T is for Technology

Small molecules play a big role inside cells. They are involved in metabolism, signaling, growth and locomotion. Yet for all their importance, little is known about how these molecules operate because their size makes them difficult to see.

UCSF's Alan Verkman is now literally bringing these molecules to light by using state-of-the-art fluorescence microscopy to construct an accurate real-time picture of a cell's interior. Rather than the static view an electron micrograph provides, Verkman's novel optical methods can directly measure the dynamics of molecules - how they move and interact with other molecules - within living cells.

In doing so, Verkman has clarified a number of previously mysterious biological processes. For example, his findings dispute the textbook notion that the interior of a cell is so crowded that molecules would be seriously hindered in their motion (he has found that they are slowed down far less than suspected). At the same time, his studies of the mobility of DNA and DNA complexes - which show how quickly DNA moves inside the cell - are likely to improve the efficiency of gene delivery in gene therapy. Verkman's lab has also developed the first fluorescent indicators to measure chloride in living cells. A defective cellular channel for chloride flow causes the lung disease known as cystic fibrosis. Not surprisingly, Verkman's group has begun looking for a way to increase the activity of the malfunctioning protein - the cystic fibrosis transmembrane conductance regulator, or CFTR.

Over time, Verkman's group has refined the fluorescent indicators into a very useful screen for drugs that might increase the function of CFTR. Using a form of green fluorescent protein that is sensitive to chloride, Verkman and his colleagues are able to monitor the fluorescence change inside the cell: if it changes quickly, then that compound is something that might activate CFTR. "For the vast majority of compounds tested, it doesn't change at all," says Verkman. "But if it does, the compound is potentially a drug for cystic fibrosis." Based on this assay, Verkman proposed to the Cystic Fibrosis Foundation that screening for drugs would be worthwhile. They agreed and funded a large multi-investigator program, including the purchase of a half-million-dollar robotic instrument to screen compounds efficiently.

Screening Strategies

The lab now has the ability to rapidly screen thousands of compounds looking for applications in cystic fibrosis and other human diseases. Verkman explains, "Our strategy is to screen 100,000 unrelated compounds one by one to see if they have a particular activity. Out of 100,000 compounds, 50 compounds might have some activity. We then look at those compounds more carefully to find those that are nontoxic and those that have the highest potency for increasing activity." Those became candidates for the development of a new drug.

Cystic fibrosis, being the most common lethal hereditary disease in the Caucasian population, is an obvious target for his lab since it has an assay for chloride. But CFTR plays multiple roles in the body, including being responsible for secretory diarrhea including that caused by cholera. An inhibitor of CFTR that works effectively in humans could prevent fluid secretion and diarrhea. Indeed, Verkman's lab found such a compound and has submitted the data for publication.

Yet another target are the aquaporins, or water channels, found in cells of organs that rapidly secrete or absorb fluid, such as the kidney, gastrointestinal tract, eye and central nervous system. Inhibitors of aquaporin might have implications in the therapy of congestive heart failure, hypertension, glaucoma and brain and spinal cord swelling.

There are many possibilities for drug discovery with a robotic assembly line that can screen more than 5,000 compounds per day. "We have high hopes."

Targeting Cancer

Researchers at UCSF have also devised a strategy to outsmart cancer - specifically through targeted agents that deliver drugs directly to cancer cells. Capitalizing on parallel advances in the areas of monoclonal antibody technology and liposome research, James Marks, an antibody engineer, John W. Park, an oncologist and cancer researcher, and other UCSF colleagues have combined their respective disciplines to create immunoliposomes: synthetic membrane-coated particles that can carry a large cargo of drugs, onto which antibody fragments are linked to recognize and target tumor cells.

Park, the current project leader, explains their rationale: Liposomes are useful because of their safety, stability and carrying capacity. More than 10,000 drug molecules can be packed into each, so liposomes can contain a very large active payload of drugs against cancer, which by itself has proven to be a useful tool against a number of different cancers.

Although these liposomes circulate for a long while and can get into tumor tissues, they don't go after the tumor cells directly. On the other hand, antibodies can target specific molecular features of tumor cells, but often do not have sufficient potency to eliminate the targeted cells. Since antibodies can provide targeting ability and liposomes can provide drug delivery, it was Park's idea that the two technologies be combined.

In creating these immunoliposomes, the scientists began with a system with which Park was very familiar - monoclonal antibodies against HER2 (he worked on Herceptin® at Genentech). HER2 is an oncogene product that is frequently produced at abnormally high levels, or overexpressed, in breast cancer. Marks created the anti-HER2 antibody currently used for the immunoliposomes by developing an improved technique of making antibodies. Instead of immunizing animals to generate antibodies, his method uses molecular biology techniques alone. He creates a library of billions of different antibodies generated from the genes of healthy human volunteers. From this pool, he can screen for the particular quality he wants - in this case, an antibody against HER2 that was very well internalized by breast cancer cells.

The UCSF researchers have completed the preclinical studies showing that their HER2-targeted immunoliposomes containing the chemotherapeutic doxorubicin can shrink even large tumors in mice, while having fewer side effects than chemotherapy alone. This technology is currently in the final phases of preclinical research as part of an exclusive licensing and development agreement between Hermes Biosciences - the biotechnology corporation created by the UCSF investigators who developed the licensed immunoliposome technology - and Alza Corporation of Mountain View, California. The UCSF group streamlined the production effort by developing a method for attaching antibody fragments to existing liposomal drugs, allowing modification of an already FDA-approved chemotherapeutic liposome (Doxil) manufactured by Alza.

The researchers were encouraged to bring their discovery quickly to clinical trials by one of their funders, the National Cancer Institute's Breast Cancer Specialized Program of Research Excellence (SPORE). "Under the SPORE mandate, we very much wanted to bring what we thought was a promising technology to clinical testing," says Park. "One always wants to do that in cancer research, to have discoveries that actually help people, but we had extra motivation that this funding really wanted it to happen soon."

Park acknowledges Christopher Benz, UCSF adjunct professor and program director at the Buck Institute for Age Research in Novato, California, as the original principal investigator of this effort. In addition, the Liposome Research Laboratory - a longtime UCSF affiliate now based at the California Pacific Medical Center Research Institute in San Francisco - which includes Keelung Hong, Dmitri B. Kirpotin, and the late Demetrios Papahadjopoulos, provided critical liposome expertise for the development of this technology.

These scientists are also working to expand their repertoire of available liposome-drug-antibody delivery systems. More than a single method of targeting breast cancer, the combination of techniques can be even more far-reaching. "We see this as a true platform technology," says Park. "It's a general-purpose way of linking antibody fragments to drug-carrying particles." One can envision a smorgasbord - antibodies with various targets and liposomes containing an assortment of drugs attacking different aspects of malignant cells - to be combined at will.

"One of the nice things about this technology is that there are a lot of drugs that work against tumors, but that also have problems, such as bioavailability, solubility and toxicity, which prevent their use," says Marks. "This process allows you to take a drug that has these problems and open the door to combinatorial therapy."

by Mitzi Baker