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Tymora Developing Extracellular Vesicle-Based Phosphoproteomic Liquid Biopsies

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NEW YORK (GenomeWeb) – Researchers at Purdue University and Tymora Analytical Operations have developed a method for blood- and urine-based phosphoproteomic analysis that they aim to use to detect and manage various cancers.

The approach, which combines a microfluidics-based capture of microvesicles and exosomes with Tymora's phosphoprotein enrichment reagents, could allow researchers to profile tumor signaling pathways via blood or urine samples, similar to how liquid biopsies make use of ctDNA analysis, said Tymora President and CTO Anton Iliuk.

Protein phosphorylation is one of the primary vehicles for cellular signaling and, as such, are a major area of focus for cancer research and drug development. Agents that inhibit kinases, the enzymes responsible for protein phosphorylation, are one of the main classes of targeted cancer therapies, and a number of research groups are exploring the use of phosphoproteomics analysis to aid selection of patient treatments and track their response to therapy.

For instance, at MD Anderson Cancer Center, researcher Gordon Mills is using reverse-phase protein arrays to profile cancer signaling pathways in tens of thousands of patients with the goal of identifying patient subtypes and therapies.

Emanuel Petricoin, co-director of the Center for Applied Proteomics and Molecular Medicine at George Mason University, is similarly employing RPPA to profile tumor signaling and guide patient therapy. In a recent interview he noted that initial results from a study funded by the breast cancer charity Side-Out Foundation indicate that using phosphoproteomics data to guide patient treatment extends progression-free survival in metastatic breast cancer patients.

And as mass spec workflows become more sensitive, they are likewise being employed for analyses of tumor signaling. Researchers at Memorial Sloan Kettering Cancer Center recently published on a mass spec method they plan to use in upcoming clinical trials to measure cancer signaling activity with the ultimate aim of guiding patient treatment.

By and large, however, such work has focused solely on analysis of tumor tissue. This presents challenges, because in some patients tumors of interest aren't accessible for biopsy, and in cases where biopsies are available, they are taken relatively infrequently.

In genomics, these limitations have led researchers and companies to pursue liquid biopsies, wherein they analyze ctDNA shed into the bloodstream by the tumor of interest. Taking a similar approach to blood-based phosphoproteomics analysis has not been feasible, however, due to the low concentration of these molecules in the bloodstream and the fact that phosphorylated proteins in blood are often rapidly de-phosphorylated by circulating phosphatases, Iliuk said.

"Most phosphoproteins are never [shed into] the blood in the first place," he noted. "And even if they are released [by a tumor] they will get de-phosphorylated almost immediately because phosphatase is a very active and abundant enzyme.

"People have tried using blood and urine samples for phosphoproteomics analysis before, but these analyses have come up short," he said.

To get around this limitation, Iliuk and his colleagues looked for phosphoproteins in circulating extracellular vesicles (EVs) like microvesicles and exosomes, which are known to contain genetic material and proteins from their cells of origin.

Various researchers are exploring exosomes as potential clinical samples, and diagnostics companies like Exosome Diagnostics and NX Prenatal are using measurements of exosome-bound RNA and proteins to detect and assess conditions including prostate cancer and preterm birth.

In a paper published earlier this year in Proceedings of the National Academy of Sciences, Iliuk and his co-authors looked at plasma EVs in 30 breast cancer patients and six healthy controls and identified 144 proteins more highly phosphorylated in the cancer patients.

Following up on four of those proteins using a targeted mass spec analysis on a Thermo Fisher Q Exactive HF instrument, they found that three of them, RALGAPA2, PKG1, and TJP2, showed significantly higher phosphorylation levels in breast cancer patients than in controls.

Iliuk cited as one key to the work a phosphoprotein enrichment method Tymora has been developing under a Phase II SBIR grant from the National Institutes of Health the company received in 2015.

Called PolyMAC, the approach uses soluble nanopolymers functionalized with metal ions to capture phosphoptides in complex mixtures. The other aspect, which Iliuk and his colleagues have developed since the work presented in the PNAS study, is an improved workflow for capturing target EVs and extracting the phosphoproteins inside.

In the PNAS study, the researchers used ultracentrifugation to collect EVs, however, Iliuk said, this approach has poor reproducibility and low rates of EV recovery.

"We've since created a microfluid-based approach where we use a microfluidics cartridge to capture the EVs," he said. This is followed by in situ extraction of the target molecules using the application of a small amount of voltage, which Iliuk said allows the researchers to access the phosphoproteins inside without degrading them in the process.

While the work is still in its early stages, Iliuk said he envisions the approach as an additional option for liquid biopsies beyond analysis of circulating tumor cells and ctDNA. He cited research using RPPA to look at protein signaling in patient tumor tissue.

"We are able to do that at the liquid biopsy stage, so we don't have to take a biopsy or wait for the tumor to appear," he said. "We can monitor the disease constantly instead of only when the biopsies are available."

Iliuk said Tymora is currently looking for commercial collaborators in oncology with which to further explore the approach in certain cancers. The company is also working on an internal project using phosphoproteomic analysis of EVs in urine for monitoring bladder cancer recurrence after surgery.

"We have found quite a bit of phosphorylation differences between bladder cancer patients and normal individuals, so we are trying to develop our own assay and move into the diagnostic area," he said.

Additionally, the company is working to commercialize its microfluidic EV capture and extraction platform as a tool for other researchers working with EVs, Iliuk said.

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