Project Grant R61CA291117

EVOLVED MOLECULES THAT DESTROY CANCER RELEVANT PROTEINS - ABSTRACT CANCER RESEARCH IS OFTEN DRIVEN BY HYPOTHESES THAT POSTULATE THAT A SPECIFIC PROTEIN IS IMPORTANT FOR THE DISEASE, HYPOTHESES OFTEN TESTED BY ADDING AN ANTIBODY TO THAT SYSTEM. THE TECHNOLOGY TO BE DEVELOPED HERE WILL CREATE NEW REAGENTS (AEGISBODIES AND AEGISCLEAVERS) THAT NOT ONLY BIND A TARGET PROTEIN (LIKE AN ANTIBODY), BUT ALSO CLEAVE IT, AT A COST 10 TO 100 FOLD LOWER THAN A SERVICE-PROVIDED ANTIBODY, IN WEEKS RATHER THAN MONTHS, AND ACROSS A RANGE OF AFFINITIES THAT SUPPORT QUANTITATIVE RESEARCH. THESE WILL BE OBTAINED BY APPLYING LABORATORY IN VITRO EVOLUTION (LIVE) TO LIBRARIES BUILT FROM AN ARTIFICIALLY EXPANDED GENETIC INFORMATION SYSTEM (AEGIS), AND DECORATED WITH PRECISELY SELECTED CHEMICAL GROUPS. AEGIS IS A BIOPOLYMER LIKE DNA, BUT WITH UP TO 12 BUILDING BLOCKS, ENHANCED FOLDING, GREATER STABILITY, AND FUNCTIONAL GROUPS THAT ASSIST IN THE CLEAVAGE REACTION, WHICH GIVES PRODUCTS WHERE AN AEGISCLEAVER IS COVALENTLY ATTACHED TO A FRAGMENT OF THE TARGET PEPTIDE. AEGIS-LIVE HAS HIGH LEVEL OF TECHNICAL READINESS BECAUSE OF PRELIMINARY STUDIES THAT CREATED (A) PLATFORMS TO MANUFACTURE AEGIS DNA, (B) A MOLECULAR BIOLOGY TO SUPPORT AEGIS-LIVE, AND (C) SEQUENCING, FOLD PREDICTION, AND OTHER TOOLS TO ANALYZE THE PRODUCTS OF AEGIS-LIVE. THE TEAM HAS PROVEN THE CANCER-RELEVANCE OF AEGIS-LIVE, CREATING AEGISBODIES THAT BIND SPECIFICALLY AND SELECTIVELY TO BREAST AND LIVER CANCER CELLS, TO CANCER-RELEVANT PROTEINS HETEROLOGOUSLY EXPRESSED ON MAMMALIAN CELL SURFACES, AND TO ISOLATED PROTEINS FROM ANTHRAX. THE FIRST CATALYTIC AEGISZYMES ARE IN HAND. TO FURTHER THIS TECHNOLOGY DEVELOPMENT, WE WILL: AIM 1. APPLY AEGIS-LIVE TO CREATE AEGISBODIES THAT BIND PROGRAMMED DEATH-LIGAND 1 (PD-L1), A RECOGNIZED TARGET FOR CANCER THERAPY. WE WILL ALSO TARGET EXTRACELLULAR PEPTIDE LOOPS IN THE PD-L1 FOLD, MADE FROM L- AND D- AMINO ACIDS. THE SECOND ARE MIRROR IMAGES OF THE NATURAL AMINO ACIDS, AND ALLOW PRODUCTION OF MIRROR IMAGE L- AEGISBODIES THAT ARE STABLE AGAINST NUCLEASE DIGESTION. WE WILL METRIC THESE FOR BINDING AFFINITIES AND STABILITY, AND VALIDATE THEIR EFFICACIES COMPARED TO CURRENT TECHNOLOGIES. AIM 2. APPLY AEGIS-LIVE TO CREATE L- AND D-AEGISCLEAVERS THAT CLEAVE PEPTIDES FROM PD-L1, AND THE ANALOGOUS SEGMENTS WHEN EMBEDDED IN THE COMPLETE PROTEIN. WE WILL METRIC AND BENCHMARK THESE FOR AFFINITY, CLEAVAGE RATES, AND STABILITY AGAINST NUCLEASES. THE NEW REAGENTS WILL BE TRANSFORMATIVE SINCE THEY DO THINGS THAT ANTIBODIES CANNOT: COVALENTLY TAG AND DESTROY TARGET PROTEINS. FURTHER, THE REAGENTS ARE EXPECTED TO BE STABLE IN BIOLOGICAL FLUIDS, INCLUDING CELL CULTURE, BLOOD, AND LIVING ANIMALS. LONGER TERM, ULTRA-STABLE AEGISCLEAVERS MAY EVEN COME TO BE DIAGNOSTIC AND THERAPEUTIC TOOLS.