Mouse pre immune or immune sera were collected prior to vac Vorinostat order cination or one week after the final boost. SALTO cells were incubated with purified Igs derived from rV neuT or V wt BALB neuT vaccinated mice followed by labeling with goat anti mouse IgG Ale a fluor 488 conjugated antibody. Figure 3, Panel B shows representative membrane staining of SALTO cells by Igs form rV neuT vaccinated mice simi larly to that of monoclonal anti Neu antibody Ab4. Con versely, Igs derived from V wt vaccinated Balb neuT mice or pre immune serum did not bind SALTO cells. Sera were employed to immunoprecipitate p185 Neu from LTR Neu or SALTO cells. Specific reactiv ity was visualized by immunoblotting of immunoprecipi tates using a Neu specific commercial antibody.

Analysis of serum reactivity taken from representative 108 pfu rV neuT and V wt vaccinated mice is depicted in Figure 3, Panel C. rV neuT vaccination was able to in duce specific anti Neu antibodies able to immunoprecipi tate the antigen from LTR Neu and SALTO cells. Specific antibodies were detected in all rV neuT vaccinated mice. Conversely, serum from V wt vaccinated mice was not able to immunoprecipitate the antigen from LTR Neu. Specific antibody response to Neu was quantitatively evaluated by ELISA. As shown in Table 2, 108 pfu rV neuT vaccinated mice developed a significantly higher titer of anti Neu antibodies than 107 pfu rV neuT and 106 pfu rV neuT vaccinated mice. No significant difference on anti Neu titer antibodies was observed between the 107 pfu rV neuT and 106 pfu rV neuT dose.

It is of note that anti Neu serum titer paralleled antitumor in vivo activity of rV neuT vaccinated mice. The administration of V wt did not result in the induction of anti Neu antibodies. E periments were then carried out to evaluate the iso type of the immunoglobulins elicited by rV neuT vac cination. As shown in Table 3, anti Neu immunoglobulins of rV neuT vaccinated Balb neuT mice were mainly of the IgG1, IgG2a and IgG2b isotype with a lesser amount of IgG3, IgM and IgA. In vitro biological activity of immune sera of rV neuT vaccinated mice ADCC, cell proliferation of BALB neuT SALTO tumor cells, receptor down regulation and induction of apoptosis in SALTO cells were analyzed using pooled sera or purified Igs from108 pfu rV neuT or AV-951 V wt vaccinated mice in order to investigate potential mechanisms of tumor inhibition by anti Neu Igs.

As shown in Figure 4, Panel A, spleen cells produced no cytoto icity in the presence of pooled sera from 108 pfu V wt vaccinated mice. Conversely, spleen cells in the presence of pooled sera from 108 pfu rV neuT vacci nated mice mediated higher ADCC at 1 10 and 1 20 dilution than sera from V wt vaccinated mice. To determine whether specific anti Neu Igs trichostatin a clinical trials were able to interfere with in vitro cell growth, SALTO cells were chron ically treated with different concentrations of purified Igs from rV neuT or V wt vaccinated mice in absence of fetal bovine serum. As shown in Figure 4, Panel B