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Measurement of Trogocytosis: Quantitative Analyses Validated with Rigorous Controls.

Ronald P Taylor1, Margaret A Lindorfer1

  • 1Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, Virginia.

Current Protocols
|October 13, 2023
PubMed
Summary
This summary is machine-generated.

Trogocytosis, the removal of antibody-bound targets by immune cells, hinders antibody-based cancer therapies like rituximab. This study details methods to analyze and potentially block this process, improving treatment efficacy.

Keywords:
antibody-drug conjugatescomplementimmune adherenceimmune complex clearancetrogocytosis

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Area of Science:

  • Immunology
  • Cell Biology
  • Cancer Therapy

Background:

  • Trogocytosis is a cellular process impacting antibody-based cancer immunotherapy.
  • The loss of CD20 targets on chronic lymphocytic leukemia (CLL) cells treated with CD20 mAbs (rituximab, ofatumumab) is a clinically observed phenomenon.
  • This target loss has been replicated in vitro, suggesting a mechanism beyond simple antibody dissociation.

Purpose of the Study:

  • To investigate the mechanism of antibody-dependent cell-mediated removal of antibody-bound targets.
  • To demonstrate and validate trogocytosis as the mechanism responsible for target loss in antibody immunotherapy.
  • To provide experimental methods for studying trogocytosis and developing strategies to counteract its adverse effects.

Main Methods:

  • Utilized flow cytometry and fluorescence microscopy to observe trogocytosis.
  • Employed fluorescently labeled monoclonal antibodies (mAbs) and various cell types, including THP-1 monocytic cell line and primary human monocytes.
  • Implemented rigorous controls to distinguish trogocytosis from simple dissociation or internalization.

Main Results:

  • Demonstrated that FcγR-expressing acceptor cells (monocytes/macrophages) actively remove and internalize both antigen and cognate IgG mAbs from donor cells.
  • Confirmed transfer of fluorescent mAbs and cell membrane fragments from donor to acceptor cells.
  • Validated the experimental setup, ruling out alternative mechanisms.

Conclusions:

  • Trogocytosis is a significant mechanism contributing to the reduced efficacy of mAb-based cancer immunotherapies, particularly in CLL.
  • This process may also underlie adverse effects like neutropenia, thrombocytopenia, and liver damage associated with antibody-drug conjugates.
  • The described methods offer a platform for developing interventions to block trogocytosis and improve therapeutic outcomes.