Science magazine, 14 April 2006
Tens of thousands of horticultural enthusiasts who attended a 1999 flower show in Holland were potentially exposed to something far more insidious than the sight and fragrance of exotic blossoms. Legionella pneumophila, the bacterium that causes Legionnaire’s disease, contaminated the water in a whirlpool spa on display and a sprinkler system that misted the flowers, which probably dispersed the bacterium widely. More than 100 people fell ill, and 18 died.
The tragedy piqued the curiosity of infectious disease specialist Annelies Verbon of the Academic Hospital Maastricht in the Netherlands. Why did a handful of people get sick, whereas most came away unharmed? Verbon, along with a research team led by immunologist Alan Aderem of the Institute for Systems Biology in Seattle, Washington, and Thomas Hawn, an infectious disease specialist at the University of Washington Medical Center in Seattle, came up with an intriguing answer. When the researchers analyzed the DNA of groups of attendees, they discovered that a higher percentage of those who fell ill carried a single mutation in the gene for an immune cell surface protein called toll-like receptor (TLR) 5. The team found that the mutation cripples the receptor, apparently preventing immune cells from recognizing the whiplike tail of L. pneumophila and rendering those with the mutation more susceptible to severe Legionnaire’s disease.
That finding, published in 2003, is part of an avalanche of research on TLRs. TLR5 is a member of a family of at least 10 human TLRs discovered within the past 7 years. Their discovery has “revolutionized immunology,” in the words of Luke O’Neill, an immunologist at Trinity College in Dublin, Ireland.
Scientists had long known that the immune system has an initial line of defense that triggers the inflammatory response and recruits B and T cells to mount an attack on invading microbes. But they didn’t know how that initial alarm was sounded. TLRs have turned out to be the key. “These are the sensors of the microbial world that turn on the immune system,” says O’Neill. And more recent evidence suggests that they also play important roles in autoimmunity and inflammatory conditions such as heart disease.
All this has caught the collective fancies of immunologists and biomedical researchers around the globe. Some 3500 papers on these immune sentinels have appeared to date, and at least nine biotech companies have compounds that stimulate or inhibit TLRs in clinical and preclinical trials for illnesses as diverse as hepatitis, cancer, asthma, and allergies. In the past year, big pharma has moved in too, with companies such as GlaxoSmithKline and Novartis buying up or inking deals with smaller firms developing TLR-based drugs. Commercial interest in TLRs is “suddenly hot,” says O’Neill, who has himself founded a company, Opsona Therapeutics in Dublin, that is now collaborating with Wyeth Pharmaceuticals on TLR-targeting drugs for inflammatory diseases.
Many more experimental TLR-based drugs are in earlier stages of development, including ones designed to treat or prevent allergies, inflammatory bowel disease, and autoimmune afflictions. Basic researchers, meanwhile, are uncovering a slew of additional drug targets as they identify the cascade of enzymes and other molecules involved in transmitting the messages sent by TLRs to the interior of the cell. In the past 3 years, researchers have unveiled 15 previously unknown proteins activated by TLRs and have now cataloged about 30 of the estimated 40 to 50 components of TLR signaling pathways, says Bruce Beutler, an immunologist at the Scripps Research Institute in San Diego, California.
Of course, many hurdles remain before most of these compounds can be marketed. And some, such as pediatric immunologist Jean-Laurent Casanova of Necker Medical School in Paris, contend that more work must be done to prove the importance of TLRs in human immunity, as most studies on these receptors involve animal models of infection. “Enthusiasm for TLRs is deserved,” cautions Casanova, “but it should be tempered.”
Weird discovery
The explosion of interest in human TLRs had humble beginnings: the 1988 discovery of a protein involved in fruit fly development. German researchers noted that flies lacking the protein looked toll, the German word for “weird” or “far out.” The insects’ bodies were disordered, with bottom body parts mixed with parts that belonged on top, and vice versa. It wasn’t until 1996 that scientists learned that the protein, Toll, had a second job, helping defend against fungi. Flies with mutated versions of the protein or its signaling partners more readily succumbed to fungal infections, according to work by Jules Hoffmann and colleagues at the National Center for Scientific Research in Strasbourg, France.
That curious finding led immunologists to wonder whether humans have a corresponding protein. Two years earlier, it turned out, Nobuo Nomura and his colleagues at the Kazusa DNA Research Institute in Chiba, Japan, had unveiled a human protein that bore strong resemblance to the fly Toll. It was later dubbed TLR1. In 1997, Yale University scientists unveiled the second human TLR (now called TLR4), and soon thereafter, researchers at DNAX in Palo Alto, California, identified five more TLR proteins in people.
A year later, Beutler and his colleagues provided the first evidence that a TLR plays a part in mammalian immunity: They found that mice with a defective version of TLR4 could survive injections of endotoxin, a cell-wall component of some bacteria that invariably kills normal mice and in humans can incite a deadly inflammatory disorder called sepsis. The work, Beutler says, “proved that TLR4 was the receptor for endotoxin.” The receptor suddenly became a hot drug target for sepsis, a killer of 750,000 Americans each year.
After that, discoveries came thick and fast. Immunologist Shizuo Akira of Osaka University in Japan and his colleagues systematically deleted the mouse genes for TLRs to reveal their specific functions. Several groups determined which compounds trigger individual receptors. Akira, in collaboration with Aderem and Hawn, for example, determined that flagellin, a protein in the flagella of bacteria, activates TLR5. Others traced out signaling pathways inside the cell that connect to the TLRs, discovering that triggering these receptors can prompt a range of immune responses. These include the release of cytokines such as interferon—a powerful antiviral agent—and other messenger molecules that stimulate the immune system’s storm troopers, T cells and antibody-producing B cells.
The unveiling of TLRs and their specific activities has given immunologists new respect for the body’s frontline defenses, known as the innate immune system. Long considered an evolutionary holdover that provides broad protection before the more specific adaptive immune system of T cells and B cells kicks in, innate immunity is turning out to wield a rather precise set of counter-measures. It is the TLRs that direct the innate immune system’s responses, and immunologists are realizing that those responses in turn guide the subsequent adaptive immune reaction that is even more targeted. “The two go hand in glove,” says O’Neill. “You wouldn’t get any adaptive response without the innate.” Adds Kleanthis Xanthopoulos, who heads Anadys Pharmaceuticals, a San Diego firm developing antiviral drugs that target TLRs: “The innate immune system had always been viewed as the ‘low-tech immune system.’ Only now do we understand that innate immunity has significant molecular specificity.”
Revving up immune responses
The more scientists learn about TLRs, the more excited they—and drug company executives—get about the potential of stimulating the receptors to combat everything from infectious diseases to cancer. Among the early targets is the hepatitis C virus. Once the virus sets up a chronic infection, it can be very difficult to dislodge: The current treatment—nearly a year of interferon injections combined with an oral antiviral—clears only about half the infections with the most resistant strain, and its side effects can be debilitating. Anadys recently tested a compound that stimulates TLR7 on 32 patients chronically infected with hepatitis C. The injectable drug lowered blood levels of the virus by 82%—a response comparable to injected interferon—in eight of the 12 patients who received the highest dose daily for a week. Only a few patients had side effects, and those were mild to moderate, Anadys scientists reported in the September 2005 issue of Hepatology. The results were promising enough to inspire Novartis to collaborate with Anadys on a pill version of the drug.
Coley Pharmaceutical Group in Wellesley, Massachusetts, a company founded by University of Iowa immunologist Arthur Krieg, has also begun clinical tests of a TLR stimulator aimed at hepatitis C. In a trial with 60 patients, a drug called Actilon that stimulates TLR9 elicited a dose-dependent release of the body’s own interferon-α without producing serious side effects, Coley scientists reported at the November 2005 meeting of the American Association for the Study of Liver Diseases. Among the 13 patients who received the highest dose levels of Actilon once or twice a week for 4 weeks, 11 achieved greater than 90% reduction in blood levels of viral RNA.
Krieg started working on TLR stimulators in the mid-1990s—but he wasn’t aware of it at the time. He discovered that short sequences of nucleotides containing cytosine (C) followed by guanine (G), which are common in viruses and bacteria, could powerfully activate B cells in mice. At about the same time, immunologist Eyal Raz and cancer biologist Dennis Carson of the University of California, San Diego (UCSD), and their colleagues observed that T cells respond to loops of bacterial DNA called plasmids, which also contain these so-called CpG sequences. In 2000, Akira’s group connected both sets of findings to the world of TLRs: The researchers reported that mice lacking TLR9 fail to generate the immune response typically produced by CpG DNA. Since then, several groups, including Krieg’s, have used CpG sequences to construct TLR stimulators for a variety of applications, including cancer treatments.
At a cancer meeting last year, Coley scientists and their collaborators reported that one such compound—a 24-base DNA sequence with four CG pairs—plus chemotherapy shrank lung tumors in 37% of 75 patients with advanced forms of lung cancer, compared to just 19% of 37 patients who received chemotherapy alone. The data also show a positive trend toward increased survival after 1 year. The results are “preliminary—but potentially very exciting,” says Trinity’s O’Neill. Pfizer, which pledged up to $515 million for the rights to develop the Coley drug last year, started large-scale trials in November.
Several companies are using TLR stimulants as adjuvants, boosting the immune system’s response to vaccines to increase their efficacy. Coley has developed a CpG-based adjuvant called VaxImmune that seems to improve the human immune response to the anthrax vaccine. GlaxoSmithKline recently bought Corixa, a Seattle-based company devoted to TLR therapeutics, for $300 million and is sponsoring large-scale trials of Corixa’s MPL, a derivative of bacterial endotoxin that stimulates TLR4, as an adjuvant in a vaccine against human papillomavirus, which causes cervical cancer. And scientists at Dynavax in Berkeley, California, are conducting large-scale human trials of a hepatitis B vaccine in which a CpG DNA sequence is linked to a surface protein from the virus. Early trials suggest that the adjuvant generates a more robust immune response in older adults—and a faster response in young adults—than does the current vaccine.
Dampening immunity
In addition to revving up immune defenses, researchers are also investigating ways of using TLRs to turn them down. One such strategy may provide faster and safer immunotherapy for allergic conditions.
Current allergy immunotherapy regimens are cumbersome, requiring weekly or monthly shots for 3 to 5 years. They can also be dangerous, causing symptoms from swelling to anaphylaxis in some patients. UCSD’s Raz and Carson have discovered that bacterial CpG DNA may offer another solution. By stimulating TLR9, such DNA spurs macrophages and other innate immune cells to kill the so-called T helper 2 (TH2) cells whose overzealous activity characterizes allergy and asthma. Following up on the UCSD team’s work, Dynavax is developing medication that may safely prevent allergies in just 6 weeks. The compound—a CpG molecule bound to DNA encoding the culprit allergen—elicits an anti-TH2 response in animals that is stronger than that from separately administered CpG DNA. The company has already sponsored some clinical tests of this strategy and at the March meeting of the American Association of Allergy, Asthma & Immunology reported promising results for ragweed-allergic adults.
A more obvious way of taming the immune system through TLRs is to block one or more of the receptors. This may be useful for inflammatory conditions such as the autoimmune disorder lupus and sepsis. Last year, for example, Seattle’s Hawn and Aderem reported that the nonworking TLR5 variant they had implicated as a risk factor in Legionnaire’s disease was about half as common in lupus patients as it was in their unaffected relatives, suggesting that inactivating TLR5 may protect people against the disorder. And researchers at Eisai Inc. in Teaneck, New Jersey, are already testing a TLR-blocking compound against sepsis. The compound, dubbed eritoran, is a molecular mimic of a portion of the endotoxin molecule; it binds with TLR4 but does not activate it. In a trial of nearly 300 hospital patients diagnosed with sepsis, a large dose of eritoran reduced the death rate by 12%, compared to a placebo, in the 80% of participants who fully complied with the regimen. In the patients at highest risk for death, a large eritoran dose reduced the death rate by 18%, Eisai announced in August. Beutler calls the results “encouraging,” given how hard it is to administer a drug soon enough to save sepsis patients. The work also shows that TLR blockers are possible. “It sets a precedent for making small molecule antagonists to other TLRs,” he says.
Hope for the heart
Drugs that hinder TLRs might one day treat heart disease as well. Accumulating evidence suggests that TLR activity contributes to atherosclerosis, perhaps by stimulating inflammation.
Hints of this role appeared in 2001 when Moshe Arditi, an infectious disease specialist at Cedars-Sinai Medical Center in Los Angeles, California, and his colleagues found that TLR4 was abundant in atherosclerotic plaques from the coronary arteries of five patients needing bypass surgery but scarce in four normal arteries. More recently, they have found that atherosclerosis-prone mice lacking the TLR4 gene developed arterial lesions that were about 25% smaller than those in the same mice with the TLR4 gene. And if such mice lacked the gene for MyD88, which is involved in the signaling of several TLRs, the atherosclerosis in their aortas dropped by nearly 60%, the researchers reported in 2004. The study “really shows that TLR4 and MyD88 play a role in the production of atherosclerosis,” says Arditi.
TLR1 and TLR2 are also implicated. These receptors have been found to be much more prevalent in plaque-pocked human arteries than in normal ones. And when immunologist Linda Curtiss and her colleagues at The Scripps Research Institute deleted the TLR2 gene in atherosclerosis-prone mice, the animals’ arterial lesions shrank by almost 50% compared to similar mice that retained the TLR2 gene. What’s more, injecting susceptible mice with a compound that activates TLR2 greatly worsened their lesions.
“It looks like the innate immune system is going to be very clearly involved in the pathogenesis of atherosclerosis,” says Arditi. If so, new treatments could include drugs that block TLR4, TLR2, and MyD88. Such drugs might cripple a person’s infection-fighting ability if delivered systemically, but administering them locally to the arteries could reduce that risk, Arditi suggests.
Lingering doubts
Despite this flurry of activity, there is still uncertainty about the receptors’ role in human immunity. There are rare cases in which people have a mutation in an enzyme in the signaling pathway shared by TLRs and the interleukin-1 (IL-1) receptor, making the receptors useless, and yet these people still seem to resist most types of viral, fungal, and parasitic infections, Necker Medical School’s Casanova and his colleagues reported in Science in 2003 (28 March 2003, p. 2076). What immune impairment these people do experience could conceivably be caused by an IL-1 pathway defect, Casanova suggests. Because there is no human genetic defect that selectively impacts the TLRs, Casanova says, “there is so far no conclusive evidence that human TLRs are critical, nonredundant players in protective immunity in infection.”
Even if TLRs are vital and unique players in human immunity, as many researchers believe, almost all of the TLR-based experimental therapies must undergo further testing. It’s also worth remembering that turning up or down the volume of the human immune system—by means of TLRs or any other method—is a risky proposition. Attempts to dampen dangerous inflammatory responses by means of TLRs could cripple the immune system and cause patients to succumb more easily to infectious diseases, for example. On the other hand, TLR-stimulating compounds could rev up the immune system too much or for too long, leading to sepsis or autoimmune diseases.
“These are huge challenges,” says Beutler. “But they could have rich payoffs.” A growing number of companies are betting on that.