Humanization of KC4G3, an anti-human carcinoma antibody.
We have previously constructed a chimeric version of KC4G3, a murine antibody that reacts with several human epithelial cancers and binds to the human breast epithelial mucin. We have now successfully humanized KC4G3 using positional consensus data, previously compiled after examining several other antibody structures, listing residues in the VH and V kappa frameworks that could influence antigen binding. We have previously showed that a fraction of the kappa chains of murine and chimeric KC4G3 migrates abnormally on SDS-PAGE most likely due to N-linked glycosylation in V kappa. The glycosylation signal has now been removed from V kappa, as a consequence of humanization. As expected, the humanized kappa chain migrates normally on SDS-PAGE. We detected no significant differences either in the affinities (1.6 x 10(9) M-1 vs. 1.4 x 10(9) M-1, respectively) or in the ability to compete for antigen binding, between the murine and the humanized antibodies. The humanized version is an IgG1, kappa immunoglobulin produced by mouse myeloma SP2/0-Ag14 cells and is designated HuKC4v2. The HuKC4v2 frameworks conform to the V kappa II and VHIII human consensus in all but six positions in V kappa and three positions in VH.
Couto
J R
JR
Cancer Research Fund of Contra Costa, Walnut Creek, California 94596.
Padlan
E A
EA
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*; D000915:Antibody Affinity; D001483:Base Sequence; D001667:Binding, Competitive / Q000276:immunology; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology
D000911:Antibodies, Monoclonal; D000912:Antibodies, Neoplasm; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins