The current approach utilized in our immune reconstitution trials for pediatric sarcomas involves the adoptive transfer of T cells derived from patients prior to the administration of cytotoxic chemotherapy. This approach is used because peripheral expansion of mature T cells represents the primary pathway for T cell regeneration in the immediate post-chemotherapy period and even limited cycles of chemotherapy induce significant T cell depletion. Unfortunately, these products are contaminated with tumor cells in approximately 50-80% of cases. Similarly, a high incidence of tumor cell contamination has been observed in peripheral blood stem cell products used extensively in the context of autologous BMT for these tumors. Because such contaminating tumor cells have been shown to contribute to tumor recurrence in other pediatric tumors, a primary goal is the elimination of contaminating tumor cells in the cellular products used in the context of these therapies. To this end, we are currently studying a monoclonal antibody in collaboration with Dr. Nai-Kong Cheung of Memorial Sloan Kettering Cancer Center. This moab (8H9) shows excellent binding to osteosarcoma, Ewing's sarcoma, neuroblastoma and rhabdomyosarcoma cell lines in our laboratory by flow cytometric analysis. In addition, we show no evidence of binding to stem cell populations (CD34+), T cells or other hematologic populations. Therefore, current work is focused on the utilization of magnetic bead selection to purge these cell populations of contaminating tumor cells. In order to document removal of tumor cells, we are utilizing quantitative polymerase chain reaction which amplifies the fusion proteins present in Ewing's sarcoma and alveolar rhabdomyosarcoma. Currently, this technique is able to detect the equivalent of one Ewing's sarcoma cell in 1,000,000 peripheral blood mononuclear cells using nested techniques. By purging the contaminated peripheral blood mononuclear populations or bone marrow populations using 8H9 mediated magnetic bead selection, we can diminish the level of contamination by 2-4 logs. We have now shown that clinical samples are typically contaminated in the range of 1:10e5-1:10e6. Therefore, we anticipate the 8H9 based purging alone will reduce contamination below detectable levels. Furthermore, when combined with CD34 selection for autologous BMT, we anticipate an approximate 5 log reduction in contaminating tumor.The results of this work were presented at the ASCO meeting by Dr. Merino in May 2000. They are currently being prepared for submission for publication and we are currently developing clinical grade moab 8H9 which may be used in clinical trials within the upcoming year.
