287 Background: Trastuzumab has been proven to be effective in the immunotherapy for Her-2/neu-positive breast cancer. However, this adoptively transferred therapeutic Ab must be continuously given to the patient with huge financial cost. Thus, it would be desirable if tumor vaccines could elicit long-lasting trastuzumab-like Ab. Methods: Constructing fusion protein of anti-mouse CD19 single chain variable fragment (anti-CD19-scFv) with human Her-2/neu extracellular domain P3-4 (P3-4) by extracting total RNA from Rat anti-mouse CD19 hybridoma (1D3) and first strand cDNA was synthesized accordingly. VH and VL were amplified using designed primers. Single chain Fv (VL-VH) was then synthesized by overlapping PCR. Then constructs were sequenced and cloned into pET-20b(+) vectors. Fragments P3-4 of extracellular domains of human Her-2/neu was cloned from pcDNA3.1-Her2 and then cloned into pET-20b(+)-scFv. Series of constructs were expressed and purified according to standard protocol and verified by Western Blot. Results: Both in vitro and in vivo studies demonstrated that fusion proteins anti-CD19-scFv and anti-CD19-scFv-P3-4 but not P3-4 could specifically bind to B cells with high affinity. Mice immunized with anti-CD19-scFv-P3-4 secreted higher titers of IgG and IgM than those from controls (p<0.05). Studies also demonstrated that sera from anti-CD19-scFv-P3-4 but not anti-CD19-scFv or P3-4 immunized mice stained with Her-2/neu expressing SKOV-3 tumor cells. These Abs also competitively inhibited trastuzumab-mediated Ag binding, suggesting that trastuzumab-like Ab responses were elicited. WT mice immunized with anti-CD19-scFv-P3-4 fusion protein then challenged with D2/F2-Her-2 mammary tumor cells showed significantly reduced tumor burden compared to those immunized with control fusion proteins (p<0.05) and had enhanced median overall survival to 45 days versus 34 days in WT mice immunized with either anti-CD19-scFv or P3-4. Conclusions: Targeting of Her-2/neu antigens to B cells stimulates Th-dependent humoral immune responses with anti tumor effect in mouse model. These findings provide a novel avenue for successful development of breast cancer vaccination strategy and help to fight for cancer.
Identification of tumor associated single-chain Fv by panning and screening antibody phage library using tumor cells
