cDNA sequence analysis of monoclonal antibody FU-MK-1 specific for a transmembrane carcinoma-associated antigen, and construction of a mouse/human chimeric antibody.
Mouse monoclonal antibody (MAb) FU-MK-1, raised against a human gastric adenocarcinoma, recognizes a transmembrane antigen, GA733-2, present on most adenocarcinomas and seems to be of potential utility for immunodiagnosis and immunotherapy of those cancers. However, an inherent problem in their in vivo application is the human anti-mouse antibody response. In this study, we cloned and sequenced the variable region genes of the heavy and light chains (V(H) and Vkappa) of FU-MK-1 using the reverse transcription-polymerase chain reaction method. Then, we constructed a mouse/human chimeric antibody, designated as Ch FU-MK-1, by fusing the FU-MK-1 V(H) and Vkappa genes to the human Cgamma1 and Ckappa genes, respectively, and by ligating the chimeric H and L chain genes to each other in a mammalian cell expression vector. The final gene construct was transfected into mouse non-Ig-producing hybridoma cells by electroporation. The Ch FU-MK-1 antibody thus prepared bound to human adenocarcinoma cells and competitively inhibited the binding of the parental FU-MK-1 to the adenocarcinoma cells. Ch FU-MK-1 also showed a potent antibody-dependent cell-mediated cytotoxicity (ADCC) with human peripheral blood mononuclear cells as effectors against the adenocarcinoma cells, indicating that this chimeric antibody seems to be suitable for in vivo therapeutic approaches.
Arakawa
F
F
First Department of Biochemistry, School of Medicine, Fukuoka University, Japan.
Yamamoto
T
T
Kanda
H
H
Watanabe
T
T
Kuroki
M
M
D000230:Adenocarcinoma / Q000276:immunology*; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics*; D000912:Antibodies, Neoplasm / Q000235:genetics*; D000951:Antigens, Neoplasm / Q000276:immunology*; D001483:Base Sequence; D001667:Binding, Competitive; D015815:Cell Adhesion Molecules / Q000276:immunology*; D003602:Cytotoxicity, Immunologic; D018076:DNA, Complementary / Q000235:genetics; D018274:Electroporation; D000071858:Epithelial Cell Adhesion Molecule; D006801:Humans; D006825:Hybridomas; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics; D007145:Immunoglobulin kappa-Chains / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013274:Stomach Neoplasms / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000912:Antibodies, Neoplasm; D000951:Antigens, Neoplasm; D015815:Cell Adhesion Molecules; D018076:DNA, Complementary; D000071858:Epithelial Cell Adhesion Molecule; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D007145:Immunoglobulin kappa-Chains; D011993:Recombinant Fusion Proteins