Scot A. Marsters

Safety and antitumor activity of recombinant soluble Apo2 ligand

TNF and Fas ligand induce apoptosis in tumor cells; however, their severe toxicity toward normal tissues hampers their application to cancer therapy. Apo2 ligand (Apo2L, or TRAIL) is a related molecule that triggers tumor cell apoptosis. Apo2L mRNA is expressed in many tissues, suggesting that the ligand may be nontoxic to normal cells. To investigate Apo2Ls therapeutic potential, we generated in bacteria a potently active soluble version of the native human protein. Several normal cell types were resistant in vitro to apoptosis induction by Apo2L. Repeated intravenous injections of Apo2L in nonhuman primates did not cause detectable toxicity to tissues and organs examined. Apo2L exerted cytostatic or cytotoxic effects in vitro on 32 of 39 cell lines from colon, lung, breast, kidney, brain, and skin cancer. Treatment of athymic mice with Apo2L shortly after tumor xenograft injection markedly reduced tumor incidence. Apo2L treatment of mice bearing solid tumors induced tumor cell apoptosis, suppressed tumor progression, and improved survival. Apo2L cooperated synergistically with the chemotherapeutic drugs 5-fluorouracil or CPT-11, causing substantial tumor regression or complete tumor ablation. Thus, Apo2L may have potent anticancer activity without significant toxicity toward normal tissues.

Genomic amplification of a decoy receptor for Fas ligand in lung and colon cancer

Fas ligand (FasL) is produced by activated T cells and natural killer cells and it induces apoptosis (programmed cell death) in target cells through the death receptor Fas/Apo1/CD95 (ref. 1). One important role of FasL and Fas is to mediate immune-cytotoxic killing of cells that are potentially harmful to the organism, such as virus-infected or tumour cells. Here we report the discovery of a soluble decoy receptor, termed decoy receptor 3 (DcR3), that binds to FasL and inhibits FasL-induced apoptosis. The DcR3 gene was amplified in about half of 35 primary lung and colon tumours studied, and DcR3 messenger RNA was expressed in malignant tissue. Thus, certain tumours may escape FasL-dependent immune-cytotoxic attack by expressing a decoy receptor that blocks FasL.

Differential hepatocyte toxicity of recombinant Apo2L/TRAIL versions.

Our findings not only provide a novel insight into the pathogenesis of the transplant-related atherosclerosis, but also point to a new therapeutic strategy that involves targeting of homing, differentiation and proliferation of putative smooth-muscle progenitor cells derived from the recipient. This is the first report demonstrating that circulating progenitor cells contribute to the development of proliferative diseases. AKIO SAIURA, MASATAKA SATA, YASUNOBU HIRATA, RYOZO NAGAI MASATOSHI MAKUUCHI Department of Surgery, University of Tokyo, Graduate School of Medicine, Tokyo, Japan, Department of Cardiovascular Medicine University of Tokyo, Graduate School of Medicine, Tokyo, Japan A.S. and M.S. supervised this study equally as senior authors Email: sata-2im@h.u-tokyo.ac.jp 1. McKay, R. Stem cells-hype and hope. Nature 406, 361–364 (2000). 2. Asahara, T. et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science 275, 964–967 (1997). 3. Yamashita, J. et al. Flk1-positive cells derived from embryonic stem cells serve as vascular progenitors. Nature 408, 92–96 (2000). 4. Carmeliet, P. One cell, two fates. Nature 408, 43–45 (2000). 5. Clarke, D.L. et al. Generalized potential of adult neural stem cells. Science 288, 1660–1663 (2000).

A novel receptor for Apo2L/TRAIL contains a truncated death domain

Apo2 ligand (Apo2L [1], also called TRAIL for tumor necrosis factor (TNF)-related apoptosis-inducing ligand [2]) belongs to the TNF family and activates apoptosis in tumor cells. Three closely related receptors bind Apo2L: DR4 and DR5, which contain cytoplasmic death domains and signal apoptosis, and DcR1, a decoy receptor that lacks a cytoplasmic tail and inhibits Apo2L function [3-5]. By cross-hybridization with DcR1, we have identified a fourth Apo2L receptor, which contains a cytoplasmic region with a truncated death domain. We subsequently named this protein decoy receptor 2 (DcR2). The DcR2 gene mapped to human chromosome 8p21, as did the genes encoding DR4, DR5 and DcR1. A single DcR2 mRNA transcript showed a unique expression pattern in human tissues and was particularly abundant in fetal liver and adult testis. Upon overexpression, DcR2 did not activate apoptosis or nuclear factor-kappaB; however, it substantially reduced cellular sensitivity to Apo2L-induced apoptosis. These results suggest that DcR2 functions as an inhibitory Apo2L receptor.

Interaction of the TNF homologues BLyS and APRIL with the TNF receptor homologues BCMA and TACI.

BLyS (also called TALL-1, THANK, or BAFF) [1] [2] [3] [4] is a member of the tumor necrosis factor (TNF) gene family that stimulates proliferation and immunoglobulin production by B cells. BLyS interacts with the TNF receptor (TNFR) homologue TACI (transmembrane activator and CAML-interactor) [5], and treatment of mice with a TACI-Fc fusion protein abolishes germinal center formation after antigenic challenge [6]. Here we report a novel interaction between BLyS and another TNFR homologue, BCMA (B cell maturation antigen) [7] [8]. Further, the TNF homologue APRIL [9], a close relative of BLyS, also bound to BCMA and TACI. BLyS or APRIL activated nuclear factor-kappaB (NF-kappaB) through TACI and BCMA, and each ligand stimulated immunoglobulin M (IgM) production by peripheral blood B cells. These results define a dual ligand-receptor system that may play an important role in humoral immunity.

Herpesvirus Entry Mediator, a Member of the Tumor Necrosis Factor Receptor (TNFR) Family, Interacts with Members of the TNFR-associated Factor Family and Activates the Transcription Factors NF-κB and AP-1

The mammalian tumor necrosis factor receptor (TNFR) family consists of 10 cell-surface proteins that regulate development and homeostasis of the immune system. Based on an expressed sequence tag, we have cloned a cDNA encoding a novel member of the human TNFR family. A closely related protein, designated HVEM (for herpesvirus entry mediator), was identified independently by another group as a mediator of herpesvirus entry into mammalian cells (Montgomery, R., Warner, M., Lum, B., and Spear, P. (1996)Cell 87, 427–436). HVEM differed from our clone by two amino acid residues, suggesting that the two proteins represent polymorphism of a single HVEM gene. We detected HVEM mRNA expression in several human fetal and adult tissues, although the predominant sites of expression were lymphocyte-rich tissues such as adult spleen and peripheral blood leukocytes. The cytoplasmic region of HVEM bound to several members of the TNFR-associated factor (TRAF) family, namely TRAF1, TRAF2, TRAF3, and TRAF5, but not to TRAF6. Transient transfection of HVEM into human 293 cells caused marked activation of nuclear factor-κB (NF-κB), a transcriptional regulator of multiple immunomodulatory and inflammatory genes. HVEM transfection induced also marked activation of Jun N-terminal kinase, and of the Jun-containing transcription factor AP-1, a regulator of cellular stress-response genes. These results suggest that HVEM is linked via TRAFs to signal transduction pathways that activate the immune response.

Identification of a mutation in the extracellular domain of the Epidermal Growth Factor Receptor conferring cetuximab resistance in colorectal cancer.

Antibodies against epidermal growth factor receptor (EGFR)—cetuximab and panitumumab—are widely used to treat colorectal cancer. Unfortunately, patients eventually develop resistance to these agents. We describe an acquired EGFR ectodomain mutation (S492R) that prevents cetuximab binding and confers resistance to cetuximab. Cells with this mutation, however, retain binding to and are growth inhibited by panitumumab. Two of ten subjects studied here with disease progression after cetuximab treatment acquired this mutation. A subject with cetuximab resistance harboring the S492R mutation responded to treatment with panitumumab.

Identification of a receptor for BLyS demonstrates a crucial role in humoral immunity

B ymphocyte stimulator (BLyS) is a member of the tumor necrosis factor (TNF) superfamily. BLyS stimulates proliferation of, and immunoglobulin production by, B cells. However, the relative importance of BLyS in physiological B cell activation is unclear. We identified a B cell receptor for BLyS through expression cloning as TACI, an orphan TNF receptor homologue of unknown function. Binding of BLyS to TACI activated signaling by nuclear factor-κB (NF-κB). In vitro soluble TACI-Fc fusion protein blocked BLyS-induced NF-κB activation in B lymphoma cells and IgM production in peripheral blood B cells. In vivo treatment of immunized mice with TACI-Fc inhibited production of antigen-specific IgM and IgG1 antibodies and abolished splenic germinal center (GC) formation. Thus, BLyS activity must play a critical role in the humoral immune response.

Isotype-Dependent Inhibition of Tumor Growth In Vivo by Monoclonal Antibodies to Death Receptor 4

To explore an approach for death receptor targeting in cancer, we developed murine mAbs to human death receptor 4 (DR4). The mAb 4H6 (IgG1) competed with Apo2L/TNF-related apoptosis-inducing ligand (DR4’s ligand) for binding to DR4, whereas mAb 4G7 (IgG2a) did not. In vitro, both mAbs showed minimal intrinsic apoptosis-inducing activity, but each triggered potent apoptosis upon cross-linking. In a colon tumor nude mouse model in vivo, mAb 4H6 treatment without addition of exogenous linkers induced apoptosis in tumor cells and caused complete tumor regression, whereas mAb 4G7 partially inhibited tumor growth. An IgG2a isotype switch variant of mAb 4H6 was much less effective in vivo than the parent IgG1-4H6, despite similar binding affinities to DR4. The same conclusion was obtained by comparing other IgG1 and IgG2 mAbs to DR4 for their anti-tumor activities in vivo. Thus, the isotype of anti-DR4 mAb may be more important than DR4 binding affinity for tumor elimination in vivo. Anti-DR4 mAbs of the IgG1 isotype may provide a useful tool for investigating the therapeutic potential of death receptor targeting in cancer.

Activation of apoptosis by Apo-2 ligand is independent of FADD but blocked by CrmA

A new member of the tumor necrosis factor (TNF) cytokine family, designated Apo-2 ligand (Apo-21) [1] or TRAIL [2], has been shown recently to induce apoptosis in various tumor cell lines; however, its biological role is unknown. Here, we show that Apo-21, activated apoptosis in T-cell-enriched cultures of peripheral blood lymphocytes stimulated by interleukin-2 (IL-2), but not in unstimulated cells. This finding suggests that, like Fas/Apo-1 ligand and TNF [3-5], Apo-2L may play a role in regulating post-stimulation apoptosis of mature lymphocytes. Studies on the mechanism of Apo-2L action demonstrated marked membrane blebbing, a hallmark of apoptosis, within a few minutes of the addition of Apo-2L to tumor cells. Ectopic expression of a dominant negative mutant of FADD, a cytoplasmic protein that mediates death signalling by Fas/Apo-1 and by TNF receptor type 1 (TNFR1) [6-9], inhibited the induction of apoptosis by anti-Fas/Apo-1 antibody, but had little effect on Apo-2L function. In contrast, expression of CrmA, a cowpox virus-derived inhibitor of the Ced-2-like proteases ICE [10] and CPP32/Yama [11,12], blocked the induction of apoptosis by either Apo-2L or anti-Fas/Apo-1 antibody. These results suggest that Apo-2L activates a rapid, FADD-independent pathway to trigger a cell-death programme that requires the function of cysteine proteases such as ICE or CPP32/Yama.