Something had to be wrong. None of the mice had cancer.
||In 1992, Leaf Huang made his first liposome for a clinical trial. It was the first step in years of research that is leading to new, more effective therapies for cancer.
John Dileo had injected the three mice with a substance he thought would prevent cervical cancer. Then he exposed them to cervical cancer.
And none of them had cancer.
Something had to be wrong because scientific tests never come back 100 percent successful.
Dileo had to talk to Leaf Huang. Dileo was earning his PhD in molecular genetics in Huang’s lab. He had to tell him that something was wrong.
This is too good to be true, Huang said. You have to try the tests again.
The sixth floor of Salk Hall is the home of Pitt's Center for Pharmacogenetics. There, Huang mentors graduate students and junior faculty members. The center started in 1999 when Pitt’s Arthur S. Levine, dean of the School of Medicine and senior vice chancellor for health sciences, asked Huang, a faculty member of the School of Medicine’s Department of Pharmacology, if he would direct a new research center in the School of Pharmacy. Since the center’s inception, National Institutes of Health funding for the entire School of Pharmacy has moved the school from 29th to sixth on the list of pharmacy schools with the most NIH research moneya feat that Huang partly attributes to faculty members he recruited. His investigators conduct so much research and win so many grants that the sixth floor is no longer big enough for all their laboratories and offices. The center focuses on studying genetics and pharmaceuticalstargeted drug delivery.
Huang has devoted his career to the study of nonviral vectors, specifically for use in targeted drug delivery and gene therapy. Nonviral vectors are agents, which are not made of viruses, used to deliver gene therapy or drugs to specific locations in the body. Many think that targeted drug delivery and gene therapy will soon be the way that diseases are treated.
Huang and others studying gene therapy have to outsmart the immune system. When scientists inject the therapy into the body, the immune system rightfully thinks it is being attacked, because a foreign agent has been introduced. Scientists have to find a way to trick the immune system to allow the gene therapy to get past the body’s natural defense.
Today, if a person is undergoing chemotherapy, the drugs don’t just attack the tumorthey also attack the surrounding tissue, indiscriminately killing cells. Maybe the cancer cells die, but maybe some of the tissue surrounding the tumor also dies. Delivering drugs to the body like this is ineffective and highly toxic. That’s why people who undergo chemotherapy treatment often get sick. In targeted drug delivery, the drug recognizes a tumor cell and only affects that tissue.
Huang first started investigating effective drug-delivery methods more than 30 years ago at Carnegie Institution of Washington located in the nation’s capital. He was a postdoctoral scholar there researching nonviral vectors called liposomes. They are naturally occurring fluid-filled pouches made mostly of phospholipids, which resemble the make-up of cell membranes. Liposomes are the most effective nonviral vectors in drug-delivery systems. Pharmaceuticals that use liposome vectors to deliver drugs are very popular, making up a $1 billion share of the drug industry, Huang says. For example, one of the most popular anticancer drugs, adriamycin, is a liposomal prescription.
But the most effective way to deliver targeted drugs is to use viral vectorsmodels that employ viruses, like a simple cold virus, as a method of transporting drugs or genes directly to the site that needs treatment. Viral vectors are effective, but they can be very potent and cause other complications. In 1999, the negative side effects of viral vector gene therapy were in the spotlight when a young man died during a University of Pennsylvania clinical trial. Pioneers in gene therapy and drug delivery research, like Huang, hope that methods of transportation that don’t use viruses will prove to be safer.
In 1990, when Huang was at the University of Tennesseewhere he had worked since 1976he and his colleagues were among the first to write a paper about drug design using liposome vectors. A year later, he and his wife packed their bags for the University of Pittsburgh. Huang was very interested in the gene therapy work being done at the University, and he thought that his ideas about drug delivery could provide a model for gene therapy delivery. Also, no one else at Pitt was studying gene therapy using nonviral vectors.
In 1992, Huang made his first liposome for a clinical trial, conducted with a collaborator at the University of Michigan. In the early 1990s, gene therapy was still experimental. Even though most of his research now focuses on the development of more effective therapies, this trial was to make sure that the liposome was safe and that it targeted the gene it was intended to affect. It worked. It was the first step in years of research that is leading to therapies using liposomes.
Huang has a powerful voice, which probably gets its authority from the sermons he gives in Mandarin at the Pittsburgh Chinese Church in Oakland, where he and his wife are founding members. He stores everything on his computer and eagerly shows images to explain what he is doing. Behind him are photographs of his family.
As a young student in Taiwan, Huang says he studied physics as an undergraduate but learned when he was a junior that he was more interested in biology and chemistry. By then, he felt it was too late to change majors. Still, the physics major tried applying to graduate biology programs in the United States. He chuckles, recalling that the schools rejected him because of his weak biology and chemistry background. Finally, he was accepted into a biophysics program at Michigan State University, a field that uses the ideas of chemistry, physics, math, and computer analysis to examine how biological systems work. His graduate work led to his postdoc at the Carnegie Institution of Washington, where he learned of liposomes.
Since 1999, Huang has been The Joseph Koslow Professor of Pharmaceutical Sciences. He currently has about 25 graduate students in his lab. He’d like even more.
“I love to work with graduate students,” Huang says. He doesn’t just share his knowledge with them; they also help to advance his knowledge and research. “I think it is imperative that any good research-oriented university should have a good graduate program.”
Dileo is one of the many graduate students who have worked with Huang. When Dileo conducted those tests on the three mice in Huang’s lab, he suspected he had done something wrong, which caused his experimental cancer vaccine to seem flawless. To identify his mistake, he injected about five more mice with the substance. His control mice didn’t get any injection. And as he did in his first test, he exposed all the mice to cervical cancer.
Within 10 days, the control group had cervical cancer. The group of mice who were injected with the experimental vaccine didn’t have any signs of cancer. Although this was a limited test, with a few mice, Dileo was in disbelief. Could this suggest a vaccine for cervical cancer?
Dileo was injecting the mice with LPD, short for liposome, protamine, and DNA. Huang first discovered that LPD was able to bypass the immune system and deliver genes or drugs to the lungs. Because the researchers knew that LPD could foil the immune system to enter the lungs, they reasoned that it might be a good nonviral vector to deliver therapies to other organs.
But, there was a side effect. If the scientists injected too much LPD, it could be fatal, causing an acute inflammation, producing massive amounts of cytokinesa substance that a cell produces during an immune response.
Mark Whitmore, another graduate student who worked in Huang’s lab, did some investigating on the cause of the potentially fatal immune response. His focus was on the CpG gene sequence, which is found in bacteria and viruses. Mammals’ immune systems recognize CpG as foreign and attack it. LPD prompted a CpG response.
“On the one hand, you get the benefit of an anticancer effect,” Whitmore says of the CpG. But he found in some cases the immune system overcompensates in its attack, flooding the body with cytokines, which creates inflammation that can cause fatalities.
Whitmore earned his PhD in pharmacology in 2000 from the University, where his thesis focused on immunology. It may seem a bit odd that someone interested in an immunology degree would work with Huang rather than an immunologist, but Whitmore says it is the way that Huang looks at gene therapy and drug delivery that makes him an appealing mentor across academic fields.
“I had experience with Leaf in class. I liked him as a teacher. He was doing gene therapy, and he was taking it from a drug delivery perspective. Gene therapy is a cutting-edge therapy, and he was dealing with the major hurdle of getting it into the body as a drug delivery problem,” Whitmore says. Since 2000, Whitmore has been at the Cleveland Clinic as a postdoctoral research fellow, and this summer he was promoted to research associate.
“I think gene therapy is doing something like a virus does,” Whitmore says. “I study the host’s defense against the virus to understand the defense in gene therapy.”
About the time that Whitmore was finishing his PhD, Dileo started researching LPD. Huang and Whitmore knew the reason that LPD successfully created an immune response was that certain cells pick up LPD. One of the types of cells drawn to LPD is a dendritic cell, which helps T cells in fighting off infection in the body. Dendritic cells are also called “antigen-presenting cells,” meaning they create a very specific target for a drug or a gene to attack. Dileo bundled an E7 viral protein in LPD because E7 is expressed in cervical cancer.
This LPD package is designed to provoke an immune response against E7, which could explain why Dileo’s mice with the LPD injection still didn’t have cervical cancer. After testing LPD three times, Huang and Dileo realized that they might have a cervical cancer vaccine in the making. It seemed exciting; yet, the researchers knew they had to be cautious. As with any lab breakthrough, there is no guarantee that it will ultimately prevail in clinical trials.
What, though, if this discovery actually leads to a therapeutic vaccine?
Conventional vaccines are great for protecting people who don’t have a disease from being susceptible to getting it, but they offer little help to those who already suffer from a disease. A therapeutic vaccine, on the other hand, is used after a person develops a disease, like cancer. If, for instance, a woman has a tumor in her cervix, the therapeutic vaccine could be used to treat the tumor, and it would later protect her from getting cervical cancer again. Huang realized that if they could come up with a therapeutic vaccine, it would surpass all existing conventional cervical cancer treatments.
“He [Dileo] took my challenge,” Huang says.
Dileo gave five mice cervical cancer tumors. Then he injected them with the LPD package. Soon, all five animals were cancer-free.
Could this be true?
Once again, Huang made Dileo repeat his trials. Another perfect test.
Dileo retested the LPD.
And once again their malignant tumors disappeared.
Well, they wondered, what if you cured the mice of the tumors, but exposed them again to cervical cancer?
The mice did not get the cancer.
The researchers were still cautious. Their minds are used to well-reasoned arguments and experiments that they try over and over again. Instant perfection isn’t usually in their lexicon.
“Anything that works so well is surprising,” Huang says. “Science usually takes many times.”
The researchers discovered that the mouse vaccine doesn’t just cause complete remission in the early stages of cervical tumors; it also eradicates them in advanced stages. This discovery has profound implications, especially for the developing world. Eighty percent of cases of cervical cancer occur in developing nations; this cancer kills about 300,000 women annually, making it the second leading cancer that kills women worldwide, according to the World Health Organization. And “it is important that our treatment can treat [advanced stages of cervical cancer], because we’re not going to save lives if it doesn’t,” Huang says.
His focus on treatment is what attracts graduate students, like Dileo and Whitmore, to his lab. No one started with the intention of finding a therapeutic vaccine for cancer.
“I am very happy it is highly reproducible,” Huang says. Many different people in Huang’s lab have been able to make the vaccine work; he no longer thinks the seemingly flawless mouse vaccine is a fluke.
“Humans could be a totally different story. I cannot say that it would work in humans. I hope it will work in humans. Then we can cure one type of cancer, and I think that is significant.”
Dileo is now working for the Mitre Corp. in northern Virginia; the company has several contracts with the U.S. Department of Defense. Before starting at this company, Dileo did postdoc work in the lab of Janey Whalen, a faculty member in Pitt’s urology department. He was trying to see if the work he did with LPD could be used in different cancer lab models, specifically prostate cancer. There had also been some hints of success in using LPD in melanoma scenarios. He is excited about the prospect that something he worked on as a graduate student could be an important breakthrough in treating cancer.
One of the reasons that Huang came to the University of Pittsburgh was the number of clinical trialstrials that test potential treatments in peoplethat are carried out here. Yet, in his years as a researcher at Pitt, he has never worked on a clinical trial based at the University. Often his collaborators are at other universities, and they carry out the clinical trials at those institutions.
He laughs, recalling upon his arrival here the promise he made to Ronald Herberman, director of the UPCI and UPMC Cancer Centersthat he would collaborate with a doctor at Pitt to conduct clinical trials here. In a few months, Pitt will have a new clinical trialHuang’s first at Pittfor a treatment for cervical cancer.
Meghan Holohan is assistant editor of Pitt Magazine.
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