Cloning and expression of functional cDNA genes of a mouse/human chimeric antibody rcM4.
OBJECTIVE
The purpose of this study was to clone and express cDNA genes of heavy and light chains of the mouse/human chimeric antibody rcM4, which recognize the sialosyl-Tn epitope of the TAG72 antigen.


METHODS
The cDNA genes of chimeric heavy-chain M4H2 and light-chain M4K4 were cloned from the cDNA library of the ccM4 transfectoma. The M4H2 and M4K4 genes were modified in polymerase chain reaction and ligated into mpSV2neo-EP-PA and mpSVgpt-EP-PA to form the heavy- and the light-chain expression vectors mpSV2neo-EP-M4H2-PA and mpSV2gpt-EP-M4K4-PA, respectively. These two expression vectors were then co-transfected into the myeloma cell line SP2/0Ag14. Transfectants were selected in media containing G418 and mycophenolic acid. The rcM4 antibody was purified from transfectant culture supernates by protein A affinity chromatography.


RESULTS
The yield of the recombinant chimeric antibody rcM4 from its culture supernates was 6 micrograms/ml. We demonstrated that the rcM4 antibody retained its binding reactivity for sialosyl-Tn and that it was able to mediate effective antibody-dependent cellular cytotoxicity to the human ovarian cancer cell line OVCAR 3 in a manner comparable to the original ccM4 antibody.


CONCLUSIONS
The cDNA genes of chimeric heavy-chains (M4H2) and light-chains (M4K4) could be functionally expressed in the SP2/0Ag14 myeloma cell line. They thus have potential utility in the construction of some novel hybrid proteins such as those containing both antitumor immunoglobulins and cytokine molecules for use in cancer immunotherapy.
Boyd
M
M
Saskatoon Cancer Center, Department of Microbiology, University of Saskatchewan, Canada.
Xiang
J
J
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D000912:Antibodies, Neoplasm / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D005803:Genes, Immunoglobulin / Q000235:genetics* / Q000276:immunology; D005822:Genetic Vectors; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D009101:Multiple Myeloma; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D014162:Transfection; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D000912:Antibodies, Neoplasm; D018076:DNA, Complementary; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D011993:Recombinant Fusion Proteins