scholarly journals 772 A potent and off-the-shelf oNK cell therapy product targets HER2+ cancer cells and resists suppressive tumor microenvironment

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A821-A821
Author(s):  
Hao-Kang Li ◽  
Ching-Wen Hsiao ◽  
Sen-Han Yang ◽  
Hsiu-Ping Yang ◽  
Tai-Sheng Wu ◽  
...  
Keyword(s):  
Tumor Microenvironment ◽  
Membrane Proteins ◽  
Cell Therapy ◽  
Nk Cells ◽  
Cancer Cells ◽  
Cell Line ◽  
Nk Cell ◽  
Binding Specificity ◽  
Cell Therapy Product

BackgroundAutologous or allogeneic natural killer (NK) cells possess efficient cytotoxicity against tumor cells without severe side effects such as CRS or graft-versus-host disease (GvHD). In addition to chimeric antigen receptor (CAR) strategy, antibody-cell conjugates (ACC) platform provides more efficient way to arm NK cells with binding specificity and enhanced potency against target cells. In this work, we develop a NK cell therapy product ACE1702, a novel NK cell line oNK conjugated with trastuzumab, and assess its potency against HER2+ solid tumors.Methods oNK cells were covalently conjugated with monoclonal antibody Trastuzumab after sublethal irradiation by our patented antibody-cell conjugates (ACC) platform to become our cryopreserved final product ACE1702 compliant with current good manufacturing practice (cGMP). Function of ACE1702 was validated by real-time xCELLigence analyzer and MTT assay in vitro. Efficacy of intraperitoneally (ip.) delivered ACE1702 was evaluated in tumor-bearing female immune compromised NSG mice. Characterization of ACE1702 was analyzed by flow cytometry.ResultsWe demonstrated that the trastuzumab-armed oNK cells, ACE1702, exerted human epidermal growth factor 2 (HER2) binding specificity and enhanced cytotoxicity against various types of cancer cells with different grade of HER2 expressions compared to control oNK cells in vitro. In vivo results in human ovarian cancer cell line SK-OV-3-bearing xenograft mouse model further supported the in vitro observations. Of note, ACE1702 also displayed a better cytotoxicity against HER2+ cancer cells than trastuzumab and its derived antibody-drug conjugate. ACE1702 also remained cytotoxicity against cancer cells in the suppressive tumor microenvironment. Characterization revealed a preferential expression of NK activation receptors, and conjugation of trastuzumab with cell membrane proteins responsible for NK activity capacitated ACE1702 with enhanced cytotoxicity. These results underscore the potency of ACE1702 in eradication of cancer cells.ConclusionsHere we introduced a novel trastuzumab-modified oNK cell product with enhanced specificity against myriad types of HER2+ cancers. Selective conjugation of trastuzumab with membrane proteins contributing to NK activation conferred ACE1702 with enhanced cytotoxicity even in the suppressive tumor microenvironment.AcknowledgementsNoneTrial RegistrationNoneEthics ApprovalThe animal study was conducted according to protocols approved by the Institutional Animal Care and Use Committee of Muragenics.ConsentNone

scholarly journals IMMU-47. PROTEIN PHOSPHATASE 2A INHIBITION IN NK CELL THERAPY FOR TREATMENT OF MEDULLOBLASTOMA

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii115-ii115
Author(s):  
Rongze Olivia Lu ◽  
Winson Ho ◽  
Brandon Chiou
Keyword(s):  
Nk Cells ◽  
Cell Line ◽  
Protein Phosphatase ◽  
Nk Cell ◽  
Protein Phosphatase 2A ◽  
T Cell Response ◽  
Intrinsic Activity ◽  
Cd107a Expression ◽  
Phosphatase 2A

Abstract Checkpoint immunotherapy (ICB) thus far has shown limited efficacy against brain tumors, such as medulloblastoma (MB). Its low mutational burden is thought to result in a paucity of neoantigen to trigger an effective T-cell response. Natural killer (NK) cells, can recognize tumor cells independently of neoantigens, making them appealing against MBs. Modulation of NK cells to enhance cytotoxicity against MBs could be a novel treatment strategy. Protein Phosphatase 2A (PP2A), a ubiquitous serine/threonine phosphatase, has been shown to inhibit IFNg and Granzyme B production by NK cells. We hypothesize that NK92, a transformed human NK cell line, has intrinsic activity against human MB cells and that inhibiting PP2A pharmacologically can enhance cytotoxicity of NK92 cells. We performed NK cytotoxicity assay and granulation assay against human MB cell line D425. We also used a small molecular inhibitor, LB100, to modulate PP2A activity in NK92. NK92 cells were co-cultured with D425, in increasing E:T (Effector:Target) ratio for 4 hours. D425 cells were pre-labeled with CellTrace Violet dye. The percentage of D425 (Violet+) cells in apoptosis (Cas3/7+) or necrosis (AAD+) were compared with different ET ratios to quantify NK mediated cell cytotoxicity. We also measured CD107a expression in NK92 to assess granulation with LB100 treatment. D425 cells were sensitive to NK92 killing. Percentage of D425 cells either apoptotic or necrotic increased with increasing ET ratio, suggesting that there was NK92 mediated cytotoxicity. Percentage of killed D425 cells ranged from 18% at baseline (without NK92) to 80% at ET ratio of 20. Inhibition of PP2A using LB100, enhanced NK92 degranulation. CD107a+ NK92 cells increased from 19% to 28% with 8uM of LB100. NK92 cells are cytotoxic against MB cells in vitro and inhibition of PP2A in NK cells can enhance their activity against MB cells.

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 RTID-07. HUMAN PLACENTAL HEMATOPOIETIC STEM CELL DERIVED NATURAL KILLER CELLS (CYNK-001) FOR TREATMENT OF RECURRENT GLIOBLASTOMA

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii194-ii195
Author(s):  
Nazanin Majd ◽  
Maha Rizk ◽  
Solveig Ericson ◽  
Kris Grzegorzewski ◽  
Sharmila Koppisetti ◽  
...  
Keyword(s):  
Cell Therapy ◽  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
In Vitro Cytotoxicity ◽  
Orthotopic Mouse Model ◽  
Hematopoietic Stem ◽  
Dismal Prognosis

Abstract Glioblastoma (GBM) is the most aggressive primary brain tumor with dismal prognosis. Recent advances of immunotherapy in cancer have sparked interest in the use of cell therapy for treatment of GBM. Active transfer of Natural Killer (NK) cells is of particular interest in GBM because NK cells are capable of exerting anti-tumor cytotoxicity without the need for antigen presentation and sensitization, processes that are impaired in GBM. CYNK-001 is an allogeneic, off-the-shelf product enriched for CD56+/CD3- NK cells expanded from placental CD34+ cells manufactured by Celularity. Here, we demonstrate in vitro cytotoxicity of CYNK-001 against several GBM lines and its in vivo anti-tumor activity in a U87MG orthotopic mouse model via intracranial administration resulting in 94.5% maximum reduction in tumor volume. We have developed a phase I window-of-opportunity trial of CYNK-001 in recurrent GBM via intravenous (IV) and intratumoral (IT) routes. In the IV cohort, subjects receive cyclophosphamide for lymphodepletion followed by 3-doses of IV CYNK-001 weekly. In the IT cohort, subjects undergo placement of an IT catheter with an ommaya reservoir followed by 3-doses of IT CYNK-001 weekly. Patients are monitored for 28-days after last infusion for toxicity. Once maximum safe dose (MSD) is determined, patients undergo IV or IT treatments at MSD followed by surgical resection and the tumor tissue will be analyzed for NK cell engraftment and persistence. We will utilize a 3 + 3 dose de-escalation design (maximum n=36). Primary endpoint is safety and feasibility. Secondary endpoints are overall response rate, duration of response, time to progression, progression free survival and overall survival. Main eligibility criteria include age ≥18, KPS ≥60, GBM at first or second relapse with a measurable lesion on ≤2mg dexamethasone. This is the first clinical trial to investigate CYNK-001 in GBM and will lay the foundation for future NK cell therapy in solid tumors.

scholarly journals Hitting More Birds with a Stone: Impact of TGF-β on ILC Activity in Cancer

2020 ◽  
Vol 9 (1) ◽  
pp. 143 ◽  
Author(s):  
Cinzia Fionda ◽  
Helena Stabile ◽  
Cristina Cerboni ◽  
Alessandra Soriani ◽  
Angela Gismondi ◽  
...  
Keyword(s):  
Tumor Microenvironment ◽  
Nk Cells ◽  
Cancer Cells ◽  
Nk Cell ◽  
Critical Role ◽  
Cell Activity ◽  
Lymphoid Cells ◽  
Receptor Expression ◽  
Nk Cell Activity ◽  
Group I

Transforming growth factor (TGF)-β is a central immunosuppressive cytokine within tumor microenvironment inhibiting the expansion and function of major cellular components of adaptive and innate immune system. Among them, compelling evidence has demonstrated that TGF-β is a key regulator of natural killer (NK) cells, innate lymphoid cells (ILCs) with a critical role in immunosurveillance against different kinds of cancer cells. A TGF-β rich tumor microenvironment blocks NK cell activity at multiple levels. This immunosuppressive factor exerts direct regulatory effects on NK cells including inhibition of cytokine production, alteration of activating/inhibitory receptor expression, and promotion of the conversion into non cytotoxic group I ILC (ILC1). Concomitantly, TGF-β can render tumor cells less susceptible to NK cell-mediated recognition and lysis. Indeed, accumulating evidence suggest that changes in levels of NKG2D ligands, mainly MICA, as well as an increase of immune checkpoint inhibitors (e.g., PD-L1) and other inhibitory ligands on cancer cells significantly contribute to TGF-β-mediated suppression of NK cell activity. Here, we will take into consideration two major mechanisms underlying the negative regulation of ILC function by TGF-β in cancer. First, we will address how TGF-β impacts the balance of signals governing NK cell activity. Second, we will review recent advances on the role of this cytokine in driving ILC plasticity in cancer. Finally, we will discuss how the development of therapeutic approaches blocking TGF-β may reverse the suppression of host immune surveillance and improve anti-tumor NK cell response in the clinic.

Immunoselection by natural killer cells of PIGA mutant cells missing stress-inducible ULBP

Blood ◽  
2006 ◽  
Vol 107 (3) ◽  
pp. 1184-1191 ◽  
Author(s):  
Nobuyoshi Hanaoka ◽  
Tatsuya Kawaguchi ◽  
Kentaro Horikawa ◽  
Shoichi Nagakura ◽  
Hiroaki Mitsuya ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Nk Cells ◽  
Natural Killer ◽  
Blood Cells ◽  
Cell Injury ◽  
Nk Cell ◽  
K562 Cells ◽  
Cytotoxic Cells ◽  
Immune Mediated

AbstractThe mechanism by which paroxysmal nocturnal hemoglobinuria (PNH) clones expand is unknown. PNH clones harbor PIGA mutations and do not synthesize glycosylphosphatidylinositol (GPI), resulting in deficiency of GPI-linked membrane proteins. GPI-deficient blood cells often expand in patients with aplastic anemia who sustain immune-mediated marrow injury putatively induced by cytotoxic cells, hence suggesting that the injury allows PNH clones to expand selectively. We previously reported that leukemic K562 cells preferentially survived natural killer (NK) cell-mediated cytotoxicity in vitro when they acquired PIGA mutations. We herein show that the survival is ascribable to the deficiency of stress-inducible GPI-linked membrane proteins ULBP1 and ULBP2, which activate NK and T cells. The ULBPs were detected on GPI-expressing but not on GPI-deficient K562 cells. In the presence of antibodies to either the ULBPs or their receptor NKG2D on NK cells, GPI-expressing cells were as less NK sensitive as GPI-deficient cells. NK cells therefore spared ULBP-deficient cells in vitro. The ULBPs were identified only on GPI-expressing blood cells of a proportion of patients with PNH but none of healthy individuals. Granulocytes of the patients partly underwent killing by autologous cytotoxic cells, implying ULBP-associated blood cell injury. In this setting, the lack of ULBPs may allow immunoselection of PNH clones.

Lenalidomide Effects on NK Function in Patients with Myelodysplastic Syndrome (MDS).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3437-3437 ◽  
Author(s):  
P.K. Epling-Burnette ◽  
Fanqi Bai ◽  
Jeffrey S. Painter ◽  
Julie Y. Djeu ◽  
Alan F. List
Keyword(s):  
Nk Cells ◽  
Cell Line ◽  
Cell Lines ◽  
Cellular Response ◽  
Nk Cell ◽  
Leukemia Cell ◽  
T Test ◽  
Leukemia Cell Line ◽  
Immunomodulatory Effects

Abstract Lenalidomide, which is a 4-amino-glutarimide analogue of thalidomide, has significant erythropoietic activity in patients with lower-risk MDS (List et al, NEJM, 351:26,2004). Although its precise target of action in MDS is not known, lenalidomide modulates cellular response to varied stimuli including inhibition of angiogenic response and endotoxin induction of inflammatory cytokines and enhancement of antigen-induced immunologic response and erythropoietin receptor signaling. Clinical investigations in multiple myeloma indicate that the immunomodulatory effects of thalidomide and lenalidomide extends to the expansion of natural killer (NK) cells. We recently found that MDS patients have defective NK function, araising in part to reduced expression of activating NK receptors (NKRs) NKp30, CD244 (2B4), and NKG2D. To determine if lenalidomide may restore NK function in MDS, we investigated the effects of in vitro treatment with lenalidomide on NK function and phenotype. Lytic function was studied using peripheral blood mononuclear cells (PBMCs) as effector cells and the leukemia cell line, K562, as a target in standard 4-hr 51Cr-release assays at 12:1 and 25:1 effector:target (E:T) ratios. Among eight MDS patient’s specimens evaluated, five patients had significant increase in tumor lysis after treatment with 1 μM lenalidomide for 72 hours (p ≤ 0.01, T-test). In similar experiments using PBMCs from normal donors, we found that NK lysis of K562 was 42% ± 15 (25:1 E:T ratio) pre-treatment which increased to 71% ± 17 (25:1 E:T ratio) after treatment, which was statistically significant (p ≤ 0.01, T-test). We also examined the in vitro effects of lenalidomide on lytic activity by the NK cell lines, NK92 and NKL, which was significantly increased after drug treatment. To discern the mechanisms of lenalidomide action in NK cell lines and normal NK cells, we evaluated NKR display by flow cytometry, and NKR function by antibody redirected cytotoxicity using the FcγR+ murine mastocytoma (P815) target cell line. Using NK92 and NKL cells, treatment with lenalidomide 1 μM for 72 hrs increased lysis by anti-NKG2D and anti-CD244 activating antibodies. We also found that NKG2D surface expression was increased on normal NK cells after lenalidomide treatment in vitro. These results suggest that some MDS patients may have improved NK function through the immunomodulatory effects of lenalidomide. The relationship between in vitro NK responsiveness to lenalidomide and in vivo hematological response warrants investigation in patients with MDS.

scholarly journals P06.09 Anti-hPSMA CAR engineered NK-92 cells: An off-the-shelf cellular therapeutic for targeted elimination of prostate cancer cells

2020 ◽  
Vol 8 (Suppl 2) ◽  
pp. A45.1-A45
Author(s):  
G Zuccolotto ◽  
A Penna ◽  
IM Montagner ◽  
D Carpanese ◽  
A Rosato
Keyword(s):  
T Cells ◽  
Cell Therapy ◽  
Cancer Cells ◽  
Cell Line ◽  
Cell Population ◽  
Tumor Escape ◽  
Tumor Models ◽  
Car T

BackgroundAdoptive cell therapy of malignant diseases takes advantages of the cellular immune system to recognize and destroy cancer cells. Despite the remarkable success in B cell malignancies after adoptive transfer of CD19 CAR T cells, CAR T cell therapy in solid tumors has shown less encouraging clinical results, above all caused by tumor escape mechanisms.In order to overcome such limitations, NK-92, a permanent and IL-2-dependent cell line with a high cytotoxicity in vitro, has been engineered in preclinical models with CAR. In this project, we exploited a CAR directed against the human antigen hPSMA that is overexpressed in prostate tumors. This project aimed at transducing NK-92 cell line to obtain a hPSMA-specific CAR NK-92 cell population, to be thereafter characterized in vitro and in vivo for antigen-specific functional activity.Materials and MethodsNK-92 cell line was transduced with a lentiviral vector (LV) carrying a CAR anti-hPSMA. The cell population obtained was then sorted and analyzed for degranulation capacity, IFNγ production and lytic activity against hPSMA+ (PC3-hPSMA, LNCaP) or hPSMA-tumor cell lines. In vivo therapeutic efficacy of CAR-transduced NK-92 was evaluated initially using Winn-Assay and than in subcutaneous and orthotopic tumor models.ResultsCAR-expressing LV efficiently transduced NK-92 cells, which in turn produced cytokines, degranulated and exerted a relevant cytotoxic upon challenge with PSMA+ prostate tumor cells, irrespective of 10 Gy γ-irradiation. In all the in vivo, tumor models CAR-transduced NK-92 shown a statistically significant inhibition of tumor growth.ConclusionsChimeric antigen receptor-engineered NK-92 could offer a valid and cost-effective alternative to primary CAR NK or T cells, in particular in cases, where a suitable donor is not available or the sophisticated infrastructure needed for cell isolation, expansion and genetic modification is missing. This work demonstrates that CAR-engineered NK-92 cells display a high and specific recognition of hPSMA+ PC both in vitro as is in vivo, and could represent an efficient strategy as a new therapeutic intervention against prostate carcinoma, thus paving the way to an Off-The-Shelf cellular therapeutic for targeted elimination of cancer cells and induction of protective antitumor immunity.Disclosure InformationG. Zuccolotto: None. A. Penna: None. I.M. Montagner: None. D. Carpanese: None. A. Rosato: None.

scholarly journals Engineered iPSC-Derived NK Cells Expressing Recombinant CD64 for Enhanced ADCC

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 10-11
Author(s):  
Kate Dixon ◽  
Robert Hullsiek ◽  
Kristin Snyder ◽  
Zachary Davis ◽  
Melissa Khaw ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cell Line ◽  
Board Of Directors ◽  
Research Funding ◽  
Nk Cell ◽  
Target Cells ◽  
Advisory Committees ◽  
High Affinity ◽  
Adcc Assay

Natural killer (NK) cells are innate cytotoxic lymphocytes. They target malignant cells via non-clonotypic receptors to induce natural cytotoxicity and also recognize tumor-bound antibodies to induce antibody-dependent cell-mediated cytotoxicity (ADCC). While ADCC by NK cells is a key mechanism of several clinically successful therapeutic monoclonal antibodies (mAbs), most patients exhibit or acquire resistance to mAb therapies. ADCC by human NK cells is exclusively mediated by the IgG Fc receptor, CD16A (FcγRIIIA). Studies have demonstrated that increasing the binding affinity between CD16A and therapeutic mAbs can augment their clinical efficacy. Given the exquisite specificity and diverse antigen detection of anti-tumor mAbs, we are interested in enhancing the ADCC potency of NK cell-based therapies for various malignancies. CD64 is the only high affinity FcγR family member and binds to the same IgG isotypes as CD16A (IgG1 and IgG3) but with > 30-fold higher affinity. CD64 (FcγRI) is normally expressed by certain myeloid cells but not by NK cells. We generated a recombinant version of this receptor consisting of the extracellular region of CD64 and the transmembrane and intracellular regions of human CD16A, referred to as CD64/16A (figure 1A). An important feature of CD64/16A is that due to its high affinity state, soluble monomeric anti-tumor mAbs can be pre-adsorbed to engineered NK cells expressing the recombinant FcγR, and these pre-absorbed mAbs can be switched or mixed for universal tumor antigen targeting (figure 1B). The engineered NK cells used in our study were derived from genetically edited and clonally derived induced pluripotent stem cells (iPSCs) through a series of stepwise differentiation stages (figure 2). Engineered iPSC-derived NK (iNK) cells can be produced in a uniform and clinically scalable manner (figure 2). In Figure 3, using an in vitro Delfia® ADCC assay, we show that iNK-CD64/16A cells mediated ADCC against SKOV3 cells, an ovarian adenocarcinoma cell line, in the presence of the anti-HER2 therapeutic mAb trastuzumab (Herceptin) or anti-EGFR1 therapeutic mAb cetuximab (Erbitux), when either added to the assay or pre-adsorbed to the iNK cells (figure 3). Considering the high affinity state of CD64, we examined the effects of free IgG in human serum on ADCC by iNK-CD64/16A cells. Using an IncuCyte® Live Cell Analysis System, ADCC was evaluated in the presence or absence of 5% human AB serum, in which free IgG was approximately 50-fold higher than the IgG saturation level of the CD64/16A receptors on iNK cells (data not shown). Despite the high levels of excess free IgG, iNK-CD64/16A cells mediated efficient ADCC when Herceptin was either added to the assay or pre-adsorbed to the cells (figure 4). ADCC assays were also performed with Raji cells, a Burkitt lymphoma cell line, as target cells and the therapeutic mAb rituximab (Rituxan). iNK-CD64/16A cells were added with or without pre-adsorbed Rituxan and the assay was performed in 10% AB serum. Again, iNK-CD64/16A cells mediated effective target cell killing in the presence of serum IgG (figure 5), demonstrating that saturating levels of free IgG did not prevent ADCC. To determine if we can further optimize the function of recombinant CD64, we engineered CD64 with the transmembrane regions of CD16A or NKG2D and signaling/co-signaling domain from CD28, 2B4 (CD244), 4-1BB (CD137), and CD3ζ (figure 6). CD64/16A signals by non-covalent association with the immunoreceptor tyrosine-based activation motif (ITAM)-containing signaling adapters CD3ζ and FcRγ found in the cell membrane, whereas the other recombinant CD64 constructs use ITAM and non-ITAM regions to mediate their signaling. The various recombinant CD64 constructs were initially expressed in NK92 cells (lacks expression of endogenous FcγRs) (figure 7). Using the Delfia® ADCC assay system, we examined the function of each recombinant CD64 construct and found all combinations are able to effectively induce ADCC (figure 8). We are in the process of generating iNK cells with these constructs and testing their ability to kill hematologic and solid tumors in vitro and in vivo. Our goal is to utilize this docking approach to pre-absorb mAbs to iNK cells for adoptive cell therapy. The mAbs would thus provide tumor-targeting elements that could be exchanged as a means of preventing tumor cell escape by selectively and easily altering NK cell specificity for tumor antigens. Figure Disclosures Lee: Fate Therapeutics, Inc.: Current Employment. Chu:Fate Therapeutics: Current Employment. Abujarour:Fate Therapeutics, Inc: Current Employment. Dinella:Fate Therapeutics: Current Employment. Rogers:Fate Therapeutics, Inc: Current Employment. Bjordahl:Fate Therapeutics: Current Employment. Miller:Fate Therapeutics, Inc: Consultancy, Patents & Royalties, Research Funding; Nektar: Honoraria, Membership on an entity's Board of Directors or advisory committees; Vycellix: Consultancy; GT Biopharma: Consultancy, Patents & Royalties, Research Funding; Onkimmune: Honoraria, Membership on an entity's Board of Directors or advisory committees. Valamehr:Fate Therapeutics, Inc: Current Employment, Current equity holder in publicly-traded company. Walcheck:Fate Therapeutics: Consultancy, Research Funding.

scholarly journals CRISPR Screens Identify Mechanisms of Natural Killer Cell Evasion across Blood Cancers

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3597-3597
Author(s):  
Olli Dufva ◽  
Khalid Saeed ◽  
Sara Gandolfi ◽  
Michal Sheffer ◽  
Jay Klievink ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cancer Cells ◽  
Cell Line ◽  
Research Funding ◽  
Hematological Malignancies ◽  
Nk Cell ◽  
Cell Cytotoxicity ◽  
Gain Of Function ◽  
A Genome ◽  
Nk Cell Cytotoxicity

Natural killer (NK) cells have been suggested to control progression and relapse in several hematological malignancies. Enhancing NK cell reactivity represents an attractive approach to improve treatment responses. However, mechanisms enabling evasion of hematologic cancer cells from NK cells are incompletely understood. To identify cancer cell-intrinsic factors enabling resistance to NK cell cytotoxicity, we conducted genome-wide CRISPR screens in a range of hematological malignancies. Cas9-expressing cancer cells from diverse hematological malignancies, including acute and chronic myeloid leukemia (AML and CML), multiple myeloma (MM), diffuse large B-cell lymphoma (DLBCL), and B cell acute lymphoblastic leukemia were infected with a genome-scale lentiviral sgRNA library and exposed to primary human NK cells. Genes essential for NK cell cytotoxicity were discovered from surviving cancer cells which were enriched with gene knockouts inducing reduced sensitivity to NK cell killing. Additional data from a gain-of-function screen using a genome-scale CRISPR activation system were generated using the MM.1S myeloma cell line. Results from the functional genomic screens were integrated with transcriptomic data from > 9,000 patients across hematological cancers, as well as multi-omics data from AML and DLBCL, and both public and in-house single-cell RNA-sequencing data from AML patients. Knockout of genes encoding components involved in antigen presentation (B2M, HLA-A, HLA-C, HLA-E, TAP1, TAP2) sensitized multiple blood cancer cell lines to NK cell cytotoxicity, consistent with the inhibitory effect of MHC I on NK cells. Furthermore, knockout of interferon and JAK-STAT signaling components sensitized cancer cells to NK cell lysis, suggesting that MHC class I induction in response to interferon promotes NK cell resistance across cancer types. Interestingly, genes and pathways whose disruption conferred resistance for NK cell-mediated lysis exhibited partial overlap but also substantial variability across blood cancer types. Knockout of NCR3LG1 (B7-H6, ligand for the NKp30 activating NK cell receptor), resulted in resistance of K562 CML cells to NK cell cytotoxicity. In contrast, disruption of genes encoding apoptotic mediators (FADD, PMAIP1, BID) and TRAIL receptors (TNFRSF10B) conferred resistance to NK cell cytotoxicity in SUDHL4 DLBCL cells. The same pathways were identified in the MM cell line MM.1S, in which knockout of FAS, CASP2, and CASP8 as well as the TRAIL receptor TNFRSF10A induced NK cell resistance. Furthermore, loss of CD48, a ligand of the non-MHC binding receptor CD244 on NK cell surface, resulted in resistance and a genome-scale CRISPR gain-of-function screen in the same cell line showed sensitization upon CD48 overexpression. A CRISPR screen in the AML cell line MOLM14 identified disruption of TNFRSF1B encoding TNFR2 as strongly conferring NK cell resistance. Interestingly, TNFRSF1B overexpression sensitized the MM cell line MM.1S to NK cell treatment in the gain-of-function screen. Integration with genomic data from patients with hematological malignancies revealed selective expression of TNFRSF1B in AML. Within AML, TNFRSF1B expression was enriched in myelomonocytic and monocytic subtypes as well as in MLL-rearranged cases represented by the MOLM14 cell line. Further dissection at the single-cell level revealed increased expression of TNFRSF1B with differentiation of AML cells along the monocytic lineage. Consistently, the less differentiated MOLM13 cell line established from the same patient as MOLM14 was resistant to NK cell killing, suggesting that a less differentiated phenotype of AML cells confers resistance to NK cell cytotoxicity through lack of TNFRSF1B expression. Our data suggest that different lineages of hematological malignancies may be susceptible to NK cells through distinct mechanisms. In some cases, this heterogeneity is governed by lineage-specific expression of susceptibility genes, such as TNFRSF1B in monocytic AML. Particularly, our data identify a mechanism of NK cell evasion in less differentiated AML cells, suggesting potential for enhancing immune clearance of AML cells through differentiating therapies. figure Disclosures Lee: Kiadis Pharma: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Mitsiades:Takeda: Other: employment of a relative ; Ionis Pharmaceuticals: Honoraria; Fate Therapeutics: Honoraria; Arch Oncology: Research Funding; Sanofi: Research Funding; Karyopharm: Research Funding; Abbvie: Research Funding; TEVA: Research Funding; EMD Serono: Research Funding; Janssen/Johnson & Johnson: Research Funding. Mustjoki:BMS: Honoraria, Research Funding; Novartis: Research Funding; Pfizer: Research Funding.

scholarly journals Effect of Vasoactive Intestinal Peptide (VIP) on NKG2D Signal Pathway and Its Contribution to Immune Escape of MKN45 Cells

2013 ◽  
Vol 2013 ◽  
pp. 1-10 ◽  
Author(s):  
Chong Wang ◽  
Xi-Jin Zhou ◽  
Yuan-yuan Li ◽  
Juan Wan ◽  
Le-ying Yang ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Nk Cells ◽  
Cancer Cells ◽  
Mononuclear Cells ◽  
Immune Escape ◽  
Nk Cell ◽  
Signal Molecules ◽  
Gastric Cancer Cells ◽  
Peripheral Blood Mononuclear

Objective. To investigate VIP effect on the cytotoxicity of NK cell to gastric cancer cellsin vitroand the relation between the effect with the NKG2D signal molecules in NK cells.Material and Methods. NK cells were purified from peripheral blood mononuclear cells (PBMC). Before and after NK cells were incubated with VIP or its antagonist (D-p-Cl-Phe6,Leu17)-VIP, we detected the cytotoxicity of NK cells to MKN45 gastric cancer cells by MTT and detected the expressions of NKG2D, DAP10, and NF-κB proteins and mRNAs in NK cells by immunocytochemistry and RT-PCR in those conditions. Then we analyzed the effect of VIP and its antagonist on the cytotocicity of NK cell to gastric cancer cells and on expressions of NKG2D, DAP10, and NF-κB signal molecules in NK cells.Results. VIP could inhibit the cytotoxicity of NK cells to MKN45 cells and could inhibit the expressions of NKG2D, DAP10, and NF-κB in NK cells. However, (D-p-Cl-Phe6, Leu17)-VIP could reverse those effects.Conclusions. The VIP inhibited the cytotoxicity of NK cell to MKN45 cells which might get through inhibiting the expressions of NKG2D signal molecules in NK cells. This may be one mechanism of gastric cancer cells escaping organism immune clearance.

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