Targeting of Epidermal Growth Factor Receptor (EGFR) - American

Bioconjugate Chem. 2009, 20, 1201–1208

1201

Targeting of Epidermal Growth Factor Receptor (EGFR)-Expressing Tumor Cells with Sterically Stabilized Affibody Liposomes (SAL) Julia Beuttler,† Miriam Rothdiener,† Dafne Mu¨ller,† Fredrik Y. Frejd,‡ and Roland E. Kontermann*,† Institut fu¨r Zellbiologie und Immunologie, Universita¨t Stuttgart, Allmandring 31, 70569 Stuttgart, Germany, Affibody AB, P.O. Box 20137, SE-161 02 Bromma, Sweden, and Unit of Biomedical Radiations Sciences, Rudbeck Laboratory, Uppsala University, SE-751 85 Uppsala, Sweden. Received February 9, 2009; Revised Manuscript Received April 16, 2009

Affibody molecules are small and stable antigen-binding molecules derived from the B domain of protein A. We applied a bivalent, high-affinity epidermal growth factor receptor (EGFR)-specific affibody molecule for the generation of targeted PEGylated liposomes. These sterically stabilized affibody liposomes (SAL) were produced by chemical coupling of the cysteine-modified affibody molecule to maleimide-PEG2000-DSPE and subsequent insertion into PEGylated liposomes. These SAL showed strong and selective binding to EGFR-expressing tumor cell lines. Binding was dependent on the amount of inserted affibody molecule-lipid conjugates and could be blocked by soluble EGF. Approximately 30% of binding activity was still retained after 6 days of incubation in human plasma at 37 °C. Binding of SAL to cells led to efficient internalization of the liposomes. Using mitoxantrone-loaded liposomes, we observed for SAL, compared to untargeted liposomes, an enhanced cytotoxicity toward EGFR-expressing cells. In summary, we show that SAL can be easily prepared from affibody molecules and thus may be suitable for the development of carrier systems for targeted delivery of drugs.

INTRODUCTION The epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein and a member of the erbB receptor tyrosine kinase family. Dysregulation of EGFR signaling has been shown to contribute to malignant transformation (1). Elevated levels of EGFR are found on different types of solid tumors, e.g., breast, ovarian, lung, head and neck, prostate, and colorectal cancers, and have been proposed as prognostic marker for disease progression and survival (2). Consequently, strategies targeting EGFR have been developed as treatment option, including monoclonal antibodies, small molecule inhibitors of EGFR signal transduction, and antibody-based immunoconjugates, e.g., immunotoxins (3). Some of these reagents have already been approved for therapy, such as monoclonal antibodies (cetuximab, panitumumab) and small molecule inhibitors (gefitinib, erlotinib) (4). Antibodies recognizing EGFR have also been applied for the targeting of drug-loaded liposomes. Liposomes are small, vesicular carrier systems consisting of one or more lipid bilayers composed of natural or synthetic lipids, e.g., phospholipids and cholesterol (5). Encapsulation of drugs into the liposomal interior or the lipid bilayer alters the drug pharmacokinetics, e.g., protects the drug from rapid elimination and degradation, and has been shown to improve efficacy and safety (6). Half-life of liposomal drugs can be drastically improved by incorporating polymers such as poly(ethylene glycol) into the lipid bilayer, thus sterically stabilizing the liposomes. Coupling of antibodies to the surface results in immunoliposomes allowing for an active targeting to tumor cells and receptor-mediated internalization (7-9). In order to avoid Fc-mediated uptake of immunoliposomes by phagocytic cells, recent applications focused on the * Corresponding author. Prof. Roland E. Kontermann, Institut fu¨r Zellbiologie and Immunologie, Universita¨t Stuttgart, Allmandring 31, 70569 Stuttgart, Germany. Tel. +49 711 685-66989. Fax. +49 711 685-67484. [email protected]. † Universita¨t Stuttgart. ‡ Affibody AB and Uppsala University.

use of antibody fragments (Fab′) and recombinant formats (e.g., scFv)1(for review, see 10). Both, anti-EGFR Fab′ and scFv molecules have been employed for the preparation of anti-EGFR immunoliposomes (11-13). Selective binding and internalization into EGFR-overexpressing tumor cells as well as selective cytotoxicity of drug-loaded anti-EGFR immunoliposomes has been demonstrated in vitro in these studies. Superiority of immunoliposomal delivery of various drugs, e.g., doxorubicin, epirubicin, and vinorelbine, over free drug and nontargeted liposomal formulations was confirmed in xenograft tumor models (12). Obviously, the antigen-binding moiety of an antibody is sufficient and advantageous to confer target specificity of immunoliposomes. Recently, various antibody mimetics based on nonimmunoglobulin scaffolds have been proposed as an alternative for antibodies (14). Several of these antibody mimetics have been developed into high-affinity antigen binders. For example, affibody molecules are small molecules (7 kDa) based on a 58 amino acid residue three-helix bundle domain from staphylococcal protein A (15-17). Anti-EGFR affibody molecules were selected from a phage display library and further affinitymaturedintomoleculeswithlownanomolaraffinity(18,19). One of these anti-EGFR affibody molecules, (ZEGFR:955)2, was shown to be efficiently internalized into EGFR-overexpressing A431 squamous carcinoma cells similar to monoclonal antibody cetuximab (20). Here, we applied a bivalent anti-EGFR affibody molecule for the generation of targeted liposomes. This was achieved by chemical coupling of the cysteine-modified affibody molecule to maleimide-PEG2000-DSPE and subsequent insertion into sterically stabilized liposomes. The resulting sterically stabilized 1 Abbreviations: EPC, egg phosphatidylcholine; Chol, cholesterol; DOPC, dioleoyl-phosphatidylcholine; Mal-PEG2000-DSPE, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)2000]; mPEG2000-DSPE, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(poly(ethylene glycol))-2000]; MTO, mitoxantrone; SAL, sterically stabilized affibody liposomes; SL, sterically stabilized liposomes; scFv, single-chain fragment variable.

10.1021/bc900061v CCC: $40.75  2009 American Chemical Society Published on Web 05/12/2009

1202 Bioconjugate Chem., Vol. 20, No. 6, 2009

affibody liposomes (SAL) showed strong and selective binding to various EGFR-expressing tumor cell lines and subsequent internalization. Furthermore, compared to untargeted drugloaded liposomes, targeting led to a selective increase in cytotoxicity of mitoxantrone-loaded SAL toward EGFRexpressing tumor cell lines. These findings demonstrate that affibody molecules are useful molecules for the generation of targeted nanocarrier systems suitable for delivery of drugs and other agents, e.g., for diagnostic purposes.

MATERIALS AND METHODS Materials. Phospholipids were purchased from Avanti Polar Lipids (Alabaster, USA) and cholesterol from Calbiochem (Gibbstown, USA). DiI and mitoxantrone were purchased from Sigma-Aldrich (Taufkirchen, Germany). Human plasma (stabilized with citrate-phosphate-dextrose solution (CPD)) was kindly provided by the blood donation center of the Katharinenhospital (Stuttgart, Germany). A PE-labeled anti-EGFR monoclonal antibody (clone EGFR.1) was purchased from BD Pharmingen (San Jose, USA). A431, MDA-MB-468, MDAMB-231, SKBR3, and MCF-7 were grown in RPMI, 10% FCS, 2 mM glutamine. Colo205, LS174T, and HEK293 were grown in RPMI, 5% FCS, 2 mM glutamine. Expression and Purification of (ZEGFR:1907)2-cys. The non his-tagged dimeric EGFR-binding affibody molecule (ZEGFR:1907)2-cys was expressed in Escherichia coli BL21(DE3) cells as previously described (21). Pelleted bacterial cells harboring (ZEGFR:1907)2-cys were suspended in 20 mM TrisHCl pH 7.1 and heat-treated at 85 °C for 3 min to disrupt the cells and release the intracellular content. The lysate was clarified by centrifugation followed by filtration and loaded on 25 mL Q Sepharose FF (GE Healthcare), previously equilibrated with 20 mM Tris-HCl pH 7.1. After wash with 20 mM Tris-HCl pH 7.1, bound protein was eluted with a linear gradient 0-0.5 M NaCl in 20 mM Tris-HCl pH 7.1. Fractions containing (ZEGFR:1907)2-cys were pooled and pH was adjusted to 8.0 by addition of 1 M Tris-HCl pH 8.0 to a final concentration of 50 mM. The C-terminal cysteine was reduced by addition of DTT to 20 mM, followed by incubation at 40 °C for 3 min. After reduction, acetonitrile (ACN) was added to a final concentration of 5% and reduced (ZEGFR:1907)2-cys was loaded on 1 mL Resource 15 RPC columns (GE Healthcare), previously equilibrated with RPC A-buffer (0.1% TFA, 5% ACN, 95% water). After wash with RPC A-buffer, bound proteins were eluted with a linear gradient 0-40% RPC B-buffer (0.1% TFA, 80% ACN, 20% water). Fractions containing pure (ZEGFR:1907)2-cys were identified by SDS-PAGE analysis and pooled. The buffer was exchanged to 10 mM ammonium hydrogen carbonate pH 8.0 using a PD-10 desalting column (GE Healthcare). Finally, (ZEGFR:1907)2-cys was lyophilized and stored at 4 °C until use. Determination of the concentration of (ZEGFR:1907)2-cys solutions was performed by measuring the absorbance at 280 nm. The purity of (ZEGFR:1907)2-cys was assessed in SDS-PAGE analysis using approximately 10 µg (ZEGFR:1907)2-cys. The identity was verified with LC/MS-analyses using an Agilent 1100 LC/MSD system, equipped with API-ESI and single quadruple mass analyzator. Determination of Protein Melting Point. The melting point of the affibody molecule was determined with the ZetaSizer Nano ZS (Malvern, Herrenberg, Germany). 50 µg of purified affibody molecule was diluted in PBS to a total volume of 500 µL and sterile filtered into a quartz cuvette. Dynamic laser light scattering intensity was measured while the temperature was increased in 1 °C intervals from 30 to 70 °C with 2 min equilibration for each temperature step. The melting point was defined as the temperature at which the light scattering intensity and size dramatically increased.

Beuttler et al.

Size Exclusion Chromatography. HPLC size exclusion chromatography of the affibody molecule was performed on a BioSep-Sec-S3000 column (Phenomenex, Torrance, CA). Mobil phase was PBS, 1 M NaCl, with a flow rate of 0.5 mL/min. The following standard molecules were employed: thyroglobulin, apoferritin, β-amylase, bovine serum albumin, carbonic anhydrase, cytochrome c, and aprotinin. Generation of Sterically Stabilized Affibody Liposomes (SAL). Affibody liposomes were generated by postinsertion of affibody molecule-conjugated micelles into preformed PEGliposomes as described previously for the generation of scFv immunoliposomes (22). In brief, liposomes composed of EPC/ Chol/mPEG2000-DSPE at a molar ratio of 6.5:3:0.5 were prepared by the film hydration-extrusion method. For binding studies, 0.3 mol % DiI was included. The lipid film was hydrated with 1 mL 10 mM Hepes buffer pH 6.7 with a final lipid concentration of 10 µmol lipid per ml buffer. The resulting multilamellar vesicle dispersion was extruded 21 times through 50 nm polycarbonate membranes using a LiposoFast extruder (Avestin, Ottawa, Canada). Mal-PEG2000-DSPE micelles were prepared dissolving dried lipid in ddH2O at 65 °C to a final concentration of 10 mg/mL. Purified affibody molecules were reduced in 2 mM tris(2-carboxyethyl)phosphine (TCEP) for 2 h at room temperature followed by removal of TCEP by dialysis against oxygen-free 10 mM Na2HPO4/NaH2PO4 buffer, 0.2 mM EDTA, 30 mM NaCl (pH 6.7) overnight at 4 °C. Micellar lipid and reduced affibody molecule were mixed at a molar ratio of 4.67:1 and incubated at room temperature for 30 min. The coupling reaction was quenched with 1 mM L-cysteine, 0.02 mM EDTA, pH 5.5. Coupling efficiency was determined by SDS-PAGE analysis and quantification of Coomassie-stained bands of free and lipid-conjugated affibody molecule. The affibody moleculecoupled micelles were inserted into preformed PEGylated liposomes by incubation at 55 °C for 30 min (0.02 mol % to 2 mol % micellar lipid in respect to liposomal lipid). Unbound affibody molecules were removed by gel-filtration using a Sepharose CL4B column (Amersham, Braunschweig, Germany). Liposome size (ζ-average) and ζ-potential were measured using a ZetaSizer Nano ZS (Malvern, Herrenberg). Generation of Drug-Loaded Affibody Liposomes. Mitoxantrone-loaded liposomes were produced by remote loading (23). Sterically stabilized liposomes (SL) composed of DOPC, Chol, and mPEG2000-DSPE (45:50:5) were prepared as described above (final conc. 100 µmol lipid/mL) using 300 mM citrate buffer (pH 4) for rehydration. After extrusion, liposomes were neutralized by adding 200 µL 0.5 M Na2HPO4 and 49 µ 2 N NaOH to 200 µL liposomes, adjusting the pH to 7.5. Liposomes were then preheated to 55 °C in a water bath for 5 min, and 120 µL mitoxantrone (10 mg/mL in water) was added resulting in a drug/lipid ratio of 0.12. After further incubation for 10 min at 55 °C and 20 min at room temperature, affibody molecules coupled to Mal-PEG2000-DSPE micelles were inserted as described above using 0.6 mol % micellar lipid in respect to liposomal lipid. Drug-loaded affibody liposomes were separated from free drug and uninserted micelles by gel filtration on a sepharose CL4B column. Encapsulated drug was quantified measuring absorption at 590 nm after lysis of liposomes with 2% Triton-X 100. Encapsulation efficiency was between 99-100%. Flow Cytometry. Cells were detached from cell culture dishes using 0.05% trypsin, 0.02% EDTA (GIBCO). Approximately 200 000 cells were incubated with DiI-labeled affibody liposomes (10 nmol lipid) in PBS containing 2% FBS, 0.02% sodium azide (PBA) for 1 h at 4 °C in the dark. After washing cells three times with PBA buffer (4 °C), cells were resuspended in 500 µL PBA buffer and analyzed by flow cytometry (Cytomics FC 500, Beckmann-Coulter). Binding of

Targeting of EGFR-Expressing Tumor Cells with SAL

PE-labeled anti-EGFR antibody was determined by incubating detached cells with antibody (diluted 1:20) in PBA buffer for 1 h at 4 °C. Cells were then washed three times with PBA as described and analyzed for binding by flow cytometry. Data were evaluated with WinMDI 2.9. In Vitro Plasma Stability. To analyze plasma stability, SAL were preincubated in the presence of 50% human plasma for up to 6 days at 37 °C in a total volume of 60 µL. Subsequently, binding of SALs to cells was analyzed by flow cytometry as described above. Internalization Studies. A431 cells were seeded onto collagen-coated coverslips (100 000 cells/well) and allowed to adhere overnight. Cells were then incubated with DiI-labeled SAL (50 nmol lipid) for 1 h at 37 °C, washed, and incubated for additional 8 h. Coverslips were washed twice with PBS, fixed with 4% PFA for 20 min at room temperature, and washed again twice with PBS. Cells were then counter-stained with Alexa-488-labeled cetuximab (0.8 µg/mL) and fixed again with 4% PFA. Nuclei were stained by subsequent incubation of cells with DAPI (1 µg/mL) for 20 min at RT. Coverslips were mounted with Moviol and analyzed by fluorescence microscopy (Zeiss Cell Observer) and confocal fluorescence microscopy (Leica TCS SP 2). Alternatively, A431 cells were grown in a 12 well plate (200 000 cells/well). The next day, DiI-labeled anti-EGFR SAL were added (50 nmol lipid) and incubated for up to 8 h. Cells were washed twice with 1 mL ice-cold PBS or 50 mM glycine, 150 mM NaCl, pH 2.7, respectively. Cells were washed again with PBS, trypsinized, and analyzed by flow cytometry as described above. In Vitro Cytotoxicity Assay. Cells were grown in 96 well plates (15 000 cells/well) for 24 h, and varying amounts of free drug or drug-loaded liposomes were added. After incubation for 20 h, wells were washed 3 times with ice-cold PBS, and viable cells were determined using an MTT ([3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]) assay. In brief, microtiter plates were incubated with 50 µL/well RPMI + 10% FCS and 5 µL/well MTT (5 mg/mL) for 2 h at 37 °C. Then, the cells were lysed by adding 45 µL/well 10% SDS, 50% N,Ndimethylformamide, pH 4.7, overnight by shaking at room temperature. Absorbance was measured at 570 and 660 nm. For analysis, the 660 nm values were subtracted as unspecific background from the 570 nm values.

RESULTS Anti-EGFR affibody (ZEGFR:1907)2-cys. SDS-PAGE analysis of purified (ZEGFR:1907)2-cys revealed a single band of 14 kDa under reducing conditions and a major band (approximately 96%) corresponding to dimeric molecules (25 kDa) under nonreducing conditions (Figure 1a). The presence of dimeric molecules was confirmed by size exclusion chromatography (SEC) (Figure 1c). Here, (ZEGFR:1907)2-cys eluted at a major peak corresponding to a molecular mass of 28 kDa. A melting point of 46 °C was determined for (ZEGFR:1907)2-cys by dynamic light scattering analysis (Figure 1b). Sterically Stabilized Anti-EGFR Affibody Liposomes. Anti-EGFR sterically stabilized affibody liposomes (SAL) were produced by coupling reduced (ZEGFR:1907)2-cys to Mal-PEG2000DSPE micelles and subsequent insertion into preformed PEGylated liposomes using 2 mol % micellar lipid in relation to liposomal lipid. Efficiency of coupling of (ZEGFR:1907)2-cys to Mal-PEG2000-DSPE micelles was determined to be 53 ( 5% (n ) 5). Postinsertion was performed at 55 °C for 30 min. Although the melting point of (ZEGFR:1907)2-cys is only 46 °C, the resulting SAL showed binding to a panel of EGFR-expressing tumor cell lines (Figure 2). For most cell lines analyzed, binding strength correlated with expression of EGFR as determined by flow cytometry analysis with an anti-EGFR monoclonal antibody.

Bioconjugate Chem., Vol. 20, No. 6, 2009 1203

Figure 1. (a) SDS-PAGE analysis of purified affibody (ZEGFR:1907)2cys. Affibody (5 µg/lane) was separated on a 20% SDS-polyacrylamide gel under nonreducing (1) and reducing (2) conditions, respectively, and stained with Coomassie blue. (b) Determination of protein melting point by dynamic light scattering (kcps, kilocounts per second). (c) Size exclusion chromatography of purified affibody (ZEGFR:1907)2-cys.

Exceptions were cell lines MDA-MB-231 and Colo 205, which expressed moderate to low levels of EGFR but did not bind anti-EGFR SAL. Binding was strongest with human squamous carcinoma cell line A431. Binding to this cell line was analyzed in more detail using anti-EGFR SAL produced by postinsertion of 0.02 to 2 mol % affibody molecule-coupled micellar lipid, corresponding to approximately 2 to 200 pmol affibody molecule/ µmol lipid. All liposomes had a size in the range 90-97 nm and a slightly negative ζ potential (Table 1). Flow cytometry showed that binding to A431 cells increases with increasing concentrations of lipid and affibody molecule density, i.e., inserted micellar lipid (Figure 3a). At the highest lipid concentration (100 nmol lipid/assay), we observed a reduction in binding for SAL prepared by insertion of 0.6 and 2 mol % micellar lipid, which was most pronounced for SAL with 2 mol % micellar lipid. Binding of anti-EGFR SAL (10 nmol lipid, 2 mol % inserted micellar lipid) to A431 was blocked by excess amounts (10 µg/mL) of soluble (ZEGFR:1907)2-cys as well as soluble EGF (EGFm1) (24) but not by an irrelevant affibody molecule, (ZTaq)2-cys (Figure 3b). Furthermore, SAL produced from (ZTaq)2-cys directed against Taq polymerase did not show any binding to A431 (Figure 3c). Stability and Internalization of anti-EGFR SAL. In order to determine stability of SAL under physiological conditions, anti-EGFR SAL (2 mol % inserted micellar lipid) were incubated with human plasma at 37 °C for up to 6 days and subsequently analyzed for cell binding by flow cytometry. Approximately 70% of the initial binding activity of the antiEGFR-SAL was retained after 1 day of incubation, which gradually decreased to a value of approximately 30% after 6 days (Figure 4). Anti-EGFR SAL were efficiently internalized as shown by flow cytometry analysis. A431 were incubated for up to 8 h at 37 °C with DiI-labeled anti-EGFR SAL and then


Beuttler et al.

Figure 2. Binding of SAL to EGFR-expressing cells. Flow cytometry analysis of binding of PE-labeled anti-EGFR monoclonal antibody (mAb) or sterically stabilized anti-EGFR affibody liposomes (SAL) to various cell lines (A431, MDA-MB-468, SKBR3, LS174T, MDA-MB-231, Colo 205, MCF-7, HEK293). 200 000 cells were incubated with 10 nmol lipid or 10 µg/mL anti-EGFR mAb, respectively, (gray, cells alone; bold line, cells incubated with antibody or SAL). Table 1. Characterization of anti-EGFR SAL inserted micelles (mol %)

affibody molecule

size (nm)

polydispersity index

ζ-potential (mV)

2.0 2.0 0.6 0.2 0.06 0.02

+ + + + +

99.6 ( 0.6 93.9 ( 1.5 96.9 ( 1.3 92.9 ( 0.6 95.1 ( 0.8 90.2 ( 0.4

0.20 0.08 0.14 0.10 0.09 0.07

-0.57 -0.04 -0.90 -2.24 -0.46 0.12

washed with PBS (pH 7.4) or acidic glycine buffer (pH 2.7). An increase of fluorescence was observed over time (Figure 5). Fluorescence of cells washed with acidic buffer, which removes liposomes bound to the cell membrane (25), was weaker than that measured for cells washed with PBS but also increased over time to approximately 90% of fluorescence of PBS-washed cells after 8 h indicating that the majority of SALs was internalized. Internalization was further shown by conventional and confocal fluorescence microscopy of A431 cells

incubated for 9 h with DiI-labeled anti-EGFR SAL (Figure 6a,d). A431 incubated with SAL at 4 °C showed only binding to the cell surface (Figure 6b). Furthermore, no binding and internalization was observed for nontargeted SL included as negative control (Figure 6c). In Vitro Cytotoxicity. In order to analyze the effects of affibody-mediated delivery of liposomal drugs, we prepared mitoxantrone (MTO)-loaded SL (MTO-SL) and SAL (MTOSAL). Because SAL prepared by insertion of 2 mol % micellar lipid show a strong reduction of binding at very high lipid concentrations, we used SAL prepared by the insertion of 0.6 mol % affibody molecule-coupled micelles for cytotoxicity assays. The size of the drug-loaded liposomes was 116 ( 5 nm for MTO-SL and 110 ( 1 nm for MTO-SAL (n ) 9). MTOSL and anti-EGFR MTO-SAL were tested for cytotoxicity on A431, MDA-MB-468, and MCF-7, the latter included as negative cell line (see Figure 2). In these experiments, cells were incubated with drug for 20 h and the viability of remaining cells



Figure 5. Internalization of anti-EGFR SAL. A431 cells were incubated with anti-EGFR SAL for up to 8 h at 37 °C, washed either with PBS or with glycine buffer (pH 2.7), respectively, and subsequently analyzed by flow cytometry (n ) 3).

Figure 3. (a) Binding to A431 cells of anti-EGFR SAL generated by insertion of different micellar-conjugated affibody (ZEGFR: 1907)2-cys (2 to 0.02 mol %) at varying lipid concentrations (1 to 100 nmol lipid per 200 000 cells). Plain liposomes (incubated with cysteine to block the maleimide group) were included as negative control. Shown is the mean fluorescence intensity (MFI) of cells incubated with liposomes and analyzed by flow cytometry (n ) 2). (b) Blocking of binding of antiEGFR SAL to A431 cells by soluble affibody (ZEGFR:1907)2-cys or a EGF variant (EGFm1) but not with an irrelevant affibody (ZTaq) (gray, cells alone; gray thin line, cells incubated with anti-EGFR SAL; bold line, cells incubated with anti-EGFR SAL + soluble proteins as indicated. (c) Flow cytometry analysis of SAL generated from control affibody ZTaq tested for binding to A431 cells at 10 nmol lipid/200 000 cells (gray, cells alone; bold line, cells incubated with SAL).

Figure 4. Stability of anti-EGFR SAL. Anti-EGFR SAL were incubated with human plasma at 37 °C for up to 6 days and subsequently analyzed for binding to A431 cells. Shown is the mean fluorescence intensity (% of untreated SAL) of cells incubated with liposomes (n ) 3).

was determined by MTT assay. All 3 cell lines were sensitive for MTO with IC50 values between 1.3-18 µM (Figure 7; Table 2). MTO-loaded liposomes (MTO-SL) showed a reduced cytotoxicity demonstrating the protective properties of encapsulation of drug into PEGylated liposomes. Compared to MTOSL, an increased cytotoxicity of MTO-SAL was observed for the two EGFR-expressing cell lines (A431, MDA-MB-468),

while no differences were found for the EGFR-negative cell line (MCF-7) (Figure 7, Table 2). Anti-EGFR SAL not loaded with drug showed no growth inhibition on MDA-MB-468 cells and only little cytotoxicity on A431 cells up to a concentration of 33 µmol lipid/mL (corresponding to the highest lipid concentration used for the MTO-loaded SAL) (Figure 8a). Furthermore, soluble (ZEGFR:1907)2-cys did not inhibit growth of MDA-MB-468 cells and was only slightly inhibitory on A431 cells up to a concentration of 6.7 µg/mL (Figure 8b).

DISCUSSION We applied a bivalent affibody molecule directed against EGFR and possessing a free cysteine residue at the C-terminus, (ZEGFR:1907)2-cys for the generation of targeted liposomes. The resulting affibody liposomes (SAL), which were sterically stabilized by incorporating mPEG into the lipid bilayer, showed selective and strong binding to a variety of EGFR-expressing tumor cell lines. Thus, coupling of the affibody molecule to Mal-PEG2000-DSPE and insertion into PEGylated liposomes does not interfere with antigen-binding activity. This is comparable to recent findings for an anti-HER2 affibody molecule, which was used for the production of targeted nanoparticle-affibody molecule bioconjugates composed of poly(D,L-lactic acid), PEG, and Mal-PEG (26). These nanoparticles also showed selective binding to HER2-expressing cell lines and efficient internalization. In another study, HER2-specific affibody moleculeconjugated thermosensitive liposomes, composed of DPPC, MalPEG2000-DSPE, and PEG2000-DSPE, were produced, however, no binding data were reported (27). Binding of anti-EGFR SAL to A431 was efficiently blocked by soluble (ZEGFR:1907)2 as well as EGF. This is in accordance with recent binding studies of the low-affinity precursor of ZEGFR:1907, (ZEGFR:955)2, where binding of the affibody molecule was blocked by EGF and antiEGFR mAb cetuximab (20). We also observed efficient internalization of anti-EGFR SAL into A431 cells, which gradually increased over time. Internalization of a bivalent, 125Ilabeled anti-EGFR affibody molecule ((ZEGFR:955)2) was recently shown to reach a maximum after 8 h, similar to that of cetuximab, while internalization of EGF occurred more rapidly with a maximum reached already after 2 h (20). Thus, antiEGFR SAL behave similarly to free affibody molecule in terms of internalization. Applying dynamic light scattering, a melting point of approximately 46 °C was determined for purified (ZEGFR:1907)2. Interestingly, the affibody molecule retained its antigen-binding activity after the postinsertion step, which was performed for 30 min at 55 °C. This indicates that the affibody molecule is


Figure 6. Internalization of anti-EGFR SAL. (a) Fluorescence microscopy of A431 cells incubated with anti-EGFR SAL (red) for 9 h at 37 °C. (b) Fluorescence microscopy of A431 cells incubated with antiEGFR SAL (red) for 1 h at 4 °C. (c) Fluorescence microscopy of A431 cells incubated with control SL (red) for 9 h at 37 °C. (d) Confocal fluorescence microscopy of a representative A431 cell as shown in (a) including sections through the z axes. In all images, fixed cells were counterstained with Alexa-488-labeled cetuximab (green) to visualize the cell membrane. Nuclei in (a-c) are stained with DAPI (blue).

capable of refolding into its native configuration after the postinsertion. The capacity to refold after denaturation is a general feature observed for affibody molecules (28, 29). In this regard, affibody molecules behave differently from antibody molecules. For example, scFv molecules with a similar melting point did not survive the postinsertion step performed at 50 °C or higher temperatures, thus were not capable of refolding into active molecules (unpublished personal data). Compared to antibodies, affibody molecules possess several further advantages for the generation of targeted liposomes and other nanocarriers. First, they do not require any disulfide bonds to assemble into stable and active molecules. Hence, a single cysteine residue can be easily introduced and used for sitedirected and defined conjugation, e.g., the formation of stable thioether bonds, without interfering with domain assembly and stability. Second, affibody molecules have been shown to possess similar selectivity and picomolar to low nanomolar affinities as antibodies (19, 30) but lack the Fc-mediated

Beuttler et al.

Figure 7. Cytotoxicity. (a) A431 (n ) 6), (b) MDA-MB-468 (n ) 6), and (c) MCF-7 (n ) 6) were incubated with free mitoxantrone (MTO), mitoxantrone-loaded liposomes (MTO-SL), or mitoxantrone-loaded anti-EGFR SAL (MTO-SAL) for 20 h, and cell viability was determined by MTT assay. Table 2. IC50 Values of Mitoxantrone and Mitoxantrone-Loaded Liposomes (in µM) cell line

free MTO

MTO-SL

MTO-SAL

A431 MDA-MB-468 MCF-7

1.3 3 18

4.7 16 >100

2.8 6.8 >100

activities, which have been shown to be involved in rapid clearance of immunoliposomes produced from whole antibodies. Finally, they can be produced at large quantities in bacteria without the need for periplasmic secretion as required for recombinant antibody molecules. Using mitoxantrone-loaded anti-EGFR SAL, we showed that binding to EGFR-expressing cells translates into increased



ACKNOWLEDGMENT The authors would like to thank doctors Per Jonasson and Nina Bandmann (Affibody AB, Stockholm, Schweden), for valuable assistance with purification of (ZEGFR:1907)2-cys, and Dr. Thomas Mu¨rdter (Dr. Margarete Fischer-Bosch Institut fu¨r Klinische Pharmakologie, Stuttgart, Germany) for providing Alexa-488-labeled cetuximab.

LITERATURE CITED

Figure 8. Effects of SAL and soluble affibody molecules on cell growth. MDA-MB-468 and A431 were incubated for 20 h with increasing concentrations of anti-EGFR SAL (a) or soluble affibody molecule (ZEGFR: 1907)2-cys (b) and viable cells were determined by MTT assay.

cytotoxicity compared to drug-loaded SL. Anti-EGFR SAL not loaded with drug were nontoxic, indicating that cytotoxicity was not mediated by the affibody molecule displayed on the liposome surface. The MTO-SAL formulation used in this study did not reach the cytotoxicity of the free drug. This is in accordance with studies for anti-EGFR immunoliposomal formulations of doxorubicin, which also reported IC50 values between those of free drug and nontargeted liposomes (13). These findings indicate that there is room for improvement, e.g., by increasing stability and thus reducing nonspecific cytotoxicity probably mediated by drug leakage. Furthermore, intracellular drug release from internalized liposomes is a subject for further improvement, e.g., by the incorporation of pH-dependent release mechanisms or by increasing fluidity of lipid bilayer through incorporation of short-chain phospholipids (31-33). In vivo efficacy of targeted liposomal drug formulations might also be influenced by other factors (34). For instance, the kind of drug can determine efficacy, as shown for anti-CD19 immunoliposomes loaded with doxorubicin and vincristine, respectively (35). Finally, the affibody molecule itself might influence efficacy of SAL. For example, affibody molecules with strong internalization capacity should result in increased intracellular delivery of the liposomal drug, as shown for anti-ErbB2 scFv immunoliposomes (36-39). In summary, we have established a method to produce targeted liposomes from cysteine-modified affibody molecules as ligands. This method should be broadly applicable to the generation of affibody liposomes and other carrier systems for targeted delivery of drugs or other reagents.

(1) Mendeson, J., and Baselga, J. (2006) Epidermal growth factor receptor targeting in cancer. Semin. Oncol. 33, 369–385. (2) Nicholson, R. I., Gee, J. M. W., and Harper, M. E. (2001) EGFR and cancer prognosis. Eur. J. Cancer 37, S9-S15. (3) Mamot, C., and Rochlitz, C. (2006) Targeting the epidermal growth factor receptor (EGFR) - a new therapeutic option in oncology? Swiss Med. Wkly. 136, 4–12. (4) Rocha-Lima, C. M., Soares, H. P., Raez, L. E., and Singal, R. (2007) EGFR targeting of solid tumors. Cancer Control 14, 295– 304. (5) Samad, A., Sultana, Y., and Aqil, M. (2007) Liposomal drug delivery systems: an update review. Curr. Drug DeliVery 4, 297– 305. (6) Drummond, D. C., Meyer, O., Hong, K., Kirpotin, D. B., and Papahadjopoulos, D. (1999) Optimizing liposomes for delivery of chemotherapeutic agents to solid tumors. Pharmacol. ReV. 51, 691–743. (7) Park, J. W., Benz, C. C., and Martin, F. J. (2004) Future directions of liposome- and immunoliposome-based cancer therapeutics. Semin. Oncol. 31 (1), 196–205. (8) Sapra, P., Tyagi, P., and Allen, T. A. (2005) Ligand-targeted liposomes for cancer treatment. Curr. Drug DeliVery 2, 369– 381. (9) Sofou, S., and Sgouros, G. (2008) Antibody-targeted liposomes in cancer therapy and imaging. Expert Opin. Drug DeliVery 5, 189–204. (10) Kontermann, R. E. (2006) Immunoliposomes for cancer therapy. Curr. Opin. Mol. Ther. 8, 39–45. (11) Mamot, C., Drummond, D. C., Greiser, U., Hong, K., Kirpotin, D. B., Marks, J. D., and Park, J. W. (2003) Epidermal growth factor receptor (EGFR)-targeted immunoliposomes mediate specific and efficient drug delivery to EGFR- and EGFRvIIIoverexpressing tumor cells. Cancer Res. 63, 3154–3161. (12) Mamot, C., Drummond, D. C., Noble, C. O., Kallab, V., Guo, Z., Hong, K., Kirpotin, D. B., and Park, J. W. (2005) Epidermal growth factor receptor-targeted immunoliposomes significantly enhance the efficacy of multiple anticancer drugs in vivo. Cancer Res. 65, 11631–11638. (13) Mamot, C., Ritschard, R., Ku¨ng, W., Park, J. W., Herrmann, R., and Rochlitz, C. F. (2006) EGFR-targeted immunoliposomes derived from the monoclonal antibody EMD72000 mediate specific and efficient drug delivery to a variety of colorectal cancer cells. J. Drug Targeting 14, 215–223. (14) Holliger, P., and Hudson, P. J. (2005) Engineered antibody fragments and the rise of single domains. Nat. Biotechnol. 23, 1126–1136. (15) Tolmachev, V., Orlova, A., Nilsson, F. Y., Feldwisch, J., Wennborg, A., and Abrahmseń, L. (2007) Affibody molecules: potential for in vivo imaging of molecular targets for cancer therapy. Exp. Opin. Biol. Ther. 7 555–568. (16) Nilsson, F. Y., and Tolmachev, V. (2007) Affibody molecules: new protein domains for molecular imaging and targeted tumor therapy. Curr. Opin. Drug DiscoVery DeV. 10, 167–175. (17) Nygren, P. Å. (2008) Alternative binding proteins: affibody binding proteins developed from a small three-helix bundle scaffold. FEBS J. 275, 2668–2676. (18) Friedman, M., Nordberg, E., Hoideń-Guthenberg, I., Brismar, H., Adams, G. P., Nilsson, F. Y., Carlsson, J., and Sta˚hl, S. (2007)

1208 Bioconjugate Chem., Vol. 20, No. 6, 2009 Phage display selection of affibody molecules with specific binding to the extracellular domain of the epidermal growth factor receptor. Protein Eng. Des. Sel. 20, 189–199. (19) Friedman, M., Orlova, A., Johansson, E., Eriksson, T. L. J., Hoïdeń-Guthenberg, I., Tolmachev, V., Nilsson, F. Y., and Sta˚hl, S. (2008) Directed evolution to low nanomolar affinity of a tumor-targeting epidermal growth factor receptor 1-binding affibody molecule. J. Mol. Biol. 376, 1388–1402. (20) Nordberg, E., Friedman, M., Go¨string, L., Adams, G. P., Brismar, H., Nilsson, F. Y., Sta˚hl, S., Glimelius, B., and Carlsson, J. (2007) Cellular studies of binding, internalization and retention of a radiolabeled EGFR-binding affibody molecule. Nucl. Med. Biol. 34, 609–618. (21) Tolmachev, V., Friedman, M., Sandstro¨m, M., Eriksson, T. L. J., Rosik, D., Hodik, M., Sta˚hl, S., Frejd, F. Y., and Orlova, A. (2009) Affibody molecules for epidermal growth factor receptor targeting in vivo: aspects of dimerization and labeling chemistry. J. Nucl. Med. 50, 274–283. (22) Messerschmidt, S. K. E., Kolbe, A., Mu¨ller, D., Knoll, M., Pleiss, J., and Kontermann, R. E. (2008) Novel single-chain Fv’ formats for the generation of immunoliposomes by site-directed coupling. Bioconjugate Chem. 19, 362–369. (23) Lim, H. J., Masin, D., Mctintosh, N. L., Madden, T. D., and Bally, M. B. (2000) Role of drug release and liposome-mediated drug delivery in govering the therapeutic activity of liposomal mitoxantrone used to treat human A431 and LS180 solid tumors. J. Pharmacol. Exp. Ther. 292, 337–345. (24) Bach, M., Ho¨lig, P., Schlosser, E., Vo¨lkel, T., Graser, A., Mu¨ller, R., and Kontermann, R. E. (2003) Isolation from phage display libraries of lysine-deficient human epidermal growth factor variants for directional coupling as targeting ligands. Protein Eng. 16, 1107–1113. (25) Lee, R. J., and Low, P. S. (1994) Delivery of liposomes into cultured KB cells via folate receptor-mediated endocytosis. J. Biol. Chem. 269, 3198–3204. (26) Alexis, F., Basto, P., Levy-Nissenbaum, E., Radovic-Moreno, A. F., Zhang, L., Pridgen, E., Wang, A. Z., Marein, S. L., Westerhof, L., Molnar, L. K., and Farokhzad, O. C. (2008) HER2-targeted nanoparticle-affibody conjugates for cancer therapy. ChemMedChem 3, 1839–1843. (27) Puri, A., Kramer-Marek, G., Campbell-Massa, R., Yavlovich, A., Tele, S. C., Lee, S.-B., Clogston, J. D., Patri, A. K., Blumenthal, R., and Capala, J. (2008) HER-2-specific affibodyconjugated thermosensitive liposomes (Affisomes) for improved delivery of anticancer agents. J. Liposome Res. 18, 293–307. (28) Orlova, A., Feldwisch, J., Abrahmseń, L., and Tolmachev, V. (2007) Update: affibody molecules for molecular imaging and therapy for cancer. Cancer Biother. Radiopharm. 22, 573–584.

Beuttler et al. (29) Arora, P., Oas, T. G., and Myers, J. K. (2004) Fast and faster: a desgined variant of the B-domain of protein A folds in 3 µsec. Protein Sci. 13, 847–853. (30) Orlova, A., Magnusson, M., Eriksson, T. L., Nilsson, M., Larsson, B., Hoideń-Guthenberg, I., Widsto¨rm, C., Carlsson, J., Tolmachev, V., Stahl, S., and Nilsson, F. Y. (2006) Tumor imaging using a picomolar affinity HER2 binding affibody molecule. Cancer Res. 66, 4339–4348. (31) Simo˜es, S., Moreira, J. N., Fonseca, C., Du¨zgunes, N., and Pedroso de Lima, M. (2004) On the formulation of pH-sensitive liposomes with long circulation times. AdV. Drug DeliVery ReV. 56, 947–965. (32) Ishida, T., Okada, Y., Kobayashi, T., and Kiwada, H. (2006) Development of pH-sensitive liposomes that efficiently retain encapsulated doxorubicin (DXR) in blood. Int. J. Pharmaceut. 309, 94–100. (33) Kawano, K., Onose, E., Hattori, Y., and Maitani, Y. (2008) Higher liposomal membrane fluidity enhances the in vitro antitumor activity of folate-targeted liposomal mitoxantrone. Mol. Pharm. 6, 98–104. (34) Sapra, P., and Allen, T. M. (2003) Ligand-targeted liposomal anticancer drugs. Prog. Lipid Res. 42, 439–462. (35) Sapra, P., Moase, E. H., Ma, J., and Allen, T. M. (2004) Improved therapeutic responses in a xenograft model of human B lymphoma (Namalwa) for liposomal vincristine versus liposomal doxorubicin targeted via anti-CD19 IgG2a or Fab’ fragments. Clin. Cancer Res. 10, 1100–1111. (36) Nielson, U. B., Kirpotin, D. B., Pickering, E. M., Hong, K., Park, J. W., Shalaby, M. R., Shao, Y., Benz, C. C., and Marks, J. D. (2002) Therapeutic efficacy of anti-ErbB2 immunoliposomes targeted by a phage antibody selected for cellular endocytosis. Biochim. Biphys. Acta 1591, 109–118. (37) Park, J. W., Kirpotin, D. B., Hong, K., Shalaby, R., Shao, Y., Nielson, U. B., Marks, J. D., Papahadopoulos, D., and Benz, C. C. (2001) Tumor targeting using anti-her2 immunoliposomes. J. Controlled Release 74, 95–113. (38) Drummond, D. C., Shao, Y., Shalaby, M. R., Nielson, U. B., Marks, J. D., Benz, C. C., and Park, J. W. (2006) Antibody targeting of long-circulating lipidic nanoparticles does not increase tumor localization but does increase internalization in animal models. Cancer Res. 66, 6732–6740. (39) Shmeeda, H., Tzemach, D., Mak, L., and Gabizon, A. (2009) Her2-targeted pegylated liposomal doxorubicin: retention of target-specific binding and cytotoxicity after in vivo passage. J. Controlled Release [Online early access] DOI: 10.1016/j.jconrel.2009.02.002. BC900061V

Targeting of Epidermal Growth Factor Receptor (EGFR) - American

Recommend Documents