Heat Shock Proteins

Fewer than 40 years ago heat shock proteins (HSPs) seemed to many researchers little more than a curiosity in Drosophila. Elizabeth Craig, professor of biomolecular chemistry at the University of Wisconsin, Madison, remembers that some scientists used to regard HSPs as "something weird that a fruit fly does." She has been studying these proteins and the genes responsible for them in yeast for more than two decades. Today HSPs are the object of intense work by scientists in the United States and abroad as a potential means of both providing vaccines to treat cancer and other diseases and increasing understanding of the immune system. HSPs as vaccines would be a novel approach to disease treatment. "They can be used as carriers, vectors, and in that regard offer a promising future," says Alberto Macario, a research physician at the New York State Health Department. Elizabeth Craig Discovery of these proteins is generally credited to Ferruccio M. Ritossa1 of the International Laboratory of Genetics and Biophysics in Naples. They are highly conserved and are expressed as a protective mechanism when Drosophila endures excessive heat. Other such proteins, which also have been dubbed chaperones, operate when there is no stress but ensure that newly synthesized proteins fold, permitting normal functioning. In the past few years, however, scientists in private companies and at universities have been working to exploit chaperones as protectors of the whole organism. A Potential Cancer Treatment Scientists break HSPs or stress proteins, as they are often called, into a series of families based on their molecular mass in kilodaltons. Among the families most actively being studied for use in vaccines are the HSP60 and 70 families. Pramod Srivastava, chairman of the Center for Immunotherapy at the University of Connecticut Health Center, has been working for more than 20 years to understand the role of HSPs and has developed HSP vaccines to treat cancer. Antigenics, the New York City-based company he founded, has begun Phase III trials of HSP cancer vaccines. Antigenics is one of the sponsors of an HSP conference scheduled for October 8-12 in Farmington, Conn. The trials are the latest step in efforts that began in 1980 to uncover the reasons behind a widely observed phenomenon: If a tumor is removed from a mouse or rat and killed, then injected as a vaccine, the animal in effect will be immunized against tumors that are transplanted into it. "I began to ask myself as a graduate student, 'What was it in the tumor that caused the immune response?'" Srivastava says. Eventually his efforts, which entailed examining various components of tumors for their immunogenic properties, led to the discovery that HSPs in the tumors produced an immune response. The next question he tackled: Why do these proteins produce an immune response in animals? "They shouldn't. They're everywhere in normal tissue," he says. What he discovered is that HSPs carry peptides, which are immunogenic. Srivastava has found receptors for HSPs on macrophages and dendritic cells, key antigen-presenting cells. "When you inject these things, they get targeted to macrophages and [dendritic cells] immediately. The peptides that are bound to the [HSPs] eventually get transferred to the macrophages and [dendritic cells], and they stimulate the T cells. That is the crucial part of the mechanism," he says. Srivastava's approach is to use autologous vaccines. He isolates HSPs from a tumor in a patient, purifies them, and injects them to fight the tumor. The result is stimulated T cells, part of the class I arm of the immune system, which attacks tumors. © 2000 Mark Winnett A New Way to Turn on the Immune System That finding, says William Welch, professor of surgery, medicine, and physiology at the University of California, San Francisco, is key in the potential to use HSP-based vaccines. "That's what people think is the important parameter for viral infections and for cancer, because those [T cells] have the ability to discriminate a cell that has a foreign antigen and kill it. It's that cellular-based arm of the immune system that really is the important one for us as it relates to keeping viral infections in check, as it relates to keeping tumors in place and eradicating them," says Welch, a founder of and scientific adviser to StressGen, a Victoria, British Columbia-based firm that is also conducting clinical trials using HSP-based vaccines. Normally, says Welch, when a foreign protein is in the bloodstream, antigen-presenting cells gobble it up, and it gives rise to antibodies. "However, it is starting to appear from research literature and StressGen science, and from Srivastava's work, that these HSPs injected into the bloodstream find their way into the class I antigen-presenting pathway. That is a huge leap; it's a brand-new concept that many scientists didn't believe was possible," asserts Welch. Injections of tumor-specific antigens in the past have elicited only "wimpy antibody responses," he says. This more robust and potentially more effective T-cell response with HSPs is triggering great excitement among immunologists, says Welch. To harness this powerful immune response, StressGen has been developing fusion proteins--combining a specific antigen with an HSP, explains Lee Mizzen, the company's director of research. Company researchers found that this combination elicited what Mizzen describes as "powerful" T-cell responses. "We've turned our attention to the type of disease in which you would require a CTL [cytotoxic lymphocyte] response. And that's why we've picked cancer and viral infections," he says. StressGen has targeted cervical and anal cancers, both of which are caused by human papillomavirus (HPV). The company has completed Phase I trials, has several Phase II trials in progress against both cancer and precancerous lesions, and is planning more trials, including some in conjunction with the National Cancer Institute. The company's immunotherapeutic, HSPE7, is a combination of a viral antigen, E7, and a heat shock protein, HSP65, from Mycobacterium bovis bacillus Calmette-Guérin. Recent preliminary Phase II data demonstrate HSPE7 activity in the treatment of HPV-related anal dysplasia. StressGen is planning a Phase III randomized, double-blind, placebo-controlled HSPE7 study. The company also is considering developing a vaccine for hepatitis B, a chronic viral infection. A TB Vaccine? Tuberculosis is another target of HSP vaccine work. Celio Silva, professor and head of the department of parasitology at the University of Sao Paulo, and his colleagues have recently reported successfully treating mice infected with the disease by injecting them with a vaccine based on the DNA for two HSPs from two different mycobacteria. The vaccinations altered the immune response "from one that is relatively inefficient and gives bacterial stasis to one that kills bacteria," the authors write.2 While HSP vaccines are exciting both basic and clinically oriented researchers, possible arising of autoimmune diseases may be a concern. Since humans possess these proteins, is there an HSP vaccine triggering an attack on the body as well as the disease? Srivastava dismisses the likelihood. "To me it's a nonconcern," he says. "We've treated a couple of hundred people so far, and there is absolutely no basis for a concern about autoimmunity," he argues. But Richard Morimoto, professor of biochemistry, molecular biology, and cell biology at Northwestern University, says of Srivastava's lack of concern about autoimmunity, "I think [that view is] silly." When it comes to the possibility of successfully treating a disease like cancer, the risk of autoimmune disease cer- tainly becomes rather minor, he argues. But "from a basic science perspective," the challenge is to modify HSPs used in vaccines so they may not present the possibility of stimulating an autoimmune response. Autoimmunity has been given a great deal of thought, says Mizzen. But "it is rare to point to a study that shows a direct causative relationship [between HSPs and autoimmunity]. What we have is a mechanism to trigger immune responses safely." He does, however, suggest that the final answer isn't in. "If you filled a room with 20 of the world's leading immunologists, I think you'd get a collection of opinions both pro and con." In spite of his concerns about autoimmunity, Morimoto sees great hope for heat shock-based vaccines. They offer an alternative to existing vaccine technology, which uses killed or attenuated organisms. "The idea that you could take a single purified protein and load into it a single peptide has a promise that is very exciting," he says. "I see it as ... promise for diseases such as cancer, but I ... find it equally important that there are some underlying basic science principles that may have a great deal of life to them." For Srivastava, the crucial answers are in the outcome of the clinical trials. "The only thing you believe is what happens in Phase III [trials]. So we don't know yet." Harvey Black (73773.2227@compuserve.com) is a freelance science writer in Madison, Wis. References 1. F.M. Ritossa, "A new puffing pattern induced by a temperature shock and DNP in Drosophila," Experimenta, 18:571-3, 1962. 2. D.B. Lowrie, "Therapy of tuberculosis in mice by DNA vaccination," Nature, 400:269-71, 1999.

Heat Shock Proteins

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