Designed tumor necrosis factor-related apoptosis-inducing ligand variants initiating apoptosis exclusively via the DR5 receptor

Abstract

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potential anticancer drug that selectively induces apoptosis in a variety of cancer cells by interacting with death receptors DR4 and DR5. TRAIL can also bind to decoy receptors (DcR1, DcR2, and osteoprotegerin receptor) that cannot induce apoptosis. The occurrence of DR5-responsive tumor cells indicates that a DR5 receptor-specific TRAIL variant will permit tumor-selective therapies. By using the automatic design algorithm FOLD-X, we successfully generated DR5-selective TRAIL variants. These variants do not induce apoptosis in DR4-responsive cell lines but show a large increase in biological activity in DR5-responsive cancer cell lines. Even wild-type TRAIL-insensitive ovarian cancer cell lines could be brought into apoptosis. In addition, our results demonstrate that there is no requirement for antibody-mediated cross-linking or membrane-bound TRAIL to induce apoptosis through DR5.

Fig. 1.

Correlation of the predicted changes in binding affinity toward DR4 and DR5 compared with the experimental results of an alanine scanning performed by Hymowitz et al. (30) (open circles) and of the DR5-selective TRAIL variants (closed circles).

Fig. 2.

Receptor binding of TRAIL and DR5-selective variants toward DR5-Ig as determined by SPR (A) or toward DR4-Ig (B). Receptor binding is calculated relative to the response of TRAIL at 250 nM.

Fig. 3.

Biological activity of TRAIL and DR5-selective variants. (A) Apoptosis-inducing activity of 100 ng/ml TRAIL in the presence of 1 μg/ml DR4 (aDR4), DR5 (aDR5), or DR4 and DR5 (+aDR4+aDR5) receptor-neutralizing antibodies in Colo205 and ML-1 cells. (B) Apoptosis-inducing activity in Colo205 cells of 100 ng/ml TRAIL or DR5-selective variants without the presence of neutralizing DR4 or DR5 antibodies (no AB) or in the presence of neutralizing antibody [aDR4, aDR5, or both (aDR4 aDR5)]. Shown is the cytotoxic potential (% cell death) of TRAIL or DR5-selective variants in Colo205 (C), ML-1 (D), and A2780 (E) and of 1, 10, or 100 ng/ml TRAIL (WT) or D269H/E195R (DE) relative to cycloheximide control (0.33 μg/ml) in BJAB cells responsive to both DR4- and DR5-mediated cell death (BJAB^wt), BJAB cells deficient for DR5 (BJAB^{DR5 DEF}), and BJAB cells deficient for DR5 stably transfected with DR5 (BJAB^{DR5 DEF}+DR5) (F) (31).

Fig. 4.

Area of interaction of TRAIL and DR4/DR5 receptor around position 269 [TRAIL (A) and D269H variant (B)] and around position 214 [TRAIL(C) and T214R variant(D)]. Red ribbons indicate a receptor, and blue ribbons indicate TRAIL. Residues in DR5 complexes are in dark green, and residues in DR4 complexes are in light green. Arg 191 and Asp 267 are key TRAIL amino acids for DR5 receptor binding in the corresponding binding pocket of the receptor, as observed in the crystal structure of TRAIL in complex with DR5.