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From Bench to Bedside: Research Shows Promise In Treatment of CancersBy Matt McGowan
This story begins in 2004, when Jeffrey Schlom, chief of the Laboratory of Tumor Immunology and Biology at the National Cancer Institute in Bethesda, Maryland, took a risk and hired an engineer with no immunology experience. Though it was a bold move, it also made sense, because the investigators in Schlom’s lab are trying to develop vaccines and immunotherapies that can be moved from the laboratory to human clinical trials in no more than 10 years. Researchers call it “bench to bedside,” and when the emphasis is on saving lives tomorrow, sometimes calculated risks are in order.
David Zaharoff, the first engineer ever to work in the tumor immunology and biology lab at the National Cancer Institute, had just finished a one-year postdoctoral fellowship at Duke University, where he had received his doctorate in biomedical engineering. While there, Zaharoff had worked on methods to improve the way pieces of DNA are delivered within the human body for gene therapy applications. Some of this investigation involved physical mechanisms, and some of it involved biomaterials.
During this period Zaharoff became interested in immune responses, a curiosity that would steer him toward the job at the National Cancer Institute. He attended numerous conferences at which investigators talked about the potential of new delivery systems for gene therapies and drugs. However, he became frustrated by the lack of research on how these new delivery systems would be recognized by the immune system.
“At these conferences, the emphasis was always on the effect of this or that drug or gene therapy, but immune response was left out,” Zaharoff says. “Every single drug or gene delivery system placed in the body will generate an immune response for better or for worse. I felt my career was going to be limited unless I understood the nature of these immune responses.”
In Schlom’s lab, Zaharoff had some catching up to do. Compared to other investigators, he entered with a limited background in immunology. But Schlom and others were patient. They gave him time to learn their field. Zaharoff describes this period as “floundering around,” but he knows it was anything but.
“I spent those years learning about immunology,” he says. “Here I was, an engineer working with a bunch of classically trained immunologists. It was a bit like learning a foreign language in a country where no one speaks English. So I studied the evolution of cancer vaccines and immunotherapies, and I realized that no one really focused on the delivery aspects of cancer vaccines.”
After two years of “floundering,” Zaharoff found his niche. He realized that cancer vaccines and immunotherapies could be made more effective if they were delivered to the right place, at the right time and at the right concentration in the human body. In short, there was a delivery design problem. As an engineer, that was exactly what he needed.
Zaharoff concentrated his efforts on developing safe and efficient means to localize therapeutic cytokines – proteins that produce an immune response – to make vaccines and immunotherapies more effective. Controlled- or extended-release delivery systems had been explored in drug delivery for decades, but these systems were unlikely to work with proteins because the systems used harsh chemicals that could destroy proteins.
One day, Zaharoff read an article about chitosan, a polysaccharide derived from the shells of crustaceans such as shrimp and lobsters. Chitosan had been evaluated for use in studies on a wound dressing, a weight-loss supplement and a nasal-delivery system. In chitosan, Zaharoff thought he might have found what he was looking for – a biomaterial that could be used to hold and slowly release cytokines nearly anywhere in the body. Chitosan satisfied his short list of criteria for an ideal delivery system. It was a biocompatible and biodegradable substance that did not require harsh organic solvents. In two early publications, Zaharoff showed that chitosan could indeed enhance immune responses and increase the effectiveness of cytokines.
Almost Fell Off the Planet
In 2006, Zaharoff began a fruitful collaboration with John Greiner, staff scientist and head of the cytokine working group within the Laboratory of Tumor Immunology and Biology. As Zaharoff focused on delivery systems in general and chitosan specifically, Greiner shared his expertise in cytokine biology.
After the initial publications on chitosan, Zaharoff and Greiner wondered if they could deliver any cytokine to any location in the body. If so, which cytokine would work, and where would they put it? Interleukin-12 (IL-12) immediately came to mind. IL-12 is a powerful cytokine that stimulates the body’s immune system to attack a range of cancerous tumors. It had been effective in animal studies, but severe toxicities in human trials ceased its commercial development. Greiner had worked with IL-12 in the early 1990s and still had some in a freezer.
“I knew that due to toxicity problems, IL-12 had killed a couple patients in clinical trials in the mid ’90s,” Zaharoff says. “Since that time, it had been out on the fringe. No one really wanted to touch it. It almost fell off the planet. But this wasn’t because IL-12 was a bad drug; we just weren’t delivering it in the right way.”
The Bladder Study
“We tried a lot of different combinations of antigens and cytokines with chitosan,” Greiner says. “In the end, IL-12 won the contest.”
While experimenting with the two substances, Zaharoff and Greiner met Benjamin Hoffman, a post-baccalaureate fellow who was “on loan” from the NCI’s Urologic Oncology Branch because he wanted to work with vaccines. Hoffman wanted to test some of the tumor immunology and biology lab’s vaccines on superficial bladder cancer. Zaharoff and Greiner decided to collaborate with him and try the chitosan/IL-12 mix on that type of cancer.
Both Greiner and Zaharoff emphasize the importance of working within a group of labs. It was this fact, more than the nature of the research itself, that led to the collaboration with Hoffman. In other words, Zaharoff and Greiner did not co-formulate chitosan/IL-12 specifically for superficial bladder cancer.
“Chitosan/IL-12 was designed for direct injections of solid tumors,” Zaharoff says. “In retrospect, it made perfect sense to instill it in a bladder.”
Each year, approximately 70,000 people in the United States are diagnosed with bladder cancer, and slightly more than 15,000 people die due to the disease.
For many decades, a drug known as BCG (bacillus of calmette-guerin, a type of bacteria) has been the standard-of-care immunotherapy for superficial bladder cancer, which leads to muscle-invasive and then metastatic bladder cancer. However, a high percentage – 20 to 30 – of patients fail initial BCG therapy, and 30 to 50 percent of patients develop recurrent tumors within five years.
Other research groups had worked with IL-12 as a potential alternative therapy for superficial bladder cancer. The drug had shown an ability to eliminate tumors, but a recent clinical study on patients with recurrent superficial bladder cancer was not successful.
Working with mouse models, Zaharoff, Greiner and Hoffman designed a study that instilled treatments of BCG, IL-12 or the chitosan/IL-12 co-formulation into the bladder using a catheter. They found that chitosan improved delivery and bio-adhesion of interleukin 12. After four treatments, 88 to 100 percent of mice treated with chitosan/IL-12 were cured, meaning the tumors were eradicated. In contrast, only 38 to 60 percent of mice treated with IL-12 alone were cured. None of the mice treated with BCG alone were cured.
“Antitumor responses following chitosan/IL-12 treatments were durable and provided complete protection from tumor re-challenge,” Zaharoff says.
These results excited Zaharoff, and he was motivated to understand the mechanisms at work. Urinary analysis showed that chitosan/IL-12 had induced multiple cytokines at levels significantly higher than either IL-12 alone or BCG. Tumor analysis following chitosan/IL-12 treatments revealed moderate to intense tumor infiltration by T cells, a group of white blood cells critical to the immune system, and macrophages, also a type of white blood cell. Bladder tissues from cured mice contained residual populations of immune cells that returned to baseline levels after several months.
What He’s Made Of
Although proud of his work at the National Cancer Institute, Zaharoff knew it was time to move on. All along, the plan was to procure an academic position at a university and establish his own lab. Having paid his dues in Schlom’s lab, Zaharoff accepted a position in the rapidly burgeoning biomedical engineering program at the University of Arkansas. He says positions at other institutions were attractive, but Arkansas offered resources and the kind of academic freedom he was seeking to build an eminent lab the way he envisioned it.
“My training phase was over,” he says. “It was time to become an investigator with my own lab and see what I’m made of.”
Without severing ties to Greiner and Schlom’s lab, Zaharoff came to the University of Arkansas in early 2009 and immediately began setting up shop in the Engineering Research Center at the Arkansas Research and Technology Park in south Fayetteville. Today the Laboratory of Vaccine and Immunotherapy Delivery occupies approximately 1,900 square feet of space in the Engineering Research Center. Inside the lab, Zaharoff directs and trains a group of researchers, from undergraduates to postdoctoral fellows, who focus on vaccines and delivery systems for several types of cancer, including lung, breast, prostate and myeloma, in addition to bladder. They are also focusing on drug addiction, specifically a new vaccine that can induce the immune system to generate drug-specific antibodies. Zaharoff intends for all of this research to reach clinical trials within five years.
A New Standard of Care
Or at least be ready for clinical trials. Zaharoff is learning how political that process can be. He and Greiner thought clinical trials testing chitosan/IL-12 on patients with superficial bladder cancer were to begin in 2010, but those studies have been pushed back. He hopes they will begin in 2011, but he understands that these decisions will be made by trial clinicians and others at the U.S. Food and Drug Administration.
When clinical trials do begin, they will treat patients whose disease has returned after an initial treatment with BCG, the current standard of care. According to Research Support and Sponsored Programs, the university department that manages and tracks all public research funds, it will mark the first time that research from the University of Arkansas – which does not have a medical school – will have reached the medical clinical trial phase.
What are his hopes and expectations for the performance of chitosan/IL-12 in clinical trials? When asked this question, Zaharoff’s response is immediate and unequivocal: “I want it to be the new standard treatment of care for superficial bladder cancer.”
The Colorectal and Pancreatic Study
Zaharoff moves on. He is not the type of person who becomes complacent and waits around for others to gauge the success of his previous work, no matter how promising. For the past year, he and Greiner have applied chitosan/IL-12 to colorectal and pancreatic tumors in mice.
Injections eradicated aggressive tumors. This study also included three types of therapy. In addition to the chitosan/IL-12 co-formulation, the researchers injected IL-12 only and combinations of chitosan with other cytokines. Administration of IL-12 alone eradicated less than 10 percent of established tumors, but the combination of chitosan/IL-12 caused complete tumor regression. Therapies with chitosan and other cytokines were not successful.
Again, the immune systems of mice were stimulated by the extended presence of chitosan/IL-12 in the tumor microenvironment. Specifically, Zaharoff found that the combination caused significant expansion of CD8+ T-cells and natural killer cells, two types of powerful immune cells. Depletion of these cells stopped anti-tumor activity. However, depletion of other important immune cells, CD4+ and Gr-1+ cells, had no impact on chitosan/IL-12’s ability to eradicate tumors.
The colorectal and pancreatic study also revealed the unique power of immunotherapy compared to other methods of treating cancer. Traditional cancer therapies, such as surgery, radiation and chemotherapy, are passive approaches that do not eliminate metastasis after a treatment is stopped. Metastasis, or the spreading of cancer from the original tumor site to other areas of the body via the circulatory or lymphatic systems, is responsible for more than 90 percent of all cancer fatalities. Zaharoff says that chitosan/IL-12 immunotherapy is an active approach that induces a massive immune response capable of “remembering” what a particular tumor cell looks like long after therapy is complete. As a result, if the tumor tries to recur in the same or distant place, the immune system will recognize it and destroy it.
This behavior – the memory and subsequent ability to stimulate the immune system as cancerous cells return – motivates Zaharoff and Greiner to test chitosan/IL-12 on metastatic cancer. In these cases, Zaharoff proposes to combine immunotherapy with surgery, to inject chitosan/IL-12 into a tumor before it is surgically removed.
Training the Immune System
With metastatic cancer in mind, Zaharoff has turned his attention to breast cancer, which claims most of its victims after the cancer has spread to other areas of the body. Zaharoff is studying a model in mice in which tumors grow and metastasize in a pattern similar to cancer progression in women. Before surgically removing tumors from female mice, the researchers tested one of several vaccines that are intended to “train” the immune system to find and destroy metastases.
“This immunotherapy is not meant to replace surgery,” Zaharoff says. “In fact, we would still like to see all breast tumors removed. But it gives us a couple of weeks to ‘train’ the immune system before the surgery and thus reduce chances for recurrence or metastasis.”
In addition to the breast cancer study, researchers in Zaharoff’s lab are also pursuing projects focused on prostate cancer, myeloma, lung cancer and drug addiction.
“By no means do we think this therapy is limited to colorectal, bladder and pancreatic cancers,” Zaharoff says. “We think this is a broadly applicable immunotherapy that may be used for the treatment of any injectable solid tumor.”
Excited to Go to Work
Sometimes the indirect route is the best way to get to where you want to be, even if you don’t know exactly where that is. At least you know where you don’t want to be. This was how it worked for David Zaharoff. He started as a mechanical engineer. After finishing his bachelor’s degree at the University of Illinois, he watched his friends go off to Detroit and other places, where they earned good money. But Zaharoff wasn’t inspired by the types of problems – “fuel efficiency, aerodynamics, stuff like that” – that they were solving.
“All my friends went to work at Ford and GM, and they’re perfectly happy,” he says. “But I couldn’t see myself designing a luggage rack on a minivan. I wanted a bigger challenge.”
Questions he wanted to answer: Why does the human body randomly develop deadly diseases? How does the body respond to different types of biomaterials? Why are most drugs taken orally if they are meant to act at very specific locations within the body? What is the ideal way to treat cancer? These kinds of questions steered him toward biomedical engineering and landed him at Duke.
“These were problems I could identify with, problems that excited me,” Zaharoff says. “Because ultimately you have to be excited to go to work. If not, it makes for a long day.”
Photos by Russell Cothren