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PDBsum entry 1olv
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References listed in PDB file
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Key reference
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Title
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Identification of the antigenic epitopes in staphylococcal enterotoxins a and e and design of a superantigen for human cancer therapy.
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Authors
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E.Erlandsson,
K.Andersson,
A.Cavallin,
A.Nilsson,
U.Larsson-Lorek,
U.Niss,
A.Sjöberg,
M.Wallén-Ohman,
P.Antonsson,
B.Walse,
G.Forsberg.
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Ref.
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J Mol Biol, 2003,
333,
893-905.
[DOI no: ]
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PubMed id
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Abstract
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Monoclonal antibodies have a potential for cancer therapy that may be further
improved by linking them to effector molecules such as superantigens. Tumor
targeting of a superantigen leads to a powerful T cell attack against the tumour
tissue. Encouraging results have been observed preclinically and in patients
using the superantigen staphylococcal enterotoxin A, SEA. To further improve the
concept, we have reduced the reactivity to antibodies against superantigens,
which is found in all individuals. Using epitope mapping, antibody binding sites
in SEA and SEE were found around their MHC class II binding sites. These
epitopes were removed genetically and a large number of synthetic superantigens
were produced in an iterative engineering procedure. Properties such as
decreased binding to anti-SEA as well as higher selectivity to induce killing of
tumour cells compared to MHC class II expressing cells, were sequentially
improved. The lysine residues 79, 81, 83 and 84 are all part of major antigenic
epitopes, Gln204, Lys74, Asp75 and Asn78 are important for optimal killing of
tumour cells while Asp45 affects binding to MHC class II. The production
properties were optimised by further engineering and a novel synthetic
superantigen, SEA/E-120, was designed. It is recognised by approximately 15% of
human anti-SEA antibodies and have more potent tumour cell killing properties
than SEA. SEA/E-120 is likely to have a low toxicity due to its reduced capacity
to mediate killing of MHC class II expressing cells. It is produced as a Fab
fusion protein at approximately 35 mg/l in Escherichia coli.
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Figure 2.
Figure 2. (a) Multiple sequence alignment of SEA, SEE,
SEA/E-18 and SEA/E-120. The five different regions A-E, which
contain all the substitutions in SEA/E-120 are indicated as
coloured boxes. The seven different peptides identified (see
Figure 1) are displayed as lines above the amino acid sequence
for SEA/E-120. Characters in bold indicate the residues that
were modified in SEA/E-120 compared to SEE. (b) Ribbon diagram
of SEA/E-120 model. The side-chains of residues G20, T21, G24
and K27 are marked in orange, side-chains of residues S34, S39,
S40, E41, K42, A44, T49 in red, side-chains of T74, A75, S78,
E79, E81, S83 and S84 are marked in green and side-chains of
residues T217, S220, T222, S223, S225 and S227 are marked in
purple. The coloured parts of the ribbon corresponds to regions
A-E with the same colours as in (a).
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Figure 3.
Figure 3. Characterisation of selected superantigen
variants. (a) Binding analysis of human anti-SEA to selected
superantigen variants using a scintillation proximity assay. In
more detail, human anti-SEA was labelled with 125I and the
direct binding of these antibodies to increasing concentrations
of C215FabSEA, C215FabSEA/E-18, -65, -97, -109, -110, -113 or
-120 on biotin conjugated anti-mouseF(ab)[2] on streptavidin PVT
beads was measured. (b) The ability to mediate T cell dependant
cytotoxicity on tumour cells by increasing concentrations of the
selected fusion proteins measured in a cytotoxicity assay
against Colo205 cells. (c) The ability of the constructs to
mediate T cell dependent killing of MHC class II expressing Raji
cells. The calculations are described in Materials and Methods.
In a clinical situation, the assay in (b) has been designed to
imitate the killing of tumor cells while the assay in (c)
reflects induction of systemic toxicity.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2003,
333,
893-905)
copyright 2003.
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