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.

scholarly journals Cord Blood Derived Natural Killer Cells Loaded with a Tetravalent Bispecific Antibody Construct (AFM13) As Off-the-Shelf Cell Therapy for CD30+ Malignancies

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 341-341
Author(s):  
Lucila Kerbauy ◽  
Mecit Kaplan ◽  
Pinaki P Banerjee ◽  
Francesca Lorraine Wei Inng Lim ◽  
Ana Karen Nunes Cortes ◽  
...  
Keyword(s):  
T Cell ◽  
Cord Blood ◽  
Nk Cells ◽  
Natural Killer ◽  
Research Funding ◽  
Bispecific Antibody ◽  
Nk Cell ◽  
Ex Vivo

Abstract Chimeric antigen receptors to redirect T cell specificity against tumor antigens have shown remarkable clinical responses against CD19+ malignancies. However, the manufacture of an engineered autologous T cell product is expensive and cumbersome. Natural killer (NK) cells provide an alternative source of immune effectors for the treatment of cancer. NK cell cytolytic function can be directed towards specific targets by exploiting their ability to mediate antibody-dependent cellular cytotoxicity (ADCC) through the NK cell Fc receptor, CD16 (FcγRIIIa). AFM13 is a tetravalent bispecific antibody construct based on Affimed's ROCK™ platform. AFM13 is bispecific for CD30 and CD16A, designed for the treatment of CD30 expressing malignancies. It binds CD16A on the surface of NK cells, thus activating and recruiting them to CD30 expressing tumor cells and mediating subsequent tumor cell killing. Since autologous NK effector function is impaired in many patients with malignancies, we propose to overcome this by the use of allogeneic NK cells in combination with AFM13. Cord blood (CB) is a readily available ("off-the-shelf") source of allogeneic NK cells that can be expanded to large, highly functional therapeutic doses. The feasibility and safety of therapy with allogeneic ex vivo expanded CB-derived NK cells have been shown by our group and others. In this study, we hypothesized that we can redirect the specificity of NK cells against CD30+ malignancies by preloading ex vivo activated and expanded CB-derived NK cells with AFM13 prior to adoptive infusion. Briefly, mononuclear cells were isolated from fresh or frozen CB units by ficoll density gradient centrifugation. CD56+ NK cells were cultured with rhIL-12, rhIL-18 and rhIL-15 for 16 hrs, followed by ex vivo expansion with rhIL-2 and irradiated (100 Gy) K562-based feeder cells expressing membrane-bound IL-21 and CD137-ligand (2:1 feeder cell:NK ratio). After 14 days, NK cells were loaded with serial dilutions of AFM13 (0.1, 1, 10 and 100 mg/ml). After washing twice with PBS, we tested the effector function of AFM13-loaded NK-cells (AFM13-NK) compared to expanded CB-NK cells without AFM13 against Karpas-299 (CD30 positive) and Daudi (CD30 negative) lymphoma cell lines by 51Cr release and intracellular cytokine production assays. AFM13-NK cells killed Karpas-299 cells more effectively at all effector:target ratios tested than unloaded NK cells (Figure 1) and produced statistically more INFγ and CD107a (P=0.0034; P=0.0031 respectively, n=4). In contrast, AFM13-NK cells and unloaded NK cells exerted similar cytotoxicity against Daudi cells. Next, we established the optimal concentration of AFM13 for loading (determined to be 100 μg/ml) and the optimal incubation time to obtain maximal activity (1 h) in a series of in vitro experiments. We also confirmed that the activity of AFM13-NK cells against Karpas-299 cells remains stable for at least 72h post-wash (Figure 2). Additionally, we characterized the phenotype of AFM13-NK vs. unloaded NK cells by flow cytometry using monoclonal antibodies against 22 markers, including markers of activation, inhibitory receptors, exhaustion markers and transcription factors. Compared to unloaded NK cells, AFM13-NK cells expressed higher levels of CD25, CD69, TRAIL, NKp44, granzyme B and CD57, consistent with an activated phenotype. We next tested the in vivo anti-tumor efficacy of AFM13-NK cells in an immunodeficient mouse model of FFluc-Karpas-299. Briefly, six groups of NOD/SCID/IL2Rγc null mice (n=5 per group) were transplanted by tail-vein injection with 1 x 10e5 FFluc-transduced Karpas cells. Group 1 and 6 received tumor alone or tumor + AFM13 and served as a control. Groups 2-4 receive Karpas FFLuc with either expanded NK cells or AFM13-NK cells (NK cells loaded with AFM13) or expanded NK cells and AFM13 injected separately. Group 5 received AFM13-NK cells without tumor. Initial studies confirm the antitumor activity of AFM13-NK cells. In summary, we have developed a novel premixed product, comprised of expanded CB-NK cells loaded with AFM13 to 'redirect' their specificity against CD30+ malignancies. The encouraging in vitro and in vivo data observed in this study, provide a strong rationale for a clinical trial to test the strategy of an off-the-shelf adoptive immunotherapy with AFM13-loaded CB-NK cells in patients with relapsed/refractory CD30+ malignancies. Disclosures Champlin: Sanofi: Research Funding; Otsuka: Research Funding. Koch:Affimed GmbH: Employment. Treder:Affimed GmbH: Employment. Shpall:Affirmed GmbH: Research Funding. Rezvani:Affirmed GmbH: Research Funding.

Assessment of PD-1 expression in human resting and activated natural killer cells and murine tumor models.

2019 ◽  
Vol 37 (8_suppl) ◽  
pp. 36-36
Author(s):  
Sean J. Judge ◽  
Cordelia Dunai ◽  
Ian R. Sturgill ◽  
Kevin M. Stoffel ◽  
William J. Murphy ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Ex Vivo ◽  
Fold Increase ◽  
Wild Type ◽  
Immunodeficient Mice

36 Background: Blockade of the PD-1/PD-L1/2 axis has revolutionized cancer therapy. Although reinvigorated PD-1+ T cells are the main effectors in the response to checkpoint blockade, the contribution of Natural Killer (NK) cells to PD-1/PD-L1 inhibition is under debate. While PD-1 has been identified on NK cells, this appears to be restricted to small populations under limited conditions. We sought to evaluate the extent of PD-1 expression in mouse and human resting and activated NK cells. Methods: Human NK cells were isolated from healthy donor PBMCs and cancer patients. Ex vivo activation and proliferation techniques included recombinant human cytokine and feeder line co-culture. Murine NK cells were isolated from splenocytes, and PBMCs from wild type and immunodeficient mice. We assessed NK cell surface markers and intracellular cytokine by flow cytometry, and gene expression by quantitative RT-PCR. Results: Over 21-days of ex vivo expansion, expression of PD-1 or PD-L1 on human NK cells was < 1% at all time points, while TIGIT+ expression increased to > 85%. Conversely, ConA stimulation of T cells increased PD-1 expression with no change in TIGIT expression. QRT-PCR demonstrated absent PD-1 expression in purified NK cells compared to a 5-fold increase in PD-1 gene expression in ConA stimulated PBMCs. PD-1/PD-L1 was also < 1% in the NK92 cell line and < 2.5% in peripheral CD56+CD3- NK cells from patients with soft tissue sarcoma (STS). NK cells from digested freshly resected STS show variable PD-1 ( < 10%) and minimal PD-L1 ( < 1%) expression with a small, but measurable population of intra-tumoral NK cells (1% of immune cells). In vivo mouse studies showed < 5% PD-1+ NK cells in spleen and tumor of CT26 tumor-bearing mice, while PD-L1+ NK cells increased in frequency from spleen (5-35%) to tumor (40-95%) in both wild type BALB/C and SCID mice. Conclusions: In contrast to prior studies, we did not observe a substantial PD-1+ population on human or murine NK cells after multiple activation strategies compared to T cells. Contrary to its application in T cells, our data suggest that PD-1 is not a useful marker for NK cell exhaustion/dysfunction. PD-L1 on NK cells may represent an important link between NK and T cell immunotherapy.

The Unique Ontogeny Of Natural Killer Cells As Revealed By Genetic Barcoding In The Nonhuman Primate Model

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 15-15
Author(s):  
Chuanfeng Wu ◽  
Jan K. Davidson-Moncada ◽  
Rong Lu ◽  
Brian Li ◽  
Allen E. Krouse ◽  
...  
Keyword(s):  
Lymph Node ◽  
Nk Cells ◽  
Natural Killer ◽  
Adoptive Transfer ◽  
Ex Vivo ◽  
Clonal Diversity ◽  
Hematopoietic Stem ◽  
Over Time

Abstract Natural killer (NK) cells are cytotoxic leukocytes defined in humans and rhesus macaques by the expression of CD56 and/or CD16, and the absence of T, B, and myeloid markers. Functionally, they are defined by lysis of tumor targets in a major histocompatibility complex (MHC)–unrestricted fashion, and production of cytokines critical to innate immunity. Ex vivo expanded NK cells are being developed as adoptive transfer therapeutics for a variety of malignancies. However, the ontogeny and hematopoietic lineage relationships of human NK cells have been difficult to study, given differences in phenotype and function between human and murine NK cells, poor NK engraftment and development in human xenografts, and restricted development of NK cells from hematopoietic stem and progenitor cells (HSPC) in vitro. In addition, several phenotypic subsets of NK cells have been described, and their relationships and relative locations within the hematopoietic hierarchy are supported by limited in vivo data. We “barcoded” individual CD34+ HSPC from 3 macaques using lentiviral vectors carrying highly diverse oligonucleotides, allowing precise quantitation of clonal contributions via low cycle PCR amplification followed by high-throughput sequencing and computational analysis (Lu et al, Nat Biotech). We quantitated barcode levels, and thus individual HSPC clonal contributions, over time and between lineages, following TBI and infusion of autologous barcoded CD34+ cells. Reconstitution of the NK compartment was clonally distinct from T, B and myeloid (My) lineages. Following reconstitution of NK, T, B and My from uni-lineage progenitors at 1-2 months(m), by 3-4 m, as expected from murine and in vitro models, multi-lineage clones began to contribute and dominate, first B/My, then B/T/My. However, NK cells remained clonally distinct through 9 m, despite overall clonal diversity and marking levels in NK lineage similar to B/T and My. Even the most abundant clones in the NK lineage were not found contributing in any significant way to B/T or My lineages. Shared B/T/My/NK clones finally began to contribute more extensively at 9-14 m. We fractionated peripheral blood NK cells into 3 NK subsets and compared them to overall PB and lymph node NK cells at 4.5-6.5 m. The majority (75-90%) of PB rhesus NK cells are found in the CD16+/CD56- PB subset (corresponding to human CD16+/CD56dim cytotoxic NK), and this subset accounted for the clonal pattern in overall NK cells (Pearson r=0.98 vs overall NK cells), and the disparity from B/T/My, with r values all <0.17 for CD16+/CD56- vs B/T or My lineages at the same time point. The minor PB (but dominant lymph node) cytokine-producing CD16-/CD56+ or CD16+/CD56+ NK subsets, previously hypothesized to be precursors for CD16+/CD56- NK cells, had clonal patterns that were more closely correlated with T/B/My lineages (r=0.37-0.62) than the CD16+/CD56- cells. The clonal correlations between the putative precursor CD16-/CD56+ cells and mature cytotoxic CD16+/CD56- cells was very low, only r=0.08. Furthermore, we expanded purified NK cells from barcoded macaques in vitro in the presence of IL-2 and irradiated EBV-LCL cells and assessed NK phenotype, function, and clonal diversity over time. 40-90X expansion was achieved by 15 days and was highly polyclonal, with 90% of the starting number of barcodes still present at the end of expansion. Clonal contribution levels between pre and post-expanded NK were highly correlated (r=0.73). CD16+/CD56+ cells became more dominant in post-exp NK cells, in contrast to the majority CD16+CD56- pre-exp. The clonal contributions in the post-exp CD16+/CD56- and CD16+/CD56+ cells correlated with each other (r=0.66), and with the starting CD16+/CD56- cells (r=0.75), but not with the starting CD16-/CD56+ (r=0.15) nor the post-exp CD16-/CD56+ cells (r=0.18). Our in vivo and in vitro results call into question the hypothesis that CD16-/CD56+ NK cells are precursors of circulating cytotoxic CD16+/CD56- NK cells, as does the complete deficiency of CD16-/CD56+ but not CD16+/CD56dim cells in GATA2 mutant patients. Our results also reveal that the dominant blood rhesus NK cell population has a distinct ontogeny in macaques, and thus potentially in humans. NK cells expanded ex vivo using EBV-LCL cells are polyclonal, and barcoding allows dissection of events during expansion, and will permit tracking studies of expanded cells following adoptive transfer. Disclosures: No relevant conflicts of interest to declare.

41BBL-Based Activation and Expansion of Autologous Natural Killer Cells Results in Enhanced Activity Against Leukemia Including ALL

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2293-2293
Author(s):  
Ekta Kapadia ◽  
Elad Jacoby ◽  
Mark Kohler ◽  
Waleed Haso ◽  
Christopher Daniel Chien ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Lymphoblastic Leukemia ◽  
Myelogenous Leukemia ◽  
Hematopoietic Stem ◽  
Enhanced Activity

Abstract Childhood leukemia is the most common pediatric malignancy. There are now excellent cure rates for these patients, however outcomes remain poor for those with refractory disease and for those who relapse after standard salvage therapies, with a disease recurrence of approximately 50%. Therefore, development of novel cellular therapies is essential to treat these refractory patients. Natural Killer (NK) cells generated from an allograft contribute to improved disease free survival after Hematopoietic Stem Cell Transplantation for leukemia when there is a KIR mismatch. This effect appears to be particularly potent in the setting of Acute Myelogenous Leukemia (AML) with less benefit demonstrated in Acute Lymphoblastic Leukemia (ALL). Preclinical studies have also suggested that activation and expansion of resting NK cells can enhance NK cell cytotoxicity and eliminate the need for KIR mismatch due to up-regulation of activating receptors. We are currently testing this approach in the clinic following a fully matched allogeneic transplant platform for leukemia. Our aim is to explore whether 41BB ligand (41BBL) and recombinant IL-15 (rIL-15) mediated ex vivo expansion of autologous NK cells results in enhanced activity against AML and ALL. The activation/expansion process may allow for the use of autologous NK cell infusions, thus eliminating the need for allogeneic NK cell donors. To test this hypothesis, we ex vivo expanded and activated NK cells derived from C57BL/6J (B6) mice using artificial Antigen Presenting Cells (aAPCs) containing 41BBL and rIL-15 for 7-14 days. NK cells were co-cultured with murine AML cells (C1498) and murine ALL cells (E2A-PBX) – both on B6 background. Controls included YAC cells (murine T-cell lymphoma cell line sensitive to NK cell killing) as well as Phorbol Myristate Acetate (PMA)/ionomycin. All cells were co-cultured for 5 hours prior to functional assessment of NK cells via CD107a degranulation. NK cells cultured with 41BBL aAPCs and rIL-15 had a 30-fold expansion in numbers (Figure 1) and an increase in purity to approximately 95-98% (NK1.1+, CD3–) by Day 7. In the absence of cytokine or aAPCs, cultured NK cells underwent rapid apoptosis. Functionally, although resting NK cells (harvested prior to assessment) expressed CD107a when cultured with YAC cells and PMA, only minimal degranulation was observed in the presence of autologous AML cells or ALL cells. In contrast, activated and expanded autologous NK cells displayed enhanced activity against ALL, AML, as well as YAC cells, while only minimal levels of CD107a were seen in the absence of targets (Figure 2). In vivo experiments with a single injection of activated and expanded NK cells did not result in prolonged survival of mice bearing either AML or ALL. Assessment of adoptively transferred NK cells demonstrated very transient persistence (<2 days) with no in vivo expansion, suggesting that repeated injections may be necessary for leukemia eradication. Future murine experiments will investigate the effect repeated injections of activated/expanded NK cells and/or the administration of rIL-15 will have on survival and leukemia eradication. In addition, the ability to activate and expand NK cells in culture provides an opportunity for lentiviral-based transduction with chimeric antigen receptor (CAR) vectors. We are currently testing this with a murine CD19 CAR. These experiments suggest that autologous activated and expanded NK cells may serve as a viable cellular therapy for pediatric patients with refractory/relapsed leukemia. As demonstrated in these in vitro experiments, autologous activated/expanded NK cells still show increased targeting of mouse AML and ALL cell lines despite the lack of KIR mismatch. Thus, they may serve as a potential platform for leukemia therapy, including ALL, which appear to be poor targets for resting NK cells. In addition, these cells demonstrate transient persistence in vivo, a potential advantage in the context of redirected cytotoxicity using CAR constructs that target antigens with broader expression in the hematopoietic compartment. Figure 1: <![if !vml]><![endif]> Figure 1:. <![if !vml]><![endif]> Figure 2: Figure 2:. Disclosures No relevant conflicts of interest to declare.

Therapeutic applications: natural killer cells in the clinic

Hematology ◽  
2013 ◽  
Vol 2013 (1) ◽  
pp. 247-253 ◽  
Author(s):  
Jeffrey S. Miller
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Adoptive Transfer ◽  
Cell Activation ◽  
Tumor Antigens ◽  
Nk Cell ◽  
Ex Vivo ◽  
T Cell Stimulation ◽  
Refractory Acute Myeloid Leukemia

Abstract Natural killer (NK) cells recognize targets stressed by malignant transformation or infection (particularly CMV). We now know that NK cells can be long-lived and remember past exposures. They become educated by interaction with MHC class I molecules to gain potent function to kill targets and produce cytokines. In the clinical setting, haploidentical NK cells can be transferred adoptively to treat cancer. Persistence and in vivo expansion of NK cells depends on lymphodepleting chemotherapy to make space for the release of endogenous IL-15. In vivo expansion is also enhanced by cytokine administration. IL-2 has been used at low doses to stimulate NK cells in vivo, but has the down side of stimulating CD25hi regulatory T cells. IL-15 is now being tested and has the advantage of avoiding inhibitory regulatory T cell stimulation. In refractory acute myeloid leukemia, leukemia clearance is correlated with the persistence and in vivo expansion of NK cells after adoptive transfer. Limitations to NK cell therapy include poor in vivo survival and lack of specificity. Monoclonal antibodies and bispecific or trispecific killer engagers to target CD16 on NK cells to enhance recognition of various tumor antigens and ADAM17 inhibition to prevent CD16 shedding after NK cell activation should promote enhanced killing of cancer with specificity. Future strategies to exploit favorable donor immunogenetics or to expand NK cells ex vivo from blood, progenitors, or pluripotent progenitors may overcome immune barriers of adoptive transfer and comparative clinical trials will be needed to test these approaches.

Influence of in vivo hypobaric hypoxia on function of lymphocytes, neutrocytes, natural killer cells, and cytokines

1993 ◽  
Vol 74 (3) ◽  
pp. 1100-1106 ◽  
Author(s):  
M. Klokker ◽  
A. Kharazmi ◽  
H. Galbo ◽  
I. Bygbjerg ◽  
B. K. Pedersen
Keyword(s):  
Immune System ◽  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Interleukin 2 ◽  
Cell Activity ◽  
Nk Cell Activity ◽  
Cd8 Cells ◽  
The Absolute

We have investigated the effects of short-term hypoxia in vivo on the human cellular immune system. Seven young healthy volunteers were placed in a decompression chamber (380 Torr) for 20 min with or without supplemental O2. The leukocyte concentration increased during hypobaric conditions because of an increased concentration of lymphocytes. The absolute and relative concentration of CD16+ natural killer (NK) cells increased markedly during hypoxia and returned to pretest values after 2 h of recovery. The NK cell activity of blood mononuclear cells (BMNC, %lysis/fixed no. of BMNC) boosted with interferon-alpha, interleukin-2 (IL-2), and indomethacin rose in parallel with unboosted NK cell activity during hypoxia. The percentage of CD4+ and CD8+ cells declined during hypoxia, whereas the absolute concentration of both CD8+ cells and CD14+ monocytes increased. Although the BMNC composition varied, the proliferative responses of BMNC after stimulation with phytohemagglutinin, purified derivative of tuberculin, and IL-2 did not change significantly. The in vitro production of interleukin-1 beta and IL-2 in supernatants obtained after stimulation of BMNC with phytohemagglutinin or lipopolysaccharide was not affected. The chemiluminescence response of neutrocytes increased 2 h after hypoxia. It was concluded that acute hypoxia induced marked alterations in the immune system and that the NK cells are especially sensitive to the hypoxic stimulus.

533 Cross-species immunogenomic analysis identifies pathways of canine natural killer cell response to cytokine therapy, and reveals convergence of activated dog and human natural killer transcriptomes

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A569-A569
Author(s):  
Alicia Gingrich ◽  
Taylor Reiter ◽  
Sean Judge ◽  
Daniel York ◽  
Mio Yanagisawa ◽  
...  
Keyword(s):  
Gene Expression ◽  
Clinical Trial ◽  
Phase I ◽  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Ex Vivo ◽  
Phase I Clinical Trial ◽  
Gene Profiles

BackgroundNatural killer (NK) cells are key effectors of the innate immune system, but major differences between human and murine NK cells impede translation. Outbred dogs offer an important link for NK-based cancer immunotherapy studies. We compared gene expression profiles of dog NK signatures in vitro and from a phase I clinical trial of inhaled IL-15, and analyzed dog, mouse and human NK cells using a novel orthologous transcriptome.MethodsWe performed differential gene expression (DGE) using resting healthy donor CD5dim NK populations and following ex vivo activation using recombinant human (rh)IL-15 or co-culture with irradiated feeder cells. Eight dogs with naturally-occurring pulmonary metastases were enrolled on a Phase I clinical trial of inhaled rhIL-15 using a 3+3 cohort design with escalating doses of inhaled rhIL-15. Blood was collected from study dogs before, during, and after therapy. We compared DGE among healthy and cancer-bearing dogs and then across mouse, dog and human NK cells in resting and activated states using ~7000 1:1 orthologous genes.ResultsDGE revealed distinct transcriptional profiles between the ex vivo resting, IL-15 and co-cultured canine NK cells. Among treated patients, hierarchical clustering revealed that in vivo NK cell transcriptional signatures grouped by individual dog, and not amount of time exposed to treatment. PCA showed in vivo profiles of the clinical responders were distinctly separate from the non-responding patients (PC1 38%, PC2 12%). Patient in vivo NK cell transcription profiles most closely resembled those of ex vivo resting NK cells and not IL-15 treated or co-culture activated (PC1 43%, PC2 19%), likely reflecting key differences in activation. In cross-species analysis, PCA showed within-species spatial clustering of resting NK cells. After activation, variance between dog and human NK cells decreased, while variance between human and mouse NK cells increased (PC1 40%, PC2 28%).ConclusionsIn this first transcriptomic sequencing of dog NK cells, we demonstrate distinct gene profiles of ex vivo activated NK cells from healthy donors compared to circulating NK cells from dogs receiving inhaled rhIL-15 on a clinical trial. Baseline in vivo NK cell profiles appear to predict response to therapy more than changes over time. We also show distinct gene profiles of NK cells across the most commonly used mouse, dog, and human NK populations, with convergence of dog and human NK cells after activation. By defining the canine NK cell DGE signatures, these data fill a gap in translational NK studies.Ethics ApprovalThe canine clinical trial study was approved by IACUC and Clinical Trials Review Board (Inhaled IL-15 Immunotherapy for Treatment of Lung Metastases, Protocol #20179).

scholarly journals Natural Killer Cells: The Linchpin for Successful Cancer Immunotherapy

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.

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 Cancer cells educate natural killer cells to a metastasis-promoting cell state

2020 ◽  
Vol 219 (9) ◽  
Author(s):  
Isaac S. Chan ◽  
Hildur Knútsdóttir ◽  
Gayathri Ramakrishnan ◽  
Veena Padmanaban ◽  
Manisha Warrier ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cancer Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Ex Vivo ◽  
Metastatic Potential ◽  
Dna Methyltransferases ◽  
In Vivo Models ◽  
Metastatic Recurrence

Natural killer (NK) cells have potent antitumor and antimetastatic activity. It is incompletely understood how cancer cells escape NK cell surveillance. Using ex vivo and in vivo models of metastasis, we establish that keratin-14+ breast cancer cells are vulnerable to NK cells. We then discovered that exposure to cancer cells causes NK cells to lose their cytotoxic ability and promote metastatic outgrowth. Gene expression comparisons revealed that healthy NK cells have an active NK cell molecular phenotype, whereas tumor-exposed (teNK) cells resemble resting NK cells. Receptor–ligand analysis between teNK cells and tumor cells revealed multiple potential targets. We next showed that treatment with antibodies targeting TIGIT, antibodies targeting KLRG1, or small-molecule inhibitors of DNA methyltransferases (DMNT) each reduced colony formation. Combinations of DNMT inhibitors with anti-TIGIT or anti-KLRG1 antibodies further reduced metastatic potential. We propose that NK-directed therapies targeting these pathways would be effective in the adjuvant setting to prevent metastatic recurrence.

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