By David N. Leff
The warts of childhood are those rough, dark little bumps that sprout mostly on hands and arms — and usually go away by themselves.
Genital warts arise mostly on the external reproductive organs of sexually active grown-ups. They rarely disappear spontaneously; recombinant interferon is the therapeutic drug of choice.
Both of these warty excrescenses are the work of one pathogen, the human papillomavirus (HPV). But its worst depredation is to cause cervical cancer, which last year killed some 5,000 women in the U.S. alone. (See BioWorld Today, March 24, 1998, p. 1, and June 3, 1996, p. 1.)
"People guesstimate," observed immunologist Marvin Siegel, "that 30 to 50 percent of us who are 'sexually active' carry HPV of some type or other. The viral strains of genital warts," he added, "are different from those usually seen in cervical cancer."
Siegel is vice president of research and development at StressGen Biotechnologies Corp., in Victoria, British Columbia. As such, he co-authored a report presented Tuesday to the 1998 conference of FASEB — the Federation of American Societies of Experimental Biology, in San Francisco.
StressGen scientist Randall Chu presented the paper, titled "[Cervical] cancer immunotherapy using adjuvant-free, fusion protein encoding Mycobacterium bovis BCG HSP65 and HPV16 E7." His account of preclinical in vivo experiments showed, Chu concluded, "that administration of the fusion protein induced cellular anti-tumor immunity, which resulted in the regression of established tumor."
"The most prevalent viral strain in cervical cancer," Siegel pointed out, "is HPV16, and probably the next most common is HPV18. Both," he added, "are associated with cancers not just in the cervix but also anus, and there's a lot of suspicious information they may also be involved in some esophageal and oral cancers as well.
"It turns out," Siegel observed, "that the anatomies of cervix and anus are identical, in terms of the squamo-columnar junction of tissue and cell types. And of course, the major population that suffers from HPV-induced anal carcinoma is the gay population.
"Some 93 percent of all cervical cancers show HPV in them," he said. "So there is a big correlation between HPV infection by strain 16 or 18 and cervical malignancy, neoplasia or dysplasia, the precancerous early-stage precursors.
"Both strains," he went on, "carry two well-known oncogenes, E6 and E7. They bind to appropriate tumor suppressors and block their activity.
"The interesting thing in human cervical cancer and its earlier stages," Siegel noted, "is that in the advanced cancers, almost all the tumors show E7, and most of them E6 as well."
Recipe For A Double-Threat Cancer Vaccine
StressGen scientists constructed their recombinant anti-tumor package from two building blocks cloned in Escherichia coli host cells: a heat-shock protein derived from the M. bovis BCG (bovine tuberculosis marker) antigen, fused to the full-length E7 oncogene gene sequence, derived from the HPV16 strain.
"Heat-shock proteins [HSP]," Siegel explained, "are great stimulators of the immune system, specifically its killer T cell arm, more than the humoral antibody arm. There's an awful lot of data," he continued, "suggesting that if you really want to eliminate tumor cells, you're going to have to educate the cytotoxic lymphocytes, the CTL killer T cells, which carry CD8 receptors on their surface."
Siegel made the point that StressGen's heat-shock/E7 oncogene fusion antigen "engendered high cellular responses. When we put that purified vaccine," he recalled, "into an animal subcutaneously, it invariably mounted a vigorous response of the cellular kind against the tumor-specific antigen."
The two in vivo experiments that Chu reported to FASEB demonstrated the vaccine's prowess at preventing and abolishing the growth of cervical tumors in mice.
"The first experiment," Siegel quipped, "is the one that is the last resort of all scoundrels when they want to find out how something they made works.
"We immunized mice subcutaneously with this HSP-E7 vaccine," he recounted, "then, after waiting two weeks, gave them a booster shot. Another two weeks later, we injected the animals at a different site with a cell line constructed to represent cervical cancer tumors. These are tumor cells called TC-1. It's a cell line that's been transfected and transformed with HVP16-E6/E7 and ras oncogene — quite a good tumor model."
Siegel continued: "When we put this TC-1 into a control animal pretreated with saline, the tumors grew 100 percent to a readily measurable size.
"In animals pretreated by that HSP-E7 immunization protocol, the tumors started to grow, and were rejected by all the mice. When we waited for 40 or 45 days, then injected the animals that were clear of cancer with an additional load of TC-1 cells — as a putative model of cancer recurrence — those animals rejected the second TC-1 tumor inoculation.
"They'd been immunized in a prophylactic sense against the tumor occurring in the first place, and then again on its repeat."
Cancer Prevention, Therapy Both Worked In Mice
So far so good, but Siegel pointed out a clinical problem with that prophylactic model: "Women usually show up for treatment when they already have the cancer. So we went on to a therapeutic experiment, giving the animals the TC-1 tumor cells first, waiting till these grew up to a size we could measure, then injecting those tumor-bearing mice at a distal site with the HSP E7 vaccine. Their tumors went away.
"Again we waited," Siegel narrated, "for 40-something days to make sure the tumors were cleared, then again injected those animals with a second round of TC-1 cells. Again the cells started to grow, and again the animals rejected them.
"So the first part of the second experiment," Siegel summed up, "is that we could therapeutically reject an already-growing tumor, and second, if it were to recur — this is a model, not true recurrence — the animal is still protected immunologically against the tumor.
"Given that scientific and experimental background," he observed, "we decided that this is a molecule worthy of going forward in development."
StressGen has hired Covance Biotechnology Services, in Research Triangle Park, N.C., to manufacture the HSP-E7 fusion vaccine, and PPD Pharmaco, of Wilmington, N.C., to handle regulatory and documentation matters needed for interaction with FDA.
"Everything is working at the time line," Siegel concluded, "where all our studies and documentation should be finished, so we can file an investigational new drug (IND) application by the end of the year." *