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Approaches for the Isolation of Biologically Functional Tumor-associated Antigens¹

Department of Molecular Immunology, Scripps Clinic and Research Foundation, La Jolla, California 92037 [R. A. R., G. S. D., S. F., M. A. P.], and Department of Oncology, University of California, Los Angeles, California 90024 [E. C. H.]

Melanoma-associated antigens were isolated from human melanoma cells in long-term tissue culture and from the spent culture fluid of these cells propagated in chemically defined, serum-free media. The 3 M KCI extracts from such cells and their concentrated spent culture media elicited specific delayed cutaneous hypersensitivity reactions in patients with malignant melanoma but not in patients with other neoplasms. HLA antigens present in these extracts could be specifically removed by ultracentrifugation in KBr at a density of 1.23 g/ml. Purification of melanoma-associated antigens was achieved by this step, followed by ionexchange chromatography and preparative isoelectric focusing on Pevikon C870.

Another approach is described for the isolation of carcionembryonic antigens from metastatic lesions with an 70% yield utilizing the least denaturing procedures, which avoid lyophilization and involve essentially 0.9% NaCl solution extraction, specific adsorption, elution from concanavalin A Sepharose, and subsequent gel-exclusion chromatography on Ultrogel AcA 22. For effective isolation of carcinoembryonic antigens freely shed from cultured cells derived from a primary colon tumor, a system was devised based on the use of Amicon hollow fiber culture units, in which cultured tumor cells were introduced in the extracapillary spaces of such a unit. The extracapillary fluid, containing carcinoembryonic antigens but no fetal calf serum components, is removed and further purified by affinity chromatography.

¹ Presented at the National Bladder Cancer Conference, November 28 to December 1, 1976, Miami Beach, Fla. This is Publication 1240 from the Department of Molecular Immunology, Scripps Clinic and Research Foundation. This work was supported by USPHS Grant CA 16069 and Contracts CB53899 and CP43277.

² Presenter.

³ Recipient of an Established Investigatorship from the American Heart Association, Inc.