What are functional assays?
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 in drug development 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 assays can measure a variety of functionalities, including the capacity of cells to secrete immune mediators such as cytokines, to proliferate in response to antigens, or to kill target cells.
The role of functional assays across the drug development process
Functional assays can be applied throughout the drug development process, from early preclinical characterization to the later stages of preclinical and clinical development.
Preclinical phase: candidate ranking and lead selection
In the preclinical phase, functional assays can help rank different candidates in vitro and select a lead for further clinical development. This can help companies save time and resources by identifying the most promising candidates early in the process.
Clinical phase: safety, efficacy, and MOA confirmation
In the clinical phase, functional assays can be utilized to evaluate the safety and efficacy of a drug and to confirm its MOA in humans. For example, they 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 antibody-dependent cell-mediated cytotoxicity (ADCC) by NK cells, or phagocytosis (ADCP) by macrophages after administration of therapeutic antibodies (see our case study on immunophenotyping). 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. They serve as a surrogate test to show how active a therapeutic candidate is and how the immune system can be engaged to achieve that effect.
Technologies used in functional assays
There are a variety of technologies that can be used to conduct functional assays like LBA platforms, Flow Cytometry, ELISpot methods, molecular biology assays, and LC-MS. In our facilities in Lyon, we mainly rely on LBA platforms, Flow Cytometry, ELISpot methods. A cell culture is often used as a starting point to demonstrate cellular cytotoxicity. Immune cells are typically co-cultured with target cells in the presence of a candidate antibody or antigen-derived vaccine to observe cytotoxicity. This can then be followed by endpoint assays such as ELISpot or Flow Cytometry, or by measuring immune mediators in the cell supernatants. In this respect, it is essential that highly skilled scientists handle the cells and place them in a state that provides consistent data on what they are supposed to do, whether that is killing, proliferating, secreting immune mediators, or activating specific intracellular pathways. This is critical for determining the effectiveness, safety, and/or MOA of a treatment (Maecker HT, et al., 2005).
Key challenges in functional assay development
Replicating in vivo conditions in vitro
Functional testing can be challenging because it involves creating, in vitro, a model expected to mimic what happens inside the body. This can introduce bias and uncertainty, which is why it is not a standard approach. To make it effective, certain assumptions must be made and the right conditions must be established, while being careful not to misinterpret the results. For example, 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 illuminate the clinical outcome.
Choosing the right readout
Another challenge in developing functional assays is the readout, which can differ depending on what you want to measure. Some sponsors may request a direct ex vivo measurement of functionality within the context of their clinical study. For example, in patients treated with a therapeutic antibody, one would like to document the antibody’s ability to increase the cytotoxicity of certain cells. However, this is often not possible and requires restimulation, that is, re-exposing the cells to the drug, in order to produce a measurable signal in the endpoint assays.
Sample quality and multi-center harmonization
Other critical criteria are good-quality samples and the harmonization of sample preparation in multi-center studies. Sample integrity is critical, as it can significantly affect the accuracy of results, a frequent challenge in functional testing.
Expertise and quality control: why experience matters in functional assay drug development
Developing functional assays requires a clear understanding of the drug candidate’s science, the relevant pathology, and the associated biomarkers, as well as 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 built over time. Clients come to us because we have expertise in complex assays and can navigate the many challenges and guidelines in the field. We have saved significant time and resources for clients in areas such as immuno-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 remain consistent, as working with cell cultures carries inherent risk. For ELISpot assays, we use reference samples that we track on control charts to detect any issues in a timely manner. In addition, we participate in external quality assessment programs at least once a year to benchmark ourselves against our peers. For example, we are part of a proficiency panel led by Duke University in the USA, which includes a network of 27 labs. By comparing results across operators using the same samples, we can ensure our team is performing at the highest level (Islam R, Vance J, Poirier M, et al. 2022).
Partner with experts for your functional assay needs
Functional assays are highly individualized and tailored to meet specific needs. This can make the development process difficult to navigate, as it is not always clear where you are heading, even when you know where you ultimately want to end up. An assay’s success depends on a thorough understanding of the product’s biology, its mechanism of action, and the sample used. It is therefore vital to have a team of experienced and skilled scientists who can closely collaborate with your teams to develop creative solutions and perform functional assays that will answer your needs.