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Antibody Engineering Strategy

Development of methods and tools for antibody engineering started with solving a challenging problem of redesigning 4E11, a broad spectrum anti-Dengue Virus (DENV) antibody that neutralized dengue serotypes DENV-1, DENV-2 and DENV-3, to also neutralize DENV-4. Several key metrics were developed to solve this problem by mining structural databases of antibody-antigen complexes. Using these metrics, the predicted pose of 4E11 antibody engaging with its target epitope surface was predicted accurately (verified later by X-ray crystallography) and this pose was used to engineer mutations on 4E11 to result in an antibody 4E5A with more than 450 fold enhancement in binding affinity to DENV-4 compared to 4E11 while retaining 4E11’s affinity to DENV-1, -2 and -3 serotypes.

We further developed our antibody design process by incorporating a network-based analysis. Both short-range and long-range interactions between non-bonded residues at the epitope-paratope interface were represented as a two-dimensional map (epitope paratope connectivity network or EPCN) and each residue was scored based on its connectivity to other residues in this interface. Using this network-based approach, the 4E5A antibody was further redesigned to generate an antibody Ab513 by introducing two mutations in HCDR1, which included a single residue deletion, that improved the EPCN and enhanced its affinity to all DENV serotypes and to make it clinically developable as a potent pan-DENV therapeutic.

The tools developed for redesigning antibodies were integrated towards developing a platform (referred to here as 3DMAbDesign) to engineer new antibodies, including the potent anti-Zika Virus antibody (ZAb_FLEP). The Antibody Engineering Methods involves selection, modification, and assembly of antibody parts (analogous to parts assembly with Lego building blocks), which comprise of distinct heavy and light chain framework regions (FWRs) and complementarity determining regions (CDRs), based on mining available antibody sequence and structural information to optimally engage with the target epitope surface. The process of engineering new antibodies using this platform involves an iterative cycle of designing and generating antibodies that were engineered from different parts and experimental screening of these designs for desired functional properties.