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

2018 ◽  
Vol 2018 ◽  
pp. 1-20 ◽  
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.

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

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.

scholarly journals Genetic Engineering of Natural Killer Cells for Enhanced Antitumor Function

2020 ◽  
Vol 11 ◽  
Author(s):  
Simone Mantesso ◽  
Dirk Geerts ◽  
Jan Spanholtz ◽  
Lucia Kučerová
Keyword(s):  
Tumor Microenvironment ◽  
Genetic Engineering ◽  
Nk Cells ◽  
Natural Killer ◽  
Solid Tumors ◽  
Nk Cell ◽  
Induced Response ◽  
Cell Therapies ◽  
Anti Tumor Activity ◽  
Cell Effector

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.

scholarly journals Characterization and Potential Applications of Dog Natural Killer Cells in Cancer Immunotherapy

2019 ◽  
Vol 8 (11) ◽  
pp. 1802 ◽  
Author(s):  
Alicia A. Gingrich ◽  
Jaime F. Modiano ◽  
Robert J. Canter
Keyword(s):  
Immune System ◽  
Nk Cells ◽  
Cancer Immunotherapy ◽  
Natural Killer ◽  
Ex Vivo ◽  
Genetic Regulation ◽  
Companion Animals ◽  
Clinical Applications ◽  
Similarities And Differences

Natural killer (NK) cells of the innate immune system are a key focus of research within the field of immuno-oncology based on their ability to recognize and eliminate malignant cells without prior sensitization or priming. However, barriers have arisen in the effective translation of NK cells to the clinic, in part because of critical species differences between mice and humans. Companion animals, especially dogs, are valuable species for overcoming many of these barriers, as dogs develop spontaneous tumors in the setting of an intact immune system, and the genetic and epigenetic factors that underlie oncogenesis appear to be similar between dogs and humans. Here, we summarize the current state of knowledge for dog NK cells, including cell surface marker phenotype, key NK genes and genetic regulation, similarities and differences of dog NK cells to other mammals, especially human and mouse, expression of canonical inhibitory and activating receptors, ex vivo expansion techniques, and current and future clinical applications. While dog NK cells are not as well described as those in humans and mice, the knowledge of the field is increasing and clinical applications in dogs can potentially advance the field of human NK biology and therapy. Better characterization is needed to truly understand the similarities and differences of dog NK cells with mouse and human. This will allow for the canine model to speed clinical translation of NK immunotherapy studies and overcome key barriers in the optimization of NK cancer immunotherapy, including trafficking, longevity, and maximal in vivo support.

Engineered natural killer cells impede the immunometabolic CD73-adenosine axis in solid tumors

2021 ◽  
Author(s):  
Andrea Marie Chambers ◽  
Kyle Byrnes Lupo ◽  
Jiao Wang ◽  
Jingming Cao ◽  
Sandra Toregrosa-Allen ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Solid Tumors ◽  
Immune Evasion ◽  
Immune Cells ◽  
Nk Cell ◽  
Purinergic Signaling ◽  
Single Agent ◽  
Antibody Specificity

Immunometabolic reprogramming due to CD73-produced adenosine is a recognized immunosuppressive mechanism contributing to immune evasion in solid tumors. Adenosine is not only known to contribute to tumor progression, but it has specific roles in driving dysfunction of immune cells, including natural killer (NK) cells. Here, we engineered NK cells to directly target the CD73-adenosine axis by blocking the enzymatic activity of CD73. In doing so, the engineered NK cells not only impaired adenosinergic metabolism driven by the hypoxic uptake of ATP by cancer cells, but also mediated killing of tumor cells due to the specific recognition of overexpressed CD73. This results in a "single agent" immunotherapy that combines antibody specificity, blockade of purinergic signaling, and killing of targets mediated by NK cells. We also showed that CD73-targeted NK cells are potent in vivo and result in tumor arrest, while promoting NK cell infiltration into CD73+ tumors and enhanced intratumoral activation.

scholarly journals Peptide:MHC dependent activation of natural killer cells through KIR2DS2 generates anti-tumor responses

2020 ◽  
Author(s):  
Pauline Rettman ◽  
Matthew D. Blunt ◽  
Berenice Mbiribindi ◽  
Rebecca Fulton ◽  
Ralf B. Schittenhelm ◽  
...  
Keyword(s):  
Immune Responses ◽  
Nk Cells ◽  
Cancer Immunotherapy ◽  
Natural Killer ◽  
Viral Infections ◽  
Killer Cell ◽  
Peptide Ligand ◽  
Dependent Manner ◽  
Human Cancers

AbstractNatural killer (NK) cells are key components of the immune response to viral infections and cancer. Their functions are controlled by activating and inhibitory killer-cell immunoglobulin-like receptors (KIR) which have MHC class I ligands. KIR2DS2 is an activating KIR, that binds conserved viral peptides in the context of HLA-C and has been associated with protective responses to both cancer and viral infections. We sought to investigate whether NK cells can be specifically activated in a peptide:MHC dependent manner to generate functional immune responses as a potential immunotherapeutic strategy.We developed a peptide-based KIR targeting DNA vaccine. Immunizing KIR-Tg mice with the vaccine construct generated in vivo peptide-specific activation of KIR2DS2-positive NK cells leading to canonical and cross-reactive peptide specific immune responses in vitro, and also in vivo inhibition of tumor growth. Using immunopeptidomics we identified that the nuclear export protein XPO1, which has been associated with a poor prognosis in many different human cancers, furnishes an HLA-C restricted cancer-associated peptide ligand for KIR2DS2-positive NK cells. We thus define a novel strategy to activate KIR in a peptide-specific manner and identify a rationale for its use in cancer immunotherapy.Significance statementNatural killer (NK) cells are known to have important roles in determining the outcomes of viral infections and cancer. The killer cell immunoglobulin-like receptors (KIR), and in particular the activating receptor KIR2DS2, have been associated with the outcome of a number of different human cancers. Specific activation of NK cells through KIR2DS2 is challenging because it shares high (>98%) sequence homology with related inhibitory KIR. We have used a peptide:MHC targeting strategy to activate NK cells through KIR2DS2 and identified a novel cancer-associated ligand for this receptor. The work provides a proof-of-concept for targeting NK cells through activating KIR as a cancer immunotherapy strategy.

scholarly journals Natural Killer Cells as Allogeneic Effectors in Adoptive Cancer Immunotherapy

Cancers ◽  
2019 ◽  
Vol 11 (6) ◽  
pp. 769 ◽  
Author(s):  
Kyle B. Lupo ◽  
Sandro Matosevic
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Cancer Immunotherapy ◽  
Natural Killer ◽  
Adoptive Transfer ◽  
Nk Cell ◽  
Human Leukocyte ◽  
Hematologic Malignancies ◽  
Published Evidence

Natural killer (NK) cells are attractive within adoptive transfer settings in cancer immunotherapy due to their potential for allogeneic use; their alloreactivity is enhanced under conditions of killer immunoglobulin-like receptor (KIR) mismatch with human leukocyte antigen (HLA) ligands on cancer cells. In addition to this, NK cells are platforms for genetic modification, and proliferate in vivo for a shorter time relative to T cells, limiting off-target activation. Current clinical studies have demonstrated the safety and efficacy of allogeneic NK cell adoptive transfer therapies as a means for treatment of hematologic malignancies and, to a lesser extent, solid tumors. However, challenges associated with sourcing allogeneic NK cells have given rise to controversy over the contribution of NK cells to graft-versus-host disease (GvHD). Specifically, blood-derived NK cell infusions contain contaminating T cells, whose activation with NK-stimulating cytokines has been known to lead to heightened release of proinflammatory cytokines and trigger the onset of GvHD in vivo. NK cells sourced from cell lines and stem cells lack contaminating T cells, but can also lack many phenotypic characteristics of mature NK cells. Here, we discuss the available published evidence for the varying roles of NK cells in GvHD and, more broadly, their use in allogeneic adoptive transfer settings to treat various cancers.

scholarly journals 215 Tissue-resident natural killer cells resembling intraepithelial ILC1 have potent anti-tumor activity in human head and neck cancer and represent a novel class of effector cells for immunotherapy

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A228-A228
Author(s):  
June Shin ◽  
Nina Horowitz ◽  
Quan Tran ◽  
Chen Chen ◽  
Uriel Moreno-Nieves ◽  
...  
Keyword(s):  
Head And Neck Cancer ◽  
Tumor Microenvironment ◽  
Nk Cells ◽  
Head And Neck ◽  
Natural Killer ◽  
Human Head ◽  
Effector Cells ◽  
Anti Tumor Activity

BackgroundNatural killer (NK) cells comprise a subset of the innate lymphoid cell (ILC) family. Although NK cells have been observed to be present in most solid tumors, their role in the protection against tumor formation in humans has been unclear. Studies have been hampered by the heterogeneity of NK cells within the tumor microenvironment (TME) and lack of information about the broader ILC subsets found in tumors. Further, there is an increasing recognition of plasticity between NK cells and other ILC family members in various disease contexts, calling for a broader examination of ILCs within solid tumors. We previously analyzed the ILC population in primary samples from human head and neck squamous cell carcinoma (HNSCC) and matched blood by single-cell RNA sequencing (scRNA-seq).1 Those studies revealed that peripheral NK cells differentiate along two divergent trajectories in the TME, resulting in different end-states: one with a hyporesponsive phenotype and another possessing potent anti-tumor activity and resembling intraepithelial ILC1s (ieILC1s).MethodsIn vitro co-culture approaches and in vivo mouse models were used to investigate the ability of peripheral NK cells to differentiate into alternate ILC states with heterogeneous functions. Cytotoxicity assays were used to assess functional activity of in vitro derived ieILC1-like cells. Adoptive cell transfer of ieILC1-like cells into tumor-bearing mice was also used to assess anti-tumor function.ResultsPeripheral human NK cells could be efficiently differentiated into ieILC1-like cells using an in vitro co-culture system. These ieILC1-like cells had enhanced natural cytotoxicity against target cells compared to conventional IL-15-activated and K562-expanded NK cells. In addition, they infiltrated the TME efficiently and were a more effective means of adoptive cell therapy against HNSCC solid tumor xenografts in vivo compared to conventional NK cells.ConclusionsOur data indicate that peripheral NK cells change cell states within the TME of HNSCC. The heterogeneity in the relative proportion of the cell states may influence host response to tumors. We identified the ieILC1-like cell state to be the phenotype with the most potent anti-tumor activity within the TME. Importantly, this cell state can be induced from peripheral donor NK cells ex vivo for differentiation into and expansion of highly active ieILC1-like cells, providing a platform for a novel class of effector cells for adoptive cell immunotherapy.AcknowledgementsThese studies were supported by the Lokey Stem Cell Research Building (SIM1) Flow Cytometry core facility for cell sorting and flow cytometric analysis and the Stanford Cancer Institute Tissue Bank for procurement of tumor samples and blood. This work was supported by funding from the National Institutes of Health (R01CA158516; R35DE030054; U54CA209971) to J.B.S.ReferenceMoreno-Nieves UY, Tay JK, Saumyaa S, Shin JH, Horowitz NB, Mohammad IA, Luca B, Mundy DC, Gulati GS, Bedi N, Chang S, Chen C, Kaplan MJ, Rosenthal EL, Holsinger FC, Divi V, Baik FM, Sirjani DB, Gentles AJ, Newman AM, Freud AG, Sunwoo JB. Landscape of ILCs in human head and neck cancer reveals divergent NK cell states in the tumor microenvironment. Proc Natl Acad Sci U S A 2021;118(28):e2101169118.Ethics ApprovalThe studies reported here were approved by the Stanford Institutional Review Board (IRB 11402) and the Stanford Administrative Panel on Laboratory Animal Care (APLAC 20547).

scholarly journals Sonazoid-Conjugated Natural Killer Cells for Tumor Therapy and Real-Time Visualization by Ultrasound Imaging

Pharmaceutics ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 1689
Author(s):  
Hyeong-Woo Song ◽  
Han-Sol Lee ◽  
Seok-Jae Kim ◽  
Ho Yong Kim ◽  
You Hee Choi ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Real Time ◽  
Solid Tumors ◽  
Ultrasound Imaging ◽  
Tumor Therapy ◽  
Real Time Control ◽  
Time Control

Various cell therapy strategies, including chimeric antigen receptor-expressing T or natural killer (NK) cells and cell-mediated drug delivery, have been developed for tumor eradication. However, the efficiency of these strategies against solid tumors remains unclear. We hypothesized that real-time control and visualization of therapeutic cells, such as NK cells, would improve their therapeutic efficacy against solid tumors. In this study, we engineered Sonazoid microbubble-conjugated NK (NK_Sona) cells and demonstrated that they were detectable by ultrasound imaging in real-time and maintained their functions. The Sonazoid microbubbles on the cell membrane did not affect the cytotoxicity and viability of the NK cells in vitro. Additionally, the NK_Sona cells could be visualized by ultrasound imaging and inhibited tumor growth in vivo. Taken together, our findings demonstrate the feasibility of this new approach in the use of therapeutic cells, such as NK cells, against solid tumors.

163 Nice: neoantigen-cytokine-chemokine multifunctional engager for NK cell immunotherapy of solid tumors

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A176-A176
Author(s):  
Xue Yao ◽  
Sandro Matosevic
Keyword(s):  
Tumor Microenvironment ◽  
Nk Cells ◽  
Solid Tumors ◽  
Immune Cells ◽  
Nk Cell ◽  
Wilms Tumor ◽  
Killer Cell ◽  
Immunosuppressive Tumor Microenvironment

BackgroundThe effectiveness of natural killer cell-based immunotherapy against solid tumors is limited by the lack of specific antigens and the immunosuppressive tumor microenvironment. To improve the clinical efficacy and specificity of NK cell therapy, we are designing, developing, and characterizing a new generation of multi-specific killer engagers, which consists of a neoantigen-targeting moiety, together with cytokine and chemokine-producing domains.MethodsTargeting a neoantigen-an antigen formed specifically in response to tumor genome mutations-enables substantially enhanced tumor specificity to be achieved. We evaluated the responsiveness of NK cells to Wilms Tumor 1 (WT1) antigen in GBM by synthesizing an antibody that is able to recognize the WT1/HLA complex. Incorporation of cytokine (namely IL-2, IL-15, and IL-21)-essential for the maturation, persistence, and expansion of NK cells in vivo-favors the proliferation and survival of NK cells in the tumor microenvironment, thereby leading to more sustained anti-tumor responses. Additionally, our data have indicated that the chemokine CXCL10 plays an important role in the infiltration of immune cells into GBM, yet the chemokine itself is expressed at low levels in GBM. Incorporation of a CXCL10-producing element into our construct further supports NK cell recruitment and may stimulate the recruitment of other immune cells. NK activation through the tri-specific engager is achieved through NKp46-mediated signaling. We are investigating the ability of the tri-functional engager to support and enhance NK cell-mediated cytotoxicity against GBM in vitro and in patient-derived GBM xenografts in vivo.ResultsWe hypothesize that taking advantage of our multi-functional engager, NK cells will exhibit, at once, superior persistence, infiltration and antitumor activity, simultaneously addressing three of the main limitations to the use of NK cells in immunotherapy of GBM and other solid tumors.ConclusionsN/AAcknowledgementsN/A

scholarly journals Nonviral genome engineering of natural killer cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Gabrielle M. Robbins ◽  
Minjing Wang ◽  
Emily J. Pomeroy ◽  
Branden S. Moriarity
Keyword(s):  
T Cell ◽  
Tumor Microenvironment ◽  
Gene Transfer ◽  
Nk Cells ◽  
Natural Killer ◽  
Genome Engineering ◽  
Cellular Therapy ◽  
Nk Cell ◽  
Cell Based Therapy ◽  
Genetically Engineer

AbstractNatural killer (NK) cells are cytotoxic lymphocytes of the innate immune system capable of immune surveillance. Given their ability to rapidly and effectively recognize and kill aberrant cells, especially transformed cells, NK cells represent a unique cell type to genetically engineer to improve its potential as a cell-based therapy. NK cells do not express a T cell receptor and thus do not contribute to graft-versus-host disease, nor do they induce T cell-driven cytokine storms, making them highly suited as an off-the-shelf cellular therapy. The clinical efficacy of NK cell-based therapies has been hindered by limited in vivo persistence and the immunosuppressive tumor microenvironment characteristic of many cancers. Enhancing NK cell resistance to tumor inhibitory signaling through genome engineering has the potential to improve NK cell persistence in the tumor microenvironment and restore cytotoxic functions. Alongside silencing NK cell inhibitory receptors, NK cell killing can be redirected by the integration of chimeric antigen receptors (CARs). However, NK cells are associated with technical and biological challenges not observed in T cells, typically resulting in low genome editing efficiencies. Viral vectors have achieved the greatest gene transfer efficiencies but carry concerns of random, insertional mutagenesis given the high viral titers necessary. As such, this review focuses on nonviral methods of gene transfer within the context of improving cancer immunotherapy using engineered NK cells.

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