Synergistic Action of the Human Inhibitory KIR Antibody IPH2102, and the Human CD38 Antibody Daratumumab to Enhance the Lysis of Primary Multiple Myeloma (MM) Cells in the Bone Marrow Mononuclear Cells (MNCs) From Myeloma Patients

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1865-1865
Author(s):  
Inger S. Nijhof ◽  
Michel de Weers ◽  
Pascale Andre ◽  
Berris van Kessel ◽  
Henk M. Lokhorst ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Monoclonal Antibody ◽  
Bone Marrow ◽  
Nk Cells ◽  
Tumor Cells ◽  
Research Funding ◽  
Mononuclear Cells ◽  
Nk Cell ◽  
Human Monoclonal Antibody ◽  
Cell Activity

Abstract Abstract 1865 Despite significant improvements in the treatment of multiple myeloma (MM), this progressive malignancy of antibody-producing clonal plasma cells is still considered incurable. New innovative treatments need to be developed to improve long term outcomes. Recent successes of CD20 antibodies in the clinical lymphoma management indicate that targeted immunotherapy can represent a powerful therapeutical strategy for hematological malignancies. Towards developing a similar strategy for MM, we have recently generated a novel human monoclonal antibody, daratumumab (DARA), which targets the CD38 molecule expressed at high levels on MM cells. We have demonstrated that DARA mediates the lysis of CD38+ MM cells via direct apoptosis, complement mediated lysis and antibody-dependent cell mediated cytotoxicity (ADCC). Natural killer (NK) cells appeared important effector cells mediating the ADCC effect. Since NK cell activity against tumor cells is regulated by the balance of signals generated by inhibitory or activating receptors of NK cells (KIRs), we now explored whether blocking the inhibitory KIRs would improve the NK cell mediated DARA dependent lysis of MM cells. Thus, we evaluated the potential benefits of combining DARA with a novel human anti KIR monoclonal antibody, IPH2102, which blocks the inhibitory KIR2DL1/2/3 receptors (HLA-C specific KIRs), and has been shown to augment NK cell function against MM cells. We recently developed FACS-based ex vivo MM cell lysis assays, in which DARA-dependent NK cell-mediated lysis of MM cells can be directly measured in bone marrow MNCs, thus without separating the malignant cells from autologous NK cells and other accessory cells. Using these, we investigated whether the addition of IPH2102 would augment the DARA dependent lysis of MM cells. As expected, DARA induced lysis of MM cells in bone marrow MNCs isolated from MM patients (n=10). Mean lysis at 10 μg/ml DARA was 27.6% (range 11.3–48.1%). IPH2102 showed little or no lysis of MM cells (at 0.3, 1, 3 and 10 μg/ml) in this setting. The combination of 10 μg/ml IPH2102 with 3 and 10 μg/ml DARA significantly enhanced cytotoxicity against primary MM tumor cells compared to DARA alone (p=0.013 and p=0.028 respectively). Mean lysis of MM tumor cells at 10 μg/ml DARA and 10 μg/ml IPH2102 was 38%. These data confirm our previous findings that NK-cell mediated killing is an important mechanism of action of DARA. We demonstrate a clear synergy between DARA and IPH2102 to achieve effective lysis of MM cells directly in the bone marrow MNC of MM patients, indicating that complementary effects may be achieved by combining IPH2102 and DARA in clinical MM management. Disclosures: Weers: Genmab: Employment. Andre:Innate Pharma: Employment. Lokhorst:Genmab: Research Funding. Parren:Genmab: Employment. Morel:Innate Pharma: Employment. Mutis:Genmab: Research Funding.

scholarly journals Combination Therapy with Daratumumab and CAR-NK Targeting CS1 for Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1342-1342 ◽  
Author(s):  
Yibo Zhang ◽  
Lichao Chen ◽  
Yufeng Wang ◽  
Xinxin Li ◽  
Tiffany Hughes ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Monoclonal Antibody ◽  
T Cell ◽  
Combination Therapy ◽  
Nk Cells ◽  
Tumor Cells ◽  
Cell Activation ◽  
Nk Cell ◽  
Car T ◽  
Ifn Γ

Abstract Daratumumab (Dara), a targeted therapy utilizing a monoclonal antibody against CD38, and its combination with other are becoming a new standard of care treatment in multiple myeloma (MM). Recently, chimeric antigen receptor (CAR) T cell immunotherapy has been successful in the clinic for the treatment of leukemia and lymphoma. Our preliminary data suggest that both CS1-CAR T cells and CS1-CAR NK cells are effective in eradicating MM cells in vitro and in vivo (Chu et al., 2014, Leukemia and Chu et al., 2014, Clinical Cancer Research). In this study, we investigated the combination therapy with Dara and CS1-CAR NK cells for the treatment of relapsed MM. We first showed that that in MM patients, CD38brightCD138─CD34─CD20+CD27+ MM cancer stem-like cells (CSCs) express CS1 at levels much higher than any other cells, and are susceptible to being eradicated by CS1-CAR NK cells. However, CD34+hematopoietic stem cells from bone marrow of healthy donors do not express CS1. These data suggest that CS1-CAR NK cells can target MM CSCs, and thus may prevent relapse of MM, as ample evidence shows that relapsed or recurrent tumor cells are derived from CSCs. We also demonstrated that CD38 is highly expressed on NK and MM cells. Dara triggered IFN-γ and GZMB expression (p< 0.01) in primary human NK cells, even in the absence of crosslinking with tumor cells. Interestingly, the increase IFN-γ expression can be validated in the CD16 (+) haNK-92 (high-affinity natural killer cells), but not in the parental NK-92 cell line. Blocking the recognition between CD16 and Dara (an IgG1 mAb) with an Fc blocking Ab completely impaired Dara-induced IFN-γ and GZMB expression, indicating that Dara-induced NK cell activation is CD16-dependent. Mechanistically, Dara significantly induced phosphorylation of NFkB and STAT1, indicating that Dara induces IFN-γ and GZMB in NK cells, which may occur through CD16 and be mediated downstream by STAT1 and NFkB. We also found that Dara failed to stimulate GZMB and IFN-γ expression in CD38(-) CD16(+) NK cells, while successful in stimulating CD38(+) CD16(+) NK cells, indicating that Dara induces NK cell activation, which requires not only the binding between CD16 and Fc fragment of Dara, but also the CD38 signaling pathway. Furthermore, we found that Dara mediated cytotoxicity of NK cells against MM cells through antibody-dependent cell-mediated cytotoxicity (ADCC) against CD38-positive (e.g., MM1.S), but not CD38-negative (e.g., U266), which can be blocked by CD16 blocking Ab. Moreover, Dara displays ADCC effects in CD16(+) NK cells but not CD16(-) NK cells. When CD16(+) NK cells were armed with the CS1-CAR, ADCC is still observed against CD38(+) MM cells at low effector to target ratios, i.e., Dara still enhances cytotoxicity of CS1-CAR NK cells, which already have enhanced cytotoxicity. We observed that Dara-induced NK cell ADCC against CD38(+) MM MM1.S cells led to increased T cell proliferation and activation in a co-culture system including dendritic cells. This effect was not observed when MM U266 cells were included as the NK cell target. Out data are consistent with that recent discovery by DiLillo and Ravetch showing that engagement of monoclonal antibody can induce an antitumor vaccine effects (David J et al., Cell, 2015). To tested Dara affects NK cell survival, immunoblotting was performed with anti-cleaved Caspase-3 and anti-cleaved PARP-1 antibodies. We demonstrated that apoptotic activity was increased in both CD16(+)NK cells (primary NK and haNK-92) and parental CD16(-)NK-92 cells treated with Dara for 24 h in a dose-dependent manner. Unlike Dara's positive effects on CD16(+) NK cells (i.e. stimulating IFN-γ production and ADCC), induction of apoptosis seems to be CD16-independent, as parental NK-92 cells, which are CD16(-), also showed an increased levels of apoptosis induced by Dara. We are testing whether the apoptosis induction is dependent on the antigen for Dara, because as mentioned above, both primary NK cells, and modified as well as unmodified NK-92 cells, that were CD38 (+). In conclusion, our study demonstrates that the combination of Dara and CS1-CAR NK cells, which target two different tumor-associated antigens, both of which have potent anti-MM efficacy, may show additive or synergistic effects; however due to the positive and negative effects of Dara on NK cells, sequential treatment rather than a concomitant treatment modality should be considered. Disclosures No relevant conflicts of interest to declare.

scholarly journals High Dimensional Immune Profiling in Smoldering Multiple Myeloma Identifies Novel Organizing Features of the Tumor Microenvironment

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4384-4384
Author(s):  
Hearn Jay Cho ◽  
Deepak Perumal ◽  
Adeeb H Rahman ◽  
Seunghee Kim-Schultze ◽  
Jennifer Yesil ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Tumor Microenvironment ◽  
Tumor Cells ◽  
Research Funding ◽  
Mononuclear Cells ◽  
Plasma Cells ◽  
High Dimensional ◽  
Integrated Analysis ◽  
Symptomatic Disease

Multiple myeloma (MM) is a malignancy of plasma cells that arises from premalignant Monoclonal Gammopathy of Undetermined Significance (MGUS) and often progresses through an asymptomatic Smoldering (SMM) phase lasting months or years before manifesting clinical symptoms warranting therapy. Current research indicates that the tumor microenvironment (TME) in the bone marrow may play a significant role in governing progression to symptomatic disease. Therefore, understanding of the interactions between malignant plasma cells and the TME in early disease states is critical in the pursuit of therapies that will prevent progression to symptomatic disease. We performed high dimensional genomic and immunologic analysis of bone marrow specimens from 73 subjects with SMM. We performed RNA-seq on the malignant plasma cells isolated by anti-CD138 magnetic bead positive selection, mass cytometry (CyTOF) and T cell receptor sequencing (TCR Seq) of CD138-depleted bone marrow mononuclear cells, and proteomics, seromic, and grand serology analysis of bone marrow plasma. These samples and assays provided a broad view of the tumor cells and the cellular and soluble components of the TME. Each of these assays identified self-organizing clusters of subjects, indicating that subgroups of SMM patients shared common characteristics in the tumor or TME populations. We then applied novel bioinformatic methods to compare data from pairs, trios, quartets, and quintets of assays to identify communities of subjects with similar immunologic and genomic characteristics. Integrated analysis of CyTOF, proteomic, and TCR Seq resolved three distinct communities with a high degree of significance. These communities shared distinct cellular and proteomic features that suggested early adaptive, activated adaptive, or innate immune characteristics. These results suggest that the continuum from MGUS to MM does not consist of a single pathway in either the tumor cells or the TME, and that complex interactions ultimately determine progression. This suite of assays (CyTOF, proteomics, and TCR Seq) may be applicable in translational and clinical studies to understand key tumor and immune determinants of SMM and lead to rationally designed therapy to replicate these conditions to prevent progression to symptomatic disease. Disclosures Cho: Genentech: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; The Multiple Myeloma Research Foundation: Employment; Takeda: Research Funding; BMS: Consultancy; Agenus: Research Funding; GSK: Consultancy. Adams:Janssen Pharmaceuticals R&D: Employment, Other: Own Stock. Parekh:Foundation Medicine Inc.: Consultancy; Celgene Corporation: Research Funding; Karyopharm Inc.: Research Funding.

634 Production and testing of a novel bispecific nanobody construct targeting NK cells and EGFR expressing malignancies

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A670-A670
Author(s):  
Elisa Toffoli ◽  
Abdolkarim Sheikhi ◽  
Roeland Lameris ◽  
Lisa King ◽  
Jurriaan Tuynman ◽  
...  
Keyword(s):  
Nk Cells ◽  
Tumor Cells ◽  
Mononuclear Cells ◽  
Nk Cell ◽  
Cell Activity ◽  
Nk Cell Activity ◽  
Peripheral Blood Mononuclear ◽  
Mutational Status ◽  
Tumor Cell Lysis

BackgroundThe ability to kill tumor cells with an acceptable toxicity profile, makes Natural Killer (NK) cells promising assets for cancer therapy. However, strategies to enhance the preferential accumulation and activation of NK cells in the tumor microenvironment would likely increase the efficacy of NK cell-based therapies.MethodsIn this study, we show a novel bispecific nanobody-based construct (biVHH) targeting both CD16A (low-affinity Fc receptor: FcRγIIIA) on NK cells and EGFR on tumors of epithelial origins.ResultsHigher levels of NK cell activity and subsequent tumor cell lysis were found in vitro in the presence of the biVHH and were dependent on the expression of both CD16A and EGFR while they were independent of the KRAS mutational status of the tumor. Increased NK cell activity was found in NK cells derived from colorectal cancer (CRC) patients when co-cultured with the biVHH and EGFR expressing tumor cells. Finally, higher levels of cytotoxicity were found against patient-derived metastatic CRC cells in the presence of the biVHH and autologous peripheral blood mononuclear cells or allogeneic NK cells.ConclusionsBased on our results, the bispecific CD16A and EGFR targeting VHH construct could be a useful tool in combination with various NK cell-based therapies.

scholarly journals Combination of Melphalan Flufenamide and Anti-PD-L1 or Anti-CD38 Antibodies Enhances Anti-Myeloma Immunity

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4357-4357
Author(s):  
Arghya Ray ◽  
Ting DU ◽  
Nina N. Nupponen ◽  
Fredrik Lehmann ◽  
Jakob Lindberg ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Board Of Directors ◽  
Tumor Cells ◽  
Research Funding ◽  
Mononuclear Cells ◽  
Nk Cell ◽  
Cytolytic Activity ◽  
Publicly Traded ◽  
Advisory Committees

Abstract Introduction Melphalan flufenamide (Melflufen; Oncopeptides AB) is a novel enzyme-activated analogue of melphalan that enables a more rapid and higher intracellular accumulation of melphalan in tumor cells than is achievable by direct exposure to equimolar doses of melphalan. Our preclinical study showed that melflufen is a more potent anti-myeloma (MM) agent than melphalan, overcomes drug-resistance, and induces synergistic anti-MM activity in combination with bortezomib, lenalidomide, or dexamethasone (Chauhan et al, Clinical Cancer Res 2013;19:3019). However, the effect of melflufen on the immunosuppressive and tumor-promoting MM-host bone marrow (BM) accessory cells such as immunologically dysfunctional plasmacytoid dendritic cells (pDCs; CD123/IL-3Rα) remains unclear. Here, we utilized our coculture models of pDCs, T-, and NK cells with autologous patient MM cells to examine whether a combination of melflufen and immune checkpoint inhibitor anti-PD-L1 Ab, or daratumumab (anti-CD38 Ab), restores anti-MM immunity. Methods MM patient BM and PB samples (N=10; obtained after informed consent), and cell lines were used for the study. Minimally cytotoxic concentration of melflufen (0.1 µM) was used to assess immune functions. CTL/NK activity assays MM CD8 + T- or NK-cells were cultured with autologous pDCs (1:10 pDC:T/NK ratio) with melflufen (0.1 μM) alone, and with anti-PD-L1 (5 μg/ml) or anti-CD38 (0.5 μg/ml) Abs for 3-5 days; cells were washed to remove the drugs, and then cultured for another 24h with pre-stained target MM cells (10:1 E/T ratio; T/NK:MM), followed by quantification of viable MM cells by flow. Results 1) Both MM tumor cells and pDCs showed higher PD-L1 and CD38 levels vs normal plasma cells; 2) Treatment of MM patient total BM mononuclear cells or purified MM cells with melflufen (0.1 µM) increased PD-L1 expression on MM cells (1.84-fold, treated vs untreated; p&lt;0.05). Importantly, treatment of MM cells with melflufen and anti-PD-L1 Abs enhanced anti-MM cytotoxicity; 3) Combination of melflufen and anti-PD-L1 Ab triggers activation of CD3 + T cells, evidenced by an increase in CD69 expression on CD3 + T cells (1.15-fold, treated vs untreated, p&lt;0.05); 4) Combination of melflufen and anti-PD-L1 Ab induced a more robust autologous MM-specific CD8 + cytotoxic T lymphocyte (CTL) activity than melflufen alone (% MM lysis: melflufen: 20%; melflufen plus anti-PD-L1 Ab: 60%; n=5; p=0.013); 5) Meflufen and anti-PD-L1 also triggered pDC-induced NK cell-mediated MM-specific cytolytic activity (p&lt;0.05); and finally, 6) Low doses of melflufen and anti-CD38 Abs enhanced pDC-induced NK cell-mediated MM-specific cytolytic activity (%Viability: melflufen: 75%; melflufen + anti-CD38 Ab: 12.5%; n=4; p=0.001). Conclusions The combination of melflufen and anti-PD-L1 increases pDC-induced T- and NK cell-mediated cytolytic activities against MM. Moreover, combined melflufen and anti-CD38 Abs modestly enhance pDC-induced NK cell-mediated MM-specific cytolytic activity. Our preclinical data suggest targeting PD-L1 in combination with melflufen as well as support an ongoing clinical trial of melflufen with anti-CD38 Abs to enhance anti-MM immunity. Disclosures Nupponen: Oncopeptides AB: Consultancy. Lehmann: Oncopeptides AB: Current Employment. Lindberg: Oncopeptides: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months, Other: Travel, Accommodations, Expenses; Camurus: Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations, Expenses; Affibody: Membership on an entity's Board of Directors or advisory committees. Gullbo: Oncopeptides AB: Consultancy. Richardson: Takeda: Consultancy, Research Funding; Celgene/BMS: Consultancy, Research Funding; Janssen: Consultancy; Sanofi: Consultancy; Protocol Intelligence: Consultancy; Karyopharm: Consultancy, Research Funding; GlaxoSmithKline: Consultancy; Regeneron: Consultancy; AstraZeneca: Consultancy; Secura Bio: Consultancy; AbbVie: Consultancy; Oncopeptides: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy, Research Funding. Chauhan: C4 Therapeutics: Current equity holder in publicly-traded company; Oncopeptides: Consultancy; Stemline Therapeutics: Consultancy. Anderson: Janssen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Identification of Molecular Markers of Tumor Cell Sensitivity and Resistance to Natural Killer Cells through Genome-Wide CRISPR Activation and CRISPR Editing Screens in Multiple Myeloma Cell Lines: Implications for Anti-Myeloma Immunotherapy

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1115-1115
Author(s):  
Sara Gandolfi ◽  
Michal Sheffer ◽  
Emily Lowry ◽  
Olga Dashevsky ◽  
Ryosuke Shirasaki ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Molecular Markers ◽  
High Risk ◽  
Tumor Cell ◽  
Nk Cells ◽  
Tumor Cells ◽  
Research Funding ◽  
Nk Cell ◽  
Death Receptor ◽  
Genome Wide

Abstract Natural killer (NK) cells represent a promising immunotherapeutic approach as they can potently kill tumor cells without triggering graft-versus-host reactions. Indeed, infusion of high numbers of NK cells, either autologous or allogeneic, after their ex vivo expansion and activation, has been feasible and safe in clinical studies. However, prior studies and early clinical trials indicate that tumor cells can exhibit decreased response to NK due to the protective effect of nonmalignant mesenchymal stromal cells; and depending on the genetic background of the tumor cells. To our knowledge, since earlier subgenome-scale RNAi-based studies, there have been no genome-wide CRISPR-based screens to identify candidate markers conferring tumor cell resistance or sensitivity to NK cells in multiple myeloma (MM). To address this void, and building on a recent loss-of-function (LOF) study by our group on solid tumors, we sought to identify genes regulating the response of MM cells to the cytotoxic activity of NK cells by conducting a genome-wide CRISPR/Cas9-based gene editing (Brunello library of sgRNA) and gene activation (Calabrese library of sgRNA) screens in MM.1S cells co-cultured with primary NK (pNK) cells (effector-to-target [E:T] ratio of 3.75:1) derived from healthy donor peripheral blood mononuclear cells (PBMCs) cultured in vitro in GMP SCGM medium with IL-2. Briefly, MM.1S cells engineered to stably express the nuclease SpCas9 (Brunello) or a catalytically inactive programmable RNA-dependent DNA-binding protein (dCas9)-VP64 (Calabrese) were also transduced with lentiviral particles for a pool of ~70,000 (Brunello library) or ~120,000 (Calabrese) sgRNAs, targeting exons of ~20,000 genes (plus non-targeting control sgRNAs), under conditions of transduction which allow for an average of no more than 1 sgRNA to be incorporated in a given cell. This allowed us to convert the initial population of MM.1S cells into heterogeneous pools in which each gene is subject to individual LOF or gain-of-function (GOF), due to Cas9-mediated editing, by only 1 sgRNA. Flow cytometry was performed to verify pNK viability, purity (CD56 and CD3), and expression of p46 receptor, surrogate marker of NK cell activity. These screens identified genes whose knock-out (Brunello sgRNA library) or activation (Calabrese sgRNA library) led to NK cells resistance or potential sensitivity. The hits observed in the current MM-oriented study exhibited, compared to our similar studies in solid tumor model, substantial gene level differences, but notable overlap at the pathway level (including death receptor pathways, NK activating pathways), which suggests that mechanisms determining tumor cell response vs. resistance to NK cells operate through modules consistent across tumors, but manifested through potentially different members of the respective pathways in different neoplasms. For instance, in this MM-oriented study, we identified that NK cell sensitivity of tumor cells is modulating by activation of several metabolic and homeostatic genes, receptor kinases, and interestingly membrane-bound proteins of the mucin family, e.g. MUC1, and MUC4, which have been reported to play a role in NK-mediated tumor killing in other types of cancer. MUC1 in particular has a clinical relevance as a small molecule inhibitor with prior preclinical studies in MM is available. Interestingly, our GOF screen identified as potential NK cell sensitizers TNFRSF10B, a death receptor related to TNFRSF10A (a hit identified in our studies in solid tumors), the putative death receptor adaptor TRADD, and the NK ligands PVR and ULBP1. Interestingly, genes such as PTEN and TP53, commonly associated with high-risk MM, didn't affect the response to NK cell, suggesting that NK cell-based therapies may potentially have a role in treatment of MM patients with high-risk clinical or biological features. In conclusion, this is the first study applying both LOF and GOF genome-wide screens to NK cell response in MM. The combination of such screens performed in parallel provide complementary and orthogonal information that allows us to identify genes that might not have been appreciated if only either LOF or GOF alone screens had been performed. We envision that the methodology and results presented herein will provide a framework towards validation of molecular markers which can help to optimize and individualize the use of NK cell-based therapy in MM. Disclosures Mitsiades: Abbvie: Research Funding; TEVA: Research Funding; EMD Serono: Research Funding; Janssen/ Johnson & Johnson: Research Funding; Takeda: Other: employment of a relative.

scholarly journals Role and Modulation of NK Cells in Multiple Myeloma

Hemato ◽  
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.

NK-Cell Reconstitution after HLA-Identical Blood and Marrow Transplantations for CML: The Recovery of NKG2D Is Inversely Correlated with NKG2A and It Promotes the GVL Effect.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4879-4879
Author(s):  
Juan Tong ◽  
Huilan Liu ◽  
Liangquan Geng ◽  
Zimin Sun ◽  
Baolin Tang ◽  
...  
Keyword(s):  
Nk Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Nk Cell ◽  
Linear Regression Analysis ◽  
Cell Activity ◽  
Target Cells ◽  
Peripheral Blood Stem Cells ◽  
Peripheral Blood Mononuclear ◽  
Blood Stem Cells

Abstract Natural killer (NK) cell alloreactivity is reported to mediate strong graft versus leukemia (GVL) effect in patients after allogeneic stem-cell transplantation. NKG2D receptors recognize human MHC class Ichain related A and B (MICA/B) and UL16-binding protein 1∼4(ULBP 1∼4) on target cells, thereby regulating NK cell activity. To examine the recovery of NKG2D, NKG2A and other receptors expression by NK cells, we used flow cytometry to evaluate samples from 11 chronic myeloid leukemia patients and their donors in the year following unmanipulated HLA completely matched peripheral blood stem cells plus bone marrow transplantation. Peripheral blood mononuclear cells from patients and their donors were tested in standard 51Cr release assays against cultured K562 targets to determine the cytotoxicity of the NK cells in the same intervals. There is no mismatched immunoglobulin-like receptor (KIR) ligand in both GVH and HVG direction. The reconstitution of KIR2DL1 (CD158a) after this transplantation protocol was very slow and these receptors didn’t reach normal value in the year and KIR2DL2 (CD158b) was much better. The NKG2D increased and the NKG2A decreased quickly at the same time after engraftment, and used linear regression analysis we demonstrated that NKG2A recovery was inversely correlated with NKG2D recovery in the year following transplantation. The ratio of NKG2D/NKG2A was directly associated with the capacity of NK-cell cytotoxicity. Thus, the reconstitution of NKG2D makes contribution to the recovery of the NK cytotoxicity. These results reveals that the NK cells generated after HLA matched blood plus bone morrow transplantation of CML patients are promoted at an immature state characterized by specific phenotypic features and enhanced functioning, having potential impact for immune responsiveness and transplantation outcome.

Phase 1/2 Study of Elotuzumab in Combination with Bortezomib in Patients with Multiple Myeloma with One to Three Prior Therapies: Interim Results.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3876-3876 ◽  
Author(s):  
Andrzej J Jakubowiak ◽  
William Bensinger ◽  
David Siegel ◽  
Todd M. Zimmerman ◽  
Jan M. Van Tornout ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Clinical Response ◽  
Board Of Directors ◽  
Research Funding ◽  
Mononuclear Cells ◽  
Phase 1 ◽  
Surface Glycoprotein ◽  
Advisory Committees ◽  
Cell Surface Glycoprotein

Abstract Abstract 3876 Poster Board III-812 Background Elotuzumab is a humanized monoclonal IgG1 antibody directed against CS1, a cell surface glycoprotein, which is highly and uniformly expressed in multiple myeloma (MM). In mouse xenograft models of MM, elotuzumab demonstrated significantly enhanced anti-tumor activity when combined with bortezomib compared to bortezomib alone (Van Rhee et al., Mol. Cancer Ther., in press, 2009). This phase 1/2 trial will determine the maximum tolerated dose (MTD), overall safety, pharmacokinetics (PK) and clinical response of elotuzumab in combination with bortezomib in patients with relapsed MM following 1-3 prior therapies. Methods The study consists of 4 escalating cohorts of elotuzumab (2.5 mg/kg to 20 mg/kg) administered on Days 1 and 11 and bortezomib (1.3 mg/m2) administered on Days 1, 4, 8 and 11 of a 21-day cycle. Patients with progressive disease at the end of Cycle 2 or 3 also receive oral dexamethasone (20 mg) on Days 1, 2, 4, 5, 8, 9, 11 and 12 of each subsequent cycle. Patients with stable disease or better at the end of 4 cycles will continue treatment for 6 or more cycles unless withdrawn earlier due to unexpected toxicity or disease progression. Key entry criteria: age ≥ 18 years; confirmed diagnosis of MM and documentation of 1 to 3 prior therapies; measurable disease M-protein component in serum and/or in urine; and no prior bortezomib treatment within 2 weeks of first dose. Results To date, a total of 16 MM patients with a median age of 64 years have been enrolled in the study. The median time from initial diagnosis of MM was 3.5 years and patients had received a median of 2 prior MM treatments. Patients have been treated in four cohorts; 3 each in 2.5, 5 and 10 mg/kg elotuzumab cohorts, and 7 in the 20 mg/kg elotuzumab cohort. No dose limiting toxicity (DLT) was observed during the first cycle of the study and the MTD was not established. Five SAEs have been reported in four patients in later treatment cycles; two events, chest pain and gastroenteritis, occurring in one patient, were considered elotuzumab-related. Other SAEs include grade 3 sepsis, vomiting, pneumonia and grade 2 dehydration. The most common AEs reported include Grade 1-3 diarrhea, constipation, nausea, fatigue, thrombocytopenia, neutropenia, anemia and peripheral neuropathy. The best clinical response (EBMT criteria) for the 16 patients who have received at least two cycles of treatment is shown in the table below. Preliminary PK analysis suggests a serum half-life of 10-11 days at higher doses (10 and 20 mg/kg). Preliminary analysis of peripheral blood mononuclear cells and bone marrow of patients on study indicates that objective responses in the study correlate well with complete saturation of CS1 sites by elotuzumab on bone marrow plasma and NK cells. Conclusions The combination of elotuzumab with bortezomib has a manageable adverse event profile and shows promising preliminary efficacy with ≥PR in 44% and ≥MR in 75% of all enrolled patients. Accrual is ongoing in the expanded 20 mg/kg cohort. Updated safety, efficacy, and PK data will be presented at the meeting. Disclosures: Jakubowiak: Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Centocor Ortho Biotech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Exelixis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Off Label Use: Bortezomib in combination with elotuzumab for the treatment of relapsed/refractory multiple myeloma. Bensinger:Millennium: Membership on an entity's Board of Directors or advisory committees. Siegel:Millennium: Speakers Bureau; Celgene: Speakers Bureau. Zimmerman:Millennium: Speakers Bureau; Centecor: Speakers Bureau. Van Tornout:BMS: Employment. Zhao:Facet Biotech: Employment. Singhal:Facet Biotech: Employment. Anderson:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3870-3870 ◽  
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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