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Antibodies That Efficiently Form Hexamers upon Antigen Binding Can Induce Complement-Dependent Cytotoxicity under Complement-Limiting Conditions.

ABSTRACT: Recently, we demonstrated that IgG Abs can organize into ordered hexamers after binding their cognate Ags expressed on cell surfaces. This process is dependent on Fc:Fc interactions, which promote C1q binding, the first step in classical pathway complement activation. We went on to engineer point mutations that stimulated IgG hexamer formation and complement-dependent cytotoxicity (CDC). The hexamer formation-enhanced (HexaBody) CD20 and CD38 mAbs support faster, more robust CDC than their wild-type counterparts. To further investigate the CDC potential of these mAbs, we used flow cytometry, high-resolution digital imaging, and four-color confocal microscopy to examine their activity against B cell lines and primary chronic lymphocytic leukemia cells in sera depleted of single complement components. We also examined the CDC activity of alemtuzumab (anti-CD52) and mAb W6/32 (anti-HLA), which bind at high density to cells and promote substantial complement activation. Although we observed little CDC for mAb-opsonized cells reacted with sera depleted of early complement components, we were surprised to discover that the Hexabody mAbs, as well as ALM and W6/32, were all quite effective at promoting CDC in sera depleted of individual complement components C6 to C9. However, neutralization studies conducted with an anti-C9 mAb verified that C9 is required for CDC activity against cell lines. These highly effective complement-activating mAbs efficiently focus activated complement components on the cell, including C3b and C9, and promote CDC with a very low threshold of MAC binding, thus providing additional insight into their enhanced efficacy in promoting CDC.

SUBMITTER: Cook EM

PROVIDER: S-EPMC4991250 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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Antibodies That Efficiently Form Hexamers upon Antigen Binding Can Induce Complement-Dependent Cytotoxicity under Complement-Limiting Conditions.

Cook Erika M EM Lindorfer Margaret A MA van der Horst Hilma H Oostindie Simone S Beurskens Frank J FJ Schuurman Janine J Zent Clive S CS Burack Richard R Parren Paul W H I PW Taylor Ronald P RP

Journal of immunology (Baltimore, Md. : 1950) 20160729 5

Recently, we demonstrated that IgG Abs can organize into ordered hexamers after binding their cognate Ags expressed on cell surfaces. This process is dependent on Fc:Fc interactions, which promote C1q binding, the first step in classical pathway complement activation. We went on to engineer point mutations that stimulated IgG hexamer formation and complement-dependent cytotoxicity (CDC). The hexamer formation-enhanced (HexaBody) CD20 and CD38 mAbs support faster, more robust CDC than their wild- ...[more]

PMID: 27474078

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Project description:The phospholipase A2 receptor (PLA2R1) is the major autoantigen in idiopathic membranous nephropathy (MN). However, the pathogenic role of anti-PLA2R1 autoantibodies is unclear. Our aim was to evaluate the in vitro cytotoxicity of anti-PLA2R1 antibodies mediated by complement. Forty-eight patients with PLA2R1-related MN from the prospective cohort SOURIS were included. Anti-PLA2R1 titer, epitope profile, and anti-PLA2R1 IgG subclasses were characterized by ELISA. Cell cytotoxicity was evaluated by immunofluorescence in HEK293 cells overexpressing PLA2R1 incubated with patient or healthy donor sera in the presence or absence of rabbit complement or complement inhibitors. Mean cytotoxicity of anti-PLA2R1 sera for HEK293 cells overexpressing PLA2R1 was 2 ± 2%, which increased to 24 ± 6% after addition of rabbit complement (p < 0.001) (n = 48). GVB-EDTA, which inhibits all complement activation pathways, completely blocked cell cytotoxicity, whereas Mg-EGTA, which only inhibits the classical and lectin pathways, highly decreased suggesting a limited role of the alternative pathway. A higher diversity of IgG subclasses beyond IgG4 and high titer of total IgG anti-PLA2R1 were associated with increased cytotoxicity (p = 0.01 and p = 0.03 respectively). In a cohort of 37 patients treated with rituximab, high level of complement-mediated cytotoxicity was associated with less and delayed remission at month 6 after rituximab therapy (5/12 vs. 20/25 (p = 0.03) in 8.5 months ± 4.4 vs. 4.8 ± 4.0 (p = 0.02)). Kaplan-Meier analysis demonstrated that high level of cytotoxicity (≥40%) (p = 0.005), epitope spreading (defined by immunization beyond the immunodominant CysR domain) (p = 0.002), and high titer of anti-PLA2R1 total IgG (p = 0.01) were factors of poor renal prognosis. Anti-PLA2R1 antibodies containing sera can induce in vitro cytotoxicity mediated by complement activation, and the level of cytotoxicity increases with the diversity and the titer of anti-PLA2R1 IgG subclasses. These patients with high level of complement-mediated cytotoxicity could benefit from adjuvant therapy using complement inhibitor associated with rituximab to induce earlier remission and less podocyte injury.

Dataset Information

Antibodies That Efficiently Form Hexamers upon Antigen Binding Can Induce Complement-Dependent Cytotoxicity under Complement-Limiting Conditions.

Publications

Antibodies That Efficiently Form Hexamers upon Antigen Binding Can Induce Complement-Dependent Cytotoxicity under Complement-Limiting Conditions.

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