An Isolated Enriched Organ-specific Cancer Neoantigen of Human Lung Cancer for Leukocyte Adherence Inhibition Assays

Abstract

Until now, measurement of human antitumor immunity to organ-specific cancer neoantigen (OSN) by the leukocyte adherence inhibition (LAI) assay depended on using crude extracts of cancer. In this study, a new method is presented to generate and to isolate a highly enriched OSN from spent medium of a lung cancer cell line, NCI-H69, grown in chemically defined medium. Production of large quantities of OSN with minimal contamination by extraneous proteins was possible. Four physicochemical steps were used to give a 1000-fold enrichment of OSN activity: anion-exchange and molecular-sieve chromatography; Blue Sepharose affinity chromatography; and finally anion-exchange high-pressure liquid chromatography. The enriched OSN isolates showed dose-response antigenicity when tested in LAI assay with leukocytes from lung cancer patients but had no antigenicity with leukocytes from control subjects or patients having malignant melanoma, colon cancer, or pancreatic cancer. Cross-reactive antigenicity was observed with leukocytes from patients with breast cancer and slight reactivity with leukocytes from bladder cancer patients. The final isolate from the four-step separation procedure as well as the isolates produced using additional separation techniques consistently had antigenicity at <10 ng in blocking LAI and 500 ng in the direct assay and showed components with molecular weights of about 62,000 ± 3,000 (SD) (p62), 40,000 ± 3,000 (p40), and 25,000 ± 1,000 (p25) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The OSN isolates on two-dimensional gels showed p40 to have microheterogeneity (seven spots), with a pl from 6.2 to 7.6, and p62 and p25 as even more basic streaks. The polypeptide bearing the antigenic determinant was not purified, although we tried to separate p62, p40, and p25 to determine whether they carried the OSN determinant. The results of this study are important in showing that an isolate of an organ-specific tumor antigen containing 5 to 13 components, as determined by highly sensitive silver stains and radiolabeled patterns on single and two-dimensional gels, can be used successfully in LAI to measure tumor immune responses.