Construction and Characterization of a Chimeric Fusion Protein Consisting of an Anti-idiotype Antibody Mimicking a Breast Cancer-Associated Antigen and the Cytokine GM-CSF

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Abstract
Anti-idiotype antibody, 11D10 mimics biologically and antigenically a distinct and specific epitope of the high molecular weight human milk fat globule (HMFG), a cancer-associated antigen present in over 90% of breast tumor samples. To augment the immunogenicity of 11D10 without the aid of a carrier protein or adjuvant, we made a chimeric 11D10-GM-CSF fusion protein for use as a vaccine. An expression plasmid for 11D10 was made by ligation of the DNA sequences of the 11D10 light-chain variable region upstream of the human κ constant region. The heavy-chain plasmid carrying GM-CSF was made by ligation of the heavy-chain variable region sequences upstream of the human γl constant region CHI fused to the DNA fragment encoding the mature GM-CSF peptide 3' to the CH3 exon. NS1 plasmacytoma cells were transfected with the light and heavy-chain vectors by electroporation. Fusion protein secreted in the culture medium was purified and was characterized by gel electrophoresis as well as by determination of the biological activity of the fused GMCSF. In nonreducing SDS-polyacrylamide gels, a single band ∼200 Kd reacted with anti-human κ, anti-human λ1 and anti-GM-CSF antibodies. In reducing polyacrylamide gels, a ∼74 kd protein reacted with antihuman λl and anti-GM-CSF antibodies. The fusion protein induced proliferation of GM-CSF dependent NFS-60 cells. These results suggest that the protein is a chimeric anti-idiotype antibody consisting of 11D10 variable domains, human κ and λl constant domains and that the GM-CSF moiety fused to the constant region λl is biologically active.

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