A Complementary Pathway

FEATUREComplement How one group of researchers brought a scientific idea to the clinic for a rare diseaseBY ISHANI GANGULI© THOM GRAVESScott Rollins was starting his graduate thesis at Oklahoma Medical Research Foundation in the late 1980s when he first heard about paroxysmal nocturnal hemoglobinuria (PNH). He learned that in PNH, red blood cells are vulnerable to attack by the complement system's terminal membrane-attack complex. Pat

Jul 1, 2006
Ishani Ganguli

How one group of researchers brought a scientific idea to the clinic for a rare disease


Scott Rollins was starting his graduate thesis at Oklahoma Medical Research Foundation in the late 1980s when he first heard about paroxysmal nocturnal hemoglobinuria (PNH). He learned that in PNH, red blood cells are vulnerable to attack by the complement system's terminal membrane-attack complex. Patients with PNH have episodes of hemolysis marked by dark urine and anemia, as well as stomach and back pain, chronic exhaustion, and an increased risk of life-threatening blood clots.

A mainstay of medical school textbooks, PNH is nonetheless rarely encountered, occurring in only 8,000 to 10,000 people in North America and Europe combined. For Rollins' graduate advisor, Peter Sims, dissecting the disease presented an interesting biological challenge, and he gave Rollins the task of isolating and characterizing the complement inhibitory protein, CD59, thought to be lacking on PNH cell surfaces.

Rollins recruited Russell Rother, a close friend in the graduate program, to work on cloning the inhibitor in platelets, which were inappropriately activated in the same disease. They published several papers defining how CD59 blocks complement and its importance in platelet activation.1,2

In 1991, he and Rother became postdocs at Yale, and they continued work on CD59 and other complement inhibitors in the lab of Al Bothwell, who had cloned the protein. There, the two met Lenny Bell, an assistant professor of medicine and pathology at the medical school. Bell had biotech yearnings, having come from a family of entrepreneurs. He and Joe Madri, a professor of pathology at Yale, would sit around Madri's office late into the night after their lab work was finished, and put in a "fantasy folder ... what we would do if we had the druthers to do it," Madri remembers. Their interest lay in xenografts; they wanted to mitigate the complement-mediated attack of these organs by engineering resistant epithelial cells. Bell started talking about the project to Steve Squinto, who was working at Regeneron at the time, and was dating one of Bell's childhood friends.

For Squinto, complement was "a bunch of letters and numbers that just made no sense ... as boring and dry a subject area as you can get." But he and Bell were quickly convinced by the business potential it held. It's "really the first line of defense against infection," Rollins says. As talk about turning CD59's inhibitory properties into a company turned earnest, it was "an easy decision" for Rollins to "take the plunge and leave academia ... this technology was everything I had worked on since graduate school, I wanted to carry it forward," he says.

In August 1991, in the middle of Hurricane Bob, Rollins, Bell, Squinto, and Madri gathered in a New Haven hotel room, sopping wet shoes and all, and decided to start a company. Madri stayed on at Yale, though continuing to consult for the company, while Bell abandoned his tenure-track position and Rollins left the prestigious Jane Coffin Childs postdoctoral fellowship, with Rother following soon after. They called the company UDEC, for Universal Donor Endothelial Cells, and took up shop in New Haven's Science Park, a half-mile from their previous labs. The run-down incubator facility was housed in a building originally constructed in the late 1800's and was decorated with little more than bird's nests, broken windows, and a solitary telephone sitting on the floor.

At the time, New Haven wasn't exactly a breeding ground for biotech startups. Bell had to do some convincing to get Yale to sell its portion of the intellectual property on CD59, which he also bought from Sims in Oklahoma. Bell set out on the road to secure venture capital from investors including Schroder Ventures Life Sciences, INVESCO Global Health Sciences Fund, Oak Investment Partners, and some high-net worth individuals. "We started the company on a shoestring of $6 million," remembers Madri.

In those early days, "we celebrated every small success, every gel that had on it what we wanted to see," Rother recalls. "It was an exciting time." They lured their scientific talent from Yale, hiring only people they knew.

Bell is now CEO of the 250-plus employee company, Squinto is executive vice president and head of research, Rollins is senior vice president of drug development and project management, and Rother is senior vice president of research. "We had a bunch of scientists who didn't know anything about running a business," Rollins says. "Lenny [Bell] was trying to learn something at that time. But we were pretty naäve."


The company's initial plan was to develop a soluble form of CD59, and use the natural complement inhibitor that is ineffective in patients with PNH to block complement in a host of diseases. Such patients have an acquired genetic defect that prevents biosynthesis of the GPI (Glycosylphosphatidylinositol) anchor proteins normally attaching CD59 and other complement inhibitors to cell surfaces.

The scientists quickly changed their company's name to Alexion, from the Greek word alexein that means to defend, which was also the original nomenclature for complement. They hoped to defend against the effects of complement, but Bell remembers that "within six months of actually starting the company, we recognized that our plan [for soluble CD59] was a failure. When you actually created it, it really didn't work very well." They made some headway with replacing CD59 in PNH cell lines using gene therapy, but this approach proved untenable. "Gene therapy is still just out of reach, not something good for a small biotech to go after for its main platform," says Rother.

CD59 seemed a dead end. But even if they couldn't manipulate the body's own mechanism for protecting against complement, they reasoned, antibodies might be used to do the same. The goal was to stop complement late in its pathway - after it had a chance to fight harmful infections but before it could attack blood cells - and in the early 1990's, high-affinity monoclonal antibody technology was on the rise. So they designed an antibody that bound to C5, which prevented C5 convertase from binding and cleaving C5, thus forming the membrane attack complex. (See Interrupting Complement) They then humanized the antibody, named eculizumab, so that it couldn't effect its own inflammatory response.

In August 1991, in the middle of Hurricane Bob, four men gathered in a New Haven hotel room and decided to start a company.

In 1993, Rother had begun collaborating with Peter Hillmen, a physician at Leeds General Infirmary in the United Kingdom, on basic research on PNH - Hillmen shared immortalized PNH cell lines with him. But the disease was little more than a model to study a number of complement-related conditions. And with the new tool in hand, they continued to set their sights broadly. They partnered with Proctor & Gamble to develop and commercialize pexelizumab - a single-chain, soluble form of the antibody - for acute cardiac conditions. And in the late 1990's, following preclinical testing and a nod from the Food and Drug Administration, they began human trials of eculizumab for rheumatoid arthritis, lupus, and nephritis (See The Paths to Market).

Eculizumab showed promising results in early trials on arthritis, among the other complex diseases, but as other drugs came out for the same indication, they quickly realized that such markets were too competitive for their small company to rely on. Rother had been looking at PNH over the years, but it wasn't until 2000 that Alexion considered the rare blood disorder as a viable primary use for the drug. Rother's continued insistence that PNH was worth pursuing and Genzyme's business success in developing orphan drugs finally convinced Bell. With no direct treatments for PNH, "there was no competition in this arena, [so] it was a setting we could excel in. And scientifically, you can't get any better than PNH," says Rollins. "They have a terminal complement inhibitor deficiency and we give them a terminal complement inhibitor back as a therapy. It's a perfect fit."

The company was continually expanding, and with 150 employees by the year 2000, had outgrown its original home. That year, it moved 14 miles north to the farming town of Cheshire, into an industrial park nestled among rolling hills, ponds, and a women's prison.


David Tomlinson woke up one morning nearly 20 years ago to find that his urine was the color of Coca Cola. The vegetable farmer from the outskirts of Leeds, UK, had been suffering from aplastic anemia for several years, but these now monthly hemolytic episodes left him debilitated. "When I was hemolyzing, I'd just sit in a chair or in bed for three or four days," he says. "I couldn't work." His son took over his family business with the help of his daughters and wife.

In Alexion's efforts to turn a profit on complement inhibition, using eculizumab to treat PNH has emerged as the company's best shot. One phase III trial has been completed successfully, with another nearly finished, and CEO Lenny Bell says they will file for approval on the drug by the end of this year.
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At a loss for the disorder's cause, hematologists told him "'that it sometimes happens,'" and "'to just put up with it,'" he says. Tomlinson would get blood transfusions every four to six weeks to replace his ruptured blood cells, but between the sessions, he'd have a hemolyzing episode for three or four days, more often if he had a cold or other infection. That's not uncommon; episodes can occur spontaneously or be triggered when an infection activates the complement pathway. Tomlinson also had a hard time swallowing food and an "awful funny feeling in the lower abdomen ... dragging you down," but "for years [doctors] told us it wasn't connected" to the episodes. Researchers now say that such pain is caused when hemoglobin spills from lysed red blood cells, binding to nitric oxide in the bloodstream and preventing nitric oxide's usual activity.

Once Tomlinson met Hillmen in the mid 1990s, he began to get some answers. Hillmen, by then a PNH expert, explained what was causing the disease and recommended that Tomlinson take the anticoagulant warfarin to prevent clots. Tomlinson continued with blood transfusions, but there was little else that even Hillmen could offer him.

As soon as he heard that his friends across the ocean had developed a promising new PNH treatment in 1999, Hillmen told Tomlinson and his other patients about eculizumab. It wasn't until May 2002, however, that reassuring safety data allowed him to bring the treatment to 11 patients through a Phase Ib trial. Hillmen, the lead clinical investigator for the trial, says they were cautious in testing the new drug on a rare disease with unusual potential complications. But many of his eculizumab patients were depending on blood transfusions as often as once every three weeks. "I thought we had nothing to lose, we were so ill, really," Tomlinson says. On the day of his first infusion, Tomlinson was in the midst of a hemolytic episode. When he woke up the next morning, "it had cleared up. It was unbelievable," he remembers. "I have not hemolyzed since that day. [I've had] no stomach ache or trouble eating since that day ... It just turned it off." Ever since, Tomlinson has gone to Leeds Infirmary every other week for a half hour of intravenous infusion. "It's just so easy now," he says. "I have much more energy." Now 63, he's back to growing cauliflower, cabbage, lettuce, and rhubarb with his family. "I could've done with [the drug] 18 years ago," he jokes.

Out of Hillman's 11 transfusion-dependent patients in the initial study, all but one are still taking eculizumab. (The exception is a woman who stopped taking the drug when her severe aplastic anemia became a treatment priority.) Three, including Tomlinson, have not had any transfusions since, and the others have slowed to a pace of one transfusion a year or less.3,4

"It's a very convincing drug," says Hillmen. "The black urine stops the day after you get the dose and it never comes back." Based on the results, and the rarity of the disease, the FDA allowed Alexion to move directly to Phase III trials, one of which is still ongoing.

Hillmen, whose work on PNH had made his Leeds clinic something of a mecca for patients with the disease, had no trouble recruiting subjects for subsequent trials. Nurses often bring the eculizumab IV pumps to their patients' homes. Other patients "travel a long way for treatment, but they're quite happy to do it," Hillmen says. One young man enrolled in a phase III trial was dependent on opiates for 12 years because of severe stomach pains associated with PNH. "Locally, people shunned him because they thought he was a druggie," says Anita Hill, Hillmen's co-clinical investigator. Now that eculizumab has taken away the patient's pain, he's kicked his morphine habit and runs a small business.


The success of eculizumab and the size of the market means that Avant Immunotherapeutics, which Rollins says was the first company to start developing a complement inhibitor, won't be applying its own inhibitor, TP10, to PNH. "I don't think it's a big enough market to compete in," says Avant president and CEO Una Ryan. TP10 prevents C3 and C5 convertase assembly and has been successful in Phase III trials for cardiac indications. It is being tested in female patients having cardiopulmonary bypass surgery, since previous trials protected only men. Ryan argues that successful treatment requires higher concentrations of eculizumab than of TP10, because to "mop up preexisting C5 ... is not very efficient." She also says eculizumab has a poor half-life compared to TP10. But the latter, a larger molecule, is a "lot more expensive to make and not as [kinetically] efficient," says Greg Stahl at Brigham and Women's Hospital in Boston and a member of Alexion's scientific advisory board.

Alexion was "pretty smart in that they went after the most important biological mediators within the complement system," Stahl says. "One of the best things about Alexion's [inhibitor] is that you leave the upstream intact."

Jun-ichi Nishimura, assistant research professor in oncology and medical transplantation at Duke University, argues that a CD8 blocker, like the small-molecule inhibitor he is working on, is an even safer bet because of the increased infection risks associated with blocking C5.

Hillmen and Hill attempted a version of Alexion's original plan, testing a CD59 substitute with a synthetic lipid anchor. When they infused membrane-targeted CD59 in mice, the PNH cells were protected against complement-mediated lysis, but the protein would insert into all cells. As a result, large doses were required for the drug to be effective and the treatment never reached human trials.5 "Eculizumab will take the market in PNH," says Hill. "We don't need another therapy. [It was] difficult to get [subjects] willing to not be on eculizumab."

Hill, whose research is funded by Alexion, is now studying the effect of eculizumab in reversing nitric oxide consumption in PNH, and she says the results have been promising so far. "Now that we have a drug that's blocking the complement cascade," Hill says she is working to find out "in more detail what effects early complement have in PNH."


Wendell Rosse, Duke University professor of medicine emeritus, cautions that although the drug is effective in curbing hemolysis and related symptoms, "eculizumab does nothing at all about the bone marrow failure aspect of PNH." Rosse, who studies the disease, explains that all PNH patients have some degree of this autoimmune-related failure. Hematopoietic cells with the PNH mutation, which are found in low quantities in all individuals, have a survival advantage over normal blood cells and thus proliferate in the bone marrow.

The predominant cause of death in patients with PNH is thrombosis, which occurs when platelets are activated by unchecked complement. It may be too early to know the treatment's efficacy in preventing blood clots, but Hill is optimistic. The major predicted side effect of the treatment was decreased protection against the bacteria that terminal complement usually fights: meningitis- and gonorrhea-causing Neisseria. "People with inherited [terminal] complement deficiency have more frequent meningococcal infections," says Hillmen. They were reminded of this the hard way, when a patient was infected in a Phase II trial for nephritis. Since then, they have been prevaccinating subjects. Rosse says his patients who lack C5 congenitally "do quite well" since most neisserial infections can be readily treated. "One girl [with C5 deficiency] had a very active social life and she had every possible form of gonorrhea," he says. "We had a hard time keeping up with her."


Following the eculizumab pilot, a placebo-controlled randomized trial with 87 subjects (TRIUMPH) closed successfully in January, while a safety extension trial with 90 subjects (SHEPHERD) is ongoing. Trial sites for the rare disease are scattered across North America, Western Europe, and Australia, with about 45 sites in 13 countries. The results so far, says Rollins, have been striking. "Patients will still have some level of anemia, though a lot of it was normalized by the drug. Some patients still get transfusions, though [the number of transfusions was] dramatically reduced," he reports. "Half the patients got no transfusions during the entire trial." Those results have yet to be published.

On message boards of online PNH support groups, members share their hopes of getting into the trials. Hill says that though recruitment has ended, they still have "patients knocking on [our] door wanting to enter." Bell says they expect to submit eculizumab, marketed as Soliris, for approval in the United States and Europe later this year.

For patients like Tomlinson, who still receive the treatment for free, the expense is a daunting prospect (visit www.the-scientist.com for an additional profile of patient Margarita Soto). Hillmen expects Soliris to cost $100,000 to $200,000 per year, compared to somewhere between $5,000 and $9,000 per year for frequent transfusions. "I certainly couldn't afford to pay for it," Tomlinson says; he hopes the British health system will cover it.

"There's really a niche population for whom eculizumab is ultimately going to be appropriate. It's a disease that even busy hematologists are going to see sporadically," says Bob Siegel, a hematologist at Hartford Hospital. Bothwell was "surprised that [the company's stock] has grown as much as it has based on PNH." Alexion, which celebrated its 10-year anniversary on the NASDAQ this year, had an initial public offering of $8.25 and is now trading in the low- to mid-$30 range. The company's market capitalization is about $1.1 billion, although its losses are upward of $4 per share. Alexion has raised $589 million in public and private equity offerings since it was formed. Bell says he's not worried about the size of the eculizumab market, since he's relying on the dramatic therapeutic outcome of the drug in patients with PNH. Though he says it's too early to tell if the orphan indication alone will buoy the company to profitability, "I think we are comfortable that the PNH opportunity is a valuable commercial opportunity," he says.

"We've studied complement to the ends of the earth," says Rosse, and "this is the first effective therapy based on our knowledge of complement that's come along. This is the first time that we've been able to control complement that's out of control."

Alexion is still trying to apply eculizumab and its single-chained version to the broad swath of complex diseases they originally had in mind. The company even acquired San Diego-based Prolifaron to bolster its antibody expertise and to expand its repertoire by developing new antibody therapeutics against cancer. So far, using the original drugs for other indications such as asthma has produced promising results, and Bell says they've "studied their antibodies in 15,000 to 20,000 patients." But they've struggled a bit in the process. The use of pexelizumab to limit complement-driven tissue damage in patients undergoing coronary artery bypass graft surgery had passed Phases I and II, but was discontinued this year after disappointing Phase III results. For now, says Bell, they "expect to divert all financial resources to support the completion of Soliris for treating PNH."

Bench-to-bedside stories like the one of eculizumab and PNH are rare, because "the contribution of the initial science" gets you only "10% of the way there," says Bell. "Humans are extremely complex."

"We focused on making CD59 a drug, and we never succeeded in doing that, but we did succeed in making a drug that replaces its function," he says. "What goes around comes around."

1. P.J. Sims et al., "Regulatory control of complement on blood platelets. Modulation of platelet procoagulant responses by a membrane inhibitor of the C5b-9 complex," J Biol Chem, 264:19228-35, 1989.
2. S.A. Rollins et al., "The complement-inhibitory activity of CD59 resides in its capacity to block incorporation of C9 into membrane C5b-9," J Immunol, 144:3478-83, 1990.
3. P. Hillmen et al., "Effect of Eculizumab on hemolysis and transfusion requirements in patients with paroxysmal nocturnal hemoglobinuria," N Engl J Med, 350:552-9, 2004.
4. A. Hill et al., "Sustained response and long-term safety of eculizumab in paroxysmal nocturnal hemoglobinuria," Blood, 106:2559-65, 2005.
5. A. Hill et al., "Protection of erythrocytes from human complement-mediated lysis by membrane-targeted recombinant soluble CD59: a new approach to PNH therapy," Blood, 107:2131-7, March 1, 2006.