DT390-triTMTP1, a Novel Fusion Protein of Diphtheria Toxin with

Nov 30, 2012 - Cancer Biology Center, Tongji Hospital, Tongji Medical College, Huazhong ... TMTP1 and diphtheria toxin, we developed a new fusion...
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DT390-triTMTP1, a Novel Fusion Protein of Diphtheria Toxin with Tandem Repeat TMTP1 Peptide, Preferentially Targets Metastatic Tumors Xiangyi Ma,† Peng Lv,‡ Shuangmei Ye,† Yiqun Zhang,† Shu Li,† Chunyi Kan,† Liangsheng Fan,† Ronghua Liu,† Danfeng Luo,† Aiping Wang,‡ Wanhua Yang,† Shuhong Yang,† Xiangyang Bai,† Yunping Lu,† Ding Ma,† Ling Xi,*,† and Shixuan Wang*,† †

Cancer Biology Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jiefang Avenue, Wuhan, Hubei 430030, People's Republic of China ‡ Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, People's Republic of China S Supporting Information *

ABSTRACT: Peptide-based therapies have emerged as one of the most promising therapeutics strategy in cancer-targeted therapy. Using our laboratory newly identified peptide TMTP1 and diphtheria toxin, we developed a new fusion protein that showed remarkable ability to target highly metastatic tumors. Fusion protein toxins were generated by fusing the first 390 amino acids of diphtheria toxin [truncated diphtheria toxin (DT390)] to different repeats of peptide TMTP1 (DT390-TMTP1, DT390-biTMTP1, and DT390triTMTP1). Efficacies of the recombinant fusion proteins on tumor growth and metastasis were evaluated in vitro and in vivo. DT390-triTMTP1 showed the most powerful toxicity against cancer, which led to tumor growth retardation or regression and prolonged survival of human prostate cancer PC-3M-1E8 subcutaneously bearing or gastric cancer MKN-45 orthotopic nude mice. Increased TUNEL and caspase-3 staining and reduced ki67 staining in tumor cells suggested that the anticancer effects of DT390-triTMTP1 were through selectively inducing apoptosis and inhibiting proliferation of cancer cells. In a murine model of human orthotopic gastric carcinoma, DT390-biTMTP1 significantly inhibited metastases to liver and spleen, while DT390-triTMTP1 not only totally suppressed metastasis but also reduced primary tumors by 66.6%. In the biodistribution test, DT390-triTMTP1 was observed to home to tumor tissue rapidly and lasted over 48 h, with only a transient appearance in liver and kidney immediately after injection. Thus, our present study provided a novel recombinant fusion protein DT390triTMTP1 with preferential targeting and high cytotoxicity, which may be a promising strategy for the targeted therapy of cancer metastasis. KEYWORDS: DT390, TMTP1, targeted therapy, tumor metastasis

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INTRODUCTION

reticuloendothelial system. Peptides, in contrast to large molecules such as antibodies, exhibit maximal efficiency, excellent tissue penetrability, and minimal toxicity and immunogenicity and are thus more apt to be accepted by patients and clinicians.4,5 Although many endogenous and exogenous peptides have been developed into clinical therapeutics, only a subset of these consists of cancer-targeting peptides. Phage display libraries have been used to identify a number of peptides that bind receptor molecules, oncoproteins,

Metastasis, the most insidious and life-threatening aspect of malignancy, is a multistep process whereby cells from the primary tumor systematically spread and colonize distant new sites. Blocking critical steps in this process could potentially inhibit tumor metastasis and dramatically improve cancer survival rates.1 In this context, the targeted delivery of bioactive agents to tumoral metastases, especially micrometastases, seems to be a particularly promising anticancer therapeutic modality.2,3 Monoclonal antibodies have been successfully utilized as cancer-targeting therapeutics and diagnostics. However, the efficacies of monoclonal antibodies are limited due to the large size of the molecules and nonspecific uptake by the © 2012 American Chemical Society

Received: Revised: Accepted: Published: 115

March 5, 2012 September 26, 2012 October 23, 2012 November 30, 2012 dx.doi.org/10.1021/mp300125k | Mol. Pharmaceutics 2013, 10, 115−126

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containing 5% CO2. Medium and serum were purchased from Invitrogen (Carlsbad, CA). Both PC-3M-1E8 and MKN-45 are cancer cells with high metastatic potential. Construction of Recombinant Prokaryotic Expression Vector. The 1.17 kb DNA fragment encoding the first 390 amino acids of the DT (DT390) was amplified by polymerase chain reaction (PCR), using a plasmid from our library containing the DT sequence as a template. Used were the following primers: DT1up, 5′-GC CAT ATG GGC GCT GAT GAT GTT-3′, Nde1 site underlined; DT1down, 5′-CTC GAG TCC TCC TTG ACG CAC CAC GTT TCC TCC AAG AAA TGG TTG CGT TTT ATG-3′, Xho1 site underlined, TMTP1 in bold, and GG linker between DT and TMTP1 in italics; DT2up, 5′-GC CAT ATG GGC GCT GAT GAT GTT-3′, Nde1 site underlined; DT2down, 5′-CTC GAG TCC TCC TTG ACG CAC CAC GTT TGA TCC TCC TCC TCC TTG ACG CAC CAC GTT-3′, Xho1 site underlined, TMTP1 in bold, and GGGGS linker between two TMTP1s in italics; DT3up, 5′-GC CAT ATG GGC GCT GAT GAT GTT-3′, Nde1 site underlined; and DT3down, 5′-CTC GAG TTG ACG CAC CAC GTT AGA ACC ACC ACC ACC TTG ACGCAC CAC GTT TGA-3′, Xho1 site underlined, TMTP1 in bold, and GGGGS linker between two TMTP1s in italics. The PCR reaction involved heating to 95 °C for 1 min followed by 30 cycles at 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 45 s, with a final 5 min, 72 °C extension. The plasmid containing the DT sequence served as the template for the DT1 and DT2 primers to produce DT390-TMTP1 and DT390-triTMTP1, respectively; the DT390-TMTP1 fusion gene served as the template for the DT3 primers to produce DT390-triTMTP1. The PCR products were digested with Nde1 and Xho1 and then purified by 0.8% agarose gel electrophoresis with ethidium bromide to obtain target DNA segments, according to the manual of the Sangon gelatin retrieving kit. Next, the target DNA was ligated into an E. coli expression vector, pET28a (+), that was cut in advance with the same enzymes mentioned above. The recombinant constructs were enzymatically digested and sequenced (Invitrogen, Shanghai, China) to verify that the fusion genes had been cloned into the vector construct and were correct in sequence. Computer-Generated 3D Structural Models of Fusion Proteins. Three-dimensional structures of proteins are crucial for understanding protein function at a molecular level. To detect whether reconstruction of DT390 with TMTP1 disturbed the structures of the catalytic and binding domains of the DT, we generated 3D structures of the fusion proteins based on the known crystal structure of wild-type DT provided in the RCSB Protein Data Bank (http://www.pdb.org, structure ID 1F0L). The structures were created using the SWISS-MODEL automated protein homology-modeling server of the Swiss Institute of Bioinformatics (http://swissmodel. expasy.org) and visualized using the highly extensible CHIMERA program. Production of the Fusion Protein Toxins. Fusion protein toxins were produced following the Novagen·pET System Manual. Briefly, the correctly recombined plasmids were transformed into the expression host BL21 (DE3) (Novagen, Madison, WI). Positive transformants were selected and cultured in 20 mL of SOB broth (2% w/v bacto-tryptone, 0.5% w/v bacto-yeast extract, 8.56 mM NaCl, and 2.5 mM KCl) supplemented with 100 μg/mL kanamycin and 34 μg/mL chloromycetin in a 50 mL flask at 37 °C with shaking at 250 rpm for 10 h. Then, the culture was diluted 1:100 with fresh

integrins, and tumor-associated carbohydrates, which could serve as many excellent attack methods for the targeted cancer treatments.6−10 As several peptides derived from phagedisplayed peptide library screenings such as RGD peptides,11 GRP peptide,12 and AID peptide13 have been developed into cancer therapeutics, the use of more specific and efficient peptide in cancer-targeting therapeutics should be further exploited. Despite tremendous progress in the design of targeted peptides, peptides that specifically recognize highly metastatic cancer and its metastatic foci are rare. In our previous study,14 we have identified a 5-amino acid peptide, TMTP1, which specifically bound to a series of highly metastatic cancer cell lines in vitro and in vivo but not to the poorly metastatic or nonmetastatic cell lines. Furthermore, TMTP1 has shown a remarkable ability to target the very early stage of occult metastasis foci, which could be exploited to detect and eradicate concealed tumor cells before they evade the host's immune surveillance and cause damage to the host. Thus, TMTP1 may be a useful target for cytotoxic drugs designed to selectively kill highly metastatic and micrometastatic cells, while sparing their normal tissue cell counterparts. One novel class of tumor therapeutics includes fusion proteins consisting of toxins conjugated to tumor-selective peptides that direct the agent to the tumor cell surface by enhanced specifically targeted delivery. A series of fusion proteins were successfully used to exhibit antitumor activity in tissue culture, in animal models, and in patients.15−23 A novel class of fusion proteins, in which the catalytic and translocation domains of diphtheria toxin (DT) are genetically fused to ligands that can selectively target malignant cells, induces cell death through a mechanism different from that of conventional chemotherapy drugs.24−26 After ligand binding and receptormediated endocytosis, the toxin translocates to the cytosol where it activates ADP-ribosylates elongation factor 2, leading to inactivation of protein synthesis and cell death.27−30 Studies of a series of internal frame deletion mutations established that the DT390 truncation of the DT was optimal for genetic fusion.31−35 The purpose of this study was to develop a novel targetspecific delivery system to introduce therapeutic toxin into highly metastatic tumor cells and micrometastatic foci. A targeted fusion toxin composed of DT390 and a single TMTP1 peptide exhibited poor activities for both the dose-dependent inhibition of cell viability and the induced apoptosis of tumor cells. Therefore, we generated fusion proteins consisting of double and triple TMTP1 coupled to the COOH-terminal region of DT390. Finally, DT390-triTMTP1 showed efficient inhibition of tumor growth and metastasis in vitro and in vivo without any apparent side effects in other organs as expected. These results provide new insights for developing new generation targeted therapies of highly metastatic cancers.

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MATERIALS AND METHODS Cell Lines. The human gastric cancer cell line MKN-45 was a kind gift from Dr. Li (Shanghai Cancer Institute, China).36 The human prostate cancer cell line PC-3M-1E8 was kindly provided by Dr. Jie Zheng (Beijing University, China).37 The human embryonic kidney cell line HEK-293 was obtained from the American Type Culture Collection. MKN-45 and PC-3M1E8 cells were maintained in RPMI-1640 with 10% fetal calf serum, while HEK-293 cells were cultivated in DMEM with 10% fetal calf serum at 37 °C in a humidified atmosphere 116

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using a FACSCalibur flow cytometer (BD Biosciences, United States). Mouse Tumor Model Experiments. Four week old BALB/c ν/ν mice were obtained from the SLAC Laboratory Animal Co. Ltd. (Shanghai, China). Tumor cells were harvested and injected subcutaneously into the posterior trunk of mice in a total volume of 100 μL (3 × 106 PC-3M1E8 cells and 2 × 106 MKN-45 cells) and allowed to grow to 4−6 mm in diameter. Then, the subcutaneous tumor was resected aseptically and cut into small fragments (2 mm3), which were implanted subcutaneously into the left posterior trunk of anesthetized mice to induce tumor growth. To assess the antitumor efficacy of fusion proteins on the subcutaneous tumors, model mice were randomly divided into five groups (n = 5 per group for PC-3M-1E8, and n = 4 per group for MKN-45) that were treated with either PBS, TMTP1, DT390-TMTP1, DT390-biTMTP1, or DT390triTMTP1 7 days after tumor implantation. TMTP1 and fusion protein toxins were ip injected at a dose of 10 μg/ injection every 3 days for 21 days. Tumor growth was evaluated every 3 days using a caliper. Tumor volume was calculated using the formula: V = 4/3 π × (d/2)2 × (D/2), where d is the minor tumor axis and D is the major tumor axis.38 The survival rate in each group of the gastric tumor subcutaneous model was evaluated at the end of the study. The mouse model of MKN-45 orthotropic gastric cancer, which has liver-specific metastasis potential, was kindly provided by Dr. Jinjun Li (Shanghai Cancer Institute, Medical College of Shanghai Jiao Tong University, Shanghai, China).39,40 In this model, fresh tumor fragments were obtained as described above. Then, healthy mice were anesthetized, their stomachs were exposed, and the serosal membrane in the middle of the curvatura gastrica major was scraped with forceps. A piece of tumor fragment (1 mm3) was fixed on the scraped site of the serosal surface with a 5-0 absorbent suture. The stomach was then returned to the peritoneal cavity, and the abdominal wall and skin were closed with 1-0 sutures. To assess the effects of fusion protein toxins on the growth and metastasis of orthotropic gastric cancer, model mice were randomly divided into five groups 7 days after gastric operation (n = 5 for each group) and then ip injected with either PBS, TMTP1, DT390-TMTP1, DT390-biTMTP1, or DT390-triTMTP1 at a dose of 10 μg/injection. Treatments were repeated every 3 days for 28 days, and then, model mice were sacrificed to resect the primary tumor, liver, spleen, and the metastatic tumor in the abdominal wall. All of the resected tissues were subjected to routine histological examination for evidence of metastasis by a pathologist. To further analyze the distribution and spread of DT390triTMTP1 after ip injection in the subcutaneous tumor model of prostate cancer (PC-3M-1E8), tumor-bearing mice were injected with 10 μg/injection fusion protein toxin once tumors had grown to a size of 0.5−1.0 cm3. Animals were sacrificed at the indicated time points, and the primary tumor, heart, liver, spleen, lung, kidney, and brain were harvested for immunohistochemistry analyses to detect the distribution of DT390triTMTP1 using the flag antibody of fusion protein toxin (His antibody). Animal experiments were approved by the Hubei Institute Animal Research Committee. All animals were bred at our animal facility according to the Chinese laboratory animal guidelines.

SOB broth. When the OD600 of the culture reached 0.8, expression of the hybrid gene was induced by the addition of 1 mM isopropyl-h-D-thiogalactopyranoside (FisherBiotech, Fair Lawn, NJ). Four hours after induction, the bacteria were harvested by centrifugation. The cell pellets were subjected to repeated freeze−thaw cycles followed by sonication on ice in lysing buffer (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 5 mM EDTA, 0.1% NaN3, and 0.5% Triton-X100 with 0.1 mM PMSF added immediately before use) to release inclusion bodies. After centrifugation and washing, the inclusion bodies were resuspended in denaturing buffer (8 M urea, 0.1 M NaH2PO4, and 0.01 Tris-Cl, pH 8.0) and then loaded into a column assembled with 50% Ni-NTA His·Bind slurry. The lysate-resin mixture was incubated at 4 °C for 1 h and washed thoroughly with buffer C (8 M urea, 0.1 M NaH2PO4, and 0.01 M Tris-Cl, pH 6.3) and buffer D (8 M urea, 0.1 M NaH2PO4, and 0.01 M Tris-Cl, pH 5.9). The protein was eluted with buffer E (8 M urea, 0.1 M NaH2PO4, and 0.01 M Tris-Cl, pH 4.5), and the fractions were analyzed by SDS-PAGE. Purified inclusion bodies were refolded with a 10-fold dilution into a redox refolding buffer (1 mM GSH and 0.2 mM GSSG), followed by incubation with stirring at 4 °C for 48 h. TMTP1 peptide was commercially obtained from Xi'an Huachen Biotech. SDS-PAGE for Identification of Refolded Protein Toxins. Concentrations and purities of renatured fusion protein toxins were determined by comparison to a BSA standard on Coomassie-stained SDS polyacrylamide gels. Cell Viability Assay. The cytotoxic effects of fusion protein toxins and TMTP1 peptide were determined with the MTT assay. Cells were cultured in 96-well plates at a density of 104/ well and incubated overnight at 37 °C with 5% CO2. Varying concentrations of fusion protein toxins and TMTP1 peptide were added to wells in triplicate and incubated for 24 h. Then, 20 μL of MTT (5 mg/mL; Sigma Chemical Co.) was added to the wells, and cells were incubated at 37 °C for 4 h, followed by the addition of 150 μL of DMSO and an additional 20 min of incubation. The cell viability was determined by measuring the optical absorbance of cells at a wavelength of 570 nm. Data are reported as percentages of corresponding controls (untreated cells). Apoptosis Assay. Cells were plated overnight on sterile glass coverslips in six-well plates, then 3 μg/mL fusion protein toxins and TMTP1 peptide were added, and samples were incubated for 5 h. Changes in cellular morphology were observed, and photographs were taken under an inverted microscope. To analyze nuclear morphology, cells were stained with Hoescht 33342 and scored for apoptotic nuclei using a confocal laser scanning microscope (The Olympus FluoView FV1000). At least 300 cells from each sample were counted. FACS Analysis. Cells were treated with 3 μg/mL fusion protein toxins and TMTP1 peptide for 24 h, and then, the apoptotic cells were stained using the Annexin V/PI Apoptosis Detection Kit (Nan Jing Key Gen Biotech Co., Jiangsu, People's Republic of China) according to the manufacturer's instructions. Briefly, cells were harvested and washed with cold PBS. Cells were resuspended in 1× binding buffer at a concentration of 1 × 106 cells/mL, and 100 μL of this solution (1 × 105 cells) was transferred to a 4 mL culture tube. After the addition of 5 μL of FITC Annexin V and 5 μL of PI, cells were gently vortexed and incubated for 15 min at room temperature (25 °C) in the dark. Prior to testing, 400 μL of 1× binding buffer was added to each tube. Apoptotic cells were sorted 117

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Figure 1. Computer-generated 3D structural models of recombinant proteins and their cytotoxicity on human cancer cell lines. (A) The structure of DT and DT390-TMTP1: a, the toxin regions of catalytic domain; t, the toxin regions of transmembrane domain; and b, receptor binding domain. (B and C) Computer-generated models of DT (B) and recombinant protein DT390-TMTP1 (C) are shown in ribbon representation. (D, I, and J) MTT assay showed different cell viability of MKN-45, PC-3M-1E8 cancer cells, and HEK-293 cells incubated with different concentrations (1−6 μg/ mL) of DT390-TMTP1 (D), DT390-biTMTP1 (I), and DT390-triTMTP1 (J) for 24 h, respectively. (E) PC-3M-1E8, MKN-45 cancer cells, and HEK293 cells were treated with different concentrations (1−6 μg/mL) of DT390-TMTP1 for 24 h and then analyzed with flow cytometry. (F) The structure of recombinant protein DT390-biTMTP1 and DT390-triTMTP1. (G and H) Computer-generated models of recombinant proteins DT390-biTMTP1 (G) and DT390-triTMTP1 (H) are shown in ribbon representation.

I × P).41 I is the staining intensity, which was evaluated according to the following criteria: 0 (no staining), 1 (weak immunostaining), 2 (moderate immunostaining), and 3 (strong immunostaining). P is the percentage of positively stained cells in each sample. Three observers, who were unaware of the origin of the tissues, independently scored the sections, and the average of their scores was used as the final score. TUNEL Assay. Murine models of MKN-45 subcutaneous gastric cancer were sacrificed 3 days after the final therapy. The primary tumors were removed, photographed, and then fixed in 3.7% paraformaldehyde to obtain paraffin-embedded sections. TUNEL staining of the tissue sections was performed according to the manufacturer's instructions of the One Step TUNEL Apoptosis Assay Kit (Nan Jing Key Gen Biotech Co., Jiangsu, People's Republic of China). Statistical Analysis. All in vitro experiments were repeated at least three times. The software package SPSS13.5 was used to analyze data. The two-tailed Student's t test was used for comparisons between groups. P < 0.05 was defined as

Histology and Immunohistochemistry. At the third day of the final treatment, mice burdened with MKN-45 subcutaneous gastric tumors were killed, and tumors were excised to determine proliferation and apoptosis by immunohistological detection of ki67 and caspase-3. All macroscopic tumors excised from mice were fixed with formalin, embedded with paraffin, and confirmed by H&E staining. Immunohistochemistry was performed according to standard procedures. Briefly, after samples were dewaxed and rehydrated, antigen retrieval was performed in a microwave oven. Endogenous peroxidase activity was inhibited with 0.3% hydrogen peroxide, nonspecific binding was blocked with normal goat serum, and sections were incubated with primary antibodies (Ki67, bs0722R; 6xHis, bs-0287R; and caspase3, bs-0081R; all from Beijing Boisynthesis Biotechnology Co.) at 4 °C overnight. The primary antibody was detected using a biotinylated antirabbit IgG. The signal was amplified by avidin−biotin complex formation and developed with diaminobenzidine followed by counterstaining with hematoxylin. Immunoreactivity was determined using the well-standardized H-Score system (H = 118

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Figure 2. Effects of recombinant proteins on proliferation in various cell lines. (A−C) Cell viability of PC-3M-1E8 (A) or MKN-45(B) and HEK293 (C) cells incubated with TMTP1 peptide and various recombinant proteins were determined by MTT assay. Data are the mean of triplicate samples (error bars ± SD) and represent the percentage of survived cells as compared with control. Representative results shown are from at least three separate experiments. (D and E) The cellular morphological changes of MKN-45, PC-3M-1E8 cancer cells, and HEK293 cells after treatment with 3 μg/mL fusion proteins and TMTP1 peptide. The morphological changes were observed under an inverted microscope (D), and cells were stained with Hoescht 33342 and scored for apoptotic nuclei using a confocal laser scanning microscope (E). Typical apoptotic nuclear morphology like pyknosis and karyolysis were obvious in MKN-45 and PC-3M-1E8 cancer cells rather than in HEK293 cells after treatment with DT390-triTMTP1 and DT390-biTMTP1.

statistically significant. All values were presented as means ± standard deviations (SDs).

protein homology-modeling server, the computer-generated 3D structural models of recombinant DT are shown in ribbon representation. We maintained the toxin regions of catalytic domain (C-domain, green) and transmembrane domain (Tdomain, blue), while the receptor binding domain (R domain) was replaced by a TMTP1 (Figure 1C), biTMTP1 (Figure 1G), or triTMTP1 (Figure 1H), which are represented as red and white ribbons. Our rearrangement did not induce conformational changes, which meant that the toxic activities of recombinant DTs remained intact. Cytotoxicity of Recombinant Proteins toward Human Cells. MKN-45, PC-3M-1E8, and HEK-293 cells were treated with different concentrations (1−6 μg/mL) of DT390-TMTP1 or control peptide TMTP1 for 24 h. MTT assays and flow cytometry were applied to measure the inhibition rates of cellular growth and apoptosis rates. As shown in Figure 1D,E, DT390-TMTP1 slightly inhibited proliferation of PC-3M-1E8 and MKN-45 cells in a dose-dependent manner. However, it exhibited less toxicity to targeted cells than other well-reported DT-derived fusion toxins.43 Furthermore, there were no significant differences in the apoptosis rates between cancer cells and normal cells. At a rather high concentration of 6 μg/ mL DT390-TMTP1, only about 20% apoptosis was observed in cancer cells, as compared to 10% in normal cells (Figure 1D). As the DT was a large molecule containing 390 amino acids and TMTP1 contained only five amino acids, the low toxicity of

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RESULTS Construction of Recombinant Plasmids. The DT390 and TMTP1 fusion protein were expressed by using the T7 promoter from pET28a, a 6× His-tag expression vector (Novagen, Madison, WI). Recombinant protein contained peptide TMTP1 and the first 390 amino acids of DT. Glutamic acid was mutated into glycine at position 218, which affected neither the proteolytically cleaved activity (amino-acid residues Arg193 and Ser194)42 nor the formation of the disulfide loop (Cys186 and Cys201)42 of DT. There was a glycine−glycine linker between DT390 and TMTP1 and a glycine−glycine− glycine−glycine−serine linker between two TMTP1 peptides. Characterization of Purified Recombinant Protein Toxins. To determine the size, concentration, and purity of each recombinant toxin, renatured fusion protein toxins were analyzed by comparison to a BSA standard on Coomassiestained SDS polyacrylamide gels. The purities of recombinant protein toxins were over 90% with molecular masses of ∼55 kDa (S1). The concentrations of fusion proteins were ∼1 mg/ mL (Figure 1 in the Supporting Information). Computer-Generated 3D Structural Models of Recombinant Protein Toxins. According to the highly extensible CHIMERA program of SWISS-MODEL automated 119

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Figure 3. Cell apoptosis induced by TMTP1 peptide and recombinant proteins. (A) Annexin V-PI double staining and flow cytometry analyzed the apoptosis rates of MKN-45, PC-3M-1E8 cancer cells, and HEK293 cells after treatment with 3 μg/mL TMTP1 peptide or recombinant proteins for 24 h. (B−D) The quantification of apoptosis rates of MKN-45 (B), PC-3M-1E8 cancer cells (C), and HEK293 cells (D) after treatment with 3 μg/ mL TMTP1 peptide or recombinant proteins for 24 h.

DT390-TMTP1 might be explained by a presumption that a single TMTP1 was unable to deliver the fusion protein to target cells. Thus, we decided to fuse DT390 with two and three tandem TMTP1 repeats. As shown in Figure 1 I,J, DT390biTMTP1 and DT390-triTMTP1 significantly increased apoptosis rates among cancer cell lines in a dose-dependent manner. Six micrograms per milliliter of DT390-biTMTP1 and DT390-triTMTP1 both achieved almost 100% apoptosis rates in PC-3M-1E8 and MKN-45 cell lines, while HEK-293 cells were mildly affected. To compare the different cytotoxities of TMTP1, DT390-TMTP1, DT390-biTMTP1, and DT390triTMTP1, 3 μg/mL peptide or fusion protein toxins were added to MKN-45, PC-3M-1E8, and HEK-293 cells. The MTT assay proved that MKN-45 and PC-3M-1E8 cells were effectively inhibited by both DT390-biTMTP1 and DT390triTMTP1 in a dose-dependent manner, and DT390-triTMTP1 showed the most powerful toxicity (Figure 2A−C). TMTP1 and DT390-TMTP1 led to inconspicuous toxic effects. In addition, toxin only control on the MKN-45 and PC-3M-1E8

cancer cell lines was also tested, and the results showed that the toxin DT-390 had no effect on the these cancer cell lines in vitro (Figure 2 in the Supporting Information). HEK-293 cells were unresponsive to any treatment. It has been proved that targeted toxins such as DT388GMCSF kill cells by inducing caspase-dependent apoptosis.44 To investigate whether MKN-45 and PC-3M-1E8 cancer cells showed typical features of apoptosis after these recombinant protein toxins treatments, we observed cellular morphological changes under an inverted microscope. As shown in Figure 2D, almost all of cancer cells (MKN-45 and PC-3M-1E8) were killed by 3 μg/mL DT390-triTMTP1 and DT390-biTMTP1 for 5 h, while no obvious morphological cellular change was observed when treated with DT390-TMTP1 or TMTP1. In addition, none of the peptide or fusion toxins resulted in morphological changes in HEK293 cells. We also used Hoescht 33342 staining to show nuclear condensation and fragmentation, characteristics of apoptosis.44 DT390-triTMTP1 and DT390-biTMTP1 led to typical apoptotic nuclear morphology 120

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Figure 4. Antitumor effects of the recombinant proteins in PC-3M-1E8 subcutaneous xenografts and MKN-45sci orthotopic gastric cancer bearing mice. (A−E) PC-3M-1E8 subcutaneous xenografts treated with PBS (A), TMTP1(B), DT390-TMTP1 (C), DT390-biTMTP1 (D), and DT390triTMTP1(E). (F) Tumor volume of MKN-45sci orthotopic gastric cancer bearing mice after treatment with TMTP1 peptide and recombinant proteins for 24 days. (G) Curve of PC-3M-1E8 subcutaneous tumor volume treated with TMTP1 peptide and recombinant proteins at different time points. (H) Kaplan−Meier survival plot of PC-3M-1E8 subcutaneous tumor-bearing mice treated with TMTP1 peptide and recombinant proteins. (I) Quantitative analysis of of MKN-45sci orthotopic gastric tumor volume after treatment with TMTP1 peptide and recombinant proteins for 24 days. (J and K) The apoptosis rates (J) and histochemistry staining (K) of MKN-45sci orthotopic gastric cancer after treatment with recombinant proteins for 24 days by TUNEL assay.

in MKN-45 and PC-3M-1E8 cells, while DT390-TMTP1 and TMTP1 did not (Figure 2E). In accordance with results observed under light microscope, none of the four treatments resulted in nuclear morphology changes in HEK293 cells. These data suggest that DT390-triTMTP1 and DT390biTMTP1 induced significant apoptosis in cancer cells, with DT390-triTMTP1 showing a more intense killing effect in targeted cells than DT390-biTMTP1. Neither treatment induced apoptosis of normal human cells. The same doses of DT390-TMTP1 and TMTP1 failed to induce an apoptotic effect in both cancer cells and normal cells. In accordance with results of Hoescht 33342, flow cytometry demonstrated no obvious apoptosis effects on HEK-293 cells among all of the tested groups, while MKN-45 and PC-3M-1E8 treated with DT390-biTMTP1 and DT390-triTMTP1 had conspicuous higher apoptosis rates than those treated with TMTP1 or DT390-TMTP1 (p < 0.05), with the most powerful effect caused by DT390-triTMTP1 (Figure 3). Treatment of MKN-45 and PC-3M-1E8 Xenografts with Recombinant Protein toxins. As shown in Figure 4A− E, DT390-biTMTP1 and DT390-triTMTP1 effectively inhibited PC-3M-1E8 subcutaneous tumor growth and prolonged the survival of nude mice. The mean tumor volumes of both DT390-biTMTP1 and DT390-triTMTP1-treated mice were significantly (P < 0.05) smaller than those of TMTP1 and DT390-TMTP1-treated mice (Figure 4G). Moreover, DT390triTMTP1 was more effective than DT390-biTMTP1 in controlling PC-3M-1E8 tumor growth and prolonging survival periods (Figure 4H). There was no death observed in mice with treatment of DT390-triTMTP1. Although the mean

tumor volume of DT390-TMTP1-treated mice appeared to be smaller than those of the PBS control group, the effects of TMTP1 and DT390-TMTP1 groups failed to exhibit statistical differences in terms of tumor volume and survival (Figure 4G). Similar results were obtained in MKN-45 orthotopic tumor bearing nude mice. DT390-biTMTP1 and DT390-triTMTP1 significantly suppressed the tumor growth as compared with TMTP1 and DT390-TMTP1 (Figure 4F,I). Treatment with TMTP1 or DT390-TMTP1 seemed to have little effect on MKN-45 orthotopic tumor growth. Treatment of MKN-45 Orthotropic Gastric Cancer with Recombinant Protein Toxins. MKN-45 orthotopic human gastric carcinoma model mice exhibit extensive growth of local and metastatic tumors.36 To determine whether recombinant protein toxins had any effect on the growth and metastasis of the established orthotropic tumor, 10 μg/mL of TMTP1, DT390-TMTP1, DT390-biTMTP1, or DT390triTMTP1 was ip injected every 3 days after orthotropic tumors were established. At the end of the 10th treatment, mice were sacrificed to resect the primary tumor, liver, spleen, and the metastatic tumor in the abdominal wall. As shown in Figure 5, the orthotropic tumor formation rate reached 100% in mice treated with PBS, TMTP1, DT390-TMTP1, and DT390biTMTP1, while that only reached 33.3% in DT390triTMTP1-treated group. Liver metastases were detected 100% in mice of control group and 66.7% in mice of TMTP1- and DT390-TMTP1-treated groups. There was 33.3% spleen metastases detected in PBS-, TMTP1-, and DT390-TMTP1-treated mice. On the other side, there was no evidence of liver and spleen metastases detected in DT390121

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Figure 5. DT390-triTMTP1 suppresses tumor metastasis of MKN-45sci orthotopic gastric xenografts in athymic mice. (A−E) The primary gastric tumor volume and distant metastatic tumor volumes including liver, spleen, and abdominal wall in the MKN-45sci orthotopic gastric nude mice after treatment with PBS (A), TMTP1(B), DT390-TMTP1 (C), DT390-biTMTP1 (D), and DT390-triTMTP1 (E). (F−I) Metastatic tissue samples were harvested and pathologically confirmed with H&E staining: (F) stomach, (G) liver, (H) spleen, and (I) abdominal wall. Table, tumor formation rates, and quantification of distant metastasis in the MKN-45sci orthotopic nude mice after treatment with the recombinant proteins and TMTP1 peptide, respectively.

biTMTP1- and DT390-triTMTP1-treated groups. Abdominal wall metastases were detected in 100% of control and TMTP1treated groups, 33.3% of the DT390-TMTP1-treated group, 66.7% of the DT390-biTMTP1-treated group, and 0% in the DT390-triTMTP1-treated group. These results indicated that DT390-biTMTP1 only tenuously inhibited the onset of primary tumor but significantly decreased the frequency of metastases to the liver, spleen, and abdominal wall. DT390triTMTP1 showed a more powerful antitumor effect in suppressing both local invasive growth and metastasis; only 33.3% of mice developed gastric primary tumor, and no distant metastasis were detected. TMTP1 and DT390-TMTP1 had mild effects on liver metastasis and abdominal wall metastasis. Dynamic Biodistribution of DT390-triTMTP1 after Systemic Administration in a Human Prostate Subcutaneous Tumor Mice Model. As the above results had shown, DT390-triTMTP1 exhibited the strongest antitumor effects among those recombinant protein toxins in vitro and in vivo. To further determine its safety in vivo, 10 μg/mL DT390triTMTP1 was ip injected, and its distribution was examined after systemic administration in the subcutaneous tumor model of prostate cancer (PC-3M-1E8). Primary tumor, heart, liver, spleen, lung, kidney, and brain were collected to detect the distribution of DT390-triTMTP1 in vivo at various time points. Immunohistochemistry staining of his-tag flag showed that DT390-triTMTP1 significantly reached a peak in the tumor 2 h after injection (P < 0.05) and persisted at a higher level for 48 h than DT390-biTMTP1 and DT390-TMTP1 (Figure 6A−E and

Figures 3 and 4 in the Supporting Information). Previous studies had proved that DT-derived immunotoxins can accumulate in liver and kidney.45,46 In our study, DT390triTMTP1 was observed to exhibit a transient accumulation in liver and kidney (Figure 6F−I). No differences in the amount of DT390-triTMTP1 were observed in heart, spleen, lung, and brain at various time points (Figures 4 and 5 in the Supporting Information). Tumor homing of DT390-triTMTP1 led to inhibition of tumor cells proliferation and induction of tumor tissue necrosis Microscopic evaluation of TUNEL-stained tumors in MKN45 orthotropic gastric cancer revealed large areas of necrosis in tumors from DT390-biTMTP1- and DT390-triTMTP1-treated mice (Figure 4J,K). Similarly, tumors treated with DT390biTMTP1 and DT390-triTMTP1-treated mice exhibited a marked reduction in Ki-67 staining (Figure 6J,L) but a distinct increase in caspase-3 staining (Figure 6K,M). Furthermore, more apoptosis rates and inhibition of proliferation were observed in the DT390-triTMTP1-treated group than in the DT390-biTMTP1-treated group. Meanwhile, apoptosis and inhibition of proliferation were hardly detected in TMTP1- and DT390-TMTP1-treated groups (Figured 4L and 6J,L). These data indicated that DT390-biTMTP1 and DT390-triTMTP1 suppressed tumor growth and metastasis in vivo by inhibiting proliferation and inducing apoptosis of tumor cells. 122

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Figure 6. Dynamic biodistribution of DT390-triTMTP1 after systemic administration in a human prostate subcutaneous tumor mice model. (A−E) DT390-triTMTP1 was accumulated in the tumor 1 h after injection and lasted at a high level for 48 h. (F−H) The quantition of DT390-triTMTP1 dynamic biodistribution in tumor (F), liver (G), and kidney (H) of PC-3M-1E8 prostate subcutaneous tumor mice model. (I) Comparison of DT390-triTMTP1 quantition between tumor and liver (P < 0.05). (J−L) The quantitive analysis (J and K) and histochemistry staining (L) of ki67 (J) and caspase 3 (K) in PC-3M-1E8 prostate subcutaneous tumor after treatment with TMTP1 peptide and the recombinant protein toxins.

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DISCUSSION A standard therapy for malignant tumor normally includes surgical ablation, radiation therapy, and chemotherapy. However, for some refractory tumors, all conventional therapies are inadequate; in situ or metastatic tumor cells can survive and lead to tumor progression or recurrence.47 Therefore, novel agents are required to efficiently and selectively kill nonresectable, early metastasis or multidrug resistant tumors. One class of such therapeutics includes fusion toxins consisting of cellular toxins fused to tumor cell targeting ligands. Several groups of fusion toxins have been proved to be effective in treating patients with malignant brain tumors (Tf-CRM107),47 malignant leukemia (DT388GMCSF),48 and T cell diseases (DAB389IL2);49 meanwhile, numerous other kinds of fusion proteins are currently being tested as anticancer agents. In our present study, the truncated DT390 was genetically fused to TMTP1, and the fusion protein DT390-TMTP1 was produced by the prokaryotic expression system. Unfortunately,

fusion protein toxin DT-TMTP1 did not show any targeted cytotoxicity to the highly metastatic PC-3M-1E8 and MKN-45 cells as expected. We initially considered that the inefficacy of DT-TMTP1 might be due to insufficient dose and less exposure time of the fusion protein toxin. However, extremely high doses (up to 50 μg/mL) and long exposure times (120 h) still did not work (data not shown). Targeting ligands of fusion protein toxins based on DT are normally composed of antibodies that selectively bind to membrane receptors. Although targeting peptides have advantages over antibodies with regard to efficiency, penetrability, toxicity, and immunogenicity, peptides have by far smaller molecular sizes than antibodies. Thus, we assumed that the inefficacy of DT-TMTP1 was because the 5-amino acid peptide TMTP1 could not direct the relatively large toxic molecular of DT390 to highly metastatic cancer cells. Thompson et al. reported that a bivalent single-chain immunotoxin A-dmDT390-bisFV (G4S) increased 7-fold 123

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toxins are toxic to liver due to nonspecific uptake of immunotoxins by hepatic cells.54 Our results showed that DT390-triTMTP1 accumulated in liver transiently (0.5 h after injection) and decreased to a rather low level 1 h after injection. Previous studies showed that administration of fusion toxins as DT390 anti-CD3sFV has caused severe and irreversible renal damage by inducing necrosis of proximal renal tubular cells.45 Our study demonstrated that although DT390-triTMTP1 transiently filtered in the kidney 0.5 h after injection, it did not accumulate later. In accordance with other studies,46 no obvious distribution of DT390-triTMTP1 was observed in heart, spleen, lung, and brain at all time points. Taken together, our data indicate that DT390-triTMTP1 showed desirable targeted anticancer effects both in vitro and in vivo. Moreover, it drastically inhibited metastasis of primary stomach tumor to liver, spleen, and abdominal wall. The biodistribution in tissues suggested that it accumulated mainly in tumor and transiently in liver and kidney and was absent in the most important organs like heart, brain, lung, and spleen, which meant it was safe for therapy. This recombinant toxin could serve as an alternative to current therapies for patients with high metastatic potential tumors.

binding potency over the monovalent A-dmDT390-sFV, which significantly increased efficacy in vitro and in vivo.50 The underlying mechanism was that the DT moiety provided steric inhibition to the FV binding domain, resulting in a reduction of FV binding affinity bioactivity.50 Accordingly, we presumed that addition of the DT390 moiety diminished the binding potency of TMTP1 by providing steric inhibition to TMTP1 binding domain. Therefore, we fused the first 390 amino acid residues of DT (truncated DT390) to two and three tandem TMTP1 peptides. Interestingly, the tandem TMTP1 repeats dramatically increased the efficacy of DT and TMTP1 fusion proteins, with DT-triTMTP1 showing the most powerful cytotoxicity against cancer cells in vitro. None of the fusion proteins were observed to affect normal cells. The underlying mechanism might contribute to bivalent and trivalent binding of DT390biTMTP1 and DT390-triTMTP1.51,52 Additional TMTP1 seemed to minimize steric hindrance of fusion protein due to the large N-terminal toxin domain. From the computergenerated 3D structural models, we observed that DT390biTMTP1 and DT390-triTMTP1 formed two and three generous arrowhead ribbons, which represented the TMTP1 regions, respectively, and mediated binding of the fusion proteins to targeted cells, while DT390-TMTP1 could hardly form an arrowhead ribbon (Figure 1B,C,G,H). In spite of the remarkable cytotoxicity of DT390-biTMTP1 and DT390-triTMTP1 in vitro, their anticancer effects in vivo should be further investigated because neutralizing antibodies against the heterogeneous DT might diminish them. In accordance with data from in vitro experiments, DT390biTMTP1 and DT390-triTMTP1 demonstrated dramatic anticancer potential in terms of significant reductions in tumor mass and prolonged survival in subcutaneously tumorbearing mice. It is possible that a part of the injected DT escapes neutralization by antitoxin in the bloodstream and reacts tightly with specific membrane receptors of the tumor cells.53 Immunohistochemical analyses uncovered that DT390biTMTP1 and DT390-triTMTP1 inhibited proliferation as well as induced apoptosis in tumor cells, which led to eradication of tumors in vivo. Metastasis represents the most lethal stage of cancer progression.36 In a previous study, we demonstrated that TMTP1 effectively targeted occult metastases or micrometastases in tumor-bearing athymic mice.14 To evaluate the antimetastasis potencies of the TMTP1 fusion toxin in vivo, recombinant toxin proteins were intermittently ip injected 7 days in a human orthotopic gastric carcinoma model. Results showed that DT390-triTMTP1 totally inhibited metastasis of primary gastric tumors to liver, spleen, and abdominal wall, while DT390-biTMTP1 could not. On the other hand, TMTP1 and DT390-TMTP1 had inconspicuous antimetastasis effects in vivo. These results showed that DT390-triTMTP1 had desirable anticancer effects and targeted specificity. Clinical uses of recombinant fusion proteins have been hampered by obvious organ toxicity or side effects.45 In the present study, biodistribution of DT390-triTMTP1 fusion protein was investigated. The distribution of DT390-triTMTP1 in important organs of the mice, including heart, liver, spleen, lung, kidney, brain, and subcutaneous tumor was determined by immunohistochemistry. Our tissue distribution studies suggested that the accumulation of the fusion toxin occurred quickly, with maxim concentration in tumor tissue 2 h after administration, and lasted at a high level for 48 h. Some fusion

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ASSOCIATED CONTENT

S Supporting Information *

Figures of SDS-PAGE of purified fusion protein DT390triTMTP1 and concentration quantition of protein DT390triTMTP1, cytotoxicity of recombinant protein DT390 on human cancer cell lines, dynamic biodistribution of different fusion toxins after systemic administration in a human prostate subcutaneous tumor mice model, biodistribution of the fusion toxins in tumor and liver tissues 2 h after systemic administration, and dynamic biodistribution of DT390-tri TMTP1 protein in various tissues of PC-3M-1E8 subcutaneous tumor-bearing mice. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Address: Cancer Biology Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jiefang Avenue, Wuhan, Hubei 430030, People's Republic of China. Tel: 86(27)83662681. Fax: 86(27) 83662779. E-mail: [email protected] (L.X.). Address: Cancer Biology Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jiefang Avenue, Wuhan, Hubei 430030, People's Republic of China. Tel: 86(27)83663180. Fax: 86(27)83662681. E-mail: [email protected] (S.W.). Author Contributions

X.M., P.L., and S.Y. contributed equally to this manuscript. Funding

Grant support: National Science Foundation of China (Nos. 81001006, 81230038, 81172468, and 30973148), Specialized Research Fund for the Doctoral Program of Higher Education of China (No. 200804871030), and the “973” Program of China (No. 2009CB521808). Notes

The authors declare no competing financial interest. 124

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