Hemophiliacs lack a necessary blood clotting factor called Factor VIII. In the first half of the twentieth century, whole blood transfusions were the only treatment for the disease. In the 1950s, blood plasma products containing Factor VIII became available. When the AIDS epidemic began in 1981, the blood supply became contaminated with HIV. Thousands of hemophiliacs were infected. Two early biotechnology companies, Genentech and Genetics Institute, Inc. raced to manufacture a safe recombinant version of the vital clotting factor.
Hemophilia is an inherited blood disorder that impairs the body’s ability to coagulate blood. The primary danger is internal bleeding, which can result in swelling, joint damage, disfigurement, and death. Symptoms range from mild to severe.
For centuries, the cause of the disease remained a medical mystery. Scottish physician Thomas Addis established the basis for modern understandings in 1911. He reasoned that since blood transfusions were therapeutic, the condition stemmed from a deficiency, the lack of a ‘factor’ that he called anti-hemophilic factor (AHF).
Two types of hemophilia are now known, A and B. Both result from the absence of a specific clotting factor in the blood. Hemophilia A, a deficiency of functional Factor VIII, occurs in about one in every 10,000 male births. Hemophilia B is due to a lack of Factor IX, present in around one in every 25,000 male births. An estimated 500,000 people suffer from hemophilia. Approximately 20,000 reside in the United States.
Hemophilias A and B are X-linked recessive genetic disorders caused by mutations in the genes that code for Factors VIII and IX. Both genes are located on the X chromosome. Hemophilia generally occurs in males because males possess only one X chromosome. If a male child inherits a defective gene, the disease manifests. It is possible but rare for hemophilia to be passed down to females (when the father is a hemophiliac and the mother is a carrier), or to occur as the result of a spontaneous mutation.
In the first half of the 20th century, hemophilia was treated with whole blood transfusions. The life expectancy of patients was about twenty-seven years. In the 1950s, doctors began treating the condition with fresh frozen plasma (FFP). Concentrations of clotting factors remained low, so FFP was also administered intravenously in hospitals, in high volume transfusions.
In the 1960s, Stanford scientist Judith Pool discovered a method for making FFP concentrates. When FFP is thawed in cold rooms, a blood factor-enriched cryoprecipitate forms. By the early 1970s, scientists had developed methods to calibrate doses, which were then packaged and sold in labeled bottles. Hemophiliacs were able for the first time to manage the condition themselves with injections. Life expectancy was extended to forty years of age. By 1980, the figure reached sixty years, but this string of spectacular improvements in the life chances of hemophiliacs was about to be erased.
Hemophilia — The Royal Disease
Hemophilia was known through much of the nineteenth and twentieth centuries as the ‘Royal Disease’ because of its prevalence in the ruling houses of Europe. The type was hemophilia B, a deficiency of Factor IX. Geneticists suspect that the original carrier was Queen Victoria, the monarch of Great Britain for sixty-three years, from 1837 to 1901, likely due to a spontaneous mutation. The disorder is apparently extinct in contemporary royal bloodlines.
The first cases of the infectious disease that came to be known as AIDS were identified in 1981, in New York and Los Angeles. Public health officials soon recognized that homosexuals and intravenous drug users in these cities and others were presenting with multiple devastating and unexplained infections, and that diagnoses were accumulating at an alarming rate. The following year, the US Centers for Disease Control (CDC) counted thousands who were affected. Hundreds had already died.
The cause and mode of transmission were mysterious. The uncertainty fueled public fears. If the pathogen was airborne, then all were at risk. Some healthcare workers refused to attend to victims of the disease. The initial reaction of many groups and individuals was to blame and shun victims. The malady was widely stigmatized as a ‘gay disease’ and a ‘drug-users disease,’ the result of lifestyle choices for which patients were responsible. The scientific community suspected a virus. A broad search was undertaken by epidemiologists and virologists.
In July 1982, the CDC reported the first diagnosed cases in hemophiliacs. Mounting evidence suggested that the pathogen was transmitted by blood. In January 1983, the American Red Cross, the American Association of Blood Banks, and the Council of Community Blood Banks issued a joint statement that acknowledged the hazard and advised member organizations against accepting donations from individuals belonging to high-risk groups.
Mandatory screening of donated blood for the presence of hepatitis B had been instituted in 1971, but patients remained at high risk of contracting a ‘non-A, non-B’ hepatitis (known today as type C). Since transfusions are life-saving measures and there was uncertainty in the 1970s and early 1980s about the course and severity of non-A, non-B infections, doctors generally judged the risk acceptable. With the emergence of the AIDS epidemic, however, this risk-benefit calculus was radically altered.
The immediate response of the gay community to disease, death, and discrimination was to organize. The first advocacy groups — Gay Men’s Health Crisis, People Living with AIDS, and the National Association of People with AIDS — appeared in 1982 and 1983. They staged highly visible protests against ‘the biomedical establishment’ — public health officials, the scientific community, pharmaceutical companies, and the FDA.
The activists complained that responses to the epidemic were slow and inadequate. They demanded increased federal support for AIDS research, broad access to experimental therapies, accelerated regulatory approvals, reduced drug prices, and opportunities to participate directly in health policy decision-making. They challenged the conflation of medical and moral agendas in public discourse, and railed against figures such as Christian evangelist Dr. Jerry Falwell who expressed the opinion that AIDS represented “the wrath of a just God against homosexuals.”
In contrast, most hemophiliacs with AIDS suffered in silence. To avoid the stigma associated with the disease, many attempted to conceal the diagnosis, but as the pathogen and its mode of transmission became known, their predicament became apparent. Due to their dependence on the blood supply, hemophiliacs were hit harder by HIV/AIDS than any other group. In addition, spouses of hemophiliacs were exposed to HIV through sexual intercourse, and the virus was passed on to many infants of infected parents. Alan Brownstein, director of the American Hemophilia Foundation, called the tragedy “staggering in its magnitude.”
Hemophiliac Ryan White contracted AIDS as a child from a blood transfusion. When his condition became known in his hometown of Kokomo, Indiana in 1985, concerned parents and teachers objected to his presence at school. Public health officials insisted that the risk of transmission was virtually non-existent, but the local school board voted to bar White from attending classes.
A legal battle ensued. The case received widespread publicity. Celebrities flocked to Indiana to support White and champion AIDS awareness. The Indiana Department of Education intervened and permitted White to return to school. Opponents secured a temporary restraining order, but a Federal Circuit Court judge overturned the ban and the Court of Appeals refused to hear further arguments.
The case moved the plight of hemophiliacs into the national spotlight. White became a symbol of the universality of AIDS, the personification of efforts to combat ignorance and fear and promote decency and compassion.
Ryan White passed away in April 1990 at the age of eighteen. In August, Congress enacted the Ryan White Comprehensive AIDS Resources Emergency (CARE) Act, which provided federal funds for the care and treatment of people living with AIDS.
A RECOMBINANT SOLUTION?
The AIDS epidemic was an important backdrop for the emergence of the commercial biotechnology industry. In the late 1970s and early 1980s, new recombinant DNA companies surveyed the commercial landscape in pharmaceuticals and drew similar conclusions regarding prospects and opportunities — the technology could revolutionize the production of therapeutic proteins. Two firms, Genentech of South San Francisco, California, and Genetics Institute, Inc. (GI) of Cambridge, Massachusetts, selected Factor VIII as a target since the plasma concentration process was crude and expensive, clotting factors were in short supply, and hemophiliacs were contracting hepatitis (and would soon be beset by HIV).
Genentech and Genetics Institute had assembled some of the world’s best gene cloners, along with top protein and nucleic acid chemists. Many of the scientists knew each other personally. Most had been recruited from the higher circles of molecular biology — a relatively closed guild of gene workers with affiliations at a limited number of leading academic institutions, places such as Cold Spring Harbor Laboratory, Caltech, Harvard, MIT, Stanford, the University of California, San Francisco, the University of Washington, and a handful of others.
Genetics Institute, Inc. was founded in 1980 by Harvard molecular biologists Mark Ptashne and Tom Maniatis. With help from investor Henry McCance of the venture capital firm Greylock Partners, they recruited Gabe Schmergel, an experienced general manager in the healthcare industry, to direct the organization as CEO. Schmergel came from Baxter International, a fifty year-old public corporation headquartered in the Chicago suburbs that specialized in blood products, including plasma concentrates. He knew the market well and saw that recombinant Factor VIII (rFVIII) would transform it.
Schmergel also knew that Genentech was interested in Factor VIII. Genentech was the original recombinant DNA company, founded in 1976. Its first commercial project was the manufacture of recombinant human insulin. That work was underwritten by Eli Lilly & Company. Sometime after, the California cloners approached Baxter to propose a partnership for the development and marketing of a genetically engineered version of Factor VIII. Schmergel was not involved in the talks, but knew of the big company’s response: “Baxter said it was blue sky and would never work.” Genentech set the project temporarily aside.
Eventually, Genentech’s grand success changed perceptions of the technology and its practical import. In 1979, the company announced that it had cloned the gene for human growth hormone. In the fall of 1980, the firm made a hugely successful public offering of stock. In 1982, as Lilly was preparing to introduce Genentech’s human insulin to the marketplace as the world’s first recombinant biotherapeutic product, Schmergel was able to persuade former colleagues at Baxter that the Factor VIII project wasn’t impossible after all. He traded marketing rights on an initial product to Baxter’s Hyland Laboratories Division in exchange for R&D support, royalties, and a contract to manufacture the protein.
When GI elected to pursue Factor VIII, Genentech quickly followed suit. David Goeddel, Genentech’s chief scientist, remembers that Bob Swanson, the company’s co-founder and CEO, was determined not to surrender the commercial prize to a competitor: “There were rumors that Genetics Institute had something on Factor VIII, so Swanson wanted me to work on it.”
As research at the two companies got underway, the AIDS crisis exploded, public health officials realized that contaminated blood products posed a grave threat to hemophiliacs, and the urgency of efforts to clone the Factor VIII gene became even more pronounced — recombinant DNA technology represented a means of producing a pure, pathogen-free version of the life-saving protein.
THE DIMENSIONS OF THE PROBLEM
In the early 1980s, Factor VIII was still not fully understood at the molecular level. Researchers remained uncertain about the identity, size, and source of the protein. Factor VIII is ordinarily bound to a molecule called von Willebrand factor (vWF) that stabilizes and protects it from degradation in the bloodstream. Only on initiation of the clotting process is Factor VIII cleaved from von Willebrand factor.
Clear evidence that Factor VIII and vWF are distinct proteins did not appear until 1979 when Edward (‘Ted’) Tuddenham, a British hematologist working in the lab of Leon Hoyer at the University of Connecticut, devised a method for separating them by immunoadsorbent chromatography, and showed that von Willebrand factor was not directly implicated in coagulation.
Researchers at Genentech and GI (and elsewhere) anticipated that cloning the Factor VIII gene would be extraordinarily difficult due to its immense size. It is now known that the gene spans 186 kilobases (kb) of DNA, with twenty-six exons (coding regions) containing instructions for the manufacture of 2,351 amino acids. By comparison, the first human gene cloned by Genentech coded for somatostatin, a peptide hormone composed of a mere fourteen amino acid residues. Genentech’s other recombinant proteins, human insulin and growth hormone, contained fifty-one and 191 amino acids, respectively. Factor VIII was gargantuan.
Genentech had used chemical methods to synthesize the somatostatin and insulin genes. Human growth hormone was produced with a ‘semi-synthetic’ method that married chemically-fabricated bits of DNA with biologically-derived complimentary DNA (cDNA) sequences. Neither approach was suitable for Factor VIII. The gene was too big. The task exceeded the capabilities of the then current state-of-the-art in DNA chemistry.
The cloners saw that it would be necessary to replicate the gene almost entirely by biological means, by isolating messenger RNA transcripts (mRNA) from cells and using an enzyme, reverse transcriptase, to synthesize new cDNA copies of the original gene. They knew how to make genes in this way, but they weren’t sure they could produce a cDNA molecule as large as Factor VIII. It had never been done.
There was another complication — the researchers could not proceed directly to this step because Factor VIII’s tissues of origin had not been identified. The protein circulates in the blood, but the whereabouts of cells that produce it were unknown. No one knew where to look for the mRNA that indicates gene expression and serves as a template for cDNA synthesis. The biology of clotting factors was only partially understood, and Factor VIII is extremely scarce, expressed at very low levels. If ever a gene was a needle in a haystack, it was this one.
Despite the degrees of difficulty, both Genentech and GI judged that the potential rewards of success outweighed the risks of failure. Steve Clark, GI’s first scientific employee, remembers deliberations: “I looked at the current literature, evaluated it, and said, ‘I don’t think we can do this. It looks too hard.’ I passed it on to others, and Jay Toole said, ‘I think we can do it.’” Ptashne was skeptical, but approved the project. Maniatis called Factor VIII “scary,” but also gave assent.
At Genentech, David Goeddel was busy trying to clone interferon. He assigned Factor VIII to Dick Lawn, and sent his best postdoc, Dan Capon, to Lawn’s lab to assist. The race was on. The commercial stakes were high, the work was demanding, and the outstanding problems were enormously complex. It was also dawning on the scientists that the outcome of the race could have life or death implications. At the end of 1982, biomedical researchers and public health officials had not yet determined the cause of AIDS, but they had recognized that blood transfusions were placing hemophiliacs in jeopardy.
The two companies adopted similar technical strategies. The first step was to find a source of purified protein that would enable ‘reverse genetics’ — working backwards from the amino acid sequences of protein chains to deduce the codons that compose DNA sequences. It wasn’t easy. Purified Factor VIII was a scarce commodity. Only very small amounts of the protein are produced in the body, and when isolated, the molecule is highly unstable, fragile, and easily degraded.
Toole began making inquiries to protein chemistry labs. He was eventually put in touch with David Fass of the Mayo Clinic in Rochester, Minnesota, who had recently used monoclonal antibodies to purify Factor VIII from the blood of hogs. Toole considered porcine Factor VIII a viable substitute for the human protein. It was widely assumed that the gene was highly conserved in both species. The molecules were likely to be very similar.
Fass agreed to provide the protein, but it was in short supply. Bob Kamen, GI’s director of research, recalls wondering “how politely to ask the Mayo to work a little faster.” The company sent John Knopf to Minnesota to assist. Knopf visited hog farms, took buckets of blood from pigs, and ran them through the purification process. GI was able to secure an adequate quantity of porcine Factor VIII with which to work.
Rod Hewick, GI’s chief protein chemist, was responsible for determining the amino acid structure of the molecule. He had worked at Caltech with Leroy Hood, Mike Hunkapiller, and Bill Dreyer to develop the first automated gas-liquid phase protein sequencer. Applied Biosystems, Inc. shipped the first commercial units in August 1982. GI purchased one, and set out to identify the more than 2,300 amino acids that compose porcine Factor VIII.
Protein chemistry work at Genentech was directed by Gordon Vehar. Vehar had reported the first purification of bovine Factor VIII — in miniscule amounts — in 1980 while working as a postdoc in the lab of biochemist Earl Davie at the University of Washington. He was unable reproduce the feat with the human protein at Genentech, and turned to Ted Tuddenham for help.
Tuddenham had moved to the Royal Free Hospital in London and devised means for purifying human Factor VIII. It took Genentech nine months to negotiate a deal with the hospital, which had already entered into a contract with a British company, Speywood, for certain rights to the molecule. Once an agreement was reached, production was meager. Tuddenham’s lab shipped purified protein at the rate of one milligram every three weeks. In all, twenty milligrams were sent from London to South San Francisco. That quantity was derived from 2,000 liters of blood collected and roughly 10,000 individual donations.
When Hewick and Vehar had partial protein sequences in hand, they designed oligonucleotide probes (relatively short pieces of single-stranded DNA) with which to screen a genomic DNA library — a permanent collection of total genomic DNA maintained in culture. The probes were configured to hybridize with complementary portions of the Factor VIII gene. They carried radioactive labels, so hybridization events could be monitored.
Because the library contained all DNA in the human genome, it was virtually guaranteed to contain the gene (albeit as a vanishingly small fraction of the total DNA). The approach was novel — for good reason. In the cloning business, combing through the entire human genome for a scarcely-expressed gene was a method of last resort. Factor VIII was a special case.
Both companies used a human genomic library that Genentech’s Dick Lawn and GI’s Ed Fritsch had established while working as postdocs in Tom Maniatis’ laboratory at Caltech in the late 1970s. Lawn and Fritsch had chopped up human DNA with restriction enzymes, separated the pieces by gel electrophoresis, and packaged them into lambda phage particles. Lambda phage is a virus that infects E. coli bacteria. The library consisted of millions of viral particles multiplying in a bacterial culture, each propagating a bit of dissected human DNA. The companies looked through it to find partial clones of the Factor VIII gene, which would then be used as probes in complementary DNA (cDNA) libraries.
cDNA libraries contain, not total genomic DNA extracted from cell nuclei, but DNA synthesized from mRNA gene transcripts recovered from cytoplasm. mRNA molecules convey instructions for protein synthesis to ribosomes, the cellular organelles on which proteins are manufactured. The presence of mRNA in a cell is evidence of gene expression — it indicates that genes are being replicated, transcribed, and translated into amino acid chains, the structural components of proteins. By the early 1970s, molecular biologists had acquired enzymatic tools that enabled them to reverse the transcription process and create cDNA replicas of genes from mRNA templates. A cDNA library is a collection of such replicas.
Obtaining a cDNA clone was necessary because the DNA in human genomic libraries is composed of interspersed exons and introns — coding and non-coding regions of genes. In 1983, neither company held out hope that bacterial or mammalian cells would modify RNA transcribed from a genomic clone in a manner permitting the expression of a functional recombinant protein (even if a full-length sequence of genomic DNA could somehow be cobbled together — that in itself would be a monumental accomplishment). cDNA clones, in contrast, are synthesized from mRNA templates and contain only uninterrupted exons. mRNA molecules are formed in enzymatic processes that cut out introns and re-splice exons into concise coding sequences.
Deriving a Clone
The gene hunters in Boston and San Francisco planned to screen the big genomic library to identify bits of the Factor VIII gene sequence. Probes that hybridized portions of the gene would then be used to screen cDNA libraries. Libraries containing traces of the Factor VIII sequence would be re-screened once more with the same probes in order to uncover either a full-length clone or a series of partial clones that would together encompass the entire gene. With a full-length cDNA clone in hand, the cloners could move on to expression, to the manufacture of the recombinant protein.
Due to the complexities of DNA transcription and translation, and the scarcity and size of Factor VIII, the search for the gene was inefficient and subject to error. Missteps were numerous and progress slow. Toole worked with Fritsch, Knopf, and John Wozney to search for Factor VIII sequences in the genomic library. “We had failure after failure after failure,” he says. “We kept getting false positives. We would take a hybridized probe, clone it out, and it would turn out to be junk.” In Lawn’s lab at Genentech, Dan Capon, Jane Gitschier, and Bill Wood encountered similar difficulties.
Small probes lacked specificity. They hybridized with DNA sequences repeated throughout the genome, and not unique to the Factor VIII gene. Longer probes were more selective, but the genetic code contains redundancies. Protein sequences can be coded in a variety of ways. The longer the targeted sequence, the larger was the battery of probes that had to be tested. Finding the right oligonucleotides was a frustrating slog. Endurance was as important as biological knowledge and analytical skill.
The teams persevered. By mid-1983, both Genentech and GI had obtained short segments of the gene. To confirm that they were X-linked, positive probes were deployed a second time to screen a library containing DNA from a rare XXXXY cell line. The line came from the cells of an individual with four X chromosomes. When Southern blot comparisons of results from Lawn and Fritsch’s library and the XXXXY library displayed expected hybridization frequency ratios (1:4), the researchers knew the sequence was X-linked and could belong to the Factor VIII gene.
From these starting points and preliminary validations, the teams laboriously assembled progressively longer genomic clones, piece by piece, and used them to probe cDNA libraries. As incremental advances accumulated, the companies began devoting greater resources to the projects. Both eventually assigned more than thirty scientists to Factor VIII cloning duties. Lawn says: “We pillaged other Genentech labs for a couple of years while the project was hot.”
The Formation of cDNA Libraries
cDNA libraries are assembled in the following steps: 1) single-stranded mRNA is extracted from cells and purified for in vitro processing; 2) reverse transcriptase is added to synthesize complimentary DNA strands; 3) RNase is employed to degrade the RNA; 4) DNA polymerase is used to replace degraded RNA with newly-synthesized DNA, creating double-stranded cDNA copies of the genes from which the original mRNA molecules were transcribed; 5) the cDNA copies are spliced into viral or plasmid vectors; 6) the vectors are deployed to transfect or transform bacteria; 7) bacterial colonies are grown up in culture, and as they multiply, the recombinant cDNA is reproduced.
When cDNA libraries are constituted, and portions of gene sequences are known, specific genes can be targeted with labeled nucleic acid probes and isolated for full sequencing or further cloning.
KEEPING SECRETS AND MAKING DISCLOSURES
At both companies, there was intense interest in what the other side was doing, and much internal speculation. The competitors looked over their shoulders constantly. Jay Toole recalls that “rumors were rampant.” Dick Lawn remembers being “nervous the entire time.” On business trips that brought him from London to California, Ted Tuddenham observed what he calls “almost paranoid secrecy” at Genentech.
Lines of communication remained open nevertheless. Molecular biologists Chris Simonsen at Genentech and Randy Kaufman at GI had worked together in Bob Schimke’s laboratory at Stanford, and had remained friends. They didn’t divulge trade secrets or report on progress, but, Kaufman says, “We talked. They were cloning Factor VIII, we were cloning Factor VIII. We generally knew what was going on.”
Lawn’s experience was similar — he went to GI to visit his former Caltech boss, Maniatis, and former benchmate, Fritsch: “We never discovered directly where we stood. It was like baseball players on opposing teams before the game — you could shake hands or chat around the batting cage, but once the game started, it was very competitive.”
Kaufman concedes that it was sometimes difficult to avoid unintended disclosures while maintaining collegial relations in the scientific community: “We had invited [Genentech scientist and later CEO] Art Levinson to give a talk at GI. It was the day that Rod Hewick’s chemistry group figured out an important amino-terminal sequence of Factor VIII. I was showing Art around. Rod saw me and shouted, ‘We got it!’ I said, ‘Rod, this is Art Levinson.’”
According to Toole, the urgency and gravity of the undertaking generated high levels of stress: “John Knopf and I would sometimes see the company’s CFO in the elevator, and he would ask ‘Have you cloned it yet?’ He had no concept of how the science worked. If we said no, he would say things like, ‘If you guys don’t succeed, the whole company is going to fail.’ John Knopf wanted to strangle him. The pressure was enormous.”
The work at the lab bench inched forward. At Genentech, Jane Gitschier developed a boot-strapping sequencing technique called ‘genome-walking,’ the fundamentals of which are still widely-employed. She worked from established sequence data to configure extended probes that would overlap and generate data on adjacent regions. The GI group devised similar techniques.
On October 28, 1983, GI filed a patent application on “preparations of recombinant DNA which code for cellular production of human and porcine Factor VIII and methods of obtaining such DNA and expression thereof in bacteria and eukaryotic cells.” The application was based on a partial clone and specified steps for deriving a full human sequence. The inventors, Jay Toole and Ed Fritsch, later amended sequence data — in a ‘continuation-in-part’ filing — in order to strengthen the claim to the gene. The original application was filed quietly in order to establish priority. On December 1, however, the company issued a press release on further developments.
Dick Lawn remembers hearing about it the next day: “Somebody called me and said, ‘There’s a New York Times article about Genetics Institute and their Factor VIII project.’ I went down to Irving Street in San Francisco, bought the paper, and started reading.” Lawn was afraid that researchers at GI had obtained the full sequence and expressed human Factor VIII. They hadn’t. They were reporting a cDNA clone that represented “a significant portion” of the human gene. “I was so relieved,” Lawn says. “GI was announcing in public that they were behind us.”
Dan Capon’s reaction was very different. He was working with genomic clones produced by Jane Gitschier and Bill Wood to screen cDNA libraries for Factor VIII mRNA. “My heart sank,” he says. “They were so far ahead of us.” Capon had constructed over fifty cDNA libraries from various cell lines, each containing an average of seven million clones. He recalls having worked out just 10 percent of the sequence, while GI had started with liver and spleen tissues and found enough Factor VIII mRNA to synthesize a larger partial clone.
Capon redoubled his efforts: “Instead of working 16 hours a day, I started working 20 hours a day.” On December 26th, he was finished — the cDNA sequence was complete. Dan Eaton, Art Levinson, Chris Simonsen, and Bill Wood pressed ahead to express the gene in mammalian cells. Genentech had used E. coli cells to produce recombinant somatostatin, insulin, and growth hormone, but the group did not expect the simple bacterial system to manufacture a functional protein as complex as Factor VIII. They tried it and found that they were right.
After a second failed attempt with Chinese hamster ovary (CHO) cells, the company achieved expression in baby hamster kidney (BHK) cells. “On 10 April 1984,” Gitschier reports, “Gordon came by Dick’s lab and left a note on Bill Wood’s desk: ‘See me. They had activity!’” Tests had shown that the recombinant protein was a viable substitute for native Factor VIII. A patent application was hastily written up and filed ten days later (setting the stage for a legal contest). The submission covered cloning, expression, and the full sequence of the human Factor VIII gene. The inventors were Dan Capon, Dick Lawn, Art Levinson, Gordon Vehar, and Bill Wood. Genentech was ahead, by a nose.
Sometime shortly after — precisely when isn’t clear — GI benefitted from a chance occurrence that eliminated a great deal of monotonous bench work in a single lucky stroke. “One night,” Toole says, “we were able to get two clones that overlapped to create a full length clone. It was pretty remarkable.” Toole checked the human clone against the known porcine sequence. It corresponded closely. “The ultimate test,” he says, “was performed in Randy Kaufman’s lab. Randy put the full-length cDNA into a monkey kidney cell line using an SV 40 vector, took out the supernatant, and showed that it had coagulation activity. It was Factor VIII. We had it. We started celebrating.”
The race to clone Factor VIII had ended in a virtual dead heat. David Goeddel says, “I’m not sure it was ever resolved who got the clone first or who got it expressed first.” Progress reports were part of a cat and mouse game for the control of intellectual properties. Genentech was first to announce a complete cDNA clone, and first to achieve expression, but by mid-1984, both companies had rFVIII, and both had patents pending. It was a photo finish.
INTERSECTIONS AND TURNING POINTS
In June 1984, Professor Luc Montagnier of the Pasteur Institute in Paris, France and Dr. Robert Gallo of the US National Institutes of Health (NIH) held a joint press conference to announce that the retroviruses they had associated with AIDS — Montagnier’s lymphadenopathy associated virus (LAV) and Gallo’s human T-lymphotropic virus (HTLV-III) — were probably the same, and likely the cause of the spreading epidemic. The pathogen was later renamed the human immunodeficiency virus (HIV). Virologists and public health officials had a target at which to aim.
In mid-August, Genentech submitted three papers to Nature — one each on Factor VIII protein structure, cloning, and gene sequence. Deputy editor Peter Newmark contacted GI and offered to publish a report on the sum of its work, too — if it could be written up within a week. “Later,” Lawn recalls, “Jay told me they stayed up night and day writing it. I think he said someone flew to London with the manuscript, I’m not sure.”
In the first week of September, Lawn and Toole attended International Congress XVI of the World Federation of Hemophilia in Rio de Janeiro. They were the main speakers on the first night of the meeting. “Sure enough,” says Toole, “we presented the same data back-to-back, on the expression of full-length Factor VIII.” The implications for the AIDS crisis were obvious, but the researchers prefaced talk about recombinant blood factors as substitutes for plasma concentrates with a disclaimer: ‘It could take years.’
B.L. Evatt of the CDC’s Division of Hematology also attended the Congress. He presented the results of experiments to deactivate LAV (HIV) in blood: the virus was readily destroyed by brief exposures to temperatures in excess of 70 degrees Celsius (158 degrees Fahrenheit).
In the second week of September, Genentech signed a clinical testing, manufacturing, and marketing agreement with Cutter Biological, a division of Bayer’s US subsidiary, Miles Laboratories, located in Berkeley, California. In exchange for cash and downstream royalties, Genentech surrendered worldwide marketing rights to rFVIII (save for certain United States and Canadian rights that would be restored to Genentech two years after the introduction of a product).
In October, the American Hemophilia Foundation’s Medical and Scientific Advisory Committee strongly recommended that physicians and hemophiliacs rely on heat-treated blood and plasma products exclusively.
On November 22, 1984, Nature ran four papers on Factor VIII — three by Genentech, and one by GI — as lead articles. Editor John Maddox hailed the cloning of the protein as “a technical triumph without parallel.”
By the end of the year, the CDC reported 7,699 confirmed cases of AIDS in the United States, and 3,665 deaths. Epidemiologists reckoned that tens of thousands more had been infected by HIV.
On March 20, 1985, the US Food and Drug Administration (FDA) licensed Abbott Laboratories to sell the first antibody test to detect HIV in serum. Blood banks began screening donated blood. By this time, heat-treatment had been broadly implemented. The AIDS epidemic raged on, but HIV transmission to hemophiliacs through blood transfusions or the use of plasma products was halted abruptly. The blood supply and blood products became virtually HIV-free.
Recombinant Factor VIII didn’t rescue hemophiliacs from exposure to HIV after all. The cavalry didn’t charge over the hill in the movie’s climactic scene. The real-life solution was prosaic and low-tech. It was also tragically belated. According to the National Heart, Blood, and Lung Institute of the NIH, half of all hemophiliacs in the United States had contracted HIV by 1985; among patients with severe forms of the disease requiring frequent blood transfusions, the figure was 90 percent.
Recombinant Factor VIII was no longer desperately needed to rescue hemophiliacs from exposure to HIV, but the AIDS epidemic had made medical professionals, the scientific community, the healthcare industry, and the government acutely aware of the vulnerability of the blood supply to unknown threats. It was widely understood that the availability of recombinant blood factors could avert similar tragedies in the future.
Nevertheless, without the urgency and demand generated by contamination of the blood supply, subsequent decisions regarding the development of rFVIII became at once more mundane and more complex. GI, Genentech, and their corporate partners believed that the recombinant product would be superior, but safety and efficacy in human beings hadn’t been demonstrated. There were concerns that recombinant proteins secreted from non-human mammalian cells — and especially proteins as large and complex as Factor VIII — would not be properly glycosylated (decorated with carbohydrates) and folded, and rendered ineffective or immunogenic as a result.
There were financial, commercial, and legal issues to sort out as well. Screening and heating made blood and plasma products more expensive to produce, but it was unclear whether recombinant methods afforded economic advantage. Making market projections was impossible. Recombinant production could help to alleviate chronic clotting factor shortages, and perhaps generate surpluses that would permit prophylactic treatments, but by the time new products had passed through clinical trials, the patient population could be wiped out by AIDS. Intellectual property positions were also difficult to assess. For the involved parties — Baxter and GI and Bayer and Genentech — the only sure thing was that litigation would be expensive.
Given the prevailing uncertainties, GI diverted resources from rFVIII to R&D on erythropoietin (EPO), a red blood cell growth-stimulating hormone used to treat anemia. According to Randy Kaufman, “The company couldn’t afford to go all out on both at the same time. It had to focus on one. EPO was chosen because the market was bigger.” GI remained committed to delivering recombinant Factor VIII to Baxter and to hemophiliacs in need, but CEO Schmergel needed to allocate materials and personnel to both projects contingently. In the shuffle, the development of Factor VIII was slowed for nearly two years.
On the West Coast, Genentech was developing two important recombinant therapeutic proteins simultaneously: Factor VIII and tissue plasminogen activator (tPA), a molecule that dissolves blood clots. Genentech needed cash, and could not afford to manufacture and market both products on its own. tPA was expected to be the greater revenue generator as a treatment to unblock coronary arteries after heart attacks and cranial arteries after strokes. The company sold the rights to rFVIII and turned its technology over to Cutter Biological. Cutter made conventional plasma concentrates and lacked expertise in the manufacture of recombinant proteins in mammalian cell cultures. The company had trouble producing a stable protein. The Genentech molecule languished for a time in pre-clinical development.
GI solved stability problems by co-expressing recombinant von Willebrand factor (VWF) with Factor VIII in CHO cells. VWF, which naturally binds to Factor VIII and protects it from disintegration, was cloned at Harvard in 1986. GI licensed and incorporated the technology. According to Randy Kaufman, who led the expression effort, the uniquely adaptable and scalable CHO cells were indispensable for high volume production of rFVIII: “We had these cells that made a boatload of Von Willebrand factor in order to make just a little usable Factor VIII, but we were able to scale it up.”
When clinical testing of the GI product got underway in March of 1987, the immunogenicity of CHO cell-derived proteins remained a worrisome possibility. Dr. Gilbert C. White conducted the first human trial at the Center for Thrombosis and Hemostasis at the University of North Carolina. He tells the story of an enrolled subject, G.M., a forty-three-year-old male: “I infused the material and noticed that G.M.’s eyes were closed and his chin was resting on his chest. I asked if he was OK, but he didn’t answer. I asked again, but no answer. Louder, I said, ‘Speak to me.’ He looked up and started making hamster noises.”
Baxter held a dinner to celebrate the first clinical trial. Toole was invited, along with patient #1, the first person to receive a therapeutic dose of rFVIII. There was an emergency. The patient, Toole explains, “felt his mouth filling up with blood. He injected himself with rFVIII and the bleeding stopped immediately. Everybody breathed a sigh of relief.”
GI’s Factor VIII patent was issued in 1989. The interference between the companies’ claims remained unresolved, and the Californians had a strong argument to test — GI had described a method for producing the Factor VIII gene, but Genentech had specified the complete gene sequence. Neither party was eager to enter into risky and potentially damaging litigation, so a royalty-free cross-licensing agreement was negotiated. The terms gave GI and Baxter and Genentech and Bayer clear paths to the marketplace, so long as the FDA approved their products.
GI’s Recombinate® was approved for sale in December 1992. Cutter Biological had moved the Genentech molecule into clinical trials in June of 1988. Its product, Kogenate®, made in BHK cells, came to market in early 1993. The race to clone the Factor VIII gene had been a sprint; the race to bring the product to market became — as is the norm in the pharmaceutical industry — a test of endurance.
Research at Genetics Institute led eventually to a ‘second generation’ Factor VIII product. In 1986, Toole’s group compared the gene sequences coding for three principal structural domains of Factor VIII molecules — A, B, and C — in hogs and human beings. The A and C portions were nearly identical, but the B sections differed considerably. “We saw,” says Toole, “a huge middle region in which the homology between the two factors was completely lost.”
The lack of conservation between species suggested that the B domain did not play a functional role in blood coagulation. Toole discovered that the entire middle region (38 percent of the molecule) could be removed without diminishing therapeutic efficacy: “It was just filler protein. I made a clone that got rid of the B domain, and it retained activity, which was a remarkable thing.” Genetics Institute published the finding in Proceedings of the National Academy of Sciences in August 1986. The modification increased the efficiency of expression and significantly reduced costs of producing the protein.
A Genentech group led by Dan Eaton and Bill Wood reported the same alteration in Biochemistry in December, but according to Dennis Kleid, one of Genentech’s senior scientists: “Bayer didn’t want to work on the B-domain deleted product even though we had given them an exclusive license to make and sell it. They never developed it.” In March 2000, the FDA approved Genetics Institute’s product under the trade name Refacto®. It was marketed by Pharmacia and Wyeth (which wholly acquired Genetics Institute in 1996).
A ‘third generation’ product followed eight years later. Serum albumin (human or bovine) was added to CHO and BHK cell culture media in the production of Recombinate, Kogenate, and Refacto, and monoclonal antibodies of murine origin were employed in immunoaffinity purification processes. In the manufacture of the third generation product, all contact with human or animal-derived proteins was eliminated. In addition, a virus-retaining filtration step was incorporated.
These measures make the latest version of recombinant Factor VIII as pure, safe, and protected against blood-borne pathogens as is possible given the current state-of-the-art in pharmaceutical technologies. The FDA approved Wyeth’s Xyntha/ReFacto AF® for sale in February of 2008.
The AIDS epidemic was a public health watershed. Blood banks, government officials, and scientists joined forces to solve the problem of HIV contamination, and, as a result, the blood supply and blood-based medical products have never been safer. Today, physicians and hemophiliacs rely on clotting factor-enriched plasma concentrates without anxiety or fear. The transmission of disease through transfusions is rare.
However, risks of contamination from known and unknown pathogens can never be eliminated entirely. For this reason, the Medical and Scientific Advisory Council (MASAC) of the National Hemophilia Foundation endorses recombinant clotting factors as first-line therapies, and most physicians prescribe them for newly-diagnosed hemophiliacs.
Approximately 40 percent of the Factor VIII used to treat hemophilia A patients is genetically-engineered. Recombinant products are more expensive, but guaranteed to be safe, and they have done much to alleviate chronic shortages of the life-saving protein. Prophylactic treatment of hemophilia A is now possible and common — rFVIII has helped thousands of patients lead normal lives. Memories of the AIDS tragedy linger, but the outlook for hemophiliacs in the US has never been brighter. Patients and their families now await the next great scientific advance — a gene therapy that can cure the disease.
The cloning of Factor VIII was a triumph for genetic engineering. It was one of the signal events that established molecular biology as a legitimate technological platform in the pharmaceutical industry. Looking back, Tom Maniatis says: “It was a huge challenge to characterize the protein, and to isolate, clone, and express this enormous gene. Making that happen was a tremendous accomplishment.” David Goeddel calls Factor VIII “the last great cloning project.” “Never again,” he says, “was a project as difficult as Factor VIII was in its time.”
All of the main participants went on to fashion distinguished careers in science and industry. Jay Toole’s experience with Factor VIII left him fascinated with human physiology. He left GI in 1987 to train in medicine at Stanford. He subsequently returned to industry as Gilead Sciences’ Senior vice-president of clinical research. After Factor VIII was shuttled off to Cutter, Dick Lawn went on to direct atherosclerosis R&D projects at Genentech. In 1993, he moved to CV Therapeutics in Palo Alto, California to manage projects in genomics and cardiovascular disease.
When Jane Gitschier’s postdoctoral stint came to an end, she moved to a faculty position at the University of California, San Francisco and carried on with investigations into the genetics of hemophilia. Bill Wood stayed at Genentech until 2008. He led a string of cloning projects, and after 1997 began working in bioinformatics. Dan Capon became involved in Genentech’s efforts to develop an HIV vaccine. In 1991, he joined newly-founded Abgenix to work on the transgenic production of human antibodies, and in 1995, started his own company, Monogram Biosciences, in South San Francisco. Gordon Vehar remained at Genentech until 2000 when he left to join Raven Biotechnologies as vice-president of research & development.
John Knopf stayed with Genetics Institute through its acquisition by American Home Products (later Wyeth), until 2002. The following year, he founded Acceleron Pharma, a biotherapeutics company, in Cambridge, MA. Knopf was appointed the firm’s CEO in 2007. Randy Kaufman eventually returned to academic science as a professor and Howard Hughes Medical Research Institute investigator at the University of Michigan Medical School. He is now a faculty member at the Sanford Burnham Medical Research Institute in La Jolla, California.
After Factor VIII, John Wozney worked on the development of GI’s successful bone morphogenetic proteins and various other projects. In 2000, he became director of basic research and pre-clinical R&D at Pfizer. Ed Fritsch spent nineteen years at GI, contributing to numerous research programs until 2000. He subsequently became a senior vice-president of research & development at Phylos in Lexington, Massachusetts. Rod Hewick continued to work in protein chemistry at GI and Wyeth for more than twenty years.
The scientific competition between Genentech and Genetics Institute for Factor VIII was relatively brief — about two years — but intense. Genentech’s Jane Gitschier has written about it: “We knew what those at Genetics Institute had been through. We felt a kindred spirit.” Dan Capon tells of running into Jay Toole many years later, in a parking lot in Foster City, California. He says he was pleased to meet “one of the few people in the world who understood what it was all about.”
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