Abstract
Effective biologic therapeutics require binding affinities that are fine-tuned to their disease-related molecular target. The ADAPT (Assisted Design of Antibody and Protein Therapeutics) platform aids in the selection of mutants that improve/modulate the affinity of antibodies and other biologics. It uses a consensus z-score from three scoring functions and interleaves computational predictions with experimental validation, significantly enhancing the robustness of the design and selection of mutants. The platform was tested on three antibody Fab-antigen systems that spanned a wide range of initial binding affinities: bH1-VEGF-A (44 nM), bH1-HER2 (3.6 nM) and Herceptin-HER2 (0.058 nM). Novel triple mutants were obtained that exhibited 104-, 46- and 32-fold improvements in binding affinity for each system, respectively. Moreover, for all three antibody-antigen systems over 90% of all the intermediate single and double mutants that were designed and tested showed higher affinities than the parent sequence. The contributions of the individual mutants to the change in binding affinity appear to be roughly additive when combined to form double and triple mutants. The new interactions introduced by the affinity-enhancing mutants included long-range electrostatics as well as short-range nonpolar interactions. This diversity in the types of new interactions formed by the mutants was reflected in SPR kinetics that showed that the enhancements in affinities arose from increasing on-rates, decreasing off-rates or a combination of the two effects, depending on the mutation. ADAPT is a very focused search of sequence space and required only 20–30 mutants for each system to be made and tested to achieve the affinity enhancements mentioned above.
Highlights
The design of superior biologic therapeutics, including monoclonal antibodies, single-domain antibodies, and engineered proteins, involves optimizing their ability to bind to disease-related molecular targets
That study was a retrospective analysis of data in the SiPMAB database [12], which contains mutational, structural and binding affinity data for antibody-antigen complexes culled from the literature
We have presented ADAPT, a combined interleaved computational and experimental protocol and workflow for affinity maturation of antibodies
Summary
The design of superior biologic therapeutics, including monoclonal antibodies, single-domain antibodies, and engineered proteins, involves optimizing their ability to bind to disease-related molecular targets (antigens). Immunization of animals [1] and phage display methods [2] are the workhorses of antibody discovery and optimization. One of the most daunting challenges in antibody sequence optimization is prioritizing the sequence space resulting from the combinatorial explosion when mutating multiple sites in complementarity determining region (CDR) loops. For CDR loops containing 60 amino acids there are about 109 different triple mutants possible. This number increases by a factor of 103 for each additional mutation site. We sought to develop a more deterministic approach for building a focused set of mutations with a high likelihood of improving binding
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