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PDBsum entry 4zfg
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Signaling protein/immune system
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PDB id
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4zfg
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Contents |
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220 a.a.
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215 a.a.
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213 a.a.
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References listed in PDB file
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Key reference
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Title
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Deep sequencing-Guided design of a high affinity dual specificity antibody to target two angiogenic factors in neovascular age-Related macular degeneration.
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Authors
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P.Koenig,
C.V.Lee,
S.Sanowar,
P.Wu,
J.Stinson,
S.F.Harris,
G.Fuh.
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Ref.
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J Biol Chem, 2015,
290,
21773-21786.
[DOI no: ]
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PubMed id
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Abstract
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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.
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