Purified Natural Killer Cells Research Articles

Natural Killer Cells from Allergic Donors Are Defective in Their Response to CCL18 Chemokine.

Natural killer (NK) cells were originally described as cytolytic effector cells, but since then have been recognized to possess regulatory functions on immune responses. Chemokines locate NK cells throughout the body in homeostatic and pathological conditions. They may also directly stimulate immune cells. CCL18 is a constitutive and inducible chemokine involved in allergic diseases. The aim of this study was to evaluate CCL18’s effect on NK cells from allergic and nonallergic donors in terms of both chemotactic and immune effects. Results showed that CCL18 was able to induce migration of NK cells from nonallergic donors in a G-protein-dependent manner, suggesting the involvement of a classical chemokine receptor from the family of seven-transmembrane domain G-protein-coupled receptors. In contrast, NK cells from allergic patients were unresponsive. Similarly, CCL18 was able to induce NK cell cytotoxicity only in nonallergic subjects. Purified NK cells did not express CCR8, one of the receptors described to be involved in CCL18 functions. Finally, the defect in CCL18 response by NK cells from allergic patients was unrelated to a defect in CCL18 binding to NK cells. Overall, our results suggest that some NK cell functions may be defective in allergic diseases.

Determination of Cytotoxic Activities Against Melanoma Cells Using Flow Cytometry.

Within the adaptive and innate immune system, effector lymphocytes known as cytotoxic T cells (CTLs) or natural killer (NK) cells play an essential role in host defense against tumor cells and virally infected cells. Here we describe a flow cytometry-based method to quantify CTLs or NK cell cytotoxic activity against melanoma cells. In this assay, spleen cells, peripheral blood mononuclear cells (PBMCs), or purified NK cell preparations are co-incubated at different ratios with a target tumor cell line. The target cells are pre-labeled with a fluorescent dye to allow their discrimination from the effector cells. After the incubation period, killed target cells are identified by a nucleic acid stain, which specifically permeates dead cells. This method is amenable to both diagnostic and research applications.

Selectively and Efficiently Expanding Natural Killer Cells in Vitro

Natural killer (NK) cells are the main innate immune cells, and have powerful antitumor activity in vitro, but NK cells based immunotherapy does not often get satisfactory results in clinical practice due to insufficient number and poor activity of NK cells.. Therefore, how to improve expansion efficiency and killing activity of NK cells in vitro is critical. Here we report an in vitro culture system culturing peripheral blood mononuclear cells (PBMC) with irradiated LCL as feeder cells in 10%FBS/RPMI1640 culture medium in the presence of 100U/ml IL-2 and 5ng/ml IL-15 for 14 days could selectively expand NK cells. With this culture system, the purity of NK cells increased from 11.5%± 7.01% to 75.3±10.9% (P< 0.01), and the expansion efficiency was about 625±63 times than the original PBMC. Compared with the fresh NK cells isolated from PBMC, there was no significant change in the expression of inhibitory receptors on the surface of expanded NK cells, while the expression of activating receptors was significantly increased (p<0.05). The cytotoxicity of expanded NK cells against leukemia cell line K562 was significantly higher than that of freshly isolated NK cells (72± 5.6% vs 8.7±2.3%, E: T=1, P<0.01), expanded NK cells also showed stronger cytotoxicity to LCL (22±3.6%, E: T=1) and primary leukemia cells (42±5.2%, E:T=1). In summary, we could selectively expand NK cells with this in vitro culture system, the expanded NK cells gained higher expression of NCRs and NKG2D receptors, and displayed greater cytotoxicity against tumor cells, therefore could be readily applied to anti-tumor treatment in clinical treatment. Figure Disclosures No relevant conflicts of interest to declare.

EVALUATION OF NATURAL KILLER CELLS USING DIFFERENT TECHNIQUES

Natural killer (NK) cells mediate spontaneous cytotoxicity toward abnormal and tumor cells and rapidly secrete numerous cytokines and chemokine`s which promote subsequent adaptive immune responses. The presented study aimed to compare between different methods for NK evaluation and determination. Ten samples of healthy individuals were collected from Al-Zahraa University hospital and evaluated for phenotypic identification for NK cells. Isolation procedure of NK was performed in Clinical Pathology Department, Hematology Unit, Flow-cytometry lab, Al-Azhar University. Protocols started with isolation of peripheral blood mononuclear cells (PBMCs) from fresh human whole blood depending on Ficoll-Paque density gradient centrifugation. Other protocol depended on flow cytometry sorting technique. All experiments were carried out in accordance with the approved guidelines. Sorting procedure using FACS Calibur machine yielded 64.97 % purity before cells were cultured, while mixed Ficoll separation of whole blood showed purity 22.74%. Through our study, it is clear to us that the percentage of purity of NK cells using the flow-cytometry sorting technique is much higher than the percentage of purity of NK cells using the separation method dependent on Ficoll-Paque density technique, Therefore, through our study, we see that it is better to rely on the flow-cytometry sorting technique using the flow cytometry device and CD56 +CD16 and CD3 antibodies in order to obtain the best purity of the NK cells (CD56+ CD16 positive /CD3 negative population) for use in medical purposes and laboratory research.

An Inverse Relationship Between c-Kit/CD117 and mTOR Confers NK Cell Dysregulation Late After Severe Injury

Major trauma-induced tissue injury causes a dysregulation of the immune system. Severe systemic inflammation occurs early after the insult. Later on, an enhanced risk for life-threatening opportunistic infections develops that culminates at the end of the first week after trauma. CD56bright Natural killer (NK) cells play a key role in the defense against infection due to their rapid release of Interferon (IFN) γ in response to Interleukin (IL) 12. NK cells are impaired in IFN-γ synthesis after severe injury due to a disturbed IL-12/IFN-γ axis. Thereby, a circulating factor mediates extrinsic suppression of NK cells. Yet unknown cell-intrinsic mechanisms manifest by day 8 after trauma and render NK cells unresponsive to stimulatory cytokines. In the present study, we investigated the origin of such late NK cell-intrinsic suppression after major trauma. Peripheral blood mononuclear cells (PBMC) were isolated from patients 8 day after severe injury and from healthy control subjects and were stimulated with inactivated Staphylococcus aureus. The expression of diverse cytokine receptors, intracellular signaling molecules, and the secretion of IFN-γ by CD56bright NK cells were examined. After stimulation with S. aureus, NK cells from patients expressed enhanced levels of c-kit/CD117 that inversely correlated with IFN-γ synthesis and IL-12 receptor (IL-12R) β2 expression. Supplementation with IL-15 and inhibition of the transforming growth factor receptor (TGF-βR) I reduced CD117 expression and increased the level of IL-12Rβ2 and IFN-γ. NK cells from patients showed reduced phosphorylation of mammalian target of rapamycin (mTOR). Addition of IL-15 at least partly restored mTOR phosphorylation and increased IL-12Rβ2 expression. The reduced mTOR phosphorylation after severe injury was cell-intrinsic as it was not induced by serum factors. Inhibition of mTOR in purified NK cells from healthy donors by rapamycin decreased the synthesis of IFN-γ. Thus, impaired mTOR phosphorylation in response to a microbial challenge contributes to the cell-intrinsic mechanisms that underlie NK cell dysregulation after trauma. Restoration of the mTOR phosphorylation capacity along with inhibition of the TGF-βRI signaling in NK cells after severe injury might improve the immune defense against opportunistic infections.

Superior Expansion and Cytotoxicity of Human Primary NK and CAR-NK Cells from Various Sources via Enriched Metabolic Pathways

Clinical success of chimeric antigen receptor (CAR) T cell immunotherapy requires the engineering of autologous T cells, which limits the broader implementation of CAR cell therapy. The development of allogeneic and universal cell products will significantly broaden their application and reduce costs. Allogeneic natural killer (NK) cells can be used for universal CAR immunotherapy. Here, we develop an alternative approach for the rapid expansion of primary NK and CAR-NK cells with superior expansion capability and in vivo cytotoxicity from various sources (including peripheral blood, cord blood, and tumor tissue). We apply a human B-lymphoblastoid cell-line 721.221 (hereinafter, 221)-based artificial feeder cell system with membrane-bound interleukin 21 (mIL-21) to propagate NK and CAR-NK cells. The expansion capability, purity, and cytotoxicity of NK cells expanded with 221-mIL-21 feeder cells are superior to that of conventional K562-mIL-21 feeder cells. RNA sequencing (RNA-seq) data show that 221-mIL-21 feeder cell-expanded NK cells display a less differentiated, non-exhausted, limited fratricidal, memory-like phenotype correlated with enriched metabolic pathways, which explains underlying mechanisms. Thus, “off-the-shelf” NK and CAR-NK cells with superior functionalities and expansion using a genetically modified 221-mIL-21 feeder cell expansion system will greatly support clinical use of NK immunotherapy.

Fully automated integrated process for depletion of T cells with or without subsequent purification of NK cells from apheresis using the CliniMACS Prodigy

Background & Aim Donor-derived natural killer (NK) cells mediate graft-versus-leukemia effects low risk in inducing alloreactive graft-versus-host-disease and are used as component of T cell-depleted donor lymphocyte infusion (DLI) or purified. As clinical grade purification of NK cells requires two semi-automated separation procedures, including multiple manual hands-on steps, we developed an integrated fully automated process enabling either preparation of T cell-depleted DLI or clinical grade purification of NK cells within a single automated process on the CliniMACS Prodigy. Methods, Results & Conclusion The novel integrated CliniMACS Prodigy LP-3-56 System uses the CliniMACS Prodigy Tubing Set 320 for an fully automated procedure with integrated labeling and washing steps for both, CD3 and CD56 separation enabled through an integrated lytic rinsing sequence allowing subsequent CD56 enrichment in the same tubing set. By users choice the depletion of CD3+ cells will be performed with or without subsequent enrichment of CD56+ cells. For both strategies target cells are prepared with user-chosen formulation solution (e. g. sodium chloride or NK MACS medium). The new application software effectively depletes 9.6 × 109 CD3+ cells, and enriches 4.5 × 109 CD56+ cells from up to 40 × 109 total white blood cells. Inhouse evaluation runs with leukapheresis products (n= 29 in total, n=17 with CD3 depletion, n=12 with CD3 depletion and CD56 enrichment) were performed. After CD3 depletion, mean log depletion of T cells was 4.0. The mean recovery of NK cells was 89% with an average NK:T ratio-increase of 9509 fold. After CD3 depletion and CD56 enrichment, mean log depletion of T and B cells was 4.2 and 2.7, respectively. Purity of NK cells was 90.7% on average with a mean recovery of 49%. The majority of non-NK cells after the process were monocytes with a mean frequency of 5.9%. Functionality of purified NK cells was proven by their ability to lyse AML cells, to secrete cytokines after co-culture, and to perform antibody dependent cell-mediated cytotoxicity (ADCC) against Raji cells, using human CD20 antibody. In summary, the integrated fully automated CliniMACS Prodigy LP-3-56 System is capable of efficiently deplete CD3+ cells with or without subsequent CD56 enrichment to purify functional NK cells from leukapheresis products and provides target cells in formulation solution. The performance verification and submission to an European notified body for CE mark are the important next steps.

Seeding density is critical for optimizing scalable feeder-free natural killer cell expansion

Background & Aim Generating clinical doses of natural killer (NK) cells is an important challenge for Chimeric Antigen Receptor (CAR)-NK cell therapies. Current manufacturing methods to expand NK cells include irradiated feeder cells. However, these methods are restrictive due to high costs, scale-up difficulties, and licensing restrictions. In this study we investigated an optimized method for the expansion of NK cells in a feeder-free system that enables scalable cell expansion for NK therapies. Specifically, we investigated the impact of cell activation methods, cell culture vessels, & cell seeding density on NK expansion and purity. Methods, Results & Conclusion Human peripheral blood mononuclear cells (PBMCs) were cultured for 10-days in either G-Rex® 24-well plates, T25 Flasks, or G-Rex 6-well plates. PBMCs were analyzed using a flow cytometer for CD3−/CD56+ NK cells prior to expansion to determine NK seeding density. The Cloudz™ NK Cell Expansion Kit or Miltenyi MACS iBeads were used to activate & expand NK cells. A half-volume media change was performed every 3 days and cytokines were refreshed at the following concentrations: 27 ng/mL IL-2, and 10 ng/mL each of IL-12, IL-18, & IL-21. After 10 days in culture the cells were analyzed for CD3−/CD56+ NK cell expansion and purity, defined as % CD3−/CD56+ cells relative to the total population. We found that the Cloudz NK Cell Expansion Kit produced the greatest NK cell expansion & purity from PBMCs after 10 days of culture (318-fold NK cell expansion and 85% purity) when used with a cytokine combination of IL-2/IL-12/IL-18/IL-21. These data represent 6 experiments utilizing 85 samples across 5 separate donors. For comparison, MACS iBeads produced an average of 177-fold expansion and 64.5% purity. While developing a scalable protocol to allow flexibility in NK cell manufacturing, we found that cell seeding density was an important variable. In the 24-well G-Rex platform, a seeding density of 60,000 NK cells/cm2 yielded the highest growth and purity after 10-days. However, 30,000 NK cells/cm2 produced the highest growth rate in the 6-well G-Rex platform over 10 days (Figure 1). These results indicate that robust expansion & high purity can be achieved using feeder-free NK cell expansion methodologies. These data suggest that seeding density is a critical factor for cell expansion under feeder-free conditions and that utilizing the G-Rex platform can enable scalable expansion for cell therapy manufacturing.

GMP-compliant, automated process for generation of CAR NK cells in a closed system for clinical use

Background & Aim Major advances have been made in harnessing natural killer (NK) cells in cancer immunotherapy in recent years. Regulated by their germ-line encoded activating and inhibitory receptors, NK cells can recognize and eliminate tumor cells rapidly without prior sensitization. Clinical evidence has shown that donor-derived NK cells have low risk in inducing graft-versus-host-disease (GvHD) as well as a reduced risk of life-threatening cytokine storms during therapy. Both properties make NK cells ideal for allogeneic application. To enhance the anti-tumor specificity and efficiency, NK cells can be further modified by chimeric antigen receptors (CARs). Promising clinical outcome of CAR-engineered NK cells has been recently reported for patients with relapsed or refractory B cell malignancies. Methods, Results & Conclusion In this study purified NK cells were cultivated with a feeder cell-free protocol including cytokines in NK MACS GMP Medium. For generation of CAR NK cells we used a Baboon endogenous virus (BaEV)-pseudotyped lentiviral vector system to efficiently transduce NK cells with various CARs and routinely obtained transduction efficiencies between 30-70%. To facilitate the clinical application of CAR NK cells, we implemented this protocol into a highly efficient, automated process to generate CAR NK cells under good manufacturing practice (GMP)-compliant conditions in a closed system by using CliniMACS Prodigy device. The process covers the complete procedure of NK cell manufacturing, including separation, activation, gene modification, and expansion/cultivation. High purity of NK cells (mean 94%) could be achieved by CD3 depletion followed by subsequent CD56 enrichment, resulting in a mean log depletion of T cells of 4.3. Inhouse evaluation runs with leukapheresis products (n=11) achieved a mean transduction efficiency of 52.0% CAR+ cells, resulting in 5.2 – 17.9 x 108 (mean 7.17 x 108) CAR+ NK cells after 14 days of culture. In summary, we developed a novel process for automated NK cell purification, transduction, and cultivation in a closed GMP-compatible system. The high level of automation enables standardized, consistent, and operator independent genetic engineering of NK cells for therapeutic applications.

Delineation and Modulation of the Natural Killer Cell Transcriptome in Rhesus Macaques During ZIKV and SIV Infections.

Natural killer (NK) cells are crucial regulators of antiviral and anti-tumor immune responses. Although in humans some NK cell transcriptional programs are relatively well-established, NK cell transcriptional networks in non-human primates (NHP) remain poorly delineated. Here we performed RNA-Seq experiments using purified NK cells from experimentally naïve rhesus macaques, providing the first transcriptional characterization of pure NK cells in any NHP species. This novel NK cell transcriptomic signature (NK RMtsig) overlaps with published human NK signatures, allowing us to identify new key signaling and transcription factor networks underlying NK cell function. Finally, we show that applying NK RMtsig to an unrelated rhesus macaque cohort infected with SIVmac251 or ZIKV can sensitively detect NK cell repertoire perturbations, thus confirming applicability of this approach. In sum, we propose this NHP NK cell signature will serve as a useful resource for future studies involving infection, disease or treatment modalities in NHP.

Natural killer cells expanded in vivoor ex vivo with IL-15 overcomes the inherent susceptibility of CAST mice to lethal infection with orthopoxviruses.

The wild-derived inbred CAST/EiJ mouse, one of eight founder strains in the Collaborative Cross panel, is an exceptional model for studying monkeypox virus (MPXV), an emerging human pathogen, and other orthopoxviruses including vaccinia virus (VACV). Previous studies suggested that the extreme susceptibility of the CAST mouse to orthopoxviruses is due to an insufficient innate immune response. Here, we focused on the low number of natural killer (NK) cells in the naïve CAST mouse as a contributing factor to this condition. Administration of IL-15 to CAST mice transiently increased NK and CD8+ T cells that could express IFN-γ, indicating that the progenitor cells were capable of responding to cytokines. However, the number of NK cells rapidly declined indicating a defect in their homeostasis. Furthermore, IL-15-treated mice were protected from an otherwise lethal challenge with VACV or MPXV. IL-15 decreased virus spread and delayed death even when CD4+/CD8+ T cells were depleted with antibody, supporting an early protective role of the expanded NK cells. Purified splenic NK cells from CAST mice proliferated in vitro in response to IL-15 and could be activated with IL-12/IL-18 to secrete interferon-γ. Passive transfer of non-activated or activated CAST NK cells reduced VACV spread but only the latter completely prevented death at the virus dose used. Moreover, antibodies to interferon-γ abrogated the protection by activated NK cells. Thus, the inherent susceptibility of CAST mice to orthopoxviruses can be explained by a low level of NK cells and this vulnerability can be overcome either by expanding their NK cells in vivo with IL-15 or by passive transfer of purified NK cells that were expanded and activated in vitro.

The Synergistic Use of IL-15 and IL-21 for the Generation of NK Cells From CD3/CD19-Depleted Grafts Improves Their ex vivo Expansion and Cytotoxic Potential Against Neuroblastoma: Perspective for Optimized Immunotherapy Post Haploidentical Stem Cell Transplantation.

Neuroblastoma (NB) is the most common solid extracranial tumor in childhood. Despite therapeutic progress, prognosis in high-risk NB is poor and innovative therapies are urgently needed. Therefore, we addressed the potential cytotoxic capacity of interleukin (IL)-activated natural killer (NK) cells compared to cytokine-induced killer (CIK) cells for the treatment of NB. NK cells were isolated from peripheral blood mononuclear cells (PBMCs) by indirect CD56-enrichment or CD3/CD19-depletion and expanded with different cytokine combinations, such as IL-2, IL-15, and/or IL-21 under feeder-cell free conditions. CIK cells were generated from PBMCs by ex vivo stimulation with interferon-γ, IL-2, OKT-3, and IL-15. Comparative analysis of expansion rate, purity, phenotype and cytotoxicity was performed. CD56-enriched NK cells showed a median expansion rate of 4.3-fold with up to 99% NK cell content. The cell product after CD3/CD19-depletion consisted of a median 43.5% NK cells that expanded significantly faster reaching also 99% of NK cell purity. After 10–12 days of expansion, both NK cell preparations showed a significantly higher median cytotoxic capacity against NB cells relative to CIK cells. Remarkably, these NK cells were also capable of efficiently killing NB spheroidal 3D culture in long-term cytotoxicity assays. Further optimization using a novel NK cell culture medium and a prolonged culturing procedure after CD3/CD19-depletion for up to 15 days enhanced the expansion rate up to 24.4-fold by maintaining the cytotoxic potential. Addition of an IL-21 boost prior to harvesting significantly increased the cytotoxicity. The final cell product consisted for the major part of CD16−, NCR-expressing, poly-functional NK cells with regard to cytokine production, CD107a degranulation and antitumor capacity. In summary, our study revealed that NK cells have a significantly higher cytotoxic potential to combat NB than CIK cell products, especially following the synergistic use of IL-15 and IL-21 for NK cell activation. Therefore, the use of IL-15+IL-21 expanded NK cells generated from CD3/CD19-depleted apheresis products seems to be highly promising as an immunotherapy in combination with haploidentical stem cell transplantation (SCT) for high-risk NB patients.

Natural Killer Cell Expansion with Autologous Feeder Layer and Anti-CD3 Antibody for Immune Cell Therapy of Hepatocellular Carcinoma.

Background:one of the promising approaches for treatment of some cancers is adoptive cell therapy using natural killer (NK) cells. Various methods have been investigated for ex vivo expansion of NK cells in large-scale, but most of them involved cancer or genetically modified cells as feeder layer and also some of them have the risk of T cell contamination and graft-versus-host disease (GVHD). Method:In this study, irradiated autologous peripheral blood mononuclear cells (PBMCs) as feeder layer with an anti-CD3 monoclonal antibody (mAb) were used. For activation and expansion of NK cells, human recombinant IL2 and IL15 were used. After co-culturing of expanded NK cells (eNKC) and isolated NK cells (iNKC) with hepatocellular carcinoma (HCC) cells, the viability of eNKC in compared to iNKC were analyzed by CCK-8 assay and degranulation of NK cells after co-culturing was assayed by measuring CD107a expression. Enzyme-Linked Immunosorbent Assay (ELISA) assay was used for the ability of NK cells to secretion of IFN-γ (interferon-γ) and TNF-α (Tumor Necrosis Factor-α) after co-culture with HCC cells. Real Time PCR analysis was used for expression of human Perforin and Granzyme B genes in the NK cells exposed to target HepG2 cells. Result:This method strongly expanded highly purified NK cells with powerful cytotoxicity against HCC cells. The expanded NK cells showed high level of expression of degranulation marker and human Perforin and Granzyme B genes, and also was secreted larger amounts of TNF-α and IFN-γ compared with fresh isolated NK cells. Conclusion:we proposed an effective method for expansion of cytotoxic NK cells using irradiated autologous PBMC as feeder layer for more successful transfer of allogeneic NK cell in immuno cell therapy of HCC.

A simple method for in vitro preparation of natural killer cells from cord blood

BackgroundCord Blood (CB) has been considered a promising source of natural killer (NK) cells for cellular immunotherapy. However, it is difficult to expand the large numbers of highly pure NK cells from CB without cell sorting and feeder cells/multiple cytokines. In this study, we try to develop a simple, safe and economical method for ex vivo expansion and purification of NK cells from CB without cell sorting and feeder cells/multiple cytokines.ResultsThe large numbers (mean: 1.59 × 1010) of highly pure (≥90%) NK cells from CB could be obtained through interleukin-2, group A streptococcus and zoledronate stimulation of mononuclear cells using the 21-day culture approach. When compared to resting NK cells, expanded NK cells were a higher expression of activating receptors CD16, NKG2D, NKp30, NKp44, NKp46 and activating markers CD62L and CD69, while the inhibitory receptors, CD158a and CD158b remained largely unchanged. In addition, these cells showed a higher concentration of IFN-γ, TNF-α and GM-CSF secretion and cytotoxicity to K562 cells and acute myeloid leukemia targets than resting NK cells.ConclusionWe develop a simple, safe and economical method to obtain high yield, purity, and functionality NK cells from CB without cell sorting and feeder cells/multiple cytokines.

Availability of NK cell expansion agent combined with recombinant IL‑2 and IL‑15 stimulation on the expansion and high‑purity of NK cells in patients with immune‑related pancytopenia invitro.

Natural killer (NK) cells are a group of large granular lymphocytes that play an important regulatory role in innate immunity and adaptive immunity. Immune‑related pancytopenia (IRP) is a type of pancytopenia resulting from bone marrow hematopoietic cells that were destroyed or suppressed by auto‑antibodies. The specific mechanism of IRP is not clear. In the present study, it was identified that the percentage of NK cells in peripheral blood lymphocytes was decreased in patients with IRP. Subsequently, high purity NK cells were extracted from 6 untreated patients with IRP using the immunomagnetic beads sorting, magnetic‑activated cell‑sorting method, which were then cultured then in RPMI‑1640 medium containing 20%FBS. NK cell expansion agents, with or without recombinant interleukin (IL)‑15, were used to amplify high‑purity NK cells on the basic of recombinant IL‑2. Expression of the activated receptors NKG2‑D typeII integral membrane protein (NKG2D) and natural killer cell p46‑related protein (NKp46), and the inhibitory receptors CD158a and NKG2‑A/NKG2‑B typeII integral membrane protein (NKG2A), in CD56+ NK cells were detected by flow cytometry before and after cell culture. It was observed that treatment with an NK cell expansion agent combined with the stimulation of recombinant IL‑2 and recombinant IL‑15 could increase the number whilst maintaining the purity of NK cells. There were no significant changes in the expression of NKG2D, NKp46, NKG2A and CD158a in patients with IRP before and after NK cell culture. This new amplification method lays a foundation for clinical NK cell immunotherapy and anti‑tumor applications.

Abstract LB-072: Activating and expanding NK cells using dissolvable alginate particles functionalized with αNKp46 and αCD2

Abstract Introduction: Generating clinical scale doses of natural killer (NK) cells is currently a challenge for manufacturing CAR-NK cell therapies. Current methods to expand NK cells include irradiated feeder cells that have been engineered to mimic dendritic cells as well as commercially available combinations of antibody & proteins for targeted NK cell expansion. These methods are restrictive due to high costs, scale-up challenges, and licensing restrictions for clinical & commercial applications. We hypothesized that using a dissolvable phase-change hydrogel conjugated with ligands targeting NK cell activation may provide a more efficient and defined alternative for NK manufacturing processes. Additionally, we investigated cytokine culture combinations that optimize the expansion of NK cells. Methods: Hydrogel microspheres (9 µm median diameter) - referred to as Cloudz™ particles - were functionalized with αNKp46 and αCD2 ligands using standard bioconjugation techniques. In this study, human peripheral blood mononuclear cells (PBMCs) were cultured for 7 days in G-Rex® 24-well plates containing NK cell-functionalized Cloudz™ particles along with various cytokine combinations. Prior to expansion, PBMCs were analyzed for CD3-/CD56+ NK cells and 6x105 NK cells were seeded into each well with a 2:1 ratio of Cloudz™ particles:NK cells. Miltenyi MACS iBeads were used as a control and were prepared according to the manufacturer instructions. During days 0-3, the cells were cultured with IL-2 (40 U/mL) along with combinations of IL-12, IL-18, and IL-21 (10 ng/mL each). After 3 days, the IL-2 concentration was increased to 200 U/mL, while the other cytokine concentrations remained constant. The cytokines and media were refreshed every 3 days with ½ media changes. After 7 days in culture the cells were analyzed by flow cytometry for CD3-/CD56+ NK cell expansion and purity. NK cell purity was defined as the percentage of CD3-CD56+ cells relative to the total population. Results: Cultures using NK cell-functionalized Cloudz™ particles combined with IL-2 resulted in a 69% NK cell purity and 29.9-fold expansion. In comparison, cultures using MACS iBeads combined with IL-2 resulted in 49% NK cell purity and a 6.8-fold expansion, suggesting that Cloudz™-based methods provide better expansion. When IL-12 was added to cultures with Cloudz™ particles, both the purity and expansion decreased (45% and 7.2-fold, respectively). When IL-12 and IL-18 were added, the expansion increased to 33.9-fold and the purity increased to 60%. The best combination was IL-2/IL-12/IL-18/IL-21 which had an 88.9-fold expansion with a 78% purity. IL-12 and IL-18 mimic the activating signals released by dendritic cells and IL-2 and IL-21 mimic the activating signals released by activated T cells. Conclusion: These data demonstrate that Cloudz™ particles functionalized with αNKp46 and αCD2 can efficiently induce NK activation and expansion with high purity and growth. In future studies, we will determine if NK cells expanded using this method show killing ability of cancer cells and persistence in in vivo models. We will also further explore the possibilities of this novel system for rapid expansion and recovery of specific NK cell subtypes. Citation Format: Christopher Johnson, Hannah Senior, Nithya Jesuraj. Activating and expanding NK cells using dissolvable alginate particles functionalized with αNKp46 and αCD2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-072.

Assessment of PD-1 expression in human resting and activated natural killer cells and murine tumor models.

36 Background: Blockade of the PD-1/PD-L1/2 axis has revolutionized cancer therapy. Although reinvigorated PD-1+ T cells are the main effectors in the response to checkpoint blockade, the contribution of Natural Killer (NK) cells to PD-1/PD-L1 inhibition is under debate. While PD-1 has been identified on NK cells, this appears to be restricted to small populations under limited conditions. We sought to evaluate the extent of PD-1 expression in mouse and human resting and activated NK cells. Methods: Human NK cells were isolated from healthy donor PBMCs and cancer patients. Ex vivo activation and proliferation techniques included recombinant human cytokine and feeder line co-culture. Murine NK cells were isolated from splenocytes, and PBMCs from wild type and immunodeficient mice. We assessed NK cell surface markers and intracellular cytokine by flow cytometry, and gene expression by quantitative RT-PCR. Results: Over 21-days of ex vivo expansion, expression of PD-1 or PD-L1 on human NK cells was < 1% at all time points, while TIGIT+ expression increased to > 85%. Conversely, ConA stimulation of T cells increased PD-1 expression with no change in TIGIT expression. QRT-PCR demonstrated absent PD-1 expression in purified NK cells compared to a 5-fold increase in PD-1 gene expression in ConA stimulated PBMCs. PD-1/PD-L1 was also < 1% in the NK92 cell line and < 2.5% in peripheral CD56+CD3- NK cells from patients with soft tissue sarcoma (STS). NK cells from digested freshly resected STS show variable PD-1 ( < 10%) and minimal PD-L1 ( < 1%) expression with a small, but measurable population of intra-tumoral NK cells (1% of immune cells). In vivo mouse studies showed < 5% PD-1+ NK cells in spleen and tumor of CT26 tumor-bearing mice, while PD-L1+ NK cells increased in frequency from spleen (5-35%) to tumor (40-95%) in both wild type BALB/C and SCID mice. Conclusions: In contrast to prior studies, we did not observe a substantial PD-1+ population on human or murine NK cells after multiple activation strategies compared to T cells. Contrary to its application in T cells, our data suggest that PD-1 is not a useful marker for NK cell exhaustion/dysfunction. PD-L1 on NK cells may represent an important link between NK and T cell immunotherapy.

S100A14 Is Increased in Activated NK Cells and Plasma of HIV-Exposed Seronegative People Who Inject Drugs and Promotes Monocyte-NK Crosstalk.

HIV-exposed seronegative people who inject drugs (HESN-PWID) have been shown to have increased natural killer (NK) cell and myeloid activation when compared with control donors. We investigated potential mechanisms maintaining NK activation by conducting quantitative proteome comparisons of NK cells from HESN-PWID subjects and control donors. Proteins upregulated in NK cells were measured in the plasma of HESN-PWID subjects by ELISA and further investigated for their ability to induce innate immune activation in vitro. The NK cell proteome comparison showed markedly higher levels of interferon-stimulated proteins and S100 proteins, including S100A14. Consistent with these results, we observed significantly higher levels of S100A14 in the plasma of HESN-PWID subjects compared with controls (P = 0.033, n = 25). In vitro, the addition of recombinant S100A14 protein significantly activated NK cells in a peripheral blood mononuclear cell mixture (P = 0.011, n = 9), but not purified NK cells alone. Treatment of purified monocytes with recombinant S100A14 protein induced secretion of TNF-alpha and led to significantly higher NK CD69 activation (P = 0.0156, n = 7) in a co-culture through a TLR4-dependent interaction. Our study identified S100A14 as a novel protein increased within NK cells and plasma of HESN-PWID subjects with the capacity to sustain NK activation through TLR4-dependent activation of myeloid cells.

Identification of Primary Natural Killer Cell Modulators by Chemical Library Screening with a Luciferase-Based Functional Assay

Natural killer (NK) cells are essential players of the innate immune response that secrete cytolytic factors and cytokines such as IFN-γ when contacting virus-infected or tumor cells. They represent prime targets in immunotherapy as defects in NK cell functions are hallmarks of many pathological conditions, such as cancer and chronic infections. The functional screening of chemical libraries or biologics would greatly help identify new modulators of NK cell activity, but commonly used methods such as flow cytometry are not easily scalable to high-throughput settings. Here we describe an efficient assay to measure the natural cytotoxicity of primary NK cells where the bioluminescent enzyme NanoLuc is constitutively expressed in the cytoplasm of target cells and is released in co-culture supernatants when lysis occurs. We fully characterized this assay using either purified NK cells or total peripheral blood mononuclear cells (PBMCs), including some patient samples, as effector cells. A pilot screen was also performed on a library of 782 metabolites, xenobiotics, and common drugs, which identified dextrometorphan and diphenhydramine as novel NK cell inhibitors. Finally, this assay was further improved by developing a dual-reporter cell line to simultaneously measure NK cell cytotoxicity and IFN-γ secretion in a single well, extending the potential of this system.

Enhancing Human NK Cell Function and Specificity for Cancer Immunotherapy

▪Natural Killer (NK) cells are cytotoxic lymphocytes capable of immune surveillance and represent an excellent source of cells for cancer immunotherapy for numerous reasons: 1) they mediate direct killing of transformed cells with reduced or absent MHC expression, 2) they can carryout antibody-dependent cell-mediated cytotoxicity (ADCC) on cells bound by appropriate antibodies via CD16, 3) they are readily available and easy to isolate from peripheral blood, 4) they can be expanded to clinically relevant numbers in vitro. Moreover, as NK cells do not cause graft versus host disease, they are inherently an off-the-shelf cellular product, precluding the need to use a patient's own NK cells to treat their cancer. In light of these attributes, NK cells have been used in many clinical trials to treat a number of cancer types; however, the results have not been as successful as other cellular based immunotherapies, such as CAR-T. In light of this, many groups have taken approaches to augment NK cell function, such as high dose IL15, CARs and Bi- or Tri-specific killer engagers. A synergistic or even alternative approach to these technologies is the use of CRISPR/Cas9-based genome editing to disrupt or manipulate the function of NK genes to improve their utility as an immunotherapeutic agent.In order to enhance the immunotherapeutic efficacy of NK cells we have implemented the CRISPR/Cas9 system to edit genes and deliver CARs. To this end, we have developed methods for high efficiency nucleic acid delivery to NK cells using electroporation. First, primary human NK cells are immunomagnetically isolated from peripheral blood mononuclear cells (PBMCs) of healthy donors. Purified NK cells are then activated and expanded using artificial antigen presenting cells (aAPCs) expressing membrane bound IL21 and 41BB for 7 days and subsequently electroporated (Figure 1A). Using this approach with EGFP encoding mRNA, we achieve high rates of transfection (>90%) and high viability (>90%) (Figure 1B).We next developed gRNAs targeting PD1, CISH, and ADAM17. PD1 is a negative regulator of NK cell function and its cognate receptor, PD-L1, is upregulated in a number of cancers. ADAM17 mediates CD16 cleavage on NK cells to negatively regulate their ability to perform ADCC. CISH is a recently described negative regulator of NK cell activation and integrates cytokine signals, including IL-15. We consistently achieved high rates (up to 90%) of gene inactivation in primary human NK cells across multiple donors (Figure 1C). Importantly, these gene edits do not affect expansion potential and are stable over several rounds of expansion (Figure 1D, E). Moreover, ADAM17 KO NK cells are highly resistant to CD16 cleavage upon activation (Figure 2A-E) and PD1 KO NK cells demonstrate significantly enhanced function against PD-L1 expressing cancer cell lines in vitro and in vivo (Figure 2F-J). These data demonstrate that high efficiency gene editing of NK cells can significantly enhance their function while maintaining in vitro expansion.In an effort to engineer NK cell specificity for cancer immunotherapy, we recently developed CAR molecules designed for use in NK cells (Li et al., 2018, Cell Stem Cell 23, 1-12). To this end, we engineered and tested 10 mesothelin CAR molecules with NK specific transmembrane domains (CD16, NKp44, NKp46, or NKG2D) and intracellular signaling domains (2B4, DAP10, DAP12, CD3ζ, and/or CD137). Utilizing several cancer models, we identified an architecture that significantly enhanced NK activation compared to T-CAR architectures (CAR4: scFv-NKG2D-2B4-CD3ζ). Moreover, NK-CAR4 cells demonstrated increased in vivo expansion, improved activity, and reduced toxicity compared to CAR-T cell therapy.In our studies to develop novel NK CARs, CARs were delivered to iPSC derived test NK cells (iNKs) using the PiggyBac transposon system. In order to deliver NK-CAR4 to peripheral blood NK cells we developed methods for high frequency, site specific integration. To this end, we utilized CRISPR/Cas9 combined with non-integrating recombinant Adeno-Associated Virus (rAAV) DNA donor for homologous recombination. Using an EGFP reporter we were able to optimize this process and deliver EGFP reporter to the AAVS1 safe harbor site with efficiencies >80% in NK cells. We are now utilizing our optimized gene editing approaches to generate multiplex edited CAR-NK cells and results from these studies will be presented. [Display omitted] DisclosuresWebber:BEAM Therapeutics: Consultancy; B-MoGen Biotechnologies: Employment, Equity Ownership. Felices:GT Biopharma: Research Funding. Moriarity:BEAM Therapeutics: Consultancy; B-MoGen Biotechnologies: Employment, Equity Ownership.