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Imaging Active Urokinase PlasminogenActivator in Prostate CancerAaron M. LeBeau1, Natalia Sevillano2, Kate Markham3, Michael B.Winter2,Stephanie T. Murphy1, Daniel R. Hostetter3, James West3, Henry Lowman3,Charles S. Craik2, and Henry F. VanBrocklin1

Abstract

The increased proteolytic activity of membrane-bound andsecreted proteases on the surface of cancer cells and in thetransformed stroma is a common characteristic of aggressivemetastatic prostate cancer. We describe here the development ofan active site-specific probe for detecting a secreted peritumoralprotease expressed by cancer cells and the surrounding tumormicroenvironment. Using a human fragment antigen-bindingphage display library, we identified a human antibody termedU33 that selectively inhibited the active form of the proteaseurokinase plasminogen activator (uPA, PLAU). In the full-lengthimmunoglobulin form, U33 IgG labeled with near-infrared fluor-ophores or radionuclides allowed us to noninvasively detectactive uPA in prostate cancer xenograft models using optical andsingle-photon emission computed tomography imaging modal-

ities. U33 IgG labeled with 111In had a remarkable tumor uptakeof 43.2% injected dose per gram (%ID/g) 72 hours after tail veininjection of the radiolabeled probe in subcutaneous xenografts.In addition, U33 was able to image active uPA in small soft-tissue and osseous metastatic lesions using a cardiac dissem-ination prostate cancer model that recapitulated metastatichuman cancer. The favorable imaging properties were the directresult of U33 IgG internalization through an uPA receptor–mediated mechanism in which U33 mimicked the function ofthe endogenous inhibitor of uPA to gain entry into the cancercell. Overall, our imaging probe targets a prostate cancer–associated protease, through a unique mechanism, allowingfor the noninvasive preclinical imaging of prostate cancerlesions. Cancer Res; 75(7); 1225–35. �2015 AACR.

IntroductionProstate cancer afflicts men in the western world at rates greater

than any other malignancy, resulting in the deaths of approxi-mately 30,000men annually (1). Androgen ablation therapy is aneffective treatment for men with hormone-sensitive prostatecancer. Despite high initial response rates, a majority of menundergo androgen ablation relapse, leading to castration-resistantprostate cancer (CRPC; ref. 2). Recent drug approvals by the FDAhave demonstrated the impact that novel therapies can have on

improving the quality and quantity of life formenwith CRPC (3).Men with metastatic CRPC, however, are still likely to die fromthis disease and therefore, better therapies are needed. The devel-opment of novel therapies and the efficient use of establishedtherapies are hindered by poor measures of response. Sensitivenoninvasive imaging probes that identify cancerous lesions andmeasure cancer cell viability posttherapy would allow physiciansto rapidly assess treatment efficacy and provide personalized carefor men suffering from CRPC.

The ability to accurately imagemetastatic prostate cancer in softtissue and bone remains an unmet clinical need. Modalities suchas CT and MRI require large anatomic changes to be effective andprovide limited information regarding the underlying tumorphysiology (4).Dynamic hyperpolarized carbon-13MRI has beenused to characterize tumor metabolism in patients with organ-confined prostate cancer, but it is not yet available for imagingmetastases (5). Radionuclide bone scans, which measure boneremodeling, are common for evaluating metastatic cancer andtherapeutic response. However, false-positives are common withbone scans because remodeling can occur from preexistingbone trauma, inflammation, and arthritis (6). The FDA-approvedmetabolic imaging tracer, 18F-fluorodexoyglucose, has met withlimited success imaging prostate cancer because of the changingmetabolic signatures at different stages of the disease (7). Othermetabolic agents, including 11C-choline, 18F-fluorocholine, 11C-acetate, 11C-methionine, 18F-1-(2-deoxy-2-fluoro-ß-L-arabino-furanosyl)-5-methyluracil; FMAU), 1-amino-3-18F-fluorocyclo-butane-1-carboxylic acid (18F-FACBC), and 18F-fluorothymidine,for measuring membrane synthesis, fatty acid transport, aminoacid transport/protein synthesis, and proliferation, respectively,

1Center for Molecular and Functional Imaging, Department of Radi-ologyandBiomedical Imaging, University of California, San Francisco,San Francisco, California. 2Department of Pharmaceutical Chemistry,University of California, San Francisco, San Francisco, California.3CytomX Therapeutics, Inc., South San Francisco, California.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

A.M. LeBeau and N. Sevillano contributed equally to this article.

Current address for A.M. LeBeau: Department of Pharmacology, University ofMinnesota Masonic Cancer Center, Minneapolis, Minnesota.

Corresponding Authors: Aaron M. LeBeau, Department of Pharmacology,University of Minnesota Masonic Cancer Center, 6-120 Jackson Hall, 321 ChurchSt. SE, Minneapolis, MN 55455-0217. Phone: 612-301-7231; Fax: 612-625-8408;E-mail: [email protected]; and Henry F. VanBrocklin, Department of RadiologyandBiomedical Imaging, University of California, San Francisco, 185Berry Street,Suite 350, San Francisco, CA 94143-2280. Phone: 415-353-4569; Fax: 415-514-8242; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-14-2185

�2015 American Association for Cancer Research.

CancerResearch

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on February 8, 2020. © 2015 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst February 11, 2015; DOI: 10.1158/0008-5472.CAN-14-2185

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continue to be investigated (8, 9). The lack of an identifiablemetabolic phenotype has led to the selective targeting of proteinsoverexpressed by prostate cancer. One such example is the FDA-approved ProstaScint, a murine antibody for single-photon emis-sion computed tomography (SPECT) imaging that binds themetalloprotease prostate-specific membrane antigen (PSMA;ref. 10). ProstaScint recognizes an intracellular epitope of PSMAallowing the antibody to only image cells that are dead orundergoing necrosis. The resulting scans are of poor quality andlimited to lymph node staging (11). Next-generation antibodiesand small molecules labeled with PET and SPECT isotopestargeting the extracellular PSMA domain have shown promise inearly human trials (12, 13).

The plasminogen activation system (PAS) is an attractive targetfor a biomarker-based imaging strategy for CRPC.Overexpressionof the PAS—which consists of the serine protease urokinaseplasminogen activator (uPA), the uPA receptor, uPAR, and uPAinhibitor, PAI-1,—has been documented in primary and meta-static prostate cancer (14–16). Central to the role of the PAS inprostate cancer is the proteolytic activity of uPA. Importantly, uPAactivity has been implicated in the formation of osteoblastic bonelesions and in prostate cancer with increased metastatic potential(14, 16, 17). Enzymatically active uPA has been isolated andcharacterized in CRPC bone metastases and in primary prostatecancer tumors (18). Upon secretion by cancer cells, uPA exists asinactive pro-uPA that is converted to its active formbyproteases inthe pericellular milieu. uPA binding to uPAR results in theaccelerated conversion of plasminogen to plasmin. Plasmin canthen directly cleave basement membrane proteins, or activateother proteases, leading to dissolution of the extracellular mem-brane. Inhibition of active uPA by PAI-1 results in the internal-ization of the uPA–uPAR–PAI-1 complex. Studies have shownthat PAI-1, independent of uPA, functions as a signalingmoleculeby interacting with cell-surface receptors (19). PAI-1 can also beinactivated by other prostate cancer–associated proteases, such ashuman kallikrein 2 (20). These studies suggest that little func-tional PAI-1 exists to inactivate the PAS, resulting in high levels ofactive uPA in prostate cancer.

Several groups have investigated uPA as a prostate cancerbiomarker in the serum and by immunohistochemical analysisof diseased tissue. Circulating levels of uPA in the serum ofprostate cancer patients have been found to directly correlatewith cancer stage and metastasis (21, 22). From studies usingprostate cancer tissue microarrays, uPA was found to be ubiqui-tously expressed inorgan-confined andmetastatic cancer (23, 24).In addition, overexpression of uPA and its inhibitor PAI-1 wasfound to be associated with aggressive cancer recurrence post-prostatectomy by IHC (25, 26). Encouraged by these data, wehypothesize that enzymatically active uPA can be selectivelytargeted for preclinical imaging in prostate cancer models. In thisreport, the development of a new technology for imaging prostatecancer centered on active uPA is detailed.Using ahuman fragmentantigen-binding (Fab) phage display library, the active-site bind-ing inhibitory antibody (termed U33) was discovered. U33 IgGwas found tobe apotent and selective inhibitor of uPA, displayingno affinity toward homologous proteases. Through a novelmechanism, we show that U33 IgG results in the internalizationof uPA by uPAR, thereby mimicking the action of its endogenousinhibitor PAI-1. Labeledwith a near-infrared (NIR) dye for opticalimaging or 111In for SPECT,U33 IgGwas used to detect active uPAin vivo in prostate cancer xenografts and in experimental mesta-

tasis models. Because of its internalization, U33 IgG demonstrat-ed high tumor retention in vivo with high signal to noise. Thesepreclinical data presented justify a clinical trial to assess the impactof U33 IgG at imaging CRPC in men.

Materials and MethodsCell culture

All cancer cells lines used in this study were purchased from theATCC and were maintained in their respective recommendedmedia, supplemented with 10% FBS, 100 U/mL penicillin, and100 mg/mL streptomycin at 37�C. The cell lines were authenti-cated using short-tandem repeat profiling provided by the vendor.The uPAR knockout cell line was generated using uPAR shRNAPlasmid (h): sc-36781-SH from Santa Cruz Biotechnology. Trans-fection was performed with a lentiviral particle according to themanufacturer's protocol. Following puromycin treatment, cloneswere selected using flow cytometry with an Alexa Fluor 488–labeled anti-uPAR antibody (27). Gene expression of the cloneused for the xenograft study was analyzed using quantitative PCR(qPCR) and flow cytometry.

Quantitative PCRRNAwas prepared from each cell line (�2� 106 cells/cell line)

using an RNEasy kit (Qiagen). Following RNA isolation, eachsamplewas treatedwithTurboDNA-free (Ambion) to remove anyresidual DNA. RNA was synthesized to cDNA using the HighCapacity RNA-to-cDNA Kit (Applied Biosystems). For each gene,the TaqMan qPCR was performed in quadruplicate usingthe TaqMan Universal PCR Master Mix (Applied Biosystems).The following TaqMan Gene Expression Assay probes wereused: uPAR—Hs00182181_m1 PLAUR, uPA—Hs01547054_m1PLAU, PAI-1 Hs01126606_m1, and 18s ribosomal 1 (referencegene) Hs03928985_g1 RN18S1. All qPCR were performed on anABI 7300 Real Time PCR system instrument. Data were analyzedusing the comparative Ct method (fold change¼ 2�DDCt; ref. 28).

HistologyImmnofluoresence was performed on prostate cancer tissue

microarrays purchased from US Biomax, Inc. (PR959). uPA wasdetected with antibody sc-14019 (Santa Cruz Biotechnology;1:100) following the manufacturer's recommendation using ananti-rabbit Alexa Fluor 488–conjugated secondary. The protocolfor antigen retrieval and staining for e-cadherin was previouslypublished (29).

Phage display panningA fully human na€�ve Fab phage display library was used to

identify inhibitory antibodies against human active uPA (30).Recombinant human uPA (R&D Systems) was immobilizedovernight in wells of a MaxiSorp flat-bottomed 96-well plate(Nunc) at 20 mg/mL in PBS (137 mmol/L NaCl, 2.7 mmol/L KCl,Na2HPO4, 10 mmol/L, KH2PO4 2 mmol/L pH 7.4). The panningwas accomplished in four rounds as described previously (31, 32).After four rounds of selection, Fab was produced from 192individual clones in a 96-well format, the Fabs that leaked intothe cell culture media were screened for binding to uPA by ELISA.Clones with a positive signal in ELISA were analyzed by BstNIrestriction analysis to identify the unique clones. Clones withunique sequence were expressed, purified, and tested for inhibi-tion of uPA.

LeBeau et al.

Cancer Res; 75(7) April 1, 2015 Cancer Research1226




IgG productionThe heavy chain and light chain variable domains of

U33 Fab sequence were cloned separately into pcDNA3.1-derived human IgG1 expression vectors, cotransfected into293 F cells (Life Technologies) cells, and selected with bothhygromycin and neomycin for 14 days. The stable cells werethen subcloned and a high U33 antibody-expressing cell line,8G4, was obtained. This cell line was expanded and grown inFreeStyle 293 medium (Life Technologies) using Wave System(GE) and supernatants were harvested after 10 to 12 days'culture. IgG was purified using a MabSelect SuRe ProteinA column (GE), and followed by preparative size exclusionchromatography using a HiPrep 16/60 Sephacryl S-200 HRcolumn (GE).

ELISAMaxiSorp plates were coated with 50 mL of 5 mg/mL recombi-

nant juman uPA (in some experiments, the zymogen, single chainuPA-pro-urokinase, or mouse uPA was used) in PBS overnight at4�C. The unbound uPA was removed and the plate was washedwith PBS and blocked with milk-PBS. Supernatants of Fab-induced cultures or serial dilutions of pure Fab ranging from 1mmol/L to 0, were added to each well and incubated for 1 hour.Wells were washed with PBS–Tween and the Fab was detectedwith anti-myc antibody conjugated to peroxidase (Roche) andTMB reagent (Pierce). The absorbance was determined at 450 nmusing a microplate reader.

Kinetic assaysHuman uPA (6.2 nmol/L) was incubated with 1 mmol/L of

Fab in assay buffer (50 mmol/L Tris pH 8.8, 0.01% Tween 20),after 1 hour of incubation at room temperature, the chromo-genic substrate Spectrozyme uPA (American Diagnostica, Inc.)at a final concentration of 50 mmol/L was added. The reactionvelocity was monitored by reading the absorbance at 405 nm.For the Ki calculation, 6.2 nmol/L of uPA was incubated withFab (0–2 mmol/L) in assay buffer at room temperature for5 hours. uPA activity was measured for each inhibitor concen-tration by addition of substrate (3.9 �500mmol/L). All datawere analyzed using GraphPad Prism software. Inhibition ofuPA bound to uPAR:uPAR was immobilized in wells of aMaxiSorp plate. uPA (2.5mg/mL) was added to uPAR-coatedplates and incubated for 1 hour. After washing, serial dilutionsof pure Fab ranging from 1 mmol/L to 16 nmol/L were addedto the wells and incubated for 1 hour. For the specificityassays, U33 IgG was used at concentrations ranging from 2to 0.010 mmol/L. Protease concentrations and fluorogenicsubstrates were used as previously described for the specificityassay (33).

InternalizationCancer cell lines (30,000 cells/well in 12-well plates in tripli-

cate) were incubated in conditioned media (protein concentra-tion of 5mg/mL)with 10nmol/L (0.1mCi) 111In-U33or 111In-A11for 0 to 4 hours at 4�C and 37�C. At the indicated time, the mediawere removed and the cells were washed with a mild acid buffer[50 mmol/L glycine, 150 mmol/L NaCl (pH 3.0)] at 4�C for 5minutes. Cells were tryspinized and pelleted at 20,000 � g for 5minutes. The supernatant (containing cell surface–bound radio-activity) and the cell pellet (containing internalized radioactivity)were counted on a Gamma counter.

Mass spectrometryProtein identification of conditioned media was carried out as

describedwith the following notable exceptions (34). Conditionedmedia samples (4 mg) were digested with trypsin (1:20 trypsin toprotein ratio). Extracted peptides were sequenced with an LTQ-FTICR mass spectrometer (Thermo Scientific). SwissProt databasesearcheswere conductedwithmass tolerances of 20ppm for parentions and 0.6 Da for fragment ions using maximum expectationvalues of 0.01 for protein and 0.05 for peptide matches.

Animal modelsThe animal work was in accordance with a University of

California, San Francisco (UCSF; San Francisco, CA) InstitutionalAnimal Care and Use Committee protocol. Six- to 7-week-oldnu/nu mice were purchased from Taconic Farms. Nude mousexenografts were generated by subcutaneous injection of each cellline (1 � 106 cells/mL; 100 mL per site/mouse). Animals forimaging and biodistribution studies had tumor volumes between100 and 350 mm3. The intracardiac dissemination model wasgenerated using the previously described method (35).

In vivo optical imagingU33 IgG was labeled with Alexa Fluor 680 and characterized

in vivo using a previously published method. Images werecollected in fluorescence mode on an IVIS 50 (Caliper/Xeno-gen) using Living Image 2.50.2 software at 24-hour intervals.Region of interest measurements were made and the fluores-cence emission images were normalized to reference imagesand the unitless efficiency was computed. For bioluminescenceimaging (BLI), the mice were injected with intraperitoneallywith D-luciferin (150 mg/kg body weight). Images wereacquired 10 minutes after the injection of D-luciferin and thetotal flux (p/s) in the region of interest was measured. For onePC3 xenograft, the tumor was removed at 72 hours and frozenin optimal cutting temperature. Blocks were cut into 8-mmsections, fixed in acetone for 10 minutes at �20�C, andmounted using ProLong Gold with DAPI. Probe localizationwas visualized in the Cy7 channel using a Nikon 6D HighThroughput Epifluorescence Microscope.

Radiolabeling and SPECT/CT imagingSPECT/CT. The chelate group for 111In, 1,4,7,10-tetraazacyclo-dodecane-1,4,7,10-tetraacetic acid N-hydroxysuccinimide ester(DOTA-NHS; Macrocyclics), was attached to lysine residues onthe IgG using a 25:1 molar excess of chelate in a 0.1 mol/LNaHCO3, pH 9.0 buffer with an antibody concentration of 6mg/mL. After 2 hours of labeling at room temperature, theantibody–DOTA conjugate was FPLC purified to removeunreacted DOTA–NHS. For 111In radiolabeling, 111InCl3 waspurchased from PerkinElmer. To radiolabel the IgG, 50 mg ofDOTA conjugate in 0.2 mol/L ammonium acetate (pH 6.0) wasincubated with 12 mL of InCl3 (2.10 mCi) in 0.1 N HCl for 60minutes at 40�C. The labeled products were purified using a PD-10 column preequilibrated with PBS buffer. Labeling efficiencyand purity of the product were determined using thin-layerchromatography. The specific activity of 111In-U33 IgG was cal-culated to be 31.6� 4mCi/mg (n¼ 4). For imaging, 2.5 to 5.0 mgof probe, corresponding to 275 to 360 mCi of activity, wereinjected into the tail vein. The mice were imaged using a GammaMedica Ideas XSPECT SPECT/C system. Reconstructed data wereanalyzed with AMIDE and AMIRA software.

Imaging Active uPA

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Biodistribution studyMice (n ¼ 4/time point) bearing PC3 and CWR22Rv1 xeno-

grafts were injected with 25 mCi (2.5 mg) of111In-U33 IgG. At 24,48, and 72 hours, the animals were euthanized for analysis inaccordance with UCSF Animal Care and Use Committee guide-lines. Blood was collected by cardiac puncture. The tumor, heart,lung, spleen, kidneys, and muscle were harvested, weighed, andcounted in an automated g-counter (Wizard2; PerkinElmer). Thepercentage of injected dose per gram (% ID/g) of tissue wascalculated by comparison with standards of known radioactivity.

ResultsIdentification of uPA in prostate cancer

qPCR determined that PAS expression was highest in theandrogen-independent metastatic prostate cancer cell lines, PC3and DU145 (Fig. 1A). Little or no expression was documented inother prostate cancer cells lines, normal prostate epithelial cells

(PrEC) and in the bladder cancer cell line TSU. Out of the two celllines that expressed the PAS, the mRNA expression of uPAR anduPA were highest in PC3 cells, while DU145 cells expressedsignificantly higher PAI-1. Data have demonstrated that over-expression of the PAS can be induced by hypoxia in breast cancermodels, and that hypoxia is common in prostate cancer (36, 37).PC3 and DU145 cells were cultured in 1.5% O2, to mimic theO2-deprived environment of prostate cancers, and the levels of thePAS were analyzed (Fig. 1B; refs. 38, 39). After 72 hours, hypoxiahad induced a2-fold increase in expressionof the PASmembers inPC3 cells. DU145 cells were less affected by hypoxia with onlyuPAR expression increased.

The presence of uPA at the protein level was next investigated intissue sections taken from subcutaneous PC3 and DU145 xeno-grafts using IHC. A commercially available antibody (sc-14019)that recognized total uPA protein (zymogen uPA, active uPA, andPAI-1 bound uPA) detected uPA in both xenograft sections withmore intense staining visible in the PC3 section (Fig. 1C). Total

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Figure 1.uPA expression in prostate cancer cell lines and prostate cancer tissue microarray sections. A, mRNA levels of uPAR (top) and uPA and PAI-1 (bottom) wereanalyzed using quantitative RT-PCR in prostate cancer cell lines and normal human PrECs. B, qRT-PCR analysis of the PAS components in PC3 and DU145 cellscultured under 1.5% O2 for 72 hours. The increase in mRNA expression is compared with the cells grown under normoxia. C, IHC staining of a PC3 xenograftsection (left) and a DU145 xenograft section (right) for total uPA protein using the antibody sc-14019. D–G, visualization of the total uPA protein in prostate cancertissue microarray sections using immunofluoresence. H&E-stained sections are shown on the left with the merged fluoresence channels on the rightwith uPA (green), E-caderin (red), and nuclei (DAPI). The sections stained are: adenocarcinoma, Gleason score 5 (2 þ 3) (D); adenocarcinoma, Gleasonscore 5 (1 þ 4) (E); adenocarcinoma, Gleason score 9 (4 þ 5) (F); bone metastasis (G).

LeBeau et al.





uPA protein was next visualized in a prostate cancer tissue micro-array using immunofluorescence (IF; Fig. 1D–G). Total uPAprotein was detected in adenocarcinomas and in osseous metas-tases with IF using the antibody sc-14019. Of the tumor sectionsfound to be positive for uPA, 70% (28 of 40) were found to haveGleason score 7 or higher. This finding mirrored an earlier reportby Cozzi and colleagues (24) that found that 76% of the uPApositive tumors were Gleason score 7 and or higher. In concor-dance with previous work, we documented that uPA was locatedin both the epithelium and stroma (14, 40).

U33 antibody developmentA human na€�ve B-cell phage display library with a diversity of

4.1 � 1010 was used to identify inhibitory antibodies againsthuman uPA. After four rounds of panning, 192 independentclones were screened by ELISA. Of these clones, 67 showed highELISA signals and 23had unique sequences. The 23 unique clonesidentified were expressed, purified, and tested for uPA inhibition.CloneU33was the only Fab that exhibited inhibitory activitywithsequence ofU33 Fab shown in Supplementary Fig. S1. The affinityand specificity of U33 Fab were determined by quantitative ELISAand fluorogenic inhibition assays using human uPA (active andzymogen) and mouse uPA. ELISA results showed that U33 Fab

bound to active uPA in a concentration-dependent manner, butnot zymogen uPA or mouse uPA (Fig. 2A). The inhibition datausing a fluorogenic substrate in Fig. 2B showed that U33 Fabinhibits more than 80% of human uPA activity and has no effecton the mouse enzyme.

Under steady-state conditions, U33 Fab possessed a Ki of 20nmol/L for soluble uPA (Supplementary Fig. S2) andwas also ableto inhibit uPA bound to uPAR (Fig. 2C). Further characterizationstudies were performed and found that U33 Fab could blockbinding of uPA to its endogenous inhibitor PAI-1 in a dose-dependent manner (Fig. 2D), but was unable to dislodge PAI-1 ina preformed uPA–PAI-1 complex (Supplementary Fig. S3). Themechanism of inhibition of uPA by U33 Fab was next investi-gated. Active uPA was pretreated with the irreversible active siteinhibitor Glu–Gly–Arg–chloromethyl ketone (CMK). Whenadded to the uPA–CMK complex, the binding of U33 Fab to uPAwas significantly decreased, suggesting that U33 Fab required afree unoccupied active site for binding and inhibition (Fig. 2E).The heavy and light chain variable domains of U33 Fab werecloned into a full-length IgG1 expression vector, cotransfectedinto 293 F cells, expressed and purified. Kinetic analysis usingdouble reciprocal plots revealed that U33 IgG was a competitiveinhibitor of uPA with a Ki of 10nmol/L (Fig. 2F). Additional

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Figure 2.Characterization of the U33 clone. A, U33 Fab binds specifically to the active formof uPA. Serial dilutions of U33 Fabwere added to uPA-coated plates and incubatedfor 1 hour. The amount of Fab bound to uPA was determined by ELISA. U33 Fab was only detected in wells coated with human active uPA. B, inhibition ofhuman uPA by U33 Fab. The proteolytic activity of human or mouse uPA was read in the absence and presence of 1 mmol/L of U33 Fab. The enzyme activity isexpressed as percentage of the uPA activity in the absence of Fab (100%). C, inhibition of uPA bound to uPAR. Serial dilutions of U33 Fabwere added to uPAR–uPA–coated plates and incubated for 1 hour and the activity of uPA was read. D, U33 Fab prevents uPA binding to PAI-1–coated plates. Serial dilutions of U33 Fab(4 mmol/L–31.2 nmol/L)were preincubated overnightwith uPA and added to PAI-1–coated plates. The amount of uPA bound to PAIwas determined by ELISA. E, U33does not bind to uPA inhibited by a CMK inhibitor. Serial dilutions of U33 (0.0625–1 mmol/L) were added to an uPA-coated plate preincubated with and without 1mmol/L CMK. U33 Fab bound to uPA was determined by ELISA. F, the Lineweaver–Burk plot demonstrating that U33 IgG is a competitive inhibitor of uPA.

Imaging Active uPA





characterization studies of U33 IgGwere performed (Supplemen-tary Fig. S4) and it was found that U33 IgG could displace thenoncovalent small-molecule inhibitor p-aminobenzamidine inthe active site of uPA when incubated with inhibited protease(Supplementary Fig. S5).

U33 IgG in vitro characterizationU33 IgG was specific for uPA when assayed against a panel of

proteases using a fluorogenic substrate inhibition assay. No cross-reactivity was observed with proteases displaying an array ofspecificities, including the prostate cancer–associated serine pro-teases hK2, PSA, and KLK4 (Fig. 3A and Supplementary Fig. S6).U33 IgGwas tested for its ability to inhibit trypsin-like proteolysisin PC3 andDU145 conditionedmedia (Fig. 3B).When incubatedwith the generic trypsin fluorogenic substrate, Z-Gly–Gly–Arg–AMC, PC3, and DU145 conditioned media showed substantialtrypsin-like activity. Addition of 100-nmol/L U33 IgG inhibitedall trypsin-like proteolytic activity. Proteomic analysis of thesecreted proteases in the conditioned media found that both cellslines had high levels of uPA (Fig. 3C). The other proteasesidentified in the conditioned media did not display activity

against the substrate, were not active at physiologic pH, or mighthave been in an inactive state such as zymogen.

U33 IgG labeledwith 111In via aDOTA chelate (111In-U33 IgG)was internalized by PC3 cells at 37�C with 72% of the totalradioactivity internalized within 120 minutes (Fig. 3D). A studyconducted at the 120-minute timepoint found that 111In-U33 IgGinternalization was dependent on the presence of active uPA anduPAR (Fig. 3E). Internalization was observed in DU145 cells butwas blocked in PC3 and DU145 cells pretreated with excesscold U33 IgG. Internalization was not observed in PC3 cells withuPAR expression knocked out. In PC3 cells expressing bothactive uPA and uPAR, 111In-U33 IgG internalization was pre-vented by the addition of an antagonistic uPAR antibody (2G10)that blocks uPA binding to uPAR. No internalization occurred inCWR22Rv1 cells and in PC3 cells treated with the isotype control111In-A11 IgG.

U33 IgG in vivo imagingEncouraged by the in vitrodata,U33 IgGwas tested for its ability

to detect active uPA in vivo using NIR optical imaging. U33 IgGlabeled with Alexa Fluor 680 (AF680-U33 IgG) allowed for the

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Figure 3.In vitro characterization studies of U33 IgG. A, specificity of U33 IgG for uPA compared with a panel of proteases. Proteases were treated with 1 mmol/L of U33 IgG inthe presence of fluorogenic substrate. B, inhibition of trypsin-like proteolytic activity in the condition media of PC3 and DU145 cells by U33 IgG. Conditioned media(4.0 mg/mL protein concentration) were incubatedwith the trypsin cleavable fluorogenic substrate Z-Gly–Gly–Arg–AMC (ex. 355 nm; em. 460 nm) at 400 mmol/L inthe presence and absence of U33 IgG. C, mass spectrometry proteomic analysis of secreted proteases from PC3 and DU145 prostate cancer cell lines. The cell linesPC3 and DU145 were cultured for 24 hours under serum-free conditions. The resulting conditioned media were digested with trypsin and the peptides weresequenced on an LTQ-FT ICR mass spectrometer followed by SwissProt database analysis. D, PC3 cellular internalization of 111In-U33 IgG at 37�C and 4�C inconditionedmedia. PC3 cells were incubatedwith 10 nmol/L of radiolabeled antibody at the indicated time points andwere washed and treatedwith an acidic bufferto remove noncovalently bound and noninternalized 111In-U33 IgG. Each time point was performed in triplicate. E, internalization of 111In-U33 IgG at the 120-minutetimepoint by the cells lines PC3, DU145, PC3 (uPAR-), andCWR22Rv1 in conditionedmedia. Blockingwas performedby adding 1mmol/L of coldU33 IgGor 1mmol/L of2G10 IgG before the media before addition of radiolabeled antibody. 111In-A11 IgG was used as the isotype control antibody for the PC3 cells.

LeBeau et al.





qualitative detection of active uPA in xenografts (Fig. 4A). Max-imum probe localization was achieved at 72 hours with the PC3xenograft demonstrating high tumor uptake and retention. Lowertumor uptake was observed in the DU145 xenograft corroborat-ing themRNA and IHC results. No probe localization was presentin the CWR22Rv1 xenograft. Cryosectioning of the PC3 tumor at72 hours, and subsequent imaging of AF680-U33 IgG withfluorescence microscopy, found probe penetration in the tumortissue (Fig. 4B). The accumulation of the probe in the tumor wasgreater than in the liver, the main clearance organ for IgG anti-bodies (Fig. 4C). Graphing the fluorescence efficiency of theregions of interest for each of the mice imaged as a function oftime highlighted the uptake kinetics and selectivity of the probe(Fig. 4D).

The clinically relevant imaging modality SPECT/CT was nextused to acquire three-dimensional (3D) tomographic data. 111In-U33 IgG localization was seen 72 hours after injection in the PC3xenograft as documented by a pronounced tumor signal in the 3Ddata reconstruction and the 2D transverse view of the fusedSPECT/CT image (Fig. 4E). In the SPECT/CT images, noticeablehepatic clearance of the probe was observed with little secondaryaccumulation in other locations. A biodistribution study of 111In-U33 IgG in the PC3 xenograft found that the probe accumulatedpreferentially over time in the tumor with a%ID/g of 43.2% at 72hours (Fig. 4F). 111In-U33 IgG had low background in vivo with a

tumor-to-blood ratio of 9.9 and a tumor-to-muscle ratio of 63.When treated with excess cold U33 IgG (200 mg) before radio-tracer injection, probe accumulationwas blocked 80%at 72hoursafter injection (Fig. 4G.). No uptake of 111In-U33 IgG was foundin the CWR22Rv1 xenograft with a %ID/g of 4.8% representingnonspecific tumor localization at 72 hours.

111In-U33 IgG was tested for its ability to detect small lesionsthat mimic human prostate cancer using a PC3 intracardiacdissemination model. The PC3 cells used for this model wereengineered to stably express luciferase and the formation ofexperimental metastatic lesions was monitored by BLI after injec-tion of luciferin. By week 6, distinct experimental metastases hadformed in the bone, brain, and lymph nodes of the mice. 111In-U33 IgG imaged a pronounced osseous lesion in the jaw of thismodel that was identified by BLI (Fig. 5A). In the 2D and 3Dreconstructed views, the lesion (11.6mm3 volume)was located inthe left mandible. The lesion was homogenized and the super-natant had marked trypsin-like proteolytic activity, when incu-bated with the fluorogenic trypsin substrate, compared withnormal control tissue extracted from the right mandible (Fig.5B). This proteolytic activity was inhibited by the addition of 100nmol/L U33 IgG. In another example, 111In-U33 IgG was able toresolve a lesion from the skull infiltrating the brain (28.3 mm3

volume) identified by BLI (Fig. 5A). Staining of the lession for Ki-67 found that the lesion was highly proliferative compared with

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Figure 4.Molecular imaging and biodistribution of U33 IgG in prostate cancer xenografts. A, NIR optical imaging of prostate cancer xenografts using AF680-U33 IgG. Micebearing PC3, DU145, or CWR22Rv1 xenografts were tail-vein injected with 2 nmol/L of AF680-U33 IgG and imaged using NIR optical imaging. The images shownare representative of n ¼ 3 mice/xenograft and were acquired 72 hours after injection. B, the resected PC3 tumor at 72 hours fluoresence intensity (left)and a tumor section demonstrating probe penetration and localization by fluoresencemicroscopy (right). C, probe fluoresence intensity (left) and localization (right)in the liver of a PC3 xenograft mouse. D, graph depicting the localization of AF680-U33 IgG as fluoresence efficiency of the tumor ROIs for the mice imagedusing NIR optical imaging. Included in the graph are the data for the mice imaged with the isotype control AF680-A11 IgG in PC3 xenografts. E, SPECT imaging with111In-U33 IgG in a PC3 xenograft model. Depicted are SPECT/CT images shown as a 3D volume rendering of the SPECT data (blue) overlaid onto surfacerendered CT data and a reconstructed transverse view using a rainbow color scale to show uptake (below). Image is representative of n ¼ 3 mice imaged with111In-U33 IgG at 72 hours after injection. Each animal for imaging received 2.5 mg of antibody corresponding to 220 mCi of activity. F, probe biodistributionwas determined by radioactivity assays in PC3 tumor-bearing mice (n ¼ 4 for each time point). Tissues were harvested at 24, 48, and 72 hours after injectionof 111In-U33 IgG (25 mCi). Probe uptake is reported as %ID/g. G, tumor uptake specificity measured at 72 hours after injection (n ¼ 4 mice for each treatment).PC3 xenograft bearing mice were treated with isotype control 111In-A11 IgG (25 mCi) and 111In-U33 IgG blocked (80% reduction) by i.v. preinjection of 200 mgof cold U33 IgG. Probe uptake in CWR22Rv1 xenografts is also depicted.

Imaging Active uPA





adjacent normal tissue (Fig. 5C). In addition to the skull-brainlesion, the 2D and 3D reconstructed views also showed thedetection of lymphnode lesions thatwere obscured by the intensesignal coming from the brain lesion in the BLI image (Fig. 5A).

DiscussionIn this article, the development of the SPECT imaging probe,

U33 IgG, is documented from its initial discovery, using a humanantibody identified from a Fab phage display library, to itspreclinical evaluation in vivo in prostate cancer models. Targetingthe active form of uPA, U33 IgG detects a serine protease foundextensively in prostate cancer. In healthy prostate tissue, the uPApromoter is epigenetically silenced by hypermethylation, result-ing in no detectable uPA in the prostate (41, 42). As prostatecancer progresses, methylation patterns change and uPA isexpressed (42). High levels of uPA protein in tumor tissue andserum have directly correlated with cancer progression, metasta-sis, and poor clinical outcome in men with prostate cancer (23–25, 43, 44). In vitro uPA expression was highest in the androgen-independent cell lines, PC3 and DU145, and expression wassignificantly increased in PC3 cells under hypoxia. Prostate cancercell lines that express androgen receptor (AR) can be induced toexpress uPAwhen treated with demethylating agents. LNCaP cellschallenged with 5-azacytidine were found to turn on uPA expres-sion, resulting in cells that were more proliferative and invasivethan the parental line in vitro and in vivo (42). Although onlyexpressed in two clonal-derived cell lines, IF found total uPA

protein was present in prostate tumors of every grade and in bothsoft tissue and osseousmetastases. These data support and furthervalidate the earlier findings attesting to the presence of uPA inboth AR-positive and AR-negative prostate cancer and also speakto the paucity of good in vitromodels that accurately reflect humandisease in prostate cancer research (23–25, 40, 45).

The development of uPA inhibitors has mainly focused on lowmolecular weight compound (46–48). The further translation ofthese molecules has been prevented by poor specificity and off-target effects. A previous attempt to develop an inhibitory anti-body for uPA gave a human monoclonal antibody with a lownanomolar affinity (49). This antibody could not, however,distinguish between active uPA and pro-uPA and lacked speciesspecificity. Studies with U33 Fab found that the antibody couldinhibit both secreted and uPAR-bound uPA in the lownanomolarrange and was specific for the active human form. U33 Fab couldnot bind to uPA inhibited by PAI-1 or displace PAI-1 from thecomplex. Inhibition studies against other proteases, includingS1A proteases associated with prostate cancer, found U33 IgGto be a specific, competitive inhibitor of uPA. Further evidencefor U33 binding to the active site was provided by use of activesite–directed uPA inhibitors. U33 IgG could displace a non-covalent small-molecule inhibitor from the S1 pocket of uPAand preincubation of uPA with a covalent CMK inhibitorblocked U33 binding. On the basis of these data, we demon-strate that U33 specifically targets active uPA in vitro and in vivowith an accuracy not seen with other uPA inhibitory antibodiesor small molecules.

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Figure 5.Imaging active uPA in the PC3 cardiacdissemination model with 111In-U33IgG. A, 2D and 3D reconstructed 111In-SPECT/CT images of 111In-U33 IgGshowing colocalization of themetastatic lesions with the BLI data.Yellow arrows, tumor location in the2D images (top). Images showing thelocalization of the anti-uPA probe toan osseousmetastatic lesion in the leftmandible of the mouse (top) andprobe localization to an infiltratingskull lesion and lymph node lesions(bottom). B, inhibition of the trypsin-like proteolytic activity by U33 IgG inthe supernatant from thehomogenized mandible lesion andcontrol tissue. Supernatant from bothhomogenates (2.5 mg/mL proteinconcentration) were assayed forproteolytic activity using Z-Gly–Gly–Arg–AMC (400 mmol/L) in thepresence and absence of 100 nmol/LU33 IgG. C, the imaged brain lesionwas fixed, sectioned, and stained forKi-67. Intense staining for Ki-67 isapparent in the cancerous lesion (top)compared with normal adjacent braintissue (bottom).

LeBeau et al.





The imaging properties of U33 IgG in vivowere characteristic ofantibody imaging probes that target membrane proteins.Although targeting a secreted protein, U33 IgG demonstratedhigh tumor uptake and retention in uPA–uPAR–expressing xeno-grafts by NIR and SPECT imaging. U33 IgG was sensitive enoughto detect small osseous and soft tissue metastatic lesions a fewmillimeters in size using SPECT/CT. Key to the success of U33 IgGas an imaging probe was its internalization through an uPAR-mediated mechanism. Internalization was blocked with excesscoldU33 IgG and not observed in PC3 cells with uPAR expressionknocked out or in PC3 cells with the uPA–uPAR binding epitopeblocked by an antibody. These results suggest an internalizationmechanism requiring active uPA and uPAR, with U33 IgG mim-icking PAI-1. Both PAI-1 and U33 IgG bind to the C-terminalprotease domain, whereas uPAR binds to the N-terminal domainof uPA. Furthermore, PAI-1 inhibition of uPA-bounduPAR resultsin internalization of the uPA–uPAR–PAI-1 complex. In vivo theinternalization mechanism of U33 IgG afforded probe accumu-lation and sequestration in tumor tissue. Internalization pre-vented the dissemination of uPA–U33 IgG complex to peripheraltissue, resulting in high tumor uptake values that increased overtime as demonstrated by the biodistribution. The internalizationof U33 IgG is in direct contrast to a recent study that targetedanother secreted protease, PSA, for PET imaging using a murineIgG antibody (89Zr-5A10; ref. 50). With no means of internali-zation or bioaccumulation, 89Zr-5A10 uptake reached its maxi-mum uptake 24 hours after injection with a low tumor-to-bloodratio. There are nopublished reports of an antibody that binds to aprotease receptor ligandmimicking the activity of the endogenousinhibitor and causing the internalization of the antibody–ligand–receptor complex.

Several research groups have imaged the PAS by targeting theuPA receptor, uPAR, using antibodies and peptides (51, 52). Thesubsequent translation of uPAR-targeted antibodies failedbecause of a lack of specificity or humanization affected theiraffinity. The data presented here document the first time the PAShas been imaged by targeting uPA instead of uPAR using aclinically relevant imaging modality. By targeting uPA instead ofuPAR, we gain a greater insight into the proteolytic activity of thecancer. Increased proteolysis is a common trait of aggressivecancer with a high metastatic potential and studies have directlycorrelated increased uPA levels with disease progression andmetastasis (22, 53). Because U33 only binds active uPA, U33directly informs about the amount of uPA activity in the system.The levels of active uPA can change based on the depletion ofactivating proteases, such as hK2orMMP-9, due to treatment. Thisdecrease in active uPA could be detected with U33—decreasedproteolytic activity can act as surrogate marker of indolence andregression.

Together, our data demonstrate that U33 is a unique, potent,and selective active site inhibitor of uPA that can be used to imageprostate cancer in preclinicalmodels.While secreted, uPA remainslargely restricted to the tumor microenvironment because of itsinteraction with uPAR. uPA that does escape into circulation isquickly inactivated by macromolecular protease inhibitors. U33IgG would not bind to complexed uPA in the blood, thus pre-

venting a disseminated imaging probe that would reduce target tonontarget ratios. Because both uPA and uPAR are expressed bycancerous prostate cells and tumor-associated stromal cells, ouruPA-targeted probehas the ability todetect the tumor cells and theabetting microenvironment with a single agent. Notably, theutility of U33 IgG is not limited to prostate cancer. uPA and theother components of the PAS are overexpressed in a myriad ofcancers ranging from ovarian to breast (54). In addition, U33 IgGhas the potential to be both a diagnostic and therapeutic agent.The internalization and clearance from the blood makes U33 IgGan ideal candidate for radioimmunotherapy. The unique mech-anism of U33 antibody accumulation in tumors expressing activeuPA and uPAR presents significant opportunities for clinicalapplications fromdiagnostic imaging to therapeutic intervention.

Disclosure of Potential Conflicts of InterestD.R.Hostetter is a senior scientist at CytomXTherapeutics andhas ownership

interest (including patents) in the same. H. Lowman reports receiving othercommercial research support from CytomX Therapeutics; has ownership inter-est (including patents) in CytomX Therapeutics; and is a consultant/advisoryboard member for the same. C.S. Craik reports receiving a commercial researchgrant from CytomX Therapeutics; has ownership interest (including patents) inStock Options; and is a consultant/advisory board member for CytomX Ther-apeutics. No potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: A.M. LeBeau, N. Sevillano, D.R. Hostetter, J. West,H. Lowman, C.S. Craik, H.F. VanBrocklinDevelopment of methodology: A.M. LeBeau, N. Sevillano, D.R. Hostetter,J. West, H. Lowman, C.S. CraikAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A.M. LeBeau, N. Sevillano, K. Markham, M.B. Winter,S.T. Murphy, D.R. Hostetter, J. West, H.F. VanBrocklinAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A.M. LeBeau, N. Sevillano, M.B. Winter, D.R. Hos-tetter, J. West, H.F. VanBrocklinWriting, review, and/or revision of themanuscript: A.M. LeBeau, N. Sevillano,M.B. Winter, D.R. Hostetter, H.F. VanBrocklinAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): A.M. LeBeau, S.T. Murphy, D.R. Hostetter,J. WestStudy supervision: A.M. LeBeau, D.R. Hostetter, J. West, C.S. Craik, H.F.VanBrocklin

AcknowledgmentsThe authors thank Fei Han, Jason Gee, Shouchun Liu, and Jeanne Flandez

of CytomX Therapeutics, Inc., for construction, expression, and purification ofU33 IgG.

Grant SupportThis work was supported by the Rogers Family Award (C.S. Craik and H.F.

VanBrocklin) and the NIH grant R01 CA128765 (C.S. Craik). A.M. LeBeau wassupported by a DOD Prostate Cancer Postdoctoral Award PC094386 and theDOD Idea Award PC111318. A.M. LeBeau is currently the Steve Wynn 2013Prostate Cancer Foundation Young Investigator.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received July 23, 2014; revised December 8, 2014; accepted January 9, 2015;published OnlineFirst February 11, 2015.

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2015;75:1225-1235. Published OnlineFirst February 11, 2015.Cancer Res Aaron M. LeBeau, Natalia Sevillano, Kate Markham, et al. CancerImaging Active Urokinase Plasminogen Activator in Prostate

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