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Aptamers Working to Carve Out Niche in Affinity Reagent Space


NEW YORK (GenomeWeb) – The problems with antibodies are well known. They can be expensive and time consuming to make. They don't exist to all the targets researchers would like to measure, and they offer limited multiplexing capabilities. Some suffer from poor specificity and high background.

Given these issues, proteomics researchers and some in the life sciences community more generally have for years been on the hunt for an alternative. Perhaps most prominently, mass spectrometry has emerged as an approach that some hope could, despite its own challenges, dislodge antibodies as the primary tool for protein detection.

Another potential antibody alternative is aptamers, nucleic acid-based reagents capable of binding proteins or other targets and which can then be readout via technologies like PCR, arrays, or next-generation sequencing.

Methods for developing and selecting these molecules were discovered around 30 years, and since then they have achieved performance levels that rival that of antibodies. Additionally, unlike antibodies, they are developed synthetically, making them faster and cheaper to create, and also offer advantages in terms of size, stability, and a lack of immunogenicity — a key point for their therapeutic potential.

Yet, despite these advantages, aptamers have thus far failed to catch on as a major research or clinical tool. One possible reason for this is that significant portions of the technology underlying aptamer development has until recently been under patent. For instance, the Systematic Evolution of Ligands by Exponential enrichment, or SELEX, method for aptamer discovery, were developed in part by Larry Gold, the chairman and founder of aptamer technology firm Somalogic, and were under patent at the company until 2014.

Nebojsa Janjic, Somalogic's chief science officer, noted in an interview last month that, in the time since those patents expired, a number of commercial and academic outfits have moved into the aptamer space.

Gold himself, though, said that a number of the foundational patents in the field were filed in 1990, and so expired around a decade ago. And, said Michael Weiner, an antibody development expert and vice president of molecular sciences at antibody firm Abcam, even if IP issues kept industry from wholeheartedly pursuing aptamer development, it's unlikely they would have dissuaded academic researchers from using the molecules.

"Academic researchers never really give a crap about IP issues," Weiner said. "So, they would have used them anyway."

Rather, he suggested that along with their potential advantages, aptamers come with downsides of their own and ultimately don't represent enough of an advance to dislodge an entrenched technology like antibodies.

"The infrastructure that exists for antibody-based testing is in the billions, maybe trillions of dollars," Weiner said. "You don't have to convince anybody to use them as an affinity reagent. Everyone has experience using antibodies."

Despite being one of the inventors of aptamer technology, Somalogic's Gold essentially agreed. One major disadvantage of aptamers is their inability to recognize denatured proteins, which makes them poorly suited to applications like western blotting or measuring proteins in formalin-fixed paraffin-embedded tissue samples. That aside, Gold said that Somalogic's aptamer technology, a modified form of the molecules called Somamers, worked as well as antibodies in basically any application where a person was trying to measure a protein with its conformation intact.

"Would Somamers work in cell sorting for example? The answer is yes," he said. "Would they work in purifying a protein drug? Yes. The applications one might reasonably do with Somamers are those where you are trying to measure or intervene on a protein with conformation."

Does he expect Somamers to dislodge antibodies as the preferred tool for such applications?

"Absolutely not," Gold said. Instead he suggested that the molecules would find their niche by carving out new applications for which antibodies are poorly suited.

David Spetzler, president and chief science officer of molecular profiling firm Caris Life Sciences, likewise said he believed aptamers wouldn't replace antibodies but would, rather, open up new applications.

While Spetzler noted that companies like Somalogic have managed to create aptamers with target binding affinities similar to that of good antibodies, he said that he hasn't seen anything significantly superior.

"There is definitely a manufacturing advantage to anything that is synthetic and chemical [like aptamers] as opposed to biological [like antibodies] in nature," he said. But [replacing antibodies] is a tough, long road, because the amount of infrastructure that exists to produce antibodies is significant."

Caris offers a variety of antibody-based immunoassays as part of its cancer profiling business, and it has no plans to move to aptamers for such applications, Spetzler said. However, aptamers are the key technology underlying its ADAPT Biotargeted System, which uses libraries of 1015 aptamers to profile circulating vesicles and molecules in patients to better detect and assess their disease state.

"Rather than have one aptamer bind to a target, if you expand that to hundreds or thousands or hundreds of thousands of aptamers, then the affinity of one single aptamer doesn't really matter that much because you have mass action on your side," Spetzler said.

The patterns of binding of these many aptamers generate profiles that can be linked to different clinical phenotypes, allowing the company to interrogate entire cellular systems, as opposed to individual molecules, he said. In this, the work is similar to efforts using massive peptide arrays being pursued by researchers like Arizona State University's Stephen Johnston and his spin-out diagnostics firm HealthTell.

Spetzler suggested, though, that aptamers might prove an even more effective molecule for such an approach due to the massive size of the libraries that can be generated.

He provided HER2-positive breast cancer as an example of the sort of condition where the ADAPT platform could prove useful.

While Herceptin treatment is the standard of care for such patients, "only about 45 percent of HER2-positive women respond well to Herceptin," Spetzler said. "This is because there can be downstream components that are continuing to hyperstimulate that HER2 pathway independent of interference with the receptor at the surface of the cell."

"Just as important," he added, "a pretty significant number of HER2-negative women respond to Herceptin, and that is because you can have phosphorylation on the HER2 receptor. So, even though you don't have a lot of these receptors, there is still hyperstimulation of that pathway or inducement of that pathway and its downstream attributes. Having the ability to query that system as a whole is really an advantage when you are trying to predict clinical outcome, and that is where we are going."

For its part, Somalogic sees multiplexed measurements of large panels of proteins as the "sweet spot" for its Somamer technology, Gold said.

Antibody-based sandwich assays like ELISAs are highly specific due to the fact that they use two different antibodies to detect their target. However, they have limited multiplexing capabilities because of the cross-reactivity of secondary antibodies with off-targets, which creates noise.

Somalogic's approach, Gold said, was to combine the two levels of specificity represented by the two antibodies in a typical sandwich assay into a single molecule. It did this by developing the molecules so that they would exhibit slow dissociation rates from their targets [on the order of an hour or more] in the case of true hits and fast dissociation [on the order of seconds] in the case of off-target binding.

This, combined with the molecules' affinity for their targets provides two-dimensions of specificity in a single molecule, which, Gold said, means they can offer highly specific detection without the noise created by a secondary detection molecule as in a sandwich immunoassay.

This, he said, has made Somamers particularly well suited for massively multiplexed protein detection and quantification panels, and Somalogic currently offers such a capability through its SomaScan platform, which has become the company's main source of revenue since the platform launched in 2012.

The current commercial version of the platform can measure 1,310 proteins, and the company maintains for internal use a custom platform it developed in collaboration with Novartis that can measure more than 5,000 proteins. Ultimately, Somalogic hopes to expand the platform to cover the full human proteome, around 20,000 targets not including variants and modified forms. The company has placed platforms in a number of labs around the world including with prominent proteomics researchers like Cedars Sinai Medical Center's Jennifer Van Eyk.

A recent SomaScan customer is Stockholm Royal Institute of Technology researcher Mathias Uhlén, who heads the antibody-based Human Protein Atlas as well as the Antibodypedia portal, which provides performance and validation information on more than 1.4 million antibodies to human proteins.

Uhlén, who said his institution will install a SomaScan platform in the coming months, noted that while he had little success with aptamers in the technology's early days, he has been impressed with the SomaScan system.

"We are, of course, interested in affinity reagents, and obviously the golden standard is antibodies, but we are very interested in alternatives," he said. "In the early days, we did work quite a lot with aptamers, but we had some problems with getting good binders."

More generally, Uhlén noted that while "a lot of people have tried aptamers, there have been relatively few success stories."

He added, though, that in their experience with the SomaScan platform thus far, he and his colleagues have found it to be "rather fantastic."

"It seems [Somalogic] has achieved something rather useful for the community," he said.

Uhlén said that he, too, saw aptamers not as a replacement for antibodies but rather as an addition to a protein analysis toolkit that includes technologies like Olink Biosciences' proximity extension assay, which uses pairs of antibodies linked to nucleic acids for read-out, conventional antibody-based immunoassays, and mass spectrometry.

"In our institute we will have all of these different technology platforms to analyze plasma [proteins]," he said. "We do think there will be a place for all of them, and the next two or three years will tell us which technology is the best for what application."