Cloning of cDNAs encoding the variable domains of antibody KC4G3 and construction of a chimeric antibody.
We have cloned and sequenced cDNAs encoding the variable regions of the light (VL) and heavy (VH) chains of monoclonal antibody KC4G3. VL belongs to group II and resulted from a V kappa-J kappa 2 recombination. VH belongs to group IIId and arose from a V-D9-JH3 recombination. The VL and VH frameworks are respectively 84% and 83% identical to the corresponding VL and VH human consensus frameworks. The deduced amino acid sequence of VL contains an asparagine-linked glycosylation site in framework 3 (N74 I75 S76). We have determined that a large fraction of the light chains are indeed glycolysated. We constructed an IgG1, kappa human/mouse chimeric antibody (by inserting the murine KC4G3 Fv-encoding cDNAs into plasmids encoding a human IgG1, kappa Fc domain) and expressed it in SP2/0-Ag14 mouse myeloma cells. This chimeric monoclonal antibody is designated ChiKC4. We have determined that the murine monoclonal antibody KC4G3 binds the human breast mucin with an affinity constant of 1.1 x 10(9) M-1. ChiKC4 binds the same antigen with an affinity constant of 1.2 x 10(9) M-1. ChiKC4 binds the carcinoma tissue sections by the ABC immunoperoxidase method in an identical manner as does KC4G3.
Couto
J R
JR
Cancer Research Fund of Contra Costa, Walnut Creek, CA 94596.
Blank
E W
EW
Peterson
J A
JA
Ceriani
R L
RL
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D000912:Antibodies, Neoplasm / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D000951:Antigens, Neoplasm / Q000276:immunology*; D015295:Antigens, Tumor-Associated, Carbohydrate; D001483:Base Sequence; D001943:Breast Neoplasms / Q000276:immunology*; D003001:Cloning, Molecular; D016384:Consensus Sequence; D018076:DNA, Complementary / Q000235:genetics; D004848:Epithelium / Q000276:immunology; D005803:Genes, Immunoglobulin / Q000235:genetics*; D005813:Genes, Synthetic; D006031:Glycosylation; D006801:Humans; D007127:Immunoglobulin Constant Regions / Q000235:genetics; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007147:Immunoglobulin Light Chains / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D007145:Immunoglobulin kappa-Chains / Q000235:genetics; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D009101:Multiple Myeloma; D009363:Neoplasm Proteins / Q000276:immunology; D011471:Prostatic Neoplasms / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D013045:Species Specificity; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D000912:Antibodies, Neoplasm; D000951:Antigens, Neoplasm; D015295:Antigens, Tumor-Associated, Carbohydrate; D018076:DNA, Complementary; D007127:Immunoglobulin Constant Regions; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D007145:Immunoglobulin kappa-Chains; D009363:Neoplasm Proteins; D011993:Recombinant Fusion Proteins; C057387:mucinous carcinoma-associated antigen