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.
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 kappa constant region. The heavy-chain plasmid carrying GM-CSF was made by ligation of the heavy-chain variable region sequences upstream of the human gamma1 constant region CH1 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 GM-CSF. In nonreducing SDS-polyacrylamide gels, a single band approximately 200 Kd reacted with anti-human kappa, anti-human lambda1 and anti-GM-CSF antibodies. In reducing polyacrylamide gels, a approximately 74 kd protein reacted with anti-human lambda1 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 kappa and lambda1 constant domains and that the GM-CSF moiety fused to the constant region lambda1 is biologically active.
Tripathi
P K
PK
Department of Internal Medicine, and The Lucille Parker Markey Cancer Center, University of Kentucky Medical Center, Lexington 40536, USA.
Qin
H
H
Bhattacharya-Chatterjee
M
M
Ceriani
R L
RL
Foon
K A
KA
Chatterjee
S K
SK
D000276:Adjuvants, Immunologic; D000595:Amino Acid Sequence; D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000235:genetics / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000951:Antigens, Neoplasm / Q000235:genetics / Q000276:immunology*; D001483:Base Sequence; D001943:Breast Neoplasms / Q000276:immunology*; D019496:Cancer Vaccines / Q000276:immunology*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005260:Female; D006017:Glycolipids / Q000235:genetics / Q000276:immunology*; D006023:Glycoproteins / Q000235:genetics / Q000276:immunology*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics / Q000276:immunology*; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics; D066292:Lipid Droplets; D051379:Mice; D018716:Molecular Mimicry; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000276:immunology
D000276:Adjuvants, Immunologic; D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D019496:Cancer Vaccines; D018076:DNA, Complementary; D006017:Glycolipids; D006023:Glycoproteins; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; C110367:milk fat globule; D016178:Granulocyte-Macrophage Colony-Stimulating Factor