Binding mechanisms of therapeutic antibodies to human CD20

Science ◽  
2020 ◽  
Vol 369 (6505) ◽  
pp. 793-799 ◽  
Author(s):  
Anand Kumar ◽  
Cyril Planchais ◽  
Rémi Fronzes ◽  
Hugo Mouquet ◽  
Nicolas Reyes
Keyword(s):  
B Cell ◽  
Rational Design ◽  
Local Concentration ◽  
Type I ◽  
Type Ii ◽  
Cell Binding ◽  
Complement Components ◽  
Cytotoxic Effects ◽  
Terminal Complexes ◽  
Cluster Of Differentiation

Monoclonal antibodies (mAbs) targeting human antigen CD20 (cluster of differentiation 20) constitute important immunotherapies for the treatment of B cell malignancies and autoimmune diseases. Type I and II therapeutic mAbs differ in B cell binding properties and cytotoxic effects, reflecting differential interaction mechanisms with CD20. Here we present 3.7- to 4.7-angstrom cryo–electron microscopy structures of full-length CD20 in complexes with prototypical type I rituximab and ofatumumab and type II obinutuzumab. The structures and binding thermodynamics demonstrate that upon binding to CD20, type II mAbs form terminal complexes that preclude recruitment of additional mAbs and complement components, whereas type I complexes act as molecular seeds to increase mAb local concentration for efficient complement activation. Among type I mAbs, ofatumumab complexes display optimal geometry for complement recruitment. The uncovered mechanisms should aid rational design of next-generation immunotherapies targeting 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.

A Reliability-Based Method for Determining Design Ice Loads on Offshore Structures

2002 ◽  
Author(s):  
X. Wu ◽  
A. T. Wang ◽  
C. E. Heuer ◽  
T. D. Ralston ◽  
G. F. Davenport ◽  
...  
Keyword(s):  
Rational Design ◽  
Offshore Structures ◽  
Type I ◽  
Type Ii ◽  
General Applicability ◽  
Ice Load ◽  
Research Company ◽  
Ice Loads ◽  
Offshore Development ◽  
Systematic Framework

This paper describes a reliability-based methodology that has been developed at ExxonMobil Upstream Research Company (URC) for determining rational design ice loads on offshore structures. The URC methodology provides a systematic framework to account for Type I (aleatory) and Type II (epistemic) uncertainties in assessing global probabilistic ice hazards. Specifically, a logic-tree based approach is developed to model Type II uncertainties in the assessment of ice hazards. Although the method has general applicability, the present work considers a wide, vertical-sided, gravity-based structure (GBS) in a dynamic, annual ice environment. Both FORM/SORM methods and Monte Carlo simulation are used in the analyses. Results obtained from this reliability-based approach indicate that the modeling of Type II uncertainties plays a significant role in quantifying the ice hazards for determining the design ice load. Further, this effort may potentially reduce over-conservatism in typical deterministic ice load calculations. The probabilistic methodology developed in this study has broad applicability and can provide a rational framework for calculating design ice loads on other types of structures for arctic offshore development.

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 Type I NKT cells protect (and type II NKT cells suppress) the host's innate antitumor immune response to a B-cell lymphoma

Blood ◽  
2008 ◽  
Vol 111 (12) ◽  
pp. 5637-5645 ◽  
Author(s):  
Gourapura J. Renukaradhya ◽  
Masood A. Khan ◽  
Marcus Vieira ◽  
Wenjun Du ◽  
Jacquelyn Gervay-Hague ◽  
...  
Keyword(s):  
T Cell ◽  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Nkt Cells ◽  
Type I ◽  
Type Ii ◽  
Nkt Cell ◽  
Tumor Bearing

Abstract Natural killer T (NKT) cells are a T-cell subpopulation known to possess immunoregulatory functions and recognize CD1d molecules. The majority of NKT cells express an invariant T-cell receptor (TCR) α chain rearrangement (Vα14Jα18 in mice; Vα24Jα18 in humans) and are called type I NKT cells; all other NKT cells are type II. In the current study, we have analyzed the roles for these NKT-cell subsets in the host's innate antitumor response against a murine B-cell lymphoma model in vivo. In tumor-bearing mice, we found that type I NKT cells conferred protection in a CD1d-dependent manner, whereas type II NKT cells exhibited inhibitory activity. Pro- and anti-inflammatory cytokines secreted by splenocytes from tumor-bearing mice correlated with tumor progression. Myeloid cells (CD11b+Gr1+) were present in large numbers at the tumor site and in the spleen of tumor-bearing type I NKT–deficient mice, suggesting that antitumor immunosurveillance was inhibited by CD11b+Gr1+ cells. Overall, these data suggest that there are distinct roles for NKT-cell subsets in response to a B-cell lymphoma in vivo, pointing to potential novel targets to be exploited in immunotherapeutic approaches against blood cancers.

A type II anti-CD20 monoclonal antibody efficiently depletes CNS B cells in a mouse model of multiple sclerosis

2021 ◽  
Author(s):  
Sabine Tacke ◽  
Rittika Chunder ◽  
Verena Schropp ◽  
Philipp Kirchner ◽  
Arif B. Ekici ◽  
...  
Keyword(s):  
Multiple Sclerosis ◽  
B Cells ◽  
B Cell ◽  
Single Cell ◽  
Total Serum ◽  
Enzyme Linked Immunosorbent Assay ◽  
Type I ◽  
Type Ii ◽  
Cns Pathology ◽  
Anti Cd20

Abstract BackgroundSuccessful therapy with anti-CD20 monoclonal antibodies (mAbs) has reinforced the key role of B cells in the immunopathology of multiple sclerosis. While treatment with currently available anti-CD20 mAbs results in rapid and robust elimination of vascular B cells, B cells residing within compartments of the central nervous system (CNS) are not well targeted. The aim of this study was to determine the effects of a novel class of anti-CD20 mAbs on vascular and extravascular CNS-infiltrating B cells in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. MethodsMale double transgenic hCD20xhIgR3 mice and male wild-type C57BL/6 (B6) mice were injected with human myelin oligodendrocyte glycoprotein (MOG)1–125 to induce chronic EAE. On days 19, 22, and 25 after immunization, the hCD20xhIgR3 mice were injected intravenously with an anti-human CD20 mAb (5 mg/kg), either rituximab (a type I anti-CD20 mAb) or obinutuzumab (a type II humanized anti-CD20 mAb). The B6 mice received a dose of the murine anti-mouse CD20 antibody 18B12. Development of EAE was assessed daily. Seven days after the last injection, mice were euthanized, splenic B-cell subsets were analyzed by flow cytometry, and differential gene expression determined by single-cell RNA sequencing. Total serum immunoglobulin (Ig)G and anti-MOG1–125 IgG titers were measured by enzyme-linked immunosorbent assay. Reduction in CNS-infiltrated CD19+ and CD3+ cells was analyzed by immunohistochemistry, and ultrastructural CNS pathology was studied by transmission electron microscopy. ResultsTreatment with either anti-CD20 mAb had no effect on the clinical course of the disease, animal weight, or serum antibody levels. Obinutuzumab was superior to rituximab in reducing both splenic and CNS-infiltrated B cells. At the single-cell level, obinutuzumab showed pronounced effects on germinal center B cells as well as on CD4+ T cells, which acquired a regulatory-gene signature. In addition, obinutuzumab had beneficial effects on spinal cord myelination. B-cell depletion rates in the 18B12/B6 model were comparable with those observed in obinutuzumab-treated hCD20xhIgR3 mice. ConclusionsOur results demonstrate differential effects of anti-CD20 mAbs on peripheral immune response and CNS pathology, with type II antibodies potentially being superior to type I in the depletion of tissue-infiltrating B cells.

Anti-Factor VIII A2 Epitopes In a Murine Bleeding Model

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1100-1100
Author(s):  
Joshua Eubanks ◽  
Wallace Hunter Baldwin ◽  
Rebecca Markovitz ◽  
Ernest T Parker ◽  
Shannon L. Meeks
Keyword(s):  
Plasma Concentration ◽  
Factor Viii ◽  
B Cell ◽  
Hemophilia A ◽  
High Dose ◽  
Type I ◽  
C2 Domain ◽  
Type Ii ◽  
Acquired Hemophilia

Abstract Up to 30% of patients with severe hemophilia A will develop inhibitory antibodies to factor VIII (fVIII inhibitors). In addition, autoimmune antibodies to fVIII can develop in non-hemophiliacs, producing acquired hemophilia A, which frequently produces life- or limb-threatening bleeding. Patients with congenital hemophilia who develop inhibitors usually have a polyclonal antibody response directed against the A2 and C2 domains of fVIII. Patients with acquired hemophilia typically have a more limited B-cell epitope response with antibodies directed against the A2 or C2 domain but not both. We have shown that within the C2 domain of fVIII antibody epitope is more important than inhibitory titer in predicting pathogenicity in a murine in vivo bleeding model. In this project we investigated the pathogenicity of a diverse panel of anti-A2 monoclonal antibodies (Mabs) in the murine in vivo bleeding model. We have previously characterized anti-A2 antibodies into groups A, AB, B, BCD, C, D, DE, and E based on the pattern of overlap on the B-cell epitopes in a competition ELISA. Table 1 shows the characteristics of the anti-A2 Mabs. Mabs were injected retro-orbitally into Exon 16 hemophilic (E16) mice at a dose of 0.5 umg per g body weight (∼ 65nM plasma concentration). Fifteen minutes later, mice were injected with B-domain deleted human fVIII at a dose of 180U/kg (∼ 2.5nM plasma concentration). In addition, a subset of Mabs has also been tested at a high dose of 360U/kg (∼5 nM). Two hours after fVIII injection, the mice were anesthetized and a 4mm tail snip was performed. Blood was collected in a tube of normal saline over 40 minutes and measured. 4A4, 2-76, and 1D4 are all high inhibitory titer, type I Mabs that produced significant bleeding with 180 U/kg fVIII when compared to control. In addition, 2-76 and 4A4 were tested at the higher dose of fVIII and significant bleeding was again seen. In comparison, the high titer type II Mab 2-54 had a similar inhibitory titer but no significant bleeding at either dose of fVIII. B94 is a type II inhibitor with a similar inhibitory profile to 2-54, but maximum inhibition is 45% as compared to 82% for 2-54. B94 also was not pathogenic at either fVIII dose tested. Both 4C7—a non-inhibitory Mab—and B25—a very low titer Mab that would be predicted to have residual fVIII activity at the Mab concentration tested—did not produce significant bleeding. The inhibitory titer alone did not predict bleeding phenotype within a diverse panel of anti-A2 Mabs. This discrepancy combined with similar findings in the C2 domain stress the importance of inhibitor properties not detected in the standard Bethesda assay in predicting response to fVIII therapy. Disclosures: No relevant conflicts of interest to declare.

scholarly journals IFN type I and type II independent enhancement of B cell TLR7 expression by natural killer cells

2012 ◽  
Vol 92 (4) ◽  
pp. 713-722 ◽  
Author(s):  
Suwan Sinha ◽  
Yuhong Guo ◽  
Suwannee Thet ◽  
Dorothy Yuan
Keyword(s):  
Natural Killer Cells ◽  
B Cell ◽  
Natural Killer ◽  
Type I ◽  
Type Ii ◽  
Killer Cells

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

Blood ◽  
2011 ◽  
Vol 117 (17) ◽  
pp. 4519-4529 ◽  
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.

scholarly journals Clinical and Molecular Characteristics of TCF3-Hlf Gene Fusion-Positive B-Cell Precursor Acute Lymphoblastic Leukemia: A Series of 24 Cases from a Large Cohort of Leukemia Patients

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2387-2387
Author(s):  
Xue Chen ◽  
Fang Wang ◽  
Yang Zhang ◽  
Xiaoli Ma ◽  
Lili Yuan ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cell ◽  
Differential Expression ◽  
Large Cohort ◽  
Type I ◽  
Type Ii ◽  
Hematopoietic Stem ◽  
Dismal Prognosis ◽  
Car T

Abstract Introduction B-cell acute lymphoid leukemia (B-ALL) with t(17;19)(q22;p13)/TCF3-HLF is very rare and has a dismal prognosis even with the application of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Due to the rarity of this fusion, only a few cases have been described in the literature. In this study, we retrospectively analyzed 24 cases with TCF3-HLF from a large cohort of B-ALL, which constituted the largest cohort of TCF3-HLF-positive ALL reported to date and reported in detail the laboratory characteristics and prognoses of this group of patients. Methods From Apr. 2012 to Feb. 2020, a total of 3287 cases were diagnosed with B-ALL in our hospital. All of them underwent afusion gene screening test including TCF3-HLF through multiplex-nested reverse transcription-PCR. Whole transcriptome sequencing (WTS) was performed using RNA extracted from the bone marrow (BM) samples by HiSeq 2500. The gene expression signature for TCF3-HLF-positive B-ALL was investigated by comparing with healthy controls, B-ALL with TCF3-PBX1/TCF3-ZNF384, and B-ALL negative for pathogenic fusion genes (Chen X et al., Blood Cancer J 2021). Results A total of 24 cases with TCF3-HLF were identified, accounting for 0.73% of all B-ALL cases. Among them, 22 (91.67%) were children (≤18 years), and 2 (8.33%) were adults. Length of follow-up varied. The endpoint of the follow-up was Jul. 1st, 2020. Overall survival (OS) was defined as the time from diagnosis to death or the time of the last follow-up. Of the 24 cases with TCF3-HLF, 8 had type I, 12 had type II, 4 had both type II and III chimera isoforms. Karyotype was available in 22 cases, 19 showed abnormal karyotypes and most of them (12/19, 63%) harbored further structural and/or numerical aberrations besides t(17;19)(q22;p13) translocation. Gene mutation screening of 58 genes was performed on 17 cases at the time of diagnosis. Eight (47%) of them showed mutations and 7 of them had mutations involving RAS signaling pathway genes (NRAS, KRAS, FLT3, and PTPN11). Immunophenotypic examination showed 12 (50%) and 19 (79%) patients exhibited aberrant expression of CD13 and CD33, only 4 patients (17%) were negative of both CD13 and CD33. Gene expression clustering revealed apparent separation of TCF3-HLF-positive cases from TCF3-PBX1/TCF3-ZNF384-positive cases and those without pathogenic fusions. The differential expression of 535 genes (up: 207, down: 328) was identified in TCF3-HLF-positive cases compared to TCF3-PBX1-positive cases. The differential expression of 471 genes (up: 281, down: 190) was identified in TCF3-HLF-positive cases compared to TCF3-ZNF384-positive cases. TCF3-HLF-positive patients displayed a significantly up-regulated expression of HLF, which was almost not expressed in other cases (Figure 1). The median OS of the 24 patients was 18.5 months (range 6-75 months). Thirteen of them underwent allogeneic HSCT (allo-HSCT) and the median OS was 23 months (range 13-75 months). Eight of them were in complete remission (CR) until the last follow-up; 2 of them relapsed after a first allo-HSCT and survived in CR after a second allo-HSCT; 3 of them died (2 died of relapse and 1 died of lung infection under CR). Eleven cases did not receive allo-HSCT, and the median OS was 9 months (range 6-29 months). Seven of them died (6 died after relapse, 1 died without achieving remission); 3 of them relapsed and re-induction was failed; only one case has survived in CR for 19 months till the last follow-up. Twelve cases underwent chimeric antigen receptor T-cells (CAR-T) therapy. Nine of them achieved CR after CAR-T therapy and bridged to allo-HSCT; one case achieved CR after CAR-T therapy but relapsed and lost opportunity for allo-HSCT; the other 2 patients achieved CR after the first application of CAR-T therapy but failed to achieve CR again by CAR-T therapy when relapsed. Conclusions We provide systematic insights into the laboratory characteristics and prognoses of B-ALL cases with TCF3-HLF in a large cohort. TCF3-HLF-positive B-ALL has a characteristic gene expression profile that differs markedly from TCF3-PBX1 and TCF3-ZNF384-positive B-ALL and shows a dismal prognosis. TC3F-HLF-positive ALL remains an incurable disease, although CAR-T therapy and allo-HSCT can improve the prognosis to some extent. Advanced therapeutic approaches, including novel drug discovery and development, are urgently required to improve the outcome of this ALL subtype. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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