Special Report: Biotechnology's Growth And Divergence

Modern biotechnology stems from the recombinant DNA technology of the 1970s, but the discipline has grown far beyond that core. The people, products, and potentials of biotechnology have mushroomed from a handful of companies focused on a select few pharmaceuticals to hundreds of firms developing products for such diverse fields as forensics, food science, and environmental cleanup. Today, biotech consumers range from megacorporations to a couple at home confirming that a child is on the way. The third International Biotechnology Expo & Scientific Conference (IBEX '90), taking place next week in San Mateo, Calif., will demonstrate how biotech has grown. This year's expo has triple the number of sessions held at the 1989 show. The first biotechnologists were not very business oriented. A decade ago, a lot of us on the science side were very starry-eyed, and the corporate world ignored us to a large extent, says William R. Sharp, executive vice president and director of business development at DNA Pharmaceuticals Inc. in Stamford, Conn. It was thought that biologists did not have a lot of corporate experience and weren't realistic about timetables and bringing products to market. But it is clear now that we are addressing very large markets, and we are more realistic about how fast the technology moves to new products. The move from research lab to biotech company required more than fascinating science. Specialists in scale-up, regulatory affairs, public relations, patent law, and strategic planning were added to the biotechs' staffs to increase the chances for corporate success. And as the applications of biotechnology expanded, scientists in other disciplines began to join these firms as well. Twelve years ago, biotech was in the hands of the molecular biologists the cloners and sequencers, says Jay George, vice president of research and development at Oncor Inc., located in Gaithersburg, Md. The industry is maturing quickly. The people who will ultimately bring to first fruit the things developed from molecular biology are chemists, physicists, and engineers. The tools of biotechnology have diverged as well. Many a molecular biologist can recall the days when extracting restriction enzymes was difficult and costly. The restriction enzyme EcoRI, for example, was expensive if ordered commercially, and purifying it from Escherichia coli was rather crude. Today, however, it is readily available from several companies. Spectrometers, separation protocols, and analytical devices have been adapted from chemistry; fermenters, microscopes, and bioreactors from microbiology. Sequencers and synthesizers have been developed for manipulation of DNA and protein. While some biotechnologists point proudly to the increasingly automated machinery of the field, others are amazed by the power of the biomolecules that those machines probe. When I think of biotech tools, I think not of the hardware, but of the elegant molecules that allow us to ask more elegant questions, says Jim Richards, director of business development at Gene-Trak Systems of Framingham, Mass. The specificity of enzymes like ribo-zymes and ligases is so marvelous. It's like using a finer pair of surgical scissors that allows you to snip very tiny pieces, as opposed to the big hunks we snipped before. Biotech's image has improved as well. In 1974, a rather uninformed populace clamored to halt the research, fearing the creation of triple-headed purple monsters, as one geneticist puts it. So in February 1975 at Asilomar, Calif., and later in Washington, D.C., scientists and lawyers struggled amid protesters chanting, We will not be cloned to create guidelines to reduce the risks of genetic engineering. Gradually, both researchers and the general public came to realize that the benefits of the technology far outweighed the risks. In 1980, the first genetically engineered drug, insulin, was introduced to an enthusiastic public. Soon came interferon, b-endorphin, and growth hormone. Later, the spread of AIDS brought recognition that drugs derived from recombinant DNA technology are actually safer than their counterparts pooled from tissue donations. The use of clotting factor to treat hemophilia, for example, has become undesirable because of the accompanying risk of AIDS. Thus, the initial trickle of biotech drugs under development became a torrent, although many are currently quagmired in a regulatory roadblock fewer than a dozen recombinant DNA-derived drugs have actually cleared all U.S. Food and Drug Administration hurdles (see story on page 32). The seeds of biotechnology were planted when James D. Watson and Francis H.C. Crick first described the double-helical nature of DNA in 1953 (Nature, 171:737-8). Their concise, one-page paper changed the field of genetics overnight. From the three-dimensional picture of DNA flowed the story of heredity, which Crick dubbed the central dogma how complementary base pairing allows the molecules of life to faithfully replicate and remain available for future use, while sending a message to the rest of the cell to manufacture proteins. Researchers began to think of ways to harness this fantastic, self-driven specificity. The idea of nucleic acid hybridization as a means to isolate and characterize specific DNA sequences was born. During the 1960s, many biochemicals key to the central dogma were identified. Synthetic messenger RNA molecules were used to decipher the genetic code (J. Molec. Biol., 13:302-24, 1965; PNAS, 47:1588-1602, 1961). The universality of that code the fact that organisms from aardvarks to zebras follow the same genetic instructions to build proteins suggested that DNA, and therefore traits, could be mixed and matched between species. But how to cut and paste DNA? At first researchers sought to chemically construct recombinant DNA molecules. But nature provided the tools, in the form of bacterial restriction endonucleases discovered by Hamilton Smith at Johns Hopkins University in 1970 to cut DNA at specific sequences. Ligating enzymes, or ligases, discovered three years earlier, could be used to paste together DNA pieces. With these long-sought implements, biotechnology was set to blast off from concept to reality. Here is what the field has become. Gel electrophoresis used to separate nucleic acids and proteins by size, charge, or other physical characteristics was teamed with restriction enzymes in the late 1970s to spawn a technique that would reverberate in a dizzying diversity of applications (Am. J. Hum. Gen., 32:314-31, 1980). Restriction fragment length polymorphism (RFLP) technology is based on the fact that DNA sequences vary from individual to individual. Therefore, restriction enzymes cut two individuals' DNA into fragments of different sizes if the DNA sequence differs at the enzyme's cutting sites. The ability to separate DNA on gels opened up a host of applications, not the least of which is DNA fingerprinting, says Karen Enz, technical support specialist for Hoefer Scientific Instruments of San Francisco. DNA fragments are displayed on gels, yielding an individualized genetic bar code that can be used directly to identify individuals, or indirectly as markers of unknown genes carried nearby on the chromosome. Markers are used to detect disease-causing genes in people and agronomic traits in plants. RFLP technology can show how closely related extinct species were to living descendants, and can reveal relationships at the DNA level among contemporary species. Perhaps the greatest testament to the power of this technology came in 1987, when a group led by Helen Donis-Keller of Collaborative Research Inc. in Bedford, Mass., published data on 393 polymorphisms, scattered among the 23 human chromosome types, providing the footholds for the Human Genome Project (Cell, 51:319-337, 1987). DNA sequencing revolutionized the study of DNA and proteins. Says Richard Cunningham, associate professor of biology at the State University of New York, Albany, Sequencing technology combined with automated DNA synthesis allows us to construct genes which we can alter in very precise ways to create engineered proteins. The latest advance in gel electrophoresis, pulsed field electrophoresis, is aiding scientists in their exploration of the human genome. Cells into which new genes have been sent through injection or electroporation, or by being shot with a gun can be cultured. Protein products of these cultured cells are collected, purified, and investigated for use in research as well as the applied sciences. The therapeutics arena has been dominated by a handful of body-chemicals-turned-pharmaceuticals. These include tissue plasminogen activator (t-PA), a clot buster; erythropoieten (EPO), a kidney protein that boosts red blood cell supplies; and immune system biochemicals, such as the interferons, interleukins, and colony stimulating factors to combat cancer. Recombinant vaccines for AIDS, malaria, and Lyme disease have been developed, and vaccines can even be combined to tailor multiple protection. The in vitro diagnostics market is perhaps the most promising of all because of the diverse possibilities borne from the base-pairing rules. A DNA probe complementary to a virus or microbe may reveal infectious disease, one complementary to a tumor virus or oncogene variant may reveal cancer, and a probe complementary to a gene or a linked marker may reveal inherited disease. By the year 2000, sales of biotech diagnostics are expected to top the $1 billion mark, with DNA probes constituting a significant portion of this total. Ask a group of molecular biologists to name the most significant biotech breakthrough in recent years, and they are likely to respond with a chorus of PCR, referring to the polymerase chain reaction. In PCR, sample DNA is treated with DNA polymerase, a DNA replicating enzyme, plus two short DNA pieces (oligonucleotide primers) that bracket a gene of interest. The polymerase copies the bracketed sequence, then copies the copies, generating millions of copies of the desired gene in just hours. PCR, as originally described by a team at Cetus Corp., of Emeryville, Calif., was limited by the use of a polymerase from E. coli. When researchers heated the polymerase in order to do PCR studies, it fell apart and became inactivated (Science, 230:1350-54, 1985). But in 1988, Taq I, a thermostabile DNA polymerase courtesy of Thermus aquaticus, a bacterial resident of deep sea thermal vents, greatly simplified the procedure, prompting Science to dub it 1989's Molecule of the Year (239:487-92, 1989). Taq I polymerase can amplify, more than 10 million-fold, DNA 50 to 2,000 bases long. PCR is revolutionizing forensics, in which samples can be very small, and prenatal diagnosis, in which time is short. Despite PCR's popularity, it is cumbersome and can replicate DNA other than the desired sequence. Several different gene amplification schemes are in the works. Some, such as the ligase chain reaction (developed by BioTechnica International, Cambridge, Mass.), QB replicase system (Gene-Trak Systems, Framingham, Mass.), and nucleic acid sequence-based amplification (Cangene Corp., Mississauga, Ontario), all use replication enzymes other than Taq I. The DNA molecule is a double helix, but only one side, the sense strand, contains the information to construct a protein. The other, antisense side, once thought to be functionless, may actually help regulate the sense strand by clamping onto complementary DNA or RNA. Many researchers are now trying to harness this built-in genetic control. Like any good idea, lots of people came up with it at about the same time, recalls Christopher Mirabelli, vice president for research at Isis Pharmaceuticals of Carlsbad, Calif. It evolved from the central dogma of how DNA specifies RNA, which specifies protein. If we could bind something selectively, it would arrest the translation of protein. Isis is one of several companies exploring applications of antisense sequences or synthetic molecules modeled after them. Antisense agents can squelch the activity of ripening enzymes in tomatoes, create new petunia petal patterns by shutting off pigment synthesis, and arrest the growth of herpes simplex virus in culture. Researchers have long attempted to generate pure antibodies, the proteins secreted by the immune system's B cells in response to a foreign biochemical (antigen). But injecting an animal with an antigen the traditional way to generate antibodies yields many different B cells, each secreting a different antibody type into the serum. A simple way to obtain a large amount of a single antibody type was needed. In 1974, Georges Kohler, working in the Cambridge, England, lab of well-known biologist Cesar Milstein, fused a mouse cancer cell with a mouse B cell. The result was a hybridoma: a hybrid of the two cell types. Like the B cell, it churned out a specific, or monoclonal, antibody; and, like the cancer cell, it was immortal. MAbs have since been widely used in both basic research and such applied fields as diagnostic imaging and drug delivery. MAbs cause the pigment change in an at-home pregnancy test and can also detect proteins released from dying muscle following a heart attack. The advent of MAbs spawned a whole new breed of biotech businesses those that mass-produce and market them, and those that custom-produce antibodies to suit a particular need. One designer antibody firm is Berkeley Antibody Co. Inc. (BabCo), headquartered in Richmond, Calif. Clients provide the antigen, and we immunize mice with it, says Kirby Bartlett, research coordinator at BabCo. High-titer mice are selected for fusion. After the fusion, screening and cloning procedures come up with the antibody for specific needs. As labs diversify in the type and scope of biotechnology research they pursue, many businesses, fueled by economic realities, are funneling their interests and consolidating their strengths. Companies are recognizing those projects that won't pan out as well as identifying corporate partners whose skills mesh with and complement their own. Throughout the biotech industry, mergers have resulted in new firms with greatly expanded capabilities: examples of such synergies are the mergers of the Boston-based Genzyme Corp. with Integrated Genetics Inc. of Framingham, Mass.; DNA Plant Technology Corp. of Cinnaminson, N.J., with Advanced Genetic Sciences of Oakland, Calif.; Berkeley, Calif.-based Xoma Corp. with Santa Monica, Calif.-based Ingene Inc.; and Calgene Inc. with Plant Genetics Inc., both of Davis, Calif. The companies that were focused early on have tended to survive intact in this ever-changing field. For example, Bio-Rad Laboratories Inc., based in Hercules, Calif., and Applied Biosystems Inc. of Foster City, Calif., carved a permanent niche by specializing in supplying key instrumentation. Applied Biosystems had the right idea, says Jay George of the Gaithersburg, Md.-based Oncor Inc. They automated tedious procedures such as oligonucleotide synthesis, extractions, and sequencing. That was all done by hand at one time. Biotechnologists have also found that concentrating on a few promising projects makes the most economic sense. Back in the early 1980s, DNA Plant Technology Corp. had its fingers in many pots protoplast fusion, somaclonal variation, clonal propagation, and monoclonal antibodies involving many species. Since then the company has abandoned those projects not likely to bear fruit. Basically, we have been able to develop three products so far, and more are coming, says Dave Evans, vice president for business development. We enter into business relationships with well-regarded companies and keep those relationships in place for a long period of time. The company markets VegiSnax to supermarkets and fast-food chains; diagnostic kits for turf grass disease to golf courses; and SnoMax, a snow enhancer acquired in the merger with Advanced Genetic Sciences, to ski resorts. Many biotech companies are finding that the pipeline of pharmaceutical products is bottlenecked maddeningly close to the homestretch. Consider the Emeryville, Calif.-based Cetus Corp. Formed in 1971, Cetus was one of the founding firms of the biotech industry. In July 1990, the firm presented data to the Food and Drug Administration's newly formed Biological Response Modifier Advisory Committee on interleukin-2 (IL-2) to treat metastatic renal cell carcinoma, a disease for which there currently is no treatment. But some of Cetus' data came from National Cancer Institute trials using IL-2 along with lymphokine-activated killer (LAK) cells. The data didn't pass FDA muster and, much to Cetus' surprise, the company was asked to redesign its clinical trials. Their stock plunged when the IL-2 application was rejected by the FDA, and it's rumored that Du Pont will buy them, says George. The FDA expects to rule on many more biotech products as the Human Genome Initiative moves ahead. The agency is gearing up to recruit people to help in the review process, according to Tom Tsakeris, director of the FDA's Division of Clinical Laboratory Devices. Biotechnology struggles onward, a field built on ingenious ideas but requiring difficult follow-throughs, facing a scientifically illiterate and skeptical public, and competing against familiar, successful products. Whether biotechnology can survive red tape and reality determine how far. But hybridomas have shortcomings. Very few cells actually fuse, and nurturing them to produce desired antibodies is often difficult. Plus, mouse-derived antibodies often elicit allergic responses in humans, yet human cells do not form hybridomas readily. Although these antibodies can be used despite the adverse reactions, the situation is far from optimal. A technique pioneered by Richard Lerner and Bill Huse at the Research Institute of Scripps Clinic and Joe Sorge at Stratagene Inc., both located in La Jolla, Calif.; Steve Benkovic at Pennsylvania State University, University Park; and others, endows E. coli cells with mammalian (mouse or human) antibody genes (The Scientist, April 2, 1990, page 22). Such a `combinatorial library' creates antibodies that nature hasn't made before. It shuffles the deck, says Benkovic. The mixing and matching of antibody genes in bacteria permits products that are more diverse than those obtained using the finicky hybridomas. With one in four people developing cancer at least once, according to the National Cancer Institute, it's no wonder that biotech is sharply keyed into this area. When the field dawned, human oncogenes cell-division regulators that, when overexpressed, lead to cancer occupied the realm of hypothesis. Today, several human oncogenes are under intense scrutiny. Moreover, Du Pont Co. of Wilmington, Del., has patented the oncomouse, an animal with a human breast cancer oncogene stitched into each of its cells. This creature will provide a way of testing treatments, enabling researchers to examine how a mammal responds. Even as oncogene action was being unraveled, other researchers pursued a different route to understanding cancer. In the mid-1980s, tumor suppressors were discovered, first for Wilms' tumor, the inherited childhood kidney cancer, and then for neurofibromatosis. These tumors grow when tumor suppressing genes malfunction. Biotech has aimed many immune system chemicals at cancer cells. A new approach, used on some forms of leukemia, is to coat magnetizable polystyrene beads with tumor-specific antibodies, then use them to cleanse the affected tissue. The beads are coated with antibodies specific for the individual and mixed with the bone marrow [extracted from the patient] to remove cancerous cells, explains Steven Moss, senior product specialist at Dynal Inc. of Great Neck, N.Y. The cancer-free bone marrow is then injected back into the patient, where it is hoped that healthy cells will proliferate. In the early 20th century, Johns Hopkins University neurobiologist R.G. Harrison showed that animal cells could be removed from a living organism and grown in a culture flask. For many years, scientists studied the biochemistry and physiology of cells grown in vitro. With the advent of genetic engineering, cell culture has taken on a whole new meaning. Today, manipulated cells from a range of organisms spew out useful biochemicals, growing amid networks of hollow fibers, between glass beads, or in alginate blobs in state-of-the-art bioreactors. Cells packed a billion per cubic centimeter are coaxed to secrete their particular products in systems that mimic the human circulatory system, supplying the raw materials for exciting new drugs and vaccines. A particularly promising cell culture system consists of army worm (Spodoptera frugiperda) cells infected with baculovirus. This virus normally produces enormous amounts of a protein called polyhedrin. If the gene for a protein of interest is spliced next to the control sequence of the polyhedrin gene, that protein is mass-produced. The result cultured cells whose volume is up to 50 percent desired protein. With $480 billion spent yearly in the U.S. on food, agriculture holds the most economic promise of any of the biotechnologies. It is also free of public dissent, to some extent, because people are less threatened by tastier melons than they are by human gene therapy. Ironically, though, plant biotech has been slower to reach fruition than other applications, largely because the tech tends to precede the bio that is, researchers can effectively manipulate plant cells without understanding the underlying mechanisms. The result a frustrating lack of control and precision. For example, a fusion of cabbage and radish cells yields a commercially useless hybrid with cabbage roots and radish leaves. Scientists do not fully understand why cultured plant cells of one species, given a certain mix of hormones and nutrients, sprout an embryo (a seed without its coat), while cells of another species treated the same way yield plantlets (small plants). Yet Plant Genetics Inc. of Davis, Calif., spent much of the 1980s wrapping such embryos in alginate coats with fertilizers, herbicides, and other such growth enhancers to create artificial seeds. This technology has not yet paid off not for lack of ingenuity, but simply because it's cheaper to plant good old-fashioned seeds. Likewise, DNA Plant Technology Corp., Cinnaminson, N.J., spent seven years perfecting its VegiSnax carrot and celery sticks, derived from unusual somaclonal variant plant-lets. At the Animal Physiology and Genetics Research Station in Edinburgh, Scotland, a ewe named Ethel is matriarch to a very special clan, whose members secrete a human clotting factor in their milk. Trans-genic corn developed at St. Louis-based Monsanto Co. glows in the dark, thanks to a firefly's gene, as do tomatoes similarly engineered by Keith Wood of the University of California, San Diego. The firefly's gene serves as a marker for other transfer genes. Transgenic organisms are an inevitable outgrowth of recombinant DNA technology. Genes are injected, electroporated, or biolistically shot into a germ cell or fertilized ovum. With luck and skill, an organism develops that retains the genetic gift and expresses it at the appropriate time and place. Transgenic organisms occupy a valuable niche in United States-based research. Understanding gene expression at its outset usually in the prenatal period is crucial to developing treatments. Because of a ban on experiments using human pre-embryos, U.S. researchers have had to make do with transgenic prenatal mice endowed with human disease genes. In Britain we have been extremely fortunate, says Nicholas Hastie, a human geneticist at the Medical Research Council's Western General Hospital in Edinburgh. Because British scientists are able to use human pre-embryos, we have done what most couldn't, he says. Thus, Hastie was able to determine how the Wilms' tumor gene fails to shut off cell division in the human kidney, while his U.S. counterparts who had actually found the gene had to resort to raising trans-genic mice to model the human situation. Future applications of somatic gene therapy include endowing endothelium (the tile-like cells lining large blood vessels and forming capillaries) with a missing gene that releases the missing chemical perhaps insulin, or a clotting factor directly into the bloodstream. Another possibility involves genetically engineering skin cells, which can then be grown to a graftable size. The skin would then be grafted back onto its original owner, where it would replace a missing protein. Early in the morning of April 19, 1987, Steven Lindow, a plant pathologist at the University of California, Berkeley, planted several thousand potato seeds in an isolated patch of land in the tiny town of Tulelake, Calif. The seeds were coated with ice-minus Pseudomonas syringae, bacteria lacking a gene promoting ice crystal formation. This technology, if successful, could significantly cut the $1.5 billion loss in frost damage sustained each year in the U.S. But a few weeks after the planting, angry Tulelake residents yanked out the feared potato seedlings. This first deliberate release experiment did not bode well for the dozens of others lined up. Yet just two years later, public reaction to biotechnology's transition from lab to land had tempered. One August eve in 1989, researchers from Cornell University, Ithaca, N.Y., sprayed a disabled form of baculovirus onto 2,750 cabbage plants at the New York State Agricultural Experimental Station in Geneva, N.Y. A few reporters showed up, but there were no triple-headed purple monsters in the next day's headlines, nor did frantic neighbors furtively tear at the plants. Since then, hundreds of projects have been readied for field testing. But their developers must first circumnavigate regulatory agencies to some, the toughest test of all. A bill now before Congress, the proposed Biotechnology Regulation and Research Integration Act, calls for a board to manage the often overlapping roles of the Environmental Protection Agency, the Department of Agriculture, and the FDA, and create separate permits for large-scale commercial and smaller-scale R&D experiments. The bill should help to unclog the backlog of biotech products awaiting field tests. When General Electric Co. of Schenectady, N.Y., introduced its oil eater bacteria in 1980, the idea of bacteria chomping up waste seemed like science fiction. Ananda Chakrabarty had combined four naturally occurring plasmids in a single bacterial cell, enabling the microbe to degrade a number of crude oil components. Throughout the 1980s, GE developed bacteria for removal of chlorines from PCBs in the Hudson River that are in use today. In 1990, bioremediation is taken quite seriously. The oddity of 10 years ago is now being intensely studied by various government agencies. The Department of Energy, for example, has established a Microbiology of Subsurface Environments program to match microbes' metabolic tastes to environmental problems. Many fish have metallothionein proteins that bind and sequester heavy metals, such as copper, cadmium, and zinc. Animal bioremediation efforts could include the release into polluted waters of fish with natural appetites for what we dub contaminants. The spectacular growth and divergence of biotechnology over the past decade can give researchers an awesome feeling of control over life. But part of capitalizing on the gifts of nature is to recognize science's limitations. Even though you can manipulate genes and do all kinds of fancy stuff, there are some things that cannot be changed, says DNA Pharmaceuticals' Sharp. It will always take nine months to gestate a human child and five to six years for a coffee tree to reach full production. So, yes, we can do things that we couldn't do before, but we need to be realistic about when a research result will translate into a product. I think that's happened now. Despite these caveats, biotech's future appears to be limited only by the extent of the human imagination. Gene-Trak Systems' Jim Richards perhaps sums it up best: The more we dig in, the more things there are to find. Ricki Lewis and Holly Ahern teach biology at the State University of New York, Albany.

Special Report: Biotechnology's Growth And Divergence

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