5 Different Kinds of Cytokine Release Assays: Weathering the Storm | CRA Post II


Guest blog post by Chris Cooper, Scientific Specialist II, Immunotoxicology & Immunology, Labcorp Early Development Laboratories Ltd

In our previous post, we outlined the dangers of Cytokine Release Syndrome (CRS) and the importance of preclinical Cytokine Release Assays (CRAs) when developing monoclonal antibodies (mAbs) that interact with the patient’s immune system. In this second post, we describe the different kinds of assays in use and how these may fit into your drug development program. An alternative type of CRA, peripheral blood mononuclear cell (PBMC) blood outgrowth endothelial cell (BOEC) co-culture, will be discussed in more detail in our next blog post.

Some of the questions you should consider when choosing the most appropriate CRA include the following:

  • Does the compound have an Fc region that can bind to neonatal Fc gamma receptors on endothelial cells?
  • Does the compound bind to more than one target?
  • Target expression healthy subjects?
  • Which cytokines should you measure, since not all cytokines are upregulated during a cytokine storm?

Whole blood assays

Whole blood assays are one of the most widely used CRAs. Their main advantage is that they closely replicate in vivo conditions. The assay format recreates immune cell subset frequencies, FcγR cellular distribution and expression levels at physiological levels, revealing links between the FcγR genotype and magnitude of cytokine release in response to mAb treatment.

The whole blood CRA is far more sensitive than the PBMC solid-phase CRA (see below) for alemtuzumab (anti-CD52 antibody) which stimulates FcγRI-mediated cytokine release. However, whole blood CRAs are not sensitive toward muromonab-CD3 and TGN1412 (anti-CD28 superagonist) cytokine release. Other studies have determined that a whole blood assay format is preferable over PBMCs for detection of cytokine release by oligonucleotides which bind to toll-like receptors.

Human PBMCs

PBMCs are composed of pure isolation of lymphocytes (T cells, B cells, NK cells) and monocytes. They are extracted from whole blood, so the assay is less physiologically relevant than whole blood CRAs, but may be more sensitive to revealing a cytokine signal.

Solid-phase CRA, which involves the co-incubation of PBMCs with mAbs that have been dry-coated onto a tissue culture plate, was shown to be predictive for the cytokine release potential of TGN1412. In the liquid phase, PBMC assays do not produce target crosslinking or FC binding. Alternative solid-phase assessments include wet-coating mAbs onto the tissue culture well plastic at a lower density to that presented via dry-coating. All the above are used to mimic physiological mAb presentation but are ultimately different to in vivo presentation with unfortunate drawbacks with regards to false positive rate and high assay background. Dry and wet coat presentations also apply to the whole blood assay.   

High Density CRA

First described by Romer et al in 2011 pre-culturing PBMC cells at high density improves the  sensitivity of the assay to detect therapeutic T-cell activation. High density PMBCs respond akin to lymph node-like cells compared to regular low density PBMC assay. This assay doesn’t initiate target crosslinking or FC binding. Although unlike regular PBMC assays, this assay system can be used to detect positive responses to TGN1412 in a soluble/liquid phase due to the cells being in a “primed” lymph node-like state.  

Human Umbilical Vein Endothelial Cell (HUVEC) PBMC Co-culture

This assay produces target cross-linking and FC binding. Endothelial cells are routinely grown from umbilical veins, which are used as the interface with PBMCs for cytokine release testing. Because these assays are made of a heterologous mixture of cells, this could result in a tissue mismatch – when cells from one donor are mixed with those of another – and that this might be responsible for some of the limitations of HUVEC/PBMC co-culture assays.

Blood Outgrowth Endothelial cell (BOEC) PBMC Co-culture

This autologous assay produces target cross-linking and FC binding.  First described by Mitchelle, et al, in 2015, this assay format is similar in concept to the HUVEC assay with a minor difference. BOECs are endothelial cells outgrown from PBMC collected from the same donor. Due to this assay being completely autologous, there are no tissue mismatch issues thus improving sensitivity of the assay. This assay system to date is the most reflective of in vitro systems to replicate the in vivo vascular microenvironment. A major benefit of this assay system over other formats is that the biologic therapeutic is presented in a more physiologically relevant manner akin to being dosed. This assay is a patented assay system and currently we are the only CRO to commercially offer it.

Typical study design

Since the absolute levels of the produced cytokines vary depending on the CRA assay used, it can be difficult to identify a true positive result. Therefore, cytokine release studies should include relevant positive and negative controls. Selection of control monoclonal antibodies depends on the mechanism of action of the product evaluated, e.g. muromonab-CD3 or TGN1412 for T-cell targeting products; alemtuzumab and/or rituximab for products with cytotoxic effector function or phytohemaggluttin for a mitogen positive control.

The study should also use blood products obtained from multiple donors (advise a minimum of 10) to enable a natural physiological mixture of samples. If target is not present in healthy donor samples, it is suggested to conduct hybrid studies using healthy donors alongside patient-derived samples. Suggestions for compound dosing selection should be selected based on the target receptor occupancy and as a minimum a high, medium and low dose should be considered.   

Challenges of current assay formats

Not all CRA platforms can discriminate between mAbs inducing mild or moderate cytokine release, nor can they be used to determine a threshold where the levels of cytokines released may be associated with serious adverse events in humans. CRAs are not yet fully predictive of cytokine release, and their accuracy is dependent on the types of antibodies being assessed. For this reason, researchers continue to search for improved CRA formats.

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Missed the first post in our series? Read our introductory post on in vitro cytokine release assays here.


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