Antigen targeting and drug delivery employing bifunctional fusion protein and bispecific antibodies

Bispecific antibodies and bifunctional fusion proteins incorporate different binding specificities in one molecule. As a unique molecular design, they can bind two different antigens for numerous applications such as drug delivery and immune system targeting. Hypothesis: most of the protein based drug and immune system targeting vehicles are not versatile to deliver multiple molecules, and thus limits potential applications. Development of bispecific antibodies and bifunctional fusion proteins that can deliver distinct classes of molecules to the target sites is a promising strategy. Objectives: three strategies were tested using bispecific antibodies and bifunctional fusion proteins for targeting dendritic cells (DC) and ovarian cancer. The first study investigated the ability to construct full length bispecific monoclonal antibodies for targeting biotinylated antigens to DC. A quadroma secreting bispecific monoclonal antibodies (bsmAb), with one paratope specific for mouse DC DEC-205 receptor and another paratope specific for biotin, was developed. Studies have shown enhanced binding and internalization of biotinylated antigen to DC. In vivo studies in mice with biotinylated ovalbumin (OVA) have shown that in the presence of bsmAb and co-stimulator anti-CD40 mAb the immune responses were augmented. Refinement of the bispecific construct incorporated a truncated streptavidin and a single chain antibody (scFv) against the DC DEC-205 receptor. The versatile delivery of biotin labeled protein, peptide, glycolipids and DNA to DC was demonstrated. The bifunctional activity against DEC-205 and biotin were characterized. In vivo targeted immune response studies in mice with several proteins, cancer peptide, gangliosides and DNA were performed. In the presence of bifunctional fusion protein and anti-CD40 mAb, immune responses were augmented in single or multiple antigen targeting. The third study investigated the development of a universal ovarian cancer cell targeting vehicle that can deliver biotinylated therapeutic drugs. A scFv that recognizes the CA125 antigen of ovarian cancer cells was fused with a core-streptavidin domain. The two distinct activities of the fusion protein were demonstrated. In the presence of fusion protein, there was enhanced binding of biotinylated antigen and liposome to ovarian cancer cells. Conclusion: these studies demonstrated the versatility of bispecific targeting systems for vaccine or therapy applications.