Genetic Engineering of Natural Killer Cells for Enhanced Antitumor Function

Natural Killer (NK) cells are unique immune cells capable of efficient killing of infected and transformed cells. Indeed, NK cell-based therapies induced response against hematological malignancies in the absence of adverse toxicity in clinical trials. Nevertheless, adoptive NK cell therapies are reported to have exhibited poor outcome against many solid tumors. This can be mainly attributed to limited infiltration of NK cells into solid tumors, downregulation of target antigens on the tumor cells, or suppression by the chemokines and secreted factors present within the tumor microenvironment. Several methods for genetic engineering of NK cells were established and consistently improved over the last decade, leading to the generation of novel NK cell products with enhanced anti-tumor activity and improved tumor homing. New generations of engineered NK cells are developed to better target refractory tumors and/or to overcome inhibitory tumor microenvironment. This review summarizes recent improvements in approaches to NK cell genetic engineering and strategies implemented to enhance NK cell effector functions.

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Approaches to Enhance Natural Killer Cell-Based Immunotherapy for Pediatric Solid Tumors

Cancers ◽

10.3390/cancers13112796 ◽

2021 ◽

Vol 13 (11) ◽

pp. 2796

Author(s):

Aicha E. Quamine ◽

Mallery R. Olsen ◽

Monica M. Cho ◽

Christian M. Capitini

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Solid Tumors ◽

Nk Cell ◽

Therapeutic Option ◽

Treatment Options ◽

Intensive Therapy ◽

Killer Cell ◽

Cell Therapies ◽

Pediatric Solid Tumors

Treatment of metastatic pediatric solid tumors remain a significant challenge, particularly in relapsed and refractory settings. Standard treatment has included surgical resection, radiation, chemotherapy, and, in the case of neuroblastoma, immunotherapy. Despite such intensive therapy, cancer recurrence is common, and most tumors become refractory to prior therapy, leaving patients with few conventional treatment options. Natural killer (NK) cells are non-major histocompatibility complex (MHC)-restricted lymphocytes that boast several complex killing mechanisms but at an added advantage of not causing graft-versus-host disease, making use of allogeneic NK cells a potential therapeutic option. On top of their killing capacity, NK cells also produce several cytokines and growth factors that act as key regulators of the adaptive immune system, positioning themselves as ideal effector cells for stimulating heavily pretreated immune systems. Despite this promise, clinical efficacy of adoptive NK cell therapy to date has been inconsistent, prompting a detailed understanding of the biological pathways within NK cells that can be leveraged to develop “next generation” NK cell therapies. Here, we review advances in current approaches to optimizing the NK cell antitumor response including combination with other immunotherapies, cytokines, checkpoint inhibition, and engineering NK cells with chimeric antigen receptors (CARs) for the treatment of pediatric solid tumors.

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799 Neoantigen-cytokine-chemokine multifunctional natural killer cell engager for immunotherapy of solid tumors

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.799 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A834-A834

Author(s):

Xue Yao ◽

Sandro Matosevic

Keyword(s):

Tumor Microenvironment ◽

Cell Therapy ◽

Nk Cells ◽

Natural Killer ◽

Solid Tumors ◽

Tumor Cells ◽

Nk Cell ◽

Killer Cell ◽

Nk Cell Therapy

BackgroundThe effectiveness of natural killer (NK) cell-based immunotherapy against solid tumors is limited by the lack of specific antigens and the immunosuppressive tumor microenvironment (TME). Glioblastoma multiforme (GBM) is one such heavily immunosuppressive tumor that has been particularly hard to target and remains without a viable treatment. The development of novel approaches to enhance the efficacy of NK cells against GBM is urgently needed. NK cell engagers (NKCE) have been developed to enhance the efficacy of NK cell therapy.MethodsTo improve the clinical efficacy of NK cell therapy, we are developing a new generation of multi-specific killer engagers, which consists of a neoantigen-targeting moiety, together with cytokine and chemokine-producing domains. Neoantigens are new antigens formed specifically in tumor cells due to genome mutations, making them highly specific tools to target tumor cells. Our engager has been designed to target Wilms' tumor-1 (WT-1), a highly specific antigen overexpressed in GBM among other solid tumors. This is done through the generation of an scFv specific targeting the complex of WT-1126-134/HLA-A*02:01 on the surface of GBM. On the NK cell side, the engager is designed to target the activating receptor NKp46. Incorporation of the cytokine IL-15 within the engager supports the maturation, persistence, and expansion of NK cells in vivo while favoring their proliferation and survival in the tumor microenvironment. Additionally, our data indicated that the chemokine CXCL10 plays an important role in the infiltration of NK cells into GBM, however, GBM tumors produce low levels of this chemokine. Incorporation of a CXCL10-producing function into our engager supports intratumoral NK cell trafficking by promoting, through their synthetic production, increased levels of CXCL10 locally in the tumor microenvironment.ResultsCollectively, this has resulted in a novel multifunctional NK cell engager, combining neoantigen-cytokine-chemokine elements fused to an activating domain-specific to NK cells, and we have investigated its ability to support and enhance NK cell-mediated cytotoxicity against solid tumors in vitro and in vivo against patient-derived GBM models. The multi-specific engager shows both high tumor specificity, as well as the ability to overcome NK cell dysfunction encountered in the GBM TME.ConclusionsWe hypothesize that taking advantage of our multi-functional engager, NK cells will exhibit superior ex vivo expansion, infiltration, and antitumor activity in the treatment of GBM and other solid tumors.

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Viral and Nonviral Engineering of Natural Killer Cells as Emerging Adoptive Cancer Immunotherapies

Journal of Immunology Research ◽

10.1155/2018/4054815 ◽

2018 ◽

Vol 2018 ◽

pp. 1-20 ◽

Cited By ~ 27

Author(s):

Sandro Matosevic

Keyword(s):

Tumor Microenvironment ◽

Gene Transfer ◽

Genetic Engineering ◽

Nk Cells ◽

Cancer Immunotherapy ◽

Natural Killer ◽

Solid Tumors ◽

Viral Vectors ◽

Vector Systems

Natural killer (NK) cells are powerful immune effectors whose antitumor activity is regulated through a sophisticated network of activating and inhibitory receptors. As effectors of cancer immunotherapy, NK cells are attractive as they do not attack healthy self-tissues nor do they induce T cell-driven inflammatory cytokine storm, enabling their use as allogeneic adoptive cellular therapies. Clinical responses to adoptive NK-based immunotherapy have been thwarted, however, by the profound immunosuppression induced by the tumor microenvironment, particularly severe in the context of solid tumors. In addition, the short postinfusion persistence of NK cellsin vivohas limited their clinical efficacy. Enhancing the antitumor immunity of NK cells through genetic engineering has been fueled by the promise that impaired cytotoxic functionality can be restored or augmented with the use of synthetic genetic approaches. Alongside expressing chimeric antigen receptors to overcome immune escape by cancer cells, enhance their recognition, and mediate their killing, NK cells have been genetically modified to enhance their persistencein vivoby the expression of cytokines such as IL-15, avoid functional and metabolic tumor microenvironment suppression, or improve their homing ability, enabling enhanced targeting of solid tumors. However, NK cells are notoriously adverse to endogenous gene uptake, resulting in low gene uptake and transgene expression with many vector systems. Though viral vectors have achieved the highest gene transfer efficiencies with NK cells, nonviral vectors and gene transfer approaches—electroporation, lipofection, nanoparticles, and trogocytosis—are emerging. And while the use of NK cell lines has achieved improved gene transfer efficiencies particularly with viral vectors, challenges with primary NK cells remain. Here, we discuss the genetic engineering of NK cells as they relate to NK immunobiology within the context of cancer immunotherapy, highlighting the most recent breakthroughs in viral vectors and nonviral approaches aimed at genetic reprogramming of NK cells for improved adoptive immunotherapy of cancer, and, finally, address their clinical status.

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116 Development of logic gated CAR-NK cells for the treatment of solid tumors

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.116 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A125-A125

Author(s):

Alba Gonzalez Junca ◽

Nicholas Frankel ◽

Marcus Gainer ◽

Alyssa Mullenix ◽

Miguel Palermo ◽

...

Keyword(s):

Colorectal Cancer ◽

Epithelial Cells ◽

Nk Cells ◽

Solid Tumors ◽

Nk Cell ◽

Lung Epithelial Cells ◽

Gene Circuits ◽

Cell Therapies ◽

Lung Epithelial ◽

Anti Tumor Activity

BackgroundCAR-based therapies have transformed the treatment of several cancers, but this progress has not translated into solid tumors. One challenge of CAR-mediated therapies for solid tumors is the lack of specific tumor-associated antigens (TAA’s) that are only expressed on cancer cells and not on healthy cells, thereby posing a risk for on-target off-tumor toxicities. This presents a unique opportunity to use Logic Gates to expand the universe of cancer targets that may be treated with CAR-based cell therapies. CEA, a widely expressed tumor antigen, found in >90% of colorectal cancer (CRC), is also expressed in healthy gastrointestinal and lung epithelial cells. Clinical experience targeting CEA resulted in severe dose-limiting toxicities,1,2 highlighting the need for healthy tissue protection. Logic-gated gene circuits can prevent off-tumor toxicities by pairing a CEA activating-CAR (aCAR) with an inhibitory-CAR (iCAR) that recognizes a safety antigen (SA) uniquely expressed in healthy epithelial cells.MethodsWe developed a bioinformatics-driven antigen paired discovery platform using single-cell transcriptomics to discover and prioritize TAA’s and pair them with SA’s that are selectively expressed on the membrane of healthy cells. TAA’s and SA’s were validated in primary cancer and healthy tissue samples using IHC. We constructed aCAR/iCAR gene circuits and tested their function in NK cells.ResultsOur bioinformatics platform identified VSIG2 to be co-expressed with CEA in healthy gastrointestinal and lung epithelial cells. IHC confirmed the expression of VSIG2 on the membrane of healthy colon (N=72 samples) and lung (N=24 samples) epithelial cells.Using our Design-Build-Test-Learn platform, we screened >250 CAR constructs targeting CEA. CAR-NK cells were generated and tested for anti-tumor activity against CRC CEA+ cells and lead candidates were selected based on NK cell performance. A single dose of CEA-CAR-NK cells had anti-tumor activity in a human CRC xenograft model, reducing tumor burden in >33% of the treated mice. We identified iCARs with different intracellular domains derived from native domains containing immunoreceptor tyrosine-based inhibitory motifs. These iCARs suppressed >50% of aCAR-mediated killing (p<0.05) and significantly reduced TNFa secretion (p<0.0005) in a SA-specific manner.ConclusionsWe are developing Logic-Gated CAR-NK cell therapies aimed at reducing on-target off-tumor toxicities, to spare healthy cells in a SA-dependent manner. SENTI-401 will focus on targeting CEA+ CRC tumors with a NOT gate that recognizes the SA VSIG2 in the colon and lungs.ReferencesParkhurst M, et al. T cells targeting carcinoembryonic antigen can mediate regression of metastatic colorectal cancer but induce severe transient colitis. Mol Ther 2011; Mar;19(3):620–6.Thistlethwaite FC, et al. The clinical efficacy of first-generation carcinoembryonic antigen (CEACAM5)-specific CAR T cells is limited by poor persistence and transient pre-conditioning-dependent respiratory toxicity. Cancer Immunol Immunother 2017 Nov;66(11):1425–1436.

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Toxoplasma gondiiInfection Drives Conversion of NK Cells into ILC1s

10.1101/642017 ◽

2019 ◽

Author(s):

Eugene Park ◽

Swapneel J. Patel ◽

Qiuling Wang ◽

Prabhakar S. Andhey ◽

Konstantin Zaitsev ◽

...

Keyword(s):

Steady State ◽

Tumor Microenvironment ◽

Toxoplasma Gondii ◽

Nk Cells ◽

Natural Killer ◽

Nk Cell ◽

Innate Lymphoid Cells ◽

Lymphoid Cells ◽

Cell Conversion ◽

Ongoing Infection

AbstractInnate lymphoid cells (ILCs) were originally classified based on their cytokine profiles, placing natural killer (NK) cells and ILC1s together, but recent studies support their separation into different lineages at steady-state. However, tumors may induce NK cell conversion into ILC1-like cells that are limited to the tumor microenvironment and whether this conversion occurs beyond this environment remains unknown. Here we describeToxoplasma gondiiinfection converts NK cells into cells resembling steady-state ILC1s that are heterogeneous and distinct from both steady-state NK cells and ILC1s in uninfected mice. Most toxoplasma-induced ILC1s were Eomes-dependent, indicating that NK cells can give rise to Eomes−Tbet-dependent ILC1-like cells that circulate widely and persist independent of ongoing infection. Moreover, these changes appear permanent, as supported by epigenetic analyses. Thus, these studies markedly expand current concepts of NK cells, ILCs, and their potential conversion.

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A natural killer cell gene signature predicts melanoma patient survival

10.1101/375253 ◽

2018 ◽

Author(s):

Joseph Cursons ◽

Fernando Souza-Fonseca-Guimaraes ◽

Ashley Anderson ◽

Momeneh Foroutan ◽

Soroor Hediyeh-Zadeh ◽

...

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Solid Tumors ◽

Immune Cell ◽

Nk Cell ◽

Killer Cell ◽

Cell Activity ◽

Human Tumors ◽

The Cancer Genome Atlas ◽

Cell Gene

AbstractAnimal models have demonstrated that natural killer (NK) cells can limit the metastatic dissemination of tumors, however their ability to combat established human tumors has been difficult to investigate.A number of computational methods have been developed for the deconvolution of immune cell types within solid tumors. We have taken the NK cell gene signatures from several tools, then curated and expanded this list using recent reports from the literature. Using a gene set scoring method to investigate RNA-seq data from The Cancer Genome Atlas (TCGA) we show that patients with metastatic cutaneous melanoma have an improved survival rate if their tumor shows evidence of greater NK cell infiltration. Furthermore, these survival effects are enhanced in tumors which have a higher expression of NK cell stimuli such as IL-15, suggesting NK cells are part of a coordinated immune response within these patients. Using this signature we then examine transcriptomic data to identify tumor and stromal components which may influence the penetrance of NK cells into solid tumors.These data support a role for NK cells in the regulation of human tumors and highlight potential survival effects associated with increased NK cell activity. Furthermore, our computational analysis identifies a number of potential targets which may help to unleash the anti-tumor potential of NK cells as we enter the age of immunotherapy.

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Human cytokine-induced memory-like NK cells preserve increased glycolysis but the glycolytic-dependence of their effector functions differ between stimuli

10.1101/2020.08.20.258731 ◽

2020 ◽

Author(s):

Iñigo Terrén ◽

Ane Orrantia ◽

Alba Mosteiro ◽

Joana Vitallé ◽

Olatz Zenarruzabeitia ◽

...

Keyword(s):

Nk Cells ◽

Cancer Immunotherapy ◽

Natural Killer ◽

Nk Cell ◽

Cellular Functions ◽

Effector Functions ◽

Cytokine Stimulation ◽

Cancer Immunotherapies ◽

Cell Effector ◽

Human Cytokine

ABSTRACTNatural Killer (NK) cells acquire memory-like properties following a brief stimulation with IL-12, IL-15 and IL-18. These IL-12/15/18-stimulated NK cells, also known as cytokine-induced memory-like (CIML) NK cells, have been revealed as a powerful tool in cancer immunotherapy due to their persistence in the host and their increased effector functions. Several studies have shown that NK cells modulate their metabolism in response to cytokine-stimulation and other stimuli, suggesting that there is a link between metabolism and cellular functions. In this paper, we have analyzed metabolic changes associated to IL-12/15/18-stimulation and the relevance of glycolytic pathway for NK cell effector functions. We have found that CIML NK cells are able to retain increased glycolytic machinery seven days after cytokine withdrawal. Furthermore, we found that glycolytic inhibition with 2-DG is stimuli-dependent and that differently affects to distinct effector functions. These findings may have implications in the design of NK cell-based cancer immunotherapies.

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M144, a Murine Cytomegalovirus (Mcmv)-Encoded Major Histocompatibility Complex Class I Homologue, Confers Tumor Resistance to Natural Killer Cell–Mediated Rejection

Journal of Experimental Medicine ◽

10.1084/jem.190.3.435 ◽

1999 ◽

Vol 190 (3) ◽

pp. 435-444 ◽

Cited By ~ 60

Author(s):

Erika Cretney ◽

Mariapia A. Degli-Esposti ◽

Eloise H. Densley ◽

Helen E. Farrell ◽

Nick J. Davis-Poynter ◽

...

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Cell Line ◽

Nk Cell ◽

Effector Function ◽

Class I ◽

Murine Cytomegalovirus ◽

Cell Effector Function ◽

Cell Effector

Until now, it has been unclear whether murine cytomegalovirus (MCMV)-encoded protein m144 directly regulates natural killer (NK) cell effector function and whether the effects of m144 are only strictly evident in the context of MCMV infection. We have generated clones of the transporter associated with antigen processing (TAP)-2–deficient RMA-S T lymphoma cell line and its parent cell line, RMA, that stably express significant and equivalent levels of m144. In vivo NK cell–mediated rejection of RMA-S-m144 lymphomas was reduced compared with rejection of parental or mock-transfected RMA-S clones, indicating the ability of m144 to regulate NK cell–mediated responses in vivo. Significantly, the accumulation of NK cells in the peritoneum was reduced in mice challenged with RMA-S-m144, as was the lytic activity of NK cells recovered from the peritoneum. Expression of m144 on RMA-S cells also conferred resistance to cytotoxicity mediated in vitro by interleukin 2–activated adherent spleen NK cells. In summary, the data demonstrate that m144 confers some protection from NK cell effector function mediated in the absence of target cell class I expression, but that in vivo the major effect of m144 is to regulate NK cell accumulation and activation at the site of immune challenge.

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Enhancing network activation in Natural Killer cells: Predictions from in silico modeling

10.1101/395756 ◽

2018 ◽

Cited By ~ 1

Author(s):

Sahak Z. Makaryan ◽

Stacey D. Finley

Keyword(s):

Nk Cells ◽

Natural Killer ◽

In Silico ◽

Intracellular Signaling ◽

Cell Activation ◽

Nk Cell ◽

Mechanistic Model ◽

Published Data ◽

Cell Therapies ◽

Network Activation

ABSTRACTNatural killer (NK) cells are part of the innate immune system and are capable of killing diseased cells. As a result, NK cells are being used for adoptive cell therapies for cancer patients. The activation of NK cell stimulatory receptors leads to a cascade of intracellular phosphorylation reactions, which activates key signaling species that facilitate the secretion of cytolytic molecules required for cell killing. Strategies that maximize the activation of such intracellular species can increase the likelihood of NK cell activation upon contact with a cancer cell, and thereby improve efficacy of NK cell-based therapies. However, due to the complexity of intracellular signaling, it is difficult to deduce a priori which strategies can enhance species activation. Therefore, we constructed a mechanistic model of the CD16, 2B4 and NKG2D signaling pathways in NK cells to simulate strategies that enhance signaling. The model predictions were fit to published data and validated with a separate dataset. Model simulations demonstrate strong network activation when the CD16 pathway is stimulated. The magnitude of species activation is most sensitive to the receptor concentration and the rate at which the receptor is deactivated. Co-stimulation of CD16 and NKG2D in silico required fewer ligands to achieve half-maximal activation than other combinations, suggesting co-stimulating these pathways is most effective in activating the species. We applied the model to predict the effects of perturbing the signaling network and found two strategies that can potently enhance network activation. When the availability of ligands is low, it is more influential to engineer NK cell receptors that are resistant to proteolytic cleavage. In contrast, for high ligand concentrations, inhibiting phosphatase activity leads to more activation. The work presented here establishes a framework for understanding the complex, nonlinear aspects of NK cell signaling and provides detailed strategies for enhancing NK cell activation.

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Natural Killer Cells: The Linchpin for Successful Cancer Immunotherapy

Frontiers in Immunology ◽

10.3389/fimmu.2021.679117 ◽

2021 ◽

Vol 12 ◽

Author(s):

Kari A. Shaver ◽

Tayler J. Croom-Perez ◽

Alicja J. Copik

Keyword(s):

T Cells ◽

Immune System ◽

Nk Cells ◽

Cancer Immunotherapy ◽

Natural Killer ◽

Nk Cell ◽

Checkpoint Inhibitors ◽

Cytotoxic T Cells ◽

Current Evidence ◽

Cell Therapies

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.

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