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CELLULAR AND MOLECULAR
Fusion Protein Induces HER2/neu Signaling and Facilitates Repair of Injured Epithelia
Department of Microbiology, Immunology and Molecular Genetics (T.-H.H., S.L.M.) and the Molecular Biology Institute (S.L.M.), University of California, Los Angeles, Los Angeles, California
Received for publication
September 9, 2005
Accepted
November 11, 2005.
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Abstract |
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genetically fused to the carboxyl terminus of a single-chain Fv (ScFv) antibody specific for the human HER2/neu (anti-HER2/neu ScFv-TNF-) forms a homotrimeric structure that retains both TNF- activity and the ability to bind HER2/neu. In contrast to anti-HER2/neu IgG3, anti-HER2/neu ScFv-TNF- induces potent HER2/neu signaling, activating the downstream mitogen-activated protein kinase (MAPK) and Akt pathways in SKBR3 cells. Activation of MAPK and Akt by anti-HER2/neu ScFv-TNF- inhibited the apoptosis of SKBR3 cells induced by actinomycin D. Remarkably, anti-HER2/neu ScFv-TNF- facilitated the repair of injured epithelia. Accelerated wound healing required binding to HER2/neu but not TNF- activity since anti-HER2/neu ScFv-TNF- (S147Y), containing a mutant TNF- with significantly decreased biological activity, demonstrated equivalent ability to facilitate wound healing and soluble HER2/neu inhibited the effect. These results suggest that trimeric anti-HER2/neu ScFv has the potential to facilitate wound healing. In addition, fusion with TNF- provides a novel approach to producing polymeric antibodies.
), with extensive homology to human epidermal growth factor (EGF) receptors (Coussens et al., 1985). The intrinsic tyrosine kinase activity of HER2/neu has been shown to trigger a network of signaling pathways culminating in responses, including cell division, differentiation, and proliferation. Abundant evidence has supported the role of this proto-oncogene in tumorigenesis with overexpression correlated with poor prognosis in cancer patients (Slamon et al., 1987). In addition, it has recently been demonstrated that HER2/neu plays an essential role in the repair of injured airway (Vermeer et al., 2003) and corneal (Xu et al., 2004) epithelia. HER2/neu is expressed on the basolateral surface of epithelial cells, and injury allows ligands of erbB receptors to bind erbB3 or erbB4, which then forms a heterodimer with HER2/neu, and the downstream signaling promotes cell proliferation and repair of the epithelial layer.
The effects induced by the tyrosine kinase activity of HER2/neu are mediated by an intricate downstream signaling cascade. Dimerization of erbB receptors activates phosphatidylinositol 3-kinase (PI3K) (Hu et al., 1992), generating PtdIns-3,4-P2, which in turn recruits and activates Akt (Zhou et al., 2000). Activated Akt phosphorylates specific targets such as pro-caspase-9 (Cardone et al., 1998) and Bad (del Peso et al., 1997), thereby promoting cell survival. In addition, activated HER2/neu associates with a Src homology 2 domain-containing protein, SHC (Meyer et al., 1994), leading to the activation of mitogen-activated protein kinase (MAPK). Activated MAPK translocates to the nucleus and activates transcription factors, thereby promoting cell growth and development (Lenormand et al., 1998). Thus, the Akt and MAPK signaling pathways play an essential role in the cell survival and proliferation induced by HER2/neu activation.
Antibodies targeting HER2/neu have been used for therapy of HER2/neu-overexpressing tumors. Herceptin is a human IgG1 recombinant antibody designed to block HER2/neu-mediated cellular proliferation. Although Herceptin has been shown to exhibit a transient and modest agonistic effect in HER2/neu activation (Scott et al., 1991), it inhibits long-term growth of HER2/neu-overexpressing breast cancer cells in vitro (Hudziak et al., 1989) and, in combination with taxanes, improves the survival of patients with HER2-positive metastatic breast cancer (Leyland-Jones et al., 2001). Although the mechanism of the antiproliferative effect of Herceptin remains unclear, preventing ligand binding to HER2/neu is believed to contribute to this effect.
Tumor necrosis factor- (TNF-), a pleiotropic cytokine secreted primarily by activated macrophages and monocytes, exhibits a wide spectrum of biological activities including promoting cytolysis of some tumor cell lines by activating apoptosis (Laster et al., 1988), enhancing the antitumor effect of dendritic cells (Candido et al., 2001) and activating host immunity against tumors (Hock et al., 1993). Although TNF- should be an effective anticancer therapeutic agent, its clinical use is hampered by its severe systemic toxicity. The use of a tumor-specific antibody as a targeting vehicle to deliver higher doses of TNF- to the tumor site is one approach for improving the therapeutic index of TNF-. Since the trimeric structure of TNF- is essential for its biological activity, it is unlikely that TNF- fused to either the amino or carboxyl terminus of the immunoglobulin heavy chain would be active. However, a single-chain Fv (ScFv) fusion with TNF- maintained both antigen-binding specificity and TNF- activity (Cooke et al., 2002).
In the present study, we constructed a fusion protein consisting of the anti-HER2/neu ScFv, C6MH3-B1, (Schier et al., 1996) and TNF- (anti-HER2/neu ScFv-TNF-) and investigated its effect on HER2/neu-overexpressing cells. Unexpectedly, the trimeric anti-HER2/neu ScFv-TNF- exhibited robust activation of HER2/neu as well as the downstream MAPK and Akt pathways in HER2/neu-overexpressing cells. In addition, it protects HER2/neu-overexpressing cells against actinomycin D-induced apoptosis and, remarkably, facilitates the repair of injured epithelia.
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Materials and Methods |
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fusion proteins were cultured in Iscove's modified Dulbecco's medium (IMDM) (Irvine Scientific, Irvine, CA) supplemented with 2 mM L-glutamine, 10 U/ml penicillin, and 10 µg/ml streptomycin (GPS) (Sigma Chemical Company, St. Louis, MO) and 5% calf serum (Atlanta Biologicals, Norcross, GA). Murine myeloma cell lines Sp2/0 (ATCC) and P3x63Ag8.653 (ATCC) and their derivatives and D2F2/E2 (kindly provided by Dr. Wei-Zen Wei, Wayne State University, Detroit, MI), a murine mammary cell line expressing human HER2/neu on the cell surface, were grown in IMDM supplemented with 10% calf serum and GPS. J-774A.1, a murine macrophage cell line (ATCC), human breast cancer cell line SKBR3 (ATCC), and L-929 fibroblasts (ATCC) were cultured in IMDM with 5% calf serum and GPS. The human colonic epithelial cell line Caco2 (ATCC) was maintained in high-glucose Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) supplemented with 5% calf serum and GPS.
Plasmid Construction. Anti-HER2/neu IgG3 with the heavy and light chain variable regions of the humanized antibody 4D5-8 (rhuMab HER2, Herceptin; Genentech, South San Francisco, CA) and the constant region of human IgG3 has been described previously (Dela Cruz et al., 2000). The heavy and light chain variable regions of C6MH3-B1, an anti-human HER2/neu ScFv kindly provided by Dr. James D. Marks (University of California, San Francisco, CA) were inserted into the human 
3 heavy chain (pAH480) and 
light chain (pAG4622) expression vectors, respectively (Coloma et al., 1992) and used to produce chimeric IgG3 of this specificity. The construction of the anti-human HER2/neu ScFv (C6MH3-B1)-TNF- fusion protein is shown in supplemental Fig. 1A. Site-directed mutagenesis of TNF- was used to create the point mutation S147Y. The construction and characterization of an antidansyl ScFv antibody has been described previously (Coloma and Morrison, 1997). The construction of anti-dansyl ScFv-TNF- is shown in supplemental Fig. 1B.
Production and Purification of Different Recombinant Proteins. Plasmids encoding anti-HER2/neu ScFv-TNF-, anti-HER2/neu ScFv-TNF-(S147Y), and anti-dansyl ScFv-TNF- were transfected to Pro-5 using the lipofectamine plus reagent (Invitrogen). Stable transfectants were selected with 1 mM histidinol (Sigma), and the highest producers were identified using an ELISA plate coated with rat anti-mouse TNF- (BD Biosciences, San Jose, CA) and detected by biotinylated rat anti-mouse TNF- (BD Biosciences). Transfectants were expanded in 150 x 25-mm tissue culture dishes (BD Biosciences) containing protein-free CHO liquid soy medium (Hyclone Laboratories, Logan, UT), and the culture supernatants were concentrated with an Amicon stirred ultrafiltration cell (Amicon, Billerica, MA). Transfectants producing anti-HER2/neu (C6MH3-B1)-IgG3 were selected and characterized as described previously (Dela Cruz et al., 2000). The anti-HER2/neu (C6MH3-B1) IgG3 antibody was purified from culture supernatants using protein G immobilized on Sepharose 4B fast flow (Sigma). Purity and integrity were assessed by Coomassie blue staining of proteins separated by SDS-PAGE. The production and purification of soluble HER2/neu have been described previously (Dela Cruz et al., 2003).
Protein Cross-Linking. Nine microliters of PBS was mixed with 10 µl of 10 µM TNF- fusion proteins and 1 µl of a freshly made solution of 6.35 mM ethylene glycolbis(succinimidylsuccinate) (EGS) (Pierce Chemical, Rockford, IL). After a 30-min incubation at room temperature, 1 µl of 1 M glycine was added, and the solution was incubated for another 30 min. Then 5 µl of 5x SDS sample buffer and 1 µl of 2-mercaptoethanol (Fisher Scientific, Hampton, NH) were added, samples were boiled at 95°C for 5 min, and 25-µl aliquots were fractionated on 12% SDS-PAGE. Proteins were visualized by Western blot using biotinylated rat anti-mouse TNF-.
FPLC. Purified TNF- fusion proteins and standard proteins were analyzed in a 0.5 M NaCl-20 mM phosphate solution, pH 6.5, using a Superose 6HR 10/30 column (Amersham Biosciences, Piscataway, NJ) at a flow rate of 0.25 ml/min. The injection volume of 100 µl contained 40 µg of protein.
Flow Cytometry Analysis. To detect the reactivity of various ScFv-TNF- fusion proteins with D2F2/E2 cells, 1 x 106 cells were incubated at 4°C for 1 h with a 10 pM concentration of the fusion protein. Cells were then reacted with biotinylated rat anti-mouse TNF- (BD Biosciences) diluted 1:35. The bound biotinylated antibodies were detected with phosphatidylethanolamine-labeled streptavidin (BD Biosciences) diluted 1:1500 and analyzed by flow cytometry using a FACScan (Becton Dickinson).
TNF- Cytotoxicity Activity. The L-929 fibroblast cell line sensitive to the cytotoxic effect of TNF- was used to quantify the biological activity of TNF-. L-929 cells were plated in a 96-well tissue culture plate (Falcon, Lincoln Park, NJ) at a density of 4 x 104 cells/well and incubated overnight at 37°C in a 5% CO2 atmosphere. Afterward, serial dilutions of different ScFv-TNF- fusion proteins or recombinant murine TNF- were added in the presence of actinomycin D (8 µg/ml, A.G. Scientific, San Diego, CA), and the plate was incubated for 24 h. Surviving adherent cells were stained with 50 µl of crystal violet (0.05% in 20% ethanol) for 10 min. The plates were washed with water, and the remaining dye was solubilized by the addition of 100 µl of 100% methanol. Plates were read on an ELISA reader at 595 nm.
MTS Assay for the Antiapoptotic Effect of Anti-HER2/neu ScFv-TNF-. SKBR3, a human breast cancer cell line that expresses a high level of HER2/neu and has an intact HER2/neu signaling cascade, was used to test the direct apoptotic or antiapoptotic effect of different anti-HER2/neu ScFv fusion proteins. SKBR3 cells were plated in a 96-well tissue culture plate at a density of 4 x 104 cells/well and incubated overnight at 37°C in a 5% CO2 atmosphere. Afterward, serial dilutions of different ScFv alone or with the indicated competitive antibodies were added in the presence of actinomycin D (4 µg/ml), and the plate was incubated overnight. For some experiments, the fusion protein-treated SKBR3 cells were incubated with U0126 (Calbiochem, San Diego, CA) and/or LY294002 (Calbiochem) in the presence of actinomycin D (4 µg/ml) overnight. Plates were then developed by addition of 20 µl of MTS solution (Promega, Madison, WI) and measured on an ELISA reader at 490 nm.
Western Blot Analysis. SKBR3 cells were treated with different fusion proteins or antibodies for the indicated times, washed with ice-cold PBS, and lysed on ice for 10 min in lysis buffer (0.125% Nonidet P-40, 0.875% Brij 97, 10 mM Tris-HCl, pH 7.5, 2 mM EDTA, 0.15 M NaCl, 0.4 mM Na3VO4, 0.4 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 2.5 µM leupeptin, and 2.5 µM aprotinin). Cell lysates were clarified at 10,000g for 10 min at 4°C. Protein samples were then boiled in sample buffer before separation on 8% SDS-PAGE gels and transfer onto polyvinylidine difluoride microporous membranes (Millipore, Billerica, MA). After blocking with 3% bovine serum albumin in 150 mM NaCl, 50 mM Tris-HCl, pH 7.6 (TBS) for 1 h at room temperature, blots were probed with the indicated primary antibodies overnight at 4°C. The blots were then washed three times at room temperature with 0.05% Tween 20 in TBS, incubated with the appropriate secondary antibodies conjugated with horseradish peroxidase (HRP), and detected by a peroxidase-catalyzed enhanced chemiluminescence detection system (ECL; Pierce). To confirm equal loading of proteins, blots that had been probed for the phosphorylated proteins were stripped and reprobed with an antibody against an appropriate control protein. For this procedure, 10 ml of stripping buffer, consisting of 2% (w/v) SDS, 62.5 mM Tris, pH 6.7, and 100 mM 2-mercaptoethanol, was added to the membrane for 15 min with constant shaking at 60°C. The membrane was then washed (6 x 5 min in TBS), blocked, and probed with the appropriate primary antibody.
Antibodies for Western Blot Analysis. Monoclonal anti-phosphotyrosine 4G10 was obtained from Upstate Biotechnology Inc. (Lake Placid, NY). Anti-HER2/neu antibody sc-284, a rabbit polyclonal antibody against the carboxyl terminus of human HER2/neu, was obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Anti-Akt, anti-phospho-Akt (Ser473), anti-p44/42 MAPK, and antiphospho-p44/42 MAPK (E10) were obtained from Cell Signaling Technology Inc. (Beverly, MA). Polyclonal HRP-conjugated rabbit anti-mouse IgG was obtained from ICN Pharmaceuticals Inc. (Aurora, OH). Polyclonal HRP-conjugated donkey anti-rabbit IgG was obtained from Amersham Biosciences.
Wounding and Measurement of Wound Repair on Polarized Epithelia. The polarized human colonic epithelial cell line Caco2 was used to demonstrate the wound healing effect initiated by the anti-HER2/neu ScFv fusion proteins. Caco2 cells were plated in a 24-well tissue culture plate (Falcon) at a density of 2.5 x 105 cells/well and incubated 72 h at 37°C in a 5% CO2 atmosphere for polarization. The bottom of a yellow tip (Fisher Scientific) was used to generate a consistent injury area on the polarized cell layer. The injured epithelium was treated with a 100 nM concentration of fusion protein or antibodies for the indicated times, and the wound was photographed at different times using a Nikon Diaphot 300 inverted microscope (numerical aperture 0.30) and a 3CCD camera (Toshiba, New York, NY). The images were acquired by Image Pro 4.1 software. The width of each wound was measured at three sites in each image, and the percentage of wound recovery was calculated by comparison with the wound width at time 0.
Statistical Analysis. Statistical analysis was performed using a two-tailed Student's t test. Results were regarded as significant if P values were 
0.05.
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and Anti-HER2/neu ScFv-TNF-(S147Y). Since a trimeric structure is required for TNF- activity, we elected to fuse the anti-HER2/neu ScFv C6MH3-B1 (Schier et al., 1996) to the amino terminus of mature murine TNF-, using a flexible [(Gly4) Ser]3 linker and a NWSHPQFEK streptavidin tag to separate the two protein moieties (Fig. 1A). An identical antibody lacking TNF- activity was constructed using TNF- with a point mutation at residue 147 (Ser
Tyr). TNF-(S147Y) has been shown to exhibit a 100-fold decrease in TNF- biological activity while still maintaining a trimeric structure (Zhang et al., 1992). In addition, we also expressed an anti-dansyl ScFv-TNF- fusion protein and recombinant murine TNF- (rTNF-).
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migrated with an apparent molecular mass of 17 kDa, and all three ScFv-TNF- fusion proteins migrated at an apparent molecular mass of 47 kDa, the expected size for the monomeric fusion protein. The double bands of the ScFv-TNF- fusion proteins were due to variability in the N-linked glycosylation of murine TNF- since the proteins secreted by tunicamycin-treated transfectants showed only a single band on SDS-PAGE (data not shown). When anti-HER2/neu ScFv-TNF- and anti-HER2/neu ScFv-TNF-(S147Y) were treated with the cross-linking agent EGS, separated by SDS-PAGE, and visualized by Western blot analysis, dimer and trimer forms were observed (Fig. 1C). Similar results are seen after cross-linking of wild-type TNF- (Van Ostade et al., 1991). FPLC analysis confirmed the trimeric structure of the anti-HER2/neu ScFv-TNF- and anti-HER2/neu ScFv-TNF-(S147Y) as both eluted in the fractions expected for the 141-kDa trimers (Fig. 1D).
Antigen Binding and Cytotoxic Activity of Anti-HER2/neu ScFv-TNF-. Both anti-HER2/neu ScFv-TNF- (Fig. 2A, panel 1) and anti-HER2/neu ScFv-TNF-(S147Y) (Fig. 2A, panel 2) bound D2F2/E2 cells, which express high levels of human HER2/neu, whereas anti-dansyl ScFv-TNF- did not, excluding the possibility that binding was through the TNF- receptor (Fig. 2A, panel 3). rTNF-, anti-HER2/neu ScFv-TNF-, and anti-dansyl ScFv-TNF- all exhibited similar cytotoxicity against L-929 (Fig. 2B), whereas, as predicted, anti-HER2/neu ScFv-TNF-(S147Y) exhibited decreased cytotoxicity. The IC50 values were 2.5, 4, and 6 pM for rTNF-, anti-dansyl ScFv-TNF-, and anti-HER2/neu ScFv-TNF-, respectively, and 300 pM for anti-HER2/neu ScFv-TNF-(S147Y). Therefore, in the fusion proteins the ScFv moiety of anti-HER2/neu ScFv-TNF- and anti-HER2/neu ScFv-TNF-(S147Y) retained its ability to bind human HER2/neu and TNF- retained its biological activity, although there was a slight reduction in specific activity.
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Anti-HER2/neu ScFv-TNF- and Anti-HER2/neu ScFv-TNF-(S147Y) Induce a Potent Antiapoptotic Effect in SKBR3 Cells via HER2/neu Binding. Although TNF- has been shown to elicit a direct cytotoxic effect in some tumors (Laster et al., 1988), it has been demonstrated that HER2/neu activation can block the apoptosis induced by TNF- by activating the Akt/nuclear factor-B pathway in HER2/neu-overexpressing tumors, including SKBR3 (Zhou et al., 2000). Treatment with actinomycin D was cytotoxic to SKBR3 cells with O.D.490 values of 0.85 and 2.0 after MTS addition for the actinomycin D-treated cells and control medium-treated cells, respectively (data not shown). Unexpectedly, in the presence of actinomycin D, anti-HER2/neu ScFv-TNF--treated SKBR3 cells survived better than cells treated with medium alone (Fig. 2C). Anti-HER2/neu ScFv-TNF-(S147Y) exhibited similar activity, indicating that it was HER2/neu binding and not the biological activity of TNF- that was responsible for this protective effect. Consistent with this conclusion, no protection against the apoptotic effect of actinomycin D was seen when SKBR3 cells were treated with rTNF- (Fig. 2C). The protection against actinomycin D-induced apoptosis is only seen with the trimeric ScFv moiety, since an IgG3 antibody with the same variable region as the anti-HER2/neu ScFv-TNF- did not exhibit this effect (Fig. 2C). In addition, increasing concentrations of anti-HER2/neu IgG3 but not anti-dansyl IgG3 abolished the antiapoptotic effect against actinomycin D induced by anti-HER2/neu ScFv-TNF- and anti-HER2/neu ScFv-TNF-(S147Y) (Fig. 2D). However, anti-HER2/neu ScFv-TNF- and anti-HER2/neu ScFv-TNF-(S147Y) as well as rTNF- and anti-HER2/neu IgG3 did not demonstrate any cytotoxic or proliferative effect in SKBR3 cells without the incubation of actinomycin D (supplemental Fig. 2). These results suggest that trimeric anti-HER2/neu ScFv initiates a cellular resistance mechanism against the apoptotic effect of actinomycin D by cross-linking HER2/neu on SKBR3 cells.
Anti-HER2/neu ScFv-TNF- and Anti-HER2/neu ScFv-TNF-(S147Y) Induced HER2/neu Signaling and Robust Activation of p44/42 MAPK (ERK1 + 2) and Akt. The addition of both anti-HER2/neu ScFv-TNF- and anti-HER2/neu ScFv-TNF-(S147Y) at concentrations of 50 and 100 nM induced a strong increase in the phosphotyrosine content of HER2/neu compared with the control by 5 min, whereas 100 nM rTNF- had little effect (supplemental Fig. 3).
The MAPK and PI3K pathways are the major signaling cascades downstream of activated ErbB receptors, including HER2/neu (Olayioye et al., 2000). Activation of these pathways has been shown to result in cellular proliferation and resistance to apoptosis in HER2/neu-expressing tumor cells (Zhou et al., 2000; Leung et al., 2004). As shown in Fig. 3A, both anti-HER2/neu ScFv-TNF- and anti-HER2/neu ScFv-TNF-(S147Y) initiated robust MAPK phosphorylation in SKBR3 cells. By 2 min, MAPK phosphorylation showed an 8-fold increase that persisted for at least 10 min in SKBR3 cells treated with either anti-HER2/neu ScFv-TNF- or anti-HER2/neu ScFv-TNF-(S147Y). In contrast, IgG3 antibodies with the variable regions of C6MH3-B1 or Herceptin and anti-dansyl ScFv-TNF- induced weak and transient MAPK phosphorylation (Fig. 3A). Anti-HER2/neu ScFv-TNF- and anti-HER2/neu ScFv-TNF-(S147Y) also induced significant phosphorylation of Akt within 30 sec (Fig. 3B). By 10 min, the Akt phosphorylation was 2.7- and 2.2-fold increased in SKBR3 cells treated with anti-HER2/neu ScFv-TNF- and anti-HER2/neu ScFv-TNF-(S147Y), respectively. Akt phosphorylation persisted in anti-HER2/neu ScFv-TNF--treated SKBR3 cells for over 60 min, whereas the increased Akt phosphorylation in anti-HER2/neu ScFv-TNF-(S147Y)-treated SKBR3 cells was barely detectable by 30 min. The Akt activation was slightly increased in the anti-HER2/neu IgG3-treated SKBR3 cells, and no increased Akt activation was observed in the anti-dansyl ScFv-TNF--treated SKBR3 cells (Fig. 3B). These results demonstrate that the trimeric anti-HER2/neu ScFv antibodies can initiate potent MAPK and Akt activation downstream of activated HER2/neu in SKBR3 cells.
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and Anti-HER2/neu ScFv-TNF-(S147Y). To further investigate whether MAPK and/or Akt activation induced by the trimeric anti-HER2/neu ScFv antibodies contributed to the resistance of SKBR3 cells to actinomycin D-induced death, actinomycin D-treated SKBR3 cells were incubated with anti-HER2/neu ScFv-TNF- or anti-HER2/neu ScFv-TNF-(S147Y) in the presence of the MAPK inhibitor U0126 and/or the Akt inhibitor LY294002 for 24 h (Fig. 4). The activation of MAPK or Akt was significantly compromised in the presence of the indicated concentration of U0126 or LY294002, respectively (supplemental Fig. 4). Although anti-HER2/neu ScFv-TNF- or anti-HER2/neu ScFv-TNF-(S147Y) significantly protected SKBR3 cells against the apoptosis induced by actinomycin D (lanes 1–3), treatment with LY294002, (lanes 4–6) or U0126 (lanes 7–9) reduced their protective effect. Importantly, the protection against actinomycin D-induced apoptosis mediated by anti-HER2/neu ScFv-TNF- or anti-HER2/neu ScFv-TNF-(S147Y) was completely abolished when both LY294002 (40 µM) and U0126 (10 µM) were present (lanes 10–12). Therefore, activation of MAPK and Akt contributes to the antiapoptotic effect induced by the trimeric anti-HER2/neu ScFv antibodies in SKBR3 cells.
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and Anti-HER2/neu ScFv-TNF-(S147Y) Facilitate the Repair of Mechanically Injured Epithelia. HER2/neu activation has been shown to participate in the repair of injured epithelia (Vermeer et al., 2003). Trimeric anti-HER2/neu ScFv also facilitates the repair of mechanical wounding in cultured human colonic epithelial cells (Fig. 5A) with HER2/neu binding required for the effect (Fig. 5B). By 29 h, the wound in anti-HER2/neu ScFv-TNF-- or anti-HER2/neu ScFv-TNF-(S147Y)-treated cells was almost completely healed, whereas the wound of the control medium-treated cells was still apparent (Fig. 5A). Analysis of six independent experiments demonstrated a statistically significant (P < 0.001) increase in the speed of wound healing in anti-HER2/neu ScFv-TNF-- and anti-HER2/neu ScFv-TNF-(S147Y)-treated cells (Table 1). In contrast, treatment with IgG3 antibodies with the variable regions of C6MH3-B1 or Herceptin did not result in a significant increase in the rate of wound healing compared with treatment with PBS (Fig. 5A; Table 1). The enhancement of wound healing required the binding of the trimeric fusion proteins to HER2/neu on the injured epithelial cells, as it was inhibited by the presence of soluble HER2/neu (Fig. 5B). The wound in anti-HER2/neu ScFv-TNF-- or anti-HER2/neu ScFv-TNF-(S147Y)-treated cells (Fig. 5B, lines 1 and 3) was almost healed within 25 h; in contrast, the rate of wound healing was significantly decreased when an excess of soluble HER2/neu was present (Fig. 5B, lines 2 and 4). These results suggest that the trimeric anti-HER2/neu ScFv antibodies but not bivalent IgG3 antibodies facilitate wound repair through the binding of HER2/neu on the injured epithelial cells.
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fused to an anti-HER2/neu ScFv containing the variable region of C6MH3-B1 (Schier et al., 1996). Surprisingly, this fusion protein, anti-HER2/neu ScFv-TNF-, exhibited a potent agonistic effect, activating HER2/neu and the downstream MAPK and PI3K signaling cascades. Remarkably, this fusion protein also facilitated the repair of injured epithelial cell monolayers. These unique effects did not require the biological activity of TNF-, since anti-HER2/neu ScFv fused to TNF- (S147Y) exhibited similar activities. The trimeric structure of anti-HER2/neu ScFv seems essential for initiating HER2/neu signaling and facilitating the repair of injured epithelia, since an IgG3 antibody with the same variable region as C6MH3-B1 did not show similar effects.
Anti-HER2/neuScFvc-TNF- offers some advantages over recombinant epidermal growth factor (rEGF), the current Food and Drug Administration-approved treatment for wound healing (Brown et al., 1989). EGF drives homodimerization of the receptor erbB1 but does not elicit a heterodimer between erbB1 and erbB2 (Ferguson et al., 2000), indicating that it facilitates wound healing only through erbB1 signaling. Because erbB2 signaling has been shown to play a critical role in wound healing in human epithelial cells (Bindels et al., 2002; Vermeer et al., 2003; Xu et al., 2004), it is likely that anti-HER2/neuScFv-TNF- will either be effective in treating wounds that are resistant to rEGF treatment or will synergize with EGF in facilitating wound healing by activating potent erbB2 signaling. In addition, in contrast to the erbB1 receptor, which is rapidly internalized and degraded upon ligand binding, thereby attenuating its signaling (Baulida et al., 1996), the erbB2 receptor is an internalization-resistant receptor even when bound by Herceptin (Hommelgaard et al., 2004). Therefore, anti-HER2/neuScFv-TNF- may be more effective than rEGF in promoting wound healing because it induces more potent and prolonged signaling. The fact that anti-HER2/neuScFv-TNF- is a trimer with three erbB2 binding sites may also contribute to more potent downstream signaling than is seen with the homodimerization of erbB1 induced by rEGF.
The anti-HER2/neu ScFv-TNF- described in the present study seems to differ in its functional properties from TNF- fused with a different anti-HER2/neu variable region (sFv23/TNF) (Rosenblum et al., 2000). sFv23/TNF exhibited modest cytotoxicity against SKBR3 cells in the absence of actinomycin D, whereas anti-HER2/neu ScFv-TNF- did not exhibit any significant cytotoxic effect on SKBR3 cells in the absence of actinomycin D. Because antibodies recognizing different epitopes on the same antigen may exhibit different effects, activation of HER2/neu signaling and the facilitation of wound repair induced by anti-HER2/neu ScFv-TNF- and anti-HER2/neu ScFv-TNF-(S147Y) may require both their unique anti-HER2/neu variable region, C6MH3-B1, and a trimeric structure.
The ERK signaling pathway, also known as the p44/42 mitogen-activated protein kinase pathway, is a major determinant in the control of cell growth and migration, and aberrantly active ERK signaling has been identified in many types of human tumors (Hoshino et al., 1999; Pola et al., 2003). ERK activation is essential for cell survival after oxidant injury (Guyton et al., 1996), and NIH 3T3 cells expressing constitutively active mitogen-activated protein kinase kinase (the immediate upstream regulator of ERK) were more resistant to oxidant toxicity (Guyton et al., 1996). In addition, the migratory and invasive activity of human neuroblastoma cells was inhibited by the ERK inhibitor PD98059 (Pola et al., 2003). In the present study, the trimeric anti-HER2/neu ScFv antibodies were found to induce robust and persistent ERK activation in HER2/neu-expressing cells even when the activity of the attached TNF- was greatly compromised. ERK activation was initiated within 30 s and, remarkably, was maintained for at least 90 min (Fig. 3A). Although ERK activation was observed in the anti-dansyl ScFv-TNF--treated SKBR3 cells, the intensity was significantly lower than that seen in cells treated with the trimeric anti-HER2/neu ScFv (Fig. 3A), indicating that the TNF- moiety of the anti-HER2/neu ScFv-TNF- was not solely responsible for the greatly enhanced ERK activation. Therefore, proliferation and migration induced by ERK activation undoubtedly makes a major contribution to the enhancement of wound repair induced by the trimeric anti-HER2/neu ScFv antibodies.
Activated ERK and Akt both contribute to the antiapoptotic effect induced by the trimeric anti-HER2/neu ScFv antibodies. The presence of inhibitors of either ERK or Akt decreased the antiapoptotic effect of the trimeric fusion proteins, but this effect was completely abolished only in the presence of both ERK and Akt inhibitors (Fig. 4). Although TNF- has been shown to induce the phosphorylation of Akt in a variety of cells (Osawa et al., 2001; Sandra et al., 2002), we did not observe significant enhancement of Akt activation in SKBR3 cells treated with the anti-dansyl ScFv-TNF- fusion protein (Fig. 3B). The anti-HER2/neu IgG3 antibodies (C6MH3-B1 variable regions and Herceptin variable regions) only induced transient and weak Akt activation compared with the trimeric anti-HER2/neu ScFv antibodies; the maximal Akt activation was 1.5-fold and nearly 3-fold for the IgG3 antibody and the trimeric anti-HER2/neu ScFv antibodies, respectively (Fig. 3B). Anti-HER2/neu ScFv-TNF- but not anti-HER2/neu ScFv-TNF-(S147Y) also induced prolonged phosphorylation of Akt. The HER2/neu signaling induced by trimeric anti-HER2/neu ScFv may sensitize SKBR3 cells to TNF- stimulation, thus resulting in the prolonged phosphorylation of Akt observed. Whereas anti-HER2/neu ScFv-TNF- induced prolonged activation of Akt compared with anti-HER2/neu ScFv-TNF-(S147Y), both exhibited a similar antiapoptotic effect against actinomycin D and facilitated the repair of injured epithelia with the same potency, indicating that the prolonged activation of Akt was not responsible for these effects.
Activation of the PI3K pathway is induced by the trimeric anti-HER2/neu ScFv antibodies as shown by the induction of phosphorylated Akt (Fig. 3B). Rac, a member of the Rho GTPases, has been shown to stimulate the migration of different types of cells (Pola et al., 2003; Weiss-Haljiti et al., 2004), with PI3K activity necessary and sufficient for Rac activation (Hawkins et al., 1995). Therefore, it is likely that Rac-induced cell migration contributed to the enhancement of wound repair.
Genetic fusion of antibody with a protein capable of forming noncovalent oligomers, for example, avidin, has been shown to be an effective method for producing a polymeric antibody (Ng et al., 2002). However, the immunogenicity of foreign proteins hampers the clinical use of these antibodies in humans. Use of a human TNF- with a point mutation at residue 87 (Tyr Ser) and lacking biological activity but maintaining its trimeric structure (Zhang et al., 1992) provides a novel strategy to construct a polymeric antibody with minimal immunogenicity.
Herceptin, a human IgG1 antibody binding to the extracellular domain of HER2/neu, has been shown to transiently induce a modest activation of HER2/neu signaling in vitro (Scott et al., 1991). In the present study, we have shown that both Herceptin and the human IgG3 antibody with the C6MH3-B1 variable region induced a similar transient and weak activation of ERK and Akt; however, the trimeric anti-HER2/neu ScFv antibodies with the C6MH3-B1 variable region initiated a potent and persistent HER2/neu signaling and exhibited an antiapoptotic effect in SKBR3 cells. Therefore, it is likely that cross-linking of multiple HER2/neu receptors is one of the critical determinants for these effects.
In summary, we have constructed and characterized a novel anti-HER2/neu ScFv fusion protein in which the ScFvs are trimerized by TNF- or TNF-(S147Y). These fusion proteins initiate robust HER2/neu signaling and, remarkably, facilitate the repair of the injured cultured epithelial cell monolayers. Unfortunately, there seems to be no animal model available for evaluating the wound healing effect of anti-HER2/neu ScFv-TNF- and anti-HER2/neu ScFv-TNF-(S147Y) in vivo. Although human HER2/neu transgenic mice are available, it is not clear whether the human HER2/neu is appropriately expressed on the epithelial cells. For use in humans the murine TNF- present in the fusion proteins would need to be replaced by human TNF-. Analysis of human TNF- has shown that a point mutation at residue 87 (Tyr Ser) exhibits undetectable TNF- biological activity while still maintaining a trimeric structure (Zhang et al., 1992). Therefore, an approach to making an effective therapeutic agent for human use could be to fuse human TNF-(Y87S) with the anti-HER2/neu ScFv. In addition, the mutant human TNF- may be used to construct more effective polymeric antibodies for clinical use.
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ABBREVIATIONS: EGF, epidermal growth factor; PI3K, phosphatidylinositol 3-kinase; MAPK, mitogen-activated protein kinase; TNF-, tumor necrosis factor-; ScFv, single-chain Fv; CHO, Chinese hamster ovary; ATCC, American Type Culture Collection; IMDM, Iscove's modified Dulbecco's medium; U0126, 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; EGS, ethylene glycolbis(succinimidylsuccinate); PAGE, polyacrylamide gel electrophoresis; ERK, extracellular signal-regulated kinase; FPLC, fast protein liquid chromatography; HRP, horseradish peroxidase; rTNF-, recombinant TNF-; rEGF, recombinant EGF; PD98059, 2-(2'-amino-3'-methoxyphenol)-oxanaphthalen-4-one.
The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material. 
Address correspondence to: Dr. Tzu-Hsuan Huang, Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Box 951489, Los Angeles, CA 90095-1489. E-mail: thhhuang{at}ucla.edu
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Akiyama T, Sudo C, Ogawara H, Toyoshima K, and Yamamoto T (1986) The product of the human c-erbB-2 gene: a 185-kilodalton glycoprotein with tyrosine kinase activity. Science (Wash DC) 232: 1644–1646.
Baulida J, Kraus MH, Alimandi M, Di Fiore PP, and Carpenter G (1996) All ErbB receptors other than the epidermal growth factor receptor are endocytosis impaired. J Biol Chem 271: 5251–5257.
Bindels EM, van der Kwast TH, Izadifar V, Chopin DK, and de Boer WI (2002) Functions of epidermal growth factor-like growth factors during human urothelial reepithelialization in vitro and the role of erbB2. Urol Res 30: 240–247.[CrossRef][Medline]
Brown GL, Nanney LB, Griffen J, Cramer AB, Yancey JM, Curtsinger LJ 3rd, Holtzin L, Schultz GS, Jurkiewicz MJ, and Lynch JB (1989) Enhancement of wound healing by topical treatment with epidermal growth factor. N Engl J Med 321: 76–79.[Abstract]
Candido KA, Shimizu K, McLaughlin JC, Kunkel R, Fuller JA, Redman BG, Thomas EK, Nickoloff BJ, and Mule JJ (2001) Local administration of dendritic cells inhibits established breast tumor growth: implications for apoptosis-inducing agents. Cancer Res 61: 228–236.
Cardone MH, Roy N, Stennicke HR, Salvesen GS, Franke TF, Stanbridge E, Frisch S, and Reed JC (1998) Regulation of cell death protease caspase-9 by phosphorylation. Science (Wash DC) 282: 1318–1321.
Coloma MJ, Hastings A, Wims LA, and Morrison SL (1992) Novel vectors for the expression of antibody molecules using variable regions generated by polymerase chain reaction. J Immunol Methods 152: 89–104.[CrossRef][Medline]
Coloma MJ and Morrison SL (1997) Design and production of novel tetravalent bispecific antibodies. Nat Biotechnol 15: 159–163.[CrossRef][Medline]
Cooke SP, Pedley RB, Boden R, Begent RH, and Chester KA (2002) In vivo tumor delivery of a recombinant single chain Fv:tumor necrosis factor-alpha fusion [correction of factor: a fusion] protein. Bioconjug Chem 13: 7–15.[CrossRef][Medline]
Coussens L, Yang-Feng TL, Liao YC, Chen E, Gray A, McGrath J, Seeburg PH, Libermann TA, Schlessinger J, et al. (1985) Tyrosine kinase receptor with extensive homology to EGF receptor shares chromosomal location with neu oncogene. Science (Wash DC) 230: 1132–1139.
Dela Cruz JS, Lau SY, Ramirez EM, De Giovanni C, Forni G, Morrison SL, and Penichet ML (2003) Protein vaccination with the HER2/neu extracellular domain plus anti-HER2/neu antibody-cytokine fusion proteins induces a protective anti-HER2/neu immune response in mice. Vaccine 21: 1317–1326.[CrossRef][Medline]
Dela Cruz JS, Trinh KR, Morrison SL, and Penichet ML (2000) Recombinant anti-human HER2/neu IgG3-(GM-CSF) fusion protein retains antigen specificity and cytokine function and demonstrates antitumor activity. J Immunol 165: 5112–5121.
del Peso L, Gonzalez-Garcia M, Page C, Herrera R, and Nunez G (1997) Interleukin-3-induced phosphorylation of BAD through the protein kinase Akt. Science (Wash DC) 278: 687–689.
Ferguson KM, Darling PJ, Mohan MJ, Macatee TL, and Lemmon MA (2000) Extracellular domains drive homo- but not hetero-dimerization of erbB receptors. EMBO (Eur Mol Biol Organ) J 19: 4632–4643.[CrossRef][Medline]
Guyton KZ, Liu Y, Gorospe M, Xu Q, and Holbrook NJ (1996) Activation of mitogen-activated protein kinase by H2O2. Role in cell survival following oxidant injury. J Biol Chem 271: 4138–4142.
Hawkins PT, Eguinoa A, Qiu RG, Stokoe D, Cooke FT, Walters R, Wennstrom S, Claesson-Welsh L, Evans T, Symons M, et al. (1995) PDGF stimulates an increase in GTP-Rac via activation of phosphoinositide 3-kinase. Curr Biol 5: 393–403.[CrossRef][Medline]
Hock H, Dorsch M, Kunzendorf U, Qin Z, Diamantstein T, and Blankenstein T (1993) Mechanisms of rejection induced by tumor cell-targeted gene transfer of interleukin 2, interleukin 4, interleukin 7, tumor necrosis factor, or interferon . Proc Natl Acad Sci USA 90: 2774–2778.
Hommelgaard AM, Lerdrup M, and van Deurs B (2004) Association with membrane protrusions makes ErbB2 an internalization-resistant receptor. Mol Biol Cell 15: 1557–1567.
Hoshino R, Chatani Y, Yamori T, Tsuruo T, Oka H, Yoshida O, Shimada Y, Ari-i S, Wada H, Fujimoto J, et al. (1999) Constitutive activation of the 41-/43-kDa mitogen-activated protein kinase signaling pathway in human tumors. Oncogene 18: 813–822.[CrossRef][Medline]
Hudziak RM, Lewis GD, Winget M, Fendly BM, Shepard HM, and Ullrich A (1989) p185HER2 monoclonal antibody has antiproliferative effects in vitro and sensitizes human breast tumor cells to tumor necrosis factor. Mol Cell Biol 9: 1165–1172.
Hu P, Margolis B, Skolnik EY, Lammers R, Ullrich A, and Schlessinger J (1992) Interaction of phosphatidylinositol 3-kinase-associated p85 with epidermal growth factor and platelet-derived growth factor receptors. Mol Cell Biol 12: 981–990.
Laster SM, Wood JG, and Gooding LR (1988) Tumor necrosis factor can induce both apoptic and necrotic forms of cell lysis. J Immunol 141: 2629–2634.[Abstract]
Lenormand P, Brondello JM, Brunet A, and Pouyssegur J (1998) Growth factor-induced p42/p44 MAPK nuclear translocation and retention requires both MAPK activation and neosynthesis of nuclear anchoring proteins. J Cell Biol 142: 625–633.
Leung DW, Tompkins C, Brewer J, Ball A, Coon M, Morris V, Waggoner D, and Singer JW (2004) Phospholipase C delta-4 overexpression upregulates ErbB1/2 expression, Erk signaling pathway and proliferation in MCF-7 cells. Mol Cancer 3: 15.[CrossRef][Medline]
Leyland-Jones B, Arnold A, Gelmon K, Verma S, Ayoub JP, Seidman A, Dias R, Howell J, and Rakhit A (2001) Pharmacologic insights into the future of trastuzumab. Ann Oncol 12 (Suppl 1): S43–S47.[Abstract]
Meyer S, LaBudda K, McGlade J, and Hayman MJ (1994) Analysis of the role of the Shc and Grb2 proteins in signal transduction by the v-ErbB protein. Mol Cell Biol 14: 3253–3262.
Ng PP, Dela Cruz JS, Sorour DN, Stinebaugh JM, Shin SU, Shin DS, Morrison SL, and Penichet ML (2002) An anti-transferrin receptor-avidin fusion protein exhibits both strong proapoptotic activity and the ability to deliver various molecules into cancer cells. Proc Natl Acad Sci USA 99: 10706–10711.
Olayioye MA, Neve RM, Lane HA, and Hynes NE (2000) The ErbB signaling network: receptor heterodimerization in development and cancer. EMBO (Eur Mol Biol Organ) J 19: 3159–3167.[CrossRef][Medline]
Osawa Y, Banno Y, Nagaki M, Brenner DA, Naiki T, Nozawa Y, Nakashima S, and Moriwaki H (2001) TNF--induced sphingosine 1-phosphate inhibits apoptosis through a phosphatidylinositol 3-kinase/Akt pathway in human hepatocytes. J Immunol 167: 173–180.
Pola S, Cattaneo MG, and Vicentini LM (2003) Anti-migratory and anti-invasive effect of somatostatin in human neuroblastoma cells: involvement of Rac and MAP kinase activity. J Biol Chem 278: 40601–40606.
Rosenblum MG, Horn SA, and Cheung LH (2000) A novel recombinant fusion toxin targeting HER-2/NEU-over-expressing cells and containing human tumor necrosis factor. Int J Cancer 88: 267–273.[CrossRef][Medline]
Sandra F, Matsuki NA, Takeuchi H, Ikebe T, Kanematsu T, Ohishi M, and Hirata M (2002) TNF inhibited the apoptosis by activation of Akt serine/threonine kinase in the human head and neck squamous cell carcinoma. Cell Signal 14: 771–778.[CrossRef][Medline]
Schier R, McCall A, Adams GP, Marshall KW, Merritt H, Yim M, Crawford RS, Weiner LM, Marks C, and Marks JD (1996) Isolation of picomolar affinity anti-c-erbB-2 single-chain Fv by molecular evolution of the complementarity determining regions in the center of the antibody binding site. J Mol Biol 263: 551–567.[CrossRef][Medline]
Scott GK, Dodson JM, Montgomery PA, Johnson RM, Sarup JC, Wong WL, Ullrich A, Shepard HM, and Benz CC (1991) p185HER2 signal transduction in breast cancer cells. J Biol Chem 266: 14300–14305.
Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, and McGuire WL (1987) Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science (Wash DC) 235: 177–182.
Van Ostade X, Tavernier J, Prange T, and Fiers W (1991) Localization of the active site of human tumour necrosis factor (hTNF) by mutational analysis. EMBO (Eur Mol Biol Organ) J 10: 827–836.[Medline]
Vermeer PD, Einwalter LA, Moninger TO, Rokhlina T, Kern JA, Zabner J, and Welsh MJ (2003) Segregation of receptor and ligand regulates activation of epithelial growth factor receptor. Nature (Lond) 422: 322–326.[CrossRef][Medline]
Weiss-Haljiti C, Pasquali C, Ji H, Gillieron C, Chabert C, Curchod ML, Hirsch E, Ridley AJ, van Huijsduijnen RH, Camps M, et al. (2004) Involvement of phosphoinositide 3-kinase gamma, Rac and PAK signaling in chemokine-induced macrophage migration. J Biol Chem 279: 43273–43284.
Xu KP, Riggs A, Ding Y, and Yu FS (2004) Role of ErbB2 in corneal epithelial wound healing. Investig Ophthalmol Vis Sci 45: 4277–4283.
Zhang XM, Weber I, and Chen MJ (1992) Site-directed mutational analysis of human tumor necrosis factor- receptor binding site and structure-functional relationship. J Biol Chem 267: 24069–24075.
Zhou BP, Hu MC, Miller SA, Yu Z, Xia W, Lin SY, and Hung MC (2000) HER-2/neu blocks tumor necrosis factor-induced apoptosis via the Akt/NF-B pathway. J Biol Chem 275: 8027–8031.
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