By David N. Leff

Try matching the following list of risk factors to their disease:

* Top-scoring middle-school athletes

* Teen-agers, around 14 or 15 years old

* Male sex

* Long-limbed, big body build

This bill of medical attainder defines pediatric osteosarcoma, a rare, inherited bone cancer of arms and legs.

"Childhood osteosarcoma is a relatively rare disease," observed pediatric oncologist Fatih Uckun, director of the Wayne Hughes Institute, in St. Paul, Minn. "There are maybe fewer than 1,000 cases in the U.S. today," he added, "which represent approximately 60 percent of primary bone neoplasms among patients under the age of 20."

As recently as the 1970s, when a young person complained of local pain in a limb, by the time X-rays had diagnosed osteosarcoma the preferred treatment was immediate and radical amputation, one joint above the tumor, to capture any occult metastases. Yet by this time the cancer often had vaulted to its favorite metastatic target tissue, the lung.

Even heroic rounds of chemotherapy served only as a delaying action, with survival limited to months or a very few years.

That picture has brightened considerably, though not enough.

"When a patient presents with osteosarcoma," Uckun recounted, "the current mode of treatment begins with presurgical chemotherapy. Its aim is to reduce tumor size to such an extent that full amputation becomes unneccesary. Instead, surgeons resect the tumor without having to cut off the whole extremity. Treatment then continues with long-term chemotherapy."

If the patient already has metastases at the time of diagnosis, a different surgical effort is called for, Uckun explained. "Osteosarcoma macrometastases are very easy to remove from the body. They're almost like bullets or stones. The surgeon goes in and with his fingers plucks them out of the lungs."

A New Form Of Adjuvant Therapy

Since 1986, Uckun and his 125-person scientific staff at the Wayne Hughes Institutte [unrelated to the Howard Hughes Medical Institute] have been perfecting a tumor-targeting monoclonal antibody linked to a potent toxin as adjuvant treatment in various cancer therapies.

The key tumor-poisoning molecule, PAP (pokeweed antiviral protein) has been in FDA-approved clinical trials since 1991, notably a nationwide Phase III study now ongoing against childhood leukemia. An HIV human study has just begun.

Next on Uckun's hit list is osteosarcoma.

He is senior author of a paper in the July issue of the journal Clinical Cancer Research. Its title is "Antitumor activity of TP3(anti-p80)-pokeweed antiviral protein immunotoxin in hamster cheek pouch and severe combined immunodeficient [SCID] mouse xenograft models of human osteosarcoma."

"To build upon our existing system," Uckun told BioWorld Today, "we used an interesting antibody and made this conjugate against bone cancer, especially since clinical trials have shown we can no longer increase the intensity of chemotherapy for this disease.

"We felt that this conjugate might be beneficial in a setting where adjuvant therapy seems to be incredibly important, since those patients then come up with micrometastases and macrometastases in the lung."

"As we reported in the journal," he went on, "our initial work with this conjugate was in a mouse model with mouse sarcoma. We found that it eliminated the ability of that tumor to metastasize to the lung. This was a mouse tumor in mice, so it was a little bit more artificial than human tumor cells. We really didn't know whether or not it would kill human osteosarcoma cells."

As a preliminary test of this question, the co-authors planted human osteosarcoma cells in the cheek pouches of Golden Syrian hamsters. "The cheek pouch is an immune-privileged site," Uckun pointed out, "so you can put in tumors as they grow, and very easily visualize their blood vessels. That model did indeed cause those tumors to shrink.

"So we went ahead and looked at SCID mice xenografted with human tumors," he continued, "and these tumors grew around their femur, around their legs. They grew very big tumors that invaded the soft tissue inside the bone, mimicking the clinical situation.

"In the TP-3/PAP system, the conjugate worked to prevent the tumor from emerging. This model allowed us to get long-term, tumor-free survival in the mouse. When we looked at good-size established tumors, it had delaying effects, but was not as effective as against smaller tumors.

"The effectiveness," Uckun went on, "seemed to be dependent on the conjugate dose and on the size of the tumor. It should be very effective in a clinical setting, in which the tumor will be removed surgically, and you would then put patients on this kind of agent to prevent emergence of tumor, and metastases to the lung."

A Different Use For Pokeweed

Uckun extracts and purifies his PAP toxin from the common, garden-variety pokeweed (Phytolacca americana). This plant is best known to immunologists as the source of pokeweed mitogen, which stimulates B and T cells.

But "pokeweed mitogen is not the same thing as pokeweed antiviral protein, PAP," Uckun pointed out. "The mitogen comes from the plant's roots, the antiviral protein from its leaves. It sits in the wall of the plant cells," he explained, "and protects them from infection by viruses.

"It's an apoptotic — suicidal — protein," he went on. "When a virus attacks a plant cell, this PAP substance kills it.

"The PAP enzyme," Uckun narrated, "works by depurinating ribosomes. In other words, what happens is that protein synthesis is inhibited enzymatically, irreversibly. It's calculated that one to 10 molecules of PAP will inactivate all the ribosome in a mammalian cell.

"In many ways," he noted, "PAP is similar to diphtheria toxin or ricin, which are also used in anti-tumor immunoconjugates. The difference is that unlike these, it does not have a cell-binding chain portion. So conjugates containing PAP bind to the target cancer cells only through their antigen portion."

As for the TP3 tumor-antigen-seeking antibody, Uckun originally obtained its cell line from its discoverer, oncologist Oyvind Bruland of the Norwegian Radium Hospital, in Oslo.

"The antibody has been used in Europe in patients for imaging purposes as a radioimmune-conjugate," Uckun said. "It's highly selective, and allows a high-sensitivity picture of osteosarcoma metastases. We acquired it from the Norwegians for therapeutic development.

"It is highly selective for osteosarcoma cells. It does not link with any other normal human tissues, except for placenta. This is no problem, as our patients are all children."

Uckun was director of the Biotherapy Institute at the University of Minnesota in 1995 when he received a call from an oncological colleague at the University of Southern California, in Los Angeles.

"What happened was," he recounted, "he had a leukemia patient who was sent home to die in Los Angeles, because there was really nothing more to do. This child came to us, received our anti-leukemia conjugate and went into complete remission.

"It turned out that the patient's father, Wayne Hughes, was a very wealthy gentleman, a billionaire, who told me basically, 'This therapy that saved my child's life should be made available in all forms to all children.' He made a major endowment, with a verbal commitment of $200 million to establish the Wayne Hughes Institute, with me as director. We already have the initial $40 million in hand.

"When I left the university," Uckun concluded, "all of the faculty members in my department, 25 of them, went with me. Then we recruited an additional hundred scientists, so we now have 125 Ph.D.s or MD-Ph.D.s at the institute, all dedicated to drug discovery." *

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