Disialoganglioside G(D2) is widely expressed among neuroblastomas, melanomas, small-cell lung carcinoma, sarcomas and brain tumors. Immunity directed against this antigen may have anti-tumor utility. Since G(D2) is poorly immunogenic, anti-idiotypic antibodies may serve as alternative tumor vaccines. Monoclonal antibody 3F8, a murine IgG3 specific for G(D2), has shown excellent tumor-targeting ability in vitro and in vivo. LOU/CN rats were immunized with 3F8 and their spleens were used in somatic-cell hybridization, using SP2/0, P3 and Y3 as fusion partners. Six anti-idiotypic (anti-id) MAbs (C2D8, Idio-2, AlG4, C2H7, C4E4, A2A6) were selected based on their reactivity with 3F8 and non-reactivity with murine IgG3 myelomas. Specificity of each anti-id was demonstrated by using various ELISA: (i) lack of direct binding to solid phase myelomas and serum proteins; (ii) inability of other myelomas to inhibit anti-id binding to 3F8; (iii) absence of cross-reactivity of other myelomas to solid-phase anti-id; (iv) lack of inhibition by anti-id of binding of other ganglioside antibodies to their antigens. Antigen specificity was further examined by inhibition of binding of 3F8 to G(D2) on immuno-thin-layer chromatography, and by inhibition of 3F8 immunostaining of neuroblastoma cell lines. These 6 antibodies were demonstrated to be distinct, in view of their cross-reactivity, fusion partners and relative strength of binding to 3F8. Anti-G(D2) antibodies were induced after immunization with these anti-id antibodies in C57B1/6 mice. These rat anti-3F8-idiotypic antibodies with exquisite specificity for anti-G(D2) antibodies may be useful in vaccine construction.
