Immunotherapy research is a rapidly expanding field in which dozens of monoclonal antibodies are being developed to treat cancer and autoimmune diseases. The mechanism of action (MOA) used by an antibody to mediate a therapeutic response must be defined in order for a candidate antibody to advance down the preclinical development pipeline. Defining the MOA is necessary to fulfilling regulatory requirements for antibodies used in clinical trials and also critical to understanding if the antibody may cause any detrimental side effects.

Therapeutic antibodies have been shown to primarily use one of six major MOAs to mediate their effect, including blockade, apoptosis, antibody dependent cellular phagocytosis (ADCP), antibody dependent cellular cytotoxicity (ADCC), complement dependent cytotoxicity (CDC) and trogocytosis. Flow cytometry is the only assay platform that can test all six of these MOAs for an experimental antibody. These assays can be customized for any antibody or cell type, which makes this approach ideal for preclinical and clinical studies.

Check out these examples of ways to analyze different mechanisms by flow cytometry: 

• Blockade is a frequently observed mechanism by which a therapeutic antibody interferes with the binding of a target cell receptor or surface molecule and blocks downstream immunological responses (e.g., immune checkpoint inhibitors). Blockade can be monitored using different flow cytometry-based approaches such as receptor occupancy assays.

• Apoptosis assays can quantified by treating target cells with an experimental antibody and measuring molecules associated with apoptosis pathways such as caspases.

• Antibody dependent cellular phagocytosis (ADCP) can be measured by enumerating antibody-coated target cells phagocytosed by macrophages.

• Antibody dependent cellular cytotoxicity (ADCC) assays can measure death of antibody-coated target cells by cytotoxic mechanisms from effector cells in PBMCs, primarily natural killer (NK) cells.

• Complement dependent cytotoxicity (CDC) can quantify the death of target cells bound with antibodies that interact with components of the complement system and activate the complement cascade, which causes target cell lysis. 

• Trogocytosis is a mechanism by which lymphocytes (B, T or NK cells) bind to antibodies on a target cell, extract the antibody-ligand complexes from the target cell membrane and transfer the complexes to the lymphocyte membrane. The transfer of the antibody complex from target cell to lymphocyte can be measured by flow cytometry.

These six MOAs trigger many different types of downstream immune responses depending on the target and effector cells involved. Consider flow cytometry if you are developing therapeutic monoclonal antibodies and need to analyze the full scope of the immune response.