Efficient Lysis of Myeloma Cells by CD16 Positive Effector Cells with Recombinant Single Chain Bispecific Antibodies Targeting HM1.24.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2752-2752
Author(s):  
Matthias Peipp ◽  
Claudia Ehlert ◽  
Matthias Staudinger ◽  
Joerg Bruenke ◽  
Georg Fey ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Bispecific Antibody ◽  
Lytic Activity ◽  
High Dose ◽  
Antigen Specificity ◽  
Single Chain ◽  
Myeloma Cells ◽  
Effector Cells ◽  
Single Chain Fv

Abstract Different therapeutic options are available for the treatment of multiple myeloma patients, but conventional chemotherapy often is not able to completely eradicate the tumor. After high dose chemotherapy, complete remission with only minimal residual disease could be achieved in many patients, but additional targeted strategies may help to eradicate residual cells and improve prognosis. Therapy with monoclonal antibodies is well established in CD20 positive B-cell lymphomas, but in contrast not many suitable target antigens are defined / expressed by multiple myeloma cells. HM1.24 a surface molecule expressed on terminally differentiated B-lineage cells represents a promising candidate antigen that is overexpressed on multiple myeloma cells. Here the development of a recombinant bispecific single chain Fv HM1.24 × CD16 antibody (tandem format) with novel features is presented. The HM1.24×CD16 bispecific antibody was expressed in 293T cells and purified to homogeneity by two-step affinity chromatography. Binding to HM1.24 and CD16 was demonstrated by immunofluorescence staining and flow cytometry with antigen positive and negative cells. The lytic activity of the bispecific HM1.24×CD16 scFv was evaluated in an antibody-dependent cellular cytotoxicity (ADCC) assay with different myeloma cell lines (RPMI 8226, INA-6, U266, JK6L) and primary patient derived cells as targets. Mononuclear cells (MNC), isolated from healthy donors served as effector cells. The bispecific HM1.24×CD16 scFv mediated efficient lysis of all tested cell lines at concentrations as low as 1 nM. In direct comparison to an HM1.24-IgG1 control molecule, the recombinant bispecific antibody demonstrated superior lytic activity at saturating concentrations and showed significant enhanced killing capacity. In conclusion, the recombinant bispecific HM1.24×CD16 retained its antigen specificity and demonstrated efficient lytic activity against patient-derived tumor cell lines and primary material. These results indicate that the bispecific antibody may be promising as a new therapeutic strategy in multiple myeloma.

scholarly journals Inhibition of Aurora-Kinases for Tailored Risk-Adapted Treatment of Multiple Myeloma

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 248-248
Author(s):  
Dirk Hose ◽  
Thierry Rème ◽  
Tobias Meißner ◽  
Jérôme Moreaux ◽  
Anja Seckinger ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Chromosomal Aberrations ◽  
Genetic Instability ◽  
Kinase Inhibitor ◽  
Aurora Kinase ◽  
Myeloma Cell ◽  
High Dose ◽  
Aurora A ◽  
Myeloma Cells

Abstract BACKGROUND. At the time of diagnosis, myeloma cells are characterized by a low proliferation rate that increases in relapse. Presence of proliferation correlates with adverse prognosis. At the same time, myeloma cells harbor a high median number of chromosomal aberrations, often associated with genetic instability. Cellular proliferation and genetic instability in turn have been associated with Aurora-kinase expression in several cancer entities, including multiple myeloma. PATIENTS AND METHODS. Expression of Aurora-A, -B and -C was assessed using Affymetrix DNA-microarrays in 784 samples including two independent sets of 233 and 345 CD138-purified myeloma cells from previously untreated patients. Chromosomal aberrations were assessed by comprehensive iFISH using a set of probes for the chromosomal regions 1q21, 6q21, 8p21, 9q34, 11q23, 11q13, 13q14.3, 14q32, 15q22, 17p13, 19q13, 22q11, as well as the translocations t(4;14)(p16.3;q32.3) and t(11;14) (q13;q32.3). Proliferation of primary myeloma cells (n=67) was determined by propidium iodine staining. The effect of the clinical Aurora-kinase inhibitor VX680 on proliferation of 20 human myeloma cell lines and survival of 5 primary myeloma cell-samples was tested. RESULTS. We found Aurora-A and -B to be expressed at varying frequencies in primary myeloma cells of different patient-cohorts, including 23% for Aurora A in our first cohort of patients treated with high dose therapy (see figure shown below). Aurora-C expression was found in testis-samples only. Myeloma cell-samples with detectable Aurora-A expression show a significantly higher proliferation rate, whereas the number of chromosomal aberrations (aneuploidy) is not higher compared to myeloma-cells with absent Aurora-A expression. The same holds true for subclonal aberrations (i.e. genetic instability). The Aurora-kinase inhibitor VX680 induces apoptosis in all myeloma cell lines and primary myeloma cell-samples tested. Presence of Aurora-A expression delineates significantly inferior event-free and overall survival in two independent cohorts of patients undergoing high-dose chemotherapy and autologous stem cell transplantation. This observation is independent of conventional prognostic factors, i.e. serum-ß2-microglobulin or ISS-stage. CONCLUSION. Aurora-kinase inhibitors (including VX680 tested here) are very active on myeloma cell lines as well as primary myeloma cells and represent a promising weapon in the therapeutic arsenal against multiple myeloma. Gene expression profiling allows the assessment of Aurora-kinase expression and thus in turn a tailoring of treatment to patients expressing Aurora-A associated with adverse prognosis. Figure Figure

scholarly journals Highly Effective Recombinant Format of a Humanized IgG-like Bispecific Antibody for Cancer Immunotherapy with Retargeting of Lymphocytes to Tumor Cells

2007 ◽  
Vol 282 (38) ◽  
pp. 27659-27665 ◽  
Author(s):  
Ryutaro Asano ◽  
Yasuhiro Watanabe ◽  
Hiroko Kawaguchi ◽  
Hidesuke Fukazawa ◽  
Takeshi Nakanishi ◽  
...  
Keyword(s):  
Antitumor Activity ◽  
Growth Inhibition ◽  
Mammalian Cells ◽  
Bispecific Antibody ◽  
Cell Phenotype ◽  
Inhibition Assay ◽  
Single Chain ◽  
Lymphokine Activated Killer Cells ◽  
Effector Cells ◽  
Single Chain Fv

We previously reported the marked in vitro and in vivo antitumor activity of hEx3, a humanized diabody (small recombinant bispecific antibody) with epidermal growth factor receptor (EGFR) and CD3 retargeting. Here, we fabricated a tetravalent IgG-like bispecific antibody with two kinds of single-chain Fv (scFv), i.e. humanized anti-EGFR scFv and anti-CD3 scFv, that contains the same four variable domains as hEx3, on the platform of human IgG1 (hEx3-scFv-Fc). hEx3-scFv-Fc prepared from mammalian cells showed specific binding to both EGFR and CD3 target antigens. At one-thousandth (0.1–100 fmol/ml) of the dose of normal hEx3, hEx3-scFv-Fc showed intense cytotoxicity to an EGFR-positive cell line in a growth-inhibition assay using lymphokine-activated killer cells with the T-cell phenotype (T-LAK cells). The enhanced antitumor effect was more clearly observed when peripheral blood mononuclear cells (PBMCs) were used as effector cells, indicating the utility of IgG-like fabrication. These results suggested that the intense antitumor activity is attributable to the multivalency and the presence of the fused human Fc, a hypothesis that was supported by the results of flow cytometry, PBMC proliferation assay, and protein kinase inhibition assay. Furthermore, the growth inhibition effects of hEx3-scFv-Fc were considerably superior to those of the approved therapeutic antibody, cetuximab, which recognizes the same EGFR antigen even when using PBMCs as effector cells. The high potency of hEx3-scFv-Fc may translate into improved antitumor therapy and lower costs of production because of the smaller doses needed.

Bone Morphogenic Protein 6: A Prognostic Factor Expressed by Normal Plasma Cells and Multiple Myeloma Cells Inhibiting Their Proliferation and Angiogenesis Induction

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2701-2701
Author(s):  
Anja Seckinger ◽  
Tobias Meißner ◽  
Jérôme Moreaux ◽  
Hartmut Goldschmidt ◽  
Axel Benner ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Overall Survival ◽  
Cell Lines ◽  
Plasma Cells ◽  
Myeloma Cell ◽  
High Dose Chemotherapy ◽  
High Dose ◽  
Myeloma Cells

Abstract BACKGROUND: Pathogenesis of multiple myeloma is partly attributed to an aberrant expression of proliferation-, pro-angiogenic and bone-metabolism modifying factors by malignant plasma-cells. AIM. Given the long and variable time-span from first diagnosis of early-stage plasma-cell dyscrasias to overt myeloma and the low proliferation rate of malignant plasma-cells, we hypothesize these to concomitantly express a novel class of anti-proliferative factors of potential prognostic relevance. Here, bone morphogenic proteins (BMPs) represent possible candidates, as they inhibit proliferation, stimulate bone formation, and have an impact on the survival of cancer patients. PATIENTS AND METHODS. We assessed expression of BMPs and its receptors by Affymetrix DNA-microarrays (n=434) including CD138-purified primary myeloma-cell-samples, normal bone-marrow plasma-cell-samples, polyclonal plasmoblasts-samples, human myeloma-cell-lines (HMCL), and whole bone-marrow. Presence and differential gene expression was determined by PANP-algorithm and empirical Bayes statistics. Event-free (EFS) and overall survival (OAS) were investigated for the 168 patients undergoing high-dose chemotherapy (HM-group) using Cox’s proportional hazard model. Findings were validated using the same strategy on an independent group of 345 patients from the Arkansas-group. For validation, quantitative real-time PCR and flow cytometry were performed. In vitro induction of angiogenesis was assessed using the AngioKit-assay. Effect of BMP6 on proliferation of HMCL was assessed by 3H-thymidine uptake. RESULTS. BMP6 is the only BMP expressed by normal- (13/14 samples) and malignant plasma-cells (228/233 samples). It is significantly lower expressed in proliferating non-malignant plasmablastic cells and human myeloma cell-lines. In vitro, BMP6 significantly inhibits proliferation of myeloma-cell-lines with an IC50 ranged from 0.08–2.15μg/ml, survival of primary myeloma-cells, and in vitro tubule formation down to the level of the negative control. High BMP6-expression in malignant plasma cells delineates significantly superior overall-survival for patients undergoing high-dose chemotherapy in both independent series of patients (n=168, P=.02 and n=345, P=.03, respectively, see below). CONCLUSION. With BMP6 we report for the first time the autocrine expression of a prognostically relevant anti-angiogenic and anti-proliferative factor and its receptors by normal and malignant plasma-cells. Figure Figure

scholarly journals An inhibitor of the EGF receptor family blocks myeloma cell growth factor activity of HB-EGF and potentiates dexamethasone or anti–IL-6 antibody-induced apoptosis

Blood ◽  
2004 ◽  
Vol 103 (5) ◽  
pp. 1829-1837 ◽  
Author(s):  
Karène Mahtouk ◽  
Michel Jourdan ◽  
John De Vos ◽  
Catherine Hertogh ◽  
Geneviève Fiol ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Growth Factor ◽  
Cell Growth ◽  
Cell Lines ◽  
Stromal Cells ◽  
Egf Receptor ◽  
Myeloma Cell ◽  
Factor Activity ◽  
Myeloma Cells ◽  
Induced Apoptosis

Abstract We previously found that some myeloma cell lines express the heparin-binding epidermal growth factor–like growth factor (HB-EGF) gene. As the proteoglycan syndecan-1 is an HB-EGF coreceptor as well as a hallmark of plasma cell differentiation and a marker of myeloma cells, we studied the role of HB-EGF on myeloma cell growth. The HB-EGF gene was expressed by bone marrow mononuclear cells in 8 of 8 patients with myeloma, particularly by monocytes and stromal cells, but not by purified primary myeloma cells. Six of 9 myeloma cell lines and 9 of 9 purified primary myeloma cells expressed ErbB1 or ErbB4 genes coding for HB-EGF receptor. In the presence of a low interleukin-6 (IL-6) concentration, HB-EGF stimulated the proliferation of the 6 ErbB1+ or ErbB4+ cell lines, through the phosphatidylinositol 3-kinase/AKT (PI-3K/AKT) pathway. A pan-ErbB inhibitor blocked the myeloma cell growth factor activity and the signaling induced by HB-EGF. This inhibitor induced apoptosis of patients'myeloma cells cultured with their tumor environment. It also increased patients' myeloma cell apoptosis induced by an anti–IL-6 antibody or dexamethasone. The ErbB inhibitor had no effect on the interaction between multiple myeloma cells and stromal cells. It was not toxic for nonmyeloma cells present in patients' bone marrow cultures or for the growth of hematopoietic progenitors. Altogether, these data identify ErbB receptors as putative therapeutic targets in multiple myeloma.

scholarly journals Inhibition of aurora kinases for tailored risk-adapted treatment of multiple myeloma

Blood ◽  
2009 ◽  
Vol 113 (18) ◽  
pp. 4331-4340 ◽  
Author(s):  
Dirk Hose ◽  
Thierry Rème ◽  
Tobias Meissner ◽  
Jérôme Moreaux ◽  
Anja Seckinger ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Chromosomal Aberrations ◽  
Cellular Proliferation ◽  
Kinase Inhibitor ◽  
Therapeutic Option ◽  
Aurora Kinase ◽  
Myeloma Cell ◽  
High Dose ◽  
Aurora A ◽  
Myeloma Cells

Abstract Genetic instability and cellular proliferation have been associated with aurora kinase expression in several cancer entities, including multiple myeloma. Therefore, the expression of aurora-A, -B, and -C was determined by Affymetrix DNA microarrays in 784 samples including 2 independent sets of 233 and 345 CD138-purified myeloma cells from previously untreated patients. Chromosomal aberrations were assessed by comprehensive interphase fluorescence in situ hybridization and proliferation of primary myeloma cells by propidium iodine staining. We found aurora-A and -B to be expressed at varying frequencies in primary myeloma cells of different patient cohorts, but aurora-C in testis cell samples only. Myeloma cell samples with detectable versus absent aurora-A expression show a significantly higher proliferation rate, but neither a higher absolute number of chromosomal aberrations (aneuploidy), nor of subclonal aberrations (chromosomal instability). The clinical aurora kinase inhibitor VX680 induced apoptosis in 20 of 20 myeloma cell lines and 5 of 5 primary myeloma cell samples. Presence of aurora-A expression delineates significantly inferior event-free and overall survival in 2 independent cohorts of patients undergoing high-dose chemotherapy, independent from conventional prognostic factors. Using gene expression profiling, aurora kinase inhibitors as a promising therapeutic option in myeloma can be tailoredly given to patients expressing aurora-A, who in turn have an adverse prognosis.

Expression of functional interleukin-15 receptor and autocrine production of interleukin-15 as mechanisms of tumor propagation in multiple myeloma

Blood ◽  
2000 ◽  
Vol 95 (2) ◽  
pp. 610-618 ◽  
Author(s):  
Inge Tinhofer ◽  
Ingrid Marschitz ◽  
Traudl Henn ◽  
Alexander Egle ◽  
Richard Greil
Keyword(s):  
Multiple Myeloma ◽  
Cell Survival ◽  
Cell Lines ◽  
Plasma Cells ◽  
Constitutive Expression ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Cytotoxic Treatment ◽  
Interleukin 15 ◽  
Induced Apoptosis

Interleukin-15 (IL-15) induces proliferation and promotes cell survival of human T and B lymphocytes, natural killer cells, and neutrophils. Here we report the constitutive expression of a functional IL-15 receptor (IL-15R) in 6 of 6 myeloma cell lines and in CD38high/CD45low plasma cells belonging to 14 of 14 patients with multiple myeloma. Furthermore, we detected IL-15 transcripts in all 6 myeloma cell lines, and IL-15 protein in 4/6 cell lines and also in the primary plasma cells of 8/14 multiple myeloma patients. Our observations confirm the existence of an autocrine IL-15 loop and point to the potential paracrine stimulation of myeloma cells by IL-15 released from the cellular microenvironment. Blocking autocrine IL-15 in cell lines increased the rate of spontaneous apoptosis, and the degree of this effect was comparable to the pro-apoptotic effect of depleting autocrine IL-6 by antibody targeting. IL-15 was also capable of substituting for autocrine IL-6 in order to promote cell survival and vice versa. In short-term cultures of primary myeloma cells, the addition of IL-15 reduced the percentage of tumor cells spontaneously undergoing apoptosis. Furthermore, IL-15 lowered the responsiveness to Fas-induced apoptosis and to cytotoxic treatment with vincristine and doxorubicin but not with dexamethasone. These data add IL-15 to the list of important factors promoting survival of multiple myeloma cells and demonstrate that it can be produced and be functionally active in an autocrine manner.

Reduced Response of IRE1α/Xbp-1 Signaling Pathway to Bortezomib Contributes to Drug Resistance in Multiple Myeloma Cells

2016 ◽  
Vol 103 (3) ◽  
pp. 261-267 ◽  
Author(s):  
Xiaoxuan Xu ◽  
Junru Liu ◽  
Beihui Huang ◽  
Meilan Chen ◽  
Shiwen Yuan ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Resistance Mechanism ◽  
Proteasome Inhibition ◽  
Myeloma Cells ◽  
Potential Marker ◽  
Rpmi 8226 ◽  
Basic Level

Purpose Proteasome inhibition with bortezomib eliminates multiple myeloma (MM) cells by partly disrupting unfolded protein response (UPR). However, the development of drug resistance limits its utility and resistance mechanism remains controversial. We aimed to investigate the role of IRE1α/Xbp-1 mediated branch of the UPR in bortezomib resistance. Methods The expression level of Xbp-1s was measured in 4 MM cell lines and correlated with sensitivity to bortezomib. LP1 and MY5 cells with different Xbp-1s level were treated with bortezomib; then pivotal UPR regulators were compared by immunoblotting. RPMI 8226 cells were transfected with plasmid pEX4-Xbp-1s and exposed to bortezomib; then apoptosis was determined by immunoblotting and flow cytometry. Bortezomib-resistant myeloma cells JJN3.BR were developed and the effect on UPR signaling pathway was determined. Results By analyzing 4 MM cell lines, we found little correlation between Xbp-1s basic level and bortezomib sensitivity. Bortezomib induced endoplasmic reticulum stress-initiated apoptosis via inhibiting IRE1α/Xbp-1 pathway regardless of Xbp-1s basic level. Exogenous Xbp-1s reduced cellular sensitivity to bortezomib, suggesting the change of Xbp-1s expression, not its basic level, is a potential marker of response to bortezomib in MM cells. Furthermore, sustained activation of IRE1α/Xbp-1 signaling pathway in JJN3.BR cells was identified. Conclusions Our data indicate that reduced response of IRE1α/Xbp-1 signaling pathway to bortezomib may contribute to drug resistance in myeloma cells.

scholarly journals Iron Causes Lipid Oxidation and Inhibits Proteasome Function in Multiple Myeloma Cells: A Proof of Concept for Novel Combination Therapies

Cancers ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 970 ◽  
Author(s):  
Jessica Bordini ◽  
Federica Morisi ◽  
Fulvia Cerruti ◽  
Paolo Cascio ◽  
Clara Camaschella ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Cell Lines ◽  
Iron Toxicity ◽  
Ammonium Citrate ◽  
Ferric Ammonium Citrate ◽  
Ferric Carboxymaltose ◽  
High Dose ◽  
Iron Loading

Adaptation to import iron for proliferation makes cancer cells potentially sensitive to iron toxicity. Iron loading impairs multiple myeloma (MM) cell proliferation and increases the efficacy of the proteasome inhibitor bortezomib. Here, we defined the mechanisms of iron toxicity in MM.1S, U266, H929, and OPM-2 MM cell lines, and validated this strategy in preclinical studies using Vk*MYC mice as MM model. High-dose ferric ammonium citrate triggered cell death in all cell lines tested, increasing malondialdehyde levels, the by-product of lipid peroxidation and index of ferroptosis. In addition, iron exposure caused dose-dependent accumulation of polyubiquitinated proteins in highly iron-sensitive MM.1S and H929 cells, suggesting that proteasome workload contributes to iron sensitivity. Accordingly, high iron concentrations inhibited the proteasomal chymotrypsin-like activity of 26S particles and of MM cellular extracts in vitro. In all MM cells, bortezomib-iron combination induced persistent lipid damage, exacerbated bortezomib-induced polyubiquitinated proteins accumulation, and triggered cell death more efficiently than individual treatments. In Vk*MYC mice, addition of iron dextran or ferric carboxymaltose to the bortezomib-melphalan-prednisone (VMP) regimen increased the therapeutic response and prolonged remission without causing evident toxicity. We conclude that iron loading interferes both with redox and protein homeostasis, a property that can be exploited to design novel combination strategies including iron supplementation, to increase the efficacy of current MM therapies.

Induction of Multiple Myeloma-Specific Cytotoxic T Lymphocytes Using HLA-A2.1-Specific CD19 and CD20 Peptides.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2477-2477
Author(s):  
Jooeun Bae ◽  
Jeff A. Martinson ◽  
Hans G. Klingemann ◽  
Steven Treon ◽  
Kenneth C. Anderson ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Lymphocytes ◽  
Cytotoxic Activity ◽  
Cell Lines ◽  
Cytotoxic T Lymphocytes ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Specific Ctls ◽  
Ifn Γ ◽  
Hla A2.1

Abstract We have identified novel CD19 and CD20 antigen-derived HLA-A2.1-specific immunogenic peptides, CD19150–158 (KLMSPKLYV) and CD20188–196 (SLFLGILSV), for generating cytotoxic T lymphocytes (CTLs) against malignant B-cell diseases. Initial testing showed that the CTLs displayed antigen-specific and HLA-A2.1-restriced cytotoxic activity against both Burkitt’s lymphoma and chronic lymphoid leukemia cell lines. The observed cytotoxic activity of the CTLs was shown to be specific to the CD19150–158 or the CD20188–196 peptides. Additionally, the CTLs displayed a distinct phenotype (majority CD69+/CD45RO+) along with a significant (p<0.05) increase in cell proliferation and IFN-γ release following re-stimulation with HLA-A2.1+/CD19+/CD20+ tumor cell lines. Based on emerging information that clonogenic myeloma cells express CD19 and/or CD20, we evaluated the activity of the CD19 and CD20 peptide specific-CTLs against several multiple myeloma cell lines. Five of 10 myeloma cell lines evaluated were HLA-A2.1-positive and expressed both CD19 and CD20 antigens. CD19 peptide specific-CTLs generated from normal donors were able to specifically lyse CD19+/HLA-A2.1+ MM cell lines (30% lysis; 10:1 E:T ratio) but did not lyse CD19−/HLA-A2.1+ or CD19+/HLA-A2.1− cell lines. Similarly, the CD20-specific CTLs generated from normal donors lysed CD20+/HLA-A2.1+ MM cell lines (25% lysis; 10:1 E:T ratio), in a manner restricted to HLA-A2.1 and specific to antigens. We next showed IFN-γ production by the CTLs after exposure to CD19+/HLA-A2.1+ or CD20+/HLA-A2.1+ MM cells. Moreover, we have demonstrated the ability to expand CD20-CTLs under serum-free culture conditions while maintaining their cytotoxic activity (28–49%). In ongoing studies, we are evaluating the ability of CD19- and CD20-specific CTLs to eliminate clonogenic myeloma cells in vitro and in vivo in a SCID mouse model of myeloma. These preclinical studies strongly suggest that immunogenic CD19 and CD20 peptide-based vaccines represent a promising immunotherapeutic approach in myeloma.

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