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

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<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<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.

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CD38 contributes to human natural killer cell responses through a role in immune synapse formation

10.1101/349084 ◽

2018 ◽

Cited By ~ 7

Author(s):

Mathieu Le Gars ◽

Christof Seiler ◽

Alexander W. Kay ◽

Nicholas L. Bayless ◽

Elsa Sola ◽

...

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Tumor Cells ◽

Synapse Formation ◽

Cell Function ◽

Nk Cell ◽

Critical Role ◽

Target Cells ◽

Immune Synapse ◽

Ifn Γ

AbstractNatural killer (NK) cells use a diverse array of activating and inhibitory surface receptors to detect threats and provide an early line of defense against viral infections and cancer. Here, we demonstrate that the cell surface protein CD38 is a key human NK cell functional receptor through a role in immune synapse formation. CD38 expression marks a mature subset of human NK cells with a high functional capacity. NK cells expressing high levels of CD38 display enhanced killing and IFN-γ secretion in response to influenza virus-infected and tumor cells. Inhibition of CD38 enzymatic activity does not influence NK cell function, but blockade of CD38 and its ligand CD31 abrogates killing and IFN-γ expression in response to influenza-infected cells. Blockade of CD38 on NK cells similarly inhibits killing of tumor cells. CD38 localizes and accumulates at the immune synapse between NK cells and their targets, and blocking CD38 severely abrogates the ability of NK cells to form conjugates and immune synapses with target cells. Thus, CD38 plays a critical role in NK cell immune synapse formation. These findings open new avenues in immunotherapeutic development for cancer and infection by revealing a critical role for CD38 in NK cell function.

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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 ◽

10.1182/blood.v114.22.3029.3029 ◽

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.

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Recent Advances to Augment NK Cell Cancer Immunotherapy Using Nanoparticles

Pharmaceutics ◽

10.3390/pharmaceutics13040525 ◽

2021 ◽

Vol 13 (4) ◽

pp. 525

Author(s):

Kwang-Soo Kim ◽

Dong-Hwan Kim ◽

Dong-Hyun Kim

Keyword(s):

Clinical Trials ◽

Nk Cells ◽

Cancer Immunotherapy ◽

Tumor Cells ◽

Therapeutic Efficacy ◽

Nk Cell ◽

Cell Cancer ◽

Immune Surveillance ◽

Cytolytic Activity ◽

Contact Frequency

Among various immunotherapies, natural killer (NK) cell cancer immunotherapy using adoptive transfer of NK cells takes a unique position by targeting tumor cells that evade the host immune surveillance. As the first-line innate effector cell, it has been revealed that NK cells have distinct mechanisms to both eliminate cancer cells directly and amplify the anticancer immune system. Over the last 40 years, NK cell cancer immunotherapy has shown encouraging reports in pre-clinic and clinic settings. In total, 288 clinical trials are investigating various NK cell immunotherapies to treat hematologic and solid malignancies in 2021. However, the clinical outcomes are unsatisfying, with remained challenges. The major limitation is attributed to the immune-suppressive tumor microenvironment (TME), low activity of NK cells, inadequate homing of NK cells, and limited contact frequency of NK cells with tumor cells. Innovative strategies to promote the cytolytic activity, durable persistence, activation, and tumor-infiltration of NK cells are required to advance NK cell cancer immunotherapy. As maturing nanotechnology and nanomedicine for clinical applications, there is a greater opportunity to augment NK cell therapeutic efficacy for the treatment of cancers. Active molecules/cytokine delivery, imaging, and physicochemical properties of nanoparticles are well equipped to overcome the challenges of NK cell cancer immunotherapy. Here, we discuss recent clinical trials of NK cell cancer immunotherapy, NK cell cancer immunotherapy challenges, and advances of nanoparticle-mediated NK cell therapeutic efficacy augmentation.

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Role and Modulation of NK Cells in Multiple Myeloma

Hemato ◽

10.3390/hemato2020010 ◽

2021 ◽

Vol 2 (2) ◽

pp. 167-181

Author(s):

Marie Thérèse Rubio ◽

Adèle Dhuyser ◽

Stéphanie Nguyen

Keyword(s):

Multiple Myeloma ◽

Monoclonal Antibodies ◽

Nk Cells ◽

Tumor Cells ◽

Nk Cell ◽

Ex Vivo ◽

Killer Cell ◽

Proteasome Inhibitors ◽

Disease Evolution ◽

Cell Functions

Myeloma tumor cells are particularly dependent on their microenvironment and sensitive to cellular antitumor immune response, including natural killer (NK) cells. These later are essential innate lymphocytes implicated in the control of viral infections and cancers. Their cytotoxic activity is regulated by a balance between activating and inhibitory signals resulting from the complex interaction of surface receptors and their respective ligands. Myeloma disease evolution is associated with a progressive alteration of NK cell number, phenotype and cytotoxic functions. We review here the different therapeutic approaches that could restore or enhance NK cell functions in multiple myeloma. First, conventional treatments (immunomodulatory drugs-IMids and proteasome inhibitors) can enhance NK killing of tumor cells by modulating the expression of NK receptors and their corresponding ligands on NK and myeloma cells, respectively. Because of their ability to kill by antibody-dependent cell cytotoxicity, NK cells are important effectors involved in the efficacy of anti-myeloma monoclonal antibodies targeting the tumor antigens CD38, CS1 or BCMA. These complementary mechanisms support the more recent therapeutic combination of IMids or proteasome inhibitors to monoclonal antibodies. We finally discuss the ongoing development of new NK cell-based immunotherapies, such as ex vivo expanded killer cell immunoglobulin-like receptors (KIR)-mismatched NK cells, chimeric antigen receptors (CAR)-NK cells, check point and KIR inhibitors.

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894 The bispecific innate cell engagers AFM13 (CD30/CD16A) and AFM24 (EGFR/CD16A) increase the fraction of tumor target-responsive NK cells and boost serial killing

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.894 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A938-A938

Author(s):

Chiara Zambarda ◽

Karolin Guldevall ◽

Chiara Zambarda ◽

Karolin Guldevall ◽

Christian Breunig ◽

...

Keyword(s):

Nk Cells ◽

Single Cell ◽

Tumor Cells ◽

Cell Biology ◽

Nk Cell ◽

Biological Evaluation ◽

Target Cells ◽

List Type ◽

Tumor Target ◽

Serial Killing

BackgroundThe use of bispecific natural killer (NK) cell engagers has emerged as a successful strategy for immune cell activation and killing of tumor cells through antibody-dependent cellular cytotoxicity (ADCC). Among these, tetravalent, bispecific innate cell engagers (ICE®) with specificity for the activating receptor CD16A selectively triggering innate responses from NK cells or macrophages represent the most clinically advanced concept. The CD30/CD16A specific ICE® AFM13, has shown efficacy in patients with CD30+ lymphomas as monotherapy1 and combination therapy with check-point inhibitors2 and most recently in combination with adoptive NK cell therapy.3 The EGFR/CD16A specific ICE® AFM24, targeting a variety of solid tumors like colorectal, or lung cancer with a unique mode of action independent of EGFR signaling inhibition, is currently evaluated in an ongoing Ph1/2a clinical study.MethodsWe used a microchip-based screening with single cell resolution4 to elucidate the dynamic responses of individual NK cells towards tumor target cells upon treatment with AFM13 or AFM24.ResultsWe found that AFM13 and AFM24 mediated potent activation of NK cells, leading to increased responsive cytotoxic NK cells and significantly increased the number of NK cells that exerted engagement with multiple target cells rendering these NK cells serial killers. Strikingly, bispecific ICE® molecules triggered stronger cytotoxic responses compared to monoclonal antibodies. One suggested strategy to boost killing by NK cells is to use molecular inhibitors or protein constructs that prevent shedding of CD16.5 However, previous results have shown that this can lead to impaired detachment from target cells, reducing the capacity for an individual NK cell to form serial contacts to target cells.6 We observed that the elevated NK cell killing induced by ICE® molecules was largely conserved when cells were treated with the shedding inhibitor Batimastat. Analysis of the functional dynamics of NK cells revealed that inhibition of CD16 shedding prevented NK cell detachment from target cells, resulting in cell cluster formation. This might strongly impact targeting of distant tumor cells by an individual NK cell thus limiting its anti-tumoral activity.ConclusionsIn conclusion, we show that both AFM13 and AFM24 increase the fraction of tumor-target responsive NK cells and boost serial killing of target cells by individual NK cells. Based on these data, ICE® molecules can be characterized as potent anti-tumoral agents leveraging the enormous potential of NK cells while maintaining crucial features of NK cell biology.AcknowledgementsWe thank members of the Önfelt lab for their valuable help and feedback.ReferencesSawas A, Elgedawe H, Vlad G, Lipschitz M, Chen P-H, Rodig SJ, et al. Clinical and biological evaluation of the novel CD30/CD16A tetravalent bispecific antibody (AFM13) in relapsed or refractory CD30-positive lymphoma with cutaneous presentation: a biomarker phase Ib/IIa study (NCT03192202). Blood 2018;132(Supplement 1):2908–2908.Bartlett NL, Herrera AF, Domingo-Domenech E, Mehta A, Forero-Torres A, Garcia-Sanz R, et al. A phase 1b study of AFM13 in combination with pembrolizumab in patients with relapsed or refractory Hodgkin lymphoma. Blood 2020. Blood 2020;136(21):2401–2409.Kerbauy LN, Marin ND, Kaplan M, Banerjee PP, Berrien-Elliott MM, Becker-Hapak M, et al. Combining AFM13, a bispecific CD30/CD16 antibody, with cytokine-activated blood and cord blood–derived NK cells facilitates CAR-like responses against CD30 + malignancies. Clin Cancer Res Epub 2021.Guldevall K, Brandt L, Forslund E, Olofsson K, Frisk TW, Olofsson PE, et al. Microchip screening platform for single cell assessment of NK cell cytotoxicity. Front Immunol 2016;7:119.Romee R, Foley B, Lenvik T, Wang Y, Zhang B, Ankarlo D, et al. NK cell CD16 surface expression and function is regulated by a disintegrin and metalloprotease-17 (ADAM17). Blood 2013;121(18):3599–608.Srpan K, Ambrose A, Karampatzakis A, Saeed M, Cartwright ANR, Guldevall K, et al. Shedding of CD16 disassembles the NK cell immune synapse and boosts serial engagement of target cells. J Cell Biol 2018;217(9):3267–83.Ethics ApprovalThis work was performed with NK cells from healthy anonymous blood donors, which requires no ethical permit according to local regulations.

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Blockade of Ubiquitin Receptor Rpn13 in Plasmacytoid Dendritic Cells Enhances Anti-Myeloma Immunity

Blood ◽

10.1182/blood-2019-122901 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3098-3098

Author(s):

Arghya Ray ◽

Yan Song ◽

Ting DU ◽

Dharminder Chauhan ◽

Kenneth C. Anderson

Keyword(s):

Dendritic Cells ◽

Cell Growth ◽

Nk Cells ◽

Tumor Cells ◽

Immune Suppression ◽

Nk Cell ◽

Plasmacytoid Dendritic Cells ◽

Cytolytic Activity ◽

Immune Checkpoints ◽

Autologous Tumor

Introduction Although proteasome inhibitor (PI) based combination therapies achieve remarkable responses multiple myeloma (MM), emergence of PI resistance is common. The mechanism(s) of PI-resistance include tumor-intrinsic factors such as mutations of the 20S proteasomal subunits, and/or tumor-extrinsic cellular components in the BM microenvironment. Interactions of BM accessory cells, immune effector cells, and tumor cells confer both drug-resistance and immune suppression in MM. For example, we showed that interactions of MM plasmacytoid dendritic cells (pDCs) with MM cells and with T/NK cells both confer immune suppression via immune checkpoints, as well as trigger MM cell growth by inducing secretion of MM cell growth factors. We recently reported that targeting proteasome-associated ubiquitin receptor Rpn13 triggers cytotoxicity and overcomes tumor-intrinsic PI-resistance in MM (Song et al, Leukemia 2016;30:1877). Here we utilized our co-culture models of patient pDCs, T cells, NK cells, and autologous MM cells to characterize the immune sequelae of Rpn13 inhibition. Methods Analysis of pDCs activation Purified patient-pDCs (n =7) were treated with Rpn13 inhibitor RA190 (0.05 µM) for 24h, followed by multicolor staining using fluorophore-conjugated Abs against pDC activation/maturation markers CD80, CD83, and CD86. Transient transfections Purified MM patient pDCs were transfected with Rpn13-siRNA using TransIT-X2 transfection Kit,and analyzed for alterations in maturation markers. CTL/NK activity assays Purified MM-BM CD8+ T- or NK-cells (n = 8) were co-cultured with autologous BM-pDCs (pDC:T/NK; 1:10 ratio) for 3 days, in the presence or absence of Rpn13 inhibitor RA190 (100 nM). After washing, cells were cultured for 24h with autologous MM cells pre-stained with CellTracker/CellTrace Violet (10 T/NK:1 MM), followed by 7-AAD staining and quantification of CTL-or NK cell-mediated MM cell lysis by FACS. Results 1) RA190 triggers significant upregulation of maturation markers CD80, CD83, and CD86 on MM-pDCs (fold change vs untreated: CD80: 1.2; p = 0.007; CD83: 2.15; p = 0.006; CD86: 1.4; p = 0.003). In contrast, bortezomib-treated pDCs showed no significant upregulation of these markers. 2) Similar to pharmacological inhibition of Rpn13 with RA190, Rpn13-siRNA increased CD80 (1.76-fold), CD83 (3.12-fold), and CD86 (2.28-fold) expression on MM pDCs (p<0.01). Of note, both RA190 and bortezomib block protein degradation via proteasome, but only RA190 activates pDCs. 3) RA190 treatment increases pDC-induced MM-specific CD8+ CTL activity, as well as NK cell-mediated cytolytic activity against autologous tumor cells, evidenced by decreased viable patient MM cells. 4) Treatment of MM-pDCs with RA190 increases expression of calnexin, a molecular chaperone protein of endoplasmic reticulum which regulates immune co-stimulatory molecules, immune-regulatory signaling, and restores the ability of pDCs to induce proliferation of MM-specific CTLs or NK cells. These findings were also confirmed using pDC cell line CAL-1. Conclusions Our prior findings showed that inhibition of UbR Rpn13 overcomes intrinsic PI-resistance in MM cells. Here we show that targeting Rpn13 also triggers anti-MM immune responses. Rpn13 blockade therefore represents a novel therapeutic approach to overcome both PI-resistance and immune suppression in MM. Disclosures Chauhan: C4 Therapeutics.: Equity Ownership; Stemline Therapeutics: Consultancy. Anderson:Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Sanofi-Aventis: Other: Advisory Board.

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CD38 deletion of human primary NK cells eliminates daratumumab-induced fratricide and boosts their effector activity

Blood ◽

10.1182/blood.2020006200 ◽

2020 ◽

Vol 136 (21) ◽

pp. 2416-2427 ◽

Cited By ~ 2

Author(s):

Meisam Naeimi Kararoudi ◽

Yuya Nagai ◽

Ezgi Elmas ◽

Marcelo de Souza Fernandes Pereira ◽

Syed Abbas Ali ◽

...

Keyword(s):

Nk Cells ◽

Cell Function ◽

Nk Cell ◽

Ex Vivo ◽

Human Monoclonal Antibody ◽

Metabolic Reprogramming ◽

Target Cells ◽

Plasma Cell Neoplasm ◽

Cd38 Expression ◽

The Impact

Abstract Multiple myeloma (MM) is a plasma cell neoplasm that commonly expresses CD38. Daratumumab (DARA), a human monoclonal antibody targeting CD38, has significantly improved the outcome of patients with relapsed or refractory MM, but the response is transient in most cases. Putative mechanisms of suboptimal efficacy of DARA include downregulation of CD38 expression and overexpression of complement inhibitory proteins on MM target cells as well as DARA-induced depletion of CD38high natural killer (NK) cells resulting in crippled antibody-dependent cellular cytotoxicity (ADCC). Here, we tested whether maintaining NK cell function during DARA therapy could maximize DARA-mediated ADCC against MM cells and deepen the response. We used the CRISPR/Cas9 system to delete CD38 (CD38KO) in ex vivo expanded peripheral blood NK cells. These CD38KO NK cells were completely resistant to DARA-induced fratricide, showed superior persistence in immune-deficient mice pretreated with DARA, and enhanced ADCC activity against CD38-expressing MM cell lines and primary MM cells. In addition, transcriptomic and cellular metabolic analysis demonstrated that CD38KO NK cells have unique metabolic reprogramming with higher mitochondrial respiratory capacity. Finally, we evaluated the impact of exposure to all-trans retinoic acid (ATRA) on wild-type NK and CD38KO NK cell function and highlighted potential benefits and drawbacks of combining ATRA with DARA in patients with MM. Taken together, these findings provide proof of concept that adoptive immunotherapy using ex vivo expanded CD38KO NK cells has the potential to boost DARA activity in MM.

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Ex Vivo Activated Natural Killer (NK) Cells from Myeloma Patients Kill Autologous Myeloma and Killing Is Enhanced by Elotuzumab

Blood ◽

10.1182/blood.v112.11.3666.3666 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3666-3666

Author(s):

Tarun K. Garg ◽

Susann Szmania ◽

Jumei Shi ◽

Katie Stone ◽

Amberly Moreno-Bost ◽

...

Keyword(s):

Bone Marrow ◽

Cell Therapy ◽

Nk Cells ◽

Nk Cell ◽

Ex Vivo ◽

K562 Cells ◽

Scid Mice ◽

Target Cells ◽

Recent Trial ◽

Nk Cell Therapy

Abstract Immune-based therapies may improve outcome for multiple myeloma (MM) by eradicating chemo-resistant disease. Our recent trial utilizing IL2 activated, killer immunoglobulin-like receptor-ligand mismatched NK cell transfusions from haplo-identical donors yielded (n) CR in 50% of patients. Unfortunately, after NK cell therapy, 2/10 patients had progressive disease, and the median duration of response for the other 8/10 patients was only 105 days (range 58–593). This may have been due to an insufficient dose of alloreactive NK cells and early rejection. Furthermore, appropriate donors were identified for only 30% of otherwise eligible patients. We therefore investigated whether NK cells from MM patients could be expanded and activated to kill autologous MM. We then examined whether pre-treatment of MM cell targets with elotuzumab, a humanized antibody to the MM tumor antigen CS1, could further enhance NK cell-mediated lysis. PBMC from 5 MM patients were co-cultured for 14 days with irradiated K562 cells transfected with 4-1BBL and membrane bound IL15 in the presence of IL2 (300U/ml) as previously described (Imai et al, Blood2005;106:376–383). The degree of NK cell expansion, NK immunophenotype, and ability to kill MM (4 hour 51Cr release assays) were assessed. To determine the ability of ex vivo expanded NK cells to traffic to bone marrow, activated NK cells were injected into the tail vein of NK cell depleted NOD-SCID mice, which were then sacrificed after 48 hours. Flow cytometry for human CD45, CD3, and CD56 was performed on cells from blood, marrow and spleen. There was an average 64-fold expansion of NK cells (range: 8–200) after 2 weeks of co-culture with K562 transfectants. Expansion of T cells was not observed. The NK cell activating receptor NKG2D, and natural cytotoxicity receptors NKp30, NKp44, and NKp46 were up-regulated following the expansion. Expanded NK cells were able to kill autologous MM (E:T ratio 10:1, average 31%, range 22–41%), whereas resting NK cells did not. Pretreatment of autologous MM cells with elotuzumab increased the activated NK cell-mediated killing by 1.7-fold over target cells pretreated with an isotype control antibody. This level of killing was similar to that of the highly NK kill-sensitive cell line K562 (Figure). Autologous PHA blasts and CD34+ stem cells were not killed. Activated human NK cells were detectable in the bone marrow of NOD-SCID mice 48 hours after injection. Ex vivo activation of NK cells from MM patients with K562 transfectants can induce killing of autologous MM and produce large numbers of NK cells for potential therapy. The addition of elotuzumab to activated NK cell therapy enhances anti-MM effects by ADCC thus invoking an additional NK cell-mediated mechanism of MM killing. Importantly, ex vivo activated NK cells traffic to the bone marrow in mice. Autologous NK cell therapy eliminates the issues related to allo-donor availability and early NK cell rejection, and could provide an option for patients refractory to chemotherapy agents. Figure Figure

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IPH2101, a Novel Anti-Inhibitory KIR Monoclonal Antibody, and Lenalidomide Combine to Enhance the Natural Killer (NK) Cell Versus Multiple Myeloma (MM) Effect.

Blood ◽

10.1182/blood.v114.22.3870.3870 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3870-3870 ◽

Cited By ~ 1

Author(s):

Don Benson ◽

Courtney E Bakan ◽

Shuhong Zhang ◽

Lana Alghothani ◽

Jing Liang ◽

...

Keyword(s):

Clinical Trial ◽

Nk Cells ◽

Tumor Cells ◽

Nk Cell ◽

Phase 1 ◽

Granzyme B ◽

Cell Activity ◽

Target Cells ◽

Nk Cell Activity ◽

Immune Complex Formation

Abstract Abstract 3870 Poster Board III-806 Background NK cell activity against tumor cells is regulated by a balance of inhibitory and activating signals mediated by receptors on NK cells that recognize inhibitory and activating ligands expressed by cancer cells. IPH2101 (1-7F9) is a novel monoclonal anti-inhibitor KIR blocking antibody that has been shown to augment NK cell function against MM targets. Moreover, lenalidomide has been shown to expand and activate NK cells in vivo and in vitro. We have previously reported that the combination of IPH2101 and lenalidomide enhances NK cell mediated cytotoxicity against MM cells compared to each agent alone (Zhang et al., AACR 2009). We expand our studies to investigate potential mechanisms for the enhancement of NK cell activity by the combination of IPH2101 and lenalidomide. Methods The effects of IPH2101 and lenalidomide alone and in combination were studied using primary human NK cells from healthy donors as well as from MM patients. The MM cell lines U266 and RPMI 8226 as well as primary tumor cells from marrow aspirates of MM patients served as target cells. The effect of lenalidomide on MM activating and inhibitory ligand expression was studied by flow cytometry. NK cell trafficking was investigated with standard transwell plate migration assay. Immune complex formation between NK cell effectors and MM tumor targets was characterized by flow cytometry in control conditions and with NK cells pre-treated with IPH2101 and lenalidomide. The effects of IPH2101 and lenalidomide were studied regarding interferon-gamma and granzyme B production by ELISPOT and target-specific cytotoxicity studies were conducted to complement effector-based assays. Results IPH2101 (30 ug/ml) significantly enhanced cytotoxicity against U266 cells and primary MM tumor cells by both purified NK cells at effector:target (E:T) ratios of 10:1 or less, and also of freshly isolated peripheral blood mononuclear cells (PBMC) at E:T ratios of 60:1 or less, from more than 10 random donors. In addition, treatment of PBMC with 5-10 μmol/L lenalidomide for 72h without interleukin (IL)-2 increased NK cell lysis of U266. Treatment of PBMC from normal donors did not enhance the expression of the NK receptors KIR, NKG2D, NCR, TRAIL, and DNAM-1. Incubation of U266 cells with lenalidomide (5 uM) for 3-5 days resulted in significant enhancement of cytotoxicity by normal donor NK cells. This was associated with upregulation of the activating ligands, MICA, ULBP-2, DR4, and CD112. Using blocking antibodies to NKG2D, TRAIL, and DNAM-1, lenalidomide enhancement of MM cell killing was abrogated indicating the importance of the modulation of the ligands to the latter receptors by lenalidomide. Although IPH2101 and lenalidomide did not significantly increase NK cell migration into normal media, migration was enhanced 2.98-fold (+/− 0.36, p < 0.05) towards U266 cell targets (n= 3, p < 0.05) and MM patient serum 3.2-fold (+/− 0.4, n=3, p < 0.05). IPH2101 and lenalidomide also led to a 2.3-fold (+/− 0.43, p < 0.05) increase in immune complex formation between NK cells and MM tumor cells. IPH2101 and lenalidomide also augmented NK cell interferon gamma production against MM (control mean 303 spots/well +/− 13 versus 525 +/− 83, n=3, p < 0.05) and granzyme B production (control mean 115 +/− 98 versus 449 +/−72, n=3, p < 0.05). Importantly, in all experiments described herein, the effects of IPH2101 and lenalidomide together were greater than either agent alone. Conclusions Taken together, our data suggest that IPH2101 and lenalidomide may exert complementary mechanisms on both effector and target cells to enhance NK cell mediated killing of MM cells. Moreover, these agents have no predicted clinical cross-toxicities. A single-agent phase 1 clinical trial of IPH2101 has shown the mAb to be safe and well tolerated in MM patients. These findings support a phase 1/2 clinical trial of IPH2101 with lenalidomide as a first dual-innate immunotherapy for patients with MM. Disclosures: Andre: Innate Pharma: Employment. Squiban:Innate pharma: Employment. Romagne:Innate Pharma: Employment.

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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 ◽

10.1182/blood.v116.21.3928.3928 ◽

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.

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