Cloning of cDNAs Encoding the Variable Domains of Antibody KC4G3 and Construction of a Chimeric Antibody

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Abstract
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 Vκ-Jκ2 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,κ human/mouse chimeric antibody (by inserting the murine KC4G3 Fv-encoding cDNAs into plasmids encoding a human IgG1,κ 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×l09 M-1. ChiKC4 binds the same antigen with an affinity constant of 1.2×109 M-1. ChiKC4 stains breast carcinoma tissue sections by the ABC immunoperoxidase method in an identical manner as does KC4G3.