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InterVenn Aims to Push Glycoproteomics Into the Clinic


NEW YORK (360Dx) – Diagnostic startup InterVenn BioSciences is working to develop mass spectrometry-based glycoproteomic tests for a variety of cancers and other conditions.

The Redwood City, California-based company's lead product is an ovarian cancer assay intended to help doctors triage pelvic masses, similar to protein tests offered by firms including Vermillion and Fujirebio.

InterVenn launched in March of 2018 and announced this week that it had raised $9.4 million in a funding round led by Genoa Ventures. The company was formed to commercialize glycoproteomic analysis methods developed by University of California, Davis professor Carlito Lebrilla and Stanford University professor Carolyn Bertozzi, both cofounders of the firm.

Protein glycosylation, in which sugars modify various amino acids, is a common post-translational modification and an important phenomenon in the biology of various diseases, including many cancers. Given this, researchers have long considered glycoproteins to have high potential as protein biomarkers, and, in fact, more than half of the cancer protein markers currently approved by the US Food and Drug Administration are glycoproteins.

Glycoproteomic analysis has been challenging, however, due to the wide variety of possible glycosylations, called glycans, and the fact that traditionally, researchers have analyzed these modifications separately from the proteins to which they are attached.

Recent developments, however, have allowed researchers to look at glycans while still attached to their host peptides, which both simplifies the analysis while also allowing for more in-depth coverage, said Lebrilla.

"For a while we looked at the glycans just by themselves," he said. "We would take the [glycoproteins] and release the glycans from the proteins. Then we would enrich [the glycans] and would do LC-MS."

This approach required upfront sample prep to first separate the glycans from the proteins they were modifying and then enrich them for mass spec analysis. Additionally, Lebrilla said, because this workflow enriched for glycans from the entire glycoproteome, samples were dominated by glycans that had been attached to the most abundant proteins, making it difficult to explore less abundant glycoproteins.

"If you take the glycans from a serum sample [and analyze them using mass spec], you're actually seeing glycans from only about the top 20 [most abundant] proteins or so," he said.

To address this issue, the researchers moved to a more targeted mass spec method analyzing specific glycopeptides with their glycans still attached. This method, Lebrilla noted, results in a more streamlined assay, as it is no longer necessary to separate glycans from their proteins and enrich them prior to mass spec analysis. Additionally, by looking at specific glycopeptides, the researchers are able to simplify their analysis while also improving their depth of coverage.

"We pretty much just inject the sample into the mass spectrometer, so it's really fast," he said. "And we can see both the glycomic changes and the changes with the specific glycan-associated proteins. So our coverage is better and the analysis is faster."

Lebrilla said that despite the widely acknowledged potential of glycoproteins as disease markers, researchers and clinicians have shied away from glycoproteomics due to its perceived complexity.

"If you take a protein with three glycosylation sites and each site has, say, 10 different glycans, which is a small number, that's 1,000 glycoforms for that protein," he said. "That's just one protein. Now you look at 1,000 proteins, and that's 1,000 times 1,000 [different potential glycoforms]. It just spirals out. So I think, perhaps, people couldn't wrap their head around those numbers and so they just didn't attempt it."

Focusing the analysis at the glycopeptide level simplifies matters, Lebrilla said, noting that while an individual glycopeptide does not reflect the full glycosylation state of a particular protein, he and his colleagues have found that glycopeptide-based data can be a sensitive indicator of disease state, distinguishing, for instance, between cancers and benign conditions, or between different cancers or even different stages of cancers.

Lebrilla's lab and InterVenn both use multiple-reaction monitoring mass spec on triple quadrupole instruments to make targeted measurements of specific glycopeptides, and Lebrilla said one key to the development of these workflows was the broadening in recent years of the mass range amenable to MRM analysis.

Previously, he said, MRM assays had trouble with glycopeptides due to the large size of some of the attached glycan molecules, but the expansion of the analysis mass window "has really opened up the ability to look at some of these compounds."

Lebrilla said that he and his colleagues have also worked to refine aspects of the mass spec process like peak annotation and prediction of LC retention times that has further improved their analyses. He said that his lab is currently able to look at around 1,000 glycopeptides in serum simultaneously.

InterVenn has focused on "industrializing" this process and "preparing it for clinical rather academic applications," said Aldo Carrascoso, the company's CEO.

Leading this effort within the company has been Lieza Danan-Leon, cofounder and CSO of InterVenn and previously the head of mass spectrometry at drug development firm Stemcentrx, now a part of AbbVie.

The company plans to use its glycoproteomic platform to explore markers for a variety of diseases including several cancers and neurological conditions like Alzheimer's disease, Carrascoso said.

It is currently collecting samples for a validation trial of its ovarian cancer test, which InterVenn plans to launch as a laboratory-developed test, likely sometime in 2021, he said.

While the company is interested in potentially developing a general population ovarian cancer screening test, Carrascoso noted the great difficulty of developing such a test, given the low prevalence of the disease. Instead, it is tackling first what he called "the lowest-hanging fruit," a test for triaging patients presenting with pelvic masses to determine whether the mass is likely cancerous, in which case they should be operated on by a gynecologic oncologist, or if it is likely benign, in which case they can be safely treated by a regular gynecologic surgeon.

This is the intended use of Vermillion's OVA1 test and Fujirebio's ROMA test. The protein biomarker CA125, which is a glycoprotein, is used for this purpose, as well.

Neither Vermillion or Fujirebio have managed much commercial success with their tests, though. In the case of Vermillion, despite a number of management changes and strategic reboots, sales of OVA1 have flatlined at around 2,000 per quarter for several years, well below the amount the company would need to sell to turn a profit.

Carrascoso said that he believed based on the performance of the InterVenn test in initial studies that it would outperform existing commercially available assays.

According to studies funded by Vermillion, OVA1 is able to distinguish between women with ovarian cancer and a benign mass with sensitivity of 92.4 percent and a specificity of 53.5 percent. Carrascoso said that in its internal work, InterVenn had seen its ovarian cancer assay perform with sensitivity and specificity in the high 90-percent range. This data has not been published in peer-reviewed form, however, making it difficult to know how the test would perform in an actual clinical environment.

Carrascoso said InterVenn's platform also appears to work well using dried blood spot samples and that the company is currently running another study evaluating its performance using dried blood spots. DBS sampling is of significant interest within clinical proteomics and clinical mass spec more generally as it could allow for more convenient patient sampling and transport of samples without the expensive cold storage and specialized shipping required for conventional blood draws.

The company also plans to use its technology in research projects for outside partners, Carrascoso said, noting that it has an active collaboration with the Mayo Clinic as well as with several US cancer centers he declined to name.