In vitro phage display in a rat beta cell line: a simple approach for the generation of a single-chain antibody targeting a novel beta cell-specific epitope.

In vitro phage display in a rat beta cell line: a simple approach
for the generation of a single-chain antibody targeting
a novel beta cell-specific epitope
S. Ueberberg & D. Ziegler & W. Schechinger &
J. W. Dietrich & S. Akinturk & H. H. Klein & S. Schneider
Received: 6 July 2009 /Accepted: 15 February 2010
# Springer-Verlag 2010
Abstract
Aims/hypothesis The aim of the present study was to
evaluate in vitro phage display in a beta cell line as a
novel strategy for the isolation of beta cell-specific agents/
biomarkers.
Methods A single-chain antibody (SCA) library was pre-
incubated with AR42J cells in order to eliminate SCAs with
exocrine binding properties. It was then panned against
INS-1 cells to select beta cell-targeted antibodies.
Results By these means, we isolated a novel antibody, SCA
B5, that binds rapidly (6.0 min) and with a 450-fold higher
specificity to beta cells relative to exocrine cells. We
estimated for SCA B5 a binding affinity in the low μmol/l
range and 858 binding sites per beta cell. Confocal microscopy
showed binding to the beta cell surface and confirmed
subsequent internalisation. Moreover, staining of rat and
human pancreatic tissue sections with SCA B5 suggests that
the target epitope is presented in pancreatic beta cells of
different origins. Infrared imaging revealed that labelling of
beta cells with tracer SCA B5 is strictly dependent on beta
cell mass. With competition assays we excluded insulin,
glutamate decarboxylase, C-peptide and islet amyloid
polypeptide as SCA B5 targets. In accordance with these
predictions, SCA B5 homed in vivo highly selectively to
normal beta cells and dysfunctional beta cells of diabetic rats.
Moreover, accumulation of radioactively labelled SCA B5 in
the pancreas was reduced by 80% after pre-injection with
unlabelled SCA B5, thereby confirming the specific uptake
in the pancreas.
Conclusions/interpretation We report a simple strategy for
the generation of an SCA targeting a novel beta cell-
specific epitope.
Keywords AR42J cells . Beta cell . Human islets .
INS-1 cells . Phage display . Single-chain antibody
Abbreviations
BRASIL Biopanning and rapid analysis of selective
interactive ligands
CAR Cell associated radioactivity
FPG Fasting plasma glucose
GAD-65 Glutamate decarboxylase
IAPP Islet amyloid polypeptide
ISPC INS-1-specific phage clone
Kd Dissociation constant
pfu Plaque-forming units
RT Room temperature
SCA Single-chain antibody
Introduction
Pancreatic beta cell mass is markedly reduced in patients
with type 1 or type 2 diabetes, most likely as a consequence
of an increased rate of beta cell apoptosis [1, 2]. Therefore,
strategies for preserving beta cell mass and allowing
accurate and non-invasive assessment of beta cell mass in
S. Ueberberg :D. Ziegler :W. Schechinger : J. W. Dietrich :
H. H. Klein : S. Schneider (*)
Department of Internal Medicine I,
Division of Endocrinology and Metabolism,
University Hospital Bergmannsheil, Ruhr-University Bochum,
Bürkle de la Camp Platz 1,
44789 Bochum, Germany
e-mail: Stephan.Schneider@ruhr-uni-bochum.de
S. Akinturk
Department of Neuroanatomy and Molecular Brain Research,
Ruhr-University Bochum,
Bochum, Germany
Diabetologia
DOI 10.1007/s00125-010-1725-9

humans are critically needed. In recent years, several
known islet- or beta cell-specific (or enriched) surface
markers (e.g. glucagon-like peptide 1), secretory vesicle
components (e.g. vesicular monoamine transporter 2) and
transporter molecules (e.g. glucose transporter 2), have
been evaluated for their suitability as potential islet-imaging
targets [3–11]. The properties of the tracer probes (e.g. IC2
antibody) and imaging techniques (e.g. positron emission
tomography) used in these studies have recently been
described in detail [3, 12–14]. However, as yet none of
these targets or tracer probes has allowed for successful
islet imaging in humans. This is primarily because of the
failure of these agents to label pancreatic beta cells with
sufficient selectivity over exocrine cells (factor >100 is
required) [7]. Thus, in order to permit the selective
detection of islet beta cells within the pancreas, novel
biomarkers and/or agents exhibiting high specificity for
beta cells are needed.
One reason for the obvious difficulties in generating
such agents is the paucity of knowledge about potential
targets that are exclusively presented on the beta cell
surface. Phage-display technology may be a suitable way to
overcome these limitations [15–17]. By applying this
approach to rats in vivo or freshly isolated rat islets in
vitro, we have recently reported the isolation of single-chain
antibodies (SCAs) highly specific for either pancreatic beta
or alpha cells [18]. However, these approaches applied
collagenase-digestion for islet isolation, which might alter
substantially the surface profile of the islet cells. Therefore,
in the present study, we assessed whether in vitro panning of
a phage-displayed SCA library in the rat beta cell line INS-1
(enhanced by pre-absorption in AR42J cells) would lead to
the isolation of SCAs targeting novel beta cell specific
biomarkers. By these means, we generated one beta cell-
specific SCA and compared its properties with those of the
recently characterised ones.
Methods
Phage library The human SCA library (Tomlinson Library
I, University of Cambridge, UK) represents 1.47×108
different SCAs fused to the pIII coat protein of a
bacteriophage. The controls were a phage clone without
an insert (Tomlinson library I), four beta cell-specific SCAs
(SCA B1–B4) and a control SCA identified previously
[18].
Cell culture Cells were grown as follows. INS-1 cells (C.
Wollheim, Geneva, Switzerland) were grown in RPMI
1640 (+ glutamine) with 10% (vol./vol.) FCS, penicillin/
streptomycin and vitamins. Alpha TC1 cells (ATCC,
Manassas, VA, USA) were cultured in DMEM (low
glucose) with 10% FCS (vol./vol.), penicillin/streptomycin,
15 mmol/l HEPES and 0.1 mmol/l non-essential amino acids.
AR42J and beta TC6 cells were grown in DMEM (high
glucose) with 10% (vol./vol.) FCS, penicillin/streptomycin
and vitamins (all from PAA, Paschingen, Austria). Cells were
detached with cold trypsin (0.05% trypsin, 0.53 mmol/l
EDTA-4Na) 12 h prior to conducting the assays, washed
with PBS and then stored in a 50 ml tube with appropriate
medium at 37°C and 5% CO2.
Library screening AR42J cells (106) were resuspended in
DMEM without FCS, transferred to a 1.5 ml tube and
incubated with phage (1012 plaque-forming units [pfu]/ml)
for 30 min at room temperature (RT). Cells were pelleted
by centrifugation (400×g for 5 min) and supernatant
fractions containing phage were incubated again with 1×
106 AR42J cells in DMEM without FCS for 30 min.
Subsequently, the phage–cell suspension was centrifuged
and the supernatant fractions containing phage were
incubated with 1×106 INS-1 cells in RPMI without FCS
for 2 h at RT before the cells were pelleted again, washed
twice with PBS and lysed by the addition of 0.5 ml PBS
containing 100 mmol/l Tris–HCl (pH 2.2). Subsequently,
TG1 bacteria were infected with the eluted phage
and plated on 2xTY agar with 1% glucose (wt/vol.) and
100 µg/ml ampicillin. After an overnight incubation at
37°C, colonies were determined to monitor the progress of
each round of library panning, and the recovered phage was
amplified for use in the next round [19]. Finally, 50 phage
clones each from panning rounds 1, 3 and 5 were randomly
selected and sent to GENterprise (Mainz, Germany) for
DNA sequencing.
Binding of phage in vitro The binding selectivity of phage
clones to cell lines (INS-1, beta TC6, AR42J and alpha
TC1) was analysed with the biopanning and rapid analysis
of selective interactive ligands (BRASIL) method [20]. For
experiments, cells (1×106) were re-suspended in appropri-
ate medium without FCS, transferred to a 1.5 ml tube and
incubated with a selected phage clone (1×109 pfu/ml) for
4 h on ice. Subsequently, the cell–phage suspension was
transferred to the top of a non-miscible organic lower
phase (n-dodecane/bromo-dodecane 1:90.8 (vol./vol.); d=
1.017 g/ml) and centrifuged at 12,000×g for 10 s at 4°C.
The tube was snap frozen in liquid nitrogen, the bottom of
the tube sliced off and the cell–phage pellet recovered and
grown as described [19]. On the following day the number
of colonies was counted. Background binding was deter-
mined in the presence of a control phage (1×109 pfu/ml)
and subtracted from each data point.
Production of SCAs HB2151 bacteria (optical density at
600 nm=0.4) were infected with 10 μl of a monoclonal
Diabetologia

phage clone and colonies were further grown in 2xTY
(100 µg/ml ampicillin and 1% [wt/vol.] glucose). Isopropyl
β-D-thiogalactoside (1 mmol/l; AppliChem, Darmstadt,
Germany) was added to induce SCA production. The
supernatant fraction containing the antibodies was purified
by metal affinity chromatography (Nunc ProPur; Nunc,
Langenselbold, Germany) and purity checked by SDS gel
electrophoresis and western blotting.
Binding properties of SCAs in vitro SCAs (100 µg protein)
were radiolabelled with iodine-125 (100 mCi/ml; Hartmann
Analytics, Braunschweig, Germany) and their binding
properties to cell lines (INS-1, beta TC6 and AR42J) were
analysed as previously described [8, 18]. Briefly, 1.5 ml
tubes containing cells (106) in 200 µl medium without FCS
were placed in an incubator (5% CO2 and 37°C) for
30 min. Subsequently, radiolabelled SCAs were added and
cells were further incubated for 10 min (or as noted).
Accumulation of radiolabel was determined by separating
the cell-associated radioactivity (CAR) from the free
radioactivity by transferring the cell suspension to a
1.0 ml tube containing a layer of oil comprising n-
dodecane/bromo-dodecane (1:90.8 [vol./vol.]; d=1.017 g/ml),
and centrifuged for 10 s at 12,000×g. The tube was snap
frozen in liquid nitrogen, the bottom of the tube sliced off
and the radioactivity of the tube containing the cell pellet
was counted in a gamma counter. Retention was determined
similarly except that after incubating the cells for 10 min in
the presence of radiolabel, cells were washed twice with
PBS and further incubated in radiolabel-free media prior to
spinning the cells through the oil layer. In order to account for
the cell-type-specific cell volume we normalised the CAR
(cpm/cell) with the average volume (in fl). The average cell
volume was determined in each experiment by a CASY 1 cell
counter (CASY; Innovatis, Reutlingen, Germany), which
produced cell volume information on the basis of cell
frequency distribution. Background binding was determined
in the presence of high concentrations of unlabelled control
SCA (100 μg) and subtracted from each data point.
In order to determine binding affinity, CAR was measured
in the presence of varying concentrations of labelled antibody
(0.1–50 μg) with or without pre-incubation of high concen-
trations of unlabelled SCAs (100 μg) at 4°C. The number of
molecules bound per cell, B, was calculated as B = CAR ×
Av/(SA × 106 cells) where Av is Avogadro’s number, and SA
is the specific activity of the labelled antibody in cpm/fmol.
The dissociation constant (Kd) and the maximal number of
molecules bound/cell (Bmax) was fitted by non-linear
regression according to the following equation B ¼ Bmax �
T= Kd þ Tð Þ where T is the concentration of free antibody in
the media (nmol/l). Non-linear fitting was performed with
the packages ‘graphics’ and ‘stats’ of the statistical program-
ming language R.
For competition assays, unlabelled SCAs (100 μg),
human insulin (100 µg; Roche, Mannheim, Germany),
human glutamate decarboxylase (rH-GAD-65; 100 µg;
Diamyd Diagnostics, Stockholm, Sweden), rat islet
amyloid polypeptide (IAPP; 100 µg; Bachem, Weil am
Rhein, Germany) or rat C-peptide (100 µg; Bachem)
was added to the cells before radiolabelled SCA B5 was
added.
Fluorescence microscopy INS-1 or AR42J cells were
seeded on culture dishes (50,000 cells/dish; Greiner,
Frickenhausen, Germany), with appropriate medium and
placed in an incubator (5% CO2 and 37°C) for 48 h. For
experiments, medium was aspirated, SCA B5 (2 µg/ml) re-
suspended in 100 µl medium without added FCS and
incubated either at 4°C or 37°C for 10 min. The culture
dishes were washed with PBS and cells fixed by adding
ice-cold 4.0% (vol./vol.) formaldehyde for 15 min. Subse-
quently, the cells were permeabilised with 0.1% Triton X-
100 for 10 min and an anti-myc fluorescently labelled
secondary IgG (2 µg/ml; AlexaFluor 488 conjugate;
Millipore) was added. Finally, cells were analysed using a
confocal microscope (Axiovert 100 M; ZEISS LSM 510
Laser Module).
Infrared imaging SCA B5 or the control SCA was labelled
with infrared dye 800 (CW Labeling Kit; Li-cor Bioscien-
ces, Lincoln, USA). Increasing amounts of INS-1 cells
(104–108) in medium containing 5.6 mmol/l glucose and
106 INS-1 cells in medium with increasing concentrations
of glucose (2.8–16.8 mmol/l) were grown on eight well
chamber slides and incubated for 48 h in a CO2 incubator at
37°C. Subsequently, cells were fixed with 4% (vol./vol.)
formaldehyde for 20 min at RT, permeabilised with 0.1%
Triton X-100 and incubated with labelled SCA B5 (1 µg/ml)
for 1 h. The signal was detected at 800 nm by an infrared
imaging system (Odyssey, Li-cor Biosciences). The
mean intensity of control cells that contain control-SCA-
labelled INS-1 cells was subtracted from the intensity of
wells containing INS-1cells labelled with SCA B5 to
correct for any background signal not related to SCA B5
staining.
In vivo biodistribution Female non-diabetic CD rats (n=5,
6 weeks old, ∼220 g) and male diabetic ZDF rats (n=5,
6 weeks old, ∼200 g, fasting plasma glucose [FPG] 5.4±
1.1 mmol/l; n=5, 9 weeks old, ∼300 g, FPG 9.0±1.8 mmol/l;
n=5, 12 weeks old, ∼300 g, FPG 18.2±2.5 mmol/l)
were purchased from Charles River (Sulzfeld, Germany).
The rats were injected intravenously with SCA B5
(100 µg protein/animal). Rats were killed 2 h after
injection and organs (as stated) were harvested and fixed
with formalin.
Diabetologia

In another experiment, 100 µg of 125I-labelled SCA B5
(specific activity 18.5 MBq) was injected intravenously into
non-diabetic CD rats (200–250 g, n=6). A blood probe was
taken 2 h post-injection, the animals were killed and the
organs (as stated) harvested and placed into test tubes.
Tubes containing tissues were weighed and counted for
radioactivity with a gamma counter. Data were calculated
as percentage injected dose per gram organ weight. For
competition experiments (n=3), unlabelled SCA B5
(100 µg/animal) was administered 15 min prior to the
radiolabelled SCA B5 antibody. Animal experiments were
approved by the local government.
Immunohistochemistry Staining of paraffin-embedded rat
and human tissue sections (5 μm) were performed as
follows. The use of human tissue was approved by the
local ethics committee. Sections were deparaffinised and
subsequently permeabilised by heating in the microwave
oven in antigen-unmasking solution pH 6 and cooling for
45 min. Blocking was done for 1 h at 24°C with PBS
containing 2% (wt/vol.) BSA. Primary and secondary
antibodies were diluted in PBS with 2% BSA. Primary
antibodies were incubated at 4°C overnight, except for
anti-insulin, anti-glucagon and anti-somatostatin antibodies
with an incubation period of 1 h at 37°C. Secondary
antibodies were incubated for 30 min at 24°C as well as
Cy2- and Cy3-conjugated streptavidin reagents. The
following primary antibodies and dilutions were used:
SCA B5, 1:200; mouse anti-c-myc, 1:200 (New England
Biolab, Frankfurt am Main, Germany); guinea pig anti-
swine-insulin, 1:400 (Dako, Carpinteria, CA, USA); mouse
anti-glucagon, 1:200 (Thermo Scientific, Rockford, IL,
USA) and rat anti-somatostatin antibody, 1:25 (Millipore
GmbH; Schwalbach; Germany). Secondary antibodies were
mouse anti-c-myc, 1:200; biotinylated anti-rabbit IgG and
biotinylated anti-mouse IgG, 1:200 (Linaris GmbH,
Wertheim-Bettingen, Germany); Cy3-conjugated goat
anti-mouse IgG, 1:200 (Jackson Immuno Research Europe,
Newmarket, UK); Cy3-conjugated goat anti-guinea pig
IgG, 1:800 (Jackson Immuno Research Europe); Cy3-
conjugated goat anti-rat IgG (heavy and light chains),
1:100 (Jackson Immuno Research Europe). The tertiary
reagent was Cy2-conjugated streptavidin, 1:200 (Jackson
Immuno Research Europe). Tissue slides were analysed
using a Zeiss Axioplan microscope.
Statistics Parametric comparisons of continuous data were
calculated with Student’s t test for unpaired data with
unequal variance. The relationship between beta cell mass
and fluorescence signal intensity was analysed by linear
regression analysis. All calculations have been performed
with KaleidaGraph 4.0.3 for Macintosh Computers (Synergy
Software, Reading, PA, USA).
Results
Generation of beta cell-targeting SCAs In order to generate
novel agents that bind specifically to beta cells, a
subtractive selection strategy was applied in which the
phage library encoding for 1.47×108 SCAs was first
incubated with AR42J cells in order to absorb phage with
exocrine binding properties. Subsequently, the supernatant
fraction from this incubation (∼1×105 pfu/ml) was incu-
bated with INS-1 cells. Over five rounds of this selection
process a substantial increase of the number of phage
bound to INS-1 cells was detected (from 1.2×102 to 1.2×
108; Fig. 1a), indicating enrichment of INS-1-cell-binding
phage. Subsequently, 50 phage clones from each of panning
rounds 1, 3 and 5 were randomly selected for DNA
sequencing. Surprisingly, all of these clones yielded
identical DNA sequence, thereby giving rise to one single
INS-1-specific phage clone (ISPC, the clone being named
ISPC5 = SCA B5; the amino acid sequence is shown in
Fig. 1b). Of note, repetitive runs of this protocol yielded
identical results.
In order to confirm the binding selectivity of the ISPC5
phage to beta cells, binding of ISPC5 to INS-1, beta TC6,
alpha TC1 and AR42J cells was tested with the BRASIL
method in vitro (Fig. 1c). For these experiments cells and
media were kept on ice to minimise post-binding events,
such as receptor-mediated internalisation. The assay
showed a 17-fold higher specificity for INS-1 or beta TC6
relative to alpha TC1 and AR42J cells for the ISPC5 phage
(p<0.0001).
Binding properties of SCAs in vitro As beta cell-specific
delivery requires removal of SCA B5 from the context of
the ISPC5 phage, HB2151 bacteria were infected with
ISPC5 phage and isopropyl β-D-thiogalactoside was added
to induce SCA B5 production. Subsequently, the superna-
tant fraction containing SCA B5 was purified by metal
affinity chromatography. Next, soluble SCA B5 was
radioactively labelled and its binding properties were tested
in INS-1, beta TC6 and AR42J cells in vitro. These
characteristics were then compared with those of the beta
cell-specific SCAs identified previously [18]. Of note, in
contrast to the BRASIL method, this assay was performed
at 37°C (except studies for binding affinity and binding
sites). By these means, a rapid binding with a binding half-
time of 6 min but no washout phase was found for SCA B5,
indicating internalisation of SCA B5 (Fig. 2a). The binding
selectivity of SCA B5 to INS-1 or beta TC6 was 450-fold
higher relative to AR42J cells (Fig. 2b) and was thus
comparable with the beta cell-specific SCAs evaluated
previously. For SCA B5, binding saturation could be
demonstrated after pre-incubation with unlabelled SCA
B5 (Fig. 2c). Importantly, competition assays revealed no
Diabetologia

binding inhibition of radiolabelled SCA B5 by pre-
incubation with unlabelled beta cell-specific SCAs identi-
fied previously (SCA B1–B4, Fig. 2c), suggesting that the
binding of these SCAs was at least at different epitopes of
the same antigen. Moreover, the addition of insulin, GAD-
65, C-peptide or IAPP did not inhibit binding of SCA B5 to
INS-1 cells, indicating that these proteins do not present the
epitope recognised by SCA B5 (Fig. 2c). Finally, SCA B5
had a slightly higher binding affinity (Kd 3.6 vs 4.1 μmol/l),
but a lower maximal number of binding sites (858 vs 7,023
molecules/cell) compared with SCA B1.
Next, binding of SCA B5 to INS-1 cells was analysed by
confocal microscopy. For this purpose, cells were grown on
culture dishes and subsequently incubated for 10 min with
SCA B5 at either 4°C or 37°C. After fixation of the cells,
SCA B5 was detected with fluorescently labelled secondary
IgG. By this means, the rapid binding of SCA B5 to the
beta cell surface and subsequent internalisation was shown
(Fig. 3). Control reactions with AR42J cells and without the
primary antibody were negative.
To determine the relationship between the number of
beta cells and imaging signal, increasing numbers of INS-
1 cells (1×104–1×108) were grown on chamber slides and
incubated for 1 h with fluorophore-labelled SCA B5. The
signal intensity was detected by an infrared scanner. By
this means, a strong correlation between the signal
A M A E V Q L L E S G G G L V Q P G G S L R L S C A A S G F T F
S S Y A M S W V R Q A P G K G L E W V S S I S S T G D S T S Y A
D S V K G R F T I S R D N S K N T L Y L Q M N S L R A E D T A V
Y Y C A K A A D S F D Y W G Q G T L V T V S S G G G G S G G G
G S G G G G S T D I Q M T Q S P S S L S A S V G D R V T I T C R A
S Q S I S S Y L N W Y Q Q K P G K A P K L L I Y G A S S L Q S G V
P S R F S G S G S G T D F T L T I S S L Q P E D F A T Y Y C Q Q T
N G A P T T F G Q G T K V E I K R A A A H H H H H H G A A E Q K
L I S E E D L N
1 × 100
1 × 101
1 × 102
1 × 103
1 × 104
1 × 105
1 × 106
1 × 107
1 × 108
1 × 109
1 2 3 4 5
Round of library panning
Bo
un
d
ph
ag
es
p
er
1
×

10
6
Bo
un
d
ph
ag
es
p
er
1
×

10
6
ce
lls
a b c
IN
S-
1
ce
lls
0
500
1,000
1,500
2,000
2,500
3,000
3,500
ISPC5 Control phage
††
Fig. 1 a Phage-library screening for beta cell-specific phage clones.
Five rounds of selection were carried out and a substantial increase in
bound phage per 1×106 INS-1 cells was observed. b The amino acid
sequence of the ISPC5 phage (SCA B5). Amino acids are shown in
one-letter codes. Positions that are diverse in the repertoire are marked
in bold. Boxes indicate the complementarity-determining region
(CDR). Heavy and light chain regions are underlined (heavy chain
shown with dashed underlining, light chain shown with dotted
underlining). At the C-terminus the SCA contains a His tag (blue)
and a c-myc tag (red) c In vitro binding of ISPC5 phage or a control
phage without an insert to INS-1 (black bars), beta TC6 (grey bars),
alpha TC1 (white bars) and AR42J cells (hatched bars). Cells and
media were kept on ice to minimise post-binding events, such as
receptor-mediated internalisation. The ISPC5 phage showed highly
specific binding to INS-1 and beta TC6 cells, whereas no binding to
alpha TC1 or AR42J cells was detected. n=6, †p<0.0001
0
5
5
15
15
10
10
20
0
10
20
30
40
0
10
20
30
40
30 60 120
Time (min)
CA
R
(cp
m/
fl)
CA
R
(cp
m/
fl)
CA
R
(cp
m/
fl)
a b c
SCA B5 Control SCA
*** ***
†
N
O
P
I
SC
A
B5
SC
A
B1
SC
A
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SC
A
B3
SC
A
B4
In
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pe
pt
id
e
IA
PP
G
AD
-6
5
Pre-incubation
Fig. 2 Binding properties of 125I-labelled SCA B5 to different
endocrine and exocrine cell lines in vitro. a Rapid binding (t½=
6.0 min) of SCA B5 to INS-1 cells (black circles), but no washout
(white circles) was detected, indicating internalisation. b Highly
selective binding of SCA B5 to INS-1 (black bars) and beta TC6
cells (grey bars) was detected (***p<0.001 vs AR42J cells [hatched
bars]). c Binding saturation to INS-1 cells was demonstrated for SCA
B5 by pre-incubation with unlabelled SCA B5 (†p<0.0001 vs cells
pre-incubated with SCA B5). In contrast, competition assays
involving pre-incubation with SCA B1–B4, insulin, C-peptide, IAPP
and GAD-65 demonstrated no inhibition of binding of SCA B5 to
INS-1 cells
Diabetologia

intensity and the number of beta cells was detectable (r2=
0.823, Fig. 4). The overall intensity has a clear tendency to
increase with increasing numbers of beta cells. In contrast,
the signal remained unchanged when 106 INS-1 cells were
incubated for 48 h with increasing concentrations of
glucose (2.8–16.8 mmol/l). These data suggest that the
labelling signal is dependent on beta cell mass but not beta
cell function.
In vivo biodistribution For translation into clinical practice,
specific binding of SCA B5 to beta cells in vivo would be
required. Therefore, SCA B5 was injected intravenously
into non-diabetic CD rats for a circulation time of 2 h.
Subsequently, the animals were killed and the pancreas and
control organs were prepared for immunohistochemical
analyses. SCA B5 was clearly identifiable in the islets and
the signal overlapped with insulin production, whereas no
co-localisation with glucagon was detectable, suggesting
that SCA B5 was binding exclusively to beta cells (Fig. 5).
Of note, no binding to control organs (liver, kidney, spleen,
lung, thyroid, stomach, brain, parotid gland; data not
shown) was detectable and experiments with the control
SCA did not reveal binding to islets or other structures.
In another set of experiments, the biodistribution of
radiolabelled SCA B5 was analysed by injection into non-
diabetic rats. Subsequently, pancreas and control organs
were harvested for detection of radioactivity. In accordance
with the in vitro predictions and the immunohistochemistry
data, SCA B5 accumulated selectively in the pancreas and
Fig. 3 Binding analyses by confocal laser microscopy. INS-1 cells
were incubated with SCA B5 for 10 min either at 4°C or 37°C. After
fixation of the cells, SCA B5 was detected with a fluorescently
labelled secondary IgG antibody. At 4°C binding to the cell surface
was shown, whereas at 37°C internalisation was detected. Control
reactions without the primary antibody or with AR42J cells were
negative. Scale bars, 10 µm
0
10
20
30
1.0 × 104 1.0 × 106 1.0 × 108
Number of cells
Glucose concentration (mmol/l)
In
te
ns
ity
%
(b
ac
kg
rou
nd
co
rre
cte
d)
2.8 5.6 16.811.0
15
10
5
0
20
25
In
te
ns
ity
%
(b
ac
kg
rou
nd
co
rre
cte
d)
a
b
Fig. 4 Imaging with SCAB5 labelled with infrared dye 800. INS-1 cells
were labelled in vitro with tracer SCA B5 and the signal intensity was
detected by an infrared imaging system. a Linear regression analyses
show a close correlation between increasing amounts of INS-1 cells
(1×104–1×108) and signal intensity (r2=823). b The signal intensity
remained unchanged when 1×106 INS-1 cells were incubated with
increasing concentrations of glucose. Each data point represents one
well of an eight well chamber slide. To correct for any background
signal not related to SCA B5 staining, the mean signal intensity of wells
containing INS-1 cells with fluorophore-labelled control SCA was
subtracted from each data point
Diabetologia

exhibited no affinity for the control tissues (Table 1).
Moreover, probe accumulation in the pancreas was reduced
by 80% after pre-injection with unlabelled SCA B5,
thereby confirming the specific uptake of SCA B5 in the
pancreas.
A valuable feature of beta cell-targeting agents would be
that they allow the fate of beta cells to be followed during
the development of diabetes. Therefore, we assessed the
ability of SCA B5 to target beta cells of ZDF rats of
different ages. As in the previous experiments, SCA B5
was injected intravenously and its presence in organs of
interest was determined by immunohistochemistry. As
shown in Fig. 6, SCA B5 was selectively identifiable
within the cytoplasm of the pancreatic beta cells of
normoglycaemic (6 weeks old), mildly hyperglycaemic
(9 weeks old) and severely hyperglycaemic (12 weeks
old) ZDF rats, whereas no binding to exocrine cells and
other control tissues (e.g. liver; data not shown) was
detectable. Of note, the SCA B5 was not identifiable in
destroyed islet beta cells of ZDF rats (data not shown),
providing further evidence that the signal is dependent on
the beta cell mass. Taken together, these results indicate that
Fig. 5 Immunofluorescence
analyses of the biodistribution of
SCA B5 or the control SCA
after intravenous injection in a
non-diabetic CD rat (n=5 rats
per group, 30–40 sections and
60–80 islets per rat). The
presence of SCA was deter-
mined by immunohistochemis-
try using an anti-myc antibody,
because the SCAs contained a
c-myc tag. In the pancreas SCA
B5 co-localised exclusively
with the insulin-producing beta
cells, whereas no binding to
glucagon-producing cells or
exocrine cells was detected
(staining in green colour for
SCA with an anti-c-myc
antibody and nuclei were stained
blue with DAPI). All images are
at ×40 magnification
Table 1 Biodistribution of 125I-labelled SCA B5 in non-diabetic rats
Tissue Biodistribution of 125I-labelled SCA B5
(% injected dose/g tissue)
Without pre-injection With pre-injection
Pancreas 5.12±1.01 0.95±0.18***
Stomach 0.69±0.24 0.57±0.17
Spleen 0.55±0.18 0.58±0.26
Kidney 0.38±0.13 0.44±0.17
Liver 0.71±0.28 0.64±0.22
Brain 0.26±0.09 0.33±0.14
Blood 0.23±0.11 0.22±0.15
Thyroid 0.27±0.15 0.31±0.11
125 I-labelled SCA B5 was injected into the tail vein of non-diabetic CD
rats (n=6) and, after 2.0 h, tissues were harvested as described in Methods
For competition experiments (n=3), unlabelled SCA B5 was injected
15 min prior to injection of the radiolabelled probe
***p<0.001 vs without pre-injection
Diabetologia

SCA B5 highly selectively targets normal beta cells as well
as dysfunctional beta cells during diabetes development.
Binding of SCA B5 to human and rat pancreatic tissue in
situ To further prove specific binding of SCA B5 to rat beta
cells and its potential applicability in humans, rat and
human pancreatic tissue samples were co-stained by
immunohistochemistry using SCA B5 as well as specific
anti-insulin, anti-glucagon and anti-somatostatin antibodies.
SCA B5 was exclusively identifiable within the islets and
co-localised with insulin-producing cells, but not with
glucagon- and somatostatin-producing cells (Fig. 7). These
data strongly suggest that SCA B5 binds to a molecule that
is produced in pancreatic beta cells of different origins.
Fig. 6 Evaluation of binding to
pancreatic beta cells of SCA
B5 after intravenous injection in
diabetic ZDF rats (n=5 rats
per group, 30–40 sections and
60–80 islets per rat). The
presence of SCAs was
determined by immunohisto-
chemistry using an anti-myc
antibody, because the SCAs
contained a c-myc tag. SCA B5
selectively co-localised with
insulin-producing beta cells and
showed no binding to other
endocrine or exocrine cells,
irrespective of the age of the
animals. DAPI stains nuclei
blue. All images are at ×20
magnification
Diabetologia

Discussion
The field of imaging technology has made tremendous
progress in recent years, allowing for the non-invasive and
accurate detection of small structures, such as specific brain
cell populations. However, as yet the lack of sufficiently
specific beta cell targets and/or tracer molecules has
prevented the successful application of these powerful
technologies to beta cell imaging in humans [13, 14].
Recently, we reported the successful isolation of beta and
alpha cell-specific SCAs by screening of a phage library on
primary rat islets in vitro and in vivo [18]. A key element of
the method used for the generation of these SCAs was the use
of collagenase. Particularly, collagenase disrupts the endothe-
lial cell lining of the intra-islet capillaries during the islet-
isolation process and thus eliminates phage bound to the
Fig. 7 Immunofluorescence
staining for binding of SCA
B5 to human (representative
pancreas from a non-diabetic
donor) and rat islets (represen-
tative pancreas from a non-
diabetic CD rat) in situ. The
presence of SCA was deter-
mined by immunohistochemis-
try using an anti-myc antibody,
because the SCAs contained a c-
myc tag. SCA B5 co-localised
with insulin, but not glucagon or
somatostatin staining. DAPI
stains nuclei blue. All images
are at ×40 magnification
Diabetologia

endothelium. This is supported by the finding that all
collagenase-based approaches generated alpha and/or beta
cell-specific agents [18, 21], whereas the approaches that
abandoned collagenase identified agents selectively targeting
the islet endothelium [22, 23]. However, collagenase alters
the surface profile of pancreatic islets, and thus we anticipate
that the identification of valuable beta or alpha cell-specific
targeting agents could be prevented.
Therefore, in this proof-of-concept study we report a
simple and very time-efficient in vitro panning strategy, in
which a phage-displayed SCA library was pre-incubated
with the rat exocrine cell line AR42J in order to eliminate
SCAs with exocrine binding properties and then panned
against the rat beta cell line INS-1 to select beta cell-
targeted antibodies. By these means, we isolated SCA B5, a
novel antibody that binds rapidly (t1/2=6.0 min) and with a
450-fold higher specificity for beta cells relative to exocrine
cells. These highly promising features are comparable with
those of the beta cell-specific SCAs (SCA B1 - SCA B4)
reported previously. However, binding of SCA B5 to beta
cells was not inhibited by these SCAs, indicating that
binding occurs at least via different binding pockets within
the same cell-surface antigen. This claim is supported by
the finding that the novel antibody SCA B5 had a slightly
higher binding affinity for beta cells compared with the
SCA B1 (both in the low μmol/l range), but a lower
maximal number of binding sites (by a factor of ten).
Confocal microscopy images provide further evidence for
binding of SCA B5 to the beta cell surface and confirm its
subsequent internalisation. In particular, the incubation of
INS-1 cells with varying concentrations of radiolabelled
SCA B5 revealed a capacity of >630,000 SCAs to be
internalised per beta cell. Moreover, staining of rat and
human pancreatic tissue sections with SCA B5 suggests
that the target epitope is produced in pancreatic beta cells of
different origins. Finally, infrared imaging of INS-1 cells
indicates that labelling of beta cells with tracer SCA B5 is
strictly dependent on beta cell mass. Taken together,
although the binding affinity of the novel antibody SCA
B5 for beta cells is below that of full antibodies or its
fragments [8], SCA B5 has promising features (i.e. required
beta cell selectivity, rapid binding and strong correlation
with beta cell mass) that qualify for specific labelling of
beta cells.
Our impression that SCA B5 represents a novel highly
promising agent was strengthened by the finding that,
following intravenous administration, SCA B5 targeted
normal beta cells of non-diabetic CD rats in vivo, as well
as dysfunctional beta cells of ZDF rats, whereas no binding
or uptake to control tissues was detected. The specificity of
this approach was confirmed by additional in vivo experi-
ments showing that more than 80% of the pancreatic
radiolabelling intensity could be inhibited by pre-
administration of unlabelled SCA B5.
Even though the method applied in this study is novel, a
theoretical concern could be that the antibodies resulting
from this panning approach might recognise a known beta
cell target that has already been explored in previous
studies. Against this, by means of various competition
assays, we could rule out that SCA B5 targets insulin,
GAD-65, C-peptide or IAPP. However, the identification of
the respective target is still highly important to estimate the
diagnostic and/or therapeutic potential of this novel SCA.
Unfortunately, as yet none of the generated SCAs has
immunoprecipitated any protein in western blotting or
classic immunoprecipitation, suggesting that the targeted
epitopes are structure sensitive.
In summary, we report a phage-panning approach in the
rat beta cell line INS-1 as a novel and simple strategy for
the generation of islet-targeted agents. The novel beta cell-
specific antibody SCA B5 generated by this approach offers
a promising perspective for non-invasive targeting of islets
(e.g. in imaging) that, however, has to be shown in future
studies. Moreover, this panning strategy applied to other
cells lines (e.g. alpha TC1) and/or performed with other
phage libraries (e.g. peptide-based) may set the route for the
development of novel agents and the identification of as yet
unknown beta or alpha cell-specific biomarkers.
Acknowledgements This work was supported by research grants of
the Ruhr-University Bochum (FoRUM to S. Schneider), the Beta cell-
Biology Consortium of the NIH (DK072473 to S. Schneider), the
Deutsche Forschungsgemeinschaft (SCHN 702/2-1 to S. Schneider)
and the European Foundation for the Study of Diabetes (EFSD/MSD
to S. Schneider and EFSD/Novartis to H. H. Klein) and the Menarini
Projektförderung (to S. Schneider) of the German Diabetes Association.
Duality of interest S. Schneider holds a patent application on the
SCAs described. The remaining authors declare that there is no duality
of interest associated with this manuscript.
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