Cloning of cDNAs encoding the variable domains of antibody BrE-3 and construction of a chimeric antibody.
BrE-3 is an IgG1,kappa murine monoclonal antibody that binds to human breast epithelial mucin and that has been shown to be promising for imaging and treatment of breast cancer. We have cloned and sequenced cDNAs encoding the variable regions of the light (VL) and heavy (VH) chains of BrE-3. VL belongs to group II and resulted from a V kappa-J kappa 1 fusion. VH belongs to group IIIc and arose from a V-D-JH3 non-conservative fusion which left little or nothing of the original D minigene. Thus, the third VH CDR contains only 4 amino-acids. We constructed an IgG1,kappa human/mouse chimeric antibody (by joining the murine variable domains to human constant domains) and expressed it in SP2/0 myeloma cells. This chimeric monoclonal antibody stains breast carcinoma tissue sections by the ABC immunoperoxidase method. Its affinity for the BrE-3 antigen is 2.68 x 10(8) M-1, which, considering the experimental error, is indistinguishable from the affinity of the original murine antibody (3.75 x 10(8) M-1). The VL and VH domains alone are respectively 73%, and 63% identical to the human V kappa II and VHIII consensus sequences. If the CDRs are excluded, these numbers become respectively 82% and 80%. Therefore, we expect the reported chimeric BrE-3 to be considerably less immunogenic to humans than the original murine antibody, while retaining the original binding properties.
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; D000912:Antibodies, Neoplasm / Q000235:genetics / Q000276:immunology; D000951:Antigens, Neoplasm / Q000276:immunology; D015295:Antigens, Tumor-Associated, Carbohydrate; D001483:Base Sequence; D001943:Breast Neoplasms / Q000276:immunology; D016384:Consensus Sequence; D004247:DNA / Q000235:genetics*; D005803:Genes, Immunoglobulin; D005813:Genes, Synthetic; D007074:Immunoglobulin G / Q000235:genetics / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000276:immunology; D007145:Immunoglobulin kappa-Chains / Q000235:genetics / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology
D000911:Antibodies, Monoclonal; D000912:Antibodies, Neoplasm; D000951:Antigens, Neoplasm; D015295:Antigens, Tumor-Associated, Carbohydrate; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D007145:Immunoglobulin kappa-Chains; D011993:Recombinant Fusion Proteins; C057387:mucinous carcinoma-associated antigen; D004247:DNA