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,1,1
* National Laboratory for Oncogenes and Related Genes, Shanghai Cancer Institute, Medical College of and
School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China; and
Medical college of Fudan University, Shanghai, China
1Correspondence: National Laboratory for Oncogenes and Related Genes, Shanghai Cancer Institute, No.25/2200, Xietu Road, Shanghai 200032, China. E-mail: yhxu@sjtu.edu.cn or nlorg{at}sh163.net
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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22 nM, but with much lower mitogenic activity than with EGF. We showed that the peptides were internalized preferentially into EGFR highly expressing cells, and they accumulated in EGFR overexpressing tumor xenografts after i.v. delivery in vivo. In gene delivery studies, GE11-conjugated polyethylenimine (PEI) vectors were less mitogenic, but still quite efficient at transfecting genes into EGFR highly expressing cells and tumor xenografts. Taken together, GE11 is a potentially safe and efficient targeting moiety for selective drug delivery systems mediated through EGFR.—Li, Z., Zhao, R., Wu, X., Sun, Y., Yao, M., Li, J., Xu, Y., Gu, J. Identification and characterization of a novel peptide ligand of epidermal growth factor receptor for targeted delivery of therapeutics.
Key Words: EGFR • peptide synthesis • peptide ligand
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. EGFR overexpression was frequently found in a wide spectrum of human tumors of epithelial origin, including non-small cell lung cancer (NSCLC), breast, head and neck (SCCHN), gastric, colorectal, esophageal, prostate, bladder, renal, pancreatic, and ovarian cancers (2)
. All these findings set EGFR as an important target for receptor-mediated delivery system of drugs and genes for cancer treatment. Its natural ligand EGF, however, has strong mitogenic and neoangiogenic activity. Therefore, it is essential to seek a substitute for EGF. Peptide ligands, which have the advantages of readily diffusible ability, low immunogenicity, and easy incorporation into certain gene delivery vectors (3)
, are being pursued as targeting moiety for selective delivery of radionuclide, cytokines, chemical drugs, and therapeutic genes to tumors (4
5
6)
. Recently, several studies reported the successful identification of peptide ligands by screening phage display libraries (7
8
9)
. We took a similar approach and isolated a novel peptide clone with high binding capacity to EGFR but low mitogenic activity. The peptide was shown to be able to mediate target-specific delivery of reporter genes to EGFR overexpressing tumor cells in vitro and in vivo. |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Phage display
Ph.D.-12TM Phage Display Peptide Library Kit was purchased from New England Biolabs Inc. (Beverly, MA, USA). Biopanning procedures were done according to the manufacturer’s instruction with certain modifications. Briefly, a 96-well plate was coated with 50 µL hEGFR (in the first round at 60 µg/mL, the next two rounds at 40 µg/mL) overnight at 4°C. Wells were washed with PBS, blocked with blocking buffer, washed three times with cold PBST (PBS, 0.05% Tween-20), then incubated with 4x1010 pfu phage peptide library Ph.D.-12 for 30 min at 4°C. Unbound phages were removed by washing 10 times with cold PBST. Bound phages were eluted with 100 µL of 0.2 M glycine-HCl (pH 2.2) and neutralized with 15 µL of 1 M Tris-HCl (pH 9.1). The elution procedure was repeated three times and the final eluate was used for amplification and titration in Escherichia coli ER2738 culture. Recovered phages were subjected for two more rounds of biopanning with hEGFR proteins. The eluate in the third round of screening was titrated and blue clones were randomly picked and amplified by infecting ER2738. Phage single-stranded DNA was eluted and purified for sequencing.
Peptide synthesis
Peptides were synthesized and purified by reverse-phase high-performance liquid chromatography with >95% purity. GE11 peptide and GEL peptide, which is GE11 peptide with a linker sequence (GGGGS)3 at the carboxyl terminal were synthesized. An irrelevant peptide, HW12 (HYPYAHPTHPSW), was also synthesized.
Phage recovery assay
Recombinant hEGF, expressed in E. coli, was gifted by Dr. Li ZP (State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences). hEGFR or BSA (at 1 µg in 50 µL 0.1 M NaHCO3 per well) were immobilized on 96-well plate overnight at 4°C. Wells were washed twice with PBS, filled with blocking buffer as mentioned above for 2 h at 4°C, and incubated at 25°C for 1 h with 1 x 109 pfu of purified phages diluted in 50 µL PBST (PBS containing 0.5% Tween-20). Wells were washed 10 times with PBST and phages were recovered by bacterial infection. The resulting eluted phages were titrated and the recovery percentage was quantified. In some cases hEGF or synthetic peptides were used to evaluate competitive inhibition of phage binding. The experiments were performed in triplicate.
Iodide labeling and binding assays
The IODO-GEN® (Sigma, St. Louis, MO, USA) was used to iodinate GE11, hEGF, or HW12 with 125I (Amersham, Piscataway, NJ, USA) following the manufacturer’s instruction. Peptide binding to hEGFR, BSA, SMMC-7721, and K562 cells were quantified. Briefly, 0.5 µg hEGFR or BSA in 50 µL 0.1 M NaHCO3 was coated in 96-well plate overnight, while SMMC-7721 cells or K562 cells were inoculated at 1 x 104 cells per well in 96-well plate and cultured overnight. The next day wells were washed three times with PBS and blocked with 200 µL PBS-BSA (PBS, 10 mg/mL BSA). About 1 x 105 cpm of 125I-labeled peptides was diluted in 100 µL PBS-BSA and added into each well. After incubation at 4°C for 1 h, wells were washed three times with PBST. As for K562 cells, liquids were removed by centrifuged at 1500 g for 5 min at 4°C. Cells were then lysed in 100 µL 0.2 N NaOH for 15 min, the wells of hEGFR or BSA were eluted with 100 µL 0.2 N glycine-HCl (pH 2.2), and the eluates were counted in Wallac 1409 Liquid Scintillation Counter (Amersham). Binding activities were also tested in the absence or presence of unlabeled GE11 or hEGF.
In vivo biodistribution
Subcutaneous tumors were produced in 4- to 6-wk-old BALB/c female athymic nude mice by injecting 5 x 106 SMMC-7721 cells into their dorsal subcutaneous space. Tumor formation was monitored until tumors reached 5 mm in size in one plane. 125I-Labeled GE11 (1 µCi, 1 µg) in a total volume of 100 µL PBS was injected through the tail vein. Mice were killed 0.5 or 4 h after injection. Tumor tissues and organs were excised and blotted dry on tissue paper. Each sample was weighed and its radioactivity was measured with Wallac 1409 Liquid Scintillation Counter. The %ID/g was calculated according to injection dose standard curve. In another set of experiments, 100 µg unlabeled GE11 was coinjected with the 125I-GE11 solution. Standard deviations were calculated by the measurements obtained from samples of three mice.
Internalization assay
FITC (Pierce, Rockford, IL, USA) was conjugated to the NH2 terminus of tested peptides. FITC-labeled peptide was purified by gel filtration with Sephadex G-25. The SMMC-7721 cells cultured on cover slices were incubated with FITC-labeled peptide at 37°C for 16 h. The slices were washed three times with PBS, then fixed by 4% paraformaldehyde and mounted by ProLong ® Gold Antifade Reagent (Molecular probes, Eugene, OR, USA). After that, the slices were examined under confocal microscope (Zeiss LSM 510, German). In some experiments, Cells were incubated with FITC-labeled peptides in the absence or presence of unlabeled GE11, hEGF, or HW12. The cells were washed with PBS three times after 10 min incubation at 37°C. Cells were visualized under a fluorescence microscope (Zeiss axioskop2, Germany).
Synthesis of GE11-linked PEI (gPEI) and EGF-linked PEI (ePEI)
PEI (1 mg ) of 22 kDa (Exgen 500, Fermentas Inc., Hanover, MD, USA) was conjugated with GE11 peptide or EGF at the molar ratio of 1:1. The conjugation procedure was largely the same as described previously (11)
. Briefly, Dithiobis (succinimidylpropionate) (DSP) (Sigma) was first conjugated with PEI, then GEL peptide or EGF. The reaction mixture was incubated for 2 h at room temperature. To remove reaction byproducts and DMSO, the mixture was dialyzed against deionized water.
Gel retardation assay
Gel retardation assays were used to determine the ability of gPEI to condense DNA. The PEI cation to DNA anion ratio (N/P ratio) is presented as the molar ratio of PEI nitrogen to DNA phosphate. pGL3 plasmid DNA (3 µg) (Promega, Madison, WI, USA) was mixed with PEI, gPEI, or ePEI at selected N/P ratios by adding the conjugates into DNA in sterile water. The resulting polyplexes were incubated at room temperature for 20 min, then subjected to electrophoresis in 0.9% agarose gel containing 0.5 µg/mL ethidium bromide. About 0.5 µg of pGL3 plasmid DNA was loaded into each well.
DNase protection assays
The ability of polyplexes to protect DNA from DNase digestion was characterized by DNase protection assays (11)
. An aliquot of 30 µL of PEI, gPEI, or ePEI solutions (in double-distilled H2O) was added to 30 µL of pGL3 plasmid DNA (0.2 µg/µL), then allowed to stand for 20 min at room temperature. Each sample was divided into two aliquots of 20 µL and placed into two separate tubes. Four units of DNase I (4 µL, Promega) or 4 µL of PBS (as the control) was added and the samples were incubated at 37°C for 15 min. Immediately after incubation, the samples were treated with 10 µL of EDTA (100 mM) at room temperature for 15 min to inactivate the enzyme. Each enzyme-inactivated sample was further divided into two aliquots of 15 µL. Heparin solution (5 µL; 10 µg/µL in H2O) or blank H2O was added into each tube and the tubes were then incubated at room temperature for 2 h to allow the complete dissociation of polyplexes by heparin. The extent of DNA degradation was evaluated using gel electrophoreses (0.9% agarose gels containing 0.5 µg/mL ethidium bromide).
MTT assay
The MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay was used for measuring cell growth as described previously (10)
. Briefly, 3 x 103 SMMC-7721 cells were seeded in each well of a 96-well plate overnight in DMEM containing 10% FBS. Cells were cultured in 100 µL of fresh medium containing 1 µg/mL GE11, HW12, hEGF, 10 µg /mL gPEI, ePEI, or PEI for 48 h. This incubation time was optimal for measuring stimulation effects of the peptides and the PEI. MTT(20 µL) solution (5 mg/mL in PBS) was added to each well and incubated for 5 h at 37°C. The solution was removed and 200 µL DMSO was added per well. After 5 min incubation at 37°C, the optical density at 570 nm was measured by using an enzyme-linked immunosorbent assay reader (Bio-Rad Model 550).
In vitro transfection studies
For transfection studies, 1.0 x 105 cells (SMMC-7721 cells) per well were seeded on 24-well plates (Falcon) and incubated for 24 h in DMEM media supplemented with 10% FBS and antibiotics at 37°C. One microgram of pGL3 plasmid DNA per well was used for all the transfection studies. Prior to transfection, the medium was removed and plates were rinsed with 0.5 mL of PBS. Polyplexes or naked DNA was then added to each well and incubated with cells in 0.5 mL serum-free media at 37°C for 4 h. After incubation, the incubation media were removed and cells were rinsed with 0.5 mL of PBS, followed by the addition of 0.5 mL of fresh medium containing 10% FBS and antibiotics. The cells were incubated for another 24 h, then lysed and assayed for luciferase activity. In some experiments, 0.5 mM GE11 peptide was added with the DNA polyplexes simultaneously to compete for the binding. All transfection experiments were performed in triplicate. To assay for the luciferase activity, the medium was removed from each well and the cells were washed with 0.5 mL/well of cold PBS. Ice-cold lysis buffer (200 µL; 100 mM Tris-HCl, 2 mM EDTA, 1% Triton X-100, pH 7.8) was added to each well and incubated on ice for 20 min. The cell lysate was then transferred to 0.5 mL centrifuge tubes and centrifuged at 13,000 rpm for 2 min. The luminescence was measured in a MiniLumat LB 9506 luminometer (EG&G BERTHOLD, Wildbad, Germany) immediately after mixing 10 µL of cell lysate with 50 µL of luciferase substrate (Promega). Relative light units were standardized for protein concentration determined by the bicinchoninic acid protein assays using bovine serum albumin standards. 1 ng luciferase corresponds to 1 x 107 RLU.
In vivo gene delivery
Athymic nude mice with SMMC-7721 tumor xenografts (5 mm in size) in their dorsal subcutaneous space were used for in vivo gene delivery. The polyplexes (containing 50 µg of pGL3 DNA) in a volume of 300 µL were injected via the tail vein. Twenty-four hours after injection, expression of luciferase was determined in the tumor as well as in lung and other tissues. In a competitive study, 100 µg free GE11 peptide was coinjected with the gPEI/DNA polyplexes. Standard deviations were calculated by the measurements obtained from samples of five mice.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A. Phage-GE11, which displayed a peptide with a sequenced of YHWYGYTPQNVI, was confirmed to bind specifically to EGFR. The number of phages bound to EGFR was 1000-fold higher than to BSA. The binding could be inhibited by both hEGF and synthetic GE11 peptide (Fig. 1B
). Phage-GE31 (displaying QIQVFHRWMTGP), however, did not have such a binding specificity (Fig. 1A
).
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To further confirm the specificity of the GE11 peptide binding to EGFR, GE11 was synthesized and labeled with 125I to evaluate its binding affinity to hEGFR protein or SMMC-7721 cells. Figure 2
A illustrated that the binding of 125I-GE11 to hEGFR proteins was significantly inhibited by excess unlabeled GE11. No significant binding was found between 125I-GE11 and BSA. As demonstrated in Fig. 2B
, binding of 125I-GE11 was evident in SMMC-7721 cells but not in EGFR-negative K562 cells. Again, the binding was efficiently inhibited by free EGF or GE11. Reciprocally, the 125I-EGF binding to SMMC-7721 cells was also blocked by GE11. On the other hand, an irrelevant peptide HW12 did not bind to SMMC-7721 cells. All these data demonstrated that GE11 could specifically bind to EGFR and implicated that the binding sites of GE11 and EGF on EGFR were partly overlapped.
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We further quantified the binding affinity of GE11 with EGFR. The saturation binding curve of [125I]GE11 to SMMC-7721 cells and the linear transformation of the data are shown in Fig. 2C, D
. Scatchard analysis revealed the existence of one population of binding sites with Kd of 22.28 ± 0.4 nM and Bmax of 2741 fmol/mL protein. This binding affinity is at least 10-fold less than that of hEGF, which bound to EGFR at a dissociation constant of 1 2 nM (12)
. A previous report showed that EGF bound both the domain I and domain III of EGFR (13)
. GE11 is a much smaller peptide containing only 12 residues, which might bind only one region of EGFR, therefore resulted in the relatively lower affinity.
In vivo biodistribution of 125I-labeled GE11
125I-Labeled GE11 was injected intravenously via tail vein in mice and the distribution of radioactivities in various tissues were counted. As shown in Fig. 3
, the radioactivity in the tumor was the highest among the tissues examined 4 h after injection, although 0.5 h after injection it was lower than those in kidney, liver, or blood. When excess unlabeled peptide was coinjected with labeled peptide, the radioactivity in tumor was dramatically reduced by 5-fold at 0.5 h and 4.5-fold at 4 h. These data suggested that GE11 had specific affinity toward this EGFR overexpressing SMMC-7721 tumor xenograft, although the dose accumulated in tumor tissues was still limited at 5.85% at 0.5 h and 3.18% at 4 h.
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GE11 internalization by EGFR overexpressing cells
To examine whether GE11 can be internalized into EGFR-expressing cells, we incubated SMMC-7721 cells or K562 cells with FITC-labeled GE11 peptide and found that the peptide was taken up efficiently by SMMC-7721 cells but not K562 cells (Fig. 4
A). In a blocking experiment, an excess amount of unlabeled GE11 or hEGF was shown to completely prevent FITC-labeled GE11 from binding to the cells, while HW12 peptide did not.
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Preparation and physicochemical characterization of GE11-conjugated PEI polyplexes
To determine the targeting ability of GE11, GEL (GE11 with a linker (GGGS)3) was conjugated with PEI. The abilities of the resulting conjugant gPEI vectors to package DNA were determined by gel retardation assay. The N/P ratio of gPEI/DNA polyplexes reflects the overall positive to negative charge ratio the DNA complex. The results in Fig. 5
A, B showed that all gPEI and ePEI (EGF conjugated with PEI) eliminated the mobility of plasmid DNA at N/P 
3, suggesting that gPEI and ePEI were capable of forming polyplexes, similar to unconjugated PEI. Maintaining the stability of plasmid DNA during formulation and delivery process is an important factor in successful gene delivery. In the current study, the stability of the gPEI polyplexes was assessed by DNase protection assay. As shown in Fig. 5C,D
after incubation with DNase I much of the plasmid DNA in gPEI and ePEI polyplexes remained intact, whereas naked DNA was completely digested. Furthermore, the protection effect was increased as the N/P ratio increased from 2 to 10.
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Mitogenic activity of peptide GE11 and gPEI
Biological activity of GE11 peptide was evaluated by examining their ability to inhibit or stimulate cell proliferation using standard MTT assays. SMMC-7721 cells were used for this purpose. The mitogenic activities of GE11 and hEGF at the concentration of 1 µg/mL was shown in Fig. 6
A. The GE11 peptide stimulates the growth of SMMC-7721 cells by 10% while hEGF resulted in 50% increase of cell growth. There is no significant increase of stimulation by GE11 even at a concentration up to 10 µg/mL. When EGFs were conjugated to PEI, ePEI maintained the ability to promote cell growth (27% cell proliferation), while GE11-conjugated PEI showed no growth stimulating activity (shown in Fig. 6B
).
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In vitro gene delivery assay
To examine the targeting specificity of GE11-conjugated PEI vectors, a gene transfection study was conducted using SMMC-7721 cells. As shown in Fig. 7
A, gPEI/pGL3, ePEI/Pgl3, or PEI/pGL3 polyplexes all mediated efficient gene transfection at N/P ratio of 10:1. There was no significant enhancement of gene delivery by either EGF-conjugated or GE11-conjugated vectors, probably because PEI by itself could transfect cells by nonspecific charge interactions. The effect of specific binding by EGF and GE11 was demonstrated more clearly, however, by the competitive binding and delivery data. The resulting luciferase activities were shown to be reduced by 7- and 2.8-fold, respectively, when excessive free GE11 peptides were coincubated with gPEI/DNA and ePEI/DNA polyplexes. No reduction of transfection efficiency was found in the PEI/DNA polyplexes in the presence of the same amount of free GE11 peptides. We also tested the transfection efficiency of the polyplexes at other N/P ratios and found that GE11 could inhibit the gene transfer of gPEI or ePEI but not that of PEI (data not shown). These data indicated that both EGF- and GE11-conjugated PEI vectors mediated gene transfection by target-specific bindings.
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Analysis of GE11 targeting after intravenous administration of the gPEI/pGL3 polyplexes
To determine the ability of GE11 to mediate targeted gene delivery in vivo, the polyplexes was administered intravenously to mice bearing subcutaneous SMMC-7721 tumors. Analysis of luciferase expression in tumors from these animals identified as much as an 18-fold increase in expression over levels obtained in tumors from animals injected with the PEI/pGL3 vector (Fig. 7B
). When the gPEI/DNA was coinjected with 100 µg free GE11 peptide, the gene expression were 5-fold lower. This demonstrated that the GE11 peptide was indeed aiding in targeting of the vector to the SMMC-7721 tumors.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Antibody such as mAb C225, which can block ligand-dependent EGFR signaling, had been used to mediate targeted immunoliposome internalization (15)
. Trastuzumab, a well-known inhibitory antibody of HER2, was also used in targeting PEI into HER2 expressing cells (11)
.
To explore the use of GE11 as a target ligand for delivery of therapeutics to cells and tumors, we prepared a PEI-based nonviral vector by conjugating GE11 as the targeting element. Two methods have been used frequently to incorporate targeting element into the PEI polyplexes. One is to use PEGylation of PEI and attach the ligand to PEG (16
17
18)
. The other is to directly conjugate PEI with ligand (11
, 19
, 20)
. A previous study has shown that tetrapeptide RGDC without a PEG spacer improved transfection efficiency of PEI in integrin-expressing Mewo cells by 1 to 2 orders of magnitude, especially at low N/P ratios, while no targeting effect could be found when using a PEG spacer (20)
. The data implicated PEG spacer is possible to shield both PEI and RGD ligand. Therefore, we adopted direct conjugation to PEI in this study. To reduce the effect of positive charge of PEI on GE11, GEL (GE11 with a linker (GGGGS)3 at the carboxylic terminus) was synthesized and conjugated to PEI by using DSP as a cross-linking agent that reacts with primary amines on the PEI and GEL molecules. DSP, a water-insoluble, homobifunctional N-hydroxysuccimide ester (NHS ester), is a thiol-cleavable, primary amine reactive reversible cross-linker (21
, 22)
. Because DSP contains a disulfide bond, bioconjugate synthesized via DSP cross-linking can potentially be cleaved intracellularly where the environment is highly reducing (23)
. This might be beneficial to the intracellular trafficking of plasmid DNA and facilitate transgene expression.
In this study, we demonstrated that EGF had significant mitogenic activities even after it was conjugated to PEI. This will certainly impede use of EGF as a targeting moiety although it has a binding affinity to EGFR at 1–2 nM. Unlike EGF-conjugated PEI vectors (ePEI), GE11-conjugated vectors (gPEI) did not have detectable mitogenic activity, and so is safer to be used in vivo. Although the resulting transduction levels in tumor cell lines were not significantly different from levels observed with PEI/DNA vector alone, the transfection mechanism would still be different. Previous studies (24
, 25)
have reported similar observations, and they hypothesized that this lack of increase was because in vitro there was no external force to increase vector-to-cell interaction as one may see in vivo after intravenous administration (26
27
28)
. On the other hand, competition studies confirmed that at least 80% of the transduction mediated by the EGFR-targeted vector could be competed away, whereas no competition was seen with the PEI-DNA vector or the other control vector formulations. These results confirmed the specificity of the GE11-EGFR interaction in cell lines and therefore favor a receptor-mediated uptake of the GE11 peptide. In addition, it was demonstrated that up to 15-fold increases in luciferase transduction occurred in tumors after intravenous administration of gPEI/DNA. The data suggest that GE11 peptide with a linker sequence (GGGGS)3, when conjugated to PEI, can mediate efficient and specific gene transfer to tumors in vivo, although the transduction levels were still somewhat less than those resulting from EGF-conjugated PEI vectors (shown in Fig. 7B
). The luciferase transduction in the tumor was the highest among the tissues tested, indicating that GE11 can mediate specific delivery of DNA complexes into tumor cells.
To our knowledge, this is the first study to identify a peptide ligand with high affinity for EGFR. We demonstrated that this peptide is capable of targeting itself or a nonviral gene delivery vectors to cancer cells in vivo. Such a novel peptide ligand of EGFR holds considerable promise for systemic delivery of therapeutics to tumor cells with EGFR overexpression. Further studies to optimize the use of this peptide are required. We are currently performing a comprehensive in vivo study on the delivery of potential therapeutics to tumors using this peptide. This paper provides a basis for further development of peptide ligand-based, EGFR-targeted cancer therapy.
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ACKNOWLEDGMENTS |
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Received for publication April 3, 2005. Accepted for publication August 19, 2005.
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DNA/HindIII marker. C, D) DNase protection assay of gPEI and ePEI respectively. Plasmid DNA alone or complexed with gPEI or pEI at different N/P ratios were exposed to DNase digestion. Lanes 13 is 
