Natural Killer Target Cells Research Articles

Dual-responsive natural killer cell nano-engagers for cancer immunotherapy via inhibiting IL-6/JAK2/STAT3 pathway

Natural killer (NK) cell-based immunotherapy is a promising approach for treating solid tumors and hematological malignancies. However, its therapeutic efficacy is limited by poor tumor infiltration and suppressive tumor microenvironment. Here, we introduce a rapid, simple, and efficient solution: bovine serum albumin-stabilized ultra-small (4 nm) NK cell nano-engagers, termed Bi2Se3@BSA nanodots (Nds). These Nds, responsive to high H2O2 levels in tumor cell environments, enhance near-infrared (NIR)-II photothermic immunotherapy. In vitro and in vivo experiments demonstrate their effectiveness in activating photothermal therapy (PTT)-induced immunogenic cell death (ICD), initiating immune cascade reactions, and stimulating immune response activation. By upregulating NKG2D, RAE-1, and secreting cytokines such as IFN-γ and TNF-α, targeted NK cell infiltration is achieved, reversing the immunosuppressive tumor microenvironment and optimizing NK cell cytotoxicity. Transcriptomic analysis confirms the association of immune response activation with the IL-6/JAK2/STAT3 signaling pathway. Notably, combining these nano-engagers with immune checkpoint inhibitors (aPD-1) significantly enhances therapeutic efficacy, facilitating tumor eradication and reducing recurrence risk. Taken together, NK cell nano-engagers inhibit the IL-6/JAK2/STAT3 signaling pathway, target NK cell activation, stimulate immune functions, and improve the immunosuppressive microenvironment. This study presents an alternative strategy for NK cell-based immunotherapy and highlights this promising tool for cancer immunotherapy.

Circumventing resistance within the Ewing sarcoma microenvironment by combinatorial innate immunotherapy

BackgroundPediatric patients with recurrent/metastatic Ewing sarcoma (ES) have a dismal 5-year survival. Novel therapeutic approaches are desperately needed. Natural killer (NK) cell number and function are low in ES patient...

PH‐Sensitive Magnetic Nanoparticle‐Mediated Natural‐Killer‐Cell‐Based Microrobots for Dual‐Targeted Delivery and Induction of Pro‐Inflammatory Macrophage Polarization

Adoptive cell therapy using natural killer (NK) cells emerges as the next‐generation cancer immunotherapy. Based on the intrinsic capability of direct cancer cell necrosis without prior education, a chemotherapeutic, free‐drug delivery, NK‐cell‐based platform can be developed. However, weak targeting of NK cells to solid tumors is observed. In addition, to boost the tumor‐killing efficiency, another immune‐regulating function should be added to the cells. Herein, a NK‐cell‐based hybrid system (NK‐Robot) conjugated with magnetic nanoparticles (MNPs) that provides 1) efficient guidance of NK cells and 2) targeted delivery of MNPs in situ is demonstrated, taking advantage of a newly designed, stimuli‐responsive polymeric linker. Thus, NK cells are allowed to target tumors under magnetic control conditions, and the liberated MNPs effectively reeducate the tumor‐associated macrophages (M2) to antitumor macrophages (M1), beneficial for the immunotherapy of tumors. In vivo experiments on BALB/c nude mice further support the finding that NK‐Robots effectively inhibit tumor growth.

CD94+ natural killer cells potentiate pulmonary ischaemia-reperfusion injury.

Pulmonary ischaemia-reperfusion injury (IRI) is a major contributor to poor lung transplant outcomes. We recently demonstrated a central role of airway-centred natural killer (NK) cells in mediating IRI; however, there are no existing effective therapies for directly targeting NK cells in humans. We hypothesised that a depleting anti-CD94 monoclonal antibody (mAb) would provide therapeutic benefit in mouse and human models of IRI based on high levels of KLRD1 (CD94) transcripts in bronchoalveolar lavage samples from lung transplant patients. We found that CD94 is highly expressed on mouse and human NK cells, with increased expression during IRI. Anti-mouse and anti-human mAbs against CD94 showed effective NK cell depletion in mouse and human models and blunted lung damage and airway epithelial killing, respectively. In two different allogeneic orthotopic lung transplant mouse models, anti-CD94 treatment during induction reduced early lung injury and chronic inflammation relative to control therapies. Anti-CD94 did not increase donor antigen-presenting cells that could alter long-term graft acceptance. Lung transplant induction regimens incorporating anti-CD94 treatment may safely improve early clinical outcomes.

KLF2 determines the susceptibility of T cells to immunoregulatory NK cells.

Natural killer (NK) cells suppress cellular and humoral immune responses via killing of T cells, resulting in diminished vaccine responses in mice and humans. Efforts to overcome this roadblock and achieve optimal immunity require an improved understanding of the molecular mediators facilitating NK cell-targeting of discrete subsets of CD4 T cells. We employed single-cell forensic victimology and CRISPR-Cas9 editing to delineate a transcriptional program uniquely responsible for the susceptibility of a subpopulation of CD4 T cells to perforin-dependent immunoregulation by NK cells. The unique vulnerability of these CD4 T cells relative to other subsets of CD4 T cells was not associated with a pattern of NK-cell-receptor ligand expression that would favor activation of NK cells. Instead, susceptible CD4 T cells were skewed toward follicular helper T cell (Tfh) differentiation and exhibited intermediate expression of Klf2 and a related suite of KLF2-target genes (e.g. S1pr1) involved in cell migration and spatial positioning. NK-cell dependent suppression of the subset of Tfh exhibiting intermediate expression of KLF2 and S1PR1 was confirmed with single-cell proteomics. CRISPR targeting of KLF2 in CD4 T cells prevented suppression by NK cells. Thus, KLF2 regulation of spatial positioning of T cells is a key determinant of NK-cell immunoregulatory function and a possible target for strategies to enhance vaccine efficacy.

The nuclear export protein XPO1 provides a peptide ligand for natural killer cells.

XPO1 (Exportin-1/CRM1) is a nuclear export protein that is frequently overexpressed in cancer and functions as a driver of oncogenesis. Currently small molecules that target XPO1 are being used in the clinic as anticancer agents. We identify XPO1 as a target for natural killer (NK) cells. Using immunopeptidomics, we have identified a peptide derived from XPO1 that can be recognized by the activating NK cell receptor KIR2DS2 in the context of human leukocyte antigen-C. The peptide can be endogenously processed and presented to activate NK cells specifically through this receptor. Although high XPO1 expression in cancer is commonly associated with a poor prognosis, we show that the outcome of specific cancers, such as hepatocellular carcinoma, can be substantially improved if there is concomitant evidence of NK cell infiltration. We thus identify XPO1 as a bona fide tumor antigen recognized by NK cells that offers an opportunity for a personalized approach to NK cell therapy for solid tumors.

Apoptotic Receptors and CD107a Expression by NK Cells in an Interaction Model with Trophoblast Cells.

Natural killer cells (NK cells) exert cytotoxicity towards target cells in several ways, including the expression of apoptosis-mediating ligands (TRAIL, FasL). In addition, NK cells themselves may be susceptible to apoptosis due to the expression of TRAIL receptors. These receptors include TRAIL-R1 (DR4), TRAIL-R2 (DR5), capable of inducing apoptosis, and TRAIL-R3 (DcR1), TRAIL-R4 (DcR2), the so-called "decoy receptors", which lack an intracellular domain initiating activation of caspases. Of particular interest is the interaction of uterine NK cells with cells of fetal origin, trophoblasts, which are potential targets for natural killer cells to carry out cytotoxicity. The aim of this work was to evaluate the expression of proapoptotic receptors and their ligands as well as CD107a expression by NK cells in a model of interaction with trophoblast cells. To evaluate NK cells, we used cells of the NK-92 line; cells of the JEG-3 line were used as target cells. The cytokines IL-1β, IL-15, IL-18, TNFα, IL-10, TGFβ and conditioned media (CM) of the first and third trimester chorionic villi explants were used as inducers. We established that cytokines changed the expression of apoptotic receptors by NK cells: in the presence of TNFα, the amount and intensity of Fas expression increased, while in the presence of TGFβ, the amount and intensity of expression of the DR5 receptor decreased. Soluble chorionic villi factors alter the expression of TRAIL and FasL by NK-92 cells, which can reflect the suppression of the TRAIL-dependent mechanism of apoptosis in the first trimester and stimulating the Fas-dependent mechanism in the third trimester. In the presence of trophoblast cells, the expression of TRAIL and DcR1 by NK cells was reduced compared to intact cells, indicating an inhibitory effect of trophoblast cells on NK cell cytotoxicity. In the presence of chorionic villi CM and trophoblast cells, a reduced number of NK-92 cells expressing DR4 and DR5 was found. Therefore, soluble factors secreted by chorionic villi cells regulate the resistance of NK cells to death by binding TRAIL, likely maintaining their activity at a certain level in case of contact with trophoblast cells.

Abstract IA007: Tumor HLA-E expression and cytomegalovirus infection modulate NK cell activity in human bladder cancer

Abstract The intersection of viral infections and cancer therapy presents a complex landscape for immunotherapy strategies, particularly in solid tumors, where treatments such as systemic chemotherapy and targeted radiation are standard. Largely unexplored in solid tumors are the effects of human herpes virus-5 (HHV-5) also known as HCMV that imprints both lifelong effects on natural killer (NK) cells as well progressive effects on T cells with aging. HCMV is an extremely successful virus as it rarely kills the host outside of settings of compromised immunity, e.g. transplantation and advanced HIV-1 infection. HCMV is not considered a comorbidity in patients with solid tumors. Our group has identified reactivated cytomegalovirus (HCMV), found in bladder tissues post treatment, including infection of monocytes, fibroblasts, and even tumor cells, suggesting a viral influence that extends beyond traditional perspectives on cancer treatment resistance and response. We have observed spread of infection in both settings of BCG-treated non-muscle-invasive bladder cancer (NMIBC) as well as MIBC treated with gemcitabine and cisplatin chemotherapy. HCMV infection leads to the development of “adaptive” NK cells that express the activating isoform of NKG2A: NKG2C, which are likely expanded in response to HCMV reactivation and have established functions for controlling virus reactivation. Our central hypothesis is that certain treatments may reactivate HCMV, potentially greatly altering the biology within the tumor microenvironment (TME) and providing a framework to exploit NK cells for immunotherapy strategies. In a cohort of NMIBC patients (N=90) receiving BCG therapy at Mount Sinai, we profiled patients’ serum for HCMV-IgG (N=47 HCMV seropositive; N=43 HCMV seronegative) and observed a significantly improved recurrence-free survival in HCMV seropositive patients (HR=0.58, p=0.03). Further, HCMV-seropositive patients with circulating NKG2C+ adaptive NK cells experienced a near perfect response to BCG therapy (p=0.0002). Strikingly, in situ imaging and RNAscope analyses of patient tumors before and after BCG therapy revealed strong presence of active viremia in tumors. Further, analysis of on-treatment bladder biopsies after tumor resection demonstrated the presence of adaptive NK cells in HCMV+ bladder tissues, but only after exposure to BCG therapy and tissue-derived adaptive NK cells expressed high levels of chemokine receptors, CXCR3 and CXCR4 as well as tissue-residency markers, suggesting a mechanisms for trafficking to the bladder and taking up residence for subsequent antitumor control against recurrence. Impact and Translation: Our findings suggest that adaptive NK cells mediate response to immunotherapies for solid tumors as well as provide preclinical models for adoptive transfer therapies targeting NK cells. Additionally, if HCMV reactivation is widespread across solid tissues in response to systemic therapies, it may prove a critical comorbidity to consider in design of treatment strategies and in clinical trials. Citation Format: Amir Horowitz. Tumor HLA-E expression and cytomegalovirus infection modulate NK cell activity in human bladder cancer [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr IA007.

Abstract 3603: NKX101, an allogeneic off-the-shelf CAR NK cell therapy targeting NKG2D-Ls, has potent anti-leukemic activity alone or in combination with Ara-C

Abstract Introduction: NKG2D-Ligands (NKG2D-Ls) are a family of immunomodulatory proteins that are upregulated on damaged or transformed cells but have limited expression on normal tissues. Natural killer (NK) cells target and eliminate NKG2D-L+ cells through NKG2D receptor binding and subsequent activation. NKX101 is an allogeneic, off-the-shelf healthy donor-derived chimeric antigen receptor (CAR) NK cell therapy candidate engineered to express an NKG2D-L targeting CAR and membrane-bound IL-15 for extended persistence. In a clinical trial, NKX101 treatment combined with modified lymphodepletion incorporating cytarabine (Ara-C) has shown promise for the treatment of relapsed/refractory (r/r) AML, including generating complete responses (CR) with MRD negativity (MRD−). To support the rationale of NKX101 combined with modified lymphodepletion, we developed assays to (i) investigate the cytotoxic activity of NKX101 against individual NKG2D-Ls, (ii) determine the effect of Ara-C pre-treatment on NKG2D-L expression on AML cell lines, and (iii) evaluate the combined anti-leukemic activity of NKX101 and Ara-C. Methods: NKX101 cells were generated from peripheral blood leukopaks from healthy donors. CHO-K1 cells were engineered to stably express individual NKG2D-Ls or transduced with empty vector controls and were combined with NKX101 cells for cytotoxicity experiments. AML cell lines were treated with increasing levels of Ara-C and evaluated for NKG2D-L expression by flow cytometry. To measure NKX101 cytotoxicity against AML cell lines, NucRed™-labeled target cells were pre-treated with Ara-C using IC50 and IC90 concentrations and co-cultured with NKX101. Specific cell killing of target cells was measured using an Incucyte® instrument for five days. Assessment of the contribution of each agent was determined using a dose-response matrix of increasing concentrations of NKX101 and Ara-C to generate specific cytotoxicity values. Combination effects were analyzed using the SynergyFinder Plus web application. Results: Expression of a single NKG2D-L was sufficient to trigger NKX101 cytotoxicity against target cell lines, further validating NKG2D-Ls as therapeutic targets. Flow cytometry analysis revealed that pre-treatment with Ara-C upregulated NKG2D-Ls on AML cell lines in a dose-dependent manner. NKX101 showed enhanced anti-leukemic activity against AML cells pre-treated with Ara-C compared to untreated controls, demonstrating that the combination can enhance NKX101 potency. Assessment of the combination of NKX101 and Ara-C using a dose-response matrix revealed that the two modalities combine to potently kill AML cells in a synergistic manner, supporting continued investigation of NKX101 CAR NK cell therapy after lymphodepletion incorporating Ara-C for the treatment of r/r AML. Citation Format: Cynthia Cho, Nikki Kimura, Jessica Sood, Damie Juat, Max Zhang, Carmel Chan, Tao Geng, Kyle Hansen, James Trager. NKX101, an allogeneic off-the-shelf CAR NK cell therapy targeting NKG2D-Ls, has potent anti-leukemic activity alone or in combination with Ara-C [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 3603.

Abstract 1338: Novel activation domains coupled to chimeric ILT receptors (CIR) enhance NK cell targeting of HLA-G+leukemic and solid tumor cells

Abstract Human leukocyte antigen-G (HLA-G) suppresses lymphoid and myeloid immune systems by binding ILT2 and ILT4 respectively and activating ITIM signaling. HLA-G is abundantly expressed in placental cytotrophoblasts to block fetal rejection. HLA-G is not expressed in most normal cells but is derepressed in in about 50% of malignancies. We engineered ILT2 and ILT4 to produce activating signals as Chimeric ILT Receptors or CIR. Preliminary results indicated robust killing of Acute Myeloid Leukemia (AML) cells by Natural Killer (NK) cells expressing either ILT2 or ILT4 CIRs. Here, we evaluated the effect of alteration of activating domains in the CIR constructs to produce signals that mimic the effect of IL-18 signaling in CIR-NK cells to enhance cytotoxicity, NK cell growth and persistence. Further, HLA-G expression and CIR-NK cell targeting of primary human cell types were rigorously screened. Activated NK cells derived from peripheral blood (2 to 5 donors) were transduced with γ-retroviruses directing expression of CIR proteins (CIR.X.Y or CIR.Y.X) where X stimulates cytotoxicity through CD3ζ, DAP10 or DAP12, and Y directs coactivation through 4-1BB, fusion of the MyD88 death domain or recruitment of endogenous MyD88 with receptor-derived TIR domains and soluble IL-15. CIR-NK cells were cocultured with GFPffluc-expressing AML and solid tumor target cell lines or normal primary human cells. NK cell proliferation and cytotoxicity of CIR-NK cells was monitored by Incucyte microscopy and supernatants were analyzed for cytokine release by ELISA. At low E:T (1:20 - 1:40) in 7-day cocultures, mock-transduced NK cells displayed innate killing activity against HLA-G+ Molm13 and Kasumi1 AML cells that was augmented by CIR constructs containing direct fusions of MyD88 and DAP10 or DAP12 (mock = 2.06E7 ± 4.53E6 vs. CIR-ILT4.MyD88.DAP10 = 1.03E7 ± 5.46E6) with 7-fold elevation of IFN-γ production. CIR constructs that recruited endogenous MyD88 signaling through the TLR2 TIR domain (4-1BB.DAP10.TLR2) also exhibited enhanced anti-HLA-G activity (9.13E6 ± 5.33E6). Against HLA-G+ HT-1376 bladder cancer cells (E:T = 1:10-1:20), CIR-NK cells also displayed enhanced tumor cell killing with enhanced NK cell growth. Conversely, CIR-enhanced killing was not observed against HLA-G− HCT-116 colorectal cancer cells. The persistence of CIR-NK cell potency after 4 weeks of expansion varied markedly with different activation domains employed. When screened against primary human cells (cardiomyocytes, hepatocytes, hepatic endothelial cells, corneal, colon and lung epithelial cells), no CIR-specific targeting was observed at high E:T ratios (1:1 and 1:5). In conclusion, CIR-NK cells exhibit potent and specific anti-tumor activity against HLA-G+ solid and leukemia cells. Alteration of CIR-NK cell intracellular signaling can optimize the potency and persistence of NK cell anti-tumor activity. Citation Format: MyLinh Duong, Jihyun Park, Raphel Ognar, Simon Wain-Hobson, Henri Bayle. Novel activation domains coupled to chimeric ILT receptors (CIR) enhance NK cell targeting of HLA-G+leukemic and solid tumor cells [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 1338.

Guardians of immunity: NK cell-mediated defense in COVID-19 and post-COVID scenarios.

The COVID-19 pandemic has left a lasting impact on global health, challenging communities, healthcare systems, and researchers worldwide. As we navigate this unprecedented crisis, this paper embarks on a multifaceted exploration of the pivotal role played by natural killer (NK) cells in the context of COVID-19. A significant portion of this paper is devoted to dissecting the nuanced role that NK cells assume in the context of COVID-19. From the initial acute infection to post-recovery immunity, NK cells emerge as critical players. We scrutinize the activation and dysregulation of NK cells during SARS-CoV-2 infection, shedding light on their potential contribution to disease severity. Moreover, we explore the fascinating landscape of post-COVID immunity, where NK cells are known to interact with adaptive immune responses, providing a foundation for long-term protection. In light of their central role, we investigate therapeutic strategies targeting NK cells in COVID-19 management, presenting an overview of current research efforts and their promise in mitigating disease progression. Lastly, we draw attention to research gaps, emphasizing the need for further investigation into NK cell dynamics during COVID-19. These gaps represent opportunities for advancing our understanding of NK cell biology and, by extension, enhancing our strategies for combating this global health crisis. This comprehensive exploration not only highlights the intricate interplay between NK cells and the COVID-19 pandemic but also underscores the importance of these innate immune warriors in shaping both the acute response and long-term immunity, ultimately contributing to the broader discourse surrounding the pandemic's pathophysiology and therapeutic approaches.

Targeting natural killer cells: from basic biology to clinical application in hematologic malignancies.

Natural killer (NK) cell belongs to innate lymphoid cell family that contributes to host immunosurveillance and defense without pre-immunization. Emerging studies have sought to understand the underlying mechanism behind NK cell dysfunction in tumor environments, and provide numerous novel therapeutic targets for tumor treatment. Strategies to enhance functional activities of NK cell have exhibited promising efficacy and favorable tolerance in clinical treatment of tumor patients, such as immune checkpoint blockade (ICB), chimeric antigen receptor NK (CAR-NK) cell, and bi/trispecific killer cell engager (BiKE/TriKE). Immunotherapy targeting NK cell provides remarkable advantages compared to T cell therapy, including a decreased rate of graft versus-host disease (GvHD) and neurotoxicity. Nevertheless, advanced details on how to support the maintenance and function of NK cell to obtain better response rate and longer duration still remain to be elucidated. This review systematically summarizes the profound role of NK cells in tumor development, highlights up-to-date advances and current challenges of therapy targeting NK cell in the clinical treatment of hematologic malignancies.

CAR-mediated targeting of NK cells overcomes tumor immune escape caused by ICAM-1 downregulation

BackgroundThe antitumor activity of natural killer (NK) cells can be enhanced by specific targeting with therapeutic antibodies that trigger antibody-dependent cell-mediated cytotoxicity (ADCC) or by genetic engineering to express chimeric...

Upregulation of HLA-E Drives Defective Natural Killer Cell Targeting in Venetoclax-Resistant Acute Myeloid Leukemia

Upregulation of HLA-E Drives Defective Natural Killer Cell Targeting in Venetoclax-Resistant Acute Myeloid Leukemia

Abstract B040: Trispecific killer engagers against IGF1R and fetal form of insulin receptor induce human natural killer cell mediated killing of hormone receptor positive breast cancer in vitro and in vivo

Abstract Patients with hormone receptor-positive (HR+) breast cancers frequently recur with metastatic disease 5-20 years after the initial diagnosis and completion of chemotherapy and prolonged treatment with hormonal therapy. Thus, patients with metastatic HR+ breast cancer need new therapeutic options. A potential mechanism of therapeutic resistance in HR+ patients is the presence of slow-growing, well-differentiated cancer cells that result in clinically detectable metastases after a prolonged latency. One way to eliminate these cancer cells is with immunotherapy (IT). Unfortunately HR+ breast cancers have not responded to current T-cell based IT. The objective of this study is to target HR+ breast cancers using a natural killer (NK) cell based approach that targets cell surface receptors in HR+ breast cancer. The type I IGF receptor (IGF1R) is expressed on HR+ breast cancers and its expression is tightly related to ER function. Endocrine therapy resistance is associated with increased expression of the A-isoform or fetal form of IR (IR-A) and depend on IR signaling. We hypothesized that a drug that would target NK cells to IGF1R or IR-A expressing HR+ cells will be effective in killing them. We generated IGF1R or IR-A specific Tri specific Killer Engagers (TriKE) that leverage expression of IGF1R and IR-A on HR+ cells to cause NK directed death. The IGF1R TriKE consists of humanized single domain antibody (VHH) sequence of a CD16 nanobody to engage and activate NK cells, a humanized VHH of a nanobody against IGF1R to target HR+ breast cancer cells, and an IL-15 molecule between them to drive NK cell expansion and survival. The IGF1R TriKE did not bind embryonal fibroblasts from IGF1R knock out mice (R- cells) but bound R-/IGF1R cells (R- cells engineered to express human IGF1R) and to IGF1R+ MCF-7 and T47D breast cancer cells indicating specific binding to IGF1R on cells. It did not cause downregulation of IGF1R levels in multiple HR+ breast cancer cells and did not inhibit signaling via IGF1R. It specifically induced NK cell degranulation, measured by surface CD107a expression and intracellular IFNγ, against IGF1R+ target breast cancer cells (MCF-7, T-47D, ZR-75-1) in a functional assay compared to the anti-IGF1R nanobody. It did not induce degranulation of NK cells against IGF1R negative cells suggesting that the functionality was specific for IGF1R expressing targets. The IGF1R TriKE enhanced cell killing of HR+ breast cancer cells as measured by cytotoxicity assays. IGF1R TriKE combined with human NK cells inhibited tumor growth of MCF-7 tumors in NSG mice compared to control groups. Human NK cells persisted in peripheral blood three weeks after final NK infusion based on flow cytometry assessment. The IR-A TriKE incorporated a novel engineered non-immunoglobulin IR-A specific peptide binder. Our preliminary data show that the IR-A TriKE degranulated NK cells against breast cancer cells overexpressing IR-A. These data indicate that this strategy could be an effective treatment for HR+ metastatic breast cancer and make them responsive to IT. Citation Format: Courtney Baar, Emily Chiu, Yvette Soignier, Behiye Kodal, Jeffrey S Miller, Martin Felices, Deepali Sachdev. Trispecific killer engagers against IGF1R and fetal form of insulin receptor induce human natural killer cell mediated killing of hormone receptor positive breast cancer in vitro and in vivo [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B040.

Structural basis for the activity and specificity of the immune checkpoint inhibitor lirilumab

The clinical success of immune checkpoint inhibitors has underscored the key role of the immune system in controlling cancer. Current FDA-approved immune checkpoint inhibitors target the regulatory receptor pathways of cytotoxic T-cells to enhance their anticancer responses. Despite an abundance of evidence that natural killer (NK) cells can also mediate potent anticancer activities, there are no FDA-approved inhibitors targeting NK cell specific checkpoint pathways. Lirilumab, the most clinically advanced NK cell checkpoint inhibitor, targets inhibitory killer immunoglobulin-like receptors (KIRs), however it has yet to conclusively demonstrate clinical efficacy. Here we describe the crystal structure of lirilumab in complex with the inhibitory KIR2DL3, revealing the precise epitope of lirilumab and the molecular mechanisms underlying KIR checkpoint blockade. Notably, the epitope includes several key amino acids that vary across the human population, and binding studies demonstrate the importance of these amino acids for lirilumab binding. These studies reveal how KIR variations in patients could influence the clinical efficacy of lirilumab and reveal general concepts for the development of immune checkpoint inhibitors targeting NK cells.

Direct Tumor Irradiation Potentiates Adoptive NK Cell Targeting Against Parental and Stemlike Cancer in Human Liver Cancer Models

Radiation therapy (RT) has been shown to effectively induce the expression of intercellular adhesion molecule-1 (ICAM-1), which is recognized by lymphocyte function-associated antigen 1 (LFA-1) expressed on natural killer (NK) cells. However, the potential synergistic antitumor immune response of tumor irradiation and administered NK cells has not been explored in intractable human liver cancers. Furthermore, NK cell targeting against both parental and cancer stemness has never been investigated. Highly activated ex vivo NK cells were administered into the human liver tumor-bearing mice. Tumor direct RT was optimized according to tumor bearing site. HepG2 and Hep3B ICAM-1 knockout cells were generated using CRISPR/CAS9. Stemness tumor spheres were generated. NK cell cytolysis against parental and tumor sphere was evaluated using flow cytometry and real-time cytotoxicity assay. A combination of adoptive NK cell therapy with RT significantly improved therapeutic efficacy over monotherapies against subcutaneous, orthotopic, and metastatic human liver tumor models. Direct tumor irradiation potentiated NK cell recognition and conjugation against liver cancer through the LFA-1/ICAM-1 axis. Suppression of immune synapse formation on NK cells using high-affinity LFA-1 inhibitors or ICAM-1 knockout liver cancer induced "outside-in" signal blocking in NK cells, resulting in failure to eliminate liver tumor despite the combination therapy. NK cells effectively recognized and targeted triple-high epithelial cell adhesion molecule+CD133+CD24+ liver cancer expressing upregulated ICAM-1 in the irradiated tumor microenvironment, which led to prevention of the initiation of metastasis, improving survival in a metastatic model. In addition, the LFA-1/ICAM-1 axis interruption between NK cells and stemness liver tumor spheres significantly diminished NK cell cytolysis. Consistent with our preclinical data, the LFA-1/ICAM-1 axis correlated with survival outcomes in patients with metastatic cancer from the The Cancer Genome Atlas databases. NK cells in combination with tumor irradiation can provide synergistic therapeutic effects for NK cell recognition and elimination against both parental and stemlike liver cancer through LFA-1/ICAM-1.

High-Throughput Screens Identify NEDD8 Inhibition As a Strategy to Augment Natural Killer Cell Cytotoxicity Against Blood Cancers

Natural killer (NK) cell-based therapies are amongst the emerging immunotherapeutic approaches that aim to target malignant cells refractory to standard therapies. Currently used strategies to augment the NK cell anti-cancer function of NK cells include cytokine treatments and the use of feeder expansion protocols for culturing NK cells. Recent studies have indicated that NK cell cytotoxicity and function may also be enhanced by certain anti-cancer drugs. However, the effects of the hundreds of available oncology drugs on NK cell function have not been systematically evaluated. Here, we perform large-scale high-throughput testing of NK cell-drug combinations using co-culture models with diverse blood cancer cells, identifying the NEDD8-activating enzyme inhibitor pevonedistat as a promising candidate for augmenting NK cell anti-cancer responses. To identify oncology drugs synergizing with NK cell-based immunotherapy, we established a high-throughput drug sensitivity and resistance testing (DSRT) platform to investigate effects of 527 approved drugs and investigational compounds on NK cell cytotoxicity against blood cancer cell lines. Overall, 10 cell lines, including acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), and multiple myeloma (MM), were screened in combination with primary expanded NK cells. Target cells were treated for 24 hours with drugs at five different concentrations both with and without NK cells. In NK cell co-culture, an effector-target (ET) cell ratio at which half of the target cells were killed was selected for experiments. Most effective compounds were then selected for further testing using multiplexed single-cell RNA sequencing (scRNA-seq) and analysis of secreted cytokines (Figure 1). Overall, the results from our DSRT platform demonstrated varying effects of different drug classes on NK cell cytotoxicity against blood cancer cell lines. In general, 5-10% of drugs had NK cell cytotoxicity-enhancing effects, 30-40% inhibited NK function and 35-50% of drugs had no effect. Similarities in results between cell lines highlighted the dependence of NK cell-drug synergistic effects on disease pathobiology. Across multiple disease models, pevonedistat (MLN4924), a selective NEDD8 activating enzyme inhibitor that has been studied as treatment for relapsed/refractory AML, showed strong synergistic effects with NK cells. For example, in OCI-AML-3 (AML), pevonedistat alone showed no effect on target cell viability, but in combination with NK cells at a 1:4 ET ratio, it resulted in 98% target cell killing at 100 nM. Similar effects were also seen in MM1.S (MM), although a higher concentration (10000 nM) of pevonedistat was required (Figure 2). Results from scRNA-seq after combination treatment with NK cells and pevonedistat revealed distinct transcriptomic changes in both NK cells and target cells when compared to controls. In both NK cells and target cells, pevonedistat caused upregulation of TXN, TALDO1, TXNRD1, NQO1, SRXN1 and SLC7A11,collectively indicating effects on the NRF2 signaling pathway. The upregulation of these genes correlated with the tested concentration of pevonedistat, with notably higher expression when treated with 1000 nM pevonedistat compared to 100 nM. Changes were also observed in the secretion of cytokines detected from the cell culture media, where concentrations of IL-1β, IL-2, IL-6, IL-8, IL-10, IFN-γ and TNF-α all increased up to 300% compared to controls. In HEL (AML) and LAMA-84 (CML), where pevonedistat had only minor effects on NK cell cytotoxicity, the expression TNF and TNFRSF1B at baseline and after pevonedistat treatment were notably lower, when compared to cell lines where the combination therapy led to stronger responses. This suggests a possible mechanism for pevonedistat-NK cell synergy, given that previous reports have demonstrated a synergy between pevonedistat and TNF-α to activate apoptosis. In conclusion, we discovered a cell context-specific synergy between pevonedistat and NK cells, which increased NK cell cytotoxicity against malignant cells in vitro. In addition, our results demonstrate the potential of using a high-throughput platform for studying NK cell-drug interactions against malignant cells, while aiming to improve treatment for hematological malignancies through combination immunotherapies.

NKX101, an Allogeneic Off-the-Shelf CAR NK Cell Therapy Targeting NKG2D-Ls, Has Potent Anti-Leukemic Activity Alone or in Combination with Ara-C

Introduction NKG2D-Ligands (NKG2D-Ls) are a family of immunomodulatory proteins that are upregulated on damaged or transformed cells but have limited expression on normal tissues. Natural killer (NK) cells target and eliminate NKG2D-L + cells through NKG2D receptor binding and subsequent activation. NKX101 is an allogeneic off-the-shelf healthy donor-derived chimeric antigen receptor (CAR) NK cell therapy candidate engineered to express an NKG2D-L targeting CAR and membrane-bound IL-15 for extended persistence. In a clinical trial, NKX101 treatment combined with modified lymphodepletion incorporating cytarabine (Ara-C) has shown promise for the treatment of relapsed/refractory (r/r) AML, including generating complete responses (CR) with MRD negativity (MRD -). To support the rationale of NKX101 combined with modified lymphodepletion, we developed assays to (i) investigate the cytotoxic activity of NKX101 against individual NKG2D-Ls, (ii) determine the effect of Ara-C pre-treatment on NKG2D-L expression on AML cell lines, and (iii) evaluate the combined anti-leukemic activity of NKX101 and Ara-C. Methods NKX101 cells were generated from peripheral blood leukopaks from healthy donors. CHO-K1 cells were engineered to stably express individual NKG2D-Ls or empty vector controls and were combined with NKX101 cells for cytotoxicity experiments. AML cell lines were treated with increasing levels of Ara-C and evaluated for NKG2D-L expression by flow cytometry. To measure NKX101 cytotoxicity against AML cell lines, NucRed™-labeled target cells were pre-treated with Ara-C using IC 50 and IC 90 concentrations and co-cultured with NKX101. Specific cell killing of target cells was measured using an Incucyte® instrument for five days. Assessment of the contribution of each agent was determined using a dose-response matrix of increasing concentrations of NKX101 cells and Ara-C to generate specific cytotoxicity values. Combination effects were analyzed using the SynergyFinder Plus web application. Results Expression of a single NKG2D-L was sufficient to trigger NKX101 cytotoxicity against target cell lines. Flow cytometry analysis revealed that pre-treatment with Ara-C upregulated NKG2D-Ls on AML cell lines in a dose-dependent manner. NKX101 showed enhanced anti-leukemic activity against AML cells pre-treated with Ara-C compared to untreated controls, demonstrating that the combination can enhance NKX101 potency. Assessment of the combination of NKX101 and Ara-C using a dose-response matrix revealed that the two modalities combine to potently kill AML cells. Conclusions In summary, we show that NKX101 potently targets and eliminates AML cells expressing NKG2D-Ls further validating NKG2D-Ls as therapeutic targets. Additionally, we show that the combination of Ara-C and NKX101 leads to increased anti-leukemic activity, supporting continued investigation of NKX101 CAR NK cell therapy after lymphodepletion incorporating Ara-C for the treatment of r/r AML.

TRAIL-conjugated liposomes that bind natural killer cells to induce colorectal cancer cell apoptosis.

Natural killer (NK) cell functionality is a strong indicator of favorable prognosis in cancer patients, making NK cells an appealing therapeutic target to prevent lymph node dissemination. We engineered liposomes that are conjugated with anti-CD335 antibodies for NK cell targeting, and the apoptotic ligand TRAIL to kill cancer cells. Liposomes were made using a thin film hydration method followed by extrusion to approximately 100 nm in diameter and conjugation of proteins via thiol-maleimide click chemistry. TRAIL/anti-CD335 liposomes successfully bound to isolated NK cells. Once piggybacked to the surface of NK cells, these "Super Natural Killer Cells" were able to more effectively kill oxaliplatin-resistant SW620 cells and metastatic COLO205 colorectal cancer cells via TRAIL-mediated apoptosis compared to NK cells alone. Importantly, Super NK cells were more effective under physiological levels of fluid shear stress found in the lymphatics. Liposome biodistribution after intravenous administration confirmed the sustained presence of liposomes within the spleen and tumor draining mesenteric lymph nodes for at least 4 days. These results demonstrate the enhanced apoptotic effects of NK cells armored with liposomal TRAIL against clinically relevant colorectal cancer cells, providing the groundwork for in vivo treatment studies in mouse models of colorectal cancer metastasis.