Chimeric “icon” finds another target

A molecule effective against tumors may also be relevant to macular degeneration.

A versatile molecule developed at Yale, already shown to destroy blood vessels in tumors, now shows promise for arresting macular degeneration, a leading cause of vision loss in the United States.

Known as an icon—short for immunoconjugate—the chimeric molecule is made, using recombinant DNA technology, from part of an antibody and a molecule that targets abnormal blood vessels. “We describe it as a synthetic antibody, because it really functions as an antibody, but its targeting mechanism is different,” said Alan Garen, Ph.D., professor of molecular biophysics and biochemistry. Garen used as a model an antibody found in camels, because it is more suitable for manipulation by recombinant technology than human antibodies. The targeting portion, fVII, draws the molecule tightly and specifically to the abnormal vessel; then the antibody portion activates an immune attack, destroying the vessel. An important feature of the icon is its flexible design that can generate an all-human molecule for clinical use.

The icon’s targeting mechanism ignores normal blood vessels and zeroes in on tissue factor (TF), a protein produced on the inner wall of abnormal but not of normal vessels. The same search-and-destroy strategy works against tumors by destroying the blood vessels that nourish them, while leaving normal tissue unharmed.

“The pleasing result is that the same mechanism can apply to two very different medical problems: cancer and macular degeneration. What links them are the unique properties of the blood vessels that are involved in both diseases,” said Garen, who collaborated on the work with Research Scientist Zhiwei Hu, Ph.D., M.D. Ophthalmologist Henry J. Kaplan, M.D., and associates at the University of Louisville performed the macular-degeneration studies, which were published in the March 4 issue of the Proceedings of the National Academy of Sciences.

Macular degeneration is the deterioration of the macula—a tiny spot at the center of the retina—resulting in blurs and blank spots in the field of vision. Abnormal blood vessels are the culprit in the wet form of the disease; they leak fluid onto the macula, damaging the cells. The Yale molecule destroys those leaky vessels without harming normal ones.

So far, the icon has been tested in mouse models of both diseases, and studies are under way at Louisville on a model of macular degeneration in pigs, whose eyes are similar to those of humans. The researchers plan to apply to the U.S. Food and Drug Administration for permission to begin clinical trials of the icon for patients with cancer and macular degeneration.

An icon could be administered in either of two ways, said Garen. “One way would be to produce it externally and administer it by injection,” either into the bloodstream or directly into the affected area. A second method, which the researchers are proposing to use in the cancer trial, involves the insertion of a gene for the molecule into an adenoviral vector that has been rendered harmless. “Then you inject the vector directly into the tumor,” said Garen. “It infects the tumor cells, and this sets up what you might call in vivo factories for producing the molecule in the body. With this method, you get continual synthesis, and it seems to be more effective.”

—Nancy Ross-Flanigan

When errant proteins stray, a cellular cowboy rides in to save the day

In the drama of life at the cellular level, proteins can be heroes or villains. When they’re wearing their white hats, proteins provide and maintain structure, act as enzymes and hormones and perform vital functions such as transporting oxygen in the blood. But they can also run amok, contributing to cancer, heart disease and inflammatory conditions such as rheumatoid arthritis.

The cellular scenario of this Wild West tale stars Protac, a molecule developed by researchers at Yale and Howard Hughes Medical Institute investigators at the California Institute of Technology. Playing the role of the sheriff galloping in to save the day, Protac rounds up rogue proteins inside cells and orchestrates the proteins’ demise. One end of the dumbbell-shaped molecule is customized to bind to the protein of interest; the other end homes in on the cell’s natural protein-degrading apparatus. “By bringing the protein into close proximity to the degradation machinery, you can target it for destruction,” said Craig M. Crews, Ph.D., an associate professor in the Department of Molecular, Cellular, and Developmental Biology with joint appointments in chemistry and pharmacology.

In a presentation at the Experimental Biology 2003 meeting in San Diego in April, Crews and co-workers showed that Protac—for protein-targeting chimera—degrades targeted proteins in intact cells. In a proof-of-concept experiment, they engineered Protac to bind to green fluorescent protein (GFP), a naturally occurring protein that gives off a bright, green glow under ultraviolet light. When Protac was added to cultured cells containing GFP, it gathered up the glowing protein and promoted its destruction. Within an hour, the cells that contained GFP had lost their fluorescence.

While Protac has potential for treating disease, its more immediate use probably will be in screening large numbers of proteins for better understanding of their functions, much as genetic screens currently are used, said Crews.

“When a genetic screen is used to study some aspect of cell biology, the process involves generating a lot of different mutants that are each defective in some gene that encodes some protein, and then looking for individuals that have a defect in the particular process that you’re interested in. The geneticist then determines which gene, and which corresponding protein function, has been altered,” said Crews. “But there are several areas of cell biology that are difficult to study using traditional genetics. So what we’d like to do is induce the loss of protein function, not by altering the underlying encoding mechanism—the DNA—but by physically inducing the degradation of particular proteins. One can imagine doing large-scale screens, knocking out every protein individually and looking for loss of particular functions. In this way, we hope to discover new, critical proteins that are required for intracellular processes. So in addition to targeting known proteins, we hope this molecule will aid in the discovery of things we don’t even know about.”

—Nancy Ross-Flanigan

Et Cetera

New axons in mice

A Yale scientist has encouraged axonal sprouting in mice by removing a protein, Nogo, that blocks the regrowth of nerve fibers in the brain and spinal cord. The research builds on previous findings by Stephen M. Strittmatter, M.D., Ph.D., the Vincent Coates Professor of Neurology.

“In the mice with a mutation that prevents Nogo A/B expression, the central nervous system is largely normal but responds to injury in a unique fashion with robust axonal sprouting and long-distance growth,” said Strittmatter, lead author of a study published in the April issue of the journal Neuron.
The researchers bred mice without the Nogo A and Nogo B proteins. In these mice new axons sprouted after a spinal cord injury, and the mice showed better recovery of locomotor function than control mice.
“Once we can demonstrate that the Nogo protein constitutes an important pathway limiting axon growth, then we can pharmacologically improve functional recovery first in animals and then in humans,” Strittmatter said.
—John Curtis

A doubling of the human genome?

A new analysis of the well-studied chromosome 22 suggests that there may be far more than the estimated 30,000 genes in the human genome. “Our study reveals twice as many transcribed bases than have been reported previously,” said first author John L. Rinn, a fourth-year graduate student, “potentially indicating there are twice as many genes in the human genome.”

Rinn and colleagues in molecular, cellular and developmental biology used advanced microarray technology to map the 34 million bases of gene-dense chromosome 22. Earlier this year in the journal Genes & Development, the team reported finding previously undiscovered sequences which were also found in the mouse genome. “This study was a proof of the principle that we can find, en masse, all the regions of a chromosome that are biologically relevant,” Rinn said. “In the future we will scale this process to tackle the entire human genome.”

—John Curtis


			Originally published in Yale Medicine, Autumn 2003. Copyright © 2003 Yale University School of Medicine. All rights reserved.