Coley Toxin's Hidden Message

From a 19th century mystery comes a potential new class of drugs: CpG oligonucleotides

By Tom Hollon

Few drug discovery stories have offered researchers as many chances for dismissive disbelief as the one William B. Coley launched with his bacterial lysate treatments for cancer. If, perhaps, he looks down from above, he's probably watching the development of immunostimulatory oligonucleotides with a keen sense of excitement and anticipation. "For who would have thought," marvels Robert L. Bratzler, CEO of Coley Pharmaceuticals Group, "that DNA, which was not supposed to have immune stimulation properties, would be so potent?"

Coley was a New York bone surgeon with an interest in cancer. When he learned of a cancer survivor who coincidentally had suffered severe skin infection caused by Streptococcus pyogenes, he wondered if the bacteria had caused the patient's tumor to regress. In the 1890s, he began injecting cancer patients with the crude bacterial preparations that became known as Coley's toxins. Coley treated nearly 900 patients, and claimed that some 40 percent achieved lengthy remissions.

For a brief period Parke Davis sold Coley toxins, but production stopped in 1953. At the time the active ingredient was thought to be lipopolysaccharide. But many who tried to repeat his results failed, and the father of immunotherapy endured decades of derision. According to history, he did it with much grace.

Courtesy Coley Pharmaceuticals Group

CpG oligonucleotides influence both antibody and cell-mediated immunity. Applications include vaccine adjuvants, taming allergic reactions, and potentiating monoclonal antibodies and cytotoxic immune cells.

In the 1980s, Japanese researchers looked anew for the active agent in Coley's toxin.¹ Working with fractions of Bacillus Calmette-Guerin (BCG), bacteria best known in connection with tuberculosis vaccination, they concluded BCG's antitumor activity was a property of its DNA. An explanation for the effect eluded everyone for more than a decade, until Arthur Krieg realized that without knowing it, he had taken up where they left off.

In 1993, Krieg, a rheumatologist, reluctantly decided to find a new research project. His research at the University of Iowa wasn't working, "and I knew as an academic that you better find something that is, or you'll soon be out of grants." Krieg was puzzled by a connection between DNA and immunity he couldn't explain. Working on B cell gene inhibition with antisense oligonucleotides, he was upset that oligos supposed to be controls were stimulating mouse B cells. There were by this time a couple of reports in the antisense literature of similar misbehavior, but no explanation. Krieg decided to find one.

"It took me two years, but I finally figured out that if you had a C followed by a G, and if the right bases were upstream and downstream, then you got immune activation." Later, in a thrilling moment of insight, he understood why. "All of a sudden, it just dawned on me that CGs are unmethylated in germ DNA, but their cytosines methylated in our own [DNA]. My oligos had been unmethylated. It's the usual way of DNA synthesis. I realized that my oligos could be mimics for germ DNA." When Krieg methylated CpG in his oligos (p stands for the phosphodiester connecting the bases), their immune stimulation ceased.

What the Japanese saw, he thought, was the destruction of tumors caught in immune responses provoked by unmethylated BCG DNA. Krieg empathized with the way the scientific community had ignored their news: "It was just inconceivable. How could the immune system possibly tell BCG DNA from human DNA? It didn't fit any of the prevailing paradigms of how the immune system worked."

Immune response to CpG explained oddities that popped up not just in laboratories, but also in some of the antisense clinical trials. Hybridon, of Cambridge, Mass., noticed the problem in 1996 in a dose escalation study of GEM 91 as a treatment for HIV. It was an encounter with what Sudhir Agrawal, the company's chief scientific officer, calls DNA's hidden message: the properties no one knew about until researchers started testing DNA as a drug. The lower doses were safe. But as they went up, recalls Agrawal, "We found flu-like symptoms like muscle ache and fever that were very difficult to correlate to DNA." Going back to the lab to find out why, they discovered GEM 91 harbored an immunostimulatory CpG.²

Once Again, Mice Aren't Men

The commotion Krieg set off by publishing his results³ was brief. Several groups verified his work with mice, but the real question was what about humans. When results with human immune cells and CpG oligos came in, they had all the sparkle and effervescence of day-old champagne. CpG's effect was nil.

Lucky once, now Krieg was lucky twice: "What I discovered, after a lot more work, was that the sequences that activate human cells are different from those that activate mouse cells." GACGTT, for instance, is a very good CpG activator motif in mouse cells; GTCGTT is very good for humans. Researchers had also been unaware how quickly human cells degrade DNA. "If you use experimental conditions that worked fine with mouse cells," he notes, "you get no results in human cells because they're simply degrading the DNA."

With this discovery, testing CpG oligos on human immune cells began in earnest. Biotech companies formed to fashion immunostimulatory oligos. Krieg, who founded Coley in 1997, is heading to Wellesley, Mass, finally going full time as its scientific director. Another company devoted to CpG oligos is Dynavax Technologies, in Berkeley, Calif. Hybridon, a third company in the field, divides its efforts between antisense drugs and CpG.

Several drugs already on the market stimulate the immune system in various ways, prominently the interferons and colony stimulating factors. Krieg asserts that oligos are entirely different: "CpG activates the immune system in the way it evolved to be activated, by detection of bacterial products. You activate a well-orchestrated symphony of responses that synergize with each other." Everything enters on cue: cells, cytokines, and actions. Toxicity is minimal. Immune factors, although great medicines, drive "an artificial, disorganized response. You have to use very high doses to get any activity, and because of that, you also get a lot of toxicity."

Scores and Players

It was clear from the outset that not all CpG oligos were alike, as antisense clinical trials proved that some CpG dinucleotides escaped notice by the immune system. Work proceeding in haste among the competitors reveals CpG oligos that do stimulate immune cells are different indeed. "The oligo sequence is like a score," says Coley Pharmaceuticals' Bratzler, who speaks of the drug screening process as listening for the players--the cytokine combinations--who read it in different cell types. Each CpG oligo has a profile of cell types affected and cytokines stimulated; and usually each leans either toward the Th1 (cell mediated) or Th2 (antibody) T helper cell pathway.⁴

Coley's CpG 7909, for instance, boosts antibody and cell-mediated responses to pathogens and cancerous cells. CpG 8916 activates natural killer (NK) cells, which can attack infected cells and NK-sensitive cancers such as melanoma. CpG 8954 stimulates interferon-alpha production. It might have use as a prodrug, providing a low toxicity way to stimulate interferon alpha against cancers and viral infections.

Hybridon too has been busy learning what happens as bases near CpG change. Building on a decade of research on antisense base chemistry, Hybridon CpG oligos (14 to 18-mers) mix natural and synthetic bases. It has several immunostimulatory YpG motifs (pyrimidine analogs in place of cytosine) and CpR motifs (purine stand-ins for guanine). Their oligos make effective immune stimulators, according to Agrawal. The harvest of Hybridon's labor is a set of oligos driving a range of immune response, dialing it up or down. The company will pursue clinical trials through joint ventures, combining its chemical know-how with partners' disease expertise and financial backing.

Dynavax's lead CpG oligo, now in Phase II, treats ragweed allergy, where the Th2 pathway is implicated. Inflammation, histamine release, and runny nose can be treated by shifting the immune response to the Th1 side, according to Andrew Gengos, who is in charge of business development at Dynavax. "We've found that CpG oligos can switch the immune system from the Th2 to Th1."

All three companies are developing CpG oligos as vaccine adjuvants. Krieg comments that these adjuvants illustrate how the simplified idea of Th1 vs. Th2 sometimes breaks down. While other adjuvants give much more of a Th2 effect, CpG adjuvant oligos recruit strong Th1 support as well. That's why he thinks they make "fabulous" adjuvants--"You get antibodies and a strong cellular immune response."

Dynavax and Coley have products in clinical trials to boost the effectiveness of hepatitis B vaccine,⁵ where current prophylactic treatment requires three vaccine injections over six months. "One market is people who travel," Gengos explains. "It takes a lot of foresight to start a series of shots before traveling, especially if it's business traveling." Cutting the number of shots would be a big advance. Besides that, he adds that not everyone is responding to the vaccine. "Sometimes the elderly can't develop a good enough response to be protective, he says. "A more amplified immune response would be an improvement."

CpG oligos with strong Th1 effects may find a place in cancer therapy. Krieg comments that the strongest Th1 stimulant he has ever seen is CpG. Activating Th1 "is exactly what you want in cancer therapy," he declares, "and something everyone's had a hard time getting. It tells the immune system, 'This is an infected cell, kill it.'" Coley's experiments with mice that have large, established tumors show that injecting CpG oligos into tumors saves mice that otherwise die. Clinical trials under way with CpG 7909 treating relapsed or refractory non-Hodgkin's lymphoma will ask if the same thing is possible in humans.

The next goal, says Agrawal, "is to find out if we can custom design oligos that induce the cytokines we choose." This requires explaining how CpG oligos can be so different, which in turn probably depends on understanding CpG receptors and their signaling transduction pathways. CpG oligos exert their effects not at the cell surface, but from inside. Two receptors have been identified, but research on how they work is only beginning.

A clearer picture also requires understanding which cells they activate directly and which indirectly. Distinguishing between the two is a knotty task, because immune cells, once activated, secrete factors that activate other cell types. So far, it's known that CpG oligos directly activate plasmacytoid dendritic cells and B cells.⁶

After all this is done, there still won't be a final answer for one question: What was Coley's toxin all about? When the sun goes down, and the day's work is done, a good question to clink beer mugs over is whether unmethylated DNA was what made Coley's toxin work. "We would like to think so," Krieg muses, "But no one ever determined what the active ingredient was. In the Japanese BCG case, the active ingredient was DNA. In mice it appears our oligonucleotides are doing the same thing, but with much less toxicity. So it's not too farfetched to speculate that the active ingredient in Coley's toxin was DNA."

Tom Hollon (thollon@...) is a freelance writer in Rockville, Md.

References

1. T. Tokunaga et al., "Antitumor activity of deoxyribonucleic acid fraction from Mycobacterium bovis BCG: Isolation, physicochemical characterization, and antitumor activity," Journal of the National Cancer Institute, 72:955-62, 1984.

2. Q. Zhao et al., "Effect of different chemically modified oligodeoxynucleotides on immune stimulation," Biochemical Pharmacology 51:173-82, 1996.

3. A.M. Krieg et al, "CpG motifs in bacterial DNA trigger direct B-cell activation," Nature, 374:546-9, 1995.

4. G. Hartmann et al., "CpG DNA: A potent signal for growth, activation, and maturation of human dendritic cells," Proceedings of the National Academy of Sciences, 96:9305-10, 1999.

5. M.J. McCluskie, et al., "CpG DNA is an effective oral adjuvant to protein antigens in mice," Vaccine, 19[7-8]:950-7, Nov. 22, 2000.

6. T. Sparwasser et al., "Bacterial CpG-DNA activates dendritic cells in vivo: T helper cell-independent cytotoxic T cell responses to soluble proteins," European Journal of Immunology 30:3591-7, 2000.