Eradication of Neuroblastoma Cells in Vitro by Monoclonal Antibody and Human Complement: Method for Purging Autologous Bone Marrow1

We describe an in vitro method which is useful for purging autologous bone marrow of neuroblastoma cells. The method utilizes a single murine monoclonal antibody 3G6 (an immunoglobulin M«)which we have previously developed against the ganglioside GD2;undiluted human complement; and unfractionated whole bone marrow at 1 x 107 nucleated cells/ml. Tumor cell clonogenic assays, Hoechst 33342 fluorescent nuclear stain, and trypan blue viability stain methods were used to assay cytotoxicity. This complement-mediated cytotoxicity technique killed 99.9-100% of neuroblastoma cell lines NMB-7, LAN-1, LAN-5, and IMR-6, while normal marrow precursor cells were not detectably damaged. The presence of normal bone marrow did not inhibit the human complement-mediated cytotoxicity. Applying the cytotoxicity method to whole unseparated bone marrow demonstrated killing of seeded neuroblastoma cells, with no gross hemolysis or cell clumping. The method did not require expensive special equipment, use of animal complement sera, or prior fractionation of the bone marrow. The average marrow nucleated cell recovery was 95%. These studies indicate that in vitro purging of autologous marrow infiltrated with neuroblas toma with monoclonal antibody 3G6 and human complement is both technically feasible and effective in eradicating residual tumor while preserving bone marrow stem cells.