Treatment of MCL Cells with Combined Rituximab and Lenalidomide Enhances NK-Mediated Synapse Formation and Cell Killing.

Abstract Abstract 1687 Poster Board I-713 Introduction Mantle cell lymphoma (MCL) is a subtype of aggressive B-cell non-Hodgkin's Lymphoma (NHL) characterized by poor prognosis and very few therapeutic options that improve survival. Lenalidomide is an immunomodulatory agent that has demonstrated significant clinical activity in the treatment of patients with MCL. A mechanism attributed to lenalidomide, which has also been demonstrated in B-CLL models, is the restoration of impaired T-cell activity to form immunological synapses, thus enhancing T-cell effector functions thereby eliminating aberrant tumor B cells. Natural killer (NK) cells are another cell type that eliminates aberrant cell types via a mechanism dependent on active formation of immunological synapses. By engaging in both direct killing and ADCC, NK cells are an important component of innate immunity eliminating transformed cell types. Here we assess the effect of lenalidomide on the ability of NK cells to form immune synapses with MCL cell lines and cell samples from MCL patients. We also evaluate whether lenalidomide-mediated immunologic activity is altered when lenalidomide is combined with rituximab, an anti-CD20 antibody shown to eliminate MCL cells through the ADCC mechanism. Methods To measure immune synapse formation, NK and JeKo-1 cells were pre-treated with DMSO or 1μM lenalidomide for 24 or 48 hrs. JeKo-1 cells (labeled with red PKH26) were incubated for 30 min with or without 10 μg/ml rituximab, and cell conjugates were fixed and stained with Phalloidin-FITC to measure mean fluorescent intensity and F-actin formation. Adenylate kinase (AK) release and 7AAD staining were used to measure NK-mediated cytotoxicity after NK and MCL cells were co-cultured at different target-to-effector ratios, and with and without rituximab. Flow cytometry was used to measure relative expression of cell surface markers CD56 (N-CAM), CD54 (I-CAM1), and NKG2D in both JeKo-1 cells and in B-cell samples from MCL patients. Results Addition of lenalidomide enhanced the formation of immunological synapses between JeKo-1 cells and NK cells, increasing the number of synapses approximately 3-fold after 24 hrs of treatment. When lenalidomide was combined with rituximab the number of synapses increased 3.5-fold. A significant increase in the number of synapses also occurred when NK cells co-cultured with MCL patient samples were treated with lenalidomide (2.5-fold increase) and lenalidomide plus rituximab (3-fold increase). Lenalidomide also augmented the intensity of F-actin at the synaptic site, which indicates more matured synapses rich with F-actin. The increased synapse-forming activity resulting from treatment with rituximab plus lenalidomide also translated into enhanced NK-mediated cytotoxicity. After 48 hours of concurrent treatment with lenalidomide and rituximab, approximately 80% of JeKo-1 cells released AK compared with approximately 45% after treatment with lenalidomide alone. Comparable cell killing data upon treatment with lenalidomide plus rituximab was obtained using a 7AAD assay. Finally, to understand the mechanism by which lenalidomide enhances the formation of immune synapses and augments the rituximab-dependent NK cell-mediated cytotoxicity in MCL cells, we studied the effect of lenalidomide on effector and target cells separately. In the NK cells, lenalidomide treatment induced F-actin polymerization and polarization, and the accumulation of perforin, which is evidence for effector cell activation. Previously we showed that lenalidomide treatment of tumor cells induces changes in actin structure and increases expression of cell surface markers, including CD54 and co-stimulatory molecules, which correlated with increased antigen presentation properties of tumor cells. Here we show that lenalidomide and rituximab induced F-actin polymerization and polarization in the JeKo-1 cells when administered individually and in combination, and resulted in slightly increased cytotoxicity. Conclusion Our results suggest that in MCL, combined use of lenalidomide and rituximab enhances NK-mediated immune synapse formation and the resultant cytotoxicity. Combining lenalidomide with rituximab may also enhance the anti-tumor immune response mediated through enhanced activity of NK cells. These studies suggest that lenalidomide plus rituximab may have clinical utility in the treatment of patients with MCL. Disclosures Gaidarova: Celgene: Employment, Equity Ownership. Corral:Celgene: Employment. Glezer:Celgene: Employment, Equity Ownership. Schafer:Celgene: Employment. Lopez-Girona:Celgene: Employment.

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Lenalidomide Alone and in Combination with Rituximab Enhances NK Cell Immune Synapse Formation in Chronic Lymphocytic Leukemia (CLL) Cells in Vitro through Activation of Rho and Rac1 GTPases.

Blood ◽

10.1182/blood.v114.22.3441.3441 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3441-3441 ◽

Cited By ~ 5

Author(s):

Svetlana Gaidarova ◽

JianWu Li ◽

Laura G Corral ◽

Emilia Glezer ◽

Peter H Schafer ◽

...

Keyword(s):

B Cells ◽

Nk Cells ◽

Synapse Formation ◽

Nk Cell ◽

Employment Equity ◽

Immune Synapse ◽

Immunological Synapses ◽

Immune Synapses ◽

Cd20 Expression ◽

Equity Ownership

Abstract Abstract 3441 Poster Board III-329 Background CLL is characterized by the progressive accumulation of monoclonal B lymphocytes. One theory to explain how CLL cells avoid elimination through immune surveillance mechanisms is through a defect in the ability of T-cells to form immunological synapses with antigen-presenting tumor B-cells (Ramsay et al JCI 2008). Lenalidomide is an immunomodulatory agent with clinical activity in the treatment of B-cell malignancies. Recent laboratory studies showed that lenalidomide not only stimulates T- and natural killer (NK)-cell-mediated ADCC, it also restores the T-cell-mediated ability to form immunological synapses with CLL tumor cells. Since NK cells also exert cytotoxicity through immune synapse formation, here we explore how lenalidomide affects NK-cell-mediated cytotoxicity mechanisms and whether this activity is altered in the presence of rituximab since published studies showed that lenalidomide-pretreated B-cells have a down-regulated surface CD20 expression. Further, we investigated the molecular events associated with immune synapse formation and the effect of lenalidomide. Methods Immune synapse formation was assessed in NK cells (from healthy donors PBMCs) co-cultured with either B-CLL cells derived from pts or with K562 cells (positive control). Cells were fixed and the ability to form synapses was assessed via immunohistochemisty co-staining for either F-actin and CD2, or F-actin and perforin (a cytolytic protein found in NK cells). Synapse formation was visualized by microscopy and measured via relative mean fluorescent intensity. Activity of RhoA, Rac1, Cdc42 were measured using Rho GTPases assay kits. Inhibition of lenalidomide-mediated immune synapse activity was assayed using the cell permeable Rho inhibitor C3 (0.5 mM). Flow cytometry was used to measure changes in surface CD20 and CD54 (ICAM-1) expression in B-CLL samples from 3 pts after treatment with lenalidomide. Results Lenalidomide induced the formation of immunological synapses between NK cells and primary B-CLL cells (p<.01) or the K562 cell line. Lenalidomide activated NK cells regardless of the presence of target cells, as measured by F-actin and perforin staining. RhoA and Rac1 were activated at the immunological synapse in the presence of lenalidomide. Inhibition of RhoA by the C3 inhibitor blocked F-actin localization, as well as perforin accumulation induced by lenalidomide at cell-cell contact sites, indicating inhibition of immune synapses and the associated cytolytic activity. This was also observed with Rac1 inhibition, but to a lesser degree than with RhoA inhibition. Functionality of formed synapses was confirmed by co-localization of F-actin and perforin at the synapse sites. 3 CLL pt samples treated ex vivo with lenalidomide demonstrated variable changes in CD20 expression: a 20-30% decrease in CD20 expression was observed in 2 B-CLL pt samples, whereas CD20 levels remained unchanged in the third. In the presence of rituximab, lenalidomide-induced synapse formation between NK cells and B-cells from CLL patients was further enhanced. This was accompanied by upregulation of costimulatory and adhesion molecule CD54 on B-CLL cells suggesting increased antigen presentation, which might contribute to the increased synapse formation. Conclusion Lenalidomide can directly activate NK-cell-mediated anti-tumor activity through enhanced formation of immune synapses via the regulation of Rho and Rac1 GTPases and the cytoskeleton. Despite some down-modulation of CD20 expression in lenalidomide-pretreated B-CLL cells, the immune synapse activity increases when lenalidomide is combined with rituximab suggesting that combining lenalidomide and anti-CD20 antibodies warrants exploration in the CLL clinical setting. Disclosures Gaidarova: Celgene: Employment, Equity Ownership. Li:Celgene: Employment. Corral:Celgene: Employment. Glezer:Celgene: Employment, Equity Ownership. Schafer:Celgene: Employment. Xie:Celgene: Employment. Lopez-Girona:Celgene: Employment.

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Lenalidomide Induces Capping of CD20 and Cytoskeleton Proteins to Enhance Rituximab Immune Recognition of Malignant B-Cells

Blood ◽

10.1182/blood.v116.21.2845.2845 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2845-2845 ◽

Cited By ~ 1

Author(s):

Svetlana Gaidarova ◽

Derek Mendy ◽

Carla Heise ◽

Sharon Lea Aukerman ◽

Tom Daniel ◽

...

Keyword(s):

Cell Surface ◽

Nk Cells ◽

Lipid Rafts ◽

Synapse Formation ◽

Nk Cell ◽

Employment Equity ◽

Immune Synapse ◽

Immune Synapses ◽

Cd20 Expression ◽

Equity Ownership

Abstract Abstract 2845 Lenalidomide is an immunomodulatory agent that has both direct tumoricidal and immunomodulatory activities which are critical for its clinical activity in the treatment of various hematologic malignancies. This activity is at least in part mediated by enhanced T-cell and NK-cell effector function to eliminate tumor B cells, attributed to restoration of impaired T-cell activity and formation of immunologic synapses. Rituximab is an anti-CD20 monoclonal antibody that is active in the treatment of B-cell lymphomas through a variety of mechanisms, including antibody-dependent cellular cytotoxicity (ADCC). Preclinical studies and early clinical trials have shown an enhancement, and potentially synergy, in antitumor activity when lenalidomide is combined with rituximab. In order to further explore the mechanistic basis of this enhancement we investigated the impact of lenalidomide and rituximab on immune synapse formation and ADCC. We have previously shown that the combined use of lenalidomide and rituximab enhances NK cell-mediated immune synapse formation and the resultant cytotoxicity, versus each agent alone. Here we evaluate the molecular events that take place on the cell surface upon exposure of JeKo-1 cells (mantle cell lymphoma) and primary B-CLL cells to lenalidomide alone or lenalidomide plus rituximab. Change in CD20 expression resulting from exposure to vehicle control (0.1% DMSO) or 1 μM lenalidomide for 30 min or 24, 48, 72 hrs was assessed using immunocytochemistry, flow cytometry and isolation of cell membrane-associated proteins followed by Western blotting. At all time points evaluated, levels of cell surface and cell membrane-associated CD20 expression were unchanged in JeKo-1 cells. However, the distribution of CD20 was dramatically altered within 30 minutes after addition of lenalidomide. CD20 redistribution was accompanied by F-actin polymerization and lipid raft aggregation associated with the polarized localization (capping) of a number of proteins including CD20, CD19 and cytoskeleton signaling molecules Rac1 and Vav1, critical regulators of immune synapse formation in effector cells. Of note, other surface proteins involved in signaling such as CD45 were not part of this capping mechanism. By 48 hours of lenalidomide treatment, the majority of JeKo-1 cells (>80%) showed continued capping of CD20. These responses were also seen in primary B-CLL cells, although the effects were variable. In addition, CD20, F-actin and lipid rafts co-localized at the immune synapses formed between JeKo-1 and NK cells treated with either 1 μM lenalidomide for 24 hrs, 0.1% DMSO for 24 hrs followed by 10 μg/ml rituximab for 30 min, or treated sequentially with 1μM lenalidomide for 24 hrs followed by 10 ug/ml rituximab for 30 min. Lenalidomide and rituximab induced similar effects on B-CLL cells and the immune synapses formed between B-CLL and NK cells. We also determined whether formation of lipid rafts and actin cytoskeleton modifications were a prerequisite for CD20 capping. Cholesterol extraction from JeKo-1cells by 5 mM methyl-β-cyclodextrin (MCD) treatment for 30 min led to complete abrogation of lenalidomide-induced capping. The polymerization of the F-actin cytoskeleton and capping of CD20 was also affected, with no impact on cell viability. In addition, MCD treatment inhibited the formation of immunologic synapses between JeKo-1 cells and NK cells treated with 1 μM lenalidomide alone and in cells co-treated with 1 μM lenalidomide and 10 μg/ml rituximab. These data are consistent with a requirement for the integrity of lipid rafts to maintain the capping of CD20 and to potentially mediate lenalidomide enhancement of ADCC by rituximab. Our results further demonstrate that lenalidomide does not down-regulate CD20, but rather induces its polarized localization at the cell surface. The capping of CD20 is accompanied by redistribution of proteins such as Vav1 and Rac1 that become part of the immune synapse complex. Therefore the capping process induced by lenalidomide appears integral to immune synapse formation and may coordinately enhance the clustering of both the CD20 antigen and the attached rituximab, potentially further enhancing its activity, which would support the clinical combination of these agents. Ongoing studies are currently examining the role of the capping process and intracellular signaling cascades in the direct tumoricidal activity of lenalidomide. Disclosures: Gaidarova: Celgene Corporation: Employment, Equity Ownership. Mendy:Celgene Corporation: Employment. Heise:Celgene Corporation: Employment. Aukerman:Celgene Corporation: Employment. Daniel:Celgene Corporation: Employment. Chopra:Celgene Corporation: Employment. Lopez-Girona:Celgene Corporation: Employment, Equity Ownership.

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Lenalidomide Treatment Enhances Immunological Synapse Formation of Cord Blood Natural Killer Cells with B Cells Derived From Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v118.21.1794.1794 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1794-1794 ◽

Cited By ~ 1

Author(s):

Dongxia Xing ◽

Alan G. Ramsay ◽

Simon Robinson ◽

Catherine M. Bollard ◽

Nina Shah ◽

...

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Nk Cells ◽

Actin Polymerization ◽

Synapse Formation ◽

Immunological Synapse ◽

Nk Cell ◽

Immune Dysfunction ◽

Lymphocytic Leukemia ◽

Immune Synapse

Abstract Abstract 1794 Immune dysfunction is a hallmark of chronic lymphocytic leukemia (CLL) including suppressed humoral and cell-mediated immune responses. The immunomodulatory agent lenalidomide has shown effective clinical activity against CLL, but its mechanism of action is poorly understood. Previous work has demonstrated that the T cell immunological synapse and functional defects in CLL can be reversed following lenalidomide treatment (J Clin Invest. 2008; 118). Polymerization of F-actin at the NK cell immunological synapse with tumor cells is required for signaling molecules to assemble and regulate NK cell activation and effector function. Confocal microscopy was used to visualize and analyze F-actin polymerization at the immune synapse between NK cells and CLL cells. The impaired immune synapse defect identified in CLL could result from not only the defects of CLL B cells but also defects in the CLL NK cells or a combination of both factors. To investigate the contribution of each factor, we examined synapse formation in experiments using CLL B cells with autologous CLL NK cells or healthy allogeneic NK cells. Conjugates formed with healthy NK cells and CLL B cells exhibited a strong band of F-actin at the immune synapse. In contrast, significantly less actin polymerization at the synapse was observed in autologous CLL NK cells and CLL B cells (P < 0.01). These results indicate CLL B cells, together with CLL NK cells contributed to the immune dysfunction in CLL. As autologous NK cell function in CLL is suppressed, we investigated the utility of CB as a potential functional source of NK cells for CLL immunotherapy. We examined the effect of lenalidomide on NK cell immune synapse function with CLL B cells acting as APCs. We demonstrated that ex vivo treatment of CLL cells with lenalidomide (500 ng/ml) for 48 hours caused a significant increase in the ability of autologous CLL NK cells to form F-actin immune synapses with CLL B cells. The same treatment of CLL B cells also significantly increased the ability of CB-NK cells to form F-actin immunological synapses with these treated CLL B cells compared to untreated CLL B cells (33.6% to 67.3%, P < 0.01, n=6). Our results also show that lenalidomide treatment of autologous NK cells from CLL patients enhanced synapse formation with treated CLL cells compared to experiments using untreated NK cells, but with reduced function compared to CB NK cells. Of note, lenalidomide treatment was shown to increase the recruitment of the signaling molecule Lck to NK cell:CLL cell synapse site, that is known to regulate lytic synapse function. Importantly, lenalidomide treatment significantly increased CB-NK killing of CLL B cells compared to untreated CLL B cells (20.5% versus 48.2%, E:T ratio of 10:1, n = 6, p < 0.001). These results provide insight into the potential mechanism of action of lenalidomide's anti-leukemic function – priming CLL tumor cells for enhanced NK cell lytic synapse formation and effector function. In addition, the data suggests that immunotherapeutic strategies utilizing a combination of CB-NK cells and lenalidomide has an enhanced clinical efficacy in CLL. Disclosures: Gribben: Roche: Honoraria; Celgene: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria; Pharmacyclics: Honoraria.

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Ex Vivo Activity of Immunotherapeutic Approaches Targeting CD38 Against Daratumumab-Resistant Multiple Myeloma Patient Samples

Blood ◽

10.1182/blood-2019-127893 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1848-1848

Author(s):

Maria Karvouni ◽

Heyue Zhou ◽

Arnika Kathleen Wagner ◽

Qiangzhong Ma ◽

Alamdar H. Baloch ◽

...

Keyword(s):

Multiple Myeloma ◽

Nk Cells ◽

Therapeutic Potential ◽

Ex Vivo ◽

Phase 1 ◽

Employment Equity ◽

Multiple Myeloma Patient ◽

Myeloma Cells ◽

Car T ◽

Equity Ownership

Background: Multiple myeloma (MM) is a plasma cell malignancy that remains incurable. The identification of CD38, a transmembrane glycoprotein overexpressed on MM cells, led to the development of target-specific therapeutics such as the FDA approved monoclonal antibody (mAb) Daratumumab (DARA). Although a valuable treatment option for refractory/relapsed (R/R) MM patients, DARA has a limited response rate of below 50%, which highlights the clinical need for novel therapeutics. Aims: Aiming to further exploit the therapeutic potential of CD38 in the MM setting, immunotherapies based on the novel anti-CD38 mAb CD38A2 were tested. Methods: For the first approach, the CD38A2 mAb -that binds to a unique, distinct from DARA's, CD38 epitope- was conjugated with either the alkylating agent Duomycin (ADC-136) or the microtubulin binder Duostatin (ADC-129). The ADCs were compared to DARA, in cultures of primary MM cells from patients refractory to DARA treatment. In a second approach, a chimeric antigen receptor (CAR) consisting of the CD38A2 scFv and the intracellular domains of CD28 and CD3ζ was used to transduce primary T and NK cells from R/R MM patients. The functionality of the CAR-T and CAR-NK cells was assessed in cytotoxicity assays against autologous myeloma cells. Results: ADC-136 demonstrated the most potent cytotoxicity against the MM cells with an IC50 of 6pM at day 6 following a single dose treatment. ADC-129 showed cell killing with an IC50 of 30pM, while DARA did not exhibit appreciable cytotoxicity. Regarding the cell therapy approach, patients' T and NK cells were effectively transduced, showing a CD38A2-CAR expression ranging between 11-68%. In functional assays, CAR-T and CAR-NK cells were assayed against autologous myeloma cells, where they exhibited an increase in target cell cytotoxicity, compared to the untransduced cells. Summary/Conclusion: Altogether, our preliminary findings demonstrate that CD38 targeting using CD38A2-based immunotherapies could be a viable therapeutic approach in R/R MM patients previously exposed to DARA. Currently, an anti-CD38 CAR-T therapy based on CD38A2 is being evaluated in Phase 1 studies in R/R MM patients by Sorrento Therapeutics, Inc. Disclosures Zhou: Sorrento Therapeutics Inc: Employment, Equity Ownership. Ma:Sorrento Therapeutics Inc: Employment, Equity Ownership. Zhu:Sorrento Therapeutics Inc: Employment, Equity Ownership. Zhang:Sorrento Therapeutics Inc: Employment, Equity Ownership. Kaufmann:Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties.

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Effects of Selectin Antagonist GMI-1070 on the Activation State of Leukocytes In Sickle Cell Patients Not In Crisis

Blood ◽

10.1182/blood.v116.21.2672.2672 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2672-2672

Author(s):

Scott I Simon ◽

Shannon Chase ◽

Sandra K Larkin ◽

Frans Kuypers ◽

Lori Styles ◽

...

Keyword(s):

Steady State ◽

Sickle Cell ◽

Fold Increase ◽

Employment Equity ◽

Blood Samples ◽

High Affinity ◽

Equity Ownership ◽

First Time ◽

Time Point

Abstract Abstract 2672 It is hypothesized that activated leukocytes play key roles in sickle cell vaso-occlusion by adhering to inflamed venules and capturing circulating platelets and sickle red blood cells. GMI-1070 is a small molecule selectin antagonist which was recently reported to reverse acute vascular occlusion in a humanized sickle cell disease (SCD) mouse model (Chang et al, Blood 2010) presumably by inhibiting E-selectin and its effects on downstream signaling of leukocyte activation. Sickle cell patients express elevated levels of soluble E-selectin (Kato et al, Brit J Haem 2005) activated polymorphonuclear neutrophils (PMN) (Lum et al Amer J Hem 2004) and platelet/monocyte aggregates (PMA) (Wun et al Clin Lab Haem 2002). In this study, the activation state of leukocytes from whole blood samples of sickle cell patients not in crisis before and after infusion of GMI-1070 was evaluated ex vivo. Isolated PMN from normal, healthy volunteers were strongly activated by binding soluble E-selectin/hIg in vitro as determined by a 7-fold increase of the integrin MAC1 (CD11b) and an 8-fold increase in expression of the high affinity form of CD18 detected by antibody 327C. Addition of GMI-1070 completely blocked upregulation of MAC1 and 327C at 50μg/ml and showed pronounced inhibition (79% MAC1; 75% 327C) at 10μg/ml. These in vitro concentrations are consistent with blood levels of GMI-1070 found in sickle cell patients 4 and 8 hours after dosing. A phase 1/2 study was conducted on 10 adult subjects with SCD at steady state. GMI-1070 was given IV at 20mg/kg as a loading dose and at 10 hours a final dose of 10mg/kg was given. Blood samples were drawn from these adults pre-infusion and at 8, 24, and 48 hours after the initial infusion. In some subjects, a blood sample was also drawn at 4 hours post infusion. Activation of PMN's in whole blood samples from subjects was assessed by upregulation of MAC-1, expression of the high affinity CD18 and the loss of CD62L due to shedding of L-selectin determined by flow cytometric analysis of cell surface labeling with fluorescently conjugated antibodies. Of 4 subjects tested, 3 showed increased surface expression of L-selectin, 3 showed decreased expression of MAC-1, and 2 showed decreased expression of high affinity CD11b at the first time point tested (4 or 8hr) after dosing with GMI-1070 suggesting an inhibition of PMN activation in these patients. A functional consequence of monocyte activation is the formation of platelet/monocyte aggregates due to expression of high affinity integrins. Platelet-monocytes aggregates (PMA) in blood were detected using anti-CD11c for monocytes and anti-CD41a for platelets. Treatment of samples with lipopolysaccharide (LPS) was used for positive controls. Intracellular IL-1β was used as a marker of activated monocytes. In 5 patients out of 6 tested with this assay, PMA in the subject's blood were decreased at the first time point after dosing (8hr). These results are consistent with an effect of GMI-1070 on inhibition of activation given its IC50 value for E-selectin (4.3μM), the blood concentration in subjects after dosing, and the serum half life (7.7hr) in steady state sickle cell adults. Conclusions: GMI-1070 significantly inhibited E-selectin-mediated activation of PMNs in vitro as determined by expression of the integrin MAC-1 and high affinity CD18 at 10μg/ml. Similar concentrations of GMI-1070 in sickle cell subjects' blood at 4 and 8 hours after dosing also resulted in a lowered activation state of PMNs identified by reduced expression of cell surface integrin molecules as well as the inhibition of shedding of L-selectin in some cases. A more functional measure of leukocyte activation is the aggregation of platelets on monocyte cell surfaces. In 5 of 6 subjects tested, GMI-1070 reduced PMA 8 hours after dosing. Thus, GMI-1070 not only inhibits E-selectin, but also blocks the expression of downstream integrin adhesion molecules that together play crucial roles in vaso-occlusion by promoting the adhesion to platelets and erythrocytes in the formation of occlusions that block blood flow. The effects of GMI-1070 on the activation state of leukocytes via the inhibition of functional adhesion molecules in steady state sickle cell subjects supports the further evaluation of treatment with GMI-1070 during vaso-occlusive episodes. Disclosures: Simon: GlycoMimetics: Research Funding. Chase:GlycoMimetics:.Kuypers:GlycoMimetics Inc.: Research Funding. Styles:GlycoMimetics: Consultancy, clinical trial sponsorship. Wun:GlycoMimetics Inc.: Consultancy, clinical trial sponsorship. Thackray:GlycoMimetics: Employment, Equity Ownership. Magnani:GlycoMimetics: Employment, Equity Ownership. Off Label Use: The drug (GMI-1070) is not approved for any clinical indication.

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CD137L Reverse the Immunological Synapse Defects of Natural Killer Cells in Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v118.21.246.246 ◽

2011 ◽

Vol 118 (21) ◽

pp. 246-246

Author(s):

Dongxia Xing ◽

Alan G. Ramsay ◽

William Decker ◽

Dean A. Lee ◽

Simon Robinson ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Nk Cells ◽

Natural Killer ◽

Myeloid Leukemia ◽

Synapse Formation ◽

Immunological Synapse ◽

Leukemic Cells ◽

Cytolytic Function ◽

Immunological Synapses ◽

Acute Myeloid

Abstract Abstract 246 Natural killer (NK) cells are an innate component of immune system that can produce a graft vs. leukemia (GVL) effect after stem cell transplantation. NK cells derived from acute myeloid leukemia (AML) patients are defective in their cytolytic function against leukemic cells. In order to better understand the mechanism of this defect, we performed functional assays examining immunological synapse formation of AML patient NK cells with autologous and allogeneic primary AML cells acting as antigen-presenting cells (APCs). Confocal microscopy was used to image and score F-actin polymerization at the immunological synapse between patient NK cells and leukemic cells. Accumulation of F-actin beneath the area of the NK: APC contact site is a hallmark of NK lytic synapses and allows signaling molecules to regulate appropriate activation and effector function. AML patient derived NK cells (AML-NK cells) formed significantly fewer synapses with autologous leukemia cells than healthy donor NK cells (12% versus 30%, n = 16. p > 0.001). Moreover, AML-NK cells were defective in their ability to recruit the key receptor NKG2D and the signaling molecule phosphotyrosine to immunological synapse contact sites. Signaling through the costimulatory ligand4-1BB-L (CD137L) has been shown to activate T cells, enhance antitumor responses and has multiple immunomodulatory effects on dendritic cells and NK cells. We postulated that AML-NK cells could be activated for enhanced cytolytic activity using artificial APCs generated to express CD137L. To test this, we setup co-culture assays using AML-NK cells and artificial CD137L-APCs before subsequent examination of immunological synapse function with AML blasts. Stimulated AML-NK cells that formed cell conjugate interactions with AML blasts, showed a significant increase in formation of immunological synapses compared to unstimulated AML-NK cells. The number of AML-NK/AML blast immunological synapses increased 16 hours after stimulation and peaked at approximately 72 hours. CD137L stimulation of AML-NK cells was also associated with increased cytotoxic function against primary AML cells (n = 6, p <0.01). Furthermore, CD137L stimulation increased recruitment of tyrosine-phosphorylated proteins at AML-NK immunological synapses compared with unstimulated control experiments (RRI 4.1 versus 2.3, n = 3, p < 0.01). Taken together, our data suggests that immune functional suppression of AML-NK cells in leukemia patients can be reversed by CD137L activation signaling, resulting in enhanced F-actin synapse formation, phosphotyrosine signaling, and cytolytic function. Thus, enhanced recruitment of signaling molecules to the NKIS may represent a novel immunomodulatory function of CD137L in the NK cell–mediated killing of AML cells. These findings should aid development of new immune based therapies for leukemia. Disclosures: Gribben: Roche: Honoraria; Celgene: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria; Pharmacyclics: Honoraria.

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Stroma-Free Ex Vivo Expanded, CD34+ Cord Blood-Derived NK Cells Retain Lytic Activity After Long-Term Engraftment in NSGS Mice

Blood ◽

10.1182/blood.v120.21.580.580 ◽

2012 ◽

Vol 120 (21) ◽

pp. 580-580

Author(s):

Mark Wunderlich ◽

Mahesh Shrestha ◽

Lin Kang ◽

Eric Law ◽

Vladimir Jankovic ◽

...

Keyword(s):

Nk Cells ◽

Research Funding ◽

Nk Cell ◽

Ex Vivo ◽

Employment Equity ◽

Cell Product ◽

Equity Ownership ◽

Cellular Therapeutics

Abstract Abstract 580 Generating a large number of pure, functional immune cells that can be used in human patients has been a major challenge for NK cell-based immunotherapy. We have successfully established a cultivation method to generate human NK cells from CD34+ cells isolated from donor-matched cord blood and human placental derived stem cells, which were obtained from full-term human placenta. This cultivation method is feeder-free, based on progenitor expansion followed by NK differentiation supported by cytokines including thrombopoietin, stem cell factor, Flt3 ligand, IL-7, IL-15 and IL-2. A graded progression from CD34+ hematopoietic progenitor cells (HSC) to committed NK progenitor cells ultimately results in ∼90% CD3-CD56+ phenotype and is associated with an average 10,000-fold expansion achieved over 35 days. The resulting cells are CD16- and express low level of KIRs, indicating an immature NK cell phenotype, but show active in vitro cytotoxicity against a broad range of tumor cell line targets. The in vivo persistence, maturation and functional activity of HSC-derived NK cells was assessed in NSG mice engineered to express the human cytokines SCF, GM-CSF and IL-3 (NSGS mice). Human IL-2 or IL-15 was injected intraperitoneally three times per week to test the effect of cytokine supplementation on the in vivo transferred NK cells. The presence and detailed immunophenotype of NK cells was assessed in peripheral blood (PB), bone marrow (BM), spleen and liver samples at 7-day intervals up to 28 days post-transfer. Without cytokine supplementation, very few NK cells were detectable at any time-point. Administration of IL-2 resulted in a detectable but modest enhancement of human NK cell persistence. The effect of IL-15 supplementation was significantly greater, leading to the robust persistence of transferred NK cells in circulation, and likely specific homing and expansion in the liver of recipient mice. The discrete response to IL-15 versus IL-2, as well as the preferential accumulation in the liver have not been previously described following adoptive transfer of mature NK cells, and may be unique for the HSC-derived immature NK cell product. Following the in vivo transfer, a significant fraction of human CD56+ cells expressed CD16 and KIRs indicating full physiologic NK differentiation, which appears to be a unique potential of HSC-derived cells. Consistent with this, human CD56+ cells isolated ex vivo efficiently killed K562 targets in in vitro cytotoxicity assays. In contrast to PB, spleen and liver, BM contained a substantial portion of human cells that were CD56/CD16 double negative (DN) but positive for CD244 and CD117, indicating a residual progenitor function in the CD56- fraction of the CD34+ derived cell product. The BM engrafting population was higher in NK cultures at earlier stages of expansion, but was preserved in the day 35- cultured product. The frequency of these cells in the BM increased over time, and showed continued cycling based on in vivo BrdU labeling 28 days post-transfer, suggesting a significant progenitor potential in vivo. Interestingly, DN cells isolated from BM could be efficiently differentiated ex vivo to mature CD56+CD16+ NK cells with in vitro cytotoxic activity against K562. We speculate that under the optimal in vivo conditions these BM engrafting cells may provide a progenitor population to produce a mature NK cell pool in humans, and therefore could contribute to the therapeutic potential of the HSC-derived NK cell product. The in vivo activity of HSC-derived NK cells was further explored using a genetically engineered human AML xenograft model of minimal residual disease (MRD) and initial data indicates significant suppression of AML relapse in animals receiving NK cells following chemotherapy. Collectively, our data demonstrate the utility of humanized mice and in vivo xenograft models in characterizing the biodistribution, persistence, differentiation and functional assessment of human HSC-derived cell therapy products, and characterize the potential of HSC-derived NK cells to be developed as an effective off-the-shelf product for use in adoptive cell therapy approaches in AML. Disclosures: Wunderlich: Celgene Cellular Therapeutics: Research Funding. Shrestha:C: Research Funding. Kang:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Law:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Jankovic:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Zhang:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Herzberg:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Abbot:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Hariri:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Mulloy:Celgene Cellular Therapeutics: Research Funding.

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Pharmacological Inhibition Of Histone Deacetylase (HDAC) 1, 2 Or 3 Have Distinct Effects On Cellular Viability, Erythroid Differentiation, and Fetal Globin (HbG) Induction

Blood ◽

10.1182/blood.v122.21.564.564 ◽

2013 ◽

Vol 122 (21) ◽

pp. 564-564

Author(s):

Jeffrey R Shearstone ◽

John H van Duzer ◽

Simon S Jones ◽

Matthew Jarpe

Keyword(s):

Bone Marrow ◽

Cell Viability ◽

Bone Marrow Cells ◽

Fold Increase ◽

Erythroid Differentiation ◽

Employment Equity ◽

Human Cd34 ◽

Ic50 Values ◽

Equity Ownership ◽

Marrow Cells

Abstract Induction of HbG is an established therapeutic strategy for the treatment of sickle cell disease (SCD), and could also be effective in treating beta-thalassemia (bT). Genetic ablation of HDAC1 or HDAC2, but not HDAC3, results in the induction of HbG expression (Bradner JE, Proc Natl Acad Sci, 2010). Furthermore, we have previously shown that selective chemical inhibitors of HDAC1 and 2 elicit a dose and time dependent induction of HbG mRNA and fetal hemoglobin (HbF) protein in cultured human CD34+ bone marrow cells undergoing erythroid differentiation (Shearstone JS, ASH Annual Meeting Abstracts, 2012). While a variety of selective HDAC inhibitors have been used successfully to induce HbF, further clinical development has been limited by variable efficacy and concerns over off-target side-effects observed in clinical trials, potentially due to inhibition of HDAC3. Additionally, it remains to be determined if HDAC1 or HDAC2 is the preferred therapeutic target. In this work we present data that investigates the effects of selective inhibitors of HDAC1, 2, or 3 on cytotoxicity, erythroid differentiation, and HbG induction in cultured human CD34+ bone marrow cells. Acetylon Pharmaceuticals has generated a library of structurally distinct compounds with a range of selectivity for each of HDAC1, 2, or 3 (Class I HDAC) as determined in a biochemical assay platform. From our initial chemical series, we identified ACY-822 as a Class I HDAC inhibitor with IC50 values of 5, 5, and 8 nM against HDAC1, 2, and 3, respectively. In contrast, ACY-1112 is 30-fold selective for HDAC1 and 2, with IC50 values of 38, 34, and 1010 nM against HDAC1, 2, and 3, respectively. Treatment of cells for 4 days with ACY-822 (1 μM) resulted in a 20-fold decrease in cell viability, while ACY-1112 (1 μM) treatment resulted in a minimal reduction in viability (1.2-fold) and a 2-fold increase in the percentage of HbG relative to other beta-like globin transcripts. This result suggests that pharmacological inhibition of HDAC3 is cytotoxic and is consistent with the therapeutic rationale for the design selective inhibitors of HDAC1 and 2. To investigate if HDAC1 or HDAC2 is the preferred therapeutic target, we utilized a second series of structurally distinct compounds. We identified ACY-957 as an HDAC1/2 selective compound biased towards HDAC1 with IC50 values of 4, 15, and 114 nM for HDAC1, 2, and 3, respectively. In contrast ACY-1071 showed balanced HDAC1 and 2 selectivity with IC50 values of 27, 24, and 247 nM for HDAC1, 2, and 3, respectively. Treatment of cells for 6 days with 1 μM of ACY-957 or ACY-1071 resulted in a 3-fold increase in the percentage of HbG relative to other beta-like globin transcripts. However, we found that ACY-957 treatment resulted in an approximately 3-fold decrease in cell viability after 6 days of treatment, while ACY-1071 treatment resulted in a minimal reduction (1.2-fold) in cell viability. Decreased cell viability observed with ACY-957 was associated with a reduction of cells positive for the erythroid differentiation markers CD71 and glycophorinA. This result is consistent with the Mx-Cre mouse model where HDAC1KO; HDAC2het had reduced numbers of erythrocytes, thrombocytes, and total bone marrow cells, while the HDAC1het; HDAC2KO was unaffected (Wilting RH, EMBO Journal, 2010). Our results suggest that compounds with a pharmacological profile of increased selectivity towards HDAC2 inhibition versus HDAC1 may be less cytotoxic and minimize effects on differentiation, while still inducing HbG in human CD34+ bone marrow cells. Disclosures: Shearstone: Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. van Duzer:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. Jones:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Jarpe:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership.

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Ex Vivo Pharmacologic Modulation in a Nutrient-Rich Medium to Accelerate Engraftment of Human Umbilical Cord Blood

Blood ◽

10.1182/blood.v124.21.3807.3807 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3807-3807

Author(s):

Corey S Cutler ◽

Daniel Shoemaker ◽

Peter Westervelt ◽

Daniel R. Couriel ◽

Sumithra Vasu ◽

...

Keyword(s):

Gene Expression ◽

Umbilical Cord Blood ◽

Ex Vivo ◽

Phase 1 ◽

Fold Increase ◽

Phase 2 ◽

Employment Equity ◽

Phase 2 Trial ◽

Equity Ownership ◽

Neutrophil Engraftment

Abstract Umbilical cord blood (UCB) offers many potential advantages as a source of hematopoietic stem cells (HSCs) for allogeneic transplantation, including ease of collection, rapid availability, flexibility of HLA-matching, lower rates of GvHD and potentially lower relapse rates. However, the low HSC content of UCB compared to other graft sources results in a prolonged time to engraftment, and higher rates of graft failure and early mortality. Pulse ex vivo exposure of HSCs to 16,16-dimethyl PGE2 (FT1050) has been demonstrated to enhance HSC engraftment potential, which could benefit clinical UCB transplant. FT1050 modulation promotes multiple mechanisms, including increased proliferation, reduced apoptosis, and improved migration and homing [North 2007&2009; Hoggatt 2009]. Improved HSC homing is mediated by induction of CXCR4 gene expression leading to increased cell surface CXCR4. Further optimization of the UCB modulation process demonstrated that incubation with 10µM FT1050 for 2 hrs at 37C resulted in a maximal biological response of the FT1050-UCB (ProHema®). A Phase 1 trial was performed to evaluate the safety of FT1050-UCB paired with an unmanipulated UCB unit in reduced-intensity double UCBT (dUCBT) [Cutler 2013]. We observed durable, multi-lineage engraftment of FT1050-UCB with acceptable safety. Earlier neutrophil engraftment was observed relative to historical controls (median 17.5 vs. 21 days (historical control), p=0.045), coupled with preferential engraftment of the FT1050-UCB unit in 10 of 12 subjects. A Phase 2 multi-center clinical trial of FT1050-UCB in adult patients undergoing dUCBT for hematologic malignancies was then initiated. Subjects are randomized 2:1 to FT1050-UCB-containing vs. standard dUCBT after high-dose conditioning. The primary endpoint is a categorical analysis of neutrophil engraftment using a pre-specified control median. Data on the initial 11 subjects, of which 8 were randomized to receive FT1050-UCB, continue to demonstrate acceptable safety with adverse events attributed to FT1050-UCB limited primarily to common infusion-related side effects. Of the 8 FT1050-UCB subjects, 1 died prior to neutrophil engraftment, with the remaining 7 subjects engrafting at a median of 28 days vs. 31 days for the 3 control subjects. With median overall follow-up of 16.1 months, 4 of 8 subjects on the FT1050-UCB arm are alive with a median survival not reached (> 11.0 months). 1 of 3 control subjects is alive with median survival of 6.0 months. During the clinical translation process, the media used during FT1050 modulation of UCB was identified as a key variable. Standard UCB washing media, consisting of a nutrient-free saline solution of low molecular weight dextran and human serum albumin (LMD/HSA), is used clinically to stabilize fragile cells post-thaw by reducing lysis. This media was used in the Phase 1 trial and to initiate Phase 2. Early during the Phase 2 trial, we identified a novel cell-stabilizing nutrient-rich formulation (NRM), containing glucose, amino acids and other HSC-supporting nutrients that promoted full FT1050 modulation of UCB and increased cell viability. The expression of key FT1050-pathway genes was significantly higher with NRM compared to intermediate levels observed with LMD/HSA. Modulation of human CD34+ (hCD34+) cells with FT1050 in NRM led to an 8-fold increase over LMD/HSA in induced CXCR4 gene expression (20-fold total), which translated to significantly increased surface CXCR4 protein. In vivo homing models demonstrated that UCB CD34+ cells modulated with FT1050 in NRM resulted in a 2.2-fold homing increase relative to vehicle (p < 0.001) compared to a 1.6-fold increase with LMD/HSA (p = 0.002), with a significant difference between the two media conditions (p = 0.04). A xenotransplantation study in NSG mice with hCD34+ cells modulated with FT1050 in either NRM or LMD/HSA demonstrated a 2-fold increase in circulating hCD45+ cells 12-weeks post-transplant with NRM (p = 0.007; unpaired t-test). These findings supported the incorporation of NRM into the FT1050-UCB manufacturing process in order to further improve its clinical engraftment potential. Enrollment of a 60-patient Phase 2 trial has been initiated that incorporates this manufacturing change. Disclosures Shoemaker: Fate Therapeutics: Employment, Equity Ownership. Rezner:Fate Therapeutics: Employment. Guerrettaz:Fate Therapeutics: Employment. Robbins:Fate Therapeutics: Employment. Medcalf:Fate Therapeutics: Employment. Wolchko:Fate Therapeutics: Employment, Equity Ownership. Ferraro:Fate Therapeutics: Employment. Multani:Fate Therapeutics: Employment.

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Pharmacokinetic and Pharmacodynamic Study of NKTR-255, a Polymer-Conjugated Human IL-15, in Cynomolgus Monkey

Blood ◽

10.1182/blood-2018-99-115638 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2952-2952

Author(s):

Takahiro Miyazaki ◽

Peiwen Kuo ◽

Mekhala Maiti ◽

Palakshi Obalapur ◽

Murali Addepalli ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

Nk Cells ◽

Cd8 T Cells ◽

Cynomolgus Monkey ◽

Memory T Cells ◽

Granzyme B ◽

Employment Equity ◽

Equity Ownership ◽

Dose Dependent

Abstract Introduction IL-15 is a common gamma chain cytokine that activates and provides a survival benefit to T-cells and NK cells and has long been recognized as having potential as an immunotherapeutic agent for the treatment of cancer. Therapeutic use of native IL-15 has been challenging due to, for example, its unfavorable pharmacokinetic and safety properties. NKTR-255 is a polymer-conjugated human IL-15 that retains binding affinity to the alpha subunit of IL-15 receptor and exhibits reduced clearance to thereby provide a sustained pharmacodynamics response. Here we investigate the biological effects of NKTR-255 in naïve cynomolgus monkey. Methods In vitro monkey whole blood was treated with NKTR255 and the percentage of pSTAT5 positive populations in each NK, CD4 T and CD8 T cells was determined by flow cytometry. In an PK/PD study, monkeys received single IV doses of 0.001, 0.003, 0.01, 0.03, or 0.1 mg/kg NKTR-255. Blood samples were collected to determine the plasma concentrations of NKTR-255 and to assess the effects of NKTR-255 on NK and CD8 T cells at multiple time points; flow cytometry was used to measure STAT5 phosphorylation, Ki-67 expression and frequency of cell populations. Granzyme B expression was assessed in NK and CD8 T cells by flow cytometry. Results NKTR-255 induced dose-dependent phosphorylation of STAT5 in monkey whole blood (EC50 values NK cells: 6.9 ng/ml, CD8 T cells: 39 ng/ml, CD4 T cells: 53 ng/ml). The half-life and clearance of NKTR-255 were 26x longer and 38x lower, respectively, than IL-15. NKTR-255 engaged the IL-15 signaling pathway, in vivo, demonstrating both robust and sustained STAT5 phosphorylation in lymphocytes. NKTR-255 drove the proliferation of total CD8 T cells and NK cells in a dose-dependent manner, with dramatic and durable increases observed in Ki67 positive population and absolute cell numbers (NK cells: 6.1 fold; CD8 T cells: 7.8 fold from baseline on day 5 at 0.1 mg/kg). These effects were strongly biased towards CD8 T cells and NK cells, with substantially less induction of CD4 T cells. The Ki67 response analyses of the T cell subpopulation revealed a higher response of memory populations than for naive T cells. Among memory T cells, effector memory T cells showed the highest response over stem cell memory T cells and central memory T cells. Finally, NKTR-255 also increased the expression of Granzyme B in both NK and CD8 T cells, concomitant with an enhancement in target cell lysis. Conclusions Nektar has generated a novel and potent molecule in NKTR-255 that not only preserves the relevant biology of IL-15, but additionally provides enhanced PK and PD properties relative to the native IL-15 cytokine. NKTR-255 is being developed as an immune-stimulatory agent to target NK and CD8 T cell biology for the treatment of cancer. Disclosures Miyazaki: Nektar Therapeutics: Employment, Equity Ownership. Kuo:Nektar Therapeutics: Employment, Equity Ownership. Maiti:Nektar Therapeutics: Employment, Equity Ownership. Obalapur:Nektar Therapeutics: Employment, Equity Ownership. Addepalli:Nektar Therapeutics: Employment, Equity Ownership. Rubas:Nektar Therapeutics: Employment, Equity Ownership. Sims:Nektar Therapeutics: Employment, Equity Ownership. Zhang:Nektar Therapeutics: Employment, Equity Ownership. Madakamutil:Nektar Therapeutics: Employment, Equity Ownership. Zalevsky:Nektar Therapeutics: Employment, Equity Ownership.

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