scholarly journals KIR 2DS4 May Influence Autologous and Cord Blood(CB) Natural Killer (NK)Cell Mediated in Vitro Cytotoxicity Against Freshly Isolated Human Bone Marrow Myeloma Plasma Cells and Cell Lines

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1920-1920
Author(s):  
Pinar Yurdakul Mesutoglu ◽  
Hasan Yalim Akin ◽  
Merve Bunsuz ◽  
Eylul Turasan ◽  
Mustafa Merter ◽  
...  
Keyword(s):  
Nk Cells ◽  
Nk Cell ◽  
Myeloma Cell ◽  
Target Cells ◽  
Advisory Committees ◽  
Cytotoxic Effects ◽  
Effector Cells ◽  
Cytotoxicity Assays

Abstract Background and Aim: NK cell effects are mediated by Killer Immunoglobulin-like Receptor (KIR) inhibition. There are few reports on the protective role of KIRs against relapse in myeloma(MM) (Gabriel et al Blood 2010, Kröger et al leukemia 2011). However, which NK cells or for whom will be more effective and the role of individual differences in this mechanism is indefinite. In this study we aimed to accomplish cytotoxicity against both MM cells and human myeloma cell lines (HMCL) and to compare the cytotoxicity achieved with CB versus autologous NK cells as well as to investigate the influence of NK cell KIR geno/phenotypes. Patients and Methods: A total of 18 patients with refractory/relapsed MM were included in this study. Cell culturing: CD138+ PC isolated from fresh bone marrow aspirates of relapsed MM patients or HMCL (U266, RPMI8226 and H929) using RosetteSep™ Human Multiple Myeloma Cell Enrichment Cocktail (StemCell Technologies) were subjected to cytotoxicity assays with CB derived or autologous PB derived NK cells expanded in the presence of IL-15 (10ng/ml) for 2-8 days. Phenotyping: Effector cells (autologous or CB-NK) were analyzed by flow cytometry using CD45, CD3, CD16, CD56, CD158a (KIR2DL1), CD158i (KIR2DS4) and CD158b1/b2 (KIR 2DL2/2DL3) monoclonal antibodies. Cytotoxicity assays: 3,3 dioctadecyloxacarbocyanine perchlorate (DiO) dye was used to distinguish target cells (PC) from effector cells (NK). PC and NK cells were mixed at an 1:10 target: effector cell ratio and inoculated with Propidium Iodide (PI) in order to counter stain the dead cells. Naive PC were used as controls. Following 120 mins of incubation, PC death was evaluated using flow cytometry. After incubation of target cells (PC) with effector cells (NK cells), cytotoxic effects were determined by subtraction of NK mediated PC death from spontaneous PC death. Results: Following IL-15 expansion NK cells were found to express the CD16+56+3- phenotype. Eighteen MM patient PCs and six different HMCL samples were used in cytotoxicity assays(Table 1). In vitro PC viability was less than 45% in four samples and could not be analyzed in the study. Following incubation of PC wo NK cells, spontaneous PC death ratio was found to be 22.6% (min-max: 3%-42.3%). Twelve cytotoxicity assays targeting six MM PC were performed with autologous and CB-derived NK cells. Autologous NK and CB NK mediated cytotoxicity median rates were found to be 19.0% (range: 1.4% - 43.6%) and 31.3% (range: 2.4% - 51.2%), respectively. Additionally, CB-NK cells were evaluated against HMCL samples (four U266, one RPMI8226 and one H929). Median cytotoxicity against HMCL was 27.4% (range: 5.6% - 52.1%). Highest cytotoxicity against MM PC (MM-9) and HMCL (H929) were achieved by CB NK cells (51.2% and 52.1 % respectively). There was no cytotoxic effect against PCs of patients MM6 and MM10 (Figure 1-2). Flow-cytometric phenotyping of effector NK cells showed consistency (10/13) with genotypes. Since only one (CB19) with 19% CD158i positivity was PCR negative, a %20 cut-off rate was chosen. Both CD158a (KIR2DL1) and CD158b1/b2 (KIR 2DL2/2DL3) were positive among all patients and no significant correlation with cytotoxic effects was detectable. However out of five high cytotoxic reactions three NK cells were 2DS4 (+). While out of eight assays which resulted with low cytotoxicity only three NK cells were positive for the activating KIR 2DS4. When we analyzed CB NK cells against HMCL U266, three CB NK cells two of which were 2DS4(+) showed high cytotoxicity, while the 2DS4(-) CB NKs did not. The only assay targeting RPMI-8226 which is known to be a NK resistant HMCL, KIR2DS4(+) (CB17) derived NK cells were not sufficiently cytotoxic. Conclusion: Allogeneic CB NK cells were able to induce higher cell kill than autologous PB-derived NK cells. KIR types of autologous NK cells were not predictive for cytotoxic efficacy. Lacking KIR2DS4 in CB NK cells was correlated with less cytotoxicity. Additionally, KIR2DS4(+) CB NK cells were found to be highly cytotoxic against both PC and HMCL. NK cells may be an effective alternative due to its profound clinical advantages such as matching for HLA or KIR ligand is not required. Furthermore, if confirmed by others, certain KIR haplotypes (ie Haplotype A and KIR2DS4 +) may offer better therapeutic potential. Acknowledgment: The study has been supported by Scientific and Research Council of Turkey (TUBITAK grant no: 115S579) and Turkish Academy of Sciences. Disclosures Beksac: Takeda: Membership on an entity's Board of Directors or advisory committees; Amgen,Janssen-Cilag,Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Loss of CD20 Expression and Exhaustion of Effector Cells Limit ADCC in CLL Patients Treated with Rituximab.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1610-1610 ◽  
Author(s):  
Berengere Vire ◽  
Justin SA Perry ◽  
Elinor Lee ◽  
Lawrence S Stennett ◽  
Leigh Samsel ◽  
...  
Keyword(s):  
Nk Cells ◽  
Effector Cell ◽  
Cell Function ◽  
Nk Cell ◽  
Cell Surface Expression ◽  
Target Cells ◽  
Effector Cells ◽  
Cell Functions ◽  
Anti Cd20

Abstract Abstract 1610 Poster Board I-636 A major mechanism how the chimeric anti-CD20 monoclonal antibody rituximab (RTX) depletes B-cells is antibody-dependent cellular cytotoxicity (ADCC). ADCC has been modeled in-vitro and in mouse models. However, investigations on ADCC directly in patients treated with RTX are scarce. Recent efforts have focused on improving ADCC through modifications in the Fc binding portion of novel antibodies or through stimulation of effector cell functions with GM-CSF. A more detailed understanding of ADCC as a therapeutic process is needed to optimize such strategies and to identify biomarkers of improved efficacy. Here we report a comprehensive analysis of ADCC in previously untreated CLL patients during the first two RTX infusions (375mg/m2) given in combination with fludarabine every 4 weeks. Following the initial infusion of RTX the absolute lymphocyte count (ALC) decreased by a median of 74% at 2h, followed by a partial recrudescence of cells so that by 24h the median decrease in ALC reached 39% (n=11). ADCC is mediated by effector cells that include NK cells, monocytes/macrophages, and granulocytes. First, we investigated changes in NK cell function: consistent with NK cell activation we found an increase in CD69 at 2, 6 and up to 24h (median 4.2-fold, p=0.005, n=10) after RTX administration and increased expression of the degranulation marker CD107a/b (median 1.9-fold, p<0.001, n=5) and down-regulation of perforin expression (median decrease 63%, p<0.001, n=5) at 4h from treatment start. Activation of NK cells is triggered by the engagement of CD16/FcγRIIIa by RTX coated CLL cells. Interestingly, CD16 expression on NK cells was rapidly lost, already apparent at 2h and maximal at 6h from the start of the RTX infusion (median decrease 82%, p=0.02, n=10) and was not completely recovered by 24h. We also found a significant decrease in expression of CD16 on granulocytes (78%, p<0.001, n=5) but an increase in monocytes (3.9-fold, p<0.001, n=5). In addition to loss of CD16, we found that the cytotoxic capacity of the effector cells was rapidly exhausted: in an oxidative-burst assay, monocytes showed a significant decrease in the production of reactive oxygen species 4h after initiation of RTX infusion (median 60% decrease, p=0.043) and at 6h from the start of the RTX infusion NK cell-mediated killing of K562 target cells was reduced by half (p<0.001, n=3). Interestingly, both the acute reaction to RTX infusions that manifest as a cytokine release syndrome and changes in effector cell function peaked during the first hours of the RTX infusion. We hypothesized that this might be due to the process of CD20 shaving, a rapid and pronounced decrease of CD20 cell surface expression modeled in-vitro and in mice as the result of a mechanism called trogocytosis that relies on the direct and rapid exchange of cell membrane fragments and associated molecules between effectors and target cells (Beum, J Immunol, 2008). First, we used western blot analysis of total CD20 protein in CLL cells and found a rapid loss of CD20 that was apparent already at 2h resulting in virtually complete loss of expression at 24h. Next, we used ImageStream technology to directly visualize ADCC interactions in-vivo. We indeed detected transfer of CD20 from CLL cells to NK cells and monocytes, resulting in complete CD20 loss in circulating CLL cells. While we detected transfer of CD20 into both cell types, monocytes were much more engaged in trogocytosis than NK cells. Consistently, 4h post RTX infusion we found a significant increase in intracellular RTX in granulocytes and monocytes using intracellular staining for human IgG. CD20 shaving appears to be of particular importance given that immunohistochemical analyses revealed that persistent disease in the bone marrow aspirates after 4 cycles of RTX treatment was mostly CD20 negative. Collectively, our results identify loss of CD20 from CLL cells by trogocytosis and exhaustion of immune effector mechanisms as limitations for anti-CD20 immunotherapy. These data identify possible avenues for improving CD20 mediated immunotherapy and characterize endpoints on which different anti-CD20 antibodies can be compared. Given that trogocytosis appears to be a common occurrence our findings likely have general importance to immunotherapy of hematologic malignancies. Disclosures No relevant conflicts of interest to declare.

CD30/CD16A Tandab AFM13-Induced Target Cell Lysis By NK-Cells Is Enhanced By CD137 Co-Stimulation and Blocking PD-1

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2747-2747 ◽  
Author(s):  
Xing Zhao ◽  
Narendiran Rajasekaran ◽  
Uwe Reusch ◽  
Jens-Peter Marschner ◽  
Martin Treder ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Nk Cells ◽  
Target Cell ◽  
Tumor Size ◽  
Target Cells ◽  
In Vivo Models ◽  
Effector Cells ◽  
Cytotoxicity Assays

Abstract Introduction: AFM13 is a CD30/CD16A bispecific tetravalent TandAb antibody that recruits and activates NK-cells by specific binding to CD16A for targeted lysis of CD30+ tumor cells. Given promising clinical activity and safety profile of AFM13 and proof-of-mechanism demonstrating dependence on the immune response, potential synergy of AFM13 and checkpoint modulators was evaluated. Methods: Efficacy of AFM13 alone or in combination with anti-CTLA-4, anti-PD-1, or anti-CD137 antibodies was assessed by in vitro cytotoxicity assays with human PBMCs or enriched NK-cells and CD30+ target cells as well as patient-derived xenograft in vivo models with autologous PBMC. To evaluate NK-cell-mediated lysis of CD30+ lymphoma cell lines, 4 hour cytotoxicity assays were performed with PBMCs or enriched NK-cells as effector cells in the presence of suboptimal concentrations of AFM13 alone, and in combination with anti-CTLA-4, anti-PD-1, or anti-CD137 antibodies. For the in vivo model tumor fragments derived from surgical specimens of newly diagnosed patients with CD30+ Hodgkin Lymphoma were xenografted (PDX) in immuno-deficient mice. After 28 days mice were reconstituted with autologous patient-derived PBMC and treated with AFM13 alone and in combination with anti-CTLA-4, anti-PD-1, or anti-CD137 antibodies weekly for a total of three weeks. Tumor size, tumor-infiltrating human lymphocytes and intra-tumoral cytokines were evaluated on day 58. Results: AFM13 as a single agent at suboptimal concentrations induced effector-to-target cell-dependent lysis of CD30+ lymphoma cells up to 40% using enriched NK-cells as effector cells in a 4 hour in vitro assay. Immune-modulating antibodies alone mediated substantially lower lysis (<25%). However, the addition of anti-PD-1 or anti-CD137 to AFM13 strongly enhanced specific lysis up to 70%, whereas the addition of anti-CTLA-4 to AFM13 showed no beneficial effect. The most impressive increase of efficacy was observed when AFM13 was applied together with a combination of anti-PD-1 and anti-CD137. In vivo, reduction of tumor growth was observed when AFM13 and anti-PD-1 were used as single agents or when AFM13 was combined with anti-CD137. Synergy was most impressive in these PDX models for the combination of AFM13 and anti-PD-1 which led to a very strong reduction of tumor size. Of note, reduction of tumor growth was strongly correlated with infiltrating NK- and T-cells and intra-tumoral cytokines. Conclusions: The combination trials performed with companion intra-tumoral assessment of lymphocytes and cytokines may enhance the efficacy of AFM13 in patients. This may be explained by a potential cross-talk between NK-cells and T-cell which was enhanced when AFM13 was used in combination with checkpoint modulators. Disclosures No relevant conflicts of interest to declare.

Romedepsin (RM), a HDACi, Significantly Increases the Expression of NKG2D Ligands, MIC A/B, in Leukemia/Lymphoma Cells (LL), in Part through the Glycogen Synthase Kinase-3 (GSK-3) Pathway, Resulting in Enhanced NK Cytotoxicity.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3027-3027
Author(s):  
Aniket Saha ◽  
Sejal Bavishi ◽  
Frances Zhao ◽  
Janet Ayello ◽  
Carmella van de Ven ◽  
...  
Keyword(s):  
Nk Cells ◽  
Nk Cell ◽  
Glycogen Synthase ◽  
Signal Transduction Pathway ◽  
Surface Expression ◽  
Glycogen Synthase Kinase 3 ◽  
Cytotoxicity Assays ◽  
Nkg2d Receptor

Abstract Abstract 3027 Poster Board II-1003 Introduction Natural killer (NK) cells recognize malignant cells through the tumor-associated expression of NKG2D-ligands, including MIC A/B, which are known to be expressed on epithelial tumors, resulting in tumor cytotoxicity (Ayello/Cairo et al, BBMT, 2006). The expression of MIC A/B on these tumors can be induced by in vitro exposure of these cells to HDACi, specifically Romedepsin (RM) (Skov et al, Cancer Res, 2005). Glycogen synthase kinase-3 (GSK-3), a constitutively active serine-threonine kinase with numerous functions including regulation of cellular differentiation, stress and apoptosis, has also been shown to be an important regulatory enzyme in the expression of MIC A/B in response to RM (Doble et al, J cell Sci 2003; Frame et al, Biochem J 2001; Skov et al, Cancer Res, 2005). Objective We sought to determine of the expression of MIC A/B in response to RM in various leukemia and lymphoma cells (LL), its influence on NK cell mediated cytotoxicity and to investigate the role of the GSK-3 pathway in the regulation of expression of MIC A/B in response to RM. Methods LL cells (106/ml, RS 4:11 [MLL-ALL], REH [pre-B cell ALL], Jurkat [T-cell ALL], Toledo [DLBCL], Ramos [Burkitt's Lymphoma]) were exposed to RM (10 ng/mL) for 24 hours, followed by FACS staining with PE-conjugated anti-MIC A/B antibody to determine surface expression of MIC A/B. Peripheral blood NK cells (CD3-/56+) were isolated via magnetic separation followed by IL-2 activation (3000IU/ml, 18 hrs). LL cells exposed to RM (generously supplied by Gloucester Pharmaceuticals) were subjected to NK cell mediated cytotoxicity assays (using an europium assay) at effector:target (E:T) ratio of 10:1, as we had previously described (Ayello/Cairo et al, BBMT 2006). LL cells were also pre-treated for 1 hour with 100mM lithium chloride (LiCl), a potent inhibitor of GSK-3 activity (Davies et al, Biochem J, 2000), to determine the role of this regulatory enzyme in the RM mediated expression of MIC A/B in these LL cells. Finally, blocking studies were also performed with anti-NKG2D receptor blockers to determine the specific role of NKG2D signal transduction pathway in NK cell mediated cytotoxicity. Results MIC A/B expression significantly increased in LL cells in response to RM ([RS4:11 0.2% vs 19.2%, p< 0.0001], [REH 0.2% vs 46%, p= 0.0003], [Jurkat 1.12% vs 44.7%, p< 0.0001], [Toledo 0.5% vs 15.8%, p=0.0001], [Ramos 0.57% vs 33.6%, p=0.0003]). In addition, the expression of MIC A/B in response to RM was inhibited when LL cells are pre-treated with LiCl (Jurkat [RM vs RM+LiCl] 85% vs 18%, p<0.0001; RS 4:11 [RM vs RM+LiCl] 82% vs 5%, p<0.0001; Ramos [RM vs RM+LiCl] 67% vs 35%, p<0.0001). Cytotoxicity assays revealed significant increases in-vitro cytotoxicty in RS 4:11, Ramos and REH cells at E:T ratio of 10:1. Mean specific release (MSR)(±SEM) was measured for four conditions in the assay: NK cells (A) vs NK cells with 10 ng/ml RM (C) vs IL-2 activated NK cells (B) IL-2 activated NK cells +10 ng/ml of RM (D). MSR for RS 4:11 10%±3.6% vs 108%±47% and 12%±4% vs 145%±45% p<0.05(A vs C, B vs D) for RS 4;11. MSR for REH was 13.2%±14.4% vs 50%±17.6% and 22%±18% vs 132%±29% p<0.01(A vs C, B vs D). MSR for Ramos was 7.6%±7.6% vs 79%±15.6% and 35%±13% vs 90%±22% p<0.05(A vs C, B vs D). NKG2D receptor-blocking experiments resulted in significant decrease in NK cell mediated cytotoxicity in REH (p<0.03) and Ramos cells (p<0.001). Conclusion Our data suggests that the surface expression of MICA/B in LL cells is significantly increased by RM leading to enhanced susceptibility for NKG2D-mediated cytotoxicity by NK cells. Furthermore, up-regulation of MICA/B in LL cells secondary to RM exposure is in part regulated by the GSK-3 signal transduction pathway. Disclosures No relevant conflicts of interest to declare.

The JAK Inhibitor Ruxolitinib Substantially Affects NK Cells in MPN Patients

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3169-3169
Author(s):  
Kathrin Schönberg ◽  
Janna Rudolph ◽  
Maria Vonnahme ◽  
Isabelle Cornez ◽  
Sowmya Parampalli Yajnanarayana ◽  
...  
Keyword(s):  
Nk Cells ◽  
Research Funding ◽  
Nk Cell ◽  
Receptor Expression ◽  
Target Cells ◽  
Jak Inhibitor ◽  
Advisory Committees ◽  
Nk Cell Differentiation

Abstract Introduction: Ruxolitinib (INCB018424) is the first JAK inhibitor approved for treatment of myelofibrosis (MF). Ruxolitinib-induced reduction of splenomegaly and symptoms control is linked to a substantial suppression of MF-associated circulating pro-inflammatory and pro-angiogenic cytokines. However, an increased rate of infections in ruxolitinib-exposed patients with MF was recently described. Natural killer (NK) cells are innate immune effector cells eliminating malignant or virus-infected cells. Thus, the aim of this project was to define in more detail the impact of JAK inhibition on NK cell biology both in vitro and in vivo. Methods: 28 patients with myeloproliferative neoplasms (MPN) with or without ruxolitinib therapy and 12 healthy donors were analyzed for NK cell frequency, NK receptor expression and function. Phenotypic and functional NK cell markers (e.g. CD11b, CD27, KIR, NKG2A, NKG2D, NKp46, CD16, granzyme B, and perforin) were analyzed by FACS. NK cell function was evaluated by classical killing assays upon stimulation with MHC class I-deficient target cells K562. Finally, a set of additional in vitro experiments (e.g. analysis of lytic synapse formation by FACS and confocal microscopy) were performed to define in more detail the characteristics and potential mechanisms of ruxolitinib-induced NK cell dysfunction. Results: In addition to our recent finding that ruxolitinib induces NK cell dysfunction in vitro (e.g. reduced killing, degranulation and IFN-γ production), we here demonstrate that NK cell proliferation and cytokine-induced receptor expression as well as cytokine signalling are drastically impaired by ruxolitinib. Interestingly, reduced killing is at least in part due to a reduced capacity to form a mature lytic synapse with target cells. The significance of the in vitrofindings is underscored by a dramatically reduced proportion and absolute number of NK cells in ruxolitinib-treated MPN patients when compared to treatment-naïve patients or to healthy controls (mean percentage of NK cell frequency: ruxolitinib-naïve MPN patients 12.63% ±1.81; healthy donors 13.51% ±1.44; ruxolitinib-treated patients 5.47% ±1.27). A systematic analysis of NK cell receptor expression revealed that the reduction of NK cells in ruxolitinib-exposed individuals is most likely due to an impaired NK cell differentiation and maturation process, as reflected by a significantly increased ratio of immature to mature NK cells. Finally, the endogenous functional NK cell defect in MPN is further aggravated by intake of the JAK inhibitor ruxolitinib. Conclusion: We here provide compelling in vitro and in vivo evidence that inhibition of the JAK/STAT-pathway by ruxolitinib exerts substantial effects on the NK cell compartment in MPN patients due to the inhibition of NK cell differentiation and NK cell key functions. Our data may help to better understand the increased rate of severe infections and complement recent reports on ruxolitinib-induced immune dysfunction. Disclosures Koschmieder: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Travel, Accomodation, Expenses Other. Brümmendorf:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Wolf:Novartis: Consultancy, Honoraria, Research Funding, Travel and Accommodation Other.

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 &gt; 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 FT538: Preclinical Development of an Off-the-Shelf Adoptive NK Cell Immunotherapy with Targeted Disruption of CD38 to Prevent Anti-CD38 Antibody-Mediated Fratricide and Enhance ADCC in Multiple Myeloma When Combined with Daratumumab

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 133-133
Author(s):  
Ryan Bjordahl ◽  
Svetlana Gaidarova ◽  
Karrune Woan ◽  
Frank Cichocki ◽  
Greg Bonello ◽  
...  
Keyword(s):  
Nk Cells ◽  
Board Of Directors ◽  
Research Funding ◽  
Pluripotent Stem Cell ◽  
Nk Cell ◽  
Target Cells ◽  
Stem Cell Line ◽  
Advisory Committees ◽  
Cell Immunotherapy

Monoclonal antibody (mAb) treatment is an effective therapeutic strategy for many cancer types, though there remains meaningful opportunity to improve mAb efficacy by optimizing the interaction with natural killer (NK) cells to enhance antibody-dependent cellular cytotoxicity (ADCC). NK cells are an ideal effector cell for combined use with tumor-targeting mAbs, as NK cells effect both innate tumoricidal capacity and ADCC. CD38-targeting mAbs, such as daratumumab, are effective in treating multiple myeloma (MM) and achieve their efficacy through multiple mechanisms, including ADCC. However, because activated NK cells express high levels of CD38, daratumumab induces NK cell depletion through fratricide, potentially reducing treatment effectiveness. Adoptive NK cell immunotherapy therefore has the potential to augment daratumumab's ADCC activity if fratricide can be reduced or prevented. FT538 is an off-the-shelf adoptive NK cell immunotherapy product candidate designed for enhanced cellular persistence and ADCC while avoiding anti-CD38 mAb induced fratricide. It is derived from induced pluripotent stem cells (iPSC) engineered to lack CD38 expression, which we have previously shown to eliminate daratumumab-induced fratricide among iPSC-derived NK cells, resulting in enhanced long-term daratumumab-mediated ADCC. FT538 is engineered to express an IL-15 receptor alpha fusion protein (IL-15RF; IL-15 tethered to IL-15 receptor α) to enhance persistence and a high-affinity non-cleavable CD16 (hnCD16, FcRγIII) to increase ADCC. To support the clinical translation of FT538, and to enable the repeatable and scalable cell production to support off-the-shelf availability of a uniform NK cell product, a clinical-grade master pluripotent stem cell line was developed. The FT538 master pluripotent stem cell line was created by reprogramming donor fibroblasts into iPSCs using our non-integrating cellular reprogramming platform, and cells were further genetically edited by targeting IL-15RF and hnCD16 to the CD38 locus. Clonal iPSC lines were generated and screened for precise knock-in and knock-out edits at the CD38 locus and a lack of off-target genome integration (15% total success rate for CD38-/-IL-15RF+CD16+). Selected engineered iPSC clones were confirmed to be free of reprogramming transgenes and to maintain genomic stability. Engineered iPSC clones were additionally tested for their NK cell differentiation potential and function, and a single clone was selected to serve as the renewable starting material for cGMP manufacturing and clinical development. Upon differentiation and expansion FT538 demonstrated a mature NK cell phenotype with expression of NK cell receptors including NKp30, NKp46, NKG2D, KIR, NKG2A, and DNAM-1. The functional impact of CD38 knockout on FT538 NK cells was confirmed in an in vitro fratricide assay, where peripheral blood (PB)-NK cells exhibited fratricide at a frequency of 33% after 3 hr culture with increasing daratumumab concentrations. In contrast, FT538 cells were entirely resistant (&lt;1% specific cytotoxicity) to daratumumab-induced fratricide. In vitro cytotoxic re-stimulation assays showed that repeat exposure of PB-NK cells to daratumumab plus MM target cells resulted in a loss of cytotoxic capacity (from 74% to 58% upon re-stimulation), and a similar effect was seen for non-engineered iPSC-derived NK cells. In contrast, FT538 NK cells maintained robust ADCC in during primary and secondary exposure to MM target cells and daratumumab. FT538 with daratumumab resulted in 86% cytotoxicity against MM target cells upon first exposure and 92% cytotoxicity upon re-stimulation, with a 20-fold increase in viable NK cells at the conclusion of the assay compared to non-engineered iPSC-derived NK cells. Additionally, the combined survival benefit of IL-15RF expression and fratricide resistance mediated by the CD38 knockout as well as the enhanced hnCD16-mediated ADCC allowed for greater cytotoxicity of FT538 against MM tumor spheroids. Together, these preclinical data support the clinical translation of FT538, an off-the-shelf adoptive NK cell immunotherapy product engineered for uniform hnCD16 and IL-15RF expression with CD38 elimination for enhanced ADCC in combination with daratumumab and other anti-CD38 mAbs for the treatment of MM. Disclosures Bjordahl: Fate Therapeutics, Inc.: Employment. Gaidarova:Fate Therapeutics, Inc: Employment. Cichocki:Fate Therapeutics, Inc: Research Funding. Bonello:Fate Therapeutics, Inc.: Employment. Robinson:Fate Therapeutics, Inc.: Employment. Ruller:Fate Therapeutics, Inc.: Employment. Pribadi:Fate Therapeutics, Inc.: Employment. Dinella:Fate Therapeutics, Inc.: Employment. Fong:Fate Therapeutics, Inc.: Employment. Huffman:Fate Therapeutics, Inc.: Employment. Chu:FATE THERAPEUTICS: Employment. Lee:Fate Therapeutics, Inc.: Employment. Abujarour:Fate Therapeutics, Inc.: Employment. Kaufman:FATE Therapeutics: Consultancy, Research Funding. Malmberg:Fate Therapeutics, Inc.: Consultancy, Research Funding; Vycellix: Consultancy, Membership on an entity's Board of Directors or advisory committees. Miller:CytoSen: Membership on an entity's Board of Directors or advisory committees; Moderna: Membership on an entity's Board of Directors or advisory committees; OnKImmune: Membership on an entity's Board of Directors or advisory committees; GT BioPharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Dr. Reddys Laboratory: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics, Inc: Consultancy, Research Funding. Valamehr:Fate Therapeutics, Inc: Employment.

scholarly journals FT576: Multi-Specific Off-the-Shelf CAR-NK Cell Therapy Engineered for Enhanced Persistence, Avoidance of Self-Fratricide and Optimized Mab Combination Therapy to Prevent Antigenic Escape and Elicit a Deep and Durable Response in Multiple Myeloma

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 4-5
Author(s):  
Jode P Goodridge ◽  
Ryan Bjordahl ◽  
Sajid Mahmood ◽  
John Reiser ◽  
Svetlana Gaidarova ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Board Of Directors ◽  
Research Funding ◽  
Nk Cell ◽  
Target Cells ◽  
Publicly Traded ◽  
Advisory Committees ◽  
Effector Cells ◽  
Exogenous Cytokine

Multiple redundancy within the spectrum of an immune response is required to prevent antigen escape or adaptation of the targeted population to host defenses. As adoptive cell therapies continue to evolve, multi-modal engineering of effector cells offers the prospect of tackling increasingly complex disease settings such as multiple myeloma (MM), where targeting of a single tumor associated antigen is frequently confounded by antigen shedding and escape variation resulting in the inability to develop a curative therapy. There are multiple advantages in expanding treatment options beyond autologous primary T and NK cells, including the use of induced pluripotent stem cells (iPSC) to derive effector cells that can be uniformly manufactured at scale from renewable starting cellular material and where precision genetic engineering can be achieved at the clonal level which can be applied sequentially in order to build multiple specificities and functional modalities. To create a platform targeted toward MM, a multiplexed edited base iPSC-derived NK (iNK) cell configuration consisting of a CD38 KO iPSC modified to overexpress a recombinant IL-15 signaling complex (IL15RF) for autonomous persistence and a functionally enhanced high-affinity, non-cleavable CD16 (hnCD16) was developed. Introduction of IL15RF enabled expansion of iNK cells without additional exogenous cytokine support during the manufacturing process and greatly improved functional persistence of iNK cells both in vitro and in various xenograft mouse models (Figure 1). To target MM in a broad and comprehensive manner, we tested our novel BCMA-CAR in combination with different myeloma targeted antibodies. In combination with hnCD16, co-expression of BCMA-CAR and IL15RF culminates in an iNK cell therapeutic, termed FT576, capable of multiantigen-specificity through combinatorial use of CAR and hnCD16 with monoclonal antibodies to tackle antigen escape. Chimerization of an anti-BCMA scFv shown to elicit higher affinity onto the CAR platform produced specific in vitro recognition of BCMA+ myeloma cells in short-term and long-term NK cell cytotoxicity assays. Specificity of the BCMA-CAR was demonstrated using NALM6 overexpressing BCMA using a short range 4H caspase assay (NALM6_BCMA EC50 14.4, NALM6wt EC50 39.1, p*&lt;0.0001). Utilizing a long range clearance assay, serial restimulation by repeated rounds of exposure to fresh MM1S MM target cells was tested, showing remarkable persistence and antigen-mediated expansion of CAR function in isolation or combined with antibody through 3 rounds of stimulation in the absence of exogenous cytokine support (Figure 2). Continuous long-range clearance assays demonstrated levels of BCMA targeting activity of FT576 alone was equivalent to primary BCMA-targeted CAR-T cells against a panel of BCMA+ target cells. Utilizing hnCD16, BCMA-CAR was tested in combination with anti-CD38 (daratumumab), anti-SLAMF7 (elotuzumab), or anti-CD19, showing synergistic increase in tumor targeting through various tumor associated antigens (TAAs). Polyfunctionality of FT576 stimulated either through CAR or ADCC was similarly measured by both Isoplexis and single cell RNA sequencing. Specificity for plasma cells was confirmed using primary bone marrow samples from either healthy donors or patients. In animal models, as a monotherapy, FT576 achieved sustained tumor control against disseminated MM1s with persistence profile suggestive of antigen mediated expansion (Figure 3). In combination with daratumumab, FT576 was able to achieve complete clearance of MM1S. Combination with other monoclonal antibodies displayed a similar response demonstrating the unique ability of FT576 to be directed to target multiple TAAs. Together, these studies demonstrate the versatility of FT576 as a highly effective multi-antigen targeting and cost-effective off-the-shelf BCMA-CAR iNK cell product and supports the rational for a first-of-kind Phase I Study as a monotherapy or in combination with therapeutic mAbs targeted to MM-associated surface antigens, driving a path towards a curative therapeutic in MM. Disclosures Goodridge: Fate Therapeutics, Inc: Current Employment. Bjordahl:Fate Therapeutics: Current Employment. Mahmood:Fate Therapeutics, Inc: Current Employment. Reiser:FATE THERAPEUTICS: Current Employment. Gaidarova:Fate Therapeutics, Inc: Current Employment. Blum:Fate Therapeutics: Current Employment. Cichocki:Fate Therapeutics, Inc: Consultancy, Patents & Royalties, Research Funding. Chu:Fate Therapeutics, Inc: Current Employment. Bonello:Fate Therapeutics, Inc: Current Employment. Lee:Fate Therapeutics, Inc.: Current Employment. Groff:Fate Therapeutics, Inc: Current Employment. Meza:Fate Therapeutics, Inc: Current Employment. Chu:Roche Holding AG: Current equity holder in publicly-traded company; Fate Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Walcheck:Fate Therapeutics: Consultancy, Research Funding. Malmberg:Vycellix: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics: Consultancy, Patents & Royalties. Miller:Vycellix: Consultancy; Onkimmune: Honoraria, Membership on an entity's Board of Directors or advisory committees; Nektar: Honoraria, Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics, Inc: Consultancy, Patents & Royalties, Research Funding; GT Biopharma: Consultancy, Patents & Royalties, Research Funding. Valamehr:Fate Therapeutics, Inc: Current Employment, Current equity holder in publicly-traded company.

scholarly journals GSK-3α Inhibition in Drug-Resistant CML Cells Promotes Susceptibility to NK Cell-Mediated Lysis in an NKG2D- and NKp30-Dependent Manner

Cancers ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1802
Author(s):  
Nayoung Kim ◽  
Mi Yeon Kim ◽  
Woo Seon Choi ◽  
Eunbi Yi ◽  
Hyo Jung Lee ◽  
...  
Keyword(s):  
Nk Cells ◽  
Nk Cell ◽  
Negative Regulator ◽  
Target Cells ◽  
Dependent Manner ◽  
Cell Functions ◽  
Cell Based Therapy ◽  
Activating Receptors ◽  
Gsk 3Β

Natural killer (NK) cells are innate cytotoxic lymphocytes that provide early protection against cancer. NK cell cytotoxicity against cancer cells is triggered by multiple activating receptors that recognize specific ligands expressed on target cells. We previously demonstrated that glycogen synthase kinase (GSK)-3β, but not GSK-3α, is a negative regulator of NK cell functions via diverse activating receptors, including NKG2D and NKp30. However, the role of GSK-3 isoforms in the regulation of specific ligands on target cells is poorly understood, which remains a challenge limiting GSK-3 targeting for NK cell-based therapy. Here, we demonstrate that GSK-3α rather than GSK-3β is the primary isoform restraining the expression of NKG2D ligands, particularly ULBP2/5/6, on tumor cells, thereby regulating their susceptibility to NK cells. GSK-3α also regulated the expression of the NKp30 ligand B7-H6, but not the DNAM-1 ligands PVR or nectin-2. This regulation occurred independently of BCR-ABL1 mutation that confers tyrosine kinase inhibitor (TKI) resistance. Mechanistically, an increase in PI3K/Akt signaling in concert with c-Myc was required for ligand upregulation in response to GSK-3α inhibition. Importantly, GSK-3α inhibition improved cancer surveillance by human NK cells in vivo. Collectively, our results highlight the distinct role of GSK-3 isoforms in the regulation of NK cell reactivity against target cells and suggest that GSK-3α modulation could be used to enhance tumor cell susceptibility to NK cells in an NKG2D- and NKp30-dependent manner.

scholarly journals Microfluidic tumor-on-a-chip model to evaluate the role of tumor environmental stress on NK cell exhaustion

2021 ◽  
Vol 7 (8) ◽  
pp. eabc2331 ◽  
Author(s):  
Jose M. Ayuso ◽  
Shujah Rehman ◽  
Maria Virumbrales-Munoz ◽  
Patrick H. McMinn ◽  
Peter Geiger ◽  
...  
Keyword(s):  
Nk Cells ◽  
Immune Cells ◽  
Molecular Mechanisms ◽  
Nk Cell ◽  
Checkpoint Inhibitors ◽  
Waste Product ◽  
Extended Period ◽  
Cell Exhaustion

Solid tumors generate a suppressive environment that imposes an overwhelming burden on the immune system. Nutrient depletion, waste product accumulation, hypoxia, and pH acidification severely compromise the capacity of effector immune cells such as T and natural killer (NK) cells to destroy cancer cells. However, the specific molecular mechanisms driving immune suppression, as well as the capacity of immune cells to adapt to the suppressive environment, are not completely understood. Thus, here, we used an in vitro microfluidic tumor-on-a-chip platform to evaluate how NK cells respond to the tumor-induced suppressive environment. The results demonstrated that the suppressive environment created by the tumor gradually eroded NK cell cytotoxic capacity, leading to compromised NK cell surveillance and tumor tolerance. Further, NK cell exhaustion persisted for an extended period of time after removing NK cells from the microfluidic platform. Last, the addition of checkpoint inhibitors and immunomodulatory agents alleviated NK cell exhaustion.

scholarly journals Medulloblastoma rendered susceptible to NK-cell attack by TGFβ neutralization

2019 ◽  
Vol 17 (1) ◽  
Author(s):  
Allison B. Powell ◽  
Sridevi Yadavilli ◽  
Devin Saunders ◽  
Stacey Van Pelt ◽  
Elizabeth Chorvinsky ◽  
...  
Keyword(s):  
Nk Cells ◽  
Transforming Growth Factor ◽  
Nk Cell ◽  
Transforming Growth Factor Β ◽  
In Vitro Models ◽  
Dominant Negative ◽  
Conditioned Media ◽  
Retroviral Transduction ◽  
Effector Cells

Abstract Background Medulloblastoma (MB), the most common pediatric brain cancer, presents with a poor prognosis in a subset of patients with high risk disease, or at recurrence, where current therapies are ineffective. Cord blood (CB) natural killer (NK) cells may be promising off-the-shelf effector cells for immunotherapy due to their recognition of malignant cells without the need for a known target, ready availability from multiple banks, and their potential to expand exponentially. However, they are currently limited by immune suppressive cytokines secreted in the MB tumor microenvironment including Transforming Growth Factor β (TGF-β). Here, we address this challenge in in vitro models of MB. Methods CB-derived NK cells were modified to express a dominant negative TGF-β receptor II (DNRII) using retroviral transduction. The ability of transduced CB cells to maintain function in the presence of medulloblastoma-conditioned media was then assessed. Results We observed that the cytotoxic ability of nontransduced CB-NK cells was reduced in the presence of TGF-β-rich, medulloblastoma-conditioned media (21.21 ± 1.19% killing at E:T 5:1 in the absence vs. 14.98 ± 2.11% in the presence of medulloblastoma-conditioned media, n = 8, p = 0.02), but was unaffected in CB-derived DNRII-transduced NK cells (21.11 ± 1.84% killing at E:T 5:1 in the absence vs. 21.81 ± 3.37 in the presence of medulloblastoma-conditioned media, n = 8, p = 0.85. We also observed decreased expression of CCR2 in untransduced NK cells (mean CCR2 MFI 826 ± 117 in untransduced NK + MB supernatant from mean CCR2 MFI 1639.29 ± 215 in no MB supernatant, n = 7, p = 0.0156), but not in the transduced cells. Finally, we observed that CB-derived DNRII-transduced NK cells may protect surrounding immune cells by providing a cytokine sink for TGF-β (decreased TGF-β levels of 610 ± 265 pg/mL in CB-derived DNRII-transduced NK cells vs. 1817 ± 342 pg/mL in untransduced cells; p = 0.008). Conclusions CB NK cells expressing a TGF-β DNRII may have a functional advantage over unmodified NK cells in the presence of TGF-β-rich MB, warranting further investigation on its potential applications for patients with medulloblastoma.

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