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: cladelor@unina.it
*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 ª2008 The Authors Journal compilation ª2008 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 nmfor Erbicin, 30 nmfor
ERB-hRNase and 7 nmfor 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 ª2008 The Authors Journal compilation ª2008 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 ª2008 The Authors Journal compilation ª2008 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 ª2008 The Authors Journal compilation ª2008 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
Req (RU)
Req/ERB-hcAb
(RU/nM)
0
20
40
60
80
Req (RU)
-50
50
150
250
350
200 250 300 350
Req (RU)
Req/Herceptin
(RU/nM)
0
50
100
150
200
250
Req (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 nM(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 nM(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 nMfor ERB-hcAb (E) and 0.5–361 nMfor 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 ª2008 The Authors Journal compilation ª2008 FEBS 4971