New Weapons Against HIV

As the AIDS pandemic enters its third decade, viral resistance is beginning to counter the success of "highly active antiretroviral treatment" (HAART), the multidrug cocktails introduced in 1996. "Viral resistance is a significant problem, particularly for patients who began therapy in the pre-protease inhibitor era and who developed resistance to multiple reverse transcriptase inhibitors," says Robert Schooley, a professor of infectious diseases at the University of Colorado Health Sciences Center and national head of the AIDS Clinical Trials Group. The need for a new pharmaceutical strategy is more compelling than ever. Several novel and diverse drug candidates, however, are on the horizon. According to the World Health Organization, at least 42 million people worldwide are HIV positive, and each day, 16,000 more become infected. Viral resistance, an inevitability of molecular evolution, arises when treatments do not totally suppress replication, so that variants can flourish. "HIV so far has been able to resist anything. There are so many viral variants that the chance that one can overcome a specific inhibition is certainly there," explains Dani Bolognesi, chief executive officer of Trimeris Corp. of Durham, N.C. "We now combine a number of different inhibitors that operate under different mechanisms so the chance that HIV replicates and spawns a variant that is resistant to all are low," A mathematical model based on the rate of increase of drug-resistant HIV infections in San Francisco predicts that by 2005, 42 percent of the city's cases will be resistant.1 The study also revealed that resistance arises more often within individuals as their resident viruses mutate, than by passing from person to person in drug-resistant forms, as is the case for antibiotic-resistant bacteria. And that means that people can't avoid resistance simply by avoiding people--it comes from within. "Our recent analysis shows that it is absolutely critical for new and better drugs to be developed and used to treat HIV. It is essential that the new drugs be extremely effective in viral suppression, which will minimize the probability of resistance," says Sally Blower, a professor in the department of biomathematics at the University of California at Los Angeles AIDS Institute and lead author of the study, which took place at the University of California, San Francisco. New approaches to HIV complement existing HAART "cocktails." The entry inhibitors zero in on the chemical choreography of HIV approaching, contacting, attaching co-receptors CD4 and CCR5, and finally fusing with and traversing the cell membrane. The excitement in the AIDS research community for these drugs is high. "The entry inhibitors can and will turn into successful drugs," enthuses John Moore, a professor in the department of microbiology and immunology at Weill Cornell Medical College in New York City, who is collaborating with several groups in developing CCR5 and attachment inhibitors. Other coming treatments amplify innate antibody defenses or realign the immune system. Enter Entry Inhibitors Farthest along in the pipeline is a drug called, for now, T-20, from Trimeris. It is a 36-amino-acid-long peptide that corresponds to a key part of gp41, the glycoproteins that anchor the gp120 spokes that emanate from HIV and contact the cell membrane. When T-20 aligns itself with the part of gp41 that extends from the viral envelope, the virus can no longer fuse with the cell. ©2001 Trimeris Inc & Hoffmann-Laroche, LtdT-20 (yellow coils) binds to matching coil segments that are part of HIV's gp41 (blue threads and red coils), preventing viral fusion with the cell membrane of the CD4+ T cell below. The road to T-20 began in 1989, when Tom Matthews, today senior vice president of research and development at Trimeris but then an associate professor of medicine and a member of Bolognesi's research group at the AIDS Center at Duke University, was looking for epitopes on the HIV envelope that might serve as the basis of a vaccine. "He came upon part of gp41 that had a unique two-dimensional helical part," recalls Bolognesi. It resembled the spring-loaded guise of the influenza virus as it infects, a loop that contorts into a coil. Because the researchers knew that this epitope in other viruses could elicit antibody production, they synthesized the key helical part and added it to T cells in culture exposed to HIV. "Tom decided to test it, not as an elicitor of an immune response, but to see if it had any antiviral activity. And lo and behold, it did! Not only was it active, it was very active. He came racing upstairs saying, 'I think we have something very worthwhile here!'" Bolognesi relates. T-20 has raced through preclinical and clinical trials.2,3 At the 13th International AIDS Conference in Durban, South Africa, in July 2000, researchers reported on a 48-week clinical trial involving 70 patients who had each failed HAART. In 23 of the 41 patients who survived the 48 weeks, T-20 cut HIV levels by 10-fold or to below the detection level. And participants could handle the daily injections. Larger scale Phase III trials, involving more than 1,000 patients at 58 centers in Europe, Australia, and North and South America, are now under way. The company plans to submit final clinical trial results to FDA by the second half of 2002. If all goes well, T-20 will debut as Enfuvirtide. It may be joined soon thereafter by a similar peptide drug from Trimeris, T-1249, that corresponds to a different part of gp41. Although T-20 is not immune to resistance, it will make new cocktails possible. "The existing drugs have an entirely different mechanism of action, so cross-resistance between the fusion inhibitors and the existing drugs is not likely," explains Kathleen Squires, a visiting associate professor in the department of medicine at the Keck School of Medicine at the University of Southern California. Just behind T-20 in the pipeline is PRO 542, a "recombinant antibody-like fusion protein" from Progenics Pharmaceuticals Inc. in Tarrytown, N.Y. PRO 542 is essentially an IgG antibody molecule whose tips have been replaced by parts of CD4.4 HIV recognizes CD4 and binds to it with gp120, presumably instead of binding to CD4 on a cell. In Phase I trials, PRO 542 vanquished traces of HIV from the bloodstream. And most exciting is that PRO 542 demonstrates synergy with T-20.5 ©2001 Trimeris Inc & Hoffmann-Laroche, LtdWithout T-20, gp41 tightly binds to co-receptors on the CD4+ T cell membrane. CCR5 emerged as a target for intervention when researchers recognized that people fortunate enough to lack it are resistant to HIV infection. "A critical step for HIV infection is the binding of a highly conserved region of the virus to a nine-amino-acid stretch of CCR5. The CCR5-binding region of HIV is among the most highly conserved portions of the virus. Therefore, we believe that the CCR5-virus interaction provides a preferred target for therapy," says Tatjana Dragic, assistant professor of microbiology and immunology at Albert Einstein College of Medicine in New York City. Progenics is also developing monoclonal antibodies against CCR5, in collaboration with F. Hoffmann-LaRoche Corp. of Nutley, N.J. Harnessing a Rare Antibody Toby Rodman, professor emeritus at Cornell University Medical College and an adjunct professor in the laboratory of molecular genetics and informatics at Rockefeller University has discovered a potential AIDS treatment in an unlikely place: cord blood. She and her colleagues have isolated B cells from cord blood and created hybridomas by immortalizing the cells with Epstein-Barr virus. Like traditional monoclonal antibodies (MAbs) made by fusing a B cell with a cancer cell, these hybridomas produce single antibody types, two of which bind to the HIV protein tat.6 Rodman had previously shown that both HIV positive and HIV negative individuals produce antibodies against tat, and that levels of this antibody fall as infection progresses. Since the antibodies are present without exposure to the virus, Rodman hypothesized, they must be innate--hence, the search in cord blood, where she found rare but real anti-tat antibodies. Targeting tat with MAbs may slow or halt the disease process. "Tat is able to cross the blood-brain barrier and therefore tat is probably directly concerned with progression to AIDS-related dementia. As long as antibodies are there, progression does not occur," Rodman explains. The hybridomas make possible unlimited supplies of the antibodies. "This could be a new treatment modality or be incorporated into a vaccine. We are now isolating a whole powerhouse of hybridomas from cord blood B cells," she adds. Perhaps even more intriguing than the clinical possibilities is the bizarre evolutionary tale that anti-tat MAbs tell--that is, why a fetus would manufacture antibodies against a virus that it has never encountered. The story starts a dozen years ago when Rodman studied protamines, the arginine-rich proteins that compact a sperm's DNA so that it can fit into a sperm head. After fertilization, the protamines are replaced with the normal histone proteins. "Twelve years ago I began studying antibodies to protamines and was systematically searching databases. I was struck by the fact that the tat database matched the database for sperm protamines," Rodman says. Protamines can cross the blood-brain barrier, and evolution may have found a way to keep them out of the brain: destroy them with antibodies. Perhaps a holdover from the past, innate antibodies that target protamines may recognize similar arginine-rich stretches of tat, providing a compelling example of what Harvard paleontologist Stephen Jay Gould terms "exaptation"--a new use for an old function. "If I last long enough, I'll actually get it written up," Rodman says, referring to her 86 years. (She was in her lab in lower Manhattan on Sept. 11, wheelchair bound due to a broken ankle, when terrorists flew two commercial airliners into the World Trade Center Towers. Her son wheeled her the 60 blocks home.) Correcting Immune Dysregulation Theoretically, HIV will eventually resist even the brave new entry inhibitors. Another approach to treating HIV infection, from Hollis-Eden Pharmaceuticals Inc. in San Diego, may solve that problem. Their drug, HE2000, is showing promise in preliminary clinical trials in Africa. HE2000 is an analog of the adrenal steroid hormone DHEA, which binds to receptors on T cells and stimulates their activity. Progression to AIDS is associated with a decline in DHEA level. HE2000 counters the inflammatory response that accompanies a hike in HIV replication rate. Because HE2000's target is the immune system and not HIV, viral resistance should not arise. So far clinical results bear that out. "By trying to control the immune system rather than directly trying to kill the virus, our drug treatment so far has not had problems with patients becoming resistant to it. Some patients have now been on therapy for more than two years only taking the drug intermittently and have not shown signs of resistance," says Bob Marsella, vice president, business development for Hollis-Eden. The clinical trials are tracking HE2000's ability to dampen the inflammatory response cytokine by cytokine. Specifically, the trials measured levels of tumor necrosis facto (TNF) alpha, cyclooxygenase-2, interleukin-16 and interleukin-6, which were 11-, 8-, 10-, and 43-fold above normal, respectively, before treatment. Following twice-daily subcutaneous injections for five days, the levels fell to near-normal. The company is also testing a tablet formulation that is held in the inner cheek until it dissolves. "Immune-based therapies are the future of HIV treatment, either novel vaccine candidates, or therapies designed to optimize immune response to keep the virus in check rather than continue to chase it with antivirals that are plagued with resistance and toxicity issues, not to mention cost," says Marsella. The idea to mimic the effect of a natural immune-regulating hormone came from the work of Gene Shearer, an investigator in the experimental immunology branch of the National Cancer Institute. Shearer suggests renaming AIDS "activated immune dysregulatory syndrome," because it is a derangement in immune system regulation, in addition to a deficit of T cells.7 The secret to HIV's success, like the key to all evolution, lies in the value of variety. With a new collection of drugs on the way, perhaps the battle will soon shift to the human side. Ricki Lewis (rickilewis@nasw.org) is a contributing editor for The Scientist. References 1. S.M. Blower et al., "Predicting the unpredictable: Transmission of drug-resistant HIV," Nature Medicine, 7:1016-20, September 2001. 2. C.T. Wild et al., "Peptides corresponding to a predictive alpha- helical domain of human immunodeficiency virus type 1 gp41 are potent inhibitors of virus infection." Proceedings of the National Academy of Sciences, 91:9770-4, 1994. 3. J.M. Kilby et al., "Potent suppression of HIV-1 replication in humans by T-20, a peptide inhibitor of gp41-mediated virus entry," Nature Medicine, 4:1302-7, 1998. 4. M.J. Jacobson et al., "Single-dose safety, pharmacology, and antiviral activity of the human immunodeficiency virus (HIV) type I entry inhibitor PRO 542 in HIV-infected adults," Journal of Infectious Diseases, 182:321-9, 2000. 5. K.A. Nagashima et al., "Human immunodeficiency virus type I entry inhibitors PRO 542 and T-20 are potentially synergistic in blocking virus-cell and cell-cell fusion," Journal of Infectious Diseases, 183:1121-5, April 1, 2001. 6. T.C. Rodman et al., "Innate natural antibodies. Primary roles indicated by specific epitopes," Human Immunology, 55[2]:87-95, 1997. 7. G.M Shearer, "HIV-induced immunopathogenesis," Immunity, 9:587-93, 1998.

New Weapons Against HIV

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