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PDBsum entry 1bj1
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Immune system
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PDB id
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1bj1
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Contents |
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213 a.a.
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218 a.a.
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94 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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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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Abstract
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BACKGROUND: Vascular endothelial growth factor (VEGF) is a highly specific
angiogenic growth factor; anti-angiogenic treatment through inhibition of
receptor activation by VEGF might have important therapeutic applications in
diseases such as diabetic retinopathy and cancer. A neutralizing anti-VEGF
antibody shown to suppress tumor growth in an in vivo murine model has been used
as the basis for production of a humanized version. RESULTS: We present the
crystal structure of the complex between VEGF and the Fab fragment of this
humanized antibody, as well as a comprehensive alanine-scanning analysis of the
contact residues on both sides of the interface. Although the VEGF residues
critical for antibody binding are distinct from those important for
high-affinity receptor binding, they occupy a common region on VEGF,
demonstrating that the neutralizing effect of antibody binding results from
steric blocking of VEGF-receptor interactions. Of the residues buried in the
VEGF-Fab interface, only a small number are critical for high-affinity binding;
the essential VEGF residues interact with those of the Fab fragment, generating
a remarkable functional complementarity at the interface. CONCLUSIONS: Our
findings suggest that the character of antigen-antibody interfaces is similar to
that of other protein-protein interfaces, such as ligand-receptor interactions;
in the case of VEGF, the principal difference is that the residues essential for
binding to the Fab fragment are concentrated in one continuous segment of
polypeptide chain, whereas those essential for binding to the receptor are
distributed over four different segments and span across the dimer interface.
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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
reprinted
by permission from Cell Press:
Structure
(1998,
6,
1153-1167)
copyright 1998.
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Secondary reference #1
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Title
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Antibody humanization using monovalent phage display.
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Authors
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M.Baca,
L.G.Presta,
S.J.O'Connor,
J.A.Wells.
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Ref.
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J Biol Chem, 1997,
272,
10678-10684.
[DOI no: ]
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PubMed id
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Figure 1.
Fig. 1. Amino acid sequences of murine A4.6.1 and humanized
A4.6.1 variants hu2.0 and hu2.10. Sequence numbering is
according to Kabat et al. (26), and mismatches are indicated by
asterisks (murine A4.6.1 versus hu2.0) or bullets (hu2.0 versus
hu2.10). Variant hu2.0 contains only the CDR sequences
(boldface) from the murine antibody grafted onto a human light
chain  subgroup
I-heavy chain subgroup III framework. hu2.10 was the consensus
humanized clone obtained from phage sorting experiments. CDRs
are defined according to the sequence definition of Kabat et al.
(26), except for CDR-H1, which is the combined sequence and^
structural (19) definition.
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Figure 3.
Fig. 3. Phagemid construct for surface display of Fab-pIII
fusions on phage. The phagemid construct is similar to that
described^ previously (31) and encodes a humanized version of
the Fab fragment for antibody A4.6.1 fused to a portion of the
M13 gene III coat protein. The fusion protein consists of the
Fab joined at the^ carboxyl terminus of the heavy chain to a
single glutamine residue^ (from suppression of an amber codon in
supE E. coli) and then the carboxyl-terminal region of the gene
III protein (residues 249-406). Transformation into F  E. coli,
followed by superinfection with M13KO7 helper phage, produces
phagemid particles in which a small proportion of these display
a single copy of the fusion protein.
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The above figures are
reproduced from the cited reference
with permission from the ASBMB
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Secondary reference #2
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Title
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Humanization of an anti-Vascular endothelial growth factor monoclonal antibody for the therapy of solid tumors and other disorders.
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Authors
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L.G.Presta,
H.Chen,
S.J.O'Connor,
V.Chisholm,
Y.G.Meng,
L.Krummen,
M.Winkler,
N.Ferrara.
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Ref.
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Cancer Res, 1997,
57,
4593-4599.
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PubMed id
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