THE SEATTLE TIMES 12/18/94
THE RAREST THING ON EARTH
THE STORY OF AN ELUSIVE BLOOD HORMONE,
A DETERMINED GROUP OF SEATTLE RESEARCHERS
AND BIOTECHNOLOGY’S POWER AND PROMISE
By TERRY MCDERMOTT
George Rathmann, a biotech-industry pioneer, tells this joke:
A man, having had too much to drink, has lost his car keys. The guy is pretty sure he lost the keys in the woods, but is looking for them in the parking lot.
Why don’t you look in the woods, somebody asks.
Well, he says, it’s too dark in the woods. I’m looking over here where it’s light.
“Science is done where you can make progress,” Rathmann says.
“It is not done taking on an assignment that you can’t even start to attack. It doesn’t do you any good to walk in the woods; it’s dark, you can’t do anything. Go back in the light and hope you find something.”
In the spring of 1989, Francoise Wendling, a French virologist, shone a bright light in the direction of a mysterious molecule that scientists had been hunting for decades.
Wendling was researching leukemia viruses when she noticed that a mutant gene in mice shared characteristics with a family of proteins involved in the production of human blood cells.
Scientists following Wendling’s work thought she might have stumbled on a clue to the whereabouts of the elusive molecule thrombopoietin, or TPO, which had been theorized in 1958 as a critical blood-clotting hormone but had never been found.
“It was like chasing a ghost,” one scientist said.
If it existed at all, thrombopoietin was rare, apparently rarer even than the very rare hormone responsible for creating red blood cells. That hormone was isolated almost a decade ago. Drugs based on it have been on the market for five years. Five billion dollars’ worth of them have been sold. The total volume of those drugs wouldn’t fill a 5-gallon bucket.
Those numbers – one hormone, one bucket, five years and $5 billion – are a definition of the promise of the biotechnology industry. They were a powerful incentive for scientists to search for thrombopoietin.
The Wendling research was “like a gun going off,” one biologist said. Between the appearance of her first paper in 1986 and June of this year, tens, perhaps hundreds, of millions of dollars were spent in a furious race to find the hormone. The race ran around the globe, from giant conglomerates like Kirin in Japan, to lone researchers in Tennessee, to obscure biotech firms like ZymoGenetics here in Seattle.
It was so difficult to find, one scientist involved in the chase said, because thrombopoietin is the most dilute substance ever isolated, which is an abstruse way of saying there isn’t very much of it.
In fact, thrombopoietin, when compared to any known substance, is the thing there is less of. It is, in other words, the rarest thing on earth.
BLOOD WORK
Ken Kaushansky, a research hematologist at the University of Washington Medical Center, was among those following Wendling’s work.
Kaushansky’s interest was more than academic. A university research scientist leads a work life of multiple personalities. Kaushansky is foremost a researcher, a detective intent on uncovering the biology of human blood. For the last 10 years, he has been studying the hormones that make blood cells grow. He is also a teacher, staffing his laboratory with graduate and postgraduate students, often physicians who want to practice science rather than medicine. He is finally a clinician, a medical doctor with patients at the adjacent university hospital.
As a clinician, Kaushansky deals with blood disorders. Patient L was typical. A middle-aged woman with a classic, aggressive breast tumor, she had a radical mastectomy last year. She then went through the usual six-month course of chemotherapy. At the time of her surgery, physicians found 16 of her 22 lymph nodes involved with the cancer, making her a prime candidate for recurrence.
In such cases, a second, intensive round of chemotherapy is recommended. The way in which chemo or radiation therapies work is to kill cells that are actively reproducing. Cancer cells reproduce constantly – that’s what makes them so dangerous – so the therapy is effective against them. Most adult human cells do not reproduce most of the time, meaning they are unaffected.
Some, however, are affected. These include hair follicles and fingernails. More seriously, they also include blood cells, including platelets, the principal clotting agents in blood.
Without hair, people are bald. Without platelets, they are dead. The cancer therapies are often held at less-than-maximum levels to allow the blood to rebuild. This dose-limiting toxicity risks not killing the cancers. Pushing the dose risks killing the patient.
Platelets and other blood cells are replenished in time, but often slowly.
By Day Nine of the new round of therapy, Patient L required platelet transfusions, which, although expensive and sometimes ineffective, are the most frequently administered form of blood transfusion, according to the Puget Sound Blood Center.
By Day 12, Kaushansky feared Patient L’s platelets were not being replenished.
By Day 20, she was still requiring platelets.
On Day 25, Patient L had what she described as the worst headache of her life. Her brain was hemorrhaging.
By that point she had received 230 units of blood. It hadn’t worked. Patient L’s hemorrhage was discovered and her life saved, but at enormous physical cost to her and economic cost to the health-care system.
Kaushansky thought he saw in the French research a way to find the elusive platelet-producing hormone, thrombopoietin, and thereby a way to prevent Patient L’s pain, others’ deaths and the costs incurred by both.
He read with particular interest Wendling’s observation that the leukemia-inducing gene she had found was a mutation of a gene for a protein known as a receptor.
A receptor protrudes from a cell wall the way a dock juts from a sea wall. Like a dock, a receptor is designed as a berth. The boat it is designed to receive is a molecule called a ligand (meaning a thing that binds). The ligand floats through the body looking for the perfect receptor to lock onto.
Wendling’s receptor, which was called MPL, was empty. There was no boat present.
“Science sort of plods along from one conclusion to the next in a very logical way,” Kaushansky said. “Sometimes, when you’re lucky, on a good day, there’s a discovery made in one field, which has merit by itself in that field, which then can cross over to a completely unrelated field and just be the key that opens the door.”
Kaushansky noticed that the normal form of the mutated receptor Wendling described was found in a type of cell that could become either a red blood cell or a megakaryocyte, a large blood cell that breaks into platelets.
“And that’s the one and one I put together, and came up with the idea. It was my guess – and it was a guess – that this might have a role in platelet production. That was the leap of faith.”
TOWN AND GOWN
Kaushansky and Don Foster, the senior molecular biologist at ZymoGenetics, had been research fellows together a decade ago. Kaushansky had since consulted with ZymoGenetics. He went to Zymo with his belief that the MPL receptor might be used to fish for thrombopoietin.
One of the differences between academic research and that carried out in the biotech industry is illustrated by the offices Kaushansky and Foster occupy.
Foster basks in the glow of sunlight as it reflects off the water outside ZymoGenetics’ new headquarters, the smartly restored City Light steam plant on the south shore of Lake Union. In summer, the lake is dotted with kayakers and sailors. If Foster looks out on certain sunny days, he might see one of his own scientists, the lanky biochemist and consummate outdoorsman, Pieter Oort, skittering across the water on his sailboard.
On the pier Zymo built as part of the steam-plant restoration, other employees might be eating lunch or feeding the ducks.
Foster’s office itself is spartan, decorated in modern, muted grays, but spacious enough for a large desk, a small round table, new bookcases and a Macintosh computer.
Kaushansky’s UW office barely fits Kaushansky. It is a narrow shotgun shack compared to Foster’s mansion on the lake. It would make a nice closet. Its one advantage is that the similarly narrow Kaushansky can reach nearly everything in the room without getting out of his chair.
If Kaushansky looks out his window, he sees not windsurfers but other wings of the sprawling UW Medical Center, the house the late Sen. Warren Magnuson built.
The science labs at the Medical Center and hundreds of others scattered like federal crumbs across the campus are rich in many things – ideas, enthusiasm, even the occasional genius – but they are often lacking resources. Students come and go. Funding is even flightier.
The head of a university lab is its chief breadwinner. Its continued existence and thus the careers of the students in it depend on the lab chief’s ability to get money, usually in the form of government grants.
This search for funds is constant. If you ask an academic scientist what was the first thing he or she did after determining a new line of inquiry, or making a discovery, or getting up in the morning, the answer is likely to be: “I applied for a grant.”
“Your main responsibility as the head of an academic lab is as a procurer of money,” said one scientist. “It’s a myth that you have academic freedom. You can do science only if somebody will give you money to do science.”
This constraint limits the scale of academic research and is a powerful cause underlying the rise of biotechnology as a commercial enterprise.
Kaushansky has five scientists in his lab. ZymoGenetics has 200.
“Often the role of biotech companies is to fund projects that don’t get funded by the official sources,” Kaushansky said. “This (the thrombopoietin project) was my idea at the basic level but I also realized I wasn’t going to be able to carry it out. I have five other academic careers to worry about and I didn’t want to put them all into something that might not turn out.”
As big as ZymoGenetics is compared to Kaushansky’s lab, it is small to the point of obscurity among its biotech competitors. Even locally, the company is known, where it is known at all, for the mock smokestacks it was forced by historic preservationists to install atop the old steam plant.
Foster saw Kaushansky’s idea as a way to push Zymo into competition with the biotech giants known to be working in the same research area. The firms with well-known expertise in blood-growth proteins included cross-town rival Immunex as well as two of the biggest and richest biotech companies in the world – Amgen and Genentech, both in California.
There are well-worn methods for finding proteins. Typically, they require what might be characterized as brute force. Zymo wasn’t big enough to be a brute. To beat the others, Foster would have to find a way to sneak up on the discovery.
“The question is who can get there quickest. Who gets there first,” said Doug Williams of Immunex. “Because anything other than first is last.”
Si Lok, a molecular biologist at ZymoGenetics, had been involved in earlier races to clone proteins. Losing was not something he liked to contemplate.
“It’s embarrassing,” he said. “It’s like coming in second in a war.”
FINANCIAL ENGINEERING
Biotechnology has existed for millennia. Every time a baker takes a loaf of bread from the oven, he’s looking at it. Every time a brewer turns sugar into beer, she practices it. But modern biotech differs in a fundamental respect. Its tools, living organisms like the baker’s yeast, have been altered in the lab. They are genetically engineered.
The ability to alter fundamental genetic composition began to emerge with the discovery of the structure of deoxyribonucleic acid by Francis Crick and James Watson in 1953. As every schoolchild should know, DNA is composed of strings of nucleic acids. If unraveled, a single strand of human DNA would be 7 feet long. Each strand contains 3 billion nucleic acid pairs. Sections of these strands, varying in length from a few dozen “base pairs” to several thousand, form genes. The 100,000 or so human genes make up about 10 percent of the DNA. The rest is so-called junk, perhaps a vestige of earlier evolutionary needs. The genes and junk are laid out along 23 pairs of long molecules called chromosomes. Every cell has copies of each chromosome and hence each gene.
All genes have one function – dictating the production of proteins. Different proteins in turn carry out all the work of cells, and thus the body. Certain proteins, for example, make muscle. Another makes hair.
Twenty years after Watson and Crick’s work, scientists discovered individual genes could be combined with one another by cutting-and-pasting techniques that use enzymes as scissors and glue. This ability to combine genes, and thus the activities they dictate, is fundamentally what is meant by genetic engineering, or recombinant genetics.
Much that was written about these techniques in the first few years after their discovery concentrated on the mad-scientist aspects of it. Would new, somehow dangerous life forms escape from laboratory benches? Would they poison the air? Take over the earth?
Within a decade, these fears largely receded under an onslaught of publicity that promised miracles from the new technology. Food would be abundant. Hunger would disappear. New wonder drugs would be created. A cure for cancer was just around the corner. Amidst such hype, biotech emerged in the late 1970s. It immediately began fulfilling some of its wild promise.
It had long been known that a lack of the protein insulin caused diabetes. In 1978, human insulin was isolated and inserted into yeast cells. Yeast cells reproduce every few hours. If some other substance can be inserted into a yeast cell and grown with it, you can make a lot of whatever that something else is.
When Genentech, a South San Francisco company, proved this could be done with insulin and done cheaply, everything seemed suddenly possible.
Genentech had been founded with a $1,000 investment in 1976. The company sold stock to the public in 1980, two years before it had any products whatsoever. The stock opened at $35 a share. It went to $89 in an hour. The two men who put up the original $1,000 suddenly had stock worth $66 million.
An industry was born. And its two fundamental traits – astonishing science and equally astonishing finances – were joined.
THE SIZZLE
Ben Hall wasn’t present at the creation of the recombinant era, but he was close by. He had been working in the general area of DNA research for 20 years. A chemist by training, he and a student, Gustav Ammerer, had begun doing recombinant experiments at the University of Washington. In early 1980, they inserted one yeast gene into another.
“No one had done that before,” Hall recalled. “We thought, well, that second gene did not have to be a yeast gene.”
Lacking access to any human genes, Hall and Ammerer began a collaboration with Genentech, which had genes, and was looking for yeast expertise. The hot molecule of the early biotech era was interferon, touted to have potential for curing everything from cancer to infertility. Genentech wanted to produce it in yeast. Hall helped do it. Although successful, Hall gained little more than publicity from the venture. He and the company parted on less than friendly terms.
Hall had by then become chairman of the UW genetics department. By unpleasant coincidence, just as his relationship with Genentech soured, so did the prospects for his department. With Ronald Reagan marching triumphantly on Washington, D.C., and the similarly budget-minded John Spellman moving into the governor’s office in Olympia, talk of cost-cutting warmed the political air. Some of the hot air proved chilly by the time it blew into Hall’s genetics lab in the J-wing of the Med Center.
“Things were pretty grim on campus,” Hall recalled. “Deans were talking about doing away with entire programs. Things were going downward. I all of a sudden became aware of commercial interests in science.
“The joke at the time was that interferon was a substance you rubbed on stockbrokers to make them sell stock. But it seemed that we had the basis for a new biotech company.”
Hall had been working with Mike Smith, a biochemist at the University of British Columbia who had invented a method of synthesizing short strings of DNA. Because the acids that compose DNA bind molecularly to specific other acids, it is possible to use short synthetic chains as probes to search out complementary swatches in larger strings of DNA. Together, Hall and Smith used Smith’s handmade probes to isolate genes.
“Mike Smith then was one of three or four guys in North America who knew how to do this kind of chemistry,” Hall said. (This preeminence was confirmed spectacularly in 1993 when Smith won the Nobel Prize in chemistry.)
Hall called Smith.
“Around the late ‘70s and early ‘80s, all of us were getting phone calls from potential investors,” Smith said. “Most of them seemed to be coming from the eastern part of North America. Young people in dark suits, mostly from Wall Street. I was sort of interested. My whole career, or most of it, was in developing technologies that could be useful in an applied way.”
Hall also contacted Earl Davie, a UW biochemistry professor regarded as one of the fathers of modern hematology. His lab was doing groundbreaking research into the nature of blood proteins. ndependently, Smith and Davie had been talking with investors in North Carolina.
“When I told them it was possible to start a company here, they were interested,” Hall said. “It was a lot easier than flying to North Carolina all the time.”
The scientists thought they might combine Davie’s blood proteins, Smith’s chemistry and Hall’s yeast work into a commercial venture.
Intermediaries put the scientists in contact with Tom Cable, a young venture capitalist in Bellevue. Cable had been instrumental in getting several high-tech firms off the ground. Cable quickly raised half a million dollars in seed money. The money was pushed into the pot without the investors’ knowing what it would be spent on. It was invested “on the sizzle,” Cable said. “I raised all that money on the phone. Five or six phone calls. Basically, I said, `Hey, this company’s getting started with two scientific superstars at the university.’ That was it.
“We were betting on Ben Hall and Earl Davie. It was pretty basic. We hoped they would identify good ideas and good people. It was relatively typical of start-ups. The industry was really young. Nobody knew where it was going. Nobody knew how long it would take to get there. Nobody knew how much money it would take.”
WAREHOUSE DAYS
Mark Murray, a young molecular biologist, was doing postdoctoral research at the Massachusetts Institute of Technology’s Center for Cancer Research when he got a call from Hall and Davie. They were starting a new company that was going to produce proteins in yeast. Was he interested?
“The company was still just an idea when they called me,” Murray recalled. “I came out. They took me to a warehouse on north Lake Union, which they hadn’t even moved into yet.”
The company was to be called Zymos, the Greek word for yeast. The name was about as much of a business plan as existed. Murray accepted a job, then returned to Cambridge to finish his research there, pack and move. By the time he got back to Seattle, another 10 scientists had been hired. A president, somebody to actually run the company, had been hired, too. And fired.
“That was a little disconcerting,” Murray said.
It also turned out to be normal. For much of its early existence, Zymos had no management. None. As it was losing presidents, the company also lost its name. Mail kept getting sent to an electronics company called XMOS, so the word Genetics was tacked on.
“It’s not like this was done on purpose,” Murray said. “This was a series of screw-ups. Earl and Ben saw this simply as a way to do more science. They had no clue about how to build a business. They weren’t even thinking of that. It was just a group of guys in the clubhouse.”
Donna Prunkard, another of the early hires, remembers: “In the first few years we didn’t even have a structure. We had a president and we had us. And some of the time we didn’t even have a president.”
The science was entirely driven by the scientists. At the time, the bacterium E. coli was the industry-standard host cell for producing recombinant proteins. Unfortunately, E. coli did not do a very good job of making complex human molecules. The scientific basis for Zymo was to replace E. coli with yeast. The test case was a protein from Earl Davie’s UW lab called Factor IX. It didn’t work. The yeast couldn’t produce it.
“Right off the bat,” Murray said, “the whole basis for the company was blown out of the water.”
The company lived hand-to-mouth doing contract research, most of it for pharmaceutical companies, known within biotech as Big Pharma. Hall worried. He and Davie argued over direction.
Hall provoked a showdown. He and Davie each had a candidate for president. Hall called a meeting of the staff and insisted the scientists decide. Many felt backed into a corner and resented Hall for pushing them there.
“Ben forced us to choose,” Murray said. “Brought us all in there and made us vote. We resented even being put in that position. So we voted for Earl’s guy.”
Hall, the principal force behind its creation, quit the company. Within two years of its founding, Zymo had lost its founder, two presidents, its name and much of its reason for being. The company continued to struggle financially after Hall left.
Biotech financing rolls in and out every year or so with great waves of investor confidence. Zymo missed every wave. The company did develop some promising product candidates. One of its contract jobs – inventing a yeast-production system for insulin – grew from an idea sketched out over a restaurant dinner into a long-term relationship with Novo Nordisk, a large Danish pharmaceutical company.
In 1988, Zymo was still floundering financially; the previous autumn’s stock-market crash had wiped out a plan to sell stock to the public. The Novo contract called for royalty payments to Zymo if it hit specified targets. A big payment was due in that year – big enough, Mike Smith said, that it became cheaper for Novo to buy the whole company. Zymo was willing.
Smith, Davie and the other original investors sold for $23 million. Zymo has since evolved into a principal Novo research center.
RUSH HOUR
For most of human history, the function of the human circulatory system was a mystery. The predominant view held that blood’s main function was to keep the body warm. William Harvey discovered in the 17th century that blood serves mainly as a transportation system of considerable complexity. The bloodstream might be imagined as a particularly crowded highway: Interstate 5, say, at rush hour on a rainy winter night.
A single drop of blood contains more than a quarter of a billion individual cells. Almost half of those cells are blood cells: red, oxygen-bearing cells; white, immune-system cells; and the tiny platelets that cause clots to form at the site of injuries.
The rest of blood is a swamp of vitamins, sugars, salts – almost everything the body needs, including the class of molecules called hormones.
The general role of hormones is to control other cells or systems in the body. They are related to one another more by function than composition. They might be proteins, amino acids or cholesterol derivatives, such as steroids. Typically, they are produced in one part of the body and travel through the blood to the cells they are designed to control.
The blood is full of these hormones and other proteins commuting to work at various job sites throughout the body. Being of often great potency, hormones make inviting targets for research. They have been a principal focus of the biotech industry since its inception.
A small, especially powerful subclass of hormones called cytokines began to emerge as particular objects of interest in the 1980s. Cytokines typically do not travel through the blood in great numbers. Most of them are made in a particular area – the bone marrow, for example – and travel only to neighboring cells to do whatever they do. Think of them as hormones that walk to work. Because they do not move around much, cytokines are hard to find if you don’t happen to know where they’re made.
Red and white blood cells and platelets all derive from identical so-called stem cells. A stem cell will become red, white or platelet depending on what hormones hit it. Over time, scientists had discovered, isolated and turned into drugs the hormones, or cytokines, that make stem cells become red cells and white cells. They had not found thrombopoietin, the hormone that was supposed to make platelets. Different people around the world looked for it for so long, in so many different ways, that many leaders in the field had concluded it didn’t exist. Thrombopoietin came to assume a kind of comic-mythic status.
No one looked longer or harder than Ted McDonald, a biochemist at the University of Tennessee. McDonald has published more than 300 papers in an academic career that dates to 1958. All but three of them concerned TPO.
“People laughed at me,” McDonald said. “We ran out of money. I couldn’t get enough, ever. I lost my grants. I couldn’t even get a postdoc to work with me. People would tell me, if this doesn’t work, you’ve shot your whole career.”
McDonald is now 64. The search, in fact, did consume his career, or, as he said recently, “99.9 percent of it.” After a moment’s pause, McDonald, thinking back on those 35 years, added:
“The other tenth of a percent was breathing.”
Kaushansky, the UW hematologist, knew the difficulties other researchers had encountered. He knew he was asking ZymoGenetics to take a gamble when he showed up with a new plan to find TPO.
As it happened, the company already had a search under way. One of Kaushansky’s UW colleagues, Gerry Roth, had been a constant source of new ways to look for TPO. He himself had been searching since 1987.
“We thought of maybe a hundred different ways to fail,” Roth said.
A small group met every couple of weeks to talk about TPO. “I used to laugh, saying I was going to my TPO meeting. It was kind of an embarrassment,” Roth said.
BAD BLOOD
The best way to find a hormone is to know where it’s made. Erythropoietin, the red-blood-cell hormone, is made in the kidney. This knowledge helped give scientists a place to look for it: urine. Amgen, George Rathmann’s old company, purified and cloned EPO in the mid-1980s. The project established the company and has paid for a sprawling campus of labs, offices and production facilities in suburban Los Angeles.
Finding it, however, was as much luck as it was scientific brilliance, said Fu Kuen Lin, the molecular biologist who discovered it. When Lin joined Amgen in 1981, the company had a single lab bench and a list of potential projects ranging from EPO to indigo dye for blue jeans.
Lin chose EPO. Who knows what blue jeans might be like today if he had picked indigo. Molecular biology was still in its infancy then, and Lin was making up much of what he did as he went along.
“Management tried to help,” Lin said. “They were running all over the world trying to line up a source of EPO. At one point Dow Chemical came to us. They said they had this urine source – the Italian army. They were collecting all the Italian army urine and they were concentrated on purifying EPO out of it.”
As Kaushansky says, “Not a pleasant task, but simpler than blood.”
Protein-purification experts regard blood as a singularly bad starting material. “There’s a lot of stuff you have to get rid of,” said Roth.
In any chemical purification process, half of the material that is the object of the purification is lost in each step, and successful purification often takes a dozen steps. This was, nonetheless, the main path by which people looked for TPO.
Zymo was working on purification when the crucial Wendling papers were published. When Kaushansky made Zymo aware of the new receptor research, Foster decided it was worth a renewed effort.
“The meetings started with two or three people,” Roth said.
“Then they increased to four or five. Then after a while I didn’t even recognize half the people walking into the room.”
The expertise in Foster’s lab is molecular biology, not protein chemistry. Foster adopted a search suited to the talents at hand. Zymo would try to find TPO not by purifying blood, as Foster suspected Amgen and Genentech would, but by a method called expression cloning. Rather than trying to isolate the material and see if it would cause the activity, Zymo would look for the activity, then try to isolate the material.
It is as if you were trying to find what kind of people scream when they hear Eddie Vedder sing. Say you suspected that teenagers wearing socks with very subtle polka dots and living in Tukwila were the only people who did this. If you were a protein chemist you would round up all the people in Tukwila and sort them, first getting rid of all the adults, then the children, then those not wearing socks, then those wearing solid-color socks, and so on until you found a 14-year-old wearing what looked like polka-dot socks. Then you’d play a Pearl Jam song and see what happened.
This could take a lot of people a very long time, and the kid might not scream. Maybe the socks were just dirty and not polka dot after all.
“With protein chemistry, if it works it always requires brute force,” Foster said. “The beauty of the molecular biological approach is if it works it doesn’t ever require brute force.”
If you were a molecular biologist, you would get in your car, pop a Pearl Jam tape into the cassette player and drive around Tukwila high schools with the stereo cranked up as high as it goes. If somebody screamed, you’d stop and see what color socks he had on.
THE LOTTERY
Andrzej Sledziewski, Zymo’s vice president for bio-pharmaceutics, subscribes to a description of the company derived from “One Flew Over the Cuckoo’s Nest.” It is called The Theory of the Staff and the Patients.
“No one knows who is which,” he said.
“Him. See him,” he said one day this fall, pointing to one of his top scientists. “He thinks he’s staff.”
Sledziewski shook his head. “Uh-uh. He’s a patient.”
When Foster set out to build his TPO team, he went straight for the patients. The first person was Si Lok, a cloning expert who had recently joined the lab. Lok is by all accounts the quirkiest scientist in the Zymo asylum. He cultivates some of this image, wearing what are alleged to be different, specified pairs of jeans for each day of the week. He has never been seen in an unwrinkled shirt.
According to company legend, when Lok first arrived at Zymo, he didn’t own a car or know how to drive one. But after a problem on the bus one night, he went out the next morning, bought a car and taught himself to drive it on the way to work. It took 45 minutes to cover a couple of miles, largely because he searched diligently for a route that included no hills or left turns.
Lok is the only Zymo scientist not tied into the company’s electronic-mail system.
“If something is important enough, they’ll come and tell me. If it’s not, I don’t want to hear about it anyway,” he said.
This self-confidence comes across as charming, not off-putting. Another scientist has labeled a project folder in his computer “IwannabelikeSi.” People happily put up with Lok’s idiosyncrasies because he is very good at what he does.
“When we got the information on the (Wendling) receptor, we asked Si to clone it, just to position us in case we wanted to try something with it,” Foster said.
As Zymo scientists began investigating TPO, they found more and more dead ends.
“Everybody worked in the lab,” Foster said. “We would meet weekly and go through our data. Discuss it. We were just getting together and sharing results, which were all bad.”
Foster is not apt to pursue ill-fated ventures simply because he starts them. His steadfast goal from the time he was a child was to become a physician. After receiving his undergraduate degree, he enrolled in medical school. He hated it, immediately and passionately.
“I woke up every morning and didn’t want the day to be,” he said. He quit school after a month.
This same willingness to make decisions, to go straight to the heart of a problem, is one of Foster’s best qualities as a scientist, according to John Forstrom, his boss.
“Essentially, I decided – with some support and some opposition – that if the other things weren’t working and if the MPL receptor was the right thing, other companies would be working on it. If this is the right thing, we better put a massive effort into it,” Foster said.
All other TPO projects were shut down. Eventually, all other molecular biology projects were shut down.
Once Lok and Joe Kuijper cloned the MPL receptor, Pieter Oort spliced it into a line of cells whose chief quality was that they thrived when fed blood-cell growth hormones; they died without the hormones. If all other hormones were taken away, only those cells that were receiving TPO would stay alive. It was a classic biological solution: The cells would select for life.
This gave Zymo a quick lab test, called an assay, for the existence of TPO. It would work like this:
The ligand half of a receptor-ligand pair is often a hormone. The receptor is on the cell surface and acts as a communication link between the inside of the cell and its surroundings. When the hormone bumps into its receptor, it adheres “like hair sticking to a comb,” Kaushansky said. This initiates some prescribed action inside the cell on which the receptor sits: for example, making another protein, or dividing, or growing; doing, in short, any of the things cells do. Nobody knows why this happens. It’s what cells do, “a black box,” Foster said.
Zymo had the receptor. The question was where to find the hormone that would bind to it and stimulate the cell. Where might TPO be made? What tissue sources should the assay be exposed to?
Blood is a homostatic system; it’s self-balancing. When it has too much of something, it sheds it. When it has too little, it makes more.
When platelets, for example, are low, hormones are called forth to make more. Theoretically, then, killing platelets should stimulate the production of TPO, which would go to work replacing the platelets. Laboratory mice were injected with platelet antibodies, killing their platelets and presumably causing TPO to go to work. The mice were then killed – sacrificed, in the industry vernacular – and from each mouse four organs were “harvested.” There was some reason to suspect any of the four organs – the kidney, liver, spleen and bone marrow – might be the place TPO was made. There was also reason to suspect none of them was the place.
Lok and Kuijper built what are called libraries of DNA samples from the four different mice organs.
The basic plan was to extract the DNA from the library samples, then combine it, one bit at a time, with the receptor cells. Theoretically, if any of the DNA samples contained the TPO gene, the gene would make the hormone, the hormone would bind to the receptor and the receptor cells would live. Otherwise they would die.
The logic of the approach was probabilistic. If you had enough DNA samples of each tissue, you would have at least one copy of every gene in the tissue. If you took enough samples, in fact, you couldn’t miss.
The literature contained numerous examples of similar approaches being used for other discovery efforts. Because TPO was thought to be especially rare, Zymo made more samples than anyone thought would be necessary – more, they thought, than anyone in history had ever made, enough samples that if one in 100,000 molecules was TPO, they would find it.
As Lok said, “It was a lottery. The more tickets you bought, the better chance you had.”
CHIMP WORK
Molecular biology is the chemistry of the very small. Many of its tools are those a 19th-century chemist might recognize. The most ubiquitous device in a modern DNA lab is nothing as sophisticated as, say, an electron microscope. It’s a long suction needle called a pipette.
Even many of the most powerful devices are not technologically complicated. Machines called polymerase chain reactors are capable of multiplying – or amplifying, as it’s called – DNA from a single molecule to more than a billion identical copies in a day. Yet a PCR machine is little more than a heat block.
Most lab work is craft, not genius. “It’s not so complicated,” Sledziewski said. “You get some of this stuff and put it together with some of that stuff and see what happens.”
“No one enjoys working on a bench,” Lok said. “Anyone who tells you they do is either lying or they mean something else. Surely, no one can actually enjoy pipetting.”
Chimp work, Anne Bell calls it. Bell led Zymo’s massive effort to prepare the TPO DNA samples, which are called “mini-preps” because a small amount of DNA is extracted from each sample. It’s a time-consuming but necessarily exacting task. Like much DNA lab work, mini-preps require precise repetition of mundane tasks: combining, then separating various compounds.
Bell, Foster, Lok and almost everyone else involved in the TPO project belong to a generation of scientists who were in college during the 1970s and were seduced by the promise of biotech, which was just then being born.
Everybody in the industry, Foster said, is “within one or two standard deviations of 40.”
Bell had planned on being a physical therapist.
“I loved the course work, but I hated the idea of helping little old ladies up and down the hall,” she said. In her junior year at Syracuse University, she took a class on restriction enzymes. It was brand-new science, “really cool,” she recalled. The thought that you could “take a cell from a frog’s toe and turn it into a frog” changed her life.
She took a job in an academic lab and eventually made her way west. She arrived in Seattle on a typically depressing December day in 1984.
“It was cool, dark, raining. I said, `Wow. This is great.’ “ She’s been at Zymo since.
Biologists tend to divide along the lines of the cells they work with. It will be said of someone that she is a bacteria person. Or a mammalian. Bell is a yeast person. “They’ll do amazing things for you,” she said.
Bell rolled her eyes when it was suggested that Foster thought she had “volunteered” to lead the mini-prep effort.
Given the competition Zymo thought it was racing against, speed was a paramount consideration in the TPO project and the DNA prep work was the biggest roadblock.
“We were all trying to duck it. It sounded like your worst nightmare come true,” she said. “Preps are one of the first things you learn in the lab. It’s difficult to be motivated to do a lot of them. It is not creative at all. The goal was to do 5,000 of them, which everybody regarded as crazy. A crazy mini-prep day was 50. At one point on this project we did 824.”
In part because of the effort involved, in part because of Bell’s personality – she wears her emotions on her sleeve; some days the sleeve gets a pretty good workout – the mini-prepping became the visible symbol of the project within the company. People passing in the hall would be pulled in to help with the most mundane tasks.
The basic process involved separating the DNA from the source tissues and inserting it into E. coli bacterium cells. The cells, each containing a single inserted gene, were grown, producing at least 600,000 copies, or clones, of the DNA from each of the four mouse tissues.
Then the clones were grown in groups of approximately 250 colonies each. The colonies were scraped off and a series of chemical treatments was used to break up the cells and release the DNA. A centrifuge removed the cell debris. The DNA was then bound to a silica-type material to extract it from the liquids. Then the silica was washed and treated, freeing the naked DNA.
“The pace was feverish,” Bell said. “I’ve never been so tired in my life. . . . As soon as we reached one goal, there’d be another.”
As if she needed further motivation, as Bell got deeper into the TPO project, her father became ill. His blood had quit making platelets. He was dying.
IN THE DARK
Different cells have different properties that make them easier or harder to deal with in different circumstances. Thousands of different cell lines are available commercially. They’re advertised in thick catalogs and range from tongue, bullfrog, to lung, buffalo.
After the naked DNA was extracted from the E. coli cells, it was inserted into hamster kidney cells, which are especially hospitable for growing foreign proteins. Proteins grown this way are secreted from the cells into the liquid medium the cells are in.
If the cells were producing TPO, it would be in the liquid.
That liquid was combined with the receptor cells and tested. Almost all of the tests, or assays, were done by one person, Pieter Oort.
Oort was born to science. His grandfather discovered the Oort asteroid belt. His father was a research meteorologist.
“He would come home, have dinner, then go to his study and work until late at night. On Saturdays, he’d go into the office. On Sundays, he’d work at home,” Oort said.
By his nature, Oort is a loner, almost an outsider in the lab.
This was emphasized during the TPO project by the physical layout of the work. While most of the rest of the molecular biology group was making the libraries and doing mini-preps in the main lab, Oort worked by himself in a separate tissue-culture lab.
Tissue culture, as its name implies, involves the growing of living cells. Because of the temperamental nature of most live things, culture work requires persistent attention. There is a care-giving quality to it. Mini-prepping is cooking. Tissue culture is baby-sitting. The cells can’t be left alone.
As the TPO project progressed, Oort began to take on his father’s work habits. Hardly a day went by that he stayed away from the lab. More precisely, hardly a night went by. Oort became nocturnal. He was seldom seen during the day; almost never out of the lab at night.
The mini-prepping was the most labor-intensive work and as such governed the pace of the project. The assay was the very end. It was where you would find, or not find, what you were looking for. It was where the pressure built.
There is in the lobby of Zymo’s offices a mixed-media piece of art called “The Ambiguity of Negation.” The scientists, who are largely pragmatic people not much into what Zymo chemist John Forstrom calls “brain-farting,” do not care much for this piece, or for any of the other abstractions collected by the company’s president, Bruce Carter, and scattered through their building.
But ambiguity and negation are woven into their work. Much of science is finding things that aren’t, doing what are called null experiments. You don’t win Nobel Prizes doing null experiments, but you learn from them. The problem is, you can never be certain exactly what.
“The good thing about this job is you’re always learning,” Bell said. “That keeps it exciting. It also makes it heartbreaking. Most of the excitement is over failure. Most things don’t work.”
Oort’s solitary habits, combined with what he was finding, which is to say, nothing, put enormous pressure on him. Nobody blamed Oort outright – he was doing what he was asked to do – but the failure to get results led some people to wonder about the quality of the assay. Oort was confident the assay worked, but without the thing it was meant to test, TPO, there was no way to test the assay itself.
The assays were read by computer. The results were data plots. Negative results would have one general shape. Positive results supposedly another. People began to wonder how much difference there would be between them. The least variation in the data was scrutinized over and over, trying to make something pop out of the background. This went on and on and yielded nothing.
Different twists were attempted. New targets were set for the mini-prep group, buying more tickets in the lottery. A total of more than 12,000 were done. At one point, the manufacturer of one of the chemicals used in the lab changed its formula unannounced. More than 4,000 preps had to be thrown out.
WEEDS
Zymo scientists assumed they were playing in a bigger league than they were used to. If we were smart enough to see the Wendling stuff, so were the big guys, is the way several people put it. But beyond assumption, rumor, and hints, there was nothing anyone could do to find out.
Foster is usually upbeat. When some project or another is described to him, his typical response is to say something the equivalent of, “No problem.”
As in: “Don, we’d like your people to find a cure for cancer.”
“Piece of cake,” he’ll say. “Do it in a day.”
Some of the scientists in his lab call such Pollyanna-ish appraisals Fosterisms. As it happens, they have the wholly unanticipated effect of making some people work very hard to make them come true.
TPO was at the other end of scientific feasibility from a piece of cake. The project, Foster said, began as a gamble and sank into a “long trough of discouragement.”
Most people in science would not be there, however, if they were pessimists. Every time the lab embarked on a new tissue source, optimism would rise. One night, Oort got what looked like a positive result on one of the assays. But when he looked closer at the culture dish, he realized it had been contaminated. The contamination was the cause of the spike. He left the data on his desk and when people noticed it the next morning, they started celebrating.
“By the time I came in they were all jumping up and down,” he said.
The frustration went on for weeks, until one night they hit a spike in the data that couldn’t be explained away. They had it.
No, they didn’t. The spike was caused by a growth hormone, but not the one they were looking for. This one, interleukin-3, had been discovered years earlier.
They discovered IL-3 several more times in the course of not discovering TPO. They came to think of it as a weed. People by this point were not inclined to find humor in their annoyance at repeated discovery of something that would have set off rockets a decade before.
Lok claims never to have lost confidence.
“You have to think the thing will work,” he said. “I just accept that as a foregone conclusion.”
One of the scientists involved early in the TPO project was Rick Holly. Holly is unusual in the industry in that he has risen to a senior scientific position without a doctoral degree, which at many companies is a requirement. Foster said Holly succeeds on sheer brilliance. He consistently has insights others regard as strokes of genius.
Holly isn’t so sure. He once wrote in his annual performance review that he was lazy and ignorant. As a consequence of these failings, he said, he tried to take shortcuts. Sometimes they worked.
Foster had asked Holly to perform a routine chore in the TPO project. He, Mark Heipel and Steve Burkhead were responsible for injecting the DNA clones into the hamster cells before the assay process.
It’s “drudgery,” Foster said. As was his habit, Holly began fooling around.
He saw two obvious ways the project could fail: One, the receptor might not be what they thought it was and so wouldn’t bind to the ligand they were looking for; or, two, they might be looking in the wrong place for the ligand.
“We had no way of knowing where it was going to be. We didn’t know which tissue to use. I started working on that aspect of it, trying to find a way around it.”
Holly decided if he couldn’t figure out where the ligand was made, he would try to make it somewhere.
All cells have copies of all genes. What a cell does depends on which genes are turned on. TPO would be made only in the cells with TPO genes activated.
One of the things that happen when cells are mutated is different sets of genes are switched on and off. The classic causes of mutations include viruses and chemicals. Holly began experimenting with chemicals.
“He does this all the time,” Foster said. “Every project he’s on. He goes off and plays.”
With Burkhead, Holly exposed some cells to a chemical that causes random mutations. If you exposed enough cells to the mutating agent, some of them might have their TPO genes turned on, he thought.
“It seemed theoretically this should work,” Holly said. “It was sort of like applying the most basic genetic idea.”
Holly and Burkhead played with the idea. Once it seemed like it should work, Holly told Foster. A new experiment was approved. Ten million cells were doused with the chemical mutagen. Cells in 24 of the pools stayed alive.
Nineteen pools produced something. Fourteen produced a hormone. One produced a hormone they couldn’t identify.
“Almost everything we do all day is working with apparently empty test tubes,” Foster said. When the cells started producing the unidentified hormone, Holly said, “within an hour you could see the cells. You could see them stretching and breaking.”
After that, isolating the gene for the hormone was straightforward. A new library of all the DNA in the pool producing the unidentified hormone was prepared. The DNA clones were again broken into 2,000 groups of 250, inserted into the hamster cells and assayed. The ligand hit twice. It had come home and Zymo had it.
They had TPO.
BURNING UP
The stakes in biotechnology are high. Said the UW’s Roth: “This stuff works. It actually helps people. It’s not just a game to see who can find it first.”
A U.S. government report recently concluded: “Genetic engineering is to modern humans what the discovery of fire was to early cave-dwellers.”
Perhaps, but biotech at this point is setting fire to itself as often as it is being warmed by the flames. The key index in most commercial undertakings is revenue. Sales, in other words. In biotech, the key index is called the burn rate, which is a measure of how quickly money is spent. That it is said to “burn” is an indication of just how fast that can be.
There are now more than 1,300 U.S. biotech companies. There are more than 100 in Washington state alone. An overwhelming majority of the 1,300 have no products. They have not been able – yet – to find or make or sell whatever it is they set out to find or make or sell. Some of them have worked for a decade without making a product.
The majority of biotech companies try to identify naturally occurring, medically useful human molecules, isolate and reproduce them, a process called cloning, then develop them into drugs. This is a time- and money-consuming enterprise. The great promise of biotech was it would nonetheless be much faster at this than traditional pharmaceutical companies.
Big Pharma was scorned by biotech pioneers as slow and clumsy.
Its traditional way of finding new drugs is to collect samples of microbes, sprinkle them on tissues and see what happens. The lack of elegance, the sheer randomness in this, compared to rationally isolating genes in a lab and reproducing them, led many people to think that biotech would somehow be easy.
That it is not has been a surprise, often an unpleasant one, especially to the people who invest in the industry.
The reasons for this are many, but in most cases boil down to two simple facts:
The human body is complex and little understood. Biotechnology has developed a set of techniques that make finding genetic sources of action in the body, if not exactly easy, then doable. Understanding the action is altogether another matter.
The case of TPO is unusual in that its biology was suspected to begin with. More typically, scientists will discover a gene and then try to figure out what it does. The only way to definitively do that is to remove the gene and see what the organism does differently.
Lee Hartwell, a UW geneticist, likens this to examining a factory with 70,000 employees, and – solely by looking at the factory’s output – trying to figure out who didn’t come to work that day.
THE NEXT ROUND
Patrick O’Hara, the head of Zymo’s computational biology lab, said later of Rick Holly’s experiment, “It was one of the most brilliant things I’ve seen here at Zymo.”
That was the predominant view within the company. Outside, some people thought Zymo had gotten really lucky. After all, one cell out of 10 million? Couldn’t they have as easily missed it?
“We could do 10 million cells a week. We would have just kept going,” Foster said. Nobody disagreed there was luck involved. Lok called it “truth, beauty and chance.” Kuijper called it “serendipity.”
Whatever the reason, Zymo had found the hormone and now had to confirm it did, indeed, create platelets.
Once a substance is isolated, it is relatively easy to grow more of it. Since the discovery, Zymo has made more TPO than had existed in nature in all of human history.
After the first small amount of it had been made, Zymo injected it into mice. Within seven days, their platelet counts quadrupled. TPO worked.
“Usually, with the initial results you sort of scratch your head, `What’s this mean?’ This was not subtle,” said Kaushansky.
ZymoGenetics published news of its discovery last June in the prestigious journal Nature. The same issue carried a paper by Genentech scientists announcing they, too, had found TPO. Shortly thereafter, Amgen and the Japanese company Kirin announced similar discoveries.
Amgen and Genentech had used Wendling’s receptor to help purify the hormone from blood. They had glued the receptor chemically to inert material in a glass column, then poured blood – lots of blood, from rats and dogs – through the column. Eventually, a bit of the hormone stuck to the receptor.
Kirin, to everyone’s utter astonishment, found the hormone by traditional chemical purification.
No one knows who was first. They have all filed patent claims and are racing to develop the hormone into a drug. The stakes are large. Often after a discovery, a company will ensure it has a secure patent before spending money on development. The TPO competition has undone the process. The company that gets a drug to market first could make billions whether a patent has been issued or not. Asked about the sequence of development and patenting, Foster said:
“There is no sequence. The race is so intense nobody’s willing to back off to see what the resolution of the patent fight is. We’re just plowing ahead.”
Decisions have been made. More millions of dollars are being spent. Some people expect the development to go much quicker than usual and clinical tests to begin sometime next year. Others aren’t so sure. By some counts, 99 percent of biotech discoveries never make it to market. Doug Williams of Immunex cautions that “everything looks good and sounds good when it gets cloned,” but most clones don’t become human drugs.
George Rathmann, the Amgen founder now running Bothell-based Icos, with no more effort than peeling playing cards off a deck, can list dozens of ways a drug-development effort can fail.
Zymo isn’t yet halfway through the deck.