Most therapeutic antibodies have a Fab’ region that binds the antigen, and an Fc region which binds to and activates various cell surface receptors and complement proteins. Both ends of the antibody together mediate various physiological effects of the drug molecule. A global focus on the development of therapeutic antibodies has driven the need for assays to interrogate the Fc effector function of new drug candidates. These assays are critical for studying the mechanism of action of the antibody drug candidate, and therefore are required for successful submission of a biologics license application (BLA) to regulatory authorities.
Specific Measurement of Target Cell Death is Needed
The Fc region of an antibody activates and recruits various immune responses that mediate killing of cells expressing the antigen bound by the Fab’ region, including antibody dependent cell-mediated cytotoxicity (ADCC). Therefore, a cell-based assay that evaluates effector cell-mediated target cell death in the presence of the antibody drug is an important component of a molecule’s development path.
Challenges of Traditional Cytotoxicity Assays
Current technologies to evaluate ADCC with immune effector cells typically utilize fluorescent dyes, such as calcein, or radioactive dyes, such as Chromium-51, to label cancer cells expressing the target antigen. An ADCC response will cause the dye to be released, and the fluorescence or radioactivity can be detected in the media.
The challenge of using membrane permeable dyes to label target cells:
- These dyes are membrane permeable and can diffuse out just as easily as they diffuse into the cell.
- The assays using these dyes can only be run for up to 4 hours and suffer from high background activities.
- Additionally, the use of a radioactive dye requires an appropriate facility, increasing the cost of the assay.
Alternatively, general cell death responses such as detection of lactate dehydrogenase (LDH) have also been used for ADCC. But these assays suffer from a lack of specificity.
Recently, there has been an advent of a reporter gene assay that can measure the activation of the immune cells upon engagement with the Fc portion of the antibody. Although this assay is highly reproducible, it does not demonstrate the true MOA of the drug, i.e., it does not demonstrate target cell death, and only goes so far as to demonstrate immune cell activation.
A New and Powerfully Accurate Cytotoxicity Assay
Here at DiscoverX, we have developed an easy way to label target cells that addresses a number of the issues described above:
- The assay measures cell death of the target cells only and, therefore, the true mechanism of action of the drug molecule.
- It is also a dye-free, non-radioactive way to detect target cell death.
The technology of the assay is quite simple:
- We express one part of our proprietary split enzyme system in the target cells, and the other part is added exogenously into the media.
- When the target cells die, the two fragments come together to create an active enzyme and generate a chemiluminescent light.
This homogeneous assay, known as the KILR (Killing-mediated Immune Lysis Reaction) cytotoxicity assay, is an easy to use, add and read assay that is compatible with almost any target cell and multiple effector cell types. This approach has enabled multiple drug development companies worldwide to conduct screens to identify antibodies that are functional for Fc effector function and, importantly, it has also been useful to create ADCC assays that can be used for lot release testing of an antibody drug.
Additional Uses Beyond Antibody Dependent Cell-mediated Cytotoxicity
Apart from the use of this assay for ADCC, our clients in the industry have also found several exciting new uses for this platform. Almost any approach where you have a co-culture of immune cells and target cells and you would like to determine target cell death will find valuable the use of this assay platform. Our partners have already demonstrated the use of this assay in the following drug development applications:
- Antibody dependent cellular phagocytosis (ADCP)
- Bispecific antibody mediated T-cell redirection
- Adoptive t-cell therapies such as CAR-T or chimerin antigen receptor t-cells
These applications are facilitated by an important feature of the KILR platform where the reporter protein is stable in the media for at least 72 hours (and maybe longer) making it possible for the first time to run a plate-based cytotoxicity assay for molecules that need a long time to kill cells. This enabling technology is now available from DiscoverX as KILR cell lines that stably express this reporter protein or as a retroviral vector that can be used to create your own cell line within the flexible confines of your lab.