This proposal outlines two closely related projects, (1) production and evaluation of monoclonal antibodies which interfere with in vitro regulation of growth and differentiation of cultured human neuroblastoma, (2) development of techniques of identification and elimination of neuroblastoma from bone marrow for autologous transplantation. The effects of humoral factors on the regulation of growth and differentiation of cultured human neuroblastoma (CHNB) cells will be examined with assays of clonal growth and morphological differentiation. Effects of the regulatory factors on phosphorylation of specific cell proteins will be assessed by labeling cells with 32PO4 in the presence of the humoral factor and separating proteins by electrophoresis. A panel of mouse monoclonal antibodies which bind to cell surface antigens of many CHNB cell lines will be made by immunizing mice sequentially with different CHNB cell lines. Effects of monoclonal antibodies on protein phosphorylation will be assayed to find mouse monoclonal antibodies which mimic or interfere with biological actions of the regulatory factors. In addition, mouse monoclonal antibodies which identify CHNB cell surface differentiation antigens will be made. The monoclonal antibodies identified will be powerful tools to investigate growth and differentiation of human neuroblastoma, and may be useful therapeutically. Some of the mouse monoclonal antibodies produced will be used to develop methods to identify and eliminate metastatic neuroblastoma cells in patient bone marrow in vitro, prior to treatment of neuroblastoma with autologous bone marrow transplantation. Transplantation with cytotoxic therapy is promising treatment for potentially fatal neuroblastoma. CHNB cells mixed with normal bone marrow will be used as a model in which to develop the techniques. One assay to detect metastatic neuroblastoma will use neuron specific enolase antiserum and/or monoclonal antibodies, binding of which will be detected by immunoperoxidase and immunofluorescent flow cytometry techniques. A second assay to identify and quantify neuroblastoma in bone marrow will be selective growth of tumor cell colonies in semi-solid medium. Mouse monoclonal antibodies will be used to selectively kill neuroblastoma by antibody dependent, complement-mediated cytotoxicity. The techniques of quantifying and eliminating CHNB cells from bone marrow in vitro will be assessed in patient bone marrow containing metastatic neuroblastoma.
