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

Abstract

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.

Materials and 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.

Conclusion: These results indicate that the nonimmunogenic 45KHscFv antibody could be advantageously used for fusing with other human functional proteins such as cytokines.