Construction and expression of two mouse-human chimeric antibodies with high specificity and affinity for carcinoembryonic antigen.
We have previously reported that a group of monoclonal antibodies (MAbs) to carcinoembryonic antigen (CEA), designated Group F MAbs, are able to discriminate CEA in tumor tissues from the CEA-related normal antigens and that CEA assay systems utilizing at least one Group F MAb show the improved cancer diagnosis. In this study, we cloned the genes coding for two Group F MAbs (F11-35 and F11-39) and deduced the amino acid sequences of the variable regions for their heavy and light chains. The variable region for the heavy chain of F11-35 contained a possible N-glycosylation site (Asn/Asp/Thr) at amino acid positions 89-91. Then, we constructed two mouse-human chimeric antibodies by using the F11-35 and F11-39 variable region genes of heavy and light chains (VH and V kappa) and human heavy and light chain constant region genes (gamma 1 and kappa) derived from a human plasma cell leukemia line (ARH77). The chimeric gene constructs were sequentially co-transfected into murine non-Ig-producing myeloma (P3-U1) or hybridoma (Sp2/0) cells by electroporation. The resulting chimeric heavy chain of F11-35 showed a slightly but significantly higher molecular weight than that of F11-39, but the molecular weights of their unglycosylated peptides synthesized in the presence of tunicamycin were similar, indicating the glycosylation at the possible N-glycosylation site in the variable region of the Ch F11-35 heavy chain. Both chimeric antibodies exhibited the same specificity and affinity for CEA as those of the parental murine hybridoma antibodies, respectively. Ascites production of Sp2/0 transfectomas is sufficiently high (600-900 micrograms/ml) for initial clinical studies with the chimeric antibodies.
Arakawa
F
F
First Department of Biochemistry, School of Medicine, Fukuoka University, Japan.
Haruno
M
M
Kuroki
M
M
Kanda
H
H
Watanabe
T
T
Misumi
Y
Y
Matsuoka
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D000915:Antibody Affinity; D001483:Base Sequence; D002272:Carcinoembryonic Antigen / Q000276:immunology*; D005803:Genes, Immunoglobulin; D005813:Genes, Synthetic; D005822:Genetic Vectors; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D007127:Immunoglobulin Constant Regions / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D009101:Multiple Myeloma; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000276:immunology* / Q000302:isolation & purification; D014162:Transfection; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D002272:Carcinoembryonic Antigen; D007127:Immunoglobulin Constant Regions; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins