Molecular identification of a human carcinoma-associated glycoprotein antigen recognized by mouse monoclonal antibody FU-MK-1

Mouse monoclonal antibody FU-MK-1, raised against a human gastric adenocarc inoma, recognizes an antigen (termed MK-1 antigen) present on the majority of carcinomas. The present study aimed to identify the MK-I molecule and to establish its relationship to other carcinoma antigens, Immunoprecipitatio n studies of human tumor cell lines revealed that FU-MIK-1 recognizes a mon omeric membrane glycoprotein with two forms, 40 kDa (major form) and 42 kDa (minor form), and with a molecular mass of 35 kDa following treatment with the N-glycosylation inhibitor tunicamycin, The partial amino acid sequence of a main fragment of the MK-1 molecule obtained by spontaneous cleavage u nder hypotonic conditions was examined, and the 17 contiguous NH2-terminal amino acids were found to be identical with residues 81-97 of the 314-resid ue GA733-2 protein [Szala et al.; Proc. Natl, Acad, Sci, USA, 87, 3542-3546 (1990)], Hence, the GA733-2 cDNA was cloned and the specificity of FU-MK-1 was confirmed using four recombinant forms of the GA733-2 antigen expresse d in COS-1 cells. Immunoprecipitation with FU-MK-1 of the cell lysate trans fected with the full-length GA733-2 cDNA revealed two bands corresponding t o those obtained from the tumor cell lines, FU-MK-I also precipitated three other recombinant proteins consisting of amino acids 1-265, 1-201, and 1-1 39 of the GA733-2 protein, respectively. Furthermore, immunoblotting analys is indicated that FU-MK-1 binds to a small fragment (6 kDa) generated from a tumor cell line under hypotonic conditions, suggesting that the FU-MK-I e pitope exists on the distal 6-kDa peptide of the extracellular domain of th e GA733-2 molecule. We thus conclude that the MK-1 antigen is the GA733-2 a ntigen, which is currently being used as a target in clinical trials with m onoclonal antibodies.