Design and engineering of epitope specific antibodies




Hyun, Jeongmin

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The knowledge of three-dimensional structures of membrane proteins aids in structure-based drug design, since about 60% of approved drug targets are known as membrane proteins. To date, chaperone-assisted protein co-crystallization that bypass the need for animal immunization is becoming an attractive method to elucidate structures of recalcitrant targets such as proteins with intrinsically disordered domains as found in membrane proteins. Here we describe antibody-engineering strategies for developing crystallization chaperones. Toward this goal, we (1) engineered EE peptide-specific single-chain variable fragment (scFv) to improve biophysical characteristics, (2) constructed synthetic single domain antibody library to be specific for targets by phage display, and (3) de novo designed FLAG peptide-specific antibodies using a novel computational method. In the first study, we converted peptide-specific scFv to antigen-binding fragment (Fab), which is the most successful format of antibody-based crystallization chaperones for integral membrane proteins so far. The larger size of Fab/EE increased the overall stability without disruption of binding affinity and extended crystal contact areas those are favorable characteristics for use as a crystal chaperone. In the second study, a 10⁶ synthetic phage display single domain antibody (sdAb) library was constructed and used to identify sdAbs binding the repeat in toxin domain of B.pertussis adenylate cyclase toxin (ACT). This protein is an intrinsically disordered calcium binding protein with no homology to any known protein structure and is a candidate vaccine antigen. From phage-based screening, we isolated three sdAbs to be used for further characterization. In the last study, we utilized an in silico approach to the design the antibodies using OptCDR that is a general computational method that employs de novo design of complementarity determining regions (CDRs) to engineer antibody-antigen interactions. Using this method, we designed CDRs binding the minimal FLAG peptide (sequence: DYKD) and isolated four antibodies with high specificity and nanomolar affinity for the DYKD. The result demonstrates that antibody specificity based on in silico design method can guide future engineering of antibody-based crystallization chaperone. Taken together, we have identified antibodies with improved binding properties and biophysical characteristics for using as crystallization chaperones without animal immunization to help guide future antibody chaperone engineering for the structural investigation of diverse target proteins.


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