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PDBsum entry 1cz8
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Immune system
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PDB id
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1cz8
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Contents |
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94 a.a.
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213 a.a.
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218 a.a.
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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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Selection and analysis of an optimized anti-Vegf antibody: crystal structure of an affinity-Matured FAB in complex with antigen.
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Authors
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Y.Chen,
C.Wiesmann,
G.Fuh,
B.Li,
H.W.Christinger,
P.Mckay,
A.M.De vos,
H.B.Lowman.
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Ref.
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J Mol Biol, 1999,
293,
865-881.
[DOI no: ]
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PubMed id
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Abstract
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The Fab portion of a humanized antibody (Fab-12; IgG form known as rhuMAb VEGF)
to vascular endothelial growth factor (VEGF) has been affinity-matured through
complementarity-determining region (CDR) mutation, followed by affinity
selection using monovalent phage display. After stringent binding selections at
37 degrees C, with dissociation (off-rate) selection periods of several days,
high affinity variants were isolated from CDR-H1, H2, and H3 libraries.
Mutations were combined to obtain cumulatively tighter-binding variants. The
final variant identified here, Y0317, contained six mutations from the parental
antibody. In vitro cell-based assays show that four mutations yielded an
improvement of about 100-fold in potency for inhibition of VEGF-dependent cell
proliferation by this variant, consistent with the equilibrium binding constant
determined from kinetics experiments at 37 degrees C. Using X-ray
crystallography, we determined a high-resolution structure of the complex
between VEGF and the affinity-matured Fab fragment. The overall features of the
binding interface seen previously with wild-type are preserved, and many contact
residues are maintained in precise alignment in the superimposed structures.
However, locally, we see evidence for improved contacts between antibody and
antigen, and two mutations result in increased van der Waals contact and
improved hydrogen bonding. Site-directed mutants confirm that the most favorable
improvements as judged by examination of the complex structure, in fact, have
the greatest impact on free energy of binding. In general, the final antibody
has improved affinity for several VEGF variants as compared with the parental
antibody; however, some contact residues on VEGF differ in their contribution to
the energetics of Fab binding. The results show that small changes even in a
large protein-protein binding interface can have significant effects on the
energetics of interaction.
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Figure 2.
Figure 2. Radiolabeled VEGF binding assay. [
125
I]VEGF was equilibrated (23 °C) with serial dilutions of unlabeled
VEGF and (a) Fab-12 or (c) Y0317. Fabs were captured with an anti-Fab antibody-coated immunosorbant plate.
Scatchard analysis (Munson & Rodbard, 1980) with a 1:1 binding model was used to calculate Kd of (b) 433 (±116)
pM for Fab-12 and (d) 19.8(±4.3) pM for Y0317.
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Figure 4.
Figure 4. Structure of the affinity-improved Y0317 Fab
in complex with VEGF. A superposition of the structure
(Muller et al., 1998a) of wild-type humanized antibody
Fab-12 (gray) in complex with VEGF (gray) is shown
with that of Fab Y0317 (green) in complex with VEGF
(yellow). (a) Overall view of the complex, including one
Fab molecule bound to one dimer of VEGF (a second
Fab molecule is bound at left in the crystal) shows that
the binding site for both antibody variants centers on
the ``80's loop'' of VEGF. (b) A view of the four CDR
changes between Fab-12 and Y0317 Fab shows that the
new D28 and T100a side-chains do not directly contact
antigen. However, H31 and Y97 form new contacts.
(c) Interactions of H97 and an associated, buried water
molecule in the Fab-12 complex, compared with those of
Y97 in the Y0317 complex.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(1999,
293,
865-881)
copyright 1999.
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Secondary reference #1
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Title
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Vegf and the FAB fragment of a humanized neutralizing antibody: crystal structure of the complex at 2.4 a resolution and mutational analysis of the interface.
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Authors
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Y.A.Muller,
Y.Chen,
H.W.Christinger,
B.Li,
B.C.Cunningham,
H.B.Lowman,
A.M.De vos.
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Ref.
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Structure, 1998,
6,
1153-1167.
[DOI no: ]
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PubMed id
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Figure 3.
Figure 3. Schematic representation of the binding epitope
of VEGF for the humanized anti-VEGF antibody. Residues buried in
the interface as seen in the crystal structure are colored red;
residues marked with yellow display a greater than 20-fold
reduction in binding affinity when changed to alanine. For
comparison, and to allow discussion of the neutralizing effect
of the antibody, residues buried in the interface between VEGF
and domain 2 of the Flt-1 receptor [16] are colored blue, and
VEGF binding determinants for KDR [15] are in green. The
position of the twofold axis of the VEGF dimer is indicated by a
black ellipse.
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The above figure is
reproduced from the cited reference
with permission from Cell Press
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