Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a typical member of the TNF family, and is a membrane protein participating in apoptosis. TRAIL is a protein consisting of 281 amino acids. The extracellular domain containing amino acids from arginine at position 114 to glycine at position 281 affects apoptosis. Three molecules of TRAIL form a structurally modified trimer. The TRAIL trimer assembles with receptors participating in cell death to induce apoptosis.
A major difference between TRAIL and other members of the TNF superfamily, such as TNF and CD95L, is their ability not to induce cell death of normal tissues. A variety of medical or pharmaceutical applications have been attempted using TNF and CD95L to induce cell death. Since TNF and CD95L proteins induce death of normal cells as well as cancer cells and over-activated immune cells, they have limited applicability. In contrast, TRAIL induces apoptosis in a wide range of cancer cells and over-activated immune cells with little effect on normal cells. This is due to the differential expression of TRAIL receptors between cell types.
Five TRAIL receptors have been identified. Among them, DR4 (TRAIL-R1) and DR5 (TRAIL-R2) are representative cell death-related receptors. When TRAIL binds to DR4 or DR5, an intracellular death domain of the receptor is activated and thus transduces apoptotic signals via various signal transduction pathways, leading to apoptotic cell death. TRAIL can also bind to DcR1, DcR2 and osteoprotegerin (OPG), which do not induce apoptosis. No marked difference has been seen in the expression levels of the cell death-inducing receptors DR4 and DR5 between normal and tumor cells. In contrast, the three other receptors not inducing apoptosis are expressed at high levels in normal cells, but are either expressed at low levels or are not expressed at all in tumor cells. Thus, in normal cells, TRAIL binds mostly to DcR1, DcR2 and OPG, which do not contain a death domain, and thereby do not induce cell death. In contrast, in cancer cells and over-activated immune cells, apoptosis is induced by the binding of TRAIL to DR4 and DR5, which contain a death domain. Such selective apoptosis induction of TRAIL seems to be a particularly attractive feature in medical or pharmaceutical applications.
TRAIL-mediated apoptosis has been observed in various types of cancer cells, including colon carcinoma, glioma, lung carcinoma, prostate carcinoma, brain tumors and multiple myeloma cells. TRAIL has been proven to have very high anticancer activity in animals. The good anticancer efficacy of TRAIL has been obtained through the use of TRAIL alone, as well as in combination with other anticancer agents, such as paclitaxel and doxorubicin, and radiotherapy. Clinical trials are currently being conducted by Genentech and Amgen using TRAIL, which has good anticancer efficacy in solid tumors. In addition to cancer, various approaches using TRAIL have been made in arthritis, an autoimmune disease, for relieving and treating arthropathy by inducing the death of overactivated immune cells. In addition to protein therapy, gene therapy has been attempted through the delivery of the TRAIL gene. TRAIL may also be useful in the treatment of T cell-mediated autoimmune disorders such as experimental autoimmune encephalomyelitis, rheumatoid arthritis and type I diabetes.
However, native TRAIL has some problems as a therapeutic. The major problem is the low trimer formation ratio of native TRAIL. TRAIL monomers do not bind to the TRAIL receptors, and thus do not induce apoptosis. In this regard, many studies have been performed with the goal of improving the trimeric structure and trimer formation ratio of TRAIL. The zinc ion has been known to play a critical role in trimerization of native TRAIL. Mutants of TRAIL have been developed based on computer analysis results. For the formation of TRAIL trimers, the most useful method appears to be the introduction of an amino acid sequence favoring trimeric folding. Such sequences include a leucine zipper motif and an isoleucine zipper motif. Henning Walczak reported the anticancer efficacy of a trimeric TRAIL derivative in which a leucine zipper motif is added to the N terminus of native TRAIL (Walczak et al., Nature Medicine, 5:157-163 (1999)). Seol reported a TRAIL derivative containing a novel isoleucine zipper motif and having good apoptotic activity (Kim et al., BBRC, 321:930-935 (2004)).
Another problem in the clinical applications of TRAIL involves cytotoxicity shown in normal cells of some tissues. Most normal cells are resistant to cytotoxicity, resulting from the expression of the various TRAIL receptors, but some hepatocytes and keratinocytes are sensitive to TRAIL-mediated cytotoxicity (Yagita et al., Cancer Sci., 95:777-783 (2004); Jo et al., Nature Medicine, 6:564-567 (2000); Zheng et al., J. Clin. Invest., 113:58-64 (2004)).
TRAIL also has a short half-life in vivo, which should be overcome for the successful clinical application of TRAIL. TRAIL has different half-lives according to the species of animals used in tests. For example, TRAIL has been reported to have a half-life of several minutes in rodents and about 30 minutes in apes (Xiang, et al., Drug Metabolism and Disposition, 32:1230-1238 (2004)). Most TRAIL is rapidly excreted via the kidneys. This short half-life is considered a drawback to the pharmaceutical usefulness of TRAIL, resulting in a need for TRAIL or derivatives thereof having an extended half-life. Other problems to be solved include the low solubility and solution stability of TRAIL.
There remains a need for biologically active, highly pure TRAIL conjugates that retain biological activity of native TRAIL having a prolonged serum half-life, increased solubility and which are not cytotoxic to healthy cells.