Novel mechanisms of action contributing to Benralizumab's potent anti-eosinophilic activity

2021 ◽  
pp. 2004306
Author(s):  
Rania Dagher ◽  
Varsha Kumar ◽  
Alan M. Copenhaver ◽  
Sandra Gallagher ◽  
Mahboobe Ghaedi ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Nk Cells ◽  
Caspase 3 ◽  
Hypereosinophilic Syndrome ◽  
Mechanisms Of Action ◽  
Tnf Receptor ◽  
Dependent Cell ◽  
Membrane Depolarisation ◽  
Eosinophil Apoptosis

Benralizumab is a humanised, anti-IL-5Rα monoclonal antibody with anti-eosinophilic activity. Lack of fucose (afucosylation) increases its affinity to CD16a and significantly enhances antibody-dependent cell-mediated cytotoxicity (ADCC) by NK cells. Although benralizumab proved clinically efficacious in clinical trials for patients with severe asthma and hypereosinophilic syndrome, in-depth characterisation of its anti-eosinophilic mechanisms of action remain elusive. Here, we further investigated the mechanisms involved in benralizumab's anti-eosinophilic activities. In the presence of NK cells benralizumab induced potent eosinophil apoptosis as demonstrated by the upstream induction of caspase 3/7 and upregulation of cytochrome C. In addition, we uncovered a previously unrecognised mechanism whereby benralizumab can induce eosinophil phagocytosis/efferocytosis by macrophages, a process called antibody-dependent cell phagocytosis (ADCP). Using live cell imaging we unravel the stepwise processes leading to eosinophil apoptosis and uptake by activated macrophages. Through careful observations of cellular co-culture assays we identified a novel role for macrophage derived TNF to further enhance benralizumab-mediated eosinophil apoptosis through activation of TNF-receptor 1 on eosinophils. TNF-induced eosinophil apoptosis was associated with Cytochrome C upregulation, mitochondrial membrane depolarisation, and increased caspase 3/7 activity. Moreover, activated NK cells were found to amplify this axis through the secretion of IFNγ, subsequently driving TNF expression by macrophages. Our data provide insights into the timely appearance of events leading to benralizumab-induced eosinophil apoptosis and suggest that additional mechanisms may contribute to the potent anti-eosinophilic activity of benralizumab in vivo. Importantly, afucosylation of benralizumab strongly enhanced its potency for all mechanisms investigated.

scholarly journals Silica nanoparticles inducing the apoptosis of spermatocyte cell through microRNA-450b-3p targeting MTCH2-mediating mitochondrial signaling pathway

2020 ◽  
Author(s):  
Guiqing Zhou ◽  
Jianhui Liu ◽  
Xiangyang Li ◽  
Yujian Sang ◽  
Yue Zhang ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Silica Nanoparticles ◽  
Caspase 3 ◽  
Reproductive Toxicity ◽  
Control Group ◽  
Caspase 9 ◽  
Protein Expressions ◽  
Underlying Mechanisms

Abstract Background: Silica nanoparticles (SiNPs) are found in environmental particulate matter and are proven to have adverse effects on fertility. The relationship and underlying mechanisms between miRNAs and apoptosis induced by SiNPs during spermatogenesis is currently ambiguous. Experimental design: The present study was designed to investigate the role of miRNA-450b-3p in the reproductive toxicity caused by SiNPs. In vivo, 40 male mice were randomly divided into control and SiNPs groups, 20 per group. The mice in the SiNPs group were administrated 20 mg/kg SiNPs by tracheal perfusion once every 5 days, for 35 days, and the control group were given the equivalent of a normal luminal saline. In vitro, spermatocyte cells were divided into 0 and 5 μg/mL SiNPs groups, after passaged for 30 generations, the GC-2spd cells in 5 μg/mL SiNPs groups were transfected with miRNA-450b-3p and its mimic and inhibitor. Results: In vivo, the results showed that SiNPs damaged tissue structures of testis, decreased the quantity and quality of the sperm, reduced the expression of miR-450b-3p, and increased the protein expressions of the MTCH2, BID, BAX, Cytochrome C, Caspase-9, and Caspase-3 in the testis. In vitro, SiNPs obviously repressed the viability and increased the LDH level and apoptosis rate, decreased the levels of the miR-450b-3p, significantly enhanced the protein expressions of the MTCH2, BID, BAX, Cytochrome C, Caspase-9, Caspase-3; while the mimic of miR-450b-3p reversed the changes induced by SiNPs, but inhibitor further promoted the effects induced by SiNPs.Conclusion: The result suggested that SiNPs could induce the spermatocyte apoptosis by inhibiting the miR-450b-3p expression to target promoting the MTCH2 resulting in activating mitochondrial apoptotic signaling pathways in the spermatocyte cells.

scholarly journals Fuzheng Qingjie Granules Inhibit Growth of Hepatoma Cells via Inducing Mitochondria-Mediated Apoptosis and Enhancing Immune Function

2016 ◽  
Vol 16 (3) ◽  
pp. 329-338 ◽  
Author(s):  
Xuzheng Chen ◽  
Zhiyun Cao ◽  
Youquan Zhang ◽  
Jinnong Li ◽  
Suqing Wang ◽  
...  
Keyword(s):  
T Cells ◽  
Cytochrome C ◽  
Immune Function ◽  
Nk Cells ◽  
Hepg2 Cells ◽  
Caspase 3 ◽  
Tumor Tissue ◽  
Hepatoma Cells ◽  
Tumor Bearing ◽  
Tnf Α

Fuzheng Qingjie (FZQJ) granules, a compound Chinese medicine, have been used as an adjuvant therapy for alimentary tract cancers. However, the underlying anticancer mechanisms are still not well understood. In the present study, HepG2 cells were treated with FZQJ-containing serum. Cell proliferation was evaluated using MTT assay. Apoptosis was analyzed using a flow cytometer. Cell ultrastructure was observed under a transmission electron microscope. The mitochondrial membrane potential (Δψ) was examined with JC-1 dye. In H22 tumor–bearing mice, CD4+ T cells, CD8+ T cells, CD3+ T cells, and natural killer (NK) cells in peripheral blood were evaluated cytometrically. Interleukin (IL)-2 and tumor necrosis factor (TNF)-α levels were measured using radioimmunoassay.The mRNA levels of Bax and Bcl-2 were examined by reverse transcription–polymerase chain reaction. The protein levels of Bax, Bcl-2, cytochrome C, caspase 3 and 9, PARP, and CD69 were examined by Western blotting. The apoptotic cells in tissues were observed using TUNEL method. Alanine transaminase (ALT), aspartate transaminase (AST), blood urea nitrogen (BUN), and creatinine (CRE) were detected by an automatic biochemical analyzer. The results showed that FZQJ-containing serum remarkably inhibited proliferation of HepG2 cells in dose- and time-dependent manners, induced HepG2 cell apoptosis and caused a decrease of Δψ. Analysis of tumor tissue showed that FZQJ-induced apoptosis was accompanied by downregulation of Bcl-2 and upregulation of Bax, release of cytochrome c, activation of caspase 3 and 9, and cleavage of PARP. In addition, FZQJ increased the percentages of CD4+ T and NK cells, the ratio of CD4+/CD8+ T cells as well as the levels of serum TNF-α. FZQJ also increased CD69 expression in tumor tissue. No hepatorenal toxicity was observed in H22 tumor–bearing mice. These results indicated that FZQJ could inhibit the growth of hepatoma cells via regulating immune function and inducing mitochondria mediated apoptosis.

Obinutuzumab (GA101) Significantly Enhances Cell Death and ADCC Compared to Rituximab Against CD20+ sensitive and Rituximab Resistant B-Cell Non-Hodgkin Lymphoma (NHL) and Lymphoblastic Leukemia (BLL)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4865-4865 ◽  
Author(s):  
Aradhana Awasthi Tiwari ◽  
Janet Ayello ◽  
Carmella van de Ven ◽  
Danielle Glassman ◽  
Anthony Sabulski ◽  
...  
Keyword(s):  
Cell Death ◽  
B Cell ◽  
Nk Cells ◽  
Cell Lines ◽  
Caspase 3 ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Dismal Prognosis

Abstract Abstract 4865 Background: Patients who relapse with CD20+ B-NHL and B cell lymphoblastic leukemia (B-LL) have a dismal prognosis, often associated with chemotherapy resistance (Cairo et al. JCO, 2012,Mils/Cairo et al. BJH,2012) and often require alternative therapeutic strategies. Rituximab (RTX) in combination with FAB 96 chemotherapy is a safe, well-tolerated treatment that is associated with > 90% EFS in children with newly diagnosed and advanced mature B-Cell NHL (Cairo M.S. et al. ASCO, 2010). Resistance to RTX, however, may predispose patients with CD20+ NHL to an increase risk of relapse and or disease progression (Barth/Cairo et al. BJH, 2012; Tsai et al. Cl. Can. Res, 2012). Obinutuzumab (GA101), a novel type II glycoengineered CD20 antibody of the IgG1 isotype, mediates enhanced cell death vs RTX and has a glycoengineered Fc region that induces significantly enhanced ADCC (Mössner et al. Bld, 2010; Niederfellner G. et al. Bld, 2011; Bologna L et al. JI, 2012). Objective: To evaluate the in-vitro efficacy of GA101 compared to RTX against RTX sensitive and resistant CD20+ B-NHL and B-LL cell lines. Methods: Raji (CD20+,ATCC, Manhass, VA), U698-M (CD20+, DSMZ, Germany), Loucy cells (CD20−) (T-ALL) (ATCC, Manhass, VA) and Raji-2R and Raji-4RH (generously supplied by M. Barth, Roswell Park Cancer Institute) were cultured in RPMI with 10% FBS and incubated with GA101 and/or RTX at 100 μg/ml for 24 hrs (n=6), 48 and 72 hrs (n=5). Cell death was evaluated by staining with AnnexinV/7AAD and flow-cytometry. Loucy cells (CD20−) were used as the negative control. The caspase 3/7 activity was measured by FAM caspase 3/7 assay kit by FLICA™ methodology. RSCL, RRCL, U698-M and Loucy were incubated with GA101 and RTX treatment for 24, 48 and 72 hrs, and caspase3/7 activity was detected by FACS using 488 nm excitation and emission filter (n=3). ADCC were performed with K562-IL-15–41BBL expanded NK cells (Ayello/Cairo et al. ASH, 2010) as well as IL-2 expanded NK cells, at 20:1 effector: target ratio (E: T, n=3) using europium release assay (Perkin-Elmer). Results: GA101 induced significantly more cell death compared to RTX in B-NHL and BLL cell lines. (Table-1) GA101 vs RTX shows a significantly increase in caspase 3/7 activity in Raji 16.92±0.84% vs 11.76±0.08% compared to Raji2R 6.7±0.62% vs 2.8±0.7%, Raji4RH 5.8±0.35% vs 2.0±0.3% and U698-M 12.54±0.44% vs 9.6±0.95% compared to Loucy 3.22±0.45% vs 2.59±0.05%, respectively, at 24 hrs of treatment (p<0.0001). GA101 vs RTX also elicited a significant increase a ADCC with K562-IL15–41BBL expanded NK cells, in Raji 73.8±8.1% vs 56.81±4.6% compared to Raji-2R 38.0±2.0% vs 21.6±1.2%, Raji-4RH 40.0±1.6% vs 0.5±1.1% and U698-M 70.0±1.6% vs 45.56±0.1%, compared to Loucy 21.67±0.48% vs 15.92±0.52%, respectively (p<0.001) at day 7.The IL-2 alone expanded Hu-NK cells demonstrated a reduction of 10–20% cytotoxicity compared to K562-IL15–41BBL Hu-NK cells at day 7 against BLL, RSCL and RRCL, in-vitro. Conclusion: Obinutuzumab compared to RTX significantly enhanced cell death, caspase3/7 activity and NK mediated ADCC in sensitive and RTX resistant B-NHL and B-LL. Obinutuzumab represents a promising candidate for treating RTX sensitive and resistant CD20+ B-Cell Lymphomas and lymphoblastic leukemia. Further studies will investigate the combination of activated NK cells or chemotherapy that may enhance or synergize with the efficacy of GA101 (Obinutuzumab) both in -vitro and in-vivo in xenografted NOD/SCID mice. Disclosures: No relevant conflicts of interest to declare.

scholarly journals CD137 stimulation enhances the antilymphoma activity of anti-CD20 antibodies

Blood ◽  
2011 ◽  
Vol 117 (8) ◽  
pp. 2423-2432 ◽  
Author(s):  
Holbrook E. Kohrt ◽  
Roch Houot ◽  
Matthew J. Goldstein ◽  
Kipp Weiskopf ◽  
Ash A. Alizadeh ◽  
...  
Keyword(s):  
B Cell ◽  
Nk Cells ◽  
Costimulatory Molecule ◽  
Host Immune System ◽  
Sequential Administration ◽  
Dependent Cell ◽  
Human Lymphoma ◽  
Anti Cd20 ◽  
Stimulation Of

Abstract Antibody-dependent cell-mediated cytotoxicity (ADCC), which is largely mediated by natural killer (NK) cells, is thought to play an important role in the efficacy of rituximab, an anti-CD20 monoclonal antibody (mAb) used to treat patients with B-cell lymphomas. CD137 is a costimulatory molecule expressed on a variety of immune cells after activation, including NK cells. In the present study, we show that an anti-CD137 agonistic mAb enhances the antilymphoma activity of rituximab by enhancing ADCC. Human NK cells up-regulate CD137 after encountering rituximab-coated tumor B cells, and subsequent stimulation of these NK cells with anti-CD137 mAb enhances rituximab-dependent cytotoxicity against the lymphoma cells. In a syngeneic murine lymphoma model and in a xenotransplanted human lymphoma model, sequential administration of anti-CD20 mAb followed by anti-CD137 mAb had potent antilymphoma activity in vivo. These results support a novel, sequential antibody approach against B-cell malignancies by targeting first the tumor and then the host immune system.

scholarly journals Lysosomes and Fas-mediated liver cell death

2007 ◽  
Vol 403 (1) ◽  
pp. 89-95 ◽  
Author(s):  
Robert Wattiaux ◽  
Simone Wattiaux-De Coninck ◽  
Jacqueline Thirion ◽  
Mańe-Christine Gasingirwa ◽  
Michel Jadot
Keyword(s):  
Cell Death ◽  
Cytochrome C ◽  
Caspase 3 ◽  
Ex Vivo ◽  
Jurkat Cells ◽  
Intermembrane Space ◽  
Lysosomal Hydrolases ◽  
Cytochrome C Reductase ◽  
Effector Caspases

A number of studies, mostly performed ex vivo, suggest that lysosomes are involved in apoptosis as a result of a release of their cathepsins into the cytosol. These enzymes could then contribute to the permeabilization of the outer mitochondrial membrane; they could also activate effector caspases. The present study aims at testing whether the membrane of liver lysosomes is disrupted during Fas-mediated cell death of hepatocytes in vivo, a process implicated in several liver pathologies. Apoptosis was induced by injecting mice with aFas (anti-Fas antibody). The state of lysosomes was assessed by determining the proportion of lysosomal enzymes (β-galactosidase, β-glucuronidase, cathepsin C and cathepsin B) present in homogenate supernatants, devoid of intact lysosomes, and by analysing the behaviour in differential and isopycnic centrifugation of β-galactosidase. Apoptosis was monitored by measuring caspase 3 activity (DEVDase) and the release of sulfite cytochrome c reductase, an enzyme located in the mitochondrial intermembrane space. Results show that an injection of 10 μg of aFas causes a rapid and large increase in DEVDase activity and in unsedimentable sulfite cytochrome c reductase. This modifies neither the proportion of unsedimentable lysosomal enzyme in the homogenates nor the behaviour of lysosomes in centrifugation. Experiments performed with a lower dose of aFas (5 μg) indicate that unsedimentable lysosomal hydrolase activity increases in the homogenate after injection but with a marked delay with respect to the increase in DEVDase activity and in unsedimentable sulfite cytochrome c reductase. Comparative experiments ex vivo performed with Jurkat cells show an increase in unsedimentable lysosomal hydrolases, but much later than caspase 3 activation, and a release of dipeptidyl peptidase III and DEVDase into culture medium. It is proposed that the weakening of lysosomes observed after aFas treatment in vivo and ex vivo results from a necrotic process that takes place late after initiation of apoptosis.

Interleukin-5 inhibits translocation of Bax to the mitochondria, cytochrome c release, and activation of caspases in human eosinophils

Blood ◽  
2001 ◽  
Vol 98 (7) ◽  
pp. 2239-2247 ◽  
Author(s):  
Grant Dewson ◽  
Gerald M. Cohen ◽  
Andrew J. Wardlaw
Keyword(s):  
Cytochrome C ◽  
Mitochondrial Membrane ◽  
Caspase 3 ◽  
Active Form ◽  
Cytochrome C Release ◽  
Interleukin 5 ◽  
Effector Caspase ◽  
Subsequent Activation ◽  
Induced Apoptosis ◽  
Eosinophil Apoptosis

The apoptosis and subsequent clearance of eosinophils without histotoxic mediator release is thought to be crucial in the resolution of airway inflammation in asthma. Interleukin-5 (IL-5) is a potent suppressor of eosinophil apoptosis. The mechanism by which IL-5 inhibits spontaneous eosinophil apoptosis was investigated. Freshly isolated eosinophils constitutively expressed the conformationally active form of Bax in the cytosol and nucleus. During spontaneous and staurosporine-induced apoptosis, Bax underwent a caspase-independent translocation to the mitochondria, which was inhibited by IL-5. Eosinophil apoptosis was associated with the release of cytochrome c from the mitochondria, which was also inhibited by IL-5. IL-5 and the cell-permeable caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-(OMe) fluoromethyl ketone (z-VAD.fmk), prevented phosphatidylserine (PS) externalization, although only IL-5 inhibited loss of mitochondrial membrane potential (ΔΨm). Peripheral blood eosinophils endogenously expressed “initiator” caspase-8 and -9, and “effector” caspase-3, -6, and -7. Spontaneous eosinophil apoptosis was associated with processing of caspase-3, -6, -7, -8, and -9. IL-5 and z-VAD.fmk prevented caspase activation in spontaneous apoptosis. The results suggest that spontaneous eosinophil apoptosis involves Bax translocation to the mitochondria, cytochrome crelease, caspase-independent perturbation of the mitochondrial membrane, and subsequent activation of caspases. IL-5 inhibits spontaneous eosinophil apoptosis at a site upstream of Bax translocation.

scholarly journals 2,5-Hexanedione mediates neuronal apoptosis through suppression of NGF via PI3K/Akt signaling in the rat sciatic nerve

2019 ◽  
Vol 39 (2) ◽  
Author(s):  
Enjun Zuo ◽  
Cong Zhang ◽  
Jun Mao ◽  
Chenxue Gao ◽  
Shuhai Hu ◽  
...  
Keyword(s):  
Sciatic Nerve ◽  
Cytochrome C ◽  
Neuronal Apoptosis ◽  
Caspase 3 ◽  
Specific Inhibitor ◽  
Control Group ◽  
Cytochrome C Release ◽  
Rat Sciatic Nerve

Abstract Because precise mechanism for 2,5-hexanedione (HD)-induced neuronal apoptosis largely remains unknown, we explored the potential mechanisms both in vivo and in vitro. Rats were intraperitoneally exposed to HD at different doses for 5 weeks, following which the expression levels of nerve growth factor (NGF), phosphorylation of Akt and Bad, dimerization of Bad and Bcl-xL, as well as the release of cytochrome c and the caspase-3 activity were measured. Moreover, these variables were also examined in vitro in HD-exposed VSC4.1 cells with or without a PI3K-specific agonist (IGF-1), and in HD-exposed VSC4.1 cells with or without a PI3K-specific inhibitor (LY294002) in the presence or absence of NGF. The data indicate that, as the concentration of HD increased, rats exhibited progressive gait abnormalities, and enhanced neuronal apoptosis in the rat sciatic nerve, compared with the results observed in the control group. Furthermore, HD significantly down-regulated NGF expression in the rat sciatic nerve. Moreover, suppression of NGF expression inhibited the phosphorylation of Akt and Bad. Meanwhile, an increase in the dimerization of Bad and Bcl-xL in mitochondria resulted in cytochrome c release and caspase-3 activation. In contrast, HD-induced apoptosis was eliminated by IGF-1. Additionally, NGF supplementation reversed the decrease in phosphorylation of Akt and Bad, as well as reversing the neuronal apoptosis in HD-exposed VSC4.1 cells. However, LY294002 blocked these effects of NGF. Collectively, our results demonstrate that mitochondrial-dependent apoptosis is induced by HD through NGF suppression via the PI3K/Akt pathway both in vivo and in vitro.

Apoptotic signaling induces hyperpermeability following hemorrhagic shock

2007 ◽  
Vol 292 (6) ◽  
pp. H3179-H3189 ◽  
Author(s):  
Ed W. Childs ◽  
Binu Tharakan ◽  
Felicia A. Hunter ◽  
John H. Tinsley ◽  
Xiaobo Cao
Keyword(s):  
Cytochrome C ◽  
Hemorrhagic Shock ◽  
Caspase 3 ◽  
Mitochondrial Cytochrome ◽  
Intrinsic Apoptotic Pathway ◽  
Intrinsic Pathway ◽  
Apoptotic Pathway ◽  
Apoptotic Signaling ◽  
Mitochondrial Cytochrome C

Hemorrhagic shock (HS) disrupts the endothelial cell barrier, resulting in microvascular hyperpermeability. Recent studies have also demonstrated that activation of the apoptotic signaling cascade is involved in endothelial dysfunction, which may result in hyperpermeability. Here we report involvement of the mitochondrial “intrinsic” pathway in microvascular hyperpermeability following HS in rats. HS resulted in the activation of the mitochondrial intrinsic pathway, as is evident from an increase in the proapoptotic Bcl-2 family member BAK, release of mitochondrial cytochrome c into the cytoplasm, and activation of caspase-3. This, along with the in vivo transfection of the proapoptotic peptide BAK (BH3), resulted in hyperpermeability (as visualized by intravital microscopy), release of mitochondrial cytochrome c into the cytoplasm, and activation of caspase-3. Conversely, transfection of the BAK (BH3) mutant had no effect on hyperpermeability. Together, these results demonstrate involvement of the mitochondrial intrinsic apoptotic pathway in HS-induced hyperpermeability and that the attenuation of this pathway may provide an alternative strategy in preserving vascular barrier integrity.

scholarly journals Venetoclax Enhances the Efficacy of Therapeutic Antibodies in B-Cell Malignancies

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4177-4177
Author(s):  
Fotini Vogiatzi ◽  
Julia Heymann ◽  
Thies Rösner ◽  
Lennart Lenk ◽  
Gunnar Cario ◽  
...  
Keyword(s):  
B Cell ◽  
Research Funding ◽  
Caspase 3 ◽  
Nk Cell ◽  
Target Cells ◽  
Therapeutic Antibodies ◽  
B Cell Malignancies ◽  
Nsg Mice ◽  
Dependent Cell

Abstract The application of antibodies is a promising option in the treatment of B-cell malignancies, including acute lymphoblastic leukemia (ALL) and B-cell Non-Hodgkin lymphoma (B-NHL). Although patient outcomes have improved by applying combinations of chemotherapy and antibodies, certain patients characterized by a high expression of anti-apoptotic Bcl-2 have a poor prognosis. These include adult B-NHL patients with "double-hit lymphomas" (DHLs) and pediatric ALL patients harboring a t(17;19) translocation. Furthermore, a substantial number of Burkitt´s lymphoma (BL) patients also express Bcl-2 even though the impact of this finding on prognosis is yet unclear. Here, we examine the role of low doses of the Bcl-2 inhibitor venetoclax (VTX, 1nM) on the efficacy of the therapeutic antibodies rituximab (CD20), daratumumab (CD38) and CD19-DE (a variant of the CD19 antibody MOR208 engineered for improved effector cell binding). Natural killer (NK)-cell mediated antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) were evaluated. The CD20-expressing DHL cell line Carnaval and patient-derived xenograft (PDX) cells from two BL patients were used as target cells for rituximab, the CD20-negative/CD38-positive DHL cell line Will-2 was used with daratumumab and three PDX samples from t(17;19) positive ALL patients were used with CD19-DE. For the assessment of ADCP, human monocyte-derived macrophages were incubated with labelled target cells and microscopy assays were performed. NK-cell mediated ADCC was not enhanced by VTX in any of our models. However, 17-37% increases in ADCP by macrophages were detected when Carnaval cells were subjected to combinations of VTX/rituximab and when Will-2 cells were treated with VTX/daratumumab as compared to VTX or antibody alone (p=0.0318/p=0.0185 and p=0.0012/p=0.0068, respectively, Figure A). When BL PDX cells were subjected to ADCP assays with VTX/rituximab, mean phagocytosis levels were also enhanced by 26.0% and 21.0% in the combination treatment group as compared to VTX (p=0.0283) and rituximab alone (p=0.0282; Figure A). ADCP assays with t(17;19) positive ALL-PDX cells and CD19-DE confirmed these results as phagocytosis was increased to similar extents in the combination group as compared to VTX (p=0.0017) or CD19-DE alone (p=0.0323) (Figure A/B). In order to exclude that our observations were due to an enhancement of apoptosis in target cells only, we measured cleaved caspase-3 with VTX, antibodies alone or the combination of both. Cleaved caspase-3 levels were equal in all groups suggesting that the addition of VTX resulted in an apoptosis-independent activation of macrophages. In order to minimize heterogeneity in ADCP assays, phagocytosis was next examined using expanded macrophages from NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice for our assays. Regardless of target cells and antibody, the combination of antibody treatment with VTX resulted in enhanced phagocytosis by murine macrophages, confirming our results. The effects of VTX on the efficacy of rituximab were finally examined in vivo. Carnaval cells were injected intravenously into NSG mice and animals treated with VTX (100 mg/kg 5 days/week by oral gavage), rituximab alone (1 mg/kg once weekly intraperitoneally) or the combination of both (n=6/group). Mice were sacrificed when mice showed clinical lymphoma or leukemia engraftment and survival differences were assessed using Kaplan-Meier log-rank statistics. Compared to control, mice treated with VTX displayed a slight survival advantage, which was more marked in mice treated with rituximab (p=0.0020/p=0.0004, respectively, Figure C), suggesting a better efficacy of rituximab than VTX as monotherapy. Most importantly, mice treated with the combination VTX/rituximab showed significantly superior survival as compared to either VTX or rituximab alone (p=0.0023/p=0.0268, respectively, Figure C), suggesting additive effects in vivo. Altogether, we show that VTX enhances the efficacy of therapeutic antibodies in models of B-cell malignancies including PDX samples. The mechanism is most likely dependent on distinct influences of VTX on macrophage activation, e. g. by myeloid immune checkpoints. Our in vivo data suggest that this combination strategy may become a promising therapeutic option for the treatment of Bcl-2 expressing B-cell malignancies in the future. Figure. Figure. Disclosures Bourquin: Amgen: Other: Travel Support. Valerius:Affimed: Research Funding. Peipp:Affimed: Research Funding.

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