Humanization of KC4G3,an Anti-Human Carcinoma Antibody

Abstract

ABSTRACT

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 V_H and V_κ frameworks that could influence antigen binding. We have previously showed that a fraction of the κ chains of murine and chimeric KC4G3 migrates abnormally on SDS-PAGE most likely due to N-linked glycosylation in V_κ. The glycosylation signal has now been removed from V_κ, as a consequence of humanization. As expected, the humanized κ chain migrates normally on SDS-PAGE. We detected no significant differences either in the affinities (1.6 ×10⁹M⁻¹vs. 1.4 × 10⁹ M⁻¹, respectively) or in the ability to compete for antigen binding, between the murine and the humanized antibodies. The humanized version is an IgG_l, κ immunoglobulin produced by mouse myeloma SP2/0-Agl4 cells and is designated HuKC4v2. The HuKC4v2 frameworks conform to the V_κII and V_HIII human consensus in all but six positions in V_κ and three positions in V_H.