Protective effects of scFvNLDC-145-neu in transgenic BALB-neuT mice. A female BALB-neuT mice (10 mice per group) were vaccinated with neu or scFvNLDC-145-neu in left hind limb on days -21 and -7. Control animals received pcDNA3.1 or PBS. On day 0, mice were inoculated s.c. with neu-expressing TUBO cells in opposite flank. Left panel, kinetics of tumor growth; Right panel, survival curve. *, P < 0.01, scFvNLDC-145-neu compared with other groups. B in a separate experiment, splenocytes obtained from the vaccinated mice described above were tested for tumor killing activity. Data represented the means of triplicate cultures. *, P < 0.01, scFvNLDC-145-neu compared with other groups. C sera from the vaccinated mice described above were assayed for neu-specific antibodies by flow cytometry. The data were represented as mean fluorescence intensity. *, P < 0.01, scFvNLDC-145-neu compared with other groups. D animals were immunized neu or scFvNLDC-145-neu twice at weeks 8 and 10. One group mice with scFvNLDC-145-neu vaccination also received CTX injection 4 days before the first vaccination. Control animals received pcDNA3.1 or PBS. Development of mammary tumors was monitored by manual examination of the mammary glands once every week. Measurable masses of > 2 mm diameter were regarded as tumors. Points, mean number of tumors in each group (tumor multiplicity; left panel) and percentage of tumor-free mice (right panel); bars, SE. All results were representative of two to three independent experiments. *, P < 0.01, scFvNLDC-145-neu /CTX compared with other groups.