NK-92MI Cell Research Articles

High-throughput proliferation and activation of NK-92MI cell spheroids via a homemade one-step closed bioreactor in pseudostatic cultures for immunocellular therapy.

The NK-92MI cell line has displayed significant promise in clinical trials for cancer treatment. However, challenges persist in obtaining sufficient cell quantities and achieving optimal cytotoxicity. The proliferation of natural killer (NK) cells involves the formation of cell aggregates, but excessively large aggregates can impede nutrient and waste transport, leading to reduced cell survival rates. In this study, a custom bioreactor was designed to mimic pseudostatic culture conditions by integrating brief mechanical rotation during a 6-h static culture period. This method aimed to achieve an optimal aggregate size while improving cell viability. The findings revealed a 144-fold expansion of 3D NK-92MI cell aggregates, reaching an ideal size of 80-150µm, significantly increasing both cell proliferation and survival rates. After 14days of culture, the NK-92MI cells maintained their phenotype during the subsequent phase of cell activation. Moreover, these cells presented elevated levels of IFN-γ expression after IL-18 activation, resulting in enhanced NK cell-mediated cytotoxicity against K562 cells. This innovative strategy, which uses a closed suspension-based culture system, presents a promising approach for improving cell expansion and activation techniques in immunocellular therapy.

Scalable Biomanufacturing Workflow to Produce and Isolate Natural Killer Cell-derived Extracellular Vesicle-based Cancer Biotherapeutics.

Natural killer cell-derived extracellular vesicles (NK-EVs) are being investigated as cancer biotherapeutics. They possess unique properties as cytotoxic nanovesicles targeting cancer cells and as immunomodulatory communicators. A scalable biomanufacturing workflow enables the production of large quantities of high-purity NK-EVs to meet the pre-clinical and clinical demands. The workflow employs a closed-loop hollow-fiber bioreactor, enabling continuous production of NK-EVs from the NK92-MI cell line under serum-free, xeno-free, feeder-free, and antibiotic-free conditions in compliance with Good Manufacturing Practices standards. This protocol-driven study outlines the biomanufacturing workflow for isolating NK-EVs using size-exclusion chromatography, ultrafiltration, and filter-based sterilization. Essential NK-EV product characterization is performed via nanoparticle tracking analysis, and their functionality is assessed through a validated cell viability-based potency assay against cancer cells. This scalable biomanufacturing process holds significant potential to advance the clinical translation of NK-EV-based cancer biotherapeutics by adhering to best practices and ensuring reproducibility.

Death receptors 4/5 mediate tumour sensitivity to natural killer cell-mediated cytotoxicity in mismatch repair deficient colorectal cancer.

Identifying the target of natural killer (NK) cells in colorectal cancer (CRC) is critical for optimising the clinical use of NK cell-mediated immunotherapy. Mismatch repair deficiency (dMMR) is associated with high immune cell infiltration and MHC Class I defects. Whether dMMR CRC responses to NK cell therapy remains unclear. MLH1, DR4, and DR5 knockout cell lines were established using CRISPR-Cas9 system. NK92-MI or NK cell isolated from BABL/C mice were used as effector cells against tumour cells. Inflammatory cytokines secretion by CRC cells was assessed via cytokine analysis. NK-cell-deficient/proficient animal models were used to validate the NK cell sensitivity. We observed that dMMR CRC cells were more sensitive to NK cell-mediated cytotoxicity than were mismatch-repair-proficient (pMMR) CRC cells. In dMMR CRC, Death receptor (DR)4/5 was upregulated and mediated sensitivity to NK cell-mediated cytotoxicity. DR4/5-mediated secretion of interleukin -12 sustained NK cell viability in dMMR CRC. NK cell depletion induced dMMR CRC tumour growth, and NK cell transfer inhibited lung metastasis of dMMR CRC with DR4/5 expression in vivo. TP53 upregulated DR4/DR5 expression in dMMR CRC. dMMR associated with increased sensitivity to NK cell-mediated cytotoxicity in CRC. DR4/DR5 sensitise dMMR CRC to NK cell-mediated cytotoxicity.

Abstract 5559: The anti-tumor efficacy and immune effects of combining of ceralasertib, an oral ATR kinase inhibitor, with imipridones, in prostate cancer treatment in vitro and in vivo

Abstract Prostate cancer is the most common cancer and the second leading cause of cancer death among men in the United States. There is an estimated 10% to 50% of cases progress to metastatic castration resistant prostate cancer (mCRPC) state within 3 years of diagnosis. mCPRC remains lethal despite therapeutic advances. There is approximately 20% of mCRPC patients present somatic DNA damage repair (DDR) gene mutations. Ceralasertib, formerly known as AZD6738, is a potent and selective orally bioavailable inhibitor of the ataxia tenlangiectasia and Rad3-related (ATR) kinase, which is involved in DNA repair in response to DNA damage and replication stress. Preclinical studies have demonstrated ATRi sensitizing alterations in DNA damage response (DDR) genes. Ceralasertib’s antitumor activity as a monotherapy in treating prostate cancer is moderate. Thus, we combined ceralasertib with imipridones (ONC201 and ONC206), which target mitochondrial caseinolytic protease P (ClpP) and the integrated stress response, resulting in enhanced antitumor efficacy in vitro. Prior data have demonstrated sensitivity of 4 prostate cancer lines to ceralasertib and imipridones monotherapies, synergistic activities with the combination treatments, and immune enhancement effects with the combination treatments when co-cultured with NK92MI cell line. We proceeded to a short term in-vivo study validating the combination treatment efficacy. Preliminary results include cytokine profiling using the Luminex 200 technology to understand treatment induced changes in the tumor microenvironment (TME) from both in vitro and in vivo studies. Our results guide novel clinical trials for effective clinical responses in mCPRC patients. Citation Format: Yutong Linda Xia, Leiqing Zhang, Maryam Ghandali, Maximilian P. Schwermann, Wafik El-Deiry. The anti-tumor efficacy and immune effects of combining of ceralasertib, an oral ATR kinase inhibitor, with imipridones, in prostate cancer treatment in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5559.

Low-molecular-weight fucoidan increases telomere length and immunostimulatory effects on NK-92 cells following inhaled anesthetic injury

Inhaled anesthetics, such as isoflurane, may cause side effects, including short-term immunosuppression and DNA damage. In contrast, low molecular weight fucoidan (LMF), derived from brown seaweed, exhibits promising immunomodulatory effects. In this study, we determined the effect of isoflurane on telomeres and examined the potential of LMF to ameliorate the harmful effects of isoflurane. Male Lewis rats, the mouse lymphoma cell line YAC-1, and the human nature killer cell line NK-92 MI were exposed to isoflurane. The relative telomere length (T/S) ratio and mRNA expression were determined by quantitative PCR. The viability assay was used to assess cell viability. In vivo, 2% isoflurane exposure, which is a clinically relevant concentration, reduced telomere length, and correlated with exposure frequency and duration. Isoflurane concentrations above 2% shortened YAC-1 telomeres, with minimal impact on cell viability. LMF pre-treatment enhanced NK-92 MI cell survival resulting from isoflurane exposure and exerted superior telomere protection compared with LMF post-treatment. Furthermore, adding LMF during isoflurane exposure resulted in a significant increase in IFN-γ, TNF-α, and IL-10 mRNA compared with the untreated group. LMF protected against isoflurane-induced telomere shortening, enhanced NK cell viability, and modulated cytokine expression, thus mitigating postoperative immune suppression and risk of tumor metastasis.

Screening and Characteristics Analysis of Polysaccharides from Orah Mandarin (Citrus reticulata cv. Orah).

This study aimed to screen out polysaccharides with the ability to activate NK cells. Ten polysaccharides (OP) were isolated from orah mandarin (Citrus reticulata cv. Orah) peel using hot-water extraction combined with the alcohol precipitation method and the ultrafiltration-membrane separation method. After measuring the effects of 10 OPs on NK-92MI cell proliferation and cytotoxicity, it was found that the polysaccharide OP5 had the highest activity in vitro. OP5 can significantly promote the proliferation of and increase the gene expression of perforin, granzyme B and IFN-γ in NK-92MI cells. Its molecular weight was between 50 and 70 kDa. The identification results of monosaccharide composition indicated that OP5 was composed of arabinose (31.52%), galacturonic acid (22.35%), galactose (16.72%), glucose (15.95%), mannose (7.67%), rhamnose (2.39%), fucose (1.41%), xylose (1.30%), glucuronic acid (0.42%) and ribose (0.27%). The sugar ring of the β-configuration was the main, and that of the α-configuration was the auxiliary. These results would provide a foundation for the functional product development of OPs.

A clinically relevant large-scale biomanufacturing workflow to produce natural killer cells and natural killer cell-derived extracellular vesicles for cancer immunotherapy.

Natural killer cell-derived extracellular vesicles (NK-EVs) have shown promising potential as biotherapeutics for cancer due to their unique attributes as cytotoxic nanovesicles against cancer cells and immune-modulatory activity towards immune cells. However, a biomanufacturing workflow is needed to produce clinical-grade NK-EVs for pre-clinical and clinical applications. This study established a novel biomanufacturing workflow using a closed-loop hollow-fibre bioreactor to continuously produce NK-EVs from the clinically relevant NK92-MI cell line under serum-free, Xeno-free and feeder-free conditions following GMP-compliant conditions. The NK92 cells grown in the bioreactor for three continuous production lots resulted in large quantities of both NK cell and NK-EV biotherapeutics at the end of each production lot (over 109 viable cells and 1013 EVs), while retaining their cytotoxic payload (granzyme B and perforin), pro-inflammatory cytokine (interferon-gamma) content and cytotoxicity against the human leukemic cell line K562 with limited off-target toxicity against healthy human fibroblast cells. This scalable biomanufacturing workflow has the potential to facilitate the clinical translation of adoptive NK cell-based and NK-EV-based immunotherapies for cancer with GMP considerations.

Oridonin enhances cytotoxic activity of natural killer cells against lung cancer

BackgroundOridonin is a Chinese herbal medicine exhibiting anti-tumor properties; however, its immune modulation capacity has yet to be elucidated. Our objective in this study was to determine whether oridonin enhances the anti-tumor activity of natural killer (NK) cells against lung cancer cells. MethodsLDH-releasing assays were used to investigate the effects of oridonin on NK-92MI cell activity against lung cancer cells. Flow cytometry and real-time PCR were used to examine the effects of oridonin on degranulation markers, cytotoxic factors, activating receptors on NK-92MI cells, and ligands in lung cancer cells. Western blot analysis provided insight into the mechanisms underlying the observed effects. ResultsOridonin enhanced the cytotoxic effects of NK-92MI cells against A549 lung cancer cells. This effect involved upregulating the expression of the degranulation marker CD107a and IFN-γ as well as activating receptors on NK cells and their ligand MICA/B. Oridonin also inhibited STAT3 phosphorylation in A549 cells and NK-92MI cells. A lung cancer mouse model confirmed the anti-tumor effects of oridonin and NK-92MI cells, wherein both treatments alone suppressed tumor growth. Oridonin was also shown to have a synergistic effect on the anti-tumor activity of NK-92MI cells. ConclusionsThe ability of oridonin to enhance the cytotoxic effects of NK cells indicates its potential as a novel therapeutic agent for the treatment of lung cancer.

Screening of immune cell activators from Astragali Radix using a comprehensive two-dimensional NK-92MI cell membrane chromatography/C18 column/time-of-flight mass spectrometry system

Astragali Radix (AR) is a clinically used herbal medicine with multiple immunomodulatory activities that can strengthen the activity and cytotoxicity of natural killer (NK) cells. However, owing to the complexity of its composition, the specific active ingredients in AR that act on NK cells are not clear yet. Cell membrane chromatography (CMC) is mainly used to screen the active ingredients in a complex system of herbal medicines. In this study, a new comprehensive two-dimensional (2D) NK-92MI CMC/C18 column/time-of-flight mass spectrometry (TOFMS) system was established to screen for potential NK cell activators. To obtain a higher column efficiency, 3-mercaptopropyltrimethoxysilane-modified silica was synthesized to prepare the NK-92MI CMC column. In total, nine components in AR were screened from this system, which could be washed out from the NK-92MI/CMC column after 10 min, and they showed good affinity for NK-92MI/CMC column. Two representative active compounds of AR, isoastragaloside I and astragaloside IV, promoted the killing effect of NK cells on K562 cells in a dose-dependent manner. It can thus suggest that isoastragaloside I and astragaloside IV are the main immunomodulatory components of AR. This comprehensive 2D NK-92MI CMC analytical system is a practical method for screening immune cell activators from other herbal medicines with immunomodulatory effects.

Natural killer cells activity against multiple myeloma cells is modulated by osteoblast-induced IL-6 and IL-10 production

BackgroundNatural killer (NK) cells are part of the innate arm of the immune system; as such NK cells can be activated rapidly to target virus-infected cells and tumor cells without prior sensitization. The human NK-92MI cell line is among the most widely used NK cell in preclinical research studies and has also been approved for clinical applications. Previous studies have shown that osteoblasts (OSB) confer drug resistance in multiple myeloma (MM) and other cancers that metastasize to the bone marrow. AimWe evaluated here how OSB, which are bone forming cells and a key cellular component of the bone marrow microenvironment, modulate the cytotoxic activity of NK-92MI cells against the MM.1S multiple myeloma cell line. MethodsThe osteoblastic niche was recapitulated with either the osteoblastic cell line hFOB 1.19 (hFOB) or primary osteoblasts (P-OSB) derived from surgical resections. Time-lapse imaging was utilized to quantify changes in MM.1S cell viability under different conditions, including: (1) Co-culture of MM.1S with NK92MI cells, (2) triple-culture of hFOB or P-OSB with MM.1S and NK-92MI, and (3) MM.1S or NK-92MI cells primed with OSB-derived supernatant. Cytokine analysis was conducted to quantify potential secreted factors associated with the protective effects of OSB. ResultsThe physical presence of OSB hindered the activity of NK-92MI cells, resulting in the increased viability of MM.1S compared to co-cultures which lacked OSB. This observation was accompanied by reduced perforin and granzyme A secretion from NK-92MI cells. Contact of OSB and NK-92MI cells also induced interleukin 6 (IL-6) and interleukin 10 (IL-10) production; two cytokines which are known to impair the NK cell immunity against MM and other cancers. OSB supernatant also conferred cytoprotection to MM.1S, suggesting a dual mechanism by which OSB may modulate both NK and MM cells. ConclusionsWe demonstrated here that OSB can negatively impact the activity of NK cells against MM. As NK cells and their chimeric antigen receptor-modified versions become more widely used in the clinic, our results suggest that understanding the role of OSB as potential immunoregulators of the NK cell-mediated cytotoxic response in the bone marrow tumor microenvironment may provide new opportunities for enhancing the effectiveness of this potent immunotherapeutic approach.

HLA-H*02:07 Is a Membrane-Bound Ligand of Denisovan Origin That Protects against Lysis by Activated Immune Effectors.

The biological relevance of genes initially categorized as "pseudogenes" is slowly emerging, notably in innate immunity. In the HLA region on chromosome 6, HLA-H is one such pseudogene; yet, it is transcribed, and its variation is associated with immune properties. Furthermore, two HLA-H alleles, H*02:07 and H*02:14, putatively encode a complete, membrane-bound HLA protein. Here we thus hypothesized that HLA-H contributes to immune homeostasis similarly to tolerogenic molecules HLA-G, -E, and -F. We tested if HLA-H*02:07 encodes a membrane-bound protein that can inhibit the cytotoxicity of effector cells. We used an HLA-null human erythroblast cell line transduced with HLA-H*02:07 cDNA to demonstrate that HLA-H*02:07 encodes a membrane-bound protein. Additionally, using a cytotoxicity assay, our results support that K562 HLA-H*02:07 inhibits human effector IL-2-activated PBMCs and human IL-2-independent NK92-MI cell line activity. Finally, through in silico genotyping of the Denisovan genome and haplotypic association with Denisovan-derived HLA-A*11, we also show that H*02:07 is of archaic origin. Hence, admixture with archaic humans brought a functional HLA-H allele into modern European and Asian populations.

Using NK-Derived Exosomes to Treat Leukemia

Natural killer cells (NK) are components of the innate immune system which play an important role in surveillance of cancer cells. Despite promising results in clinical trials, the use of NK-based therapies is limited due to safety issues. Exosomes are nanosized extracellular vesicles secreted by all types of cells. In recent years, exosomes have emerged as a powerful, natural therapeutic tool due to their low immunogenicity, low cytotoxicity, prolonged bioavailability and superior targeting ability. Since exosomes are known to carry cargo that reflects their cell of origin, we were prompted to test whether NK-derived exosomes (NK exo) maintain the anti-leukemia capacity of NK cells. To test the feasibility of this approach we undertook a set of preliminary experiments and evaluated the anti-leukemic potential of NK exo in-vitro on a wide variety of leukemic cell lines and patient-derived samples while also striving to assess their killing potential in-vivo using a murine xenograft model . This study aims to provide the pre-clinical evidence needed to test the NKexo approach in future clinical studies with the intention of ultimately developing an acellular “off-the-shelf” product to treat leukemia.Initially, we isolated NK exo from the engineered NK92MI cell line by ultracentrifugation and demonstrated the presence of large amounts of 100-200nm cap-shaped particles by means of nano-tracking-analysis and transmission-electron-microscopy. We then showed that these particles express the exosomal-characteristic CD63- and CD81- and NK-characteristic CD56-biomarkers. Next, we evaluated the effect of NK exo on CML (K562), T-ALL (Jurkat), B-ALL (UoC) and AML (HL-60 and KG1a) cell lines. NK exo treatment initiated a time- and dose-dependent reduction in viability and induction of apoptosis in all cell lines tested. The effect of NK exo exposure on cell viability and apoptosis was reduced in the presence of the pan-caspase inhibitor Z-VAD-FMK suggesting that NK exo exert their cytotoxic effect in a caspase-dependent manner. In addition, exposure to NK exo led to a significant increase in apoptosis in treatment naive AML, CLL and ALL patient-derived mononuclear cells. Moreover, we witnessed a significant reduction in colony formation of mononuclear cells harvested from treatment naive CML and AML patients in the presence of NK exo. While leukemia cells were targeted and severely affected by NK exo, healthy B-cells remained unaffected, indicating a selective effect. The selective trait of NK exo was also confirmed via an exosome uptake assay which demonstrated that NK exo were taken up specifically by leukemic cells but not by healthy B-cells.In an in-vivo xenograft model for AML; NOD-SCID IL2Rgama null (NSG) mice were engrafted with the well characterized human AML cell line HL60, and were then intravenously injected with NK exo. Our preliminary results indicate that NK exo treatment improves overall mice survival indicating an anti-leukemic effect of NK exo in-vivo.In this “proof-of-concept” study, we show that NK exo, similar to their cell-of-origin, have a strong and selective anti-leukemia effect. To provide the pre-clinical evidence needed to test the NK exo approach in future clinical studies, we are currently continuing to test the anti-leukemic potential of NK exo in-vivo on a xenograft murine model. Our preliminary results propose the use of NK exo as a novel therapeutic platform to combat leukemia. More broadly, since NK cells target a wide range of cancer cells, not just leukemia cells, and since we have shown that NK exo maintain the anti-leukemic capacity of their donor cells, this approach may become applicable in other hematological and solid malignancies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Influence of Galectin-9 Treatment on the Phenotype and Function of NK-92MI Cells in the Presence of Different Serum Supplements.

Galectins are one of the critical players in the tumor microenvironment–tumor crosstalk and the regulation of local immunity. Galectin-9 has been in the limelight in tumor immunology. Galectin-9 possesses its multiplex biological functions both extracellularly and intracellularly, plays a pivotal role in the modulation of adaptive and innate immunity, and induces immune tolerance. NK-92MI cell lines against different malignancies were extensively studied, and recently published trials used genetically chimeric antigen receptor-transfected NK-92MI cells in tumor immunotherapy. Besides the intensive research in tumor immunotherapy, limited information is available on their immune-checkpoint expression and the impact of checkpoint ligands on their effector functions. To uncover the therapeutic potential of modulating Galectin-9-related immunological pathways in NK-cell-based therapy, we investigated the dose-dependent effect of soluble Galectin-9 on the TIM-3 checkpoint receptor and NKG2D, CD69, FasL, and perforin expression of NK-92MI cells. We also examined how their cytotoxicity and cytokine production was altered after Gal-9 treatment and in the presence of different serum supplements using flow cytometric analysis. Our study provides evidence that the Galectin-9/TIM-3 pathway plays an important role in the regulation of NK cell function, and about the modulatory role of Galectin-9 on the cytotoxicity and cytokine production of NK-92MI cells in the presence of different serum supplements. We hope that our results will aid the development of novel NK-cell-based strategies that target Galectin-9/TIM-3 checkpoint in tumors resistant to T-cell-based immunotherapy.

Radioiodine labeling and in vivo trafficking of extracellular vesicles

Biodistribution and role of extracellular vesicles (EVs) are still largely unknown. Reliable tracking methods for EVs are needed. In this study, nuclear imaging using radioiodine were developed and applied for tracking EVs derived from cell lines. EVs were obtained from supernatant of thyroid cancer cell (Cal62) and natural killer cells (NK92-MI) using sequential ultracentrifuges. Sulfosuccinimidyl-3-(4-hydroxypheynyl) propionate were labeled to membrane of Cal62 and NK92-MI cell derived EVs, then the EVs were labeled with radioiodine (I-131 and I-125) using pre-coated iodination tubes (RI-EVs). In vivo gamma camera images were obtained after intravenous injection of the RI-EVs, and ex vivo biodistribution study was also performed. EVs were labeled with radioiodine and radiochemical purity of the RI-EV was more than 98%. Results of nanoparticle tracking analysis and electron microscopy showed that there was no significant difference in EVs before and after the radioiodine labeling. After intravenous injection of RI-EVs to mice, gamma camera imaging well visualized the real-time biodistribution of the RI-EVs. RI-EVs were mainly visualized at liver, spleen, and lung. Nuclear imaging system of EVs derived from thyroid cancer and NK cells using radioiodine labeling of the EVs was established. Thus, this system might be helpful for in vivo tracking of EVs.

Modulated Electro-Hyperthermia Facilitates NK-Cell Infiltration and Growth Arrest of Human A2058 Melanoma in a Xenograft Model.

Modulated electro-hyperthermia (mEHT), induced by 13.56 MHz radiofrequency, has been demonstrated both in preclinical and clinical studies to efficiently induce tumor damage and complement other treatment modalities. Here, we used a mouse xenograft model of human melanoma (A2058) to test mEHT (~42°C) both alone and combined with NK-cell immunotherapy. A single 30 min shot of mEHT resulted in significant tumor damage due to induced stress, marked by high hsp70 expression followed by significant upregulation of cleaved/activated caspase-3 and p53. When mEHT was combined with either primary human NK cells or the IL-2 independent NK-92MI cell line injected subcutaneously, the accumulation of NK cells was observed at the mEHT pretreated melanoma nodules but not at the untreated controls. mEHT induced the upregulation of the chemoattractant CXCL11 and increased the expression of the matrix metalloproteinase MMP2 which could account for the NK-cell attraction into the treated melanoma. In conclusion, mEHT monotherapy of melanoma xenograft tumors induced irreversible heat and cell stress leading to caspase dependent apoptosis to be driven by p53. mEHT could support the intratumoral attraction of distantly injected NK-cells, contributed by CXCL11 and MMP2 upregulation, resulting in an additive tumor destruction and growth inhibition. Therefore, mEHT may offer itself as a good partner for immunotherapy.

PET Imaging of the Natural Killer Cell Activation Receptor NKp30.

Redirecting the immune system in cancer treatment has led to remarkable responses in a subset of patients. Natural killer (NK) cells are innate lymphoid cells being explored as they engage tumor cells in different mechanisms compared with T cells, which could be exploited for treatment of nonresponders to current immunotherapies. NK cell therapies are monitored through measuring peripheral NK cell concentrations or changes in tumor volume over time. The former does not detect NK cells at the tumor site, and the latter is inaccurate for immunotherapies because of pseudoprogression. Therefore, new imaging methods are required as companion diagnostics for optimizing immunotherapies. Methods: In this study, we developed and completed preclinical in vivo validation of 2 antibody-based PET probes specific for NKp30, an activation natural cytotoxicity receptor expressed by human NK cells. Quantitative, multicolor flow cytometry during a variety of NK cell activation conditions was completed on primary human NK cells and the NK92MI cell line. Human renal cell carcinoma (RCC) tumors were stained for the NK cell receptors CD56, NKp30, and NKp46 to determine expression on tumor-infiltrating NK cells. An NKp30 antibody was radiolabeled with 64Cu or 89Zr and evaluated in subcutaneous xenografts and adoptive cell transfer mouse models. Results: Quantitative flow cytometry showed consistent expression of the NKp30 receptor during different activation conditions. NKp30 and NKp46 costained in RCC samples, demonstrating the expression of these receptors on tumor-infiltrating NK cells in human tumors, whereas tumor cells in one RCC sample expressed the peripheral NK marker CD56. Both PET tracers showed high stability and specificity in vitro and in vivo. Notably, 89Zr-NKp30Ab had higher on-target contrast than 64Cu-NKp30Ab at their respective terminal time points. 64Cu-NKp30Ab delineated NK cell trafficking to the liver and spleen in an adoptive cell transfer model. Conclusion: The consistent expression of NKp30 on NK cells makes it an attractive target for quantitative imaging. Immunofluorescence staining on human RCC samples demonstrated the advantages of NKp30 targeting versus CD56 for detection of tumor infiltrating NK cells. This work advances PET imaging of NK cells and supports the translation of imaging agents for immunotherapy monitoring.

Interactions of natural killer cells and pancreatic cancer cells in simulated microgravity

Abstract One of the major concerns of long-duration spaceflight is the exposure of astronauts to space radiation and the potential for cancer development. Natural killer (NK) cells continuously survey the body for such abnormalities and upon detection are activated to produce pro-inflammatory cytokines as well as cytotoxins which induce apoptosis of the cancer cells. This study employed the rotary cell culture system to generate a simulated microgravity (SMG) environment in order to assess its effect upon the growth of human cancer cells and their susceptibility to lysis by NK cells. We found that SMG diminishes the numbers of both pancreatic cancer cells and NK92MI, a human NK cell line, over a 7 d culture period. Additionally, SMG NK92MI cells had a ~50% decrease in cytotoxicity against PANC-1 pancreatic cancer cells. Surprisingly, a long-term SMG culture of up to 14 d resulted in complete restoration of NK92MI cell cytotoxic function. Interestingly, SMG cultured NK92MI cells produced more IFN-gamma than Static NK cells throughout the time periods assessed. In comparison, 7 d culture of pancreatic cancer cells in SMG led to a ~50% reduction in the ability of either Static or SMG NK92MI cells to induce apoptosis in the SMG cultured cancer cells. Therefore, over a two-week period in SMG culture, NK92MI cells were able to adapt to the environment and recover cytotoxic function, however, pancreatic cancer cells cultured in SMG acquired resistance to lysis by the NK cells. Hence, cancer cells developing in astronauts in microgravity might have a greater potential to resist NK cell control than in patients here on Earth which upon elucidation may aid in the identification of relevant new tumor targets for therapeutics.

Abstract B05: Intestinal microbiota mediates pancreatic carcinogenesis through modulation of tumor infiltrating natural killer cells

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is the 3rd leading cause of cancer-related death in the United States. Our group and others have demonstrated that the intestinal microbiota accelerates pancreatic carcinogenesis. The relationship of intestinal bacteria, immune response, and PDAC development is unclear. To that end, we investigated the role of intestinal bacterial soluble factors in modulating the immune environment of PDAC and its progression. Methods: Mice intestinal microbiota was depleted with wide-spectrum antibiotics. The human PDAC cell line, L3.6pl, was heterotopically implanted into Rag1-/- mice, while the syngeneic murine PDAC cell line, Pan02, was orthotopically implanted into the pancreas of C57Bl/6 mice. Tumor and pancreas infiltrated natural killer (NK) cells were quantitated by flow cytometry. In vivo NK cell depletion was attained by intraperitoneal injection of anti-Asialo-GM1 antibody twice weekly. Germ-free (GF) and conventionally housed Rag1-/- mice stool was cultured and bacteria-free supernatant extracted. The ability of these bacteria-free supernatants to regulate NK-92mi cell cytotoxicity and migration was tested in vitro by flow cytometry and a Transwell assay, respectively. Results: Compared to microbiota-intact Rag1-/- mice, microbiota depletion yielded 74% smaller tumors (p<0.05) with a 1.5-fold increase in PDAC infiltrating NK cells (p<0.001). Confirmation by immunohistochemistry demonstrated a 6.5-fold increase in NK cell tumor infiltration in microbiota-depleted mice (p<0.0001). Notably, a 3-fold increase of intrapancreatic NK cells in GF Rag1-/- mice versus conventionally housed mice was noted, suggesting that bacteria regulate immune cell trafficking. Antibody-mediated NK cell depletion with concomitant microbiota depletion increased PDAC tumor growth, negating the antitumor phenotype of microbiota depletion alone in both immunodeficient Rag1-/- (2.8-fold increase; p<0.05) and immunocompetent C57Bl/6 mice (2.4-fold increase; p<0.05). Quantitative PCR revealed 9-fold higher IFN-gamma gene expression in PDAC xenografts of microbiota-depleted mice versus microbiota-intact (p<0.05). Cell-free stool bacteria culture supernatant from conventionally housed Rag1-/- mice, but not from GF mice, inhibited NK-92mi cell migration by 26% (p<0.05) and cytotoxicity against L3.6pl cells by 51% (p<0.01). Compared to NK-92mi cells exposed to cultured stool supernatant from GF Rag1-/- mice, SPF cultured stool supernatant resulted in decreased gene expression associated with activation/recruitment of NK cells including FASLG, CCL18, IL-13, CXCR2, CCL4, and increased expression associated with inhibition of NK cell activity including CCR1 and IL-6. Conclusion: These findings suggest that intestinal bacteria modulate PDAC development through suppression of NK cell recruitment and activation, a phenomenon potentially mediated by yet to be identified small molecules. Citation Format: Qin Yu, Mark Beveridge, Ryan Thomas, Christian Jobin. Intestinal microbiota mediates pancreatic carcinogenesis through modulation of tumor infiltrating natural killer cells [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr B05.

Abstract A67: Augmented antitumor activity of NK-92 MI cells expressing a chimeric antigen receptor targeting human mesothelin

Abstract Adoptive natural killer (NK) cell immunotherapy using primary NK cells has been challenging, to date. The use of an ”off-the -shelf” NK cell line where multiple genetic changes can be introduced has advantages. In this study, we explored the use of the NK-92MI cell line expressing a chimeric antigen receptor (CAR) targeting human mesothelin, the current target in our human CAR-T cell therapy trials. The NK-92MI cell line (which makes its own IL-2) was purchased from ATCC and transduced with a lentivirus encoding GFP (vector control) or an anti-human mesothelin scFv-41BB-CD3 ζ second-generation CAR (MesoCAR). Addition of IL-21 enhanced transduction. The transduced cells (MesoCar-NK-92MI) were sorted by flow cytometry to achieve an expression level of >98%. The NK markers NKp30, NKp44, NKG2D, 2B4, and CD56 were highly expressed on the Car-NK-92MI cell line (from 75% to 99.9%). To test the antitumor effects in vitro, three types of human tumor lines transduced to express human mesothelin were tested. Lung cancer (A549Meso), mesothelioma (EMMeso), and head and neck cancer (SSC15Meso) cells were exposed to vector control NK-92MI or MesoCar-NK-92MI cells for 22 hours at varying E:T ratios. The supernatants were collected for detection of cytokines and granzyme B. We also determined the killing function of MesoCar-NK-92MI cells after irradiation of the cells at 10 Gy. To test in vivo activity, NSG mice were injected in the flank with 5 million EMMeso cells. After tumors were established, each mouse was intravenously injected with 10 million vector control NK-92MI cells or MesoCar-NK-92MI cells with a boost injection of 5 million effectors on days 4, 8, and 11. Tumor volumes were assessed over time and mice were sacrificed on day 18. In vitro killing results showed augmented antitumor effect in MesoCar-NK-92MI compared to vector control NK-92MI. Irradiated MesoCar-NK-92MI cells still possessed good antitumor activity with only 32% loss of activity on average. Increased cytokine and granzyme B secretion was also seen with the MesoCar-NK-92MI cell line (with or without irradiation) compared to NK-92MI cells. In animal studies, irradiated MesoCar-NK-92MI cells significantly inhibited tumor growth by an average of 59% with no obvious toxicity or tumor growth in other organs. The non-CAR-expressing NK-92MI cells had significantly less antitumor activity. In conclusion, use of an irradiated NK-92MI cell line expressing a CAR targeting human mesothelin could become a safe and possible “off-the-shelf” adoptive cell therapy that could be used alone or in combination with CAR-T cells or other treatments. Citation Format: Jing Sun, Patrick Woodruff, Soyeon Kim, Estela Noguera-Ortega, Evguenia Arguiri, Maria Liousia, Steven Maceko, Edmund Moon, Steven Albelda. Augmented antitumor activity of NK-92 MI cells expressing a chimeric antigen receptor targeting human mesothelin [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A67.

The Anti-Leukemia Effect of Natural Killer-Derived Exosomes

Circulating NK cells are the effector arm of the innate immune system. As such, they recognize transformed cells as "non-self" and kill them. Numerous ex vivo studies have demonstrated the ability of NK cells to kill allogeneic leukemia cells. Based on these in vitro studies, several past and ongoing clinical trials have shown allogenic NK cells have a strong anti-leukemia effect. However, the use of NK cells as an "off the shelf" product is limited since they, like most cellular products, induce an allo-reactive immunogenic response which limits efficacy and increases toxicity. Exosomes are nano scaled extracellular vesicles that are released by various types of cells including NK cells. Since the exosomal cargo reflects, in part, the molecular makeup of its cell of origin, we hypothesized that NK-derived exosomes maintain the anti-leukemia effect of their cell of origin. To test this hypothesis, we exposed leukemia cells to NK-derived exosomes and tested their ability to eliminate them. As a source for NK-derived exosomes we used the NK-92MI cell line. This is a genetically altered cell line which constitutively expresses IL-2 that augments the cytotoxic activity of the cells and is routinely used in clinical trials. We cultured these cells in exosome free medium for 48 hours and extracted the exosomes by ultracentrifugation. Nanoparticle analyzing system showed that the pellet was enriched with the typical ~100nm sized vesicles and these particles were visualized by electron microscopy. Western immunoblotting confirmed that these particles express CD63, a known exosomal biomarker. Subsequently, we subjected CML K562, ALL Jurkat and AML HL-60 to NK-92MI-Exo stained with PKH-26 and showed by flow cytometry that these cells uptake the exosomes in a dose- and time- dependent manner. As controls, we used exosomes derived from the human embryonic kidney 293 (HEK-293) cell line. LDH release assay showed a marked increase in LDH activity in NK-92MI-Exo exposed leukemia cells but not in HEK-293-Exo exposed leukemia cells across all cell lines tested. Similarly, flow cytometry of leukemia cells double stained for annexin/PI showed that the rate of apoptosis was markedly increased in NK-92MI-Exo exposed leukemia cells but not in HEK-293-Exo exposed leukemia cells. Together, these assays indicate that NK-92MI-Exo are cytotoxic to various leukemia cell lines and that this effect is specific to NK-derived exosomes. Encouraged by our preliminary results, we harvested leukemia cells from 4 patients with acute myeloid leukemia (AML), 4 patients with acute lymphoblastic leukemia (ALL), 4 patients with chronic lymphocytic leukemia (CLL) and normal B-cells from healthy volunteers and exposed them to NK-92MI-Exo. Similar to the cell line results, we found a marked cytotoxic effect to NK-92MI-Exo on these leukemia cells but not on normal B-cells from healthy individuals whereas HEK-293-Exo had only a minimal or no effect on primary leukemia cells. Clonal assay on peripheral blood sample from 2 CML patients showed that the number of colony forming unit -granulocyte, erythrocyte, megakaryocyte monocyte (CFU-GEMM) is significantly reduced following exposure to NK-92MI-Exo, suggesting that these exosomes target leukemia progenitor cells. Proteomics analysis of NK-92MI-Exo revealed that similar to NK cytotoxic granules, the exosomal cargo include granozymes known to induce apoptosis of target cells but unlike NK cytotoxic granules the exosomal cargo does not include perphorines, known to perforate target cell membrane. Taken together our data suggests that NK-derived exosomes have a potent cytotoxic effect across a wide range of leukemia cells. This effect is specific to NK-exosomes. Furthermore, NK-derived exosomes preferentially kill leukemia cells but not normal cells. We also show that these exosomes are toxic to leukemia progenitor cells. Proteomics analysis suggested that NK-cytotoxic granules and exosomes use different strategies to enter target-cells. Whether NK-derived exosomes may become a-cellular therapeutic strategy to combat leukemia remains to be determined. Figure Disclosures No relevant conflicts of interest to declare.