Natural Killer Cell Tumor Research Articles

A Pump-Free Microfluidic Co-Culture System for Investigating NK Cell-Tumor Spheroid Interactions in Flow Conditions.

Natural killer (NK) cells are pivotal in immunotherapy due to their potent tumor-targeting capabilities. However, accessible in vitro 3D dynamic models for evaluating Tumor Infiltrating Natural Killer Cells (TINKs) remain scarce. This study addresses this gap by developing a novel pump-free microfluidic chip to investigate the interactions between NK-92 cells and prostate DU 145 tumor spheroids. The platform facilitates the separation of free NKs and TINKs for subtype characterization. The design integrates multiple planes with a multi-layer paper scaffold to accommodate tumor spheroids, allowing NK-92 cells to traverse Matrigel-coated barriers that mimic the extracellular matrix. The dual-channel pump-free device enables unidirectional circulation of NK-92 cells, allowing analysis of tumor spheroid movement and NK-92 cell interactions under flow conditions. Results demonstrate continuous fluid circulation in the dual-channel device by rocking the platform at tilt angles of 21° and 15°. Tumor spheroids showed enhanced migration under flow conditions compared to static culture. Although spheroids did not recruit significantly more NK-92 cells under flow conditions, CD56 and CD16 receptor expression on IL-2-activated free NK-92 cells and tumor-infiltrating NK-92 cells matched in vivo patterns in dynamic cultures. These findings suggest that tumor cells and fluid dynamics significantly influence NK cell subtypes. This pump-free microfluidic platform is a functional tool for simulating and studying immune cell-tumor interactions, providing valuable insights into NK cell dynamics with tumor spheroids in physiologically relevant environments.

Engineered Platelet for In Situ Natural Killer Cell Activation to Inhibit Tumor Recurrence.

Natural killer (NK) cells offer profound advantages against tumor recurrence due to their unique immunological behavior. NK cell therapies associated with the antibody-dependent cell-mediated cytotoxicity (ADCC) effect have made remarkable progress while being limited by insufficient antibody binding and the exhausted state of NK cells in the postsurgical immunosuppressive microenvironment. Leveraging the adherence of PLT to tumor cells, we developed an exogenously implanted platelet (PLT)-based NK cell-driven system (PLT-IgG-IL15) to improve the identifiability of residual tumors with IgG antibody labeling for NK cells catching and engaging, which consequently restored the ADCC effect and promoted the recovery of their killing function. Furthermore, interleukin-15 (IL-15) participated in the augmentation of NK cell function. Collectively, PLT-IgG-IL15 served as an NK cell tumor cell engager as well as an NK cell charger, achieving a <40% recurrence rate in mouse tumor models.

Click Chemistry-Mediated Polymannose Surface-Engineering of Natural Killer Cells for Immunotherapy of Triple-Negative Breast Cancer.

Natural killer (NK) cells, serve as the frontline defense of the immune system, and are capable of surveilling and eliminating tumor cells. Their significance in tumor immunotherapy has garnered considerable attention in recent years. However, the absence of specific receptor-ligand interactions between NK cells and tumor cells hampers their selectivity, thereby limiting the therapeutic effectiveness of NK cell-based tumor immunotherapy. Herein, this work constructs polymannose-engineered NK (pM-NK) cells via metabolic glycoengineering and copper-free click chemistry. Polymannose containing dibenzocyclooctyne terminal groups (pM-DBCO) is synthesized and covalently modified on the surface of azido-labeled NK cells. Compared to the untreated NK cells, the interactions between pM-NK cells and MDA-MB-231 cells, a breast tumor cell line with overexpression of mannose receptors (MRs), are significantly increased, and lead to significantly enhanced killing efficacy. Consequently, intravenous administration of pM-NK cells will effectively inhibit the tumor growth and will prolong the survival of mice bearing MDA-MB-231 tumors. Thus, this work presents a novel strategy for tumor-targeting NK cell-based tumor immunotherapy.

InVivo PET Imaging of 89Zr-Labeled Natural Killer Cells and the Modulating Effects of a Therapeutic Antibody.

Natural killer (NK) cells can kill cancer cells via antibody-dependent cell-mediated cytotoxicity (ADCC): a tumor-associated IgG antibody binds to the Fcγ receptor CD16 on NK cells via the antibody Fc region and activates the cytotoxic functions of the NK cell. Here, we used PET imaging to assess NK cell migration to human epidermal growth factor receptor 2 (HER2)-positive HCC1954 breast tumors, examining the influence of HER2-targeted trastuzumab antibody treatment on NK cell tumor accumulation. Methods: Human NK cells from healthy donors were expanded ex vivo and labeled with [89Zr]Zr-oxine. In vitro experiments compared the phenotypic markers, viability, proliferation, migration, degranulation, and ADCC behaviors of both labeled (89Zr-NK) and unlabeled NK cells. Female mice bearing orthotopic human breast HCC1954 tumors were administered 89Zr-NK cells alongside trastuzumab treatment or a sham treatment and then scanned using PET/CT imaging over 7 d. Flow cytometry and γ-counting were used to analyze the presence of 89Zr-NK cells in liver and spleen tissues. Results: 89Zr cell radiolabeling yields measured 42.2% ± 8.0%. At an average specific activity of 16.7 ± 4.7 kBq/106 cells, 89Zr-NK cells retained phenotypic and functional characteristics including CD56 and CD16 expression, viability, migration, degranulation, and ADCC capabilities. In vivo PET/CT studies indicated predominant accumulation of 89Zr-NK cells in the liver and spleen. Ex vivo analyses of liver and spleen tissues indicated that the administered human 89Zr-NK cells retained their radioactivity invivo and that 89Zr did not transfer to cells of murine soft tissues, thus validating this 89Zr PET method for NK cell tracking. Notably, 89Zr-NK cells migrated to HER2-positive tumors, both with and without trastuzumab treatment. Trastuzumab treatment was associated with an increased 89Zr-NK cell signal at days 1 and 3 after injection. Conclusion: In vitro, 89Zr-NK cells maintained key cellular and cytotoxic functions. In vivo, 89Zr-NK cells trafficked to HER2-postive tumors, with trastuzumab treatment correlating with enhanced 89Zr-NK infiltration. This study demonstrates the feasibility of using PET to image 89Zr-NK cell infiltration into solid tumors.

The CNS relapse in T-cell lymphoma index predicts CNS relapse in patients with T- and NK-cell lymphomas

AbstractLittle is known about risk factors for central nervous system (CNS) relapse in mature T-cell and natural killer cell neoplasms (MTNKNs). We aimed to describe the clinical epidemiology of CNS relapse in patients with MTNKN and developed the CNS relapse In T-cell lymphoma Index (CITI) to predict patients at the highest risk of CNS relapse. We reviewed data from 135 patients with MTNKN and CNS relapse from 19 North American institutions. After exclusion of leukemic and most cutaneous forms of MTNKNs, patients were pooled with non-CNS relapse control patients from a single institution to create a CNS relapse–enriched training set. Using a complete case analysis (n = 182), including 91 with CNS relapse, we applied a least absolute shrinkage and selection operator Cox regression model to select weighted clinicopathologic variables for the CITI score, which we validated in an external cohort from the Swedish Lymphoma Registry (n = 566). CNS relapse was most frequently observed in patients with peripheral T-cell lymphoma, not otherwise specified (25%). Median time to CNS relapse and median overall survival after CNS relapse were 8.0 and 4.7 months, respectively. We calculated unique CITI risk scores for individual training set patients and stratified them into risk terciles. Validation set patients with low-risk (n = 158) and high-risk (n = 188) CITI scores had a 10-year cumulative risk of CNS relapse of 2.2% and 13.4%, respectively (hazard ratio, 5.24; 95% confidence interval, 1.50-18.26; P = .018). We developed an open-access web-based CITI calculator (https://redcap.link/citicalc) to provide an easy tool for clinical practice. The CITI score is a validated model to predict patients with MTNKN at the highest risk of developing CNS relapse.

Abstract 1334: Evaluation of NK cell combination therapies in mouse models of cancer

Abstract Adoptive immunotherapy with natural killer (NK) cells has emerged as a promising innovative approach against hematological malignancies and solid tumors. To enhance the functional aspects of NK cells and to achieve stronger, sustained anti-tumor responses, combination strategies are under development. They are focused on optimization of NK cell potency, persistence and tumor targeting using monoclonal antibodies, multi-specific NK cell engagers and immune modulators like cytokines. SAR445419 is a universal off-the-shelf allogeneic human NK cell (hNK) product made using Sanofi’s proprietary platform with potential broad application across hematologic and solid tumor indications. We evaluated the therapeutic potential of these NK cells in combination with internal assets and standard of care treatments in preclinical mouse models. The studies were performed in immunodeficient NOG mouse strains transgenic for hIL-15, supporting long-term engraftment of functional hNK cells. We demonstrated the therapeutic advantage of NK cell combination with an anti-CD20 monoclonal antibody (mAb) in a disseminated lymphoma model. We also reported prolonged lifespan of leukemia-bearing animals treated with NK cells and pegenzileukin, a PEGylated non-alpha rhIL-2 variant engineered to preferentially bind to the IL-2 βγ receptors and promote NK cell function. The anti-tumor response correlated with pegenzileukin-induced increase in NK cell numbers in the blood. A combination of CD38KO NK cells with an anti-CD38 mAb was also investigated in a model of multiple myeloma (MM). FcγR-KO immunodeficient mice were used in these studies to limit the confounding contribution of residual murine immune cells engaging with the Fc-competent therapeutic antibody and to specifically detect hNK-cell mediated activity. The comparison of CD38KO versus WT NK cell numbers in the blood of animals treated with an anti-CD38 mAb showed a significant decrease in CD38-expressing NK cell count that was mitigated by the genetic deletion of CD38. Obtained results suggest that CD38KO NK cells are resistant to anti-CD38 mAb-mediated fratricide and provide a rationale for the combination of CD38KO NK cells with anti-an CD38 mAb against MM. Overall, these preclinical in vivo studies have a pivotal role in the evaluation of NK cell combination therapies against cancer and reveal their therapeutic potential. Citation Format: Katarzyna Franciszkiewicz, Pauline Rettman, Beatrice Gauthier, Laurence Noblet, Celine Nicolazzi, Andre Kunert, Rob Igarashi, Loreley Calvet, Valeria Fantin, Sukhvinder Sidhu. Evaluation of NK cell combination therapies in mouse models of cancer [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 1334.

Abstract 5786: Epstein-Barr virus nuclear antigen 1 (EBNA1)-targeting probes stimulate interferon-β production by nasopharyngeal carcinoma (NPC) cells to enhance the natural killer cell-mediated cytotoxicity against NPC

Abstract Introduction: The prevailing histologic subtype of nasopharyngeal carcinoma (NPC) in the endemic regions is non-keratinizing carcinoma which shows almost consistent association with latent infection by Epstein-Barr virus (EBV). Previously our group developed a series of Epstein-Barr virus nuclear antigen 1 (EBNA1)-targeting probes, which can disrupt the EBV latency maintained by EBNA1, and such lytic induction could alert the immune cells. Methods: Natural killer (NK) cells of the innate immunity play a significant role in controlling EBV infection. In this study, we investigated whether our EBNA1 probes (ZRL5P4 &amp; L2P4) could activate NK cells to eliminate NPC. Results: Studies of clinical specimens indicated that tumor infiltration of the cytotoxic NK cell subset is a favorable prognostic marker for NPC patients. Furthermore, the cytotoxicity of circulating NK cells derived from NPC patients is equally well the cells from healthy individuals. If those patients’ NK cells can be attracted to infiltrate into the tumors, the tumor burden should be relieved. Indeed, the NPC xenograft with the ZRL5P4 treatment showed heavy mouse NK cell infiltration and tumor size reduction. The essential role of those mouse NK cells was demonstrated. Next, we showed that ZRL5P4 could induce the production of a soluble factor by NPC cells to stimulate the NK cell cytotoxicity. Such NK cell activity was verified in a metastasis NPC model. Mechanistically, the EBNA1 probe treatment could induce the expression of EBV early lytic gene Rta and EBNA1 itself. Both Rta and EBNA1 are known transcription factors to drive the expression of LMP1, the NF-κB signaling was then activated, which turned on the production of the well-known NK cell activation cytokine IFN-β. Conclusion: Taken together, our results demonstrated that our EBNA1 probes can mobilize NK cells to the tumor sites and activate their anti-tumor activity via the EBV reactivation and stimulation of IFN-β production. Citation Format: Luo Chen, Xiao Han Liu, Hei Tung Yuen, William C. Cho, Ka-Leung Wong, Hong Lok Lung. Epstein-Barr virus nuclear antigen 1 (EBNA1)-targeting probes stimulate interferon-β production by nasopharyngeal carcinoma (NPC) cells to enhance the natural killer cell-mediated cytotoxicity against NPC [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 5786.

Safety and efficacy of SNK01 (autologous natural killer cells) in combination with cytotoxic chemotherapy and/or cetuximab after failure of prior tyrosine kinase inhibitor in non-small cell lung cancer: non-clinical mouse model and phase I/IIa clinical study

BackgroundChoosing treatments for epidermal growth factor receptor (EGFR)-mutated non-small cell lung cancer (NSCLC) patients with osimertinib resistance is challenging. We evaluated the safety and efficacy of SNK01 (autologous natural killer...

Crosstalk Between NK Cell Receptors and Tumor Membrane Hsp70-Derived Peptide: A Combined Computational and Experimental Study.

Natural killer (NK) cells are central components of the innate immunity system against cancers. Since tumor cells have evolved a series of mechanisms to escape from NK cells, developing methods for increasing the NK cell antitumor activity is of utmost importance. It is previously shown that an ex vivo stimulation of patient-derived NK cells with interleukin (IL)-2 and Hsp70-derived peptide TKD (TKDNNLLGRFELSG, aa450-461) results in a significant upregulation of activating receptors including CD94 and CD69 which triggers exhausted NK cells to target and kill malignant solid tumors expressing membrane Hsp70 (mHsp70). Considering that TKD binding to an activating receptor is the initial step in the cytolytic signaling cascade of NK cells, herein this interaction is studied by molecular docking and molecular dynamics simulation computational modeling. The in silico results showed a crucial role of the heterodimeric receptor CD94/NKG2A and CD94/NKG2C in the TKD interaction with NK cells. Antibody blocking and CRISPR/Cas9-mediated knockout studies verified the key function of CD94 in the TKD stimulation and activation of NK cells which is characterized by an increased cytotoxic capacity against mHsp70 positive tumor cells via enhanced production and release of lytic granules and pro-inflammatory cytokines.

Master regulator: p53's pivotal role in steering NK-cell tumor patrol.

The p53 protein, encoded by TP53, is a tumor suppressor that plays a critical role in regulating apoptosis, cell cycle regulation, and angiogenesis in tumor cells via controlling various downstream signals. Natural killer (NK) cell-mediated immune surveillance is a vital self-defense mechanism against cancer and other diseases, with NK cell activity regulated by various mechanisms. Among these, p53 plays a significant role in immune regulation by maintaining the homeostasis and functionality of NK cells. It enhances the transcriptional activity of NK cell-activating ligands and downregulates inhibitory ligands to boost NK cell activation and tumor-killing efficacy. Additionally, p53 influences NK cell cytotoxicity by promoting apoptosis, autophagy, and ferroptosis in different tumor cells. p53 is involved in the regulation of NK cell activity and effector functions through multiple pathways. p53 also plays a pivotal role in the tumor microenvironment (TME), regulating the activity of NK cells. NK cells are critical components of the TME and are capable of directly killing tumor cells. And p53 mutates in numerous cancers, with the most common alteration being a missense mutation. These mutations are commonly associated with poor survival rates in patients with cancer. This review details p53's role in NK cell tumor immunosurveillance, summarizing how p53 enhances NK cell recognition and tumor destruction. We also explore the potential applications of p53 in tumor immunotherapy, discussing strategies for modulating p53 to enhance NK cell function and improve the efficacy of tumor immunotherapy, along with the associated challenges. Understanding the interaction between p53 and NK cells within the TME is crucial for advancing NK cell-based immunotherapy and developing p53-related novel therapeutics.

Chidamide As Maintenance in Peripheral T-Cell Lymphoma for Patients in Response after Induction Therapy: A Single Center Retrospective Study

Background Peripheral T-cell lymphoma (PTCL) is a set of heterogeneous mature T- and natural killer cell neoplasms, most of which are associated with poor prognosis. PTCL patients usually experience frequent relapses. To improve the poor clinical outcomes of PTCL, novel agents that target various pathways have been studied and developed. Histone deacetylase (HDAC) inhibitors are among the most significant improvements made in recent years. Chidamide, a novel benzamide class of HDAC inhibitor, showed significant efficacy in relapsed/refractory PTCL patients in previous studies. But the role chidamide of as a maintenance therapy in PTCL patients after first-line therapy is still unknown. Methods In this retrospective, single-center, single-arm study, a total of 342 newly diagnosed PTCL patients were reviewed in the First Affiliated Hospital, Zhejiang University School of Medicine (Zhejiang, China) from August 2014 to May 2022. Patients with natural killer/T-cell lymphoma, mycosis fungoides were not enrolled. After excluding patients with insufficient clinical data, with other concurrent tumor, received other anti-tumor agents during maintenance therapy, or underwent sequential transplantation, there were 35 PTCL patients achieved objective response after first-line therapy, and received chidamide as maintenance therapy. The primary endpoint of this study was progress-free survival (PFS), overall survival (OS), and adverse events (AEs). Response and progression data were evaluated according to the 2016 Lugano Classification lymphoma response criteria. Adverse events were evaluated according to the National Cancer Institute Common Terminology Criteria for AEs (NCI-CTCAE) version 4.0. Survival outcomes were assessed with Kaplan-Meier method. Results 35 PTCL patients were enrolled, including angioimmunoblastic T-cell lymphoma (AITL) (13, 37.1%), anaplastic large-cell lymphoma (ALCL) (2, 5.7%), PTCL-NOS (20, 57.1%). There were 22 (62.9%) males and 13 (37.1%) females, 16 (45.7%) patients were over 65 years old, 32 patients (91.4%) had clinical stage III-IV, 9 patients (25.7%) had B symptoms, 8 patients (22.9%) had ECOG score ≥2, 15 patients (42.9%) with IPI score ≥3-5, 18 (51.4%) achieved complete remission (CR) and 17 (48.6%) achieved partial remission (PR) after induction chemotherapy. The median follow-up time was 35.2 months (range 13.0 to 107.7). The median duration of chidamide maintenance therapy was 17.7 months (range 2.4 to 98.0). 22 (62.9%) patients received 20mg chidamide twice a week and 13 (37.1%) used 30mg twice a week . The median PFS and median OS were not reached. The 3-year PFS and OS were 64.9% and 89.2%. Patients who achieved CR after induction chemotherapy had a better prognosis trend. Among the 18 patients who received maintenance therapy after CR, there was no death and the 3-year PFS was 76.4%. Patients with B symptoms had a worse prognosis (P&amp;lt;0.05), the median PFS was 22.5 months, the median OS was not reached, and the 3-year overall survival rate was 75%. The 3-year PFS and OS of the remaining 26 patients without B symptoms were 78.5% and 94.1%, respectively. The most common grade 3-4 AEs were hematological toxicities, including neutropenia(20.0%), anemia(8.6%), thrombocytopenia(5.7%), mainly in 30mg group, as detailed in Table 1. Adverse events usually occurred during the first 6 weeks, most of with were manageable by supportive care and dose modification, none of the adverse reactions leaded to death. There was no statistical difference in PFS and OS between the 30mg and 20mg groups under current sample size. Conclusions Chidamide maintenance is effective and may prolong the PFS and OS for PTCL patients with manageable safety profile. It is an appropriate therapeutic option as up-front consolidation therapy for PTCL patients with complete or partial response after first-line therapy. Larger-sample, prospective, multicenter cohort study is still needed to further confirm the conclusion.

Analysis of Treatment Outcome in Large Granular Lymphocytic Leukemia: A Retrospective Study from India

Large granular lymphocytic (LGL) leukemia is a rare and indolent lymphoproliferative disorder that belongs to mature T and natural killer (NK) cell neoplasms, as per the World Health Organization classification. This article assesses the response to immunosuppressive therapy. In this retrospective study, various cases of chronic lymphoproliferative disorders (CLPDs) evaluated and treated in two tertiary care settings were screened and taken for subanalyses. After fulfilling the criteria for LGL leukemia, cases were further assessed for presenting features and response to treatment. Statistical analysis was performed using the Statistical Package for the Social Sciences, version 23. Out of 384 cases of CLPDs analyzed, 14 cases of LGL leukemia were identified (3.64%) and subjected to further analysis. There were six males (42.85%) and eight females (57.14%) (M: F = 1:1.33) with an age group ranging from 42 to 82 years. Thirteen cases (92.85%) were T-LGL type, and one case belonged to NK-LGL type (7.14%). Anemia was the most common presentation (92.85% of cases), followed by lymphocytosis (85.71% of cases) and neutropenia (78.57% of cases). Four patients (28.57%) presented with thrombocytopenia, and two patients presented with pancytopenia (14.28%). Splenomegaly was seen in two patients (14.28%), and lymphadenopathy in one patient (7.14%). One patient (7.14%) had rheumatoid arthritis, and one (7.14%) had recurrent chest infections. Out of the 14 cases, 5 (35.71%) attained complete remission, 5 (35.71%) attained partial remission, 2 patients were resistant to first-line treatment, and 2 patients were closely followed with observation only. The overall treatment response rate was 83.33%. This study highlights the excellent response rates with immunosuppressive therapy in a rare lymphoproliferative disorder.

NK cells in peripheral blood carry trogocytosed tumor antigens from solid cancer cells.

The innate immune lymphocyte lineage natural killer (NK) cell infiltrates tumor environment where it can recognize and eliminate tumor cells. NK cell tumor infiltration is linked to patient prognosis. However, it is unknown if some of these antitumor NK cells leave the tumor environment. In blood-borne cancers, NK cells that have interacted with leukemic cells are recognized by the co-expression of two CD45 isoforms (CD45RARO cells) and/or the plasma membrane presence of tumor antigens (Ag), which NK cells acquire by trogocytosis. We evaluated solid tumor Ag uptake by trogocytosis on NK cells by performing co-cultures in vitro. We analyzed NK population subsets by unsupervised dimensional reduction techniques in blood samples from breast tumor (BC) patients and healthy donors (HD). We confirmed that NK cells perform trogocytosis from solid cancer cells in vitro. The extent of trogocytosis depends on the target cell and the antigen, but not on the amount of Ag expressed by the target cell or the sensitivity to NK cell killing. We identified by FlowSOM (Self-Organizing Maps) several NK cell clusters differentially abundant between BC patients and HD, including anti-tumor NK subsets with phenotype CD45RARO+CD107a+. These analyses showed that bona-fide NK cells that have degranulated were increased in patients and, additionally, these NK cells exhibit trogocytosis of solid tumor Ag on their surface. However, the frequency of NK cells that have trogocytosed is very low and much lower than that found in hematological cancer patients, suggesting that the number of NK cells that exit the tumor environment is scarce. To our knowledge, this is the first report describing the presence of solid tumor markers on circulating NK subsets from breast tumor patients. This NK cell immune profiling could lead to generate novel strategies to complement established therapies for BC patients or to the use of peripheral blood NK cells in the theranostic of solid cancer patients after treatment.

Abstract 2902: Off-the-Shelf CAR-NK cells engineered to express calibrated release IL-15 exhibit enhanced persistence and anti-tumor activities

Abstract Chimeric antigen receptor (CAR) natural killer (NK) cell therapy is an attractive immunotherapy strategy due to potential for increased anti-tumor activity and favorable safety. Allogeneic donor derived NK cells have shown promising clinical outcomes in both hematological and solid tumor malignancies but have a short lifespan in the absence of cytokine support. Interleukin (IL)-15 is a pleiotropic cytokine that promotes the survival, proliferation, and cytotoxicity of NK cells. In this study, we engineered human peripheral blood-derived NK cells to co-express a CAR and calibrated release (cr) IL-15 and studied how crIL-15 affected CAR-NK cell functions. Senti Bio has designed the calibrated release technology to simultaneously produce membrane bound and secreted cytokines, such as IL-15 and IL-12, to provide autocrine and paracrine activity. We further validated crIL-15 activity in vitro and demonstrated enhanced NK cell persistence, tumor killing and autocrine activity in CAR NK cells as well as paracrine stimulation of neighboring T cells and NK cells via pSTAT5 activation. Additionally, we examined in vivo persistence and biodistribution of NK cells engineered with crIL-15 and a nanoluc reporter. crIL-15 NK cells were intravenously (i.v.) injected into NSG mice. These NK cells were detected in the lung, femur, spleen, liver and stomach 1 day post injection by bioluminescence imaging and polymerase chain reaction (PCR) and lasted up to 20 days in the lung. In addition, crIL-15 increased CAR-NK cells persistence in a dose-dependent manner compared to unengineered NK cells. Injection of higher CAR-NK cell numbers (15× 106 vs 7.5× 106 CAR+ cells per mouse) or multiple NK dosages (1 vs 3 doses) both contributed to prolonged persistence. Finally, we studied how crIL-15 affected CAR-NK cell anti-tumor function in acute myeloid leukemia (AML) xenograft models. MV4-11-Fluc AML tumor cells were co-injected with engineered NK cells expressing a FLT3 and/or CD33 bivalent activating CAR, a EMCN inhibitory CAR and crIL-15 into NSG-Tg (Hu-IL15) mice via i.v. injection. Our data showed that these CAR-NK cells significantly reduced MV4-11 tumor burden and prolonged mouse survival compared to unengineered NK cells. The improved anti-tumor functions correlated with IL-15 levels produced by the CAR-NK cells. In conclusion, our results demonstrated that crIL-15 can improve persistence and anti-tumor activity of CAR-NK cells. Citation Format: Chen-Ting Lee, Michelle Hung, Andrew Banicki, Wenqi Song, Niran Almudhfar, Deepika Kaveri, Priscilla Wong, Lawrence Naitmazi, Marcela Guzman, Alice Lam, Gozde Yucel, Timothy Lu, Alba G. Junca, Brian Garrison, Philip Lee. Off-the-Shelf CAR-NK cells engineered to express calibrated release IL-15 exhibit enhanced persistence and anti-tumor activities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2902.

Escape from NK cell tumor surveillance by NGFR-induced lipid remodeling in melanoma.

Metastatic disease is a major cause of death for patients with melanoma. Melanoma cells can become metastatic not only due to cell-intrinsic plasticity but also due to cancer-induced protumorigenic remodeling of the immune microenvironment. Here, we report that innate immune surveillance by natural killer (NK) cells is bypassed by human melanoma cells expressing the stem cell marker NGFR. Using in vitro and in vivo cytotoxic assays, we show that NGFR protects melanoma cells from NK cell-mediated killing and, furthermore, boosts metastasis formation in a mouse model with adoptively transferred human NK cells. Mechanistically, NGFR leads to down-regulation of NK cell activating ligands and simultaneous up-regulation of the fatty acid stearoyl-coenzyme A desaturase (SCD) in melanoma cells. Notably, pharmacological and small interfering RNA-mediated inhibition of SCD reverted NGFR-induced NK cell evasion in vitro and in vivo. Hence, NGFR orchestrates immune control antagonizing pathways to protect melanoma cells from NK cell clearance, which ultimately favors metastatic disease.

Targeting immune checkpoints: how to use natural killer cells for fighting against solid tumors.

Natural killer (NK) cells are unique innate immune cells that mediate anti-viral and anti-tumor responses. Thus, they might hold great potential for cancer immunotherapy. NK cell adoptive immunotherapy in humans has shown modest efficacy. In particular, it has failed to demonstrate therapeutic efficiency in the treatment of solid tumors, possibly due in part to the immunosuppressive tumor microenvironment (TME), which reduces NK cell immunotherapy's efficiencies. It is known that immune checkpoints play a prominent role in creating an immunosuppressive TME, leading to NK cell exhaustion and tumor immune escape. Therefore, NK cells must be reversed from their dysfunctional status and increased in their effector roles in order to improve the efficiency of cancer immunotherapy. Blockade of immune checkpoints can not only rescue NK cells from exhaustion but also augment their robust anti-tumor activity. In this review, we discussed immune checkpoint blockade strategies with a focus on chimeric antigen receptor (CAR)-NK cells to redirect NK cells to cancer cells in the treatment of solid tumors.

Metformin increases natural killer cell functions in head and neck squamous cell carcinoma through CXCL1 inhibition

BackgroundMetformin slows tumor growth and progression in vitro, and in combination with chemoradiotherapy, resulted in high overall survival in patients with head and neck cancer squamous cell carcinoma (HNSCC) in...

Multi-spectral immunofluorescence evaluation of the myeloid, T cell, and natural killer cell tumor immune microenvironment in chordoma may guide immunotherapeutic strategies.

Chordoma is a rare, invasive, and devastating bone malignancy of residual notochord tissue that arises at the skull base, sacrum, or spine. In order to maximize immunotherapeutic approaches as a potential treatment strategy in chordoma it is important to fully characterize the tumor immune microenvironment (TIME). Multispectral immunofluorescence (MIF) allows for comprehensive evaluation of tumor compartments, molecular co-expression, and immune cell spatial relationships. Here we implement MIF to define the myeloid, T cell, and natural killer (NK) cell compartments in an effort to guide rational design of immunotherapeutic strategies for chordoma. Chordoma tumor tissue from 57 patients was evaluated using MIF. Three panels were validated to assess myeloid cell, T cell, and NK cell populations. Slides were stained using an automated system and HALO software objective analysis was utilized for quantitative immune cell density and spatial comparisons between tumor and stroma compartments. Chordoma TIME analysis revealed macrophage infiltration of the tumor parenchyma at a significantly higher density than stroma. In contrast, helper T cells, cytotoxic T cells, and T regulatory cells were significantly more abundant in stroma versus tumor. T cell compartment infiltration more commonly demonstrated a tumor parenchymal exclusion pattern, most markedly among cytotoxic T cells. NK cells were sparsely found within the chordoma TIME and few were in an activated state. No immune composition differences were seen in chordomas originating from diverse anatomic sites or between those resected at primary versus advanced disease stage. This is the first comprehensive evaluation of the chordoma TIME including myeloid, T cell, and NK cell appraisal using MIF. Our findings demonstrate that myeloid cells significantly infiltrate chordoma tumor parenchyma while T cells tend to be tumor parenchymal excluded with high stromal infiltration. On average, myeloid cells are found nearer to target tumor cells than T cells, potentially resulting in restriction of T effector cell function. This study suggests that future immunotherapy combinations for chordoma should be aimed at decreasing myeloid cell suppressive function while enhancing cytotoxic T cell and NK cell killing.

Targeting myeloid suppressive cells revives cytotoxic anti-tumor responses in pancreatic cancer

SummaryImmunotherapy for cancer that aims to promote T cell anti-tumor activity has changed current clinical practice, where some previously lethal cancers have now become treatable. However, clinical trials with low response rates have been disappointing for pancreatic ductal adenocarcinoma (PDAC). One suggested explanation is the accumulation of dominantly immunosuppressive tumor-associated macrophages and myeloid-derived suppressor cells in the tumor microenvironment (TME). Using retrospectively collected tumor specimens and transcriptomic data from PDAC, we demonstrate that expression of the scavenger receptor MARCO correlates with poor prognosis and a lymphocyte-excluding tumor phenotype. PDAC cell lines produce IL-10 and induce high expression of MARCO in myeloid cells, and this was further enhanced during hypoxic conditions. These myeloid cells suppressed effector T and natural killer (NK) cells and blocked NK cell tumor infiltration and tumor killing in a PDAC 3D-spheroid model. Anti-human MARCO (anti-hMARCO) antibody targeting triggered the repolarization of tumor-associated macrophages and activated the inflammasome machinery, resulting in IL-18 production. This in turn enhanced T cell and NK cell functions. The targeting of MARCO thus remodels the TME and represents a rational approach to make immunotherapy more efficient in PDAC patients.

TEM8 Tri-specific Killer Engager binds both tumor and tumor stroma to specifically engage natural killer cell anti-tumor activity

BackgroundThe tumor microenvironment contains stromal cells, including endothelial cells and fibroblasts, that aid tumor growth and impair immune cell function. Many solid tumors remain difficult to cure because of tumor-promoting...