June 5, 2001 DURHAM, N.C. – In findings that may enhance efforts to starve tumors, Duke University Medical Center researchers say they have generated antibodies in rabbits that inhibit the same cellular target as angiostatin and actually surpass the natural protein's ability to prevent cell growth in the laboratory.
The researchers said they will have to develop "humanized" antibodies – ones that won't be attacked by patients' immune systems – before any clinical application of their findings is possible.
Angiostatin appears to be one of the most important molecules in controlling angiogenesis, the complex process of creating new blood vessels, the scientists said. While angiostatin and drugs impacting other angiogenesis pathways are already being tested in patients, the researchers said the new understanding of the process may lead to improved attempts to cut off blood flow to tumors or to replenish normal tissues such as the heart.
The researchers had already shown that angiostatin binds to an enzyme called ATP-synthase that they found on the cell surface, but until now they did not know whether this binding actually caused angiostatin's effects. The new study, funded by Duke University, is the first proof that ATP-synthase does, in fact, act as a receptor for angiostatin, the researchers reported in Tuesday's issue of the Proceedings of the National Academy of Sciences.
"In this study we've shown for the first time that the cell-surface ATP-synthase enzyme retains its normal activity – creating ATP – and that it is inhibited by angiostatin and the antibodies we've generated," said first author Tammy Moser, research associate in the department of pathology at Duke University Medical Center. "This is a major new observation."
The Duke scientists, led by Moser, reported in 1999 that angiostatin binds to ATP-synthase on the surface of endothelial cells, the cells that line blood vessels. Previously, ATP-synthase was known only to exist inside cells, and its only known role was as an energy-producing molecule.
"The bottom line is that the proliferation of new blood vessels is energy dependent," said Dr. Salvatore Pizzo, the study's principal investigator and chair of pathology at Duke. "If you didn't need ATP-synthase on the cell surface, then inhibiting it wouldn't change proliferation."
The researchers have now shown that the cell-surface ATP-synthase is identical to that inside the cell's powerhouses – tiny internal sacs called mitochondria. They also reported that angiostatin controls blood vessel cell growth by interfering with the enzyme's normal function, turning adenosine diphosphate (ADP) into adenosine triphosphate (ATP), which stores energy the cell can use to power other processes.
"The ATP-synthase enzyme is used as a receptor in its active form by angiostatin," said Moser. "This represents a new biological paradigm – that the receptor function is targeted, not just its structure."
The goal of angiogenesis-based therapies is to control the growth of new blood vessels, which ultimately depends on the proliferation of endothelial cells. In cancer, anti-angiogenesis treatment would stop vessel growth to starve a tumor, while in heart disease and other circulatory conditions, blood vessel growth caused by angiogenesis-enhancing treatments could help restore blood flow to oxygen-starved tissues.
"The fundamental difference between targeting the angiostatin receptor compared to other angiogenesis mechanisms, such as the vascular endothelial growth factor (VEG-F) receptor, is that the body compensates for those other pathways," explained Pizzo, also a member of the Duke Comprehensive Cancer Center. "The beauty of targeting the angiostatin receptor – and we now know that's what ATP-synthase is – is that it doesn't matter what drive creates angiogenesis, you can shut it down."
The new study is the first to show that antibodies that recognize ATP-synthase can mimic angiostatin's effects, the researchers said. "Our identification of surface ATP-synthase as angiostatin's binding site was greeted with much skepticism in 1999, and at the time we speculated that the enzyme served as angiostatin's receptor," Pizzo said. "With this paper, no one will be able to say that ATP-synthase is not angiostatin's receptor or that this enzyme is not a target for therapy development."
Antibodies are an attractive alternative for therapy because they are easier to make in a functional form than a complex protein like angiostatin, the researchers said. There may be non-protein small molecules that mimic angiostatin, but unlike angiostatin or the antibodies, they could get into a cell and shut down the vital mitochondrial ATP-synthase as well as the growth-controlling surface enzyme.
The researchers created antibodies for their laboratory experiments by exposing rabbits to the subunits of recombinant human ATP-synthase (alpha and beta) that angiostatin binds. Because the rabbit's immune system perceives the enzyme subunits as foreign, the animal develops antibodies that recognize and bind to those specific proteins. "Recombinant" means that the human enzyme was produced by using E. coli bacteria as an ATP-synthase factory.
To prove that angiostatin targets ATP-synthase's normal function, Moser developed a laboratory test that measures the enzyme's rate of ATP production, in collaboration with co-author Dennis Cheek of the Duke University School of Nursing. Moser used this test to measure the effects on ATP-synthase of angiostatin, the alpha and beta-subunit antibodies and control antibodies.
Even without isolating the most potent rabbit antibodies, the researchers found that purified serum from the exposed rabbits reduced the activity of endothelial cells' surface ATP-synthase by roughly 60 percent, while angiostatin reduced ATP production by about 81 percent. The "polyclonal" rabbit antibodies also slowed growth of cultured endothelial cells up to twice as much as angiostatin. Serum from rabbits not exposed to the ATP-synthase did not affect cell growth rates or ATP production.
The scientists added that studies in animal models of blood vessel growth support their laboratory findings, but those results have not yet been published.
Other authors on the study are co-first author Daniel Kenan, Timothy Ashley, Julie Roy, Michael Goodman and Uma Misra of the department of pathology at Duke.
Duke University will hold the rights to these discoveries through a pending patent application.
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