Abstract
Affinity maturation is a powerful technique in antibody engineering for the in vitro evolution of antigen binding interactions. Key to the success of this process is the expansion of sequence and combinatorial diversity to increase the structural repertoire from which superior binding variants may be selected. However, conventional strategies are often restrictive and only focus on small regions of the antibody at a time. In this study, we used a method that combined antibody chain shuffling and a staggered-extension process to produce unbiased libraries, which recombined beneficial mutations from all six complementarity-determining regions (CDRs) in the affinity maturation of an inhibitory antibody to Arginase 2 (ARG2). We made use of the vast display capacity of ribosome display to accommodate the sequence space required for the diverse library builds. Further diversity was introduced through pool maturation to optimize seven leads of interest simultaneously. This resulted in antibodies with substantial improvements in binding properties and inhibition potency. The extensive sequence changes resulting from this approach were translated into striking structural changes for parent and affinity-matured antibodies bound to ARG2, with a large reorientation of the binding paratope facilitating increases in contact surface and shape complementarity to the antigen. The considerable gains in therapeutic properties seen from extensive sequence and structural evolution of the parent ARG2 inhibitory antibody clearly illustrate the advantages of the unbiased approach developed, which was key to the identification of high-affinity antibodies with the desired inhibitory potency and specificity.
Highlights
In antibody engineering, affinity maturation is a method of directed molecular evolution used to improve the affinity and binding interactions of an antibody to its antigen
All six complementarity-determining regions (CDRs) were included in the mutagenesis scheme and optimized in parallel to maximize the sequence space from which variants with improved affinities may emerge, and later recombined
Individual clones were sampled from the outputs of each CDR group, and tested as scFvs in periplasmic extracts [21] using a homogeneous time-resolved fluorescence-based epitope competition (EC) assay [22] in parallel with a functional screen, the enzyme inhibition assay (EIA)
Summary
Affinity maturation is a method of directed molecular evolution used to improve the affinity and binding interactions of an antibody to its antigen. This is often done to fulfill the required potency of biotherapeutics in vivo. In vitro affinity maturation mimics this process through the introduction of sequence diversity into a candidate antibody to produce libraries of mutational variants, and subsequent selections using display methods, such as phage or ribosome display, to find higher-affinity binders. The nature and magnitude of the epitope expansion was extraordinary and unlikely to have been produced through conventional affinity-maturation methods This innovative approach demonstrates broad applicability to the optimization of candidate therapeutic antibodies, even those less amenable to CDRH3 targeting
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