Abstract 581: Bone marrow-derived B-cell hybridomas from neuroblastoma patients generate antibodies that bind to patients’ own tumors

Today antibody therapy is considered to be one of the most important and successful strategies to treat a variety of cancers. For example, the addition of antibodies such as Herceptin and Avastin to a chemotherapy regimen has shown improved survival in the treatment of breast cancer and colorectal cancer, respectively. Main problems with this kind of therapy are that many patients are not candidates because their tumors do not overexpress the drug target and that patient develop resistance to the targeted drug. A method to rapidly develop different sets of therapeutic antibodies would greatly contribute to the field of targeted anticancer therapy. This work evaluated the feasibility of using residual clinical material from pediatric neuroblastoma patients to generate antibodies to autologous tumor. Neuroblastoma is the most common extracranial solid tumor in children, accounting for 8-10% of all childhood cancers. Most patients with neuroblastoma are young and commonly present with metastatic disease. Bone marrow aspirate from neuroblatoma patients was the source material for the mononuclear cells and the tumor cells used in present study. Tumor cells were cultured and xenograft tumors were produced in mice. Hybridomas were generated by electrofusion of stimulated bone marrow mononuclear cells with plasmacytoma P3×63.Ag8.653 under hypo-osmolar condition using Eppendorf Multiporator/Helix chamber. Following hypoxanthine-aminopterin-thymidine (HAT) selection and monoclonal distribution, the culture supernatants were assayed for immunoglobulin secretion by ELISA. The supernatants from the positive clones were evaluated by immunofluorescence microscopy for binding to cultured neuroblastoma cells and neuroblastoma xenograft tissue sections derived from the same patient from which the hybridomas were generated. The results demonstrated that multiple hybridomas of bone marrow mononuclear cells secreted monoclonal antibodies that bound autologous neuroblastoma cells. Further evaluation of the tumor-binding antibodies on a panel of normal human tissues showed no binding to most of the tissues in the panel. Successful outcome of these experiments demonstrate the feasibility of generating human monoclonal antibodies from residual marrow specimens that bind autologous neuroblastoma cells. However, it remains to be determined whether these antibodies are bioactive and whether this approach will be generally applicable in more patients with neuroblastoma. It may be conc