Figure 3

Target antigen-dependent bioactivity of scFv–scTRAIL. (a) Bioactivity on target antigen-negative cells. 2 × 10⁴ SKW6.4 cells/well were seeded in 96-well plates. The next day, cells were treated with serial dilutions of the fusion proteins in the presence or absence of anti-FLAG M2 antibody (2 μg/ml). (b) Bioactivity of scTRAIL versus scFv–scTRAIL. 5 × 10⁴ Colo205 or 2 × 10⁴ HT1080 cells/well were seeded in 96-well plates. The following day, cells were treated with 2.5 μg/ml cycloheximide and challenged in duplicates with increasing concentrations of scTRAIL and scFv–scTRAIL, respectively. After over night incubation, cell viability was determined by MTT assay or KV staining. Results from three to five independent experiments are shown (mean±S.D.). (c) Bioactivity of scFv–scTRAIL compared to ‘KillerTRAIL’: Same assay as in b but incubation with serial dilutions of scFv–scTRAIL or ‘KillerTRAIL’. (d, e) Target antigen-dependent apoptosis induction of scFv–scTRAIL: Same assay as in b with scFv–scTRAIL titrated in the presence or absence of anti-ErbB2 (FRP5, 5 μg/ml) (d) or with scFv–scTRAIL (0.05 nM) in the presence or absence of recErbB2 (10 μg/ml) (e). (f) Contribution of the scFv domain in the scFv–scTRAIL to apoptosis induction. Same assay as in b with scFv–scTRAIL titrated in the presence or absence of neutralizing anti-TRAIL antibodies (2E5, 1 μg/ml). (a, c–f) Results from three independent experiments are shown (mean±S.D.). (b, d) Statistical analysis was carried out with unpaired t-test, two-tailed test, ^**0.0026 <P>0.0098, ^***P<0.0001