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.

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

Blood ◽  
2010 ◽  
Vol 115 (25) ◽  
pp. 5191-5201 ◽  
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.

Inhibition of PIM Kinases in Diffuse Large B-Cell Lymphoma Cells Targets MYC-Dependent Transcriptional Program, Increases CD20 Expression and Augments the Efficacy of Anti-CD20 Antibodies

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 33-34
Author(s):  
Maciej Szydlowski ◽  
Filip Garbicz ◽  
Ewa Jabłońska ◽  
Patryk Górniak ◽  
Beata Pyrzynska ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Raji Cells ◽  
Pim Kinases ◽  
Large B Cell Lymphoma ◽  
Cd20 Antibody ◽  
Anti Cd20 ◽  
Large B Cell

R-CHOP immunochemotherapy remains standard frontline therapy for newly diagnosed diffuse large B-cell lymphoma (DLBCL) patients. However, this therapy is ineffective in approximately 1/3 of patients, underscoring the need for better treatment modalities. Targeting DLBCL oncogenic drivers is a promising strategy to improve the treatment efficacy and outcome. Although MYC transcription factor is one of the key oncogenes in DLBCL development, direct MYC targeting strategies have been largely ineffective, highlighting the need for other, indirect approaches. For example, MYC expression is stabilized by PIM serine-threonine kinases, indicating that PIM inhibition might be a rational approach to indirectly target MYC. In this study, we assessed the PIM-MYC relationship and the consequences of PIM inhibition in DLBCL. We first evaluated the expression of PIM1-3 and MYC proteins in 57 DLBCL diagnostic sections by immunohistochemistry. In this series, 70.17% of specimens were positive for at least one PIM isoform and 84.22% cases were MYC-positive. 100% of cases with high MYC expression (MYC present in ≥30% of the cells, n=35) were PIM-positive, whereas 86,36% of cases with undetectable or low MYC expression (MYC detected in ≤20% of cells, n= 22) were PIM-negative (Fisher's exact test, p<0.0001). Since the coexpression of MYC and PIMs highlights the functional link between these proteins in DLBCLs, we evaluated the expression of PIM kinases in cell lines following siRNA-mediated MYC knockdown or treatment with MYC-MAX dimerization inhibitor, 10058F4. The genetic or chemical MYC inhibition markedly decreased PIM1-3 expression in six GCB and ABC cell lines. Likewise, knockdown of all three PIM isoforms decreased MYC levels, attenuated proliferation and induced apoptosis. Similarly, PIM blockade with SEL24/MEN1703, a novel pan-PIM/FLT3 inhibitor tested currently in clinical trial in AML patients and exhibiting favorable safety profile, decreased the expression of multiple MYC-dependent genes. To assess the MYC role in PIM inhibitor-mediated toxicity, we generated DHL4 cells expressing degradation-resistant MYC_T58A mutant. MYC_T58A expression partially protected cells from PIM inhibitor-induced proliferation arrest and apoptosis, indicating that the inhibitor's toxicity is at least partially mediated by MYC depletion. The MS4A1 gene, encoding CD20 surface antigen and rituximab target, is regulated by an upstream promoter containing potential MYC-binding sites (E-boxes). MYC association to these regions was confirmed in chromatin immunoprecipitation assays. As expected, in SEL24/MEN1703-treated cells, MYC occupancy at the MS4A1 promoter markedly decreased. To determine the consequences of MYC binding to the MS4A1 promoter, we assessed CD20 levels in a lymphoblastoid cell line carrying tetracycline-regulated (tet-off) MYC. MYC repression markedly elevated transcript and surface CD20 levels in a time-dependent manner, reaching 17.3-fold (transcript) and 3.82-fold (surface) inductions at 96 h. Consistently, the pan-PIM inhibitor decreased MYC expression in DHL4 and RAJI cells and resulted in increased surface CD20 levels up to 3.72-fold of baseline. In cells expressing the MYC_T58A mutant, PIM inhibition did not increase CD20 level, indicating that PIM kinases modulate CD20 surface expression via MYC. Importantly, PIM inhibitors increased CD20 levels also in primary, patient-derived DLBCL cells. These data suggest that indirect MYC targeting via PIM inhibition would lead to increased rituximab activity. Indeed, in PIM inhibitor-treated DHL4 and RAJI cells, rituximab triggered higher complement-dependent toxicity. Likewise, PIM inhibitor potentiated rituximab-dependent uptake of DHL4 and DHL6 cells by human monocyte-derived macrophages in antibody-dependent cellular phagocytosis assay. Taken together, we characterize a PIM-MYC regulatory circuit promoting DLBCL growth and resistance to anti-CD20 antibody. We also demonstrate that PIM inhibition exhibits pleiotropic effects that combine direct cytotoxicity with increased surface CD20 levels and increased susceptibility to anti-CD20 antibody-based therapies. Study supported by Foundation for Polish Science (POIR.04.04.00-00-5C84/17-00), Polish National Science Centre (2016/22/M/NZ5/00668 and 2017/26/D/NZ5/00561) and Ministry of Science and Higher Education in Poland (iONCO) grants. Disclosures Golas: Ryvu Therapeutics: Current Employment. Green:KDAc Therapeutics: Current equity holder in private company. Tomirotti:Menarini Ricerche: Current Employment. Brzózka:Ryvu Therapeutics: Current Employment. Juszczynski:Ryvu Therapeutics: Other: member of advisory board.

CBL-C137, a Curaxin and Potent Suppressor Of NF-Kb Activity and Modulator Of p53 Function Is Active In Rituximab-Sensitive Or Resistant Activated B-Cell (ABC) Diffuse Large B-Cell Lymphoma (DLBCL) Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1836-1836
Author(s):  
Christopher N. Ibabao ◽  
Cory Mavis ◽  
Jenny Gu ◽  
Myron S. Czuczman ◽  
Francisco Hernandez
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Cycle Arrest ◽  
Clinical Models ◽  
Cd20 Antibody ◽  
Anti Cd20

Abstract The identification of critical signaling pathways necessary for the development, maintenance and progression of specific subtypes of DLBCL are necessary in order to develop novel therapeutics against them. Increased NFkB activity and p53 deregulation contribute to either the pathogenesis of some types of DLBCL (i.e. activated B-cell [ABC] subtype) or to rituximab and/or chemotherapy resistance in B-cell lymphoma. Optimal targeting of NFkB activity is an attractive therapeutic strategy that has been evaluated in pre-clinical and clinical models for over a decade with variable degrees of success. CBL-C137 is a novel and potent member of the curaxin family, capable of modulating p53 and NFkB activity and inducing cell death in several solid tumor cancer models. It has never been previously studied in lymphoid malignancies. In order to study and define the therapeutic potential of curaxins in B-cell lymphoma, we evaluated CBL-C137 in lymphoma pre-clinical models. A panel of rituximab sensitive or resistant lymphoma cell lines representing the two most common subtypes of DLBCL (i.e. ABC-DLBCL and germinal center B-cell [GCB] DLBCL) were exposed to CBL-C137 (0.5-16mM). Changes in cell viability; cell cycle distribution; apoptosis and p53/ NFkB p65 expression were evaluated by measuring ATP content, flow cytometry, and Western blotting, respectively. Subsequently, GCB- or ABC-DLBCL cells were exposed to CBL-C137 alone or in combination with various chemotherapy agents or other available (but less selective) NFkB inhibitors (i.e. lenalidomide or Ibrutinib) for 24 or 48 hrs. Changes in cell viability were determined using the cell titer glo assay. In addition, we conducted standardized 51Cr release assays on cells previously exposed to either CBL-C137 or DMSO to investigate the effects of NFkB inhibition on rituximab (or other anti-CD20) antibody-associated complement mediated cytotoxicity (CMC) and antibody dependent cellular cytotoxicity (ADCC). CBL-C137 induced dose- and time- dependent cell death in ABC-DLBCL greater than in GCB-DLBCL cell lines. The IC50 for CBL-C137 in ABC-DLBCL (rituximab/chemotherapy sensitive or resistant cells) ranged from 1.36 to 2.77mM. In contrast rituximab/chemotherapy GCB-DLBCL cells exhibited the highest IC50 (11.91mM, 95% C.I 7.1-19.8mM). In sensitive DLBCL cells, CBL-C137 induced both apoptosis and cell cycle arrest in G1 phase. Moreover, in vitro exposure to CBL-C137 decreased p53 and p65 in sensitive cells. CBL-C137 increased Lenalidomide, but not Ibrutinib, anti-lymphoma activity in the conditions tested. Finally, CBL-C137 did not affect rituximab or other anti-CD20 antibody-associated ADCC or CMC in DLBCL cells. Our data suggest that CBL-C137 is active in DLBCL pre-clinical models, primarily in ABC-DLBCL cell lines. In sensitive cells, CBL-C137 modulates p53 and NFkB activity and promotes death and/or cell cycle arrest. Ongoing studies are aimed to further define the anti-tumor effects of CBL-C137 in combination with other small molecules inhibitors targeting directly or indirectly NFkB activity in lymphoma. (Research, in part, supported by a NIH grant R01 CA136907-01A1 awarded to Roswell Park Cancer Institute and The Eugene and Connie Corasanti Lymphoma Research Fund) Disclosures: Czuczman: Genetech, Onyx, Celgene, Astellas, Millennium, Mundipharma: Advisory Committees Other.

Targeting B-Cell Malignancies With Anti-CD20-Interleukin-21 Fusokine

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 377-377 ◽  
Author(s):  
Shruti Bhatt ◽  
Daxing Zhu ◽  
Xiaoyu Jiang ◽  
Seung-uon Shin ◽  
John M Timmerman ◽  
...  
Keyword(s):  
Cell Death ◽  
B Cell ◽  
Nk Cells ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Cd20 Antibody ◽  
Anti Cd20

Abstract The anti-CD20 antibody rituximab has revolutionized the treatment for B cell non-Hodgkin lymphomas (NHLs). However, rituximab has limited effectiveness as a single agent in some NHL subtypes and its clinical efficacy is compromised by acquired drug resistance. As a result, many patients still succumb to NHLs. Hence, strategies that enhance the activity of anti-CD20 antibody may improve patient outcome. Interleukin-21 (IL21), a member of the IL2 cytokine family, exerts diverse regulatory effects on natural killer (NK), T and B cells. IL21 has been reported to possess potent anti-tumor activity against a variety of cancers not expressing IL21 receptor (IL21R) through activation of the immune system and is in clinical trials for renal cell carcinoma and metastatic melanoma. We have recently reported that apart from immuno-stimulatory effects, IL21 exerts direct cytotoxicity on IL21R expressing diffuse large B cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) cell lines and primary tumors both in vitro as well in vivo (Sarosiek et al Blood 2010; Bhatt et al AACR 2013). Herein we designed a fusion protein comprising IL21 linked to the N-terminus of anti-CD20 antibody (αCD20-IL21 fusokine) to improve efficacy of its individual components and prolong IL21 half-life. We have verified the expression of full length fusion protein and demonstrated that αCD20-IL21 fusokine retained binding ability to its individual components; CD20 and IL21R, as analyzed by immunofluorescence and flow-cytometry analyses. Similar to our previous study of IL21 in DLBCL, treatment of B cell lymphoma cell lines with fusokine lead to phosphorylation of STAT1 and STAT3, upregulation of cMYC and BAX and downregulation of BCL-2 and BCL-XL, implying the activation of IL21R dependent signaling to trigger cytotoxic effects. In vitro, direct cell death induced by αCD20-IL21 fusokine in DLBCL (RCK8, WSU and Farage) and MCL (Mino, HBL2 and SP53) cell lines was markedly increased compared to its individual components (IL21 and parent αCD20-IgG1 antibody). More importantly, fusokine treatment resulted in cell death of MCL cell lines (L128, G519 and UPN1) that were found to be resistant to IL21 alone treatment. Furthermore, treatment of freshly isolated primary NHL cells with the αCD20-IL21 fusokine also exhibited a 40-50% increase in direct cell death compared to its individual components. Previous studies reported that IL21 enhances antibody-dependent cellular cytotoxicity (ADCC) of therapeutic antibodies by activation of NK cells. ADCC assays using chromium release with purified human NK cells demonstrated that ADCC induced by the parent antibody was enhanced in the presence of IL21 while IL21 alone had minimal effect on the lysis of Raji, Daudi, and Jeko1 target cells. Notably, αCD20-IL21 fusokine demonstrated increased ADCC activity in comparison to parent antibody plus IL21 in Raji, Daudi and Jeko-1 cells (p<0.001, p<0.005 and p<0.001, respectively). Similar results were obtained in primary MCL tumor cells. Consistent with this finding, fusokine treatment resulted in enhanced activation of the NK cells as assessed by CD69 upregulation and CD16 downregulation using flow-cytometry. Complement dependent cytotoxicity (CDC) of the fusokine was similar to the parent antibody and rituximab in Raji cells. Studies analyzing in vivo effects of the fusokine are in progress and will be presented at the meeting. These data strongly suggest that together with direct apoptotic potential, an anti-CD20 IL21 fusokine retains the ability to trigger indirect cell killing mediated via activation of immune effector cells. These dual effects may give remarkable advantage to the fusokine over existing anti-CD20 antibodies for the treatment of NHL tumors. Collectively, our study demonstrates that anti-tumor effects of IL21 and anti-CD20 antibodies can be enhanced by conjugation of IL21 with anti-CD20 antibody that may serve as a novel anti-lymphoma therapy. Disclosures: Rosenblatt: Seattle Genetics, Inc.: Research Funding.

scholarly journals Imaging the mechanisms of anti-CD20 therapy in vivo uncovers spatiotemporal bottlenecks in antibody-dependent phagocytosis

2021 ◽  
Vol 7 (8) ◽  
pp. eabd6167
Author(s):  
Capucine L. Grandjean ◽  
Zacarias Garcia ◽  
Fabrice Lemaître ◽  
Béatrice Bréart ◽  
Philippe Bousso
Keyword(s):  
B Cell ◽  
Cell Lymphoma ◽  
Critical Path ◽  
B Cell Lymphoma ◽  
Antibody Dependent Cellular Cytotoxicity ◽  
Fluorescent Reporter ◽  
Cd20 Antibody ◽  
Anti Cd20 ◽  
Partial Depletion

Anti-CD20 antibody (mAb) represents an effective strategy for the treatment of B cell malignancies, possibly involving complement activity, antibody-dependent cellular cytotoxicity and phagocytosis (ADP). While ADP by Kupffer cells deplete circulating tumors, mechanisms targeting non-circulating tumors remain unclear. Using intravital imaging in a model of B cell lymphoma, we establish here the dominance and limitations of ADP in the bone marrow (BM). We found that tumor cells were stably residing in the BM with little evidence for recirculation. To elucidate the mechanism of depletion, we designed a dual fluorescent reporter to visualize phagocytosis and apoptosis. ADP by BM-associated macrophages was the primary mode of tumor elimination but was no longer active after one hour, resulting in partial depletion. Moreover, macrophages were present at low density in tumor-rich regions, targeting only neighboring tumors. Overcoming spatiotemporal bottlenecks in tumor-targeting Ab therapy thus represents a critical path towards the design of optimized therapies.

Targeted anti-cancer therapy using rituximab, a chimaeric anti-CD20 antibody (IDEC-C2B8) in the treatment of non-Hodgkin's B-cell lymphoma

1997 ◽  
Vol 25 (2) ◽  
pp. 705-708 ◽  
Author(s):  
D. R. Anderson ◽  
A. Grillo-López ◽  
C. Varns ◽  
K. S. Chambers ◽  
N. Hanna
Keyword(s):  
Cancer Therapy ◽  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Anti Cancer ◽  
Cd20 Antibody ◽  
Anti Cd20

scholarly journals Diffuse Large B-cell Lymphoma Arising in a Patient with Neurofibromatosis Type I and in a Patient with Neurofibromatosis Type II

2006 ◽  
Vol 208 (2) ◽  
pp. 169-176 ◽  
Author(s):  
Osamu Dohi ◽  
Masahito Hatori ◽  
Ryo Ichinohasama ◽  
Masami Hosaka ◽  
Sho Hashimoto ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Neurofibromatosis Type ◽  
Type I ◽  
Type Ii ◽  
Neurofibromatosis Type I ◽  
Large B Cell Lymphoma ◽  
Neurofibromatosis Type Ii ◽  
Large B Cell
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