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Abstract
Therapeutic and diagnostic applications of monoclonal antibodies often require careful selection of binders that recognize specific epitopes on the target molecule to exert a desired modulation of biological function. Here we present a proof-of-concept application for the rational design of an epitope-specific antibody binding with the target protein Keap1, by grafting pre-defined structural interaction patterns from the native binding partner protein, Nrf2, onto geometrically matched positions of a set of antibody scaffolds. The designed antibodies bind to Keap1 and block the Keap1-Nrf2 interaction in an epitope-specific way. One resulting antibody is further optimised to achieve low-nanomolar binding affinity by in silico redesign of the CDRH3 sequences. An X-ray co-crystal structure of one resulting design reveals that the actual binding orientation and interface with Keap1 is very close to the design model, despite an unexpected CDRH3 tilt and VH/VL interface deviation, which indicates that the modelling precision may be improved by taking into account simultaneous CDR loops conformation and VH/VL orientation optimisation upon antibody sequence change. Our study confirms that, given a pre-existing crystal structure of the target protein-protein interaction, hotspots grafting with CDR loop swapping is an attractive route to the rational design of an antibody targeting a pre-selected epitope.
Highlights
With very limited disclosure of successful examples of computational antibody design, especially lacking in structural validation of designed antibodies’ binding mode with antigen, the computational design of high-affinity antibodies targeting precise epitopes remains an elusive problem[12]
We used a computational approach to design rationally orthosteric antibodies targeting the pre-defined epitopes exemplified by a proof-of-concept Keap1–Nrf[2] interaction
Such antibodies can interact through the designed residues, conformational changes at the interface may lead to reorientation of the binding pose or missing the targeted epitopes completely, as observed in the previous reported design of Prd-Pdar protein complex using dock-and-design strategy[32]
Summary
With very limited disclosure of successful examples of computational antibody design, especially lacking in structural validation of designed antibodies’ binding mode with antigen, the computational design of high-affinity antibodies targeting precise epitopes remains an elusive problem[12]. Keap[1] is an intracellular drug target, that is not accessible to antibodies for therapeutic purposes, the Keap1-Nrf[2] interaction features a concave binding surface surrounded by a six-blade β-propeller structure and readily identifiable hotspot residues (ETGE motif) from Nrf[2] that serve as reference patterns to guide the design of novel antibodies targeting the Nrf[2] binding site to block the cognate Keap1-Nrf[2] interaction This enables prioritisation of antibody scaffold binding configurations that present the desired binding patches on the antigen to the CDR loops, reducing the complexity of rational design of antibodies to block the Keap1-Nrf[2] interaction by mimicking and displacing the binding partner Nrf[2]. Comparing with direct use of the recombinant cognate protein binders as therapeutic agents to block the target protein-protein interactions, designing antibodies to precisely occlude the cognate protein’s binding site is more medically beneficial because of antibodies’ typically higher affinities and well-known Fc-mediated effector functionalities and long-acting efficacy in circulation[14,15]
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