TRAIL

Apo2L/TRAIL Avi Ashkenazi* Department of Molecular Oncology, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080-4918, USA * corresponding author tel: 650-225-1853, fax: 650-225-6443, e-mail: [email protected] DOI: 10.1006/rwcy.2000.05012.

SUMMARY Apo2L/TRAIL is a member of the TNF ligand family that is closely related to FasL. Many tissues express the Apo2L/TRAIL mRNA, and T cells upregulate Apo2L/TRAIL expression upon T cell receptor stimulation. The distinct biologic function of Apo2L/ TRAIL has yet to be defined. There is evidence that Apo2L/TRAIL might play a role in T cell activationinduced cell death (AICD), in target-killing by cytotoxic lymphocytes and macrophages, and perhaps in the immune-privilege of certain tumors. Apo2L/ TRAIL binds to a complex system of receptors: DR4 and DR5 have death domains that signal apoptosis, whereas DcR1, DcR2, and OPG lack functional death domains and can inhibit apoptosis induction by the ligand. Apo2L/TRAIL potently activates apoptosis in a wide variety of tumor cell lines, but it is not cytotoxic towards most normal cell types studied so far. Experiments in mouse models of cancer suggest that Apo2L/TRAIL might be useful as an anticancer agent that kills tumor cells without damaging normal tissues.

BACKGROUND The members of the tumor necrosis factor (TNF) gene superfamily modulate diverse biological functions, including cell differentiation, proliferation, and apoptosis (Gruss and Dower, 1995). Most TNF family ligands are expressed as type 2 transmembrane proteins, some of which are processed by specific proteases into soluble homotrimeric cytokines. The sequence homology between TNF and its known relatives occurs primarily in the C-terminal extracellular domain (ECD). The crystal structures of TNF

and lymphotoxin  indicate that this region contains a series of 10 strands that form the basic fold of the ECD. TNF-related ligands bind to specific receptors that belong to the TNF receptor (TNFR) gene superfamily (Smith et al., 1994). Most TNFR family members are type 1 transmembrane proteins; their common structural feature is the presence of 2±6 cysteine-rich domains in the extracellular portion. The crystal structure of the complex between lymphotoxin- and the ECD of TNFRI reveals that three receptor molecules bind to the homotrimeric ligand, each docking in the groove between two ligand subunits (Banner et al., 1993). TNFR family proteins fall into two subgroups on the basis of intracellular sequence: receptors that contain death domains (a 70 amino acid homophilic protein interaction motif), and receptors that lack death domains. The receptors that have death domains, dubbed `death receptors', trigger cellular responses by engaging specific adapter molecules such as TRADD (TNFR-associated death domain) or FADD (Fasassociated death domain) through homophilic death domain interactions (Ashkenazi and Dixit, 1998). TNFR family members that lack death domains signal by engaging specific adaptor molecules called TRAFs (TNFR-associated factors). The best characterized death receptor and ligand systems (Simonet et al., 1997) are those of Fas/Apo1/ CD95 with Fas ligand (FasL), and the p55 TNF receptor (TNFRI) with TNF. There is a growing interest in a third, more recently discovered apoptosis-inducing ligand, called Apo2 ligand (Apo2L) or TNF-related apoptosis-inducing ligand, which is structurally related to FasL and TNF. Much of the interest stems from the existence of an unusually complex system of death and decoy receptors that interact with this ligand (Ashkenazi and Dixit, 1998, 1999).

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Discovery Investigators (Wiley et al., 1995; Pitti et al., 1996) identified expressed sequence tags (ESTs) that showed homology to the TNF ligand family. On the basis of the ESTs, a full-length cDNA was isolated that encoded a previously unknown member of the TNF family. The novel protein, which showed highest homology to Fas/Apo1 ligand, was designated Apo2L (Pitti et al., 1996) or TRAIL (Wiley et al., 1995).

Alternative names Alternative names are TL2 (Tan et al., 1997) and TNFSF10.

Structure The human Apo2L/TRAIL cDNA predicts a polypeptide of 281 amino acids. Hydropathy analysis suggests that Apo2L/TRAIL has the topology of a type 2 transmembrane protein, with a 15±17 amino acid-long N-terminal cytoplasmic region, followed by a transmembrane region of 21±23 residues, and an extracellular C-terminal region. The protein has one potential N-linked glycosylation site (Asn109). Alignment of the extracellular region with that of other family members suggests that the strand region starts around amino acid 120 (Figure 1). FACS staining of cells, transfected with the full-length Apo2L/TRAIL cDNA tagged by a C-terminal Myc epitope, with anti-Myc antibody indicates that the molecule is expressed at the cell surface as a type 2 protein (Pitti et al., 1996). FACS staining of cells that express endogenous Apo2L/TRAIL with polyclonal antiligand antibodies also indicates cell surface expression (Mariani and Krammer, 1998a, 1998b), as does immunohistochemical staining of tissue sections with monoclonal anti-Apo2L/TRAIL (Ashkenazi et al., 1999). The full-length Apo2L/TRAIL protein migrates on SDS-PAGE gels as a 32±33 kDa protein (Mariani and Krammer, 1998a, 1998b). There is evidence that cells can release Apo2L/ TRAIL in soluble form, apparently through proteolytic processing of the ECD. First, supernatants from phytohemagglutinin (PHA)-stimulated human Jurkat T cells contain cytotoxic activity that can be blocked partially by a neutralizing monoclonal anti-Apo2L/ TRAIL antibody (Martinez-Lorenzo et al., 1998). Second, the surface expression of Apo2L/TRAIL on Jurkat cells increases upon treatment with inhibitors of cysteine proteases (Mariani and Krammer, 1998a).

Third, Chinese hamster ovary cells transfected with an expression plasmid encoding full-length Apo2L/ TRAIL release a soluble form of the ligand that migrates on SDS-PAGE gels under reducing conditions as a 24 kDa protein band (Figure 2).

Main activities and pathophysiological roles The biologic functions of Apo2L/TRAIL are not fully understood; however, the ligand's ability to trigger apoptosis in a variety of transformed cell lines suggests that it may be a physiologic modulator of apoptosis. Apo2L/TRAIL also is capable of activating the proinflammatory transcription factor NFB; however, in comparison to TNF, it is a very weak NFB activator (Sheridan et al., 1997; Ashkenazi et al., 1999). Several lines of evidence suggest that Apo2L/TRAIL may play a role in the AICD of peripheral T cells, a process in which FasL plays a central role (Nagata, 1997). First, a subset of peripheral T cells acquires sensitivity to Apo2L/TRAIL-induced apoptosis after a 3-day stimulation by IL-2 (Marsters et al., 1996) or a 5-day stimulation by IL-2 and PHA (Snell et al., 1997). Second, Apo2L/TRAIL mRNA expression is induced upon the stimulation of CD4+ and CD8+ peripheral blood T cells with PHA or with the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) and the calcium ionophore ionomycin (Jeramias et al., 1988; Screaton et al., 1997; MartinezLorenzo et al., 1998). Third, neutralizing anti-Apo2L/TRAIL antibody reduces the cytotoxicity of the supernatants of PHAstimulated peripheral blood mononuclear cells or Jurkat T cells towards unstimulated T cells (MartinezLorenzo et al., 1998). In addition, T cells from HIVinfected patients show increased sensitivity to Apo2L/ TRAIL, which suggests a potential role for the ligand in the killing of virus-infected cells (Jeramias et al., 1998). There is evidence also for the involvement of Apo2L/TRAIL in target-killing by CD4+ cytotoxic T lymphocytes (CTLs) and by natural killer (NK) cells, both of which express Apo2L/TRAIL mRNA (Thomas and Hersey, 1998; Zamani et al, 1998). Further evidence suggests that Apo2L/TRAIL is involved in tumor cell killing by macrophages (Griffith et al., 1999). Human astrocytic brain tumors, but not normal glia, express Apo2L/TRAIL mRNA (Rieger et al., 1998), as do several cell lines derived from other types of cancer (Ashkenazi et al., 1999); this expression

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Figure 1 Alignment of the putative extracellular strand region of Apo2L/TRAIL with that of other TNF family members. The strand regions are marked above with lines and letters.

suggests a potential involvement of Apo2L/TRAIL in the immune-privilege of tumors. The apparent involvement of Apo2L/TRAIL in T cell AICD, in target-killing by cytotoxic lymphocytes, and in the immune-privilege of tumors suggests that this ligand resembles FasL not only in sequence, but also in certain functions. Recent work suggests that Apo2L/TRAIL, but not FasL or TNF, plays a key role in the killing of dendritic cells by autologous CD4+ CTLs (Wang et al., 1999), suggesting a potentially unique role for Apo2L/TRAIL in modulating the immune system.

GENE AND GENE REGULATION Published information on the structure of the Apo2L/ TRAIL gene is not yet available. The PHAstimulation of peripheral blood T cells induces

transcription of the Apo2L/TRAIL gene (Screaton et al., 1997; Martinez-Lorenzo et al., 1998).

Accession numbers Human mRNA: HSU37518, HSU57059 Mouse mRNA: MMU37522

Chromosome location Human chromosome 3q26 (Wiley et al., 1995).

Relevant linkages Apo2L/TRAIL receptors Osteoprotegerin FasL TNF

506 Avi Ashkenazi Figure 2 Western blot analysis of soluble Apo2L/TRAIL shed by Chinese hamster ovary cells after transfection with cDNA encoding the full-length protein. Cell supernatants were collected 24 hours after transfection, resolved by SDS-PAGE, and analyzed by immunoblot with monoclonal antiApo2L/TRAIL antibody 5C2.

PROTEIN

Accession numbers Human protein: GEN13977 Human protein: P_W19777 Mouse protein: GEN13978

Sequence 60

See Figure 3.

Description of protein 42 30 22 17

Cells and tissues that express the gene Northern blot analyses with probes that are based upon the Apo2L/TRAIL cDNA reveal a single mRNA transcript of 2 kb (Wiley et al., 1995; Pitti et al., 1996). The Apo2L/TRAIL message is present constitutively in many tissues, including fetal lung, liver, and kidney, and adult spleen, prostate, thymus, ovary, small intestine, colon, PBLs, heart, placenta, lung, skeletal muscle, and kidney. In situ hybridization analysis indicates Apo2L/TRAIL mRNA expression in human astrocytic brain tumors and in glial cancer cell lines (Rieger et al., 1998). In addition, the stimulation of CD4+ and CD8+ peripheral blood T cells with PHA or with PMA and ionomycin induces expression of the Apo2L/TRAIL mRNA (Screaton et al., 1997; Jeramias et al., 1998; MartinezLorenzo et al., 1998). Stimulation of macrophages with IFN induces cell surface expression of the Apo2L/TRAIL protein (Griffith et al., 1999).

Apo2L/TRAIL is a 281 amino acid, type 2 transmembrane protein, with a calculated molecular weight of 32.5 kDa and an isoelectric point of 7.63. The fulllength protein migrates as a 32±33 kDa band on SDSPAGE gels (Mariani and Krammer, 1998a, 1998b); a shed form of the protein migrates as a band of approximately 24 kDa (see Figure 2). Several recombinant soluble versions of the protein have been generated. A polyhistidine-tagged soluble form of Apo2L/TRAIL (amino acids 114±281) forms homotrimers and is biologically active (Pitti et al., 1996; Ashkenazi et al., 1999). In contrast, a Flag-epitopetagged soluble form of the protein (amino acids 95± 281) is poorly active, and requires oligomerization by anti-Flag antibody for potent biologic activity (Wiley et al., 1995; Walczak et al., 1999). A fusion protein, containing the same Apo2L/TRAIL amino acids (95± 281) fused N-terminally to a modified leucine-zipper that promotes trimerization, forms trimers and is biologically active (Walczak et al., 1999).

Important homologies The C-terminal portion of the Apo2L/TRAIL ECD shows 28% identity to the FasL ECD, 23% identity to the ECDs of TNF and lymphotoxin , and 22% identity to the ECD of lymphotoxin (see Figure 1). The loop that connects the first and second putative strands is substantially longer in the ECD of Apo2L/TRAIL ECD than in other family members. The murine Apo2L/TRAIL protein is 291 amino acids long, and it shows 65% identity to the human protein (Wiley et al., 1995).

Posttranslational modifications Human and murine Apo2L/TRAIL each have one potential N-linked glycosylation site (Asn109 and

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Figure 3 Amino acid sequence of Apo2L/TRAIL. Sequence MAMMEVQGGPSLGQTCVLIVIFTVLLQSLCVAVTYVYFTNELKQMQDKYSKSGIA CFLKEDDSYWDPNDEESMNSPCWQVKWQLRQLVRKMILRTSEETISTVQEKQQ NISPLVRERGPQRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGHSFL SNLHLRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQMVQYIYKYTSYPDPIL LMKSARNSCWSKDAEYGLYSIYQCGIFELKENDRIFVSVTNEHLIDMDHEASFFG AFLVG

Asn52 respectively). Apo2L/TRAIL is made as a type 2 transmembrane protein; a soluble form that contains the ECD can be released proteolytically by cells that express the full-length protein (see Description of protein).

CELLULAR SOURCES AND TISSUE EXPRESSION

Cellular sources that produce Apo2L/TRAIL appears to be expressed constitutively on the surface of mouse and human tumor cells of T and B origin, including mouse EL4 T cells and A20 B cells, and human Jurkat T cells and Bjab B cells (Mariani and Krammer, 1998a). Freshly isolated murine splenocytes, concanavalin A/IL-2-activated T cells, and lipopolysaccharide-activated B cells were analyzed by surface staining with anti-Apo2L/TRAIL antibody (Mariani and Krammer, 1998b). Activated, but not resting, CD3+ cells expressed Apo2L/ TRAIL. Freshly isolated B220+ cells displayed surface Apo2L/TRAIL and CD95L that were retained following activation. In CD3+ TCR cells with the CD4+ or CD8+ phenotype, restimulation with PMA and ionomycin or an agonistic anti-CD3 monoclonal antibody induced a significant upregulation of surface Apo2L/TRAIL and CD95L. Apo2L/TRAIL upregulation was inhibited by cyclohexamide and by cyclosporin A.

Eliciting and inhibitory stimuli, including exogenous and endogenous modulators See Gene and gene regulation.

RECEPTOR UTILIZATION Apo2L/TRAIL interacts with four cell surface receptors that form a distinct subgroup within the TNFR gene superfamily (for a review, see Ashkenazi and Dixit, 1998, 1999). Death receptor 4 (DR4) (Pan et al., 1997) and DR5 (Pan and Dixit, 1997; Sheridan et al., 1997) (also called TRICK2, TRAIL-R2 or KILLER) (Screaton et al., 1997; Walczak et al., 1997; Wu et al., 1997) have cytoplasmic death domains and signal apoptosis. Decoy receptor 1 (DcR1) (Sheridan et al., 1997) (also called TRID, TRAIL-R3, or LIT) (Degli-Esposti et al., 1997a; Mongkolsapaya et al., 1998; Pan et al., 1998a), is a phospholipid-anchored cell surface protein that lacks a cytoplasmic tail. DcR2 (Marsters et al., 1997) (also called TRAIL-R4 or TRUNDD) (Degli-Esposti et al., 1997b; Pan et al., 1998b) has a substantially truncated death domain that does not signal apoptosis induction. Upon overexpression, DcR1 or DcR2 inhibits apoptosis induction by Apo2L/TRAIL. Apo2L/TRAIL binds also to a secreted, soluble member of the TNF receptor family, osteoprotegerin (OPG) (Simonet et al., 1997; Emery et al., 1998). The binding affinity of Apo2L/ TRAIL for OPG is 3±5-fold lower than the affinity for DR5 or DcR1 (Emery et al., 1998). OPG is not closely related to the other four Apo2L/TRAIL receptors, and it binds also to another TNF family member, OPGL (also called RANKL or TRANCE).

IN VITRO ACTIVITIES

In vitro findings The main in vitro biologic activity of Apo2L/TRAIL so far reported is the induction of caspase-dependent apoptosis in tumor cell lines from various tissue origins (Wiley et al., 1995; Pitti et al., 1996; Mariani et al., 1997; Snell et al., 1997; Jeramias et al., 1998;

508 Avi Ashkenazi Thomas and Hersey, 1998). There is also evidence that Apo2L/TRAIL can induce apoptosis in a subset of activated T cells (Marsters et al., 1996; Snell et al., 1997; Martinez-Lorenzo et al., 1998), and in a subset of T cells from HIV-infected children (Jeramias et al., 1998). In addition, Apo2L/TRAIL is capable of activating NFB; the magnitude of this activation is much less than that of activation by TNF, and it requires a substantially higher concentration of Apo2L/TRAIL relative to TNF (Sheridan et al., 1997; Ashkenazi et al., 1999). Apo2L/TRAIL is also capable of inducing the activation of c-Jun N-terminal kinase (JNK): in HeLa cells, but not in Kym-1 cells, the activation can be blocked by the caspase inhibitor zVAD-fmk, which suggests the involvement of caspase-dependent and caspaseindependent mechanisms in this effect (Muhlenbeck et al., 1998). More than half of the 60 tumor cell lines in a panel developed by the US National Cancer Institute, which contains cell lines derived from leukemia, melanoma, non-small cell lung cancer, colon cancer, central nervous system cancer, ovarian cancer, renal cancer, prostate cancer, and breast cancer, showed sensitivity to Apo2L/TRAIL (Ashkenazi et al., 1999). In contrast, several normal cell types were resistant in culture to Apo2L/TRAIL (Ashkenazi et al., 1999; Walczak et al., 1999), with the exception of fetal astrocytes (Walczak et al., 1999). Neutralizing anti-Apo2L/TRAIL antibodies attenuate the cytotoxic activity of supernatants from activated T cells towards unstimulated T cells (Martinez-Lorenzo et al., 1998), which implicates Apo2L/TRAIL in the AICD of T lymphocytes. This notion gains further support from the observation that blocking anti-Apo2L/TRAIL antibody inhibits the AICD of T cells from HIV-infected patients (Katsikis et al., 1997). Soluble Apo2L/TRAIL receptors in the form of an Fc-fusion protein (immunoadhesin) inhibit the killing of target tumor cells by macrophages, implicating the ligand in mediating the cytotoxic activity of macrophages (Griffith et al., 1999).

Regulatory molecules: Inhibitors and enhancers Extracellular, as well as intracellular, mechanisms may modulate apoptosis induction by Apo2L/TRAIL. Extracellularly, three decoy receptors that belong to the TNFR gene superfamily can bind to Apo2L/ TRAIL and inhibit the ligand's apoptosis-inducing activity: these are DcR1, DcR2, and OPG (Ashkenazi

and Dixit, 1998; Emery et al., 1998). Intracellularly, the c-FLIP molecule, which blocks apoptosis signaling by FasL and TNF, is also capable of inhibiting apoptosis induction by Apo2L/TRAIL (Thome et al., 1997). Certain chemotherapeutic drugs, including inhibitors of transcription or translation and DNAdamaging agents, sensitize cancer cell lines to the cytotoxic activity of Apo2L/TRAIL (Keane et al., 1999; Mori et al., 1999). In addition, IL-1 protects transformed keratinocyte cell lines against apoptosisinduction by Apo2L/TRAIL (Kothny-Wilkes et al., 1998).

Bioassays used Apo2L/TRAIL bioactivity can be conveniently assayed in standard cytotoxicity or cell viability assays. Alternatively, Apo2L/TRAIL bioactivity can be assayed by measuring apoptosis induction, on the basis of parameters such as DNA fragmentation, or phosphatidylserine-flip, as measured by FACS staining with annexin V (Pitti et al., 1996). Apoptosis can also be assayed on the basis of caspase activation, measured by the cleavage of synthetic caspase substrates, or by the cleavage of cellular caspase substrates such as poly-ADP ribose polymerase (PARP) or caspase 3.

IN VIVO BIOLOGICAL ACTIVITIES OF LIGANDS IN ANIMAL MODELS

Species differences Studies on a limited number of cell lines indicate that human Apo2L/TRAIL is cytotoxic towards murine cell lines similarly to murine Apo2L/TRAIL, and that murine Apo2L/TRAIL is cytotoxic towards human cell lines similarly to human Apo2L/TRAIL (Wiley et al., 1995; Mariani et al., 1997; Walczak et al., 1999).

Endogenous inhibitors and enhancers The mRNA for Apo2L/TRAIL is expressed constitutively in many tissues, as are the mRNAs for DR4 and DR5; hence, there may be endogenous mechanisms that control cellular sensitivity to the cytotoxic action of Apo2L/TRAIL. One such mechanism may

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involve the expression of the decoy receptors DcR1 and DcR2 in normal tissues. Because DcR1 and DcR2 are expressed infrequently and generally at low levels in cancer cell lines, it is unlikely that they regulate the sensitivity to the ligand in transformed cells. Whether the decoys are important inhibitors of Apo2L/TRAIL cytotoxicity in normal tissues that express DcR1 or DcR2 more abundantly remains to be investigated. Consistent with the latter hypothesis is the observation that T cell activation downregulates DcR1 expression, perhaps as a mechanism for regulating the sensitivity to Apo2L/TRAIL during AICD (Mongkolsapaya et al., 1998). In addition, the treatment of human umbilical cord endothelial cells with phosphatidylinositol phospholipase C sensitizes the cells to apoptosis induction by Apo2L/TRAIL, consistent with the removal of GPI-anchored DcR1 from the cell surface (Sheridan et al., 1997). Another mechanism to regulate the sensitivity of cells to Apo2L/TRAIL may involve the expression of antiapoptotic factors such as c-FLIP (Thome et al., 1997) or BclXL (Mori et al., 1999). It is also possible that there is regulation of the Apo2L/TRAIL protein itself at the level of translation, cellular transport, and/or proteolytic processing. Further, in certain tumor cell lines, activation of the p53 tumor suppressor upregulates the expression of DR5 mRNA, perhaps increasing sensitivity to Apo2L/TRAIL (Wu et al., 1997).

The leucine-zipper Apo2L/TRAIL fusion protein exhibited a distribution half-life of 1.3 hours and an elimination half-life of 4.8 hours after intravenous injection in mice (Walczak et al., 1999). The native sequence form of recombinant soluble Apo2L/ TRAIL exhibited a pharmacologic half-life of 0.5 hours in cynomolgus monkeys (Ashkenazi et al., 1999).

IN THERAPY

Toxicity

Preclinical ± How does it affect disease models in animals?

The systemic injection of up to 0.5 mg of the human leucine-zipper Apo2L/TRAIL fusion protein and 1 mg of the corresponding murine fusion protein in mice had no detectable adverse effects on viability, tissue integrity, or blood cell count (Walczak et al., 1999). Intravenous injection of the native sequence form of recombinant soluble Apo2L/TRAIL at up to 10 mg/kg/day for 7 days in cynomolgus monkeys showed no evidence of toxicity using a comprehensive set of clinical tests. Apo2L/TRAIL did not affect body weight, body temperature, blood pressure or heart rate. There were no Apo2L/TRAIL-associated changes in leukocyte count, liver enzyme activity, coagulation profiles, serum chemistry or urine analysis. Histologic evaluation of liver, lung, heart, brain, kidney, ovary, testis, spleen, bone marrow, and lymph nodes did not reveal Apo2L/TRAIL-related adverse changes. In particular, there was no morphologic evidence of hepatotoxicity or lymphotoxicity, and bone marrow sections displayed normal cellularity and progressive maturation of all lineages (Ashkenazi

Apo2L/TRAIL has been studied in mouse-based xenograft models of human cancer (Ashkenazi et al., 1999; Walczak et al., 1999). In nude mice, systemic treatment with a leucine-zipper-fused Apo2L/TRAIL protein begun shortly after the subcutaneous injection of tumor cells substantially reduced tumor incidence (Walczak et al., 1999). Treatment with the fusion protein after solid tumors had begun to form reduced tumor volume and in some cases eliminated the tumors if started within a few days, delaying tumor progression if started later. Histologic examination indicated that the systemic injection of the fusion protein in tumor-bearing mice induced apoptosis in tumor epithelial cells (Walczak et al., 1999). In another study, a native sequence version of recombinant soluble Apo2L/TRAIL was investigated (Ashkenazi et al., 1999). Repeated intravenous

injections of the protein in nonhuman primates did not cause detectable toxicity to the tissues and organs examined. Apo2L/TRAIL exerted cytostatic or cytotoxic effects in vitro on 32 of 39 cell lines from colon cancer, lung cancer, breast cancer, kidney cancer, brain cancer, and skin cancer. The treatment of athymic mice with Apo2L/TRAIL shortly after tumor xenograft injection markedly reduced tumor incidence. The Apo2L/TRAIL treatment of mice bearing solid tumors induced tumor cell apoptosis and tumor regression, suppressed tumor progression, and improved survival. Apo2L/TRAIL cooperated synergistically with the chemotherapeutic drugs 5-fluorouracil and CPT-11 at causing substantial tumor regression or complete tumor ablation. These studies suggest that Apo2L/TRAIL may turn out to be useful as an anticancer agent that induces apoptosis in tumors while sparing normal tissues.

Pharmacokinetics

510 Avi Ashkenazi et al., 1999). These data suggest that, unlike its relatives TNF and FasL, Apo2L/TRAIL is not significantly toxic in experimental animal models; careful testing will, however, be needed to evaluate the safety of this molecule in human beings.

References Ashkenazi, A., and Dixit, V. M. (1998). Death receptors: signaling and modulation. Science 281, 1305±1308. Ashkenazi, A., and Dixit, V. M. (1999). Apoptosis control by death and decoy receptors. Curr. Opin. Cell Biol. 11, 255± 260. Ashkenazi, A., Pai, R., Fong, S., Leung, S., Lawrence, D., Marsters, S., Blackie, C., Chang, L., McMurtrey, A., Hebert, A., DeForge, L., Khoumenis, I., Lewis, D., Harris, L., Bussiere, J., Koeppen, H., Shahrokh, Z., and Schwall, R. (1999). Safety and anti-tumor activity of recombinant soluble Apo2 ligand. J. Clin. Invest. 104, 155±162. Banner, D. W., D'Arcy, A., Janes, W., Gentz, R., Schoenfeld, H. J., Broger, C., Loetscher, H., and Lesslauer, W. (1993). Crystal structure of the soluble human 55 kd TNF receptor-human TNF beta complex: implications for TNF receptor activation. Cell 73, 431±435. Degli-Esposti, M., Smolak, P. J., Walczak, H., Waugh, J., Huang, C. P., Dubose, R. F., Goodwin, R. G., and Smith, C. A. (1997a). Cloning and characterization of TRAIL-R3, a novel member of the emerging TRAIL receptor family. J. Exp. Med. 186, 1165±1170. Degli-Esposti, M. A., Dougall, W. C., Smolak, P. J., Waugh, J. Y., Smith, C. A., and Goodwin, R. G. (1997b). The novel receptor TRAIL-R4 induces NF-B and protects aginst TRAILmediated apoptosis, yet retains an incomplete death domain. Immunity 7, 813±820. Emery, J. G., McDonell, P., Burke, M. C., Deen, K. C., Lyn, S., Silverman, C., Dul, E., Appelbaum, E. R., Eichman, C., DiPrinzio, R., Dodds, R. A., James, I. E., Rosenberg, M., Lee, J. C., and Young, P. R. (1998). Osteoprotegerin is a receptor for the cytotoxic ligand TRAIL. J. Biol. Chem. 273, 14363± 14367. Griffith, T., Wiley, S., Kubin, M., Sedger, L., Maliszewski, C., and Fanger, N. (1999). Monocyte-mediated tumoricidal activity via the tumor necrosis factor-related cytokine, TRAIL. J. Exp. Med. 189, 1343±1353. Gruss, H. J., and Dower, S. K. (1995). Tumor necrosis factor ligand superfamily: involvement in the pathology of malignant lymphomas. Blood 85, 3378±3404. Jeramias, I., Herr, I., Boehler, T., and Debatin, K. M. (1998). TRAIL/Apo-2-ligand-induced apoptosis in human T cells. Eur. J. Immunol. 28, 143±152. Keane, M. M., Ettenberg, S. A., Nau, M. M., Russel, E., and Lipkowitz, S. (1999). Chemotherapy augments TRAIL-induced apoptosis in breast cell lines. Cancer Res. 59, 734±741. Katsikis, P. D., Garcia-Ojeda, M. E., Torres-Roca, J. F., Tijoe, I. M., Smith, C. A., and Herzenberg, L. A. (1997). TNF-related apoptosis-inducing ligand can mediate activationinduced T cell death in HIV infection. J. Exp. Med. 186, 1365± 1372. Kothny-Wilkes, G., Kulms, D., Poppelmann, B., Luger, T. A., Kubin, M., and Schwartz, T. (1998). Interleukin-1 protects transformed keratinocytes from tumor necrosis factor-related apoptosis-inducing ligand. J. Biol. Chem. 273, 29247±29253.

Mariani, S., and Krammer, P. (1998a). Differential regulation of TRAIL and CD95 ligand in transformed cells of the T and B lymphocyte lineage. Eur. J. Immunol. 28, 973±982. Mariani, S., and Krammer, P. (1998b). Surface expression of TRAIL/Apo2 ligand in activated mouse T and B cells. Eur. J. Immunol. 28, 1492±1498. Mariani, S. M., Matiba, B., Armandola, E. A., and Krammer, P. H. (1997). Interleukin 1-b-converting enzyme related proteases/caspases are involved in TRAIL-induced apoptosis of Myeloma and leukemia cells. J. Cell. Biol. 137, 221±229. Marsters, S., Pitti, R., Donahue, C., Ruppert, S., Bauer, K., and Ashkenazi, A. (1996). Activation of apoptosis by Apo-2 ligand is independent of FADD but blocked by CrmA. Curr. Biol. 6, 750±752. Marsters, S. A., Sheridan, J. P., Pitti, R. M., Huang, A., Skubatch, M., Baldwin, D., Yuan, J., Gurney, A., Goddard, A. D., Godowski, P., and Ashkenazi, A. (1997). A novel receptor for Apo2L/TRAIL contains a truncated death domain. Curr. Biol. 7, 1003±1006. Martinez-Lorenzo, M. J., Alava, M. A., Gamen, S., Kim, J. K., Chuntharapai, A., Pineiro, A., Naval, J., and Anel, A. (1998). Involvement of Apo2 ligand/TRAIL in activation-induced death of Jurkat and human peripheral blood T cells. Eur. J. Immunol. 28, 2714±2725. Mongkolsapaya, J., Cowper, A., Xu, X. N., Morris, G., McMichael, A., Bell, J. I., and Screaton, G. R. (1998). Lymphocyte inhibitor of TRAIL: a new receptor protecting lymphocytes from the death ligand TRAIL. J. Immunol. 160, 3±6. Mori, S., Murakami-Mori, K., Nakamura, S., Ashkenazi, A., and Bonavida, B. (1999). Sensitization of AIDS-Kaposi's sarcoma cells to Apo2 ligand-induced apoptosis by actinomycin D. J. Immunol. 162, 5616±5623. Muhlenbeck, F., Haas, E., Schwenzer, R., Schubert, G., Grell, M., Smith, C., Scheurich, P., and Wajant, H. (1998). TRAIL/ Apo2L activates c-Jun NH2-terminal kinase (JNK) via caspase-dependent and caspase-independent pathways. J. Biol. Chem. 273, 33091±33098. Nagata, S. (1997). Apoptosis by death factor. Cell 88, 355±365. Pan, G., and Dixit, V. M. (1997). An antagonist decoy receptor and a new death domain-containing receptor for TRAIL. Science 277, 815±818. Pan, G., O'Rourke, K., Chinnaiyan, A. M., Gentz, R., Ebner, R., Ni, J., and Dixit, V. M. (1997). The receptor for the cytotoxic ligand TRAIL. Science 276, 111±113. Pan, G., Bauer, J. H., Haridas, V., Wang, S., Liu, D., Yu, G., Vincenz, C., Aggarwal, B., Ni, J., and Dixit, V. M. (1998a). Identification and functional characterization of DR6, a novel death domain-containing TNF receptor. FEBS Lett. 431, 351± 356. Pan, G., Ni, J., Yu, G. L., Wei, Y. F., and Dixit, V. M. (1998b). TRUNDD, a new member of the TRAIL receptor family that antagonizes TRAIL signaling. FEBS Lett. 424, 41±45. Pitti, R. M., Marsters, S. A., Ruppert, S., Donahue, C. J., Moore, A., and Ashkenazi, A. (1996). Induction of apoptosis by Apo-2 Ligand, a new member of the tumor necrosis factor receptor family. J. Biol. Chem. 271, 12697±12690. Rieger, J., Naumann, U., Glaser, T., Ashkenazi, A., and Weller, M. (1998). Apo2 ligand: a novel lethal weapon against malignant glioma? FEBS Lett. 427, 124±128. Screaton, G. R., Mongkolsapaya, J., Xu, X. N., Cowper, A. E., McMichael, A. J., and Bell, J. I. (1997). TRICK2, a new alternatively spliced receptor that transduces the cytotoxic signal from TRAIL. Curr. Biol. 7, 693±696.

Apo2L/TRAIL Sheridan, J. P., Marsters, S. A., Pitti, R. M., Gurney, A., Skubatch, M., Baldwin, D., Ramakrishnan, L., Gray, C., Baker, K., Wood, W. I., Goddard, A. D., Godowski, P., and Ashkenazi, A. (1997). Control of TRAIL-induced apoptosis by a family of signaling and decoy receptors. Science 277, 818±821. Simonet, W. S., Lacey, D. L., Dunstan, C. R., Kelley, M., Chang, M. S., Luthy, R., Nguyen, H. Q., Wooden, S., Bennet, L., Boone, T., Shimamoto, G., DeRose, M., Elliot, R., Colombero, A., Tan, H. L., Trail, G., Sullivan, J., Davy, E., Bucay, N., and Boyle, W. J. (1997). Osteoprotegerin: a novel secreted protein involved in the regulation of bone density. Cell 89, 309±319. Smith, C. A., Farrah, T., and Goodwin, R. G. (1994). The TNF receptor superfamily of cellular and viral proteins: activation, costimulation, and death. Cell 76, 959±962. Snell, V., Clodi, K., Zhao, S., Goodwin, R., Thomas, E. K., Morris, S. W., Kadin, M. E., Cabanillas, F., Andreeff, M., and Younes, A. (1997). Activity of TNF-related apoptosisinducing ligand (TRAIL) in haematological malignancies. Br. J. Haematol. 99, 618±624. Tan, K., Harrop, J., Reddy, M., Young, P., Terrett, J., Emery, J., Moore, G., and Truneh, A. (1997). Characterization of a novel TNF-like ligand and recently described TNF ligand and TNF receptor superfamily genes and their constitutive and inducible expression in hematopoietic and non-hematopoietic cells. Gene 204, 35±46. Thomas, W. D., and Hersey, P. (1998). TNF-related apoptosisinducing ligand (TRAIL) induces apoptosis in Fas ligand-resistant melanoma cells and mediates CD4 T cell killing of target cells. J. Immunol. 161, 2195±2200. Thome, M., Schneider, P., Hofmann, K., Fickenscher, H., Meinl, E., Neipel, F., Mattmann, C., Burns, K., Bodmer, J., Schroter, M., Scaffidi, C., Krammer, P., Peter, M., and Tschopp, J. (1997). Viral FLICE-inhibitory proteins (FLIPs)

511

prevent apoptosis induced by death receptors. Nature 386, 517±521. Walczak, H., Degli-Esposti, M. A., Johnson, R. S., Smolak, P. J., Waugh, J. Y., Boiani, N., Timour, M. S., Gerhart, M. J., Schooley, K. A., Smith, C. A., Goodwin, R. G., and Rauch, C. T. (1997). TRAIL-R2: a novel apoptosis-mediating receptor for TRAIL. EMBO J. 16, 5386±5397. Walczak, H., Miller, R. E., Ariail, K., Gliniak, B., Griffith, T. S., Kubin, M., Chin, W., Jones, J., Woodward, A., Le, T., Smith, C., Smolak, P., Goodwin, R. G., Rauch, C. T., Schuh, J. C. L., and Lynch, D. H. (1999). Tumoricidal activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo. Nature Med. 5, 157±163. Wang, J., Zheng, L., Lobito, A., Chan, F., Dale, J., Sneller, M., Yao, X., Puck, J., Straus, S., and Lenardo, M. (1999). Inherited human caspase 10 mutations underlie defective lymphocyte and dendritic cell apoptosis in autoimmune lymphoproliferative syndrome type II. Cell 98, 47±58. Wiley, S. R., Schooley, K., Smolak, P. J., Din, W. S., Huang, C. P., Nicholl, J. K., Sutherland, G. R., Davis-Smith, T., Rauch, C., Smith, C. A., and Goodwin, R. G. (1995). Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity 3, 673±682. Wu, G. S., Burns, T. F., McDonald, E. R., Jiang, W., Meng, R., Krantz, I. D., Kao, G., Gan, D. D., Zhou, J. Y., Muschel, R., Hamilton, S. R., Spinner, N. B., Markowitz, S., Wu, G., and El-Deiry, W. (1997). KILLER/DR5 is a DNA damage-inducible p53-regulated death receptor gene. Nature Genet. 17, 141±143. Zamani, L., Ahmad, M., Bennet, I. M., Azzoni, L., Alnemri, E. S., and Perussia, B. (1998). Natural killer (NK) cell-mediated cytotoxicity: differential use of TRAIL and Fas ligand by immature and mature primary human NK cells. J. Exp. Med. 188, 2375± 2380.