuity Pathway Analysis, we first investigated the expression of NK activation and inhibitory ligands in varied lymphoma cells. The bNHL cell lines, SUDHL10 (DLBCL), L540, L428 (Hodgkin lymphoma), HF1 (follicular), Raji (Burkitt's), and Mino (mantle cell) were purchased from ATCC and maintained in RPMI1640 medium. aNK and CD19.TaNK were supplied by NantKwest, Inc and were maintained in Myelocult supplemented with recombinant human IL-2 (500IU/ml). NK cell mediated cytotoxicity was determined using lactate dehydrogenase (LDH) release glucose-6-phosphate dehydrogenase (G6PD) release (aCella-tox assay). Briefly, 10,000 target bNHL cells were co-cultured with effector NK cells, at clinically relevant effector to target ratios (E:T 1:1-10:1) for 4 hours, and the supernatant was assayed for LDH or G6PD release. Percent cytotoxicity was determined based on the experimental levels of LDH or G6PD release from NK mediated bNHL cell lysis compared to maximum LDH or G6PD release from target cells. To determine if resistance to anti-CD20 antibodies would interfere with sensitivity to CD19.TaNK therapy, rituximab and obinutuzumab resistant bNHL cell lines (SUDHL4, SHUDHL10, and Raji) were established; cells were exposed to incremental increasing concentrations of antibody drugs (5-20μg/ml) over a period of 8 weeks. CD19, CD20 and CD30 expression in bNHL cells was determined by flow cytometry. Additionally, the efficacy of primary NK cells were determined against CD20 monoclonal antibody sensitive and resistant cell lines utilizing droplet microfluidics based assessment. Results: We observed that bNHL cell lines expressed a multitude of ligands associated with stimulating NK cell activity, while expression of inhibitory ligands was minimal. This indicates that NK cell interaction with bNHL cells is predicted to lead to overall robust antitumor immune response (Figure). Using LDH and G6PD release assays in bNHL cell lines, we observed increased cytolytic activity in an E:T ratio dependent manner, with Raji and L428 cells being the most sensitive to CD19.TaNK at 1:1 E:T ratio. Development of resistance to anti-CD20 antibodies (rituximab and obinutuzumab) resulted in significantly decreased down regulation of CD20, but not CD19 or CD30, as detected by flow cytometry. After direct contact with primary NK cells, we observed that rituximab resistant SUDHL10 cells were poorly sensitive (7%), while in rituximab sensitive cells, there was 22% cell loss. Moreover, at 4 hours using CD19.TaNK therapy (1:5 ratios), there was marked cytolytic activity with consistent high LDH release seen across all bNHL cell lines without differences noted regardless of rituximab or obinutuzumab resistance (ie, SUDHL4, SHUDHL10, and Raji). These results were further confirmed using live cell video microscopy measuring the cytolytic activity of CD19.TaNK versus bNHL cells. Conclusion: We identified that bNHL cells contain high expression levels of NK activation ligands and low amounts of inhibitory ligands and that CD19.TaNK immunotherapy had potent single-agent anti-tumor activity against a spectrum of bNHL cells. Furthermore, CD19.TaNK maintained high cytolytic activity in bNHL cells resistant to standard CD20 antibody therapy, which were poorly sensitive to innate NK cells. Ongoing analyses include systems biology studies to determine potential biologic mechanisms of activity of CD19.TaNK therapy as well as well as to help guide optimum combinatorial therapy. Figure Expression of NK activation and inhibitory ligands in lymphoma cells. Figure. Expression of NK activation and inhibitory ligands in lymphoma cells. Disclosures Boissel: NantKwest, Inc.: Employment. Evens:Takeda: Other: Advisory board.

Quantitative HLA Class I Expression and KIR-Mismatch between NK-Cell-Donor and Patient Predict NK Cell Mediated Lysis of Leukemic Cells from Children with Acute Lymphatic Leukemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5205-5205
Author(s):  
Matthias M. Pfeiffer ◽  
Michael Schumm ◽  
Klaus Dietz ◽  
Tobias Feuchtinger ◽  
Rupert Handgretinger ◽  
...  
Keyword(s):  
Nk Cells ◽  
Adhesion Molecules ◽  
Nk Cell ◽  
Hla Class I ◽  
Class I ◽  
Leukemic Cells ◽  
Specific Lysis ◽  
Lymphatic Leukemia ◽  
Hla Class I Molecules ◽  
The Impact

Abstract Relapses represent a major problem after transplantations in children with ALL. Natural Killer (NK) cells have been shown to exert remarkable Graft versus Leukemia effects after mismatched stem cell transplantation in myeloic leukemia, whereas the efficacy against lymphatic leukemia is still unclear. We therefore measured intensity of HLA class I expression on leukemic blasts by quantitative FACS analysis and investigated the impact of quantitative HLA class I expression, of several adhesion molecules and of KIR-mismatch on NK cell mediated lysis of the leukemic blasts from 21 pediatric patients with ALL. Expression of HLA class I molecules differed widely from patient to patient (range 5000–500000) and was reduced in comparison to B cells from healthy donors in 70% of cases. NK cells killed leukemic blasts very heterogeneously but a clear association between number of HLA class I molecules per cell and specific lysis (range 13–98%) was found (r2=0.68, p<0.0001). For the subgroup of leukemic blasts without KIR-mismatch, this association was even stronger (r2=0.98), whereas a weak association was found for the subgroup with KIR-mismatch, since most of these targets were lysed more efficiently than one could expect according to HLA class I expression alone. KIR-mismatch alone (t-test, p=0.45) as well as different patterns of adhesion molecules (ICAM1-3, LFA1/3) and CD95 had no significant influence. However, a multivariate model taking both HLA expression and KIR-ligand-mismatch into account, provided an even stronger association (r2=0.87 p<0.0001) for the whole group. Lysis was mainly dependent on HLA class I expression and in addition on KIR-mismatch for these leukemic cells. Assessment of HLA expression on leukemic blasts and KIR-receptor-ligand-mismatch between donor and recipient may be valuable to define patients who will benefit most from a NK mediated GvL effect.

KIR Incompatible NK Cells Effectively Lyse Osteosarcoma Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4905-4905
Author(s):  
David C. Delgado ◽  
Heather Hardin ◽  
Dan Webster ◽  
Ken B. DeSantes ◽  
Aimen F. Shaaban
Keyword(s):  
Flow Cytometry ◽  
Stem Cell ◽  
Nk Cells ◽  
Cell Lines ◽  
Nk Cell ◽  
Osteosarcoma Cell ◽  
Osteosarcoma Cells ◽  
Receptor Ligand ◽  
Variable Expression ◽  
Ligand Expression

Abstract Recurrent or metastatic pediatric solid malignancies, including osteosarcoma, carry a dismal prognosis despite modern multi-modality treatment approaches. Although the success of KIR (Killer Immunoglobulin-Like Receptor) incompatible, haploidentical stem cell transplantation has been documented in hematological malignancies in adults and children, this approach has not been thoroughly examined in pediatric solid tumors. In this study, we evaluated the potential for KIR-incompatible lysis of osteosarcoma cells, in vitro. We hypothesized that the killing of osteosarcoma cell targets could be predicted by the degree of inhibitory KIR receptor/ligand mismatch with NK cell effectors. To test this hypothesis, healthy donor NK cells were isolated by magnetic bead sorting and their KIR phenotype determined by flow cytometry. Consistent with previous studies, donor NK cells exhibited a high prevalence of all three relevant inhibitory KIRs (KIR2DL1, KIR2DL2/KIR2DL3, KIR3DL1). Conversely, examination of three established osteosarcoma cell lines (HOS, SaOS, and U2OS) demonstrated significant variability in cell surface HLA class I expression, by flow cytometry. QRTPCR analysis of these cell lines revealed variable expression of the three known inhibitory KIR ligands (HLA-C groups 1 and 2, HLA-Bw4). This variable KIR-ligand expression allowed evaluation of NK-mediated lysis of targets with varying KIR receptor-ligand incompatibility. Following a 12-hour incubation of donor NK cells in IL-2, lysis of osteosarcoma targets was measured in an annexin V flow cytometric assay. Osteosarcoma cell lines that expressed fewer KIR ligands consistently showed greater susceptibility to NK-mediated cytotoxicity. These findings were consistent using NK effectors from different donors. Additionally, we observed that at high passage number (>20), SaOS cells demonstrated down-regulation of KIR ligand expression. These changes correlated with increased lysis by the same donor NK cells. Our findings suggest: Variable expression of KIR ligands in osteosarcoma cell lines allows potential susceptibility to KIR-incompatible, NK cell-mediated lysis; The killing of osteosarcoma cells by NK cells can be predicted by the degree of receptor/ligand mismatch; and During expansion, osteosarcoma cells may alter expression of KIR ligands, resulting in increased or decreased susceptibility to lysis by KIR-incompatible NK cells. Further studies are needed to explore the utility of KIR-incompatible, haploidentical stem cell transplantation for patients with high-risk osteosarcoma.

Sign in / Sign up

Export Citation Format

Share Document