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).

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.

scholarly journals Autologous Activated and Expanded Natural Killer Cells Are Safe and Clinically Actives in Multiple Myeloma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1856-1856
Author(s):  
Alejandra Leivas ◽  
Antonio Pérez-Martínez ◽  
María Jesús Blanchard ◽  
Estela Martín Clavero ◽  
Dario Campana ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Multiple Myeloma ◽  
Phase I ◽  
Nk Cells ◽  
Disease Progression ◽  
Natural Killer ◽  
Peripheral Blood ◽  
Nk Cell ◽  
Phase I Clinical Trial ◽  
End Products

Abstract Multiple myeloma (MM) remains an incurable disease, despite that it has had a huge increase in survival in part due to new drugs as proteasome inhibitors and immunomodulatory drugs; however new therapeutic venues are required. Immune-based therapies are having an important relevance to control cancer, and are a new therapeutic armamentarium. Natural killer (NK) cells have an important role as natural control of tumor cells; based on that, NK cell infusions could be a novel treatment strategy to treat MM. By co-culture with the genetically modified cell line K562-mb15-41BBL it is possible to expand ex vivo large numbers of activated NK (NKAE) cells from MM patients. NK cell therapy has some challenges to be answered in real clinical practice: Could they be used out of transplantation setting? Could they be used with other anti-myeloma drugs? Could they be infused and expanded several times? To answer these questions we have designed a phase I clinical trial to make multiple infusions of autologous NKAE cells together with anti-myeloma drugs bortezomib or lenalidomide in MM (NCT02481934). Five MM patients on 2nd or later relapse have been enrolled in this phase I clinical trial to date. To activate and expand NK cell, peripheral blood mononuclear cell (PBMCs) were co-cultured with K562-mb15-41BBL cells and 100 IU/ml IL-2. We collected 200 ml of peripheral blood (PB) from patients every cycle (n=4) to produce autologous NKAEs under GMP conditions and cells were harvested on day 14 and 21 for infusions. Four cycles of pharmacological treatment with 2 infusions of 7.5x106 autologous NKAEs/kg on day 1 and 8 of each cycle were performed. NKAEs purity and T regulatory cells (Treg) were analyzed by flow cytometry. NK cells presence in PB was also assessed by PB smear examination before and after each infusion. Serum cytokines concentration was determined by cytometric bead assay. Safety of NKAE end products was verified by real time-PCR of c-MYC and telomerase on NKAE from the 2th and 3rd week of expansion. BCR-ABL PCR studies were performed on NKAE cultures and on PB samples from the patients after treatment. Three patients received lenalidomide-based treatment and 2 bortezomib-based treatment. Patients received a total of 35 NKAEs infusions. We have not observed any serious toxicity attributable to NKAE infusion. Two patients had grade II neutropenia, which did not require dose adjustment. The 5 MM patients enrolled had 23% (±11%) NK cells of PBMCs. We collected a mean of 21x106 NK cells from PB. After 1 week NKAEs number increased x13 with 71% of NKAEs, at 2nd week the fold of NKAE cells expansion was x30 with a purity of 92%. We collected 550x106 (±50x106) NKAEs from culture for the first infusion. At 3rd week NKAEs number increased 45 times (fig.1.A). NKAEs infusion was completely safe; expression of c-Myc and telomerase was not altered in NKAE end products. The expression of BCR-ABL disappeared from cultures after the first week, and was undetectable in PB after NKAE therapy. Contamination of autologous T cells on NKAE end products was not significant; less than 4%. NKAE cells were detectable on PB after infusions; percentage of PB NK cells increased a mean of 5% and expression of activatory receptors NKp30 and NKG2D and apoptosis ligands TRAIL and FasL increased on PBMCs after infusion. PB smear showed an increase fold of activated circulating lymphocytes change of x3.8 (p<0.05). There was no variation on Treg CD4+CD25+CD127- during therapy. Serum levels of IFN-γ increased progressively until the 7th day of cycle and IL-10 levels showed an increase at the end of cycle. Patient 01 achieved a partial response and maintained it for 13 months after NKAEs infusion. Patient 02 started NKAEs infusion while in relapse and, achieved stable disease, which was maintained for 9 months before disease progression. Of note, bone marrow infiltration by MM plasma cells decreased at least 50% at the end of NKAE treatment in these two patients. Patient 03 had disease progression 2 months after stopping treatment due to unrelated toxicity. Patients 04 and 05 recently finished NKAEs treatment and achieved disease stabilization 4 months after the first NKAE infusion (fig.1.B). Clinical-grade NKAEs can be obtained from MM patients undergoing treatment, and multiple infusions of NKAEs are feasible without toxicity. NKAEs showed clinical anti-myeloma activity. These results warrant further development of NKAEs infusion as a treatment modality for MM. Disclosures Lahuerta: Janssen Cilag, Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees.

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.

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.

scholarly journals Comparative Immunogenomics of Canine Natural Killer Cells as Immunotherapy Target

2021 ◽  
Vol 12 ◽  
Author(s):  
Alicia A. Gingrich ◽  
Taylor E. Reiter ◽  
Sean J. Judge ◽  
Daniel York ◽  
Mio Yanagisawa ◽  
...  
Keyword(s):  
Gene Expression ◽  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Species Analysis ◽  
Cell Gene Expression ◽  
Immunotherapy Target ◽  
Multiple Conditions

Natural killer (NK) cells are key effectors of the innate immune system, but major differences between human and murine NK cells have impeded translation. Outbred dogs offer an important link for studies of NK biology and immunotherapy. We analyzed gene expression of putative NK populations from healthy dogs and dogs with naturally-occurring cancers examining differential gene expression across multiple conditions, including steady-state, in vitro activation with cytokines and co-culture, and in vivo activation with inhaled IL-15 in dogs receiving IL-15 immunotherapy. We also compared dog, mouse and human CD3-NKp46+ NK cells using a novel orthologous transcriptome. Distinct transcriptional profiles between NK populations exist between conditions and in vitro versus in vivo treatments. In cross-species analysis, canine NK cells were globally more similar to human NK cells than mice. These data define canine NK cell gene expression under multiple conditions and across species, filling an important gap in translational NK studies.

scholarly journals Autologous NK-cell-enrichment: preclinical setting phase, Shiraz experience

2020 ◽  
Author(s):  
Somayeh Rezaeifard ◽  
yuji Heike ◽  
Junichi Masuyama ◽  
Alireza rezvani ◽  
Reza vojdani ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Cell Therapy ◽  
Phase I ◽  
Nk Cells ◽  
Peripheral Blood ◽  
Nk Cell ◽  
Ex Vivo ◽  
University Hospital ◽  
Healthy Individuals ◽  
Therapy Regimen

Abstract Background: NK cell therapy has proven to be a promising approach for treatment of hematological malignancies and solid tumors. Masuyama et al. have recently introduced a new method for ex-vivo autologous NK cell expansion (Osaki method); resulting in the production of ample active NK cells for a promising cell therapy regimen. In order to start clinical trial phase I at Shiraz University of medical Sciences in collaboration with Masuyama clinic and St. Luck's International University Hospital, this preclinical setting study aimed to evaluate the proliferative efficacy of the method, the activation status of expanded autologous NK cells and the likely unwanted contamination of the final cell product.Methods: PBMCs were isolated from 30 ml of 5 healthy individuals' peripheral blood transferring directly to the specified initial culture bag containing antibodies for CD3, CD52 as well as IL-2 cytokine. The cells were cultured for 14-17 days in incubators; during which the cell received condition media, and underwent several passages into bigger culture bags. All the procedure was carried out in the clean room and associated facilities. Results: Our results indicated that NK cells were expanded 510-fold in average (range 200-1100 fold), and the purity of NK cells per whole lymphocytes exceeded 68%. The expanded cells were highly lytic as indicated by in-vitro cytotoxic assay; with strong expression of NKG2D and CD16. The prepared final cell products were negative for HCV, HBV, HIV, Mycoplasma and endotoxin. Conclusion: In the preclinical setting phase, large numbers of activated and un-contaminated NK cells from 30 ml of healthy individuals' peripheral blood were successfully generated. The method seems to provide ample clean cell product with no contamination; suitable to be infused back to the patients in phase I clinical trial.

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.

scholarly journals Autologous Natural Killer Cell-enrichment for Immune Cell Therapy: Preclinical Setting Phase, Shiraz Experience

2021 ◽  
Author(s):  
Somayeh Rezaeifard ◽  
Yuji Heike ◽  
Jun-Ichi Masuyama ◽  
Alireza Rezvani ◽  
Reza Vojdani ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Cell Therapy ◽  
Phase I ◽  
Nk Cells ◽  
Natural Killer ◽  
Peripheral Blood ◽  
Nk Cell ◽  
Healthy Individuals ◽  
Cell Product ◽  
Nk Cell Therapy

Natural killer (NK) cell therapy has proven to be a promising approach for the treatment of malignancies. Osaki method for ex-vivo autologous NK cell expansion has been recently introduced in Japan. To start clinical trial phase I at Shiraz University of Medical Sciences in collaboration with the Japanese group, this preclinical setting study aimed to evaluate the proliferative efficacy of the method, the activation status of expanded autologous NK cells, and the likely unwanted contamination of the final cell product. Peripheral blood mononuclear cells (PBMCs) were isolated from 5 healthy individuals' peripheral blood and transferred directly to the specified initial culture bag containing anti-CD52 and anti-CD3 and Interleukin (IL)-2. The cells were cultured for 14-17 days in an incubator, during which the cells received condition media, and underwent several passages into bigger culture bags. All the procedures were carried out in a cleanroom and associated facilities. Before and after activation PBMCs were analyzed for their phenotype and cytotoxic activity; using flow cytometry and cytokine release assay. Our results indicated that NK (CD3-CD16+/-CD56+) cells were expanded 510-fold on average (range 200-1100 fold), and the purity of NK cells per whole lymphocytes exceeded 68%. The expanded cells were highly lytic as indicated by in-vitro cytotoxic assay, with a strong expression of Natural killer group 2 member D (NKG2D) and CD16. The prepared final cell products were negative for HCV, HBV, HIV, mycoplasma, and endotoxin. In the preclinical phase, large numbers of activated and un-contaminated NK cells from healthy individuals' peripheral blood were successfully generated. The method seems to provide ample clean cell product with no contamination and has the potential to be used for NK cell therapy in future clinical trials, suitable to be infused back to the donors in phase I clinical trial.

138 In vivo localization of genetically engineered natural killer cells against glioblastoma using PET imaging

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A151-A151
Author(s):  
Yeonhee Yun ◽  
Jiao Wang ◽  
Karen Pollok ◽  
Tony Sinn ◽  
Randy Brutkiewicz ◽  
...  
Keyword(s):  
Mr Imaging ◽  
Nk Cells ◽  
Natural Killer ◽  
Pet Imaging ◽  
Nk Cell ◽  
Ex Vivo ◽  
Killing Activity ◽  
Gamma Counting

BackgroundGlioblastoma (GBM) is a deadly brain malignancy with a dismal prognosis. While immunotherapy holds great promise for GBM treatment, most have failed due to a suppressive tumor microenvironment (TME). Antigen heterogeneity and adenosine signaling are two immunosuppressive mechanisms in GBM. The CD73-adenosine axis plays a multifaceted role in GBM pathogenesis and drives the dysfunction of NK cells in GBM TME.1,3 Our NKG2D-chimeric antigen receptor (CAR)-natural killer (NK) cells have shown anti-tumor activity when combined with CD73 blockade in vivo.2 To further extend the potency of these cells against GBM and address antigen heterogeneity in GBM, we combined the local blockade of CD73 with multi-antigen-targeting engineered NK cells. In order to improve treatment assessment, PET/MR imaging was employed to enable detailed, non-invasive assessment of tumor progression. Imaging assessment of adoptively-transferred CAR- NK cells was also developed to determine the fate of NK cell delivery to the tumor site over time.MethodsWe generated multifunctional engineered NK (E-NK) cells that express an anti-CD73 scFv, which is cleavable by GBM-associated proteases, an NKG2D-CAR, as well as a GD2 CAR, which can actively target the GD2 antigen overexpressed on GBM (Figure 1A). For E-NK cell radiolabeling, zirconium-89 (89Zr, ½ life = 78 Hr) radiotracer was attached covalently to the E-NK cell surface via conjugation with DFO-Bz-NCS in a range of doses from 50–600 µCi.ResultsAn optimal balance between labeling efficiency and cell viability was attained at 120 µCi 89Zr resulting in 39% labeling efficiency and 46% cell viability over for 48 hours. After labeling, the NK cells maintained their in vitro killing activity against GBM cells (figure 1B). The 89Zr labeled E-NK cells were administered intravenously in mice containing intracranial GBM10 tumors at week 5 post-implant. PET imaging was performed at 1 and 2 days later and gamma imaging ex vivo at 4 days. Free 89Zr was visible diffusely throughout the body with low levels in the brain. The majority of 89Zr labeled E-NK cell groups localized to the lungs with detectable activity elsewhere in various organs (figure 1C and 1D).Abstract 138 Figure 1PET imaging and gamma counting of the engineered NK cellsFigure 1 (A) Multifunctional, responsive CAR constructs; (B) In vitro killing activity against GBM43 cells after co-incubation with 89Zr labeled NK cells at an E:T ratio of 10 for 4 h with LDH assay (N=3); (C) & (D) In vivo PET imaging and ex vivo gamma counting with 89Zr at week 5 in 10 mice during 4 days, GBM intracranial implantation to NSG male mouse, 89Zr, 89Zr + NK cell, or 89Zr + E NK cell (7 × 106 cells with 500 µCi) was administered through intravenous injection, Qimage was used for the PET/MRI co-registration and analysisConclusionsWe generated multifunctional E-NK cells which showed the improved killing of GBM cells using novel targeting approaches, including the blockade of CD73-mediated adenosinergic signaling. We also optimized E-NK cell radiolabeling with 89Zr for GB10 therapy in vitro and in vivo fate mapping against a xenograft of patient-derived GBM.AcknowledgementsWe gratefully acknowledge the Walther Oncology Embedding Program, Indiana University Simon Cancer Center, and In Vivo Therapeutics Core.ReferencesWang J, Matosevic S. NT5E/CD73 as correlative factor of patient survival and natural killer cell infiltration in glioblastoma. J Clin Med 2019;8(10):1526.Wang J, Lupo KB, Chambers AM, Matosevic S. Purinergic targeting enhances immunotherapy of CD73+ solid tumors with piggyBac-engineered chimeric antigen receptor natural killer cells. J Immunother Cancer 2018;6(1):136.Yan A, Joachims ML, Thompson LF, Miller AD, Canoll PD, Bynoe MS. CD73 promotes glioblastoma pathogenesis and enhances its chemoresistance via A2B adenosine receptor signaling. J Neurosci 2019;39(22):4387.Flink J, Muzi M, Peck M, Krohn K. Multimodality brain tumor imaging: mr imaging, PET, and PET/MR imaging. J Nucl 2015;5(10):1554–1561.

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