Detection of Antibody-Dependent Cell-Mediated Cytotoxicity—Supporting Antibodies by NK-92-CD16A Cell Externalization of CD107a: Recognition of Antibody Afucosylation and Assay Optimization

Judith Cruz Amaya, Bruce Walcheck, Julie Smith-Gagen, Vincent C. Lombardi, Dorothy Hudig

Research output: Contribution to journalArticlepeer-review

Abstract

Antibody-dependent cell-mediated cytotoxicity (ADCC) by natural killer (NK) lymphocytes eliminates cells infected with viruses. Anti-viral ADCC requires three components: (1) antibody; (2) effector lymphocytes with the Fc-IgG receptor CD16A; and (3) viral proteins in infected cell membranes. Fc-afucosylated antibodies bind with greater affinity to CD16A than fucosylated antibodies; individuals’ variation in afucosylation contributes to differences in ADCC. Current assays for afucosylated antibodies involve expensive methods. We report an improved bioassay for antibodies that supports ADCC, which encompasses afucosylation. This assay utilizes the externalization of CD107a by NK-92-CD16A cells after antibody recognition. We used anti-CD20 monoclonal antibodies, GA101 WT or glycoengineered (GE), 10% or ~50% afucosylated, and CD20-positive Raji target cells. CD107a increased detection 7-fold compared to flow cytometry to detect Raji-bound antibodies. WT and GE antibody effective concentrations (EC50s) for CD107a externalization differed by 20-fold, with afucosylated GA101-GE more detectable. The EC50s for CD107a externalization vs. 51Cr cell death were similar for NK-92-CD16A and blood NK cells. Notably, the % CD107a-positive cells were negatively correlated with dead Raji cells and were nearly undetectable at high NK:Raji ratios required for cytotoxicity. This bioassay is very sensitive and adaptable to assess anti-viral antibodies but unsuitable as a surrogate assay to monitor cell death after ADCC.

Original languageEnglish (US)
Article number44
JournalAntibodies
Volume12
Issue number3
DOIs
StatePublished - Sep 2023

Bibliographical note

Funding Information:
This research was supported in part at UNR by grants from the Nevada IDeA Network of Biomedical Research Excellence (NIH GM103440), a Robert E. Dickenson Scholarship (JCA), a UNR Foundation Award, and an NIH P30 for the Cytometry Center (NIH P30 GM110767). Support to BW at the University of Minnesota includes NIH R01CA203348.

Publisher Copyright:
© 2023 by the authors.

Keywords

  • ADCC
  • antibody-dependent cell-mediated cytotoxicity
  • CD107a
  • CD16A
  • fucosylation
  • GA101
  • NK-92 cells

PubMed: MeSH publication types

  • Journal Article

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