type i collagen targeted pet probe for pulmonary fibrosis ...pulmonary fibrosis is scarring of the...

SC I ENCE TRANS LAT IONAL MED I C I N E | R E S EARCH ART I C L E

PULMONARY F I BROS I S

1Athinoula A. Martinos Center for Biomedical Imaging, Department of Radiology,Massachusetts General Hospital and Harvard Medical School, Charlestown, MA02129, USA. 2Division of Thoracic Surgery, Massachusetts General Hospital andHarvard Medical School, Boston, MA 02114, USA. 3Division of Pulmonary andCriticalCare Medicine, Massachusetts General Hospital and Harvard Medical School, Boston,MA 02114, USA. 4Department of Pathology, Massachusetts General Hospital andHarvard Medical School, Boston, MA 02114, USA. 5Biogen, Cambridge, MA 02142,USA. 6Division of Surgical Oncology, Massachusetts General Hospital and HarvardMedical School, Boston, MA 02114, USA.*These authors contributed equally to this work.†Present address: School of Medicine, Vanderbilt University, Nashville, TN 37232, USA.‡Present address: Department ofMolecular Biotechnology andHealth Sciences, Centerof Excellence for Preclinical Imaging, University of Turin, Turin, Italy.§Present address: College of the Environment and Life Sciences, University ofRhode Island, Kingston, RI 02881, USA.¶Corresponding author. Email: [email protected] (P.C.); [email protected] (M.L.)

Désogère et al., Sci. Transl. Med. 9, eaaf4696 (2017) 5 April 2017

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Type I collagen–targeted PET probe for pulmonaryfibrosis detection and staging in preclinical modelsPauline Désogère,1* Luis F. Tapias,2* Lida P. Hariri,3,4 Nicholas J. Rotile,1 Tyson A. Rietz,1†

Clemens K. Probst,3 Francesco Blasi,1‡ Helen Day,1§ Mari Mino-Kenudson,4 Paul Weinreb,5

Shelia M. Violette,5 Bryan C. Fuchs,6 Andrew M. Tager,3 Michael Lanuti,2¶ Peter Caravan1¶

Pulmonary fibrosis is scarring of the lungs that can arise from radiation injury, drug toxicity, environmental orgenetic causes, and for unknown reasons [idiopathic pulmonary fibrosis (IPF)]. Overexpression of collagen is ahallmark of organ fibrosis. We describe a peptide-based positron emission tomography (PET) probe (68Ga-CBP8)that targets collagen type I. We evaluated 68Ga-CBP8 in vivo in the bleomycin-induced mouse model of pulmo-nary fibrosis. 68Ga-CBP8 showed high specificity for pulmonary fibrosis and high target/background ratios indiseased animals. The lung PET signal and lung 68Ga-CBP8 uptake (quantified ex vivo) correlated linearly (r2 =0.80) with the amount of lung collagen in mice with fibrosis. We further demonstrated that the 68Ga-CBP8 probecould be used to monitor response to treatment in a second mouse model of pulmonary fibrosis associatedwith vascular leak. Ex vivo analysis of lung tissue from patients with IPF supported the animal findings. Thesestudies indicate that 68Ga-CBP8 is a promising candidate for noninvasive imaging of human pulmonary fibrosis.

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INTRODUCTIONIdiopathic pulmonary fibrosis (IPF) is a specific form of progressive,fibrosing interstitial pneumonia of unknown etiology that primarilyaffects older adults. In most cases, it is a relentlessly progressive diseasethat results in dyspnea and functional decline until death (1, 2). Cur-rently, there is no treatment able to reverse fibrosis in this disease (3).

Despite efforts to establish precise, universally acknowledged diag-nosis criteria for IPF, its ascertainment remains a challenge—althoughhigh-resolution computed tomography (HRCT) scanning can aid di-agnosis of the disease (4). Patients with suspected IPF that showatypical features on HRCT images usually require a surgical lungbiopsy to confirm the diagnosis (5, 6); however, many of these patientshave physiological impairments and comorbidities that make biopsy arisky procedure.

These challenges highlight the need for the development and valida-tion of diagnostic and prognostic markers specific to IPF to guide treat-ment decisions. The ability to recognize fibrosis noninvasively, forexample, with innovativemolecular imaging techniques, could substan-tially improve management of patients with fibrotic lung disease. Inrecent years, collagen degradationmarkers (7), activatedmacrophages(8, 9), fibroblasts (10, 11), and integrins avb6 (12–15) and avb1 (16)have been studied as diagnostic and prognostic markers. Pulmonary18F-fluorodeoxyglucose (FDG) uptake has also been reported to be a

predictor of global health score and lung physiology in patients withIPF (17–19); 18F-FDG, however, lacks specificity for IPF.

We set out to develop an approach based on direct molecular im-aging of type I collagen as a noninvasive means of detecting, as well asstaging, pulmonary fibrosis. Fibrosis, regardless of its cause or location,is characterized by excess deposition of collagens, primarily type I col-lagen, and other extracellular matrix proteins in the parenchyma (20).Histological proof of fibrosis is predicated on collagen staining. Wepreviously described a 16–amino acid disulfide-bridged cyclic peptidethat was identified via phage display and that recognized and bound totype I human collagen. This peptide was functionalized with threegadolinium diethylenetriamine pentaacetic acid chelates to providemagnetic resonance (MR) signal enhancement, and the resultingprobe (EP-3533) showed excellent ability to detect and stage diseasein preclinical models of cardiac (21, 22), hepatic (23–25), and pulmo-nary fibrosis (26).

We have next sought to develop an analogous positron emissiontomography (PET) probe. PET is a quantitative modality that provideshigher spatial resolution than other nuclear imaging techniques inhumans and that could be used to quantify pulmonary fibrosis.PET is an accepted method that is already frequently used forinvestigating lung pathology. Equally important is the growinginstalled base of PET-CT and PET-MR systems. In the absence of lungarchitectural distortion (which may not be present early in fibroticlung diseases), fibrosis may be indistinguishable by HRCT from otherpathological processes that attenuate x-ray signals, such as inflamma-tion. By fusing collagen-specific PET with HRCT, however, we hopedto be able to determine whether specific opacities and patternsidentified by HRCT are the result of fibrosing disease (27). In ad-dition, the development of PET molecular imaging probes offers ashorter and more economical path to clinical translation than doesthat of MR imaging (MRI) probes because the low microgram massdose required for PET allows for an abbreviated preclinical safetyand toxicology package to be submitted to regulators to initiate hu-man studies.

Here, we describe a peptide-based, collagen-targeted PET probe,68Ga-CBP8, and demonstrate its ability to detect and quantify pul-monary fibrosis in two mouse models of disease, as well as show its

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capacity to monitor therapeutic responses. We also evaluated thecapacity of the probe to detect fibrosis in lung tissue samples fromIPF patients.

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RESULTS68Ga-CBP8 is a type I collagen specific PET probe with rapidrenal clearanceWe synthesized a type I collagen–targeted probe, 68Ga-CBP8, andan inactive negative control probe, 68Ga-CBP12. 68Ga-CBP8 and68Ga-CBP12 are isomers that differ only in the chirality of the cysteineat the 13th amino acid position from the C terminus (fig. S1). Thepeptide precursors of both probes were conjugated to tris(tert-butyl)ester–protected 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid(NODAGA) chelators. We then deprotected the chelating carboxylategroups and purified the peptide conjugates. Radiolabeling was per-formed under standard conditions, was complete in 5 min, and gavehigh radiochemical yields without the need for high-performanceliquid chromatography (HPLC) purification (fig. S2). Specific activ-ities ranged from 324 to 462 GBq/mmol. We estimated type I collagenaffinity by incubating increasing concentrations of nonradioactive Gaprobes with either type I rat or type I human collagen (21). The disso-ciation constant (Kd) for Ga-CBP8 binding to type I human collagenwas 2.1 ± 0.1 mM and 4.6 ± 0.5 mM for rat collagen, whereas the controlcompound Ga-CBP12 showed weaker binding to human (Kd = 42 ±5 mM) and rat (Kd = 50 ± 10 mM) collagens (figs. S3 and S4).

Mice were injected intravenously with about 3.7 MBq of either68Ga-CBP8 or 68Ga-CBP12, and the change in the decay-correctedsignal in the heart was measured by PET as a function of time. Bothprobes showed similar biexponential clearance, and we estimated sim-ilar blood half-lives from fits to the data (32.9 ± 4.5 min for 68Ga-CBP8and 30.4 ± 3.0 min for 68Ga-CBP12; P = not significant, n = 5 foreach probe). Urine was collected at 150 min after probe administra-tion and injected onto an analytical HPLC column, and the resultswere compared with standards of pure probe. Both probes were>90% intact in urine, indicating that the probes are highly stable invivo (fig. S5).

68Ga-CBP8 specifically binds collagen in the bleomycinmouse model of pulmonary fibrosisA single transtracheal instillation of bleomycin (BM) (2.5 U/kg) resultsin histopathology consistent with pulmonary fibrosis. Two weeks afterinstillation, all of the BM-injuredmice had histopathological findings ofsubstantial pulmonary fibrosis, including excessive interstitial deposi-tion of collagen as demonstrated by Sirius Red staining, and destructionof lung architecture; sham animals, which received transtracheal saline,showed no signs of pulmonary disease (Fig. 1A). The BM-injured lungsexhibited a lymphoplasmacytic infiltrate along with excessive collagendeposition as visualized with Sirius Red in the areas of fibrosis, with apredominant subpleural and peribronchial distribution. Lungs fromsham animals had preserved alveolar architecture, with no excess colla-gen deposition or alveolar infiltration.

As expected, disease progressed in a stepwise fashion as determinedby histological Ashcroft scoring of lung fibrosis (28). Seven days afterinstillation of BM, most animals had moderate fibrous thickening ofalveolar or bronchiolar walls without damage to lung architecture(Ashcroft score, 2.3 ± 0.3; range, 2 to 3) (Fig. 1B). Disease progressedwith formation of fibrous bands, small fibrous masses, or large fibrousareas with definite damage to lung structure (Ashcroft score, 5.8 ± 0.4;


range, 4 to 7 at day 14). The amount of lung tissue affected by the disease(measured with Sirius Red staining) also increased with time: 9% of thearea of tissue sections from the right lung of BM-injured mice wasaffected by disease at day 7, whereas this increased to 28% at day 14(Fig. 1C). Sham animals exhibited histologically normal lung tissuesections. Consistently, collagen deposition [as determined by analysisof hydroxyproline (29) content in the left lung] increased progressivelywith time (Fig. 1D).

To determine whether the 68Ga-CBP8 signal was an accurate ref-lection of disease progression, we compared mice injured with BM atdays 7 and 14 after instillation. Ex vivo measurement of probe uptakeinto the lung increasedwith disease progression in the BM-injuredmice[0.028±0.005percent injecteddoseper lung (% ID/lung) at day7; 0.083±0.008% ID/lung at day 14] (Fig. 1, E and F). Additionally, there was astrong correlation between probe uptake in the lung and lung hydroxy-proline content (r2 = 0.80, P < 0.0001) (Fig. 1G).

To demonstrate the specificity of 68Ga-CBP8, we compared itsuptake into lung with that of the isomer 68Ga-CBP12, in which one ofthe cysteine amino acids was changed from L- to D-chirality (figs. S6 to S8).68Ga-CBP8 accumulated specifically in fibrotic lungs of BM-treated micebut not in the healthy lungs of control mice, whereas 68Ga-CBP12 was notpreferentially taken up into lungs of BM-treated mice compared tocontrols (Fig. 2A). There were no significant differences in hydroxy-proline content between the BM-treated mice that received 68Ga-CBP8and those that received 68Ga-CBP12 (Fig. 2B). The control probe68Ga-CBP12 showed no preferential uptake in the lungs of BM-injuredmice compared to control mice, as determined by ex vivo and PET dataanalysis (Fig. 2, C and D). The distribution of 68Ga-CBP12 in otherorganswas similar to that of 68Ga-CBP8 (Fig. 2E, figs. S6 to S8, and tableS1). These results show the in vivo specificity of 68Ga-CBP8 for type Icollagen, which allows the noninvasive detection and monitoring ofprogression of pulmonary fibrosis in the BM-treated mouse model.

68Ga-CBP8 detects and stages disease in a mouse model ofpulmonary fibrosis associated with enhancedvascular permeabilityIPF is generally thought to result when a lung-injuring environmentalstimulus is experienced by a personwith a genetic predisposition to pul-monary fibrosis. In the context of such a genetic predisposition, fibrosisis thought to result from aberrant or exaggerated wound-healing re-sponses to a relatively common and/or mild lung injury, which wouldbe well tolerated and repaired without fibrosis by most people (30, 31).A growing number of mouse models are trying to capture this impor-tant “gene-by-environment”nature of pulmonary fibrogenesis (32).Weused a low-dose BM vascular leak (LDBVL) model that combines theS1P receptor functional antagonist fingolimod (FTY720) to disruptendothelial barrier function with low-dose BM to inducemild lung in-jury (33). Sustained exposure to FTY720 causes increased vascularleak and intra-alveolar coagulation after lung injury, which also leadsto an exaggerated fibrotic response to a low dose of BM challenge (33).

One group ofmice (LDBVL) was treated with a low dose of BM andthe vascular leak agent FTY720 (n = 9). A second group (LDB) (n = 4)received a low dose of BM, and the third group (FTY) (n = 6) receivedthe vascular leak agent only. FTY720was administered intraperitoneallyto the mice at 1 mg/kg three times a week (Fig. 3A). The three groupswere imaged at day 14 after BM or vehicle instillation. After imaging,the mice were euthanized, and the right lung was taken for histo-pathology analysis and the left lung was taken for biochemical analysis.The LDBVL mice showed an average fibrosis score of 6 (±0.2; range,

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4 to 7), the LDB mice showed an average fibrosis score of 1.5 (±0.9;range, 0 to 3), whereas all FTY mice showed a score of 0 (Fig. 3B).Less than 1% of the total area of tissue sections from the right lungwasaffected by disease in LDB animals (measured by Sirius Red staining),whereas 18% of the total area was affected by disease in the LDBVLgroup (Fig. 3C). There was no area affected by disease in the rightlungs of the FTY animals upon histological examination. Figure 3Dshows twice as much hydroxyproline in the left lung of LDBVL


animals compared with LDB and FTYmice (116.9 ± 4.9 mg for LDBVL, 59.1 ±4.5 mg for LDB, and 49.2 ± 2.6 mg forFTY; P < 0.0001). There was no signifi-cant difference in hydroxyproline contentin the left lung betweenLDBandFTYani-mals (P = 0.4057).

Consistently, PETquantification showeda significantly higher signal in lungs ofLDBVL mice (0.98 ± 0.07% ID/cc) com-pared to lungs of FTY animals (0.30 ±0.02% ID/cc; P < 0.0001) and LDB ani-mals (0.33 ± 0.02% ID/cc; P < 0.0001)(Fig. 3E). There was no significant dif-ference in lung signal measured by PETimage analysis between the FTY and theLDB group (P = 0.9156). Ex vivo quanti-fication of 68Ga-CBP8 uptake revealedfive times more uptake in the LDBVLgroup than in the FTY group (P < 0.0001)and three times more in the LDBVL groupcompared to the LDB group (P < 0.0001)(Fig. 3F and fig. S9). The difference inlung uptake between the LDB and theFTY groups was not statistically signifi-cant (P = 0.8202). Hydroxyproline con-tent was strongly correlated with % ID/cc(r2 = 0.86,P < 0.0001) (Fig. 3G) and% ID/lung (r2 = 0.93, P < 0.0001) (Fig. 3H).These results demonstrate the in vivospecificity of 68Ga-CBP8 for detection ofexcess collagen deposition in a secondmodel of pulmonary fibrosis.

In a separate group of LDBVL mice,we performed a test-retest study (table S2).Reproducibility was evaluated in sevenLDBVL mice by scanning each mousetwice, with an interval of 24 hours be-tween scans, 13 days after BM instillation.Paired t tests revealed no statistically sig-nificant difference between the values of% ID/cc between test and retest for bothlung and muscle regions of interest (P =0.8262). Reproducibility was calculatedby using the % ID/cc of the first scann-ing segment in lung or muscle, as 100 ×[% ID/cc (test)−% ID/cc (retest)]/% ID/cc(test).Within-subject test-retest variabil-ity was <12% in the lungs and <7% inmuscle. The intraclass correlation co-efficient, which is a measure of the relia-

bility of within-subject data relative to between-subject data, was 0.93for lung analysis and 0.99 for muscle analysis (34). Reproducible mea-surements inmouse lungs are challenging to obtain because of the var-iation in lung density within individual mice. Breathing rate andposition of the subject in the scanner are also factors influencing re-producible measurement in the lungs (35). Nevertheless, our test-retestexperiment illustrates that quantitation of fibrosis with 68Ga-CBP8can yield reproducible results.

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Fig. 1. Probe 68Ga-CBP8 monitors disease progression in the BM mouse model of pulmonary fibrosis. (A) Repre-sentative images of lung tissue stainedwith hematoxylin and eosin (H&E) and Sirius Red (magnification, ×10; scale bar, 300 mm)for sham and BM-treated mice at days 7 and 14 after BM instillation; higher-magnification views are also displayed (×40;scale bar, 60 mm). (B to D) Disease progresses in a stepwise fashion as determined by histological Ashcroft scoring of lungfibrosis (B), by histological quantification of the area affected by disease (C), and by hydroxyproline analysis (D). (E) Ex vivolunguptake of 68Ga-CBP8 increaseswith disease progression in the BM-treatedmice: 5-fold higher in BMmice 14 days afterinstillation than in sham animals and 1.5-fold higher in BM mice 14 days after instillation than in BM mice 7 days afterinstillation. (F) Correlation of ex vivo 68Ga-CBP8 lung uptake and Ashcroft score. (G) Correlation between ex vivo lunguptake of 68Ga-CBP8 and lung hydroxyproline. Data were analyzed with one-way analysis of variance (ANOVA), followedby post hoc Tukey tests with two-tailed distribution. For (B) to (F), data are displayed as box plots, with the dark band insidethe box representing the mean, the bottom and top of the box representing the first and third quartiles, and the whiskersrepresenting theminimum andmaximum values. For all of the experiments shown in this figure: n = 11 for sham, n = 4 forBM-treated mice at day 7, and n = 7 for BM-treated mice at day 14.

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68Ga-CBP8 PET allows monitoring of the response toantifibrotic therapy in miceWe monitored the antifibrotic effects of 3G9, a murine antibody tar-geted to integrin avb6, with

68Ga-CBP8 PET. The humanized versionof 3G9, STX-100 (Biogen), has successfully completed phase 1 clinicaltesting and is now under investigation in a phase 2 clinical trial of IPF(ClinicalTrials.gov identifier: NCT01371305). Integrin avb6 is a key ac-tivator of transforming growth factor–b (TGF-b) signaling in the lung(12, 13), and specific targeting of avb6 with 3G9 can partially prevent


pulmonary fibrosis in mice without sys-temic disruption of other homeostaticroles of TGF-b (36, 37).

Mice injured with a low dose of BM andthe vascular leak agent were treated withthe anti-avb6 antibody 3G9 (LDBVL +3G9 group) or the irrelevant isotypic anti-body 1E6 (LDBVL + 1E6 group) (37). Here,a control group of mice received the 3G9antibody only (3G9 group). FTY720 wasadministered intraperitoneally to themice at 1 mg/kg three times a week start-ing on day 0. Both 3G9 (anti-avb6 anti-body) and the control antibody 1E6 wereinjected into mice intraperitoneally threetimes per week at a concentration of1 mg/kg starting on day 0. PET-CT imag-ing, biodistribution, and hydroxyprolineanalysis were performed on LDBVL +3G9, LDBVL + 1E6, and 3G9mice 14 daysafter the initiation of administration of thedifferent compounds (n = 11, 11, and 5,respectively), and data were compared tothose for the LDBVL group (n = 9) shownin Fig. 3.

Treatment with the therapeutic anti-body 3G9 reduced pulmonary fibrosisin the LDBVL group as assessed by his-tology (Fig. 4A) and by hydroxyprolinequantification (Fig. 4C). LDBVL miceshowed an average fibrosis score of 6(±0.2; range, 4 to 7), whereas LDBVL +3G9 mice had an average score of 4(±0.3; range, 1 to 6), LDBVL + 1E6 miceshowed an average fibrosis score of 6(±0.3; range, 5 to 7), and mice treatedwith 3G9 alone had histologically normallungs with a score of 0. Fourteen percentof the total area of right lung tissuesectionswas affected by disease in LDBVLmice, whereas only 7% was affected forthe LDBVL + 3G9 group and 20% forLDBVL+1E6 animals. Therewas no lungarea affected by disease in 3G9-treatedanimals. Hydroxyproline analysis of theleft lung showed significantly lower valuesin lungs of LDBVL+ 3G9mice comparedto those of LDBVL and LDBVL + 1E6mice (83.8 ± 2.7 mg per left lung forLDBVL + 3G9 mice, 116.5 ± 4.9 mg per

left lung for LDBVL mice, and 108.9 ± 3.5 mg per left lung for LDBVL +1E6 mice). There were no significant differences in hydroxyprolinecontent between LDBVL + 3G9–treated and 3G9-treatedmice or be-tween LDBVL and LDBVL + 1E6 mice.

Similar effects of 3G9 treatmentwere also seen by lung PET imaging,where the lung PET signal was similar to what was seen in control miceand significantly lower than what was observed in the fibrotic group(Fig. 4B). Quantification of the 68Ga-CBP8 PET signal showed 1.5 timeshigher lung uptake in the LDBVL animals that received the irrelevant

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Fig. 2. 68Ga-CBP8 is specifically takenupby fibrotic but not healthy lungs (BMmodel). (A) Representative fused PET-CT images show specific accumulation of 68Ga-CBP8 (left images) in fibrotic but not in control lungs; 68Ga-CBP12 (rightimages) showed no preferential uptake in the lungs of BM-treatedmice compared to control mice. Grayscale image showsCT image, and color scale image shows PET image from integrated data 50 to 80 min after probe injection. (B) Hydroxy-proline level was significantly higher in fibrotic BM-treated than in sham-treated animals. (C) PET activity values for 68Ga-CBP8and 68Ga-CBP12 in sham- and BM-treated mice (50 to 80 min after injection). Data are expressed as percent injecteddose per cubic centimeter of tissue (% ID/cc). Data show significantly higher uptake in fibrotic lungs with 68Ga-CBP8.(D) Ex vivo uptake of 68Ga-CBP8 and 68Ga-CBP12 in lungs from sham- and BM-treated mice 150 min after injectionexpressed as % ID/lung. (E) Distribution of 68Ga-CBP8 and 68Ga-CBP12, expressed as percent injected dose per gram oftissue (% ID/g) (mean ± SE) in various organs, was similar except in fibrotic lungs. (Inset) Same data as in (E) expressed as %ID/lung. Data were analyzed using one-way ANOVA, followed by post hoc Tukey tests with two-tailed distribution. Forall of the experiments shown in this figure: n = 4 for sham injected with 68Ga-CBP12, n = 4 for BM-treatedmice injectedwith 68Ga-CBP12, n = 11 for sham injected with 68Ga-CBP8, and n = 7 for BM-treated mice injected with 68Ga-CBP8. For(B) to (D), data are displayed as box plots, with the dark band inside the box representing themean, the bottom and top ofthe box representing the first and third quartiles, and the whiskers representing the minimum and maximum values.

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antibody (fibrotic mice; LDBVL + 1E6, 1.00 ± 0.08% ID/cc) than inmice of the LDBVL group treated with the anti-avb6 antibody(LDBVL + 3G9, 0.63 ± 0.06% ID/cc; P = 0.0017) (Fig. 4D). Organuptake measured by ex vivo analysis was not significantly differentamong groups, except for the lungs where there was three times moreuptake in the LDBVL + 1E6 group than in the LDBVL + 3G9 group(P=0.0004) and six times higher uptake in the LDBVL+1E6 group thanin the 3G9-treated group (P < 0.0001) (Fig. 4E and fig. S10). Lung uptakeof 68Ga-CBP8 was not statistically different between the LDBVL + 3G9group and the 3G9-treated group (P = 0.4524). These results illustratethat 68Ga-CBP8 enables noninvasivemonitoring of in vivo responses totherapeutic interventions in animals affected by pulmonary fibrosis.

Probe 68Ga-CBP8 allows monitoring of collagen content inhuman IPF lung samplesTo determine whether our findings could be translated to human tis-sues, we evaluated the binding of the probe 68Ga-CBP8 to fresh humanlung tissues from three IPF patients undergoing pneumonectomybefore lung transplantation (figs. S11 to S13). Lung tissues werecollected fresh within 1 hour of resection. Three sampling areas weredefined in the lung of the patient [the superior upper lobe (S1), the lat-eral upper lobe (S2), and the lower lobe (S3)], and chunks of tissuesweresampled in these three areas.We incubated samples from S1, S2, and S3(70 to 100 mg) with 68Ga-CBP8 for 2 hours (n = 5 per area) and quan-tified the probe uptake. Once 68Ga-CBP8 radioactivity had decayed, weincubated the samples with 68Ga-CBP12 for 2 hours and quantified68Ga-CBP12 uptake. The uptake of 68Ga-CBP8 in human lung samples


showed differences in collagen concentra-tion among lobes in the three IPF patientsand heterogeneity of disease progressionamong patients (Fig. 5A). 68Ga-CBP12uptake was significantly lower than 68Ga-CBP8 uptake in all samples. We per-formed histological analyses on samplesjust adjacent to those used for probe in-cubation (Fig. 5B). Uptake of 68Ga-CBP8in IPF human lung tissue samplesincreased linearly with increasing fibro-sis, asmeasured by Sirius Red quantifica-tion of the area affected by disease (r2 =0.94, P < 0.0001) (Fig. 5C). The 68Ga-CBP12 signal did not change with colla-gen concentration in IPF human tissuesamples, demonstrating the specificity of68Ga-CBP8 for collagen.

DISCUSSIONFibrosis, regardless of its cause or location,is characterized by excess deposition ofcollagens, primarily type I collagen, andother extracellular matrix proteins in theparenchyma (20). Histological proof offibrosis is predicated on collagen staining.Here, we developed a method for directmolecular PET imaging of type I collagenas a noninvasive means of detecting andmonitoring pulmonary fibrosis. Themajoradvantages of PET are that it enables in

vivo visualization ofmolecular physiological processes in real timewhileat the same time allowing quantification bymeasuring regional concen-tration of the radiation source. Themass dose of 68Ga-CBP8 that wouldbe required for administration is extremely low (<100 mg per humansubject) and, like most PET probes, is very safe because of the ultralowdose. Targeted PET probes typically have affinities in the low nanomolarrange, but such probes typically bind sparse targets like cell surface re-ceptors present at nanomolar concentrations. Our probe 68Ga-CBP8 haslowmicromolar affinity to type I collagen, but this is sufficient for quan-tification because collagen is present in tens of micromolar concentra-tions during pathological fibrosis.

To generate our probe, we modified a known collagen-specificpeptide with a chelator so that it binds the 68Ga ion. Gallium-68 is apositron-emitting radioisotope that is produced from a 68Ge/68Gagenerator system, and therefore 68Ga radiopharmacy does not requirean on-site cyclotron, similar to 99Mo/99mTc-based radiopharmacy. Thechelation reaction to introduce 68Ga is quantitative, driven by the highthermodynamic stability of the resultant 68Ga complex. The 68-minhalf-life of 68Ga permits facile production of the probe, and the shorthalf-life results in low radiation exposure to the subject. The radio-chemistry is amenable to kit formulation and/or automation, with a lowbarrier to clinical translation. There has been a tremendous increase in thenumber of clinical studies with 68Ga probes over the past year around theworld, including within the United States (38).

We demonstrated that PET imaging with our collagen-targetedprobe, 68Ga-CBP8, is sensitive enough to detect pulmonary fibrosisin the commonly used BM mouse model. Our study showed that

FTYLDB

LDBVL0

40

80

120

160 P < 0.0001

Hyd

roxy

pro

line

(μ

g/l

un

g)

P = 0.4057

FTYLDB

LDBVL0.0

0.5

1.0

1.5

% ID

/cc

P < 0.0001

P = 0.9156

P < 0.0001

FTYLDB

LDBVL0.00

0.03

0.06

0.09

0.12

% ID

/lu

ng

P < 0.0001

P = 0.8202

P < 0.0001

FTYLDB

LDBVL0

2

4

6

8

Ash

cro

ft s

core

P < 0.0001

30 60 90 120 1500.0

0.5

1.0

1.5

r2 = 0.86P < 0.0001

LDBVLLDBFTY

Hydroxyproline (μg/lung)

% ID

/cc

FTYLDB

LDBVL0

12

24

36

48

Sir

ius

Re

d s

tain

ing

(%

to

tal a

ffe

cte

d a

rea)

P = 0.0014

30 60 90 120 1500.00

0.04

0.08

0.12

Hydroxyproline(μg/lung)

% ID

/lu

ng

r2 = 0.93P < 0.0001

LDBFTY

LDBVL

A B C D

E F G H

Vascular leak agentthree IP injections/week

Day 14 = Imaging + sacrifice

Day 0

= Low-dose BMinjection

Fig. 3. 68Ga-CBP8 detects and stages disease in a mouse model of pulmonary fibrosis with enhanced vascular per-meability. (A) Treatment scheme for the LDBVL group, whichwas treatedwith a low dose of BM (0.1 U/kg) and the vascularleak agent FTY720. (B toF) Micewere treatedwith a very lowdose of BM (LDB), the vascular leak agent FTY720 (FTY), or both,administered together (LDBVL). The LDBVL group, but not the LDBor FTYgroup, exhibited a fibrotic response as determinedby histological Ashcroft scoring of lung fibrosis (B), by Sirius Red staining (% total area affected by disease) (C), and byhydroxyproline analysis (D). Data show significantly higher uptake of 68Ga-CBP8 in the lungs of LDBVL animals comparedto FTY or LDB animals, as quantified by PET data (% ID/cc) (E) and by ex vivo uptake (% ID/lung) (F). (G and H) Correlationsbetween hydroxyproline levels and % ID/cc (G) and % ID/lung (H). Data were analyzed using one-way ANOVA, followed bypost hoc Tukey testswith two-tailed distribution. For all of the experiments shown in this figure: n=6 for the FTYgroup, n=4for the LDB group, and n=9 for the LDBVL group. For (B) to (F), data are displayed as box plots, with the dark band inside thebox representing the mean, the bottom and top of the box representing the first and third quartiles, and the whiskersrepresenting the minimum and maximum values.

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68Ga-CBP8 provided significantly enhanced PET signal in the lungs offibrotic mice compared with control mice. Nonspecific uptake in thesurrounding tissues (such as muscle, heart, and bone) was similar andlow in both fibrotic and control mice. The highest off-target accumu-lation was in the kidney, but the short half-life of gallium will limit theamount of radiation exposure (39).

Whenwe expanded the 68Ga-CBP8 study to includemice at an earlystage of fibrosis, we observed a strong correlation of lung uptake of theprobewith hydroxyproline content, indicating the ability of the probe tostage pulmonary fibrosis. The control probe, 68Ga-CBP12, showedsimilar low uptake in the lungs of both fibrotic and control mice, dem-


onstrating that the result observed with68Ga-CBP8 was a result of specific colla-gen binding.

We further validated the probe in a sec-ondmodel of pulmonary fibrosis withmicein which a host response to injury was ex-aggerated by increasing the animals’ vascu-lar permeability. Here, a low-dose BMchallenge plus a vascular leak–inducingagent produces robust fibrosis, whereasthe low-dose BM challenge alone causesonly a mild, self-limited injury in controlmice (33, 40). This model captures an im-portant aspect of IPF pathogenesis, inwhich fibrosis is thought to result froman aberrant or exaggerated host responsesto relatively common and/or mild envi-ronmental injuries, whichmaybewell tol-erated and repaired in most people. Here,PET imaging of collagen with 68Ga-CBP8successfully identified animals with pul-monary fibrosis and, additionally, de-tected blunted disease progression inanimals treated with 3G9, a murine anti-body targeted to integrin avb6 that inter-feres with TGF-b activation, leading toreduced TGF-b signaling.

Our data raise the possibility that68Ga-CBP8 more readily/avidly bindsnewly formed collagen in active diseasethan it does in established, mature colla-gen. For instance,we see relatively lowup-take of 68Ga-CBP8 in bone or skin, whichare rich in type I collagen. The lungs them-selves are rich in collagen, and by directbiochemical measurement, lung collagenincreases two- to threefold inmousemodelsof pulmonary fibrosis. We nevertheless seea sixfold higher probe uptake in fibroticmouse lungs compared to control lungs,suggestive of higher binding to the col-lagen newly formed during fibrosis. Sim-ilar observations were made in othermodels of fibrosis with an analogous MRIprobe (21–26).

There are two factors that could con-tribute to the specificity of the probe fornewly formed collagen and active fibrosis.

The first relates to the structural nature of type I collagen. A single col-lagen molecule (tropocollagen) is made of three polypeptide strandsthat assemble into a microfibril triple helix. In normal tissue, these mi-crofibrils interdigitate, are cross-linked, and form stable fibrils (41).Therefore, most of the collagen monomers within these fibrils are in-accessible to a molecular probe like 68Ga-CBP8 because most mono-mers will be inside the fibril and the probe can only access the fibrilsurface. However, in active fibrosis, collagen production is up-regulated,and new collagen is not yet fully organized into stable fibrils. As aresult, collagen monomers are more accessible to the probe. If con-firmed, this property may augment the ability of 68Ga-CBP8 to provide

0

% ID/ccLDBVL LDBVL + 3G9

B

H&E

3G9/cc

33GG99 LDBVL + 3G9

3G950

80

110

140

170P = 0.2281

P < 0.0001P < 0.0001

P < 0.0001

0.0

0.5

1.0

1.5

2.0

% ID

/cc

P = 0.6590

P = 0061P = 0.0017

P = 0.0007

0.00

0.05

0.10

0.15

0.20%

ID/l

un

gP = 0.4524

P = 0.0298

P = 0.0004

P < 0.0001C D E

=5LDBVL + 1E6

LDBVL

LDBVL + 3

G9

LDBVL + 1

E6

3G9

LDBVL

LDBVL + 3

G9 3G9

LDBVL

LDBVL + 3

G9

LDBVL + 1

E6

LDBVL + 1

E6

Hyd

roxy

pro

line

(μ

g/l

un

g)

3G9 LDBVL LDBVL + 3G9 LDBVL + 1E6A

Sirius Red

Fig. 4. 68Ga-CBP8 PET allows monitoring of the response to antifibrotic therapy in mice. (A) Representative imagesof lung tissue stainedwith H&E and Sirius Red (magnification, ×10; scale bar, 300 mm) formice treatedwith 3G9 only (n= 5),formice treatedwith a lowdose of BM and FTY720 (LDBVL; n= 9), for LDBVLmice receiving 3G9 (LDBVL + 3G9; n= 11), andfor LDBVL mice receiving 1E6 (LDBVL + 1E6; n = 11); higher-magnification views are also displayed (×40; scale bar, 60 mm).(B) Representative fused PET-CT images show specific accumulation of 68Ga-CBP8 in the lungs ofmice from the LDBVL andLDBVL + 1E6 groups but low PET signal in the lungs of the control 3G9 or treated LDBVL + 3G9 groups. Grayscale imageshows CT image, and color scale image shows PET image from integrated data 50 to 80 min after probe injection. (C) Hy-droxyproline content was significantly higher in animals from the LDBVL and LDBVL + 1E6 groups than in those from the3G9 and LDBVL + 3G9 groups. (D and E) Similar effects were seen for quantitative PET data (D) and ex vivo uptake in thelungs. Data were analyzed using one-way ANOVA, followed by post hoc Tukey tests with two-tailed distribution. For (C) to(E), dataaredisplayedasboxplots,with thedark band inside thebox representing themean, thebottomand topof theboxrepresenting the first and third quartiles, and the whiskers representing the minimum andmaximum values. For all of theexperiments shown in this figure: n=5 for the 3G9group, n=9 for the LDBVL group,n=11 for the LDBVL+ 3G9group, andn = 11 for the LDBVL + 1E6 group.

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amuch-needed assessment of “disease activity” in IPF and other fibroticdiseases.

Whereas HRCT can accurately measure an IPF patient’s disease se-verity at the time of evaluation, this imagingmodality and other currentlyavailable measures to monitor IPF patients provide little informationabout disease activity (that is, how likely that patient’s fibrosis is toworsenin the upcoming weeks and months). If the ability of 68Ga-CBP8 molec-ular imaging to assess disease activity in pulmonary fibrosis patients isvalidated, it could improve patient care and clinical research in IPF inseveral important ways. Progress in IPF patient care and clinical researchhas been hampered by the substantial heterogeneity of the disease. Al-though IPF has a very poor prognosis overall, its clinical course in differ-ent patients is highly variable (3). Whereas some patients have activedisease that quickly progresses to respiratory failure, others have mini-mally active disease that remains clinically stable for long periods of time(3). The ability to distinguish IPF patients who are worsening rapidlyfrom those who are remaining stable would enable clinicians to individ-ualize patient care plans. Being able to make this distinction would alsoallow clinical trials of new IPF therapies to enroll subjects more likely toprogress during the period of observation, improving the ability of thesetrials to detect treatment effects. Additionally, because current assess-


ments of IPF usually take 6 or 12 monthsto showmeaningful changes, being able toassess disease activity would enable fasterassessments of patient responses to newtherapies.

68Ga-CBP8 has moderate affinity forcollagen (2.1 mM), which may also resultin better sensitivity to fibrosis. For instance,we measured 50 mg of hydroxyproline inthe left lung of control mice. Assuming thatall the hydroxyproline arises from collagenand that it comprises ~13% of collagen, thisresults in a collagen monomer concentra-tion of ~10 mM, which rises in excess of25 mM in fibrotic lungs. Thus, in normallung, the collagen concentration is at or be-low (depending on accessibility to the colla-gen) the Kd of the probe, which results inpoor uptake.However, in fibrosis, the targetconcentration is in excess of the Kd, result-ing in increased probe uptake and the ob-served collagen concentration–dependentuptakeof theprobe.The results of our studysuggest that 68Ga-CBP8 PETmay be a use-ful complement to HRCT for noninvasivecharacterization of pulmonary fibrosis.HRCT provides a good measure of fibroticburden, whereas 68Ga-CBP8 may highlightregions where fibrosis is more active.

An important limitation of the currentstudy is that BM fibrosis in the animallacks important features of the humandisease. The slow and irreversible pro-gression of fibrosis seen in IPF patientsis not reproduced in the BM model.Chronic diseases are notoriously difficultto model. IPF is particularly complicatedbecause the etiology andnatural history of

the disease is unclear, and no single trigger is known that is able to in-duce “IPF” in animal models.

To assess probe performance in human tissues, we incubated humanIPF lung tissue samples with 68Ga-CBP8 and were able to correlateprobe uptake with histological findings of IPF in these samples. 68Ga-CBP12 was not sensitive to collagen changes in IPF human tissuesamples, which demonstrates the specificity of 68Ga-CBP8 for collagen.Although this pilot study was limited to three patients, the data provideproof-of-principle evidence of the potential of 68Ga-CBP8 PET as animaging agent for pulmonary fibrosis in humans. The human tissue datasupport the hypothesis that the probe is more sensitive to newer, lessorganized collagen. We found that uptake in a region of the lung thatwas characteristic of dense, established subpleural fibrosis with micro-scopic honeycomb change was higher than in a region reflective of end-stage fibroelastosis with cystic honeycomb change. This distinctionsuggested that as fibrosis becomes end stage, a larger fraction of thematrix is elastin and the collagen is more organized and less availablefor binding to our probe. The barrier to translate our collagen-bindingPET probe into clinic is very low. Unlike other imaging modalities (forexample, MRI and optical), the low mass dose required for PET allowsclinical translation under the U.S. Food andDrug Administration (FDA)

Upper

Mid

dle

Lower

0

1

2

3

% ID

/g

CBP12

CBP8

P = 0.0004

P < 0.0001

P < 0.0001

Upper

Mid

dle

Lower

0

2

4

6

8

% ID

/g

CBP12

CBP8

P = 0.0328

P < 0.0001

Upper

Mid

dle

Lower

0

1

2

3

% ID

/g

CBP12

CBP8

P =0.0196

P = 0.0012

P = 0.0005

0 20 40 600

2

4

6

8

% ID

/g

Sirius Red staining (% total area affected)

CBP8(r2 = 0.94, P < 0.0001)

CBP12

Patient 1 Patient 2 Patient 3A

B C

1 mm1 mm

Early stage f ibrosis Late stage f ibrosis

Fig. 5. Probe 68Ga-CBP8 signal reflects changes in collagen concentration in human IPF lung tissues. (A) Uptake of68Ga-CBP8 in human lung samples taken from the upper, middle, and lower lobes of explanted lung of IPF patients. Theuptake is defined as% ID/g lung tissue (mean±SE). Datawere analyzedusing one-wayANOVA, followedbypost hoc Tukeytests with two-tailed distribution. For patient 1, n = 5 (upper lobe), n = 5 (middle lobe), and n = 6 (lower lobe). For patient2,n= 8 (upper lobe), n=6 (middle lobe), andn=6 (lower lobe). For patient 3,n=5 (upper lobe), n=5 (middle lobe), andn=9 (lower lobe). (B) Representative images of Sirius Red staining of collagen showing early stage of IPF (left, lung sectiontaken from the upper lobe of patient 2) characterized by thickening of the alveolar septa and knot-like formation and latestageof fibrosis (right, lung section taken from the lower lobeof patient 2) characterizedby a denser deposition of collagen.(C) Correlation of 68Ga-CBP8 (n=9) and 68Ga-CBP12 (n= 9) uptake in lung tissue samples from IPF patientswith histologicalstaining of collagen, as measured by quantification of % collagen staining (Sirius Red) in the area affected by disease (for68Ga-CBP8, r2 = 0.94, P < 0.0001).

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exploratory investigational new drugmechanism on the basis of a singlehigh-dose acute toxicity study in rodents.

Recently, the FDA approved two different therapeutic agents forthe treatment of IPF in the United States, pirfenidone and nintedanib,largely based on results from the CAPACITY (42), ASCEND (43), andINPULSIS trials (44). Both drugs slow functional decline and diseaseprogression and reduce long-term mortality in patients with mild tomoderate IPF, highlighting the importance of early diagnosis and ini-tiation of treatment. The usefulness of these drugs in other forms ofpulmonary fibrosis is starting to be evaluated, including scleroderma-associated interstitial lung disease, chronic hypersensitivity pneumonitis,and radiation pneumonitis. If current antifibrotic therapy can slowfunctional decline and disease progression in fibrotic lung diseases, thenearly detection of pulmonary fibrosis of any cause and initiation oftreatment could benefit patients across this broad category of lung dis-eases. Because lung architectural distortion may be absent early in thecourse of fibrotic lung diseases, HRCT often cannot distinguish fibrosisfrom other pathological processes in the lung. In this regard, our resultsmay represent a consistent and reliable noninvasive method to detectexcess collagen deposition in the lungs, even early in the course of pul-monary fibrosis. This ability of 68Ga-CBP8 molecular imaging, as wellas the potential application to assess the rate of progression, or diseaseactivity of pulmonary fibrosis, suggests that translation of this approachto human will benefit patients with IPF and other fibrotic lung diseases.


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MATERIALS AND METHODSStudy designThe objectives of this study were to address the clinically unmet need ofnoninvasive tools to diagnose IPF at early stage, to quantify and stagefibrosis, and tomonitor disease progression. These goals were addressedby (i) evaluating the efficacy and specificity of the probe to detect andstage disease in an established animal model for pulmonary fibrosis(BM), (ii) evaluating the probe efficacy to detect disease and its capacitytomonitor treatment response in a secondmodel of pulmonary fibrosisusing mice in which a host response to injury has been exaggerated byincreased vascular permeability, (iii) and initiating a pilot study totranslate these findings using human IPF tissue samples.

All experiments were performed in accordance with the NationalInstitutes of Health Guide for the Care and Use of Laboratory Animals,with theARRIVE (Animal Research: Reporting of InVivo Experiments)guidelines (45), and were approved by the institution’s animal care anduse committee. Evaluation of 68Ga-CBP8 and 68Ga-CBP12 uptakemea-surements in animals was performed in a nonblinded fashion. Authorswere blinded for histology analysis. Because 68Ga-CBP8 microPET im-aging is a new imaging technology, it is difficult to estimate sample sizewith adequate power.Ann=4 to 13was selected for thesewell-controlledmodels with a low (<10%) error in consecutive studies (table S2). Bindingstudies were conducted using three independent experiments. No sam-ples or animals were excluded from data analyses.

Fresh lung tissue samples were collected from explanted IPF lungsobtained from three IPF patients undergoing transplantation. The Part-nersHumanResearchCommittee Institutional ReviewBoard approvedthis study. Informed consent was obtained from the patients.

Synthesis of precursors and nonradioactive standardsAll chemicals were purchased commercially and used without furtherpurification. The cyclic disulfide peptide precursors of CBP8 andCBP12 [cPep(8) and cPep(12)] were custom-made by American


Peptide. The tri-tert-butyl–protected activated ester NODAGA-N-hydroxysuccinimide [(tBu)3NODAGA-NHS]was synthesized in-housefollowing a publishedprocedure (46) starting from1,4,7-triazacyclononane(CheMatech).(tBu)3NODAGA-cPep(x).Three equivalents of (tBu)3NODAGA-NHS were added to solution ofcPep(x) (x = 8, 12) in 1 ml of dimethylformamide. The pH of eachsolution was adjusted to 6.5 using diisopropylethylamine, and the mix-tures were stirred at room temperature for 24 hours. (tBu)3NODAGA-cPep(8) and (tBu)3NODAGA-cPep(12) were purified separately byreversed-phase semipreparative purification [Dynamax HPLC system,Phenomenex Luna C18 column (5 mm; 250 mm × 21.2 mm), water/acetonitrile gradient (85:15, 60:40 in 30 min) containing 0.1% (v/v) tri-fluoroacetic acid (TFA); the absorbance at 280 nm was monitored fordetection]. The two products had a purity of >98%, as determined byliquid chromatography–mass spectrometry (LC-MS) [Agilent 1100 Se-ries apparatus with an LC/MSD trap and Daly conversion dynode de-tector with ultraviolet (UV) detection at 220, 254, and 280 nm;Phenomenex Kinetex C18 column (2.6 mm; 100 mm × 4.6 mm);water/acetonitrile gradient (95:5, 5:95 in 10 min) containing 0.1%(v/v) formic acid]. (tBu)3NODAGA-cPep(8): molecular weight forC193H285N37O43S2. MS(ESI) calculation: 970.0 [(M + 4H)/4]4+; found:969.9. (tBu)3NODAGA-cPep(12):molecularweight forC193H285N37O43S2.MS(ESI) calculation: 970.0 [(M + 4H)/4]4+; found: 969.7.NODAGA-cPep(x).In two separate reaction vessels, about 20 mg of (tBu)3NODAGA-cPep(x) (x = 8, 12) was dissolved in a 1-ml solution of TFA,methanesulfonicacid, 1-dodecanethiol, and H2O (92:3:3:2). Each reaction mixture wasstirred for 2 hours. Cold diethyl ether was added to precipitate out thesolids. The mixtures were centrifuged, and the supernatant was re-moved. The solids were washed with diethyl ether and dried to givethe product as white solids. NODAGA-cPep(8) and NODAGA-cPep(12)were purified by reversed-phase semipreparative purification. The twoproducts had a purity of >98%, as determined by LC-MS. NODAGA-cPep(8): molecular weight for C157H213N37O43S2. MS(ESI) calcu-lation: 843.8 [(M + 4H)/4]4+; found: 843.4. NODAGA-cPep(12):molecular weight for C157H213N37O43S2. MS(ESI) calculation: 843.8[(M + 4H)/4]4+; found: 843.5.69/71Ga-NODAGA-cPep(8).Three milligrams of NODAGA-cPep(8) were dissolved in 2 ml ofsodium acetate buffer (20 mM, pH 4.1). A sample of 296 ml of a 3 mM69/71Ga(NO3)3 was added in the vessel, and the reaction mixture wasstirred at 60°C for 20 min. 69/71Ga-NODAGA-cPep(8) was purifiedby reversed-phase semipreparative HPLC. The product had a puri-ty of >98%, as determined by LC-MS. Molecular weight forC157H210GaN37O43S2. MS(ESI) calculation: 860.3 [(M + 4H)/4]4+;found: 860.5.69/71Ga-NODAGA-cPep(12).FivemilligramsofNODAGA-cPep(12)weredissolved in 2ml of sodiumacetatebuffer (20mM,pH4.1).Asampleof1.5mlof a3mM69/71Ga(NO3)3was added in the vessel, and the reaction mixture was stirred at 60°Cfor 20 min. 69/71Ga-NODAGA-cPep(12) was purified by reversed-phasesemipreparativeHPLC.The product had a purity of >98%, as determinedby LC-MS.Molecular weight for C157H210GaN37O43S2. MS(ESI) calcula-tion: 860.3 [(M + 4H)/4]4+; found: 860.5.

Radiosynthesis68GaCl3 was obtained from a SnO2-based

68Ge/68Ga generator(iThemba LABS). 68GaCl3 [10 mCi, in 0.5 ml of HCl (0.6 M)] was

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diluted with 200 ml of pH 5 sodium acetate (3 M) to reach pH 4.1. Asample of 180 ml of the 68GaCl3 solution was combined to 5 ml of a0.1mM(NODAGA)-cPep(8) or (NODAGA)-cPep(12) solution [in so-dium acetate (pH 4.1)], and the reactionmixture was heated at 60°C for5 min and purified by Sep-Pak C18 cartridge (Waters) to remove anyradiometal impurities (germanium-68 breakthrough). The radiochemi-cal purity of the final solution of CBP8 and CBP12 was ≥99%, asdetermined by radio-HPLC analysis [Agilent 1100 Series HPLC unitwith a Carroll/Ramsey radiation detector with a silicon PIN photodiodeand with UV detection at 254 nm, Phenomenex Luna C18 column(2.6 mm; 100 mm × 4.6 mm), water/acetonitrile gradient (95:5, 5:95in 10 min) containing 0.1% (v/v) TFA]. Specific activities ranged from324 to 462 GBq/mmol.

Collagen bindingBinding isotherms were obtained using the cold version of the probes(69/71Ga-CBP8 or 69/71Ga-CBP12) by following a method previouslyreported (21).

Animal model and probe administrationIn the standard-dose BM model (47), pulmonary fibrosis was inducedin 10-week-old male C57/BL6 mice (20 to 28 g; Charles River Labora-tories) by transtracheal administration of BM (2.5 U/kg) in 50 ml ofphosphate-buffered saline (PBS) under direct vision using a small cer-vical incision. In the low-dose BMmodel associated with a vascular leakagent (FTY720) (33), adult male C57Bl/6 mice, 7 to 8 weeks old (~25 g;Charles River Laboratories), were administered a single intratrachealdose of BM at 0.1 U/kg (low dose), in a total volume of 50 ml of sterilesaline. FTY720was administered intraperitoneally to themice at 1mg/kgthree times a week. To effect treatment in the low-dose BM, vascularleak model, the therapeutic murine antibody 3G9 (anti–avb6-blockingantibody) and 1E6 (matched isotype control antibody) were used (48).All administrations of FTY720 and antibodies were initiated on day 0,~30 min before BM challenge, and continued throughout the durationof the experiments.

Small-animal PET-CT imaging and analysisAnimals were placed in a small-animal PET/SPECT/CT scanner(Triumph, TriFoil Imaging), equipped with inhalation anesthesia andheating pad. Each animal was anesthetized with isoflurane (4% for in-duction, 1 to 1.5% for maintenance in medical air). After placement ofan in-dwelling catheter in the femoral vein for probe administration,mice were positioned in the PET-CT and probe was given as a bolus.Dynamic imaging was performed for 120 min for sham and BM mice(day 14) injected with 68Ga-CBP8 or 68Ga-CBP12. Static imaging wasperformed on mice treated with BM (day 7), FTY alone, LDB, LDBVL,LDBVL+ 3G9, and LDBVL+ 1E6, and acquisitions were collected 50minafter 68Ga-CBP8 injection for a duration of 30 min. A whole-body CTwas obtained either immediately before or immediately after the PETacquisition, the mice were then euthanized at 150 min after injection,and the organs were taken for biodistribution analysis. Instrument cal-ibration was performed with phantoms containing small knownamounts of radioactivity. Isotropic (0.3-mm) CT images were acquiredover 6 min with 512 projections with three frames per projection (expo-sure time per frame, ~200 ms; peak tube voltage, 70 kV; tube current,177 mA). PET and CT images were reconstructed using the LabPETsoftware (TriFoil Imaging), and the CT data were used to provide atten-uation correction for the PET reconstructions. The PET data were re-constructed using a maximum-likelihood expectation-maximization


algorithm run over 30 iterations to a voxel size of 0.5 × 0.5 × 0.6mm3.For the pharmacokinetic analyses, the PET data were reconstructedin 1-min (first 10 frames), 3-min (next 10 frames), and 10-min (last 8to 10 frames) intervals out to 120 min after injection. ReconstructedPET-CT data were quantitatively evaluated using AMIDE softwarepackage (49). For each PET scan, volumes of interest (VOIs) weredrawn over major organs on decay-corrected whole-body coronalimages. The radioactivity concentration within organs was obtainedfrom mean pixel values within the VOI and converted to counts permilliliter per minute and then divided by the injected dose (ID) to ob-tain an imagingVOI-derived percentage of the injected radioactive doseper cubic centimeter of tissue (%ID/cc). Sizeof theVOIwas about65mm3

for the lungs, 53 mm3 for the heart, and 20 mm3 for the muscle.

Biodistribution protocolThe left lung, blood, urine, heart, liver, left rectus femorismuscle, spleen,small intestine, kidneys, and left femur bone were collected from allanimals. The tissues were weighed, and radioactivity in each tissuewasmeasured on a gammacounter (Wizard2AutoGamma, PerkinElmer).Tracer distribution was presented as % ID/gram for all organs. Theradioactivity in the lung was reported as % ID/lung.

Human lung tissues were collected within 1 hour of resection.Samples were weighed (70 to 100 mg per sample), cut in smallpieces, and introduced in an Eppendorf tube containing a solution of68Ga-CBP8 (50 to 75 kBq) in PBS (1 ml). The mixture was shakenfor 2 hours at room temperature. After centrifugation, supernatant(sup-1) was removed and kept for further analysis, and tissue sampleswere washed with 1 ml of PBS; this procedure was repeated twice foreach sample.Wash solutions (wash-1 andwash-2) were kept for furtheranalysis. Activities in tissue samples and in solutions sol-1, wash-1, andwash-2 were measured on a gamma counter. Activity in the tissue waspresented as% lung uptake, defined by activity in the tissuemeasured bygamma counter divided by total of activity (from tissue, sup-1, wash-1,and wash-2).

Ex vivo analysisMouse lung tissues (right lungs) were inflated and fixed with 10% for-malin, embedded in paraffin, and cut into 5-mm sections. Tissuesections were stained with H&E and Picrosirius Red with a counter-stain of Fast Green (Fig. 4A). Images were acquired using a NikonTE2000 microscope (×10 and ×40 magnification). Sirius Red–stainedsections were analyzed by a pathologist, who was blinded to the study,to score the amount of lung disease according to the method ofAshcroft et al. (28). In addition, % Sirius Red was quantified fromthe histology images using ImageJ as per our standard procedures (26).Human lung tissues were obtained just adjacent to fresh tissues for probeincubation and were fixed in 10% formalin, embedded in paraffin,and cut into 5-mm sections. Tissue sections were stained with H&E,Picrosirius Red, Masson trichrome, and Verhoeff’s elastic stain. Slideswere scanned using a digital slide scanner (Aperio CS2, Leica Biosystems).

Quantification of collagenHydroxyproline in tissue was quantified by HPLC analysis of tissueacid digests, as previously described. Hydroxyproline is expressed asamount per lung (50).

StatisticsUnless otherwise noted, the results are expressed as means ± SEM(with n = 4 to 13 mice per group). Statistical analyses were performed

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with GraphPad Prism 7 software. A one-way ANOVA, followed bypost hoc Tukey tests with two-tailed distribution, was used for analyz-ing the data between different groups. A P value of <0.05 wasconsidered significant.

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SUPPLEMENTARY MATERIALSwww.sciencetranslationalmedicine.org/cgi/content/full/9/384/eaaf4696/DC1Fig. S1. Structures of the collagen-binding probe CBP8 and the control probe CBP12 (L-4,4′-biphenylalanine).Fig. S2. Radio-HPLC of pure 68Ga-CBP8 and 68Ga-CBP12 were compared with the UV-HPLC ofthe cold gallium complexes.Fig. S3. Binding of Ga-CBP8 (black circles) and Ga-CBP12 (white circles) to type I rat tailcollagen at 37°C.Fig. S4. Binding of Ga-CBP8 (black triangles) and Ga-CBP12 (white triangles) to type I humancollagen at 37°C.Fig. S5. Radio-HPLC of pure 68Ga-CBP8 and 68Ga-CBP12 was compared with urine analysis 150 minafter probe injection.Fig. S6. Representative CT, PET, and fused PET-CT from integrated data 50 to 80 min after 68Ga-CBP8injection of a sham (n = 11) and a BM-treated (day 14; n = 7) mouse.Fig. S7. Time-activity curves of lung and muscle from dynamic PET imaging in sham and BM-treated mice after 68Ga-CBP8 and 68Ga-CBP12 injection.Fig. S8. Time-activity curves of heart and muscle from dynamic PET imaging in sham and BM-treated mice after 68Ga-CBP8 injection.Fig. S9. Ex vivo uptake of 68Ga-CBP8 in blood, lung, heart, liver, muscle, spleen, small intestine,kidneys, and bone 2.5 hours after probe injection in FTY, LDB, and LDBVL mice.Fig. S10. Ex vivo uptake of 68Ga-CBP8 in blood, lung, heart, liver, muscle, spleen, smallintestine, kidneys, and bone 2.5 hours after probe injection in 3G9, LDBVL, LDBVL + 3G9, andLDBVL + 1E6 mice.Fig. S11. Representative images of lung tissue stained with Sirius Red and most recent CT scanof patient 1 before transplant.Fig. S12. Representative images of lung tissue stained with Sirius Red and most recent CT scanof patient 2 before transplant.Fig. S13. Representative images of lung tissue stained with Sirius Red and most recent CT scanof patient 3 before transplant.Table S1. Lung-to-tissue ratio of 68Ga-CBP8 and 68Ga-CBP12 from ex vivo analysis of BM-treated mice (day 14) 2.5 hours after injection.Table S2. Original individual subject-level data from all experiments.

uest on October 1, 2020

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Acknowledgments: We thankO.M. Evbuomwanand J. Hooker for use of the 68Ge/68Ga generator;H. Chen, P. Waghorn, D. Dos Santos Ferreira, and H. Diyabalanage for assistance during animalsetup; S. E. Conley for scanning slides of human lung tissues; M. K. Selig for performing the electronmicroscopy imaging; and the Wellman Center for Photomedicine Photopathology core for thework on thehuman lung tissues.Funding: This studywas supportedby theNationalHeart and Lungand Blood Institute (R01HL116315). P.D. was supported by the Harvard/Massachusetts GeneralHospital (MGH) Nuclear Medicine Training program, which is funded by the U.S. Department ofEnergy (DE-SC0008430). We acknowledge instrumentation support from the National Center forResearch Resources (microPET/SPECT/CT system; RR029495) and the Office of Director(ICP-QQQ, OD010650). Author contributions: P.C. initially conceptualized this project andsupervised the entire project. P.D., L.F.T., M.L., A.M.T., L.P.H., and P.C. conceived the experiments.P.D. developed the synthesis and synthesized the radioactive probes. P.D., L.F.T., N.J.R., T.A.R.,C.K.P., F.B., and H.D. performed the animal experiments. P.D., L.F.T., B.C.F., and N.J.R. analyzed thedata. P.W. provided the antibodies. M.M.-K., B.C.F., and L.P.H. analyzed histological data.L.P.H. enabled the experiment with human tissues. P.D. wrote the manuscript draft, which wasthen further refined by P.C. All other authors reviewed and edited the intermediate and finalversions of the manuscript. Competing interests: P.C. has equity in Collagen Medical, thecompany that holds the patent rights to the peptides used in these probes. P.C. and B.C.F. arepaid consultants to Collagen Medical. S.M.V. and P.W. are employees of Biogen. P.C. and P.D.are inventors on patent/patent application (WO 2015196208) submitted byMGH that covers the68Ga-CBP8 probe. S.M.V. and P.W. are inventors on patent/patent application (US 7,465,449)submitted by Biogen and the University of California, San Francisco, that covers anti-avb6antibody 3G9. Data and materials availability: The patent on the 68Ga-CBP8 probe does notrestrict the research or noncommercial use of the probe technique. The 68Ga-CBP8 probe canbe obtained using the synthetic procedures described in the paper. Anti-avb6 antibodies wereprovided by Biogen to the investigators at MGH under a material transfer agreement. These antibodiesare available to other researchers under a material transfer agreement with Biogen.

Submitted 12 February 2016Resubmitted 20 October 2016Accepted 16 March 2017Published 5 April 201710.1126/scitranslmed.aaf4696

Citation: P. Désogère, L. F. Tapias, L. P. Hariri, N. J. Rotile, T. A. Rietz, C. K. Probst, F. Blasi, H. Day,M. Mino-Kenudson, P. Weinreb, S. M. Violette, B. C. Fuchs, A. M. Tager, M. Lanuti, P. Caravan,Type I collagen–targeted PET probe for pulmonary fibrosis detection and staging in preclinicalmodels. Sci. Transl. Med. 9, eaaf4696 (2017).

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preclinical modelstargeted PET probe for pulmonary fibrosis detection and staging in−Type I collagen

Lanuti and Peter CaravanBlasi, Helen Day, Mari Mino-Kenudson, Paul Weinreb, Shelia M. Violette, Bryan C. Fuchs, Andrew M. Tager, Michael Pauline Désogère, Luis F. Tapias, Lida P. Hariri, Nicholas J. Rotile, Tyson A. Rietz, Clemens K. Probst, Francesco

DOI: 10.1126/scitranslmed.aaf4696, eaaf4696.9Sci Transl Med

idiopathic pulmonary fibrosis, where higher probe uptake correlated with regions of increasing fibrosis.mouse models of bleomycin-induced pulmonary fibrosis and in samples of human lungs from patients withcollagen, an extracellular matrix protein present in fibrotic tissues. The probe detected fibrotic lung tissue in two Désogère and colleagues developed an imaging probe for positron emission tomography that detects type Iand skin, among other organs, it remains difficult to visualize and diagnose noninvasively. To address this,

Although fibrosis is known to play a role in the progression of multiple diseases, affecting heart, lung, liver,Focusing on fibrosis

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http://stm.sciencemag.org/content/9/384/eaaf4696#BIBL

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type i collagen targeted pet probe for pulmonary fibrosis ...pulmonary fibrosis is scarring of the...

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