Drug developers turn to the new and established immunoassay platforms to measure and identify protein biomarkers.
Imagine what life would be like without the antibody. Without the antibody, vertebrates would be incapable of defending their bodies against microbial invasion. Without the antibody, vertebrates (and humans!) would surely have become extinct millions of years ago. But vertebrates did evolve the ability to produce antibodies in response to antigenic challenge. So this is a moot point.
So, instead, imagine that the antibody had never been discovered. Imagine studying proteins without an antibody. Specific protein identities would be difficult, if not impossible, to decipher. Protein detection in biological fluids or in cells would be an absolute nightmare. Without an antibody to capture or detect a protein, the immunoassay would not exist.
There are many different types of immunoassays, each with their own official-sounding name. And the immunoassay platform is applied the world over, from the immunodiagnostic assays performed in the clinical laboratory to the immunofluorescence microscopy protocol performed in the drug discovery lab. And, in fact, the use of immunoassays for quantitative and qualitative measurements of protein biomarkers in biological fluids—assays customarily performed in clinical laboratories—are being used with increasing frequency in drug discovery labs.
Biomarker, first.
Drug discovery, next.
For example, in the Department of Immunology and Inflammation at SRI International, Menlo Park, Calif., senior director Annalisa D’Andrea, PhD, primarily uses an immunoassay platform that is an enzyme-linked immunosorbent assay (ELISA). Using several different detectors, each with a different purpose, D’Andrea and her colleagues are able to achieve high throughput ELISA. The high throughput nature of these ELISAs is only possible through automation. And as anyone that has ever performed a manual immunoassay can attest, performing manual ELISA is as tedious as trying to hit a golf ball out of a sand trap.
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Although D’Andrea’s lab does perform classical ELISA based on colorimetric detection, their most recently-acquired detector, a Meso Scale Discovery (Gaithersburg, Md.) instrument, works on a different principle—electrochemiluminescence. “[The MSD] is very good. It has a very low background because it is based on electrical charges,” says D’Andrea, who adds that the MSD instrument is the primary one used for drug discovery research in her lab.
In D’Andreas’s lab, the main thrust for drug discovery is inflammatory diseases, for example, multiple sclerosis and inflammatory bowel disease. And for that purpose, they implement the immunoassay to define those biomarkers that will aid in the drug discovery process. The primary biomarkers in this case are inflammatory cytokines. On the subject of cytokine biomarkers used for drug discovery, D’Andrea explains her reasoning: “If we have to define if a therapeutic intervention is affecting the mechanism of action of a drug, we will do the biomarker screening for cytokines. Or, if we want to define if one of these cytokines is a marker of a disease state, then we will pass the samples through the machine to see if the same cytokines are consistently affected in these patients. And the crucial part right now is to define what a biomarker for a specific disease is and to define the drug discovery around it.”
Animal platforms
But to find disease-specific biomarkers, researchers must first develop disease models in which the effects of experimental induction of pathophysiology on the expression of such biomarkers can be tested. Melior Discovery, Inc. in Exton, Pa., is an in vivo pharmacology-based company whose primary work is in drug repositioning, but also includes pharmacological profiling of preclinical compounds. Whether they are repositioned or preclinical stage compounds, the pharmacological properties of all of these compounds are determined using Melior’s proprietary multiplexed theraTRACE mouse model platform. The platform spans 12 different therapeutic areas and 35 animal models covering these areas, many of which utilize ELISA-based immunoassays. Test compounds are tested for activity in multiple therapeutic indications in these animal models. The theraTRACE platform allows optimal pharmacological profiling of a test compound utilizing a small number of animals.
“These compounds are tested for multiple therapeutic indications when dosed into groups of mice. The ways that we quantify these assays range from physiological—to behavioral—to the biochemical. The biochemical measurements include these immunoassay-based biomarkers,” says Michael S. Saporito, PhD, vice president of research at Melior. The biomarkers include cytokines, hormones, and other markers associated with the specific disease state being investigated; these biomarkers are measured using commercially available, as well as Melior-developed immunoassays.
One of the models Melior runs is a septic shock model, which is used to determine the effectiveness of new drug candidates against septic shock. A typical experiment with this model involves injecting lipopolysaccharide—an inducer of septic shock-like symptoms—into two groups of mice: treatment and placebo. The test agent is then injected into the treatment group and blood levels of multiple cytokine biomarkers, including tumor necrosis factor alpha (TNF-?) and interleukin 6 (IL-6), are measured using ELISA-based immunoassays. “What we are looking for in that specific assay is a reduction in those cytokines by the test agent. So anything that reduces those cytokine levels may be considered to be a positive hit in our screening platform and may be worthy of further investigation,” says Saporito, who explains that positive hits are then investigated in follow-up studies in additional animal models of inflammatory disease.
There are also several depression models in Melior’s multiplex animal model platform, one of which is the stress-induced corticosterone model. This depression model is based on the fact that sustained, elevated blood levels of cortisol (the physiological equivalent of which in mice is corticosterone) are associated with chronic depression in humans. “We can induce mild stress in mice and elevate corticosterone levels. In these instances, we’re looking at drugs that can reduce the corticosterone levels. So anything that reduces those corticosterone levels may be an indication that that compound may be useful in a disease such as chronic long-term depression or major depression,” explains Saporito. Results of these assays are also compared to, and integrated with, those from additional depression models from Melior’s platform.
Building immunoassays
Once a disease-specific biomarker is discovered, then an immunoassay needs to be developed to measure it. As director of R&D for Millipore Corporation, St. Louis, Mo., Jehangir Mistry, PhD, focuses on developing biomarker immunoassays for commercialization. The focus of his group is to develop immunoassays that measure biomarkers involved metabolic diseases, cardiovascular disease, as well as bone and cartilage metabolism, inflammatory diseases, neuroscience, and cancer. The biomarker immunoassays that Millipore has developed are for research purposes only, encompassing drug discovery research as well as basic biomedical research into disease mechanisms.
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Mistry’s group develops their immunoassays based on three different platforms: ELISA, radioimmunoassay, and Luminex’s xMap technology; the latter technology allows them to multiplex. According to Mistry, Millipore used these assay platforms to develop immunoassays for disease-specific biomarkers because they are the most common immunoassays available and they are very robust assays. “These technologies are established, but the way we develop our assays is proprietary, meaning the way we develop our antibodies and the way we formulate those assays with buffers and coating solutions and other proprietary reagents we develop. That is our IP,” says Mistry.
To determine for which bio-markers it should develop assays, Millipore applies a thorough planning process. And that process starts with information-gathering. “Our marketing group and the R&D scientists scour the literature, talk to customers in academia and in pharmaceutical/biotech companies, and attend scientific meetings. Afterwards, we basically compile a database of targets that are requested by our customers and through our own literature search. And we assign priorities and resources to get them developed and commercialized,” says Mistry.
According to Mistry, there are many challenges Millipore faces when trying to develop an immunoassay. “The techniques have been established. And the platforms have been established. But it’s the development of assays to new molecules becomes challenging because many times these molecules are protein and they are quite complex, i.e., they may exist in various molecular forms,” he says. Some of the processes he mentions include posttranslational modifications of proteins (cleavage, phosphorylation, etc.) and conservation of protein sequence across species. Major problems arise when trying to develop high affinity, high specificity antibodies against such proteins. And therein lies the challenge, because without high quality antibodies, the immunoassay may not have desired sensitivity and specificity for measurement of a target protein.
In summary, immunoassays have been used to detect, identify, and quantify protein biomarkers for more than two decades, especially in clinical laboratories, and now these diagnostic assays are being used with increasing frequency in drug discovery labs as well.
This article was published in Drug Discovery & Development magazine: Vol. 11, No. 10, October, 2008, pp. 24-28.
Filed Under: Drug Discovery
