Development of single chain antibodies to P185 tumor antigen

ABSTRACT

The human heavy [VH] and light [VL] chain variable genes are amplified and randomly assembled together and cloned into the minor coat protein gene [g3p] of M13 bacteriophage. The resulting library of scFv is expressed on the phage as g3p fusion protein. The high affinity specific scFv antibodies can be selected against a key antigen using panning process. Our aim was development of scFv antibodies against P185 tumor antigen by recombinant phage antibody system and panning process. Antibody engineering technology was applied to lymphocyte mRNA of a non-immune donor and a scFv library was constructed. The library was panned against an immunodominant epitope of P185 which its reactivity had been tested with sera from breast cancer patients. DNA fingerprinting of the scFvs selected the predominated clones. These were then screened by ELISA. A large library including high repertoires of VH and VL was constructed. DNA fingerprinting differentiated a number of clones. After selection against the immunodominant epitope of P185, nine clones were differentiated including two predominated scFv antibodies. The predominated antibodies were bound to the corresponding epitope and produced a positive ELISA. The high affinity P185 specific scFv antibodies which were originated from human genes and bound specifically to the P185 epitope are valuable clinical agents and have the potential to be used in cancer immunotherapy in which P185 overexpression and metastasis occurs