Purification from a human lung cancer cell line of a water soluble molecule mediating leukocyte adherence inhibition for patients with lung cancer.
Soluble lung tumor activity as determined by LAI2 was enriched by physicochemical methods from chemically - defined spent medium of a lung cancer cell line (NCI-H69). To identify the polypeptide carrying the antigenic determinant, splenic lymphocytes of BALB/c mice were immunized with the enriched isolate and hybridized with mouse plasmacytoma cells. Eight hybrids were cloned successfully and produced MAbs that immunoprecipitated principally a single chain of Mr 40,000 (p40) as well as minor chains of Mr 25,000 (p25) and Mr 13,000 (p13) which were probably degradation products of p40. On 2D gels, p40 was composed of 7 spots with a p1 of 6.3 to 7.6, which was not altered by neuraminidase digestion. Affinity chromatography with MAb anti-p40 absorbed p40 and LAI activity. The bound and recovered fraction was enriched for p40 and LAI activity. Affinity-purified p40 also contained the previously identified p25 and p13 as well as a Mr 32,000 peptide (p32). MAb anti-p40 was directed to a common framework determinant on p40 since MAb anti-p40 bound to cancer cells from other organs. The comparatively lung cancer organ-specific determinant recognized by leukocytes from lung cancer patients was not recognized by the MAb. Affinity-purified p40 triggered LAI for leukocytes from patients with lung cancer but not for leukocytes from control subjects or patients with colon cancer or malignant melanoma in rigorous blind testing. Crossreactivity was observed with leukocytes from patients with breast cancer. LAI activity of affinity-purified p40 seems unlikely to result from an unidentified impurity. Thus a p40 molecule has been purified that is expressed on the membranes of lung cancer cells and triggers immunologically-mediated LAI.
D000911:Antibodies, Monoclonal / Q000096:biosynthesis; D000951:Antigens, Neoplasm / Q000302:isolation & purification*; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods; D000090266:Chromobox Protein Homolog 5; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007957:Leukocyte Adherence Inhibition Test; D007962:Leukocytes / Q000276:immunology*; D008175:Lung Neoplasms / Q000276:immunology*; D008565:Membrane Proteins / Q000302:isolation & purification; D009363:Neoplasm Proteins / Q000302:isolation & purification*; D011233:Precipitin Tests; D012995:Solubility
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; C000715594:CBX5 protein, human; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D000090266:Chromobox Protein Homolog 5