Technologies in Cancer Research: Progress at the Molecular Level
Building on advances in genetics and genomics, researchers have started to delve into the molecular bases of cancers. The work promises to make cancer a treatable chronic condition rather than a tough-to-manage acute disease.
by Peter Gwynne and Gary Heebner




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CONTENTS

Even though it kills fewer individuals than heart disease, cancer has long ranked as Public Enemy No. 1 in the view of the general public and many health professionals. According to the World Health Organization, physicians currently diagnose 10 million new cases of cancer each year. Statistical trends indicate that this number will double by 2020. Given the fact that one in three people in the developed world will get the disease and one in four will die as a result, it comes as no surprise that the big C is the condition most feared by the public.

In the past few years a flurry of advances in several fields of life science has promised to reduce that fear. The advances offer the prospect of more effective and efficient detection, diagnosis, and treatment of cancers.

Much of the progress has occurred in molecular biology laboratories. There, techniques of molecular and cell biology, genetics, and genomics have converged to reveal how the disease develops at the levels of gene and molecule. As a result, says Robert Weinberg, professor of biology at the Massachusetts Institute of Technology and a member of the school's Whitehead Institute, "Cancer research will no longer represent a grab bag collection of complex, apparently chaotic phenomena. Instead it will soon become a logical discipline able to explain the genes and proteins driving malignant cell proliferation in terms of a small number of underlying principles."

Complementing that understanding of cancer-causing processes at the molecular level is recent work on the nature of tumors. "I think that one of the most important areas in cancer research is the understanding of tumors not just as collections of autonomous cancer cells but as organs," explains Richard Klausner, director of the National Cancer Institute (NCI). "We're beginning to realize that the molecular processes within tumor cells are influenced by the complex organ that is the tumor. These tumors represent complicated development of a complex multicellular entity."

The fundamental work has also helped researchers to stratify cancers more knowledgeably than in the past. The new technology of functional genomics offers the prospect of stratifying malignancies in ways that may prove far superior to traditional pathologies involving microscopy. The research has shown, for example, that not all breast cancers have the same molecular origin. Some may resemble certain forms of colon cancer more than they do other breast cancers. "Breast cancer and colon cancer describe the geography of cancers," explains Randy Scott, chairman of the board of Incyte Genomics. "But when we stratify these diseases and name them properly, these diseases will lose their geographic names."

TARGETED TREATMENT

That stratification points the way to better targeted treatment of cancers. Instead of blasting tumors with radiation, chemical agents, or surgical excision, all of which destroy plenty of healthy tissue along with the tumors, the pharmaceutical industry is developing drugs that will interfere only with the molecular and genetic processes that initiate the development of cancers. "At present therapies are anatomically based," points out Paul Workman, director of the CRC Centre for Cancer Therapies at The Institute of Cancer Research in Sutton, near London, United Kingdom. "I like to think that we'll move towards a medical treatment for cancer that will be genetically driven. We have the vision that a patient will have a complete gene structure and expression profile from a tumor biopsy. Then some cocktail of individualized therapy will be designed for the patient."

Individualized therapies can take several forms. Monoclonal antibodies have already moved into the medical mainstream in the form of cancer drugs Rituxan, codeveloped by biotechnology firm Genentech and IDEC Pharmaceuticals to treat non-Hodgkins lymphoma, and Herceptin, created by Genentech alone as a therapy for breast cancer. Further down the line are angiogenesis agents, designed to prevent tumors from creating new blood vessels and to attack their existing vessels. The concept of cancer vaccines has also drawn significant interest, along with some skepticism. Canada has already approved the first cancer vaccine, a compound called Melacine produced by Seattle company Corixa. And gene therapy has appeared on the horizon as a means of treating cancers, although with little indication of success so far.

The new approaches to cancer therapy will cause a significant change in the experience of patients under treatment by detecting cancers earlier than is possible today. "Today we only treat advanced states of cancer," says Les Hughes, head of discovery research for cancers and infectious disease at AstraZeneca Pharmaceuticals. "We'll now be able to treat patients earlier in the disease stage."

The treatments will also cause patients less distress. "We're entering a world in which you'll see less toxic therapies with a greater emphasis on the patients' quality of life," says Paul Maddon, chairman and CEO of Progenics Pharmaceuticals, Inc. And because tomorrow's cancer drugs will be less toxic than today's, adds Karol Sikora, vice president of global clinical research, oncology, for pharmaceutical company Pharmacia, "they'll take longer to work. Cancer will become a disease to be treated over five years or so rather than six months."

The new treatments will alter the ways in which pharmaceutical companies take on the search for new cancer drugs. The idea of a blockbuster drug that will treat a variety of cancers will go out of the window. In its place will come a philosophy of developing a range of narrowly targeted therapies. "Drugs considered 'successful' in oncology may treat as few as 10 percent of cancer patients," explains Gerard Kennealey, vice president of clinical research, oncology, for AstraZeneca Pharmaceuticals. "We're convinced that we can do that and maintain viability for the company."

Several pharmas agree with Kennealey. At present, says Sikora, "We have a lot of interesting ideas in the lab but only a very few drugs to try out on patients. After three to five years we will see a huge release of new drugs for clinical trials. The problem then will be choosing the best ones for investment."

CAUSES OF CANCER

The great promise of effective new therapies stems from the past decade of scientific research on the fundamental nature of cancer. As they become cancerous, cells take on abnormal forms in which they seem to divide continuously. In the later stages of cancer those dividing cells metastasize, invading parts of the body beyond their original locations. Researchers have implicated genetic predisposition, chemicals, and viruses in the list of possible causes for cancer. They remain unsure about the actual mechanism of this disease. However, they generally agree that understanding the processes of cell signaling and regulation of cell division are critical to finding cures for cancer.

Clinicians believe that only a relatively small percentage of the tumors they see is genetically influenced or inherited via a mutated gene from one parent or both. One of these well-studied genes, BRCA1, strongly predicts a woman's risk of developing breast or ovarian cancer. Genetic screening for this and other cancer-related genes has become a relatively simple procedure for some ethnic groups. It requires only a very small sample of the patient's serum or blood. DNA from the sample is amplified by polymerase chain reaction and subsequently analyzed to determine if any of the "cancer" mutations are present in the particular gene.

In contrast to genetically influenced tumors, scientists believe that the vast majority of tumors seen in the clinic result from external factors that induce mutations in individuals' DNA. External factors such as smoking, diet, exercise, stress, and radiation may affect these forms of cancer. Researchers have identified several genes that, when mutated, change the development of a cell from its normal life cycle to one that results in the formation of cancerous tissue or cells. Now that most of the human genome has been sequenced the number of mutations linked to cancer will certainly increase.

Cancer research currently focuses most strongly on biochemical pathways operating in normal and cancer cells and the ways in which they control cell division and cell death (one form of which is known as apoptosis). Every day researchers are identifying new molecules and new pathways for cell regulation and cell growth. As they identify more steps in these pathways they will provide potential new targets for therapeutic intervention to this disease.

THE BASIC TOOLS

Early cell culture studies demonstrated that attached cells grown in culture exhibit contact inhibition. In other words, they grow on a surface until they reach confluence and then stop dividing. Obviously they must communicate with each other to coexist in such an environment. Several companies supply cell culture media and reagents that permit scientists to standardize these research efforts and produce much more predictable results. Not surprisingly, culturing cells from living organisms such as mice has moved from art to a much more scientific procedure. Major suppliers of cell culture media include BioWhittaker, Invitrogen Corporation, and Sigma Chemical Company.

Commercial cell culture media usually require serum replete with undefined growth-promoting factors. Because these media introduce significant variability from lot to lot of serum, scientists find it difficult to analyze what factors cause cells to divide or become senescent. To overcome that problem laboratories can now buy serum-free (or defined) media that can sustain the growth of even some of the most finicky cells in culture. The defined nature of these media allows researchers to add specific growth factors to examine their effect on normal cells. In addition to offering defined media, most commercial suppliers will custom-develop and produce serum-free media for use in specific cell culture systems.

Microscopes have long allowed scientists to examine and discover the inner workings of cells grown invitro or isolated from diseased tissue. In the early years the light microscope established a new field of medicine—pathology—that enabled scientists to study structural changes in cells and the relationship of those changes to the diagnosis of diseases such as cancer. Carl Zeiss, Leica, Nikon, and Olympus, among other manufacturers, have designed light microscopes for research and clinical use for many years.

Today, light microscopes and fluorescence microscopes remain two of the most essential tools for clinical pathologists. But while they permit researchers to see structural changes in cancer cells, microscopes cannot reveal what causes cells to become cancerous or the ways in which the inner workings of a cancer cell differ from those of normal cells. To answer those conundrums and reach rapid diagnoses of several diseases, including cancer, scientists must complement their microscopy with biochemical assays.

RULES OF PROLIFERATION

The earliest use of such assays made it clear that cancer involves not only the morphological changes visible to microscopy but also significant molecular changes in a cell's DNA. The finding that certain mutations in a cell activate tumor producing genes and inhibit tumor suppressor genes has been pivotal in understanding what happens in a cell as it changes from normal health to a state of malignancy in which it threatens to destroy its host organism.

Scientists have suggested that several essential changes in a cell's metabolism collectively determine the change from normalcy to malignancy. "In my lab we're trying to work out the rules that determine how many different changes are required to convert a normal human cell into a cancer cell," says MIT's Robert Weinberg. "I have the idea that there is a common set of rules that will influence how many types of cells in the body become cancerous. When we understand the rules we'll know how genes act in concert to create malignancy."

Weinberg guesses that research teams will be able to spell out the rules within a year. Meanwhile they know that factors involved in tumorigenesis include the cell's ability to produce its own growth factors, an insensitivity to growth inhibitors, the ability to escape apoptosis (programmed cell death), immortality, sustained angiogenesis, and the ability to invade tissue and metastasize. Each of these steps represents a breach of the defense mechanisms designed to prevent uncontrolled cell growth and tumorigenesis.

While our understanding of the signaling pathways within cancer cells is rapidly evolving, some researchers are shifting their focus to another, equally important, problem: studying the interactions between cancer cells and the normal cell types that have been recruited into the tumor mass. "Many labs, my own included, are now trying to understand how breast cancers develop by learning how different cells in a tumor speak to each other," explains Weinberg.

Researchers have established that normal tissues maintain their state of homeostasis through multiple signaling systems, among them soluble growth inhibitors in cells and insoluble growth inhibitors in the extracellular matrix and on the surfaces of neighboring cells. These regulators keep normal cells in a quiescent or differentiated state. Systems that inhibit cell division involve the retinoblastoma (pRB) and other related proteins. Potential cancer cells apparently break out of these systems. Disruption of the pRB pathway leads to production of E2F proteins. That allows continuous cell division to occur as the cells become resistant to the growth inhibiting factors that operate along this pathway.

Once started, cancer cells keep proliferating via two mechanisms. Grown invitro, normal cells need a ready supply of exogenous growth factors to sustain continued cell division. In contrast, growing tumor cells depend far less on such exogenous factors. They may produce their own growth factors or stimulate cells nearby to produce the needed factors for their continued division. One example of this occurs in sarcomas. By producing their own tumor growth factor, they eliminate the need for other cells to help out in the growth of the malignant cells.

Equally important is the ability of cancer cells to persuade normal cells to help them grow. "When one looks at a tumor one sees several types of cells coexisting and collaborating to make the cancer grow," Weinberg points out. "They include normal cells that have been co-opted and are used by the tumor cells to aid and abet their proliferation. These heterotypic interactions are as important as interactions between cancer cells. I work with a faith that they will be able to give us a set of the common rules that regulate the process."

DEFYING DEATH

Cancer cells keep growing because they can avoid apoptosis, the process of programmed cell death that permits normal cells to discard extra cells or those that have become defective. Understood only recently, the machinery that can trigger apoptosis is operative in most if not all cells, just waiting to be activated by some physiological event. Once activated the process removes unnecessary, damaged, or aged cells. When a cell undergoes apoptosis its morphology changes. The cell shrinks and becomes more dense. At the same time several significant changes take place at the molecular level, including fragmentation of DNA, activation of a group of enzymes called caspases, and a decrease in the transmembrane potential of mitochondria.

Cancer cells can acquire resistance to apoptosis in several ways. One common mechanism involves mutation of the p53 tumor suppressor gene. That mutation inactivates the p53 tumor suppressor protein, a key component of the DNA damage detection systems that can induce apoptosis in normal cells. Inactivation of similar proteins can also reduce the ability of aberrant cells to undergo apoptosis.

Life scientists know that cells in culture will go through cycles of growth and division only a finite number of times before they stop dividing and become senescent. The expression in cells of introduced proteins that inactivate the pRB or p53 tumor suppressor proteins enables these cells to continue dividing until they reach a stage at which they experience chromosome breakdown and massive cell death. Very few cells escape death at this stage, termed crisis. The few that do—fewer than one in a million—have solved the problem of maintaining chromosomal structure and have acquired the ability to grow indefinitely. Such cells are considered immortalized. Virtually all types of malignant cells appear to be immortalized.

Cell mortality is linked to telomeres, the ends of chromosomes that consist of several thousand short, repeating DNA sequences. Each time a cell replicates, every one of its chromosomes loses several of these terminal DNA sequences. Scientists believe that this shortening results from an inability of DNA polymerases to replicate the 3' ends of chromosomal DNA completely during the synthesis phase of cell division. Because normal cells cannot repair the ends, they eventually enter the crisis state. Most types of malignant cells, however, possess an internal system that uses the telomerase enzyme to maintain and repair their telomeres. That enables them to grow and divide indefinitely.

GROWTH FACTORS

Another factor critical to sustaining the growth and homeostasis of tissue is angiogenesis, the formation of new blood vessels in growing tissue. Tumor cells appear to stimulate angiogenesis by affecting the balance of inducers and inhibitors of this process. The use of specialized mouse models will be key in assigning specific functions to each of these regulators and to understanding the molecular basis of their activity. Several providers, including Eurogentec, Lexicon Genetics, and Taconic, have developed mouse model systems to explore the effects of knocking out specific genes and to determine their functions in complex living systems. These companies allow researchers and pharmaceutical companies access to very sophisticated and specialized technology without the large setup costs and learning curves required to develop such services in-house.

The ability to invade other tissues and metastasize allows cancer cells to escape from the primary tumor mass and find new ground to inhabit and regions to colonize in which the supply of nutrients and the amount of space available for growth are not so limited. This process of spreading throughout the body of the host involves several classes of proteins that enable cells to anchor to their surroundings. They include cell adhesion molecules (CAMs) and molecules that link cells to ECM substrates known as integrins. Changes in the expression of CAMs seem to play a key role in invasion and metastasis. Invasive or metastatic cells are also marked by changes in integrin expression. And extracellular proteases sometimes help to stimulate the spread of cancer cells.

One other key attribute of cancer cells is the instability of their DNA. Several systems in normal cells watch for aberrations. If they detect any, the systems keep them in check by steering the cell into apoptosis. As a result, DNA mutations are very rare. The fact that cancers appear in humans at a significant frequency suggests that our DNA is relatively susceptible to mutations during the course of our lifetimes. Research teams have implicated malfunctions of the safeguarding systems for DNA in this susceptibility. For example, the p53 tumor suppressor protein responds to DNA damage by halting the cell cycle to allow for either DNA repair or, if the damage is serious, apoptosis. The blockage of that protein that occurs in cancer cells prevents the repair and permits mutations to spread.

MOLECULAR TOOLS

Several areas of research have helped life scientists to reach their current level of understanding of carcinogenesis and to use the understanding to develop drugs to combat the disease. They include the elucidation of signal transduction pathways, genomics, combinatorial chemistry, high throughput screening, and bioinformatics. "It's a combination of molecular biology, genetics, and genomics," says NCI's Klausner. "There are some wonderful and interesting technological developments that signify a switch in our ability to study biologic processes."

For cells, cancer represents the antithesis of controlled homeostasis. A single mutation in the genome of an organism can profoundly affect cell regulation and thereby upset the delicate balance among many biochemical pathways. So when they select drug targets for cancer therapy, pharmaceutical researchers focus on those pathways most often deregulated in cancers. These include the receptor tyrosine kinase/ras/ raf/MAP kinase pathway that participates in the proliferation of cells, the cyclin-dependent kinase/RB/E2F pathway that is involved in the cell cycle, and the p53 stress response pathway.

Calbiochem, ICN Biomedicals, and Sigma-Aldrich, among other companies, provide researchers with broad lines of the basic compounds and reagents they need to study these pathways and the cellular functions of signal transduction (ST). These vendors have made it possible for life scientists to access a more standardized supply of biochemical tools to study cellular function while benefiting from the convenience of buying many products from a single supplier.

Other companies have entered the ST market niche, offering reagents and kits specifically designed for use in this discipline. These vendors, who include Alexis Corporation, Biomol, and BD Biosciences-PharmMingen, can offer expertise and technical support for scientists new to ST research. Still other companies, among them Chemicon International, Oncogene Research, SantaCruz, and Upstate Biotechnology, have focused on specific lines of products for ST research, such as antibodies.

Recent advances in the field of genomics have helped life scientists to develop a detailed understanding of how genes and the proteins they produce can change a normal cell and cause it to progress to a cancerous state. These key functional proteins are the basis for rational drug discovery using small molecules that can attack the affected biochemical pathways responsible for cancer.

Structural and functional genomics have led to the identification of many new cancer targets that researchers can use in their efforts to develop novel drugs that will act as effective and potent therapeutic agents. The use of combinatorial and computational chemistry coupled with high throughput screening systems has allowed chemists to create and screen thousands of samples in no more time than they would have taken just a few years ago to screen a few dozen samples for anti-cancer activity. "We have been aggressively profiling tumors for several years to characterize and analyze markers for pharmas," says Incyte's Scott. "We're now striving to go deeper into genomics for early stage drug development applications."

CHEMISTRY, MOLECULAR BIOLOGY, AND INFORMATICS

As chemists and molecular biologists discover more targets and create more potential drugs, or leads, to affect these targets, they encounter the tyranny of volume. The number of samples they need to process overwhelms traditional approaches. So assay miniaturization and high-volume screening become critical. "Alongside the breakthrough in genomics have come breakthroughs in high throughput sequencing, combinatorial chemistry, and structural biology," comments Paul Workman of the Centre for Cancer Therapies.

High throughput screening (HTS) systems consist of automated instruments designed to handle, prepare, and process many samples. The instruments help to screen cell lines for the presence of specific genes, proteins, or other markers, all of them essential in identifying potential drugs for cancer treatment. The ability of HTS and UHTS (ultra-high throughput systems) to screen thousands of compounds each day has two positive effects on the drug discovery process. It improves the efficiency and productivity of laboratories, thereby keeping sample processing from becoming a serious bottleneck in the process of drug discovery. And because the systems can be run on a micro scale they conserve precious samples and reduce the cost of screening protocols. Key manufacturers of HTS systems include Beckman Coulter, Eppendorf, LJL Biosystems, and TECAN.

Naturally occurring substances extracted from plants have long acted as the major source of drugs for treating cancer. Now another source of anti-cancer drugs is emerging: small molecules synthesized by organic chemists. Combinatorial chemistry has enabled researchers to design and produce families of related compounds that have good probabilities of interacting with targets in a cell's biochemical pathway.

Molecular biologists can take the synthetic process further by altering a cell's genes to enable that cell to produce a new protein that it couldn't create before it was genetically engineered. These new genes ultimately produce new proteins, which affect the biochemical activities of a cell. Gene Therapy Systems, Inc., based in San Diego, designs, develops, and commercializes gene therapy products and other molecular biology reagents for life scientists working on gene therapy. The company has the goal of accelerating and enhancing new discoveries in gene delivery systems for gene therapy research as well as aiding the use of vaccines for cancer. The tools include transfection products and plasmid DNAs.

Scientists at Gene Therapy Systems have developed a high throughput technology to generate tens, hundreds, or even thousands of individual genes that can be used directly in functional invitro and invivo assays. Currently the established way to produce a transcriptionally active gene is to clone it into a plasmid expression vector, expand it in bacteria, and extract and purify it by column chromatography. This process, which usually takes several days to complete for each gene, is too slow and labor intensive to accomplish when hundreds or thousands of genes are being considered. Genes can be amplified in a very high throughput fashion using PCR, but typical PCR products are not transcriptionally active until they get cloned into an expression vector.

"People working in the field might be interested in making hundreds or thousands of functional genes at a time," says chief scientific officer Philip Felgner. "Our TAP Express system allows investigators to use PCR to accomplish this." The PCR products can be used directly in invitro transfection assays or injected directly into animals. A project that would have consumed months or years with the old technology can now be accomplished easily in a single day." For applications to vaccines, Felgner continues, "investigators interested in identifying protective, immunogenic antigens from complex microorganisms or tumor cells can now use TAP Express to generate PCR fragments that can be injected directly into animals." The protein produced as a result could protect the animal or patient against a specific microbe or cancer.

Handling the vast amounts of scientific data that emerge from high throughput systems and combinatorial chemistry experiments threatens to create its own roadblock in drug discovery. Here bioinformatics makes its appearance. Software specialists and the occasional life scientist have developed powerful computer programs to help researchers organize and analyze structural and functional data. Scientific teams mine the data to identify similarities and differences in the sequences of genes and proteins within individual species and between different species.

Databases of DNA and protein sequences and of other functional data often exist on different platforms. That complicates the researcher's job of comparing data. In recent months DNASTAR, Genomic Solutions, Oxford Molecular Group, and other scientific software companies have improved programs for analyzing and comparing sequences that have made data analysis more routine than in the past.

GOING WITH THE FLOW

The recent advances in cancer research at the molecular level promise to have an early impact on diagnosing the disease. "Diagnostics will be vital," asserts Pharmacia's Karol Sikora. "I predict that in the future the type of tumor won't matter. What will matter will be molecular characteristics. So we'll have very good molecular diagnostics."

NCI's Richard Klausner foresees a bright future for the field. "For some time the diagnosis of cancer has been slowly evolving to include molecular diagnostics," he says. "Our expectation is that this will undergo a rapid expansion." NCI plans to fund consortia that will search for new classifications of cancers based on their molecular characteristics. Methods will include high throughput screening, arrays and gene chips, and proteomics. "These not only classify tumors but have prognostic significance," says Klausner.

Flow cytometry provides a good example of the promise of molecular diagnostics. It has had particular application in studying cell surface markers in general and, recently, apoptosis in particular. "Cell surface markers allow physicians to stratify patients to determine treatments," says Steve Koester, manager for advanced technology at Beckman Coulter, Inc. "We're right in the middle of a revolution in cytometry. We have a whole plethora of things coming down the pike to break open diagnostics. They will change the way that diagnostic medicine is applied to cancer in the future."

Flow cytometry has become a valuable tool in apoptosis research relevant to cancer because it offers rapid and accurate measurements of cell constituents and cell functions. This technique has the ability to take measurements in many cells in a very short time and also to describe a population of cells based on one or more particular parameters of interest. Life scientists often use flow cytometry to identify and quantify such proteins as Bcl-2/Bax members, detected using immunocytochemistry.

"Our Epics XL benchtop analyzer can tell if there is an abnormal DNA content in the nucleus of cells; many solid tumors have abnormally high or low amounts of DNA in their cells," says Grant Howes, marketing manager for flow cytometry at Beckman Coulter, one of the companies specializing in production of flow cytometers. Some of these instruments require dedicated staff to operate them, but offer the advantage that one can measure several parameters at a time using dyes that are excited at different wavelengths.

Antibodies tagged with labels such as fluorescein and other molecules that allow scientists to visualize them find broad use to identify and locate specific proteins in or on a cell. Antibody-based probes are ideal for identifying specific cell populations based on differences in their cell surface proteins or markers. They can also be used for histochemical applications in which a cell is fixed in paraffin and sections of it stained with antibody for a specific molecule. These tagged cells can be identified using microscopy, fluorescent readers, or flow cytometers. Molecular Probes provides many of the fluorescent labels used with antibodies.

Several assays for diagnosing cancer have already reached the market, many of them based on antibodies that detect the presence of tumor antigens. The reliability of these antibody-based assays has improved over time, although issues remain with nonspecific binding and cross-reactivity in these systems. One company very active in cancer diagnostics, Zymed Laboratories, Inc., produces its own antibodies for cancer diagnostics and supplies them to several market leaders in the field as well.

THE THERAPEUTIC PROMISE

The ultimate value of understanding cancer at the molecular level will arise in the development of treatments. "The most promising and exciting approaches to cancer represent the ability to direct therapy toward defined targets," explains Klausner. "We're seeing immunologic approaches coming into their own as molecularly targeted reagents such as human monoclonal antibodies are approved."

Monoclonal antibodies designed to attack cancer at the molecular level can complement traditional cancer treatments such as chemotherapy. "We believe that adding antibodies to standard therapy can dramatically influence the outcome of treatment," says Gwen Fyfe, senior director for medical affairs, oncology, at Genentech. "One weakens the cancers and the other gives the final blow, although it's unclear which does which." Genentech has led the way in this area of treatment with its co-development of Rituxan and its pioneering of Herceptin. Herceptin's success in treating metastatic breast cancer has been linked to overexpression of the HER-2 allele in patients with the disease.

Those drugs represent just a start for antibody therapy. At the research level, says Leonard Presta, a staff scientist in research at Genentech, "our major target is to refine the mechanisms of Rituxan and Herceptin. That research includes looking at signal pathways." Meanwhile Genentech and several other companies have developed new antibody therapies that are in various stages of patient testing. According to Presta, more than 400 are in clinical trials worldwide for all diseases, including cancer. "This has very quickly become a growth area," adds Fyfe. "It's likely that antibodies will become a standard treatment for metastatic cancer within 10 years."

Pharmaceutical companies have shown similar interest in cancer vaccines. "We're on the cusp of a new paradigm with cancer in which, once you're diagnosed with the disease, you'll get a vaccine just like the one you had as a child for polio or measles," says Progenics Pharmaceuticals' Paul Maddon.

The treatments are not vaccines in the traditional sense. Rather, they are therapeutic agents that may stimulate the immune system in such a way that it can more effectively fight off the disease already in the body. As such, they should be applied as soon as cancer is diagnosed. "The vast majority of cancer patients find themselves in this position," explains Maddon. "They have the initial diagnosis but the cancer has not yet spread. These patients need therapies that will allow them to maintain a good quality of life and prevent relapse of the cancer." Not only do vaccines that augment the immune system have the potential to provide enhanced therapeutic benefit. They also create fewer side effects for the patient. Progenics has several cancer vaccines undergoing clinical trials. Notably its GMK cancer vaccine has become the first to enter Phase III clinical trials.

Several researchers have started to develop cancer vaccines using Eppendorf's cell electrofusion technology. "The system is used to fuse tumor cells with dendritic cells, a kind of antigen presenting immune cells," explains Eppendorf's product manager Kurt Lucas. "It transfers both DNA and membranes. We use very short pulses — a microsecond rather than a millisecond. One of the advantages of using electrofusion is that the technique can work on very small numbers of cells compared with the less efficient, chemically induced cell fusion."

So far researchers have applied the technique mainly to patients with kidney cancers. "The cancers need to be slow-growing," explains Lucas. "And it's best if the patients have not had radiation treatment or chemotherapy, because they need relatively strong immune systems."

CUTTING OFF THE BLOOD SUPPLY

As tumors grow, they require more vascularization of their mass to provide nutrients and eliminate cellular waste. Tumors develop their circulatory systems through angiogenesis, which involves actively recruiting and incorporating blood vessels from nearby normal tissue. So in theory compounds specific to tumor cells that inhibit this process could shrink or eliminate tumors without damaging normal tissue.

"Angiogenesis is where the major effort in small molecules is," says AstraZeneca's Les Hughes. "The key is that you have to have the effect clearly on the tumor and not on the normal tissue." Hughes's company has over 15 products in preclinical and clinical development for a range of cancers that includes breast, colorectal, lung, and gastric tumors. Genentech is targeting angiogenesis via a monoclonal antibody directed at the VEGF gene itself. Genentech is studying its anti-VEGF antibody in breast, colorectal, and non-small cell lung cancer.

Gene therapy shows promise as a treatment for cancer, and progress is being made in both preclinical and clinical research. Thousands of patients have been given various gene therapy treatments, mostly directed against cancer, and several potential products are in late stage clinical trials. "We're still in the early stage," admits Felgner of Gene Therapy Systems. "Many technical enhancements need to be identified and implemented to improve invivo gene delivery." Gene Therapy Systems has a technology that may contribute to this end. "Our PNA Dependent Gene Chemistry enables investigators to chemically modify genes in order to improve their function and invivo bioavailability," says Felgner. The company is looking for licensing partners to accelerate the development of this technology.

As clinical trials of molecularly influenced cancer therapies prove successful, fresh problems emerge. "There's an awful lot of regulatory pressures beyond clinical protocols," says Shawn Smith, senior business development manager for the GIBCO Cell Culture division of Invitrogen Corporation. "Companies are paying a lot of attention to the manufacture of new diagnostics and therapeutics. As items migrate out of trials onto the market, there's a change from scientific to business thinking."

Concern in Europe over the relationship between bovine spongiform encephalitis—the so-called "mad cow disease"—and human Creutzfeld-Jacob disease has forced pharmaceutical companies to develop processing methods that avoid animal products. In helping biotechnology and pharmaceutical companies to set up manufacturing operations, says Smith, "we have to develop bioprocessing systems that are much more sophisticated." He adds a caution. "Only a small handful of the hundreds of cancer therapies in clinical research will successfully reach phase III trials, let alone get to the market," he says.

Nevertheless, as scientists improve their understanding of the molecular mechanisms of cellular function and malfunction, the treatment of cancer will take on a more scientific and logical approach. "We can foresee the therapeutic Holy Grail," says NCI's Richard Klausner. "That is the ability to identify the specific molecular machinery of cancer, to validate the importance of the altered machinery, and then to design a drug that will specifically affect that molecular machinery."


Peter Gwynne is a freelance science writer based on Cape Cod, Massachusetts, U.S.A. Gary Heebner is president of Cell Associates, a scientific marketing firm in Chesterfield, Missouri, U.S.A.

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www.corixa.com

DNASTAR
www.dnastar.com

Eppendorf, AG
www.eppendorf.com

Eurogentec, SA
www.eurogentec.be

Gene Therapy Systems, Inc.
www.genetherapysystems.com

Genentech, Inc.
www.genentech.com

Genomic Solutions
www.genomicsolutions.com

ICN Biomedicals
www.icnbiomed.com

IDEC Pharmaceuticals
www.idecpharm.com

Incyte Genomics, Inc.
www.incyte.com

Institute of Cancer Research (UK)
www.icr.ac.uk

Invitrogen Corporation
www.invitrogen.com

Leica
www.leica.com

Lexicon Genetics, Inc.
www.lexgen.com

Life Technologies, Inc. (GIBCO/BRL)
www.lifetech.com

LJL Biosystems, Inc.
www.ljlbio.com

Massachusetts Institute of Technology
www.mit.edu

Molecular Probes, Inc.
www.probes.com

National Cancer Institute
www.nci.nih.gov

Nikon, Inc.
www.nikonusa.com

Olympus America, Inc.
www.olympus.com

Oncogene Research
www.calbiochem.com

Oxford Molecular Group
www.oxmol.co.uk

Pharmacia Corporation
www.pharmacia.com

Progenics Pharmaceuticals, Inc.
www.progenics.com

Santa Cruz Biotechnology, Inc.
www.scbt.com

Sigma-Aldrich Corporation
www.sigma-aldrich.com

Taconic
www.taconic.com

TECAN US, Inc.
www.tecan.com

Upstate Biotechnology, Inc.
www.upstatebiotech.com

World Health Organization
www.who.int

Zymed Laboratories, Inc.
www.zymed.com
Note: Readers can find out more about the companies and organizations listed by accessing their sites on the World Wide Web (WWW). If the listed organization does not have a site on the WWW or if it is under construction, we have substituted its main telephone number. Every effort has been made to ensure the accuracy of this information. The companies and organizations in this article were selected at random. Their inclusion in this article does not indicate endorsement by either AAAS or Science nor is it meant to imply that their products or services are superior to those of other companies.
This article was published
as a special advertising supplement
in the 16 March issue of Science