Cloning and sequencing of variable region cDNAs of a novel human monoclonal antibody to carcinoembryonic antigen, and generation of a single chain variable fragmented antibody.
BACKGROUND
Taking into consideration immunogenicity in humans, human antibodies and their derivatives have potential advantages for cancer immunotherapy and/or gene therapy over those from different species such as mouse. Recently, we generated 22 human monoclonal antibodies (HmAbs) specific for human carcinoembryonic antigen (CEA) using KM mouse, which carries human antibody genes. In the present study, we tried to clone the variable (V) region genes of the C2-45 HmAb, with the highest affinity for CEA and to generate a human single-chain variable fragmented (scFv) antibody.


METHODS
Using RT-PCR methods, we cloned and sequenced cDNAs encoding V regions of C2-45, and constructed an scFv gene including a signal sequence for secreting into the periplasmic space of E. coli.


RESULTS
Soluble scFv antibody, designated 45KHscFv, was secreted in the periplasmic space and purified by Ni2+ affinity column from the supernatant of E. coli after osmotic shock. When analyzed by SDS-PAGE, the 45KHscFv antibody showed a band corresponding to a calculated molecular weight of 30 kDa. The 45KHscFv antibody also retained a high reactivity with CEA in ELISA using immobilized CEA.


CONCLUSIONS
These results indicate that the nonimmunogenic 45KHscFv antibody could be advantageously used for fusing with other human functional proteins such as cytokines.
Shibaguchi
Hirotomo
H
Department of Biochemistry, Fukuoka University School of Medicine, Fukuoka, 814-0180, Japan.
Kuroki
Masahide
M
Kuroki
Motomu
M
Badran
Adel
A
Hachimine
Ken
K
Kinugasa
Tetsushi
T
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification; D001483:Base Sequence; D002272:Carcinoembryonic Antigen / Q000276:immunology*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D008969:Molecular Sequence Data; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000911:Antibodies, Monoclonal; D002272:Carcinoembryonic Antigen; D018076:DNA, Complementary; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region