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From Black-eyed Peas to Nanotechnology


Photo by Russell Lee

The Steinmetz Lab advances medicine and materials through molecular engineering of bio-inspired nanotechnologies.

Plants are frequently used in biomedical research. One of the most publicized recent success stories is the development of ZMapp™, an experimental therapy to treat Ebola Virus Disease. Created by a team of scientists, the intravenous treatment is composed of three monoclonal antibodies manufactured in Nicotiana benthamiana, commonly known as Australian tobacco.

Nicole Steinmetz, an associate professor of biomedical engineering and the George J. Picha Designated Professor in Biomaterials at the Case Western Reserve University School of Medicine, also uses Australian tobacco to push frontiers in medicine and materials through molecular engineering of biology-inspired nanotechnologies. However, there’s a twist. “Many labs use nanotechnology for human or plant health applications,” says Steinmetz. “Our unique angle is that we use plant viruses.”

Founded in 2010, the Steinmetz Lab studies and applies plant viruses generated from Australian tobacco and black-eyed peas to manufacture nanoparticles to cure diseases. “The interest lies in the nanometer size scale because these materials can navigate through the body in ways other materials can’t,” says Steinmetz, principal investigator in the lab.

Nanoscale self-assembly has been mastered in nature with atomic precision. Rather than synthesize nanoparticles in the lab, researchers in the Steinmetz Lab use biology to make nanomaterials for them. Viruses also offer distinct characteristics that the researchers capitalize on: They have naturally evolved to deliver cargos to cells and tissues. “Through structure-function studies, we are beginning to understand how to tailor these materials appropriately for applications in medicine and biotechnology,” says Steinmetz.

The team in the Steinmetz Lab includes two research professors, a lab manager and more than a dozen postdoctoral fellows and graduate students. Their work is organized into three interconnected research thrusts:

  • Drug delivery and immunotherapies
  • Molecular imaging for diagnosis and prognosis
  • Synthetic virology approaches toward novel materials

A Focus on Plant Viruses

 
Once grown to the appropriate size, the plants are infected with the virus by putting a drop on the leaf and rubbing it in.   

Steinmetz’s interest in bio-inspired approaches to fighting disease was sparked early on. In the late 1990s, she was a biology student at the Ruhr University Bochum in Germany majoring in biology. “I enjoyed learning about biology and how it works, but I always wanted to do something more applied — use biology to make a new material, cure a disease or make a vaccine,” she says.

Because of her interests in applied biology, Steinmetz continued her studies in molecular biotechnology at the RWTH Aachen University in Germany. While there, she heard a lecture by Dr. Ulrich Commandeur. “He was using plant viruses to produce antibodies in plants to make them more accessible to the developing world,” recalls Steinmetz. “You could eliminate the cold chain and produce pharmaceuticals in plants on site. That was really cool!” Shortly afterward, Steinmetz joined the Commandeur Lab to start her diploma (equivalent to a master’s thesis) in molecular farming with plant viruses.

Plant viruses are the center — the nucleus — of all the work done in the Steinmetz Lab. Two common viruses the researchers utilize are tobacco mosaic virus and cowpea mosaic virus. In a new lab in the basement of the Biomedical Research Building on the School of Medicine’s campus, researchers grow a couple hundred Australian tobacco and black-eyed pea plants.

“Once they grow to a certain size, we infect them by putting a drop of the virus on the leaves and rubbing it in. It’s very simple,” says Frank Veliz, lab manager of the Steinmetz Lab. “We continue to water the plants and let them grow. Then, over time, the plants start to show a mosaic pattern on the leaves.” At that point, the leaves are harvested, undergo a purification process and are ready for use in the main facility of the Steinmetz Lab on the third floor of the Biomedical Research Building.

Whether for drug delivery, immunotherapy or molecular imaging, the basic concept remains the same. “The whole idea is that you want a cargo — either your drug or your contrast agent — to specifically deliver to the site of the disease,” says Steinmetz. Researchers create a hollow nanotube, akin to an incredibly small paper towel roll. The interior is solvent accessible, so a drug or contrast agent in solution can freely diffuse on the inside. Chemistries are then used to trap the drug or contrast agent on the inside. The exterior of the nanotube is modified to provide direction on where it should go within the body — what Steinmetz calls “molecular ZIP codes.”

One of the potential applications for this technology is risk stratification for cardiovascular disease. Non-invasive imaging techniques can help improve survival and quality of life, as well as reduce healthcare costs. While MRI is beneficial, it’s limited in its ability to distinguish between diseased and healthy tissue of similar signal intensities. Researchers at the Steinmetz Lab developed molecularly targeted nanoscale contrast agents carrying large payloads of the clinically approved agent Gd-DOTA. The contrast agent, which uses the nucleoprotein components of the tobacco mosaic virus, exhibits a relaxivity five orders of magnitude higher than current clinical agents.

Using plant virus nanotubes, the team identified novel biomarkers and their peptide ligands to differentiate between vulnerable and stable plaque. Ultimately, this could help physicians decide which patients require treatment and what treatment is most suitable.

A Promising Approach to Immunotherapy

Steinmetz says the most exciting research area in the lab right now — and the one that’s closest to translation — is cancer immunotherapy. “The contemporary approach in nanomedicine is to take a nanocarrier, put a drug inside and deliver the drug to the site of the disease to help position the chemotherapy more toward the tumor,” she says. “Ours is a completely different approach. We seek to interact with the body’s immune system and train it to recognize the tumor.”

The immune system defends the body against cancer by sending T-cells to find and kill tumor cells. Over time, however, the tumor shuts down the immunity cycle and tumors become immunosuppressive. The Steinmetz Lab recently demonstrated that virus-like particles (VLPs) from plants induce a potent anti-tumor immune response when introduced into the tumor microenvironment after tumors are established.

The researchers have termed their proteinaceous plant virus-derived nanocarriers an “in situ vaccine.” When injected directly into the tumor, the therapy manipulates tumors to overcome local tumor-mediated immunosuppression and subsequently stimulate systemic anti-tumor immunity to treat metastases. It is both a cancer treatment and a preventive measure against future tumor growth. The Steinmetz Lab has demonstrated efficacy of the in situ vaccine in melanoma, ovarian, colon, glioma and breast tumor models.

In collaboration with Steven N. Fiering, professor of microbiology and immunology, and P. Jack Hoopes, a veterinarian and professor of surgery and radiation oncology at the Dartmouth Geisel School of Medicine, Steinmetz has tested the therapy on four dogs with highly malignant oral melanoma. The dogs were treated with standard clinical radiation therapy (RT) and intra-tumor VLP. Radiation therapy alone provides an average disease-free survival of nine months in these patients. Currently none of four RT/VLP treated melanomas has recurred locally or metastasized. The ongoing average disease-free survival is now at 12 months, and the tumor/peri-tumor influx of cytotoxic immune cells is more than three-fold greater than is seen with radiation alone.

“We are just scratching the surface with immunotherapy,” says Steinmetz. “We have something that works in large animals, so we want to push this toward a clinical trial. But there’s still a lot of research to be done.” Her team is further studying the molecular mechanism and how to make it more potent. They are also looking at combining the therapy with other drugs, formulating it into slow release devices and trying different routes of injection.

A Bright Future

The work done in the Steinmetz Lab is being expanded to another facility on the Case Western Reserve campus, the new Center for Bio-Nanotechnology. Steinmetz is director of the center, which opened in July. “The idea is to bring different principal investigators and labs together to build on different technologies to attack the same problems,” she says. While her team utilizes plant viruses, others have made advancements with different materials. Together, researchers ranging from nanotechnologists to cell biologists and clinicians can work on common goals.

Such collaboration appeals to researchers in the Steinmetz Lab. “What I really like about our lab is that we work well together,” says Veliz. “We collaborate not just with each other, but with other labs on campus for a lot of our projects. We turn to people with the required expertise.”

The work done by Steinmetz has garnered attention — and funding. Earlier this year, she received three new major grants from the National Institutes of Health to further develop her immunotherapy approach, as well as to further study drug-delivery systems for patients living with triple-negative breast cancer and those at risk for serious blood clots. The two R01 awards and one U01 award total more than $8.5 million.

“If this therapy really translates well in human patients – the immunotherapy in particular – not only will we have a treatment, but also a vaccine that would prevent recurrence of the disease,” says Steinmetz. “That’s what I’m most excited about.”