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Monoclonal antibodies specific for human IgM Fc receptor inhibit ligand-binding activity.

ABSTRACT: A panel of six different murine hybridoma clones secreting IgG monoclonal antibodies (MAbs) specific for the human IgM Fc receptor (FcμR) was generated. All MAbs specifically precipitated a major protein of ∼60 kDa from membrane lysates of FcμR-bearing, but not FcμR-negative, cells as did IgM-ligands. Pre-incubation of membrane lysate of FcμR-bearing cells with these MAbs completely removed the ∼60 kDa IgM-reactive protein. By using recombinant human/mouse chimeric FcμR proteins, the epitope recognized by HM7 and HM10 MAbs was mapped to the Ig-like domain of human FcμR, whereas the other MAbs recognized the stalk region. Pre-incubation of FcμR(+) cells with the Ig-like domain-specific MAbs, but not with others, markedly inhibited subsequent IgM-ligand binding. A similar, but much weaker, inhibition was also observed when the incubation order was reversed. When FcμR(+) cells were simultaneously incubated with both IgM-ligands and MAbs, HM7 MAb efficiently competed with IgM for FcμR binding. Unlike control Jurkat cells, FcμR-bearing cells were resistant to apoptosis induced by agonistic IgM anti-Fas MAb (CH11); however, addition of the HM7 MAb inhibited the interaction of the Fc portion of CH11 MAb with FcμR, thereby promoting apoptosis of FcμR-bearing Jurkat cells. The variable regions of the HM7 MAb were composed of Ighv14-3, Ighd1-2, and Ighj2 for the γ2b heavy chain and Igk3-4 and Igkj2 for the κ light chain. These findings suggest that HM7 MAb efficiently blocks the ligand-binding activity of FcμR.

SUBMITTER: Kubagawa Y

PROVIDER: S-EPMC4278172 | biostudies-literature | 2014 Dec

REPOSITORIES: biostudies-literature

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Monoclonal antibodies specific for human IgM Fc receptor inhibit ligand-binding activity.

Kubagawa Yoshiki Y Honjo Kazuhito K Kang Dong-Won DW Kubagawa Hiromi H

Monoclonal antibodies in immunodiagnosis and immunotherapy 20141201 6

A panel of six different murine hybridoma clones secreting IgG monoclonal antibodies (MAbs) specific for the human IgM Fc receptor (FcμR) was generated. All MAbs specifically precipitated a major protein of ∼60 kDa from membrane lysates of FcμR-bearing, but not FcμR-negative, cells as did IgM-ligands. Pre-incubation of membrane lysate of FcμR-bearing cells with these MAbs completely removed the ∼60 kDa IgM-reactive protein. By using recombinant human/mouse chimeric FcμR proteins, the epitope rec ...[more]

PMID: 25545208

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Project description:Both non-immune "natural" and antigen-induced "immune" IgM are important for protection against infections and for regulation of immune responses to self-antigens. The roles of its Fc receptor (FcµR) in these IgM effector functions have begun to be explored. In the present study, by taking advantage of the difference in IgM-ligand binding of FcµRs of human (constitutive binding) and mouse (transient binding), we replaced non-conserved amino acid residues of human FcµR with mouse equivalents before establishment of cell lines stably expressing mutant or wild-type (WT) receptors. The resultant eight-different mutant FcµR-bearing cells were compared with WT receptor-bearing cells for cell-surface expression and IgM-binding by flow cytometric assessments using receptor-specific mAbs and IgM paraproteins as ligands. Three sites Asn66, Lys79-Arg83, and Asn109, which are likely in the CDR2, DE loop and CDR3 of the human FcµR Ig-like domain, respectively, were responsible for constitutive IgM binding. Intriguingly, substitution of Glu41 and Met42 in the presumed CDR1 with the corresponding mouse residues Gln and Leu, either single or more prominently in combination, enhanced both the receptor expression and IgM binding. A four-aa stretch of Lys24-Gly27 in the predicted A ß-strand of human FcµR appeared to be essential for maintenance of its proper receptor conformation on plasma membranes because of reduction of both receptor expression and IgM-binding potential when these were mutated. Results from a computational structural modeling analysis were consistent with these mutational data and identified a possible mode of binding of FcµR with IgM involving the loops including Asn66, Arg83 and Asn109 of FcµR interacting principally with the Cµ4 domain including Gln510 and to a lesser extent Cµ3 domain including Glu398, of human IgM. To our knowledge, this is the first experimental report describing the identification of amino acid residues of human FcµR critical for binding to IgM Fc.

Project description:The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein is the main target for neutralizing antibodies. These antibodies can be elicited through immunization or passively transferred as therapeutics in the form of convalescent-phase sera or monoclonal antibodies (MAbs). Potently neutralizing antibodies are expected to confer protection; however, it is unclear whether weakly neutralizing antibodies contribute to protection. Also, their mechanism of action in vivo is incompletely understood. Here, we demonstrate that 2B04, an antibody with an ultrapotent neutralizing activity (50% inhibitory concentration [IC50] of 0.04 μg/ml), protects hamsters against SARS-CoV-2 in a prophylactic and therapeutic infection model. Protection is associated with reduced weight loss and viral loads in nasal turbinates and lungs after challenge. MAb 2B04 also blocked aerosol transmission of the virus to naive contacts. We next examined three additional MAbs (2C02, 2C03, and 2E06), recognizing distinct epitopes within the receptor binding domain of spike protein that possess either minimal (2C02 and 2E06, IC50 > 20 μg/ml) or weak (2C03, IC50 of 5 μg/ml) virus neutralization capacity in vitro. Only 2C03 protected Syrian hamsters from weight loss and reduced lung viral load after SARS-CoV-2 infection. Finally, we demonstrated that Fc-Fc receptor interactions were not required for protection when 2B04 and 2C03 were administered prophylactically. These findings inform the mechanism of protection and support the rational development of antibody-mediated protection against SARS-CoV-2 infections. IMPORTANCE The ongoing coronavirus disease 2019 (COVID-19) pandemic, caused by SARS-CoV-2, has resulted in the loss of millions of lives. Safe and effective vaccines are considered the ultimate remedy for the global social and economic disruption caused by the pandemic. However, a thorough understanding of the immune correlates of protection against this virus is lacking. Here, we characterized four different monoclonal antibodies and evaluated their ability to prevent or treat SARS-CoV-2 infection in Syrian hamsters. These antibodies varied in their ability to neutralize the virus in vitro. Prophylactic administration of potent and weakly neutralizing antibodies protected against SARS-CoV-2 infection, and this effect was Fc receptor independent. The potent neutralizing antibody also had therapeutic efficacy and eliminated onward aerosol transmission. In contrast, minimally neutralizing antibodies provided no protection against infection with SARS-CoV-2 in Syrian hamsters. Combined, these studies highlight the significance of weakly neutralizing antibodies in the protection against SARS-CoV-2 infection and associated disease.

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Monoclonal antibodies specific for human IgM Fc receptor inhibit ligand-binding activity.

Publications

Monoclonal antibodies specific for human IgM Fc receptor inhibit ligand-binding activity.

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