scholarly journals 880 Tetravalent, bispecific innate cell engager AFM24 enhances macrophage mediated tumor cell phagocytosis

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A922-A922
Author(s):  
Sheena Pinto ◽  
Susanne Wingert ◽  
Jens Pahl ◽  
Armin Beez ◽  
Sabrina Purr ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Nk Cells ◽  
Signaling Pathway ◽  
Tumor Cells ◽  
Cell Lines ◽  
Treatment Option ◽  
Nk Cell ◽  
Wide Spectrum ◽  
Egfr Signaling Pathway ◽  
Egfr Expression

BackgroundEnabling innate immunity holds promise to provide a treatment option for patients suffering from various kinds of malignancies. Innate cell engagers (ICE®) derived from the ROCK® (redirected optimized cell killing) platform have demonstrated to induce antibody dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP) via bivalent targeting of a unique epitope on CD16A of NK cells and macrophages, respectively. Previously published preclinical and clinical data of ICE® molecules show promising efficacy and safety as monotherapy, as a combination therapy with immuno-oncology drugs such as PD-1/PD-L1, and in combination with adoptive NK cell transfer. AFM24 is a tetravalent bispecific epidermal growth factor (EGFR)- and CD16A-binding ICE® for enhanced targeting and killing of EGFR+ tumor cells currently in clinical development. In contrast to approved EGFR-targeting antibodies, AFM24 does not inhibit the signalling pathway of the EGFR but utilizes this receptor merely as an ”anchor” to direct NK cells and macrophages to attack tumor cells via ADCC and ADCP.MethodsADCP assays were performed with monocyte-differentiated macrophages from healthy donor PBMCs. Target tumor cells were labelled and co-cultures with macrophages, AFM24 and control antibodies. FACS analysis and live-cell imaging (IncuCyte®) were used to measure ADCP events.ResultsWe show that AFM24 enhances macrophage mediated tumor cell phagocytosis i.e., ADCP of tumor cell lines with varying levels of EGFR expression and irrespective of EGFR signaling pathway mutations. The ability of AFM24 to enhance ADCP was further demonstrated in patient-derived xenograft cell lines from various EGFR+ tumor indications. Assays with myeloid-derived suppressor cells, natural killer cells and other immune modulators were designed to address the activity of our ICE® in the context of the suppressive nature of the tumor microenvironment.ConclusionsWe report the ability of our ICE® to enhance ADCP, which might be instrumental to their efficacy, especially in tumors enriched with macrophages. In addition, due to its novel mechanism of action, AFM24 may overcome limitations of existing EGFR-targeting agents, such as dose limiting toxicity, and/or intrinsic or acquired resistance of the tumor. Consequently, AFM24 may be a potential future treatment option for a wide spectrum of patients including those that do not respond or are resistant to current EGFR-directed therapies that inhibit the signaling pathway.

scholarly journals Chemiluminescence response of human natural killer cells. I. The relationship between target cell binding, chemiluminescence, and cytolysis.

1982 ◽  
Vol 156 (2) ◽  
pp. 492-505 ◽  
Author(s):  
S L Helfand ◽  
J Werkmeister ◽  
J C Roder
Keyword(s):  
Tumor Cell ◽  
Nk Cells ◽  
Natural Killer ◽  
Tumor Cells ◽  
Cell Lines ◽  
Target Cell ◽  
Nk Cell ◽  
Target Antigen ◽  
Target Cells ◽  
Tumor Cell Lines

The binding of tumor cells or fetal fibroblasts to human natural killer (NK) cells led to a rapid chemiluminescence response within seconds of target-effector interaction. The degree of chemiluminescence was dependent on the concentration of NK-enriched lymphocytes or target cells, and plasma membrane vesicles from K562 also induced a chemiluminescence response. Mild glutaraldehyde treatment of effector cells abrogated their ability to generate chemiluminescence, whereas K562 target cells treated in the same way were almost fully able to induce a chemiluminescence response to NK-enriched lymphocytes. These results show a directionality of response with NK as the responders and tumor cells as the stimulators. A survey of eight different tumor cell lines and fetal fibroblast lines revealed a striking correlation (r greater than 0.93, P less than 0.001) between the ability of a given line to bind to NK-enriched lymphocytes, induce chemiluminescence, and to be lysed. Three differentiated sublines of K562 grown in butyrate and cloned induced little chemiluminescence compared with the K562 parent, and they were selectively resistant to NK-mediated binding and cytolysis. In addition, treatment of K562 cells with higher concentrations of glutaraldehyde for longer periods led to varying degrees of target antigen preservation, as measured in cold target competition assays and in conjugate formation. The degree of NK target antigen preservation correlated directly with the ability of the cells to induce chemiluminescence (r greater than 0.95). The degree of NK activation was also important because interferon-pretreated effectors generated more chemiluminescence upon stimulation with K562 or MeWo targets. Monocytes or granulocytes did not contribute to the chemiluminescence induced by NK-sensitive targets. Some NK-resistant tumor cell lines were sensitive to monocyte-mediated cytolysis and also induced chemiluminescence in monocytes but not NK cells. These results show that the target structures recognized by the NK cell may play a role in NK activation because the degree of chemiluminescence was directly proportional to the ability of a given target cell line to bind to the NK cell and to be lysed.

36 Molecular events regulating solid tumor cell responses to natural killer cells

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A37-A37
Author(s):  
Michal Sheffer ◽  
Constantine Mitsiades
Keyword(s):  
Tumor Cell ◽  
Antigen Presentation ◽  
Nk Cells ◽  
Tumor Cells ◽  
Cell Lines ◽  
Solid Tumor ◽  
Nk Cell ◽  
Tumor Cell Lines ◽  
Cell Responses ◽  
Genome Scale

BackgroundNatural killer (NK) cells exhibit potent activity in pre-clinical models of diverse hematologic malignancies and solid tumors and 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.MethodsTo systematically define molecular features in human tumor cells which determine their degree of sensitivity to human allogeneic NK cells, we quantified the NK cell responsiveness of hundreds of molecularly-annotated ‘DNA-barcoded’ solid tumor cell lines in multiplexed format (PRISM; Profiling Relative Inhibition Simultaneously in Mixtures approach),1 correlating cytotoxicity scores for each cell line with the CCLE transcriptional data2 (RNA-seq), to reveal genes that are associated with resistance or sensitivity to NK cells. In addition, we applied genome-scale CRISPR-based gene editing screens in several solid tumor cell lines to interrogate, at a functional level, which genes regulate tumor cell response to NK cells.3 4Figure 1 schematically depicts the two screens.ResultsBased on these orthogonal studies, NK sensitive tumor cells tend to exhibit high levels of the NK cell-activating ligand B7-H6 (NCR3LG1); low levels of the inhibitory ligand HLA-E; microsatellite instability (MSI) status; high transcriptional signature for chromatin remodeling complexes and low antigen presentation machinery genes. Treatment with HDAC inhibitor reduced the sensitivity of SW620 colon cancer cells, increased antigen presentation machinery, including HLA-E, and reduced B7-H6. Importantly, transcriptional signatures of NK cell-sensitive tumor cells correlate with immune checkpoint inhibitor resistance in clinical samples. Widespread analysis of CCLE transcriptional signatures revealed that cell lines with mesenchymal-like program tend to be more sensitive to NK cells, compared with epithelial-like cell lines. Indeed, mesenchymal tumors tend to have lower expression of antigen presentation machinery in both CCLE and TCGA.Abstract 36 Figure 1Overview of PRISM and CRISPR studies a, Schematic depiction of PRISM study. b, Schematic depiction of CRISPR screens. c, Histogram of gene fold changes (z-scores). Listed are selected genes with most prominent p-values across more than one screen.ConclusionsThis study provides a comprehensive map of mechanisms regulating tumor cell responses to NK cells, with implications for future biomarker-driven applications of NK cell immunotherapies. The integration of PRISM and CRISPR identified potential regulators of tumor cell response to NK cell, which upon further validation, may serve as biomarkers in future NK cell-based studies. Moreover, NK cells may complement T-cells, killing tumor cells that do not respond to immune checkpoint inhibitors.AcknowledgementsThis work was supported by Stand Up To Cancer (SU2C) Convergence 2.0 Grant; SU2C Phillip A. Sharp Award for Innovation in Collaboration; Claudia Adams Barr Program in Innovative Basic Cancer Research; Human Frontier Science Program Fellowship; and Leukemia and Lymphoma Society Scholar Award.ReferencesYu C, et al., High-throughput identification of genotype-specific cancer vulnerabilities in mixtures of barcoded tumor cell lines. Nat Biotechnol 2016. 34(4): p. 419–23.Ghandi M, et al. Next-generation characterization of the cancer cell line encyclopedia. Nature 2019. 569(7757): p. 503–508.Doench JG, et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol 2016. 34(2): p. 184–191.Shalem O, et al. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 2014;343(6166): p. 84–87.

JAK1 and JAK2 Modulate Tumor Cell Susceptibility To Natural Killer (NK) Cells Through Regulation Of PDL1 Expression

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3472-3472
Author(s):  
Roberto Bellucci ◽  
Allison Martin ◽  
Davide Bommarito ◽  
Kathy S. Wang ◽  
Gordon J Freeman ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Nk Cells ◽  
Tumor Cells ◽  
Cell Lines ◽  
Primary Tumor ◽  
Nk Cell ◽  
Cell Lysis ◽  
Cell Killing ◽  
Tumor Cell Lines ◽  
Ifn Γ

Abstract NK cells are the primary effectors of the innate immune response against infections pathogens and malignant transformation through their efficient cytolytic activity and cytokine secretion. Nevertheless, tumor cells have developed mechanisms to evade innate immune surveillance and the molecular basis for target resistance to NK cell-mediated lysis is not yet completely understood. To identify novel pathways that modulate tumor cell resistance to NK cells, we previously developed a cell-cell interaction based screening approach using a large sub-set of a lentiviral shRNA library containing multiple independent shRNAs targeting more than 1,000 human genes. Using this approach we found that silencing JAK1 and JAK2 significantly increased secretion of INF-γ from NK cells and increased tumor cell susceptibility to NK cell lysis. To examine the role of the JAK signaling pathway in the modulation of tumor cell susceptibility to NK lysis, we analyzed down-stream signaling pathways in several cell lines (IM9, U937, K562, RPMI, MM1S KM12BM) and primary tumor cells (AML, MM, ALL). In the absence of NK cells, silencing JAK1 or JAK2 did not affect the basal activation of STAT proteins (STAT1(pY701), STAT1(pS727), STAT3(pY705), STAT3(pS727), STAT4(pY693), STAT5(pY694), STAT6(pY641)) or AKT(pS473) and ERK1/2(pT202/pY204) or expression of activating or inhibitory ligands on tumor cells. Because JAK1 and JAK2 transduce signals downstream of the IFN-γ receptor, we hypothesized that JAKs may play a role in tumor cell evasion of NK cell activities such as cytolysis and IFN-γ secretion. To test this hypothesis we pre-incubated various tumor cell lines or primary tumor cells with activated NK supernatant or recombinant human IFN-γ. Tumor cell activation in this fashion resulted in activation of STAT1 (pSTAT1(pY701)) but none of the other STATs, ERK or AKT. As expected, STAT1 activation was blocked when JAK1 or JAK2 were silenced or inhibited by a JAK inhibitor. Silencing of STAT1 with 2 independent shRNAs also resulted in increased tumor susceptibility to NK cell cytolysis in 3 different tumor cell lines tested. To confirm that IFN-γ secreted by activated NK cells induced resistance in tumor cell targets we used a blocking IFN-γ antibody (D9D10). 10μg/ml D9D10 completely blocked STAT1 phosphorylation and in different experiments using U937, IM-9, KM12BM, MM1S and RPMI we found that D9D10 significantly increased specific NK target cell lysis by 51.8%, 78.5%, 25.1%, 20.6% and 28.5% compared to IgG1 isotype controls. Similar results were obtained whit different primary tumor cells. To determine whether IFN-γ stimulation affected expression of ligands involved in NK cell recognition of tumor cells, we analyzed the effect of activated NK supernatant or IFN-γ on the expression of MHC Class I, β2M, HLA-C, HLA-A2, NKG2D, NKP44, NKP46, NKP30 ligands using chimeric FC proteins, MICA/B, DNAM-1 ligands (CD112, CD155), 2B4 ligand (CD48), TRAIL ligands (TRAIL-R1, TRAIL-R2), Fas ligand (CD95) and PD1 ligands (PDL1, PDL2, B7H3, B7H4). The basal expression of these ligands varied among the various tumor cell lines or primary tumors tested but the only ligand that was significantly up-regulated in every tumor sample tested was PDL1. PDL1 expression by tumor cells is known to inhibit T cell immunity. To test whether increased levels of PDL1 could also inhibit NK cell killing, we co-cultured primary NK cells with U937, IM9, KM12BM, RPMI, K562, MM1S, primary MM, AML and ALL cells with or without 10μg/ml anti-PDL1 antibody (recombinant mab with Fc mutated to eliminate FcR-mediated effects). Blocking PDL1 significantly increased NK cell killing of U937, IM9, KM12BM, RPMI, MM, AML and ALL (p=0.03, p=0.02, p=0.03, p=0.005, p=0.009, p=0.03 and p=0.02 respectively). NK cell killing activity did not further increase when a JAK inhibitor was added to the co-culture. These results show that NK cell secretion of IFN-γ results in IFN receptor signaling and activation of JAK1, JAK2 and STAT1 in the tumor cell targets, followed by rapid up-regulation of PDL1 expression and increased resistance to NK cell lysis. Blockade of JAK pathway activation prevents subsequent PDL1 up-regulation resulting in increased susceptibility of tumor cells to NK cell activity suggesting that JAK pathway inhibitors may work synergistically with other immunotherapy regimens by eliminating IFN-induced PDL1 mediated immunoinhibition. Disclosures: Freeman: Bristol-Myers-Squibb/Medarex: Patents & Royalties; Roche/Genentech: Patents & Royalties; Merck: Patents & Royalties; EMD-Serrono: Patents & Royalties; Boehringer-Ingelheim: Patents & Royalties; Amplimmune: Patents & Royalties; CoStim Pharmaceuticals: Patents & Royalties; Costim Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees.

scholarly journals NK cells promote cancer immunoediting through tumor-intrinsic loss of interferon-stimulated gene expression

2020 ◽  
Author(s):  
Yung Yu Wong ◽  
Luke Riggan ◽  
Edgar Perez-Reyes ◽  
Christopher Huerta ◽  
Matt Moldenhauer ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Nk Cells ◽  
Tumor Cells ◽  
Cell Lines ◽  
Nk Cell ◽  
Metastatic Cancer ◽  
Cancer Immunoediting ◽  
Ifn Γ

AbstractNatural killer (NK) cells are innate lymphocytes that constantly patrol host tissues against transformed cells in a process known as cancer immunosurveillance. Previous evidence in mice has demonstrated that NK cell-derived IFN-γ can promote immunoevasion by sculpting the immunogenicity of developing tumors in a process known as cancer immunoediting. This process involves the elimination of highly immunogenic “unedited” tumor cells followed by the eventual escape of less immunogenic “edited” tumor cell variants that are able to escape recognition or elimination by the immune system. Here, we show that NK cell-edited fibrosarcomas decrease the expression of 17 conserved IFN-γ-inducible genes compared to unedited tumor cells. High expression of 3 of these identified genes (Psmb8, Trim21, Parp12) in human tumor samples correlates with enhanced survival in breast cancer, melanoma, and sarcoma patients. While NK cell-edited fibrosarcomas displayed resistance to IFN-γ growth suppression in vitro, functional knockouts of individual interferon stimulated genes (ISGs) were not required for outgrowth of unedited tumor cell lines in vitro and in vivo compared to complete loss of IFN signaling. Furthermore, knockout of IFN-γ-intrinsic signaling via deletion of Ifngr in edited B16 F10 and E0771 LMB metastatic cancer cell lines did not impact host survival following lung metastasis. Together, these results suggest that unedited tumors can be selected for decreased IFN-γ signaling to evade immune responses in vivo, and as a consequence, tumor-extrinsic IFN signaling may be more important for potentiating durable anti-tumor responses to advanced solid tumors.

Expression of rat complement control protein Crry on tumor cells inhibits rat natural killer cell–mediated cytotoxicity

Blood ◽  
2002 ◽  
Vol 100 (9) ◽  
pp. 3304-3310 ◽  
Author(s):  
Theresa A. Caragine ◽  
Masaki Imai ◽  
Alan B. Frey ◽  
Stephen Tomlinson
Keyword(s):  
Tumor Cell ◽  
Nk Cells ◽  
Natural Killer ◽  
Tumor Cells ◽  
Cell Lines ◽  
Nk Cell ◽  
Human Tumor ◽  
Mammary Adenocarcinoma ◽  
Tumor Cell Lines ◽  
Human Tumor Cell

Abstract Crry is a rodent membrane–bound inhibitor of complement activation and is a structural and functional analog of the human complement inhibitors decay-accelerating factor and membrane cofactor protein. We found previously that expression of rat Crry on a human tumor cell line enhances tumorigenicity in nude rats. In this study, we investigated the effect that rat Crry expressed on tumor cells has on rat cell–mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC). The expression of rat Crry on the surface of different human tumor cell lines inhibited ADCC mediated by rat natural killer (NK) cells. C3 opsonization is known to enhance NK cell–mediated cytolysis, and a potential mechanism for Crry-mediated inhibition of NK cell lysis is through Crry modulation of C3 deposition on target cells. However, the transfection of tumor cell lines with Crry enhanced their resistance to NK cell–mediated lysis in the absence of exogenous complement. The resistance of Crry-expressing tumor cells to NK cell–mediated ADCC could be reversed by treatment with anti–Crry F(ab)2. In addition, anti–Crry F(ab)2 enhanced the susceptibility of 13762 rat mammary adenocarcinoma cells (that endogenously express Crry) to ADCC mediated by allogeneic rat NK cells in the absence of added complement. We found no evidence that rat NK cells were a source of complement for target cell deposition during the in vitro cytolysis assay. These data suggest a novel function for rat Crry in tumor immune surveillance that may be unrelated to complement inhibition.

Enhancement of Tumor Cell Susceptibility to NK Cell Activity Through Inhibition of the PI3K Signaling Pathway.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2152-2152
Author(s):  
Davide Bommarito ◽  
Allison Martin ◽  
Maria-Dorothea Nastke ◽  
Jerome Ritz ◽  
Roberto Bellucci
Keyword(s):  
Tumor Cell ◽  
Nk Cells ◽  
Tumor Cells ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Nk Cell ◽  
Cell Activity ◽  
Hematologic Malignancies ◽  
Target Cells ◽  
Tumor Susceptibility

Abstract Abstract 2152 NK cells are the primary effectors of the innate immune response against cells that have undergone malignant transformation. Unlike T cells, NK cells recognize their targets through a complex array of activating and inhibitory receptors, which regulate the intensity of the NK response against individual cells. Tumor cells have developed ways to escape killing by NK cells through a variety of mechanisms that are poorly understood. Using shRNA libraries, we previously demonstrated that several common signaling pathways modulate susceptibility of various tumor cells to NK cell activity. In this study we focused on one of the PI3K sub-units (PI3KCB, p110β isoform) that was identified in this genetic screen. The PI3K pathway has been linked to diverse cellular functions including cell growth, proliferation, survival and intracellular trafficking but PI3K signaling has not previously been associated with susceptibility to NK cells. To examine the mechanisms of PI3KCB involvement in tumor susceptibility to NK cell lysis, we tested 4 tumor cell lines representing different hematologic malignancies: IM9-multiple myeloma, U937-acute myeloid leukemia, K562-chronic myeloid leukemia and Jurkat-acute T cell leukemia. Stable cell lines were established expressing 4 independent shRNAs (P1, P2, P3, P4-shRNA) and 2 irrelevant shRNAs as controls. Western blot and RT-PCR showed different silencing activity of individual PI3KCB-shRNAs with efficient silencing of PI3KCB by 3 of 4 shRNAs in IM9 (P1, P2, P4-shRNA), 2 of 4 in K562 and U937 (P1, P2-shRNA) and 2 of 4 in Jurkat (P2, P4-shRNA). No PI3KCB gene silencing or protein down regulation was observed with P3-shRNA that was used as an additional negative control. PI3KCB silencing did not affect the proliferative capacity of the 4 tumor cell lines tested over a 5 day period. When compared with irrelevant shRNAs or P3-shRNA, effective silencing of PI3KCB in IM-9, K562 and U937 induced increased IFNγ secretion when target cells were incubated with primary purified NK effector cells and 2 different human NK cell lines (NKL and NK-92). Silencing of PI3KCB in Jurkat target cells did not affect IFNγ secretion by NK cells. To determine whether increased IFNγ secretion by NK cells correlated with increased lytic activity, we incubated target cells with NK effectors overnight and assessed cytotoxicity using AnnexinV/7AAD. Lysis of IM9-PI3KCB-P1, P2 and P4-shRNA was increased by 10%-15% compared to irrelevant controls (P=0.04, P=0.04 and P=0.03 respectively); lysis of U937-PI3KCB-P1 and P2-shRNA was increased by 20% and 22% (P=0.02 and P=0.04 respectively); and lysis of K562-PI3KCB-P1 and P2-shRNA was increased by 10% and 20% (P=0.04 and P=0.04, respectively). Correlating with IFNγ assays, increased lysis was not detected for the Jurkat-PI3KCB-silenced cell lines. To define mechanisms responsible for increased susceptibility to NK cells, we examined the expression of several activating/inhibitory ligands on the membrane of PI3KCB-silenced tumor cells. Flow cytometry demonstrated increased expression of MICB (NKG2D ligand) and CD48 (2B4 ligand) in IM9-PI3KCB-KO lines; increased expression of MICA, MICB, CD48 and CD112/CD115 (DNAM-1 ligands) in K562-PI3KCB-KO lines; and increased expression of CD48, CD112 in U937-PI3KCB-KO lines. Reduced expression of MHC class I was also found in IM9 and U937 lines after PI3KCB silencing. Importantly, none of the activating/inhibitory ligands tested were modulated in Jurkat cells after PI3KCB silencing. Co-culture experiments with NK cells and blocking antibodies targeting NKG2D significantly reduced NK IFNγ secretion against IM9-PI3KCB-KO and K562-PI3KCB-KO targets when compared to controls. In contrast, DNAM-1 blocking in U937-PI3KCB-KO produced a uniform decrease of IFNγ secretion in U937-PI3KCB-KO and controls suggesting that in this target, modulation of other NK ligands could also be involved. These data demonstrate that the beta sub-unit of PI3K (PI3KCB) can modulate tumor susceptibility in different hematologic malignancies and that this effect is, at least in part, due to modulation of several activating/inhibitory ligands. These findings identify a new and important role of PI3KCB in modulating tumor cell susceptibility to NK cells and open the way to future combined target immunotherapies. Disclosures: No relevant conflicts of interest to declare.

Lenalidomide Strongly Enhances Natural Killer (NK) Cell Mediated Antibody-Dependent Cellular Cytotoxicity (ADCC) of Rituximab Treated Non-Hodkin’s Lymphoma Cell Lines In Vitro.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3714-3714 ◽  
Author(s):  
Lei Wu ◽  
Peter Schafer ◽  
George Muller ◽  
David Stirling ◽  
J. Blake Bartlett
Keyword(s):  
Nk Cells ◽  
Tumor Cells ◽  
Cell Lines ◽  
Nk Cell ◽  
Dependent Manner ◽  
Activation Markers ◽  
Early Results ◽  
Ifn Γ ◽  
Dose Dependent

Abstract Lenalidomide (Revlimid® is approved for the treatment of transfusion-dependent patients with anemia due to low- or intermediate-1-risk MDS associated with a del 5q cytogenetic abnormality with or without additional cytogenetic abnormalities, and in combination with dexamethasone is for the treatment of multiple myeloma patients who have received at least one prior therapy. Encouraging early results suggest a potential for clinical efficacy in B cell non-Hodgkin’s lymphoma (NHL). Potential mechanisms of action include anti-angiogenic, anti-proliferative and immunomodulatory activities. Lenalidomide has been shown to enhance Th1-type cytokines and T cell and NK cell activation markers in patients with advanced cancers. Furthermore, lenalidomide has been shown to enhance rituximab-mediated protection in a SCID mouse lymphoma model in vivo. We have utilized an in vitro ADCC system to assess the ability of lenalidomide to directly enhance human NK cell function in response to therapeutic antibodies, such as rituximab (chimeric anti-CD20 mAb). Isolated NK cells produced little or no IFN-γ in response to IgG and/or IL-2 or IL-12. However, pre-treatment of NK cells with lenalidomide greatly enhanced IFN-γ production by NK cells in a dose-dependent manner. In a functional ADCC assay, NHL cell lines (Namalwa, Farage & Raji) were pre-coated with rituximab and exposed to NK cells pre-treated with lenalidomide in the presence of either exogenous IL-2 or IL-12. After 4 hours in culture the viability of the tumor cells was assessed. Lenalidomide consistently and synergistically increased the killing of tumor cells in a dose-dependent manner and up to >4-fold compared to rituximab alone. Rituximab alone had only a small effect in this model and there was no killing of cells in the absence of rituximab. The presence of either exogenous IL-2 or IL-12 was required to see enhanced killing by lenalidomide. In cancer patients lenalidomide has been shown to increase serum IL-12 levels and is also known to induce IL-2 production by T cells in vitro. Potential mechanisms for enhanced ADCC include increased signaling through NK FCγ receptors and/or IL-2 or IL-12 receptors. However, we found that these receptors are unaffected by lenalidomide, although downstream effects on NK signaling pathways are likely and are being actively investigated. In conclusion, we have shown that lenalidomide strongly enhances the ability of rituximab to induce ADCC mediated killing of NHL cells in vitro. This provides a strong rationale for combination of these drugs in patients with NHL and CLL.

543 Natural killer cells restrict the growth of liver metastases in nude hosts

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A579-A579
Author(s):  
Alexandra Quackenbush ◽  
Pepper Schedin
Keyword(s):  
Breast Cancer ◽  
Tumor Cell ◽  
Liver Metastases ◽  
Nk Cells ◽  
Natural Killer ◽  
Cancer Patients ◽  
Tumor Cells ◽  
Mammary Tumor ◽  
Nk Cell ◽  
Cell Depletion

BackgroundCancer patients with liver metastases have limited treatment options, especially as only 15–20% are eligible for curative-intent surgical resection.1 Unfortunately, liver metastases also seem to be poorly responsive to immune checkpoint inhibitors (ICI)].2 3 It could be that the unique immunological hallmarks of the liver, including resident macrophages and significant numbers of NK and NKT cells, create a tumor microenvironment that is best suited to alternative forms of immunotherapy that do not rely exclusively on ICI.MethodsWe investigated how the presence of T, natural killer (NK), and NKT cells impact overt liver metastases using a model in which tumor cells are delivered to the liver via intraportal injection to hosts that were either wiltype, nude, or nude with NK-depletion. NK cell depletion was achieved via administration of anti-asialo GM1 antibody 2 days before tumor cell injection and for the duration of the experiment until endpoint at 6 weeks post tumor cell injection, with NK cell depletion confirmed by flow cytometry. Tumors were assessed histologically.ResultsUsing the portal vein model in female nulliparous mice, overt liver metastasis incidence was about 30% across 2 different mammary tumor cell lines. The incidence rose to 80–100% when tumor cells were delivered to hosts in the post-wean window (referred to as involution hosts), mirroring increased breast cancer metastasis to the liver observed in postpartum breast cancer patients.4 Conversely, when tumor cells were delivered to nude hosts, either nulliparous or involution stages, the incidence of metastases dropped to 0–10%. Importantly, tumor cells injected into the mammary gland of nude mice grew robustly with 100% take. Nude hosts lack T cells and NKT cells; however, NK cells are present. Furthermore, the liver is enriched for NK cells, whilst the mammary gland has few NK cells.5 We hypothesized that NK cells, when in the background of T- and NKT-cell depletion (i.e. nude host), restrict outgrowth of mammary tumor cells in the liver. Six weeks after portal vein injection of mammary tumor cells to nude hosts we find increased incidence of metastasis in the NK-depleted group compared to isotype control, as well as increased number of metastases per mouse.ConclusionsOur data suggest that NK cells play an important role in controlling liver metastases in nude hosts, and that NK activity in wild type hosts is insufficient to control liver metastases. Increasing NK cell cytotoxic activity could be an effective immunotherapy strategy to control liver metastases.ReferencesNordlinger B, Sorbye H, Glimelius B, Poston GJ, Schlag PM, Rougier P, Bechstein WO, Primrose JN, Walpole ET, Finch-Jones M, et al: Perioperative FOLFOX4 chemotherapy and surgery versus surgery alone for resectable liver metastases from colorectal cancer (EORTC 40983): long-term results of a randomised, controlled, phase 3 trial. Lancet Oncol 2013;14(12):1208–1215.Bilen MA, Shabto JM, Martini DJ, Liu Y, Lewis C, Collins H, Akce M, Kissick H, Carthon BC, Shaib WL, et al: Sites of metastasis and association with clinical outcome in advanced stage cancer patients treated with immunotherapy. BMC Cancer 2019;19(1):857.Topalian SL, Hodi FS, Brahmer JR, Gettinger SN, Smith DC, McDermott DF, Powderly JD, Sosman JA, Atkins MB, Leming PD, et al: Five-year survival and correlates among patients with advanced melanoma, renal cell carcinoma, or non-small cell lung cancer treated with nivolumab. JAMA Oncol 2019.Goddard ET, Hill RC, Nemkov T, D’Alessandro A, Hansen KC, Maller O, Mongoue-Tchokote S, Mori M, Partridge AH, Borges VF, et al: The rodent liver undergoes weaning-induced involution and supports breast cancer metastasis. Cancer Discov 2017;7(2):177–187.Shi FD, Ljunggren HG, La Cava A, Van Kaer L. Organ-specific features of natural killer cells. Nat Rev Immunol 2011;11(10):658–671.

scholarly journals Trispecific Antibodies for Selective CD16A-Directed NK-Cell Engagement in Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4513-4513 ◽  
Author(s):  
Thorsten Gantke ◽  
Michael Weichel ◽  
Uwe Reusch ◽  
Kristina Ellwanger ◽  
Ivica Fucek ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Nk Cells ◽  
Tumor Cells ◽  
Cell Lines ◽  
Effector Cell ◽  
Nk Cell ◽  
Surface Antigens ◽  
Model System ◽  
Effector Cells ◽  
Dual Targeting

Abstract Development of antibody scaffolds to directly engage cytotoxic effector cells such as T-cells for therapeutic applications is limited by the scarcity of surface antigens which are expressed exclusively on tumor cells and show limited or no expression on non-malignant cells. We have therefore designed a novel antibody format to selectively retarget effector cell cytotoxicity to tumor cells co-expressing two surface antigens. NK-cells play an important role in the innate immune response to multiple myeloma (MM) and are known to contribute to the efficacy of novel therapeutics. We, therefore, utilized a MM-based model system to generate proof-of concept data demonstrating antibody-mediated NK-cell retargeting to cell lines co-expressing two MM-expressed surface antigens with increased selectivity ('dual-targeting'). B-cell maturation antigen (BCMA/CD269) is widely considered to be a promising target antigen for antibody-based therapies of MM due to its almost universal expression on patient myeloma cells and its restricted surface expression on cells outside of the haematological lineage. However, low levels of expression on healthy tissue, including skin, has been reported, which could account for potential side effects associated with BCMA-targeted antibody therapies due to effector cell activation in these organs. To increase selectivity of antibody-induced, effector cell-mediated cytotoxicity towards malignant tissue, we developed a trispecific antibody format capable of selectively engaging NK-cells through bivalent binding to CD16A (FcγRIIIa) and monovalent binding to both BCMA and CD200, a second MM-expressed surface antigen found in the majority of MM patients. Using an in vitro model system, we demonstrated that binding to BCMA+/CD200+ cell lines and the resulting increase in avidity leads to preferential lysis of antigen double-positive cells compared with antigen single-positive cells. These data suggest that dual-targeting may increase the therapeutic window compared to approaches targeting only one antigen, thereby improving safety of BCMA-directed antibody therapeutics for MM. In addition to the MM-based model system used here, the novel trispecific antibody scaffolds we have developed may be adapted to alternative target combinations within MM or in other tumor indications. Moreover, they could be used to target phenotypically distinct tumor cell clones to induce deeper and more prolonged antitumor responses. Consequently, dual-targeting of effector cells to tumors using the described antibody technology could also be applied to increase safety of T-cell engaging antibodies in the absence of exclusively tumor-expressed target antigens. Disclosures Gantke: Affimed GmbH: Employment. Weichel:Affimed GmbH: Employment. Reusch:Affimed: Employment, Patents & Royalties: Patents. Ellwanger:Affimed GmbH: Employment. Fucek:Affimed GmbH: Employment. Griep:AbCheck s.r.o.: Employment. Molkenthin:AbCheck s.r.o.: Employment. Kashala:Affimed Inc.: Employment. Treder:Affimed: Employment.

Tumoricidal Potential of Native Human Blood Dendritic Cells: Direct Tumor Cell Killing and Activation of NK Cell-Mediated Cytotoxicity.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 449-449
Author(s):  
Marc Schmitz ◽  
Senming Zhao ◽  
Yvonne Deuse ◽  
Knut Schaekel ◽  
Martin Bornhaeuser ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Nk Cells ◽  
Cell Lines ◽  
Human Blood ◽  
Nk Cell ◽  
Tnf Alpha ◽  
Tumor Cell Lines ◽  
Specific Lysis ◽  
Tumoricidal Activity ◽  
Ifn Gamma

Abstract Dendritic cells (DCs) are characterized by their unique capacity for primary T cell activation, providing the opportunity of DC-based cancer vaccination protocols. Recently, we defined a novel major subset of human blood DCs by using the monoclonal antibody M-DC8 which recognizes a carbohydrate modification of P selectin glycoprotein ligand-1 (PSGL-1) selectively expressed on these cells (Immunity2002;17:289-301). In addition to a marked capacity to activate tumor-reactive cytotoxic T cells M-DC8+ DCs efficiently mediate antibody-dependent cytotoxicity (Blood;2002;100:1502-1504). In the present study, we analyzed the capacity of M-DC8+ DCs to kill tumor cells in the absence of antibodies and to enhance the tumor-directed cytotoxicity of NK cells. To determine whether M-DC8+ DCs exhibit tumoricidal activity, DCs were isolated at high purity (>93%) from the blood of healthy donors by immunomagnetic separation. These cells were cultured for 6 h in the presence or absence of 200 U/ml interferon (IFN)-gamma. Subsequently, DCs were coincubated with four chromium-labeled tumor cell lines and two normal cell lines for 18 h. Whereas unstimulated DCs demonstrated only moderate tumor-directed cytotoxicity (specific lysis: 7–13%), IFN-gamma-stimulated M-DC8+ DCs displayed potent killing of each of these tumor cell lines (specific lysis: 27–35%). Only a marginal cytotoxic effect was seen when normal human cells such as lung fibroblasts or endothelial cells were used as targets. When evaluating the cytotoxic effector mechanisms FACS analysis and ELISA assays revealed that IFN-gamma-stimulated M-DC8+ DCs secreted a high amount of tumor necrosis factor (TNF)-alpha induced by direct cell-to-cell contact with the different tumor cell lines. This effect was already observed after 3 h of cocultivation. Interestingly, no significant induction of TNF-alpha was detected during contact of M-DC8+ DCs with the normal human cell lines. These results suggest that tumor-associated surface molecules are important for the observed increase of TNF-alpha production in M-DC8+ DCs. Inhibition experiments with neutralizing antibodies clearly demonstrated that tumor cell-induced TNF-alpha play an important role in tumor-directed cytotoxicity mediated by M-DC8+ DCs. To investigate whether M-DC8+ DCs enhance the tumoricidal activity of NK cells freshly isolated DCs were cultured for 6 h in the presence or absence of IFN-gamma. Thereafter, DCs were coincubated with highly enriched (>90%) NK cells. The cytotoxic potential of the stimulated NK cells was tested towards various tumor cell lines in a 4 h chromium release assay. We observed a two- to threefold increase of NK cell-mediated cytotoxicity towards all analyzed tumor cell lines by IFN-gamma-stimulated M-DC8+ DCs. In addition, transwell experiments demonstrated that this triggering effect was mainly dependent on cell-to-cell contact. In conclusion, our data provide evidence that a major subpopulation of circulating human blood DCs exhibits efficient tumoricidal activity and clearly enhances NK cell-mediated tumor-directed cytotoxicity. The capacity of DCs to induce tumor-specific T cell responses and to kill tumor cells either directly or by activating NK cells points to the pivotal role of DCs in triggering the innate and adaptive immune response against tumors.

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