By David N. Leff
The AIDS virus doesn't fight fair.
An HIV-1 virion, after invading its victim's bloodstream, then breaks, enters and infects the cells that spearhead the body's immune defenses against it. Antiviral drugs can't effectively follow the invaders into these intracellular strongholds. Instead, they have to play catch as catch can with the virus as its proliferating progeny exit the cells it has infected.
In principle, gene therapy could cut these infective hordes off at the pass, by planting genes encoding anti-HIV monoclonal antibodies inside the enemy's target cells. In practice, reported efforts along these lines, observed molecular biologist Bernard Malfroy, "will require extensive clinical development to become a useful treatment to inhibit HIV-1."
As an alternative to this "tremendously sophisticated and complex genetic-engineering approach," he proposes "a technology that is extremely straightforward, very practical and that works * namely, HIV-specific, intracellular immunotherapy."
Malfroy is president and CEO of Eukarion Inc., in Medford, Mass. His concept made its bow in print late last month in the Journal of Acquired Immunodeficiency Syndromes and Human Retrovirology, dated March 1997. He is senior author of the paper, titled "A lipidated anti-Tat antibody enters living cells and blocks HIV-1 viral replication." Its lead author is molecular biologist William Cruikshank, at Boston University School of Medicine.
Unlike the viral surface antigens targeted by most experimental monoclonal antibodies, Malfroy told BioWorld Today, "Tat is a regulatory gene product of HIV which is expressed inside the infected cells."
Lipidation is Eukarion's proprietary process for making cells permeable to neutralizing monoclonal antibodies programmed to destroy the antigens that infect them — in this case, Tat. Besides penetrating cell membranes, Malfroy pointed out, "lipidated antibodies cross capillary barriers and the blood-brain barrier."
Explaining the potential clinical applications, he said: "Monoclonal antibodies work very well in models of tumor growth in mice. Yet they are almost always disappointing in human clinical studies. One reason is that antibodies, when injected into the bloodstream, do not cross the capillary walls, do not reach the tumors."
Testing Proof-Of-Concept In Vitro
Aside from some in vivo pharmacokinetic studies of lipidated antibodies in mice, Eukarion's research of this technology has been limited to in vitro tests of the antibodies' ability to kill HIV inside infected cells. As reported in their just-published Journal article, these experiments included fresh isolates from HIV-positive patients as well as infected laboratory cell lines, such as HeLa cells.
"Once it entered the infected cells," Malfroy recounted, "the lipidated anti-Tat antibody bound to the Tat protein and blocked its function. This led to suppressed viral replication and, concomitantly, to greatly improved cell survival."
Thus, intracellular uptake of lipidated anti-Tat antibodies was 32.8 percent of HIV-infected cells, but only 4.8 percent of uninfected ones. In another experiment, treatment of infected cell cultures with the lipidated antibody preserved cell viability at day 13 by about 90 percent. By comparison, HIV-infected cells that received no treatment had lost more than 75 percent of their number by day 13.
"The fact of the matter," Malfroy observed, "is that what we have done is a proof-of-concept which is much more wide-ranging than HIV alone."
He foresees for lipidated antibodies a broader therapeutic panoply of disease entities.
"These are dictated by the availability of immunogenic targets," Malfroy pointed out. "For example, hepatitis B has such a target, which is well identified by molecular biologists and virologists. Another example," he continued, "is the mutated ras oncogene in various cancers. The ras mutations are intracellular, and there is nothing more specific than antibodies to detect point mutations. One could easily envision lipidating specific antibodies against the intracellular domain of this mutated ras."
Wanted: Research And Development Partners
To reduce such visions to practice, Malfroy said, "I definitely hope that we will find partners who might want to follow up on our HIV results and develop the core technology. We believe that can be of tremendous interest to a number of companies that are focused on antibody-based therapy." (See BioWorld Today, Feb. 19, 1997, p. 2.)
Along that research and development path there is one obvious obstacle * immunogenicity.
"A potential limitation to the use of lipidated antibodies for immunotherapy," the Journal paper cautioned, "is the extent to which they are immunogenic . . . . [This] has not been determined . . . ."
Malfroy said he is "right now doing in vivo experiments in immunogenicity, but I would rather talk about that on another occasion."
To outfit their monoclonals with the ability to penetrate cells, escape from blood vessels and enter the brain, Eukarion links the antibodies' carbohydrate moiety to lipids — fat molecules.
"The lipid," Malfroy explained, "is chemically linked to an amine. It's the same mild chemistry that's used to link peroxidase to antibodies. As a result, we know that we do not damage the antibody, in the sense of modifying its affinity for its epitope."
Medical virologist Joseph Sidrosky, who teaches pathology at Harvard Medical School, told BioWorld Today: "This lipidated antibody method certainly deserves to be explored. But it has major problems. One is delivery: Would it have to be given parenterally? Another: Might it be immunogenic?
"These hurdles," he concluded, "may be overcome some day, but right now that technology is in the same situation as intracellular gene therapy of antibodies." *