Using proteomic cell array technology developed by UK-based biotech firm Retrogenix, an international research team has identified the cell membrane target of a protein expressed in severe malaria – a discovery that may help better understand the disease and identify new treatments.
Though Retrogenix has been working for several years with major pharmaceutical companies and other academic groups, the study, presented in a letter published earlier this month in Nature, is one of the first to share data on a specific target identified using the company's platform, according to Co-founder and Managing Director Jim Freeth.
"Generally, most of our work has been with drug companies where they have molecules, particularly antibodies or also protein ligands like in the Nature study, but they don't know exactly what these [molecules] are targeting," Freeth told ProteoMonitor this week.
"We haven't been able to disclose any of that data, so this study, which was really more about further understanding how the malaria parasite sticks to the brain, was really the first one really that we've been able to publish," he said.
In the study, the researchers, led by authors at the University of Copenhagen, used Retrogenix's array to identify the binding target of a member of the P. falciparum erythrocyte membrane protein 1, or PfEMP1, family.
Severe forms of childhood malaria, some of which see parasite-infected blood cells bind to blood vessels in the brain, are associated with expression of specific members of the PfEMP1 family. But it has not been known what these proteins target, how that interaction causes the parasite to bind to endothelial cells, or how that binding may influence severe disease symptoms.
In the Nature study, the team screened a recombinant PfEMP1 family member associated with severe disease against Retrogenix arrays expressing 2,505 human plasma membrane proteins. The group identified one significant hit — endothelial protein C receptor, which is involved with regulating blood coagulation and the inflammatory response — as a potential binding partner.
Retrogenix's technology is unlike standard protein arrays in that instead of spotting proteins onto a plate, the company spots slides with expression vectors and then grows human cells on top of them.
"Each vector encodes a different full-length human membrane protein," Freeth explained. "So when we grow human cells over these spotted vectors, the ones that sit directly over a vector take it from underneath and over-express that particular protein."
"Unlike protein arrays, we are expressing full-length proteins in the context of the cell," he said.
According to Freeth, Retrogenix now has panels covering 3,500 human plasma membrane proteins.
While the company can spot up to 1000s of vectors on a single array, they have settled on dividing its 3,500 targets into 10 slides, each containing two duplicate sets of cells expressing 384 proteins, or 768 total targets.
"This allows us to have better sensitivity, to look for micromolar interactions between a molecule and its receptor," Freeth said.
In the Nature study, the Copenhagen team followed up its Retrogenix array work with further experiments in the lab looking at parasites from non-immune children with severe malaria symptoms in Tanzania to confirm that EPCR is the likely target of PfEMP1, and that this interaction is involved in the binding of P. falciparum-infected erythrocytes to endothelial cells.
The research explains an important biological mechanism in severe forms of malaria, and, according to the study authors, could inform more effective treatment for children with this severe disease form.
“Under normal conditions, EPCR plays a crucial role in regulating blood clotting, inflammation, cell death, and the permeability of blood vessels. The discovery that parasites bind and interfere with this receptor's normal function may help us explain why severe symptoms of malaria develop," Thomas Lavstsen, one of the study's two first authors, said in a statement.
According to the group's results, by binding with EPCR, malaria-infected cells may interfere with some of the normal activity of the receptor — influencing things like blood coagulation, inflammation, and the integrity of blood vessels.
"The results … open new avenues for studies of malaria pathogenesis, and possibilities for the development of new adjunct therapy and vaccines," the group wrote.
Though academic studies like the Nature work are important to Retrogenix, Freeth said the company's commercial strategy is mainly centered on pharmaceutical research.
While Retrogenix has not been able to disclose most of its pharmaceutical partnerships, he said, the company has made public two ongoing collaborations — one with MedImmune, the biologics research and development arm of AstraZeneca, and another with the Swedish antibody drug discovery firm BioInvent.
Retrogenix has not been able to publish detailed data from its pharma work, but, Freeth said, the company estimates that its overall success rate in identifying the target of a particular molecule has been above 60 percent overall.
"We think we have about 60 percent of the known human plasma membrane proteome [covered], so a success rate of 60-65 percent is as good as we could have hoped for," Freeth said. "That's been really consistent in all of the studies we've done to date, and we expect our success rate to improve as we have more and more proteins represented in our panels."
"We've been told by [our customers] that using other techniques, like immunoprecipitation, or mass spec, or standard protein arrays, their success rate has been only around the 10 percent mark," he added.
Since the publication of the Nature letter, Freeth also said several other academic groups have approached Retrogenix, interested in using the technology for similar disease studies.
Additionally, he said the firm expects work from another academic collaboration looking at the target of a different ligand to be published soon in a peer-reviewed journal.