Measurement of Trogocytosis: Quantitative Analyses Validated with Rigorous Controls

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.