SUPPLEMENTAL DATA MATERIALS AND METHODS Peptide synthesis, radiolabelling and radiochemical analysis Solid phase synthesis of peptides was carried out on using ABI 433A peptide synthesizer, on a preloaded Thr(tBu)-Wang resin, using N-Fmoc amino acids and HBTU as the coupling reagent (Merck Chemicals). Amino acids had the following protection groups Arg(Pbf), Lys(Boc) Gln (Trt) Asp(OtBu)and Asn(Trt). In order to biotinylate DTPA-A20FMDV2, biotinyl-lysine was incorporated at position two in place of the usual alanine. Following chain assembly, DTPA-tetra(tBu ester) (4eq) (Macrocyclics) was coupled to the N terminal of the protected peptidyl-resin using HOBT/DIC activation and overnight agitation. Following reaction, and washing of the peptidyl-resin, the peptide was cleaved from the resin and side chain protecting groups removed by treatment with 92.5% trifluoroacetic acid, 2.5% ethanedithiol, 2.5% triisopropyl silane, and 2.5% H 2 O. After 2 h, the resin was removed by filtration and peptides were precipitated with diethyl ether on ice. Peptides were isolated by centrifugation, then dissolved in H 2 O and freeze dried overnight. The crude peptides were analysed by Agilent 1100 reverse phase HPLC and electrospray mass spectroscopy. Peptides were purified by reverse phase HPLC to > 90% purity on an Aquapore ODS 20 micron 250 x 10 mm column and lyophilised. During synthesis and purification, use of glassware was avoided. Peptide stock solutions were prepared in metal-free water and standard concentrations were confirmed spectrophometrically using absorption at 215nm. Briefly, 111 In-InCl 3 (58 µl, 15-30 MBq in 0.1mM HCl) and ammonium acetate (15 µl, 1 M, ph 5.5) were mixed in a polypropylene vial. The peptide conjugate DTPA-A20FMDV2 or DTPA-A20FMDV2ran (5 µl. 1mg/ml in sterile water) was added, the solution was vortexed
briefly and incubated for 30 minutes at 25 o C, after which time the reaction was quenched with ethylenediaminetetraacetic acid ([EDTA] 4 µl, 0.1M). Radiochemical purity was determined by reverse-phase HPLC on LC Module 1 (Waters, Hertfordshire, UK) connected to sodium-iodide detector (5-µm Jupiter 300 column, 250 x 4.6 mm inner diameter, Phenomenex; solvent A, 0.1% trifluoroacetic acid [TFA] in HPLC grade water; solvent B, 0.1% TFA in acetonitrile; flow rate, 1 ml/min; gradient, 5% solvent B for 5 min, then change to 60% solvent B over 20 min, then change to 100% solvent B over 3 min, and change back to 5% solvent B over 5 min). Radiochemical purity was greater than 95% at a specific activity of 16 MBq/nmol with no evidence of fragmentation or aggregation during the labelling process as indicated by sharp single peak (see Supplemental Fig. 1) therefore no further purification was done. Any un-reacted 111 In-InCl 3 formed a complex with EDTA eluting off first with t R of 3 min which formed 3-5% impurity. The RP-HPLC elution time for 111 In-DTPA-A20FMDV2 was 13.08 min and 111 In-DTPA-A20FMDVran 12.25 min. Instant thin-layer chromatography was carried out using reverse phase plates (Whatman MKC18 Silica Gel 60 A, 200 mm, 1.0 x 7.0 cm) with mobile phase (25mM EDTA in 0.1 M ammonium acetate, ph 5.5); Rf for both radiolabelled peptides 111 In-DTPA-A20FMDV2 and 111 In-DTPA-A20FMDVran was 0.252 and 111 In-EDTA was 0.585. Endotoxin content in the final sample was analysed to be less than 17.5 EU (endotoxin unit) using an Endosafe - Portable Testing System (Charles River Laboratories, L Arbresle Cedex, France) with use of FDA-licensed disposable cartridges for accurate endotoxin testing.
Supplemental Table 1 PCR primer sequences Gene Sequence Murine itgb6 Sense 5 - TCTGAGGATGGAGTGCTGTG -3 Antisense 5 - GGCACCAATGGCTTTACACT -3 Murine hprt Sense 5 -TGAAAGACTTGCTGGAGATGTCA-3 Antisense 5 -CCAGCAGGTCAGCAAAGAACT-3 Human ITGB6 Sense 5 -AAACGGGAACCAATCCTCTGT-3 Antisense 5 -GCTTCTCCCTGTGCTTGTAGGT-3 Human β2m Sense 5 -AATCCAAATGCGGCATCT-3 Antisense 5 -GAGTATGCCTGCCGTGTG-3
Supplemental Figure 1 HPLC profiles of 111 In-DTPA-A20FMDV (A) and 111 In-DTPA-A20FMDVran (B) peptides showing the retention times at 13.08 and 12.25 minutes respectively.
Supplemental Figure 2 Kinetics and biodistribution of 111 In-labelled DTPA-A20FMDV2 peptide in mice instilled with saline 28 days prior to imaging. Mice were injected intravenously with the αvβ6-
specific 111 In- DTPA-A20FMDV2 peptide and subjected to NanoSPECT-CT analysis 1 hour (1-h) and 3 hours (3-h) later. The bladder is masked in each of the scans. Images of a representative mouse are shown.
Supplemental Figure 3 NanoSPECT-CT analysis of 111 In-DTPA-A20FMDV2ran peptide binding in the lungs of mice instilled intratracheally with saline (A) and bleomycin-treated (B) mice 28 days earlier. Mice were injected intravenously with peptide and imaged 1 hour later. Images of a representative mouse are shown.
Supplemental Figure 4 Coronal NanoSPECT-CT scans of the upper torso showing binding of 111 In- DTPA-A20FMDV2 in the lungs, oral cavity and submandibular glands of animals instilled intratracheally with bleomycin. Mice received IgG2a isotype control (A) or an αvβ6 blocking antibody (B) (2.5mg/Kg) 24 hours prior to imaging, and binding was assessed one hour after intravenous injection of the radiolabelled peptide. Images of a representative mouse are shown.
Supplemental Figure 5 Axial NanoSPECT-CT scans of 111 In- DTPA-A20FMDV2 peptide binding within the lungs of bleomycin (A&B) or saline-treated (C&D) animals. Peptide uptake was assessed in each individual animal at both 2 weeks (A&C) and 4 weeks (B&D) after intratracheal instillation of bleomycin or saline. Images of representative mice are shown.
Supplemental Figure 6 Immunohistochemical analysis of lung αvβ6 levels (brown), 28 days after treatment with intratracheal saline (A) or bleomycin (B). Images from representative mice are shown. Scale bar is 20µm.
Supplemental Figure 7 Hydroxyproline (A) and itgb6 mrna (B) levels were measured in lungs from mice treated with bleomycin (n=6/group) or saline (n=4/group) for 28 days. ITGB6 mrna expression was assessed in lung samples from IPF patients compared with non-fibrotic lungs (C; n=3/ group). Data are presented as mean ± SEM. (**** P< 0.0001; * P<0.05). Correlation of 111 In-DTPA- A20FMDV2 binding within the lungs at 28 days post saline (open circles; n=4) or bleomycin (closed circles; n=6) treatment with itgb6 mrna expression (D).
Supplemental Video 1 3D reconstruction of NanoSPECT-CT images of the thorax visualizing binding of 111 In- DTPA-A20FMDV2 peptide within saline-treated lungs. Mice instilled intratracheally with saline 28 days prior to NanoSPECT-CT imaging were injected intravenously with the αvβ6 binding peptide, 111 In-DTPA-A20FMDV2. NanoSPECT-CT scans were performed 1 hour later. The reconstruction of a representative mouse is shown. Supplemental Video 2 3D reconstruction of NanoSPECT-CT images of the thorax visualizing binding of 111 In- DTPA-A20FMDV2 peptide within bleomycin-treated lungs. Mice instilled intratracheally with bleomycin 28 days prior to NanoSPECT-CT imaging were injected intravenously with the αvβ6 binding peptide, 111 In- DTPA-A20FMDV2. NanoSPECT-CT scans were performed 1 hour later. The reconstruction of a representative mouse is shown. Supplemental Video 3 3D reconstruction of NanoSPECT-CT images of the thorax visualizing binding of 111 In- DTPA-A20FMDVran control peptide within saline-treated lungs. Mice instilled intratracheally with saline 28 days prior to NanoSPECT-CT imaging were injected intravenously with the control peptide, 111 In- DTPA-A20FMDVran. NanoSPECT-CT scans performed 1 hour later. The reconstruction of a representative mouse is shown. Supplemental Video 4 3D reconstruction of NanoSPECT-CT images of the thorax visualizing binding of 111 In- DTPA-A20FMDVran control peptide within bleomycin-treated lungs. Mice instilled intratracheally with bleomycin 28 days prior to NanoSPECT-CT imaging were injected intravenously with the control peptide, 111 In- DTPA-A20FMDVran. NanoSPECT-CT scans were performed 1 hour later. The reconstruction of a representative mouse is shown. Supplemental Video 5 3D reconstruction of NanoSPECT/CT images of the upper torso visualizing binding of 111 In- DTPA-A20FMDV2 peptide within bleomycin-treated animals in the presence of isotype control antibody (2.5mg/Kg) 24 hours before peptide administration. NanoSPECT-CT scans were performed 1 hour after the intravenous injection of the radiolabelled peptide. The reconstruction of a representative mouse is shown.
Supplemental Video 6 3D reconstruction of NanoSPECT-CT images of the upper torso visualizing binding of 111 In- DTPA-A20FMDV2 peptide within bleomycin-treated animals in the presence of antiαvβ6 blocking antibody (2.5mg/Kg) administered 24 hours prior to imaging, NanoSPECT-CT scans were performed 1 hour after the intravenous injection of the radiolabelled peptide. The reconstruction of a representative mouse is shown.