Superior Efficacy of the Novel Type II, Glycoengineered CD20 Antibody GA101vs. the Type I CD20 Antibodies Rituximab and Ofatumumab

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3925-3925 ◽  
Author(s):  
Sylvia Herter ◽  
Inja Waldhauer ◽  
Tina Otz ◽  
Frank Herting ◽  
Sabine Lang ◽  
...  
Keyword(s):  
Cell Death ◽  
Type I ◽  
Type Ii ◽  
B Cell Depletion ◽  
Employment Equity ◽  
Direct Cell ◽  
Equity Ownership ◽  
Cd20 Antibody ◽  
Dose Dependent ◽  
Cd20 Antibodies

Abstract Abstract 3925 GA101 is Type II, glycoengineered CD20 monoclonal antibody currently in PhII/III clinical trials. We have previously shown that GA101 mediates superior in vitro and in vivo activity compared to the Type I CD20 antibody rituximab. By epitope mapping and crystallography we have shown that GA101 recognizes CD20 in a unique way that is different from Type I CD20 antibodies and have proposed that this may be the basis for the Type II character of GA101. Here we compare for the first time GA101 with rituximab, the standard of care in various clinical settings in NHL and B-CLL in combination with chemotherapy, as well as with the Type I CD20 antibody ofatumumab, which was recently approved for treatment of B-CLL patients refractory to fludarabine and alemtuzumab. The following assays were used to compare the three anti-CD20 antibodies: i) Binding to NHL cell lines Z138 (MCL, ca. 60.000 CD20 binding sites per cell) and SU-DHL4 (DLBCL, ca. 1 Mio CD20 binding sites per cell) assessed by FACS, ii) Cell death induction, detected by AxV/PI staining and FACS, on a panel of NHL cell lines, iii) Antibody dependent cellular cytotoxicity mediated by PBMNCs as effector and Z138, SU-DHL4 as target cells (ADCC, LDH release assay); iv) Complement dependent cytotoxicity with Z138, SU-DHL4 as target cells (CDC, LDH release assay) and v) B-cell depletion (assessed by FACS) in whole blood from healthy donors. Dose-dependent anti-tumoral activity was assessed in a s.c. SU-DHL4 NHL xenograft model in Scid beige mice. Survival experiments in a disseminated Z138 MCL model are ongoing and an update on the results will be included as part of the poster presentation. Ofatumumab (“Arzerra”) was purchased from a local pharmacy, GA101 and rituximab were obtained from Hoffmann La Roche AG, Basel. First, binding studies confirmed that GA101 shows half-maximal binding to NHL cells relative to rituximab and ofatumumab, a known property of Type II CD20 antibodies. EC50 values of binding were comparable indicating that GA101, rituximab and ofatumumab have apparent binding affinities in the low nanomolar range on NHL cells independent of the level of CD20 expression. Second, the three CD20 antibodies were compared for their induction of direct cell death as measured by AxV/PI staining. Overall, GA101 mediated superior direct cell death induction compared to rituximab and ofatumumab utilizing a panel of NHL cell lines of different origins. Immune effector-related mechanisms of action were subsequently compared by ADCC and CDC assays. GA101, a glycoengineered antibody with enhanced affinity for FcgRIIIa, was found to exhibit up to 100-fold higher ADCC potency than rituximab and ofatumumab on Z138 and SU-DHL4 cells. CDC, as expected for a Type II CD20 antibody was ca. 10 to 1,000 less potent compared to the Type I antibodies rituximab and ofatumumab. In order to integrate the different mechanisms of action (direct cell death, ADCC, CDC), autologous ex vivo B-cell depletion assays with whole blood from healthy donors containing natural immune effector cells, human complement and physiological concentrations of human immunoglobulins were performed. These studies showed that GA101 was more potent in terms of EC50 values and more efficacious in terms of absolute B-cell depletion when compared to rituximab and ofatumumab. Finally, the dose-dependent effects of the three CD20 antibodies was studied on the growth of s.c. SU-DHL4 DLBCL xenografts in SCID beige mice. GA101 induced a dose-dependent anti-tumoral effect including complete tumor remission and was superior to the Type I antibodies rituximab and ofatumumab at saturating antibody doses. In summary, the preclinical data presented herein demonstrate that the Type II, glycoengineered CD20 antibody GA101 is differentiated from the Type I CD20 antibodies rituximab and ofatumumab by its superior overall activity supporting its further clinical investigation. Of note, in contrast to previous publications, in this series of assays no superior preclinical activity of ofatumumab was observed when compared to rituximab. Disclosures: Herter: Roche: Employment, Patents & Royalties. Waldhauer:Roche: Employment. Otz:Roche: Employment. Herting:Roche: Employment, Patents & Royalties. Lang:Roche: Employment. Nicolini:Roche: Employment. Römmele:Roche: Employment. Friess:Roche: Employment, Patents & Royalties. Van Puijenbroek:Roche: Employment. Bacac:Roche: Employment. Weidner:Roche: Employment, Equity Ownership. Gerdes:Roche: Employment, Equity Ownership, Patents & Royalties. Umana:Roche: Employment, Equity Ownership, Patents & Royalties. Klein:Roche: Employment, Equity Ownership, Patents & Royalties.

Crystal Structure Analysis Reveals That the Novel Type II Anti-CD20 Antibody GA101 Interacts with a Similar Epitope as Rituximab and Ocrelizumab but in a Fundamentally Different Way.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3726-3726 ◽  
Author(s):  
Gerhard J Niederfellner ◽  
Alfred Lammens ◽  
Manfred Schwaiger ◽  
Guy Georges ◽  
Kornelius Wiechmann ◽  
...  
Keyword(s):  
Research Funding ◽  
Type I ◽  
The Novel ◽  
Type Ii ◽  
Employment Equity ◽  
Equity Ownership ◽  
Cd20 Antibody ◽  
Anti Cd20 ◽  
Point Mutagenesis ◽  
Cd20 Antibodies

Abstract Abstract 3726 Poster Board III-662 CD20 is a specific cell surface marker found on normal as well as malignant B cells. Rituximab, a monoclonal antibody directed against CD20, has a major impact on treatment of malignant lymphomas. Although all therapeutic CD20 antibodies are directed against the two relatively small extracellular loops of CD20, such antibodies can be classified into Type I CD20 antibodies like Rituximab, Ofatumumab or Ocrelizumab or Type II CD20 antibodies like the novel glycoengineered humanized CD20 antibody GA101 or the murine antibody Tositumumab. Type I and Type II antibodies differ significantly in their mode of action and mechanisms of killing malignant B-cells. The molecular basis of this is not understood. We use data from epitope mapping, X-ray crystallography, isothermal titration calorimetry, and point mutagenesis i) to accurately map the epitopes of different anti-CD20 antibodies, in particular GA101, and ii) to compare the molecular interactions involved in their binding. Although the epitope regions of these antibodies largely overlap, the crystal structure shows that GA101 binds CD20 in a completely different orientation from Rituximab, Ocrelizumab and Ofatumumab and that its binding also involves a larger surface area. In agreement with predictions based on the crystallographic data, point mutagenesis of single amino acid residues confirmed that exchanges at certain positions in CD20 affect binding of Rituximab and GA101 differently. Our data suggest that engagement of CD20 by these antibodies favors different conformations of CD20, which could form the molecular basis for the observed differences in cellular signals triggered by the respective antibodies. Disclosures: Niederfellner: Roche: Employment. Schwaiger:Roche: Employment. Georges:Roche: Employment. Wiechmann:Roche: Research Funding. Franke:Roche: Research Funding. Schaefer:Roche: Employment. Jenewein:Roche: Employment. Slootstra:Pepscan: Employment, Patents & Royalties. Moessner:Glycart: Employment, Equity Ownership, Patents & Royalties. Umana:Glycart: Employment, Equity Ownership, Patents & Royalties. Hopfner:Roche: Research Funding. Klein:Roche: Employment, Equity Ownership, Patents & Royalties.

Protein Tomography Analysis Shows That CD20 Molecules Bound by Different Type I and Type II Anti-CD20 Antibodies Are Organized in Different Protein Complexes.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4782-4782
Author(s):  
Gerhard J Niederfellner ◽  
Annika Braennstroem ◽  
Frida Lindstrom ◽  
Magnus Jansson ◽  
Marika Lundin ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Large Fraction ◽  
Type I ◽  
Type Ii ◽  
Employment Equity ◽  
Downstream Signaling ◽  
Equity Ownership ◽  
Anti Cd20 ◽  
Tomography Analysis ◽  
Cd20 Antibodies

Abstract Abstract 4782 Ramos cells were labeled with Type I (Rituximab and Ocrelizumab) and Type II (B1/H299 and GA101) anti-CD20 antibodies and then fixed and stained with marker gold labeled 2ary antibodies. Protein complexes connected to marker gold were analyzed by Protein TomographyTM at SIDEC and the corresponding 3D-structures visualized (∼100 structures per antibody). Less than 5 % of the refined structures were dimeric. While most (60 – 90%) CD20 molecules were present in tetramers or even higher order defined complexes, a sizeable proportion was also engaged in large protein networks (11 -38%). For the multimeric complexes, we could clearly distinguish between extended (or open) and ring-like (or closed) conformations. Although cells had been labeled with an excess of antibody, as suggested by FACS binding curves, the antibodies bound CD20 monovalently in most refined structures. Bivalent binding was overall more prevalent with Rituximab and Ocrelizumab than with B1 and GA101 (1/3 vs 1/6 structures). The proportion of CD20 molecules present in ringlike complexes was higher for the Type II than for the Type I antibodies. In co-localization experiments, Rituximab-CD20 and GA101-CD20 complexes were also found to only partially colocalize, while a large fraction of the two antibodies were found in separate cell surface compartments. These findings suggest that the different antibodies favor different CD20 conformations that seem to be associated with different protein complexes and might form the basis for initiation of different downstream signaling processes. Disclosures: Niederfellner: Roche: Employment. Braennstroem:SIDEC: Employment. Lindstrom:SIDEC: Employment. Jansson:SIDEC: Employment. Lundin:SIDEC: Employment. Schaefer:Roche: Employment. Mundigl:Roche: Employment. Umana:Glycart: Employment, Equity Ownership, Patents & Royalties. Klein:Roche: Employment, Equity Ownership, Patents & Royalties.

GA101, a Novel Humanized Type II CD20 Antibody with Glycoengineered Fc and Enhanced Cell Death Induction, Exhibits Superior Anti-Tumor Efficacy and Superior Tissue B Cell Depletion In Vivo.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2348-2348 ◽  
Author(s):  
Pablo Umana ◽  
Moessner Ekkehard ◽  
Bruenker Peter ◽  
Klingner Gabriele ◽  
Puentener Ursula ◽  
...  
Keyword(s):  
Cell Death ◽  
B Cells ◽  
Lymph Nodes ◽  
B Cell ◽  
Cell Depletion ◽  
Type Ii ◽  
B Cell Depletion ◽  
Cynomolgus Monkeys ◽  
High Affinity ◽  
Cd20 Antibody

Abstract GA101 is a novel monoclonal antibody of IgG1 type which binds with high affinity and selectivity to the extracellular domain of the human CD20 antigen on B cells. In contrast to rituximab which is a chimeric antibody and recognizes a type I epitope, GA101 is humanized and recognizes a type II epitope which is also localized in the extracellular loop of CD20. The recognition of the type II epitope together with a modification of the elbow hinge region results in enhanced direct non-caspase dependent cell death induction, and concomitant reduction in CDC upon binding to CD20. In addition, using GlycoMab technology, the Fc-region of GA101 was glycoengineered to contain bisected, afucosylated carbohydrates. As a result GA101 has increased affinity for the low and high affinity FcγRIIIa receptor expressed on natural killer cells, macrophages and monocytes. Consequently, GA101 mediated a 5–50 fold enhanced induction of effector cell mediated ADCC. In B-cell depletion assays with whole blood from healthy donors, an assay combining all mechanisms of action, GA101 was significantly more potent and efficacious in depleting B cells than rituximab. In preclinical NHL testing these properties translated into superior anti-tumoral efficacy of GA101 in direct comparison to rituximab against a number of aggressive NHL xenograft models. In cynomolgus monkeys the induction of B cell depletion mediated by GA101 and subsequent B cell recovery were investigated. GA101 induced complete, rapid and long-lasting B cell depletion both in peripheral blood and in lymphoid tissue e.g. spleen and lymph nodes. The efficacy of GA101 (10 and 30 mg/kg) at depleting B cells in different lymphoid tissues of cynomolgus monkeys was compared with that of rituximab (10 mg/kg) following 2 i.v. doses administered on days 0 and 7. Notably, GA101 showed statistically superior depletion of total B cells from lymph nodes compared to Rituximab from day 9 to 35 onwards with B cell numbers decreased by over 95%. These results demonstrated that GA101 was more efficacious at depleting B cells from lymph nodes and spleen of cynomolgus monkeys compared to rituximab. Compared to existing antibodies, GA101 constitutes the first type II CD20 antibody engineered for increased ADCC with significantly enhanced efficacy in a variety of preclinical models. Based on these data it is assumed that the combination of the recognition of a type II epitope together with improved ADCC potency might translate into superior efficacy in the clinical treatment of CD20 positive malignant diseases.

Survival Pathways Stabilized By Proteasome Inhibition Reveal PIM2 As a Novel Target For Potentiating The Anti-Myeloma Activity Of Carfilzomib

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4440-4440
Author(s):  
Tracey Lin ◽  
Eric Lowe ◽  
Alana Lerner ◽  
Christopher J. Kirk ◽  
Shirin Arastu-Kapur
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Cell Lines ◽  
Proteasome Inhibition ◽  
Myeloma Cell ◽  
Protein Accumulation ◽  
Employment Equity ◽  
Survival Pathways ◽  
Equity Ownership ◽  
Dose Dependent

In recent years, new agents for multiple myeloma treatment (e.g., proteasome inhibitors) have become more efficacious, yet nearly all patients eventually relapse and develop refractory disease. Growing evidence suggests that clonal heterogeneity in multiple myeloma may constitute the basis for treatment resistance. Therefore, a multi-pronged approach with novel agents is needed to increase the efficacy of standard therapy and prevent or overcome resistance to standard treatments. We have undertaken a research effort to discover novel targets that potentiate the anti-tumor effects of proteasome inhibition in myeloma cells. We hypothesized that proteins that are stabilized in tumor cells following proteasome inhibition likely constitute components of both pro-apoptotic and pro-survival pathways. A mass spectrometry approach, referred to as UbiScan®, was employed to determine the identity and levels of cellular proteins modified with ubiquitin. MM cell lines (U266 and NCI-H929) were treated with either carfilzomib (CFZ) or bortezomib (BTZ) for 1 hour and the ubiquitome was profiled at 1 and 3 hours after culture in drug-free media. A concentration of 125 nM was chosen in order to reflect physiologically relevant drug and target inhibition levels and to induce cell death in ∼80% of cells after 48 hours. Approximately 400 proteins showed similar increases in ubiquitination with CFZ or BTZ. One of these proteins was PIM2, a serine/threonine proto-oncogene required for plasma cell proliferation that is highly expressed in multiple myeloma cell lines. We determined that ubiquitination on PIM2 was occurring at lysine 61, which is known to be associated with proteasomal degradation. Four hours after exposure to CFZ, PIM2 ubiquitination increased 34.6 and 24.9 fold in U266 and H929 cells, respectively, and similar changes were measured following BTZ treatment. Western blot analysis of CFZ-treated cells showed a dose-dependent accumulation of total PIM2 protein, confirming that the increase in ubiquitination correlated with protein accumulation. Next, we employed a siRNA-mediated knockdown approach to study the role of PIM2 in proteasome inhibitor mediated-cell death. Knockdown of PIM2 caused a 20% - 50% decrease in viability in both myeloma cell lines. When CFZ was added 48 hours after siRNA transfection, a significant and dose-dependent decrease in viability was observed, suggesting a synergistic interaction. Based on these results, we tested the combination of CFZ and (Z)-5-(4-propoxybenzylidene)thiazolidine-2,4-dione (PIM1/2 inhibitor), which is known to inhibit both PIM1 and PIM2. The PIM1/2 inhibitor decreased levels of phosphorylation on 4E-BP1, a downstream target, confirming its activity in cells. Chemical inhibition of PIM2 potentiated the effect of CFZ in both MM cell lines. These data suggest that the combination of targeting PIM2 and the proteasome will be efficacious in the treatment of multiple myeloma. Disclosures: Lin: Onyx Pharmaceuticals, Inc.: Employment. Lowe:Onyx Pharmaceuticals, Inc.: Employment, Equity Ownership. Lerner:Onyx Pharmaceuticals, Inc.: Employment, Equity Ownership. Kirk:Onyx Pharmaceuticals: Employment, Equity Ownership. Arastu-Kapur:Onyx Pharmaceuticals, Inc.: Employment, Equity Ownership.

Combination of the glycoengineered Type II CD20 antibody obinutuzumab (GA101) and The novel Bcl-2 selective Inhibitor GDC-0199 Results in superior In Vitro and In Vivo Anti-tumor activity in models Of B-Cell Malignancies

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4412-4412 ◽  
Author(s):  
Deepak Sampath ◽  
Sylvia Herter ◽  
Frank Herting ◽  
Ellen Ingalla ◽  
Michelle Nannini ◽  
...  
Keyword(s):  
Cell Death ◽  
Single Agent ◽  
Employment Equity ◽  
Direct Cell ◽  
Xenograft Models ◽  
Equity Ownership ◽  
Anti Cd20 ◽  
Agent Treatment

Introduction Obinutuzumab (GA101) is a novel glycoengineered type II, anti-CD20 monoclonal antibody induces a high level of direct cell death. As a result of glycoengineering, GA101 has increased affinity for FcgRIIIa on effector cells resulting in enhanced direct cell death and ADCC induction. GA101 is currently in pivotal clinical trials in CLL, indolent NHL and DLCBL. ABT-199 (GDC-0199) is a novel, orally bioavailable, selective Bcl-2 inhibitor that induces robust apoptosis in preclinical models of hematological malignancies and is currently in clinical trials for CLL, NHL and MM. Based on their complementary mechanisms of action involving increased apoptosis (GDC-0199) or direct cell death (GA101) the combination of anti-CD20 therapy with a Bcl-2 inhibitor has the potential for greater efficacy in treating B lymphoid malignancies. Experimental Methods The combination of GA101 or rituximab with GDC-0199 was studied in vitro utilizing assays that measure direct cell death induction/apoptosis (AxV/Pi positivity) on WSU-DLCL2, SU-DHL4 DLBCL and Z138 MCL cells by FACS and the impact of Bcl-2 inhibition on ADCC induction. In vivo efficacy of the combination of GA101 or rituximab and GDC-0199 was evaluated in SU-DHL4 and Z138 xenograft models. Results GA101 and rituximab enhanced cell death induction when combined with GDC-0199 in SU-DHL4, WSU-DLCL2 and Z138 cell lines. When combined at optimal doses an additive effect of the two drugs was observed. GDC-0199 did not negatively impact the capability of GA101 or rituximab to induce NK-cell mediated ADCC. Combination of GDC-0199 and GA101 induced a greater than additive anti-tumor effects in the SU-DHL4 and Z138 xenograft models resulting in tumor regressions and delay in tumor regrowth when compared to monotherapy. Moreover, continued single-agent treatment with GDC-0199 after combination with GA101 resulted in sustained in vivo efficacy in the SU-DHL4 model. Conclusions Our data demonstrate that the combination of GA101 with GDC-0199 results in enhanced cell death and robust anti-tumor efficacy in xenograft models representing NHL sub-types that is comparable to the combination of rituximab with GDC-0199. In addition, single-agent treatment with GDC-0199 following combination with GA101 sustains efficacy in vivo suggesting a potential benefit in continued maintenance therapy with GDC-0199. Collectively the preclinical data presented here supports clinical investigation of GA101 and GDC-0199 combination therapy, which is currently in a phase Ib clinical trial (clinical trial.gov identifier NCT01685892). Disclosures: Sampath: Genentech: Employment, Equity Ownership. Herter:Roche: Employment. Herting:Roche: Employment. Ingalla:Genentech: Employment. Nannini:Genentech: Employment. Bacac:Roche: Employment. Fairbrother:Genentech: Employment, Equity Ownership. Klein:Roche Glycart AG: Employment.

scholarly journals Reduction of Mitochondrial Membrane Potential Leads to Enhancement of Type-II CD20-Antibody Cytotoxicity in Diffuse Large B-Cell Lymphoma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1763-1763 ◽  
Author(s):  
Emma Vilventhraraja ◽  
Tetyana Klymenko ◽  
Jennifer Edelmann ◽  
John Gribben ◽  
Andrejs Ivanov
Keyword(s):  
Oxidative Phosphorylation ◽  
Cell Lines ◽  
Mitochondrial Function ◽  
Type I ◽  
Type Ii ◽  
Antibody Treatment ◽  
Respiratory Capacity ◽  
Cd20 Antibody ◽  
Anti Cd20 ◽  
Cd20 Antibodies

Abstract Diffuse Large B Cell Lymphoma (DLBCL) is the most prevalent non-Hodgkin lymphoma (NHL) in adults. Since the addition of the Type I anti-CD20 antibody Rituximab to chemotherapy, the overall survival of NHL patients has improved dramatically compared to the pre-Rituximab era. DLBCL however, has the worst survival rates out of all NHLs with an average 5-year survival of 55%. Unfortunately 40% of all DLBCL patients relapse within 2 years, and those that relapse or have refractory disease tend not to respond well to antibody-based salvage therapies. Since the discovery and utilisation of Rituximab, many have tried to enhance the efficacy of anti-CD20 antibodies in order to improve first-line treatment of DLBCL, leading to the evolution of Type II humanised anti-CD20 antibodies. The complete biological role of CD20 remains unclear, however it has been shown to act as part of an ion channel complex that is a component of the store operated calcium (Ca2+) system. This complex has the ability to facilitate mitochondrial membrane permeabilisation, resulting in reduced mitochondrial function. In order to investigate the effect of Type I- and Type II- anti-CD20 antibodies on mitochondrial function, we established a panel of 4 DLBCL cell lines. We used the XF Seahorse Mito Stress Test to reveal bioenergetic profiles of the cell lines before and after treatment with a panel of Type I and Type II anti-CD20 antibodies (2 Type-I and 2 Type-II anti-CD20 antibodies for each cell line). Basal oxidative phosphorylation (OxPhos), ATP production, and maximal and spare respiratory capacity of each sample were calculated as a measure of mitochondrial function. Next we used Metformin, a well-established inhibitor of oxidative phosphorylation to reduce the mitochondrial membrane potential (MMP) across our panel of cell lines. We confirmed MMP reduction by staining cells with JC-1, a chameleon dye used as an indicator of MMP and analysed samples using flow cytometry. We then used the XF Seahorse Mito Stress Test, this time to assess how combining each CD20-antibody with an OxPhos inhibitor effects mitochondrial function (10 conditions for each cell line). Finally, we used the same conditions to conduct clonogenic survival assays to see whether cytotoxicity of Type-I or Type-II anti-CD20 antibodies could be enhanced. We have observed that treatment with anti-CD20 antibodies results in a significant increase in the maximal respiratory capacity of our panel of cell lines. Conversely, pharmacological inhibition of oxidative phosphorylation causes a significant reduction in basal oxidative phosphorylation as well as a reduction in the maximal respiratory capacity of the cell lines in our panel. We also show that treatment combining an OxPhos inhibitor with either Type-I or Type-II CD20-antibodies prevents the increase in maximal respiratory capacity observed with CD20-antibody treatment alone. When analysing the clonogenic survival of cell lines we have found that only the cytotoxicity of Type-II anti-CD20 antibodies is enhanced by simultaneously treating cell lines with Metformin. We also used Annexin V/PI staining to assess cell death and show that inhibiting oxidative phosphorylation in conjunction with CD20-antibody treatment does not result in a significant increase in cell death across our panel of cell lines. Our data indicate for the first time that when cells are treated with CD20-antibodies they increase their maximal mitochondrial respiratory capacity to compensate for reduced basal mitochondrial function. We also show that inhibition of oxidative phosphorylation disables the cells from being able to compensate for the reduced mitochondrial function that is caused by CD20-antibody treatment. Importantly our data show that the reduction of mitochondrial function caused by combining Metformin with Type-II CD20 antibodies leads to a significant reduction in clonogenicity. We believe that understanding the mechanism of the inhibition of mitochondrial function will allow us to establish effective treatment combinations to significantly improve the efficacy of anti-CD20 antibody therapy in DLBCL. Disclosures No relevant conflicts of interest to declare.

scholarly journals STEM-09. INTRINSIC INTERFERON SIGNALING REGULATES THE CELL DEATH OF GLIOBLASTOMA STEM CELLS

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii198-ii198
Author(s):  
Sabbi Khan Khan ◽  
Emmanuel Martinez-Ledesma ◽  
Sandeep Mittal ◽  
Kaitlin Gandy ◽  
Kristin Alfaro-Munoz ◽  
...  
Keyword(s):  
Cell Death ◽  
Treatment Resistance ◽  
Primary Brain Tumor ◽  
The Cancer Genome Atlas ◽  
Cytokine Signaling ◽  
Type I ◽  
Type Ii ◽  
Mechanistic Investigation ◽  
Differential Cell ◽  
Stat1 Signaling

Abstract Glioblastoma (GBM) is the most common, highly aggressive, and lethal primary brain tumor in adults. Interferon (IFN)-mediated signal transducer and activator of transcription 1 (STAT1) signaling contributes to various aspects of stemness, cell death, cytokine signaling in immune and non-immune cells. However, the role of IFN/STAT1 signaling in stemness, cell death and treatment resistance in GBM is unclear. This study aimed to investigate the cancer cell-intrinsic IFN/STAT1 signaling and its role in cell proliferation, stemness, and apoptosis. By using the metagene scores for type I and type II IFN-responsive genes, we evaluated basal IFN/STAT1 signaling in The Cancer Genome Atlas (TCGA) and in patient-derived cohorts of stem-like cells (GSCs) RNA expression datasets. In-silico analyses were further validated for the constitutive IFN signaling in a subset of GSCs using qPCR, WB and ELISA assays. We employed pharmacological activators and/or inhibitors of IFN/STAT1 signaling in GSCs to study its role in stemness and cell death. We found differential cell-intrinsic type I and type II IFN-signaling markers in GSCs and GBM tumors. High IFN-signaling is associated with mesenchymal phenotype and poor survival outcomes. Acute and chronic GSC exposure to recombinant IFNs reversibly activated both type I and II IFN-signaling in GSCs. IFN-β exposure induced apoptosis in intrinsically high IFN/STAT1-signaling GSCs, but not in the low IFN/STAT1-signaling GSCs. In summary, our findings demonstrate that GBM exhibit differential cell-intrinsic IFN-signaling, and basal IFN/STAT1 is a key factor for IFN-β-mediated cell death in GSCs. However, further mechanistic investigation of intrinsic IFN signaling in GBM, particularly in the stem cell compartment is needed.

Ketamine, an N -methyl-d-aspartate Receptor Antagonist, Inhibits the Spinal Neuronal Responses to Distension of the Rat Urinary Bladder

2002 ◽  
Vol 96 (6) ◽  
pp. 1410-1419 ◽  
Author(s):  
Pablo J. Castroman ◽  
Timothy J. Ness
Keyword(s):  
Spinal Cord ◽  
Urinary Bladder ◽  
Spinal Dorsal Horn ◽  
Type I ◽  
Type Ii ◽  
Neuronal Responses ◽  
Spinal Dorsal ◽  
Aspartate Receptor ◽  
Dose Dependent ◽  
Intravenous Ketamine

Background The effect of ketamine as a treatment of visceral pain is not known. The current study investigated the effect of ketamine on spinal dorsal horn neurons excited by urinary bladder distension (UBD). The effect of other clinically available N-methyl-D-aspartate receptor antagonists on these responses was also studied. Methods Extracellular recordings of neurons located in the L6-S2 spinal dorsal horn of cervical spinal cord-transected, decerebrate female rats were obtained. Cutaneous receptive fields of neuronal units excited by UBD were characterized for responses to segmental noxious and nonnoxious stimuli. Nonsegmental noxious stimuli were also applied, and neurons were classified as type I (inhibited) and type II (noninhibited) by the stimulus. The effect of intravenous ketamine (1, 3, and 10 mg/kg), dextromethorphan (5 mg/kg), and memantine (16 mg/kg) on neuronal responses of these units was measured. Results Spontaneous and evoked neuronal activity to UBD was reduced in a dose-dependent fashion by ketamine. Responses to nonnoxious cutaneous stimuli were also significantly reduced after treatment. Dextromethorphan inhibited neuronal activity evoked by UBD in type I neurons. A similar selective effect of treatment on type I versus type II neurons was observed after intravenous ketamine and memantine. Conclusions Intravenous ketamine produces dose-dependent inhibition of the spinal cord neuronal responses evoked by UBD. All three N-methyl-D-aspartate receptor antagonists showed selective effects on spinal cord neurons subject to counterirritation. This neurophysiologic evidence supports a spinally mediated analgesic effect of ketamine in this model of urinary bladder nociception, an effect likely caused by N-methyl-D-aspartate receptor antagonism.

Carfilzomib Exerts Anti-Neoplastic Activity in Waldenstrom Macroglobulinemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4916-4916
Author(s):  
Antonio Sacco ◽  
Aldo M. Roccaro ◽  
Monette Aujay ◽  
Hai Ngo ◽  
Feda Azab ◽  
...  
Keyword(s):  
Cell Lines ◽  
Low Grade ◽  
Dependent Manner ◽  
Caspase 9 ◽  
Employment Equity ◽  
Waldenström Macroglobulinemia ◽  
Waldenstrom Macroglobulinemia ◽  
Synergistic Cytotoxicity ◽  
Equity Ownership ◽  
Dose Dependent

Abstract Abstract 4916 Introduction Proteasome inhibition represents a valid therapeutical approach in several tumors and its use has been validated in Waldenstrom's macroglobulinemia (WM), where single-agent Bortezomib has been successfully tested in phase 2 clinical trials. Nevertheless, a significant fraction of patients relapse, or develop significant toxicity due to high toxicity in non-transformed cells. Therefore preclinical evaluation of new proteasome inhibitor with a more selective inhibition of neoplastic cells is needed in order to increase efficacy and improve patient outcome. We tested Carfilzomib, a tetrapeptide epoxyketone selective inhibitor of the chymotrypsin-like activity of the immunoproteasome and constitutive proteasome in WM. Methods WM and IgM secreting low-grade lymphoma cell lines (BCWM.1, MEC1, RL) were used. Expression of imunoproteasome and constitutive proteasome subunits (beta1, beta2, beta5; LMP2, MECL1, LMP7) were detected primary WM cells and cell lines by an ELISA-based assay. Cytotoxicity and DNA synthesis were measured by thymidine uptake and MTT, respectively. Cell signaling and apoptotic pathways were determined by Western Blot. Determination of the additive or synergistic effect of drugs combination was calculated using the CalcuSyn software based on the Chou-Talalay method. Results Primary CD19 bone-marrow derived WM cells express higher level of the immunopreoteasome as compared to the constitutive proteasome. Carfilzomib inhibited the chymotrypsin-like activity of both the immunoproteasome (LMP7) and the constitutive proteasome (beta5) and in WM cells, in a dose-dependent manner; leading to inhibition of proliferation (IC50: 5nM; 48h) and induction of cytotoxicity (IC50: 7.5nM; 48h) in WM cells. Carfilzomib mediated apoptosis in WM by increasing PARP-, caspase-9- and -3-cleavage; as well as by inducing activation of c-jun-N-terminal kinase and ER-stress in a dose-dependent manner. Moreover, combination of Carfilzomib and bortezomib induced synergistic cytotoxicity in WM cells, as shown by enhanced PARP-, caspase-9- and -3-cleavage; and synergy in inhibiting the chymotrypsin-like activity of the immunoproteasome and constitutive proteasome. Conclusion Taken together, these findings provide the pre-clinical rational for testing Carfilzomib in Waldenstrom Macroglobulinemia. Disclosures Aujay: Proteolix: Employment, Equity Ownership. Demo:Proteolix: Employment, Equity Ownership. Ghobrial:Millennium: Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.

Differential Patterns of Gene Expression and CD20 Localisation by the Type II Anti-CD20 Antibody Obinutuzumab (GA101) Compared with the Type I Antibody Rituximab Suggests Binding to Functionally Distinct CD20 Subpopulations on B-Cell Lymphoma Cell Lines,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3721-3721
Author(s):  
Gerhard Niederfellner ◽  
Olaf Mundigl ◽  
Alexander Lifke ◽  
Andreas Franke ◽  
Ute Baer ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Mechanisms Of Action ◽  
Type I ◽  
Type Ii ◽  
Cd20 Antibody ◽  
Anti Cd20

Abstract Abstract 3721 The anti-CD20 antibody rituximab has become central to the treatment of B-cell malignancies over the last decade. Recently, it has been shown that anti-CD20 antibodies can be divided into two types based on their mechanisms of action on B cells. Rituximab is a type I antibody that redistributes CD20 into lipid rafts and promotes complement-dependent cytotoxicity (CDC), while the type II, glycoengineered antibody GA101 has lower CDC activity but higher antibody-dependent cellular cytotoxicity and direct cell death activity. In preclinical studies GA101 was superior to rituximab in B-cell killing in vitro, depletion of B cells from whole blood, and inhibition of tumour cell growth in lymphoma xenograft models. GA101 is currently being evaluated in Phase II/III trials, including comparative studies with rituximab. To investigate the differences in direct effects of GA101 and rituximab on B-cell lymphoma signaling, we have analysed the effects of antibody binding on gene expression in different B-cell lines using a GeneChip Human Genome U133 Plus 2.0 Array (Affymetrix). Rituximab and GA101 rapidly induced gene expression changes in SUDHL4 and Z138 cells, including regulation of genes associated with B-cell-receptor activation such as EGR2, BCL2A1, RGS1 and NAB2. The effects on gene expression differed markedly between different cell lines and between the two antibodies. SUDHL4 cells showed pronounced changes in the gene expression pattern to rituximab treatment, while Z138 cells, which represent a different B-cell stage, showed less pronounced changes in gene expression. The reverse was true for GA101, suggesting not only that the signaling mediated by CD20 differs in different cell lines, but also that in a given cell line the two types of antibodies bind CD20 molecules with different signaling capacity. For each cell line, gene expression induced by other type I antibodies (LT20, 2H7, MEM97) was more like rituximab and that induced by other type II antibodies (H299/B1, BH20) was more like GA101 in terms of the number of genes regulated and the magnitude of changes in expression. Unbiased hierarchical clustering analysis of gene expression in SUDHL4 could discriminate type I from type II antibodies, confirming that the two classes of antibody recognised CD20 complexes with inherently different signalling capacities. By confocal and time-lapse microscopy using different fluorophores, rituximab and GA101 localised to different compartments on the membrane of lymphoma cells. GA101/CD20 complexes were relatively static and predominantly associated with sites of cell–cell contact, while rituximab/CD20 complexes were highly dynamic and predominantly outside areas of contact. These findings suggest that type II antibodies such as GA101 bind distinct subpopulations of CD20 compared with type I antibodies such as rituximab, accounting for the differences in mechanisms of action and anti-tumour activity between these antibodies. Disclosures: Niederfellner: Roche: Employment. Mundigl:Roche: Employment. Lifke:Roche: Employment. Franke:Roche: Employment. Baer:Roche: Employment. Burtscher:Roche: Employment. Maisel:Roche: Employment. Belousov:Roche: Employment. Weidner:Roche: Employment. Umana:Roche: Employment, Patents & Royalties. Klein:Roche: Employment, Equity Ownership, Patents & Royalties.

Sign in / Sign up

Export Citation Format

Share Document