Enhancing natural killer cells proliferation and cytotoxicity using imidazole-based lipid nanoparticles encapsulating interleukin-2 mRNA.

mRNA applications have undergone unprecedented applications-from vaccination to cell therapy. Natural killer (NK) cells are recognized to have a significant potential in immunotherapy. NK-based cell therapy has drawn attention as allogenic graft with a minimal graft-versus-host risk leading to easier off-the-shelf production. NK cells can be engineered with either viral vectors or electroporation, involving high costs, risks, and toxicity, emphasizing the need for alternative way as mRNA technology. We successfully developed, screened, and optimized novel lipid-based platforms based on imidazole lipids. Formulations are produced by microfluidic mixing and exhibit a size of approximately 100 nm with a polydispersity index of less than 0.2. They are able to transfect NK-92 cells, KHYG-1 cells, and primary NK cells with high efficiency without cytotoxicity, while Lipofectamine Messenger Max and D-Lin-MC3 lipid nanoparticle-based formulations do not. Moreover, the translation of non-modified mRNA was higher and more stable in time compared with a modified one. Remarkably, the delivery of therapeutically relevant interleukin 2 mRNA resulted in extended viability together with preserved activation markers and cytotoxic ability of both NK cell lines and primary NK cells. Altogether, our platforms feature all prerequisites needed for the successful deployment of NK-based therapeutic strategies.

NK-92 cells activated by IL-2 inhibit the progression of endometriosis in vitro.

BACKGROUND: Interleukin (IL)-2 is a key cytokine capable of modulating the immune response by activating natural killer (NK) cells. This study was recruited to explore the therapeutic potential of IL-2-activated NK-92 cells in endometriosis in vitro. METHODS: Ectopic endometrial stromal cells (EESCs) were isolated and co-cultured with IL-2-activated NK-92 cells at varying effector-to-target (E:T) ratios (1:0 [Control], 1:1, 1:3, and 1:9). The viability, cytotoxicity, and cell surface antigen expression of IL-2-activated NK-92 cells were assessed. The viability, apoptosis, invasion, and migration ability of EESCs co-cultured with NK-92 cells at different ratios were evaluated. The apoptosis-related proteins, invasion and migration-related proteins as well as MEK/ERK pathway were examined via western blot. Each experiment was repeated three times. RESULTS: IL-2 activation enhanced NK-92 cytotoxicity in a concentration-dependent manner. Co-culturing EESCs with IL-2-activated NK-92 cells at E:T ratios of 1:1, 1:3, and 1:9 reduced EESC viability by 20%, 45%, and 70%, respectively, compared to the control group. Apoptosis rates in EESCs increased in correlation with the NK-92 cell proportion, with the highest rate observed at a 1:9 ratio. Moreover, EESC invasion and migration were significantly inhibited by IL-2-activated NK-92 cells, with a 60% reduction in invasion and a 50% decrease in migration at the 1:9 ratio. Besides, the MEK/ERK signalling pathway was down-regulated in EESCs by IL-2-activated NK-92 cells. CONCLUSION: IL-2-activated NK-92 cells exhibit potent cytotoxic effects against EESCs. They promote EESC apoptosis and inhibit viability, invasion, and migration through modulating the MEK/ERK signalling pathway.

Endometriosis is a common chronic systemic disease affecting approximately 190 million women worldwide. However, clinical treatments for endometriosis remain challenging due to the scarcity of high-quality scientific evidence and conflicting available guidelines. This research was designed to explore whether interleukin (IL)-2 affected the progression of endometriosis by modulating endometrial stromal cell apoptosis and natural killer (NK) cell-mediated cytotoxicity, thereby providing new therapeutic methods for endometriosis.

NK-92 cells labeled with Fe3O4-PEG-CD56/Avastin@Ce6 nanoprobes for the targeted treatment and noninvasive therapeutic evaluation of breast cancer.

Adoptive cellular immunotherapy as a promising and alternative cancer therapy platform is critical for future clinical applications. Natural killer (NK) cells have attracted attention as an important type of innate immune regulatory cells that can rapidly kill multiple adjacent cancer cells. However, these cells are significantly less effective in treating solid tumors than in treating hematological tumors. Herein, we report the synthesis of a Fe3O4-PEG-CD56/Avastin@Ce6 nanoprobe labeled with NK-92 cells that can be used for adoptive cellular immunotherapy, photodynamic therapy and dual-modality imaging-based in vivo fate tracking. The labeled NK-92 cells specifically target the tumor cells, which increases the amount of cancer cell apoptosis in vitro. Furthermore, the in vivo results indicate that the labeled NK-92 cells can be used for tumor magnetic resonance imaging and fluorescence imaging, adoptive cellular immunotherapy, and photodynamic therapy after tail vein injection. These data show that the developed multifunctional nanostructure is a promising platform for efficient innate immunotherapy, photodynamic treatment and noninvasive therapeutic evaluation of breast cancer.

Optimizing glutamine concentration enhances ex vivo expansion of natural killer cells through improved redox status.

Amino acids are vital components of the serum-free medium that influence the expansion and function of NK cells. This study aimed to clarify the relationship between amino acid metabolism and expansion and cytotoxicity of NK cells. Based on analyzing the mino acid metabolism of NK-92 cells and Design of Experiments (DOE), we optimized the combinations and concentrations of amino acids in NK-92 cells culture medium. The results demonstrated that NK-92 cells showed a pronounced demand for glutamine, serine, leucine, and arginine, in which glutamine played a central role. Significantly, at a glutamine concentration of 13 mM, NK-92 cells expansion reached 161.9 folds, which was significantly higher than 55.5 folds at 2.5 mM. Additionally, under higher glutamine concentrations, NK-92 cells expressed elevated levels of cytotoxic molecules, the level of cytotoxic molecules expressed by NK-92 cells was increased and the cytotoxic rate was 68.42%, significantly higher than that of 58.08% under low concentration. In view of the close relationship between glutamine metabolism and intracellular redox state, we investigated the redox status within the cells. This study demonstrated that intracellular ROS levels in higher glutamine concentrations were significantly lower than those under lower concentration cultures with decreased intracellular GSH/GSSG ratio, NADPH/NADP+ ratio, and apoptosis rate. These findings indicate that NK-92 cells exhibit improved redox status when cultured at higher glutamine concentrations. Overall, our research provides valuable insights into the development of serum-free culture medium for ex vivo expansion of NK-92 cells.

Suspension culture promoted the expansion of NK-92 cells ex vivo by enhancing the expression of IL-2 receptor.

Vigorous ex vivo expansion of NK-92 cells is a pivotal step for clinical adoptive immunotherapy. Interleukin-2 (IL-2) is identified as a key cytokine for NK-92 cells, and it can stimulate cell proliferation after binding to the IL-2 receptor (IL-2R). In this work, the differences in IL-2 consumption and IL-2R expression were investigated between the two culture modes. The results showed that suspension culture favored ex vivo expansion of NK-92 cells compared with static culture. The specific consumption rate of IL-2 in suspension culture was significantly higher than that in static culture. It was further found that the mRNA levels of the two IL-2R subunits remained unchanged in suspension culture, but the proportion of NK-92 cells expressing IL-2Rß was increased, and the fluorescence intensity of IL-2Rß was remarkably enhanced. Meanwhile, the proportion of cells expressing IL-2R receptor complex also increased significantly. Correspondingly, the phosphorylation of STAT5, a pivotal protein in the downstream signaling pathway of IL-2, was up-regulated. Notably, the expression level and colocalization coefficient of related endosomes during IL-2/IL-2R complex endocytosis were markedly elevated, suggesting the enhancement of IL-2 endocytosis. Taken together, these results implied that more IL-2 was needed to support cell growth in suspension culture. Therefore, the culture process was optimized from the perspective of cytokine utilization to further improve the NK-92 cell's expansion ability and function. This study provides valuable insight into the efficient ex vivo expansion of NK-92 cells.

Manufacturing CAR-NK against tumors: Who is the ideal supplier?

Chimeric antigen receptor-natural killer (CAR-NK) cells have emerged as another prominent player in the realm of tumor immunotherapy following CAR-T cells. The unique features of CAR-NK cells make it possible to compensate for deficiencies in CAR-T therapy, such as the complexity of the manufacturing process, clinical adverse events, and solid tumor challenges. To date, CAR-NK products from different allogeneic sources have exhibited remarkable anti-tumor effects on preclinical studies and have gradually been applied in clinical practice. However, each source has advantages and disadvantages. Selecting a suitable source may help maximize CAR-NK cell efficacy and increase the feasibility of clinical transformation. Therefore, this review discusses the development and challenges of CAR-NK cells from different sources to provide a reference for future exploration.

Recruiting In Vitro Transcribed mRNA against Cancer Immunotherapy: A Contemporary Appraisal of the Current Landscape.

Over 100 innovative in vitro transcribed (IVT)-mRNAs are presently undergoing clinical trials, with a projected substantial impact on the pharmaceutical market in the near future. Τhe idea behind this is that after the successful cellular internalization of IVT-mRNAs, they are subsequently translated into proteins with therapeutic or prophylactic relevance. Simultaneously, cancer immunotherapy employs diverse strategies to mobilize the immune system in the battle against cancer. Therefore, in this review, the fundamental principles of IVT-mRNA to its recruitment in cancer immunotherapy, are discussed and analyzed. More specifically, this review paper focuses on the development of mRNA vaccines, the exploitation of neoantigens, as well as Chimeric Antigen Receptor (CAR) T-Cells, showcasing their clinical applications and the ongoing trials for the development of next-generation immunotherapeutics. Furthermore, this study investigates the synergistic potential of combining the CAR immunotherapy and the IVT-mRNAs by introducing our research group novel, patented delivery method that utilizes the Protein Transduction Domain (PTD) technology to transduce the IVT-mRNAs encoding the CAR of interest into the Natural Killer (NK)-92 cells, highlighting the potential for enhancing the CAR NK cell potency, efficiency, and bioenergetics. While IVT-mRNA technology brings exciting progress to cancer immunotherapy, several challenges and limitations must be acknowledged, such as safety, toxicity, and delivery issues. This comprehensive exploration of IVT-mRNA technology, in line with its applications in cancer therapeutics, offers valuable insights into the opportunities and challenges in the evolving landscape of cancer immunotherapy, setting the stage for future advancements in the field.

High-free Fatty Acid Treatment Induced Anti-inflammatory Changes in a Natural Killer (NK) Cell Line.

Background: Natural killer (NK) cells play a role in the pathogenesis of various metabolic diseases related to obesity. While our initial findings have indicated a potential involvement of NK cells in the pathogenesis of type 2 diabetes mellitus, the precise mechanism underlying NK cell-mediated development of this form of diabetes remains inadequately comprehended. Objective: To investigate the impact and the underlying mechanism of high glucose and elevated levels of free fatty acids (FFAs) on immune and inflammatory responses and oxidative stress in NK92 cells. Methods: In this experiment, the CCK8 cytotoxicity assay was used to select the 44.4 mM and 1.5 mM concentrations of high glucose and high FFAs, respectively, to treat NK92 cells for 4 days. The concentrations of superoxide dismutase (SOD) and glutathione (GSH) were determined using a biochemical analyzer. Intracellular reactive oxygen species (ROS) levels, cytokines concentrations (TNF-α, IFN-γ, IL-6, and IL-10), and the expression levels of intracellular molecules (perforin and granzyme B) were assessed by flow cytometry. Results: The number of NK92 cell clumps was significantly reduced in the high-FFA (HF) group. In addition, the production of ROS and levels of cytokines (TNF-α, IFN-γ, IL-6, and IL-10) significantly decreased in the HF group but showed no significant change in the high-glucose (HG) group. This observation was consistent with the expression levels of perforin and granzyme B that decreased in the HF group. Conclusion: High FFAs induced morphological changes and serious damage to oxidative stress and inflammatory response in NK92 cells.

Development, methodological evaluation and application of a cell-based TRF assay for analysis of ADCC activity.

Interaction of an antibody with its FcγR plays an important role in effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC). Nowadays altered ADCC activity of an antibody can be achieved by utilizing an effective glyco-engineering strategy, which often involves changes of sugar moieties in Fc part of the antibody, thereby affecting its receptor binding with effector cells. We aimed to construct a cell-based time-resolved fluorescence (TRF) assay for the evaluation of ADCC activity triggered by the antibody drug Trastuzumab (anti-HER2) and T-DM1. The assay was initiated by incubating 2,2':6',2 "-Terpyridine-6,6"-dicarboxylic acid (TDA)-labeled target SK-BR3 cells with the testing antibodies and engineered NK-92 effector cells. After incubation, the target cells were lysed to detect TDA released into the supernatant. Together with added Eu, the TDA in the supernatant formed a stable chelate of EuTDA with high-intensity fluorescence. The ADCC activity was then determined by measuring the fluorescence of EuTDA. Consequently, the method demonstrated good accuracy, precision, linearity, and specificity over methodological assessment and compared well with the Luciferase release assay in terms of the agreement of the achieved results. Using the developed assay, we evaluated the ADCC activity of two glyco-engineered anti-HER-2 antibody-drug conjugates (ADCs) and the results showed that antibody Fc glycosylation modifications influenced antibody ADCC activity to varying degrees. In conclusion, the present assay is able to accurately assess the ADCC activity induced by Trastuzumab (anti-HER2) and T-DM1, and a similar methodology can be applied to other therapeutic antibodies during drug development to help screen for antibodies with desirable ADCC activity.

Detection of Antibody-Dependent Cell-Mediated Cytotoxicity-Supporting Antibodies by NK-92-CD16A Cell Externalization of CD107a: Recognition of Antibody Afucosylation and Assay Optimization.

Antibody-dependent cell-mediated cytotoxicity (ADCC) by natural killer (NK) lymphocytes eliminates cells infected with viruses. Anti-viral ADCC requires three components: (1) antibody; (2) effector lymphocytes with the Fc-IgG receptor CD16A; and (3) viral proteins in infected cell membranes. Fc-afucosylated antibodies bind with greater affinity to CD16A than fucosylated antibodies; individuals' variation in afucosylation contributes to differences in ADCC. Current assays for afucosylated antibodies involve expensive methods. We report an improved bioassay for antibodies that supports ADCC, which encompasses afucosylation. This assay utilizes the externalization of CD107a by NK-92-CD16A cells after antibody recognition. We used anti-CD20 monoclonal antibodies, GA101 WT or glycoengineered (GE), 10% or ~50% afucosylated, and CD20-positive Raji target cells. CD107a increased detection 7-fold compared to flow cytometry to detect Raji-bound antibodies. WT and GE antibody effective concentrations (EC50s) for CD107a externalization differed by 20-fold, with afucosylated GA101-GE more detectable. The EC50s for CD107a externalization vs. 51Cr cell death were similar for NK-92-CD16A and blood NK cells. Notably, the % CD107a-positive cells were negatively correlated with dead Raji cells and were nearly undetectable at high NK:Raji ratios required for cytotoxicity. This bioassay is very sensitive and adaptable to assess anti-viral antibodies but unsuitable as a surrogate assay to monitor cell death after ADCC.

Development of FAP-Targeted Chimeric Antigen Receptor NK-92 Cells for Non-Small Cell Lung Cancer.

OBJECTIVES: Over the past two decades, great progress has been made in advancing the early detection and multimodal treatment of non-small cell lung cancer (NSCLC). However, overall cure rates and survival rates of NSCLC are still not satisfactory, and research into new therapies is needed. This study attempted to construct human Fibroblast Activation Protein-Chimeric Antigen Receptor Natural killer (NK)-92 cells (hFAP-CAR-NK-92 cells) and explore their potential therapeutic effects in NSCLC. METHODS: Immunohistochemistry analysis was carried out to examine fibroblast activation protein (FAP) and Gasdermin E (GSDME) expression in clinical specimens of lung adenocarcinoma and squamous cell carcinoma tissue. Then the engineered hFAP-CAR-NK-92 cells efficiency was determined in vitro with lactate dehydrogenase (LDH) cytotoxicity assay and the cell morphology of A549, H226, and cancer-related fibroblast (CAF) was observed by electron microscopy. After the co-culture of target cells and effect cells, flow cytometry was employed for examining the CD107a expression in the effect cells, and western blotting was conducted for the cleavage levels of Caspase 3 and GSDME proteins in the target cells. The safety and efficacy of hFAP-CAR-NK-92 cells adoptive transfer immunotherapy in a tumor-bearing mouse were evaluated. RESULTS: Clinical studies have shown FAP positivity in patients with NSCLC. Compared with A549 or H226 cells alone, FAP expression was notably raised in A549+CAF cells or H226+CAF cells in nude mice, respectively (p < 0.05). The killing efficiency of K562 cells was not significantly different between hFAP-CAR-NK-92 and NK-92 cells (p > 0.05). The hFAP-CAR-NK-92 cells presented a higher killing efficiency against the hFAP-target (A549-hFAP, H226-hFAP and CAF-hFAP) cells than the NK-92 cells (p < 0.05). The degranulation of CD107a and cleavage levels of GSDME and Caspase 3 protein in the hFAP-CAR-NK-92 group were higher than those in the NK-92 group (p < 0.05). The 300 nM Granzyme B also induced pyroptosis in hFAP- or GSDME-positive cells (p < 0.05). In vivo experiments revealed that hFAP-CAR-NK-92 cells inhibited tumor progression of hFAP-positive NSCLC (p < 0.05). CONCLUSIONS: In this study, we successfully constructed hFAP-CAR-NK-92 cells and confirmed that hFAP-CAR-NK-92 cells could target hFAP-positive NSCLC to inhibit the progression of NSCLC by activating the Caspase-3/GSDME pyroptosis pathway.

CAR-NK Cells Targeting HER1 (EGFR) Show Efficient Anti-Tumor Activity against Head and Neck Squamous Cell Carcinoma (HNSCC).

(1) Background: HNSCC is a highly heterogeneous and relapse-prone form of cancer. We aimed to expand the immunological tool kit against HNSCC by conducting a functional screen to generate chimeric antigen receptor (CAR)-NK-92 cells that target HER1/epidermal growth factor receptor (EGFR). (2) Methods: Selected CAR-NK-92 cell candidates were tested for enhanced reduction of target cells, CD107a expression and IFNγ secretion in different co-culture models. For representative HNSCC models, patient-derived primary HNSCC (pHNSCC) cell lines were generated by employing an EpCAM-sorting approach to eliminate the high percentage of non-malignant cells found. (3) Results: 2D and 3D spheroid co-culture experiments showed that anti-HER1 CAR-NK-92 cells effectively eliminated SCC cell lines and primary HNSCC (pHNSCC) cells. Co-culture of tumor models with anti-HER1 CAR-NK-92 cells led to enhanced degranulation and IFNγ secretion of NK-92 cells and apoptosis of target cells. Furthermore, remaining pHNSCC cells showed upregulated expression of putative cancer stem cell marker CD44v6. (4) Conclusions: These results highlight the promising potential of CAR-NK cell therapy in HNSCC and the likely necessity to target multiple tumor-associated antigens to reduce currently high relapse rates.

The effects of KIR2DL4 stimulated NK-92 cells on the apoptotic pathways of HER2 + /HER-breast cancer cells.

Natural killer (NK) cells are immune cells that have attracted significant attention due to their cytotoxic properties. They are believed to be highly effective in cancer therapy. In this study, anti-KIR2DL4 (Killer cell Immunoglobulin like Receptor, 2 Ig Domains and Long cytoplasmic tail 4) was used to stimulate the NK-92 activator receptor to increase their cytotoxicity on breast cancer cell lines. Unstimulated and stimulated NK-92 cells (sNK-92) were cocultured with breast cancer (MCF-7 and SK-BR-3) and normal breast (MCF-12A) cell lines at 1:1, 1:5, and 1:10 (Target:Effector) ratios. The most effective cell cytotoxicity ratio (1:10) was used in the immunostaining and western blot assays to evaluate apoptosis pathway proteins. The sNK-92 cells showed higher cytotoxic activity on breast cancer cells than NK-92 cells. sNK-92 cells had a selective significant cytotoxicity effect on MCF-7 and SK-BR-3 cells but not MCF-12A cells. While sNK-92 cells were effective at all cell concentrations, they were most effective at a 1:10 ratio. Immunostaining and western blots showed significantly higher BAX, caspase 3, and caspase 9 protein levels in all breast cancer cell groups cocultured with sNK-92 than with NK-92 cells. NK-92 cells stimulated with KIR2DL4 showed elevated cytotoxic activity. The cytotoxic activity of sNK-92 cells on breast cancer cells is via apoptosis pathways. However, their effect on normal breast cells is limited. While the obtained data contains only basic information, additional clinical studies are needed to provide a basis for a new treatment model.

Cytotoxicity of CD19-CAR-NK92 cells is primarily mediated via perforin/granzyme pathway.

Chimeric antigen receptors (CARs) have improved cancer immunotherapy in recent years. Immune cells, such as Natural killer cells (NK-cells) or T cells, are used as effector cells in CAR-therapy. NK92-cells, a cell line with known cytotoxic activity, are of particular interest in CAR-therapy since culturing conditions are simple and anti-tumor efficacy combined with a manageable safety profile was proven in clinical trials. The major pathways of immune effector cells, including NK92-cells, to mediate cytotoxicity, are the perforin/granzyme and the death-receptor pathway. Detailed knowledge of CAR-effector cells' cytotoxic mechanisms is essential to unravel resistance mechanisms, which potentially arise by resistance against apoptosis-inducing signaling. Since mutations in apoptosis pathways are frequent in lymphoma, the impact on CAR-mediated cytotoxicity is of clinical interest. In this study, knockout models of CD19-CAR-NK92 cells were designed, to investigate cytotoxic pathways in vitro. Knockout of perforin 1 (Prf1) and subsequent abrogation of the perforin/granzyme pathway dramatically reduced the cytotoxicity of CD19-CAR-NK92 cells. In contrast, knockout of FasL and inhibition of TRAIL (tumor necrosis factor-related apoptosis-inducing ligands) did not impair cytotoxicity in most conditions. In conclusion, these results indicate the perforin/granzyme pathway as the major pathway to mediate cytotoxicity in CD19-CAR-NK92 cells.

Optimization of transduction conditions of recombinant adeno-associated virus into NK92 cells / 中国药科大学学报

@#To improve the transduction efficiency of recombinant adeno-associated virus (rAAV) in NK92 cells, the number of cells, concentration of IL-2 in the medium, and serotype and dosage of rAAV were explored to optimize cell state and viral transfection conditions.Then, zinc chloride (ZnCl2), chloroquine, polyvinyl alcohol (PVA) and genistein with different concentration were added separately during transfection to further improve the viral transduction efficiency.The results showed that, at cell number of 5 × 105, the expression efficiency of enhanced green fluorescent protein (EGFP) was relatively high.When the IL-2 concentration was 1 000 IU/mL, NK92 cells were most suitable for virus transfection. The transduction efficiency of different serotypes of rAAV in NK92 cells was rAAV6, rAAV2 and rAAV9 in descending order.Pretreatment of NK92 cells with genistein could significantly increase the viral transduction efficiency, while the addition of other reagents had no significant effect.Through the optimization of the above conditions, the transduction efficiency of rAAV to NK92 cells could be significantly improved, which provided evidence for functional genetic modification of NK92 cells by rAAV.

An Innovative PTD-IVT-mRNA Delivery Platform for CAR Immunotherapy of ErbB(+) Solid Tumor Neoplastic Cells.

Chimeric antigen receptor (CAR) immunotherapy includes the genetic modification of immune cells to carry such a receptor and, thus, recognize cancer cell surface antigens. Viral transfection is currently the preferred method, but it carries the risk of off-target mutagenicity. Other transfection platforms have thus been proposed, such the in vitro transcribed (IVT)-mRNAs. In this study, we exploited our innovative, patented delivery platform to produce protein transduction domain (PTD)-IVT-mRNAs for the expression of CAR on NK-92 cells. CAR T1E-engineered NK-92 cells, harboring the sequence of T1E single-chain fragment variant (scFv) to recognize the ErbB receptor, bearing either CD28 or 4-1BB as co-stimulatory signaling domains, were prepared and assessed for their effectiveness in two different ErbB(+) cancer cell lines. Our results showed that the PTD-IVT-mRNA of CAR was safely transduced and expressed into NK-92 cells. CAR T1E-engineered NK-92 cells provoked high levels of cell death (25-33%) as effector cells against both HSC-3 (oral squamous carcinoma) and MCF-7 (breast metastatic adenocarcinoma) human cells in the co-incubation assays. In conclusion, the application of our novel PTD-IVT-mRNA delivery platform to NK-92 cells gave promising results towards future CAR immunotherapy approaches.

Genistein Sensitizes Human Cholangiocarcinoma Cell Lines to Be Susceptible to Natural Killer Cells.

Cholangiocarcinoma (CCA) is a lethal bile duct cancer, which has poor treatment outcomes due to its high resistance to chemotherapy and cancer recurrence. Activation of aberrant anti-apoptotic signaling pathway has been reported to be a mechanism of chemoresistance and immune escape of CCA. Therefore, reversal of anti-apoptotic signaling pathway represents a feasible approach to potentiate effective treatments, especially for CCA with high chemoresistance. In this study, we demonstrated the effects of genistein on reactivation of apoptosis cascade and increase the susceptibility of CCA cells to natural killer (NK-92) cells. Genistein at 50 and 100 µM significantly activated extrinsic apoptotic pathway in CCA cells (KKU055, KKU100, and KKU213A), which was evident by reduction of procaspase-8 and -3 expression. Pretreatment of CCA cells with genistein at 50 µM, but not NK-92 cells, significantly increased NK-92 cell killing ability over the untreated control, suggesting the ability of genistein to sensitize CCA cells. Interestingly, genistein treatment could greatly lower the expression of cFLIP, an anti-apoptotic protein involved in the immune escape pathway, in addition to upregulation of death receptors, Fas- and TRAIL-receptors, in CCA cells, which might be the underlying molecular mechanism of genistein to sensitize CCA to be susceptible to NK-92 cells. Taken together, this finding revealed the benefit of genistein as a sensitizer to enhance the efficiency of NK cell immunotherapy for CCA.

STING agonist cGAMP enhances anti-tumor activity of CAR-NK cells against pancreatic cancer.

Activation of the stimulator of interferon gene (STING)-mediated innate immune response has been suggested as a promising therapeutic strategy for cancers. However, the effects of STING agonist on natural killer (NK) cell-mediated anti-tumor responses in pancreatic cancer remains unknown. Herein, we evaluated the effects of a classical STING agonist cyclic GMP-AMP (cGAMP) on NK cells in pancreatic cancer. We found that cGAMP could directly activate NK cells and enhance the sensitivity of pancreatic cancer cells to NK cell cytotoxicity, suggesting that cGAMP may become a potential adjuvant for NK cell therapy. In addition, combination of CAR-NK-92 cells targeting mesothelin and cGAMP displayed greater antitumor efficacy by inhibiting tumor growth and prolonging survival of the mouse model of pancreatic cancer. These results suggest that the combination of a STING agonist and NK cells may become a novel immunotherapy strategy for pancreatic cancer.

CAR NK-92 cells targeting DLL3 kill effectively small cell lung cancer cells in vitro and in vivo.

Small cell lung cancer (SCLC) is characterized by a high relapse rate, drug tolerance, and limited treatment choices. Chimeric antigen receptor (CAR)-modified NK cells represent a promising immunotherapeutic modality for cancer treatment. However, their potential applications have not been explored in SCLC. Delta-like ligand 3 (DLL3) has been reported to be overexpressed in SCLC and may be a rational target for CAR NK immunotherapy. In this study, we developed DLL3-specific NK-92 cells and explored their potential in the treatment of SCLC. A coculture of DLL3+ SCLC cell lines with DLL3-CAR NK-92 cells exhibited significant in vitro cytotoxicity and cytokine production. DLL3-CAR NK-92 cells induced tumor regression in an H446-derived pulmonary metastasis tumor model under a good safety threshold. The potent antitumor activities of DLL3-CAR NK-92 cells were observed in subcutaneous tumor models of SCLC. Moreover, obvious tumor-infiltrated DLL3-CAR NK-92 cells were detected in DLL3+ SCLC xenografts. These findings indicate that DLL3-CAR NK-92 cells might be a potential strategy for the treatment of SCLC.

Bispecific killer cell engager with high affinity and specificity toward CD16a on NK cells for cancer immunotherapy.

Introduction: The Fc region of monoclonal antibodies (mAbs) interacts with the CD16a receptor on natural killer (NK) cells with "low affinity" and "low selectivity". This low affinity/selectivity interaction results in not only suboptimal anticancer activity but also induction of adverse effects. CD16a on NK cells binds to the antibody-coated cells, leading to antibody-dependent cell-mediated cytotoxicity (ADCC). Recent clinical data have shown that the increased binding affinity between mAb Fc region and CD16a receptor is responsible for significantly improved therapeutic outcomes. Therefore, the objective of this study was to develop a bispecific killer cell engager (BiKE) with high affinity and specificity/selectivity toward CD16a receptor for NK cell-based cancer immunotherapy. Methods: To engineer BiKE, a llama was immunized, then high binding anti-CD16a and anti-HER2 VHH clones were isolated using phage display. ELISA, flow cytometry, and biolayer interferometry (BLI) data showed that the isolated anti-CD16a VHH has high affinity (sub-nanomolar) toward CD16a antigen without cross-reactivity with CD16b-NA1 on neutrophils or CD32b on B cells. Similarly, the data showed that the isolated anti-HER2 VHH has high affinity/specificity toward HER2 antigen. Using a semi-flexible linker, anti-HER2 VHH was recombinantly fused with anti-CD16a VHH to create BiKE:HER2/CD16a. Then, the ability of BiKE:HER2/CD16a to activate NK cells to release cytokines and kill HER2+ cancer cells was measured. As effector cells, both high-affinity haNK92 (CD16+, V176) and low-affinity laNK92 (CD16+, F176) cells were used. Results and discussion: The data showed that the engineered BiKE:HER2/CD16a activates haNK92 and laNK92 cells to release cytokines much greater than best-in-class mAbs in the clinic. The cytotoxicity data also showed that the developed BiKE induces higher ADCC to both ovarian and breast cancer cells in comparison to Trazimera™ (trastuzumab). According to the BLI data, BiKE:HER2/CD16 recognizes a different epitope on CD16a antigen than IgG-based mAbs; thus, it provides the opportunity for not only monotherapy but also combination therapy with other antibody drugs such as checkpoint inhibitors and antibody-drug conjugates. Taken together, the data demonstrate the creation of a novel BiKE with high affinity and specificity toward CD16a on NK cells with the potential to elicit a superior therapeutic response in patients with HER2+ cancer than existing anti-HER2 mAbs.