New Hope in Finding the Magic Bullet

For this paper, Jim Kling interviewed David Lynch, senior staff scientist at Immunex Corp. in Seattle, Wash. Data from the Web of Science (ISI, Philadelphia) show that Hot Papers are cited 50 to 100 times more often than the average paper of the same type and age. H. Walczak, R.E. Miller, K. Ariail, B. Gliniak, T.S. Griffith, M. Kubin, W. Chin, J. Jones, A. Woodward, T. Le, C. Smith, P. Smolak, R.G. Goodwin, C.T. Rauch, J.C.L. Schuh, D.H.T. Lynch, "Tumoricidal activity of tumor necrosis factor related apoptosis-inducing ligand in vivo," Nature Medicine, 5:157-63, February 1999. (Cited in 161 papers) Soon after tumor necrosis factor (TNF) was discovered in 1985,1 the gene encoding this molecule was cloned.2 TNF generated much excitement because, as its name implies, TNF is deadly to tumor cells. Indeed, the hopes of many were raised that a powerful new cancer therapeutic had been discovered. "TNF was thought to be a magic bullet that would cure cancer, because it's very good at killing tumor cells," says David Lynch, a senior staff scientist at Immunex Corp., Seattle, Wash. "But it is also toxic to many different types of normal cells." TNF and some of its brethren, including FasL (CD95L) and Lta, function normally as powerful mediators and regulators of immune responses in vivo. However, when administered at pharmacologic doses, they caused extreme hepatotoxicity, which prevented their general use as cancer therapeutics. Still, those discoveries touched off a major effort to find similarly active molecules with lower toxicity, and in the mid-1990s, researchers at Immunex and Genentech, Inc. of San Francisco, Calif., independently discovered just such a molecule, the TNF-related apoptosis-inducing ligand (TRAIL).3,4 Recently, the two companies agreed to co-develop this molecule (now known as TRAIL/Apo-2L) for the treatment of cancer. The trouble with TRAIL/Apo-2L is that, as a Type II transmembrane protein, it does not adopt the biologically active trimer conformation outside the cell membrane. In the Hot Paper published by Lynch and his colleagues at Immunex, the authors reported creating a soluble form of the trimer by using an isoleucine zipper attached to the amino-terminal end of the extracellular portion of the protein. A product of yeast, isoleucine zippers rapidly trimerize, dragging whatever is attached to them along for the ride and causing them to adopt a similar trimer conformation. With this manipulation, the researchers could produce large quantities of the molecule and perform the studies to determine its biological activity. They found that TRAIL/Apo-2L sends a potent apoptotic signal to a broad range of transformed cell lines, but does little harm to normal cells. "We've looked at 77 different tumors in vitro, and two-thirds to three-quarters of the cell lines we've tested show sensitivity to TRAIL/Apo-2L," says Lynch. As it turned out, leucine zippers were only the first step. "There is a slightly different form [which uses zinc molecules to achieve the trimer] that we're developing for use in the clinic along with our colleagues at Genentech," Lynch explains. During their efforts in protein engineering they discovered that "if just the extracellular domain of TRAIL/Apo2L is expressed and purified in the presence of zinc, those zinc molecules form a coordination complex at the amino portion of the extracellular domain, and that also stabilizes the trimer form."5 Part of the reason for developing an alternative form is that the yeast origin of leucine zippers might elicit an immune response from patients. "That might not be a bad thing; one could make arguments for it being beneficial to have antibodies generated against the zipper," says Lynch. But bringing a product through the process of testing and approval is difficult enough without unnecessary variables. "Using the [newer] form, where we have no non-human sequences, we've minimized the generation of an immune response against the therapeutic," comments Lynch. The paper probably received the attention it did because this was the first report demonstrating the therapeutic potential of this molecule, reasons Lynch. "For the first time, we were able to eliminate human tumor xenografts from SCID mice in the absence of the toxic side effects that TNF and FasL cause. It really opens up a window for development of a member of the TNF family as a therapeutic. To my knowledge, there is no other biological that has as profound and wide-ranging an effect on tumors as TRAIL-Apo2L." Immunex is now engaged in exploring the manufacturing issues and other hurdles in preparation for clinical 6/25/2001 development. Jim Kling can be contacted at jkling@nasw.org. References 1.B.B. Aggarwal et al., "Human tumor necrosis factor: Production, purification, and characterization," Journal of Biological Chemistry, 260[4]:2345-54, 1985. 2.D. Pennica et al., "Cloning and expression in Escherichia coli of the cDNA for murine tumor necrosis factor," Proceedings of the National Academy of Sciences, U.S.A., 82[18]:6060-4, 1985. 3. .R. Wiley et al., "Identification and characterization of a new member of the TNF family that induces apoptosis," Immunity, 3:673, 1995. 4. R.M. Pitti et al., "Induction of apoptosis by Apo-2 ligand, a new member of the tumor necrosis factor family of molecules," Journal of Biological Chemistry, 271[22]:12687-90, 1996. 5.S.G. Hymowitz et al., "A unique zinc-binding site revealed by a high-resolution X-ray structure of homotrimeric Apo2L/TRAIL," Biochemistry, 39[4]:633-40, 2000.

New Hope in Finding the Magic Bullet

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