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Quantification and Multiparameter Characterization of Phagocytosis and Cell Toxicity by Flow Cytometry.

The revolution in biological drug development continues to advance our approaches to precision medicine and shape the global drug market. Biologics are now being used in the treatment of a wide range of chronic and rare diseases, with many more are in the pipeline. Regulatory authorities now require mechanism of action (MOA) studies for all new investigational new drug (IND) filings, and this has resulted in many investigators looking to flow cytometry solutions for this aspect of their research. Understanding the MOA is critical in exploring the rational design of single or combination therapies for enhanced immune activity.

Although antibody-based therapeutics bind to their target cells via their variable domain regions, it is the engagement of effector functions through the Fc region of the antibody that triggers:


Antibody-Dependent Cell Phagocytosis (ADCP)



Antibody-Dependent Cell Cytotoxicity (ADCC)



Completement-Dependent Cytotoxicity (CDC)

These Fc regions bind to the Fc gamma receptors (FcγRs) that are located on the surface of immune cells and complement factors. FcγRs recognize and bind IgG-coated target cells, and this interaction results in the internalization of the cargo and activates several downstream response pathways. There are various forms of Fc gamma receptors with a range of affinities to IgG, that display either inhibitory or activating functions and are expressed in different combinations on the surface of immune cells. Understanding the dynamics of the interactions of antibody-based drugs with their targets is at the heart of MOA studies, and flow cytometry applications can help profile these biological activities and decipher the relevant biological systems that are being engaged before efficacy models are explored.

Profiling Cellular Responses to Antibody Binding Using Flow Cytometry

Most flow cytometry MOA methods utilize fluorescent probes/labels to distinguish target cells from effector cells and measure cell viability as an indicator of effector function activities. Typically, all three different Fc- related activities are examined (ADCC, ADCP, and CDC) for biological drug IND filings.


ADCP (Antibody Dependent Cell Phagocytosis)

The mechanism by which antibody opsonized target cells activate the FcRs on the surface of macrophages to induce phagocytosis.


ADCC (Antibody Dependent Cell Cytotoxicity)

The functional evaluation of the engagement and activity of effector cells against cancer cells, or infected cells bridges both the adaptive and innate immune responses.


CDC (Complement-Dependent Phagocytosis)

The mechanism by which cells coated by antibodies activate the complement pathway, resulting in cell death.

The Mediation of Immune Effector Functions Through Antibody Fc Domains

Although antibodies bind to their antigen targets through their variable domains, the Fc domains of antibodies also bind to Fc receptors (FcRs) expressed on the surface of immune cells such as Natural Killer cells and macrophages (resulting in ADCC and ADCP respectively), or through Fc binding to effector molecules that initiate the complement cascade (CDC). One key determinant of Fc-mediated functions is the isotype of the antibody which determines which type of FcR is engaged; but subclass, glycosylation profiles, and antigen specificity all influence the capacity of an antibody to interact with specific FcRs.


Antibody-Dependent Cell Phagocytosis (ADCP)

Phagocytosis is a fundamental mechanism within the innate immune system and an effective mechanism for the internalization of cells as a source of antigens for presentation on both MHC class I and II molecules. Phagocytosis commences with the binding and recognition of foreign particles by surface receptors on phagocytes (For example: macrophages, dendritic cells, and neutrophils) which results in the engulfing of the particle into the cells within a phagosome structure. This fuses with a lysosome, to form a phagolysosome. The internal environment of the phagolysosome is incredibly harsh with a low internal pH and packed hydrolytic enzymes (proteases, cathepsins, lysosomes, and lipases), reactive oxygen species, and reactive nitrogen species to overwhelm the invading pathogen. Various reagents have been developed for the detection of the diverse array of phagocytosed particles by flow cytometry. ADCP assays can be performed using reporter cells lines, or primary cells such as monocytes and macrophages.

Antibody-Dependent Cell Cytotoxicity (ADCC)

The activity of many monoclonal antibody (mAb) therapeutics is mediated through the process of antibody-dependent cell-mediated cytotoxicity which enhances the immune system’s response to dysfunctional cells such as tumor cells. ADCC activity is a measure of drug potency, efficacy, and stability but can also be used to assess the risks of unwanted side effects. ADCC refers to the cyto-lytic activity of effector cells with cytotoxic potentials such as Natural Killer cells, macrophages, and granulocytes. In this instance the antibody functions as a link between the target (bound by the variable antibody domain) and effector cells (bound through the Fc antibody domain); once bound, the effector cell secretes cytolytic molecules to eliminate the target cell. ADCC assays are widely used to screen mAbs for potent anti-cancer activities, this includes the assessment of the glycosylation state of the drug that can affect ADCC, along with CD16 polymorphism and antigen expression levels of target cells.

Fc-FcγR interactions or antibody-FcγR interactions are all being applied to anti-viral and anti-tumor drug development initiatives, to leverage the stimulatory role of FcγR in clinical applications.

Applications of ADCP and ADCC assays:

Primary cell ADCP is used to assess the biocompatibility of biosimilars

MOA of mAb therapeutics

Stability testing and release assays for mAb therapeutics

Tumor Biology and Oncology

Complement-Dependent Cytotoxicity (CDC)

Complement-dependent cytotoxicity is a mechanism of target cell lysis that is mediated through the activation of the complement system- a complex mechanism of over 20 small proteins within the serum that work together to target and ultimately lyse cells that are opsonized by antibodies. CDC is an arm of the immune response that eliminates foreign pathogens and aberrant cells that might otherwise cause illness. The process can be leveraged to target cancer cells through the binding of therapeutic mAbs to cancer-specific antigens.

There are certain considerations when engineering therapeutic mAb for CDC since certain isotypes display stronger CDC activity than others. As a rule of thumb, IgG3 displays the strongest capacity for CDC, compared with IgG1, IgG2, and IgG4 with diminishing CDC capacity, although engineering specific amino acids into the hinge and CH2 antibody domains can also improve CDC activity for therapeutic mAb. Antibody Glycoforms are also influential in CDC activity. In all of these examples, flow cytometry-based assessment of CDC activity is an important component of the drug development process.

CDC assays utilize either pooled normal serum or normal serum complement that is combined with the target cells and healthy PBMCs. The source of complement is an important consideration to ensure that it is compatible with the target cells.

The FlowMetric team has developed CDC approaches for a number of clinical applications such as safe dose ranges for cytotoxic drugs and the assessment of xeno-transplants. Our fit-for-purpose approach to assay development will ensure rapid and sensitive assessment of CDC.

Flow Cytometry-Based Mechanism of Action Assays Support Applications Across Many Areas of Drug Development

Drug safety/toxicity – quantify safe and effective dosing with minimal cytotoxic effects.

New Drug Design through modeling and assessment of drug-target interactions.

Patient Stratification responders versus non-responders – biomarker/phenotypic assessment of drug targets open the door for safer clinical trials.

Enhanced Dosing – understanding the cytotoxicity of drugs enables us to understand the dosing requirements more clearly for normal versus pathogenic applications.

Rational Enhanced Combination Therapy Design – knowledge of the mechanism of action, particularly with respect to immune-modulating drugs, enables research to fully explore and exploit combination therapies, with enhanced safety and efficacy.

The FlowMetric Advantage

Flow cytometry is a powerful tool in drug development and enables researchers to identify and evaluate the cell types and specific pathways that are influential in the mode of drug action. Complex, multiparameter flow cytometry panels can be customized to monitor receptor occupancy overtime, describe MOA, and support pharmacodynamic studies.

The FlowMetric team has expertise in the design of robust, high-performance panels to examine immune responses and evaluate the pharmacological effects of your drugs on their targets. Collectively, these work to accelerate your drug development initiatives and support clinical filings with regulatory authorities.

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