Partial purification of organ-specific neoantigens from human colon and breast cancer by affinity chromatograhy with human tumour-specific γ-globulin

Abstract

Organ-specific neoantigens (TA) shed from the tumours of patients with metastatic breast or colon cancer and which had filtered into the urine were partially purified by a combination of physicochemical methods and affinity chromatography. TA activity of the isolated materials was monitored by the blocking Tube LAI assay. Urinary protein was precipitated by 80% saturated ammonium sulphate. Albumin was removed by affinity chromatography with blue Sepharose CL-6B. Affinity columns of human IgG were prepared from sera of patients whose leucocytes were LAI+ to the breast- or colon-cancer extracts. The anti-breast-TA affinity column bound the TA in the urine of patients with metastatic breast cancer but not that of patients with metastatic colon cancer. The TA in urine of patients with metastatic colon cancer was bound by the anti-colon-TA affinity column. Analysis by SDS PAGE revealed that the isolates with and without TA activity were composed mostly of urinary protein which had bound nonspecifically to the human IgG affinity columns. With an affinity column of anti-NHS and Protein A, some of the contaminants were removed, to reveal SDD PAGE unique bands at about 38,000 and 12,000 mol. wt in the isolate with breast-TA activity. Rabbit antisera, raised to the material that had bound nonspecically to the anti-breast-TA affinity column, were used as an anti-nonspecific affinity column to remove the contaminants in the isolates from the affinity columns of anti-breast TA and anti-colon TA. After passage through the anti-nonspecific affinity column, the material that contained the putative breast or colon cancer TA revealed a unique band at about 38,000-40,000 mol. wt and residual fine bands at about 25,000-30,000 mol. wt. Both the control material and material with TA activity had similar bands at about 25,000 and 50,000 mol. wt. The specific activity of the putative colon or breast TAs, as measured by the blocking Tube LAI assay, was increased from about 30 to 5000-10,000 u/mg, a 125-400-fold enrichment.