Blockade of the TIGIT-CD155/CD112 axis enhances functionality of NK-92 but not cytokine-induced memory-like NK cells toward CD155-expressing acute myeloid leukemia.

TIGIT is an alternative checkpoint receptor (CR) whose inhibition promotes Graft-versus-Leukemia effects of NK cells. Given the significant immune-permissiveness of NK cells circulating in acute myeloid leukemia (AML) patients, we asked whether adoptive transfer of activated NK cells would benefit from additional TIGIT-blockade. Hence, we characterized cytokine-induced memory-like (CIML)-NK cells and NK cell lines for the expression of inhibitory CRs. In addition, we analyzed the transcription of CR ligands in AML patients (CCLE and Beat AML 2.0 cohort) in silico and evaluated the efficacy of CR blockade using in vitro cytotoxicity assays, CD69, CD107a and IFN-γ expression. Alternative but not classical CRs were abundantly expressed on healthy donor NK cells and even further upregulated on CIML-NK cells. In line with our finding that CD155, one important TIGIT-ligand, is reliably expressed on AMLs, we show improved killing of CD155+-AML blasts by NK-92 but interestingly not CIML-NK cells in the presence of TIGIT-blockade. Additionally, our in silico data (n = 671) show that poor prognosis AML patients rather displayed a CD86low CD112/CD155high phenotype, whereas patients with a better outcome rather exhibited a CD86high CD112/CD155low phenotype. Collectively, our data evidence that the complex CR ligand expression profile on AML blasts may be one explanation for the intrinsic NK cell exhaustion observed in AML patients which might be overcome with adoptive NK-92 transfer in combination with TIGIT-blockade.

Fatty acids promote the expansion of NK-92 cells in vitro by improving energy metabolism.

Natural killer-92 cells (NK-92 cells) need to be efficiently expanded by serum-free culture in vitro to meet clinical requirements. Fatty acids mainly provide substrates for energy production, which is of crucial importance to meet the energy demands of highly proliferating cells. This study optimized the medium (EM) for NK-92 cells by designing an experiment to expand cells efficiently. EM, an in-house designed chemically defined serum-free medium, was used as the basal medium. Fatty acids as additive ingredients were screened and optimized by the experimental design method. Three additives, arachidonic acid, myristic acid and palmitoleic acid, were screened; therefore, the optimized medium was named EM-FA. The total cell expansion of NK-92 cells in EM-FA was 72.61±11.95-fold on day 8, which was significantly higher than the 28.55±8.67-fold expansion in EM. To explore the mechanism by which fatty acids promote NK-92 cell expansion, the cell growth kinetics and metabolic characteristics in EM-FA were analyzed. The results showed that NK-92 cells in EM-FA were rapidly expanded while maintaining their cell phenotype and cytotoxicity and enhancing the oxygen consumption rate and mitochondrial function. Fatty acids promoted ATP production to elevate the energy flux for better cell expansion. This study developed an expansion strategy of NK-92 cells in vitro to facilitate their clinical application. KEY POINTS: • Arachidonic acid, myristic acid and palmitoleic acid in serum-free medium were optimized by experimental design to enable the rapid expansion of NK-92 cells in vitro. • Fatty acids upregulated oxidative phosphorylation levels and improved the energy metabolism of NK-92 cells.

Augmented anti-tumor activity of NK-92 cells expressing chimeric receptors of TGF-ßR II and NKG2D.

The capacity of natural killer (NK) cells to kill tumor cells without specific antigen recognition provides an advantage over T cells and makes them potential effectors for tumor immunotherapy. However, the efficacy of NK cell adoptive therapy can be limited by the immunosuppressive tumor microenvironment. Transforming growth factor-ß (TGF-ß) is a potent immunosuppressive cytokine that can suppress NK cell function. To convert the suppressive signal induced by TGF-ß to an activating signal, we genetically modified NK-92 cells to express a chimeric receptor with TGF-ß type II receptor extracellular and transmembrane domains and the intracellular domain of NK cell-activating receptor NKG2D (TN chimeric receptor). NK-92 cells expressing TN receptors were resistant to TGF-ß-induced suppressive signaling and did not down-regulate NKG2D. These modified NK-92 cells had higher killing capacity and interferon γ (IFN-γ) production against tumor cells compared with the control cells and their cytotoxicity could be further enhanced by TGF-ß. More interestingly, the NK-92 cells expressing TN receptors were better chemo-attracted to the tumor cells expressing TGF-ß. The presence of these modified NK-92 cells significantly inhibited the differentiation of human naïve CD4+ T cells to regulatory T cells. NK-92-TN cells could also inhibit tumor growth in vivo in a hepatocellular carcinoma xenograft tumor model. Therefore, TN chimeric receptors can be a novel strategy to augment anti-tumor efficacy in NK cell adoptive therapy.

Phase 1 clinical trial of adoptive immunotherapy using "off-the-shelf" activated natural killer cells in patients with refractory and relapsed acute myeloid leukemia.

BACKGROUND AIMS: Activated NK cells (aNK) generated by expansion of a human interleukin-2-dependent NK cell line (NK-92) were shown to mediate strong anti-leukemia activity. This phase 1 study evaluated feasibility, safety, and activity of aNK cells adoptively transferred to patients with refractory/relapsed acute myeloid leukemia (AML). In addition, effects of these aNK cells on the patient's immune system were evaluated. METHODS: Two cell-dose levels (1 × 109 cells/m2 and 3 × 109 cells/m2) were used. One treatment course consisted of two infusions of the same cell dose, each cell infusion delivered 24 h apart. The aNK cells were administered in the outpatient setting. RESULTS: Seven patients with refractory/relapsed AML were treated with a total of 20 aNK cell infusions. None of the 7 patients experienced dose-limiting toxicities during the aNK cell administration or during 21 days of the post-infusion observation period. No grade 3-4 toxicities (probable or definite) related to aNK cell infusions occurred. Activity was transient in 3 of 7 patients. No significant changes in the patient's lymphocyte counts, subsets frequency, phenotype or activity were observed post-infusion. Cell dose-dependent effects in the plasma levels of several cytokines were observed. DISCUSSION: The trial demonstrated the safety and feasibility of adoptive cell therapy with "off-the-shelf" aNK cells in patients with refractory/relapsed AML. These data provide the foundation for future combination immunotherapy trials and for the optimization of aNK cell based therapies in patients with AML.

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.

Microfluidics-assisted fabrication of natural killer cell-laden microgel enhances the therapeutic efficacy for tumor immunotherapy.

Recently, interest in cancer immunotherapy has increased over traditional anti-cancer therapies such as chemotherapy or targeted therapy. Natural killer (NK) cells are part of the immune cell family and essential to tumor immunotherapy as they detect and kill cancer cells. However, the disadvantage of NK cells is that cell culture is difficult. In this study, porous microgels have been fabricated using microfluidic channels to effectively culture NK cells. Microgel fabrication using microfluidics can be mass-produced in a short time and can be made in a uniform size. Microgels consist of photo cross-linkable polymers such as methacrylic gelatin (GelMa) and can be regulated via controlled GelMa concentrations. NK92 cell-laden three-dimensional (3D) microgels increase mRNA expression levels, NK92 cell proliferation, cytokine release, and anti-tumor efficacy, compared with two-dimensional (2D) cultures. In addition, the study confirms that 3D-cultured NK92 cells enhance anti-tumor effects compared with enhancement by 2D-cultured NK92 cells in the K562 leukemia mouse model. Microgels containing healthy NK cells are designed to completely degrade after 5 days allowing NK cells to be released to achieve cell-to-cell interaction with cancer cells. Overall, this microgel system provides a new cell culture platform for the effective culturing of NK cells and a new strategy for developing immune cell therapy.

Next-Generation CEA-CAR-NK-92 Cells against Solid Tumors: Overcoming Tumor Microenvironment Challenges in Colorectal Cancer.

Colorectal carcinoma (CRC) presents a formidable medical challenge, demanding innovative therapeutic strategies. Chimeric antigen receptor (CAR) natural killer (NK) cell therapy has emerged as a promising alternative to CAR T-cell therapy for cancer. A suitable tumor antigen target on CRC is carcinoembryonic antigen (CEA), given its widespread expression and role in tumorigenesis and metastasis. CEA is known to be prolifically shed from tumor cells in a soluble form, thus hindering CAR recognition of tumors and migration through the TME. We have developed a next-generation CAR construct exclusively targeting cell-associated CEA, incorporating a PD1-checkpoint inhibitor and a CCR4 chemokine receptor to enhance homing and infiltration of the CAR-NK-92 cell line through the TME, and which does not induce fratricidal killing of CAR-NK-92-cells. To evaluate this therapeutic approach, we harnessed intricate 3D multicellular tumor spheroid models (MCTS), which emulate key elements of the TME. Our results demonstrate the effective cytotoxicity of CEA-CAR-NK-92 cells against CRC in colorectal cell lines and MCTS models. Importantly, minimal off-target activity against non-cancerous cell lines underscores the precision of this therapy. Furthermore, the integration of the CCR4 migration receptor augments homing by recognizing target ligands, CCL17 and CCL22. Notably, our CAR design results in no significant trogocytosis-induced fratricide. In summary, the proposed CEA-targeting CAR-NK cell therapy could offer a promising solution for CRC treatment, combining precision and efficacy in a tailored approach.

Combined treatment with anti-PSMA CAR NK-92 cell and anti-PD-L1 monoclonal antibody enhances the antitumour efficacy against castration-resistant prostate cancer.

BACKGROUND: The chimeric antigen receptor NK-92 (CAR NK-92) cell targeting the prostate-specific membrane antigen (PSMA) has shown antitumour effects in castration-resistant prostate cancer (CRPC). However, the expression changes of programmed death ligand 1 (PD-L1) and its mechanisms on CAR NK-92 and CRPC cells and the effect of the anti-PD-L1 monoclonal antibody (mAb) on PD-L1 expressed on CAR NK-92 cells remain unknown. METHODS: Human dendritic cells and CD8+ T cells were acquired from blood samples of healthy donors and cocultured with C4-2 cells. Changes in PD-L1 expression were detected by flow cytometry. Differential gene expressions were investigated by RNA sequence analysis, while the regulation of PD-L1 molecular signaling was explored using western blotting. In vitro cytotoxicity was evaluated using the Cell Counting Kit-8 assay and the bioluminescent intensity (BLI) of green fluorescent protein-labelled C4-2 cells. CRPC growth in vivo was monitored using callipers and BLI in male NOD/SCID mice subcutaneously injected with C4-2 cells and treated intravenously with anti-PD-L1/PD-1 mAb, CAR NK-92 or cocultured CD8+ T cells. RESULTS: Significantly upregulated expression of PD-L1k was observed in cocultured C4-2 and CAR NK-92 cells. In addition, upregulation of PD-L1 expression was dependent on interferon-γ in C4-2 cells, while it was dependent on direct cell-to-cell interaction via the NK group 2 member D/ phosphatidylinositol 3-kinase/AKT pathway in CAR NK-92 cells. The anti-PD-L1 mAb directly acted on PD-L1 expressed on CAR NK-92 cells and augmented the cytotoxicity of CAR NK-92 cells against C4-2 and CRPC cells from one patient in vitro. Anti-PD-L1 mAb significantly enhanced the antitumour effect of CAR NK-92 cells against CRPC cells in vivo when compared to treatment with CAR NK-92 cells or combined with anti-PD-1 mAb in the absence or presence of cocultured CD8+ T cells. CONCLUSION: Combined treatment with CAR NK-92 and anti-PD-L1 mAb improved the antitumour efficacy against CRPC, which is of extraordinary translational value in the clinical treatment of CRPC.

Anti-PSMA CAR-engineered NK-92 Cells: An Off-the-shelf Cell Therapy for Prostate Cancer.

Prostate cancer (PCa) has become the most common cancer among males in Europe and the USA. Adoptive immunotherapy appears a promising strategy to control the advanced stages of the disease by specifically targeting the tumor, in particular through chimeric antigen receptor T (CAR-T) cell therapy. Despite the advancements of CAR-T technology in the treatment of hematological malignancies, solid tumors still represent a challenge. To overcome current limits, other cellular effectors than T lymphocytes are under study as possible candidates for CAR-engineered cancer immunotherapy. A novel approach involves the NK-92 cell line, which mediates strong cytotoxic responses against a variety of tumor cells but has no effect on non-malignant healthy counterparts. Here, we report a novel therapeutic approach against PCa based on engineering of NK-92 cells with a CAR recognizing the human prostate-specific membrane antigen (PSMA), which is overexpressed in prostatic neoplastic cells. More importantly, the potential utility of NK-92/CAR cells to treat PCa has not yet been explored. Upon CAR transduction, NK-92/CAR cells acquired high and specific lytic activity against PSMA-expressing prostate cancer cells in vitro, and also underwent degranulation and produced high levels of IFN-γ in response to antigen recognition. Lethal irradiation of the effectors, a safety measure requested for the clinical application of retargeted NK-92 cells, fully abrogated replication but did not impact on phenotype and short-term functionality. PSMA-specific recognition and antitumor activity were retained in vivo, as adoptive transfer of irradiated NK-92/CAR cells in prostate cancer-bearing mice restrained tumor growth and improved survival. Anti-PSMA CAR-modified NK-92 cells represent a universal, off-the-shelf, renewable, and cost-effective product endowed with relevant potentialities as a therapeutic approach for PCa immunotherapy.

Matrine inhibits the growth of natural killer/T-cell lymphoma cells by modulating CaMKIIγ-c-Myc signaling pathway.

BACKGROUND: C-Myc overexpression is associated with poor prognosis and aggressive progression of natural killer/T-cell lymphoma (NKTCL). Matrine, a main alkaloid of the traditional Chinese herb Sophora flavescens Ait, has been shown to inhibit cellular proliferation and induce apoptosis of various cancer cells. The present study investigated the effects and possible mechanisms of matrine inhibiting the growth of natural killer/T-cell lymphoma cells. METHODS: The effects of matrine on the proliferation, apoptosis and expression of apoptotic molecules, STAT3, LMP1, RUNX3, EZH2 and activation of CaMKIIγ/c-Myc pathway were examined in cultured NKTCL cell line NK92 cells. RESULTS: In cultured NK92 cells, matrine inhibited the proliferation in a dose and time dependent manner. The IC50 value of matrine was 1.71 mM for 72 h post exposure in NK92 cells. Matrine induced apoptosis with decreased Bcl-2 expression and the proteasome-dependent degradation of c-Myc protein in NK92 cells. c-Myc protein half-life in NK92 was reduced from 80.7 min to 33.4 min after matrine treatment, which meant the stability of c-Myc was decreased after matrine exposure. Furthermore, we found that matrine downregulated c-Myc phosphorylation at Ser62 together with the inhibition of CaMKIIγ, a key regulator of c-Myc protein in NKTCL. The downregulation of c-Myc transcription by matrine was mediated through LMP1 inhibition. We also observed that anti-proliferative activity of matrine was irrelevant to STAT3, RUNX3 and EZH2. CONCLUSIONS: The results of the present study indicated that matrine inhibits the growth of natural killer/T-cell lymphoma cells by modulating LMP1-c-Myc and CaMKIIγ-c-Myc signaling pathway.

Boosting Natural Killer Cell-Mediated Targeting of Sarcoma Through DNAM-1 and NKG2D.

Sarcomas are malignancies of mesenchymal origin that occur in bone and soft tissues. Many are chemo- and radiotherapy resistant, thus conventional treatments fail to increase overall survival. Natural Killer (NK) cells exert anti-tumor activity upon detection of a complex array of tumor ligands, but this has not been thoroughly explored in the context of sarcoma immunotherapy. In this study, we investigated the NK cell receptor/ligand immune profile of primary human sarcoma explants. Analysis of tumors from 32 sarcoma patients identified the proliferative marker PCNA and DNAM-1 ligands CD112 and/or CD155 as commonly expressed antigens that could be efficiently targeted by genetically modified (GM) NK cells. Despite the strong expression of CD112 and CD155 on sarcoma cells, characterization of freshly dissociated sarcomas revealed a general decrease in tumor-infiltrating NK cells compared to the periphery, suggesting a defect in the endogenous NK cell response. We also applied a functional screening approach to identify relevant NK cell receptor/ligand interactions that induce efficient anti-tumor responses using a panel NK-92 cell lines GM to over-express 12 different activating receptors. Using GM NK-92 cells against primary sarcoma explants (n = 12) revealed that DNAM-1 over-expression on NK-92 cells led to efficient degranulation against all tested explants (n = 12). Additionally, NKG2D over-expression showed enhanced responses against 10 out of 12 explants. These results show that DNAM-1+ or NKG2D+ GM NK-92 cells may be an efficient approach in targeting sarcomas. The degranulation capacity of GM NK-92 cell lines was also tested against various established tumor cell lines, including neuroblastoma, Schwannoma, melanoma, myeloma, leukemia, prostate, pancreatic, colon, and lung cancer. Enhanced degranulation of DNAM-1+ or NKG2D+ GM NK-92 cells was observed against the majority of tumor cell lines tested. In conclusion, DNAM-1 or NKG2D over-expression elicited a dynamic increase in NK cell degranulation against all sarcoma explants and cancer cell lines tested, including those that failed to induce a notable response in WT NK-92 cells. These results support the broad therapeutic potential of DNAM-1+ or NKG2D+ GM NK-92 cells and GM human NK cells for the treatment of sarcomas and other malignancies.