A Novel FACS Assay for Absolute Quantification of CD20 on B-Cells and Determination of CD20 Occupancy in Whole Blood Samples Shows a Higher Binding Strength of Obinutuzumab (GA101) to CD20 on B-Lymphocytes

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4611-4611
Author(s):  
Drifa Beroual ◽  
Danièle Boulay-Moine ◽  
Carole Badoil ◽  
Christian Klein ◽  
Maxime Moulard
Keyword(s):  
B Cells ◽  
B Lymphocytes ◽  
Cell Lines ◽  
Whole Blood ◽  
Absolute Number ◽  
Type Ii ◽  
Human Mabs ◽  
Cd20 Antibody ◽  
The Absolute ◽  
Anti Cd20

Abstract Abstract 4611 Introduction. The absolute quantification of CD20 on malignant B-lymphocytes on patients who already received anti-CD20 MAbs is technically challenging, but may play a role in identifying patients who will respond to subsequent treatment or help define patients that are rituximab-refractory due to decreased expression of CD20. We have developed a new quantitative flow cytometry assay to scrutinize the absolute number of receptors on cell lines and B-lymphocytes in whole blood (WB) samples as well as target occupancy. This assay allows us to accurately determine the number of human MAbs bound per target cell on various cell subsets. CD20 density on B-lymphocytes in WB and binding properties of the type II CD20 antibody obinutuzumab (GA101) and the classical Type I CD20 antibody Rituximab were tested on samples from healthy volunteers. Acidic striping of bound human anti-CD20 MAbs was explored to enable the set up of a trustworthy CD20 occupancy assay. Methods. Binding of anti-CD20 mAbs rituximab (RTX) and GA101 to CD20 on normal and malignant B-cells was assessed using the Human IgG Calibrator (BioCytex, F) on EDTA anticoagulated WB samples and cell lines to determine the CD20 density. In parallel, the murine MAb (Clone B9E9) was tested using Cellquant Calibrator (BioCytex, F). CD20 density and apparent Kd, a relative measure of affinity, for each MAb was calculated from the data obtained with whole blood samples. Both the total number of CD20 molecules and the CD20 bound fraction were appraised by spiking the sample with anti-CD20 MAbs at saturation and acid striping. Results. The absolute number of anti-CD20 MAbs bound to CD20 on B-cells in WB samples was between 58,000 to 123,000 molecules per cell as determined by GA101 and RTX, respectively. The apparent CD20 quantitation on cell lines was approx. 2- to 3-fold lower for GA101 which is a characteristic and reported property of type II CD20 antibodies. The apparent Kd of GA101 (4.1 to 9.9 nM) was approximately 2-fold lower than that of RTX (10.5 to 22.7 nM) on several different lymphoma-derived cell lines (Daudi, Raji, Ramos and MEC-1). On B-lymphocytes from healthy volunteers the Kd were 1.2 and 3.5 nM for GA101 and RTX, respectively. Furthermore, CD20-bound GA101 could only be detached from its target CD20 using acid-striping assay at pH < 2.0 and appropriate ionic strength whereas RTX could be stripped at pH 2.5. Binding of anti-CD20 MAbs to acid-treated B-lymphocytes was found unaffected. Conclusion. The density of RTX bound to CD20 on B-lymphocytes in whole blood is similar to previous published data obtained from radioimmunoassays and using 125I-MAb (Teeling et al, Blood, 2006). In contrast the number of the Type II CD20 antibody GA101 bound to B-lymphocytes in whole blood was approximately half in comparison to RTX as previously reported (Moessner et al, Blood, 2010). The data further confirmed a higher apparent binding affinity of GA101 for CD20 with Kd values comparable to those reported previously using 125I-MAb. In summary, the new Human IgG Calibrator is a fast and accurate technique that can be used to evaluate the CD20 density and occupancy using human MAbs in complex matrix including whole blood. The test can be implemented in pharmacodynamic clinical trials and may allow for further understanding of complex pharacodynamic relationships. Disclosures: Beroual: BioCytex: Employment. Boulay-Moine:BioCytex: Employment. Badoil:BioCytex: Employment. Klein:Roche: Employment, Equity Ownership, Patents & Royalties. Moulard:BioCytex: Consultancy, Employment.

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 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.

The Type II anti‐CD20 Antibody Obinutuzumab (GA101) is More Effective than Rituximab at Depleting B Cells and Treating Disease in a Murine Lupus Model

2020 ◽  
Author(s):  
Anthony D. Marinov ◽  
Haowei Wang ◽  
Sheldon I. Bastacky ◽  
Erwin van Puijenbroek ◽  
Thomas Schindler ◽  
...  
Keyword(s):  
B Cells ◽  
Type Ii ◽  
Murine Lupus ◽  
Cd20 Antibody ◽  
Anti Cd20

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.

Case 38

2020 ◽  
pp. 261-266
Author(s):  
Andrew Woodhouse
Keyword(s):  
B Cells ◽  
T Lymphocytes ◽  
Lymphoproliferative Disease ◽  
Solid Organ ◽  
Post Transplant ◽  
Barr Virus ◽  
Cd20 Antibody ◽  
Epstein Barr ◽  
Anti Cd20 ◽  
Lymphoid Proliferation

Post-transplant lymphoproliferative disease (PTLD) is a disorder of lymphoid proliferation seen in recipients of solid organ or haematopoietic transplants as a consequence of immunosuppression. A spectrum of disease is recognized ranging from non-malignant polyclonal proliferation of B cells to monoclonal proliferation of B or T lymphocytes which have features in common with lymphomas. Epstein–Barr virus (EBV) is associated with a majority of cases although it is not a universal feature. Treatment with anti-CD20 antibody in addition to reduction in immunosuppression has become the most common treatment approach.

scholarly journals Effect of anti-CD20 monoclonal antibody, Rituxan, on cynomolgus monkey and human B cells in a whole blood matrix

Cytometry ◽  
2003 ◽  
Vol 52A (2) ◽  
pp. 101-109 ◽  
Author(s):  
Yulia Vugmeyster ◽  
Kathy Howell ◽  
Anahid Bakshl ◽  
Clarissa Flores ◽  
Eleanor Canova-Davis
Keyword(s):  
Monoclonal Antibody ◽  
B Cells ◽  
Whole Blood ◽  
Cynomolgus Monkey ◽  
Human B Cells ◽  
Anti Cd20

Human neutrophils mediate trogocytosis rather than phagocytosis of CLL B cells opsonized with anti-CD20 antibodies

Blood ◽  
2017 ◽  
Vol 129 (19) ◽  
pp. 2636-2644 ◽  
Author(s):  
Rut Valgardsdottir ◽  
Irene Cattaneo ◽  
Christian Klein ◽  
Martino Introna ◽  
Marina Figliuzzi ◽  
...  
Keyword(s):  
B Cells ◽  
Human Neutrophils ◽  
Key Points ◽  
Cd20 Antibody ◽  
Anti Cd20 ◽  
Cd20 Antibodies

Key Points Human neutrophils mediate trogocytosis rather than phagocytosis of CD20-antibody–opsonized CLL B cells. Trogocytosis is induced more effectively by rituximab compared with obinutuzumab.

scholarly journals Human Herpesvirus 8 Infects and Replicates in Primary Cultures of Activated B Lymphocytes through DC-SIGN

2008 ◽  
Vol 82 (10) ◽  
pp. 4793-4806 ◽  
Author(s):  
Giovanna Rappocciolo ◽  
Heather R. Hensler ◽  
Mariel Jais ◽  
Todd A. Reinhart ◽  
Amarendra Pegu ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
B Lymphocytes ◽  
Cell Lines ◽  
Transmembrane Domain ◽  
Primary Cultures ◽  
Human Herpesvirus ◽  
Endocytic Pathway ◽  
Human Herpesvirus 8 ◽  
Herpesvirus 8

ABSTRACT Human herpesvirus 8 (HHV-8) is the etiological agent of Kaposi's sarcoma, primary effusion lymphoma, and some forms of multicentric Castleman's disease. Although latent HHV-8 DNA can be detected in B cells from persons with these cancers, there is little information on the replication of HHV-8 in B cells. Indeed, B cells are relatively resistant to HHV-8 infection in vitro. We have recently shown that DC-SIGN, a C-type lectin first identified on dendritic cells (DC), is an entry receptor for HHV-8 on DC and macrophages. We have also demonstrated previously that B lymphocytes from peripheral blood and tonsils express DC-SIGN and that this expression increases after B-cell activation. Here we show that activated blood and tonsillar B cells can be productively infected with HHV-8, as measured by an increase in viral DNA, the expression of viral lytic and latency proteins, and the production of infectious virus. The infection of B cells with HHV-8 was blocked by the pretreatment of the cells with antibody specific for DC-SIGN or with mannan but not antibody specific for xCT, a cystine/glutamate exchange transporter that has been implicated in HHV-8 fusion to cells. The infection of B cells with HHV-8 resulted in increased expression of DC-SIGN and a decrease in the expression of CD20 and major histocompatibility complex class I. HHV-8 could also infect and replicate in B-cell lines transduced to express full-length DC-SIGN but not in B-cell lines transduced to express DC-SIGN lacking the transmembrane domain, demonstrating that the entry of HHV-8 into B cells is related to DC-SIGN-mediated endocytosis. The role of endocytosis in viral entry into activated B cells was confirmed by blocking HHV-8 infection with endocytic pathway inhibitors. Thus, the expression of DC-SIGN is essential for productive HHV-8 infection of and replication in B cells.

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