Common Cold Research: A Cross-Continental Collaboration

Immunologist Steven D. Marlin sits in a plush office at Boehringer Ingelheim Pharmaceuticals in Ridgefield, Conn., joking with two of his collaborators, Vincent J. Merluzzi and Donald E. Staunton. The three are recalling the serendipitous events that linked independent research projects and spurred the development of a genetically engineered human protein that prevents major cold viruses from infecting cells in vitro. Marlin and Merluzzi, working in collaboration with investigators at the Ernst Boehringer Institute in Vienna and Staunton at the Harvard University-affiliated Center for Blood Research in Boston, were the first to engineer a soluble form of the intercellular adhesion molecule-1 (ICAM-1) - a protein found in cell membranes that many cold viruses use as a receptor to infect cells. Through their combined efforts, these researchers found that soluble ICAM-1, when added to cell cultures in sufficient concentrations, acts as a decoy to bind and immobilize the cold virus. Their findings were published this past spring in Nature (344:70-2, 1990). Creating soluble ICAM-1 was something these researchers had planned - but not for rhinovirus. "Our interest in making soluble ICAM-1 was conceived long before anyone made the ICAM-1/rhinovirus connection," says Marlin. "We were going to apply the same decoy principle to inflammatory diseases, which is a project that has nothing to do with virology." Marlin had been working on a larger project started at Boehringer in 1985 by fellow immunologist and ICAM-1 discoverer Robert Rotlein. Since ICAM-1 was originally identified as a molecule involved in the body's inflammatory response, the aim of the anti-inflammatory project was to develop agonists, like soluble ICAM-1, to prevent the inflammatory response common in diseases like arthritis. The research also may prove useful in preventing organ transplant rejection. In a lab directly across the hall, Merluzzi, an immunologist with an interest in virology, was developing rhinovirus cultures for experiments completely unrelated to ICAM-1. While he had been involved in testing ICAM-1 antibodies for anti-inflammatory action in vitro, for the past few years he had been collaborating with Wolfgang Somergruber, a biochemist at Ernst Boehringer Institute (a sister corporation of Boehringer Ingelheim). Their aim was to identify compounds that would inhibit important viral proteases, preventing the cold virus from multiplying after it infected the cell. (Unlike soluble ICAM-1, which prevents the virus from entering the cell, Merluzzi and Somergruber were working on disabling a specific enzyme, involving a later stage of the viral infection's cycle.) These two distinct lines of research came together in March 1989, when Marlin and colleagues and researchers at Molecular Therapeutics Inc., of West Haven, Conn., published back-to-back articles in Cell identifying ICAM-1 as the cell receptor for many cold viruses (J.M. Greve, G. Davis, A.M. Meyer, et al., Cell, 56:839-47, 1989; and D.E. Staunton, V.J. Merluzzi, R. Rothlein, et al., Cell, 56:849-53, 1989). While Greve and associates made the initial connection in an unrelated investigation at Molecular Therapeutics, the Marlin team was well positioned to act on the findings. "There were other groups that had been working with rhinoviruses for quite some time, but we were in this unique situation," says Marlin. "We had the rhinovirus cultures and all the molecular tools to work with ICAM-1 - the monoclonal antibodies, the cloned gene, and the purified proteins - so we were able to make the same observation." Genetically engineering a soluble form of ICAM-1 and testing whether it would prevent the virus from binding to cells became the next obvious step, say team members. While the group was always interested in making soluble ICAM-1, the rhinovirus connection gave Marlin and colleagues a reason to accelerate that part of the project. Although Marlin had the necessary expertise in the biochemistry of ICAM-1 and Merluzzi had already prepared rhinovirus cultures, they still needed molecular geneticists for the complicated task of cloning ICAM-1, engineering a soluble but functionally identical protein product, and ultimately developing a cell line that produced large volumes of the soluble molecule. To meet these needs and complete the team, Marlin relied on the group's close ties with Timothy Springer at the Center for Blood Research and Merluzzi's contacts with his collaborators in Vienna. Both Rothlein and Marlin were postdocs in Springer's lab at the Dana Farber Cancer Institute before moving to Boehringer. When Marlin left in 1987, Staunton, an immunologist, picked up on trying to clone ICAM-1 from gene fragments. When the pace at Boehringer quickened on developing soluble ICAM-1, Marlin and colleagues "carved out" different parts of the project, which allowed the Vienna group and Staunton to do part of the work. "Don engineered the mutation that made soluble ICAM-1 and proved that this mutation would work," says Marlin. They then passed the clone to Somergruber and Christian Stratowa, a molecular geneticist also at the Ernst Boehringer Institute, enabling Somergruber and Stratowa to develop a cell line that would make large quantities of the soluble protein. "That's a long, laborious piece of work that took months and months to complete because of its complexity," says Marlin. In the meantime, Staunton and Marlin were working on very small amounts of soluble ICAM-1 to develop the optimal purification method. By the time the cell line was complete, it was shipped from Vienna to Marlin's lab for purification and then tested by Merluzzi. Given the complexity of the project, the time difference, and the distances involved, their collaborations went smoothly. "We visited Vienna once or twice and made a lot of conference calls throughout the project," says Marlin. "They speak English, so things worked out quite well," he says. The Vienna group agrees. "It was the perfect collaboration," says Somergruber. Although the time difference prevented the Viennese researchers from contacting their U.S. collaborators until afternoon, electronic mail and facsimile messages made this encumbrance a very minor concern. Moreover, the slow nature of their research project provided a distinct advantage. "You have to keep in mind that the steps involved in making a cell line didn't take one or two days to complete but weeks and months. So when we had results, it wasn't necessary to contact the U.S. group that minute," Somergruber says. But in addition to their ability to overcome the communications problems, the researchers say, a lot of hard work, excellent planning, and good working relationships went into making their project a success. "We do a lot of science together and a lot of talking, which makes a difference," says Merluzzi. "Having a lot of support for the project from our management was also important, because it involved international resources and several labs working together," adds Marlin. Although the scientists can't predict when the results of their collaborative effort will produce a "cure" for the common cold or for inflammatory disease, the two original research projects on inhibiting cold virus proteases and preventing the anti-inflammatory response are still very much in progress. And, in addition to investigating these areas, the researchers will also pursue the soluble ICAM-1 approach to preventing rhinovirus infection. While all were surprised and delighted with ICAM-1's connection with rhinovirus, the scientists are clear about where their research priorities lie. Stratowa sums the group's feelings this way: "The more important project is to develop soluble ICAM-1's anti-inflammatory and immunosuppressive activity. There are so many people with organ transplants who have problems with rejection. If soluble ICAM-1 could lessen organ rejection, many lives would be saved." Carole F. Gan is a freelance science writer based in Philadelphia.

Common Cold Research: A Cross-Continental Collaboration

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