ENGINEERING OF ANTIBODY BINDING SITES TO TUMOR ANTIGENS

IN THIS RESEARCH, PROTEIN ENGINEERING IS BEING UTILIZED TO GENERATE THE MINIMAL COMBINING SITE OF ANTIBODIES, THE SO-CALLED FV, WHICH COMPRISES VH AND VL DOMAINS. IN PHASE I WE SYNTHESIZED GENES CODING FOR THE VH AND VL DOMAINS OF A SPECIFIC ANTIDIGOXIN ANTIBODY. THE BIOSYNTHETIC VH AND VL ARE BEING CLONED AND REASSEMBLED TO YIELD THE INTACT BINDING SITE. IN PHASE II, BASIC METHODOLOGY AND GENETIC ELEMENTS WILL BE DEVELOPED FOR PRODUCTION OF BIOSYNTHETIC ANTIBODY BINDING DOMAINS OF INTERCHANGEABLE SPECIFICITY, WHICH WILL ULTIMATELY BE DESIGNED TO BIND TUMOR ANTIGENS. VH AND VL GENES MADE FOR PHASE I WILL BE REFASHIONED SO THAT HYPERVARIABLE REGIONS WILL COPY THOSE OF AN ANTIPROTEIN ANTIBODY. THIS IS INTENDED TO ALTER SPECIFICITY WITHOUT THE NEED TO REMAKE CONSTANT FRAMEWORK OF THE FV. ATTEMPTS WILL BE MADE TO FUSE VH AND VL GENES INTO A SINGLE FV GENE VIA A LINKER WHICH WILL BE COMPATIBLE WITH NORMAL REFOLDING AND RECOMBINATION OF COMPONENT DOMAINS. THIS SINGLE POLYPEPTIDE CHAIN BINDING SITE WOULD BE USEFUL FOR IMMUNOTARGETING OF BIOLOGICALLY ACTIVE PROTEINS. FOR EXAMPLE, A SINGLE POLYPEPTIDE CHAIN COULD BE CLONED WHICH IS BIFUNCTIONAL, COMPRISING AN FV DOMAIN THAT BINDS A TUMOR ANTIGEN AND CYTOTOXIC DOMAIN THAT CONSISTS OF A RICIN A SUBUNIT. SUCH PROTEINS WOULD BE IDEAL THERAPEUTIC AGENTS FOR SELECTIVE CHEMOTHERAPY.