Preventive DNA vaccination against CEA-expressing tumors with anti-idiotypic scFv6.C4 DNA in CEA-expressing transgenic mice.

Carcinoembryonic antigen (CEA) is expressed during embryonic life and in low level during adult life. Consequently, the CEA is recognized by the immune system as a self-antigen and thus CEA-expressing tumors are tolerated. Previously, we constructed a single chain variable fragment using the 6.C4 (scFv6.C4) hybridoma cell line, which gave rise to antibodies able to recognize CEA when C57/Bl6 mice were immunized. Here, the scFv6.C4 ability to prevent the CEA-expressing tumor growth was assessed in CEA-expressing transgenic mice CEA2682. CEA2682 mice immunized with the scFv6.C4 expressing plasmid vector (uP/PS-scFv6.C4) by electroporation gave rise to the CEA-specific AB3 antibody after the third immunization. Sera from immunized mice reacted with CEA-expressing human colorectal cell lines CO112, HCT-8, and LISP-1, as well as with murine melanoma B16F10 cells expressing CEA (B16F10-CEA). Cytotoxic T lymphocytes (CTL) from uP/PS-scFv6.C4 immunized mice lysed B16F10-CEA (56.7%) and B16F10 expressing scFv6.C4 (B16F10-scFv6.C4) (46.7%) cells, against CTL from uP-immunized mice (10%). After the last immunization, 5 × 105 B16F10-CEA cells were injected into the left flank. All mice immunized with the uP empty vector died within 40 days, but uP/PS-scFv6.C4 vaccinated mice (40%) remained free of tumor for more than 100 days. Splenocytes obtained from uP/PS-scFv6.C4 vaccinated mice showed higher T-cell proliferative activity than those from uP vaccinated mice. Collectively, DNA vaccination with the uP-PS/scFv6.C4 plasmid vector was able to give rise to specific humoral and cellular responses, which were sufficient to retard growth and/or eliminate the injected B16F10-CEA cells.

The idiotype (Id) cascade in mice elicited the production of anti-R24 Id and anti-anti-Id monoclonal antibodies with antitumor and protective activity against human melanoma.

Gangliosides have been considered as potential targets for immunotherapy because they are overexpressed on the surface of melanoma cells. However, immunization with purified gangliosides results in a very poor immune response, usually mediated by IgM antibodies. To overcome this limitation, we immunized mice with R24, a monoclonal antibody (mAb) that recognizes the most tumor-restricted ganglioside (GD3); our goal was to obtain anti-idiotype (Id) antibodies bearing the internal image of GD3. Animals produced anti-Id and anti-anti-Id antibodies. Both anti-Id and anti-anti-Id antibodies were able to inhibit mAb R24 binding to GD3. In addition, the anti-anti-Id antibodies were shown to recognize GD3 directly. Anti-Id and anti-anti-Id mAb were then selected from two fusion experiments for evaluation. The most interesting finding emerged from the characterization of the anti-anti-Id mAb 5.G8. It was shown to recognize two different GD3-expressing human melanoma cell lines in vitro and to mediate tumor cell cytotoxicity by complement activation and antibody-dependent cellular cytotoxicity. The biological activity of the anti-anti-Id mAb was also tested in a mouse tumor model, in which it was shown to be a powerful growth inhibitor of melanoma cells. Thus, activity of the anti-anti-Id mAb 5.G8 matched that of the prototypic anti-GD3 mAb R24 both in vitro and in vivo. Altogether, our results indicate that the idiotype approach might produce high affinity, specific and very efficient antitumor immune responses.