Biomarkers by definition indicate some state or process that generally occurs at a spatial or temporal distance from the marker itself, and it would not be an exaggeration to say that biomedicine has become infatuated with them: Where to find them, when they may appear, what form they may take, and how they can be used to diagnose a condition or predict whether a therapy may be successful.
Biomarkers are on the agenda of many if not most industry gatherings, and in cases such as Oxford Global’s recent “Biomarker Congress” and the GTC “Biomarker Summit”, they hold the naming rights. There, some basic principles were built upon, amended, and sometimes challenged.
In oncology, for example, biomarker discovery is often predicated on the premise that proteins shed from a tumor will traverse to and persist in, and be detectable in, the circulation. By quantifying these proteins—singularly or as part of a larger “signature”—the hope is to garner information about the molecular characteristics of the cancer that will help with cancer detection and personalization of the treatment strategy.
Yet this approach has not yet turned into the panacea that was hoped for. Bottlenecks exist in affinity reagent development, platform reproducibility, and sensitivity. There is also a dearth of understanding of some of the fundamental principles of biomarker biology that we need to know the answers to, said Parag Mallick, Ph.D., whose lab at Stanford University is “working on trying to understand where biomarkers come from.” And sometimes, too, accepted wisdom just isn’t so.
For example, there are dogmas saying that circulating biomarkers come solely from secreted proteins. But Dr. Mallick’s studies indicate that fully 50% of circulating proteins may come from intracellular sources or proteins that are annotated as such. “Right now we don’t understand the processes governing which tumor-derived proteins end up in the blood.”
Other seemingly obvious questions include “how does the size of a tumor affect how much of a given protein will be in the blood?”—perhaps the tumor is necrotic at the center, or it’s hypervascular or hypovascular. “The problem is that these are highly nonlinear processes at work, and there is a large number of factors that might affect the answer to that very simple question,” he pointed out.
Their research focuses on using mass spectrometry and computational analysis to characterize the biophysical properties of the circulating proteome, and relate these to measurements made of the tumor itself.
“We’ve observed that the proteins that are likely to first show up and persist in the circulation, on average, are more stable than proteins that don’t,” Dr. Mallick said. “This is something that people qualitatively suspected, but now we can quantify how significant the effect is.”
The goal is ultimately to be able to build rigorous, formal mathematical models that will allow something measured in the blood to be tied back to the molecular biology taking place in the tumor. And conversely, to use those models to predict from a tumor forward to what will be found in the circulation. “Ultimately, the models will allow you to connect the dots between what you measure in the blood and the biology of the tumor.”
Bound for Affinity Arrays
Affinity reagents are the main tools for large-scale protein biomarker discovery. And while this has tended to mean antibodies (or their derivatives), other affinity reagents are demanding a place in the toolbox.
Among these are Affimers, a type of affinity reagent being developed by Avacta. Affimers consist of a biologically inert, biophysically stable protein scaffold containing three variable regions into which distinct peptides are inserted. The resulting three-dimensional surface formed by these peptides interacts and binds to proteins and other molecules in solution, much like the antigen-binding site of antibodies.
Unlike antibodies, Affimers are relatively small (13 KDa), non-post-translationally modified proteins that can readily be expressed in bacterial culture. They may be made to bind surfaces through unique residues engineered onto the opposite face of the Affimer, allowing the binding site to be exposed to the target in solution. “We don’t seem to see in what we’ve done so far any real loss of activity or functionality of Affimers when bound to surfaces—they’re very robust,” said CEO Alastair Smith, Ph.D.
Avacta is taking advantage of this stability and its large libraries of Affimers to develop very large affinity microarrays for drug and biomarker discovery. To date they have printed arrays with around 20–25,000 features, and Dr. Smith is “sure that we can get toward about 50,000 on a slide,” he said. “There’s no real impediment to us doing that other than us expressing the proteins and getting on with it.”
Customers will be provided with these large, complex “naïve” discovery arrays, readable with standard equipment. The plan is for the company to then “support our customers by providing smaller, bespoke, arrays with the Affimers that are binding targets of interest to them,” Dr. Smith foretold. And since the intellectual property rights are unencumbered, Affimers in those arrays can be licensed to the end users to develop diagnostics that can be validated as time goes on.
Around 20,000-Affimer discovery arrays were recently tested by collaborator Professor Ann Morgan of the University of Leeds with pools of unfractionated serum from patients with symptoms of inflammatory disease. The arrays “rediscovered” elevated C-reactive protein (CRP, the clinical gold standard marker) as well as uncovered an additional 22 candidate biomarkers. Some of the latter, when combined with CRP, appear able to distinguish between different diseases such as rheumatoid arthritis, psoriatic arthritis, SLE, or giant cell arteritis.
Sometimes biomarkers are used not to find disease but to distinguish healthy human cell types, with perhaps the most obvious examples being found in flow cytometry and immunohistochemistry. These widespread applications, however, are difficult to standardize, being subject to arbitrary or subjective gating protocols and other imprecise criteria.
Epiontis instead uses an epigenetic approach. “What we need is a unique marker that is demethylated only in one cell type and methylated in all the other cell types,” explained CBO and founder Ulrich Hoffmueller, Ph.D. Each cell of the right cell type will have two demethylated copies of a certain gene locus, allowing them to be enumerated by quantitative PCR.
The biggest challenge is finding that unique epigenetic marker. To do so they look through the literature for proteins and genes described as playing a role in the cell type’s biology, and then look at the methylation patterns to see if one can be used as a marker, Dr. Hoffmueller said. They also “use customized Affymetrix chips to look at the differential epigenetic status of different cell types on a genomewide scale.”
The company currently has a panel of 12 assays for 12 immune cell types. Among these is an assay for regulatory T (Treg) cells that queries the Foxp3 gene—which is uniquely demethylated in Treg even though it is transiently expressed in activated T cells of other subtypes. Also assayed are Th17 cells, “which are especially tricky to detect by flow cytometry because the cells have to be stimulated in vitro,” he pointed out.