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J Cancer Res Clin Oncol
DOI 10.1007/s00432-014-1884-z
ORIGINAL ARTICLE – CANCER RESEARCH
In vitro effects and ex vivo binding of an EGFR‑specific
immunotoxin on rhabdomyosarcoma cells
Judith Niesen · Hannes Brehm · Christoph Stein · Nina Berges · Alessa Pardo ·
Rainer Fischer · Andre ten Haaf · Stefan Gattenlöhner · Mehmet K. Tur ·
Stefan Barth
Received: 30 October 2014 / Accepted: 19 November 2014
© Springer-Verlag Berlin Heidelberg 2014
IT was confirmed on formalin-fixed paraffin-embedded tis-
sue samples from two RMS patients.
Results We confirmed the specific binding of 425(scFv)-
ETA′ to RMS cells in vitro and ex vivo. Both the IT and
the corresponding imaging probe were rapidly internal-
ized. The IT killed EGFR+ RMS cells in a dose-depend-
ent manner, while showing no effect against control cells.
It showed specific apoptotic activity against one selected
RMS cell line.
Conclusions This is the first study showing the promising
therapeutic potential of a recombinant, EGFR-targeting,
ETA′-based IT on RMS cells. We confirmed the selective
killing with IC50 values of up to 50 pM, and immunohisto-
chemical staining confirmed the specific ex vivo binding to
primary RMS material.
Keywords Immunotoxin · EGFR · scFv ·
Rhabdomyosarcoma · Pseudomonas exotoxin A
Introduction
Rhabdomyosarcoma (RMS) is the most common pediatric
soft tissue sarcoma with about 4.5 cases per million chil-
dren per year (Ray and Huh 2012). There are three main
subtypes: embryonal (ERMS), the more aggressive alveo-
lar (ARMS) and the rare pleomorphic (PRMS). ERMS is
the most common subtype and accounts for approximately
70 % of all cases and occurs most often in children under
10 years. It has a better prognosis than ARMS and shows a
heterogeneous mutation pattern, with diverse gain-of-func-
tion and loss-of-function mutations, but the most prominent
mutation is loss of heterozygosity at 11p15.5 which leads to
the overexpression of several other genes (Davicioni et al.
2009). ARMS, which occurs more often in young adults,
Abstract
Purpose Rhabdomyosarcoma (RMS) is a rare and aggres-
sive soft tissue sarcoma with limited treatment options and
a high failure rate during standard therapy. New therapeu-
tic strategies based on targeted immunotherapy are there-
fore much in demand. The epidermal growth factor recep-
tor (EGFR) has all the characteristics of an ideal target. It
is overexpressed in up to 80 % of embryonal RMS and up
to 50 % of alveolar RMS tumors. We therefore tested the
activity of the EGFR-specific recombinant immunotoxin
(IT) 425(scFv)-ETA′ against EGFR+ RMS cells in vitro
and ex vivo.
Methods We tested the specific binding and internaliza-
tion behavior of 425(scFv)-ETA′ in RMS cell lines in vitro
by flow cytometry, compared to the corresponding imag-
ing probe 425(scFv)-SNAP monitored by live cell imaging.
The cytotoxic activity of 425(scFv)-ETA′ was tested using
cell viability and apoptosis assays. Specific binding of the
Judith Niesen and Hannes Brehm shared first authorship.
J. Niesen · C. Stein · R. Fischer · S. Barth (*)
Fraunhofer Institute for Molecular Biology and Applied Ecology
IME, 52074 Aachen, Germany
e-mail: stefan.barth@ime.fraunhofer.de
H. Brehm · C. Stein · N. Berges · A. Pardo · S. Barth
Department of Experimental Medicine and Immunotherapy,
Institute of Applied Medical Engineering, University Hospital
RWTH Aachen, Aachen, Germany
R. Fischer
Institute of Molecular Biotechnology (Biology VII), RWTH
Aachen University, Aachen, Germany
A. ten Haaf · S. Gattenlöhner · M. K. Tur
Department of Pathology, Justus-Liebig University, Giessen,
Germany
J Cancer Res Clin Oncol
1 3
involves a recurrent chromosomal translocation t(2;13)
(q35;q14) generating the strongly expressed oncogene
PAX3-FKHR. Less frequent translocations include PAX7-
FHKR, PAX3-NCOA1 and PAX3-NCOA2 (Sumegi et al.
2010; Zanola et al. 2012). PRMS is rare, adult-specific and
has a complex mutation pattern (Jain et al. 2010). RMS is
still diagnosed and classified using histological methods,
but several studies have shown that gene expression pro-
files should also be considered for risk stratification and
treatment (Missiaglia et al. 2012; Williamson et al. 2010).
Despite recent improvements that have increased the 5-year
overall survival rate from 20 to 70 %, the clinical outcome
for adolescent ARMS patients and patients with relapsed
and metastatic RMS is still poor, with 20–30 % survival
(Ray and Huh 2012; Simon-Keller et al. 2013). Typical
treatment involves multi-agent chemotherapy, which may
be combined with radiotherapy and resection of the tumor
depending on the condition of the patient and site of the
tumor (Hawkins et al. 2014). Therefore, more effective and
targeted treatment options would be beneficial especially
for high-risk patients.
An ideal immunotherapeutic target, such as the epider-
mal growth factor receptor (EGFR), combines overexpres-
sion at the tumor site with little or no expression in healthy
tissue (Azemar et al. 2000; Bruell et al. 2003). The EGFR
or ErbB1 is a member of the ErbB family, which includes
HER-2/ErbB2, HER-3/ErbB3 and HER-4/ErbB4 (Koefoed
et al. 2011). EGFR is one of the best-defined target anti-
gens for cancer treatment, and therefore, a variety of thera-
peutic approaches and drug-development platforms are
based on this receptor (Tebbutt et al. 2013). EGFR signal-
ing is activated by the binding of ligands such as epidermal
growth factor (EGF) and transforming growth factor alpha
(TGFα). These regulate cell growth, proliferation, differ-
entiation and survival. The EGFR is a receptor tyrosine
kinase comprising an extracellular domain, a hydrophobic
transmembrane domain and an intracellular domain. The
latter is phosphorylated after ligand binding and receptor
dimerization, activating the downstream signaling cascade
(Yewale et al. 2013). EGFR signaling is often hyperac-
tive in tumor cells due to mutations that increase ligand
or receptor expression, confer constitutive receptor activ-
ity or allow cross-talk with other receptors. This causes
uncontrolled proliferation and is associated with resistance
to chemotherapy, poor prognosis and a greater likelihood
of metastasis (Bruell et al. 2003; Koefoed et al. 2011; Ped-
ersen et al. 2010). Like many other tumors, RMS is char-
acterized by the overexpression of EGFR, HER-2 and/or
HER-3 (De Giovanni et al. 1996; Ricci et al. 2002), and
studies have shown that EGFR is expressed and active in
31–76 % of ERMS and 16–50 % of ARMS tumors (Armi-
stead et al. 2007; Cen et al. 2007; Ganti et al. 2006; Wachtel
et al. 2006). Because EGFR is a tumor-associated surface
antigen that is rarely expressed in normal tissues, it repre-
sents an ideal target for RMS immunotherapy (Mendelsohn
2002; Yewale et al. 2013).
Targeted cancer therapeutics, such as antibody-based
immunotoxins (ITs), are representing an important field of
increasing interest, comprise a targeting component such as
a full-size monoclonal antibody (mAb) or fragment thereof,
and a cytotoxic agent based on small-molecule drugs, radi-
oisotopes or toxins derived from fungi, plants or bacteria
(Wayne et al. 2014; Weidle et al. 2014). Although mAbs
are specific, their size (~150 kDa) can limit tumor penetra-
tion; therefore, single-chain fragment variable (scFv) deriv-
atives (~30 kDa) comprising only the VH − VL sequences
of the parent mAb are more versatile (Ahmad et al. 2012;
Bruell et al. 2005). Recombinant ITs are fusion proteins
that usually combine scFvs with truncated toxins (Bruell
et al. 2003; Singh et al. 2007).
Here, we used a truncated version of Pseudomonas aer-
uginosa exotoxin A (ETA′) which has been used success-
fully as part of ITs against hematologic and solid tumors
(Becker and Benhar 2012). The natural cell-binding domain
of ETA is replaced with a ligand or scFv that binds to a
tumor surface antigen, so that the toxin is internalized and
can kill the cell (Antignani and Fitzgerald 2013). We used
the EGFR-specific IT 425(scFv)-ETA′, which has shown
cytotoxicity against human pancreatic cancer cells in vitro
and in vivo in a xenograft tumor model (Bruell et al. 2003,
2005; Pardo et al. 2012). To our knowledge, only Ricci
et al. (2002) have reported the targeting of EGFR+ RMS
cells using an IT, although this was an indirect approach
in which a primary specific murine mAb binding to EGFR
and a secondary F(ab′)2 anti-mouse Ig was linked to
saporin-S6 via a disulfide bond. Treatment with this indi-
rect IT induced apoptosis and inhibited protein synthesis in
the RMS cell line RD, with an IC50 of 950 pM (Ricci et al.
2002).
We compared the 425(scFv)-ETA′ IT with the corre-
sponding imaging probe 425(scFv)-SNAP which was cre-
ated using SNAP-tag technology. The SNAP-tag derived
from the human DNA-repair enzyme alkylguanine-DNA
alkyltransferase (hAGT) binds specifically, rapidly and
covalently to substrates that contain O(6)-benzylguanine
(BG). The self-labeling reaction involves the irreversible
transfer of an alkyl group to a cysteine residue. The advan-
tage of the SNAP-tag technology is the quantitative and
specific labeling of the antibody fragments without affect-
ing their antigen-binding properties. Any scFv can thus be
combined with a variety of BG-labeled fluorophores using
this simple and rapid covalent reaction (Amoury et al.
2013; Hussain et al. 2013; Kampmeier et al. 2009).
Here, we show for the first time the successful and prom-
ising in vitro and ex vivo effect of a recombinant EGFR-
specific IT and the corresponding preclinical imaging
J Cancer Res Clin Oncol
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probe against RMS cell lines and RMS tumor samples from
patients.
Materials and methods
Bacterial strains and plasmids
The expression plasmids were prepared using E. coli Dh5α
(F− endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG
Φ80dlacZΔM15 Δ(lacZYA-argF)U169, hsdR17(rK
− mK
+),
λ–). The molecular cloning steps were carried out as pre-
viously described (Green and Sambrook 2012). The IT
425(scFv)-ETA′ was generated previously (Bruell et al.
2003, 2005) and was assembled in the vector pMT, derived
from pET27b (Novagen, Wisconsin, USA) for bacte-
rial expression (Bruell et al. 2003; Tur et al. 2003). The
425(scFv)-SNAP construct was cloned in pMS, a modi-
fied version of pSecTag2/HygroB vector for mammalian
expression (Stocker et al. 2003).
Cell lines and culture conditions
Cell lines were obtained from the American Type Culture
Collection (RMS: TE-671, RD; human embryonic kidney
cells: HEK293T), German Collection of Microorganisms
and Cell Cultures (human histiocytic lymphoma: U937) or
were kindly provided by Ewa Koscielniak (Olgahospital,
Stuttgart, Germany; RMS: FL-OH1). The cell lines were
cultured in RPMI 1640 (Gibco Invitrogen, Carlsbad, USA)
supplemented with 10 % heat-inactivated fetal bovine
serum (Biochrom AG, Berlin, Germany), 2 mM l-glu-
tamine, 100 U/ml penicillin and 0.1 mg/ml streptomycin
(Invitrogen, San Diego, USA).
HEK293T cells were transfected with Roti®-Fect
according to the manufacturer’s protocol (Carl Roth
GmbH, Karlsruhe, Germany). Transfected HEK293T cells
were cultivated in RPMI 1640 medium as described above,
supplemented with 100 µg/ml Zeocin™ (Invitrogen, San
Diego, USA). All cell lines were cultivated at 37 °C in a
humidified environment at 5 % CO2 in a 95 % air incubator
using the recommended media.
Production and purification of the IT and the SNAP-tag
imaging probe
The IT was cloned into a pMT vector as described above.
It was expressed under stress conditions in E. coli BL21
(DE3) cells (F– ompT gal dcm lon hsdSB(rB
− mB
−) λ(DE3
[lacI lacUV5-T7 gene 1 ind1 sam7 nin5])) purchased from
Novagen (Wisconsin, USA) as described elsewhere (Barth
2002; Barth et al. 2000).
The scFv-SNAP construct was cloned in the pMS vector
and expressed as described earlier (Kampmeier et al. 2009;
Stocker et al. 2003). Both proteins were purified using
IMAC as previously described (Hristodorov et al. 2013).
The protein samples were separated by SDS-PAGE (12 %
acrylamide) and detected by staining with Coomassie bril-
liant blue. The separated IT was transferred to a nitrocellu-
lose membrane and specifically detected using the in-house
produced murine ETA′-specific mAb TC-1 and an alkaline
phosphatase (AP)-labeled mouse-specific mAb (Green and
Sambrook 2012; Laemmli 1970). The SNAP-tagged pro-
tein was labeled with a fluorophore prior to SDS-PAGE
(Kampmeier et al. 2009, 2010), and the protein was visual-
ized using the CRi Maestro imaging system (Perkin Elmer,
Waltham, MA, USA) and the appropriate filter sets. Protein
concentrations were determined by densitometry after gel
staining compared to bovine serum albumin (BSA) stand-
ards using AIDA Image Analyzer Software (v.4.27.039).
Flow cytometry and internalization assays
Specific cell binding was evaluated by flow cytometry. We
incubated 4 × 105 cells with 1 µg of 425(scFv)-ETA′ in
100 µl PBS for 30 min on ice. The cells were then washed
and incubated for further 30 min with TC-1, diluted 1:100
in 100 µl PBS. As a positive control, we used a commer-
cially available human EGFR-specific rabbit mAb (Sino
Biological Inc., Beijing, China) diluted 1:10 in 100 µl PBS.
The IT was detected by incubating cells for 30 min on ice
with a FITC-labeled goat anti-mouse mAb (Dianova, Ham-
burg, Germany) diluted 1:100 in 50 µl PBS. For detection
of the control, antibody cells were incubated with a FITC-
labeled goat-anti-rabbit polyclonal antibody diluted 1:50
in 50 µl PBS (Sigma-Aldrich, Taufkirchen, Germany).
The cells were analyzed using a BD FACSVerse (Becton–
Dickinson, Heidelberg, Germany) and the corresponding
software.
The rate of IT internalization was measured as described
by Cizeau et al. (2009). We incubated 2 × 105 cells/ml
with 500 nM 425(scFv)-ETA′ for 30 min at 4 °C. After
washing, the cells were incubated for different periods of
time (15, 30, 60, 120 and 240 min) in cell culture medium
under normal culture conditions. We used the TC-1 mAb,
as described above, and a PE-labeled goat anti-mouse mAb
diluted 1:100 in 50 µl PBS for detection. The results are
presented as a percentage of the mean fluorescence inten-
sity. The mean fluorescence intensity of the non-internaliz-
ing 4 °C control was set to 100 %. The number of expressed
cell surface antigens was determined using the Qifikit® kit
(Dako, Hamburg, Germany). Flow cytometry was carried
out according to the manufacturer’s protocol using the anti-
human EGFR mAb.
J Cancer Res Clin Oncol
1 3
Confocal microscopy
Internalization of the 425(scFv)-SNAP probe was visual-
ized by confocal microscopy. The 425(scFv)-SNAP con-
struct was coupled to BG-SNAP-Surface® Alexa Fluor®-
488 (New England BioLabs, Schwalbach, Germany) as
described elsewhere (Kampmeier et al. 2009, 2010). We
added 100 nM of the labeled protein to 2 × 105 cells/ml
seeded 24 h earlier in 8-well chamber slides (Thermo Sci-
entific, Waltham, MA, USA). The cells were incubated at
37 °C for 120 min. After a washing step in PBS (only for
the RD cell line), the nucleus was counterstained with bis-
benzimide Hoechst 33342 (Sigma-Aldrich, Taufkirchen,
Germany) diluted 1:1,000 in PBS. Internalization was ana-
lyzed using a LEICA TCS SP8 confocal microscope and
the corresponding software (Leica Microsystems GmbH,
Wetzlar, Germany).
Colorimetric XTT cell proliferation assay
The cytotoxic activity of 425(scFv)-ETA′ against three
different RMS cell lines (RD, FL-OH1 and TE-671) was
determined using a cell proliferation assay based on XTT
as previously described (Schiffer et al. 2013). Briefly,
5 × 103 cells were plated in 96-well plates and incubated
with different concentrations of the IT for 72 h under nor-
mal culture conditions as described above. Untreated cells,
the non-toxic 425(scFv)-SNAP and a non-binding Mock-
ETAʹ construct were used as controls. After 72 h, we added
50 µl XTT/phenazine methosulfate (Serva and Sigma,
Steinheim, Germany) to the cells, and the samples were
incubated for additional 2–4 h under the same conditions.
The absorbance was measured at 450 and 630 nm as a ref-
erence wavelength using an Epoch Microplate Spectropho-
tometer (Biotek, Bad Friedrichshall, Germany). All experi-
ments were carried out in duplicates or triplicates.
The required IT concentration to achieve a 50 % reduc-
tion in protein synthesis (IC50) relative to the untreated con-
trol cells was calculated using GraphPad Prism 5 software
(GraphPad Software, La Jolla, CA, USA).
AnnexinV/PI Apoptosis assay
The induction of apoptosis was measured by AnnexinV/
PI staining (Stahnke et al. 2008). We incubated 1 × 105
RD cells/well with 30 nM 425(scFv)-ETA′ in a 24-well
plate for 48 h under the conditions described above. An
equimolar amount of the non-binding ETA′-based IT
(Mock-ETA′) was used as a negative control. Cells were
also incubated with PBS and camptothecin as negative
and positive controls, respectively. The EGFR– cell line
U937 was used as control cell line. After incubation, the
cells were harvested (RD cells were detached by washing
in PBS followed by trypsin/EDTA treatment), washed
with 1 × AnnexinV binding buffer (15 mM NaCl, 1 mM
HEPES, 0.5 mM KCl, 0.2 mM CaCl2, pH 7.4) and stained
with 450 µl cell culture supernatant from AnnexinV-
EGFP-expressing HEK293T cells and 50 µl 10 × Annex-
inV binding buffer for 10 min at room temperature. The
cells were re-suspended in 1 × AnnexinV binding buffer
containing 1 µg/ml propidium iodide (PI) and analyzed by
flow cytometry using BD FACSVerse (Becton–Dickinson,
Heidelberg, Germany).
Ex vivo binding to human tumor tissue
Formalin-fixed and paraffin-embedded (FFPE) tis-
sue sections from RMS tumor samples representing one
ARMS patients and one ERMS patient were equilibrated
in xylene (2 × 5 min), 100 % isopropanol (2 × 5 min),
96 % isopropanol (1 × 5 min) and 70 % isopropanol
(1 × 5 min) and finally washed in PBS for 5 min. After-
ward, the slides were heated in a 600 W microwave for
5 min in citrate buffer (10 mM sodium citrate, 0.05 %
Tween 20, pH 6.0) and allowed to cool for 30 min at
room temperature before washing in PBS. The slides
were incubated for 1 h at room temperature in blocking
solution (PBS + 1 % (v/v) goat serum), washed once in
PBS and incubated for 24 h at 4 °C with either 1 µg of
recombinant 425(scFv)-ETA′ or an EGFR-specific mAb
(cetuximab/Erbitux®, Merck). Slides previously incu-
bated with the IT were also incubated for further 24 h at
4 °C with TC-1 (diluted 1:40 in PBS + 1 % (v/v) goat
serum) followed by a washing step. The secondary anti-
bodies, either an AP-labeled goat anti-mouse mAb, called
GAMAP, (Southern Biotech, Germany) for the detection
of the IT or an AP-labeled goat-anti-human mAb (Sigma-
Aldrich, Taufkirchen, Germany) for the detection of the
EGFR-specific mAb (Cetuximab/Erbitux®, Merck, Darm-
stadt, Germany), both diluted 1:50 in PBS + 1 % (v/v)
goat serum, were added to the slides, which were then
incubated for 24 h at 4 °C and washed twice in PBS and
once in 0.1 M Tris–HCl (pH 8.5). To generate a red stain-
ing, the AP activity was detected using naphthol AS-BI
phosphate (sodium salt, 50 mg/100 ml; Sigma-Aldrich,
Taufkirchen, Germany) as substrate and new fuchsin
(10 mg/100 ml; Sigma-Aldrich, Taufkirchen, Germany)
as chromogen dissolved in 0.1 M Tris–HCl (pH 8.5).
Endogenous AP activity was inhibited by adding 0.35 mg/
ml levamisole (Sigma-Aldrich, Taufkirchen, Germany)
to the reaction mixture. Finally, we counterstained the
slides with ready-to-use hematoxylin and eosin solutions
according to the manufacturer’s protocol (Sigma-Aldrich,
Taufkirchen, Germany). The slides were analyzed under
a Leica DMR-HC light microscope (Leica, Wetzlar, Ger-
many) using the corresponding Leica QWin software.
J Cancer Res Clin Oncol
1 3
Primary tissue samples were obtained during routine clin-
ical practice at the University Hospital Giessen, in accord-
ance with the principles of the Declaration of Helsinki.
Results
Expression and purification of 425(scFv)-ETA′
The IT 425(scFv)-ETA′ was expressed in E. coli BL-21
(DE3) under osmotic stress in the presence of compatible
solutes (Barth et al. 2000). The imaging probe 425(scFv)-
SNAP was expressed in HEK293T cells. Both proteins
were purified by Ni–NTA affinity chromatography using
the His-tag. The proteins were separated by SDS-PAGE
and detected by immunoblotting using a specific mAb
for the IT (Fig. 1, lane 2) or by coupling the fluorophore
SNAP-Surface® Alexa Fluor®-488 to 425(scFv)-SNAP and
visualizing the conjugate using the Maestro CRi-Imaging
system (Fig. 1, lane 4). Both proteins showed the expected
electrophoretic mobility, corresponding to ~70 kDa for
425(scFv)-ETA′ and ~48 kDa for 425(scFv)-SNAP (Fig. 1,
lanes 1 and 3). The yield of 425(scFv)-ETA′ was ~1.5 mg
per liter of bacterial culture, and the yield of 425(scFv)-
SNAP was ~21 mg per liter of cell culture supernatant.
Binding of 425(scFv)-ETA′ to RMS cell lines
The binding of 425(scFv)-ETA′ to RMS cell lines was eval-
uated by flow cytometry. The purified IT 425(scFv)-ETA′
bound to all three RMS cell lines but not to the control cell
line U937 (Fig. 2). An EGFR-specific mAb was used as a
positive control and showed specific binding to all EGFR+
RMS cell lines but not to the EGFR− cell line U937
(Fig. 2). The amount of EGFR was measured using the
Qifikit® kit. The expression profile was similar for all three
RMS cell lines with ~9,413–13,741 receptors expressed on
the surface (Table 1).
Internalization of the 425(scFv) constructs by RMS cell
lines
ITs must be internalized rapidly for greatest efficacy, so we
investigated the internalization behavior of both 425(scFv)
constructs by flow cytometry for the IT (Cizeau et al. 2009)
and live cell imaging for the SNAP-tag construct. Flow
cytometry showed that ~50 % of 425(scFv)-ETA′ was
internalized within 20 min by RD and FL-OH1 cells and
within 40 min by TE-671 cells. After ~240 min, nearly all
of the IT was internalized by all three RMS cell lines. The
4 °C non-internalization control was set as 100 % in the
evaluation (Fig. 3a).
For the corresponding SNAP-Tag construct, internali-
zation behavior was visualized by confocal microscopy
using SNAP-surface® Alexa Fluor®-488 coupled to the
SNAP-tagged 425(scFv) protein. The fluorophore-labeled
425(scFv)-SNAP construct was efficiently internalized
within 120 min by the RMS cell line RD, resulting in the
intracellular accumulation of the fluorescence signal at
37 °C but not at 4 °C (Fig. 3b). The EGFR− cell line U937
is demonstrated in Fig. 3c. No signal could be detected after
incubation of the 425(scFv)-SNAP construct; the counter-
staining of the nucleus was done as for the RD cell line.
The viability of RMS cells is reduced by 425(scFv)-ETA′
The ability of 425(scFv)-ETA′ to inhibit the growth of
EGFR+ cell lines in vitro has been demonstrated with other
tumor cell lines (Bruell et al. 2003, 2005). Here, we used an
XTT assay to confirm that 425(scFv)-ETA′ also inhibited
the growth of all three RMS cell lines in a dose-dependent
manner. The IT was added in decreasing concentrations to
the target cells, and the proliferation was measured in com-
parison with untreated cells after 72 h of incubation. As
shown in Fig. 4, 425(scFv)-ETA′ showed specific toxicity
toward the EGFR+ RMS cell lines but not to the EGFR−
cell line U937. The non-binding ETA′-construct (Mock-
ETA′) inhibited the cell proliferation only at a 100x higher
concentration as used for the 425(scFv)-ETA′ construct.
The 425(scFv)-SNAP imaging probe does not inhibit the
proliferation of the RMS cell line RD, demonstrated exem-
plarily. The IC50 values were in the picomolar range and
are summarized in Table 1.
Fig. 1 The enrichment of the IT 425(scFv)-ETA′ was determined by
sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-
PAGE) followed by Coomassie brilliant blue staining (lane 1). West-
ern blot analysis of 425(scFv)-ETA′ was carried out to verify protein
identity (lane 2) using a mouse anti-ETA′ mAb (TC-1) and a goat
anti-mouse AP-labeled secondary mAb. A pre-stained protein marker
was used. Lanes 3 and 4 show the SDS-PAGE of the 425(scFv)-
SNAP construct. Approximately 500 ng of SNAP-Surface® Alexa
Fluor®-488 labeled scFv-SNAP-tag fusion protein was separated
by SDS-PAGE, visualized by a CRi Maestro Imaging System using
Maestro software and the blue filter set (500–720 nm) to prove the
functionality of the SNAP-tag (lane 4) and stained with Coomassie
brilliant blue (lane 3). Expected size of 425(scFv)-SNAP is ~48 kDa
and of 425(scFv)-ETA′ ~70 kDa
J Cancer Res Clin Oncol
1 3
The apoptotic impact of 425(scFv)-ETA′ in one selected
RMS cell line
An AnnexinV/PI assay was used to determine whether the
observed inhibitory effect of 425(scFv)-ETA′ reflects its
pro-apoptotic activity (Fig. 5). The concentration of 30 nM
IT and the incubation time of 48 h were chosen to capture
the RMS cell line RD within the early- and late-apoptotic
stages. Both populations (early apoptosis in the lower right
corner and late apoptosis in the upper right corner) increased
Fig. 2 Specific cell-binding
activity of the recombinant IT
425(scFv)-ETA′ and a com-
mercially available rabbit anti-
EGFR monoclonal antibody
(mAb-EGFR) was analyzed by
flow cytometry, using the TC-1
anti-ETA′ antibody and a FITC-
labeled goat anti-mouse second-
ary mAb for the detection of
the IT and a FITC-labeled goat
anti-rabbit Ab for the mAb-
EGFR (FL-1 fluorescence
channel/FITC). The solid curve
illustrates the binding of mAb-
EGFR, and the dashed curve
represents the binding of the IT
with a twice as high concentra-
tion. Both curves are shown
against the background control
(filled gray curve). U937 was
used as EGFR− cell line
Table 1 The EGFR expression level and the IC50 values of 425(scFv)-ETA′ on different RMS cell lines
The IC50 values indicate the concentrations of 425(scFv)-ETA′ required to achieve the reduction of protein synthesis by 50 % in comparison
with the untreated control. The values are derived from the colorimetric XTT assays shown in Fig. 4. The EGFR expression on the RMS cell
lines was determined using the Qifikit® kit. The data represent three independent experiments and are presented as mean ± SD. An unspecific
cytotoxic effect was measured by Mock-ETA′ on all cell lines tested 100 times higher protein concentrations. For 425(scFv)-SNAP no cytotoxic
effect was determined on the cell line RD. Its unspecific cytotoxicity was not determined (n.d.) on other cell lines
Cell line RD FL-OH1 TE-671 U937
EGF-R expression 10,410 (±2,655 SD) 9,413 (±2,626 SD) 13,741 (±4,377 SD) –
IC50 [nM]
425(scFv)-ETA′
0.63 0.05 0.68 –
IC50 [nM]
Mock-ETA′
279.9 16.99 60.13 No effect
425(scFv)-SNAP No effect n.d. n.d. n.d.
J Cancer Res Clin Oncol
1 3
significantly compared to the controls, i.e., the buffer only
and Mock-ETA′ samples (Fig. 5b). Mock-ETA′ did not
induce apoptosis compared to the buffer control, whereas
the positive control camptothecin induced apoptosis in both
the EGFR+ cell line RD and the EGFR− cell line U937. The
ITs did not induce apoptosis in the control cell line U937.
Fig. 3 Internalization behavior of 425(scFv)-ETA′ and the cor-
responding 425(scFv)-SNAP into RMS cell lines. In A the three
RMS cell lines RD, FL-OH1 and TE-671 were incubated with 500
nM of the IT 425(scFv)-ETA′ for 30 min on ice, followed by incu-
bations steps at 37 °C for different points of time (15, 30, 60, 120
and 240 min). One control tube was incubated constantly at 4 °C.
For detection, the murine ETA′-specific mAb TC-1 and a PE-labeled
goat anti-mouse mAb were used. For evaluation, the 4 °C control
was set as 100 %. Datasets were normalized using GraphPad Prism
5 software (GraphPad Software). Additionally the internalization of
425(scFv)-SNAP was visualized via live cell imaging using a confo-
cal microscope shown in (b) and (c). In (a), RD cells were incubated
with 100 nM of 425(scFv)-SNAP coupled to SNAP-Surface® Alexa
Fluor®-488. Internalization was monitored after 120 min at 37 °C.
The non-internalization control was incubated at 4 °C. In Fig. 3 C,
the control cell line U937 is demonstrated after incubation with the
425(scFv)-SNAP coupled to SNAP-Surface® Alexa Fluor®-488 at
37 °C as before. No internalization signal could be measured (last
picture); the nucleus was counterstained as for the RD cells. The
first picture in C demonstrates the white light picture of the cell, the
second one the overlay with the counterstained nucleus, the third the
counterstained nucleus in the appropriated fluorescence channel and
the fourth one demonstrated the cell in the fluorescence channel for
the SNAP-Surface® Alexa Fluor®-488-dye
J Cancer Res Clin Oncol
1 3
Ex vivo targeting of EGFR in primary material from RMS
patients
We also tested the binding of 425(scFv)-ETA′ to FFPE
primary material from RMS patients. Successful binding
was shown for the IT (Fig. 6c, g) and the EGFR-specific
positive control mAb Cetuximab/Erbitux® (Fig. 6i) to sec-
tions representing two different RMS patients. The sections
in Fig. 6a–d are classified as ARMS and those in Fig. 6e–i
as ERMS. Specific binding was confirmed by new fuchsin
and hematoxylin staining. No signal was detected in the
negative control stained only with the detection antibodies
(Fig. 6b, f). The specific binding of the IT and the mAb is
shown in J-L with a higher magnification (objective: 40×).
Discussion
The limited therapeutic options available for RMS consist
of multi-agent chemotherapy, radiotherapy and surgical
resection if appropriate given the tumor site and condi-
tion of the patient (Bisogno et al. 2012; Weiss et al. 2014).
However, the treatment failure rate is high, and new thera-
peutic options are needed urgently especially for relapsed
or metastatic RMS. Immunotherapy using antibody-based
ITs is a promising approach now that third-generation ITs
based on scFv-toxin fusion proteins have demonstrated
success in clinical trials. These are made by recombinant
DNA techniques, combining the scFv of a mAb and tox-
ins lacking their cell-binding domain (Becker and Benhar
2012). Pseudomonas exotoxin A is often used as a com-
ponent of ITs (Weldon and Pastan 2011) because it can be
produced rapidly and efficiently in E. coli, and mutations
that improve its stability and reduce its immunogenicity do
not affect its cytotoxicity (Allen 2002; Becker and Benhar
2012). ITs incorporating scFv-based targeting components
are less immunogenic than full-length antibodies because
most human anti-mouse mAbs are directed against the Fc
domain of used therapeutic mAbs. In addition, the lack of
an Fc domain prevents the binding of the IT to Fc-receptor-
expressing cells. Therefore, it is not rapidly cleared from
the bloodstream or causes undesired cytotoxic effects.
(Thorpe et al. 2003). Furthermore, the smaller size of scFv-
based ITs (~70 kDa) allow more efficient tumor penetra-
tion, which is beneficial for the treatment of solid tumors
such as RMS (Colcher et al. 1998; Yokota et al. 1992).
ITs can also be combined with conventional chemo-
therapy, immunosuppression or radioimmunoconjugates. A
combination of different agents can limit the immunogenic-
ity of each component and increase the overall cytotoxic-
ity (Singh et al. 2012; Wayne et al. 2014). For example,
BL22 is an IT comprising a scFv directed against CD22
Fig. 4 The in vitro cellular cytotoxicity of recombinant 425(scFv)-
ETA′ to RMS cell lines is shown. The three RMS cell lines RD,
FL-OH1 and TE-671 were incubated with serial dilutions of sterile
425(scFv)-ETA′ in RPMI medium. As control, the cells were incu-
bated with serial dilutions of a non-specific Mock-ETA′ as well as
425(scFv)-SNAP as a scFv-control without any toxic compound
(425(scFv)-SNAP was shown exemplarily on RD cells). The cyto-
toxicity assays were performed in duplicates/triplicates at least two
times. Nonlinear regression was performed using GraphPad Prism
5 software (GraphPad Software). The error bars represent standard
error of means. No unspecific effects of the IT or the Mock-ETA′
could be demonstrated by using the control cell line U937
J Cancer Res Clin Oncol
1 3
and a version of Pseudomonas exotoxin A known as PE38.
This has been combined with the macrolide lactone bry-
ostatin 1 for the treatment of low-grade and high-grade B
cell lymphoma, with a more potent effect than each agent
alone (Biberacher et al. 2012). Wei et al. (2000) demon-
strated that pre-treatment with a radioimmunoconjugate
improved the effect of an IT in vivo against human B-cell
lymphoma. ITs combined with radioimmunoconjugates are
particularly useful for advanced metastatic cancer and large
tumors because ITs kill individual tumor cells following
internalization, whereas radioimmunoconjugates can kill
also surrounding tumor cells and can penetrate several cell
diameters thus helping to eliminate minimal residual dis-
ease (Ghetie et al. 1994; Wei et al. 2000).
Until recently, RMS has rarely been targeted using
ITs. We lately show the successful killing by an αMCSP-
ETA′ IT which specifically recognizes CSPG4 on RMS
cell lines (Brehm et al. 2014). Gattenlohner et al. (2010)
Fig. 5 Apoptotic effects of 425(scFv)-ETA′ on one selected RMS
cell line are shown. RD and U937 (control cell line, EGFR−) cells
were incubated with 30 nM 425(scFv)-ETA′ for 48 h in three inde-
pendent experiments. Dot plots of RD and U937 cells demonstrate
the population of early- and late-apoptotic cells (lower and upper
right corner). The bar chart in B illustrates the sum of early-apop-
totic and late-apoptotic cells. Error bars represent the standard devia-
tion. Differences in cell death relative to the untreated control reach-
ing statistical significance (***p ≤ 0.001) are indicated by asterisks.
camptothecin was used as an apoptosis inducing positive control
J Cancer Res Clin Oncol
1 3
achieved promising results using a recombinant scFv-ETA′
IT (scFv35-ETA′) targeting the fetal acetylcholine receptor,
and Ricci et al. (2002) have targeted RMS indirectly via
EGFR, but to our knowledge, there are no previous reports
of EGFR-specific recombinant single polypeptide chain
ITs for RMS. Ricci et al. (2002) showed that an indirect IT
approach (the application of a murine mAb recognizing the
EGFR followed by a secondary F(ab′)2 anti-mouse immu-
noglobulin chemically linked via an artificial disulfide bond
to saporin-S6) can inhibit the growth of RMS cells with an
IC50 of ~950 pM. The IC50 ranges we achieved with our
direct IT approach are better (50–680 pM depending on the
cell line, Table 1) and the single recombinant polypeptide
would be safer and more practicable in the clinic. Another
approach is the conjugation of saporin to cetuximab using
streptavidin, which can be enhanced by photochemical
internalization (Yip et al. 2007). Likewise, a combination
treatment with cetuximab and the cytotoxic agent actino-
mycin D shows antitumor activity in different RMS cell
lines (Yamamoto et al. 2013). There are different EGFR-
specific ITs published, but not specifically for RMS (Wels
et al. 2004; Chandramohan and Bigner 2013).
The purpose of this study was to evaluate the EGFR-
targeting IT 425(scFv)-ETA′ (Bruell et al. 2003, 2005;
Pardo et al. 2012) for its effect against RMS cells in vitro
and ex vivo. The cell-binding domain of ETA was removed
to generate the truncated version ETA′, which was com-
bined with an EGFR-specific scFv to allow the direct tar-
geting of EGFR+ tumor cells and cell killing by receptor-
mediated internalization (Pastan et al. 2007; Weldon and
Pastan 2011). We confirmed specific binding of the IT to
EGFR+ RMS cells, as expected because the homodimeric
425(scFv) has the same binding characteristics as the
parental mAb as measured using a BIAcore instrument
(Muller et al. 1998). The 425 mAb is presumed to bind to
the external domain of the human EGFR, recognizing an
epitope containing amino acid residues G460/S461 which
is close to the EGF-binding site (Kamat et al. 2008; Murthy
Fig. 6 Immunohistochemical staining of primary tumor tissue with
the IT 425(scFv)-ETA′ and an EGFR-specific mAb is demonstrated.
Tumor biopsies were taken from two different RMS patients. One
patient (a–d) was classified as ARMS and the other (e–i) as ERMS.
h, e Staining of tumor specimens demonstrating the presence of
tumor cells in the biopsies of both patients (a, e). b, f Demonstrate
a representative micrograph for the negative control of new fuch-
sin staining with the detection antibodies only (TC-1/GAMAP). c, g
Represent the micrograph of tumor section stained with new fuchsin
substrate using the 425(scFv)-ETA′ IT detected with TC-1/GAMAP.
d, h Demonstrate the processed samples (background subtracted),
examples of red-stained EGFR+ clusters of tumor cells are indi-
cated by arrows. The positive binding of the EGFR-specific anti-
body cetuximab (Erbitux®) is shown for one selected micrograph in
(i) (size 100 µm, objective: ×10). The micrographs j–l were taken
with a higher magnification (size 50 µm, objective: ×40). [j ARMS
425(scFv)-ETA′, k ERMS 425(scFv)-ETA′ and l ERMS EGFR-spe-
cific antibody cetuximab (Erbitux®)]
J Cancer Res Clin Oncol
1 3
et al. 1987). The binding and subsequent internalization of
ITs is necessary for cytotoxic activity. We confirmed the
specific binding and internalization of 425(scFv)-ETA′ in
three different RMS cell lines, with 50 % of the IT inter-
nalized within 20–40 min depending on the cell line. Spe-
cific internalization was confirmed for the cell line RD
after 120 min, using the corresponding imaging probe
425(scFv)-SNAP labeled with SNAP-Surface® Alexa
Fluor®-488. These internalization results support the data
reported for other scFv-based ITs (Cizeau et al. 2009).
ITs are promising anti-cancer therapeutics because only a
few molecules need to be taken up by cancer cells in order
to kill the cell, because the toxin acts in a catalytic man-
ner (Allen 2002). We tested the cytotoxicity of 425(scFv)-
ETA′ against RMS cells and calculated IC50 values in the
picomolar range, which is 2.5-fold better for the cell lines
FL-OH1 and RD than the values reported for scFv35-ETA′
(Gattenlohner et al. 2010). This reflects the two- to threefold
higher level of EGFR compared to the fetal acetylcholine
receptor, the target antigen of scFv35-ETA′. Bachran et al.
(2010) showed a correlation between EGFR expression and
the sensitivity of cervical carcinoma cells toward EGFR-
specific immunotoxins. Our data likewise show that RD and
FL-OH1 cells express EGFR at two- to threefold the level of
the fetal acetylcholine receptor and have a two- to threefold
lower IC50 value for 425(scFv)-ETA′ compared to scFv35-
ETA′. The cell line TE-671 did not fit this profile because the
EGFR was expressed at threefold the level of the fetal ace-
tylcholine receptor, but the IC50 value was 50 times lower.
Gattenlohner et al. (2010) have shown with the ETA′-based
IT (scFv35-ETA′) also a higher IC50 on TE-671 compared
to RD and FL-OH1, which is comparable to our observa-
tion with an EGFR-specific ETA′-based IT. Furthermore,
we were able to demonstrate a reduction in cell viability due
to the induction of apoptosis, which is the common mecha-
nism of ETA′-based ITs (Kreitman 2006; Pastan et al. 1992;
Pastan and FitzGerald 1991). The effect of the Mock-ETA′ at
a about 100× higher concentration as used for the specific IT
was also shown by others (Gattenlohner et al. 2010).
Our immunohistochemistry results confirmed the spe-
cific binding of 425(scFv)-ETA′ to primary material from
human RMS patients. These findings correlate with other
studies dealing with EGFR expression on RMS primary
material (Ganti et al. 2006; Wachtel et al. 2006).
EGFR expression correlates with tumor treatment resist-
ance (Chong and Janne 2013). Different clinically validated
resistance mechanisms to EGFR-targeted mAbs or small-
molecule inhibitors are known in patients with various can-
cer types, e.g., lung adenocarcinoma. These mechanisms
could involve EGFR mutations that promote drug resist-
ance or the activation of bypass signaling pathways such as
KRAS (Chong and Janne 2013). Furthermore, many differ-
ent tumors are treated with EGFR-specific therapeutics, but
such approaches are often inefficient in monotherapy (Camp
et al. 2005; Sequist and Lynch 2008). For example, EGFR
promotes resistance to radiotherapy due to nuclear shut-
tling and activation of the DNA-repair enzyme DNA pro-
tein kinase (DNA-PK) in certain types of cancer (Dittmann
et al. 2010). Mutations of the intracellular kinase domain
can also reduce the efficacy of tyrosine kinase inhibitors
such as erlotinib and gefitinib (Yun et al. 2008). Ganti et al.
(2006) did not detect mutations of the EGFR in the tested
RMS biopsies, but erlotinib does not inhibit tumor growth
in a genetically engineered ARMS mouse model (Abraham
et al. 2011). The efficacy of ITs is independent of intracel-
lular tyrosine kinase mutations as long as the extracellular
binding region is unaffected (Ratti and Tomasello 2014).
Furthermore, the existence of cancer stem cells (CSCs) with
the capacity for self-renewal and tumor bulk progression
has been reported for RMS (Walter et al. 2011). Mazzo-
leni et al. (2010) proved that EGFR is required to maintain
the CSC phenotype in glioblastomas. If the EGFR is also
required for CSC maintenance in RMS, efficient CSC-tar-
geting therapies would be likely to achieve complete remis-
sion and reduce the likelihood of a relapse. Therefore, the
application of an IT in RMS therapy could be an interesting
and promising approach.
In summary, we are the first to demonstrate the in vitro
efficacy of a recombinant IT targeting the EGFR on dif-
ferent RMS cell lines. The IC50 values were much better
than those reported for an indirect IT approach (Ricci et al.
2002). In preparation for in vivo experiments, we have
shown that the IT can also bind to primary tumor mate-
rial. Although our data need further preclinical in vivo
confirmation, 425(scFv)-ETA′ appears to be a promising
candidate for clinical development and eventual deploy-
ment as an RMS treatment option to complement current
approaches.
Acknowledgments Christoph Stein was supported by the INTER-
REG IV A project Microbiomed. We would like to thank Rado-
slav Mladenov for his help with the tissue sections. We thank Dr.
Agnieszka Weinandy (University Hospital Aachen, Neurosurgery
Clinic, Aachen, Germany) for providing cetuximab, and we also thank
Dr. Richard M. Twyman for the critical reading of the manuscript.
Conflict of interest None.
Ethical standard Primary tissue samples were obtained during rou-
tine clinical practice at the University Hospital Giessen approved by
the appropriate ethics committee, in accordance with the principles
and the ethical standards of the Declaration of Helsinki.
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