NEWS

Targeted Toxins Begin to Live Up to Early Expectations

Tracy Thompson

Early results of a phase I study of the targeted toxin BL22 look promising: 11 of 16 patients with chemotherapy-resistant hairy cell leukemia had complete remissions lasting up to 18 months, most without major side effects. More importantly, the results, combined with other recent successes, have scientists cautiously optimistic that immunotoxins may be beginning to fulfill the promise that has been associated with them for decades.

BL22 is the result of more than 15 years of work by researchers who are attempting to design cancer therapeutics using recombinant DNA techniques, but the concept of linking a toxin to some sort of binding element that would seek out a cancer cell has been talked about for decades. In the last 20 years, monoclonal antibodies, recombinant DNA, and molecular biology techniques have progressed to make design of these molecules possible.

Logical Progression

The standard treatments for cancer—chemotherapy and radiation—kill dividing cells indiscriminately. Side effects caused by killing healthy, dividing cells in the patient are common, and occasionally the dose that is sufficient to kill cancer cells harms the patient. Also, cancer cells can develop resistance to standard therapies, leading to cancers that return and are untreatable.

Targeted toxins may be able to elude these pitfalls. "Immunotoxins seem to embody a design feature that has been missing from radio-chemotherapy efforts," said Edward Sausville, M.D., associate director of the National Cancer Institute’s Developmental Therapeutics Program.

Targeted toxins, therefore, emerged as an ingenious idea that seems simple enough: Make a molecule that will go specifically to cancer cells and deliver a cell-killing toxin. But developing such a molecule was a lot more complicated than anyone could have envisioned, Sausville said. "The positive feature is that the developments that have occurred have occurred in a very logical and stepwise fashion and illustrate that once you apply science to drug design you are able to ultimately deal with each of these issues."

The first hurdle to overcome was the choice of a toxin. Ira Pastan, M.D., chief of NCI’s Laboratory of Molecular Biology, along with David FitzGerald, Ph.D., and Mark Willingham, M.D., chose Pseudomonas exotoxin. "Pseudomonas [exotoxin] was a great toxin to be used in this way because it’s very active, it’s easy to make, and a fair amount was known about it, including its structure," said Pastan.



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Dr. Ira Pastan

 
Ellen Vitetta, M.D., director of the Cancer Immunobiology Center at the University of Texas Southwestern Medical Center at Dallas, chose ricin. Ricin is a powerful toxin produced by plants. Both ricin and Pseudomonas exotoxin are very potent and both work on a step of protein synthesis found in all animal cells.



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Dr. Ellen Vitetta

 
"It’s kind of a convergence in evolution," said Pastan. By halting protein synthesis, the toxins trigger apoptosis—programmed cell death. "There is something very special that these bacterial and plant toxins have learned, and I don’t know what it is," said Pastan. "The toxins have developed to be potent and essentially there are no resistance factors in animal cells other than the fact that the protein is foreign to the animal cells and is recognized by the immune system."

The first attempts at creating an immunotoxin were fairly simple: attach the toxin to an antibody. One piece of the Pseudomonas exotoxin structure contains a binding site that will bind to and destroy all cells. Pastan and colleagues attached the antibody to this structure hoping to destroy the toxin’s natural binding and replace it with the antibody’s targeted binding.

"This didn’t ever work very well," said Pastan. "So we thought we would delete the binding domain and take the rest of it and couple it to the antibody." They found that the resulting immunotoxin, LMB-1, elicited partial responses in breast and colon cancers in a phase I study. But LMB-1 was difficult to make, and they found that it was very large and therefore did not penetrate tumors very well.

Two more discoveries helped overcome these challenges. Pastan’s group learned that a small piece of an antibody can be made that contains only the recognition portion of the antibody. They also found that they could create Pseudomonas exotoxin minus its natural binding domain. They then fused the antibody portion to the toxin portion (see diagram). "These were our first recombinant toxins," said Pastan. "We made a whole bunch of them." With design features cloned together, they could produce large amounts easily.



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The diagram above illustrates how a gene encoding a recombinant immunotoxin is produced. The Fv of the antibody is cloned as a single-chain Fv and fused to the toxin gene from which domain Ia (the binding domain) is deleted. To increase stability, the single-chain Fv may be converted to a disulfide linked Fv.

 
They had produced a targeted toxin that was smaller and worked better, but it was not very stable. The logistics of drug use—storing it in the freezer, thawing it, and taking it to the patient—require that the immunotoxin be stable, and it is also important that it stay intact and active for many hours after injection. Further work improved the stability of the recombinant product.

Better Drugs Through Learning

Meanwhile, Vitetta found that immunotoxins have a crippling side effect in patients: they caused vascular leak syndrome. Vitetta suspects that the targeted toxins bind to the endothelial cells in the blood vessels causing them to leak serum into the surrounding tissues. Vascular leak syndrome can cause edema, low serum albumin, and low blood pressure, which sometimes requires intravenous fluid replacement and close monitoring of the patient and often necessitates discontinuation of the immunotoxin therapy. This side effect was limiting the dose that Vitetta could give to patients being treated with her ricin immunotoxin.

Vitetta noticed that some patients responded more severely than others. She found that patients who had been treated with radiotherapy tended to experience vascular leak syndrome, but those treated with chemotherapy did not. "Radiotherapy predisposes patients to vascular leak syndrome because it damages the vascular endothelium," said Vitetta. "When we remove those patients we haven’t found a maximum tolerated dose [for the immunotoxin]."

Vitetta now seeks to learn which part of the immunotoxin causes vascular endothelium damage. "We have created 15 different mutations in our immunotoxin and are in the final stages of screening them for activities," said Vitetta. She hopes to find a mutant that retains the ability to kill cancer cells but reduces the occurrence of vascular leak syndrome in radiotherapy-treated patients. "Half the battle is understanding what the problems are," Vitetta said.

The First Glimmers

Building on work in the field of immunotoxins, clinical investigation of Ontak® (denileukin diftitox) began in 1992. Ontak is a fusion protein containing the active portions of the diphtheria toxin and interleukin 2. IL-2 binds to the CD25 component of the IL-2 receptor. CD25 is found on the surface of many leukemias and lymphomas.

Ontak had been shown to delay tumor onset, extend overall survival, and result in long-term tumor-free survivorship in mouse models of lymphoma. Of course, it will kill only those cancer cells that display Ontak’s target, the CD25 component of the IL-2 receptor. Potential patients are screened with an immunohistochemical assay to detect the presence of CD25 on their cancer cells before treatment.

In a phase III clinical trial of Ontak involving 71 patients with late-stage cutaneous T-cell lymphoma who did not respond to standard therapies, tumor burden was reduced by at least half in 30% of the patients. In seven patients, the disease became undetectable. Some of these results were apparent within 6 weeks and lasted up to 2 years.

The outcome of the phase III trial resulted in accelerated approval of Ontak in the United States. The U.S. Food and Drug Administration issued a license for Ontak in February 1999, and Ligand Pharmaceuticals Inc., San Diego, currently distributes it.

Now, BL22 is showing similar early success. "If the phase I results can be reproduced in a phase II trial, we may be able to proceed to applying for FDA approval," said Robert Kreitman, M.D., a clinical researcher from NCI, who presented the phase I study results of BL22 at this year’s American Association for Cancer Research annual meeting.

Even with these positive findings, there is still much to do to further the field of targeted toxins. "There is clear evidence of activity in a number of different constructs. And certainly the Pastan-Kreitman drugs are very exciting opportunities," said Sausville.

"Although these things are called magic bullets and continue to have a lot of specificity, there are a lot of potential problems," he added. "They don’t have as great a degree of selectivity as one might want. There are actually a large number of other types of toxins that have activity to varying degrees. They may have a different therapeutic index."

One remaining challenge is to overcome the body’s natural defense. The immune system recognizes that the immunotoxin is a foreign protein and often mounts an attack. It produces human anti-mouse or human anti-human antibodies to the targeted toxin. Pastan is now trying to reduce this problem using molecular techniques to block the portions of the immunotoxin detected by the body’s immune system.

Pastan’s team also continues to search for targets that are present on cancer cells but not present on normal cells.

"[To avoid toxicity to the patient] you really, really need to have noteworthy separation and indeed absence of your target from normal tissue if you want to maximize the effect," Sausville said.

Although questions remain, the progress is undeniable.

"We have to find the right diseases that respond, the doses that have therapeutic windows, and convince the [pharmaceutical] companies to take on our expensive phase III trials," Vitetta said. "But you can rightly feel that there is reason for optimism."



             
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