Development and Characterization of the Recombinant Human VEGF

Article pubs.acs.org/bc

Development and Characterization of the Recombinant Human VEGF-EGF Dual-Targeting Fusion Protein as a Drug Delivery System Jia-Je Li,□,†,‡ Keng-Li Lan,□,†,§,∥ Shun-Fu Chang,⊥ Ya-Fen Chen,# Wen-Chun Tsai,† Pei-Hsun Chiang,† Meng-Han Lin,¶ Wolfgang B. Fischer,¶,● Yi-Sheng Shih,▽ Sang-Hue Yen,†,∥ Ren-Shyan Liu,†,▲ Yeou-Guang Tsay,#,⬡ Hsin-Ell Wang,*,†,‡,● and Cheng Allen Chang*,†,¶,●,■ †

Department of Biomedical Imaging and Radiological Sciences, §Institute of Traditional Medicine, #Institute of Biochemistry and Molecular Biology, ¶Institute of Biophotonics, ●Biophotonics & Molecular Imaging Research Center (BMIRC), ⬡Proteomics Research Center, and ■Biomedical Engineering Research and Development Center, National Yang-Ming University, Taipei, Taiwan, 112 ‡ Program in Molecular Medicine, National Yang-Ming University and Academia Sinica, Taipei, Taiwan, 112 ∥ Department of Oncology Medicine, ▽Division of Gastroenterology, Department of Medicine and ▲Department of Nuclear Medicine and National PET/Cyclotron Center, Taipei Veterans General Hospital, Taipei, Taiwan, 112 ⊥ Department of Medical Research and Development, Chang Gung Memorial Hospital-Chiayi Branch, Chiayi 613, Taiwan S Supporting Information *

ABSTRACT: The design, preparation, as well as structural and functional characterizations of the recombinant fusion protein hVEGF-EGF as a dual-functional agent that may target both EGFR (R: receptor) and angiogenesis are reported. hVEGF-EGF was found to bind to EGFR more strongly than did EGF, and to bind to VEGFR similarly to VEGF. Mass spectrometry measurements showed that the sites of DTPA (diethylenetriaminepentaacetic acid) conjugated hVEGF-EGF (for radiolabeling) were the same as those of its parent hEGF and hVEGF proteins. All DTPA-conjugated proteins retained similar binding capacities to their respective receptors as compared to their respective parent proteins. In vitro cell binding studies using BAEC (a bovine aortic endothelial cell) and MDA-MB-231 (a human breast cancer) cells expressing both EGFR and VEGFR confirmed similar results. Treating BAEC cells with hVEGF-EGF induced remarkable phosphorylation of EGFR, VEGFR, and their downstream targets ERK1/2. Nevertheless, the radiolabeled 111In-DTPA-hVEGF-EGF showed cytotoxicity against MDA-MB231 cells. Pharmacokinetic studies using 111In-DTPA-hVEGF-EGF in BALB/c nude mice showed that appreciable tracer activities were accumulated in liver and spleen. In all, this study demonstrated that the fusion protein hVEGF-EGF maintained the biological specificity toward both EGFR and VEGFR and may be a potential candidate as a dual-targeting moiety in developing anticancer drugs.

Received: September 21, 2015 Revised: October 29, 2015 Published: November 2, 2015 © 2015 American Chemical Society

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DOI: 10.1021/acs.bioconjchem.5b00509 Bioconjugate Chem. 2015, 26, 2481−2496

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Bioconjugate Chemistry

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INTRODUCTION One hallmark of a majority of human epithelial cancers is the functional activation of growth factors and receptors in the epidermal growth factor receptor (EGFR) family. Upon binding to EGFR, epidermal growth factor (EGF) causes downstream signals to promote cell growth, proliferation, and differentiation. Thus, the signaling pathways governed by the EGF-EGFR axis play central roles in cancer cell proliferation, survival, metastasis, and angiogenesis.1 Different EGFR antagonists are currently available for the treatment of at least four metastatic epithelial cancers: non-small-cell lung cancer, squamous-cell carcinoma of the head and neck, colorectal cancer, and pancreatic cancer. Two classes of EGF-EGFR drugs have been successfully tested and are now in clinical use, i.e., anti-EGFR monoclonal antibodies and small-molecule EGFR tyrosine kinase inhibitors. The former, such as cetuximab and panitumumab, bind to the extracellular domain of EGFR and compete for receptor binding by occluding the ligand-binding region, thereby blocking ligand-induced EGFR tyrosine kinase activation. The latter, such as erlotinib and gefitinib, compete with ATP to bind to the intracellular catalytic domain of EGFR tyrosine kinase and thus inhibit EGFR autophosphorylation and downstream signaling.1 On the other hand, it has been well accepted that angiogenesis, i.e., the formation of new blood vessels,2,3 plays an important role in many diseases including age-related macular degeneration, atherosclerosis, rheumatoid arthritis, and cancer.4−7 Numerous treatment modalities are currently under development to treat these angiogenesis-dependent diseases. The regulation of angiogenesis requires a balance between pro- and anti-angiogenic factors. A number of the growth factor receptor family signaling pathways, including VEGFR (vascular endothelial growth factor receptor), Her-2 (human epidermal growth factor receptor 2), PDGFR (platelet-derived growth factor receptors), and c-Met (a proto-oncogene that encodes a protein known as hepatocyte growth factor receptor, HGFR), are also linked to angiogenesis and tumor cell proliferation. The VEGF−VEGFR axis appears to be one of the most important players in this balance. Upon binding to its receptors, i.e., VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1), VEGF stimulates the proliferation, migration, and survival of endothelial cells. It is known that VEGFR1 binds one form of VEGF (i.e., VEGFA) with higher affinity (Kd = 15 pM) than does VEGFR2 (Kd = 750 pM); however, the induced tyrosine kinase activity upon VEGFR1 binding is weaker than that upon VEGFR2 binding.8 Some tumor cells were also reported to express VEGFR1 to regulate tumor migration and invasion.8,9 Tumor endothelial cells proliferate at a significantly higher rate than does quiescent normal endothelium, due in part to the abundant availability of growth factors in the tumor-associated microenvironment.10 New tumor-associated vessels are structurally and functionally distinct from their normal counterparts, particularly in its tortuous and leaky vasculature, which causes increased interstitial pressure and hypoxia. Additionally, the upregulation of surface molecules such as E-selectin, endoglin, integrin-αvβ3, and VEGFR enhance the adhesion and migration of endothelial cells.11,12 Therapies targeting these receptors exert their anticancer activities partially through the inhibition of the secretion of angiogenic growth factors. Given the critical role of angiogenic factors in tumor progression and metastasis, a wide array of novel therapeutic agents are currently being developed in both academia and industry. For example, small molecular agents such as sunitinib and sorafenib and anti-VEGF antibodies such as bevacizumab have been developed for clinical use.13,14

These inhibitors of EGF−EGFR and VEGF−VEGFR signaling have achieved significant success in treating various cancers. However, many unresolved issues of cancer chemotherapy remained which included poor selectivity of chemotherapy drug, drug resistance, and unstable and higher variability genetics of tumor cells.15 One characteristic of these receptor tyrosine kinase and angiogenesis inhibitors is that they function by inhibiting the growth of cancer cells (cytostatic) instead of killing the cancer cells (cytotoxic).16 For example, it is known that in contrast to cytotoxic reagents such as chemotherapy drugs, these cytostatic angiogenesis inhibitors cannot efficiently attack the wellestablished tumor blood vessels that are often observed in latestage tumors. In recent years, an alternative novel drug-delivery approach has arisen to target the vasculature or endothelial cells of tumors in the wake of the success of anti-angiogenic therapy. Given that the endothelial cells of tumor vasculature are genetically more stable and with a much lower tendency to develop drug-resistance compared to tumor cells, and that they are readily accessible to drugs delivered, neovasculature-targeted drug delivery appears to be an intriguing approach in cancer therapy. Many studies involving the modifications of VEGF or EGF with chemodrugs, fluorophores,17 radionuclides,18−22 toxic proteins,23,24 and nanoparticles25 to serve as tumor targeted therapeutic or diagnostic agents have been published to remedy these problems. Nevertheless, there are rare studies reported to combine both proteins to develop dual-targeting carriers (vide infra).26,27 Based on the vital roles of VEGF and EGF in tumor growth, this study aims to develop a dual-targeting recombinant fusion protein vehicle hVEGF-EGF and to evaluate its chemical, biological, and pharmacokinetic characteristics as well as potential avidity. In contrast to foreign antibodies or proteins, because both EGF and VEGF are endogenous proteins, the fusion protein hVEGF-EGF may be safer with lower possibility of immunogenicity.28,29 Because both EGF and VGEF have high binding affinities toward their respective receptors with good internalization abilities, hVEGF-EGF can be armed with chemodrugs, radionuclides, or nanoparticles to serve as dualtargeting theranostic agents. These theranostic agents may kill tumor cells more effectively as compared to the cytostatic drugs that target EGFR or VEGFR only, leading to shorter treatment time and fewer side effects such as destruction of intestine mucous membrane, diarrhea, and dermatitis.30 Specifically, if cell-killing radionuclides such as 111In (Auger electrons), 90Y, 177 Lu, 131I (β particles), 123Bi, and 225Ac (α particles) are attached to the labeled fusion protein for targeting therapy, the resulting fusion proteins are expected to be cytotoxic against cells with drug resistance.31 The bystander effect32,33 exerted by the radionuclides could also kill unbound nearby tumor cells in the tumor microenvironment. A preliminary account of some initial results of this paper was presented at the 2012 SNM Annual Meeting.34

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RESULTS Cloning, Expression, and Purification of hEGF, hVEGF, and hVEGF−EGF. The coding sequence of the fusion protein, hVEGF−EGF, was cloned into a yeast expression vector, pPICZαA. The constructs were designed in such a way that an α-secreting signaling peptide would be present to assist in the secretion of the desired proteins from the yeast strain, P. pastoris, after cleavage of this signal peptide. At the C-terminus, c-myc and hexa-histidine (myc-his6) tags were added for convenient protein detection and purification. Schematic representations of hEGF,

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Figure 1. Schematic diagram and purification of proteins. (A) Genes encoding hEGF, hVEGF, and hVEGF-EGF were cloned into the yeast vector, pPCIZ-αA, and digested with the restriction enzymes, KpnI and XbaI. (B) Respective purified hEGF, hVEGF, and hVEGF-EGF proteins with reduction (lanes 1, 3, and 5) and without reduction (lanes 2, 4, and 6) treatments were characterized on 15% SDS-PAGE gels by staining with Coomassie blue.

that of nonreducing condition and more closed to recombinant hEGF molecular weight (10.8 kDa) due to protein denaturation to linear structure without intramolecular disulfide linkages (Table 1). Specific and Competition Binding Assays of hVEGFEGF Fusion Protein to EGFR and VEGFR2. The dual-specific receptor binding ability of purified hVEGF-EGF to EGFR and VEGFR2 was examined in vitro. The specific binding was plotted against the total concentration of the added proteins, and the results were analyzed by nonlinear regression using GraphPad Prism Software. Purified human hEGF and hVEGF were used as positive controls, and these proteins demonstrated high affinity binding to EGFR and VEGFR2 with Kd values of 20.2 ± 6.6 and 2.2 ± 1.2 nM, respectively (Figure 2A and B). The binding of the fusion protein hVEGF-EGF to EGFR (Kd = 3.4 ± 0.8 nM) was stronger than single protein hEGF, while its binding to VEGFR2 (Kd = 2.3 ± 0.8 nM) was similar to that of hVEGF (Figure 2A and B). The binding curves of hEGF, hVEGF, and hVEGF-EGF to purified EGFR and VEGFR simultaneously immobilized on an ELISA plate showed that the binding maximum (Bmax) of hVEGF-EGF was higher than those of hEGF and hVEGF (Figure 2C). For the competition binding assay, hVEGF-EGF could only be partially competed away by EGF or VEGF alone from plates that were simultaneously coated with EGFR and VEGFR (Figure 2D). These results indicate that the conjugation of these two growth factors with a linker peptide maintained their receptor binding activities, and specifically had a positive avidity with respect to EGFR binding. In Vitro Binding of DTPA-hEGF, DTPA-hVEGF, and DTPA-hVEGF-EGF Proteins to Their Corresponding Receptors. DTPA-hEGF and DTPA-hVEGF-EGF bound to purified human EGFR on an ELISA plate with Kd values 35.2 ± 13.8 nM and 3.6 ± 0.5 nM, respectively (Figure 2E). The binding affinity of DTPA-hEGF was lower than that of hEGF (Kd = 20.2 ± 6.6 nM) indicating that the presumptively DTPA modified lysine residue of hEGF (vide infra) may be close to the EGFR binding site and resulted in diminished binding affinity to EGFR. On the other hand, the enhanced avidity of DTPAhVEGF-EGF to EGFR was maintained. The determined Kd values of DTPA-hVEGF and DTPA-hVEGF-EGF to purified human VEGFR2 (Flk-1) were 2.3 ± 1.2 and 2.8 ± 0.8 nM, respectively (Figure 2F), which were similar to those of unmodified parent proteins.

hVEGF, and hVEGF-EGF in the pPICZ-αA vector are shown in Figure 1A. hVEGF and hEGF are connected with the PGGGG linker. The host yeast strain, wild-type X-33 P. pastoris, was transformed with the plasmids, and high-expressing colonies were selected with Zeocin. Proteins secreted into the medium were then loaded onto a nickel-resin affinity column and eluted with a high concentration of imidazole. The typical yield of hVEGF-EGF was approximately 2−5 mg/L from yeast culture medium, whereas the yields of both hEGF and hVEGF were approximately 10−20 mg/L. The purified proteins were confirmed in Coomassie stained gels (Figure 1B). The preservation of VEGFR binding by VEGF and VEGF-EGF were anticipated by the results of SDS−PAGE under nonreducing conditions, which indicated its dimerization (Mr of 35 and 49 kDa) in solution, as required for receptor binding. SDS−PAGE under reducing conditions showed that these dimers dissociated into monomers (Mr of 17.5 and 24.5 kDa, Table 1). The binding of Table 1. Predicted Molecular Weights of hEGF, hVEGF, and hVEGF-EGF Recombinant Proteins Were Calculated by Elemental Compositions Based on the Designed Sequence with Disulfide Linkage Information protein hEGF hVEGF hVEGFEGF

elemental compositiona

average MW (Da)

amino acid sequence number of main portion

C462H699N141O145S8 C748H1162N224O234S15 C1051H1609N311O328S22

10804.9 17517.7 24554.5

EGF: N16 to E67 VEGF: D16 to N131 VEGF: D16 to N131 EGF: N137 to E187

a

Elemental composition is based on the designed sequence with disulfide linkage information.

the VEGF-EGF fusion protein dimer to the VEGFR is critical for cell signaling. Note that due to different degree of glycosylation the protein migrates as multiplicity of bands (35.0−37.0, 49.0−51.0, 17.5−19.5, and 24.5−26.5 kDa). After enzymatic deglycosylation under denaturating conditions, the apparent molecular masses of both hVEGF-EGF and hVEGF were decreased as compared to that of the undigested VEGF protein (SI Figure S1). The result confirmed that the hVEGF-EGF and hVEGF containing N-glycoslation sites were glycoslated by yeast. hEGF has been reported to contain three intramolecular disulfide bonds to stabilize its folded state.35 Under reducing conditions, SDS−PAGE showed that the band was smaller than 2483


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Figure 2. In vitro binding assays of the hEGF, hVEGF, hVEGF-EGF, DTPA-hEGF, DTPA-hVEGF, and DTPA-hVEGF-EGF proteins to their corresponding receptors. (A) hEGF and hVEGF-EGF bind to purified human EGFR on an ELISA plate with Kd values of 20.2 ± 6.6 and 3.4 ± 0.8 nM, respectively. (B) hVEGF and hVEGF-EGF bind to purified human VEGFR2 (Flk-1) with Kd values of 2.2 ± 1.2 and 2.3 ± 0.8 nM, respectively. (C) Binding of hVEGF-EGF to its receptors in the presence of both EGFR and VEGFR. The binding of hEGF, hVEGF, and hVEGF-EGF to purified EGFR and VEGFR simultaneously immobilized on an ELISA plate was examined in vitro using an HRP-tagged anti-his6 antibody. The binding maximum (Bmax) of hVEGF-EGF was higher than those of hEGF and hVEGF. (D) Competition binding assay of hVEGF-EGF in the presence of both EGFR and VEGFR. Purified EGFR and VEGFR were individually or simultaneously immobilized on ELISA plates. hVEGF-EGF was competed away by serial concentrations of EGF and VEGF, and its residual binding was examined using an HRP-tagged anti-His6 antibody. hVEGF-EGF could be partially competed away by EGF or VEGF alone from plates that were simultaneously coated with EGFR and VEGFR. (E) DTPA-hEGF and DTPA-hVEGFEGF bind to purified human EGFR on an ELISA plate with Kd values of 35.2 ± 13.8 and 3.6 ± 0.5 nM, respectively. (F) DTPA-hVEGF and DTPAhVEGF-EGF bind to purified human VEGFR2 (Flk-1) with Kd values of 2.3 ± 1.2 and 2.8 ± 0.8 nM, respectively.

Mass Spectrometric Characterization of DTPA-hEGF, DTPA-hVEGF, and DTPA-hVEGF-EGF. Two mass spectrometric methods were used to examine the sites and efficiency of the DTPA conjugation of these three proteins. Intact protein analysis of DTPA-hEGF showed that the measured monoisotopic masses36 of the two major ion clusters were 11 167.07 and 11 542.21 Da, corresponding to hEGF conjugation with one and two DTPA moieties, respectively (Figure 3). The mass errors of these two molecules were as small as 4.5−5.2 ppm. The quantitative MS analyses revealed that ∼79% and ∼19% of hEGF were singly and doubly modified by DTPA, respectively. To further determine which residues were modified with DTPA, the recombinant proteins were subjected to in-gel digestion with

Asp-N or trypsin and analyzed with LC-MS/MS. All DTPAmodified peptide candidates were identified based on two properties. First, they were closely eluted with their unmodified counterparts. Second, they had a mass increase corresponding to the mass of the DTPA modifier, i.e., 375.128 Da. Our results yielded some candidate peptides with the DTPA conjugation (Tables 2 and 3). For DTPA-hEGF, we found three Asp-N peptides, specifically, 1SMNSRGPAGRLGSVPNS17, 61DLKWWELRHAGGGQLE76, and 66ELRHAGGGQLEQK78 (Table 2). We also determined the percentages of proteins with DTPA modification at each site, which suggested that ∼78%, ∼11%, and ∼8% of the N-terminus, Lys-63, and Lys-78 sequences, respectively, were modified (Table 2). For DTPA-hVEGF, two DTPA-modified 2484


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Figure 3. Average mass spectrum of DTPA-EGF acquired in the Orbitrap. The mass spectrum is enlarged over the indicated mass range to see the details of ion clusters, whose observed charge states (Z) are signified at the top. The arrows indicate the signal of hEGF conjugated with one and two DTPA moieties, respectively, whose most abundant m/z and Z are also marked. The detailed information is described in the upper right inserted table. The theoretical monoisotopic masses (Mmi) of intact hEGF and DTPA molecule are 10 791.90 and 393.14 Da, respectively.

Table 2. Summary of Identified DTPA-Modified Peptides after AspN Digestion unmodified hEGF hEGF hEGF hVEGF hVEGF-EGF hVEGF-EGF

modified

DTPA-modified peptidea

putative site

Z

RTb

m/z

RT

m/z

ΔM (ppm)

relative intensityc (%)

SMNSRGPAGRLGSVPNS17 DLKWWELRHAGGGQLE76 66 ELRHAGGGQLEQK78 109 EMSFLQHNKCE119 109 EMSFLQHNKCE119 197 EQKLISEE204

N-ter K63 K78 K117 K117 K199

3 2 2 2 2 2

5.3 9.0 2.8 18.2 18.1 17.7

562.948 947.985 711.876 735.829 735.829 488.254

6.9 9.4 6.0 18.2 18.1 17.2

687.992 1135.548 899.446 923.389 923.392 675.817

−0.1 −1.7 −6.8 −0.7 0.4 −0.9

∼78 ∼11 ∼8 ∼60 ∼8 ∼2

protein name 1

61

a

The putatively DTPA-modifided lysine or N-terminus is underlined. bRT indicates the retention time of LC-MS analysis. cPeptide quantitation is dependent on the ratio of relative intensity of modified and unmodified peptides.

Table 3. Summary of Identified DTPA-Modified Peptides after Tryptic Digestion unmodified DTPA-modified peptidea

protein name hVEGF hVEGF hVEGF-EGF hVEGF-EGF

99

117

IKPHQGQHIGEMSFLQHNK IKPHQGQHIGEM*SFLQHNK117 99 IKPHQGQHIGEMSFLQHNK117 99 IKPHQGQHIGEMbSFLQHNK117 99

modified

putative site

Z

RTc

m/z

RT

m/z

ΔM (ppm)

relative intensityd (%)

K100 K100 K100 K100

3 3 3 3

17.9 16.5 17.8 16.4

743.718 749.048 743.717 749.047

19.6 18.5 19.1 18.1

868.760 874.089 868.759 874.091

0.6 −1.6 0.0 0.7

∼1 ∼3 ∼2 ∼10

a

The putatively DTPA-modifided lysine or N-terminus is underlined. bIndicates methionine oxidation. cRT indicates the retention time of LC-MS analysis. dPeptide quantitation is dependent on the ratio of relative intensity of modified and unmodified peptides.

excess EDTA at 25 °C for 20 min, the radiochemical purity of In-DTPA-hEGF, 111In-DTPA-hVEGF, and 111In-DTPAhVEGF-EGF remained at >91%, indicating that the labeling was mediated mainly by the DTPA and not by the amino acid residues that formed a weak chelating pocket. The in vitro stabilities of 111In-DTPA-EGF, 111In-DTPA-EGF, and 111InDTPA-hVEGF-EGF after incubation in PBS at 4 °C for 24 h were all greater than 90%. The in vitro stability of 111In-DTPAEGF, 111In-DTPA-EGF, and 111In-DTPA-hVEGF-EGF after incubation in human serum at 37 °C for 24 h were approximately 60%. Cellular Uptake and Binding Specificity of 111In-DTPAVEGF-EGF. The cellular uptake of 111In-DTPA-hVEGF-EGF was affected by the densities of EGFR and VEGFR on cell membrane, and the accumulation of radioactivities reached 56.6 ± 4.3, 15.9 ± 1.7, 0.8 ± 0.1 and 1.0 ± 0.1%AD/106 cells after 8 h of incubation, respectively (Figure 4A). These results correlated

peptides were detected. One is within an Asp-N peptide, 109 EMSFLQHNKCE119, with Lys-117 as the putative modification site (Table 2), and the other is a tryptic peptide, 99IKPHQGQHIGEMSFLQHNK117, with Lys-100 as the putative modification site (Table 3). The local DTPA modification status of Lys-117 and Lys100 was estimated to be ∼4% and ∼60%, respectively. For DTPAhVEGF-EGF, two Asp-N peptides, 109EMSFLQHNKCE119 and 197 EQKLISEE204 (Table 2), and one tryptic peptide, 99IKPHQGQHIGEMSFLQHNK117, were found (Table 3). These three sites corresponded to Lys-100 (12%) and Lys-117 (8%) of hVEGF and Lys-78 (2%) of hEGF. Notably, these sites were only weakly modified (2−12% of sites). Preparation, Characterization, and in Vitro Stability of 111 In-DTPA-hEGF, 111In-DTPA-hVEGF, and 111In-DTPAhVEGF-EGF. The 111In-DTPA-EGF, 111In-DTPA-VEGF, and 111 In-DTPA-hVEGF-EGF proteins were all prepared with high radiochemical purity (>98%). After incubation with 1000-fold

111

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Figure 4. Binding specificity and cellular uptake of 111In-DTPA-VEGF-EGF in various cells. (A) The effect of EGFR and VEGFR density on the cellular uptake of 111In-DTPA-hVEGF-EGF incubated with cancer and endothelial cells at 37 °C. The radioactivity accumulation in various cells correlated with the EGFR+VEGFR density on the cell membrane (n = 4). Radioactivity uptake was expressed as % administered dose (AD). Values are shown as the mean ± SD. EGFR binding or VEGFR binding was blocked with excess unlabeled (cold) hEGF or hVEGF in (B) BAEC, (C) MDA-MB-231, and (D) MDA-MB-468 cells. The data are presented as the mean ± SD (n = 3). The binding was suppressed, indicating its specificity. (*, p < 0.05 as compared to 111In-DTPA-VEGF-EGF).

unlabeled hEGF or hVEGF to cells to saturate the EGFR or VEGFR demonstrated that the binding of 111In-DTPA-VEGFEGF to the EGFR or VEGFR could be partially inhibited by receptor saturation. It was observed that roughly 30% of the binding of 111In-DTPA-VEGF-EGF was blocked by unlabeled hEGF and 40% blocked by unlabeled hVEGF in BAEC cells (Figure 4B). On the other hand, 90% of the binding of 111InDTPA-VEGF-EGF was blocked by unlabeled hEGF, but only 20% was blocked by unlabeled hVEGF in MDA-MB-231 cells (Figure 4C). These results indicated that 111In-DTPA-VEGF-EGF could bind to both EGFR and VEGFR expressed on the plasma membrane for both BEAC and MDA-MB-231 cells. Moreover, for the MDA-MB-231 cells, the binding to EGFR by 111In-DTPAVEGF-EGF dominates over binding to VEGFR. For MDA-MB-468 cells with only a high level EGFR but not VEGFR, the binding of 111 In-DTPA-VEGF-EGF could only be blocked by unlabeled hEGF but not by unlabeled hVEGF (Figure 4D). Functional Assays of hEGF, hVEGF, and hVEGF-EGF. BAEC cells express both EGFR and VEGFR and are appropriate for use in VEGFR and EGFR signaling studies. Treatment of BAEC cells with hVEGF-EGF fusion protein (20 nM) for 15 min produced a marked increase in both EGFR and VEGFR phosphorylation, similar to that separately treated with hEGF and hVEGF (Figure 5).

well with the numbers of EGFR in the MDA-MB-468 cells (1 × 106 receptors/cell), MDA-MB-231 (5 × 105 receptors/cell), MCF-7 (1 × 104 receptors/cell), and BAEC cells, respectively (Table S1). Note that the initial concentration of 111In-DTPAhVEGF-EGF in each well was 10 nM (10 pmol in 1 mL medium) which was relatively high as compared to the number of receptors for the cells studied. For the MDA-MB-468 cells with high EGFR expression, the free 111In-DTPA-hVEGF-EGF concentration declined to ∼4 nM after 4 h of incubation (i.e., ∼60% uptake), and a dynamic equilibrium may have been reached for the free and 111In-DTPA-hVEGF-EGF taken up (Figure 4A). Considerably less radioactivity was retained in cells with medium and low EGFR expression, i.e., MDA-MB-231, MCF-7, and BAEC. Ideally, the cellular uptake of 111In-DTPA-hVEGF-EGF should correlate with the total expressed levels of EGFR and VEGFR in the cells, but because the VEGFR levels for these cell lines were either relatively lower or not present as compared to their EGFR levels, the apparent uptake of the hVEGF-EGF fusion protein was correlated mainly to the expressed EGFR level. The in vitro binding specificity of 111In-DTPA-VEGF-EGF to MDA-MB-231, MDA-MB-468, and BAEC cells is shown in Figure 4B−D. Note that BAEC and MDA-MB-231 cells express both EGFR and VEGFR. Addition of a large amount (i.e., 20-fold excess) of 2486


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VEGF (p = 0.05) but only slightly higher than 111In-DTPA-EGF (p = 0.34). Pharmacokinetics, Biodistribution, and SPECT/CT Imaging Studies of 111In-DTPA-hVEGF-EGF in BALB/c Nude Mice. The radioactivity−time curves of blood after i.v. injection of 111In-DTPA-hEGF, 111In-DTPA-hVEGF, and 111InDTPA-hVEGF-EGF in BALB/c nude mice are shown in Figure 7A. The pharmacokinetic parameters derived from the curves using a two-compartment model are summarized in Table 4. The t1/2α of 111In-DTPA-VEGF-EGF was 0.17 h, which was longer than the t1/2α of 111In-DTPA-EGF (0.09 h) but smaller than the t1/2α of 111In-DTPA-VEGF (0.24 h). The t1/2β of 111In-DTPA-VEGFEGF was 20.35 h, longer than the values for 111In-DTPA-EGF (9.30 h) and 111In-DTPA-VEGF (9.63 h) in nude mice. The AUC(o→t) values of the radioactivity in the blood after the i.v. administration of 111In-DTPA-hVEGF-EGF is 10.5 h*%ID/mL. The accumulated radioactivities in various tissues of the mice up to 24 h after injection of 111In-DTPA-hVEGF-EGF (3.7 MBq, 7.7 μg) are shown in Table 5. Note that the liver and spleen accumulated higher radioactivities than all other tissues at all time points. Consistent with the pharmacokinetic studies, the concentrations of radioactivity in the blood decreased rapidly from 1.46 ± 0.30%ID/g at 1 h after injection to only 0.80 ± 0.57%ID/g at 24 h after injection (Table 5). To confirm the distribution of 111 In-DTPA-hVEGF-EGF, the animal SPECT/CT imaging of BALB/c nude mice was assessed at 1, 4, and 24 h after an administration of 111In-DTPA-hVEGF-EGF (18.5 MBq/0.1 mL). Significant radioactivity accumulations in liver and spleen were observed in the SPECT/CT animal images and the results were consistent with the findings in the biodistribution study (Figure 7B). Molecular Modeling of Potential EGF and VEGF Interactions and Realization of DTPA Conjugation Sites. The program Zdock was employed to predict the protein rigidbody docking modes whose scoring function was designed specifically for globular proteins including pairwise shape complementary, desolvation, and electrostatics terms. Zdock has been found to be successful for the CAPRI Challenge which is a blind test of protein−protein docking algorithms that predict the complex structure from the crystal structures of the interacting proteins. In our present study, the proteins EGF and VEGF are qualified for the prediction. In a hVEGF-EGFcontaining aqueous solution, there are chances that the proteins will form oligomers. In addition, varying the length of the connection between VEGF and EGF within the hVEGF-EGF construct, could lead to “self-association” of the two proteins. At this stage the potential association of VEGF and EGF is investigated using a docking approach. The time of diffusion toward and around the proteins is minimized by screening the interaction surfaces in 10° intervals in all directions. A potential binding site of EGF with the dimer of VEGF is identified using available crystal structures from each, VEGF and EGF, both interacting with their respective receptors in a docking approach (Figure 8A). The best binding pose of the two proteins is so that the site of EGF which binds to EGFR (blue area compared to red area) is unaffected by VEGF (Figure 8B). On the other hand, the binding site of VEGF with its receptor is largely occupied by EGF (Figure 8C, green area compared to red area). The interface binding energy from the best docking pose is calculated to be Einterface = −296.9 kcal/mol.37 Thus, interaction of the two proteins affects the binding of one of them to its respective receptor. The binding pose supports the strong binding avidity of hVEGF-EGF to EGFR, and the partially blocked binding of the fusion construct to VEGFR. Potential DTPA conjugation sites,

Figure 5. EGFR and VEGFR-2 signaling stimulated by hEGF, hVEGF, and hVEGF-EGF in BAEC cells. hEGF and hVEGF-EGF induced EGFR phosphorylation and hVEGF and hVEGF-EGF induced VEGFR phosphorylation in BAEC cells. Western blot analyses of BAEC cells incubated with or without 20 nM hEGF, hVEGF, and hVEGF-EGF for 15 min. Total protein samples were probed with anti-phospho-EGFR, anti-phospho-VEGFR, anti-phospho-ERK1/2, and anti-ERK2 antibodies to determine the phosphorylation status of downstream proteins in the EGFR and VEGFR signaling cascades.

The phosphorylation of downstream targets of EGFR and VEGFR (ERK1/2) was also markedly increased after treatment with hEGF, hVEGF, and hVEGF-EGF, respectively (Figure 5). These results demonstrated that the hVEGF-EGF fusion protein retained the functional activities of both EGF and VEGF. Cytotoxicity of 111In-DTPA-hEGF, 111In-DTPA-hVEGF, and 111In-DTPA-hVEGF-EGF in MDA-MB-231 Cells. VEGF promotes breast cancer progression by inducing angiogenesis via VEGF receptors on endothelial cells, but also signals directly through receptors such as VEGFR-1 (Flt-1) on tumor cells. MDA-MB-231 tumor cells express both EGFR and VEGFR. After a 24-h coincubation with each of 111In-DTPA-hEGF, 111InDTPA-hVEGF, and 111In-DTPA-hVEGF-EGF (5.2 MBq/mL, 43 nM), significant reductions of the respective survival fractions (SF) to 0.60 ± 0.23, 0.67 ± 0.10, and 0.51 ± 0.14 as compared to that of 111In-acetate (SF, 0.79 ± 0.09) were observed in MDAMB-231 cells (the respective p values were 0.02, 0.06, and 0.002 as compared to 111In-acetate Figure 6). 111In-DTPA-hVEGFEGF exhibited significantly higher cytotoxicity than 111In-DTPA-

Figure 6. Cytotoxicity of 111In-DTPA-hEGF, 111In-DTPA-hVEGF, and 111 In-DTPA-hVEGF-EGF in MDA-MB-231 cells. Clonogenic assays were performed on MDA-MB-231 cells incubated with 111In-acetate (5.2 MBq/mL), 111In-DTPA-hEGF, 111In-DTPA-hVEGF, and 111InDTPA-hVEGF-EGF (5.2 MBq/mL, 43 nM) for 24 h. A significant decrease in SF was observed in cells exposed to 111In-DTPA-hVEGFEGF (0.51 ± 0.14) compared with untreated cells. Error bars indicate the SEM of the mean SF. (*, p < 0.05 as compared to 111In-acetate; # , p < 0.05 as compare to 111In-DTPA-hVEGF). 2487


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Figure 7. Pharmacokinetics and animal SPECT/CT images of 111In-DTPA-hVEGF-EGF in BALB/c nude mice. (A) Radioactivity−time curves of blood sampled from BALB/c nude mice after the i.v. injection of 111In-DTPA-hEGF, 111In-DTPA-hVEGF, and 111In-DTPA-hVEGF-EGF. The data are presented as the mean ± SEM (n = 3 at each time point). The inset is the expanded plot for data between time 0.0 and 2.0 h. (B) Animal SPECT/CT images of BALB/c Nude mice at 1, 4, and 24 h after intravenous injection of 18.5 MBq/0.1 mL of 111In-DTPA-hVEGF-EGF. The animals were under isoflurane anesthesia and the image acquisition time was 30 min. The arrow indicated the radioactivity was highly accumulated in the liver and spleen.

confirmed that their binding abilities were maintained. Specifically, there is an enhanced avidity with respect to EGFR binding. However, binding of hVEGF-EGF with VEGFR is not enhanced compared to that of VEGF to its receptor, in contrast to binding of hVEGF-EGF and EGF to EGFR. One possible explanation for the enhanced EGFR binding with the fusion protein is that the VEGF portion in the fusion protein can form a dimer as found in the purified form, and this dimer-formation allows two EGF units to be in the syn-direction which results in an enhanced EGFR binding due to at least a statistical effect. On the other hand, the natural binding of VEGF to VEGFR is in the form of homodimer which should not result in enhanced binding as expected. In addition to the normal specific binding of the hVEGF-EGF dimer to EGFR and VEGFR, there are also potential nonspecific binding or steric repulsion effects which lead to minor enhanced or reduced bindings, respectively. Through our molecular simulation studies, it is suggested that EGF could interact with VEGF upon free diffusion in the presence of the flexible linker so that the binding site of VEGF to VEGFR would be partially blocked.37 This scenario could explain the experimentally found non-enhanced hVEGF-EGF binding affinity toward VEGFR if minor nonspecific binding enhancement is present to offset the partial-blocking effect. On the other hand, in the docked pose, the EGF binding site for EGFR is unaffected and would allow for hVEGF-EGF binding toward EGFR. Kampmeier reported that the position of different proteins in the fusion protein may influence their biological activity.38 In that study, EGF could maintain its biological activity at C-terminal

Table 4. Estimated Pharmacokinetic Parameters Derived from the Radioactivity−Time Curves of Blood Samples from BALB/c Nude Mice after i.v. Administration of 111In-DTPAhEGF, 111In-DTPA-hVEGF, and 111In-DTPA-hVEGF-EGFa parameter

unit

VEGF-EGF

EGF

VEGF

T1/2α T1/2β Cl AUC(0→t) AIC

h h mL/h h*%ID/mL

0.17 20.35 9.51 10.51 −43.95

0.09 9.30 12.26 8.16 −21.83

0.24 9.63 5.02 19.91 −11.91

a Accuracy of fits was assessed by Akaikes Information Criterion (AIC). Pharmacokinetic parameters including half-life (T1/2), Cmax, total body clearance (Cl) and the area under the curve (AUC) were determined using WinNonlin software v 5.3.1 (Pharsight Corp., Mountain View, CA) for a two-compartment model.

such as Lys-63 and Lys-78 at EGF and Lys-100 and Lys-117 at VEGF, are not interfering with the docking site (Figure 8D,E,F). A more comprehensive report is published elsewhere.37

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DISCUSSION General Structural, Binding and Cellular Uptake Properties of hVEGF-EGF and 111In-DTPA-hVEGF-EGF. In this study, we described a novel recombinant hVEGF-EGF fusion protein and used 111In-DTPA metal chelates to label the protein to be employed as a radioactive theranostic agent. This protein interacts specifically with EGFR and VEGFR. The in vitro specific binding assays of both hVEGF-EGF and DTPAconjugated hVEGF-EGF to purified EGFR and VEGFR 2488


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Bioconjugate Chemistry Table 5. Biodistribution of 111In-DTPA-hVEGF-EGF after Intravenous Injection in BALB/c Nude Micea organ

1h

4h

8h

24 h

Blood Heart Lung Liver Stomach S.I. L.I. Pancreas Spleen Kidney Muscle Bladder Urine Feces

1.46 ± 0.30 0.30 ± 0.10 2.27 ± 0.63 13.43 ± 2.33 0.36 ± 0.12 0.67 ± 0.50 0.50 ± 0.22 0.23 ± 0.12 21.63 ± 2.55 1.12 ± 0.29 0.25 ± 0.18 0.53 ± 0.16 16.65 ± 12.03 0.17 ± 0.21

1.06 ± 0.29 0.26 ± 0.03 1.48 ± 0.31 15.10 ± 2.91 0.39 ± 0.10 0.44 ± 0.06 0.34 ± 0.22 0.19 ± 0.06 21.55 ± 10.81 1.22 ± 0.13 0.42 ± 0.35 0.71 ± 0.31 1.24 ± 0.48 1.05 ± 0.32

1.04 ± 0.12 0.31 ± 0.05 1.88 ± 0.84 15.22 ± 1.90 0.59 ± 0.17 0.76 ± 0.18 0.40 ± 0.13 0.26 ± 0.04 20.14 ± 5.30 1.77 ± 0.13 0.21 ± 0.06 0.56 ± 0.02 1.64 ± 0.40 3.57 ± 1.11

0.80 ± 0.57 0.25 ± 0.06 0.58 ± 0.26 12.75 ± 1.13 0.53 ± 0.20 0.55 ± 0.29 0.42 ± 0.17 0.34 ± 0.07 14.37 ± 8.04 2.50 ± 0.39 0.18 ± 0.05 0.47 ± 0.10 1.06 ± 0.22 0.80 ± 0.29

Values were presented as percent injected dose per gram of organ (%ID/g, mean ± SD, n = 4 at each time point). S.I.: small intestine, L.I.: large intestine.

a

Figure 8. Molecular modeling of potential EGF and VEGF interaction, and reference crystal structures of EGF−EGFR and VEGF−VEGFR complexes. (A) Predicted binding pose of EGF (1JL9, orange) and VEGF (1VPF, purple). (B) EGF structure taken from crystallized EGF-EGFR complex (1IVO). (C) VEGF structure taken from VEGF−VEGFR complex (3V2A). Blue area marks EGF-EGFR binding site, red area marks EGF-VEGF binding site, and green area marks VEGF−VEGFR binding site. Proteins are shown in either ribbon or surface mode using MOE software (www.chemcomp.com). Graphical representation of the lysine residues Lys-63 and Lys-78 of EGF (both in red colored van der Waals representation), which could be DTPA conjugation sites, within the EGF-EGFR complex (D). Lue-62 of EGF is shown in green spheres. The binding sites at the interface between EGF and EGFR are encircled. Respective lysine residues, Lys-100 and Lys-117 of VEGF (both in red) within the VEGF−VEGFR2 (E) and VEGF−VEGFR1d2 (F) complex, are shown in van der Waals representation. The backbones of the EGF-EGFR crystal structure are shown in orange (EGF) and blue (EGFR). The backbones of the VEGF and VEGFR are shown in purple (VEGF) and green (VEGFR). The pictures are made with Pymol.

activities under the standard storage conditions for at least two months. It is worth noting that DTPA conjugated radiolabeled recombinant proteins may not be the most stable ligand for radiolabeling. Brouwers et al. performed a comparison of the performance of the cyclic diethylenetriaminepentaacetic acid anhydride (cDTPA), isothiocyanatobenzyl-DTPA (SCN-BzDTPA), or 1,4,7,10-tetraazacyclododecanetetraacetic acid (DOTA) ligands as chelates to radiolabel mAb cG250 with 88 Y. It was observed that SCN-Bz-DTPA-conjugated and DOTA-conjugated radiolabeled antibodies were more stable than cDTPA-conjugates in plasma at 37 °C.40 Thus, for even more stable radiolabeled metal hVEGF-EGF bioconjugates, other ligands such as DOTA derivatives might be considered in the future. DTPA-Conjugation Sites and Binding Properties. To predict whether DTPA modification affects the protein structures and protein−receptor binding of the modified

position rather than at N-terminal position. In our study, when we changed the orientation of VEGF and EGF to generate hEGF-VEGF fusion protein (i.e., EGF at N-terminal position), the hEGF-VEGF lost their biological activities toward both EGFR and VEGFR completely (unpublished data). The hVEGF-EGF fusion protein under nonreducing conditions is a disulfide linked homodimer via presumably the VEGF portion with glycosylation, as confirmed by our present electrophoresis and enzymatic deglycosylation studies. Because EGF is known to exist as a monomer, the formation of (hVEGFlinker-EGF)-dimer is most likely via the hVEGF moiety; however, its exact structure remains to be delineated. Also, it has been reported that glycosylation does not affect VEGF−VEGFR binding capability;39 the presence of glycosylation might be preferred for future applications as it is similar to its native bioform. Studies of the hVEGF-EGF expressed in E. coli without glycosylation are underway for comparison and will be reported elsewhere. All proteins studied thus far maintained their normal 2489


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expressing EGFR and also endothelial cells expressing VEGFR, these results thus suggest a promising new treatment for EGFRoverexpressing and high angiogenesis human cancers. Pharmacokinetics and Biodistribution Studies. Reilly et al. reported the 111In-DTPA-hEGF pharmacokinetic data administered intravenously to mice using a three-compartment model with the respective t1/2α and t1/2β values 0.10 and 0.61 h.45 Their half-life of the long terminal phase (i.e., t1/2γ) could not be accurately determined. In our hands, the respective t1/2α, t1/2β, and t1/2γ values of 111In-DTPA-hEGF in mice using a similar study protocol and a three-compartment model were 0.06, 0.89, and 15.66 h which were not very different from those of Reilly’s (Supporting Information Table S2). However, if the pharmacokinetic data of all three proteins, i.e., 111In-DTPA-hEGF, 111InDTPA-hVEGF, and 111In-DTPA-hVEGF-EGF were considered, our blood time-activity curves were better described by a twocompartment pharmacokinetic model because the AIC (Akaike’s Information Criterion) values were significantly lower for both 111 In-DTPA-hVEGF and 111In-DTPA-hVEGF-EGF proteins (Table 4). Interestingly, our AIC value (−21.83) for the 111InDTPA-hEGF protein using a two-compartment model was indeed slightly higher than that using a three-compartment model, i.e., −22.86. This might be the reason that the previous report by Reilly et al. chose a three-compartment model to analyze their pharmacokinetic data. From our pharmacokinetic data in mice using a twocompartment model, it was observed that 111In-DTPA-hEGF, 111 In-DTPA-hVEGF, and 111In-DTPA-hVEGF-EGF all were excreted rather quickly. The distribution t1/2α value (0.17 h) for 111 In-DTPA-hVEGF-EGF was in between those of 111In-DTPAhEGF and 111In-DTPA-hVEGF, i.e., 0.09 and 0.23 h, respectively. The excretion t1/2β value (20.35 h) for 111In-DTPA-hVEGF-EGF was longer than those of 111In-DTPA-hEGF and 111In-DTPAhVEGF, i.e., 9.30 and 9.63 h, respectively. Thus, the fusion protein hVEGF-EGF may have the potential advantages of maintaining similar stability and increasing bioavailability as compared to its component proteins EGF and VEGF. Note that the t1/2α value of 111In-DTPA-VEGF (0.24 h) was shorter than that reported by Kim and Burgess using a rat model with 14Clabeled VEGF, i.e., 1.20 h,46 which may be caused by differences in blood circulation rates and the drug clearance mechanisms of mouse and rat. On the other hand, the t1/2β values, i.e., 9.63 h vs 8.89 h, were similar. Biodistribution studies of 111In-DTPA-VEGF-EGF in BALB/c mice revealed that a large proportion of the injected dose was rapidly accumulated in the liver and spleen which was higher than those previously reported for the liver uptake of 111In-DTPAhEGF47 in athymic mice. This was likely attributable to the moderate levels of EGFR expressed by these tissues and was consistent with 111In-DTPA-VEGF-EGF binding EGFR more strongly than EGF.48,49 Note that liver expresses moderate to high levels of EGFR (approximately 105 receptors/cell). In the presence of a 100-fold excess of unlabeled EGF, the liver sequestration of 125I-EGF decreased from 99% to 24% after portal administration of 125I-EGF to rats.48 The rapid clearance of the labeled protein from the bloodstream may be attributable to a hepatic transport mechanism.48 In 2001, Hattori et al. reported that after multiple injection of VEGF to mice, their spleens became swollen.50 In 2008, Chen et al. also found that the radio- and VEGF-labeled quantum dot (QD), i.e., 64Cu-DOTA-QD-VEGF, was accumulated more in the spleen of mice.51 Thus, it is likely that the accumulation of a

proteins, we inspected the positions of putative DTPA-modified lysine residues in the three-dimensional crystal structures of both EGF−EGFR and VEGF−VEGFR.41,42 Based on the crystallographic data, C-shaped EGFR wrapped around its ligand, and all of the EGFR domains, may contribute to the interaction with EGF, but domain I and domain III are particularly important for the ligand−receptor interface. While domain I harbors interaction site 1, domain III contains the remaining two sites41 (Figure 8D). It is noteworthy that the N-terminus of hEGF, which is highly modified by DTPA-labeling, is not involved in the interaction with EGFR. Meanwhile, the other modified residue, Lys-63, is located next to Leu-62, which has been hypothesized to serve as part of site 3. Because the percentage of proteins with this modification was only 11%, we speculate that its effect on the hEGF activity should not be prominent. Similarly, the effect of the Lys-78 modification might be minor due to its 8% population. In contrast, homodimeric VEGF was reported to bind its receptor, Flt-1, mainly through hydrophobic interactions with domain II of the extracellular domain of the receptor42 (Figure 8E). Based on our crystallographic model, DTPA modification at Lys100 or Lys-117 does not seem to impose any effects on VEGF− VEGFR interaction. Because the fusion protein hVEGF-EGF exhibited the same DTPA modification sites as hEGF or hVEGF, its capacity to bind receptors should remain largely unchanged, similar to those of its parent proteins. In Vitro Cytotoxicity Studies. Western blotting analysis proved that the hVEGF-EGF fusion protein could induce the phosphorylation of EGFR, VEGFR, and downstream ERK1/2 which might lead to tumor cell proliferation. In one way, the effective binding to targeted tumor receptors demonstrated its potential drug carrier efficacy. On the other hand, the resulting signal transduction must be overcome to achieve cytotoxicity. To circumvent this problem, we have introduced 111In-DTPA chelates to the fusion protein. 111In is a radionuclide that is commonly used for single photon emission computed tomographic (SPECT) imaging and Auger electron emission for radiotherapy due to its long physical half-life of 2.81 days with 172 and 247 keV gamma emissions. These properties make 111In a good radionuclide for both diagnosis and therapy. Thus, despite the possibility of tumor cell proliferation with this kind of signal transduction, the 111In-DTPA-conjugated hVEGF-EGF fusion proteins could emit Auger electrons to tumor cells and cause cell damage. This result is similar to Cai’s approach43 in the cytotoxic study of 111In-DTPA-EGF. The 111In-DTPA-fusion protein becomes more cytotoxic as compared with 111In-DTPAhVEGF and 111In-DTPA-hEGF. However, the differences in cytotoxicity are not large presumably because the number of VEGFR on MDA-MB231 cell surface is much smaller than EGFR, consistent with the above-discussed results of competition binding and cellular uptake studies. Another reason is that the cells were incubated with 43 nM of 111In-DTPA-hEGF and 111 In-DTPA-hVEGF-EGF, a concentration which approximately reaches saturated binding to EGFR on the MDA-MB-231 cells. Although the binding activity of hVEGF-EGF fusion protein is better than that of hEGF, the killing effect of Auger electrons emitted from either 111In-DTPA-hEGF or 111In-DTPA-hVEGFEGF may be leveling off due to the saturation of EGFR on the cell surface, and thus results in similar cytotoxicity between these two treatment groups. Note that most normal tissues express very low levels of EGFR (98%. To confirm that the chelating was stable, an aliquot of each radiolabeled product was incubated with 1000-fold molar excess EDTA for 20 min and then analyzed using ITLC with 0.1 M sodium citrate (pH 5.5). In Vitro Binding of the hEGF, hVEGF, hVEGF-EGF Proteins, and Their DTPA Conjugates to Their Corresponding Receptors. Purified recombinant human EGFR and VEGFR-2 extracellular domain/Fc Chimera were diluted in PBS (0.5 μg/mL) and immobilized on a 96-well ELISA plate by incubation at 4 °C overnight. The wells coated with individual or both receptors were washed three times with PBST (phosphate buffer saline with 0.05% Tween 20 solution) followed by blocking with the addition of 300 μL 1% BSA in PBS at room temperature for a minimum of 1 h. Then, the plate was washed again with PBST before the addition of increasing concentrations (0−2 μg/mL) of His6-tagged human VEGF, EGF, or VEGFEGF. The ligands and receptors were incubated at room temperature for 2 h, and the plate was then washed twice with PBST followed by the addition of 100 μL mouse anti-His6-HRP monoclonal antibody that was diluted in 1% BSA (1:1000). After incubation at room temperature for 1 h, the plate was washed 2492


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solution. The number of colonies in each dish was counted using light microscopy. The plating efficiency was determined by dividing the number of colonies formed in the control dishes by the number of cells seeded. The surviving fraction (SF) was calculated by dividing the number of colonies by the number of cells seeded and then multiplying by the plating efficiency. Ethics Statement in Relation to Animal Studies. BALB/c Nude mice (Female BALB/cAnN.Cg-Foxn1nu/CrlNarl) were purchased from National Laboratory Animal Center of Taiwan. The animal studies were performed in strict accordance with the recommendations presented in the Guide for the Care and Use of Laboratory Animals of the National Laboratory Animal Center. The animal experiment protocol was approved by the Institutional Animal Care and Use Committee of the National Yang-Ming University, Taipei, Taiwan (Permits Number: 990801). The imaging studies were performed under 1% to 3% isoflurane anesthesia. All animals were sacrificed by carbon dioxide narcosis and all efforts were made to minimize suffering. Biodistribution and Pharmacokinetics of 111In-DTPAhVEGF-EGF in BALB/c Nude Mice. The BALB/c Nude mice were intravenously injected with 3.7 MBq/0.1 mL of 111InDTPA-hVEGF-EGF. The mice were sacrificed at 1, 4, 8, and 24 h after the administration of the radio-drugs, blood samples were taken, and the organs of interest were excised. The tissue samples were weighed, and their activity was counted using a Wallac 1470 Wizard Gamma counter. The biodistribution data are expressed as percent injected dose per gram of tissue (%ID/g). The pharmacokinetics of 111In-DTPA-hVEGF-EGF (3.7 MBq/100 pmol protein/0.1 mL) in BALB/c nude mice after intravenous administration was studied in a similar way as previously described.55 Pharmacokinetic parameters including half-life (T1/2), Cmax, total body clearance (Cl), and the area under the curve (AUC) were determined using WinNonlin software version 5.3.1 (Pharsight Corp., Mountain View, CA). Animal SPECT/CT Imaging of 111In-DTPA-hVEGF-EGF in BALB/c Nude Mice. The animal SPECT (single-photon emission computed tomography) images were acquired using a multipinhole collimator (N5F75A10) with an FOV of 66.10 mm2. A total of 32 projections were acquired in a 60 × 60 acquisition matrix with a minimum of 8000 counts per projection for SPECT imaging. SPECT images were reconstructed using an ordered subset expectation maximization algorithm (five iterations and eight subsets). The acquisition of SPECT images was followed by CT (computed tomography) images acquisition (X-ray source: 50 kVp, 0.28 mA; 512 projections). The co-registration of microSPECT/CT images was performed using VIVID (Volumetric Image Visualization, Identification and Display) software (based on Amira 4.1 platform). The CT images were also reconstructed using the Feldkamp cone−beam algorithm for filtered backprojection in an image volume of 512 × 512 × 512 with an image resolution of 0.08 mm. The CT data were not corrected for scatter or beam hardening. VIVID software was also used for the image fusion of SPECT and CT images. After registration, the image of SPECT/CT had 256 × 256 × 256 voxels in an isotropic 0.24 mm voxel size. Static imaging was conducted for about 30 min at 1, 4, and 24 h post-intravenous injection of 18.5 MBq/0.1 mL of 111In-DTPA-hVEGF-EGF in BALB/c nude mice. Molecular Simulation Docking Approach on Potential EGF and VEGF Interactions and Realization of DTPA Conjugation Sites. The coordinates of EGF (1JL9) and VEGF (1VPF) as well as the structures of EGF (1IVO) and VEGF (3V2A) bound to their respective receptors were taken from the

an Orbitrap MS survey scan were selected for MS/MS spectra acquisition with LTQ linear ion trap MS. All MS/MS data were analyzed using the TurboSEQUEST algorithm (Thermo Finnigan, USA). An in-house program for protein modification mapping was used to identify DTPA-modified peptides. The criteria for positive identification of DTPA-modified peptides included elution time close to that of the unmodified counterpart and a difference in hypothetical mass smaller than 10 ppm. In Vitro Stability Studies. The stabilities of 111In-DTPAhEGF, 111In-DTPA-hVEGF, and 111In-DTPA-hVEGF-EGF were assayed in vitro by diluting the proteins in PBS or serum to an activity concentration of 1.85 MBq/mL. Serum samples were analyzed at 0, 1, 4, 8, and 24 h by instant thin-layer chromatography with 0.1 M sodium citrate (pH 5.5). Cellular Uptake and Blocking Tests of 111In-DTPAhVEGF-EGF. Tumor cells with high, medium, and low EGFR expression, i.e., MDA-MB-468, MDA-MB-231, and MCF-7,53 respectively, and endothelial cells (BAEC) were used. Both endothelial cells and MDA-MB-231 cells express EGFR and VEGFR.9,23,54 The cells were incubated with a medium containing 111In-DTPA-hVEGF-EGF (0.037 MBq/10 pmol/mL). After incubation at 37 °C for 0.5, 1, 2, 4, and 8 h, the cells were washed, harvested, and transferred to counting tubes. The level of radioactivity in each tube was counted using a Wallac 1470 Wizard Gamma counter. For blocking tests, the cells were incubated with the medium containing 111In-DTPA-hVEGFEGF (0.037 MBq/10 pmol/mL) and with or without 20-fold excess of hEGF and hVEGF to block the binding sites of EGFR and VEGFR. After incubation at 37 °C for 8 h, the cells were washed, harvested, and transferred to counting tubes. The level of radioactivity in each tube was counted using a gamma scintillation counter (Wallac 1470 Wizard gamma counter; GMI, Inc., Ramsey, MN). The results were normalized according to the nonblocked group of each cell types and present as cell associated radioactivity (%). EGFR and VEGFR2 Signaling Induced by hEGF, hVEGF, and hVEGF-EGF in BAEC Cells. hEGF, hVEGF, and hVEGFEGF (20 nM each) were added to BAEC cells for 15 min before the cells were harvested. Cells were washed in ice-cold phosphate-buffered saline (PBS) and lysed in RIPA lysis buffer containing complete mini-EDTA−free protease inhibitor cocktail tablets. The lysates were resolved by SDS−polyacrylamide gel electrophoresis. Proteins were transferred to Immobilon-P transfer membranes, blocked with 10% nonfat milk in Trisbuffered saline with Tween 20, and incubated with anti-phosphoEGFR, anti-phospho-VEGFR2, anti-ERK1/2, and anti-phosphoERK1/2 antibodies at 4 °C overnight. After 3 washes, the primary antibodies were detected with horseradish peroxidaseconjugated secondary antibodies. The bands were visualized using enhanced chemiluminescence. Cytotoxicity Assay of 111In-DTPA-hEGF, 111In-DTPAhVEGF, and 111In-DTPA-hVEGF-EGF in MDA-MB-231 Cells. MDA-MB-231 cells were incubated with 10 mL of DMEM with or without 111In-acetate (5.2 MBq/mL) or 111InDTPA-hEGF, 111In-DTPA-hVEGF, and 111In-DTPA-hVEGFEGF (5.2 MBq/mL, 43 nM) for 24 h before being fixed for clonogenic assays. An incubation time of 20 h was chosen to allow the evaluation of the combined effects of continuing DNA damage and DNA repair after protracted exposure to 111InDTPA-conjugated proteins. For the clonogenic assay, the cells were harvested by trypsinization and seeded in triplicate into a 10-cm culture dish containing L15 medium. After culture at 37 °C for 14 d, the cells were stained with 0.5% crystal violet 2493


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Core/NRPGM, National Science Council, Taiwan, is also gratefully acknowledged.

Protein Databank (www.pdb.org). A fast Fourier transform based, rigid-body molecular docking algorithm, ZDOCK 2.3.2,56 was used to screen VEGF and EGF assembly poses. The search process was performed by spinning on the axis through the center of the two proteins. The VEGF protein was fixed during the sampling. The translation of the EGF protein corresponding to the best geometric match between the two proteins was retained. The scoring function includes pairwise shape complementary, desolvation, and electrostatic terms especially defined for globular proteins. A complete sampling over the entire surface with default parameters in 10° rotational sampling intervals was performed. Overall, 2000 conformations were generated. After an energy minimization of 5 steps steepest decent and conjugated gradient the potential energy was calculated with the AMBER94 force field.57 Estimate interface energy (Einter) for the VEGF-EGF complex was calculated as the difference between the energy of the complex and the energy of the individual structures on the bases of the AMBER94 force field57 in vacuum using MOE software (www.chemcomp.com).

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ABBREVIATIONS DTPA, diethylene triamine pentaacetic acid; EDTA, ethylenediaminetetraacetic acid; ECM, extracellular matrix; EGF, epidermal growth factor (EGF); VEGF, Vascular endothelial growth factor; ERK1/2, extracellular-signal-regulated kinases 1/2; HRP, horseradish peroxidase

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.5b00509. Enzymatic removal of N-linked oligosaccharides from hVEGF-EGF and hVEGF (Figure S1); expression levels of EGF receptor and VEGF receptor in MDA-MB-468, MDA-MB-231, MCF-7 and BAEC cells (Table S1); Estimated pharmacokinetic parameters derived from the radioactivity-time curves of blood samples from BALB/c nude mice after i.v. administration of 111In-DTPA-hEGF, 111 In-DTPA-hVEGF, and 111 In-DTPA-hVEGF-EGF (Table S2) (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail address: [email protected]. Tel: +886-2-28267215; Fax: +886-2-28201095. *E-mail address: [email protected]. Tel: +886-2-28267199; Fax: +886-2-28201093. Author Contributions □

REFERENCES

Jia-Je Li and Keng-Li Lan contributed equally to this paper.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by Grants from the Ministry of Science and Technology of Taiwan (NSC 99−2627-M-010−001, NSC 99−2627-M-010−004, NSC 100−2627-M-010−001, NSC 100−2627-M-010−004, NSC 101−2627-M-010−001, NSC 101−2627-M-010−004, NSC 102−2627-M-010−001, NSC 102−2627-M-010−002, MOST 103−2627-M-010−001, and MOST 103−2627-M-010−002). This work also was supported by Grants from the Veterans General Hospitals University System of Taiwan Joint Research Program (VGHUST104-G7−4−1, VGHUST104-G7−4−2, and VGHUST104-G7−4−3). The authors thank the staff of the Institute of Nuclear Energy Research (Taoyuan, Taiwan), who kindly provided the radionuclide and excellent technical assistance. The Molecular and Genetic Imaging 2494


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