Immunoglobulin Fc-fused, neuropilin-1-specific peptide shows efficient tumor tissue penetration and inhibits tumor growth via anti-angiogenesis

    Immunoglobulin Fc-fused, neuropilin-1-specific peptide shows efficient tumor tissue penetration and inhibits tumor growth via anti-angiogenesis Ye-Jin Kim, Jeomil Bae, Tae-Hwan Shin, Se Hun Kang, Moonkyoung Jeong, Yunho Han, Ji-Ho Park, Seok-Ki Kim¡ce:sup¿b¡/ce:sup¿ Yong-Sung Kim PII: DOI: Reference:

S0168-3659(15)30062-6 doi: 10.1016/j.jconrel.2015.08.016 COREL 7796

To appear in:

Journal of Controlled Release

Received date: Revised date: Accepted date:

28 March 2015 14 July 2015 6 August 2015

Please cite this article as: Ye-Jin Kim, Jeomil Bae, Tae-Hwan Shin, Se Hun Kang, Moonkyoung Jeong, Yunho Han, Ji-Ho Park, Seok-Ki Kim¡ce:sup¿b¡/ce:sup¿ Yong-Sung Kim, Immunoglobulin Fc-fused, neuropilin-1-specific peptide shows efficient tumor tissue penetration and inhibits tumor growth via anti-angiogenesis, Journal of Controlled Release (2015), doi: 10.1016/j.jconrel.2015.08.016

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Immunoglobulin Fc-fused, neuropilin-1-specific peptide shows efficient tumor tissue penetration and inhibits tumor growth via anti-angiogenesis

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Ye-Jin Kima,d, Jeomil Baea,d, Tae-Hwan Shina, Se Hun Kangb, Moonkyoung Jeongc, Yunho Hanc, Ji-Ho Parkc, Seok-Ki Kimb and Yong-Sung Kima,* a

Department of Molecular Science and Technology, Ajou University, Suwon 443-749, Korea Molecular Imaging & Therapy Branch, National Cancer Center, Goyang 410-769, Korea c Department of Bio and Brain Engineering & Center of Optics for Health Science, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 305-701, Korea

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These authors contributed equally to this work.

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Corresponding author : Yong-Sung Kim, Ph.D., Dept. of Molecular Science and Technology, Ajou University, 206 Worldcup-ro, Yeongtong-gu, Suwon 443-749, Korea Tel: +82-31-219-2662; Fax: +82-31-219-1610; E-mail: [email protected]

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Supplementary information: 7 supplementary figures, 2 supplementary tables, and a supplementary materials and method.

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Abstract Neuropilin-1 (NRP1) receptor, involved in vascular endothelial growth factor (VEGF)mediated vascular permeability and tumor angiogenesis, is targeted by peptides that bind to its VEGF-binding site. However, these peptides also cross-react with the structurally related receptor, NRP2. Here, we describe an immunoglobulin Fc-fused peptide, Fc-TPP11, which specifically binds to the VEGF-binding site of NRP1 with approximately 2 nM affinity, but negligibly to that of NRP2. Fc-TPP11 triggered NRP1-dependent signaling, enhanced vascular permeability via vascular endothelial (VE)-cadherin downregulation, and increased paracellular permeability via E-cadherin downregulation in tumor tissues. Fc-TPP11 also significantly enhanced the tumor penetration of co-injected anti-cancer drug, doxorubicin, leading to the improved in vivo anti-tumor efficacy. Fc-TPP11 was easily adapted to the fulllength anti-epidermal growth factor receptor (EGFR) monoclonal antibody (mAb) cetuximab (Erbitux), cetuximab-TPP11, exhibiting more than 2-fold improved tumor penetration than the parent cetuximab. Fc-TPP11 exhibited a similar whole-body half-life to that of intact Fc in tumor bearing mice. In addition to the tumor-penetrating activity, Fc-TPP11 suppressed VEGF-dependent angiogenesis by blocking VEGF binding to NRP1, thereby inhibiting tumor growth without promoting metastasis in the mouse model. Our results show that NRP1-specific, high-affinity binding of Fc-TPP11, is useful to validate NRP1 signaling, independent of NRP2. Thus, Fc-TPP11 can be used as a tumor penetration-promoting agent with anti-angiogenic activity or directly adapted to mAb-TPP11 format for more potent anticancer antibody therapy.

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Keywords: Antibody therapy; tumor penetration; tumor targeting, Neuropilin-1; Fc-fusion peptide; anti-angiogenesis

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1. Introduction The therapeutic efficacy of anti-cancer drugs such as small-sized chemical agents and antibody therapeutics targeting solid tumors is often limited by poor accumulation in and dispersal throughout the tumor tissues [1-4]. Therefore, it is necessary to develop “promoter agents” that help improve tumor accumulation and penetration of conjugated or co-injected drugs, to increase the therapeutic index of anti-cancer therapy [4, 5]. Neuropilin-1 (NRP1) and its homologue NRP2, expressed on the surface of several normal cell types including endothelial cells, are often overexpressed on malignant tumor cells [6, 7]. NRP1 and 2 have been targeted to develop tumor penetration peptides as promoter agents [8, 9]. Due to the structural similarities of their extracellular domains, NRP1 and NRP2 (NRP1/2) have many ligands in common, such as those belonging to Class III Semaphorin (Sema3) and vascular endothelial growth factor (VEGF) families [7]. NRP1 is an essential receptor for VEGF- and Sema3A-induced vascular permeability, through activation of the NRP1/VEGF receptor 2 (VEGFR2) and NRP1/Plexin A1 receptor complexes, respectively [7, 10]. Further, NRP1/2 modulate epithelial cell-cell junction barrier permeability in response to Sema3 family ligand signaling [11]. Exploiting the NRP-dependent signaling, Ruoslahti and colleagues developed the prototype NRP1/2-targeting iRGD peptide that improved tumor tissue penetration of coadministered drugs including anti-cancer chemical agents and monoclonal antibodies (mAbs) [12, 13]. Our group has previously reported the NRP1/2-targeting peptide A22p; a fusion of this peptide with the carboxy (C)-terminus of a conventional mAb heavy chain (mAb-A22p), 2

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enhanced tumor tissue penetration by loosening the cell junctions between both, endothelial and epithelial cells [14]. In addition to tumor-penetrating peptides, NRP1/2 has also been targeted for anti-cancer peptides because NRP1/2 play essential roles in angiogenesis as well as tumor cell invasion and proliferation, as co-receptors of VEGFRs following stimulation with VEGF isoforms, primarily VEGF165 (hereafter referred to as VEGF, unless otherwise specified) [8, 15]. Some NRP-binding peptides blocked VEGF/NRP1 interactions to inhibit VEGF-mediated angiogenesis, thereby exerting anti-tumor activity [16-18]. NRP-targeting peptides with tumor-penetration and/or anti-angiogenesis activities, such as RPARPAR [13], activated iRGD (RGDR) [12], and A22p [14] peptides, bind to the arginine-binding pocket (or VEGF-binding site) on the b1 domain of NRP1/2 via the Cterminal basic sequence motif (R/K)XX(R/K) where X represents any amino acid [6, 7]. To bind and exert their biological functions, this basic sequence motif at the free C-terminus in these ligands and peptides must be exposed, with a strict requirement for the C-terminal Arg (or rarely Lys) residue [8, 19]; this requirement is referred to as the “C-end rule” (CendR) [12, 13]. Like most VEGF and Sema3 ligands [7], however, these CendR peptides [14, 20] and other NRP1-binding peptides [17, 21] simultaneously bind to both, NRP1 and NRP2 because the basic arginine-binding pocket of NRP1/2 is highly conserved. NRPs are differentially expressed, with NRP1 found primarily in arterial endothelial cells, whereas NRP2 expression is localized to venous and lymphatic endothelium [7]. Further, both, NRP1 and NRP2 are often overexpressed in various tumor cells, with NRP1 more preferentially overexpressed than NRP2, and therefore, each is implicated in different aspects of tumor pathogenesis [6, 7]. For example, anti-NRP1 antibodies reduced in vivo tumor growth [22], whereas anti-NRP2 antibodies had no effect on primary tumor growth, instead they inhibited tumor-associated lymphangiogenesis and lung metastasis [23]. In this context, the peptides specifically binding to the VEGF-binding pocket of NRP1-b1 domain, but not NRP2-b1 domain, might be valuable to specifically exploit NRP1-mediated signaling, ruling out any possible undesirable effects mediated by NRP2 engagement. Here, we describe a NRP1-specific peptide, TPP11, which selectively binds to the VEGF-binding site of the NRP1-b1 domain, but not to that of NRP2-b1 domain. We demonstrate its potential application as TPP11 fused to the C-terminus of immunoglobulin Fc (Fc-TPP11), as a potent, tumor-penetrating molecule. We found that Fc-TPP11 exhibited NRP1-mediated tumor homing and tissue penetration in mice bearing human tumor xenografts. Fc-TPP11 also inhibited VEGF-mediated angiogenesis by blocking VEGF binding to NRP1. Furthermore, TPP11-fusion to the anti-EGFR mAb cetuximab (Erbitux®), cetuximab-TPP11, exhibited enhanced tumor accumulation. Here, we present the Fc-fused, NRP1-specific peptide, Fc-TPP11, and its detailed mechanism of increasing tumor accumulation and penetration as well as anti-angiogenesis activity.

2. Materials and methods 2.1. Fc-peptide library construction and screening. The gene encoding the Fc region of human IgG1, including the hinge-CH2-CH3 regions (residues 225–447 in EU number), a 15residue (G4S)3 linker, and the A22p peptide were sub-cloned in-frame with the C-terminus of Aga2 in the yeast surface-display plasmid pCTCON [24]. The yeast surface-displayed Fcfusion peptide library was constructed by randomizing the C-terminal, 18 residues of A22p 3

ACCEPTED MANUSCRIPT peptide with a degenerate NHB (ATGC/ACT/TCG) codon, as described in Fig. 1A and the Supplementary methods. The library transformation and library screening against biotinylated proteins were performed as previously described [24].

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2.2. Construction, expression and purification of Fc-peptides and antibodies. DNA fragment encoding the CH3-(G4S)3-peptide region was sub-cloned into BsrGI/HindIII sites of the modified pcDNA3.4 vector [14] for the expression of Fc-fused peptides or cetuximab heavy chain (VH to CH3) for the expression of cetuximab-TPP11. All plasmids were confirmed by sequencing. Fc-peptides, cetuximab, and cetuximab-TPP11 were produced and purified as previously described [14, 25].

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2.3. Xenograft tumor models. All animal experiments were approved by the Animal and Ethics Review Committee of Ajou University and performed in accordance with the guidelines established by the Institutional Animal Care and Use Committee [14]. For the tumor models, 4-week-old, female, BALB/c, athymic, nude mice (NARA bio, Korea) weighing 15–20 g were used. For the tumor growth inhibition studies, FaDu cells (5 × 106 cells/mouse) were inoculated subcutaneously (SC) in the right thigh. When the mean tumor volume reached ~ 50 – 60 mm3 (after 3–5 days of growth), mice were randomized into groups, and agents (Fc proteins, antibodies or doxorubicin (DOX) (Sigma-Aldrich)), as specified in the Figure legend, were administered intravenously (IV) via the tail vein, in a dose/weight-matched fashion. Tumor volume (V) was estimated by the formula V = L × W2/2, where L and W are the long and short lengths of the tumor, respectively [24].

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2.4. Immunofluorescence microscopy of tumor tissues. A single dose of the Fc or Fcpeptides (10 mg/kg) or antibodies (1.25 mg/kg) was injected IV into mice bearing A431 tumor xenografts with an average diameter of ~ 7–8 mm [14]. Tumor tissues were harvested 15 h post-injection. Tumor tissue cryosectioning and immunofluorescence staining were performed as previously described [14, 26]. Briefly, the extracted tumors were fixed in 4% paraformaldehyde overnight at 4°C, cryoprotected in 30% sucrose for 24 h, and then frozen in OCT embedding medium (Tissue-Tek). For immunofluorescence staining, 10-µm thick cryosections were prepared and incubated with blocking solution (2% bovine serum albumin [BSA] in phosphate-buffered saline [PBS]) for 1 h at 25°C. Tissue sections were stained with fluorescein isothiocyanate (FITC)-conjugated, anti-human IgG for 1.5 h at 25°C in the dark, to detect Fc proteins, and then washed 3 times with PBST (PBS containing 0.1% Tween20) for 10 min. The following procedure was followed for vessel and pericyte staining of tumor tissues in FaDu-xenograft mice: after incubation in blocking solution for 1 h at 25°C, tissue sections were labeled with rat anti-mouse CD31 mAb, rabbit anti-human/mouse/rat NRP1 (Abcam), or mouse anti-α-smooth muscle actin (α-SMA) mAb (Sigma Aldrich) at 4°C overnight, washed 3 times with PBST, and then stained with a goat anti-rat TRITCconjugated antibody (Millipore), goat anti-rabbit rhodamine isothiocyanate (TRITC)conjugated antibody (Sigma Aldrich), or a goat anti-mouse FITC-conjugated antibody (Sigma Aldrich) at 25°C for 1.5 h. Tissue sections were then washed 3 times with PBST and then mounted on slides with VECTASHIELD mounting medium, stained with DAPI, and examined under a Zeiss LSM710 confocal microscope with ZEN software (Carl Zeiss). Areas positively stained for Fc-peptides and antibodies in the acquired fluorescence images of each tissue were quantified using ImageJ. 4

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2.5. Ex vivo tumor penetration assay. The assay was performed as previously described [14]. Briefly, subcutaneous FaDu tumor xenografts (approximately 250 mm3) were excised from the mice and incubated with PBS, Fc, or Fc-TPP11 (200 μg/mL) in minimal essential media with 1% BSA for 2.5 h at 37°C. After extensive washing with PBS, the tumors were fixed in PBS containing 4% paraformaldehyde and sectioned for immunostaining to detect Fc proteins with a FITC-conjugated anti-human IgG Fc antibody. To quantify the penetration depth of Fc-TPP11, the plot profile of fluorescence intensity versus distance from tumor edge to center was generated by Image J software. Penetration depth was defined as the distance at which fluorescence intensity drops to <5% of the maximum intensity at tumor edge [27].

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2.6. In vitro transendothelial permeability assay. Permeability across endothelial cell monolayers was assessed using human umbilical vein endothelial cells (HUVECs) as previously described [14]. Briefly, HUVECs or siRNA-transfected HUVECs (1 × 105 cells/well) were seeded into a 12-well Transwell chamber (0.4-μm pore size; Corning Costar) and incubated for 3 days to form mature monolayers. After 4 h of serum starvation, proteins (VEGF [1.3 nM ≈ 50 ng/mL], Fc [1 μM], Fc-A22p [1 μM], or Fc-peptides [100 nM]) were added to the upper chamber. After 30 min of incubation, 50 μg FITC-conjugated dextran (approximately 40 kDa, 1 mg/mL; Sigma) was added to the upper chamber. After 30 min, the fluorescence of samples from the bottom chamber was measured in triplicate using a fluorescence plate reader (Molecular Devices) and the data were normalized to the PBStreated samples.

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2.7. Quantification of DOX in tumors. Female, BALB/c, athymic, nude mice bearing preestablished FaDu tumor (~ 1 cm in diameter) were injected IV with PBS, Fc, or Fc-TPP11 (2.5 mg/kg), combined with 10 mg/kg free DOX. After 1 h of circulation, the mice were perfused through the heart with PBS containing 1% BSA, and tumors were collected (n = 3 per group). For immunohistochemistry, tumor tissue cryosection and immunofluorescence staining were performed as previously described [14]. DOX in tumor tissues was extracted as previously described [12]. Briefly, the tumors were homogenized in 1% sodium dodecyl sulfate and 1 mM H2SO4 in water. DOX was then extracted by adding 2 ml chloroform/isopropyl alcohol (1:1, v/v) to the homogenized samples, followed by vortex mixing and freeze/thaw cycles. The DOX-containing organic phase was separated by centrifugation at 3000 ×g for 10 min at room temperature. DOX concentration in the organic phase was measured by fluorescence using an excitation wavelength of 485 nm and an emission wavelength of 528 nm and then quantified by fitting the fluorescence from each sample to a free DOX standard curve after subtracting autofluorescence from PBS-treated controls [28]. 2.8. Pharmacokinetics. We determined pharmacokinetic profiles of Fc or Fc-TPP11 by measuring whole-body radioactivity using single-photon emission computed tomography (SPECT) [29, 30]. Fc and Fc-TPP11 were labeled with 125I using the chloramine-T method, as described previously [29, 30]. Six-week-old female BALB/c athymic nude mice were fed Lugol’s solution for 2 weeks to block thyroid uptake before protein injection and injected subcutaneously with FaDu cells (5 × 106 cells/mouse) in the right thigh. The FaDu tumor xenografts were allowed to grow until they reached ~400 mm3 in volume, and then the mice 5

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were injected IV with 18.5 MBq (500 μCi/60 μg per mouse) of 125I-labeled protein in PBS via the tail vein. Whole-body radioactivity was measured at 0, 0.5, 1, 2, 4, 6, 12, 24, 48, 72, and 96 h after injection with SPECT (NanoSPECT, Bioscan) [29]. The data were reported as the mean percentage of remaining whole-body radioactivity, with the initial radioactivity at injection set at 100%. Pharmacokinetic parameters were calculated by two-phasic nonlinear regression analysis using GraphPad PRISM.

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2.9. Matrigel plug assay. Subconfluent A431 cells were harvested, washed twice, and resuspended in PBS at 7.5 × 107 cells/mL [31]. Aliquots of the cells (0.1 mL) were mixed with 0.4 mL of growth factor-reduced Matrigel (BD Biosciences, 356231) with or without Fc or Fc-TPP11 (10 mg/kg), and the mixture was immediately injected SC, into the right thigh of nude mice. The mice were sacrificed at Day 9 after implantation and the Matrigel plugs were removed. Preparation of the Matrigel plug sections and immunofluorescence staining of the cryosections were carried out as described above. All vessels within the Matrigel plugs were photographed and the area that stained positive for vessels was quantified using ImageJ.

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2.10. Tumor metastasis model. 4T1 murine breast cancer cells (1 × 106) were orthotopically implanted in the fourth mammary fat pad of 7-week-old female BALB/c mice [32]. Once the 4T1 tumors reached ~ 50 mm3 in volume, the mice were injected IV with PBS, Fc, or FcTPP11 (20 mg/kg) every 3 days for 21 days. Each therapeutic cohort included 5–6 mice. Primary tumor volumes and mouse body weights were measured as described above. To assess lung metastasis, the lungs were excised at 21 days post-treatment. For India-ink staining, mice from each therapeutic cohort were sacrificed immediately after the India ink was injected through the trachea. The lungs were then excised, washed once in PBS, and fixed in Fekete’s solution (100 mL 70% alcohol, 10 mL formalin, and 5 mL glacial acetic acid) at 4°C for 3 days. Tumor metastasis sites subsequently appeared as white nodules on the surface of black lungs and were visually counted. To quantify metastasis nodules and weights, the lungs were excised, washed once in PBS, weighed, and then the nodules on the surface of the lungs were visually counted. For hematoxylin and eosin (H&E) staining, the lungs were fixed overnight in 4% formaldehyde, dehydrated with 30% sucrose for 12 h, and frozen in OCT compound. The lung tissues were sliced into 10-µm thick sections with a Cryostat (Leica), and stained with H&E following the standard protocol. All lung sections were examined using an optical microscope (Nikon). All animal studies were approved by the Korea Advanced Institute of Science and Technology (KAIST) Committee on Animal Care. 2.11. Statistical analysis. Data represent the mean ± SD of at least 3 independent experiments performed in triplicate, unless otherwise specified. Comparisons of data from tests and controls were analyzed for statistical significance by a 2-tailed, unpaired Student’s t-test using Excel (Microsoft). A P-value less than 0.05 was considered statistically significant. 2.12. Supplementary materials and methods. Details of reagents; cell lines; Fc-peptide library construction; screening of the Fc-peptide library against NRP1-b1b2; construction, expression and purification of Fc-peptides and antibodies; western blotting and immunoprecipitation; quantification of Western blotting analysis; ELISA; competitive binding assay; surface plasmon resonance (SPR); RNA interference; immunofluorescence

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ACCEPTED MANUSCRIPT microscopy of cells; wound healing assay; in vitro Transwell invasion assay are detailed in the Supplementary Materials and methods.

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3. Results

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3.1. Generation of NRP1-specific, Fc-fused peptides The crystal structures of the NRP1-b1 domain, complexed with VEGF showed that the C-terminal CendR motif of –KPRR and the upstream ~ 10 residues of VEGF specifically interact with residues in and around the arginine-binding pocket of the NRP1-b1 domain [33]. We reasoned that peptides with approximately 14–18 residues can specifically discern the arginine-binding pocket between NRP1 and NRP2. Following this rationale, we constructed a yeast surface-displayed, Fc-fused peptide library in which the C-terminal 18 residues of A22p were randomized with a degenerate codon based on the A22p template sequence (Fig. 1A). The A22p peptide contains 22 residues derived from the C-terminal basic region of Sema3A (Fig. 1B) and binds to both, NRP1 and NRP2 [14]. Screening using the Fc-peptide fusion format was intended to generate molecules with bivalent NRP1-engaging ability, similar to the homodimeric VEGF and Sema3A ligands, to mimic their biological activity and for direct extension to an mAb-peptide fusion format [14]. Screening of the yeast library with a biotinylated NRP1-b1b2 protein was followed by a round of magnetic-activated cell sorting (MACS) and 3 rounds of fluorescence-activated cell sorting (FACS) in the presence of NRP2-b1b2 as a competitor to deplete NRP2-b1b2 binders. We isolated 3 unique high-affinity NRP1 binders, designated as Fc-TPP1 (18 residues), Fc-TPP8 (11 residues), and Fc-TPP11 (14 residues) (Fig. S1A and B). Surprisingly, all 3 high-affinity peptides were shorter than the parent A22p (Fig. 1B), which can be attributed to the use of NHB degenerated codon with 2.98% stop codon frequency and/or to the synthesis error of the oligonucleotides used for library construction. Nonetheless, the Cterminal sequence of the peptides complied with the CendR sequence motif of (R/K)XX(R/K), though TPP11 showed a slightly deviated C-terminal sequence of –TPRR, similar to Tuftsin (TKPR) [34]. The isolated peptides were expressed as Fc-fusion homodimers in HEK293F mammalian cell cultures (Fig. 1B) and purified in the correctly assembled form, without nonnative oligomers (Fig. S1C and D), with purification yield of approximately 32–38 mg/L culture, comparable to that of Fc alone (Table S1). Although the parent Fc-A22p binds to both, NRP1 and NRP2 with similar affinity (dissociation constant (KD) = ~ 62 nM), the newly isolated Fc-peptides selectively bound to NRP1, showing a higher affinity from approximately 65-fold to 1,000-fold to NRP1 (KD ≈ ~ 1.7–4 nM) than to NRP2 (Fig. 1C, Table S2). All 3 Fc-peptides co-localized with cell-surface-expressed NRP1 on HUVECs at 4°C and were internalized with NRP1 at 37°C (Fig. S2A), suggesting that the isolated Fcpeptides specifically bind to NRP1 to trigger NRP1-mediated endocytosis [14, 35]. In our biological activity evaluation of the 3 Fc-peptides, Fc-TPP11 exhibited the highest NRP1associated activities including vascular permeability (Fig. 1D), VE-cadherin downregulation in endothelial HUVECs, and E-cadherin downregulation in epithelial FaDu cells (Fig. S2B and C). Fc-TPP11 exhibited higher biological activities than Fc-A22p, even at 10-fold lower concentrations. Knockdown of NRP1 by siRNA treatment completely blocked Fc-TPP11induced vascular permeability (Fig. 1D), demonstrating that this activity is NRP1-dependent. Therefore, we chose Fc-TPP11 for further analysis. 7

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Fig. 1. Generation and characterization of NRP1-specific Fc-peptides with tumor penetration activity. A. Schematic diagram of the yeast surface-displayed Fc-fused peptide library. The Cterminal 18 residues of the peptide library were randomized with the degenerate codon of NHB (ATGC/ACT/TCG) (indicated as X) based on the backbone of the 22-residue A22p sequence [14]. The peptide library was constructed in the Fc-fusion format by linking it to the C-terminus of human IgG1 Fc (residues 225–447, EU number) via a 15-residue (G4S)3 linker. B. Schematic diagram of the purified Fc-fusion peptides Fc-TPP1, Fc-TPP8, and Fc-TPP11 isolated from HEK293F cultures. The sequences of isolated peptides, denoted as TPP1, TPP8, and TPP11 are shown, for comparison to the parent A22p peptide. C. Direct enzyme-linked immune sorbent assay (ELISA) to determine the binding specificity of Fc (10 nM) and Fc-peptides (10 nM) to plate-coated NRP1-b1b2 and NRP2b1b2. D. Permeability across an HUVEC or siRNA-transfected HUVEC monolayer was assessed by FITC-dextran passage after the cells were stimulated for 30 min with VEGF (1.3 nM ≈ 50 ng/mL), Fc (1 μM), Fc-A22p (1 μM), or Fc-peptides (100 nM). Data shown represent the mean ± SD of the foldincrease in permeability, compared to that in PBS-treated cells. *P < 0.05, **P < 0.01, ***P < 0.001 vs. Fc-stimulated cells. E. Competitive ELISA of CendR RPARPAR peptide (10 μM) or VEGF (5 nM) for binding to plate-coated NRP1-b1b2 protein in the presence of increasing concentrations of the synthesized TPP11 peptide. F and G. Immunofluorescence staining images showing the distribution of intravenously injected Fc, Fc-A22p, or Fc-TPP11 (10 mg/kg, ~ 200 μg/mouse) in relation to blood vessels (F) and co-localized with NRP1 (G) within the tumor tissue in A431 xenograft mice. After 15 h of circulation, tumor tissues were excised and stained for human Fc (FITC, green) and CD31 8

ACCEPTED MANUSCRIPT (TRITC, red) (F) or human/mouse NRP1 (TRITC, red) (G). Blue represents DAPI staining. The areas in the white boxes were magnified for better visualization. Image magnification, 200×; scale bar, 100 μm. In F, the bar graph shows the quantification of the Fc-positive area (green), as analyzed by ImageJ software. Error bars, show the SD of 4 fields per tumor (n = 3 per group). *P < 0.05 vs. Fc.

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To evaluate the binding specificity of the TPP11 peptide without an N-terminal Fc fragment, the TPP11 peptide was synthesized. The TPP11 peptide alone also bound to NRP1, but with much lower affinity than Fc-TPP11 (Fig. S3). However, it did not cross-react with NRP2 or VEGFR2. Furthermore, the synthesized TPP11 peptide efficiently competed with VEGF and a CendR peptide (RPARPAR) [13] for binding to NRP1 (Fig. 1E), demonstrating that the TPP11 peptide specifically binds to the VEGF-binding site in the NRP1-b1 domain.

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3.2. Fc-TPP11 homes onto tumor vessels and extravasates into tumor tissues by downregulating NRP1-mediated VE-cadherin The tumor homing and penetration activities of NRP1-selective Fc-TPP11 was compared with those of Fc-A22p, by injecting IV into mice bearing pre-established human head and neck FaDu tumors, which express both, NRP1 and NRP2 [14]. After 15 h of circulation, tumors were excised and subjected to immunofluorescence analysis. About 3-fold and 2-fold higher Fc-TPP11 fluorescence was detected on and around the CD31-stained tumor blood vessels than in animals injected with intact Fc and Fc-A22p, respectively (Fig. 1F), indicating that Fc-TPP11 homed onto tumor vessels and penetrated into the extravascular tumor parenchyma more efficiently than Fc-A22p. Fc-TPP11 showed a high degree of co-localization with NRP1 throughout the tumor tissues (Fig. 1G), indicating that the improved tumor homing and penetration is mediated by NRP1 targeting, similar to that observed with the CendR peptides [12-14]. The co-localization of Fc-TPP11 with NRP1 on mouse blood vessel also suggests that TPP11 peptide cross-reacts with mouse NRP1, which was expected, because mouse and human NRP1 share high sequence homology (93%), with 100% sequence conservation of the arginine-binding pocket in the b1 domain [20, 36]. NRP1 functions as a co-receptor with VEGFR2 or Plexin A1 in VEGF- or Sema3Amediated vascular permeability, respectively, by downregulating VE-cadherin, a main adherent junction protein that acts as an endothelial barrier [37]. To understand the molecular mechanism(s) underlying Fc-TPP11-triggered vascular permeability, we investigated the molecules involved in the Fc-TPP11-mediated downregulation of VE-cadherin. NRP1knockdown by siRNA completely blocked the VE-cadherin downregulation induced by FcTPP11 or VEGF (Fig. 2A, Fig. S4). However, knockdown of VEGFR2 and Plexin A1 negligibly affected Fc-TPP11-induced VE-cadherin downregulation. Knockdown of VEGFR2, but not Plexin A1 or NRP2, abolished VEGF-mediated VE-cadherin downregulation, which is consistent with previous studies [10, 37]. We also assessed the involvement of GIPC1/synectin, a cytosolic protein that binds to the cytoplasmic PSD95/Dlg/ZO-1 (PDZ) domain-binding motif of NRP1 (C-terminal SEA sequence) upon VEGF activation to transduce downstream signaling [38]. Contrary to our expectation, GIPC1/synectin-knockdown did not affect Fc-TPP11-mediated VE-cadherin downregulation, whereas it did abolish the effect of VEGF (Fig. 2A). We then analyzed the molecular components that interact with Fc-TPP11 by immunoprecipitating Fc-TPP11 from the cell lysates of Fc-TPP11-treated HUVECs. NRP1, but not NRP2, VEGFR2, or Plexin A1, coimmunoprecipitated with Fc-TPP11 (Fig. 2B). Unlike with VEGF, Fc-TPP11 did not activate VEGFR2 in HUVECs (Fig. 2C). These results imply that, unlike VEGF, Fc-TPP11 9

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downregulates VE-cadherin levels in endothelial cells to increase vascular permeability by engaging NRP1, but not VEGFR2.

Fig. 2. Fc-TPP11 enhances vascular permeability in endothelial cells and paracellular permeability in tumor tissues. A. Western blot analysis of VE-cadherin levels in HUVECs that were transiently transfected with the indicated siRNAs (scrambled siRNA was used as control) for 24 h, serum-starved for 4 h, and then treated with basal medium (control), VEGF (2.6 nM), Fc (100 nM), or Fc-TPP11 (100 nM) for 10 min. B. Western blot analysis of an anti-human Fc antibody immunoprecipitation (IP) of cell lysates from HUVECs treated for 10 min with medium (control), Fc (50 nM), or Fc-TPP11 (50 nM). Equal precipitates were analyzed and β-actin was used as a loading control. C. Western blots showing VEGFR2 activation levels in the lysates of HUVECs that were serum-starved for 4 h and then treated with medium (control), VEGF (2.6 nM), Fc (100 nM), or FcTPP11 (100 nM) for 10 or 30 min. D. Western blots showing E-cadherin levels in FaDu and A431 cells that were transiently transfected with an NRP1 siRNA or a scrambled siRNA (control) for 30 h and then treated with basal medium (control), Fc (100 nM), or Fc-TPP11 (100 nM) for 10 min. E. Ex vivo tumor penetration assays of Fc-TPP11. Subcutaneous FaDu tumor xenografts were excised and incubated with PBS, Fc or Fc-TPP11 (200 μg/mL ≈ 4 μM) for 2.5 h at 37°C. The sections were stained for Fc proteins (FITC, green) and with DAPI (blue). Image magnification, 200×; scale bar, 10

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100 μm. The bar graph shows the quantification of the Fc-positive area (green), as analyzed by ImageJ software. Error bars, ± SD of 4 fields per tumor (n = 3 per group). ***P < 0.001. Fluorescence line profile from tumor surface to the center was analyzed to determine the penetration depth of FcTPP11 by Image J software (3 fields per tumor, n = 3 tumors per group). Representative three profiles of Fc-TPP11 were shown, comparing with the controls. To prevent the overlap of line profiles, the data of Fc-TPP11-1 and Fc-TPP11-2 moved up by 60 and 30 unit in Y axis, respectively. F. Western blot analysis of ex vivo tumor tissue lysates (n = 2) prepared in F to analyze the amount of E-cadherin. In (A, C, D, F), the number below the panel indicates relative value of band intensity of proteins compared to the band intensity in ‘control’ after normalization of the band intensity to that of β-actin for each sample. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control.

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3.3. Fc-TPP11 penetrates ex vivo tumor tissues through NRP1-mediated E-cadherin downregulation E-cadherin is a main adherent junction protein in epithelial cells that limits paracellular permeability in tissues [11]. Fc-TPP11 efficiently downregulated E-cadherin in FaDu and A431 cells in an NRP1-dependent manner (Fig. 2D). In an ex vivo tumor penetration assay, which can exclude tissue penetration through blood vessels, within 2 h, FcTPP11 actively penetrated multiple cell layers deep into excised FaDu tumor tissues, showing the penetration depth of 201 ± 29 μm (Fig. 2E). The tumor tissues treated with Fc-TPP11 exhibited significantly decreased E-cadherin levels compared with those treated with Fc (Fig. 2F). These results indicated that Fc-TPP11 increases paracellular tissue permeability by opening epithelial junctions via downregulation of E-cadherin.

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3.4. Fc-TPP11 enhances anti-tumor effect of co-injected drug by promoting the tumor tissue penetration The ability of Fc-TPP11 to induce tumor vascular permeability and paracellular tissue permeability was further assessed in vivo by the tumor distribution of anti-cancer drug, doxorubicin (DOX) co-injected IV with Fc-TPP11 into mice bearing FaDu tumors. Coadministration of Fc-TPP11 (2.5 mg/kg) resulted in a wider and deeper penetration of DOX into extravascular tumor tissue (Fig. 3A) with ~ 2.3-fold higher tumor accumulation of DOX, as compared to that with DOX injected alone or co-injected with Fc (Fig. 3B). Combined treatment of DOX with Fc-TPP11 (2.5 mg/kg) more substantially retarded tumor growth of FaDu xenografts (Fig. 3C), resulting in ~ 2-fold reduction in tumor weight at the end of treatment (Fig. S5), compared with tumors treated with DOX alone. However, treatment of Fc-TPP11 alone did not exhibit any anti-tumor activity (Fig. 3C), indicating that the enhanced anti-tumor activity of the combined therapy resulted from the improved tumor tissue penetration of DOX by co-injection of Fc-TPP11. 3.5. Cetuximab-TPP11 penetrates into tumor tissues more significantly than cetuximab To expand the Fc-TPP11 fusion to a full-length mAb fusion, the TPP11 peptide was fused to the C-terminus of the heavy chain of anti-EGFR mAb, cetuximab, generating cetuximab-TPP11 (Fig. 3D). Cetuximab-TPP11 was expressed well in the correctlyassembled form in HEK293F cultures, showing production levels similar to that of the parent mAb cetuximab (Table S1, Fig. S6). We compared the tumor homing and tissue penetration of cetuximab-TPP11 to that of cetuximab by administering a single IV injection into mice harboring EGFR-overexpressing A431 tumor xenografts and then assessing the immunofluorescent staining of tumor sections after 3 h of circulation. Cetuximab-TPP11 11

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exhibited a greater extent of tumor homing and more distant spread from the blood vessels (~ 2-fold farther) than cetuximab (Fig. 3E).

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Fig. 3. Enhanced anti-tumor efficacy of free DOX co-injected with Fc-TPP11 (A-C) and increased tumor tissue penetration of TPP11-fused mAb, cetuximab-TPP11 (D, E). A. Representative images of the distribution of free DOX (10 mg/kg, ~ 200 μg/mouse) co-injected IV with PBS, Fc or Fc-TPP11 (2.5 mg/kg, ~ 50 μg/mouse) in FaDu tumor xenograft mice (n = 3 per group). After 1 h of circulation, tissues were excised and stained for blood vessels with an anti-CD31 (green) and the native fluorescence (red) was used to detect DOX. Image magnification, 200×; scale bar, 100 μm. B. DOX concentration in the tumor tissues was quantified using free DOX standard curve. Error bars, ± SD (n = 4 per group). **P < 0.01. C. Tumor growth was analyzed by measuring tumor volume during the indicated treatments in FaDu tumor bearing mice. After pre-establishing FaDu tumors with a volume of ~ 50 mm3, mice (n = 7 per group) were injected IV every 2 days with PBS, DOX (3 mg/kg, ~ 60 μg/mouse), Fc-TPP11 (2.5 mg/kg, ~ 50 μg/mouse), or DOX (3 mg/kg) plus Fc-TPP11 (2.5 mg/kg) (black arrowheads). *P < 0.05. D. Schematic diagram of cetuximab-TPP11, in which the TPP11 peptide was fused via a 15-residue (G4S)3 linker to the C-terminus of the Fc heavy chain. E. Representative images of the distribution of IV injected cetuximab or cetuximab-TPP11 (1.25 mg/kg) in relation to blood vessels in A431 xenograft mice. After 3 h of circulation, tissues were excised and subjected to immunofluorescence staining to visualize antibodies (anti-human Fc, green) and blood vessels (anti-CD31, red). Blue represents DAPI staining. Image magnification, 200×; scale bar, 100 μm. The bar graph shows the quantification of the antibody-positive areas (green), as assessed by ImageJ software. Error bars, ± SD of 4 fields per tumor (n = 2 per group).

3.6. Pharmacokinetics of Fc-TPP11 We determined the effects of TPP11 fusion on the pharmacokinetic profiles of the parent Fc fragment. Whole-body radioactivity monitoring of a single intravenously injected dose of 125I-Fc and 125I-Fc-TPP11 in BALB/c mice with FaDu tumors revealed the typical biphasic clearance profiles of Fc proteins: an initial rapid clearance phase and a later slow serum clearance phase determined by interactions with neonatal Fc receptor (FcRn) in serum [39]. During the initial phase, 125I-Fc-TPP11 showed slightly faster whole body clearance rate (T1/2α = 9.0 ± 0.9 h) than 125I-Fc (T1/2α = 11.1 ± 1.4 h) (Fig. 4A), most likely due to 12

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additional degradation after NRP1-mediated intracellular internalization, as has been observed for a human anti-NRP1 mAb [40]. However, in the serum elimination phase, 125IFc-TPP11 showed a prolonged half-life (T1/2β = 66.3 ± 14.5 h) comparable to that of the parent 125I-Fc (T1/2β = 56.4 ± 15.5 h) (Fig. 4A), suggesting that the TPP11 fusion does not compromise the serum half-life of the parent Fc. The above pharmacokinetic parameters were in good agreement with those (T1/2α = 9.1 ± 0.3 h, T1/2β = 62.2 ± 6.0 h) of human Fc in mice, determined by blood sampling [39]. The systemic tissue distribution and tumor uptake of 125IFc-TPP11 were also evaluated by whole-body SPECT images obtained from the pharmacokinetic studies (Fig. 4A). At 4 h post-injection, tumor uptake of 125I-Fc-TPP11 was slightly higher than that of 125I-Fc and the enhanced tumor accumulation of 125I-Fc-TPP11 was maintained until 72 h, without notable preferential accumulation in normal organs and tissues, as compared to that of 125I-Fc (Fig. 4B). This result indicated the ability of the NRP1targeting TPP11 peptide to enhance the tumor homing and accumulation of Fc-TPP11.

Fig. 4. Pharmacokinetic profiles and SPECT images of Fc-TPP11. A. Pharmacokinetic profiles of 125 I-Fc and 125I-Fc-TPP11 (500 μCi/60 μg per mouse) were examined by measuring whole-body radioactivity after IV injection into mice bearing FaDu tumors (n = 4 per group). B. Representative whole-body SPECT images of FaDu tumor xenograft-bearing mice at 0, 4, 12, 24, and 72 h after injection of 125I-Fc or 125I-Fc-TPP11, as described in (A). The location of the tumor is indicated by the dashed white circles.

3.7. Fc-TPP11 suppresses VEGF-mediated angiogenesis as well as the migration and invasion of endothelial cells by inhibiting formation of the VEGF/NRP1/VEGFR2 complex VEGF binds to NRP1 and VEGFR2 to form the VEGF/NRP1/VEGFR2 complex, which is critical for VEGF signaling-mediated functions, such as endothelial cell migration and angiogenesis [41]. Like the synthesized TPP11 peptide (Fig. 1E), Fc-TPP11 efficiently competed with VEGF for NRP1 binding (Fig. 5A). Accordingly, we reasoned that Fc-TPP11 13

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could interfere with VEGF-mediated signaling by competing with VEGF for NRP1 binding. Indeed, Fc-TPP11 significantly inhibited VEGF-induced tube formation of HUVECs (Fig. S7A) and dramatically suppressed blood vessel formation in an in vivo Matrigel plug assay using VEGF-secreting A431 carcinoma cells (Fig. 5B). Fc-TPP11 also inhibited VEGFmediated migration of HUVECs, as assessed by a wound healing assay (Fig. 5C). Further, VEGF-induced invasion of HUVECs was also significantly inhibited in the presence of FcTPP11 in a Matrigel-coated Transwell assay (Fig. 5D). In contrast, Fc-TPP11 alone did not promote wound healing and invasion of HUVECs (Fig. S7B and C). Next, we assessed the effect of Fc-TPP11 on VEGF-stimulated activation of VEGFR2 and the downstream signaling molecules associated with cell motility and angiogenesis, including Src, FAK, and p38, in HUVECs [41, 42]. Compared with Fc, FcTPP11 remarkably reduced the VEGF-induced activation of VEGFR2 and the downstream signaling molecules Src, FAK, and p38 (Fig. 5E). We also tested the effect of Fc-TPP11 on VEGF/NRP1/VEGFR2 complex formation. As previously reported [22], NRP1 coimmunoprecipitated with VEGFR2 from HUVEC lysates following VEGF stimulation (Fig. 5F). This interaction was reduced in the presence of Fc-TPP11, suggesting that Fc-TPP11 inhibits the formation of the VEGF/NRP1/VEGFR2 complex, thereby inhibiting VEGFmediated signaling.

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3.8. Fc-TPP11 shows in vivo anti-tumor efficacy through its anti-angiogenesis activity The anti-angiogenesis activity of Fc-TPP11 prompted us to determine whether FcTPP11 alone can exert anti-tumor effects in mice bearing FaDu carcinoma xenografts. Since the relatively low dosage (2.5 mg/kg) of Fc-TPP11 did not show any anti-tumor activity, we injected Fc-TPP11 at the higher dosage (20 mg/kg). Indeed, systemic administration of FcTPP11 significantly suppressed the growth of FaDu xenografts, reducing the average tumor weight to less than 48% of that in the PBS- or Fc-treated controls at the end of the treatment period (Fig. 6A). The body weight of mice treated with Fc-TPP11 did not differ significantly from that of the controls during the treatment period (Fig. 6B). Tumors were excised at the end of treatment, and the tissue sections were stained with anti-CD31 and anti-αSMA antibodies to assess blood vessel formation and pericyte association, respectively [22]. PBSand Fc-treated tumors exhibited unorganized, thick vessels in very close association with pericytes (Fig. 6C). In contrast, Fc-TPP11-treated tumors showed more than a 2-fold decrease in vascular density and poor association of pericytes with vessels as compared to the controls, shown by the vascular density quantification and the pericyte/vessel ratios, respectively (Fig. 6C). Western blots of the tumor tissue lysates revealed markedly reduced levels of phosphorylated VEGFR2, Src, and p38 (Fig. 6D) in Fc-TPP11-treated tumors, consistent with the in vitro inhibitory effects of Fc-TPP11 on VEGF-mediated signaling in HUVECs (Fig. 5E). Compared with the PBS- and Fc-treated tumors, Fc-TPP11-treated tumors showed significant E-cadherin downregulation, but no changes in the levels of fibronectin, Ncadherin, and vimentin (Fig. 6D), mesenchymal transition markers involved in tumor invasion and metastasis [43].

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Fig. 5. Fc-TPP11 competes with VEGF for NRP1 binding, thereby inhibiting tumor angiogenesis and VEGF-induced migration and invasion of HUVECs. A. Competitive ELISA to measure the binding of Fc-TPP11 (3 nM) to plate-coated NRP1-b1b2 protein in the presence of increasing concentrations of VEGF. B. Representative photos of blood vessels in excised Matrigel plugs (top) and images of CD31-stained tumor sections (bottom) from mice carrying VEGF-secreting A431 cells after the indicated treatments. Cells were injected with either Fc or Fc-TPP11 (10 mg/kg, ~ 200 μg/mouse) SC into the right thigh of nude mice and grown for 9 days before analysis. Image magnification, 200×; scale bars, 100 μm. . The bar graph shows the quantification of the CD31stained vessel areas, as assessed with ImageJ software. Error bars, ±SD of 4 fields per tumor (n = 2 per group). **P < 0.01. C and D. Representative images and quantification of wound healing (C) and invasion (D) of HUVECs induced for 18 h (C) or 12 h (D) with medium (control) or VEGF (0.5 nM) with or without Fc (1 μM) and Fc-TPP11 (1 μM). The bar graphs show the mean ± SD. Image magnification, 100×. *P < 0.05, **P < 0.01. E. Western blot analyses of VEGF-stimulated signaling in HUVECs that were starved for 4 h (control) and then stimulated with VEGF (1.3 nM) with or without Fc (100 nM) and Fc-TPP11 (100 nM) for 10 or 30 min. The number below the panel indicates the quantified relative band intensity of phosphoproteins after normalization of the band intensity to that of the corresponding total proteins as the loading controls. *P < 0.05, (Fc + VEGF) 10 min vs. (Fc-TPP11 + VEGF) 10 min. †P < 0.05, (Fc + VEGF) 30 min vs. (Fc-TPP11 + VEGF) 30 min. F. Western blot analysis of anti-VEGFR2 antibody immunoprecipitation (IP) of cell lysates from 15

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HUVECs that were treated with medium (control) or VEGF (1.3 nM) with or without Fc (100 nM) and Fc-TPP11 (100 nM) for 10 min. Equal precipitates were analyzed, and β-actin was used as a loading control.

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Fig. 6. Fc-TPP11 inhibits in vivo tumor growth through its anti-angiogenesis activity. A. Tumor growth was analyzed by measuring tumor volume (left) during treatment and tumor weight (right) at the end of the treatment in Fc- or Fc-TPP11 (20 mg/kg, ~ 400 μg/mouse) in FaDu tumor bearing mice. After pre-establishing FaDu tumors with a volume of ~ 60 mm3, mice (n = 6 per group) were injected IV every 3 days with PBS, Fc, or Fc-TPP11 (black arrowheads). *P < 0.05. B. Weight of mice measured during the treatments described in A. C. Representative images showing blood vessels (red, CD31) and pericytes (green, α-SMA) in tumors excised from mice at Day 18 after the treatment described in A. The bars on the right show the quantification of vessel density and the pericyte/vessel ratio, as the mean ± SD from 3 different fields of each tumor (n = 3). Image magnification, 200×; scale bar, 100 μm. *P < 0.05, **P < 0.01. D. Western blot analysis of tumor tissue lysates prepared at Day 18 after the treatment described in A to analyze NRP1-related signaling molecules (VEGFR2, Src, and p38) and the amount of E-cadherin, vimentin, N-cadherin, and fibronectin. As indicated 2 independent tumor tissue samples (sample 1 and sample 2) were analyzed. The number below the panel indicates relative value of band intensity of proteins compared to the band intensity in ‘control’ after normalization of the band intensity to that of β-actin for each sample. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control.

3.9. Fc-TPP11 does not promote tumor metastasis 16

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The enhanced vascular and paracellular permeability induced by Fc-TPP11 in tumors may facilitate the intravasation of tumor cells and subsequent metastatic invasion into distal tissues [43]. Therefore, we investigated the effect of Fc-TPP11 on the frequency of tumor metastasis in an orthotopic mouse model of breast cancer metastasis. 4T1 mouse metastatic breast cancer cells, which express high levels of NRP1 (Fig. 7A), were orthotopically implanted in the fourth mammary fat pad of BALB/c mice [32]. The treatments were initiated after the primary tumors reached an average volume of 50 mm3 without lung metastasis. Mice were injected IV with PBS, Fc, or Fc-TPP11 every 3 days for 21 days, and the lungs were excised to assess distant metastasis to the lungs. Fc-TPP11 treatment significantly reduced primary tumor growth as compared to that with PBS and Fc treatment (Fig. 7B), without any difference in body weight (Fig. 7C). Intriguingly, the number of metastatic nodules at the surface of the lungs in Fc-TPP11-treated mice was only slightly lower than that in Fc-treated animals (20 ± 5.8 vs. 31 ± 6.8 metastatic nodules per mouse), but was significantly lower than that in PBS-treated mice (48 ± 6.6) (Fig. 7D and E). Images of H&E stained lungs also supported the reduced distant metastasis to the lung in Fc-TPP11-treated mice (Fig. 7E).

Fig. 7. Fc-TPP11 inhibits primary tumor growth without promoting tumor metastasis. A. NRP1 expression levels in 4T1 cells was examined by western blot, and compared to that in HUVECs, FaDu, A431, and NRP1-negative T47D cells. B. Relative tumor volume of 4T1 mouse metastatic breast tumors in different treatment groups (n = 5–6 per group, **P < 0.01, ***P < 0.001 by two-way ANOVA). After pre-establishing orthotopic 4T1 tumors with a volume of ~ 50 mm3, mice were injected IV with PBS, Fc, or Fc-TPP11 (20 mg/kg, ~ 400 μg/mouse) (black arrowheads) every 3 days. C. Mouse weight was measured during the treatments described in B. D. Quantification of lung metastasis nodules at 21 days post-treatment (n = 5–6 per group, *P < 0.05 by one-way ANOVA). E. Photos of India-ink-stained lungs (top) and microscopic images of H&E-stained lung slices (bottom) at 21 days post-treatment. The control indicates lungs collected from untreated mice. Arrows point to tumor metastasis sites. Scale bars, 300 μm. Data represent mean ± SEM in B and D and mean ± SD in C. 17

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Fig. 8. Schematic diagram showing the tumor homing, tumor tissue-penetration, and antiangiogenesis mechanism of Fc-TPP11. (1) Fc-TPP11 homes to the vasculature of NRP1overexpressing tumors, leading to tumor homing of Fc-TPP11. (2) In tumor vessels, some portions of Fc-TPP11 enhance vascular permeability by inducing NRP1-mediated VE-cadherin downregulation, leading to the increased extravascular transport of other portions of blood-borne Fc-TPP11 or coinjected drug (e.g., DOX) into the tumor parenchyma. (3) In perivascular tumor tissues, some portions of Fc-TPP11 reduce the cell junction barrier by inducing NRP1-mediated E-cadherin downregulation, resulting in enhanced spreading of other portions of Fc-TPP11 or coinjected drug (e.g., DOX) into the inside of the tumor tissues. (4) The penetrated Fc-TPP11 competes with tumor-derived VEGF for NRP1 binding, which inhibits VEGF-mediated signaling and exerts an antiangiogenesis effect. (5) When FcTPP11 is reformatted into mAbTPP11 (e.g., cetuximab-TPP11), mAb-TPP11 distributed widely within tumor tissues targets the cognate antigens of the parent mAb, such as EGFR for cetuximab, thus inhibiting tumor growth.

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4. Discussion It was previously reported that CendR peptides bind to the VEGF-binding site of NRP1 and NRP2 without receptor specificity [13, 14, 20, 21]. In this study, we successfully generated an Fc-fused peptide, Fc-TPP11, that binds to the VEGF binding site of NRP1 with approximately 1,000-fold higher selectivity (KD = ~ 1.7 nM) than the closely related NRP2 (KD = ~ 1.6 μM). The NRP1-selective, high-affinity binding Fc-TPP11 showed NRP1dependent signaling, including enhanced vascular permeability through VE-cadherin downregulation in HUVECs and increased paracellular permeability through E-cadherin downregulation in tumor tissues, even at a ~ 10-fold lower concentration than the parent NRP1/2-cross-reactive Fc-A22p (KD = ~ 63 nM) [14]. This property of Fc-TPP11 also allowed the co-injected free DOX drug to efficiently extravasate and penetrate into tumor parenchyma, thereby potentiating the anti-tumor efficacy of the co-administered anti-cancer drug. Apart from NRP1-mediated signaling, Fc-TPP11 also served as a blocker of VEGF binding to NRP1 by sharing the same arginine-binding pocket of NRP-b1 domain. Fc-TPP11 as a single agent, suppressed VEGF-dependent endothelial cell migration and vessel tube formation in vitro, decreased neovascularization in vivo, and slowed tumor growth in mouse models. To our knowledge, this is the first report of a tumor-penetrating peptide that selectively binds to the VEGF-binding site of NRP1 with negligible binding to the 18

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homologous receptor, NRP2. NRP1-specific, high-affinity binding of Fc-TPP was very useful to determine NRP1 receptor function, independent from the homologous receptor, NRP2, as well as its role as the co-receptor of VEGFRs and others. NRP1 plays a critical role in VEGF- and Sema3A-induced vascular permeability, wherein NRP1 acts as a co-receptor of VEGFR2 and Plexin A1, respectively [7, 10]. However, in this study, the NRP1-selective Fc-TPP11 efficiently downregulated VE-cadherin even when VEGFR2 or PlexinA1 receptor was knocked down (Fig. 2), suggesting that NRP1 can function, independently of its role as a co-receptor for VEGFRs, to transduce the signal for VE-cadherin downregulation in endothelial cells and E-cadherin downregulation in tumor cells for enhancing vascular permeability and paracellular permeability in tumor tissues [14]. Due to its pivotal role in VEGF-mediated vascular remodeling and angiogenesis as part of the NRP1/VEGFR2 complex [44], NRP1 has been targeted in the development of VEGF-NRP1 binding antagonists. NRP1-targeting antibodies and peptides interfere with VEGF binding to NRP1, diminish NRP1/VEGFR2 complex formation, and thereby inhibit VEGF-induced migration, invasion, and angiogenesis [16-18, 22, 45, 46]. Likewise, in HUVECs, Fc-TPP11 efficiently competed with VEGF for binding to the NRP1-b1 domain and thereby inhibited VEGF-induced NRP1/VEGFR2 complex formation, which lead to reduced VEGFR2 phosphorylation and activation of the downstream molecules Src, FAK, and p38 (Fig. 5). Activation of Src, FAK, and p38 is involved in enhanced cell motility during angiogenesis [42]. Accordingly, the inhibitory effect of Fc-TPP11 on Src, FAK, and p38 activation seems to explain how Fc-TPP11 inhibits endothelial cell migration, thereby inhibiting tumor angiogenesis, similar to the effect of an NRP1-blocking antibody [22]. Interestingly, Fc-TPP11 treatment reduced the number of tumor blood vessels and decreased pericyte association (Fig. 6), indicating immature vessel normalization, as was observed with an anti-NRP1 antibody [22]. Vessel normalization is inversely correlated with vascular permeability [47]. Thus, we speculate that the increased vascular permeability caused by FcTPP11-induced VE-cadherin downregulation may hamper the association of pericytes with vessels, thereby leading to immature vessel normalization. During tumor invasion and metastasis, tumor cells typically lose the epithelial marker E-cadherin and gain mesenchymal markers, such as vimentin, N-cadherin, and fibronectin [43]. However, Fc-TPP11 treatment only downregulated E-cadherin, and did not upregulate mesenchymal markers in tumor tissues in mice bearing FaDu carcinoma xenografts (Fig. 6D). Furthermore, as shown in a 4T1 orthotopic metastatic breast tumor model, Fc-TPP11 slightly reduced distant metastasis to the lung (Fig. 7), indicating that Fc-TPP11 selectively reduced the epithelial barrier in tumor tissues without promoting invasion and metastasis. The prototypic tumor-penetrating peptide iRGD also inhibited spontaneous metastasis in mice [46]. Some reports have shown that VEGF-induced angiogenesis promotes tumor invasion and metastasis because it allows malignant cells to escape into the blood circulation and settle in distal tissues [45, 48]. These results suggest that, despite its ability to enhance vascular and paracellular tissue permeability, Fc-TPP11 did not promote tumor metastasis through its antiangiogenic activity mediated by NRP1 blocking. A longer serum-half life is a beneficial property of tumor-penetration promoting agents for their co-administered or conjugated drugs [1, 49]. The TPP11 fusion to Fc did not compromise the serum half-life of Fc (Fig. 4A). Thus the long serum half-life of Fc-TPP11 offers a distinct advantage over other tumor-penetrating peptides and proteins. Further, FcTPP11 platform will be greatly useful to generate potent, solid tumor-targeting mAbs in the 19

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mAb-TPP11 format, by providing the parent mAbs with the advantage of tumor homing and tissue penetration, as shown by the example of cetuximab-TPP11, which exhibited ~ 2-fold enhanced tumor accumulation and spread within tumor tissues, as compared to the parent mAb, cetuximab. Furthermore, as we observed an enhanced tumor delivery and anti-tumor efficacy of the anti-cancer drug, DOX using co-injected Fc-TPP11, Fc-TPP11 is a promising promoter agent increasing the therapeutic index of co-injected chemical drugs. NRP1 is overexpressed in tumor-associated blood vessels and in many types of tumor cells; however, it is also expressed at low levels in the vessels and epithelial cells of normal tissues [50]. Thus, Fc-TPP11 might enhance tissue penetration of co-injected chemical drugs into normal tissues in addition to the targeted tumors, raising a concern of the systemic cytotoxicity. In the whole-body SPECT imaging of tumor-bearing mice, however, Fc-TPP11 showed improved accumulation in tumor tissues without notable preferential distribution in normal tissues, including brain, compared with the control Fc (Fig. 4B). Thus, we speculate that adverse side effects of Fc-TPP11 on normal tissues and organs would be minimal. Nonetheless, any toxicity of Fc-TPP11 alone or its combination with other drugs remains to be determined.

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5. Conclusion Fig. 8 proposes the mechanism of action of the NRP1-specific Fc-TPP11 platform as a tumor-homing, tumor tissue-penetrating, and anti-angiogenesis agent. Overexpression of NRP1 in tumor-associated vessels and tumor cells allows Fc-TPP11 to simultaneously target both, tumor blood vessels and tumor tissues, leading to tumor homing and subsequent tumor penetration by Fc-TPP11. Thus, the bivalent binding format of Fc-TPP11 mimics the natural ligands for NRP1 engagement, resulting in more effective NRP1-mediated signaling than the synthesized, monomeric TPP11 peptide. Fc-TPP11 also enhanced accumulation and penetration of co-injected chemical drug, DOX, into extravascular tumor parenchyma, suggesting that Fc-TPP11 could be used as a promoter agent to improve extravasation and tumor-penetration of anti-cancer drugs or tumor-imaging agents. Finally Fc-TPP11 can be easily adapted to mAb-TPP11 format for more potent solid tumor-targeting antibody therapy. Acknowledgements This study was supported by grants from the Mid-career Researcher Program (2013R1A2A2A01005817), the Pioneer Research Center Program (2014M3C1A3051470), and the Converging Research Center Program (2009-0093653) of the National Research Foundation, funded by the Korean government.

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