Preparation and in-vitro bioactivity of a novel superantigen conjugate targeting bladder carcinoma

Superantigens have shown potent effects against bladder tumours by inducing Vbeta-specific T-lymphocyte proliferation and massive cytokine release but therapeutic benefit is compromised by cytotoxicity towards non-malignant cells and hypotoxicity to major histocompability complex (MHC) II-negative tumour cells. We are therefore interested in a conjugate preparation of a monoclonal antibody (MAb)-superantigens conjugate for which these drawbacks would be resolved.

The Fab fragment of the anti-bladder carcinoma MAb BDI-1 was conjugated to one member of the staphylococcal enterotoxin A (SEA) superantigen using the chemical conjugating reagent, N-succinimidyl 3-(2-pyridyldithio) propionate.

After HPLC purification through a Superdex-200 gel column, another peak with a molecular mass of 250 KDa was observed before Fab and SEA were eluted. Indirect immunocytochemical analysis and immunofluorescence tests showed that the cell membranes of most human bladder cancer cells were positively stained only by the conjugate, confirming the ability of the conjugate to target human bladder carcinoma. Peripheral blood mononuclear cell proliferation and cytokine release were similar with the conjugate and SEA. Cytotoxicity targeting in MHC II-negative bladder cancer cell lines, evaluated by flow cytometry, showed significant differences between the conjugate and SEA, whereas there was no difference in the Lovo colon cancer cell line.

These findings indicate the conjugate of SEA protein and BDI-1 Fab fragment was prepared successfully and targeted bladder carcinoma in vitro.