Tumor-penetrating peptide for systemic targeting of Tenascin-C

Extracellular matrix in solid tumors has emerged as a specific, stable, and abundant target for affinity-guided delivery of anticancer drugs. Here we describe the homing peptide that interacts with the C-isoform of Tenascin-C (TNC-C) upregulated in malignant tissues. TNC-C binding PL3 peptide (amino acid sequence: AGRGRLVR) was identified by in vitro biopanning on recombinant TNC-C. Besides TNC-C, PL3 interacts via its C-end Rule (CendR) motif with cell-and tissue penetration receptor neuropilin-1 (NRP-1). Functionalization of iron oxide nanoworms (NWs) and metallic silver nanoparticles (AgNPs) with PL3 peptide increased tropism of systemic nanoparticles towards glioblastoma (GBM) and prostate carcinoma xenograft lesions in nude mice (eight and five-fold respectively). Treatment of glioma-bearing mice with proapoptotic PL3-guided NWs improved the survival of the mice, whereas treatment with untargeted particles had no effect. PL3-coated nanoparticles were found to accumulate in TNC-C and NRP-1-positive areas in clinical tumor samples, suggesting a translational relevance. The systemic tumor-targeting properties and binding of PL3-NPs to the clinical tumor sections, suggest that the PL3 peptide may have applications as a targeting moiety for the selective delivery of imaging and therapeutic agents to solid tumors.

The file includes: Table S1. T7-displayed peptides from round 4 in vitro biopanning on TNC-C.

Supplementary Materials and Methods
Cloning, expression, and purification of TNC-C Vector Construction. A pF1K (Promega, # FXC00319) derivative plasmid containing full-length cDNA of TNC was used for PCR amplification of cDNA region of TNC-C with Phusion Hot Start II High-Fidelity DNA Polymerase (Thermo Fisher Scientific Inc # F-537L; primer pairs: 5′-CTCCTCTCATATGGAGGCCCTGCCCCTTC -3′ and 5′-CAGACACTCGAGTTATCATGTAACAATCTC -3′ for domain TNC-C. NdeI and XhoI restriction sites underlined). The fragments were cloned in the pET28a+ plasmid for TNC-C expression as an N-terminally His-tagged protein.
Expression and purification of TNC-C. The pET28a+TNC-C plasmid was used for transformation of E. coli BL21 Rosetta 2 (DE3) pLysS cells (Novagen, #70956). The protein expression was induced by addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) (Sigma, # I6758) to 0.5 mM final concentration and the bacteria were cultured at 18 o C for 16h. Cells were collected by centrifugation, resuspended in ice-cold IMAC buffer (25mM Tris-HCl, 400mM NaCl, 25mM imidazole pH 8, containing EDTA-free protease inhibitor cocktail and DNase I) and lysed by sonification (Bandelin Sonopuls HD 2070, Germany). The cleared bacterial lysate was purified using HiTrap IMAC HP columns (GE Healthcare # 17-0920-05) on ÄKTA purification system (GE Healthcare), and the eluate was dialyzed against PBS using 3.5 kDa cutoff 3mL Slide-A-Lyzer Dialysis Cassettes (Thermo Scientific #66330). TNC-C concentration was determined by bicinchoninic acid assay (Thermo Scientific #23227), and the purity of the proteins was assessed by SDS-PAGE. Mass spectrometry and de novo peptide sequencing were used to confirm the size and sequence of the purified proteins. His-tagged NRP-1 b1b2 domain was expressed and purified as described (Teesalu et al., 2009).

Antibody production
Single-chain antibodies: The cDNA sequences encoding TNC-C-G11-scFV were retrieved from US patent application EP2157102 A1, and synthetic DNA fragments were cloned in the pET28a+ expression plasmid. Recombinant antibodies were expressed in E. coli BL21 Rosetta 2 (DE3) pLysS (Novagen, #70956) cells and purified using Protein A GraviTrap Sepharose (GE Healthcare # 28-9852-54), followed by affinity purification on immobilized TNC-C. The purified antibodies were analyzed by SDS-PAGE. Pull-down assays and ELISA were used to verify the interaction of the purified antibodies with the target TNC-C domain.

Clone No. Peptide sequence
Repeats TNC-C binding (fold G7 control phage)  Table S1. T7-displayed peptides from round 4 in vitro biopanning on TNC-C. Random phage clones from round 4 of selection on TNC-C were subjected to Sanger sequencing of the peptide-encoding segment of the genome. The table shows peptide sequences of the 38 sequenced clones, and quantitation of binding of individual peptide phages to TNC-C (fold binding of control heptaglycine phage). RGRLXR motif (7 total repeats) is shown in italic, RGRLR motif (18) is underlined, and RLXR motif (12) is indicated in bold.    S4. PL3 peptide phages binding to TNC-C and NRP1. The selected peptide PL3-phage binds to immobilized TNC and NRP1 b1b2, but not to a control protein FN EDB. Phage binding is expressed fold over control phage displaying heptaglycine (G7) peptide. NRP1 binding peptide RPARPAR used as a positive control.

Figure. S10. Anti TNC-C antibody validation on surgical explants of human glioma tissues.
The snap-frozen individual patient glioma tissues were sectioned and incubated with the anti-TNC-C mouse antibody (α-TNC-C), or α-TNC-C antibody (10 µg) is pre-incubated of TNC-C protein (60µg) before staining, or only secondary mouse α-alexa647 antibody. The tissue sections were examined by confocal microscopy. The top panel shows α-TNC-C antibody staining. The middle panel shows staining of α-TNC-C antibody is pre-blocked with recombinant TNC-C protein and reveals its specificity. The bottom panel shows secondary antibody staining control. Scale bar, 100 μm for all panels, tissues were stained for the anti-TNC-C mouse (red) and nucleus (DAPI, blue).

Figure. S11. Experimental tumor therapy with PL3-guided NWs extends survival of glioblastoma.
Mice bearing s.c. U87-MG mice were treated with 5 mg/kg of D(KLAKLAK)2-NWs, PL3-NWs, and PL3-D(KLAKLAK)2-NWs, or control PBS (N=6 mice/group). At the endpoint of the study (tumor volume >2000 mm 3 ), the mice were sacrificed by perfusion, and organs and tumors were collected. The mean survival of the different treatment groups was calculated. N = 6 mice/group; Error bars, mean ± SEM; Statistical analyses were performed using paired non-parametric test (Wilcoxon matched-pairs signed-rank test) or Friedman test in GraphPad Prism (*p< 0.05).      The Ni-NTA Magnetic Agarose Beads (QIAGEN, Hilden, Germany) were coated with His-6X tagged TNC-C (300 µg/100 µl beads) at room temperature for 1 h in 400µl of PBS. The TNC-C beads were washed 3 times with washing buffer (PBS, 0.05% NP-40, 1% BSA), followed by incubation with FAM-PL3 peptide (200 µg in 400µl in washing buffer) at room temperature for 2 h. The unbound peptides were removed by rinsing 6 times with washing buffer, and the bound FAM-PL3/TNC-C complex was eluted with 1ml of PBS containing 500mM Imidazole and 0.1% NP40. To measure the dequenching effect, the FAM-PL3/TNC-C complex was treated with trypsin (2.5mg/mL) for 4 h at 37°C. The intensity of fluorescence of the untreated and trypsintreated complex was measured by VICTOR Multilabel Plate Reader (PerkinElmer) at 488/516nm. Student's unpaired two-tailed t-test; the error bars: mean ± SD; ** p ˂ 0.01, N=3.