MONOSPECIFIC AND BISPECIFIC SINGLE-CHAIN ANTIBODY FRAGMENTS FOR CANCER-THERAPY

Especially when dealing,vith solid cancers, single-chain antibody frag ments (scFvs) have a lot of advantages. Due to their small size (27 kD a), these proteins clear more rapidly from the blood, and penetrate fa ster and deeper into tissues, than whole antibodies. Furthermore, the lack of constant regions ensures that they are not retained in tissues such as the liver and kidney. This reduces possible toxic side-effect s. Single-chain construction is normally done by polymerase chain reac tion (PCR). To decrease the overall cost of oligonucleotide primer syn thesis, time-consuming primer design, multiple PCR reactions and indiv idual PCR optimization, we designed a universal single-step overlap ex tension PCR protocol using hybridoma cDNA as a template. To overcome t he lack of effector function, bispecific scFvs, consisting of an scFv produced against a tumour-associated antigen fused to a T cell marker- specific scFv, are being created, starting from already assembled scFv , by means of two additional PCR reactions. In this paper we describe both PCR methods that were successfully used to create scFvs against t he human transferrin receptor, the human interleukin-3 receptor, the h uman CD3 molecule, a breast tumour-associated antigen and an anti-tran sferrin-anti-CD3 bispecific scFv.