A Human scFv Antibody against TRAIL Receptor 2 Induces Autophagic Cell Death in Both TRAIL-Sensitive and TRAIL-Resistant Cancer Cells

Abstract

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) induces apoptotic cell death in a variety of tumor cells without significant cytotoxicity on normal cells. However, many cancer cells with apoptotic defects are resistant to treatment with TRAIL alone, limiting its potential as an anticancer therapeutic. Here, we report on the tumoricidal activity of a human single-chain fragment variable, HW1, which specifically binds to TRAIL receptor 2 (TR2) without competing with TRAIL for the binding. HW1 treatment as a single agent induces autophagic cell death in a variety of both TRAIL-sensitive and TRAIL-resistant cancer cells, but exhibits much less cytotoxicity on normal cells. The HW1-induced autophagic cell death was inhibited by an autophagy inhibitor, 3-methyladenine, or by RNA interference knockdown of Beclin-1 and Atg7. We also show that the HW1-mediated autophagic cell death occurs predominantly via the c-Jun NH 2 -terminal kinase pathway in a caspase-independent manner. Analysis of the death-inducing signaling complex induced by HW1 binding to TR2 exhibits the recruitment of TNF receptor-associated death domain and TNF receptorassociated factor 2, but not Fas-associated death domain, caspase-8, or receptor-interacting protein, which is distinct from that induced by TRAIL. Our results reveal a novel TR2-mediated signaling pathway triggering autophagic cell death and provides a new strategy for the elimination of cancer cells, including TRAIL-resistant tumors, through nonapoptotic cell death. [Cancer Res 2007;67(15):7327-34]

A nucleic acid-hydrolyzing antibody penetrates into cells via caveolae-mediated endocytosis, localizes in the cytosol and exhibits cytotoxicity

“…These results indicate that cellular mRNAs were sensitive to endocytosed 3D8 scFv in vivo, compared to rRNAs. Intense degradation of rRNA (28S and 18S) was observed when rRNA purified from cells was incubated with 3D8 scFv (0.1 mM) in a test tube, but not when incubated with HW1 scFv, which is an irrelevant scFv purified from bacterial cells [23] using exactly the same procedures (Fig. 8D), confirming that 3D8 scFv possessed the RNase activity.…”

“…The prolonged incubation of cells after 3D8 scFv internalization exhibited cytotoxicity. Figure 7 shows the cytotoxicity of 3D8 scFv in human cancer cell lines of HeLa, HCT116, and U87MG when incubated with various concentrations of 3D8 scFv (5 -40 mM) for 48 h. Cell viability was determined by MTT assay [23]. Compared to that of the untreated cells, the viability of three cell lines treated with 10 and 40 mM 3D8 scFv was decreased 50 -60 % and~20 %, respectively (Fig.…”

“…RNA cleavage assays with purified 3D8 scFv. 3D8 scFv (0.1 mM) or HW1 scFv (0.1 mM) purified from bacterial expression [17,23] was incubated with total cellular RNA (1 mg) extracted from HeLa cells, in TBS buffer (50 mM Tris-Cl, pH 7.5, 50 mM NaCl, pH 7.5) at 37 8C for 2 h prior to detection of hydrolysis. RNase A (1 U) was used as a positive control in the buffers provided by the manufacturer (New England Biolabs).…”

“…Cells seeded at density of 5 10 3 cells/well in 96-well plates were cultured overnight and then treated with 0 -40 mM of 3D8 scFv for 48 h. Cell viability was determined using a colorimetric MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide]-based Cell Growth Determination kit (Sigma) [23].…”

“…Cells (3 10 5 cells/well) were seeded in six-well plates, grown overnight, and then treated with 3D8 scFv under conditions as indicated in the figure legends. The Western blotting was then performed as described previously [23]. The appropriate secondary antibodies conjugated to horse radish peroxidase were used for developing chemiluminescence (Amersham Pharmacia Biotech).…”