The induction of immunogenic cell death by type II anti-CD20 monoclonal antibodies has mechanistic differences compared with type I rituximab

Abstract Abstract 3979 Although treatment results for Chronic Lymphocytic Leukemia (CLL) have improved considerably over the last decade, unfortunately a curative treatment is still not available. In part this might be due to the interaction of CLL cells with their micro-environment in lymph nodes, spleen and bone marrow. Micro-environment derived signals are not only capable of driving proliferation of CLL cells, but can also induce resistance of CLL to cytotoxic drugs. Previously we have shown that in vitro CD40 stimulation of peripheral blood derived CLL cells can to a certain extent mimic the lymph node microenvironment and result in resistance to cytotoxic drugs (Kater AP et al. Br J Haematol 2004;127:404; Smit LA et al. Blood 2007;109:1660.; Hallaert DY et al. Blood 2008;112:5141). At present it is not known whether sensitivity of CLL cells to CD20 monoclonal antibodies (mAbs) is modulated by micro environmental stimuli. Therefore in the present study we investigated anti-CD20 mediated cell death of CD40-stimulated CLL cells from 17 CLL patients. We observed that in sharp contrast with the response towards cytotoxic drugs, CD40 stimulation sensitizes CLL cells to cell death mediated by anti-CD20 mAbs. In CD40-stimulated CLL cells, cell death induced by both Rituximab (a type I anti-CD20 mAb) and GA101 (a novel type II anti-CD20 mAb) (Moessner E et al. Blood, 2010;127:404, 115(22):4393-402) is increased. Both anti-CD20 mAbs induce a non-apoptotic, caspase- and p53-independent rapid cell death, but interestingly the mechanism of Rituximab and GA101-induced cell death appears to be different. Rituximab-induced cell death is dependent on extracellular Ca2+ and ROS production and CD40 stimulation sensitizes CLL cells by increasing basal ROS production. In contrast, GA101 induces cell death via a lysosome-dependent mechanism and CD40 stimulation sensitizes CLL cells by increasing the lysosomal volume of the cell. Moreover, in contrast to Rituximab, GA101 induces cell death in the absence of a secondary crosslinking mAb. Combination of GA101 with fludarabine, chlorambucil, bortezomib or bendamustine shows additive effects and results in strong cell death of CD40-stimulated CLL cells, even in p53 dysfunctional CLL cells. Our findings not only provide a rationale for combining cytotoxic drugs and anti-CD20 monoclonal antibodies, but also show that GA101 is a potent promising new anti-CD20 mAb for the treatment of CLL. Disclosures: Klein: Roche: Employment, Equity Ownership, Patents & Royalties.

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Obinutuzumab (GA101) More Potently Engages Phagocytic-Lineage Cells Resulting In Enhanced Monocyte and Macrophage Activity When Compared To Rituximab and Ofatumumab

Blood ◽

10.1182/blood.v122.21.5136.5136 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5136-5136 ◽

Cited By ~ 2

Author(s):

Sylvia Herter ◽

Christian Klein ◽

Pablo Umana ◽

Marina Bacac

Keyword(s):

Cell Death ◽

Tumor Cell ◽

Nk Cell ◽

Antibody Activity ◽

Type I ◽

Type Ii ◽

Therapeutic Antibodies ◽

Tumor Cell Death ◽

Macrophage Activity ◽

Anti Cd20

Abstract Therapeutic antibodies possess several clinically relevant mechanisms of action including cell death induction, perturbation of tumor cell signaling, activation of complement dependent cytotoxicity (CDC), antibody dependent cellular cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP) and induction of adaptive immunity. Obinutuzumab (GA101) is a novel humanized, glycoengineered Type II anti-CD20 monoclonal antibody engineered for displaying enhanced FcγRIIIa (CD16) binding affinity and characterized by stronger induction of ADCC and direct tumor cell death when compared to wild-type, Type I anti-CD20 antibodies rituximab and ofatumumab. In light of the important role of phagocytic lineage cells in the mechanism of action of therapeutic antibodies, we compared GA101, rituximab and ofatumumab for their ability to trigger FcγR-dependent monocyte and macrophage effector functions. We show that, due to glycoengineering, GA101 displays superior CD16-dependent binding to monocytes, M1 and M2c macrophages in presence of nonspecific, competing, human endogenous IgGs, a situation that more closely mimics physiological conditions. Subsequently, GA101 more strongly engages monocytes and macrophages and leads to significantly higher elimination of CD20-expressing tumor cells as shown by assays detecting total antibody activity (ADCP, ADCC and direct effects). In support of the stronger GA101 activity, higher nitric-oxide (NO) levels are also detected in supernatants of tumor/macrophage co-cultures treated with antibody. Taken together, our data show that in addition to stronger NK-cell mediated ADCC and direct cell death induction due to Type II CD20 binding, GA101 more potently engages phagocytic-lineage cells resulting in enhanced monocyte and macrophage activity under conditions that more closely resemble physiological settings. Disclosures: Herter: Roche: Employment. Klein:Roche Glycart AG: Employment. Umana:Roche: Employment, Equity Ownership. Bacac:Roche: Employment.

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Novel type II anti-CD20 monoclonal antibody (GA101) evokes homotypic adhesion and actin-dependent, lysosome-mediated cell death in B-cell malignancies

Blood ◽

10.1182/blood-2010-07-296913 ◽

2011 ◽

Vol 117 (17) ◽

pp. 4519-4529 ◽

Cited By ~ 193

Author(s):

Waleed Alduaij ◽

Andrei Ivanov ◽

Jamie Honeychurch ◽

Eleanor J. Cheadle ◽

Sandeep Potluri ◽

...

Keyword(s):

Cell Death ◽

B Cell ◽

Actin Polymerization ◽

Type I ◽

Type Ii ◽

B Cell Malignancies ◽

Therapeutic Success ◽

Wide Range ◽

Anti Cd20

Abstract The anti-CD20 mAb rituximab has substantially improved the clinical outcome of patients with a wide range of B-cell malignancies. However, many patients relapse or fail to respond to rituximab, and thus there is intense investigation into the development of novel anti-CD20 mAbs with improved therapeutic efficacy. Although Fc-FcγR interactions appear to underlie much of the therapeutic success with rituximab, certain type II anti-CD20 mAbs efficiently induce programmed cell death (PCD), whereas rituximab-like type I anti-CD20 mAbs do not. Here, we show that the humanized, glycoengineered anti-CD20 mAb GA101 and derivatives harboring non-glycoengineered Fc regions are type II mAb that trigger nonapoptotic PCD in a range of B-lymphoma cell lines and primary B-cell malignancies. We demonstrate that GA101-induced cell death is dependent on actin reorganization, can be abrogated by inhibitors of actin polymerization, and is independent of BCL-2 overexpression and caspase activation. GA101-induced PCD is executed by lysosomes which disperse their contents into the cytoplasm and surrounding environment. Taken together, these findings reveal that GA101 is able to potently elicit actin-dependent, lysosomal cell death, which may potentially lead to improved clearance of B-cell malignancies in vivo.

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Ceramide participates in lysosome-mediated cell death induced by type II anti-CD20 monoclonal antibodies

Leukemia & Lymphoma ◽

10.3109/10428194.2014.981179 ◽

2015 ◽

Vol 56 (6) ◽

pp. 1863-1868 ◽

Cited By ~ 2

Author(s):

Yuzhi Liu ◽

Ling Shu ◽

Jingjing Wu

Keyword(s):

Cell Death ◽

Monoclonal Antibodies ◽

Type Ii ◽

Anti Cd20

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Faculty Opinions recommendation of Novel type II anti-CD20 monoclonal antibody (GA101) evokes homotypic adhesion and actin-dependent, lysosome-mediated cell death in B-cell malignancies.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.718490075.793497237 ◽

2014 ◽

Author(s):

Stephan Stilgenbauer

Keyword(s):

Monoclonal Antibody ◽

Cell Death ◽

B Cell ◽

Type Ii ◽

B Cell Malignancies ◽

Anti Cd20 ◽

Homotypic Adhesion

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

Neuro-Oncology ◽

10.1093/neuonc/noaa215.826 ◽

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.

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Biomarkers of Radiotherapy-Induced Immunogenic Cell Death

Cells ◽

10.3390/cells10040930 ◽

2021 ◽

Vol 10 (4) ◽

pp. 930

Author(s):

Rianne D. W. Vaes ◽

Lizza E. L. Hendriks ◽

Marc Vooijs ◽

Dirk De Ruysscher

Keyword(s):

Cell Death ◽

Immune Responses ◽

Tumor Cells ◽

Immunogenic Cell Death ◽

Type I ◽

Future Studies ◽

Regulated Cell Death ◽

Molecular Patterns ◽

Damage Associated Molecular Patterns ◽

Potential Biomarkers

Radiation therapy (RT) can induce an immunogenic variant of regulated cell death that can initiate clinically relevant tumor-targeting immune responses. Immunogenic cell death (ICD) is accompanied by the exposure and release of damage-associated molecular patterns (DAMPs), chemokine release, and stimulation of type I interferon (IFN-I) responses. In recent years, intensive research has unraveled major mechanistic aspects of RT-induced ICD and has resulted in the identification of immunogenic factors that are released by irradiated tumor cells. However, so far, only a limited number of studies have searched for potential biomarkers that can be used to predict if irradiated tumor cells undergo ICD that can elicit an effective immunogenic anti-tumor response. In this article, we summarize the available literature on potential biomarkers of RT-induced ICD that have been evaluated in cancer patients. Additionally, we discuss the clinical relevance of these findings and important aspects that should be considered in future studies.

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Antigenic modulation limits the effector cell mechanisms employed by type I anti-CD20 monoclonal antibodies

Blood ◽

10.1182/blood-2014-07-588376 ◽

2015 ◽

Vol 125 (12) ◽

pp. 1901-1909 ◽

Cited By ~ 43

Author(s):

Thomas R. W. Tipton ◽

Ali Roghanian ◽

Robert J. Oldham ◽

Matthew J. Carter ◽

Kerry L. Cox ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Natural Killer Cell ◽

Natural Killer ◽

Effector Cell ◽

Killer Cell ◽

Type I ◽

Antigenic Modulation ◽

Effector Mechanism ◽

Key Points ◽

Anti Cd20

Key Points Antigenic modulation significantly impacts natural killer cell and macrophage ability to mediate Fc γ receptor-dependent killing. hIgG1 mAbs are unable to elicit natural killer–mediated ADCC in the mouse, supporting ADCP as the dominant effector mechanism.

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Antigenic modulation limits the efficacy of anti-CD20 antibodies: implications for antibody selection

Blood ◽

10.1182/blood-2010-01-263533 ◽

2010 ◽

Vol 115 (25) ◽

pp. 5191-5201 ◽

Cited By ~ 209

Author(s):

Stephen A. Beers ◽

Ruth R. French ◽

H. T. Claude Chan ◽

Sean H. Lim ◽

Timothy C. Jarrett ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Type I ◽

Type Ii ◽

Antigenic Modulation ◽

Lymphatic Leukemia ◽

Human B Cells ◽

Anti Cd20

Abstract Rituximab, a monoclonal antibody that targets CD20 on B cells, is now central to the treatment of a variety of malignant and autoimmune disorders. Despite this success, a substantial proportion of B-cell lymphomas are unresponsive or develop resistance, hence more potent anti-CD20 monoclonal antibodies (mAbs) are continuously being sought. Here we demonstrate that type II (tositumomab-like) anti-CD20 mAbs are 5 times more potent than type I (rituximab-like) reagents in depleting human CD20 Tg B cells, despite both operating exclusively via activatory Fcγ receptor–expressing macrophages. Much of this disparity in performance is attributable to type I mAb-mediated internalization of CD20 by B cells, leading to reduced macrophage recruitment and the degradation of CD20/mAb complexes, shortening mAb half-life. Importantly, human B cells from healthy donors and most cases of chronic lymphatic leukemia and mantle cell lymphoma, showed rapid CD20 internalization that paralleled that seen in the Tg mouse B cells, whereas most follicular lymphoma and diffuse large B-cell lymphoma cells were far more resistant to CD20 loss. We postulate that differences in CD20 modulation may play a central role in determining the relative efficacy of rituximab in treating these diseases and strengthen the case for focusing on type II anti-CD20 mAb in the clinic.

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