Clinical Scale Preparation of B Lymphoblastoid Cell Line-Tumor Cell Hybridoma Cells for Vaccination in Patients with Relapsed Ewing’s Sarcoma and Neuroblastoma.

Despite improvements in survival for pediatric patients with cancer, for those with recurrent, high risk Ewing’s sarcoma (ES) or neuroblastoma (NB) treatment options are limited. Tumor-specific antigens in these malignancies are poorly characterized, and expression of human leukocyte antigen (HLA) and co-stimulatory molecules may be down-regulated, imparing a cellular immune response. Several groups have studied the use of tumor cell-dendritic cell (DC) hybridoma vaccines in murine tumor models as well as in phase I clinical trials in human cancer. One problem in implementing these vaccine strategies on a clinical scale is the requirement for repeat blood collections or leukapheresis to expand a limited number of DC. B lymphoblastoid cell lines (BLCL) are effective APC expressing HLA as well as key co-stimulatory molecules, and can be rapidly expanded from relatively few peripheral blood mononuclear cells. We have studied the ability of BLCL to be fused to a series of NB and ES cell lines using polyethylene glycol, and the ability to propagate these hybrid cells long term in culture. Using monoclonal antibodies specific for tumor markers (anti-GD2 and CD56 for NB), BLCL (CD19), and by labeling ES tumor cells with fluorescent dye prior to fusion, 3–11% of the resulting cell products represented true fusion cells. This was confirmed in studies in which BLCL transduced to express green fluorescent protein (GFP) were fused with adherent tumor cells, resulting in a population of large green adherent cells with abnormal morphology. Dual fluorescent staining and flow cytometry of green fluorescent dye labeled tumor cells and unlabelled BLCL consistently revealed a mixed population of unfused BLCL, unfused tumor cells, and cells with irregular morphology expressing both CD19 and green fluorescent dye. Despite these findings, these hybrid cells could not be expanded in long term culture, and adherent hybrid cells did not survive in cell culture over 2 weeks. In addition, these hybrid cells could not be selected when tumor cells and BLCL were transduced with different antibiotic resistance genes and then subjected to selection media. Despite the inability to selectively expand tumor cell-BLCL hybridomas for long-term culture, the choice of BLCL as an APC for vaccine permits the preparation of relatively large numbers of fusion cells for serial vaccination or for in vitro stimulation of antigen specific CTL.