Cloning and expression of the human tumor-specific antibody GM4.
The human monoclonal antibody GM4 was generated by fusing pooled lymphocytes from cancer patients with the lymphoblastoid cell line SHFP-1. Immunohistochemical staining of tumor and normal tissue indicated that this human IgG4 antibody preferentially reacted with melanomas and neuroblastomas. In this study, we demonstrate that GM4 recognizes a "vimentin-like" peptide sequence that we have termed AgGM4. To generate a recombinant derivative of this human antibody, we isolated and expressed the complete heavy and light chain genes. The entire coding sequence for both the heavy and light chains was isolated by RT-PCR using a set of degenerate 5' signal sequence specific primers and a 3' constant region primer. High level antibody synthesis and secretion was achieved in Chinese hamster ovary (CHO) cells using a vector designed to maximize expression. Western blot and FACS analysis indicated recombinant GM4 reacted with human tumor cell lines and AgGM4 in a manner similar to the antibody produced by the hybridoma cell line, demonstrating that the specificity of the antibody was not altered during molecular cloning.
Nasoff
M S
MS
Novopharm Biotech, Inc., San Diego, CA 92121-4701, USA.
Gu
M
M
Galindo
J
J
He
X M
XM
Mukerjee
S
S
McKnight
M
M
Glassy
M C
MC
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000276:immunology / Q000627:therapeutic use*; D061067:Antibodies, Monoclonal, Humanized; D000912:Antibodies, Neoplasm / Q000235:genetics* / Q000276:immunology; D001483:Base Sequence; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary / Q000737:chemistry; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D006825:Hybridomas / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015183:Restriction Mapping; D014407:Tumor Cells, Cultured; D014746:Vimentin / Q000276:immunology*
D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D000912:Antibodies, Neoplasm; D018076:DNA, Complementary; D014746:Vimentin; C109796:gm4 antibody