scholarly journals Cytokine-induced memory-like natural killer cells for cancer immunotherapy

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Mubin Tarannum ◽  
Rizwan Romee
Keyword(s):  
Natural Killer Cells ◽  
Nk Cells ◽  
Natural Killer ◽  
Viral Infections ◽  
Nk Cell ◽  
Contact Hypersensitivity ◽  
Cellular Immunotherapy ◽  
Clinical Activity ◽  
Antigen Specificity ◽  
Killer Cells

AbstractNatural killer cells are an important part of the innate immune system mediating robust responses to virus-infected and malignant cells without needing prior antigen priming. NK cells have always been thought to be short-lived and with no antigen specificity; however, recent data support the presence of NK cell memory including in the hapten-specific contact hypersensitivity model and in certain viral infections. The memory-like features can also be generated by short-term activation of both murine and human NK cells with cytokine combination of IL-12, IL-15 and IL-18, imparting increased longevity and enhanced anticancer functionality. Preclinical studies and very early clinical trials demonstrate safety and very promising clinical activity of these cytokine-induced memory-like (CIML) NK cells, making them an attractive cell type for developing novel adoptive cellular immunotherapy strategies. Furthermore, efforts are on to arm them with novel gene constructs for enhanced tumor targeting and function.

scholarly journals Allotype-specific processing of the CD16a N45-glycan from primary human natural killer cells and monocytes

Glycobiology ◽  
2020 ◽  
Vol 30 (7) ◽  
pp. 427-432
Author(s):  
Kashyap R Patel ◽  
Jacob T Roberts ◽  
Adam W Barb
Keyword(s):  
Natural Killer Cells ◽  
Nk Cells ◽  
Natural Killer ◽  
Viral Infections ◽  
Nk Cell ◽  
Cell Surface Receptor ◽  
Mass Spectrometry Analysis ◽  
Healthy Human ◽  
Killer Cells ◽  
Spectrometry Analysis

Abstract Fc γ receptor IIIa/CD16a is an activating cell surface receptor with a well-defined role in natural killer (NK) cell and monocyte effector function. The extracellular domain is decorated with five asparagine (N)-linked glycans; N-glycans at N162 and N45 directly contribute to high-affinity antibody binding and protein stability. N-glycan structures at N162 showed significant donor-dependent variation in a recent study of CD16a isolated from primary human NK cells, but structures at N45 were relatively homogeneous. In this study, we identified variations in N45 glycan structures associated with a polymorphism coding for histidine instead of leucine at position 48 of CD16a from two heterozygous donors. It is known that H48 homozygous individuals suffer from immunodeficiency and recurrent viral infections. A mass spectrometry analysis of protein isolated from the primary natural killer cells of individuals expressing both CD16a L48 and H48 variants demonstrated clear processing differences at N45. CD16a H48 displayed a greater proportion of complex-type N45 glycans compared to the more common L48 allotype with predominantly hybrid N45-glycoforms. Structures at the four other N-glycosylation sites showed minimal differences from data collected on donors expressing only the predominant L48 variant. CD16a H48 purified from a pool of monocytes similarly displayed increased processing at N45. Here, we provide evidence that CD16a processing is affected by the H48 residue in primary NK cells and monocytes from healthy human donors.

scholarly journals The aryl hydrocarbon receptor is required for the maintenance of liver-resident natural killer cells

2016 ◽  
Vol 213 (11) ◽  
pp. 2249-2257 ◽  
Author(s):  
Luhua H. Zhang ◽  
June Ho Shin ◽  
Mikel D. Haggadone ◽  
John B. Sunwoo
Keyword(s):  
Natural Killer Cells ◽  
Aryl Hydrocarbon Receptor ◽  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Memory Function ◽  
Contact Hypersensitivity ◽  
Killer Cells ◽  
Aryl Hydrocarbon

A tissue-resident population of natural killer cells (NK cells) in the liver has recently been described to have the unique capacity to confer immunological memory in the form of hapten-specific contact hypersensitivity independent of T and B cells. Factors regulating the development and maintenance of these liver-resident NK cells are poorly understood. The aryl hydrocarbon receptor (AhR) is a transcription factor modulated by exogenous and endogenous ligands that is important in the homeostasis of immune cells at barrier sites, such as the skin and gut. In this study, we show that liver-resident NK (NK1.1+CD3−) cells, defined as CD49a+TRAIL+CXCR6+DX5− cells in the mouse liver, constitutively express AhR. In AhR−/− mice, there is a significant reduction in the proportion and absolute number of these cells, which results from a cell-intrinsic dependence on AhR. This deficiency in liver-resident NK cells appears to be the result of higher turnover and increased susceptibility to cytokine-induced cell death. Finally, we show that this deficiency has functional implications in vivo. Upon hapten exposure, AhR−/− mice are not able to mount an NK cell memory response to hapten rechallenge. Together, these data demonstrate the requirement of AhR for the maintenance of CD49a+TRAIL+CXCR6+DX5− liver-resident NK cells and their hapten memory function.

scholarly journals Nonstochastic Coexpression of Activation Receptors on Murine Natural Killer Cells

2000 ◽  
Vol 191 (8) ◽  
pp. 1341-1354 ◽  
Author(s):  
Hamish R.C. Smith ◽  
Hubert H. Chuang ◽  
Lawrence L. Wang ◽  
Margarita Salcedo ◽  
Jonathan W. Heusel ◽  
...  
Keyword(s):  
Natural Killer Cells ◽  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Inhibitory Receptors ◽  
Antigen Receptors ◽  
Killer Cells ◽  
Histocompatibility Complex ◽  
Nk Cell Cytotoxicity

Murine natural killer cells (NK) express lectin-like activation and inhibitory receptors, including the CD94/NKG2 family of receptors that bind Qa-1, and the Ly-49 family that recognizes major histocompatibility complex class I molecules. Here, we demonstrate that cross-linking of NK cells with a new specific anti–Ly-49H mAb induced NK cell cytotoxicity and cytokine production. Ly-49H is expressed on a subset of NK cells and can be coexpressed with Ly-49 inhibitory receptors. However, unlike Ly-49 inhibitory receptors, Ly-49H is not detectable on naive splenic CD3+ T cells, indicating that Ly-49H may be an NK cell–specific activation receptor. In further contrast to the stochastically expressed Ly-49 inhibitory receptors, Ly-49H is preferentially expressed with the Ly-49D activation receptor, and expression of both Ly-49H and Ly-49D is augmented on NK cells that lack receptors for Qa-1 tetramers. On developing splenic NK1.1+ cells, Ly-49D and Ly-49H are expressed later than the inhibitory receptors. These results directly demonstrate that Ly-49H activates primary NK cells, and suggest that expression of Ly-49 activation receptors by NK cells may be specifically regulated on NK cell subsets. The simultaneous expression of multiple activation receptors by individual NK cells contrasts with that of T cell antigen receptors and is relevant to the role of NK cells in innate immunity.

scholarly journals Fewer circulating natural killer cells 28 days after double cord blood transplantation predicts inferior survival and IL-15 response

2016 ◽  
Vol 1 (3) ◽  
pp. 208-218 ◽  
Author(s):  
Rachel J. Bergerson ◽  
Robin Williams ◽  
Hongbo Wang ◽  
Ryan Shanley ◽  
Gretchen Colbenson ◽  
...  
Keyword(s):  
Natural Killer Cells ◽  
Cord Blood ◽  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Cord Blood Transplantation ◽  
Killer Cells ◽  
Cell Numbers ◽  
Blood Transplantation ◽  
Key Points

Key Points Low numbers of reconstituting NK cells at D+28 after dUCBT are associated with inferior DFS. Patients with low NK cell numbers at D+28 have reduced phosphorylation of STAT5 upon IL-15 stimulation and less Eomes expression.

Circulating CD56 Bright Natural Killer Cells after Allogeneic Stem Cell Trasnplantation Express Functional TRAIL; Implication for a Novel Target of Immunotherapy.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5403-5403
Author(s):  
Sumiko Takao ◽  
Takayuki Ishikawa ◽  
Katsuyuki Ohmori ◽  
Atsumi Ishida ◽  
Takashi Uchiyama
Keyword(s):  
Stem Cell ◽  
Natural Killer Cells ◽  
Healthy Volunteers ◽  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Killer Cells ◽  
Concanamycin A ◽  
Group 2 ◽  
Group 1

Abstract Relapse of malignancies remains to be one of the major problems after allogeneic stem cell transplantation (allo-SCT). It is well-recognized that natural killer cells (NK-cells) are predominated in early phase of immune reconstitution after allo-SCT, and several studies demonstrated that CD56 bright CD16 negative (CD56++CD16−) NK-cells, which account for only a few percentage of peripheral NK-cells in healthy individuals, constitute a large subset of NK-cells at this phase. Although CD56++CD16− NK-cells possess unique ability to proliferate and produce proinflammatory cytokines in response to monokines or IL-2, they have been regarded to be less cytotoxic and unfavorable for graft-versus leukemia effects. To verify this issue, we compared the frequency of peripheral CD56++ NK-cells among total NK-cells with subsequent relapse in 25 allo-SCT recipients. Although the ratio of CD56++ NK-cells was gradually decreased as the increased duration between phlebotomy and allo-SCT, we could divide these patients into two groups. Group 1 was consisted of patients who showed consistently elevated ratio of CD56++ NK-cells, and the remainder was categorized into group 2. The relapse after allo-SCT was seen in 1 out of 8 patients in group 1, whereas it was documented in 5 out of 17 patients in group 2. This finding suggested that CD56++ NK-cells might also have a role in preventing relapse. We have found that peripheral CD56++CD16− NK-cells from patients after allo-SCT consistently expressed TNF-related apoptosis-inducing ligand (TRAIL), although its expression was faintly detectable on circulating NK-cells from healthy volunteers. As reported, stimulation with IL-2 or IL-15 resulted in the increased expression of TRAIL on NK-cells from healthy volunteers as well as the recipients of allo-SCT. However, its expression was always stronger in the CD16- subset than CD16+ in both groups. Cultivation of purified NK-cells from healthy volunteers with 0.5 nM of IL-2 for more than 2 weeks resulted in the expansion of both NK-cell subsets, and after sorting into CD16− and CD16+ NK-cells, cytotoxic assays against Jurkat were performed in the presence or absence of concanamycin A, neutralizing anti-Fas antibody, and neutralizing anti-TRAIL antibody. Cytotoxicity was more prominent in the CD16− subset than CD16+, and blocking study revealed that TRAIL expressed on CD16− NK-cells was strongly involved in the killing of Jurkat. We could not detect TRAIL-mediated cytotoxicity in the CD16+ subset, because the expression of TRAIL was much lower in the CD16+ subset than CD16−. Next, NK-cells purified from allo-SCT recipients and healthy volunteers were overnight cultured with 0.5 nM of IL-2 and their cytotoxicity against Jurkat was examined. NK-cells from patients who received allo-SCT within 3 months and those from healthy volunteers showed equivalent cytotoxicity. In patients who showed increased ratio of CD56++CD16− NK-cells, TRAIL was strongly expressed on overnight cultured CD56++CD16− NK cells, and TRAIL-mediated cytotoxicity was also detected. In murine models, TRAIL has been reported to exert strong graft-versus-tumor effects without causing GVHD. As CD56++CD16− NK cells readily express functional TRAIL on cytokine stimulation, and they usually reconstituted shortly early after allo-SCT, they may become promising targets for immunological intervention.

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.

Jagged2 promotes the development of natural killer cells and the establishment of functional natural killer cell lines

Blood ◽  
2005 ◽  
Vol 105 (9) ◽  
pp. 3521-3527 ◽  
Author(s):  
Sarah L. DeHart ◽  
Marc J. Heikens ◽  
Schickwann Tsai
Keyword(s):  
Natural Killer Cells ◽  
Nk Cells ◽  
Natural Killer ◽  
Cell Lines ◽  
Nk Cell ◽  
Killer Cell ◽  
Interleukin 2 ◽  
Lymphoid Cells ◽  
Killer Cells ◽  
Hematopoietic Stem

AbstractEmerging evidence indicates that Notch receptors and their ligands play important roles in the development of T cells and B cells. However, little is known about their possible roles in the development of other lymphoid cells. Here we demonstrate that Jagged2, a Notch ligand, stimulates the development of natural killer (NK) cells from Lin- Sca-1+ c-kit+ hematopoietic stem cells. Our culture system supports NK cell development for 2 to 3 months, often leading to the establishment of continuous NK cell lines. The prototype of such cell lines is designated as KIL. KIL depends on interleukin-7 for survival and proliferation and is NK1.1+ CD3- TCRαβ- TCRδγ- CD4- CD8- CD19- CD25+ CD43+ CD45+ CD49b- CD51+ CD94+ NKG2D+ Mac-1-/low B220- c-kit+ perforin I+ granzyme B+ Notch-1+, and cytotoxic. Like normal natural killer cells, the T-cell receptor-β loci of KIL remain in the germ-line configuration. In response to interleukin-2, KIL proliferates extensively (increasing cell number by approximately 1010-fold) and terminally differentiates into adherent, hypergranular NK cells. Our findings indicate that Jagged2 stimulates the development of natural killer cells and the KIL cell line preserves most properties of the normal NK precursors. As such, KIL provides a valuable model system for NK cell research.

scholarly journals Natural killer cells: origin, phenotype, function

2021 ◽  
Vol 23 (6) ◽  
pp. 1207-1228
Author(s):  
E. V. Tyshchuk ◽  
V. A. Mikhailova ◽  
S. A. Selkov ◽  
D. I. Sokolov
Keyword(s):  
Bone Marrow ◽  
Natural Killer Cells ◽  
Nk Cells ◽  
Natural Killer ◽  
Immunological Synapse ◽  
Nk Cell ◽  
Cell Receptor ◽  
Killer Cells ◽  
Activated State

Natural killer cells (NK) are innate immune lymphocytes produced in the bone marrow. Isolation of NK cells as a separate population of lymphocytes is related to discovery of their ability to induce the death of tumor cells without prior sensitization. In this review, an attempt was made to systematize the numerous data on the biology of NK cells presented in the literature. The authors consider the stages of NK cells` differentiation from a common lymphoid progenitor (CLP) in the bone marrow, describe two functionally different populations of mature NK cells – CD56brightCDl6- and CD56dimCD16+. In addition, the role of cytokines and chemokines in the development of NK cells is discussed. The review includes data on the spectrum of molecules expressed by NK cells: adhesion molecules (LFA-1, LFA-2, LFA-3; αMβ2, αXβ2, L-selectin, VLA-4, VLA-5; PECAM-1; CEACAM-1), cytokine receptors (IL-1R, IL-2ra, IL-2Rb/IL-2Rc, IL-6Rα, IL-7Ra, IL-8R, IL-10R, IL-12Rβ1, IL-15ra, IL-18R, IL-21ra, IFNGR2, TGFBR, c-Kit, CXCR1, CXCR3, CXCR4, CCR4, CCR5, CCR6, CCR7, IChemR23, CX3CR1), as well as receptors that regulate the activity of NK cells (LILRB1, LILRB2, LILRB4; KIR2DL1-5; KIR2DS1-5; KIR3DL1-3; KIR3DS1; NKG2A, NKG2C, NKG2D; Siglec7, Siglec9; CD16; NKRP-1; TIGIT; TACTILE; NKp30, NKp44, NKp46, NKp80; LAIR-1; PD-1; TIM-3; 2B4; TLR1-9). The authors also examine the mechanisms of implementing cytotoxic activity by NK cells, including cytotoxicity, via expression of MHC-I-specific receptors, CD16 Fc receptors, receptors and ligands of apoptosis (Fas-FasL and TRAIL-TRAILR) as well as other receptors. The review describes in detail the structure of immunological synapse between the NK cell and target cell, receptor interactions, and the role of the cytoskeleton in its formation. The data are summarized on the variants of exocytosis of lytic granules by NK cells, including complete or partial fusion of vesicles with the plasma membrane, exocytosis of vesicles containing perforin and FasL, and the formation of microvesicles containing granzyme B. The review also describes data on ability of NK cells to maintain activated state for a long time, as well as to maintain contact with several targets at the same time. In addition to the functions inherent in natural killers as cells of innate immunity, the authors point out their ability to exhibit the features of cells of adaptive immunity. In general, a variety of mechanisms that regulate the activity of NK cells may complement the specific functions of lymphocytes, thus making the immune system more efficient.

Therapeutic Effects of ALT-803, an IL-15 Superagonist, in Combination with Anti-CD20 Chimeric Antigen Receptor Modified Expanded Natural Killer CELLS Against Pediatric Burkitt Lymphoma (BL)

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3085-3085 ◽  
Author(s):  
Yaya Chu ◽  
Fangyu Lee ◽  
Janet Ayello ◽  
Brian Hang ◽  
Melanie Zhang ◽  
...  
Keyword(s):  
Natural Killer Cells ◽  
Nk Cells ◽  
Natural Killer ◽  
Peripheral Blood ◽  
Burkitt Lymphoma ◽  
Nk Cell ◽  
Target Cells ◽  
Killer Cells ◽  
Anti Cd20

Abstract Background: The outcome for children with Burkitt lymphoma (BL)has improved significantly but for patients who relapse, the prognosis is dismal due to chemo-immunotherapy resistance (Cairo et al, JCO, 2012, Cairo et al, Blood, 2007). NK cells are bone marrow-derived cytotoxic lymphocytes that play a major role in the rejection of tumors. A variety of activating and inhibitory receptors on the NK cell surface are engaged to regulate NK cell activities and to discriminate target cells from other healthy 'self' cells. However, NK therapy is limited by several factors, including small numbers of active NK cells in unmodified peripheral blood, lack of tumor targeting specificity, and multiple mechanisms of tumor escape of NK cell immunosurveillance. Our group has successfully modified expanded peripheral blood Natural Killer cells (exPBNK) with an anti-CD20 CAR to target rituximab sensitive/resistant CD20+ BL cells in vitro and in NSG mice (Chu/Cairo, et al, Can Imm Res 2015). However, the short lifespan/persistence of adoptively transferred NK cells has limited the therapeutic efficacy. ALT-803 (Altor BioScience Corporation) is a superagonist of an IL-15 variant bound to an IL-15Rα-Fc fusion with enhanced IL-15 biological activity (Zhu et al. 2009 J Immunol), longer half-life and increased potency (Han, et al. Cytokine. 2011). It is currently in several clinical trials in patients with variety of cancers such as refractory indolent non-Hodgkin's lymphoma (NCT02384954). Objective: We hypothesize that ALT-803, IL-15 superagonist complex, promotes exPBNK persistence and significantly enhances the cytotoxicity of anti-CD20 CAR exPBNK against CD20+ BL. Method: PBMCs were expanded with lethally irradiated K562-mbIL21-41BBL cells (Dean Lee et al, PLoS One, 2012). CD56+ CD3- exPBNK cells were isolated using Miltenyi NK cell isolation kit. Anti-CD20-4-1BB-CD3 ζ mRNA (CAR mRNA) was producedin vitro and nucleofected into exPBNK as we have previously described (Chu/Cairo, et al, Can Imm Res 2015). ALT-803 was provided by Altor BioScience Corporation. ExPBNK cells were cultured with 0.35ng/ml or 3.5ng/ml ALT-803. NK proliferation was monitored with MTS assays. NK receptors expression and cytotoxicity were examined by flow cytometry (Chu/Cairo, et al, ASH 2014). NK resistant BL cells Raji and Daudi were used as target cells. Results: % CD56+ CD3- PBNK cells were significantly increased compared to media alone at day 14 (mean 81.85% vs 14.91%, n=3, p<0.001) when co-cultured with the irradiated feeder cell K562-mbIL21-41BBL. The absolute NK numbers were enhanced with irradiated K562-mbIL21-41BBL cells as feeders compared to IL-2 alone after normalized to the INPUT NK cell numbers (mean 2247 fold±293.7 vs 0.516 fold±0.225, n=3, p<0.001) at day 14. Different doses of ALT-803 or IgG were added to the culture medium of purified expanded exPBNK. Proliferation assays were performed at day 3, 7,11, and 17. ALT-803 significantly promoted exPBNK proliferation and persistence compared to IgG in vitro in a dose-dependent manner (A490 reading at 3.5ng/ml dose: ALT803 vs IgG=0.3383+0.009 vs 0.0987+0.0007, P<0.0001 at d17). And ALT-803 significantly enhanced exPBNK cytotoxicity against NK resistant BL cells: Raji (ALT803 vs IgG= 49.54%+2.7% vs 5.99+0.34%, p<0.001, E:T=10:1) and Daudi (ALT803 vs IgG= 63.73%+3.09% vs 2.58+1.96%, p<0.001, E:T=10:1). It also maintained the highcytoxicity of exPBNK at d4, d10 and d18 against Raji (E:T=10:1, d4 vs d10 vs d18=62.07% vs 49.54% vs 61.47%) and against Daudi (E:T=10:1, d4 vs d10 vs d18=76.02% vs 63.73% vs 55%) by maintaining the activating receptors expression such as NKp30, NKp44, and NKp46. Further-more, we demonstrated ALT-803 significantly enhanced the cytotoxicity of anti-CD20 CAR modified exPBNK against Raji (CAR vs MOCK= 81.19%+0.35% vs 66.19+0.94%, p<0.001, E:T=10:1) and Daudi (CAR vs MOCK= 91.41%+0.45% vs 80.56+1.07%, p<0.001, E:T=10:1) compared to mock modified exPBNK. ALT-803 also significantly enhanced the cytotoxicity of anti-CD20 CAR modified exPBNK against NK resistant BL cells: Raji and Daudi compared to anti-CD20 CAR modified exPBNK maintained in medium without ALT803 (Fig.1). Conclusions: ALT-803 maintained the cytotoxicity of exPBNK and in vitro persistence and significantly enhanced anti-CD20 CAR exPBNK cytotoxicity against pediatric NK resistant BL. The in vivo effect of ALT-803 on CAR exPBNK using humanized NSG models is under investigation. Disclosures Wong: Altor BioScience Corporation: Employment, Other: stockholder of Altor Bioscience Corporation. Lee:Intrexon, Ziopharm, Cyto-Sen: Equity Ownership.

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