Project Grant R43GM149009

T-CELL RECEPTOR MIMIC AFFINITY REAGENT GENERATION USING AN IN VIVO NOVEL IMMUNOGEN STRATEGY - ABSTRACT CURRENT THERAPEUTIC ANTIBODIES TARGET THE CELL SURFACE AND WITHIN THE EXTERNAL CELLULAR ENVIRONMENT. YET INTRACELLULAR PROTEINS MAKE UP HALF OF THE PROTEOME. HAVING A FACILE MEANS OF TARGETING THOSE INTRACELLULAR PROTEINS FOR THE GENERATION OF THERAPEUTIC ANTIBODIES WOULD BE HIGHLY DESIRABLE. TARGETING MHC COMPLEXED WITH NEOANTIGENS FROM INTRACELLULAR PROTEINS WOULD GREATLY ALLOW THE GENERATION OF T CELL RECEPTOR MIMIC (TCRM) ANTIBODIES FOR DEVELOPMENT AS THERAPEUTIC DRUGS. WE HAVE DEVELOPED A UNIQUE IMMUNE-TARGETING STRATEGY THAT WE CALL 'EPIVOLVE' THAT CAN BE USED TO MAKE 'SITE DIRECTED' ABS BY LEVERAGING THE MODULARITY OF THE COMPLEMENTARITY DETERMINING REGION (CDR)-H2 OF AN AB. EPIVOLVE FUNCTIONS THROUGH THE 'EVOLUTION' OF AN AB PARATOPE USING METHODS THAT ARE DESCRIBED IN THE PROPOSAL. THE ADVANTAGES OF THE IN VIVO METHOD PROPOSED INCLUDE THE ISOLATION OF ABS THAT HAVE A HIGH AFFINITY (IN THE LOW PICOMOLAR RANGE) FOR THE PEPTIDE:MHC COMPLEX. THIS IS BECAUSE ABS CAN UNDERGO MULTIPLE AG CHALLENGES AND AFFINITY MATURATION VIA SOMATIC HYPER-MUTAGENESIS IN VIVO TO GENERATE ANTIBODIES WITH SINGLE DIGIT PM TO HUNDREDTH NM. IN VIVO METHODS CAN ALSO TAKE ADVANTAGE OF TRANSGENIC ANIMALS FOR DERIVING HUMAN ABS. AFFINITIES OF TCRM ABS PRODUCED THROUGH PHAGE DISPLAY TEND TO LIE IN THE MODERATE NANOMOLAR RANGE (50-300 NM) AND REQUIRE FURTHER IN VITRO PROTEIN ENGINEERING. FOR THIS PROPOSAL, ABBRATECH WILL APPLY ITS NOVEL SITE-DIRECTED EPIVOLVE TECHNOLOGY ALONG WITH AN IMPROVED RABBIT SINGLE B CELL SORTING PLATFORM FOR THE EFFICIENT DISCOVERY OF TCRM ABS. THE EPIVOLVE METHOD OVERCOMES TOLERANCE. AND ONE OF OUR GOALS IS TO GENERATE ANTIBODIES IN AS LITTLE AS 5 WEEKS. HIGH-AFFINITY, NEOANTIGEN:MHC-SPECIFIC TCRM ABS HAVE PROVEN DIFFICULT TO PRODUCE IN LARGE NUMBERS BY EITHER TRADITIONAL PHAGE OR HYBRIDOMA APPROACHES. ENHANCED TECHNOLOGIES FOR THE GENERATION OF TCRM ABS WITHIN A SHORT PERIOD OF TIME OFFER EXCITING OPPORTUNITIES TO ACCELERATE FUTURE TCRM AB DISCOVERY.