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(Received for publication, December 2, 1994)
From the
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
We have constructed, expressed, and purified a fusion protein,
HAR-TX 
2, consisting of heregulin-2 fused to a
binding-defective form of Pseudomonas exotoxin A, PE40. The
fusion protein was found to induce receptor tyrosine phosphorylation in
CEM cells transfected with HER4 alone or in combination with HER2 but
not in cells transfected with HER2 or HER1 alone. The phosphorylation
of receptor tyrosines was both dose-dependent and saturable in amounts
similar to those shown to be active for native heregulin. HAR-TX 2
was specifically cytotoxic toward a variety of carcinoma cell lines in
the ng/ml range. However, some tumor cell lines were found to be
insensitive to the cytotoxic action of the fusion protein even at >2
µg/ml. Relative amounts of HER4, HER3, and HER2 were determined on
seven cell lines sensitive and four cell lines insensitive to HAR-TX
2. All lines that express HER4 were killed by HAR-TX 2, while
none lacking HER4 were affected. HAR-TX 2 was able to bind to and
signal via tyrosine phosphorylation in cell lines that co-express HER2
and HER3 in the absence of HER4 without inducing cytotoxicity. Thus
HAR-TX 2 may prove to be a useful reagent for the targeting and
elimination of HER4-positive tumor cells.
Heregulin is part of a family of ligands that have structural
homology with epidermal growth factor (EGF) (
)and has been
shown to specifically bind to the protein product of the HER4 gene,
HER4/p
(1) . Recent data indicate that
heregulin also binds directly to HER3 (2) but that HER3 with
HER2 forms a high affinity receptor for heregulin(3) .
Heregulin has also been shown to bind to HER4 with 7-fold greater
affinity than to HER3(4) . Heregulin was originally purified
from conditioned medium of the human breast tumor line MDA-MB-231 as a
variety of related forms and was postulated to bind directly to
HER2(5) . To date, a variety of isoforms have been isolated
that are members of the heregulin family. They include acetylcholine
receptor-inducing activity, glial growth factor, gp30, and
p75(6, 7, 8, 9) . Neu
differentiation factor (NDF), a rat homologue of heregulin, has also
been identified(10) .
To determine whether heregulin would
be an effective ligand for the development of a targeted cytotoxic
molecule, a gene fusion encoding heregulin 2 with the hydrophilic
leader sequence of amphiregulin (AR) at its amino terminus (for
purification purposes) and a binding-defective form of Pseudomonas exotoxin A (11) was constructed and expressed in Escherichia coli. Following refolding and purification, HAR-TX
2 was tested for direct binding to tumor cells and for the ability
to induce tyrosine phosphorylation of receptors in cells transfected
with various HER family members. The cell-killing activity of HAR-TX
2 was measured against tumor cells and correlated with their
expression levels of HER4, HER3, and HER2 as determined by image
analysis with antibodies to each receptor.
2-Ig came
from Dr. J.-M. Colousco, both of Bristol-Myers Squibb (Seattle, WA).
Breast carcinoma cell lines BT474, MDA-MB-453, T47D, SKBR3, and MCF-7,
LNCaP prostate carcinoma, CEM T cell leukemia, and the ovarian
carcinoma cell line SKOV3 were purchased from ATCC (Rockville, MD). The
H3396 breast carcinoma line and the L2987 lung carcinoma line were
established at Bristol-Myers Squibb, Seattle, WA. BT474 and T47-D cells
were cultured in Iscove's modified Dulbecco's medium
supplemented with 10% fetal bovine serum (FBS) and 10 µg/ml
insulin. MCF-7, H3396, LNCaP, and L2987 were cultured in Iscove's
modified Dulbecco's medium supplemented with 10% FBS. SKBR3 and
SKOV3 cells were cultured in McCoy's medium supplemented with 10%
FBS, and the MDA-MB-453 cells were cultured in Dulbecco's
modified Eagle's medium supplemented with 10% FBS and 0.5%
nonessential amino acids. AU565 human breast carcinoma cells were
obtained from the Cell Culture Laboratory, Naval Biosciences Laboratory
(Naval Supply Center, Oakland, CA) and were cultured in RPMI 1640
supplemented with 15% FBS. CEM transfectants (1) (CEM/HER1,
CEM/HER2, CEM/HER4, and CEM/HER2-HER4) were cultured in RPMI
supplemented with 10% FBS and 500 µg/ml G418.
2 Expression
Plasmid
2 encoded in pSE 8.4 was transformed into E. coli BL21 (
DE3) and expressed by fermentation in T
broth containing 100 µg/ml ampicillin at 37 °C until A
= 4.8 followed by induction with 1
mM IPTG. The cells were harvested after 90 min by
centrifugation and frozen at -70 °C. The cell pellet was
thawed and suspended in 4 °C solubilization buffer (50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 1 µg/ml leupeptin, 2
µg/ml aprotinin, 1 µg/ml pepstatin A, 0.5 mM phenylmethylsulfonyl fluoride) containing 1% Tergitol by
homogenization and sonication. The suspension was pelleted by
centrifugation and washed three times with solubilization buffer
containing 0.5% Tergitol (first wash), 1 M NaCl (second wash),
and buffer alone (third wash). The final cell pellet was dissolved in
6.5 M guanidine HCl, 0.1 M Tris-HCl (pH 8.0), 5
mM EDTA, sonicated, and refolded by rapid dilution (100-fold)
into 0.1 M Tris-HCl (pH 8.0), 1.3 M urea, 5 mM EDTA, 1 mM glutathione, and 0.1 mM oxidized
glutathione at 4 °C. Refolded HAR-TX 2 protein was diluted
2-fold with 50 mM sodium phosphate (pH 7.0) and applied to a
cationexchange resin (POROS 50 HS; PerSeptive Biosystems, Cambridge,
MA) equilibrated in the same buffer. HAR-TX 2 protein was eluted
at 400-450 mM NaCl in 50 mM sodium phosphate
(pH 7.0), and fractions were analyzed using SDS-polyacrylamide gel
electrophoresis and Coomassie Blue staining. Final purification of
pooled fractions was performed by chromatography using Source 15S
cation-exchange media (Pharmacia Biotech Inc.) equilibrated with 50
mM sodium phosphate (pH 6.0). HAR-TX 2 fusion protein was
eluted with a gradient of 0-1 M NaCl in the same buffer
and analyzed by SDS-polyacrylamide gel electrophoresis.
10
cells) were stimulated
with indicated amounts of HAR-TX 2 for 5 min at room temperature.
The cells were pelleted and resuspended in 0.1 ml of lysis buffer (50
mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM MgCl
, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% sodium
dodecyl sulfate, 1 mM sodium ortho-vanadate) at 4
°C. Insoluble material was pelleted for 30 s at 10,000 g, and samples were analyzed by SDS-polyacrylamide gel
electrophoresis and Western blot using anti-phosphotyrosine antibodies
(4G10, ICN, Irvine, CA; and PY20, Upstate Biotechnology, Lake Placid,
NY) followed by 
I-labeled sheep anti-mouse IgG,
F(ab`) fragment (DuPont NEN).
cells/ml) in growth medium were added to 96-well
flat bottom tissue culture plates (0.1 ml/well) and incubated at 37
°C for 16 h. Cells were incubated with HAR-TX 2 for 48 h at 37
°C and washed twice with phosphate-buffered saline followed by
addition of 200 µl/well of 1.5 µM calcein AM
(Molecular Probes Inc., Eugene, OR). The plates were incubated for 40
min at room temperature, and the fluorescence was measured using a
fluorescence concentration analyzer (Baxter Healthcare Corp.,
Mundelein, IL) at excitation/emission wavelengths of 485/530 nm.
Calcein AM is membrane-permeable and virtually nonfluorescent. When it
is hydrolyzed by intracellular esterases, an intensely fluorescent
product, calcein, is formed. The percentage of cytotoxicity was
calculated as described previously(15) . Competitive
cytotoxicity assays were done by co-incubation of heregulin 2-Ig
(0.002-5.0 µg/ml) with HAR-TX 2 (50 ng/ml) on LNCaP and
MDA-MB-453 cells. Chimeric L6 (huIgG1) (16) was used as an
isotype-matched control for the competition assay.
The staining procedure was as
follows. Cells were fixed in 10% neutral buffered formalin for 60 min
at room temperature, washed with HO, rinsed with
Tris-buffered saline (0.05 M Tris, 0.15 M NaCl, pH
7.6), and blocked with 10% goat serum (for HER2) or rabbit serum (for
HER3 and HER4) in 0.1% bovine serum albumin/Tris-buffered saline for 15
min. Next, primary, secondary, and tertiary reagents were incubated for
30, 20, and 15 min, respectively, at room temperature and with
Tris-buffered saline washing between steps. Final detection was
achieved using Cellular Analysis Systems red chromagen (Becton
Dickinson, Elmhurst, IL) for 4 (HER2), 8-10 (HER3), and
10-12 min (HER4) at room temperature. Counterstaining was
performed with Cellular Analysis Systems DNA stain protocol (Becton
Dickinson).
I-HAR-TX 2 Binding
Experiments2 (100 µg) was labeled with IODO-GEN
(Pierce) in phosphate-buffered saline for 15 min at room temperature
and was purified through PD10 (Pharmacia) chromatography. The specific
activity of the I-HAR-TX 2 was determined to be 6.6
µCi/µg. Adherent tumor cells were gently removed from flasks by
incubation for 10 min at 37 °C with 2 mM EDTA in
phosphate-buffered saline. Receptor binding reactions were performed at
4 °C for 16 h with agitation using 1.67 10 cells/100-µl reaction volumes in the presence or absence of a
200-fold excess of unlabeled HAR-TX 2. Cell-bound I-HAR-TX 2 was separated from unbound by
centrifugation through a 50:50 mixture of dibutyl phthalate and dioctyl
phthalate at 4 °C, and the radioactivity present in both
supernatant and cell pellet was determined using a 
-counter.
22 expression plasmid, encoding the
hydrophilic leader sequence from AR, heregulin 2, and PE40, under
the control of the IPTG-inducible T7 promoter, was constructed as
described under ``Experimental Procedures'' and is
diagrammatically shown in Fig. 1A. The AR leader sequence
was added to the N terminus of heregulin to facilitate purification as
was previously demonstrated with ligand alone (1) (Fig. 1B).
Figure 1:
A, schematic diagram of the expression
plasmid (pSE 8.4) encoding HAR-TX 2; B, amino acid
sequence of the chimeric HAR 2 ligand composed of the AR leader
sequence and rat heregulin 2.
HAR-TX 2 fusion protein
contained in E. coli inclusion bodies was denatured and
refolded as described under ``Experimental Procedures'' and
applied to cation-exchange chroma-tography on a POROS HS column. The
major protein band migrating at 51 kDa corresponded to HAR-TX 2 (Fig. 2, lane2). The column flow-through from
POROS HS contained only small amounts of HAR-TX 2 (Fig. 2, lane3). POROS HS chromatography resulted in >50%
purity of HAR-TX 2 (Fig. 2, lane4).
Further purification, to >95% purity, was achieved by chromatography
using Source 15S cation-exchange resin (Fig. 2, lane5). The monomeric nature of purified HAR-TX 2 was
determined by nonreducing SDS-polyacrylamide gel electrophoresis (Fig. 2, lane6), which showed the same
migration pattern as under reducing conditions (Fig. 2, lane5).
Figure 2:
Purification of HAR-TX 2 fusion
protein. SDS-polyacrylamide gel electrophoresis (4-20%) stained
with Coomassie Brilliant Blue is shown. Lane1,
molecular mass standards; lane2, refolded HAR-TX
2 (concentrated 20 times); lane3, POROS HS
flow-through (concentrated 20 times); lane4, POROS
HS eluate; lane5, Source 15S eluate (pure HAR-TX
2, 2 µg); lane6, 2 µg HAR-TX 2 (lanes1-5 were reduced; lane6 was nonreduced).
2 induced receptor
phosphorylation of HER4 as previously demonstrated for
heregulin(2) . CEM cells expressing different HER family
members were analyzed by phosphotyrosine immunoblots following exposure
to HAR-TX 2. HAR-TX 2 (10 nM) induced tyrosine
phosphorylation in CEM cells expressing HER4 either alone or together
with HER2 but not in cells expressing only HER2 or HER1 (Fig. 3A). This demonstrates that HER4 and not HER2 or HER1
is sufficient for tyrosine phosphorylation of receptor in response to
HAR-TX 2. The fact that HAR-TX 2 does not induce tyrosine
phosphorylation in CEM cells transfected with HER1 shows that the
hydrophilic leader sequence from amphiregulin does not affect the
specificity of the heregulin moiety in its selective interaction
between receptor family members. HAR-TX 2-mediated phosphorylation
of receptors in CEM cells co-expressing HER2 and HER4 was found to be
both dose-dependent and saturable (Fig. 3, B and C).
Figure 3:
Tyrosine phosphorylation in CEM cells
expressing HER4 is induced by HAR-TX 2. A, CEM cells
co-expressing HER4 (H4) and HER2 (H2), HER4 alone,
HER2 alone, or HER1 (H1) alone were incubated in the presence
(+) or absence(-) of 10 nM HAR-TX 2,
solubilized, and immunoblotted with anti-phosphotyrosine mAb (PY20).
The arrow indicates phosphorylated receptor. Molecular mass
standards are in kDa. B, CEM cells co-expressing HER4 and HER2
were incubated in the presence of HAR-TX 2 (0.2-137
nM) and treated as in A. C, plot of
radioactive counts found in each receptor band from B as
determined by a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) versus the amount of added HAR-TX
2.
2 against Tumor
Cells2 was determined
against a variety of human cancer cell lines. AU565 and SKBR3 breast
carcinomas and LNCaP prostate carcinoma were sensitive to HAR-TX 2
with EC
values of 25, 20, and 4.5 ng/ml, respectively (Fig. 4A), while SKOV3 ovarian carcinoma cells were
insensitive to HAR-TX 2 (EC
>2000 ng/ml). Addition
of heregulin 2-Ig to LNCaP cells reduced the cytotoxic activity of
HAR-TX 2 in a dose-dependent fashion (Fig. 4B). In
contrast, cL6, a chimeric mouse-human antibody with a nonrelated
specificity but matching human Fc domains(16) , did not inhibit
the HAR-TX 2 cytotoxic activity. Thus, the cytotoxic effect of
HAR-TX 2 was due to specific heregulin-mediated binding. Similar
findings were observed using MDA-MB-453 cells (data not shown).
Figure 4:
A,
cytotoxic activity of HAR-TX 2 on tumor cell lines. Cell killing
of LNCaP (
), AU565 (
), SKBR3 (
), and SKOV3 (
)
cells was determined following a 48-h incubation with HAR-TX 2 by
quantification of fluorescent calcein cleaved from calcein AM. B, competitive cytotoxicity of HAR-TX 2 with heregulin
2-Ig. LNCaP cells were co-incubated with 50 ng/ml HAR-TX 2
and competed with either heregulin 2-Ig () or L6-Ig
() at concentrations ranging from 2 to 5000 ng/ml. The data
represent the mean of triplicate assays.
2-mediated Cytotoxicity2-mediated
killing with EC values ranging from 1 to 125 ng/ml. MCF-7
cells displayed the lowest detectable levels of HER4 and were found to
be the least sensitive (EC = 125 ng/ml) of the
cells that did respond. Four cell lines were found to be devoid of any
detectable levels of HER4 on their surface, and each of these was
completely resistant to the toxic effects of HAR-TX 2. Three of
these lines, L2987, SKOV3, and H3396, display both HER2 and HER3 in the
absence of HER4. Thus, the expression of HER3, even in the presence of
HER2, was not sufficient to render tumor cells sensitive to HAR-TX
2.
2 to MCF-7, MDA-MB-453, and H3396
Cells2 for three
tumor cell lines was determined. Iodinated HAR-TX 2 specifically
bound to MCF-7 and MDA-MB-453 cells, both expressing cell surface HER2,
HER3, and HER4. High and low affinity sites were present as determined
by Scatchard analysis of the binding results for MCF-7 (K
= 125 pM, K
= 2.1 nM) and for MDA-MB-453 (K = 88 pM, K = 1.25
nM) (Fig. 5). H3396 cells, co-expressing cell surface
HER2 and HER3 and insensitive to HAR-TX 2, also displayed high and
low affinity sites (K = 300 pM, K = 3.3 nM). Thus, cells
expressing HER2 and HER3, in the presence or absence of HER4, form both
high and low affinity heregulin binding sites. This demonstrates that
while HAR-TX 2 binding to tumor cells is essential for
cytotoxicity, by itself it is not sufficient.
Figure 5:
Direct binding of I-HAR-TX
2 to tumor cells. Saturation binding (insets) and
Scatchard plots are shown for MCF-7 (A), MDA-MB-453 (B), and H3396 (C) breast carcinoma cells. Cells were
incubated at 4 °C with increasing amounts of radiolabeled HAR-TX
2 for 16 h in the presence (nonspecific bound) or absence (total
bound) of a 200-fold excess of unlabeled HAR-TX 2. Specific
binding was calculated by subtracting the nonspecific radioactivity
from the total cell-bound radioactivity. The average of triplicate
counts is shown.
2 Induces Tyrosine Phosphorylation in Tumor
Cells That Are Resistant to Cytotoxic Effects2 binds to H3396
cells that co-express HER2 and HER3. Despite binding, cells expressing
HER2 and HER3 (L2987, H3396, and SKOV3) were insensitive to HAR-TX
2. Direct interaction via signaling of H3396 and L2987 cells with
the fusion protein was determined by phosphotyrosine immunoblots
following exposure to HAR-TX 2. HAR-TX 2 was found to induce
tyrosine phosphorylation in both tumor cell types (Fig. 6)
similarly to that previously seen in COS-7 cells transfected with HER2
and HER3(3) . SKOV3 cells were found to be constitutively
phosphorylated in the presence or absence of heregulin (data not
shown), and no further activation was observed. However, previous
studies reported that heregulin does not bind to these
cells(20) . Thus, despite direct binding (H3396) and
phosphotyrosine signaling via HAR-TX 2 (H3396 and L2987), these
cells are insensitive to the cytotoxic effects of the fusion protein.
Figure 6:
Tyrosine phosphorylation in tumor cells
expressing HER3 (L2987) or co-expressing HER2 and HER3 (H3396) is
induced by HAR-TX 2. Cells were incubated in the presence (+)
or absence(-) of HAR-TX 2 (10 nM), solubilized, and
immunoblotted with anti-phosphotyrosine mAb (PY20). The arrow indicates phosphorylated receptor.
We have constructed and characterized a ligand-toxin fusion
protein, HAR-TX 2, that kills a variety of carcinoma cells that
express HER4. HAR-TX 2 was produced in E. coli by
expressing a gene fusion encoding a chimeric form of heregulin 2
and a binding mutant form of PE. The fusion protein was isolated as
insoluble material, denatured, refolded, and purified by
cation-exchange chromatography.
HAR-TX 2 induced tyrosine
phosphorylation in CEM cells expressing either HER4 alone or
co-expressing HER4 and HER2 but not in cells expressing either HER1 or
HER2 alone (Fig. 3). These data confirm earlier findings that
heregulin can induce tyrosine phosphorylation in cells expressing cell
surface HER4 in the presence or absence of HER2 but not in cells
expressing HER2 alone(1) . The phosphotyrosine activity of
HAR-TX 2 was dose-dependent and saturable (Fig. 3, B and C) and reached 50% maximal phosphorylation at
approximately 10 nM. This amount is comparable with that used
in other reports describing the phosphotyrosine activity of
heregulin(2, 3, 5) .
HAR-TX 2 was
effective at killing breast and prostate cancer cells (Fig. 4A). HAR-TX 
, containing heregulin ,
was also produced. It was found to bind >10-fold less well to
MDA-MB-453 cells, and it was >10-fold less cytotoxic to tumor cells
including MDA-MB-453, BT474, and SKBR3 cells (data not shown). Because
of these results, we focused our efforts on characterizing the in
vitro activities of HAR-TX 2.
Measurements of HER2, HER3,
and HER4 by image analysis have allowed us to gain a more accurate
understanding of the expression levels of these receptors on the cell
surface of tumor cell lines and to correlate their expression with
sensitivity to the heregulin-based toxin fusion protein. The tumor cell
lines that were sensitive to HAR-TX 2, including MDA-MB-453,
LNCaP, and T47D, co-expressed HER2, HER3, and HER4 (Table 1).
L2987, SKOV3, and H3396 cells, which co-express HER2 and HER3 but do
not express HER4, were insensitive to the cell-killing activities of
HAR-TX 2. Therefore, the expression of HER2 and HER3 is not
sufficient for HAR-TX 2-mediated killing.
It has been shown
that heregulin can bind to either HER3 or HER4 in transfected cell
lines (1, 2, 4) and that COS-7 cells
co-transfected with HER2 and HER3 bind heregulin with a higher affinity
than do cells transfected with HER3 alone(3) . Radiolabeled
HAR-TX 2 bound directly to MCF-7, MDA-MB-453, and H3396 breast
carcinoma cells, demonstrating the presence of both high and low
affinity sites with K
values ranging from 88
pM to 3.3 nM (Fig. 5). The high affinity
binding site identified on MCF-7 cells (125 pM) was similar to
that previously reported for heregulin (105 pM)(5) .
In addition we also report the presence of a low affinity site on MCF-7
cells (2.1 nM). HAR-TX 2 can also induce tyrosine
phosphorylation in H3396 cells and L2987 cells (Fig. 6), which
co-express HER3 and HER2. Thus, in MCF-7 and MDA-MB-453 cells, which
express HER2, HER3, and HER4, direct binding and cell killing are found
following incubation with HAR-TX 2. In contrast, H3396 and L2987
cells, which co-express HER2 and HER3, are insensitive to HAR-TX 2
despite direct binding (H3396) and signaling (H3396 and L2987) via the
fusion protein.
The expression of HER4 correlates with sensitivity
to HAR-TX 2. This may be due in part to the higher affinity of
heregulin for HER4, thereby maximizing uptake of the toxin.
Alternatively, HER4 may also be associated with distinct signals
directing internalization and/or subcellular trafficking, such that
PE40 reaches its target in the cytosol. In addition to the expression
of HER4, this process may require HER2, HER3, and/or some other
cellular components or HER family members whose identity is still
unknown. Regardless, our results demonstrate that heregulin-based
fusion proteins can be used to kill tumor cells that express HER4 and
suggest that such proteins may prove useful for the in vivo targeting of HER4-positive tumors.
)2,
heregulin/amphiregulin leader sequence-PE40 fusion protein; NDF, neu
differentiation factor; FBS, fetal bovine serum; CHO, Chinese hamster
ovary; AR, amphiregulin; IPTG,
isopropyl-1-thio--D-galactopyranoside; mAb, monoclonal
antibody; CAS, cellular analysis system.2-Ig.
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