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.
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 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.
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