BIO ENGINEERED DECOY PROTEIN MAY STOP CANCER FROM SPREADING

A team of Stanford researchers has developed a protein therapy that disrupts the process that causes cancer cells to break away from original tumor sites, travel through the blood stream and start aggressive new growths elsewhere in the body.

This process, known as metastasis, can cause cancer to spread with deadly effect.

"The majority of patients who succumb to cancer fall prey to metastatic forms of the disease," said Jennifer Cochran, an associate professor of bioengineering who describes a new therapeutic approach in Nature Chemical Biology.

Today doctors try to slow or stop metastasis with chemotherapy, but these treatments are unfortunately not very effective and have severe side effects.

The Stanford team seeks to stop metastasis, without side effects, by preventing two proteins -- Axl and Gas6 -- from interacting to initiate the spread of cancer.

Axl proteins stand like bristles on the surface of cancer cells, poised to receive biochemical signals from Gas6 proteins.

When two Gas6 proteins link with two Axls, the signals that are generated enable cancer cells to leave the original tumor site, migrate to other parts of the body and form new cancer nodules.

To stop this process Cochran used protein engineering to create a harmless version of Axl that acts like a decoy. This decoy Axl latches on to Gas6 proteins in the blood stream and prevents them from linking with and activating the Axls present on cancer cells.

In collaboration with Professor Amato Giaccia, who heads the Radiation Biology Program in Stanford's Cancer Center, the researchers gave intravenous treatments of this bioengineered decoy protein to mice with aggressive breast and ovarian cancers.

Mice in the breast cancer treatment group had 78 percent fewer metastatic nodules than untreated mice. Mice with ovarian cancer had a 90 percent reduction in metastatic nodules when treated with the engineered decoy protein.

"This is a very promising therapy that appears to be effective and non-toxic in pre-clinical experiments," Giaccia said. "It could open up a new approach to cancer treatment."

Giaccia and Cochran are scientific advisors to Ruga Corp., a biotech startup in Palo Alto that has licensed this technology from Stanford. Further preclinical and animal tests must be done before determining whether this therapy is safe and effective in humans.

Greg Lemke, of the Molecular Neurobiology Laboratory at the Salk Institute, called this "a prime example of what bioengineering can do" to open up new therapeutic approaches to treat metastatic cancer.

"One of the remarkable things about this work is the binding affinity of the decoy protein," said Lemke, a noted authority on Axl and Gas6 who was not part of the Stanford experiments.

"The decoy attaches to Gas6 up to a hundredfold more effectively than the natural Axl," Lemke said. "It really sops up Gas6 and takes it out of action."

Directed Evolution

The Stanford approach is grounded on the fact that all biological processes are driven by the interaction of proteins, the molecules that fit together in lock-and-key fashion to perform all the tasks required for living things to function.

In nature proteins evolve over millions of years. But bioengineers have developed ways to accelerate the process of improving these tiny parts using technology called directed evolution. This particular application was the subject of the doctoral thesis of Mihalis Kariolis, a bioengineering graduate student in Cochran's lab.

Using genetic manipulation, the Stanford team created millions of slightly different DNA sequences. Each DNA sequence coded for a different variant of Axl.

The researchers then used high-throughput screening to evaluate over 10 million Axl variants. Their goal was to find the variant that bound most tightly to Gas6.

Kariolis made other tweaks to enable the bioengineered decoy to remain in the bloodstream longer and also to tighten its grip on Gas6, rendering the decoy interaction virtually irreversible.

Yu Rebecca Miao, a postdoctoral scholar in Giaccia's lab, designed the testing in animals and worked with Kariolis to administer the decoy Axl to the lab mice. They also did comparison tests to show that sopping up Gas6 resulted in far fewer secondary cancer nodules.

Irimpan Mathews, a protein crystallography expert at the SLAC National Accelerator Laboratory, joined the research effort to help the team better understand the binding mechanism between the Axl decoy and Gas6.

Protein crystallography captures the interaction of two proteins in a solid form, allowing researchers to take X-ray-like images of how the atoms in each protein bind together. These images showed molecular changes that allowed the bioengineered Axl decoy to bind Gas6 far more tightly than the natural Axl protein.

Next steps

Years of work lie ahead to determine whether this protein therapy can be approved to treat cancer in humans. Bioprocess engineers must first scale up production of the Axl decoy to generate pure material for clinical tests. Clinical researchers must then perform additional animal tests in order to win approval for and to conduct human trials. These are expensive and time-consuming steps.

But these early, hopeful results suggest that the Stanford approach could become a non-toxic way to fight metastatic cancer.

Glenn Dranoff, a professor of medicine at Harvard Medical School and a leading researcher at the Dana-Farber Cancer Institute, reviewed an advance copy of the Stanford paper but was otherwise unconnected with the research. "It is a beautiful piece of biochemistry and has some nuances that make it particularly exciting," Dranoff said, noting that tumors often have more than one way to ensure their survival and propagation.

Axl has two protein cousins, Mer and Tyro3, that can also promote metastasis. Mer and Tyro3 are also activated by Gas6.

"So one therapeutic decoy might potentially affect all three related proteins that are critical in cancer development and progression," Dranoff said.

ENGINEERED INSULIN COULD OFFER BETTER DIABETES CONTROL

For patients with diabetes, insulin is critical to maintaining good health and normal blood-sugar levels. However, it's not an ideal solution because it can be difficult for patients to determine exactly how much insulin they need to prevent their blood sugar from swinging too high or too low.

MIT engineers hope to improve treatment for diabetes patients with a new type of engineered insulin. In tests in mice, the researchers showed that their modified insulin can circulate in the bloodstream for at least 10 hours, and that it responds rapidly to changes in blood-sugar levels. This could eliminate the need for patients to repeatedly monitor their blood sugar levels and inject insulin throughout the day.

"The real challenge is getting the right amount of insulin available when you need it, because if you have too little insulin your blood sugar goes up, and if you have too much, it can go dangerously low," says Daniel Anderson, the Samuel A. Goldblith Associate Professor in MIT's Department of Chemical Engineering, and a member of MIT's Koch Institute for Integrative Cancer Research and Institute for Medical Engineering and Science. "Currently available insulins act independent of the sugar levels in the patient."

Anderson and Robert Langer, the David H. Koch Institute Professor at MIT, are the senior authors of a paper describing the engineered insulin in this week's Proceedings of the National Academy of Sciences. The paper's lead authors are Hung-Chieh (Danny) Chou, former postdoc Matthew Webber, and postdoc Benjamin Tang. Other authors are technical assistants Amy Lin and Lavanya Thapa, David Deng, Jonathan Truong, and Abel Cortinas.

Glucose-responsive insulin

Patients with Type I diabetes lack insulin, which is normally produced by the pancreas and regulates metabolism by stimulating muscle and fat tissue to absorb glucose from the bloodstream. Insulin injections, which form the backbone of treatment for diabetes patients, can be deployed in different ways. Some people take a modified form called long-acting insulin, which stays in the bloodstream for up to 24 hours, to ensure there is always some present when needed. Other patients calculate how much they should inject based on how many calories they consume or how much sugar is present in their blood.

The MIT team set out to create a new form of insulin that would not only circulate for a long time, but would be activated only when needed -- that is, when blood-sugar levels are too high. This would prevent patients' blood-sugar levels from becoming dangerously low, a condition known as hypoglycemia that can lead to shock and even death.

To create this glucose-responsive insulin, the researchers first added a hydrophobic molecule called an aliphatic domain, which is a long chain of fatty molecules dangling from the insulin molecule. This helps the insulin circulate in the bloodstream longer, although the researchers do not yet know exactly why that is. One theory is that the fatty tail may bind to albumin, a protein found in the bloodstream, sequestering the insulin and preventing it from latching onto sugar molecules.

The researchers also attached a chemical group called PBA, which can reversibly bind to glucose. When blood-glucose levels are high, the sugar binds to insulin and activates it, allowing the insulin to stimulate cells to absorb the excess sugar.

The research team created four variants of the engineered molecule, each of which contained a PBA molecule with a different chemical modification, such as an atom of fluorine and nitrogen. They then tested these variants, along with regular insulin and long-acting insulin, in mice engineered to have an insulin deficiency.

To compare each type of insulin, the researchers measured how the mice's blood-sugar levels responded to surges of glucose every few hours for 10 hours. They found that the engineered insulin containing PBA with fluorine worked the best: Mice that received that form of insulin showed the fastest response to blood-glucose spikes.

"The modified insulin was able to give more appropriate control of blood sugar than the unmodified insulin or the long-acting insulin," Anderson says.

The new molecule represents a significant conceptual advance that could help scientists realize the decades-old goal of better controlling diabetes with a glucose-responsive insulin, says Michael Weiss, a professor of biochemistry and medicine at Case Western Reserve University.

"It would be a breathtaking advance in diabetes treatment if the Anderson/Langer technology could accomplish the translation of this idea into a routine treatment of diabetes," says Weiss, who was not part of the research team.

New alternative

Giving this type of insulin once a day instead of long-acting insulin could offer patients a better alternative that reduces their blood-sugar swings, which can cause health problems when they continue for years and decades, Anderson says. The researchers now plan to test this type of insulin in other animal models and are also working on tweaking the chemical composition of the insulin to make it even more responsive to blood-glucose levels.

"We're continuing to think about how we might further tune this to give improved performance so it's even safer and more efficacious," Anderson says.

The research was funded by the Leona M. and Harry B. Helmsley Charitable Trust, the Tayebati Family Foundation, the National Institutes of Health, and the Juvenile Diabetes Research Foundation.

FROM THE NOSE TO KNEE ENGINEERED CARTILAGE REGENERATES JOINTS

Human articular cartilage defects can be treated with nasal septum cells. Researchers at the University and the University Hospital of Basel report that cells taken from the nasal septum are able to adapt to the environment of the knee joint and can thus repair articular cartilage defects. The nasal cartilage cells' ability to self-renew and adapt to the joint environment is associated with the expression of so-called HOX genes. The scientific journal Science Translational Medicine has published the research results together with the report of the first treated patients.

Cartilage lesions in joints often appear in older people as a result of degenerative processes. However, they also regularly affect younger people after injuries and accidents. Such defects are difficult to repair and often require complicated surgery and long rehabilitation times. A new treatment option has now been presented by a research team lead by Prof. Ivan Martin, professor for tissue engineering, and Prof. Marcel Jakob, Head of Traumatology, from the Department of Biomedicine at the University and the University Hospital of Basel: Nasal cartilage cells can replace cartilage cells in joints.

Cartilage cells from the nasal septum (nasal chondrocytes) have a distinct capacity to generate a new cartilage tissue after their expansion in culture. In an ongoing clinical study, the researchers have so far taken small biopsies (6 millimeters in diameter) from the nasal septum from seven out of 25 patients below the age of 55 years and then isolated the cartilage cells. They cultured and multiplied the cells and then applied them to a scaffold in order to engineer a cartilage graft the size of 30 x 40 millimeters. A few weeks later they removed the damaged cartilage tissue of the patients' knees and replaced it with the engineered and tailored tissue from the nose. In a previous clinical study conducted in cooperation with plastic surgeons and using the same method, the researchers from Basel recently already successfully reconstructed nasal wings affected by tumors.

Surprising Adaption

The scientists around first author Dr. Karoliina Pelttari were especially surprised by the fact that in the animal model with goats, the implanted nasal cartilage cells were compatible with the knee joint profile; even though, the two cell types have different origins. During the embryonic development, nasal septum cells develop from the neuroectodermal germ layer, which also forms the nervous system; their self-renewal capacity is attributed to their lack of expression of some homeobox (HOX) genes. In contrast, these HOX genes are expressed in articular cartilage cells that are formed in the mesodermal germ layer of the embryo.

"The findings from the basic research and the preclinical studies on the properties of nasal cartilage cells and the resulting engineered transplants have opened up the possibility to investigate an innovative clinical treatment of cartilage damage," says Prof. Ivan Martin about the results. It has already previously been shown that the human nasal cells' capacity to grow and form new cartilage is conserved with age. Meaning, that also older people could benefit from this new method, as well as patients with large cartilage defects. While the primary target of the ongoing clinical study at the University Hospital of Basel is to confirm the safety and feasibility of cartilage grafts engineered from nasal cells when transplanted into joint, the clinical effectiveness assessed until now is highly promising.