SINGLE-CHAIN FV-PEPTIDE FUSION INHIBITOR OF FIBRIN CLOTS

GIVEN THE MAJOR IMPORTANCE OF CARDIOVASCULAR DRUGS TO PUBLIC HEALTH IN THIS COUNTRY, IT IS REMARKABLE THAT THERE ARE STILL NO CLINICALLY EFFECTIVE AGENTS THAT ACT DIRECTLY ON THE FIBRIN DURING THE CLOTTING PROCESS. SUCH DRUGS WOULD HOLD THE POSSIBILITY OF AVOIDING MANY OF THE SIDE EFFECTS OF HEPARIN OR THROMBOLYTIC THERAPY. THE STRATEGY CALLS FOR THE DEVELOPMENT OF A NEW CLASS OF DRUGS THAT HOLD PROMISE FOR DIRECTLY INTERRUPTING THE FORMATION OF FIBRILS BY FUBRIN MONOMERS THROUGH THE ACTION OF TARGETED PEPTIDE INHIBITORS. WHILE PROGRESS IN THE BIOCHEMISTRY OF FIBRIN POLYMERIZATION HAS PERMITTED IDENTIFICATION OF PEPTIDE SEQUENCES RESPONSIBLE FOR INTERCHAIN ASSOCIATION, THE INHIBITORY ABILITY OF THESE PEPTIDES ALONE IS NOT CLINICALLY RELEVANT, AS THEIR AFFINITY FOR INTACT FIBRIN IS TOO LOW. THE RECENT ADVANCES IN ANTIBODY ENGINEERING OFFER AN ELEGANT SOLUTION TO THIS PROBLEM, THROUGH THE USE OF A TARGETING DOMAIN CONSISTING OF A SINGLE- CHAIN FV (SFV), THE MINIMAL INTACT ANTIBODY BINDING SITE. THE LONG-TERM GOAL OF THIS SBIR PROJECT, PLANNED FOR PHASE II, IS TO GENETICALLY FUSE AN APPROPRIATE SFV TO AN INHIBITORYPEPTIDE SEQUENCE AT THE DNA LEVEL, AND TO DEVELOP SUCH PEPTIDE-SFV DUSION PROTEINS AS TRUE THERAPEUTIC AGENTS. PHASE I EFFORTS WILL TEST THE HYPOTHESIS THAT TARGETED PEPTIDES CAN ENHANCE THE INHIBITORY CAPACITY OF SPECIFIC INHIBITOR PEPTIDES; PEPTIDE OF INTEREST WILL BE CHEMICALLY CONJUGATED TO THE FAB' OF AN ANTI-FIBRIN POLYCLONAL ANTIBODY AVAILABLE TO DR. KLOCZEWIAK, AND THIS CONJUGATE WILL BE TESTED FOR ITS CAPACITY TO INHIBIT FIBRIN POLYMERIZATION. GROUNDWORK WILL ALSO BE LAID FOR CLONING OF APPROPRIATE FV REGION(S) BY PROTEOLYTICALLY GENERATING ANTI-FIBRIN FV FRAGMENTS FROM DIFFERENT MEMBERS OF A PANEL OF ANTI-FIBRIN IGMS AVAILABLE AT CREATIVE BIOMOLECULES. FINALLY, INITIAL ANALYSIS OF THESE FV FRAGMENTS WILL BE CONDUCTED, WHICH FOR GOOD CANDIDATES WILL ALSO INVOLVE PEPTIDE CONJUGATION AND INHIBITION ASSAYS. THE DATA FROM THESE STUDIES WILL PERMIT A CLEAR FORMULATION OF A PHASE II PROJECT THAT CAN TAKE FULL ADVANTAGE OF OUR EXPERTISE IN THE ENGINEERING OF SFV FUSION PROTEINS.