scholarly journals Anti-Metastatic Benefits Produced by Hyperthermia and a CCL3 Derivative

Cancers ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 1770
Author(s):  
Liqiu Ma ◽  
Ryosuke Kambe ◽  
Tomoko Tsuchiya ◽  
Shiro Kanegasaki ◽  
Akihisa Takahashi
Keyword(s):  
Combination Therapy ◽  
Tumor Growth ◽  
Lung Metastases ◽  
Single Amino Acid ◽  
Tumor Growth Inhibition ◽  
Adenocarcinoma Cells ◽  
High Frequency Waves ◽  
The Right ◽  
Improve Patient ◽  
Treated Site

Significant numbers of malignant tumor cells that have spread to surrounding tissues and other distant organs are often too small to be picked up in a diagnostic test, and prevention of even such small metastases should improve patient outcomes. Using a mouse model, we show in this article that intravenous administration of a human CCL3 variant carrying a single amino acid substitution after mild local hyperthermia not only induces tumor growth inhibition at the treated site but also inhibits metastasis. Colon26 adenocarcinoma cells (1 × 105 cells/mouse) were grafted subcutaneously into the right hind leg of syngeneic BALB/c mice and after nine days, when tumor size reached ~11 mm in diameter, the local tumor mass was exposed to high-frequency waves, by which intratumoral temperature was maintained at 42 °C for 30 min. Mice received the CCL3 variant named eMIP (2 μg/mouse/day) intravenously for five consecutive days starting one day after heat treatment. We found that tumor growth in eMIP recipients after hyperthermia was inhibited markedly but no effect was seen in animals treated with either hyperthermia or eMIP alone. Furthermore, the number of lung metastases evaluated after 18 days was dramatically reduced in animals receiving the combination therapy compared with all other controls. These results encourage future clinical application of this combination therapy.

scholarly journals Highly Differentiated Anti-CD47 Antibody, AO-176, Potently Inhibits Hematologic Malignancies Alone and in Combination

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1844-1844
Author(s):  
John Richards ◽  
Myriam N Bouchlaka ◽  
Robyn J Puro ◽  
Ben J Capoccia ◽  
Ronald R Hiebsch ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Combination Therapy ◽  
Tumor Growth ◽  
Growth Inhibition ◽  
Tumor Cells ◽  
Tumor Growth Inhibition ◽  
Employment Equity ◽  
Equity Ownership

AO-176 is a highly differentiated, humanized anti-CD47 IgG2 antibody that is unique among agents in this class of checkpoint inhibitors. AO-176 works by blocking the "don't eat me" signal, the standard mechanism of anti-CD47 antibodies, but also by directly killing tumor cells. Importantly, AO-176 binds preferentially to tumor cells, compared to normal cells, and binds even more potently to tumors in their acidic microenvironment (low pH). Hematological neoplasms are the fourth most frequently diagnosed cancers in both men and women and account for approximately 10% of all cancers. Here we describe AO-176, a highly differentiated anti-CD47 antibody that potently targets hematologic cancers in vitro and in vivo. As a single agent, AO-176 not only promotes phagocytosis (15-45%, EC50 = 0.33-4.1 µg/ml) of hematologic tumor cell lines (acute myeloid leukemia, non-Hodgkin's lymphoma, multiple myeloma, and T cell leukemia) but also directly targets and kills tumor cells (18-46% Annexin V positivity, EC50 = 0.63-10 µg/ml) in a non-ADCC manner. In combination with agents targeting CD20 (rituximab) or CD38 (daratumumab), AO-176 mediates enhanced phagocytosis of lymphoma and multiple myeloma cell lines, respectively. In vivo, AO-176 mediates potent monotherapy tumor growth inhibition of hematologic tumors including Raji B cell lymphoma and RPMI-8226 multiple myeloma xenograft models in a dose-dependent manner. Concomitant with tumor growth inhibition, immune cell infiltrates were observed with elevated numbers of macrophage and dendritic cells, along with increased pro-inflammatory cytokine levels in AO-176 treated animals. When combined with bortezomib, AO-176 was able to elicit complete tumor regression (100% CR in 10/10 animals treated with either 10 or 25 mg/kg AO-176 + 1 mg/kg bortezomib) with no detectable tumor out to 100 days at study termination. Overall survival was also greatly improved following combination therapy compared to animals treated with bortezomib or AO-176 alone. These data show that AO-176 exhibits promising monotherapy and combination therapy activity, both in vitro and in vivo, against hematologic cancers. These findings also add to the previously reported anti-tumor efficacy exhibited by AO-176 in solid tumor xenografts representing ovarian, gastric and breast cancer. With AO-176's highly differentiated MOA and binding characteristics, it may have the potential to improve upon the safety and efficacy profiles relative to other agents in this class. AO-176 is currently being evaluated in a Phase 1 clinical trial (NCT03834948) for the treatment of patients with select solid tumors. Disclosures Richards: Arch Oncology Inc.: Employment, Equity Ownership, Other: Salary. Bouchlaka:Arch Oncology Inc.: Consultancy, Equity Ownership. Puro:Arch Oncology Inc.: Employment, Equity Ownership. Capoccia:Arch Oncology Inc.: Employment, Equity Ownership. Hiebsch:Arch Oncology Inc.: Employment, Equity Ownership. Donio:Arch Oncology Inc.: Employment, Equity Ownership. Wilson:Arch Oncology Inc.: Employment, Equity Ownership. Chakraborty:Arch Oncology Inc.: Employment, Equity Ownership. Sung:Arch Oncology Inc.: Employment, Equity Ownership. Pereira:Arch Oncology Inc.: Employment, Equity Ownership.

scholarly journals 572 Combination therapy of exoSTING, exoIL-12 activates systemic anti-tumor immunity

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A601-A601
Author(s):  
Katherine Kirwin ◽  
Su Chul Jang ◽  
Christine Sia ◽  
Kevin Dooley ◽  
Tong Zi ◽  
...  
Keyword(s):  
T Cell ◽  
Combination Therapy ◽  
Tumor Growth ◽  
Lung Metastases ◽  
Cell Infiltration ◽  
Therapeutic Window ◽  
List Type ◽  
T Cell Infiltration ◽  
Animal Care ◽  
Anti Tumor Activity

BackgroundEngineered exosomes are emerging as a novel therapeutic modality for cancer immunotherapy. Leveraging cell type specific delivery, tumor restricted pharmacology and compartmental dosing, exosome-based immunotherapy can elicit a tumor specific immune response that may not be achievable with other traditional drugging modalities. Pre-clinical studies have shown that exosomes loaded with a STING agonist (exoSTINGTM) or engineered to express the cytokine interleukin-12 (exoIL-12TM) can substantially improve potency and selectivity resulting in improved therapeutic window [1,2]. Both exoSTING and exoIL-12 are currently in clinical trials in cancer patients. Utilizing a combination strategy involving exoSTING and exoIL-12, we demonstrate the development of potent systemic anti-tumor responses in both injected and non-injected tumors.Methods exoSTING exosomes are engineered to overexpress PTGFRN, an abundant exosome surface protein, and loaded ex vivo with a proprietary STING agonist. exoIL-12 exosomes are engineered to overexpress functional IL-12 attached via fusion to PTGFRN. In these studies, exoSTING and exoIL-12 were dosed intratumorally into one flank tumor into mice bearing dual flank subcutaneous MC38 or B16F10 tumors, or B16F10 single flank subcutaneous tumors with B16F10 lung metastases. T-cell infiltration in the non-injected tumor was monitored by histopathology.ResultsIn the checkpoint therapy refractory B16F10 melanoma dual flank tumor model, exoSTING/exoIL-12 combination provided 93% and 78% tumor growth inhibition (TGI) in both the injected and non-injected tumors, respectively, whereas monotherapy of exoSTING or exoIL-12 provided modest anti-tumor activity (44% and 48% TGI) in the non-injected tumors, respectively. In a MC38 subcutaneous CRC model, the addition of anti-PD-1 checkpoint inhibitor further enhanced anti-tumor activity with 100% TGI (7/7 CR) in injected and non-injected tumors. The tumor free animals were refractory to tumor re-challenge demonstrating immunological memory. A dosing schedule optimization experiment showed that same day dosing of exoSTING and exoIL-12 significantly inhibited the tumor growth in the non-injected tumors. In a lung metastasis model, the triple combination also showed potent anti-tumor effect in decreasing distal lung metastases when dosed intratumorally into the subcutaneous tumors. Subsequent imaging and histology studies demonstrated enhanced T cell infiltration in the non-injected subcutaneous tumor with the combination therapy.ConclusionsBy combining both exosome immunotherapies with a checkpoint blockade, we are able to elicit systemic anti-tumor immune immunity in both injected and non-injected tumors.ReferencesJang SC, Economides KD, Moniz RJ, et al. ExoSTING, an extracellular vesicle loaded with STING agonists, promotes tumor immune surveillance. Commun Biol. 2021;4(1):497.Lewis ND, Sia CL, Kirwin K, et al. Exosome surface display of IL12 results in tumor-retained pharmacology with superior potency and limited systemic exposure compared with recombinant IL12. Mol Cancer Ther. 2020;20(3):523-534.Ethics ApprovalAll animals were maintained and treated at the animal care facility of Codiak Biosciences in accordance with the regulations and guidelines of the Institutional Animal Care and Use Committee (CB2020-001).

scholarly journals A molybdenum oxide-based degradable nanosheet for combined chemo-photothermal therapy to improve tumor immunosuppression and suppress distant tumors and lung metastases

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Na Qiu ◽  
Xiaoye Yang ◽  
Yanan Zhang ◽  
Jicheng Zhang ◽  
Jianbo Ji ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Molybdenum Oxide ◽  
Lung Metastasis ◽  
Photothermal Therapy ◽  
Lung Metastases ◽  
Tumor Growth Inhibition ◽  
Inhibition Rate ◽  
Model Drug

AbstractMolybdenum oxide (MoOx) nanosheets have drawn increasing attention for minimally invasive cancer treatments but still face great challenges, including complex modifications and the lack of efficient accumulation in tumor. In this work, a novel multifunctional degradable FA-BSA-PEG/MoOx nanosheet was fabricated (LA-PEG and FA-BSA dual modified MoOx): the synergistic effect of PEG and BSA endows the nanosheet with excellent stability and compatibility; the FA, a targeting ligand, facilitates the accumulation of nanosheets in the tumor. In addition, DTX, a model drug for breast cancer treatment, was loaded (76.49%, 1.5 times the carrier weight) in the nanosheets for in vitro and in vivo antitumor evaluation. The results revealed that the FA-BSA-PEG/MoOx@DTX nanosheets combined photothermal and chemotherapy could not only inhibit the primary tumor growth but also suppress the distant tumor growth (inhibition rate: 51.7%) and lung metastasis (inhibition rate: 93.6%), which is far more effective compared to the commercial Taxotere®. Exploration of the molecular mechanism showed that in vivo immune response induced an increase in positive immune responders, suppressed negative immune suppressors, and established an inflammatory tumor immune environment, which co-contributes towards effective suppression of tumor and lung metastasis. Our experiments demonstrated that this novel multifunctional nanosheet is a promising platform for combined chemo-photothermal therapy. Graphical Abstract

scholarly journals A cocktail of antibodies to multiple tumor-specific neoantigens increases binding and inhibits tumor growth

2019 ◽  
Author(s):  
Girja S. Shukla ◽  
Yu-Jing Sun ◽  
Stephanie C. Pero ◽  
David N. Krag
Keyword(s):  
Tumor Growth ◽  
Polyclonal Antibodies ◽  
Stage Iv ◽  
Single Amino Acid ◽  
Tumor Growth Inhibition ◽  
Specific Cell ◽  
Variable Binding ◽  
Multiple Tumor ◽  
Variable Expression ◽  
Antibody Cocktail

Abstract Background: Antibodies that target a single tumor antigen fail to cure stage IV cancer patients due to tumor heterogeneity resulting in variable expression of antigen. Tumor cells with insufficient binding of antibody will not undergo antibody induced cytotoxicity. We describe targeting multiple tumor-specific antigens that resulted in homogeneous dense binding to mouse melanoma cells and significant tumor growth inhibition. Methods: Surface-related tumor-specific mutations on B16-F10 cells were identified. Peptides containing the single amino acid mutation were synthesized for 9 different neoantigens. Rabbits were vaccinated with each of these peptides and high affinity polyclonal antibodies to each peptide were obtained. The 9 antibodies were combined as a cocktail and mice with implanted B16-F10 cells were treated with and without PD1 inhibitor. Results: Even a single dose of the antibody cocktail inhibited tumor growth and prolonged survival. PD1 inhibitor alone had little effect on tumor growth. The antibody cocktail plus PD1 inhibition increased tumor response and 4 doses of the cocktail completely prevented tumor growth in 50% of the mice. Complete responses were durable. The complete responders were highly resistant to tumor re-challenge at 6 months. No adverse events were identified in the antibody treated mice. Conclusions: Multiple tumor-specific cell surface-related neoantigens were abundant in B16-F10 cells. Antibodies to 9 of these neoantigens had variable binding but when combined had dense homogeneous binding. Even one dose of this cocktail of 9 antibodies improved survival and when multiple does were combined with PD1 inhibition 50% of the mice were rendered permanently tumor free.

Sunitinib inhibits PD-L1 expression in osteosarcoma by targeting STAT3 and remodels the immune system in tumor-bearing mice

2020 ◽  
Author(s):  
Xian Liang Duan ◽  
Jian Ping Guo ◽  
Fan Li ◽  
Chao Xiu ◽  
Hua Wang
Keyword(s):  
Immune System ◽  
Combination Therapy ◽  
Tumor Growth ◽  
Immune Checkpoint Inhibitors ◽  
Immune Cell ◽  
Checkpoint Inhibitors ◽  
Lung Metastases ◽  
Migration And Invasion ◽  
Osteosarcoma Cells ◽  
Tumor Bearing

Aim: Exploring the mechanisms of the combination therapy using VEGFR-TKI and immune checkpoint inhibitors might be useful to control the development of osteosarcoma. Materials & methods: The expression of PD-L1 and STAT3 in osteosarcoma were determined with western blot. Proliferation, migration and invasion were determined with CCK-8 and Transwell assays. Lung metastases, tumor growth, survival and immune cell populations were performed in tumor-bearing mice. Results: Sunitinib reduced the expression of PD-L1 by inhibiting the activation of STAT3 and suppressed the migration and invasion in osteosarcoma cells. Combination therapy reduced lung metastases, tumor growth, improved survival and reverse tumor microenvironment in tumor-bearing mice. Conclusion: Sunitinib inhibits PD-L1 expression by targeting STAT3 and remodels the immune system in tumor-bearing mice.

Pulmonary Vascular Versus Right Ventricular Function Changes During Targeted Therapies of Pulmonary Hypertension – An Argument for Upfront Combination Therapy?

2012 ◽  
Vol 8 (3) ◽  
pp. 209
Author(s):  
Wouter Jacobs ◽  
Anton Vonk-Noordegraaf ◽  
◽  
Keyword(s):  
Combination Therapy ◽  
Right Ventricle ◽  
Right Ventricular ◽  
Pulmonary Vascular Resistance ◽  
Vascular Resistance ◽  
Meta Analysis ◽  
Right Heart Failure ◽  
Functional Class ◽  
Pulmonary Vasculature ◽  
The Right

Pulmonary arterial hypertension is a progressive disease of the pulmonary vasculature, ultimately leading to right heart failure and death. Current treatment is aimed at targeting three different pathways: the prostacyclin, endothelin and nitric oxide pathways. These therapies improve functional class, increase exercise capacity and improve haemodynamics. In addition, data from a meta-analysis provide compelling evidence of improved survival. Despite these treatments, the outcome is still grim and the cause of death is inevitable – right ventricular failure. One explanation for this paradox of haemodynamic benefit and still worse outcome is that the right ventricle does not benefit from a modest reduction in pulmonary vascular resistance. This article describes the physiological concepts that might underlie this paradox. Based on these concepts, we argue that not only a significant reduction in pulmonary vascular resistance, but also a significant reduction in pulmonary artery pressure is required to save the right ventricle. Haemodynamic data from clinical trials hold the promise that these haemodynamic requirements might be met if upfront combination therapy is used.

Identification of 2-Fluoropalmitic Acid as a Potential Therapeutic Agent Against Glioblastoma

2020 ◽  
Vol 26 (36) ◽  
pp. 4675-4684 ◽  
Author(s):  
Shabierjiang Jiapaer ◽  
Takuya Furuta ◽  
Yu Dong ◽  
Tomohiro Kitabayashi ◽  
Hemragul Sabit ◽  
...  
Keyword(s):  
Combination Therapy ◽  
Drug Screening ◽  
Therapeutic Agent ◽  
Gelatin Zymography ◽  
Therapeutic Agents ◽  
Drug Candidate ◽  
Potential Therapeutic Agent ◽  
Sphere Formation ◽  
Improve Patient

Background: Glioblastomas (GBMs) are aggressive malignant brain tumors. Although chemotherapy with temozolomide (TMZ) can extend patient survival, most patients eventually demonstrate resistance. Therefore, novel therapeutic agents that overcome TMZ chemoresistance are required to improve patient outcomes. Purpose: Drug screening is an efficient method to find new therapeutic agents from existing drugs. In this study, we explored a novel anti-glioma agent by drug screening and analyzed its function with respect to GBM treatment for future clinical applications. Methods: Drug libraries containing 1,301 diverse chemical compounds were screened against two glioma stem cell (GSC) lines for drug candidate selection. The effect of selected agents on GSCs and glioma was estimated through viability, proliferation, sphere formation, and invasion assays. Combination therapy was performed to assess its ability to enhance TMZ cytotoxicity against GBM. To clarify the mechanism of action, we performed methylation-specific polymerase chain reaction, gelatin zymography, and western blot analysis. Results: The acyl-CoA synthetase inhibitor 2-fluoropalmitic acid (2-FPA) was selected as a candidate anti-glioma agent. 2-FPA suppressed the viability and stem-like phenotype of GSCs. It also inhibited proliferation and invasion of glioma cell lines. Combination therapy of 2-FPA with TMZ synergistically enhanced the efficacy of TMZ. 2-FPA suppressed the expression of phosphor-ERK, CD133, and SOX-2; reduced MMP-2 activity; and increased methylation of the MGMT promoter. Conclusion: 2-FPA was identified as a potential therapeutic agent against GBM. To extend these findings, physiological studies are required to examine the efficacy of 2-FPA against GBM in vivo.

581 Effects of acoustic immune priming with low-intensity focused ultrasound (LOFU) and trabectedin on a murine model of osteosarcoma

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A616-A616
Author(s):  
Shannon Keisling
Keyword(s):  
Radiation Therapy ◽  
Heat Shock ◽  
Combination Therapy ◽  
Murine Model ◽  
Heat Shock Response ◽  
Cell Activation ◽  
Pulmonary Metastases ◽  
Lung Metastases ◽  
Therapy Group ◽  
Shock Response

BackgroundOsteosarcoma is the most common primary bone tumor and has a peak incidence in adolescence. The prognosis for recurrent and metastatic disease is poor and over one-third of patients with localized disease at presentation will recur after treatment with metastases. LOFU produces non-lethal, transient mechanical and thermal stress to cause protein misfolding, endoplasmic reticulum stress, and induction of the heat shock response (refs). Trabectedin is directly tumoricidal through inhibiting transcription and DNA repair, modulates the tumor microenvironment by selectively depleting M2 macrophages, and inhibits the transcription factor heat shock factor 1 (HSF1) (refs). We hypothesized that combination therapy would synergistically intensify the unfolded protein response and heat shock response to facilitate antigen presenting cell activation and efficient presentation to cytotoxic T cells. To examine this, experiments are being conducted to investigate the effect of LOFU in combination with trabectedin and/or radiation therapy (RT) in a murine model of osteosarcoma.MethodsPalpable (<5 mm) subcutaneous K7M2 murine osteosarcoma tumors in BALB/c mice were treated with a) LOFU, b) trabectedin (intravenous (IV) or intratumoral (IT)), c) LOFU + trabectedin, and d) radiation. Tumor growth (ANOVA (Kruskal-Wallis) with Dunn’s test for multiple comparisons), pulmonary metastases (Fisher’s exact test) and survival (Kaplan-Meier) were measured and analyzed in GraphPad Prism.ResultsMean tumor volume in the combination therapy group (428 mm3) was less than nontreated controls (887 mm3), LOFU alone (670 mm3), trabectedin alone (1218 mm3, p=0.0386). Radiation therapy resulted in complete ablation of the tumors. None of the combination therapy mice had grossly detectable lung metastases at time of death but metastases were present in the trabectedin only (20%), LOFU only (50%), and control (50%) groups (not statistically significant).ConclusionsCombination therapy with trabectedin and LOFU yielded smaller tumor size and fewer pulmonary metastases compared to individual therapies alone.

245 Human TLR8 knock-in mice potentiate immunotherapy responses of MC38 syngeneic tumors

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A264-A264
Author(s):  
Shanshan Qi ◽  
Hongjuan Zhang ◽  
Ruilin Sun ◽  
Annie An ◽  
Henry Li ◽  
...  
Keyword(s):  
T Cells ◽  
Tumor Growth ◽  
Cancer Immunotherapy ◽  
Tumor Regression ◽  
Tumor Growth Rate ◽  
Tumor Growth Inhibition ◽  
List Type ◽  
Rna Recognition ◽  
Ethical Guidelines ◽  
Ifn Γ

BackgroundToll-like receptors (TLRs) serve critical roles in mediating innate immune responses against many pathogens. However, they may also bind to endogenous ligands and lead to the pathogenesis of autoimmunity. Although TLR8 belongs to the same TLR family as TLR7, its role in inflammation and tumor progression is not yet fully understood due to the lack of suitable animal models. In humans, both TLR7 and TLR8 recognize single-stranded self-RNA, viral RNA, and synthetic small molecule agonists.1, 2 However, mouse Tlr8 is non-functional due to the absence of 5 amino acids necessary for RNA recognition. In order to create a mouse model with functional TLR8, we replaced exon 3 of mouse Tlr8 with human TLR8, therefore developing a hTLR8 knock-in (KI) model. Both heterozygous and homozygous hTLR8 KI mice are viable with inflammatory phenotypes, i.e. enlarged spleens and livers, and significantly higher IL-12 p40 levels under TLR8 agonist treatment. In this study, we evaluated the potential use of hTLR8 mice for cancer immunotherapy studies.MethodshTLR8 mice, together with naïve C57BL/6 mice, were inoculated with MC38 syngeneic tumor cells. Tumor bearing mice were grouped at a mean tumor volume of approximately 100 mm3 for treatment with PBS or 10 mg/kg anti-PD-1 (RMP1-14) antibody. At the efficacy endpoint, spleens and tumors were collected for flow cytometry profiling.ResultsAnti-PD-1 treatment of MC38 tumors in naïve C57BL/6 led to moderate tumor growth inhibition (TGI = 54%). Interestingly, anti-PD-1 treatment showed improved efficacy in hTLR8 mice (TGI = 79%), including 2/10 tumors with complete tumor regression. In comparison, non-treated MC38 tumor growth rate was slower in hTLR8 mice than in naïve mice. Anti-PD-1 treated hTLR8 mice also had significantly increased IFN-γ and TNF-a positive CD4+ T cells in the spleen, along with higher numbers of differentiated effector T cells. In addition, hTLR8 mice have activated dendritic cells and macrophages, acting as critical steps in initiation of the inflammatory process, with higher levels of pro-inflammatory cytokines, such as IL-6, IFN-γ, TNF-a, and IL-1β, which may promote Th1 priming and differentiation of T cells into IFN-γ or TNF-a producing cells.ConclusionshTLR8 mice offer a great tool to model cancer immunotherapy in an inflammatory/autoimmunity prone background. Moreover, hTLR8 mice can be effectively used to shift a ‘cold’ tumor phenotype to ‘hot’ tumors in a syngeneic setting.Ethics ApprovalAnimal experiments were conducted in accordance with animal welfare law, approved by local authorities, and in accordance with the ethical guidelines of CrownBio (Taicang).ReferencesKugelberg E. Making mice more human the TLR8 way. Nat Rev Immunol 2014;14:6.Guiducci C, Gong M, Cepika A-M, et al. RNA recognition by human TLR8 can lead to autoimmune inflammation. J Exp Med 2013;210:2903–2919.

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