Endogenous Natural Killer Cells Research Articles

Abstract 2690: The role of PCNA tyrosine phosphorylation in evading innate immune surveillance

Abstract Cancer cells are continually exposed to intrinsic and extrinsic proliferative stresses that can send the genome stability awry. The aberrant DNA metabolism, evolving through deregulated replicative growth in cancer cells, is increasingly recognized as a major mechanism bridging the crosstalk between the immune microenvironment and tumor tissue, thereby either promoting or suppressing tumor progression. However, the triggering and regulation of these mechanisms remain largely unknown. Proliferating cell nuclear antigen (PCNA) and its homologs are the evolutionarily conserved central components of the DNA replication machinery. In eukaryotic cells, PCNA forms homotrimeric rings encircling the DNA double helix to coordinate the complex processes of DNA replication and damage repair, directed at least in part by differential post-translational modifications of the PCNA protein. In cancer cells, growth signal-stimulated phosphorylation at tyrosine 211 of PCNA (pY211-PCNA) is known to play an important function for active proliferation. Here, we demonstrate that inhibition of pY211 disrupts the processivity of replication forks. This disruption triggers single-stranded DNA (ssDNA) production catalyzed by the MRE11 nuclease, activating the cGAS-STING axis to launch an anti-tumor immune response by natural killer (NK) cells. We further show that the pY211 regulates site-specific post-translational modifications of MRE11, and loss of pY211 promotes the endonuclease mode of MRE11. This causes ssDNA generation in the cytosol, subsequently inducing type I interferon-activated cytotoxicity by NK cells and fostering an anti-tumor immunity that abrogates distant metastasis. Mechanistically, the pY211 determines the recruitment of the writer or eraser of the modification on MRE11, thus tuning the nucleolytic modes of MRE11 and, consequently, ssDNA generation. Therapeutic measures promoting MRE11-dependent ssDNA enhance the response of triple-negative breast cancer to cytotoxic killing mediated by endogenous or allogenic NK cells in mice. In breast cancer patients, the level of cytosolic ssDNA is inversely correlated with the expression of the eraser and pY211-PCNA, which is associated with poor overall survival in cancer patients. Our studies shed new light on the shaping of the tumor immune microenvironment and demonstrate the potential to develop therapeutic approaches that proactively leverage genomic instability and immune activation to target malignant tumors. Citation Format: Chuan-Chun Lee, Wan-Rong Wu, Yi-Chun Shen, Feng-Chi Chung, Yuan-Liang Wang, You-Zhe Lin, Chih-Hao Lu, Wei-Chung Cheng, Han Chang, Liang-Chih Liu, Ji-An Liang, Chang- Fang Chiu, Mien-Chie Hung, Shao-Chun Wang. The role of PCNA tyrosine phosphorylation in evading innate immune surveillance [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 2690.

Abstract 5235: Combination STING agonist and NK cellular therapy in patient derived organotypic tumor spheroids

Abstract Background: Increasing the immunogenicity of solid tumors may potentiate durable anti-tumor immunity. Targeting innate pattern recognition receptor signaling is a promising strategy to repolarizing the suppressive tumor immune microenvironment (TME). A potential synergistic approach is the addition of cellular therapy. Tumor models expressing high levels of endogenous STING, but where the pathway is not active, were considered for evaluating STING agonism as a potential intervention. Additionally, prior work has demonstrated that STING induction is toxic to T cells; however, natural killer (NK) cells are unaffected, due to their differential regulation of autophagy. Here we investigate the immunomodulatory properties of STING agonism alone and in combination with NK cells using short-term microfluidic culture of fresh patient-derived organotypic tumor spheroids (PDOTS). Methods: Surgical cases from Brigham and Women’s Hospital under an IRB-approved protocol were studied. PDOTS were generated as previously described. Ex vivo response was assessed by fluorescent live/dead imaging, immunofluorescence (IF), multiplexed cytokine array, and single cell RNA sequencing (scRNAseq). Baseline immune phenotypes were analyzed by FACS from single cells isolated during tumor sample preparation. Results: Twelve explants were studied. Two specimens were excluded from viability assessment due to excess of fibrotic material and low viability. Using a cut-off of -40%, three cases were characterized as responders (R) and seven as non-responders (NR). We observed significant induction of CXCL10 in all samples treated with STING agonist. IFN-β, IFN-γ, TNF-α, and MIP1-α were also consistently induced by STING agonist treatment. More detailed analysis of two samples by scRNAseq revealed a strong interferon stimulated genes (ISG) signature in STING agonist conditions; the cellular source of CXCL10 secretion in STING agonist treatment was tumor cells and fibroblasts. We also observed profound changes in myeloid biology, indicating that these cells polarized to a more pro-inflammatory and antigen-presenting phenotype with STING agonism. Interestingly, NK-based cell therapy added to PDOTS cultures displayed better persistence and enhanced effector function relative to endogenous NK cells, with and without STING agonism. Conclusions: Using tumor explants and a variety of orthogonal techniques, we investigated the response to STING agonism and NK cellular therapy ex vivo. Our results indicate that STING agonism remodels the TME, creating a more immune-permissive environment. STING agonism induces secretion of chemoattractant CXCL10 by tumor cells and fibroblasts. STING agonism does not abrogate NK cell effector function and therefore, may synergize with NK cell therapies. Citation Format: Patrick H. Lizotte, Elena Ivanova, Sung Park, Nathaniel Spicer, Sophie Kivlehan, Iliana Gjeci, Stefan Kiesgen, Vicky Appleman, LeeAnn Talarico, Michael Y. Tolstorukov, Raphael Bueno, David A. Barbie, Cloud P. Paweletz. Combination STING agonist and NK cellular therapy in patient derived organotypic tumor spheroids [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 5235.

Improved characterization and translation of NK cells for canine immunotherapy.

The field of cancer immunology has seen a meteoric rise in interest and application due to the discovery of immunotherapies that target immune cells, often leading to dramatic anti-tumor effects. However, successful cellular immunotherapy for solid tumors remains a challenge, and the application of immunotherapy to dogs with naturally occurring cancers has emerged as a high yield large animal model to bridge the bench-to-bedside challenges of immunotherapies, including those based on natural killer (NK) cells. Here, we review recent developments in the characterization and understanding of canine NK cells, a critical springboard for future translational NK immunotherapy research. The characterization of canine NK cells is exceptionally pertinent given the ongoing challenges in defining them and contextualizing their similarities and differences compared to human and murine NK cells compounded by the limited availability of validated canine specific reagents. Additionally, we summarize the current landscape of the clinical and translational literature employing strategies to capitalize on endogenous and exogenous NK cell immunotherapy in canine cancer patients. The insights regarding efficacy and immune correlates from these trials provide a solid foundation to design and test novel combinational therapies to enhance NK cell activity with the added benefit of motivating comparative work to translate these findings to human cancers with extensive similarities to their canine counterparts. The compilation of knowledge from basic canine NK phenotype and function to applications in first-in-dog clinical trials will support the canine cancer model and enhance translational work to improve cancer outcomes for both dogs and humans.

Targeting SLC3A2 Sensitizes AML Cells Towards NK Cell-Mediated Killing

Acute myeloid leukemia (AML) is an aggressive hematological malignancy with poor prognosis; hence, new therapeutic strategies are urgently needed. Natural Killer (NK) cells play a key role in tumor immune surveillance, but their anti-tumor activity in AML is often attenuated due to immunosuppressive effects of the malignant cells. Thus, strategies to restore NK cell function has therapeutic potential by boosting endogenous NK cells. In particular, the identification of cell surface proteins on AML cells that inhibit NK cell-mediated killing may reveal new targets for antibody-based therapies. To identify such targets, we performed a pooled CRISPR screen directed to 1389 cell surface genes in Mono-mac-6 (MM6), a human AML cell line. The MM6 cells were co-cultured with primary human NK cells for four days in media supplemented with the cytokines IL-2 and IL-15. Among the top hits were several genes coding for MHC class I molecules, which are known negative regulators of NK cells, demonstrating that the screen was robust. Notably, the screen also identified that SLC3A2 disruption sensitized the MM6 cells towards NK cell-mediated killing. SLC3A2 encodes the heavy chain of the transmembrane protein CD98 (CD98hc), which plays a key role in integrin signaling, regulation of intracellular calcium and the transport of L-type amino acids. CRISPR-mediated deletion of SLC3A2 in MM6 cells resulted in a two-fold downregulation of the transcripts of several inhibitory ligands of NK cells, including HLA-A, HLA-B, HLA-C and HLA-E. Upon co-culture, loss of SLC3A2 expression in MM6 cells induced an upregulation of the degranulation marker CD107a on NK cells (p<0.01), resulting in increased killing of the AML cells. CD98hc consists of two functional domains - one responsible for integrin signaling and the other responsible for amino acid transport. To identify which of these processes affect the sensitivity of AML cells towards NK cell-mediated killing, we performed rescue experiments by overexpression of mutated SLC3A2 cDNAs following SLC3A2 knockdown in MM6 cells. Overexpression of SLC3A2 wild-type or an integrin signaling deficient SLC3A2 cDNA rescued the inhibitory effect on NK cells. In contrast, the SLC3A2 variant that lacked the amino acid transportation domain failed to inhibit NK cells. These findings suggest that it is the amino acid transportation function of SLC3A2 that regulates the sensitivity of AML cells to NK cells. To validate these findings, we cultured MM6 cells in media deprived of three key amino acids (leucine, isoleucine and phenylalanine) transported across the plasma membrane by CD98. Consistent with our previous findings, culturing the MM6 cells in the amino acid deprived media resulted in a two-fold downregulation of HLA class I molecules (p<0.001) accompanied by an increased killing by NK cells. To assess the clinical relevance of these findings, we measured CD98hc expression on AML patient samples and corresponding normal bone marrow cells. CD98 levels were about 1.8-fold higher (p<0,001) in the AML samples (n = 30) compared to normal bone marrow cells (n = 5). Treating the AML patient cells with a monoclonal antibody targeting CD98 resulted in a 1.63-fold increase (p<0.0001) in NK cell-mediated killing. Corresponding treatments using normal bone marrow cells resulted in a 1.36-fold (p<0.01) increase in NK cell-mediated killing. Intriguingly, the CD98 antibody also negatively affected the viability (1.41-fold, p<0.0001) of AML patient cells in the absence of NK cells, this effect was not observed on normal bone marrow cells. Taken together, we here performed CRISPR screening on AML cells co-cultured with NK cells and identified SLC3A2 as a novel regulator of NK cells. Mechanistically, it is the amino acid transport function of SLC3A2 that regulate the sensitization of AML cells towards NK cell killing. Targeting of CD98 using a monoclonal antibody selectively increased NK cell-mediated killing of AML patient cells compared to normal bone marrow cells. These findings highlight CD98 as a new promising target on AML that boost NK cell-mediated tumor immune surveillance.

H2-K1 on MLL-AF9 Leukemia Cells Facilitates the Escape of NK Cell-Mediated Immune Surveillance

Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults, and prognosis is poor; 5-year survival approximately 30%. Recently, there is an emerging recognition of the innate immune system, in particular Natural killer (NK) cells and macrophages, for immune surveillance against AML, but the mechanistic basis for this is mostly unknow. Identifying how AML cells escape immune surveillance by NK cells may translate into new treatment opportunities for AML patients. To identify new therapeutic opportunities, we recently performed an in vivo CRISPR/Cas9 screen targeting 961 cell surface genes using the MLL-AF9 AML mouse model. One of the top leukemia stem cell dependencies in the bone marrow niche was H2-K1, an ortholog of human HLA-A, a classical MHC class-I molecule. In validation experiments, we observed a seven-fold depletion (p<0.001) of H2-K1 sgRNA-expressing c-Kit +Cas9 + leukemia cells in the bone marrow. In contrast, genetic disruption of H2-K1 did not impact the growth and survival of MLL-AF9 leukemia cells in culture. Given the known suppressive role of MHC class-I molecules for immune cells, we speculated that H2-K1 mayprovide inhibitory signals that counteract immune-surveillance mechanisms in the bone marrow niche. To test this hypothesis, we depleted NK cells and macrophages prior to transplantation of the c-Kit + MLL-AF9 leukemia cells. Macrophage depletion by clodronate liposomes did not affect the in vivo expansion of H2-K1 sgRNA-expressing leukemia cells demonstrating that macrophages were not suppressed by H2-K1 on the leukemia cells. In contrast, NK1.1 antibody-mediated depletion of NK cells fully rescued the depletion of H2-K1 sgRNA-expressing leukemia cells in vivo. These findings suggest that H2-K1 expression on MLL-AF9 leukemia cells inhibits NK cells in this model. Consistent with these findings, H2-K1 knockdown in leukemia cells triggered a two-fold increase of INFγ production (p<0.01) in the NK cells, accompanied by augmented apoptosis of the leukemia cells (p<0.01). Given that NK cells have been shown to be dysfunctional in AML patients, we next explored whether leukemia development affects NK cell maturation. The expansion of leukemia cells in the mice skewed NK cells towards a M1 (CD27 +CD11b -) state and decreased the level of the more cytotoxic M2 (CD27 +CD11b +) and M3 (CD27 -CD11b +) populations. H2-K1 disruption in the leukemic cells restored the level of M2 and M3 NK cell population in the bone marrow. Notably, restoration of matured NK cell populations was accompanied by an increased expression of NKG2D, an activating receptor, indicating a more cytotoxic state of the NK cells. In line with these findings, ablation of H2-K1 in leukemia cells induced JAK/STAT and NF-κβ signaling in the NK cells. Taken together, our study identifies that H2-K1 on MLL-AF9 leukemia stem cells facilitates immune evasion by suppressing NK cells. H2-K1 alters the maturation and activation of NK cells in the bone marrow niche. These findings increase our understanding of how leukemia cells escape immune surveillance and suggest that the identification of corresponding mechanisms in human AML could pave the way for new therapies that boost the endogenous NK cells by restoring immune surveillance mechanisms.

IMMU-55. THERAPEUTIC WINDOW ENABLING ERADICATION OF RESIDUAL GLIOMA STEM CELLS BY INTRACRANIAL NK CELL AND EXTRACELLULAR VESICLE-MEDIATED THERAPY FOLLOWING TEMOZOLOMIDE-INDUCED TUMOUR DEPOPULATION

Abstract Glioblastoma (GBM) is an incurable brain cancer, where dismal outcomes result from disease recurrence driven by tumour-initiating glioma stem cells (GSCs). GSCs survive and expand in the brain after surgery, radiation and temozolomide (TMZ) amidst weak immune and natural killer (NK) cell surveillance. NK cell-mediated killing of GBM cells occurs at effector to target rations precluding effective eradication as enforced by clinical trials with autologous NK cells in an adjuvant setting. Here we explore in a human GSC xenograft model whether tumor depopulation using high dose Temozolomide (TMZ) could create a window of curative opportunity for endogenous or exogenous NK cells. We observed that while subcutaneous (sc) xenografts of patient derived GSCs are infiltrated by endogenous functional (NCR1+) NK cells in SCID mice, the corresponding intracranial (ic) tumours remained NK cell depleted. In NK-deficient NSG mice, neither site contained functional NK cells. Notably, while TMZ caused near complete regression of GSC xenografts in both strains of mice and both locations (sc and ic), this was followed by recurrence of drug resistant lesions in all cases except NK-infiltrated sc SCID xenografts. This effect was obliterated by anti-sialo GM1 treatment. To explore whether direct delivery of NK cells into depopulated intracranial tumours would change the lethal course of the disease, mice were injected with GSCs ic, and upon tumour formation were treated with a sequence of TMZ (systemically), and two days later with irradiated NK92MI. Remarkably, this combined therapy completely obliterated recurrent disease. To assess whether NK92MI cells could be replaced by their derived extracellular vesicles (NK-EVs), the latter were injected i.c. post TMZ in GSC xenograft bearing mice. A single injection of NK-EVs resulted in tumour eradication in some but not all mice. Thus, chemotherapy-dependent tumour depopulation may create a unique window of opportunity for curative NK-mediated immunotherapy in GBM.

Humanized mouse models with endogenously developed human natural killer cells for in vivo immunogenicity testing of HLA class I-edited iPSC-derived cells

Human induced pluripotent stem cells (hiPSCs) genetically depleted of human leucocyte antigen (HLA) class I expression can bypass T cell alloimmunity and thus serve as a one-for-all source for cell therapies. However, these same therapies may elicit rejection by natural killer (NK) cells, since HLA class I molecules serve as inhibitory ligands of NK cells. Here, we focused on testing the capacity of endogenously developed human NK cells in humanized mice (hu-mice) using MTSRG and NSG-SGM3 strains to assay the tolerance of HLA-edited iPSC-derived cells. High NK cell reconstitution was achieved with the engraftment of cord blood-derived human hematopoietic stem cells (hHSCs) followed by the administration of human interleukin-15 (hIL-15) and IL-15 receptor alpha (hIL-15Rα). Such “hu-NK mice” rejected HLA class I-null hiPSC-derived hematopoietic progenitor cells (HPCs), megakaryocytes and T cells, but not HLA-A/B-knockout, HLA-C expressing HPCs. To our knowledge, this study is the first to recapitulate the potent endogenous NK cell response to non-tumor HLA class I-downregulated cells in vivo. Our hu-NK mouse models are suitable for the non-clinical evaluation of HLA-edited cells and will contribute to the development of universal off-the-shelf regenerative medicine.

A Killer Disarmed: Natural Killer Cell Impairment in Myelodysplastic Syndrome.

Myelodysplastic syndrome (MDS) treatment remains a big challenge due to the heterogeneous nature of the disease and its ability to progress to acute myeloid leukemia (AML). The only curative option is allogeneic hematopoietic stem cell transplantation (HSCT), but most patients are unfit for this procedure and are left with only palliative treatment options, causing a big unmet need in the context of this disease. Natural killer (NK) cells are attractive candidates for MDS immunotherapy due to their ability to target myeloid leukemic cells without prior sensitization, and in recent years we have seen an arising number of clinical trials in AML and, recently, MDS. NK cells are reported to be highly dysfunctional in MDS patients, which can be overcome by adoptive NK cell immunotherapy or activation of endogenous NK cells. Here, we review the role of NK cells in MDS, the contribution of the tumor microenvironment (TME) to NK cell impairment, and the most recent data from NK cell-based clinical trials in MDS.

Natural Killer T and Natural Killer Cell-Based Immunotherapy Strategies Targeting Cancer.

Both natural killer T (NKT) and natural killer (NK) cells are innate cytotoxic lymphoid cells that produce inflammatory cytokines and chemokines, and their role in the innate immune response to tumors and microorganisms has been investigated. Especially, emerging evidence has revealed their status and function in the tumor microenvironment (TME) of tumor cells. Some bacteria producing NKT cell ligands have been identified to exert antitumor effects, even in the TME. By contrast, tumor-derived lipids or metabolites may reportedly suppress NKT and NK cells in situ. Since NKT and NK cells recognize stress-inducible molecules or inhibitory molecules on cancer cells, their status or function depends on the balance between inhibitory and activating receptor signals. As a recent strategy in cancer immunotherapy, the mobilization or restoration of endogenous NKT or NK cells by novel vaccines or therapies has become a focus of research. As a new biological evidence, after activation, effector memory-type NKT cells lasted in tumor-bearing models, and NK cell-based immune checkpoint inhibition potentiated the enhancement of NK cell cytotoxicity against cancer cells in preclinical and clinical trials. Furthermore, several new modalities based on the characteristics of NKT and NK cells, including artificial adjuvant vector cells, chimeric antigen receptor-expressing NK or NKT cell therapy, or their combination with immune checkpoint blockade have been developed. This review examines challenges and future directions for improving these therapies.

Expansion, persistence, and efficacy of donor memory-like NK cells infused for posttransplant relapse.

BackgroundResponses to conventional donor lymphocyte infusion for postallogeneic hematopoietic cell transplantation (HCT) relapse are typically poor. Natural killer (NK) cell–based therapy is a promising modality to treat post-HCT relapse.MethodsWe initiated this ongoing phase I trial of adoptively transferred cytokine-induced memory-like (CIML) NK cells in patients with myeloid malignancies who relapsed after haploidentical HCT. All patients received a donor-derived NK cell dose of 5 to 10 million cells/kg after lymphodepleting chemotherapy, followed by systemic IL-2 for 7 doses. High-resolution profiling with mass cytometry and single-cell RNA sequencing characterized the expanding and persistent NK cell subpopulations in a longitudinal manner after infusion.ResultsIn the first 6 enrolled patients on the trial, infusion of CIML NK cells led to a rapid 10- to 50-fold in vivo expansion that was sustained over months. The infusion was well tolerated, with fever and pancytopenia as the most common adverse events. Expansion of NK cells was distinct from IL-2 effects on endogenous post-HCT NK cells, and not dependent on CMV viremia. Immunophenotypic and transcriptional profiling revealed a dynamic evolution of the activated CIML NK cell phenotype, superimposed on the natural variation in donor NK cell repertoires.ConclusionGiven their rapid expansion and long-term persistence in an immune-compatible environment, CIML NK cells serve as a promising platform for the treatment of posttransplant relapse of myeloid disease. Further characterization of their unique in vivo biology and interaction with both T cells and tumor targets will lead to improvements in cell-based immunotherapies.Trial RegistrationClinicalTrials.gov NCT04024761.FundingDunkin’ Donuts, NIH/National Cancer Institute, and the Leukemia and Lymphoma Society.

Roles of natural killer cells in immunity to cancer, and applications to immunotherapy.

Great strides have been made in recent years towards understanding the roles of natural killer (NK) cells in immunity to tumours and viruses. NK cells are cytotoxic innate lymphoid cells that produce inflammatory cytokines and chemokines. By lysing transformed or infected cells, they limit tumour growth and viral infections. Whereas T cells recognize peptides presented by MHC molecules, NK cells display receptors that recognize stress-induced autologous proteins on cancer cells. At the same time, their functional activity is inhibited by MHC molecules displayed on such cells. The enormous potential of NK cells for immunotherapy for cancer is illustrated by their broad recognition of stressed cells regardless of neoantigen presentation, and enhanced activity against tumours that have lost expression of MHC class I owing to acquired resistance mechanisms. As a result, many efforts are under way to mobilize endogenous NK cells with therapeutics, or to provide populations of ex vivo-expanded NK cells as a cellular therapy, in some cases by equipping the NK cells with chimeric antigen receptors. Here we consider the key features that underlie why NK cells are emerging as important new additions to the cancer therapeutic arsenal.

Harnessing natural killer cells for the treatment of multiple myeloma

Multiple myeloma (MM) is hematologic malignancy that is associated with profound immune alterations. Myeloma cells are susceptible to killing by natural killer (NK) cells but acquire the ability to elude NK cell surveillance by avoiding recognition and suppressing NK cell function. Major advances in the treatment of multiple myeloma have been achieved by effective new drugs that redirect NK cells and enhance their function. Despite significant progress, myeloma remains incurable and novel treatment approaches are required. Strategies to take advantage of the intrinsic antitumor properties of NK cells to treat MM represent a novel immunotherapeutic approach. One such strategy is adoptive NK cell therapy that consist of infusions of NK cells that have been propagated ex vivo. Adoptive NK cell therapy encompasses contemporary genetic engineering strategies such as chimeric antigen receptor (CAR)-engineered NK cells. An alternative approach involves the use of pharmacologic agents to enhance NK cell activity against myeloma. NK cell-modulating therapies can be used to bolster the function of endogenous NK cells or to enhance the efficacy of adoptively infused NK cells. Here, we review the mechanistic complexities influencing NK cell activity in MM and highlight a variety of innovative NK cell-based strategies being developed for the treatment of MM.

Bi-specific and Tri-specific NK Cell Engagers: The New Avenue of Targeted NK Cell Immunotherapy.

Natural killer (NK) cell-mediated cancer immunotherapy has grown significantly over the past two decades. More recently, multi-specific engagers have been developed as cancer therapeutics to effectively arm endogenous NK cells to more potently induce specific cytolytic responses against tumor targets. This review explores the bi- and tri-specific NK/tumor engagers that are emerging as a new generation of immunotherapeutics. These molecules vary in configuration, but they typically have small molecular weights and domains that engage specific tumor antigens and NK cell-activating receptors such as CD16, NKp30, NKp46, and NKG2D. They have demonstrated compelling potential in boosting NK cell cytotoxicity against specific tumor targets. This highly adaptable off-the-shelf platform, which in some formats also integrates cytokines, is poised to revolutionize targeted NK cell immunotherapy, either as a monotherapy or in combination with other effective anti-cancer therapies.

Exploring natural killer cell immunology as a therapeutic strategy in lung cancer.

Cytotoxic immune cells are key in the control of tumor development and progression. Natural killer (NK) cells are the cytotoxic arm of the innate immune system with the capability to kill tumor cells and surveil tumor cell dissemination. As such, the interest in harnessing NK cells in tumor control is increasing in many solid tumor types, including lung cancer. Here, we review the pre-clinical models used to unveil the role of NK cells in immunosurveillance of solid tumors and highlight measures to enhance NK cell activity. Importantly, the development of NK immunotherapy is rapidly evolving. Enhancing the NK cell response can be achieved using two broad modalities: enhancing endogenous NK cell activity, or performing adoptive transfer of pre-activated NK cells to patients. Numerous clinical trials are evaluating the efficacy of NK cell immunotherapy in isolation or in combination with standard treatments, with encouraging initial results. Pre-clinical studies and early phase clinical trials suggest that patients with solid tumors, including lung cancer, have the potential to benefit from recent developments in NK cell immunotherapy.

Innate immune cells in the tumor microenvironment.

The tumor immune microenvironment (TIME) is a complex ecosystem that contains adaptive and innate immune cells that have tumor-promoting and anti-tumor effects. There is still much to learn about the diversity, plasticity, and functions of innate immune cells in the TIME and their roles in determining the response to immunotherapies. Experts discuss recent advances in our understanding of their biology in cancer as well as outstanding questions and potential therapeutic avenues.

Natural Killer Cells: The Linchpin for Successful Cancer Immunotherapy.

Cancer immunotherapy is a highly successful and rapidly evolving treatment modality that works by augmenting the body’s own immune system. While various immune stimulation strategies such as PD-1/PD-L1 or CTLA-4 checkpoint blockade result in robust responses, even in patients with advanced cancers, the overall response rate is low. While immune checkpoint inhibitors are known to enhance cytotoxic T cells’ antitumor response, current evidence suggests that immune responses independent of cytotoxic T cells, such as Natural Killer (NK) cells, play crucial role in the efficacy of immunotherapeutic interventions. NK cells hold a distinct role in potentiating the innate immune response and activating the adaptive immune system. This review highlights the importance of the early actions of the NK cell response and the pivotal role NK cells hold in priming the immune system and setting the stage for successful response to cancer immunotherapy. Yet, in many patients the NK cell compartment is compromised thus lowering the chances of successful outcomes of many immunotherapies. An overview of mechanisms that can drive NK cell dysfunction and hinder immunotherapy success is provided. Rather than relying on the likely dysfunctional endogenous NK cells to work with immunotherapies, adoptive allogeneic NK cell therapies provide a viable solution to increase response to immunotherapies. This review highlights the advances made in development of NK cell therapeutics for clinical application with evidence supporting their combinatorial application with other immune-oncology approaches to improve outcomes of immunotherapies.

APOLLO: A phase I study of adaptive memory natural killer (NK) cells in recurrent ovarian cancer.

6044 Background: Human cytomegalovirus (CMV) infection induces a subset of long-lived CD57+NKG2C+ adaptive NK cells that exhibit enhanced antibody-dependent cellular cytotoxicity and resistance to tumor-suppressive mechanisms. We developed a 7-day culture process using a GSK3 inhibitor and IL-15 to manufacture modulated adaptive NK cells (FATE-NK100) from CMV+ haploidentical donors for adoptive transfer. The phase I Apollo trial tests the maximum tolerated dose/maximum feasible dose (MTD/MFD) of FATE-NK100 administered intraperitoneally (IP) to treat platinum-sensitive or -resistant recurrent ovarian, fallopian tube, and primary peritoneal cancer. Methods: FATE-NK100 via IP port was tested using 3 dose cohorts ([DC]; 1 × 107 cells/kg; >1 × 107 cells/kg to ≤3 × 107 cells/kg; or >3 × 107 to ≤10 × 107 cells/kg) after lympho-conditioning with fludarabine 25 mg/m2 IV and cyclophosphamide 300 mg/m2 IV on days −6 and −5. After FATE-NK100 infusion on day 0, rhIL-2 at 6 million IU was given IP 3 times a week for 6 doses for in vivo NK activation. IP fluid and peripheral blood were collected regularly until response assessment (day 28). Patients with stable disease or better were eligible for retreatment. Pre- and post-treatment tumor biopsies were collected. Results: Nine patients were treated with no dose-limiting toxicities (DLTs) to date. Retreatment based on clinical benefit was performed on 3 patients (33%), 2 following stable disease (DC 2) and 1 with partial remission (48% tumor reduction, DC 3). IP samples were collected for PK and functional analysis. FATE-NK100 product was detected by flow cytometry in 5 of 6 patients with evaluable samples (range 4.8%–91.2% donor NK cells at day +5-7). Retreatment samples were available in 1 patient, where FATE-NK100 persisted to day +21, demonstrating that repeated IP dosing did not accelerate clearance of the donor NK cells. In that same patient, measurement of NK cell CD107a degranulation or IFNg production in response to K562 targets demonstrated sustained enhanced in vivo function of FATE-NK100 compared to endogenous patient NK cells (e.g. at Day +12 CD107a+ NK were 39.0% vs. 22.5% cycle 1, and 40.3% vs. 18.2% retreatment cycle 2, and IFNg+ NK were 12.3% vs. 5.9% cycle 1, and 2.4% vs. 0.2% retreatment cycle 2). Conclusions: IP delivery of FATE-NK100 is safe, with clinical benefit in 3/9 patients treated. The allogeneic product cells persist and have enhanced function compared to patient NK cells for up to 21 days, even after retreatment. This phase I study in recurrent/refractory ovarian cancer shows promise for IP NK cell delivery. Clinical trial information: NCT00652899.

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.

NK cells for cancer immunotherapy.

Natural killer (NK) cells can swiftly kill multiple adjacent cells if these show surface markers associated with oncogenic transformation. This property, which is unique among immune cells, and their capacity to enhance antibody and T cell responses support a role for NK cells as anticancer agents. Although tumours may develop several mechanisms to resist attacks from endogenous NK cells, ex vivo activation, expansion and genetic modification of NK cells can greatly increase their antitumour activity and equip them to overcome resistance. Some of these methods have been translated into clinical-grade platforms and support clinical trials of NK cell infusions in patients with haematological malignancies or solid tumours, whichhave yielded encouraging results so far. The next generation of NK cell products will be engineered to enhance activating signals and proliferation, suppress inhibitory signals and promote their homing to tumours. These modifications promise to significantly increase their clinical activity. Finally, there is emerging evidence of increased NK cell-mediated tumour cell killing in the context of molecularly targeted therapies. These observations, in addition to the capacity of NK cells to magnify immune responses, suggest that NK cells are poised to become key components of multipronged therapeutic strategies for cancer.

Immune Monitoring of CD34+ Placental Cell Derived Natural Killer Cell Therapy (PNK-007) in Phase I Study of Multiple Myeloma

Background: Natural Killer (NK) cells are innate immune lymphocytes with cytotoxic function and are critical for immune surveillance. Unlike T cells which rely on antigen-specific responses, NK cells recognize transformed cells through a variety of NK cell-specific receptor-ligand interactions. Clinical studies have highlighted the therapeutic potential of NK cell-based therapies and Celularity has developed a GMP procedure for generating PNK-007: an off-the-shelf and allogeneic NK cell product culture-expanded and differentiated from human placental CD34+ stem cells. PNK-007 was evaluated as a single-dose allogeneic cell therapy in a phase I dose-escalation study for multiple myeloma (MM) patients receiving autologous stem cell transplant (ASCT) (NCT02955550). 12 of 15 patients received subcutaneous IL-2 every 48 hours for 10 days following PNK-007 cell infusion while 3 patients in a separate cohort did not receive IL-2. Here, we report translational studies evaluating post-treatment immune reconstitution, minimal residual disease (MRD) detection at 10-5 threshold, and serum profiling of cytokines and chemokines. Methods: Patient bone marrow aspirate was collected for EuroFlow MRD assessment and immune phenotyping by flow cytometry at baseline, 90-100 days, 6 months and 1 year post-ASCT. Peripheral blood was collected at baseline, then weekly for six weeks following PNK-007 infusion, at 6 months and at 1 year post-ASCT. PNK-007 persistence, leukocyte populations, and HLA antibody panels were determined by flow cytometry. Serum was analyzed by Luminex for panel cytokines, chemokines and soluble cytokine receptors. All patients and PNK-007 drug product were typed for HLA and killer-cell immunoglobulin-like (KIR) genotype. Results: PNK-007 infusion did not interfere with immune reconstitution kinetics post-ASCT. Cohorts receiving PNK infusion 14 days post-ASCT had already recovered white blood cell counts to normal levels. One cohort receiving 3x107 PNK cells/kg 7 days post-ASCT showed an immune deficient environment (0.05x103 ± 0.004x103 leukocytes/ml, n=3), but recovered their white blood cell counts by day 21 (6.8x103 ± 1.8x103 leukocytes/ml, n=3). Using a validated Euro-flow MRD assay, 4/15 patients were MRD(-) at pre-ASCT baseline, and by day 90, 10/15 pts were MRD(-). PNK-007 persistence was not detected in patients by flow cytometry with the earliest timepoint tested being 7 days post-infusion. Panel HLA serum antibodies were not detected at any timepoint indicating the absence of alloantibodies. Withholding IL-2 administration in one cohort allowed us to evaluate its potential effects on NK cell recovery and immune reconstitution. We found that subcutaneous IL-2 q.o.d. for 10 days following PNK-007 infusion did not affect recovery kinetics and concentration of endogenous NK cells. However, these patients showed a 5-10 fold increase in serum soluble IL-2RA from baseline and elevated regulatory T cell (Treg) count in the blood versus baseline (167 ± 107 Treg/mL vs. 61 ± 33 Treg/mL, p=0.0075). Patients not receiving IL-2 saw no change in serum soluble IL-2RA or blood Treg level from baseline. CD4 and CD8 T cells from all patients retained their ability to become activated in response to ex vivo stimulation and favored release of IL-2 and IFNg. T cell effector function was maintained in all patients post-ASCT but was lost in a subset of patients at 1 year. Serum analysis showed low levels of free IL-15 at the time of PNK dosing despite the transient lymphodepleted state associated with ASCT. Cytokines associated with myeloid inflammation and T cell immunity in the month post dosing were within normal homeostatic level. Serum TGFβ was significantly lower at the day of PNK infusion compared to normal homeostatic levels. Conclusion: Translational data from a Phase I study of PNK-007 in post-ASCT MM established that dosing up to 3x107 cells/kg at 14 or 7 days post-transplant did not impair engraftment or immune reconstitution. EuroFlow MRD assessment of the bone marrow showed conversion of 6/11 patients from MRD(+) to MRD(-) by day 90 post transplant. The administration of IL-2 in this clinical study did not appear to benefit NK cell reconstitution but instead favored soluble IL2RA antagonism and increased systemic levels of Treg. These results demonstrate the feasibility of allogeneic NK cell therapy in the MM + ASCT setting and will help inform the design of further clinical studies. Disclosures Van Der Touw: Celularity, Inc.: Employment, Patents & Royalties. Kang:Celularity, Inc.: Employment, Patents & Royalties. Stout:Celularity, Inc.: Employment. Voskinarian-Berse:Celularity, Inc.: Employment. Rousseva:Celularity, Inc.: Employment. Hariri:Celularity Inc: Employment. Zhang:Celularity Inc: Employment.