Enhancing Natural Killer Cell-Mediated Lysis of Lymphoma Cells By Combining Therapeutic Antibodies with CD20-Specific Immunoligands Engaging NKG2D or NKp30

Abstract Antibody-dependent cell-mediated cytotoxicity (ADCC) represents a major effector function of many therapeutic antibodies. Thus, enhancing ADCC is a promising approach to further improve antibody therapy. Here, the CD20-specific immunoligands ULBP2:7D8 and B7-H6:7D8, which engage the stimulatory NK cell receptors natural killer group 2 member D (NKG2D) and NKp30, respectively, were compared for their abilities to boost ADCC in an attempt to design an effective antibody combination strategy. The immunoligands are designed as single chain molecules, with a single chain fragment variable (scFv) of the CD20 antibody 7D8 fused to UL16-binding protein (ULBP) 2 or B7 homologue 6 (B7-H6), which are ligands of the activating NK cell receptors NKG2D and NKp30, respectively. By binding to lymphoma cells the immunoligands designated as ULBP2:7D8 and B7-H6:7D8 mimicked an induced self phenotype and thereby triggered NK cells to kill lymphoma and leukemia cells. Both immunoligands augmented ADCC by NK cells synergistically when combined with the lymphoma-directed antibodies rituximab or daratumumab recognizing CD20 and CD38, respectively. Antibody combinations with ULBP2:7D8 resulted in higher cytotoxicity (up to 10-fold lower EC50-values) in comparison to combinations with B7-H6:7D8, which in individual experiments failed to boost ADCC. Thus, NK cells were triggered more efficiently when NKG2D rather than NKp30 was co-ligated together with FcγRIIIA. Although a combination of ULBP2:7D8 and B7-H6:7D8 produced synergistic effects, no significant improvements were obtained by combining the three agents rituximab, B7-H6:7D8 and ULBP2:7D8. Enhancement of ADCC by the immunoligands was also achieved when NK cells from lymphoma or leukemia patients were analyzed as effector cells. ULBP2:7D8 in particular increased lysis not only of allogeneic but also of autologous tumor cells. In summary, co-targeting of NKG2D was more effective in promoting NK cell-mediated ADCC than co-ligation of NKp30 and may represent a promising approach to further enhance the efficacy of therapeutic antibodies. Based on these results we propose a ‘dual-dual-targeting’ concept by co-targeting of two surface antigens on tumor cells and concomitant engagement of two different activating NK cell receptors. Disclosures van de Winkel: Genmab BV: Employment, Patents & Royalties. Parren:Genmab: Employment, Equity Ownership.

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Natural killer cell receptors: new biology and insights into the graft-versus-leukemia effect

Blood ◽

10.1182/blood-2002-02-0350 ◽

2002 ◽

Vol 100 (6) ◽

pp. 1935-1947 ◽

Cited By ~ 352

Author(s):

Sherif S. Farag ◽

Todd A. Fehniger ◽

Loredana Ruggeri ◽

Andrea Velardi ◽

Michael A. Caligiuri

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Nk Cell ◽

Killer Cell ◽

Natural Cytotoxicity ◽

Receptor Ligand ◽

Cell Receptors ◽

Nk Cell Receptors ◽

Ligand Interactions ◽

Nk Receptor

AbstractNatural killer (NK) cells have held great promise for the immunotherapy of cancer for more than 3 decades. However, to date only modest clinical success has been achieved manipulating the NK cell compartment in patients with malignant disease. Progress in the field of NK cell receptors has revolutionized our concept of how NK cells selectively recognize and lyse tumor and virally infected cells while sparing normal cells. Major families of cell surface receptors that inhibit and activate NK cells to lyse target cells have been characterized, including killer cell immunoglobulinlike receptors (KIRs), C-type lectins, and natural cytotoxicity receptors (NCRs). Further, identification of NK receptor ligands and their expression on normal and transformed cells completes the information needed to begin development of rational clinical approaches to manipulating receptor/ligand interactions for clinical benefit. Indeed, clinical data suggest that mismatch of NK receptors and ligands during allogeneic bone marrow transplantation may be used to prevent leukemia relapse. Here, we review how NK cell receptors control natural cytotoxicity and novel approaches to manipulating NK receptor-ligand interactions for the potential benefit of patients with cancer.

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Potential Role of Hsp70 and Activated NK Cells for Prediction of Prognosis in Glioblastoma Patients

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.669366 ◽

2021 ◽

Vol 8 ◽

Author(s):

Dominik Lobinger ◽

Jens Gempt ◽

Wolfgang Sievert ◽

Melanie Barz ◽

Sven Schmitt ◽

...

Keyword(s):

Nk Cells ◽

Tumor Cells ◽

Nk Cell ◽

Human Tumor ◽

Serum Levels ◽

Hsp70 Expression ◽

Cell Receptors ◽

Nk Cell Receptors ◽

Dismal Prognosis ◽

Prediction Of Prognosis

Despite rapid progress in the treatment of many cancers, glioblastoma remains a devastating disease with dismal prognosis. The aim of this study was to identify chaperone- and immune-related biomarkers to improve prediction of outcome in glioblastoma. Depending on its intra- or extracellular localization the major stress-inducible heat shock protein 70 (Hsp70) fulfills different tasks. In the cytosol Hsp70 interferes with pro-apoptotic signaling pathways and thereby protects tumor cells from programmed cell death. Extracellular Hsp70 together with pro-inflammatory cytokines are reported to stimulate the expression of activatory NK cell receptors, recognizing highly aggressive human tumor cells that present Hsp70 on their cell surface. Therefore, intra-, extracellular and membrane-bound Hsp70 levels were assessed in gliomas together with activatory NK cell receptors. All gliomas were found to be membrane Hsp70-positive and high grade gliomas more frequently show an overexpression of Hsp70 in the nucleus and cytosol. Significantly elevated extracellular Hsp70 levels are detected in glioblastomas with large necrotic areas. Overall survival (OS) is more favorable in patients with low Hsp70 serum levels indicating that a high Hsp70 expression is associated with an unfavorable prognosis. The data provide a first hint that elevated frequencies of activated NK cells at diagnosis might be associated with a better clinical outcome.

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Highly functional natural killer (NK) cells as predictive biomarkers associated with long response to castration in newly diagnosed metastatic prostate cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2014.32.4_suppl.95 ◽

2014 ◽

Vol 32 (4_suppl) ◽

pp. 95-95

Author(s):

Christine Pasero ◽

Gwenaelle Gravis ◽

Mathilde Guerin ◽

Palma Rocchi ◽

Jeanne Thomassin ◽

...

Keyword(s):

Prostate Cancer ◽

Nk Cells ◽

Natural Killer ◽

Metastatic Prostate Cancer ◽

Nk Cell ◽

Predictive Biomarkers ◽

Newly Diagnosed ◽

Cell Receptors ◽

Nk Cell Receptors ◽

Activating Receptors

95 Background: Immunotherapy is now investigated as a promising alternative treatment for patients (pts) with metastatic prostate cancer (PC). Natural killer (NK) cells are powerful effector cells with antitumoral activity and their role have been explored in solid tumors but not yet in prostate cancer. NK cell cytotoxicity is regulated by a balance between activating and inhibitory receptors. Here, we performed a restrospective study to evaluate the link between NK cells and the time of castration response in newly diagnosed PC patients with metastases. Methods: Newly diagnosed metastatic PC pts were divided according the time of castration response, with an 18-months cutoff value: 18 pts with long castration response (LCR, median = 64.6 months), and 14 pts with short castration response ([SCR] median = 11.2 months), with a median overall survival of 97.7 months and 33.8 months respectively. Circulating NK cells from these patients were studied by flow cytometry to evaluate the expression of activating receptors and the NK cell functionality. Results: We observed thatNK cells from LCR pts express higher levels of the maturation marker CD57 (43.3% vs. 23.3% positive cells, p= 0.002), the receptor CD16 involved in cytotoxicity (29,124 vs. 16,806 MFI, p= 0.02), and the activating receptors NKp46 and NKp30 (17.5 vs. 11.4 RMFI, p= 0.0146 , and 10.9 vs. 6.3 RMFI, p = 0.0128 respectively) than NK cells from SCR pts. This suggests that LCR pts have powerful NK cells. Indeed, NK cells from LCR pts are highly efficient in CD107 functional assay than NK cells from SCR pts (28.9% vs. 19.4%, p =0.002). In vitro blocking experiments show that NKp46 is precisely one of the NK cell receptors involved in the NK-mediated recognition of prostate tumor cells, thus higher expression of NKp46 would help to control PC progression. Conclusions: Together our results show for the first time that efficient NK cells are associated to a long response to castration and prolonged survival in newly diagnosed metastatic PC. NK cell receptors might be useful as predictive biomarkers in metastatic PC, to help in stratification of patients and to design NK cell–based immunotherapeutic strategies for PC.

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Hybrid resistance to EL-4 lymphoma cells. I. Characterization of natural killer cells that lyse EL-4 cells and their distinction from marrow-dependent natural killer cells.

Journal of Experimental Medicine ◽

10.1084/jem.150.3.531 ◽

1979 ◽

Vol 150 (3) ◽

pp. 531-547 ◽

Cited By ~ 51

Author(s):

V Kumar ◽

E Luevano ◽

M Bennett

Keyword(s):

Natural Killer Cells ◽

Nk Cells ◽

Natural Killer ◽

Tumor Cells ◽

Nk Cell ◽

Nk Activity ◽

Spleen Cells ◽

Lymphoma Cells ◽

Hybrid Resistance

Natural killer (NK) cells from nonimmunized mice capable of lysing EL-4 (C57BL/6 strain H-2b) tissue culture-adapted lymphoma cells have been analyzed and compared with NK cells which lyse YAC-1 (A-strain, H-2a) lymphoma cells. A correlation was seen in the ability of inbred and B6D2F1 mice to reject C57BL/6 (B6) bone-marrow grafts and the ability of their spleen cells to lyse EL-4 cells in vitro. This suggests that hybrid or hemopoietic histocompatibility antigens, (Hh-1b), relevant in the rejection of B6 stem cells may also be the relevant target structures for the anti-EL-4 NK cells. Certain features of these NK cells are similar to the NK cells reactive against YAC-1 cells. Both types of NK cells are present in athymic nude mice, are not affected by treatment with anti-immunoglobulin plus complement, and are not depleted by techniques that remove macrophages. NK activity against both targets is stimulated 3 d after injection of Corynebacterium parvum, and 24 h after challenge with polyinosinic:polycytidylic acid. Hydrocortisone acetate and cyclophosphamide lead to reduction of NK activity within 2-3 d after administration. However, the anti-YAC and anti-EL-4 NK reactivities differed in several important respects. Treatment of mice with 89Sr, the bone-seeking isotope, to deplete marrow-dependent cells, depleted the anti-YAC-1 but not anti-EL-4 cell functions. Anti-EL-4 NK cells were unaffected by silica particles in vivo or in vitro; the NK cells reactive to EL-4 cells matured functionally much earlier in life (5 d of age) and the function did not decline with age. Irradiated mice reconstituted with syngeneic marrow or spleen cells developed functional NK cells against EL-4 targets before they developed anti-YAC-1 NK cells in their spleen. Thus anti-EL-4 NK cells that express hybrid resistance in vitro appear to differ from anti-YAC-1 NK cells and do not require an intact marrow microenvironment for functional differentiation. Despite differences in the NK-cell types involved in the lysis of YAC-1 and EL-4 cells, these two tumor cells share certain common determinants. This was ascertained both by cold competition and by utilization of YAC-1 and EL-4 cell monolayers as immunoadsorbents. We conclude that Hh-1b is the common antigen present in EL-4 and YAC-1 cells, because B6D2F1 anti-B6 (anti-Hh-1b) cytotoxic T lymphocytes lysed both the tumor cells. Our data suggest that Hh-1b antigen is recognized by both types of NK cells, but that additional determinants must be present on YAC-1 cells. Two models of NK cell lysis compatible with the data are presented.

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Cytokine-Induced Memory-Like NK Cells with High Reactivity against Acute Leukemia Blasts and Solid Tumor Cells Suitable for Adoptive Immunotherapy Approaches

Cancers ◽

10.3390/cancers13071577 ◽

2021 ◽

Vol 13 (7) ◽

pp. 1577

Author(s):

Matteo Tanzi ◽

Michela Consonni ◽

Michela Falco ◽

Federica Ferulli ◽

Enrica Montini ◽

...

Keyword(s):

Acute Leukemia ◽

Nk Cells ◽

Tumor Cells ◽

Cell Function ◽

Nk Cell ◽

Autologous Tumor ◽

Lymphoid Leukemia ◽

Hematopoietic Stem ◽

Solid Malignancies

The limited efficacy of Natural Killer (NK) cell-based immunotherapy results in part from the suboptimal expansion and persistence of the infused cells. Recent reports suggest that the generation of NK cells with memory-like properties upon in vitro activation with defined cytokines might be an effective way of ensuring long-lasting NK cell function in vivo. Here, we demonstrate that activation with IL-12, IL-15 and IL-18 followed by a one-week culture with optimal doses of Interleukin (IL-2) and IL-15 generates substantial numbers of memory-like NK cells able to persist for at least three weeks when injected into NOD scid gamma (NSG) mice. This approach induces haploidentical donor-derived memory-like NK cells that are highly lytic against patients’ myeloid or lymphoid leukemia blasts, independent of the presence of alloreactive cell populations in the donor and with negligible reactivity against patients’ non-malignant cells. Memory-like NK cells able to lyse autologous tumor cells can also be generated from patients with solid malignancies. The anti-tumor activity of allogenic and autologous memory-like NK cells is significantly greater than that displayed by NK cells stimulated overnight with IL-2, supporting their potential therapeutic value both in patients affected by high-risk acute leukemia after haploidentical hematopoietic stem cell transplantation and in patients with advanced solid malignancies.

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799 Neoantigen-cytokine-chemokine multifunctional natural killer cell engager for immunotherapy of solid tumors

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.799 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A834-A834

Author(s):

Xue Yao ◽

Sandro Matosevic

Keyword(s):

Tumor Microenvironment ◽

Cell Therapy ◽

Nk Cells ◽

Natural Killer ◽

Solid Tumors ◽

Tumor Cells ◽

Nk Cell ◽

Killer Cell ◽

Nk Cell Therapy

BackgroundThe effectiveness of natural killer (NK) cell-based immunotherapy against solid tumors is limited by the lack of specific antigens and the immunosuppressive tumor microenvironment (TME). Glioblastoma multiforme (GBM) is one such heavily immunosuppressive tumor that has been particularly hard to target and remains without a viable treatment. The development of novel approaches to enhance the efficacy of NK cells against GBM is urgently needed. NK cell engagers (NKCE) have been developed to enhance the efficacy of NK cell therapy.MethodsTo improve the clinical efficacy of NK cell therapy, we are developing a new generation of multi-specific killer engagers, which consists of a neoantigen-targeting moiety, together with cytokine and chemokine-producing domains. Neoantigens are new antigens formed specifically in tumor cells due to genome mutations, making them highly specific tools to target tumor cells. Our engager has been designed to target Wilms' tumor-1 (WT-1), a highly specific antigen overexpressed in GBM among other solid tumors. This is done through the generation of an scFv specific targeting the complex of WT-1126-134/HLA-A*02:01 on the surface of GBM. On the NK cell side, the engager is designed to target the activating receptor NKp46. Incorporation of the cytokine IL-15 within the engager supports the maturation, persistence, and expansion of NK cells in vivo while favoring their proliferation and survival in the tumor microenvironment. Additionally, our data indicated that the chemokine CXCL10 plays an important role in the infiltration of NK cells into GBM, however, GBM tumors produce low levels of this chemokine. Incorporation of a CXCL10-producing function into our engager supports intratumoral NK cell trafficking by promoting, through their synthetic production, increased levels of CXCL10 locally in the tumor microenvironment.ResultsCollectively, this has resulted in a novel multifunctional NK cell engager, combining neoantigen-cytokine-chemokine elements fused to an activating domain-specific to NK cells, and we have investigated its ability to support and enhance NK cell-mediated cytotoxicity against solid tumors in vitro and in vivo against patient-derived GBM models. The multi-specific engager shows both high tumor specificity, as well as the ability to overcome NK cell dysfunction encountered in the GBM TME.ConclusionsWe hypothesize that taking advantage of our multi-functional engager, NK cells will exhibit superior ex vivo expansion, infiltration, and antitumor activity in the treatment of GBM and other solid tumors.

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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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TIM-3 Expression Is Downregulated on Human NK Cells in Response to Cancer Targets in Synergy with Activation

Cancers ◽

10.3390/cancers12092417 ◽

2020 ◽

Vol 12 (9) ◽

pp. 2417 ◽

Cited By ~ 1

Author(s):

Tram N. Dao ◽

Sagar Utturkar ◽

Nadia Atallah Lanman ◽

Sandro Matosevic

Keyword(s):

T Cell ◽

Nk Cells ◽

Natural Killer ◽

Interferon Gamma ◽

Nk Cell ◽

Ex Vivo ◽

Cell Dysfunction ◽

Nk Cell Receptors ◽

Ifn Γ

Among natural killer (NK) cell receptors, the T-cell immunoglobulin and mucin-containing domain (TIM-3) has been associated with both inhibitory and activating functions, depending on context and activation pathway. Ex vivo and in vitro, expression of TIM-3 is inducible and depends on activation stimulus. Here, we report that TIM-3 expression can be downregulated on NK cells under specific conditions. When NK cells are exposed to cancer targets, they synergize with stimulation conditions to induce a substantial decrease in TIM-3 expression on their surface. We found that such downregulation occurs following prior NK activation. Downregulated TIM-3 expression correlated to lower cytotoxicity and lower interferon gamma (IFN-γ) expression, fueling the notion that TIM-3 might function as a benchmark for human NK cell dysfunction.

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Enhancing Natural Killer (NK) Cell Mediated Killing of Non-Hodgkin's Lymphoma.

Blood ◽

10.1182/blood.v114.22.2706.2706 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2706-2706 ◽

Cited By ~ 4

Author(s):

Shivani Srivastava ◽

Hailin Feng ◽

Shuhong Zhang ◽

Jing Liang ◽

Patrick Squiban ◽

...

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Tumor Cells ◽

Mhc Class I ◽

Median Survival ◽

Nk Cell ◽

Normal Donor ◽

Natural Cytotoxicity ◽

Raji Cells ◽

Effector Cells

Abstract Abstract 2706 Poster Board II-682 Follicular lymphoma is incurable with the current chemo- or chemoimmunotherapy with median survival of 8–12 years. Relapse free survival after each subsequent therapy steadily decreases, resulting in an expected median survival of 4.5 years following initial relapse. Hence new treatment strategies are needed. Natural killer (NK) cells are important effector cells in mediating the anti-lymphoma effect of rituximab. Indeed, antibody-dependent cell-mediated cytotoxicity (ADCC) is a major mechanisms of action of rituximab with NK cells being important effector cells. However, in addition to ADCC, NK cells also exert natural cytotoxicity against tumor cells, which is modulated by a balance of inhibitory and activating signals through NK cell receptors. NK cell function is inhibited when their inhibitory killer immunoglobulin-like receptors (KIR) are ligated by their cognate MHC class I antigens on tumor targets. The novel agent IPH2101 (1-7F9) is a fully human monoclonal antibody directed against KIR2DL receptor that blocks the interaction of KIR with its HLA-C ligands breaking NK cell tolerance to autologous tumor cells. We investigated whether the combination of the IPH2101and Rituximab will augment the NK cell mediated cytotoxicity against CD20+ lymphoma targets as compared to rituximab alone. Raji cells are human CD20+ Burkitt lymphoma cell line cells that expresses HLA-A*03,- (ligand to inhibitory KIR3DL2); -B*71[Bw6] (no inhibitory KIR-Ligand) and -Cw*03,w*04 (group 1 and 2 of HLA-C ligands to inhibitory KIR2DL2/3 and KIR2DL1), and were chosen for study because they have HLA-C antigens that ligate the inhibitory KIR2DL2/3 and KIR2DLI receptors, making them a good target to test our hypothesis of inhibiting inhibitory KIR. NK cells were isolated from normal donor PBMC (peripheral blood mononuclear cells) with the Miltenyi NK isolation Kit. Using LDH release based cytotoxicity assay, we show (Figure 1) that the treatment of target Raji cells with Rituximab significantly enhanced natural cytotoxicity of the purified NK cells against Raji cells. IPH2101alone treatment of NK cells also significantly enhanced the cytotoxicity of Raji cells, however, the combination of IPH2101treated NK cells against Rituximab treated Raji cells significantly enhanced cytotoxicity beyond that observed with each agent alone. Effector: Target (E:T) ratios of 14:1 or less, from more than 5 random donors showed similar results indicating a synergistic, or at least and additive effect ( representative experiment shown Figure 1) . In these experiments purified NK cells were treated with 30ug/ml of IPH2101for 30 min and Raji targets were treated with 0.1-30ug/ml of Rituximab for 30 min. NK cells in the presence or absence of IPH2101were co-cultured with Raji cells in the presence or absence of Rituximab for 4 hour in a 96 well plate. NK cytotoxicity was assessed with an LDH release based assay. Our results suggest that there is a positive cooperation between natural cytotoxicity mediated through KIR-MHC blockade and that mediated by ADCC. Indeed, wee have shown that the blockade of KIR-MHC class I interaction by anti-KIR blocking antibody (IPH2101) augments the cytotoxicity of freshly isolated normal donor NK cells against CD20+ lymphoma cell lines as compared to rituximab alone, providing a rationale for the clinical investigation of the combination of IPH2101 (1-7F9) and rituximab in non-Hodgkin's lymphoma Disclosures: Squiban: Innate pharma: Employment.

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Pluripotent Stem Cell-Derived Human Natural Killer Cells with Potent Anti-Multiple Myeloma Activity,

Blood ◽

10.1182/blood.v118.21.4034.4034 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4034-4034

Author(s):

David A. Knorr ◽

Zhenya Ni ◽

Allison Bock ◽

Vijay G. Ramakrishnan ◽

Shaji Kumar ◽

...

Keyword(s):

Stem Cells ◽

Multiple Myeloma ◽

Stem Cell ◽

Nk Cells ◽

Natural Killer ◽

Tumor Cells ◽

Adoptive Immunotherapy ◽

Target Cell ◽

Peripheral Blood ◽

Nk Cell

Abstract Abstract 4034 Natural Killer (NK) cells are lymphocytes of the innate immune system with anti-viral and anti-cancer activity. Over the past decade, they have gained interest as a promising cellular source for use in adoptive immunotherapy for the treatment of cancer. Most notably, NK cells play an important role in the graft-vs-tumor effect seen in allogeneic hematopoietic stem cell transplantation (allo-HSCT), and a better understanding of NK cell biology has translated into improved transplant outcomes in acute myelogenous leukemia (AML). Small studies have demonstrated a role for NK cell activity in multiple myeloma (MM) patients receiving allo-HSCT. Investigators have also utilized haplo-identical killer immunoglobulin-like receptor (KIR) mismatched NK cells for adoptive immunotherapy in patients with multiple myeloma (MM). Our group has focused on the development of NK cells from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) as a novel starting source of lymphocytes for immunotherapy. We have previously demonstrated potent anti-tumor activity of hESC-derived NK cells in vitro and in vivo against a variety of different targets. We have also shown that iPSC-derived NK cells from a variety of different somatic cell starting sources posses potent anti-tumor and anti-viral activity. Here, we demonstrate hESC- and iPSC-derived NK cell development in a completely defined, feeder-free system that is amenable to clinical scale-up. These cultures contain a pure population of mature NK cells devoid of any T or B cell contamination, which are common adverse bystanders of cellular products isolated and enriched from peripheral blood. Our cultures are homogenous for their expression of CD56 and express high levels of effector molecules known to be important in anti-MM activity, including KIR, CD16, NKG2D, NKp46, NKp44, FasL and TRAIL. We have now tested the activity of hESC- and iPSC-derived NK cells against MM tumor cells in order to provide a universal source of lymphocytes for adoptive immunotherapy in patients with treatment refractory disease. We find that similar to peripheral blood NK cells (PB-NK), hESC- and iPSC-derived NK cells are cytotoxic against 3 distinct MM cell lines in a standard chromium release cytotoxicity assay. Specifically, activated PB-NK cells killed 48.5% of targets at 10 to 1 effector to target ratios, whereas hESC (46.3%) and iPSC (42.4%) derived NK cells also demonstrated significant anti-MM activity. Also, hESC- and iPSC-derived NK cells secrete cytokines (IFNγ and TNFα) and degranulate as demonstrated by CD107a surface expression in response to MM target cell stimulation. When tested against freshly isolated samples from MM patients, hESC- and IPSC-derived NK cells respond at a similar level as activated PB-NK cells, the current source of NK cells used in adoptive immunotherapy trials. These MM targets (both cell lines and primary tumor cells) are known to express defined ligands (MICA/B, DR4/5, ULBP-1, BAT3) for receptors expressed on NK cells as well as a number of undefined ligands for natural cytotoxicity receptors (NCRs) and KIR. As these receptor-ligand interactions drive the anti-MM activity of NK cells, we are currently evaluating expression of each of these molecules on the surface of both the effector and target cell populations. Not only do hESC- and iPSC-derived NK cells provide a unique, homogenous cell population to study these interactions, they also provide a genetically tractable source of lymphocytes for improvement of the graft-vs-myeloma effect and could be tailored on a patient specific basis using banks of hESC-or iPSC-derived NK cells with defined KIR genotypes for use as allogeneic or autologous effector cells. Disclosures: No relevant conflicts of interest to declare.

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