What is Cytokine Storm?
A cytokine storm is a potentially lethal adverse effect of some immunomodulatory therapies. It is caused when a pro-inflammatory cytokine feedback loop is created by a treatment. Since the TGN-1412 clinical trial disaster, regulatory bodies have been more diligent about requiring certain classes of novel therapeutics to be assessed for their potential to trigger a cytokine storm.
How We Test
Our cytokine release assay, which can be customized to meet the demands of your program, utilizes primary human or NHP cells, sourced fresh (less than 6 hours old) to ensure maximum viability and function. The cells are incubated under positive control (anti-CD3, clone OKT3), carrier control (PBS or other) and test conditions for 24 – 72 hours. The supernatants can then be assessed for pro-inflammatory cytokines using Luminex™ technology, and the cells themselves assessed for proliferation and activation markers by flow cytometry.
Fresh, primary peripheral blood cells are reported in the scientific literature to be the best choice for assessing cytokine storm risk. As with all of our assays, we use the most suitable and most translatable cells. With access to fresh blood from humans and NHPs the studies in our lab are designed for the most successful outcome.
Fresh PBMCs are the cells of choice for cytokine release assays evaluating cytokine storm risk. These cells may be derived from normal donors and naïve-treated NHPs. Within these PBMC populations, it is the T cell population that releases IL-2, TNFα, and IFNγ, as well as multiple other cytokines.
Our cytokine release assay uses fresh PBMCs derived from humans and NHPs are cultured in the presence of both control and test antibodies. The antibodies may be presented to the cells in multiple forms so as to evaluate possible arrangements that could induce a cytokine storm. The cultures may be incubated for 24-72 hours, after which supernatants are harvested and tested for pro-inflammatory cytokines.
In response to antibodies, T cell proliferation may be evaluated by flow cytometry. From the same cultures, supernatants may be harvested and assessed for cytokine production using multiple technologies including Luminex™. Typically, pro-inflammatory cytokines are measured, but depending on the test antibody the evaluation of other cytokines may be of interest.
Traditionally, NHPs have been used to test new antibodies prior to clinical trials. In some circumstances antibodies, such as TGN-1412, did not have a deleterious effect on NHPs, but caused a massive cytokine storm in human clinical trials. For these reasons, many antibodies are screened ex vivo on both human and NHP samples.
New antibodies are being developed to target a host of diseases including multiple types of cancers, and auto-immune diseases. However, the risk of cytokine release syndrome is always a concern with this compound class. For these reasons the FDA requires extensive screening to prevent clinical cytokine storm.
Many researchers have shifted their focus to work on an antiviral to combat the novel strain of coronavirus. Antivirals should be tested to determine off-target toxicity effects, as they may generate a cytokine storm or hypercytokinemia. While inflammation is an essential part of an effective immune response, an overactive immune response like a cytokine storm, leads to healthy tissue being damaged.
Features of our in-vitro model for assessing biotherapeutic-induced cytokine release
Relevant Human Primary Cell Co-Culture System
- Uses pre-qualified human PBMCs cultured on a HUVEC monolayer
- Use of normal human primary cells allows for a more relevant system for predicting cytokine release syndrome and cytokine storm than in vivo NHP models due to nuanced immunological differences between species
- Multiple PBMC donors can be tested upon request, including donors with specific disease conditions or HLA types
Uses the Soluble Form of the Biotherapeutic Agent
- More relevant native conformation than if plastic-immobilized or artificially cross-linked
- Allows for a better dose response curve than if plastic-immobilized
- Core Panel Tested: IL-2, IL-6, TNFα and IFNγ
- Additional cytokines can be measured upon request
- Measured by ELISA or Luminex™
Relevant Positive and Negative Controls
- Positive control: anti-CD3 clone OKT3
- Negative control: humanized anti-CD20
- Clients can also send their samples blinded and include their own controls
For more information, download the poster describing this co-culture assay system that was presented at the 2014 Society of Toxicology conference.