4zfg | pdb_00004zfg
Dual-specificity Fab 5A12 in complex with Angiopoietin 2
Structural highlights
FunctionANGP2_HUMAN Binds to TEK/TIE2, competing for the ANGPT1 binding site, and modulating ANGPT1 signaling. Can induce tyrosine phosphorylation of TEK/TIE2 in the absence of ANGPT1. In the absence of angiogenic inducers, such as VEGF, ANGPT2-mediated loosening of cell-matrix contacts may induce endothelial cell apoptosis with consequent vascular regression. In concert with VEGF, it may facilitate endothelial cell migration and proliferation, thus serving as a permissive angiogenic signal.[1] [2] [3] [4] Publication Abstract from PubMedThe development of dual targeting antibodies promises therapies with improved efficacy over mono-specific antibodies. Here, we engineered a Two-in-One vascular endothelial growth factor (VEGF)1 /Angiopoietin 2 (Ang2) antibody with Dual action Fab (DAF) as a potential therapeutic for neovascular age-related macular degeneration (NV AMD). Crystal structures of the VEGF/Ang2 DAF in complex with its two antigens showed highly overlapping binding sites. In order 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. Further, 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 specific binding surface with comparable properties to individual high affinity mono-specific antibodies. Deep sequencing-guided design of a high affinity dual-specific antibody to target two angiogenic factors in neovascular age-related macular degeneration.,Koenig P, Lee CV, Sanowar S, Wu P, Stinson J, Harris SF, Fuh G J Biol Chem. 2015 Jun 18. pii: jbc.M115.662783. PMID:26088137[5] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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