In work published recently in Nature Structural & Mechanical Biology, scientists at Fred Hutchinson Cancer Research Center described self-assembling, donut-shaped protein nanoparticles designed from scratch. The team showed that the nanoparticles, which can act as scaffolds for the organization and display of biomolecules currently used in various clinical and research applications, could someday enhance these applications by simplifying biomolecule production or by enabling scientists to introduce entirely new biomolecules.
“We have developed a novel molecular scaffold that can display many copies of different types of cool proteins in a variety of numbers and organizations,” said Dr. Colin Correnti, a Hutch protein scientist who spearheaded the efforts to make the circular proteins self-assemble and to test drive potential uses of the scaffolds. Correnti joined the protein scaffolds’ original engineers, Hutch computational biologist Dr. Phil Bradley and Hutch structural biologist Dr. Barry Stoddard, to form a centerwide, interdisciplinary team.
The engineered molecular scaffolds, made up of a repeated protein motif, are called circular tandem-repeat proteins, or cTRPs. In the latest study, the team showed they could create large cTRPs that self-assemble by breaking them into six repeat-long modules with molecular “staples” on both ends. To demonstrate a real-world application, the team attached various proteins that are used in the study and manufacture of engineered anti-cancer immune cells. They showed that using the cTRPs instead of the standard molecular tool could potentially help streamline immunotherapy production — just one of the many applications the team envisions for their nano-circle scaffolds.
“We’ve basically created an endless idea-generation machine,” Correnti said. “Any biomolecule that you want to click on to a cTRP particle, you can. Really, there's not another platform out there that allows you to do that.”
The team's cTRPs can also be produced in mammalian cells rather than bacterial cells, which widens the scope of potential cargo options (as well as simplifying their production). One application has been licensed to a biotech company.
Though now poised for use in real-world applications, the self-assembling cTRPs that Bradley, Stoddard and Correnti developed sprung from basic curiosity about proteins and the relationship between their amino acid sequence and their final form. Several years ago, Bradley was developing computational methods to predict the structure of a naturally occurring TRP while Stoddard was working to visualize its structure directly.
“This all started out because Phil was fundamentally interested in whether he could predict the structure, or even actually design a particularly interesting type of folded protein,” Stoddard said. “It could not have been more a basic and curiosity-driven project.”
Why design a custom protein? You may need it to perform a function that doesn’t exist in nature.
“Designed proteins can often be, in some way, superior to naturally occurring proteins unless the naturally occurring protein already does what you want done,” Bradley said. And because protein designers can choose the most ideal characteristics for their proteins, designed proteins are often very stable and easily produced by cells.
Bradley realized he could adapt the computational tools he was developing to predict structure to design his own TRP. Stoddard jumped at the chance to put Bradley’s principles into practice and test how closely such an engineered protein would adhere to the structure predicted on the computer.
They choose to design circular proteins because of their natural stability and symmetry. In 2015, Bradley and Stoddard demonstrated that they could indeed design circular proteins out of repeating subunits, and they published the work in the journal Nature. Next, they wanted to refine the approach and begin moving toward practical applications.
“There are lots of biological processes where multiple copies of a protein have to interact with multiple copies of their target,” Stoddard said. “It occurred to us that if you attached functional protein cargo at symmetric repeating points around the periphery of these things, that that could be really interesting.”
A scaffold that carried multiple copies of a protein would offer a big advantage over Y-shaped proteins known as an antibodies, molecular workhorses used across the biological sciences. Each arm of an antibody recognizes a target. This makes antibodies an important biological tool, but it means that each antibody can only interact with two targets at a time. Antibodies’ natural limitations also constrain the kinds of questions that scientists can address, Correnti said. If successful, the designed scaffolds could broaden scientists’ horizons.
Stoddard and Bradley secured support from the Hutch’s Evergreen Fund, which is designed to support projects with great potential for commercialization. This helped them garner more funding from the National Institutes of Health and go full-steam ahead. They decided to test the cTRPs’ ability to improve the manufacture of engineered anticancer immune cells, the Hutch’s specialty.
“We just got much bolder and much more biologically focused,” Stoddard said.
The cTRPs started life as a single long protein made up of 24 repeats. To generate them, Stoddard and Bradley encoded the protein sequence in DNA, which allowed cells containing that DNA code to churn out the cTRPs like any other protein.
Correnti wondered if they could break the 24 repeat-long protein into smaller modules, which could then each be tagged with a cargo. The first attempts failed: The modules just didn’t want to join up.
Bradley applied his computational expertise and pinpointed several areas where the team could add what amounted to molecular staples at both ends of each module. It worked; the new modules easily stapled themselves together into rings.
The next step was testing the utility of the modules to act as scaffolds for biologically important proteins. First, they successfully showed that the modules snapped together when attached to a simple, well-characterized protein with an equally well-characterized target. The team then began testing more-complex proteins that could potentially be used to improve immunotherapy development or production.
Many molecular and biological tools used to characterize and manufacture cancer-killing T cells, the immune cells that form the basis for all the currently Food and Drug Administration-approved cell-based immunotherapies, are difficult and time-consuming to make, Correnti said.
The team sought to make an easier-to-produce version of an MHC tetramer, a molecular complex routinely used to study T cells. Right now, MHC tetramers are produced via an arduous, multistep process that involves expressing the MHC protein in bacteria, purifying it and encouraging it to refold properly before attaching four of them to a separate, four-pronged (or tetrameric) protein known as streptavidin.
Using the cTRP scaffold, the researchers found they could condense this into one easy step by grafting the gene for the MHC protein onto the gene encoding the cTRP component. Mammalian cells easily produced the tetrameric cTRP modules — perfectly folded right from the get-go. When the team tested other proteins commonly used to trigger T cells to multiply, cTRP-based tetramers worked just as well as tetramers produced the old-fashioned way.
By breaking the nano-circles into modules, the team has also expanded their utility, Correnti said. Now cTRPs can be adapted to a researcher’s scientific needs by including anywhere from two to six modules. And the ability to produce them in mammalian cells instead of bacterial cells expands the variety of proteins that could theoretically be attached to the cTRPs.
Right now, the team is working to test as many new cargos as possible, and is hoping that other researchers come up with new cargos and new uses for the donuts. They’re also testing different ways of connecting cargo to scaffold and modifications that could further enhance the usefulness of cTRPs.
Stoddard said the results highlighted the power of interdisciplinary research, drawing on expertise in computational and structural biology to create a molecule with translatable potential.
“We essentially built this molecular tool kit that we can apply to a lot of different types of problems,” Correnti said. “The ideal situation that you want for any technology is for people to find really interesting uses for things that we haven't even thought of.”
The National Institutes of Health and Fred Hutch’s Evergreen Fund. supported this work. Fred Hutch and its scientists who contributed to these discoveries may stand to benefit from their commercialization.
Sabrina Richards, a staff writer at Fred Hutchinson Cancer Center, has written about scientific research and the environment for The Scientist and OnEarth Magazine. She has a Ph.D. in immunology from the University of Washington, an M.A. in journalism and an advanced certificate from the Science, Health and Environmental Reporting Program at New York University. Reach her at firstname.lastname@example.org.
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