PRODUCTION AND CHARACTERIZATION OF A RECOMBINANT ANTI-MUC1 SCFV REACTIVE WITH HUMAN CARCINOMAS

Recombinant single-chain fragments (scFv) of the murine anti-MUC1 mono clonal antibody C595 have been produced using the original hybridoma c ells as a source of variable heavy (V-H)-and variable light (V-L)-chai n-encoding antibody genes. The use of the polymerase chain reaction (P CR), bacteriophage (phage) display technology and gene expression syst ems in E. coli has led to the production of soluble C595 scFv. The scF v has been purified from the bacterial supernatant by peptide epitope affinity chromatography, leading to the recovery of immunoreactive C59 5 scFv, which was similar in activity to the C595 parent antibody. Ana lysis by DNA sequencing, SDS-PAGE and Western blotting has demonstrate d the integrity of the scFv, while ELISA, FACScan analysis, fluorescen ce quenching, quantitative immunoreactivity experiments and immunohist ochemistry confirm that the activity of the scFv compares favourably w ith that of the parent antibody. The retention of binding activity to MUC1 antigen on human bladder and breast carcinoma tissue specimens il lustrates the potential application of this novel product as an immuno diagnostic and immunotherapeutic reagent.