Comprehensive interrogation of a minimalist synthetic CDR-H3 library and its ability to generate antibodies with therapeutic potential.

We have generated large libraries of single-chain Fv antibody fragments (>10(10) transformants) containing unbiased amino acid diversity that is restricted to the central combining site of the stable, well-expressed DP47 and DPK22 germline V-genes. Library WySH2A was constructed to examine the potential for synthetic complementarity-determining region (CDR)-H3 diversity to act as the lone source of binding specificity. Library WySH2B was constructed to assess the necessity for diversification in both the H3 and L3. Both libraries provided diverse, specific antibodies, yielding a total of 243 unique hits against 7 different targets, but WySH2B produced fewer hits than WySH2A when selected in parallel. WySH2A also consistently produced hits of similar quality to WySH2B, demonstrating that the diversification of the CDR-L3 reduces library fitness. Despite the absence of deliberate bias in the library design, CDR length was strongly associated with the number of hits produced, leading to a functional loop length distribution profile that mimics the biases observed in the natural repertoire. A similar trend was also observed for the CDR-L3. After target selections, several key amino acids were enriched in the CDR-H3 (e.g., small and aromatic residues) while others were reduced (e.g., strongly charged residues) in a manner that was specific to position, preferentially occurred in CDR-H3 stem positions, and tended towards residues associated with loop stabilization. As proof of principle for the WySH2 libraries to produce viable lead candidate antibodies, 114 unique hits were produced against Delta-like ligand 4 (DLL4). Leads exhibited nanomolar binding affinities, highly specific staining of DLL4+ cells, and biochemical neutralization of DLL4-NOTCH1 interaction.

Affinity maturation of a humanized rat antibody for anti-RAGE therapy: comprehensive mutagenesis reveals a high level of mutational plasticity both inside and outside the complementarity-determining regions.

Antibodies that neutralize RAGE (receptor for advanced glycation end products)-ligand interactions have potential therapeutic applications in both acute and chronic diseases. We generated XT-M4, a rat anti-RAGE monoclonal antibody that has in vivo efficacy in an acute sepsis model. This antibody was subsequently humanized. To improve the affinity of this antibody for the treatment of chronic indications, we used random and targeted mutagenesis strategies in combination with ribosome and phage-display technologies, respectively, to generate libraries of XT-M4 variants. We identified a panel of single-chain Fv antibody fragments (scFv's) that was improved up to 110-fold in a homogeneous time-resolved fluorescence competition assay against parental XT-M4 immunoglobulin G (IgG). After reformatting to bivalent scFv-Fc fusions and IgGs, we observed similar gains in potency in the same assay. Further analysis of binding kinetics as IgG revealed multiple variants with subnanomolar apparent affinity that was dictated primarily by improvements in the off-rate. All variants also had improved binding to cell surface-expressed human RAGE, and all retained, or had improved, apparent affinity for mouse RAGE. F100bL in V(H) (variable region of the heavy chain) complementarity-determining region 3 (CDR3) was one of a number of key mutations that correlated with affinity improvements and was independently identified by both mutagenesis strategies. Random mutagenesis coupled with ribosome display and high-throughput screening revealed an unexpectedly high level of mutational plasticity across the whole length of the humanized scFv, suggesting greater scope for structural optimization outside of the primary antigen-combining site defined by V(H) CDR3 and V(kappa) CDR3. In summary, our comprehensive mutagenesis approach not only achieved the desired affinity maturation of XT-M4 but also defined multiple mutational hotspots across the antibody sequence, provided an insight into the specificity-determining residues of the antibody paratope, and identified additional sites within the CDR loops where human germ-line amino acids may be introduced without affecting function.