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EC Gives New Consortium $3.8M to Develop High-throughput Technology for Biomarker Detection


The European Commission has provided a new consortium €3 million ($3.8 million) to develop a high-throughput cancer biomarker-research platform.

Called Proactive, the initiative so far has six partners, led by Olink Bioscience, whose proximity ligation assay technology will be built out as part of the effort. The EC is providing funding to the initiative for three years as part of its 7th framework program.

The goal of the initiative is to create a technology platform that can detect cancer biomarkers in small amounts of blood, a fluid rich with information that has, nonetheless, confounded researchers because of its complexity.

While Proactive will be basing its work on colorectal cancer biomarkers, an official said that the technology developed from it will have applications to other diseases.

"At the end of the project, what we will have built is a world leading platform for [doing] biomarker research in biobanked samples," Simon Fredriksson, CSO of Olink and project coordinator, told ProteoMonitor recently. "The infrastructure that we build can then be applied to new markers, new disease types … and it will be offered as a service to the research community."

The other members of Proactive are Innova Biosciences in the UK; Integromics in Spain; Fujirebio Diagnostics in Sweden; the University of Copenhagen; and Uppsala Academic Hospital.

In its work, Proactive members will assay known markers for colorectal cancer with new markers discovered as part of the initiative, Fredriksson said. "Large numbers" of such markers will then be analyzed within the same sample.

"A lot of people have found [a] marker from this sample, and another marker of interest in another sample, but we will combine these into panels, and hopefully be able to achieve higher detection sensitivity and specificity through this panel-based approach," he said.

Fredriksson estimated that there are between 500 and 1,000 "very interesting" biomarkers for which antibodies are commercially available. Proactive will narrow that list down to about 200 biomarkers, which will then be analyzed for their efficacy as indicators for colorectal cancer.

According to Nils Brünner, a professor of biomedicine at the University of Copenhagen, prospective markers will be judged against two markers that have previously been identified as markers for colorectal cancer — carcinoembryonic antigen and tissue inhibitor of metalloproteinases type I, which was discovered by Brünner and colleagues.

"What we are aiming at now is to find markers that will add value to these two," he said. Brünner will be organizing the sample collections for Proactive.

In a statement, the consortium said that where once the biomarker field was interested in "reactive diagnostics," it is now shifting toward early-stage disease detection when disease is most treatable. For a heterogeneous disease such as cancer, diagnostic tests need to have both high sensitivity and specificity, and multiple-marker tests "are essential for success," it said. And in order to develop such diagnostics, high-throughput technology that allows for the analysis of multiple low-abundant biomarkers is necessary.

That is where Proactive comes into the picture.

Fredriksson said that the goal of the consortium is to develop a technology infrastructure and then pilot a project in colorectal cancer detection, but "not to actually revolutionize colorectal cancer detection.

"We want to build a technology pipeline that we then can utilize to do lots of interesting biomarker work with."

That technology will be based on Olink's PLA platform. While the company has several iterations of the technology, including a commercially available one, one of the goals of Proactive is to build out the plexing capability of the technology. Currently, PLA has 10-plex capability, but by the end of the project, that should increase to 50-plex, Fredriksson said.

The colorectal cancer project will take place in three stages. At each stage, assays will be tested in pilot biomarker projects aimed at improving the detection of colorectal cancer in plasma samples. In the first stage, which is already taking place, four 8-plex panel assays are being tested. In the second stage, anticipated to begin in about a year, four 30-plex panel assays will be tested, followed by four 50-plex assays, two years from now.

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During the ramp-up, Olink designed and validated sequence sets that could then be coupled to target-specific antibodies for the protein-to-DNA conversion reactions.

Increasing the Plex

For Olink, Proactive is a chance to improve its PLA platform, which the company has been developing since its inception in 2004 [See PM 11/05/04].

"It's part of our plan to develop our capabilities to become a biomarker research provider," Fredriksson said, "and this is a perfect opportunity for us to pilot our technology together with our partners and use these funds to build our capacity to even higher levels to become a leading provider of these types of services."

The PLA technology is based on proximity probes containing oligonucleotide-labeled antibodies designed to bind to proteins. The probes form amplifiable tag sequences upon ligation when they are brought in proximity.

Because the protein targets are represented as DNA signatures, they can be amplified and analyzed by other methods such as PCR-based techniques.

One version of the technology is commercially available under the Duolink name for the detection of protein-protein interactions in fixed cells and tissues.

For its work with Proactive, however, the company will be using a version in which proteins will be detected by binding of the PLA probes — antibodies coupled to DNA strands — in solution in a homogeneous reaction, which are then converted to a PCR amplicon.

The potential of Olink's technology was what drew Brünner's interest in Proactive, he said.

"We have these huge tissue and blood repositories that fit extremely well to test the concept" of the PLA technology, he said. "For me, it's a natural link to link the basic research that they are doing in Olink … with our repositories so we can immediately test out the concept."

Brünner's lab also builds its own ELISAs, and the PLA "is just another way to do ELISA, but now you can do multiplexing."

As part of the initiative, the participants also will be looking to develop multivariate data analysis and data management tools, reagents, and manufacturing procedures, Fredriksson said.

The consortium chose to work with blood despite its complexity, which has flummoxed many researchers. Blood has a wide dynamic range, at least 10 orders of magnitude, but the 10 most common proteins in blood account for 90 percent of all proteins found in blood, masking the remaining 10 percent that may contain the most important disease-related information.

According to Fredriksson, one of the advantages of its PLA technology is that it can find things in blood that mass spectrometry, for instance, cannot. Mass spectrometry is the dominant technology in biomarker discovery and detection, but has a sensitivity of only about 1 nanomolar of concentration, Fredriksson said. In contrast, PLA has a sensitivity going below the picomolar level "which means we can look at putative markers which are present in plasma 10,000 times lower in concentration than any mass spectrometry-based study can look at," he added.

"We will be enabling the analysis, for the first time, of hundreds of markers in small sample-volumes, and what that will bring to the biomarker world will be very exciting," Fredriksson said.