Deep Sequencing-guided Design of a High Affinity Dual Specificity Antibody to Target Two Angiogenic Factors in Neovascular Age-related Macular Degeneration

Abstract

The development of dual targeting antibodies promises therapies with improved efficacy over mono-specific antibodies. Here, we engineered a Two-in-One VEGF/angiopoietin 2 antibody with dual action Fab (DAF) as a potential therapeutic for neovascular age-related macular degeneration. Crystal structures of the VEGF/angiopoietin 2 DAF in complex with its two antigens showed highly overlapping binding sites. To achieve sufficient affinity of the DAF to block both angiogenic factors, we turned to deep mutational scanning in the complementarity determining regions (CDRs). By mutating all three CDRs of each antibody chain simultaneously, we were able not only to identify affinity improving single mutations but also mutation pairs from different CDRs that synergistically improve both binding functions. Furthermore, insights into the cooperativity between mutations allowed us to identify fold-stabilizing mutations in the CDRs. The data obtained from deep mutational scanning reveal that the majority of the 52 CDR residues are utilized differently for the two antigen binding function and permit, for the first time, the engineering of several DAF variants with sub-nanomolar affinity against two structurally unrelated antigens. The improved variants show similar blocking activity of receptor binding as the high affinity mono-specific antibodies against these two proteins, demonstrating the feasibility of generating a dual specificity binding surface with comparable properties to individual high affinity mono-specific antibodies.

Keywords: angiopoietin2 (Ang2); antibody engineering; cooperativity; crystal structure; deep mutational scanning; deep sequencing; drug discovery; dual specificity; neovascular age-related macular degeneration; vascular endothelial growth factor (VEGF).

FIGURE 1.

Structural characterization of angiopoietin 2 and VEGF binding by DAF 5A12. Crystal structures of 5A12 Fab in complex with RBD of human Ang2 (A) or VEGF (B). A top down view of 5A12 antigen-binding sites for Ang2 or VEGF. The surface representation of the heavy chain is colored blue and the light chain is green. Structural paratopes or residues that are 5 Å or less away from Ang2 or VEGF are colored orange or red, respectively, and show an extensive overlap. C, comparison of the epitope of 5A12 (circled with black line) and Tie2 (red lines) on hAng2. D, comparison of the epitope of 5A12 (black line) and the parental antibody G6 (red line) on hVEGF. E, top view onto the binding site of 5A12 in the hVEGF and hAng2 bound state in comparison with the loop conformations of G6 in the antigen-free form and the hVEGF-bound form. Plasticity in the Cα backbone can be observed in the CDR-H2 and CDR-H3. F, CDR-H2 loop conformations of 5A12 in the hVEGF- and hAng2-bound state in comparison with G6 in the antigen-free form and the hVEGF-bound form. The CDR-H2 loop adopts the same conformation in hVEGF-bound state of G6 and 5A12, whereas the CDR-H2 loop conformation in the hAng2-bound state of 5A12 is the same as observed for the antigen-free form of G6.

FIGURE 2.

Enrichments for single mutations from selection of the heavy chain 3NNK and 1NNK library are similar. The log2 enrichment ratios obtained from the 1NNK and 3NNK heavy chain library designs panned against Ang2 (A) and VEGF (B) are compared in a scatter plot. The mutations are colored according to the CDR in which they are located.

FIGURE 3.

A, single mutation ER for all randomized CDR positions from Ang2 and VEGF panning. The heat maps show the ER for 1040 mutations in the heavy (left) and light chain (right) CDRs obtained from Ang2 (upper panel) and VEGF panning (lower panels). The line plot shows the solvent accessibility for each scanned position of 5A12 Fab as an unbound Fab based on the crystal structure of the 5A12 Fab in the Ang2-bound (orange line) or the VEGF-bound form (magenta line). B, 5A12 functional paratopes for Ang2 or VEGF binding as mapped by a deep mutational scan are viewed at the same orientation as the 5A12 structural paratopes in Fig. 1, C and D. The means of ER of all mutations at every mutated CDR position were calculated and mapped on the structures color-coded similarly as the heat map.

FIGURE 4.

Deep mutational scan of 5A12 reflects the different role of CDR-H2 for binding its two antigens. A, 5A12 (schematic representation, green and blue) in binding hAng2 (surface representation, light blue) does not involve the CDR-H2 loop (cartoon with electron density) while using CDR-H2 for hVEGF binding (surface representation, gray) (B). Detailed view of the CDR-H2 loop of 5A12 with the respective electron density (carved at 2 Å for better visibility) in the Ang2-bound (C) and VEGF-bound (D) conformation is shown. Discernible residues are labeled. Heat map representation of the log2 enrichment ratios of CDR-H2 residues scanned by selection of heavy chain 3NNK library for Ang2 (E) or VEGF (F) binding. Asterisks mark the wild type residues.

FIGURE 5.

Potential mutations for improving dual binding function. A, correlation between the enrichment (as log₂ER, y axis) for 25 selected single mutations (15 in HC and 10 in LC) based on the deep sequencing data set and the improvement of Ang2- (red) or VEGF- (blue) relative affinity determined with phage competition ELISA (as log₂(IC_{50 (mutant)}/IC_{50 (wild type)}, x axis). Many mutants were selected because they may improve binding affinity based on the positive ERs. The enrichment derived from HC 3NNK and HC 1NNK libraries or LC 3NNK libraries were compared against relative affinity change. The Pearson's correlation (ρ) is shown. A comparison of the enrichment (log₂ER) derived from the hVEGF-sorted versus hAng2-sorted HC-3NNK library (B) or LC-3NNK library (C) is shown.

FIGURE 6.

Identification of affinity-improving, synergistic mutation pairs using a combination of enrichment, epistasis, and structural analysis. The Circos plot visualizes the highest enriched position pairs as calculated from sequencing data obtained from the hAng2-sorted HC 3NNK libraries (A) and LC 3NNK libraries (B). For this visualization, the enrichment of mutation pairs at the same CDR positions were summed up. The circular segments represent CDR positions, and the ribbons, connecting two positions, represent a position pair. The width of ribbon represents the sum of enrichment at this position. At the end of each ribbon, the amino acids are listed that form the mutation pairs at the positions connected by the ribbon. The histogram at the outer layer of the Circos plot shows the Cα-Cα distance between two residues in pairs. The ribbons of the position pairs are highlighted in pink when they contain fold-stabilizing mutations (see C and D). Positions are colored orange when they contain affinity improving mutation pairs (see main text for details). The correlation between the log2 ER of a given mutation in heavy chain (C) or light chain (D) libraries and the partner potentiation score from Ang2 panning is shown in a scatterplot. The melting temperature of selected Fab variants is shown relative to the parental 5A12. Triplet repeats of T_m measurement are highly reproducible with differences less than 0.2 °C. E, fold change in affinity (K_d) for hAng2 and hVEGF as measured with BIAcore of selected double mutation pairs and the comprising single mutations. Error bars represent standard error of the mean from three independent measurements. F, locations of synergistic mutation pairs identified using double mutation enrichment analysis are mapped on the structure of 5A12_Ang2. The heavy chain is colored red, and the light chain is colored blue. Pairs are marked by spheres and connected by an orange line.

FIGURE 7.

Affinity improved variants with sub-nanomolar dual affinity show similar blocking activity as mono-specific antibodies. A, positions in the heavy (red) or light chain (blue) of 5A12, which have been mutated to generate higher affinity variants, are shown as spheres. B, dual-specific affinity matured variants of 5A12 with various mutation combination indicated and their affinity as the average K_d, k_on, and k_off values from three independent BIAcore SPR experiments (with errors less than 50%). C, in vitro receptor blocking assay to compare the blocking of hAng2 binding to Tie2 as well as of hVEGF to VEGFR1 by 5A12, the high affinity 5A12 variants, and the mono-specific hAng2 and hVEGF antibody G5.5 and G6.31, respectively.