Project Grant R43AI167147

METHOD FOR THE ISOLATION OF ANTIBODIES WITH FUNCTIONAL ACTIVITY AGAINST CELL SURFACE TARGETS - "METHOD FOR THE ISOLATION OF ANTIBODIES WITH FUNCTIONAL ACTIVITY AGAINST CELL SURFACE TARGETS" ABSTRACT WE HAVE DERIVED A METHOD FOR ISOLATING FUNCTIONAL MABS AGAINST CELL SURFACE RECEPTORS THAT ARE SEPARATELY EITHER AGONISTS, ANTAGONISTS, AND PARTIAL AGONISTS. WE TERMED THE METHOD DIRECTED LIGAND BINDING (DLB). AND IN DLB'S FIRST ITERATION (DLB1) WE HAVE SUCCESSFULLY USED IT FOR PEPTIDE- AND PROTEIN LIGANDS. USING DLB1 THESE LIGANDS WERE GENETICALLY INCORPORATED INTO THE COMPLEMENTARITY DETERMINING REGION (CDR) OF AN APPROPRIATE PHAGE DISPLAY SCFV LIBRARY. AT ITS ESSENCE DLB IS A MEANS TO DIRECT OR BIAS THE INITIAL BINDING OF A SUBTRACTED (TO REMOVE NON-SPECIFIC- AND IRRELEVANT BINDING) PHAGE DISPLAYED AB LIBRARY TOWARD A TARGET CELL SURFACE RECEPTOR BINDING SITE BY INCORPORATING THAT RECEPTOR'S LIGAND OR INHIBITOR INTO THE AB LIBRARY ITSELF. AND THEN RELY ON THE INCREASED BINDING ABILITY, VIA THE INTERACTION OF BOTH AN INDIVIDUAL MAB'S CDRS PLUS THE COVALENTLY ATTACHED LIGAND TO TRANSIENTLY STABILIZE THE AB::RECEPTOR COMPLEX TO WITHSTAND THE INCREASING STRINGENCY OF THE PHAGE BIOPANNING WASHING CYCLES USED TO REMOVE ANY WEAK BINDERS. IN THE DLB1 METHOD, WE GENETICALLY ENCODED THE LIGAND INTO THE AB'S CDR. BUT THAT LIMITED US TO PEPTIDE AND PROTEIN LIGANDS. WE NOW WANT TO APPLY THE DLB METHOD FOR THE ENZYMATIC CONJUGATION OF LIGANDS INTO AN APPROPRIATELY MODIFIED CDR OF A COMPLEMENTING LIBRARY. WE REFER TO THIS NEW METHOD AS DLB2.0. THE SIGNIFICANT ADVANTAGES OF THE DLB2.0 METHOD ARE SEVERAL FOLD, INCLUDING: (1) THE SAME LIBRARY CAN BE USED FOR MANY SCREENS, AND (2) THE MEANS FOR TESTING MABS DERIVED FROM DLB2.0 WITH AND WITHOUT ATTACHED LIGAND IS TRIVIAL; JUST OMIT THE LIGAND ATTACHMENT STEP BEFORE ANY SCREEN. THE PROPOSED STUDY USES SEVERAL GPCR TARGETS AS THE MODEL AND FUNCTIONALITY IN A CELL-BASED REPORTER ASSAY AS THE APPLICATION. REGARDING THE COMMERCIAL APPLICATION, WE ARE MAINLY FOCUSED ON THE METHOD. IT IS TRUE THAT THE TARGETS WE HAVE CHOSEN TO DEVELOP THE METHOD, IN SOME CASES, DO NOT HAVE EXCEPTIONAL COMMERCIAL VALUE. WHEN THE DLB2.0 GENERATES APPROPRIATE IGGS WE INTEND TO PARTNER AND COLLABORATE WITH STRONG ACADEMIC, AND PHARMACEUTICAL- AND BIOTECH COMPANIES BY PROVIDING THEM WITH THE IGG CLONES AND/OR PURIFIED IGG PROTEINS SO THAT THEY CAN PERFORM THE STRUCTURAL AND BIOLOGICAL STUDIES NEEDED TO BOTH BETTER UNDERSTAND AND UTILIZE THE MABS WE DEVELOP. AT ABBRATECH INTEND TO PROVIDE CUSTOM SERVICES TO COMMERCIAL ENTITIES (PHARMACEUTICAL AND BIOTECHNOLOGY COMPANIES) USING THE DATA OBTAINED FROM THIS WORK, INCLUDING ANY COLLABORATIVE RESULTS, AS A DEMONSTRATION OF THE POWER OF DLB2.0.