Báo cáo khoa học: Differential binding of human immunoagents and Herceptin to the ErbB2 receptor ppt

Differential binding of human immunoagents and
Herceptin to the ErbB2 receptor
Fulvia Troise
1,
*, Valeria Cafaro
1,
*, Concetta Giancola
2
, Giuseppe D’Alessio
1
and
Claudia De Lorenzo
1
1 Dipartimento di Biologia Strutturale e Funzionale, Universita
`
di Napoli Federico II, Italy
2 Dipartimento di Chimica, Universita
`
di Napoli Federico II, Italy
ErbB2 (HER2 ⁄ neu) is a proto-oncogene of the erbB
family of tyrosine kinase receptors [1]. It encodes a
185 kDa transmembrane protein, which comprises an
extracellular domain (ECD) and an intracellular tyro-
sine kinase activity. Although no natural ligand has
been identified for this receptor, it has been ascer-
tained that its overexpression is associated with
various carcinomas, in particular with human breast
cancer [2]. As ErbB2 overexpression is involved in the
progression of the malignancy, and is a sign of a poor
prognosis, ErbB2 is a valid target of therapeutic inter-
vention.

However, when ErbB2 is overexpressed, not all of
the ErbB2-ECD protein is embedded in the membrane
of malignant cells; a fraction of ErbB2-ECD is proteo-
lytically removed from the receptor [3] and shed as a
soluble protein in the sera of breast cancer patients [4].
Herceptin [5], a humanized anti-ErbB2 IgG1, has
been proven to be an essential tool in the immunother-
apy of breast carcinoma. However, some ErbB2-posi-
Keywords
binding affinity; ErbB2; herceptin;
immunoRNase; immunotherapy
Correspondence
C. De Lorenzo, Dipartimento di Biologia
Strutturale e Funzionale, Universita
`
di Napoli
Federico II, Via Cinthia, 80126 Naples, Italy
Fax: +39081679159
Tel: +39081679158
E-mail:
*These authors contributed equally to this
work
(Received 9 June 2008, revised 29 July
2008, accepted 1 August 2008)
doi:10.1111/j.1742-4658.2008.06625.x
Overexpression of the ErbB2 receptor is associated with the progression of
breast cancer, and is a sign of a poor prognosis. Herceptin, a humanized
antibody directed to the ErbB2 receptor, has been proven to be effective in
the immunotherapy of breast cancer. However, it can result in cardiotoxicity,
and a large fraction of breast cancer patients are resistant to Herceptin treat-

ment. We have engineered three novel, fully human, anti-ErbB2 immuno-
agents: Erbicin, a human single-chain antibody fragment; ERB-hRNase, a
human immunoRNase composed of Erbicin fused to a human RNase;
ERB-hcAb, a human ‘compact’ antibody in which two Erbicin molecules
are fused to the Fc fragment of a human IgG1. Both ERB-hRNase and
ERB-hcAb strongly inhibit the growth of ErbB2-positive cells in vivo. The
interactions of the Erbicin-derived immunoagents and Herceptin with the
extracellular domain of ErbB2 (ErbB2-ECD) were investigated for the first
time by three different methods. Erbicin-derived immunoagents bind soluble
extracellular domain with a lower affinity than that measured for the native
antigen on tumour cells. Herceptin, by contrast, shows a higher affinity for
soluble ErbB2-ECD. Accordingly, ErbB2-ECD abolished the in vitro anti-
tumour activity of Herceptin, with no effect on that of Erbicin-derived immu-
noagents. These results suggest that the fraction of immunoagent neutralized
by free extracellular domain shed into the bloodstream is much higher for
Herceptin than for Erbicin-derived immunoagents, which therefore may be
used at lower therapeutic doses than those employed for Herceptin.
Abbreviations
EDIA, Erbicin-derived immunoagent; ErbB2-ECD, extracellular domain of ErbB2 receptor; ERB-hcAb, human compact antibody against ErbB2
receptor; ERB-hRNase, human anti-ErbB2 immunoRNase with Erbicin fused to human pancreatic RNase; ITC, isothermal titration
calorimetry; RU, response unit; scFv, single-chain antibody fragment; SPR, surface plasmon resonance.
FEBS Journal 275 (2008) 4967–4979 ª 2008 The Authors Journal compilation ª 2008 FEBS 4967
tive carcinomas are resistant to the inhibitory effect on
growth of Herceptin [6], and, in other patients, the
resistance of malignant cells is induced at a later stage
in treatment [7]. Furthermore, it has been found that
Herceptin can lead to cardiotoxicity in a significant
fraction of treated patients [8,9]. An alternative
approach to the use of Herceptin in immunotherapy
has been promoted, based on the administration of

Herceptin combined with other antibodies directed to
the ErbB2 receptor [10,11]. A prerequisite for this
strategy is that the latter antibodies are directed to epi-
topes on ErbB2-ECD different from that recognized
by Herceptin.
Based on these considerations, we instituted a search
for novel immunoagents directed to epitopes different
from that recognized by Herceptin, with no cardiotoxic
side-effects and able to fulfil the therapeutic need of
Herceptin-unresponsive patients. This led us to the
production of a novel, fully human, anti-ErbB2 single-
chain antibody fragment (scFv), isolated from a large
phage display library through a double selection strat-
egy performed on live cells. This scFv, named Erbicin
[12], specifically binds to ErbB2-positive cells, inhibits
receptor autophosphorylation and is internalized by
target cells. Erbicin was used to construct human anti-
ErbB2 immunoagents by two different strategies. The
first was based on Erbicin fused to an RNase, i.e. a
pro-toxin, as RNase becomes toxic only when Erbicin
promotes its internalization in target cells. An immun-
oRNase, denoted as ERB-hRNase (Erbicin-human-
RNase), was produced by the fusion of Erbicin to
human pancreatic RNase [13].
The second strategy aimed to produce a therapeutic
reagent with an increased half-life, prolonged tumour
retention and an ability to recruit host effector func-
tions. Erbicin was thus fused to the Fc region from a
human IgG1 to obtain an immunoglobulin-like anti-
body version [14,15]. The engineered antibody was

denoted as ERB-hcAb (human anti-ErbB2-compact
antibody) because of its ‘compact’ size (100 kDa) com-
pared with the full size (155 kDa) of a natural IgG.
Both Erbicin-derived immunoagents (EDIAs) were
found to selectively and strongly inhibit the growth of
ErbB2-positive cells, both in vitro and in vivo. How-
ever, to define and implement the antitumour potential
of Erbicin and EDIAs, we deemed it essential to study
their interaction with ErbB2. To determine and evalu-
ate quantitatively their affinity for ErbB2, we used
recombinant ErbB2-ECD as a homogeneous, soluble
antigen. With this aim, three different analytical meth-
ods were employed: ELISA, surface plasmon reso-
nance (SPR) and isothermal titration calorimetry
(ITC). The results obtained with Erbicin and EDIAs
were compared with the results obtained with Hercep-
tin. Furthermore, we determined and compared the
affinity values of Herceptin and EDIAs for the free
ECD structured within the whole receptor molecule,
natively inserted into the cell membrane, with the val-
ues measured using isolated ECD.
We found that EDIAs bound soluble ECD with an
affinity lower than that of Herceptin. However, the
novel EDIAs bound ErbB2 exposed on the cell surface
with a higher affinity than that of Herceptin [13,14].
These results indicate that the fraction of immunoagent
neutralized by free ECD shed into the bloodstream,
and hence lost to immunotherapy, could be much
higher for Herceptin than for the novel immunoagents.
Results

Production and characterization of ErbB2-ECD
The cDNA coding for ErbB2-ECD was stably trans-
fected in the 293 cell line. The encoded protein was
expressed as a secretion product in the culture med-
ium, as revealed by western blotting (Fig. 1A) and
immunoprecipitation analyses performed (see Experi-
mental procedures) with ERB-hcAb or Herceptin as
anti-ErbB2 agent (see Fig. 1B). The final yield of
ErbB2-ECD, purified by affinity chromatography (see
Experimental procedures), was 12 mgÆL
)1
of medium.
The protein was analysed by SDS-PAGE, followed
by Coomassie staining and western blotting with Her-
ceptin or ERB-hcAb (Fig. 1C). Its molecular size was
about 80 kDa, as expected.
Analysis by ELISA of the interactions of EDIAs
and Herceptin with soluble ErbB2-ECD
ELISA sandwich assays were performed to determine
the ability of Erbicin and EDIAs to recognize soluble
ErbB2-ECD. Herceptin fixed on the microplate was
used to capture ErbB2-ECD, which, in turn, could inter-
act with the anti-ErbB2 immunoagents. The affinity of
ERB-hcAb or Herceptin for ErbB2-ECD was measured
by ELISA on ECD directly coated to the wells.
The results are given in Table 1 as apparent binding
constants, measured from the binding curves as the
concentrations corresponding to half-maximal sat-
uration.
The values obtained (50 nm for Erbicin, 30 nm for

ERB-hRNase and 7 nm for ERB-hcAb) were found to
be higher than those obtained with ErbB2 embedded
in ErbB2-positive cells [13,14]. Thus, these data indi-
cate that the immunoagents have a higher affinity for
ErbB2-ECD when it is inserted in the cell membrane.
Binding of human immunoagents to ErbB2 F. Troise et al.
4968 FEBS Journal 275 (2008) 4967–4979 ª 2008 The Authors Journal compilation ª 2008 FEBS
Interestingly, the lower binding affinity of EDIAs
for soluble ErbB2-ECD was not shared by Herceptin,
which displayed a high affinity for soluble ErbB2-ECD
(0.1 nm), about 50-fold higher than that determined
when Herceptin was tested with ErbB2-ECD expressed
on live cells (see Table 1). These findings can be
explained by the fact that parent Erbicin was selected
from a phage library using ErbB2-ECD inserted
into ErbB2-positive cells, whereas, for the isolation of
Herceptin, free, soluble ECD was used [16].
ELISA sandwich assays with Herceptin as a
capture agent have been performed to confirm that
Erbicin and the novel immunoconjugates recognize,
on ErbB2-ECD, an epitope different from that selected
by Herceptin, as reported previously [17].
However, this type of assay was carried out for
Erbicin and the immunoRNase only, as the peroxi-
dase-conjugated anti-His IgG1 capable of revealing
scFv and Erb-hRNase is unaffected by the presence of
Herceptin; it was not performed with Erb-hcAb, as the
anti-human secondary IgG serum fraction, used for its
detection, could not discriminate between Erb-hcAb
and Herceptin. Thus, for Erb-hcAb and Herceptin, the

assays were performed on ECD directly immobilized
on the plate.
We then tested whether soluble ErbB2-ECD could
affect the binding of anti-ErbB2 immunoagents to
ErbB2-positive cells by performing ELISA with
ERB-hcAb or Herceptin in the absence or presence of
free ECD. Each antibody was tested at increasing
concentrations, with soluble ECD added either in
equimolar amounts or in a 10-fold molar excess to the
number of receptor molecules on the cell membrane
[18]. As a control, parallel assays were carried out in
the absence of ErbB2-ECD.
As shown in Fig. 2A, the binding curves obtained
for ERB-hcAb in the absence or presence of soluble
ECD were found to be superimposable. This finding
suggests that the binding ability of ERB-hcAb to
ErbB2-positive cells is unaffected by the presence of
soluble ECD. In contrast, the binding of Herceptin to
ErbB2-positive cells (Fig. 2B) was strongly reduced by
ECD used at a 1 : 1 ratio with the receptor number,
and fully inhibited with a 10-fold molar excess of
ECD. These results, in line with those described above
on the high affinity of Herceptin for soluble ECD,
indicate that, for Herceptin, there is a favourable com-
petition of soluble ErbB2-ECD over ECD on the cell
membrane, whereas there is no detectable competition
in the case of ERB-hcAb.
Effects of soluble ErbB2-ECD on the cytotoxicity
of ERB-hcAb and Herceptin
On the basis of the results discussed above, the

antitumour effects of ERB-hcAb and Herceptin on
1
A
B
C
34
ERB-hcAb
(100 kDa)
1
Herceptin
(155 kDa)
2
80 kDa
ECD
(80 kDa)
ECD
(80 kDa)
134
80 kDa
2
2
Fig. 1. Detection of ErbB2-ECD expression. (A) Western blotting
analyses from conditioned medium of transfected 293 cells, with
Herceptin as primary antibody followed by horseradish peroxidase-
conjugated anti-human (Fc-specific) IgG serum fraction. Lane 1,
negative control (medium from non-transfected 293 cells); lanes
2–4, conditioned medium produced by various selected clones. (B)
Immunoprecipitation analyses of ErbB2-ECD from 293 cell condi-
tioned medium with ERB-hcAb (lane 1) or Herceptin (lane 2). (C)
SDS-PAGE analyses of purified ErbB2-ECD. Lane 1, molecular

weight standards; lane 2, ErbB2-ECD eluted from immunoaffinity
chromatography stained with Coomassie blue; lanes 3 and 4, wes-
tern blot analyses of the sample in lane 2 using ERB-hcAb and Her-
ceptin, respectively, as anti-ErbB2-ECD immunoagents.
Table 1. Relative affinity of Erbicin, EDIAs and Herceptin for solu-
ble ErbB2-ECD, as measured by ELISAs. Previous data [13,14]
obtained with ErbB2-positive cells are also shown.
K
D
(apparent) (nM)
ErbB2-ECD
ErbB2-positive
cells
Erbicin 50 5
ERB-hRNase 30 4.5
ERB-hcAb 7 1
Herceptin 0.1 5
F. Troise et al. Binding of human immunoagents to ErbB2
FEBS Journal 275 (2008) 4967–4979 ª 2008 The Authors Journal compilation ª 2008 FEBS 4969
ErbB2-positive cells were tested in the absence or pres-
ence of soluble ErbB2-ECD. Antibodies were incu-
bated with soluble ECD, added at a concentration of
20 nm (eight-fold molar excess over antibodies), which
was chosen on the basis of ELISA conditions in which
Herceptin binding to ErbB2-positive cells was fully
inhibited (Fig. 2B).
As shown in Fig. 2C, ERB-hcAb inhibited the
growth of SKBR3 cells similarly in the absence or
presence of soluble ErbB2-ECD. In contrast, soluble
ErbB2-ECD completely abolished the antitumour

activity of Herceptin.
These results indicate that, in the presence of soluble
ECD, ERB-hcAb preserves its cytotoxic power on
ErbB2-positive cells, whereas Herceptin does not exert
cytotoxic activity because of its high affinity for solu-
ble ECD. ECD is capable of neutralizing antibody
binding to the cells, in agreement with previously
reported data [19].
Analyses by SPR of the interactions of EDIAs and
Herceptin with ErbB2-ECD
To compare the binding properties of Erbicin, EDIAs
and Herceptin with ErbB2-ECD using a direct meth-
odology based on physicochemical principles, SPR
analyses were carried out. The experimental system
consisted of ErbB2-ECD (a monovalent ligand) cova-
lently immobilized on the chip surface, with monova-
lent (Erbicin or ERB-hRNase) or bivalent (Herceptin
or ERB-hcAb) analytes injected and flowing over the
sensor chip.
The kinetic constants for monovalent Erbicin and
ERB-hRNase were obtained by fitting the curves with
a 1 : 1 interaction model. Similar binding curves were
recorded for these immunoagents (see Fig. 3A,B),
with almost identical association rate constants, but
slightly different dissociation rate constants (see
Table 2). Erbicin, with a k
d
value of 6.16 · 10
)3
s

)1
,
dissociated from ErbB2-ECD 1.5 times faster than did
ERB-hRNase (k
d
= 4.12 · 10
)3
s
)1
). This indicated a
higher stability for the ERB-hRNase ⁄ ErbB2-ECD
complex with respect to the Erbicin ⁄ ErbB2-ECD com-
plex, with equilibrium K
D
values of 46.7 and 27.2 nm,
respectively. The significant difference in K
D
values
could be clearly ascribed to the lower dissociation rate
constant measured for the ERB-hRNase ⁄ ErbB2-ECD
complex. It should be noted that the data were in very
good agreement with those reported above from the
ELISA experiments (see Table 1).
The possibility was considered that the higher stabil-
ity of the ERB-hRNase ⁄ ErbB2-ECD complex might
be caused by aspecific electrostatic interactions
between the positively charged RNase linked in the
immunoconjugate and the negatively charged carb-
oxymethyl-dextran matrix of the SPR chip. Thus, the
kinetic analyses of the ERB-hRNase ⁄ ErbB2-ECD

complex were repeated in the presence of soluble
carboxymethyl-dextran as an added quencher. How-
ever, identical constants were measured for the
1.5
2
A
B
C
0
0.5
1
0 4 8 1012141618
2
2.5
Protein concentration (nM)
0
0.5
1
1.5
Absorbance (450 nm) Absorbance (450 nm)
40
0246810
Protein concentration (nM)
10
20
30
0
Cell growth inhibition (%)
Control
Herceptin

Herceptin + ECD
ERB-hcAb
ERB-hcAb + ECD
26
Fig. 2. Effects of soluble ErbB2-ECD on the binding and cytotoxic-
ity of ERB-hcAb and Herceptin. Binding curves of ERB-hcAb (A)
and Herceptin (B) to SKBR3 cells obtained by ELISA performed in
the absence (open symbols) or presence (filled symbols) of soluble
ECD. Soluble ECD was added at a ratio of 1 : 1 (filled squares) or
10 : 1 (filled circles) to the number of receptor molecules on the
cell membrane. (C) Antitumour activity of ERB-hcAb and Herceptin
on SKBR3 cells determined in the absence or presence of soluble
ECD.
Binding of human immunoagents to ErbB2 F. Troise et al.
4970 FEBS Journal 275 (2008) 4967–4979 ª 2008 The Authors Journal compilation ª 2008 FEBS
ERB-hRNase ⁄ ErbB2-ECD complex in the presence or
absence of soluble carboxymethyl-dextran (Table 2).
This indicates that the higher stability of the
ERB-hRNase ⁄ ErbB2-ECD complex is not caused by
simple coulombic interactions with the non-immune
moiety, but by its specific structural features which
120
60
Response (RU)
80
40
0
40
20
0

0 100 200 300 400 500
Response (RU)
160
80
A B
C D
E F
60
0 100 200 300 400 500
Time (s) Time (s)
Response (RU)
120
40
80
0
40
20
0
0 100 200 300 400 500
Response (RU)
6
7
100
120
0 100 200 300 400 500
450
550
300
350
400

Time (s) Time (s)
0
1
2
3
4
5
0 20 40 60 80 100 120
R
eq
(RU)
R
eq
/ERB-hcAb
(RU/n
M)
0
20
40
60
80
R
eq
(RU)
-50
50
150
250
350
200 250 300 350

R
eq
(RU)
R
eq
/Herceptin
(RU/n
M)
0
50
100
150
200
250
R
eq
(RU)
0 50 100 150 200 250 300 350 400
[ERB-hcAb] (nM)
0 50 100 150 200 250 300 350 400
[Herceptin] (nM)
Fig. 3. Determination by SPR of the binding between anti-ErbB2 immunoagents and ErbB2-ECD. Representative sensorgrams (jagged grey
lines) recorded for Erbicin (A), ERB-hRNase (B), ERB-hcAb (C) and Herceptin (D). Smooth black lines represent the global fits of the sensor-
grams to a 1 : 1 bimolecular interaction model. Erbicin was passed across the surface (500 RU of ErbB2-ECD) at concentrations of 10.9–
350 n
M (A) and ERB-hRNase at concentrations of 8.4–269 nM (B). Soluble ErbB2-ECD was passed over ERB-hcAb (density, 202 ± 4 RU) or
Herceptin (density, 1151 ± 5 RU), each captured by Protein A, at concentrations of 14.6–470 n
M (C) and 22.7–728 nM (D). Binding isotherms
of ERB-hcAb (E) and Herceptin (F) to immobilized ErbB2-ECD (500 RU). The equilibrium binding data (R
eq

) were measured directly on the
sensorgrams obtained by subsequent injections of analytes, and represent the mean of two determinations. The analysed concentrations
were 7.6–356.6 n
M for ERB-hcAb (E) and 0.5–361 nM for Herceptin (F). The equilibrium binding data were fitted to a two-site binding hyper-
bola (R
2
= 0.994 and R
2
= 0.999 for ERB-hcAb and Herceptin, respectively). The insets show the Scatchard analysis of the binding data. The
calculated constants from these plots (K
D1
and K
D2
) are listed in Table 2.
Table 2. Affinity and rate constants for ErbB2-ECD ⁄ ligand interactions determined by SPR.
k
a
(M
)1
Æs
)1
)
a
K
d
(s
)1
)
a
K

D
(nM)
a
K
D1
(nM)
b
K
D2
(nM)
c
Erbicin (1.33 ± 0.13) · 10
5
(6.16 ± 0.42) · 10
)3
46.7 ± 5.5
ERB-hRNase (1.50 ± 0.18) · 10
5
(4.12 ± 0.84) · 10
)3
27.2 ± 2.5 24
ERB-hRNase
d
1.61 · 10
5
4.47 · 10
)3
27.8
ERB-hcAb (1.77 ± 0.13) · 10
4

(4.35 ± 0.09) · 10
)4
24.7 ± 2.4 31 5.6
Herceptin (7.25 ± 2.41) · 10
3
(6.50 ± 1.12) · 10
)5
9.4 ± 1.5 8.9 0.1
a
The reported constants are average values obtained from three independent analyses using different biosensors, sample preparations and
ligand densities on the flow cell surfaces. The equilibrium dissociation constants (K
D
) were calculated from the relationship: K
D
= k
d
⁄ k
a
.
b
Equilibrium dissociation constants for the 1 : 1 complexes, calculated from the Scatchard plot analyses.
c
Apparent affinity constants for
the bivalent complexes, calculated from the Scatchard plot analyses.
d
Reported data were measured in the presence of soluble carboxym-
ethyl-dextran added to the sample at a final concentration of 5 mgÆmL
)1
.
F. Troise et al. Binding of human immunoagents to ErbB2

FEBS Journal 275 (2008) 4967–4979 ª 2008 The Authors Journal compilation ª 2008 FEBS 4971
lead to tighter binding in the complex, a result also
obtained for the other Erbicin-derived immunoconju-
gate, ERB-hcAb (see below).
We then studied, by equilibrium SPR analyses, the
interactions of bivalent Herceptin and ERB-hcAb,
each endowed with two identical antigen binding sites,
with immobilized ErbB2-ECD. In this experimental
approach, the analytes were expected to have the
potential to bind either to a single site or bivalently.
The ratio between the monovalent and bivalent
complexes would be dependent on the relative
concentrations of ligand and analyte flowing over the
surface.
Equilibrium binding responses (R
eq
) were determined
directly at increasing analyte concentrations. The anal-
yses of the equilibrium binding data were carried out
using Scatchard plots with the binding models
described by Junghans [20] to count receptors and
other cell surface molecules with bivalent analytes
(IgG). The binding isotherms and corresponding Scat-
chard plots are shown in Fig. 3. Biphasic Scatchard
plots were obtained, which are consistent with the
bivalent binding model.
The constants calculated from these plots, listed in
Table 2, highlight the different binding behaviour as a
function of analyte concentration. At higher antibody
concentrations (about 45–360 nm for ERB-hcAb and

100–360 nm for Herceptin), the K
D1
constants were 31
and 8.9 nm for ERB-hcAb and Herceptin, respectively.
The maximum binding responses (B
max1
), which are
proportional to the moles of bound antibody, were
116 and 336 response units (RU) for ERB-hcAb and
Herceptin, respectively. At lower analyte concentra-
tions (about 8–45 nm for ERB-hcAb and 0.5–10 nm
for Herceptin), the K
D2
constants were 5.6 and 0.1 nm
for ERB-hcAb and Herceptin, respectively, and the
maximum binding responses (B
max2
) were 76 and
298 RU for ERB-hcAb and Herceptin, respectively.
These data can be interpreted by surmising that, at
low concentrations, the antibody can simultaneously
bind two receptor molecules. This reflects the high
affinity of the antibody for the receptor, as marked by
a low K
D2
constant. It should be noted (see Table 2)
that the K
D2
values are virtually identical to the appar-
ent binding constants determined by ELISA (7 and

0.1 nm for ERB-hcAb and Herceptin, respectively,
compared with 5.6 and 0.1 nm, respectively). Indeed,
the antibody concentration range explored by ELISA
was very similar to that examined by SPR.
At high antibody concentrations, the crowding of
antibody molecules on the immobilized ErbB2-ECD
renders it difficult to obtain simultaneous binding of
antibody to two receptor molecules, and the binding is
mainly monovalent; this is reflected in the low affinity
with a higher K
D1
constant.
The analysis of the maximum binding values of the
bivalent analytes, calculated from the Scatchard plots
at low and high analyte concentrations (see below),
supports these hypotheses. If bivalent binding is
achieved, the maximum binding response (B
max2
)
should be one-half of the maximum binding response
expected for monovalent binding (B
max1
). For
ERB-hcAb, B
max2
(76 RU) was indeed about one-half
of the B
max1
value (116 RU), in good agreement with
the above-mentioned hypothesis.

Furthermore, as a control experiment, a Scatchard
plot analysis of the equilibrium binding data of
ERB-hRNase was carried out (data not shown). This
immunoagent is a monovalent analyte with a molec-
ular weight of 46 kDa, about half of the ERB-hcAb
molecular weight (100 kDa), and showed binding to
immobilized ECD in a 1 : 1 ratio.
In this case, the expected B
max
value should be simi-
lar to B
max2
determined for ERB-hcAb bivalent bind-
ing. The calculated B
max
value for ERB-hRNase was
found to be 84 RU, very similar to the value calcu-
lated for the bivalent binding of ERB-hcAb
(B
max2
= 76 RU), in line with the hypothesis formu-
lated above. The K
D
value calculated from the Scat-
chard plot for ERB-hRNase was 24 nm (Table 2), in
very good agreement with that determined by SPR
kinetic experiments (27.2 nm).
However, the B
max2
value for the bivalent binding of

Herceptin (298 RU) was higher than the expected one-
half value of B
max1
(1 ⁄ 2B
max1
= 168 RU). This differ-
ence could be ascribed to the lack of equilibrium
response data at very low Herceptin concentrations
(< 0.5 nm), required to accurately define the Scat-
chard plot for bivalent binding.
These results, in line with those predicted using the
binding models described by Junghans [20], indicate
that bivalent binding typically dominates over mono-
valent binding up to very high antibody concentra-
tions.
To further test this hypothesis, different ECD densi-
ties were immobilized on the chip surface for analysis
of the antibody affinity at equilibrium. However, when
a low ECD density (260–340 RU) was used, it was not
possible to record the equilibrium responses at anti-
body concentrations close to the K
D
value; when a
higher ECD density (1500–1800 RU) was used, equi-
librium responses were recorded, but the maximum
antibody binding values (900–1300 RU) were too high
to be reliable.
It may be of interest that the equilibrium binding
analyses carried out by SPR may be applied to deter-
Binding of human immunoagents to ErbB2 F. Troise et al.

4972 FEBS Journal 275 (2008) 4967–4979 ª 2008 The Authors Journal compilation ª 2008 FEBS
mine both affinity constants: the equilibrium dissocia-
tion constant (K
D1
) for the 1 : 1 complex, an intrinsic
property of the binding site, and the apparent affinity
constant (K
D2
) for the bivalent complex, dependent on
steric features.
It has been reported that, in mammary carcinomas,
in which ErbB2 is overexpressed, the antitumour
action of Herceptin can be neutralized in part by bind-
ing to soluble ErbB2-ECD, which is proteolytically
cleaved and shed into the patients’ sera [19]. Given
the interest in the binding properties of anti-ErbB2
immunoagents (ERB-hcAb and Herceptin) to soluble
ErbB2-ECD, a different SPR system was implemented
by performing assays on the antibodies trapped on the
sensor chip (see Experimental procedures), with soluble
ErbB2-ECD freely passed over the chip.
The kinetic constants for association (k
a
) and disso-
ciation (k
d
) were determined. Figure 3C,D shows the
binding curves used to determine k
a
and k

d
(see
Table 2) for the complexes of ERB-hcAb or Herceptin
with ErbB2-ECD. These analyses highlight the differ-
ent kinetic behaviour for the two antibodies. Herceptin
binds ErbB2-ECD with a relatively low value of k
a
(7.25 · 10
3
m
)1
Æs
)1
), about three-fold lower than the
value determined for the ERB-hcAb ⁄ ErbB2-ECD com-
plex (1.77 · 10
4
m
)1
Æs
)1
). With regard to the dissocia-
tion step, the Herceptin ⁄ ErbB2-ECD complex was
found to be much more stable, with a k
d
value of
6.5 · 10
)5
s
)1

, about one order of magnitude lower
than the k
d
value of the ERB-hcAb ⁄ ErbB2-ECD
complex (4.35 · 10
)4
s
)1
). The calculated equilibrium
dissociation constants (K
D
) for Herceptin ⁄ ErbB2-ECD
and ERB-hcAb ⁄ ErbB2-ECD 1 : 1 complexes were
9.4 and 24.7 nm per binding site, respectively.
Interestingly, these K
D
values were very similar to the
K
D1
values determined by equilibrium SPR analyses at
high antibody concentrations, when only a single
antigen binding site interacts with ErbB2-ECD (see
Table 2).
The kinetic constants for the association (k
a
) and
dissociation (k
d
) phases, determined by SPR, are clo-
sely correlated with the bivalent affinity enhancements

(avidities) relative to the monovalent interactions
(intrinsic affinities).
The avidity is usually considered as a measure of the
resistance of antibody ⁄ antigen complexes to dissociation
after dilution [21]. As the stability of immunocomplexes
is mainly the result of a low dissociation rate constant
value (k
d
), it could be predicted that the increase in
affinity of bivalent complexes in comparison with mono-
valent complexes should be inversely proportional to
the dissociation rate constant value (k
d
). Therefore, the
Herceptin ⁄ ErbB2-ECD complex with a dissociation rate
constant (k
d
) of 6.5 · 10
)5
s
)1
, about one order of mag-
nitude lower than the k
d
value of the ERB-hcAb ⁄
ErbB2-ECD complex (4.35 · 10
)4
s
)1
), is expected to

exhibit a larger increase in affinity when bivalent
binding is allowed. This is in line with the findings
that the apparent affinity constant for the Herceptin ⁄
ErbB2-ECD bivalent complex (K
D2
= 0.1 nm) is 100-
fold lower than the equilibrium dissociation constant
(K
D1
= 9.6 nm) for the monovalent complex, whereas
the apparent affinity constant for the ERB-hcAb ⁄
ErbB2-ECD bivalent complex (K
D2
= 5.6 nm) is only
five-fold lower than the equilibrium dissociation con-
stant (K
D1
= 24.7 nm) for the monovalent complex.
Together, these data confirm once again that
Herceptin binds to soluble ErbB2-ECD with a higher
affinity than does ERB-hcAb, so that, in vivo, the frac-
tion of Herceptin strongly sequestered into this immu-
nocomplex may not be available for interactions with
cell-embedded ErbB2.
Analyses by ITC of the interactions of soluble
EDIAs and Herceptin with soluble ErbB2-ECD
Given the intriguing results obtained by studying the
binding of free, soluble ErbB2-ECD to anti-ErbB2
immunoagents, we studied these interactions by ITC,
another analytical tool firmly based on physicochemical

principles. Using this methodology, a ligand is gradually
titrated against a macromolecule, thus evolving or tak-
ing up measurable heat. In the ITC experimental set-up,
both the macromolecules, in our case ErbB2-ECD and
the immunoagents, are free in solution.
Figure 4 shows the results of calorimetric titrations
for the interactions of the immunoagents (Erbicin,
EDIAs, Herceptin) with ErbB2-ECD. Exothermic heat
pulses were observed after each injection of immuno-
agent into the ErbB2-ECD solution (see insets in
Fig. 4). The integration of the heat produced per injec-
tion as a function of time, and conversion to per mole
of immunoagents, gave the corresponding binding iso-
therms (see Fig. 4). The data, plotted as a function of
the molar ratio, indicate a binding stoichiometry of
1 : 1 for ERB-hRNase and Erbicin and of 0.5 : 1 for
ERB-hcAb and Herceptin, i.e. each identical antigen
binding site binds one molecule of ErbB2-ECD. The
binding constants (K
b
; converted for comparison to
K
D
) and enthalpy changes (D
b
H°), obtained by stan-
dard equations, led to the thermodynamic parameters
summarized in Table 3. Their inspection reveals K
D
values close to those obtained with ELISA and SPR

(see Tables 1, 2) for the binding of monovalent Erbicin
and ERB-hRNase. With regard to the complexes with
bivalent ERB-hcAb and Herceptin, the K
D
values were
F. Troise et al. Binding of human immunoagents to ErbB2
FEBS Journal 275 (2008) 4967–4979 ª 2008 The Authors Journal compilation ª 2008 FEBS 4973
higher than those obtained with ELISA, but close to
those determined by SPR, based on either the kinetic
constants or equilibrium measurements analysed using
the Scatchard equation. In particular, they were close
to the K
D1
values calculated at high ligand concentra-
tions when monovalent binding prevails (see Table 2).
As shown by the D
b
H° values in Table 3, binding is
driven by a favourable binding enthalpy, but opposed
by an unfavourable binding entropy change, D
b
S°.
It is of interest that, of the studied systems, Erbicin
shows the lowest affinity for ErbB2-ECD, in agree-
ment with ELISA and SPR results, and the interaction
is characterized by the lowest enthalpy change.
This indicates a lower number of non-covalent
interactions on binding of EDIAs (ERB-hcAb and
ERB-hRNase), as also revealed by ELISA and SPR
(see above). However, the unfavourable D

b
S° value
recorded for these interactions indicates a greater dec-
rease in conformational stability on complex formation.
When an attempt was made to study the interactions
of the immunoagents with live cells, i.e. with ErbB2
inserted on the cell membrane, it was verified that the
experimental system could, in principle, be used, with
stoichiometric values identical to those obtained with
ErbB2-ECD and immunoagents in solution. However,
surprising results were found: the D
b
H° values were
about 100-fold higher than those measured with solu-
ble receptor and ligand, and the binding constants
were about 1000-fold higher.
To verify whether the very high D
b
H° and affinity
constants could be related to events occurring on inter-
nalization of the immunoagents, the experiments were
–100
–50
0
A B
C D
0 2000 4000 6000 8000 10 000
0
1
2

3
0.0 0.5 1.0 1.5 2.0 2.5 3.0
–350
–300
–250
–200
–150
Time (s)
Power (µJ·s
–1
)
0.15
0.20
0.25
–60
–30
0
[Erbicin]/[ECD]
[ERB-hRNase]/[ECD]
2000 4000 6000 8000 10 000
–0.15
–0.10
–0.05
0.00
0.05
0.10
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0
–150
–120
–90

Time (s)
Power (µJ·s
–1
)
Power (µJ·s
–1
)
–100
–80
–60
–40
–20
0
0.6
0.9
1.2
1.5
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8
–180
–160
–140
–120
[ERB-hcAb]/[ECD]
0
0 2000 4000 6000 8000 10 000
–0.3
0.0
0.3
Time (s)
–120

–100
–80
–60
–40
–20
0.3
0.6
0.9
1.2
1.5
Power (µJ·s
–1
)
kJ·mol
–1
kJ·mol
–1
kJ·mol
–1
kJ·mol
–1
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4
–160
–140
[Herceptin]/[ECD]
0 2000 4000 6000 8000 10 000
–0.3
0.0
Time (s)
Fig. 4. Determination by ITC of the binding interactions between anti-ErbB2 immunoagents and ErbB2-ECD (ECD). Binding isotherms are

shown for the titration of ErbB2-ECD with Erbicin (A), ERB-hRNase (B), ERB-hcAb (C) and Herceptin (D). The raw data are shown in the
insets.
Table 3. Thermodynamic parameters of Erbicin, EDIAs and Herceptin for soluble ErbB2-ECD by ITC assays.
nK
b
⁄ 10
7
(M
)1
) K
D
(nM) D
b
H° (kJÆmol
)1
) TD
b
S° (kJÆmol
)1
) D
b
G° (kJÆmol
)1
)
Erbicin ⁄ ErbB2-ECD 1.0 1.3 ± 0.3 77 ± 19 )139 ± 8 )260 ± 4 )44 ± 11
ERB-hRNase ⁄ ErbB2-ECD 1.0 4.8 ± 1.2 21 ± 5 )300 ± 18 )95 ± 20 )40 ± 10
ERB-hcAb ⁄ ErbB2-ECD 0.5 2.2 ± 0.6 45 ± 11 )175 ± 11 )133 ± 16 )48 ± 11
Herceptin ⁄ ErbB2-ECD 0.5 8.4 ± 2.1 12 ± 3 )150 ± 6 )105 ± 5 )45 ± 11
Binding of human immunoagents to ErbB2 F. Troise et al.
4974 FEBS Journal 275 (2008) 4967–4979 ª 2008 The Authors Journal compilation ª 2008 FEBS

repeated using, as immunoagent, an anti-ErbB2 mono-
clonal which was not internalized (anti-ErbB2 N28), or
by testing cells poisoned to inhibit endocytosis. Very
similar values were obtained. This indicates that the
surprising values are not a result of the internalization
process.
As an alternative, we concluded that the interactions
of anti-ErbB2 immunoagents with ErbB2 on live cells
could not be interpreted as simple ligand ⁄ receptor
interactions. It has been anticipated in recent reports
that ligand binding to cell receptors may trigger higher
order events in the membrane of targeted cells. In
these events, directly stimulated receptors and other
seemingly unrelated receptors and effectors are
engaged in the formation of complex networks and
receptor mosaics [22], and may induce membrane
bending and remodelling [23]. An ErbB signalling net-
work was proposed [24] after an analysis at the sys-
tems level, with ErbB2 as an amplifier of the network
[25]. Thus antibody binding, which mimics ligand
binding, may set off events beyond binding, which are
demonstrated by a higher binding affinity and a high
heat production.
Discussion
The novel antitumour immunoagents Erbicin, ERB-
hcAb and ERB-hRNase have most, if not all, the
features that make an immunoagent a valid, precious
tool for anticancer immunotherapy: (a) they are all of
human origin, which strongly decreases, if not elimi-
nates, the risks of an immune response; (b) they are

directed to a cell receptor, such as ErbB2, which is
minimally present in non-malignant cells, but overex-
pressed in many carcinomas, especially in breast cancer
cells; (c) they selectively kill ErbB2-positive cells, both
in vitro and in vivo; (d) their size, smaller than that of
immunoglobulins, should favour penetration in solid
tumours; however, in the case of ERB-hcAb and ERB-
hRNase, it should also allow for a prolonged half-life
in the bloodstream.
Binding to a cell-embedded tumour-associated anti-
gen is the first key step in the mechanism of antitu-
mour immunoagents. Thus, we directed our attention
to studying the binding properties of the novel EDIAs,
as well as Herceptin, an immunoagent successfully
employed in the therapy of breast cancer. Further-
more, the availability of soluble ErbB2-ECD enabled
us to describe, for the first time, the binding of these
immunoagents to the isolated, free ECD of ErbB2. In
addition, for the first time, the binding study was con-
ducted not only using a semiquantitative methodology,
such as that based on ELISA, previously used to
measure Herceptin binding [14], but also using quanti-
tative methods based on physicochemical principles,
such as SPR and ITC.
The main results of this study can be summarized as
follows.
1. For the first time, extensive and conclusive infor-
mation is reported on the relative affinity and binding
kinetics of the EDIAs and Herceptin for soluble or
cell-linked ErbB2. 2. The results were validated by the

use of three independent methodologies, ELISA, SPR
and ITC, which gave coherent results. 3. The binding
of Erbicin to ErbB2-ECD was found to be enhanced
and stabilized by the linking of Erbicin scFv to either
an RNase or the Fc antibody fragment, as in ERB-
hRNase and ERB-hcAb, respectively. This was
revealed by the higher binding affinity of the Erbicin
immunoconjugates with respect to that of free Erbicin
scFv. 4. The novel EDIAs display a binding affinity
towards soluble ErbB2-ECD which is lower than that
measured for ECD embedded in the membrane of
ErbB2-positive cells. Herceptin, by contrast, shows a
higher affinity for soluble ErbB2-ECD. Furthermore,
binding of ERB-hcAb to cancer cells and its antitu-
mour activity are not affected by soluble ECD,
whereas the same properties of Herceptin are strongly
inhibited. As soluble ECD is proteolytically released
from the surface of ErbB2-overexpressing cancer cells,
and is detected in the serum of patients with advanced
breast cancer, a fraction of Herceptin is neutralized in
these patients by serum ECD, and hence its cell-direc-
ted antitumour action is reduced [19]. It has been
reported that free, soluble ECD can induce resistance
to the growth inhibitory activity of anti-ErbB2 anti-
bodies [26], can neutralize their activity and affect their
pharmacokinetics, thus leading to resistance to immu-
notherapy [27]. However, the use of immunoagents
with a low affinity for soluble ECD, such as the Erbi-
cin-based immunoagents, will allow for lower thera-
peutic doses to be used compared with those needed

for Herceptin-based therapy. 5. A binding study car-
ried out by ITC on anti-ErbB2 immunoagents tested
directly on live cells revealed that the association of
the immunoagents with the receptor inserted into live
cells cannot be interpreted as a simple ligand ⁄ receptor
interaction. Antibody binding, just like ligand binding,
triggers higher order events which engage other mem-
brane receptors and effectors in the formation of com-
plex networks and receptor mosaics. These data
strongly imply that ITC on live cells and high-affinity
antibodies to ErbB2, a critical receptor for which no
specific ligand has yet been found, could be employed
in a systems biology approach to unravel the physio-
logical significance of the cell receptor.
F. Troise et al. Binding of human immunoagents to ErbB2
FEBS Journal 275 (2008) 4967–4979 ª 2008 The Authors Journal compilation ª 2008 FEBS 4975
Experimental procedures
Cell lines and antibodies
The 293 cell line (human embryonic kidney) was cultured
in DMEM (Gibco Life Technologies, Paisley, UK). The
SKBR3 cell line (human breast cancer) was cultured in
RPMI 1640 (Gibco Life Technologies). The media were
supplemented with 10% heat-inactivated fetal bovine
serum, 50 UÆmL
)1
penicillin and 50 lgÆmL
)1
streptomycin
(all from Gibco Life Technologies). All the cell lines were
obtained from the American Type Culture Collection and

cultured at 37 °Cina5%CO
2
atmosphere.
The antibodies used were as follows: Herceptin (Genen-
tech, South San Francisco, CA, USA); horseradish peroxi-
dase-conjugated anti-His IgG1 (Qiagen, Valencia, CA,
USA); horseradish peroxidase-conjugated goat anti-human
affinity-isolated IgG1 (Fc-specific; Sigma, St Louis, MO,
USA). Erbicin, ERB-hRNase and ERB-hcAb were pre-
pared as described previously [12–14]. The anti-ErbB2 N28
monoclonal was a generous gift from Michael Sela (Weiz-
man Institute of Science, Rehovot, Israel).
Production of ErbB2-ECD
ErbB2-ECD, the extracellular domain of ErbB2 (residues
1–624), was stably expressed and secreted by the 293 cell
line. The culture medium of 293 cells, before and after
transfection, was analysed: (a) by 8% SDS-PAGE and wes-
tern blotting with Herceptin followed by horseradish perox-
idase-conjugated anti-human (Fc-specific) IgG serum
fraction; (b) by immunoprecipitation assays carried out by
the incubation of 10 mL aliquots of 293 cell conditioned
medium with 10 lgÆmL
)1
of Herceptin or ERB-hcAb in
NaCl ⁄ P
i
for 3 h at 4 °C. The immune complexes were then
collected by adsorption to protein A-Agarose (Sigma) for
1 h at 4 °C. After washing with NaCl ⁄ P
i

, the proteins were
released by boiling in loading buffer [28], and run using 8%
SDS-PAGE, followed by immunoblotting assays as
described above.
Protein purification
ErbB2-ECD, secreted by transfected 293 cells, was purified
from the culture medium by immunoaffinity chromatogra-
phy with the AKTA Purifier system (GE Healthcare, Amer-
sham Bioscience AB, Uppsala, Sweden). The affinity
column was prepared by coupling 8 mg of Herceptin to
1.5 g of CNBr-activated Sepharose 4B Fast Flow (GE
Healthcare). The antibody was immobilized to agarose via
a secondary amine according to the manufacturer’s instruc-
tions. The resulting 4 mL column was loaded with 10 mL
of 10-fold concentrated conditioned medium, washed with
three volumes of 10 mm Tris ⁄ HCl, pH 7.4 and eluted with
50 mm glycine pH 3.0 containing 1 m NaCl. The collected
fractions were immediately neutralized with a 1 : 10 volume
of 1 m Tris ⁄ HCl pH 8.0.
The purity of the preparation was evaluated by 8% SDS-
PAGE, followed by Coomassie staining or western blotting
analyses with either Herceptin or ERB-hcAb as primary
antibody, followed by horseradish peroxidase-conjugated
anti-human IgG1 (Fc-specific) mAb.
ELISA
The affinity of Erbicin or ERB-hRNase for soluble
ErbB2-ECD was measured by an ELISA sandwich assay.
A 96-well plate was coated with 5 lgÆmL
)1
of Herceptin in

NaCl ⁄ P
i
(Sigma), kept overnight at 4 °C and blocked
for 1 h at 37 °C with 5% BSA (Sigma) in NaCl ⁄ P
i
.To
the plate, rinsed with NaCl ⁄ P
i
, a solution of purified
ErbB2-ECD in NaCl ⁄ P
i
(5 lgÆmL
)1
) was added. After 1 h
at room temperature, the plate was washed, and increasing
concentrations of purified ERB-hRNase or Erbicin (50–
500 nm) were added in ELISA buffer (NaCl ⁄ P
I
–BSA 1%)
in triplicate wells, and incubated for 2 h at room tempera-
ture with a blank control of NaCl ⁄ P
i
. After rinsing with
NaCl ⁄ P
i
, an anti-His horseradish peroxidase-conjugated
IgG1 was added in ELISA buffer. After 1 h at room
temperature, the plate was rinsed with NaCl ⁄ P
i
, and bound

immunoagents were detected using 3,3¢,5,5¢-tetramethyl-
benzidine as a substrate (Sigma).
The product was measured at 450 nm using a microplate
reader (Multilabel Counter Victor 3, Perkin Elmer,
Cologno Monzese, Italy).
The affinity of ERB-hcAb or Herceptin antibodies for
ErbB2-ECD was measured as follows. A 96-well plate was
coated with 5 lgÆmL
)1
of purified ECD in NaCl ⁄ P
i
and left
overnight at 4 °C. After blocking as above, increasing
concentrations of ERB-hcAb (10–60 nm ) or Herceptin
(0.1–10 nm) were added in ELISA buffer for 2 h at room
temperature. The plate was rinsed with NaCl ⁄ P
i
and an
anti-human (Fc-specific) horseradish peroxidase-conjugated
IgG serum fraction was incubated for 1 h, and detected as
described above. The reported affinity values are the means
of at least three determinations (standard deviation, £ 5%).
ELISAs with ErbB2-positive cells were carried out
on SKBR3 cells as described previously [14]. ERB-hcAb
(1–16 nm) or Herceptin (1–8 nm) was tested in the presence
or absence of soluble ErbB2-ECD, added either in equimo-
lar amounts or in a 10-fold molar excess to the ErbB2
receptor number on SKBR3 cells [18].
Cytotoxicity assays
ErbB2-positive cells were treated as described previously

[14] with ERB-hcAb or Herceptin at concentrations of
2.5 nm in the absence or presence of soluble ErbB2-ECD
(20 nm). Cell growth inhibition was reported as the percent-
age of cell survival reduction induced by the treatment with
Binding of human immunoagents to ErbB2 F. Troise et al.
4976 FEBS Journal 275 (2008) 4967–4979 ª 2008 The Authors Journal compilation ª 2008 FEBS
the immunoagents alone or combined with ECD, with
respect to control untreated cultures. Typically, standard
deviations were below 10%.
SPR analyses
The SPR [29] analyses were performed at 25 °C on a BIA-
core X instrument (Biacore AB, Uppsala, Sweden),
equipped with research-grade CM5 sensor chips (Biacore
AB). The running buffer was HBS-EP (10 mm Hepes,
0.15 m NaCl, 3.4 mm EDTA and 0.005% surfactant P20 at
pH 7.4). Coupling reagents, N-hydroxysuccinimide, 1-ethyl-
3-(3-dimethylaminopropyl)-carbodiimide hydrochloride,
ethanolamine hydrochloride and HBS-EP running buffer
were purchased from Biacore AB. Soluble carboxymethyl-
dextran sodium salt was obtained from Fluka (Buchs SG,
Switzerland) and Protein A from Staphylococcus aureus was
purchased from GE Healthcare.
To investigate the binding properties of Herceptin and
ERB-hcAb to the soluble ECD of the ErbB2 receptor, a
capture method was chosen. Herceptin and ERB-hcAb
were captured by Protein A from Staphylococcus aureus
immobilized on the surface of a CM5 sensor chip using
standard amine coupling chemistry. Typically, 5000 RU of
Protein A were captured [30]. Herceptin (2 lgÆmL
)1

)or
ERB-hcAb (1 lgÆmL
)1
) was injected onto the chip at a flow
rate of 30 lLÆmin
)1
, giving typically a response of 800–
1200 RU for Herceptin and 100–200 RU for ERB-hcAb.
ErbB2-ECD (10–950 nm) was passed over the immobilized
antibodies at a constant flow rate of 30 lLÆmin
)1
, and asso-
ciation and dissociation phases were recorded for 200 and
300–600 s, respectively. The sensor surface was regenerated
by 30 s injections of 10 mm glycine–HCl, pH 2.2, at the
end of each binding cycle.
To measure the binding properties of Erbicin and ERB-
hRNase to the extracellular domain of ErbB2, ECD was
immobilized onto the surface of sensor chip CM5 using the
standard amine coupling chemistry described previously
[17]. Typically, 500 RU of ErbB2-ECD were immobilized
onto the sensor surface. Binding curves were recorded by
injecting Erbicin (5–700 nm) or ERB-hRNase (3–700 nm)
over the immobilized ErbB2-ECD at a constant flow rate
of 30 l LÆmin
)1
. Association and dissociation phases were
recorded for 200 and 300 s, respectively. At the end of each
detection, the sensor surface was regenerated by injecting
10 lLof10mm NaOH. A second set of binding curves

was recorded for ERB-hRNase in the presence of soluble
carboxymethyl-dextran at a final concentration of
5mgÆmL
)1
.
The rate constants of the interactions described above
were calculated by non-linear analysis of the association
and dissociation curves using SPR kinetic evaluation soft-
ware (package BIAevaluation 3.2, Biacore AB), fitting data
to the 1 : 1 Langmuir binding model. Values of v
2
for the
fits were £ 0.8, indicating good fits. The equilibrium dissoci-
ation constants (K
D
) were calculated from the values of
the association rate constant k
a
and dissociation rate
constant k
d
, according to the thermodynamic relationship
K
D
= k
d
⁄ k
a
. Standard deviations were obtained from three
independent analyses using different biosensors, sample

preparations and ligand densities on the flow cell surfaces.
Surface plasmon resonance was also employed to carry out
equilibrium binding analyses of Herceptin and ERB-hcAb,
as bivalent analytes, and ERB-hRNase, as a monovalent
analyte, to ErbB2-ECD. For this purpose, Herceptin,
ERB-hcAb or ERB-hRNase was passed over ErbB2-ECD
(500 RU) immobilized on a CM5 sensor chip [17]. Increas-
ing concentrations of Herceptin (0.5–361 nm), ERB-hcAb
(7.6–356.6 nm) or ERB-hRNase (22–700 nm) were injected
at 5 lLÆmin
)1
. The equilibrium responses (R
eq
) for each
concentration were determined by subsequent injections of
analytes over the chip. The equilibrium responses (the
mean of two determinations) were plotted versus the
analyte concentrations to obtain the binding isotherms.
Data were fitted using a non-linear regression analysis
with GraphPad Prism version 4.0 (GraphPad Software,
San Diego, CA, USA). For Herceptin and ERB-hcAb, the
two-site binding model was used to fit the data.
Affinity constants were calculated from Scatchard plots
[31] of the equilibrium binding data, obtained by plotting
the equilibrium responses (R
eq
) ⁄ analyte concentration ratio
versus the equilibrium responses (R
eq
). The maximum bind-

ing responses (B
max
) were calculated from the x intercepts.
ITC analyses
ITC measurements were performed as described previously
[32]. Briefly, titrations were performed using a CSC 4200
calorimeter from Calorimetry Sciences Corporation (Provo,
UT, USA) with a cell volume of 2 mL. The concentration
of ErbB2-ECD in the cell was about 30 lm , and the immu-
noagent (Erbicin, ERB-hRNase, ERB-hcAb, Herceptin)
concentration in the syringe was about 3 lm. For each
titration, 10 lL aliquots of immunoagents in NaCl ⁄ P
i
solu-
tion were injected into the ErbB2-ECD solution in NaCl ⁄ P
i
at 400 s intervals. Binding curves involved the addition of
25 injections. The heat of dilution of the immunoagents
into the solvent was measured in a separate experiment.
The data were integrated, corrected for the heats of dilu-
tion, normalized for concentration and analysed assuming a
model based on a single set of identical independent bind-
ing sites, using the Bindwork software supplied with the
instrument, which provided the stoichiometry of binding (n
ligand : protein), the change in enthalpy (DH) and the bind-
ing constant K
b
.
For the experiments with immunoagents binding directly
to ErbB2-positive cells, SKBR3 cells were grown to

half-confluence, left overnight in the absence of serum and
collected from the plate by cell dissociation solution. After
equilibration in a buffer containing NaCl ⁄ P
i
with 3% BSA,
F. Troise et al. Binding of human immunoagents to ErbB2
FEBS Journal 275 (2008) 4967–4979 ª 2008 The Authors Journal compilation ª 2008 FEBS 4977
2.5 · 10
5
cells, corresponding to 0.83 pmol of ErbB2 recep-
tor [18], were diluted to 2 mL in buffer and introduced into
the sample compartment of the instrument. The immuno-
agents were added by successive injections as above, up to
a total of 1.66 pmol. The data were treated as described
above for the experiments with soluble proteins. In the
experiments in which cell endocytosis was blocked, cells
were treated, before their introduction in the apparatus
sample chamber, with 2-deoxy-glucose (50 mm) and sodium
azide (10 mm) for 2 h at 37 °C.
Acknowledgements
We thank Richard P. Junghans for critical reading of
the manuscript and for suggestions. This work was
financially supported by the Associazione Italiana per
la Ricerca sul Cancro, Italy, Ministero dell’Universita
`
e della Ricerca, Italy and Biotecnol, S.A., Portugal.
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