Tuning the specificity of a Two-in-One Fab against three angiogenic antigens by fully utilizing the information of deep mutational scanning.


Monoclonal antibodies developed for therapeutic or diagnostic purposes need to demonstrate highly defined binding specificity profiles. Engineering of an antibody to enhance or reduce binding to related antigens is often needed to achieve the desired biologic activity without safety concern. Here, we describe a deep sequencing-aided engineering strategy to fine-tune the specificity of an angiopoietin-2 (Ang2)/vascular endothelial growth factor (VEGF) dual action Fab, 5A12.1 for the treatment of age-related macular degeneration. This antibody utilizes overlapping complementarity-determining region (CDR) sites for dual Ang2/VEGF interaction with KD in the sub-nanomolar range. However, it also exhibits significant (KD of 4 nM) binding to angiopoietin-1, which has high sequence identity with Ang2. We generated a large phage-displayed library of 5A12.1 Fab variants with all possible single mutations in the 6 CDRs. By tracking the change of prevalence of each mutation during various selection conditions, we identified 35 mutations predicted to decrease the affinity for Ang1 while maintaining the affinity for Ang2 and VEGF. We confirmed the specificity profiles for 25 of these single mutations as Fab protein. Structural analysis showed that some of the Fab mutations cluster near a potential Ang1/2 epitope residue that differs in the 2 proteins, while others are up to 15 Å away from the antigen-binding site and likely influence the binding interaction remotely. The approach presented here provides a robust and efficient method for specificity engineering that does not require prior knowledge of the antigen antibody interaction and can be broadly applied to antibody specificity engineering projects.

Submission Details

ID: U5gUocdV3

Submitter: Shu-Ching Ou

Submission Date: Nov. 30, 2018, 3:44 p.m.

Version: 1

Publication Details
Koenig P;Sanowar S;Lee CV;Fuh G,MAbs (2017) Tuning the specificity of a Two-in-One Fab against three angiogenic antigens by fully utilizing the information of deep mutational scanning. PMID:28585908
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
99.1 A Angiopoietin-2 O15123 ANGP2_HUMAN
93.3 A Angiopoietin-2 A0A8J8 ANGP2_CANLF
91.9 A Angiopoietin-2 O77802 ANGP2_BOVIN
91.9 A Angiopoietin-2 Q9BDY7 ANGP2_PIG
100.0 H Angiopoietin-2 P01857 IGHG1_HUMAN
90.9 H Angiopoietin-2 P01860 IGHG3_HUMAN
91.1 H Angiopoietin-2 P01861 IGHG4_HUMAN
100.0 L Angiopoietin-2 P01834 IGKC_HUMAN
91.2 L Angiopoietin-2 A0A0C4DH73 KV112_HUMAN
91.2 L Angiopoietin-2 P01611 KVD12_HUMAN
91.1 L Angiopoietin-2 P0DP09 KV113_HUMAN
91.1 L Angiopoietin-2 A0A0B4J2D9 KVD13_HUMAN
90.1 L Angiopoietin-2 P01601 KVD16_HUMAN