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Exosome Diagnostics Taking Aim at Protein Analysis With Release of Shahky System

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NEW YORK (GenomeWeb) – With the first placements of its Shahky exosome analysis instrument, Exosome Diagnostics is moving into protein detection.

To date, the company has focused on the measurement of exosomal nucleic acids along with cell-free DNA, but the development of the Shahky system will allow it to explore protein detection as a tool for both its pharma customers and internal diagnostic development efforts.

One of the first instruments has gone to the lab of Hakho Lee, director of the biomedical engineering program at Massachusetts General Hospital's Center for Systems Biology, and developer of the nanoplasmonic sensing technology underlying the Shahky device. Lee and his MGH colleague Hyungsoon Im are using the device to further develop a test for the early detection of pancreatic cancer they described in a 2017 paper in Science Translational Medicine.

Exosome Dx also announced last year an agreement with Merck covering drug development in oncology and other disease areas that includes access to the Shahky system.

According to Exosome Dx CEO John Boyce, the company sees pharma as the initial sales driver for the platform with clinical applications to follow after it secures the necessary regulatory clearances.

"Initially, the Shahky platform will derive significant revenue from pharmaceutical companies, and within a number of years, the company aims to achieve regulatory clearance to allow the system to serve patients in the clinic," he said.

"We are seeing a very high demand from pharmaceutical companies for the development of companion diagnostic signatures for therapeutics," Boyce added, noting that the system's rapid turnaround time and point-of-care suitability will "allow expedited patient enrollment in clinical trials, as the patient can be run on site at the time of enrollment."

He said the company expects those same qualities will ultimately allow clinicians to use a version of the platform for in-office patient testing.

Exosome Dx has been interested in exosomal protein detection for years, but had challenges identifying a technology capable of reliable protein detection.

"In working with our collaborators and customers, in every instance of data that we reviewed, the data that was gleaned yielded results where the signal-to-noise wasn’t reliable enough," Boyce said.

In the first place, he noted, the exosome isolation methods used by the firm's collaborators were typically time-consuming and low-yielding, taking up to a day to perform and yielding more than a hundredfold less target analytes than the company is able to achieve with its existing cell free DNA/exosomal RNA isolation methodology.

Additionally, Boyce said, the ELISA and Western blot-based approaches used for analysis of the isolated exosomal proteins were not sensitive enough to achieve reliable results.

"The amount of exosomes you can extract from patient samples is really small, so conventional assays like ELISA or Western blotting can only look at a few markers on a sample, but, of course, if you want to improve your diagnostic accuracy, it's better to look at many different markers," said Lee, echoing Boyce's comments.

To address this challenge, Lee and his colleagues developed a nano-plasmonic assay, called nPLEX, that uses transmission surface plasmon resonance to measure exosome proteins captured on chip-based nanohole arrays functionalized with antibodies to proteins of interest. Initially described in a 2014 paper published in Nature Biotechnology, the assay is the basis of the Shahky system.

According to Lee, the nPLEX assay is compatible with a variety of upfront exosome isolation methods. Boyce said that Exosome Dx has developed a proprietary surface chemistry that allows researchers to place biofluids like patient blood directly on the chip and obtain results in four to six minutes.

Boyce said that "the Shahky technology is an order of magnitude more sensitive than ELISA and two orders of magnitude more sensitive than Westerns," and added that the company is continuing to work on the platform's consumables and instrumentation with the aim of further boosting sensitivity.

Boyce noted that the flexibility of the instrument's chip-based system would make it useful through the biomarker development workflow from discovery through to clinical application. The device can handle chips ranging from 12 to 1,000 elements with each element containing one analyte-antibody pairing. In the case of a discovery experiment, that would allow researchers to screen for 300 proteins in triplicate to look for disease-linked signatures. Or, in the case of a validation experiment, the platform could measure a three-protein signature across 300-plus samples.

Boyce said that another application the company saw great demand for was screening large numbers of antibodies against a particular protein.

He added that the company plans to offer both custom panels for its research customers, as well as off-the-shelf panels covering various signal transduction pathways. It also plans to provide diagnostic chips for specific diseases to clinicians upon regulatory approval, Boyce said.

Exosomes are extracellular vesicles thought to be present in all body fluids. Shed by cells, their molecular make-up reflects that of their cell of origin. This has made them an area of growing interest in liquid biopsy research, the idea being that it might be easier to measure proteins or nucleic acids in exosomes derived from, for instance, cancer cells, than to detect cancer-linked nucleic acids or protein circulating freely in patient blood or urine.

Exosome DX offers two exosome-based tests, its ExoDx Prostate, which measures three exosomal RNA markers in urine to assess the risk of high-grade prostate cancer in patients being considering for prostate biopsy; and its ExoDx Lung(ALK) test, which analyzes exosomal RNA in blood to look for EML4-ALK fusions used for guiding therapy in non-small cell lung cancer.

Its move into exosomal protein analysis follows a number of other research groups and firms. For instance, last year researchers at Peregrine Pharmaceuticals using technology developed at the University of Texas Southwestern found they were able to distinguish between patients with and without cancer based on the levels of the protein phosphatidylserine present in their exosomes.

That followed a study by Arizona State University researchers that found that the protein marker EphA2 could be used to identify exosomes shed by pancreatic cancers and to distinguish between pancreatic cancer patients and both healthy controls and patients with benign disease like pancreatitis.

Outside the cancer space, Louisville, Kentucky-based diagnostics firm NX Prenatal is developing a proteomic test for assessing women's risk of delivering prematurely that uses multiple-reaction monitoring mass spec to measure exosome-bound proteins linked to preterm birth.

MGH's Lee is using the Shahky system to further develop the early detection test for pancreatic cancer he and his colleagues developed using an early version of the technology. In the 2017 STM paper presenting the test, the researchers found that a signature of five exosomal proteins could distinguish between pancreatic cancer cases and healthy and disease controls with a sensitivity of 86 percent and a specificity of 81 percent.

Lee said he and his colleagues are also using the system for analysis of other cancers in hopes of obtaining more rigorous statistics around exosomal proteins that may be diagnostically or prognostically useful.

His lab is also continuing to work on technical development of the platform, he said.

"One of our research projects with Exosome Diagnostic is around how to make the chips [more] robust and reliable for consumers," he said. "That involves chip design and ways to immobilize the antibodies that are used for exosome capture, and also testing whether these chips can be shipped overseas or to other places without losing their activity."

Lee is also working to develop the device for measurement of protein post-translational modifications like phosphorylation. He noted that this is particularly challenging given that such modified proteins are present at even lower concentrations than typical proteins.

Other researchers have had some success in this regard. Last year, researchers at Purdue University and Tymora Analytical Operations combined microfluidics-based capture of microvesicles and exosomes with phosphoprotein enrichment reagents and mass spec to identify 144 proteins more highly phosphorylated in the exosomes of breast cancer patients than healthy controls.

Boyce said eventually Exosome Dx will use the Shahky system for combined protein and nucleic acid analysis.

"The initial system rollout will use consumables solely for exosomal protein analysis, and in the future, consumable revisions will allow for both protein and nucleic acid analysis," he said. "The Shahky instrument is engineered with the intention of running both analytes simultaneously, resulting in a chip that allows for exosomal protein plus exosomal RNA plus cfDNA."