doi:10.1016/j.jmb.2003.09.009 J. Mol. Biol.

(2003) 333, 893–905

Identification of the Antigenic Epitopes in

Staphylococcal Enterotoxins A and E and Design of a
Superantigen for Human Cancer Therapy
Eva Erlandsson, Kerstin Andersson, Anders Cavallin, Anneli Nilsson
Ulla Larsson-Lorek, Ulf Niss, Annelie Sjöberg, Marie Wallén-Öhman
Per Antonsson, Björn Walse and Göran Forsberg*
Active Biotech Research AB Monoclonal antibodies have a potential for cancer therapy that may be
Box 724, 220 07 Lund, Sweden further improved by linking them to effector molecules such as superanti-
gens. Tumor targeting of a superantigen leads to a powerful T cell attack
against the tumour tissue. Encouraging results have been observed pre-
clinically and in patients using the superantigen staphylococcal entero-
toxin A, SEA. To further improve the concept, we have reduced the
reactivity to antibodies against superantigens, which is found in all indi-
viduals. 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 impor-
tant 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 anti-
bodies 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.
q 2003 Elsevier Ltd. All rights reserved.
*Corresponding author Keywords: superantigen; antigenicity; genetic engineering; cancer; therapy

Introduction against cancer.1 Except for problems with immuno-

Antibody based cancer therapies have been
investigated for two decades. A high number of
different antibodies have been studied in clinical
trials, but due to several challenges only a few anti-
bodies have become important tools in the fight
Present address: A. Cavallin, Department of Molecular
Biology, AstraZeneca, Mölndal, Sweden; P. Antonsson,
Teknopol AB, Ideon, 223 70 Lund, Sweden.
Abbreviations used: SEA, staphylococcal enterotoxin
A; SEE, staphylococcal enterotoxin E; MHC, major
histocompatibility complex; aa, amino acid.
E-mail address of the corresponding author:
goran.forsberg@activebiotech.com
genicity of non-human antibodies, the main
obstacles have been associated with poor tumour
uptake, difficulties to find antigens that are selec-
tively expressed on a large number of tumour
cells and finally a low cytotoxic potency of anti-
bodies. Several different ways to potentiate anti-
body therapies have been investigated such as
radio-labelling or fusion to cytotoxic molecules.2
We have developed a slightly alternative approach
by fusing the superantigen staphylococcal entero-
toxin A, SEA, to tumour reactive antibody frag-
ments.3 The conceptual idea behind this fusion
protein is to stimulate and direct potent effector
cells of the immune system against the tumour.
Bacterial superantigens, activate a large fraction

0022-2836/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.
894 Antigenic Regions in SEA and SEE

Figure 1. Reverse phase HPLC analysis of peptide fragments recognised by human anti-SEA from a pepsin digest of
SEA/E-18. A partial digest of the superantigen was passed over a column with immobilized anti-SEA. The fragments
were identified both before and after affinity chromatography using reversed phase high performance liquid chroma-
tography coupled to a mass spectrometer (MS). The relative abundance is defined as intensity recorded by the MS.
Fragments found in the digest at the same retention time both before and after affinity purification were considered
as positives. The molecular masses (theoretical/measured) for the individual peptides were; aa32– 54 (1381.2/1380.8),
aa68– 87 (1096.6/1097.0), aa170– 199 (1099.5/1099.3), aa171– 210 (1445.7/1445.3), aa187– 231 (1740.9/1741.2), aa210–
224 (925.0/925.2).

of the T-cell population by their binding to the T
cell receptor, TCR Va4 or Vb chain.5,6 Despite a
relatively low sequence similarity, this family of
proteins appears to share the same three-dimen-
sional structure.7 SEA belongs to the group of the
most extensively characterised superantigens.8,9
Upon presentation on MHC class II, SEA stimulate
T cells to divide and secrete cytokines.10,11 Interest-
ingly, only a couple of superantigen molecules
need to be bound to a cell to generate a potent T
cell stimulation.12 Certain superantigens, like SEA,
have two distinct MHC class II binding sites13,14
that enable them to cross-link MHC class II
molecules on antigen presenting cells and this
lead to secretion of inflammatory cytokines.15
Fusion proteins between antibody moieties and
SEA trigger cytotoxic T cells to kill tumour cells
in the absence of MHC class II molecules.3,16 In
addition, activated T cells produce tumouricidal
and pro-inflammatory cytokines, counteracting the
problems of tumor heterogeneity and macro-
molecular uptake.17,18 In murine tumour models,
the anti-tumour effects of Fab-SEA fusion proteins
are clearly dependent on targeting of superantigen
activity to the tumour site.3,19 In contrast, dose-
limiting systemic immune activation is largely due
to MHC class II-dependent targeting of super-
antigen activity to the spleen and other lymphoid
tissues.20,21 In preclinical models, the FabSEA
fusion proteins have shown potent effects against
colorectal cancer, lymphoma, melanoma and non-
small cell lung cancer.3,19,22 – 24
A fusion protein consisting of the C242Fab
moiety and wild-type SEA has been investigated
in clinical trials of colorectal and pancreatic
cancer.25 Even though encouraging results were
obtained, this protein showed toxic effects and
caused fever and hypotension. The most likely
reason for this is that the product accumulated in
MHC class II containing organs, instead of only in
the tumour tissue. Most residues in SEA that inter-
act with MHC class II have been identified13,14,26
Antigenic Regions in SEA and SEE 895

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 identi-
fied (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).

and this has given insight into the molecular
mechanisms causing toxicity. Based on these find-
ings a novel fusion protein, 5T4FabSEAD227A, with
100– 1000-fold lower affinity for MHC class II19
has been designed for later clinical studies. Despite
the reduced binding to MHC class II and decreased
toxicity, this product still has very powerful T
cell activating properties. However, there is also
another complication due to that all individuals
have preformed antibodies against SEA which
makes dosing complex. The 5T4FabSEAD227A fusion
protein is currently in clinical trials and, although
less pronounced compared to the wild-type con-
struct, toxicity and anti-SEA antibodies may be
limiting factors for the therapy.
Here we have mapped the major antigenic sites
896
Figure 3 (legend opposite)
in SEA and we present a novel generation of syn-
thetic superantigens with reduced seroreactivity
and toxicity. The approach was based on iterative
engineering using experimental data and model-
ling. Taking advantage of this novel generation of
superantigens, the clinical development of super-
antigen therapy of cancer may be substantially
facilitated.
Results and Discussion
Identification of antibody epitopes in SEA/E-18
To simplify the clinical use of bacterial super-
antigens, one potentially very useful approach is
Antigenic Regions in SEA and SEE
to limit the impact of pre-existing antibodies
against them. When non-human proteins have
been investigated clinically, previously described
methods have been to use truncated or PEG-ylated
products.27 In this study, we decided to remove the
antigenic epitopes by amino acid replacements.
When evaluating alternative superantigens, it
was found that SEE has a lower antibody reactivity
than SEA even though the homology is higher than
80%. Unexpectedly, SEE differs from SEA because
it cannot mediate T cell dependant killing of MHC
class II negative tumour cells when fused to a
tumour reactive Fab.28 A potential reason is dif-
ferences in affinity for human TCR and therefore
chimeric constructs of SEA and SEE were investi-
gated. When introducing the corresponding amino
Antigenic Regions in SEA and SEE 897

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.

acid residues from SEA in four positions of the
TCR-binding region of SEE, the antitumour prop-
erties of SEA were retained.28 These substitutions;
Arg20Gly, Asn21Thr, Ser24Gly and Arg27Lys in
SEE (region A in Figure 2(a)), in combination with
Asp227Ala which decreases affinity for MHC class
II, resulted in the chimeric molecule SEA/E-18
(Figure 2).28 The antibody reactivity, which is
approximately 50% lower compared to SEA
(Figure 3(a); Table 1), is almost identical to SEE.
SEA/E-18 has retained the highly cytotoxic proper-
ties of SEA (Figure 3(b); Table 1) and chimeric
constructs have a higher structural stability than
SEA and SEE.29
To further reduce the ability of human anti-SEA
antibodies to recognise SEA/E-18, its antibody
binding epitopes were determined. Peptide frag-
ments from a partial pepsin digest of either SEA
(data not shown) or SEA/E-18 were captured
using immobilised anti-SEA antibodies. These
peptide sequences were identified using LC – MS
(Figure 1) and potential antigenic areas were loca-
lised in the amino acid sequence (Figure 2(a)).
Notably, most of the recovered peptides were
located in the two distinct N-terminal and
C-terminal regions known to be interaction sites
to MHC class II molecules.13,14,30 A comparative
homology model of SEA/E-18 (data not shown),
based on the SEA structure,30,31 was used to locate
the surface exposed residues within the identified
peptides. The following residues were identified
as exposed and potential candidates as antibody
binding epitopes: Glu34, Lys35, Glu39, Asn40,
Lys41, Glu42, Asp44, Asp45, Glu49 (all belonging
to region B), Lys74, Asp75, Asn78, Lys79, Lys81,
Lys83, Lys84 (region C), Asp173, His187, Ser189,
Glu190 (region D), Gln204, Lys217, Asn220,
Glu222, Asn223, His225 and Asp227 (region E)
(Table 1).
These residues were subsequently substituted to
reduce antibody binding. During this iterative
engineering procedure, the binding level to
human antibodies, biological activity and pro-
duction yields were determined. Computer models
of novel superantigen variants were continuously
made to confirm and compare the results acquired.
Specifically the influence of side-chains was studied
and substitutions affecting protein stability were
identified. The results were used in the design of
new analogues with improved properties. To fol-
low the impact of the engineering process, data on
representative variants is shown in Figure 3.
Evaluation of anti-SEA reactivity within the
different superantigen variants
The importance of the identified residues for
antibody recognition was initially evaluated by
one to six simultaneous substitutions in SEA/
E-21, a variant of SEA/E-18 with the replacements
His187Ala, Ser188Thr (Table 1). Thereby variants
containing replacements in regions D and E were
constructed and these were SEA/E-62 (Lys217Thr,
Asn220Ala, Glu222Thr, Asn223Ala, His225Ala),
Table 1. Summary of binding to human anti-SEA, potency to mediate T cell killing of tumour cells or MHC class II positive cells and production levels in E. coli of a large numbers of the various
superantigens investigated
B C D E
Region
Chimera Anti-
Yield SEA Colo205 Raji
E34 K35 E39 N40 K41 E42 D44 D45 E49 K74 D75 N78 K79 K81 K83 K84 D173 H187 S188 S189 E190 Q204 K217 N220 E222 N223 H225 D227 (mg/l) (%) (%) (%)
SEA 203 100 100 1000
SEE 69.3 47 0 1000
SEA/ A 40.0 44 100 100
E-18
SEA/ A T A 26.1 43 20 100
E-21
SEA/ A T T A T A A A 2.0 34 100 20
E-62
SEA/ A T T S T S S S 68.0 39 100 100
E-97
SEA/ A T D A A 21.6 42 100 20
E-63
SEA/ K E K S E K A T A 28.0 30 20 20
E-64
SEA/ S S S E K A T A 43.0 29 33 17
E-108
SEA/ E E E E A T A 3.4 24 100 20
E-65
SEA/ E E E E A T R A 2.9 23 100 100
E-90
SEA/ S S E E A T A 13.8 26 100 100
A-83
SEA/ E E S S A T A 14.0 26 100 100
E-84
SEA/ A A T E E E E A T D A A 24.5 23 100 0
E-85
SEA/ A A T A 39.0 41 20 100
E-68
SEA/ A A T A T A 66.0 35 100 20
E-74
SEA/ A T E E E E A T D A A 18.0 20 100 10
E-91
SEA/ T A S A T A 73.0 38 100 100
E-75
SEA/ S E K A A T E E E E A T D A A 21.0 19 100 0
E-93
SEA/ A A T D A R T S T S S S 6.0 34 100 10
E-107
SEA/ A A T D A T T S T S S S 20.0 39 20 12
E-113
SEA/ S S S E K A T T A S E E S S A T A 34.0 21 100 1
E-109
SEA/ S S S E K A T T A S E E S S A A T D A T T S T S S S 1.3 14 2 0.20
E-110
SEA/ S S S E K A T T A S E E S S A A T T S T S S S 1.7 15 17 1
E-115
SEA/ S S S E K A T T A S E E S S A S T T S T S S S 2.4 15 20 0.20
E-118
SEA/ S S S E K A T T A S E E S S A T T S T S S S 15.0 16 100 1.70
E-119
SEA/ S S S E K A T T A S E E S S T S T S S S 35.0 16 100 1.40
E-120

As a comparison, the biological activities are shown as relative figures, which have been assigned to be 100% for C215FabSEA/E-18 both against Colo205 cells and the MHC class II expressing Raji
cells. The Bmax value for evaluating the seroreactivity is expressed as percentages of C215FabSEA. The yields are defined as full-length protein recovered after purification, as described in Materials
and Methods.
Antigenic Regions in SEA and SEE
SEA/E-63 (Ser189Asp, Glu190Ala) and SEA/E-68
(Asp173Ala). Similarly, SEA/E-64 (Glu34Lys,
Lys35Glu, Glu39Lys, Asn40Ser, Lys41Glu,
Glu42Lys), SEA/E-65 (Lys79Glu, Lys81Glu,
Lys83Glu, Lys84Glu), SEA/E-74 (Asp44Ala,
Asp45Ala, Glu49Thr) and SEA/E-75 (Lys74Thr,
Asp75Ala, Asn78Ser) with replacements in regions
B and C, were constructed (Table 1). To evaluate
antigenicity, a scintillation proximity assay was
used. The reactivity for some of the variants was
also measured by ELISA using a pool of human
IgG and similar results were obtained in both
assays (results not shown). Generally, the modified
variants were recognised by the antibodies to a
lesser extent compared to SEA/E-18 (Table 1). The
most substantial reduction in binding was caused
by the substitutions made in SEA/E-65. Only
approximately 55% of the antibodies binding
SEA/E-18 bound SEA/E-65 (Figure 3(a)), which
show that Lys 79, 81, 83 and 84 in region C are
parts of major antigenic epitopes.
The antibody binding was further reduced when
the variants were combined, as in SEA/E-91 com-
posed of SEA/E-63, SEA/E-65 and a modified
SEA/E-74 (with wild-type Asp45) (Table 1). As
exemplified by SEA/E-109 (Table 1), most of the
antibodies recognised epitopes in regions B and
C. These epitopes surround the N-terminal binding
site for MHC class II, which is a relatively con-
served low affinity MHC class II binding site
within the superantigen family. The variant SEA/
E-110 that has most of the identified residues
replaced, binds less than 30% of the antibodies
recognising SEA/E-18 and approximately 14% of
the antibodies binding to wild-type SEA. However,
this particular superantigen was produced at a low
level in Escherichia coli and mediated killing of
tumour cells less efficiently than SEA/E-18 (Table
1). Therefore, further engineering to increase the
production yield was carried out as described
below.
In conclusion, since human anti-SEA antibodies
are polyclonal in their nature, many antigenic
areas of the protein need to be modified in the
design of superantigens with decreased antibody
reactivity. Most antigenic regions were located in
areas around the N-terminal MHC class II binding
site of SEA/E-18. The residues substituted in the
variants SEA/E-64, SEA/E-65, SEA/E-62 and
SEA/E-74, each resulted in 10 to 20% reduction of
the anti-SEA reactivity (Table 1).
Evaluation of biological function within the
different superantigen variants
The superantigens were primarily designed for
tumour therapy and thus it was necessary to
avoid replacements decreasing the ability to
mediate T cell cytotoxicity. The ability to mediate
cytotoxicity against tumour cells (Figure 3(a)) and
MHC class II expressing cells (Figure 3(c)) was
therefore measured for all new superantigen
variants. The latter can most likely be correlated to
899
toxicity in humans and for ideal clinical use, this
activity should be low to allow high doses of
product without causing severe side-effects. How-
ever, some of this activity is probably needed to
get initial T cell activation.
To determine anti-tumour activity, Colo205 cells
expressing the antigen GA733-2 which is reactive
with the C215 antibody, were used.32 Most of the
initial set of superantigen variants had the same
potency to kill tumour cells as SEA/E-18 (Table 1).
The exceptions were SEA/E-75 with the replace-
ments Lys74Thr, Asp75Ala and Asn78Ser (region
C) which had a tenfold decreased potency and
SEA/E-64, with the replacements Glu34Lys,
Lys35Glu, Glu39Lys, Asn40Ser, Lys41Glu and
Glu42Lys (region B), which had fivefold decreased
potency compared to SEA/E-18 (Table 1). The
decreased activity of SEA/E-64 was to some extent
caused by the substitution Lys35Glu and conse-
quently in preceding constructs residue 35 was
lysine. The decreased activity in SEA/E-75 was
only observed in this variant, in combination with
further substitutions, for example in SEA/E-109
full activity was detected (Table 1). The activity
against MHC class II expressing cells was
unchanged in SEA/E-75 compared to SEA/E-18.
The most likely explanation is that the modifi-
cations affects the three-dimensional structure in
different ways (destabilisation of the a3 helix in
the N-terminal domain) in related constructs and
changes affecting binding to the TCR have a bigger
influence on cytotoxicity against the tumour cells.
All the investigated superantigen variants con-
tain a substitution of residue Asp227 to alanine or
serine. This substitution is known to reduce the
affinity to MHC class II more than 100 times by
destroying a zinc binding site which is crucial for
a high affinity interaction with MHC class II
molecules.14 The majority of the initial variants
SEA/E-62, SEA/E-63, SEA/E-64, SEA/E-65 and
SEA/E-74 showed a further twofold decreased
activity against MHC class II positive cells, in com-
parison with SEA/E-18. However, combining all
the N-terminal substitutions as in SEA/E-109,
resulted in further reduction, compared to SEA/
E-18. This contrasts SEA/E-97, with only C-termi-
nal substitutions, which induce similar cytotoxic
activity against MHC class II positive cells as
SEA/E-18. This indicates that within SEA/E-109
and especially within region B, additional residues
have been changed that bind to MHC class II. One
of these residues is Asp45, while others like resi-
dues 40– 42 and 79 –84 only affects MHC class II
reactivity in certain combinations and do not
interact directly with MHC.
Gln204 is an exposed residue in a region that is
likely to interact with the T cell receptor.28,33 Since
this residue also is a candidate for antibody
recognition, two different replacements were
investigated, Gln204Arg and Gln204Thr. Notably,
Gln204Thr resulted in a decreased biological
activity, while Gln204Arg did not affect the activity
at all (Table 1).
900
In conclusion, substitutions of Gln204 and the
three substitutions Lys74Thr, Asp75Ala and
Asn78Ser, were under some circumstances
influencing the activity against tumour cells nega-
tively. One explanation for this is that Gln204 may
interact with or stabilise the interaction to the T
cell receptor, while residues 74, 75 and 78 are far
away from the TCR binding site controlling struc-
tural stability of the TCR Vb interacting b10 –a5
and aN – a2 loops. The remaining replacements
were more or less affecting the activity against
MHC class II expressing cells (Table 1). However,
the replacement that caused the greatest reduction
were Asp45Ala in SEA/E-74, 85 and 93. This resi-
due is next to Gln46 that interact with Gln18 on
the a chain of MHC class II26 and Asp45 is there-
fore likely to participate in the MHC class II low
affinity interaction site on SEA. It should be
pointed out that these residual substitutions only
affects the biological activities in combination with
certain other replacements. This suggests that
small changes in the three-dimensional structure
can lead to drastic changes in the abilities of
these molecules to interact with T cells or antigen
presenting cells.
Replacements affecting the production levels
As indicated above, several substitutions on the
superantigen surface resulted in decreased levels
of production in E. coli. Many combinations of
such replacements were not even possible to pro-
duce. At least four different mechanisms, such as
poor thermodynamic properties, destroyed fold-
ing pathways, poor secretion properties or newly
introduced proteolytic sites, can explain the
decreased production levels of multi-substituted
superantigen variants. A consequence of the
engineering is also the introduction of structural
modifications and in order to predict these and
guide the design, new computer models were
made for several of the new superantigen variants.
Hereby, the specific residues that caused reduced
yields within poorly produced variants were
identified and improved variants with either wild-
type residues or other more suitable substitutions
were designed (Table 1).
The strategy with the expression system used
was to obtain the products in the growth med-
ium. Analysis of production levels were per-
formed on growth medium as well as whole cell
lysates. However, in no case an increased intra-
cellular or periplasmic accumulation of the pro-
ducts were detected. The lowest levels of
leakage to growth medium were observed with
the original variants SEA/E-18 and SEA/E-21,
with 60– 70% of the total product found in the
medium. Previous studies have shown that
specific residues in the antibody moiety strongly
affects the leakage from periplasm to growth
medium.34,35 Based on the results in this study,
such leakage appears to be less affected by modifi-
cations in the superantigen molecule.
Antigenic Regions in SEA and SEE
In the initial set of superantigen variants, SEA/
E-62 and SEA/E-65 were produced at around
2 mg/l, which is approximately a tenfold lower
level than the original construct. In SEA/E-65, the
residues Lys79, Lys81, Lys83 and Lys84 within
region C had all been replaced by Glu residues.
However, alternative replacements were found to
increase the yields and SEA/E-84 with serine
residues in position 83 and 84 and Glu residues in
positions 79 and 81 (Table 1) were produced at
four times higher levels while the antibody reac-
tivity was only slightly higher than SEA/E-65
(Table 1). The production level of SEA/E-62
with the replacements Lys217Thr, Asn220Ala,
Glu222Thr, Asn223Ala, His225Ala and Asp227Ala
within region E was approximately 2 mg/l. How-
ever, by replacing the four alanine substitutions
with serine residues, resulting in SEA/E-97, a
yield of 68 mg/l was obtained (Table 1).
The yields of variants carrying combinations of
replacements were far from predictable. When
combining SEA/E-65 with other variants, such as
SEA/E-63 and modified SEA/E-74, as in SEA/
E-91 the production level was increased to 18 mg/
l (Table 1). However, some combinations resulted
in much lower production levels than expected.
For instance, SEA/E-110, a combination of SEA/
E-109 and SEA/E-113, was produced at 1.3 mg/l,
while the others were produced at 34 mg/l and
20 mg/l, respectively. The production level of
SEA/E-110 was however increased to 35 mg/l
when removing the substitutions Asp173Ala,
His187Ala, Ser188Thr, Ser189Asp, Glu190Ala and
Gln204Thr within region D and thus creating
SEA/E-120 (Table 1).
In conclusion, to substitute antigenic residues
while at least retaining the level of production in
E. coli, the following residues Lys83, Lys84,
Asn220, Asn223, His225 and Asp227 should prefer-
entially be serine, since other modifications
affected the yield. Any substitution examined of
the residues Lys35, Asp173, His187, Ser188,
Ser189, Glu190 and Gln204, resulted in decreased
production levels. Thus, these residues are likely
to be important for the overall protein stability.
There are structural explanations for these find-
ings. Lys35 is located in the middle of the b1 strand
and its side-chain makes important contacts with
residues located in the a4 helix and in the b4
strand. The side-chain carboxyl group of Asp173
forms a hydrogen bond to the backbone carbonyl
group of Gly177 and to the side-chain amide
group of Gln180. This hydrogen bond pattern is
probably important for the stabilisation of the
TCR Vb interacting a4– b9 loop. The residues
His187, Ser188, Ser189, Glu190 are all part of the
b9 strand and the b9 –b10 loop. The b9 strand
forms together with the b12 strand part of the
b-sheet that constitute the high affinity MHC class
II binding site.36 Substitutions at this site and
especially at the positions 187 and 188 probably
destabilise important contacts with the b12 strand.
The side-chain of Gln204 makes an important
Antigenic Regions in SEA and SEE
hydrogen bond to Thr21 that connects the TCR Vb
interacting loops.
Design of a novel superantigen variant
In order to design an optimal superantigen vari-
ant, all favourable substitutions were combined
leading to the SEA/E-120 construct (Figure 2).
Design criteria were: (1) a maximally decreased
anti-SEA reactivity; (2) 10 –100 fold reduced
activity against MHC class II expressing cells with-
out reduction in the tumour killing potency of
the superantigen. Finally the product should be
possible to produce at high levels in E. coli.
First, all favourable modifications in the C-termi-
nal region, i.e. residues Asp173Ala, Ser189Thr,
Glu190Ala, Lys217Thr, Asn220Ser, Glu222Thr,
Asn223Ser, His225Ser and Asp227Ser (regions D
and E) together with Gln204Thr were assembled
forming superantigen variant SEA/E-113. This
variant exhibited a significant reduction in anti-
SEA reactivity and acceptable levels of expression
but showed a somewhat decreased biological
activity (Table 1). Similarly, all favourable substi-
tutions in the N-terminal region, Glu34Ser,
Glu39Ser, Asn40Ser, Lys41Glu, Glu42Lys,
Asp44Ala, Glu49Thr, Lys74Thr, Asn78Ser,
Lys79Glu, Lys81Glu, Lys83Ser and Lys84Ser
(regions B and C) were assembled into SEA/
E-109. A most significant decrease in anti-SEA
reactivity was observed for this superantigen
variant along with a high level of expression
and even an improved biological activity profile
(Table 1). However, when creating the combi-
nation of these two variants SEA/E-113 and
SEA/E-109 in SEA/E-110, there was an unex-
pected loss of both yield and biological function
(Table 1). The biological potency was fully recov-
ered when the wild-type residues Ser189, Glu190
and Gln204 were used again in SEA/E-115
(Table 1), but production levels were still at a
low level. Molecular modelling of this variant
suggested that residues Asp173, His187 and
Ser188, could be important for the stabilisation
of the fold. Several different combinations were
made to evaluate these residues, resulting in
SEA/E-118, SEA/E-119 and SEA/ E-120 (Table
1). Best production yield was obtained for SEA/
E-120 with wild-type residues in all three pos-
itions. Together with formerly made SEA/ E-21,
SEA/E-74, SEA/E-97, SEA/E-108 and SEA/E-
109, these were the only superantigen variants
reaching expression levels of more than 20 mg/l
(Table 1). No significant differences in biological
activity or antibody reactivity were observed
between the variants SEA/E-118, 119 and 120.
Thus, by careful genetic engineering to reduce
the binding of human anti-SEA to synthetic super-
antigens, a novel product, SEA/E-120, was
designed. This product has improved properties
for human therapy and can be produced at high
levels in E. coli.
901
Implications for clinical use
Antibody-superantigen fusion proteins have
been studied in a number of experimental models
of cancer and have shown to have promising thera-
peutic properties.3,19,24 In human patients, the con-
cept has been studied in more than 200 patients.
The first product consisted of the Fab moiety of
the monoclonal antibody C242 fused to SEA.24
Encouraging observations were made in patients
with advanced pancreatic cancer and colorectal
cancer.25,37 However, due to systemic toxicity, this
product could only be given in very low doses
and therefore SEA variants with decreased binding
to MHC class II were investigated. A new product
containing the Asp227Ala variant of SEA fused to
the monoclonal antibody 5T438 performed much
better, at least judged by preclinical studies.19 This
product is currently studied in patients with cancer
in lung and breast, but preformed antibodies and
toxicity still has to be considered (J. D. Cheng, C.
Langer, S. Aamdal, F. Robert, L. R. Engelhardt, O.
Fernberg, J. Schiller, G. Forsberg, R. K. Alpaugh,
L. M. Weiner, J. S. Babb & A. Rogatko, unpublished
results).
To develop a new generation superantigens for
clinical studies, the experience has focused our
attention on preformed antibodies against SEA
which makes dosing complex. The levels of anti-
SEA can differ more than 100-fold between
individuals39 and the antibodies are also polyclonal
in their character. To minimise these problems, we
mapped the antibody binding sites of pooled
human IgG and all measurements have been per-
formed against an IgG pool from thousands of
donors rather than on IgG from individuals.
Approximately 85% of the antibodies binding the
previous products are no longer affecting the
novel superantigen product, SEA/E-120. Prelimi-
nary analysis also shows that the variation in anti-
body levels against SEA/E-120 is much lower
between individuals compared to wild-type
superantigens. Thus, SEA/E-120 presents several
advantages. Most likely, dosing will be simpli-
fied, since the product can be administrated
without initial considerations of antibody levels.
Also, the toxicity is likely to be lower. This is
important, since higher doses can then be used
and this may be necessary for optimal tumour
uptake.
In patients, Fab-SEA constructs will most likely
be given for repeated cycles. It has been shown
that these constructs are immunogenic and in
some patients very high titres of antibodies against
SEA are formed. This is likely caused by a secon-
dary B cell response. At this stage it is too early to
make predictions of the immunogenic properties
of SEA/E-120 in humans.
An additional issue for superantigen-based thera-
pies is induction of T cell unresponsiveness. It has
been shown that this phenomena is dose depend-
ent.12 The relevance of such findings for tumor
therapy is unknown, but T cell unresponsiveness
902
may be less of a problem by combing superantigen
therapy with interleukin 2.40,41
To conclude, we believe that superantigens like
SEA/E-120 have a much bigger potential for cancer
therapy than those previously investigated
clinically. A fusion protein between the 5T4
antibody and SEA/E-120 is currently in early
clinical development.
Materials and Methods
Vector constructs and mutagenesis
Genes coding for the different superantigen variants
were made using a polymerase chain reaction (PCR)
based method. For the sub-cloning procedure, the plas-
mid pUC19 (GIBCO BRL Life Technologies, Middlesex,
UK) was used and all plasmids were prepared using the
QIAprep Spin Midiprep Kit Protocol (QIAGEN, Hilden,
Germany). The PCR reactions were performed on Gene
Amp PCR system 2400 (Perkin Elmer, Wellesley, MA)
with Taq DNA polymerase and appropriate PCR
buffer containing 15 mM MgCl2 (Roche Molecular Bio-
chemicals, Basel, Switzerland). After overnight cleavage
with appropriate restriction enzymes, the PCR products
and vectors were purified using electrophoresis in a 1%
agarose gel (GIBCO BRL Life Technologies) containing
0.5 mg/ml ethidium bromide (Sigma-Aldrich, Steinheim,
Germany) in TAE buffer (Sigma-Aldrich). The DNA con-
taining fragment was excised from the gel and extracted
using the CONSERTe Rapid Gel Extraction System
(GIBCO BRL Life Technologies). Vector and insert were
ligated (T4DNA ligase, Roche Molecular Biochemicals)
at room temperature for three to four hours. The ligation
mixture was transformed into the E. coli strain DH5a
(GIBCO BRL Life Technologies) according to instructions
enclosed with the cells. Positive clones were verified
using DNA sequencing. Correct sequences were cleaved
out with Rsr II/HindIII at 37 8C overnight and ligated in
the expression vector to code for a product fused to the
Fab moiety of the tumour reactive antibody C21532
according to.34 The construct was finally electroporated
into the E. coli K12 strain UL635 (xyl-7, ara-14, T4R,
DompT).
Identification of human anti-SEA binding regions
Regions recognised by human anti-SEA were identi-
fied from a pepsin-digest of SEA or SEA/E-18, which is
a chimeric variant of SEA and SEE, (Figures 1 and 2),
previously described as SEE/A-A28 with the substitution
Asp227Ala. Each superantigen was incubated with 0.5%
(w/w) pepsin in 10 mM HCl, 150 mM NaCl for 60
minutes at 37 8C. The peptide mixture was neutralised
with 2 M Tris – HCl (pH 8.0) and applied on a 1 ml
HiTrap column (Amersham Pharmacia Biotech, Uppsala,
Sweden) with immobilised human anti-SEA (obtained
from pooled human IgG (Gammanorm, Pharmacia,
Stockholm, Sweden). PBS (8.1 mM Na2HPO4, 1.5 mM
KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.3) was used
as washing buffer and the antibody binding fragments
were eluted using 0.1 M acetic acid (pH 3.0). The frag-
ments were identified both before and after purification
using high performance liquid chromatography coupled
to a mass spectrometer. The chromatography was carried
out on a C18 column (2 mm £ 250 mm) (VYDAC,
Hesperia, CA) using a linear gradient from 10% to 60%
Antigenic Regions in SEA and SEE
acetonitrile in 0.1% triflouroacetic acid over 30 minutes
at 40 8C. Mass determination was carried out using elec-
trospray mass spectrometry (LCQ, Thermo Finnigan,
San Jose, CA). Fragments found in the digest at the
same retention time both before and after affinity purifi-
cation were considered as specific binders.
Molecular modelling
A comparative computer model of SEA/E-18 was
generated using the X-ray crystal structures of SEA
(1ESF;30 and 1SXT;31) and SED42 as templates. The
sequence identity of SEA/E-18 to SEA and SED is 83%
and 55%, respectively. Model building was performed
using the HOMOLOGY module supplied with the
INSIGHTII software (Accelrys, San Diego). The SEA/
E-18 sequence was threaded over structural conserved
regions (SCRs) determined from the SEA and SED struc-
tures. The 1SXT co-ordinates for SEA were used as tem-
plate except for the first nine residues in the N terminus
where co-ordinates from 1ESF were used. The regions
between the SCRs were in most cases flexible loop areas
and were built using SEA as template except for residues
Gln19, Ile140, Asp141, Lys142, Ser189, Gly191, Asp200,
Pro206, Asp207 and Leu224, which were built using
SED as template. Some areas within the SCRs showed
greater sequence similarity with SED and were therefore
built using SED as structural template (Ile37, Glu49,
Asn50, Thr51, Leu52, Ser195 and Thr218). Due to the
fact that SEA was used as structural template for most
of the residues in SEA/E-18 no problems with overlap-
ping side-chains occurred. Splice points before and after
the SCRs were repaired as follows. First the mutated
side-chains were relaxed and then energy minimisation
and molecular dynamics simulations were used to relax
all side-chains within the SCRs using standard protocols
in HOMOLOGY. Loop areas were relaxed one at a time
using 1000 steps of energy minimisation followed by
1000 steps of molecular dynamics. This refinement
protocol was applied first on the loop side-chains and
then on all atoms in the loop. For all simulations the
CVFF force field43 with a force constant of 100 kcal/Å2
were used using a time step of 2 fs. The final model was
tested for bad regions using the PROSTAT module in
INSIGHTII. Models of new superantigen variants were
constructed using the SEA/E-18 model as template. The
specific amino acid residues were replaced and the most
favourable side-chain conformation was selected using
a simple steric-hindrance search followed by a short
energy minimisation. The root mean square distance
between Ca atoms for residues 10-233 in 1SXT and in
the SEA/E-18 and the SEA/E-120 models are 0.43 Å
and 0.38 Å, respectively.
Culturing and purification
The superantigen variants were expressed as fusion
proteins in the E. coli K12 strain UL635 using a pre-
viously described34 production plasmid with an induc-
able Lac UV-5 promoter and a kanamycin resistance
gene. Bacteria from frozen stock solution were incubated
at 25 8C for 22– 24 hours in shaker flasks containing (per
liter) 2.5 g of (NH4)2SO4, 3 g of KH2PO4, 2 g of K2HPO4,
0.5 g of sodium citrate, 1 g of MgSO4·7H2O, 0.05 g of
kanamycin and 12 g of glucose monohydrate. To 1 l
medium, 1 ml of trace element solution44 without
Na2MoO4·2H2O was added. The cells were grown to an
A620 of 2 –3 and 10 ml of the cultivation medium was
Antigenic Regions in SEA and SEE
used to inoculate a 1 l fermenter (Belach Bioteknik,
Bromma, Sweden) with a starting volume of 800 ml.
The fermenter medium contained (per liter) 2.5 g of
(NH4)2SO4, 9 g of KH2PO4, 6 g of K2HPO4, 0.5 g of
sodium citrate, 1 g of MgSO4·7H2O, 0.05 g kanamycin,
23.1 g of glucose monohydrate and 0.1% (v/v) of the
trace element solution above. The pH was kept constant
at pH 7.0 by titration of 25% NH3, the aeration was 1 l/
minute and the temperature 25 8C. During batch phase
the dissolved O2 was kept at 30% by regulating the agita-
tion from 400 rpm to 2000 rpm and during the fed-batch
by regulating the feed of 60% (w/v) glucose. Product
formation was induced when A620 was 45 by adding
0.1 mM isopropyl-b-D -thiogalactopyranoside. After culti-
vaton the cells were removed by centrifugation at 6000g
for 45 minutes at 4 8C. The clarified medium was stored
at 2 20 8C prior to purification. The product levels in
clarified growth medium or total cell lysates after freeze
thawing, were determined by an ELISA.34
DNA in the medium was precipitated by 0.19%
polyethyleneimine (w/v) in 0.2 M NaCl (pH 7.4) slowly
added with a peristaltic pump at a flow rate of 12 ml/
min. After centrifugation at 7500g for 30 minutes, the
supernatant was collected. It was applied on a 60 ml
Protein-G Sepharose Fast Flow column (Amersham
Pharmacia Biotech, Uppsala, Sweden) using a flow rate
of 14 ml/min. The column was washed using PBS and
elution was performed with 100 mM acetic acid, 0.025%
Tween 20 (pH 3.0). The eluted product was collected
and the pH was carefully adjusted to 1.5 units below
the calculated isoelectric point with filtrated 1 M NaOH
and diluted four times with 0.025% Tween 20. Degraded
variants were then removed using ion-exchange chroma-
tography. The ionic strength of the sample was adjusted
to 2 mS/cm and the column used was a SP-Sepharose-
HP, Hiload 16/10 (Amersham Pharmacia Biotech). The
column was equilibrated and washed with 10 mM
sodium acetate, 0.025% Tween 20 (pH 5.0) at a flow rate
of 4.0 ml/minute and elution was performed using a
linear gradient from 0% to 55% 100 mM sodium
acetate, 400 mM NaCl, 0.025% Tween 20 (pH 5.0) over
50 minutes.
Anti-SEA reactivity
The reactivity between the superantigen variants
and human anti-SEA was measured in a scintillation
proximity assay (SPA). Streptavidin coated PVT-beads
(Amersham Biosciences, Uppsala, Sweden) at 3 mg/ml
in PBS (DPBSS w/o Ca2þ and Mg2þ, BioWhittaker) were
pre-incubated on a rocking machine for 30 minutes at
room temperature with 3 mg of biotin conjugated F(ab)2
fragments from anti-mouse IgG (Jackson Laboratories,
Bar Harbor, MN) per mg beads. Beads, 150 mg/well,
were then added to an OptiPlate microtiter plate
(Packard Instruments, Greve, Denmark) with C215Fab
conjugated superantigens in a 1:2 dilution series, and
incubated for two hours on a rocking platform at room
temperature. The dilutions were made with PBS contain-
ing 1% bovine serum albumin (Sigma-Aldrich, Stock-
holm, Sweden) and the highest final concentration of
the superantigens in the wells were 40 nM. Finally the
bead suspension were incubated for three hours on a
rocking platform at room temperature with 1 nM 125I
labelled human anti-SEA antibodies affinity purified
from a commercial IgG pool (Gammanorm, Pharmacia,
Stockholm, Sweden). The plates were kept in the dark
during incubation and left standing still for 30 minutes
903
before the amount of b-scintillation was measured in a
Top-Count (Packard Instruments). From the saturation
curves the cpm values at saturation (cpmsat.) were
obtained and the percentage anti-SEA reactivity for a
specific superantigen variant was calculated as 100 £
cpmsat. (superantigen)/ cpmsat. (SEAwt).
Biological function
The ability of the various superantigen constructs to
mediate T cell dependant killing of C215 positive
Colo205 cells and MHC class II positive Raji cells
(American Type Culture Collection, Rockville, MD)
were measured in standard four hour 51Cr-release
assays.45 The cells were labelled with 51Cr, diluted in
R10 medium to 50,000 cells/ml and added to V-shaped
microtiter wells. As effector cells, a SEA reactive human
T cell line, prepared as described,45 was used at an effec-
tor to target ratio of 45:1 for the Colo205 cells and 30:1
for the Raji cells. Superantigen variants were diluted
with R10 medium and added in concentrations from
1029 – 10216 M for the Colo 205 cells and from 1027 –
10214 M for the Raji cells. Supernatants were collected
and the release of 51Cr was measured in a TopCount
Microplate scintillation counter (Packard Instruments).
The percentage of specific cytotoxicity was calculated as
100 £ [(cpm experimental release 2 cpm background
release)/(cpm total release 2 cpm background release)].
Protein Data Bank accession codes
The atomic coordinates have been deposited in the
Protein Data Bank under accession codes 1OLV and
1OLW for SEA/E-18 and SEA/E-120, respectively.
Acknowledgements
We are grateful for the help and input from
Ingegerd Andersson, Cecilia Forsberg, Lena
Nielsen, Christine Valfridsson, Niels-Jörgen
Skartved, Vicky Avery, Robert Persson, Mats
Nilsson, Leif Svensson, Ann-Charlotte Johansson
and Karin Petersson.
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Edited by I. Wilson