Production and Characterization of Two Human Glioma Xenograft-localizing Monoclonal Antibodies1

Multiple fusions following immunization of athymic mice with the extensively characterized human glioma cell line D-54 MG resulted in the selection of several antibodies (Mabs) highly reactive with tumors of neuroectodermal origin and unreactive with normal nervous system tissue. Two Mabs, C12 and D12, which localized specifically to tumors in athymic mouse-human glioma xenograft paired label localization assays, are IgG3 antibodies; both bind readily to staphylococcal protein A in column purification and radioimmunoprecipitation procedures. Both iodinate via the chloramine-T method yielding I2*l-immunoreactive product by direct cell surface radioimmunoassay and absorption assay. By indirect cell surface radioimmunoassay, a cultured cell line panel consisting of 17 gliomas, 3 medulloblastomas, 2 neuroblastomas, 2 melanomas, and 2 fetal and 2 adult brain-derived cell lines was examined; the two Mabs were highly similar but distinct in their reactivity profiles. Each was positive with >47% of the gliomas tested (C12, 9 of 17; D12, 8 of 17); and with 1 of 3 medulloblastomas, 1 of 2 melanomas, and cell lines derived from 12and 16-week-gestation human fetal brain. No reactivity was observed with neuroblastoma or adult brain-derived cell lines or with neutral glycolipids and gangliosides extracted from D-54 MG xenografts or human glioma cell lines. Notable extraneuroectodermal reactivity included that of Mab D12 for splenic trabeculae and the spermatids and Sertoli cells in the testes. Following immunoprecipitation of ('Hjleucine labeled cell membrane preparations, Mabs C12 and D12 have consis tently yielded unique bands in the M, 180,000 and M, 88,000 regions respectively. When used in paired label localization experiments in s.c. D-54 MG xenograft-bearing athymic mice, Mabs C12 and D12 demon strate similar localization patterns, attaining peak localization indices at day 3 (D12) or 4 (C12); the maximum percentage of injected Mab bound to tumor ranged from 57°(D12) to 8% (C12). The peak tumor/normal brain localization ratios (167-181) attained by these Mabs at days 1-2 followed by their rapid clearance suggest that these Mabs are potentially useful imaging and therapeutic agents for further investigation.