Ex Vivo IL-2 Expansion of CB-NK Cells Promotes Synergistic LFA-1 and CD2 Engagement at the NK Cell Lytic Immune Synapse; Implications for Adoptive CB-NK Cell Therapy in Acute Myeloid Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3029-3029
Author(s):  
Dongxia Xing ◽  
Alan G. Ramsay ◽  
William Decker ◽  
Sufang Li ◽  
Simon Robinson ◽  
...  
Keyword(s):  
Cord Blood ◽  
Nk Cells ◽  
Synapse Formation ◽  
Immunological Synapse ◽  
Nk Cell ◽  
Ex Vivo ◽  
Immune Therapy ◽  
Cytolytic Activity ◽  
Lymphocyte Function ◽  
Immune Synapse

Abstract Abstract 3029 Poster Board II-1005 Donor peripheral blood (PB) natural killer (NK) cell have shown clinical promise in cancer immunotherapy. Tightly regulated receptor signaling between NK cells and susceptible tumor cells is essential for NK cell-mediated cytotoxicity. Umbilical cord blood (CB) represents an important alternative source of NK cells for adoptive immune therapy. We first demonstrated that cord blood (CB) derived NK cells have poor cytolytic activity and deficiency in the formation of the F-actin immunological synapse with HLA class I deficient target K562 cells and primary AML blasts compared to PB-NK cells. In this study, we explored the cellular mechanism of these dysfunctions. We hypothesized that adhesion and signaling molecules may be defective in unmanipulated CB NK cells. Activating receptor Both CD2 and the integrin lymphocyte function-associated antigen (LFA-1) play important roles in both T lymphocyte and NK cell immune synapse formation and their trafficking to the immune synapse regulates both T and NK cell function. We now show that unmanipulated CB NK cells exhibit reduced LFA-1 mediated adhesion to mobilized ICAM-1 compared to IL-2 expanded CB NK cells (CB NK 29.7+/- 3.2 %, vs expanded CB NK 78.5+/- 6.1%, n=6). Moreover, unmanipulated CB-NK cells demonstrated reduced surface expression of CD2, and high affintyLFA-1 detected by the specific antibody (MHM24). There was decreased recruitment of CD2 and LFA-1 to the NK cell immune synapse site as quantified by confocal microscope analysis (RRI CD2 CB NK 2.02 vs PB NK 4.98, n=3). Furthermore, defective LFA-1 trafficking lead to a decrease in downstream cytotoxic granules that traffic to the immunological synapse as demonstrated by decreased perforin trafficking to the CB-NK synapse site (> 60% reduction).We next wanted to confirm that CD2 or LFA-1 play a role in restoring the immune synapseformation for IL-2 expanded CB NK cells. We incubated expanded CB NK cells with blocking antibodies specific for LFA-1 or CD2 prior to conjugation to the K562 target cells. After CD2 or LFA-1 blocking there was decreased synapse formation, with a resultant decrease in cytotoxic function. When monoclonal antibodies against both CD2 and LFA-1 were used there was significant blockade of the formation of the immune synapse, and a marked reduction of CB NK cell cytolytic activity (Mean specific lysis of K562 targets at E:T ratio 20:1 was 81% IgG control vs 22% with anti-CD2; and 29% with anti-LFA-1, n=6, P<0.001). This data shows that CD2 and LFA-1 are defective in unmanipulated CB NK cells resulting in impaired immune synapse formation. In contrast, ex vivo IL-2 expansion of CB-NK cells enhanced lytic synapse formation with the synergistic repair of CD2 and LFA-1 localization and activity. We believe our results provide important mechanistic insights for the potential use of IL-2 expanded CB-derived NK cells for adoptive immune therapy in leukemia. Disclosures No relevant conflicts of interest to declare.

Ex Vivo Expansion of Cord Blood Natural Killer Cells Overcomes Impaired Immune Synapse Formation and Effector Function in Acute Myeloid Leukemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2905-2905
Author(s):  
Dongxia Xing ◽  
Alan G Ramsay ◽  
John G. Gribben ◽  
William K Decker ◽  
Jared K Burt ◽  
...  
Keyword(s):  
Nk Cells ◽  
Tumor Cells ◽  
Synapse Formation ◽  
Nk Cell ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Cytolytic Activity ◽  
Target Cells ◽  
Immune Synapse ◽  
Low Cytotoxicity

Abstract Natural killer (NK) cells play an important role in host immunity by eradicating pathogen-infected and tumor cells. Peripheral blood-derived NK cell therapy has shown promise in clinical trials for acute myeloid leukemia. Cord blood (CB) is another potentially rich source of NK cells. However NK cells isolated directly from CB have poor cytolytic activity. We investigated the mechanism for the low cytolytic activity of CB NK cells and whether the defect could be overcome by ex vivo expansion. NK cell killing of the target cells is achieved by formation of a mature immune synapse, followed by secretion of lytic granules containing perforin and granzymes. F-actin polymerization at the NK-tumor cell conjugates is a hallmark of immune synapse formation which can be detected and quantitated by confocal microscopy. We hypothesized that CB NK cells exhibit low cytotoxicity against leukemia target cells due to a defect in the formation of the immune synapse. We confirmed that unmanipulated CB NK cells exhibit low cytotoxicity against AML blasts (5% at an E:T ratio 20:1) in comparison to peripheral blood (PB) NK cells (35% at an E:T ratio 20:1). Evaluation of the natural cytotoxicity receptors (NCRs) showed normal expression of the NKp30 and the NK46 receptors. We then investigated whether this poor cytolytic capability is accompanied by poor immune synapse formation with tumor cells. Surprisingly, both CB and PB NK cells expressed comparable levels of perforin and demonstrated similar perforin polarization to the immune synapse. F-actin polarization was observed in only 12% (range 9– 21%) of the CB NK cell/K562 conjugates versus 85% (range 68–87%) of PB NK cell/K562 conjugates (P&lt;0.001). Remarkably, this impairment could be reversed by ex vivo expansion of CB NK cells with rIL-2. Expanded CB NK cells formed an increased percentage of immune synapses with K562 tumor cells 65% (range 60–71%) and primary human AML blasts 48% (range 39 –55%) which was comparable to the levels generated with PB NK cells. The present data reinforce the conclusion that formation of the activating NK cell immune synapse is required for cytotoxic activity. Furthermore, natural cytotoxicity receptors (NCR) and Kir receptors including KIR2DL1/S1 and KIR2DL2 were preserved on the expanded CB NK population. Moreover, the expanded CB NK cells were able to lyse AML targets in vitro (29% at 20:1 E:T ratio). Finally, we demonstrated that ex vivo expanded CB NK cells efficiently kill human AML in an NOD/SCID null mouse model. A 50 % reduction in AML tumor burden was documented in comparison to control groups by 6 weeks post NK infusion (P&lt;0.05). Our results suggest that ex vivo expansion of CB NK cells is a feasible and potentially effective strategy for the treatment of AML.

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

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1794-1794 ◽  
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.

Genetically Reengineered K562 Cells Significantly Expand Cord Blood (CB) Natural Killer (NK) Cells Ex-Vivo Expanded with Increased NK Cell Activation and Cytolytic Activity Both In-Vitro and In-Vivo: Potential Use In Adoptive Cellular Immunotherapy (ACI)

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3928-3928
Author(s):  
Michele Levin ◽  
Janet Ayello ◽  
Frances Zhao ◽  
Andrew Stier ◽  
Lauren Tiffen ◽  
...  
Keyword(s):  
Nk Cells ◽  
Nk Cell ◽  
Ex Vivo ◽  
Granzyme B ◽  
K562 Cells ◽  
Cytolytic Activity ◽  
Scid Mice ◽  
Activation Marker

Abstract Abstract 3928 Background: NK cells play a role in reducing relapse in hematological malignancy following AlloSCT (Dunbar et al, Haematologica, 2008). NK cell limitations include lack of tumor recognition and/or limited numbers of viable and functional NK cells (Shereck/Cairo et al, Ped Bld Can, 2007). NK ACI provide safe and effective therapy against tumor relapse; yet NK cells are limited to specific cancer types and not all patients demonstrate optimal response (Ruggieri et al. Science, 2002; Ljunggren et al. Nat Rev Immuno, 2007). To circumvent these limitations, methods to expand and activate PBMNCs with genetically engineered K562 cells expressing membrane bound IL-15 and 41BB ligand (K562-mbIL15-41BBL [modK562]; Imai/Campana et al, Blood, 2005) have shown to significantly increase NK cells in number and maintain heterogeneous KIR expression (Fusaki/Campana et al BJH, 2009). We have shown that CB NK cells can be activated/expanded and exhibit enhanced cytolytic activity when cultured in a cytokines/antibody cocktail (Ayello/Cairo et al, BBMT, 2006; Exp Heme, 2009). Objective: To evaluate CBNK expansion, activation, cytolytic mechanism and function against Burkitt lymphoma (BL) tumor target and its influence on NK cell mediated in-vitro and in-vivo cytotoxicity in NOD-SCID mice following stimulation with modK562 cells (generously supplied by D.Campana, St Jude's Children's Hospital, Memphis, Tx). Methods: Following 100GY irradiation, modK562cells were incubated 1:1 with CBMNCs in RPMI+IL-2 (10IU/ml) for 7 days in 5%CO2, 37°C. NK activation marker (LAMP-1), perforin and granzyme B were determined by flow cytometry. Cytotoxicty was determined via europium assay at 20:1 E:T ratio with Ramos (BL) tumor targets (ATCC). The mammalian expression construct (ffLucZeo-pcDNA (generously supplied by L.Cooper, MD, PhD) was transfected to BL cells using lipofectin and selected by zeocin for stable transfection. Six week old NOD-SCID mice received 5×106 BL cells subcutaneously. Upon engraftment, xenografted NOD-SCID mice were divided in 5 groups: injected with PBS (control), BL only, 5×106 wildtype (WT) K562 expanded (E) CBNK cells, modK562 expanded (E) CB NK cells (5×106) and modK562 expanded (E) CBNK cells (5×107). Ex-vivo ECBNK cells were injected weekly for 5 weeks and xenografted NOD-SCID mice were monitored by volumetric measurement of tumor size (Tomayko/Reynolds, Can Chemother Pharmac, 1989), bioluminescent imaging (Inoue et al Exp Heme, 2007) and survival. The survival distribution for each group was estimated using the Fisher exact test. Results: On Day 0, NK cells (CD56+/3-) population was 3.9±1.3%. After 7 days, modK562 expanded CBNK cells was significantly increased compared to WTK562 and media alone (72±3.9 vs 43±5.9 vs 9±2.4%, p<0.01). This represented a 35-fold or 3374±385% increase of the input NK cell number. This was significantly increased compared to WTK562 (1771±300%, p<0.05). ModK562 ECBNK cells demonstrated increased perforin and granzyme B expression compared to WTK562 (42±1.5 vs 15±0.5%,p<0.001; 22±0.5 vs 11±0.3%,p<0.001, respectively). Cytotoxicity was against BL tumor targets was significantly increased (42±3 vs 18±2%,p<0.01), along with NK activation marker expression, CD107a (p<0.05). At 5 weeks, in-vivo studies demonstrated increased survival of NOD-SCID mice receiving both 5×106 and 5×107 modK562 ECBNK cells when compared to those with no treatment (p=0.05, p=0.0007, respectively). There was no difference in survival when comparing mice that received 5×106 vs 5×107 modK562 ECBNK cells (p=0.0894) at 5 weeks. Tumor volume of mice receiving either dose of modK562 ECBNK cells was significantly less than those receiving WTK562 ECBNK cells (1.92±0.57 and 0.37±0.05 vs 3.41±0.25, p=0.0096 and p=0.0001, respectively). Conclusions: CBMNCs stimulated and expanded with modK562 cells results in significant expansion of CBNK cells with enhanced in-vitro cytotoxicity, significant receptor expression of NK activation marker (LAMP-1), and perforin and granzyme B. Furthermore, modK562 ECBNK cells leads to increased survival and lower tumor burden of NOD-SCID mice xenografted with BL. Future directions include modK562 ECBNK cells to be genetically modified to express chimeric antigen receptor CD20 (MSCV-antiCD20-41BB-CD3 ζ) against CD20+ hematologic malignancies for future studies to evaluate whether targeting enhances in-vitro and in-vivo cytotoxicity. Disclosures: No relevant conflicts of interest to declare.

Influence Of DNA Methyltransferese Inhibitors On The Anti-Leukemic Effect Of Umbilical Cord Blood Derived NK Cells Against Acute Myeloid Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4496-4496
Author(s):  
Harry Dolstra ◽  
Jeannette Cany ◽  
Anniek B. van der Waart ◽  
Marleen Tordoir ◽  
Basav Nagaraj Hangalapura ◽  
...  
Keyword(s):  
Cord Blood ◽  
Nk Cells ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Nk Cell ◽  
Ex Vivo ◽  
Dnmt Inhibitors ◽  
Nsg Mice ◽  
Drug Concentrations

Natural killer (NK) cell-based immunotherapy is a promising adjuvant, relatively non-toxic therapy approach for AML. However, further improvement of NK cell-based therapy is needed to increase the clinical effect. In this regard, NK cells generated ex vivo from hematopoietic progenitor cells (HPC) may have significant clinical benefits over enriched NK cells from adult donors, including the ability to choose an appropriate killer-cell immunoglobuline-like receptor (KIR)-ligand or KIR B haplotype alloreactive donor, as well as the capacity to reach high therapeutic dosages. Previously, we reported a GMP-compliant, cytokine/heparin-based culture protocol for the ex vivo generation of highly active NK cells from CD34+ HPC isolated from cryopreserved umbilical cord blood (UCB) units. Expansion in closed, large-scale bioreactors yields a clinically relevant dose of NK cells with high purity and cytolytic activity against AML cells in vitro. Currently, a clinical phase I trial with these HPC-NK cells is ongoing in our hospital. Trafficking studies in NOD/SCID/IL2Rgnull (NSG) mice demonstrated that these HPC-NK cells migrate to the bone marrow (BM) as well as to lymphoid organs where in vivo expansion and maturation can take place. Analysis of the chemokine receptor expression profile of UCB-NK cells matched in vivo findings. Particularly, a firm proportion of UCB-NK cells functionally expressed CXCR4, what could trigger BM homing in response to its ligand CXCL12. In addition, high expression of CXCR3 and CCR6 supported the capacity of UCB-NK cells to migrate to inflamed tissues via the CXCR3/CXCL10-11 and CCR6/CCL20 axis. Importantly, a single HPC-NK cell infusion combined with supportive IL-15 administration was shown to efficiently inhibit growth of K562 leukemia cells implanted in the femur of NSG mice, resulting in significant prolongation of mice survival. Furthermore, we investigated whether modulation by the DNA methyltransferase (DNMT) inhibitors Azacytidine (Aza) and Decitabine (Deci) could further potentiate the antileukemic effect of HPC-NK cells against AML cells. In concordance with previous reports, we observed a dose-dependent effect of Aza and Deci on the growth of the AML cell lines THP1 and KG1a. In subsequent NK cell killing assays, we used clinical relevant low drug concentrations to pre-treat AML cells that did not affect HPC-NK cell viability and cytolytic function. Interestingly, increased killing of pre-treated THP1 and KG1a cells by HPC-NK cells could be observed, which was correlated with an increase in the NKG2D ligand ULBP2, the DNAM-1 ligands CD112 and CD155 as well as TRAIL-R2. Notably, maintenance of low-dose DNMT inhibitors during the KG1a/NK co-culture resulted in pronounced AML growth inhibition. To examine the effect of DNMT inhibitors in vivo, THP1.LucGFP-bearing NSG mice were treated with increasing dose of both agents, which were administered according to current standard protocols applied in humans. Data indicated that treatment with Aza or Deci at dosage equivalent in human to 12.5 and 5 mg/m2 respectively was well tolerated with minimal and/or transient weight loss, and efficiently reduced the progression of THP-1.LucGFP cells in vivo. Currently, we explore whether HPC-NK cells and DNMT inhibitors can work together to combat AML in our xenograft models. These preclinical studies may provide a rationale to investigate the possible additive and/or synergistic anti-AML effects of adoptive HPC-NK cell transfer in combination with these DNMT inhibitors in AML patients. Disclosures: Tordoir: Glycostem Therapeutics: Employment. Spanholtz:Glycostem Therapeutics: Employment.

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

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

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

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 ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3441-3441 ◽  
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.

Dramatic Reduction of Chronic Lymphocytic Leukemia (CLL) Cells Following Adoptive Transfer of Cord Blood (CB) Natural Killer (NK) Cells Using CB-Engrafted NOD-SCID IL2Rγnull (NSG) Mice as a Model.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2370-2370 ◽  
Author(s):  
Dongxia Xing ◽  
Alan G. Ramsay ◽  
William Decker ◽  
Sufang Li ◽  
Simon Robinson ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
Cord Blood ◽  
Nk Cells ◽  
Adoptive Transfer ◽  
Ex Vivo ◽  
Immune Therapy ◽  
Control Group ◽  
Chimeric Mice ◽  
Post Infusion

Abstract Abstract 2370 Poster Board II-347 Adoptive transfer of NK cells in human B-CLL has been limited by the lack of suitable animal models to test the clinical efficacy of this immune therapy strategy. Primary patient B-CLL cells are difficult to engraft in NOD-SCID mice as these mice lack an immune microenvironment that provides essential accessory cells for tumor development. In the current study, we utilize a novel 2-step engraftment protocol using NOD-SCID γnull mice (NSG). Firstly, human cord blood (CB) derived CD34+ stem cells were engrafted to generate humanized chimeric mice capable of supporting B-CLL cells. By week 12, the human engraftment level reached 30% to 60% as detected in peripheral blood. Secondly, these mice were infused with human primary B-CLL cells labeled with CFSE. Our results show that following tail vein injection of 5E7 primary patient CLL cells, chimeric mice exhibited proliferation of CFSE+CD5+ B cells in the spleen and bone marrow, with disease development resembling human CLL. This allowed us to investigate the efficacy of ex vivo IL-2 expanded CB NK cell therapy using this novel mouse model system. As early as 24 hours post-infusion of IL-2 expanded human CD56+CD3- CB NK cells (10E7 per mouse) there was a detectable reduction of CD5+ leukemia cells in the peripheral circulation of CLL-engrafted mice by flow cytometry analysis. Moreover, by day 7 there was a dramatic 99.5 % reduction of CD5+ B-CLL cells in the blood, bone marrow, and spleen of experimental animals compared to the non-treated control group (P<0.001). Of note, expanded CB-NK cells exhibited high anti-leukemic specificity as healthy B cells from the original CB transplant were spared post-infusion. Overall, our studies suggest that the chimeric NSG mouse has utility as an in vivo model for testing immunotherapeutic strategies in aggressive B-CLL. Here, our results highlight the strong anti-leukemic response of infused ex vivo expanded CB-NK cells. Disclosures: No relevant conflicts of interest to declare.

Impaired Natural Killer Cells Immune Synapse Formation in Acute Myeloid Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2663-2663
Author(s):  
Dongxia Xing ◽  
Alan G. Ramsay ◽  
William Decker ◽  
Sufang Li ◽  
Simon Robinson ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Nk Cells ◽  
Natural Killer ◽  
Immune Suppression ◽  
Direct Contact ◽  
Myeloid Leukemia ◽  
Synapse Formation ◽  
Nk Cell ◽  
Immune Synapse ◽  
Acute Myeloid

Abstract Abstract 2663 Poster Board II-639 Natural killer (NK) cells are an important component of the innate immune surveillance of tumor cells. Defective NK cell function has been correlated with poor prognosis in acute myeloid leukemia (AML). It is well established that NK cell-mediated cytolytic activity is significantly diminished in AML patients; the mechanisms of this hypo-function are not well understood. Identifying mechanisms of tumor-induced immune suppression of lymphocytes function will aid the development of effective immunotherapeutic strategies. In the present study we examined the molecular basis for impaired NK cell responses in AML and demonstrate impaired NK cell immunological synapse formation. Confocal microscopy was used to visualize F-actin polymerization at the immune synapse between CD56+ CD3- NK cells and autologous AML blasts. We identified a significant reduction in formation of the NK cell immune synapse (NKIS) (p<0.001) from AML patients compared healthy donors (> 70% reduction). This defect was induced by direct tumor contact since NK cell defects were induced in healthy NK cells when they were co-cultured (in direct contact) for 48 hr with allogeneic AML blasts, but not with healthy allogeneic monocytes (P < 0.01). In control transwell co-culture experiments, where the NK cells and AML blast were not in direct contact, we did not observe the induced defect. We examined the molecular nature of the AML blast induced defect by quantifying recruitment of a number of these NK cell adhesion and cytoskeletal signaling proteins to the immune synapse. Following primary co-culture with AML blasts, healthy NK cells showed significantly reduced recruitment of integrin LFA-1, CD2, Lck, WASP, and tyrosine-phosphorylated protein to the NK-AML target interactions synapse (P < 0.001). These studies demonstrate a role for the tumor induced immune suppression of NK cells and will aid in the development of immunotherapeutic approaches targeting AML. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Rap1b facilitates NK cell functions via IQGAP1-mediated signalosomes

2010 ◽  
Vol 207 (9) ◽  
pp. 1923-1938 ◽  
Author(s):  
Aradhana Awasthi ◽  
Asanga Samarakoon ◽  
Haiyan Chu ◽  
Rajasekaran Kamalakannan ◽  
Lawrence A. Quilliam ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cell Activation ◽  
Synapse Formation ◽  
Nk Cell ◽  
Scaffolding Protein ◽  
Microtubule Organizing Center ◽  
Chemokine Production ◽  
Immune Synapse ◽  
Cell Functions ◽  
Organizing Center

Rap1 GTPases control immune synapse formation and signaling in lymphocytes. However, the precise molecular mechanism by which Rap1 regulates natural killer (NK) cell activation is not known. Using Rap1a or Rap1b knockout mice, we identify Rap1b as the major isoform in NK cells. Its absence significantly impaired LFA1 polarization, spreading, and microtubule organizing center (MTOC) formation in NK cells. Neither Rap1 isoform was essential for NK cytotoxicity. However, absence of Rap1b impaired NKG2D, Ly49D, and NCR1-mediated cytokine and chemokine production. Upon activation, Rap1b colocalized with the scaffolding protein IQGAP1. This interaction facilitated sequential phosphorylation of B-Raf, C-Raf, and ERK1/2 and helped IQGAP1 to form a large signalosome in the perinuclear region. These results reveal a previously unrecognized role for Rap1b in NK cell signaling and effector functions.

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