NK-92-CD16A cell externalization of CD107a for detection of antigen-specific antibodies able to support ADCC: sensitive discrimination of antibody a-fucosylation
Year: 2023
Type: conference-abstract
Abstract: Antibody-dependent cell-mediated cytotoxicity (ADCC) by natural killer (NK) lymphocytes kills cells infected with enveloped viruses. Antiviral ADCC has three components: antigen-specific IgG1 or IgG3; effector cells with CD16A that recognize the Fc of IgG; and antigenic proteins in the plasma membranes of infected cells. Afucosylated antibodies support ADCC better than fucosylated antibodies; afucosylated fractions vary among individuals. Current assays that detect afucosylated antibodies to specific viruses involve antigen-specific antibody purification and mass spectrometry. We are developing clinical bioassays to detect antibodies that will support ADCC and assess their extents of afucosylation. Our pilot assay uses externalization of CD107a by NK-92-CD16A cells, this’ occurs when NK cells recognize cell-bound antibodies. We used two humanized monoclonal antibodies on CD20, GA101 WT or glycoengineered GE, 10% or ~50% afucosylated, with Raji cells. Cellular CD107a externalization was more sensitive than AF-647 donkey anti-IgG to detect total cell-bound antibody. The effective concentrations (EC50s) of the two antibodies for CD107a externalization differed by >15-fold. The more afucosylated GA101-GE was more detectable, at 0.2 ng/ml (=/< 700 Abs/Raji cell). CD107a externalization increased with excess antibody-bearing cells. Short 37 °C incubation (40 minutes) optimized detection of ADCC over intrinsic NK activity. Controls of GA101-GE alone and 56 °C heated human serum lacked effects on CD107a externalization. The assay appears compatible with formaldehyde treatment before NK cell staining. This sensitive bioassay appears suitable for assess serum samples for antiviral antibodies that will support ADCC. UNR Foundation C.E. Hudig Fund.
Source: The Journal of Immunology
Institutions: University of Nevada, Reno, University of Minnesota
Language: English
Subfield: Immunology
Field: Immunology and Microbiology
Domain: Life Sciences
Open Access status: bronze
OpenAlex ID: W4385695677