Detection of Amino-terminal Extracellular Domain of Somatostatin Receptor 2 by Specific Monoclonal Antibodies and Quantification of Receptor Density in Medulloblastoma

Abstract

Somatostatin receptor 2 (SSTR2) is expressed by most medulloblastomas (MEDs). We isolated monoclonal antibodies (MAbs) to the 12-mer ₃₃QTEPYYDLTSNA₄₄, which resides in the extracellular domain of the SSTR2 amino terminus, screened the peptide-bound MAbs by fluorescence microassay on D341 and D283 MED cells, and demonstrated homogeneous cell-surface binding, indicating that all cells expressed cell surface–detectable epitopes. Five radiolabeled MAbs were tested for immunoreactive fraction (IRF), affinity (KA) (Scatchard analysis vs. D341 MED cells), and internalization by MED cells. One IgG₃ MAb exhibited a 50–100% IRF, but low KA. Four IgG_2a MAbs had 46–94% IRFs and modest KAs versus intact cells (0.21–1.2 × 10⁸ M⁻¹). Following binding of radiolabeled MAbs to D341 MED at 4°C, no significant internalization was observed, which is consistent with results obtained in the absence of ligand. However, all MAbs exhibited long-term association with the cells; binding at 37°C after 2 h was 65–66%, and after 24 h, 52–64%. In tests with MAbs C10 and H5, the number of cell surface receptors per cell, estimated by Scatchard and quantitative FACS analyses, was 3.9 × 10⁴ for the “glial” phenotype DAOY MED cell line and 0.6–8.8 × 10⁵ for four neuronal phenotype MED cell lines. Our results indicate a potential immunotherapeutic application for these MAbs.