Targeted Cancer Therapy Using Fusion Protein of TNFÎ± and Tumor

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Targeted cancer therapy using a fusion protein of TNF# and a tumor-associated fibronectin-specific aptide Hyungsu Jeon, Daejin Kim, Minsuk Choi, Sukmo Kang, Jin Yong Kim, Sunghyun Kim, and Sangyong Jon Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00520 • Publication Date (Web): 02 Oct 2017 Downloaded from http://pubs.acs.org on October 8, 2017

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Molecular Pharmaceutics

Targeted cancer therapy using a fusion protein of TNFα and a tumorassociated fibronectin-specific aptide †

†

†

†

§

Hyungsu Jeon , Daejin Kim , Minsuk Choi , Sukmo Kang , Jin Yong Kim , Sunghyun ‡

Kim*, , Sangyong Jon*, †

†

KAIST Institute for the BioCentury, Department of Biological Sciences, Korea Advanced

Institute of Science and Technology, 291 Daehak-ro, Daejeon 34141, South Korea. ‡

Korea Institute of Ceramic Engineering and Technology, Center for Convergence

Bioceramic Materials, 202 Osongsaengmyeong 1-ro, Cheongjusi, Chungcheongbuk-do, South Korea. §

Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science

and Technology, 291 Daehak-ro, Daejeon 34141, South Korea.

*Corresponding authors Tel.: +82-42-350-2634; Fax: +82-42-350-4450; E-mail: [email protected] (S. Jon). Tel: +82-43-913-1512; Fax: +82-43-913-1598; E-mail: [email protected] (S. Kim).

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Abstract Tumor necrosis factor-α has shown potent antitumor effects in preclinical and clinical studies. However, severe side effects at less than therapeutic doses have limited its systemic delivery, prompting the need for a new strategy for targeted delivery of the protein to tumors. Here, we report a fusion protein of mouse tumor necrosis factor (TNF)-α (mTNFα) and a cancertargeting, high-affinity aptide, and investigate its therapeutic efficacy in tumor-bearing mice. A fusion protein consisting of mTNFα, a linker, and an aptide specific to extra domain B of fibronectin (APTEDB), designated mTNFα-APTEDB, was successfully produced by expression in Escherichia coli. mTNFα-APTEDB retained specificity and affinity for its target, EDB. In mice bearing EDB-overexpressing fibrosarcomas, mTNFα-APTEDB showed greater efficacy in inhibiting tumor growth than mTNFα alone or mTNFα linked to a non-relevant aptide, without causing an appreciable loss in body weight. Moreover, in vivo antitumor efficacy was further significantly increased by combination treatment with the chemotherapeutic drug, melphalan, suggesting a synergistic effect attributable to enhanced drug uptake into the tumor as a result of TNFα-mediated enhanced vascular permeability. These results suggest that a fusion protein of mTNFα with a cancer-targeting peptide could be a new anticancer therapeutic option for ensuring potent antitumor efficacy after systemic delivery.

Keywords: Aptides, Cancer therapy, Combination therapy, Extra domain B of fibronectin, Tumor necrosis factor α.


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1. Introduction Tumor necrosis factor-α (TNFα), a pleiotropic pro-inflammatory cytokine produced by various immune cells, adopts a homotrimeric, biologically active form under physiological conditions.1, 2 As a therapeutic molecule, TNFα acts through a complex mechanism to exert potent antitumor effects, mainly through tumor vascular destruction attributable to induction of coagulant factors, TNFα receptor-mediated tumor endothelial cell damage, and reduction of integrin αvβ3 (angiogenic marker) expression.3-6 In addition, TNFα exerts direct cytotoxic effects on tumor cells by inducing T-cell–mediated immune responses.7,

8

Furthermore,

several studies have reported that TNFα increases tumor vascular permeability and reduces interstitial fluid pressure in the tumor mass, resulting in enhanced chemotherapeutic drug penetration.9-13 However, despite its high therapeutic potential, the systemic use of TNFα has been limited by its unacceptable side effects, such as hypotension, liver toxicity, shock and bowl necrosis, which reduce its systemically tolerable dose to a level significantly lower than the effective dose.9 For this reason, TNFα, as an antitumor biologic agent, is used only for local therapy such as isolated limb perfusion, which enables localized delivery of the highly toxic drug to the tumor while decreasing its systemic toxicity.14-16 To date, combination therapy with TNFα and a chemotherapeutic drug based on the isolated limb perfusion procedure has proven effective against non-resectable, high-grade sarcomas and melanomas, and metastatic liver tumors in the clinic.15 Among the many strategies for developing systemically available TNFα therapeutics

17-23

,

one attractive approach is the targeted delivery of TNFα to the tumor vasculature with the aid of cancer-targeting ligands.17, 18, 22, 24 Of the numerous tumor vessel-associated antigens, extra domain B of fibronectin (EDB) has been considered a promising biomarker and target because it is specifically overexpressed in tumor-associated blood vessels, extracellular matrix, and cancer cells.25-27 Interestingly, several in vivo histological analyses have shown that EDB is highly conserved across different species (e.g., human, rat, mouse), and its expression is generally undetectable in normal tissues of healthy adult individuals, except under limited situations such as tissue remodeling and wound healing.26, 27 To exploit this preferential expression of EDB in the tumor environment, researchers have developed a fusion protein of an EDB-specific small antibody fragment (scFv), termed L19, and TNFα that selectively accumulates around the tumor vasculature and shows potent therapeutic efficacy in preclinical animal studies.18 Recently, we reported a platform technology, termed ‘aptides’, that enables screening and selection of a novel class of high-affinity peptides against various biological targets.28, 29 We



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have shown that an aptide specific for EDB (APTEDB) can be used as a cancer-targeting ligand, demonstrating the selective delivery of iron oxide nanoparticles, liposomes, conventional anticancer drugs to EDB-overexpressing tumor sites.30-32 Furthermore, a structural analysis has revealed that APTEDB tightly binds EDB in a unique sequential binding mode that is markedly different from that of the L19 antibody.33 Hence, our previous findings suggest that APTEDB, a peptide considerably smaller than the L19 antibody, may have potential for use as a new class of targeting ligands that enable delivery of cytotoxic TNFα to EDBoverexpressing tumors. In this study, we report a fusion protein of mouse TNFα (mTNFα) and an EDB-specific aptide, designated mTNFα-APTEDB, for targeted cancer therapy. Preparation, characterization, and therapeutic evaluations of mTNFα-APTEDB fusion protein in vitro and in vivo are presented. Finally, the in vivo antitumor efficacy of the fusion protein in combination with the chemotherapeutic drug, melphalan, is also investigated.

2. Materials and Methods 2.1. mTNFα, mTNFα-APTEDB, and mTNFα-APTSCR expression constructs Wild-type mTNFα, and mTNFα-APTEDB and mTNFα-APTSCR fusion proteins, where APTEDB and

APTSCR

denote

the

amino

HSCSSPIQGSWTWENGKWTWKGIIRLEQQP

acid

sequence and

HASDRNGSGTGENGKGTGKGLHEQSD, respectively, were prepared using recombinant DNA technology. The mTNFα gene (amino acids 81–235) was synthesized by Bioneer (Daejeon, Korea), digested with NdeI and BamHI, and cloned into the pET28b prokaryotic expression vector (Novagen, Darmstadt, Germany). The genes encoding the fusion proteins mTNFα-APTEDB and mTNFα-APTSCR were generated by interposing an oligonucleotide encoding two aptides and a soluble linker (GSEGSEGEG)2 between the 3’-end of mTNFα and the 5’-end of two aptide genes. The oligonucleotides were created via a polymerase chain reaction (PCR) process without template DNA using the following primer pairs (Genotech, Daejeon, Korea): linker, 5’-AAG GAT CCG GCT CTG AAG GCT CTG AAG GCG AAG GTG GCT-3’ (forward) and 5’-TGA ATT CAC CTT CGC CTT CAG AGC CTT CAG AGC CAC CTT-3’ (reverse); APTEDB, 5’-AGA ATT CCA TAG CTG TAG TTC TCC TAT TCA GGG ATC TTG GAC ATG GGA AAA CGG AAA-3’ (forward) and 5’-AAT AAG CTT TAA AGG CTG TTG CTC CAA CCT AAT AAT TCC CTT CCA TGT CCA TTT TCC G-3’ (reverse); APTSCR, 5’-AAG AAT TCC ATG CGA GCG ATC GTA ACG GAT CTG GCA


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CAG GCG AAA ACG GAA A-3’ (forward) and 5’-AAT AAG CTT TTA ATC GCT CTG TTC ATG CAG TCC CTT GCC TGT GCC TTT TCC GTT TTC G-3’ (reverse). Underlined regions represent introduced restriction enzyme sites. Primer pairs were denatured once at 95 °C, annealed at 30 °C, and extended at 72 °C. The synthetic linker gene was digested with BamHI and EcoRI, and the two synthetic aptide genes were digested with EcoRI and HindIII, after which the genes were sequentially inserted into a pET28b vector containing a His6tagged mTNFα gene. All restriction enzymes were purchased from New England Biolabs (Ipswich, MA, USA).

2.2. Purification of recombinant proteins The pET28b expression vectors for mTNFα, mTNFα-APTEDB, and mTNFα-APTSCR were individually transformed into Escherichia coli strain BL21 (DE3) (Stratagene, La Jolla, CA, USA). After overnight induction with 1 mM isopropyl β-D-thiogalactopyranoside (IPTG; LPS Solution, Daejeon, Korea) at 18 °C, cells were lysed and centrifuged to pellet cell debris. His6-tagged recombinant proteins in the resulting supernatants were purified by affinity chromatography using Ni-NTA affinity agarose resin (ELPIS Biotech, Daejeon, Korea) in a gravity-flow column (Bio-Rad, Hercules, CA, USA). Unbound proteins were removed by washing the column with Tris buffer (20 mM Tris, 30 mM NaCl, 20 mM imidazole, pH 7.4), and bound proteins were eluted with Tris buffer (20 mM Tris, 30 mM NaCl, 150 mM imidazole, pH 7.4). Recombinant proteins were further resolved by size-exclusion gel filtration using a Superdex 200 column (GE Healthcare, Little Chalfont, UK), pre-equilibrated with Tris buffer (20 mM Tris, 30 mM NaCl, pH 7.4). The N-terminal His tag was cleaved with thrombin (Sigma-Aldrich, St. Louis, MO, USA), and final recombinant proteins were rechromatographed on a Superdex 200 column. Endotoxins were removed using a high capacity endotoxin removal spin column (Pierce, Waltham, MA, USA).

2.3. Enzyme-linked immunosorbent assay (ELISA) Biotinylated EDB was prepared as previously described 28, and basic fibroblast growth factor (bFGF) was obtained from Chungbuk National University (Chungbuk, Korea). Streptavidin (10 µg/mL; NEB), bFGF, fibronectin (R&D Systems, MN, USA), bovine serum albumen (BSA; Bovogen, Melbourne, Australia) and skim milk (Acumedia, Lansing, MI, USA) were directly immobilized on a 96-well plate (Corning, NY, USA) and incubated overnight at 4 °C. Thereafter, biotinylated EDB (10 µg/mL) was added and captured by pre-immobilized streptavidin, followed by incubation of the plate with phosphate-buffered saline (PBS)



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containing 2% skim milk for 2 h at room temperature. After incubating each well with 10 µg/ml of mTNFα, mTNFα-APTEDB or mTNFα-APTSCR (diluted in a 2% skim milk solution) for 1 h at room temperature to allow binding, the plate was washed six times with PBS containing 0.1% Tween-20. Bound mTNFα was detected by incubating first with rabbit antimTNFα antibody (Ab34674; Abcam, Cambridge, MA, USA) and then with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (Ab6721; Abcam, Cambridge, MA, USA), diluted 1:1000 and 1:5000, respectively, in a 2% skim milk solution. HRP activity was measured using tetramethylbenzidine (TMB) as a substrate (BD Biosciences, Franklin Lakes, NJ, USA) with monitoring of absorbance at 450 nm.

2.4. Western blot mTNFα, mTNFα-APTEDB, and mTNFα-APTSCR were resolved by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) on 15% gels, electrotransferred to a PVDF (polyvinylidene difluoride) membrane (Millipore, Darmstadt, Germany), and blocked with 5% BSA in PBS containing 0.1% Tween-20 for 2 h at room temperature. The membrane was incubated first with rabbit anti-mTNFα antibody overnight at 4 °C, and then with HRPconjugated goat anti-rabbit secondary antibody, diluted 1:1000 and 1:5000, respectively, in the same 5% BSA solution. Immunoreactive proteins were detected using enhanced chemiluminescence (ECL) reagents (ELPIS Biotech, Daejeon, Korea) and visualized using a ChemiDoc XRS+ system (BioRad, Hercules, CA, USA).

2.5. Stability test mTNFα-APTEDB at a concentration of 8 µg/20 µL in Tris buffer (20 mM Tris, 30 mM NaCl, pH 7.4) was diluted with PBS or Balb/c mouse serum at a 1:1 volume ratio and then incubated for 0, 1, 4, 8, 12, 16 and 24 h at room temperature. Each sample obtained at the predetermined time was mixed with 5X SDS loading buffer and boiled at 95 °C for 10 min. The resulting solution was loaded onto a 15% SDS-PAGE gel. Samples treated with mouse serum were further analyzed by Western blotting.

2.6. Affinity measurements The binding affinity of mTNFα-APTEDB was measured using a BIACORE 3000 system (GE Healthcare, Little Chalfont, UK), and the interaction of mTNFα-APTEDB with EDB was analyzed on a SA sensor chip (GE Healthcare, Little Chalfont, UK) using PBS as a running buffer. Biotinylated EDB was captured on the target channel of an SA chip at a flow rate of 5


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µL/min; the reference channel of the SA chip was injected with running buffer. Different concentrations of mTNFα-APTEDB were then injected at a flow rate of 30 µL/min in running buffer. Kinetic data were analyzed using BIA evaluation 2.1 software (GE Healthcare, Little Chalfont, UK).

2.7. Cell viability assays The cytotoxicity of mTNFα, mTNFα-APTEDB and mTNFα-APTSCR against WEHI-164 mouse fibrosarcoma cells (Korea Cell Line Bank, Seoul, Korea) was tested. Briefly, WEHI-164 cells in RPMI-1640 medium containing 10% (v/v) fetal bovine serum and 1% (v/v) penicillinstreptomycin (Welgene, Gyeongsangbuk-do, Korea) were cultured on 96-well cell culture plates (Corning, NY, USA) at 1 × 104 cells/well in a humidified CO2 incubator at 37 °C. After reaching ~60% confluence, cells were treated with different concentrations of mTNFα, mTNFα-APTEDB or mTNFα-APTSCR in serum-containing media for 24 h. After washing with D-PBS, cell viability was determined by WST-1 assay using the EZ-Cytox cell viability assay kit (Itsbio, Seoul, Korea), as described by the manufacturer. The IC50 value was calculated from nonlinear regression plots using GraphPad prism 7.0 (GraphPad Software, La Jolla, CA, USA).

2.8. Mice Five-week-old Balb/c mice were purchased from Orient Bio (Gyeunggi-do, Korea) and maintained in cages housed in a clean, temperature-controlled room with a 12 h (light)/12 h (dark) cycle. The care, handling, treatment, and sacrificing of mice were carried out in accordance with experimental animal guidelines approved by KAIST Institutional Animal Care and Use Committee.

2.9. Pharmacokinetics mTNFα or mTNFα-APTEDB (0.12 mg/kg) was intraperitoneally injected into 6-wk-old Balb/c mice. At predetermined time points (0, 5, 10, 20, 40, 60 and 90 min), 50 µL of blood was collected by retro-orbital bleeding into a Microtainer serum-separator tube (BD Biosciences, Franklin Lakes, NJ, USA). Blood samples were allowed to stand at room temperature for 10 min and then were centrifuged at 10,000 rpm (9,950 g) for 10 min to collect plasma (supernatant). Each plasma sample was diluted with PBS, and the concentration of mTNFα or mTNFα-APTEDB in the plasma was analyzed using an mTNFα ELISA kit, according to



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manufacturer’s protocols (R&D Systems, Minneapolis, MN, USA). Pharmacokinetic parameters were calculated using a one-compartment open model.

2.10. Antitumor experiments An allograft tumor model was created by subcutaneously implanting 5 × 105 WEHI-164 cells into 6-wk-old Balb/c mice. All antitumor experiments were performed after tumor volumes had reached ~100 mm3. Tumor dimensions were measured with a digital caliper (Mitutoyo, Kanagawa, Japan), and tumor volume was calculated as (short dimension)2 × (long dimension) × 0.5. Tumor-bearing mice were randomly divided into the following four groups (n = 5 mice/group): control (saline), mTNFα, mTNFα-APTEDB, and mTNFα-APTSCR. For experimental groups, mice were intraperitoneally injected four times on an every-other-day schedule with mTNFα, mTNFα-APTEDB or mTNFα-APTSCR at a dose of 0.12 mg/kg, diluted in 100 µL of normal saline (Choongwae Pharmaceutical, Seoul, Korea); the control group received 100 µL of normal saline only. For combination therapy, melphalan (Sigma-Aldrich) was reconstituted at 4 mg/mL in dimethyl sulfoxide before use, diluted in normal saline (200 µg/mL), and intraperitoneally injected into mice at a dose of 4.8 mg/kg. One day before melphalan treatment, mTNFα or mTNFα-APTEDB (0.12 mg/kg) was also intraperitoneally injected into mice in each group (n = 7 mice/group). Both tumor volumes and body weights were measured at 2-d intervals. At the end of monitoring, tumor-bearing mice were sacrificed, and tumors were excised for further analysis.

2.11. TUNEL assay Tumor tissues were excised from mice, formalin-fixed, and embedded in paraffin. Then, paraffin-embedded tissues were sectioned onto Histobond adhesive microscope slides (Marienfeld-Superior, Lauda-Königshofen, Germany). The deparaffinized and rehydrated tissue sections were unmasked by a heat-induced antigen retrieval in 10 mM sodium citrate buffer (pH 6.0) and stained for apoptotic cells by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay using an ApoBrdU DNA fragmentation assay kit (BioVision, Milpitas, CA, USA) according to manufacturer’s instructions.

2.12. Ex vivo staining of tumor tissues When subcutaneously injected tumors had reached a volume of ~100 mm3, mice were intraperitoneally injected with mTNFα or mTNFα-APTEDB (0.12 mg/kg). After 30 min, tumors were excised, formalin-fixed, paraffin-embedded, and sectioned onto Histobond ACS Paragon Plus Environment

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adhesive microscope slides. The sectioned tumor tissues were deparaffinized, rehydrated with proteinase K solution (20 µg/mL in Tris-EDTA, pH 8.0) for antigen retrieval, and blocked with 10% goat serum for 1 h at room temperature. Thereafter, tissue slides were incubated first with a rabbit anti-mTNFα primary antibody (1:50) overnight at 4 °C and subsequently an Alexa 647-conjugated donkey anti-rabbit IgG secondary antibody (1:200) (ab150075; Abcam, Cambridge, MA, USA) for 1 h. After each step, slides were washed twice with PBS for 5 min each. Finally, slides were mounted with Vectashield mounting medium containing 4',6diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA, USA).

2.13. Statistical analysis The data are presented as mean ± standard error of the mean (SEM). Statistical significance of differences among groups was calculated by one-way analysis of variance (ANOVA) with post hoc Tukey’s test using SPSS for Windows version 18 (Chicago, IL, USA). A p-value < 0.05 was considered statistically significant for differences among experimental groups.

3. Results & discussion 3.1. Design and preparation of mTNFα-APTEDB and mTNFα-APTSCR recombinant proteins Unlike antibody-TNFα fusion proteins 18-21, which are produced using expensive mammalian cell culture techniques, our TNFα-aptide fusion protein harnesses a cost-effective bacterial production system. A structural depiction of the fusion protein for targeted delivery of mTNFα to EDB-expressing tumors is shown in Figure 1A. As the C- terminus of mTNFα is known to interact with its receptor, it is reasonable to introduce the cancer-targeting ligand to the N-terminus of mTNFα like the case of L19 antibody-TNFα.18 However, we were not able to introduce the aptide ligand to the N-terminus of mTNFα because of problem with the protein expression in E. coli. Instead, we introduced it to the C-terminus of mTNFα using a highly soluble and flexible linker (GSEGSEGEG)2 to minimize loss of its biological activity. Recombinant proteins were prepared by constructing separate plasmid DNAs encoding mTNFα, mTNFα-APTEDB and mTNFα-APTSCR (a non-targeting, control fusion protein containing a scrambled-sequence aptide), and cloning each into the pET28b vector (Figure 1B). The three recombinant proteins, constructed with N-terminal His6 tags to facilitate initial purification using Ni-NTA agarose affinity chromatography, were overexpressed in E. coli and then sequentially purified by affinity and size-exclusion chromatography. Recombinant



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proteins were subjected to a second size-exclusion chromatography step after cleaving with thrombin to remove the His tag. Thereafter, chromatography fractions containing TNFα with a molecular weight corresponding to the homotrimer, the biologically active form of TNFα, were collected (Figure S1) and analyzed by SDS-PAGE. These analyses confirmed that monomeric mTNFα, mTNFα-APTEDB, and mTNFα-APTSCR migrated at the expected sizes of 17.7, 23.2 and 22.8 kDa, respectively (Figure 1C and Figure S1), indicative of successful expression and purification. Next, the stability of purified mTNFα-APTEDB was examined. As shown in Figure S2, mTNFα-APTEDB showed little degradation upon incubation in PBS or in the presence of 50% mouse serum for up to 24 h, indicative of high biological stability of the fusion protein.

Figure 1. Schematic representation and construction of mTNFα-APTEDB. (A) Predictive homotrimeric structure of mTNFα-APTEDB, formed by noncovalently linked monomeric mTNFα-APTEDB. (B) Schematic depiction of mTNFα-APTEDB and mTNFα-APTSCR constructs in the pET28b E. coli expression vector. (C) SDS-PAGE analyses confirming the identity and molecular weights of the three monomeric recombinant proteins.

3.2. Assessment of specificity, affinity, and biological activity of mTNFα-APTEDB We next examined whether the aptide retains its specificity and affinity for EDB in the context of mTNFα-APTEDB. ELISAs using an anti-mTNFα antibody revealed that mTNFαAPTEDB exhibited specific binding to the target EDB, and did not bind other proteins tested,


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including normal fibronectin (Figure 2A), indicating preservation of the EDB-specificity of the fusion protein. As expected, neither mTNFα nor mTNFα-APTSCR bound to any of the proteins tested, including EDB. We next measured the binding affinity of mTNFα-APTEDB for EDB by flowing different concentrations of the fusion protein over an EDB-immobilized streptavidin chip using a surface plasmon resonance (SPR)-based instrument (BIACORE 3000 system). These SPR analyses revealed an association rate constant (ka) of ~5.98 × 104 M-1S-1 and a dissociation rate constant (kd) of ~3.59 × 10-4 M-1S-1, yielding an equilibrium binding constant (Kd) of ~6 nM (Figure 2B), which is slightly stronger than the original APTEDB (~16 nM). These results clearly indicate that APTEDB retains its original specificity and affinity for the target EDB after being fused to mTNFα, and thus should be capable of delivering the therapeutic protein to EDB-overexpressing tumors in vivo. To assess the relative anticancer activity of mTNFα-APTEDB, we first performed in vitro cell viability tests by exposing WEHI-164 mouse fibrosarcoma cells, an EDB-positive and mTNFα-sensitive cancer cell line

12, 18

, to different concentrations of each recombinant

protein for 24 h. As shown in Figure 2C, the anticancer activity of mTNFα-APTEDB and mTNFα-APTSCR was decreased by 3.87- and 3.84-fold, respectively, compared with that of mTNFα. These findings show that the fusion protein largely retains the biological activity of mTNFα and is highly potent in killing EDB-positive cancer cells in vitro.



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Figure 2. Characterization of the specificity, affinity, and biological activity of mTNFαAPTEDB. (A) ELISAs were carried out on a 96-well plate using EDB and the non-specific proteins: fibronectin (FN), streptavidin (STR), bFGF, BSA, and skim milk. EDB was biotinylated and captured on streptavidin-coated wells, and then each well was incubated with each of the three recombinant proteins (mTNFα, mTNFα-APTEDB and mTNF-APTSCR). mTNFα-APTEDB bound specifically to the EDB domain, showing no appreciable binding to any of the other test proteins, including normal fibronectin, whereas mTNFα and mTNFAPTSCR did not bind any of the proteins tested. (B) SPR sensorgrams obtained for biotinylated-EDB–immobilized BIAcore SA chips upon treatment with different concentrations (80, 100, 200, 350, and 450 nM) of mTNFα-APTEDB. A kinetic analysis performed using BIA evaluation software yielded a Kd for mTNFα-APTEDB binding to EDB of 6 nM. (C) In vitro anticancer activity of mTNFα, mTNFα-APTEDB, and mTNFα-APTSCR. WEHI-164 cells were incubated with each of the three recombinant proteins for 24 h at 37 °C, after which cell viability and IC50 values were determined by WST-1 assay.

3.3. Pharmacokinetics of mTNFα-APTEDB The circulation half-life of mTNFα-APTEDB in blood was measured and compared with that of mTNFα after intraperitoneal injection. Their plasma concentrations were quantified using an ELISA kit based on a standard curve prepared from known concentrations of mTNFα. As shown in Figure 3A, mTNFα-APTEDB was rapidly eliminated from the blood circulation,


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showing a half-life (t1/2) of ~0.275 h, a value similar to that of mTNFα (~0.269 h). Also, areaunder-the curve (AUC0-∞) values for both mTNFα-APTEDB and mTNFα were comparable (0.9105 µg·h/mL vs. 0.8733 µg·h/mL), suggesting that the fusion of APTEDB to mTNFα does not affect the pharmacokinetic profile, and thus the safety margin, of the original mTNFα.

Figure 3. Pharmacokinetics, in vivo targeting, and antitumor activity of mTNFα-APTEDB in a WHEI-164 mouse fibrosarcoma model. (A) Plasma concentration of mTNFα at various time points in BALB/c mice (n = 5 mice/group). For antitumor efficacy tests, recombinant proteins (0.12 mg/kg) were intraperitoneally injected at the indicated days, as denoted by black arrows. Tumor size (B) and body weight changes (C) were monitored every other day. Data are presented as mean tumor volume ± SEM (n = 5 mice/group; *p < 0.05 vs. mTNFα; ** p < 0.01 vs. mTNFα-APTSCR and saline) and body weight ± SEM (n = 5 mice/group; *p < 0.05 and ** p < 0.01 vs. saline). (D) Representative confocal microscopic images of tumor tissue sections from mice treated with mTNFα-APTEDB or mTNFα. Tumor tissues were harvested 30 min after injecting each protein, and tumor sections were immunostained with an antimTNFα antibody, followed by Alexa 647-conjugated secondary antibody (red); nuclei were counterstained with DAPI (n = 3 mice/group). Scale bar = 50 µm.

3.4. Antitumor activity of mTNFα-APTEDB in vivo



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The in vivo therapeutic efficacy of mTNFα-APTEDB was evaluated in an EDB-positive, TNFα-sensitive mouse fibrosarcoma (WEHI-164 cell) allograft model

12,

18

. Four

intraperitoneal (i.p.) injections of mTNFα-APTEDB, mTNFα-APTSCR, or mTNFα at a dose of 0.12 mg/kg were administered every other day to tumor-bearing mice. As shown in Figure 3B and Figure S3, mTNFα-APTEDB significantly inhibited tumor growth, exhibiting much greater antitumor efficacy (73%) relative to the saline-treated group than did other controls, including mTNFα (34%) and mTNFα-APTSCR (10%), which showed marginal growth retardation. Notably, despite its two-fold lower anticancer activity than mTNFα in vitro, mTNFα-APTEDB exhibited substantially improved antitumor efficacy compared with mTNFα in vivo, indicating that the tumor-targeting ability of APTEDB more than compensated for the modest reduction in intrinsic efficacy. This result suggests that mTNFα-APTEDB delivered to the target tumor site can freely interact with its receptor and exert anticancer effect as similar as mTNFα. Moreover, mice treated with mTNFα-APTEDB showed no appreciable loss in body weight relative to saline-treated control mice, whereas mTNFα-treated mice showed appreciable body weight loss during the treatment period (p < 0.05) (Figure 3C), indicating that mTNFα-APTEDB has a better safety profile than native mTNFα at the same dose. To directly assess the relative targeting ability of mTNFα-APTEDB, we evaluated tumor accumulation of TNFα, 30 min after injection of mTNFα or mTNFα-APTEDB, by immunostaining excised tumor tissue sections for TNFα, as described in Materials and Methods. Representative confocal microscopic images of tumor sections (n = 3 mice/group) showed more intense red fluorescence signals corresponding to mTNFα in tumors from mice treated with mTNFα-APTEDB than in those treated with mTNFα (Figure 3D), indicating greater tumor accumulation (i.e., targeting) of the fusion protein. On the basis of these collective observations, we speculate that the greater antitumor efficacy and lower systemic toxicity of mTNFα-APTEDB is attributable to the APTEDB-mediated targeted delivery of mTNFα to the tumor site. 3.5. Combination therapy of mTNFα-APTEDB with a chemotherapeutic drug In addition to its direct antitumor activity, TNFα, at appropriate doses, has been shown to increase tumor vascular permeability and reduce interstitial fluid pressure, resulting in enhanced penetration of chemotherapeutic drugs

10-13

. Encouraged by our in vivo results, we

investigated potential synergistic therapeutic effects of mTNFα-APTEDB in combination with a chemotherapeutic drug. For this study, melphalan, a DNA alkylating agent that induces apoptosis, was chosen as a combination drug because combination therapy with TNFα and


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melphalan through isolated limb perfusion is currently undergoing clinical trials for treatment of bulky melanoma metastasis and soft tissue sarcoma

15

. For combination therapy, WEHI-

164 tumor-bearing mice were divided into four groups: saline, melphalan only (4.8 mg/kg), mTNFα (0.12 mg/kg) + melphalan (4.8 mg/kg), and mTNFα-APTEDB (0.12 mg/kg) + melphalan (4.8 mg/kg). Melphalan was administered 24 h after mTNFα-APTEDB injection to allow time for induction of tumor vascular permeability, the same injection schedule used in L19-mTNFα–based combination therapy targeting the same antigen (EDB) and tumor model as ours

7, 18

. The combination of mTNFα-APTEDB with melphalan effectively suppressed

tumor growth relative to the saline-treated control group, exhibiting much greater antitumor efficacy (88%) compared with other groups, including the combination of mTNFα with melphalan (55%) and melphalan alone (33%) (Figure 4A). Notably, in some mice (4 of 7), tumors became quiescent and did not grow at all after treatment with mTNFα-APTEDB + melphalan (Figure 4C). All groups of mice showed slight body weight loss relative to salinetreated controls until day 14, but gradually began to gain weight after injections ceased (Figure 4B). Moreover, TUNEL analyses of tumor tissue sections after therapy clearly revealed massive apoptosis in tumor tissues of mice treated with the mTNFα-APTEDB + melphalan combination compared with other groups (Figure 4D). Taken together, these results suggest that targeted delivery of mTNFα-APTEDB to the tumor vasculature not only destroys the tumor mass, but also enhances tumor vascular permeabilization, facilitating tumor uptake of melphalan and thereby resulting in a dramatic regression of tumor mass. However, further research will be required to quantify the effects of mTNFα-APTEDB on vascular permeability and drug penetration.



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Figure 4. Synergistic antitumor activity of mTNFα-APTEDB in combination with melphalan in a WEHI-164 fibrosarcoma model. mTNFα, mTNFα-APTEDB (0.12 mg/kg) and/or melphalan (4.8 mg/kg) were intraperitoneally injected on the indicated schedules. Next, (A) tumor size and (B) body weight changes were measured every 2 d. (C) Photograph of tumors excised from mice on day 29. (D) Confocal microscopic images of TUNEL assays performed on tumor tissue. Data are presented as mean tumor size ± SEM (n = 7 mice/group; *p < 0.05 vs. mTNFα + melphalan; **p < 0.01 vs. melphalan; ***p < 0.001 vs. saline).

4. Conclusions In this study, we developed a new targeted-delivery format for TNFα biologics by fusing TNFα with a small, high-affinity aptide targeting the tumor vasculature-associated EDB domain. The fusion protein, mTNFα-ATPEDB, retained high specificity and affinity for the EDB domain, showed little degradation for up to 24 h, and exerted strong anticancer activity against a mouse sarcoma cell line. Importantly, monotherapy with mTNFα-APTEDB showed much greater antitumor efficacy in EDB-positive mouse sarcoma-bearing mice than did mTNFα monotherapy, with little apparent systemic toxicity. The efficacy of mTNFα-APTEDB was further significantly enhanced when combined with melphalan chemotherapy. Unlike the large molecular weight fusion protein L19-TNFα (~150 kDa), mTNFα-ATPEDB is relatively ACS Paragon Plus Environment

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small (~60 kDa), and exhibited rapid clearance—almost the same rate as that for free mTNFα—resulting in minimal potential systemic toxicity. In addition, mTNFα-ATPEDB is produced through an E. coli expression system, which is more convenient and economical than mammalian cell-based production. Like L19-TNFα, which is currently entered into clinical trials for patients with advanced solid cancers34, we anticipate that the new, targeted TNFα-delivery format presented here could represent another option for systemic TNFα therapy and could be further applied to targeted delivery of other therapeutic proteins.

Supporting information Purification of recombinant proteins; stability of mTNFα-APTEDB; tumor size in monotherapy.

Acknowledgements This work was supported by a Global Research Laboratory (NRF-2012K1A1A2045436) through the National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT & Future Planning.

The authors declare no competing financial interest.

References 1. Wang, X.; Lin, Y., Tumor necrosis factor and cancer, buddies or foes? Acta Pharmacol Sin 2008, 29 (11), 1275-88. 2. Jones, E. Y.; Stuart, D. I.; Walker, N. P. C., Structure of tumour necrosis factor. Nature 1989, 338 (6212), 225-228. 3. Ruegg, C.; Yilmaz, A.; Bieler, G.; Bamat, J.; Chaubert, P.; Lejeune, F. J., Evidence for the involvement of endothelial cell integrin alphaVbeta3 in the disruption of the tumor vasculature induced by TNF and IFN-gamma. Nat Med 1998, 4 (4), 408-14. 4. Sato, N.; Goto, T.; Haranaka, K.; Satomi, N.; Nariuchi, H.; Mano-Hirano, Y.; Sawasaki, Y., Actions of tumor necrosis factor on cultured vascular endothelial cells: morphologic modulation, growth inhibition, and cytotoxicity. J Natl Cancer Inst 1986, 76 (6), 1113-21. 5. Stoelcker, B.; Ruhland, B.; Hehlgans, T.; Bluethmann, H.; Luther, T.; Männel, D. N., Tumor Necrosis Factor Induces Tumor Necrosis via Tumor Necrosis Factor Receptor Type 1Expressing Endothelial Cells of the Tumor Vasculature. The American Journal of Pathology 2000, 156 (4), 1171-1176. 6. Zhang, Y.; Deng, Y.; Wendt, T.; Liliensiek, B.; Bierhaus, A.; Greten, J.; He, W.; Chen, B.; Hach-Wunderle, V.; Waldherr, R.; Ziegler, R.; Mannel, D.; Stern, D. M.; Nawroth, P. P., Intravenous somatic gene transfer with antisense tissue factor restores blood flow by reducing tumor necrosis factor-induced tissue factor expression and fibrin deposition in mouse meth-A sarcoma. J Clin Invest 1996, 97 (10), 2213-24.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

7. Balza, E.; Mortara, L.; Sassi, F.; Monteghirfo, S.; Carnemolla, B.; Castellani, P.; Neri, D.; Accolla, R. S.; Zardi, L.; Borsi, L., Targeted delivery of tumor necrosis factor-alpha to tumor vessels induces a therapeutic T cell-mediated immune response that protects the host against syngeneic tumors of different histologic origin. Clin Cancer Res 2006, 12 (8), 257582. 8. Johansson, A.; Hamzah, J.; Payne, C. J.; Ganss, R., Tumor-targeted TNFalpha stabilizes tumor vessels and enhances active immunotherapy. Proc Natl Acad Sci U S A 2012, 109 (20), 7841-6. 9. Van Hauwermeiren, F.; Armaka, M.; Karagianni, N.; Kranidioti, K.; Vandenbroucke, R. E.; Loges, S.; Van Roy, M.; Staelens, J.; Puimege, L.; Palagani, A.; Berghe, W. V.; Victoratos, P.; Carmeliet, P.; Libert, C.; Kollias, G., Safe TNF-based antitumor therapy following p55TNFR reduction in intestinal epithelium. J Clin Invest 2013, 123 (6), 2590-603. 10. Folli, S.; Épèlegrin, A.; Chalandon, Y.; Yao, X.; Buchegger, F.; Lienard, D.; Lejeune, F.; Mach, J.-P., Tumor-necrosis factor can enhance radio-antibody uptake in human colon carcinoma xenografts by increasing vascular permeability. International Journal of Cancer 1993, 53 (5), 829-836. 11. de Wilt, J. H. W., Tumour necrosis factor alpha increases melphalan concentration in tumour tissue after isolated limb perfusion. British Journal of Cancer 2000, 82 (5), 10001003. 12. Hemmerle, T.; Probst, P.; Giovannoni, L.; Green, A. J.; Meyer, T.; Neri, D., The antibody-based targeted delivery of TNF in combination with doxorubicin eradicates sarcomas in mice and confers protective immunity. Br J Cancer 2013, 109 (5), 1206-13. 13. Kristensen, C. A.; Nozue, M.; Boucher, Y.; Jain, R. K., Reduction of interstitial fluid pressure after TNF-alpha treatment of three human melanoma xenografts. Br J Cancer 1996, 74 (4), 533-6. 14. Alexander, H. R., Jr.; Fraker, D. L.; Bartlett, D. L., Isolated limb perfusion for malignant melanoma. Semin Surg Oncol 1996, 12 (6), 416-28. 15. Grunhagen, D. J.; de Wilt, J. H.; ten Hagen, T. L.; Eggermont, A. M., Technology insight: Utility of TNF-alpha-based isolated limb perfusion to avoid amputation of irresectable tumors of the extremities. Nat Clin Pract Oncol 2006, 3 (2), 94-103. 16. Nooijen, P. T.; Manusama, E. R.; Eggermont, A. M.; Schalkwijk, L.; Stavast, J.; Marquet, R. L.; de Waal, R. M.; Ruiter, D. J., Synergistic effects of TNF-alpha and melphalan in an isolated limb perfusion model of rat sarcoma: a histopathological, immunohistochemical and electron microscopical study. Br J Cancer 1996, 74 (12), 1908-15. 17. Curnis, F.; Sacchi, A.; Borgna, L.; Magni, F.; Gasparri, A.; Corti, A., Enhancement of tumor necrosis factor alpha antitumor immunotherapeutic properties by targeted delivery to aminopeptidase N (CD13). Nat Biotechnol 2000, 18 (11), 1185-90. 18. Borsi, L.; Balza, E.; Carnemolla, B.; Sassi, F.; Castellani, P.; Berndt, A.; Kosmehl, H.; Biro, A.; Siri, A.; Orecchia, P.; Grassi, J.; Neri, D.; Zardi, L., Selective targeted delivery of TNFalpha to tumor blood vessels. Blood 2003, 102 (13), 4384-92. 19. Bauer, S.; Adrian, N.; Williamson, B.; Panousis, C.; Fadle, N.; Smerd, J.; Fettah, I.; Scott, A. M.; Pfreundschuh, M.; Renner, C., Targeted bioactivity of membrane-anchored TNF by an antibody-derived TNF fusion protein. J Immunol 2004, 172 (6), 3930-9. 20. Lyu, M. A.; Rosenblum, M. G., The immunocytokine scFv23/TNF sensitizes HER2/neu-overexpressing SKBR-3 cells to tumor necrosis factor (TNF) via up-regulation of TNF receptor-1. Mol Cancer Ther 2005, 4 (8), 1205-13. 21. Bauer, S.; Oosterwijk-Wakka, J. C.; Adrian, N.; Oosterwijk, E.; Fischer, E.; Wuest, T.; Stenner, F.; Perani, A.; Cohen, L.; Knuth, A.; Divgi, C.; Jager, D.; Scott, A. M.; Ritter, G.; Old, L. J.; Renner, C., Targeted therapy of renal cell carcinoma: synergistic activity of cG250TNF and IFNg. Int J Cancer 2009, 125 (1), 115-23.


Page 18 of 20

Page 19 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


22. Tandle, A.; Hanna, E.; Lorang, D.; Hajitou, A.; Moya, C. A.; Pasqualini, R.; Arap, W.; Adem, A.; Starker, E.; Hewitt, S.; Libutti, S. K., Tumor vasculature-targeted delivery of tumor necrosis factor-α*. Cancer 2009, 115 (1), 128-139. 23. Cai, W.; Kerner, Z. J.; Hong, H.; Sun, J., Targeted Cancer Therapy with Tumor Necrosis Factor-Alpha. Biochemistry insights 2008, 15-21. 24. Neri, D.; Bicknell, R., Tumour vascular targeting. Nat Rev Cancer 2005, 5 (6), 436446. 25. Borsi, L.; Balza, E.; Bestagno, M.; Castellani, P.; Carnemolla, B.; Biro, A.; Leprini, A.; Sepulveda, J.; Burrone, O.; Neri, D.; Zardi, L., Selective targeting of tumoral vasculature: comparison of different formats of an antibody (L19) to the ED-B domain of fibronectin. Int J Cancer 2002, 102 (1), 75-85. 26. Menrad, A.; Menssen, H. D., ED-B fibronectin as a target for antibody-based cancer treatments. Expert Opinion on Therapeutic Targets 2005, 9 (3), 491-500. 27. Kaspar, M.; Zardi, L.; Neri, D., Fibronectin as target for tumor therapy. International Journal of Cancer 2006, 118 (6), 1331-1339. 28. Kim, S.; Kim, D.; Jung, H. H.; Lee, I. H.; Kim, J. I.; Suh, J. Y.; Jon, S., Bio-inspired design and potential biomedical applications of a novel class of high-affinity peptides. Angewandte Chemie 2012, 51 (8), 1890-4. 29. Kim, S.; Kim, D.; Lee, Y.; Jeon, H.; Lee, B.-H.; Jon, S., Conversion of Low-Affinity Peptides to High-Affinity Peptide Binders by Using a β-Hairpin Scaffold-Assisted Approach. ChemBioChem 2015, 16 (1), 43-46. 30. Park, J.; Kim, S.; Saw, P. E.; Lee, I. H.; Yu, M. K.; Kim, M.; Lee, K.; Kim, Y. C.; Jeong, Y. Y.; Jon, S., Fibronectin extra domain B-specific aptide conjugated nanoparticles for targeted cancer imaging. J Control Release 2012, 163 (2), 111-8. 31. Saw, P. E.; Kim, S.; Lee, I.-h.; Park, J.; Yu, M.; Lee, J.; Kim, J.-I.; Jon, S., Aptideconjugated liposome targeting tumor-associated fibronectin for glioma therapy. Journal of Materials Chemistry B 2013, 1 (37), 4723. 32. Kim, H.; Lee, Y.; Lee, I. H.; Kim, S.; Kim, D.; Saw, P. E.; Lee, J.; Choi, M.; Kim, Y. C.; Jon, S., Synthesis and therapeutic evaluation of an aptide-docetaxel conjugate targeting tumor-associated fibronectin. J Control Release 2014, 178, 118-24. 33. Yu, T. K.; Shin, S. A.; Kim, E. H.; Kim, S.; Ryu, K. S.; Cheong, H.; Ahn, H. C.; Jon, S.; Suh, J. Y., An Unusual Protein-Protein Interaction through Coupled Unfolding and Binding. Angew Chem Int Edit 2014, 53 (37), 9784-9787. 34. Spitaleri, G.; Berardi, R.; Pierantoni, C.; De Pas, T.; Noberasco, C.; Libbra, C.; Gonzalez-Iglesias, R.; Giovannoni, L.; Tasciotti, A.; Neri, D.; Menssen, H. D.; de Braud, F., Phase I/II study of the tumour-targeting human monoclonal antibody-cytokine fusion protein L19-TNF in patients with advanced solid tumours. J Cancer Res Clin Oncol 2013, 139 (3), 447-55.



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Targeted Cancer Therapy Using Fusion Protein of TNFÎ± and Tumor

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