Understanding the interactions between drugs and biological systems is critical for the success of a new drug. One key tool in this process is functional assays.

Functional assays are customized assays that evaluate the impact of drugs on the functionality of cells. They test for a drug’s specific biological mechanism – or mode of action (MOA) – that leads to its therapeutic effect. These can measure a variety of functionalities. This can be either the capacity of cells to secrete immune mediators, such as cytokines, or to proliferate in response to antigens. They can also measure the capacity of cells to kill target cells.

Functional assays can be applied throughout the drug development process, from the early preclinical characterization of a product to the later stages of preclinical and clinical development. In the preclinical phase, functional assays can help ranking different candidates in vitro and select for a lead for further clinical development. This can help companies save time and resources by identifying the most promising candidates early in the development process. In the clinical phase, functional assays can be utilized to evaluate the safety, efficacy and confirm the MOA of the drug in humans. For example, functional assays can evaluate the capacity of cells to secrete cytokines in response to antigens after vaccination by CD4+ or CD8+ T-cells, or to modulate direct or via antibody-dependent cell-mediated (ADCC) cytotoxicity by NK cells or phagocytosis (ADCP) by macrophages after administration of therapeutic antibodies (check our case study on immunophetyping). The use of functional assays can vary depending on the product, but they are particularly important for companies developing immunotherapies, vaccines, or cell and gene therapies. It’s a kind of surrogate test to show how active your therapeutic candidate is and how you can use the immune system to do that.

There are a variety of technologies that can employ functional assays. Here in Lyon, we use LBA platforms, Flow Cytometry and ELISpot methods. A cell culture is often used as a starting point to demonstrate cellular cytotoxicity. Immune cells are usually cocultured with target cells in the presence of a candidate antibody or antigen-derived vaccines to observe cytotoxicity. This can then be followed by endpoint assays such as ELISpot or Flow cytometry or measuring immune mediators in the cell supernatants. In this respect, it is important that highly skilled scientists can handle the cells and put them in a state that provides consistent data on what they are supposed to do, either kill or proliferate or secrete immune mediators or activate specific intracellular pathways. This is important for determining the effectiveness, safety and/or MOA of a treatment.

Functional testing can be challenging because it involves creating in vitro, a model expected to mimic what happens inside the body. This can lead to bias and uncertainty, which is why it is not a standard approach. To make it effective, we must make certain assumptions and create the right conditions while also being careful not to misinterpret the results. For example, just observing a natural killer cell destroying a target cell in the lab does not necessarily mean the same thing is happening in a cancer patient’s body. However, when combined with clinical observations and other biomarkers (using imaging or immunohistochemistry techniques), it can help understand the clinical outcome.

Another challenge in developing functional assays is the readout, which can differ depending on what you want to measure. Some sponsors may ask for a direct ex vivo measurement of functionality in the context of their clinical study. For example, in patients treated with a therapeutic antibody, one would like to document the ability of the antibody to increase the cytotoxicity of certain cells. However, this is often not possible and requires restimulation, i.e., reexposing the cells to the drug, to document and see something in the endpoint assays that you use to measure.

Other critical criteria are good quality samples and harmonization of sample preparation in multi-center studies. Sample integrity is critical as it can affect the accuracy of results, which is often a challenge in functional testing. For more information on the standardization of sample preparations, please check out the latest KCAS blog on this topic.

Thus, developing functional assays can be challenging and requires a clear understanding of the science of the drug candidate, pathology, biomarker and a high level of expertise and experience in the field. In Lyon, we heavily focus on developing know-how, a combination of experience and knowledge over time. Customers come to us because we have expertise in tricky assays and can navigate the many traps and guidelines in the field. We have saved significant time and resources for our clients in areas such as immune-oncology and vaccines.

It is crucial to take advantage of that experience not to start from a white page because it can be extremely tricky.

We also ensure our methods stay consistent, as working with cell cultures carries some risk. For ELISpot assays, we use reference samples that we track on control charts to detect any problem in time. In addition, we participate in external quality assessment programs, at least once a year, to compare ourselves with our peers. For example, we are part of a proficiency panels led by Duke University in the USA including a network of 27 labs. By comparing results among operators using the same samples, we can ensure our team is performing at the highest level. Functional assays are very individualized and tailored to meet specific needs. This can make the development process difficult to navigate, as it’s not always clear where you are heading, even if you know where you ultimately want to “end up.” The assay’s success depends on a good understanding of the biology of the product, its mechanism of action and the sample used. Therefore, it is vital to have a team of experienced and skilled scientists who can closely interact with your teams to develop creative solutions and perform functional assays that will answer your needs. Let Active Biomarkers plot the course for your functional assays.