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Identification of a M_r 40,000 Polypeptide from Colorectal Cancer Which Expresses Organ-specific Cancer Neoantigen Activity As Determined by Leukocyte Adherence Inhibition¹

Montreal General Hospital Research Institute, Montreal, Quebec, Canada H3G 1A4

Human cancers express organ-specific neoantigens (OSNs) that elicit immune responses in the tumor host. Leukocyte adherence inhibition, an in vitro assay that detects the antitumor immunity, was used to monitor the purification of the OSN from serum-free spent medium of tissue-cultured colon cancer cell lines (HCT-15 and SW-620). A monoclonal antibody (anti-p40) directed to a cross-reactive framework determinant of M_r 40,000 (p40) cell surface polypeptide, which was a principal component of the enriched isolate with OSN activity, was used to monitor the purification of p40 by enzyme-linked immunosorbant assay. About 50 liters of spent medium were generated from 20 m² of cells, collected, concentrated, and then separated by anion exchange, molecular-sieve, and blue-Sepharose affinity chromatography. OSN and p40 activity coisolated. p40 was then purified by monoclonal antibody anti-p40 affinity chromatography. The affinity-purified fraction was enriched for both p40 and leukocyte adherence inhibition activity that was specific for leukocytes from colon cancer patients in blind leukocyte adherence inhibition assays. When affinity-purified p40 from colon and lung cancers was tested blind in a criss-cross fashion with leukocytes from colon and lung cancer patients, the positive responses were to the appropriate p40. The homologous colon cancer p40 molecule showed size and considerable charge microheterogeneity (pI 6.3 to 7.6). Affinity-purified p40 and OSN coisolated on hydrophobic interaction and hydroxylapatite high-pressure liquid chromatography. Note that not all colon cancer OSN activity was bound by the anti-p40 affinity column. However, unbound OSN activity also eluted from hydrophobic interaction high-pressure liquid chromatography at the same time as affinity-purified p40, and residual p40 activity was detected by enzyme-linked immunosorbant assay. The results indicate that a p40 glycoprotein from the cell membrane of colon cancer cells coisolates with fractions having OSN activity. Impurities do not seem to account for the OSN activity. The OSN epitope on the M_r 40,000 molecule is recognized by leukocytes from colon cancer patients and is distinct from the cross-reactive framework determinant recognized by mouse monoclonal antibody anti-p40.

¹ This investigation was supported by USPHS Grant 5 ROI CA31976-03 awarded by the National Cancer Institute, Department of Health and Human Services.

² To whom requests for reprints should be addressed, at the Montreal General Hospital, 1650 Cedar Avenue, Montreal, Quebec H3G 1A4, Canada.

Received 6/27/85. Revised 11/13/85. Accepted 12/31/85.