Applications of ImmunoPET: Using I-Anti-PSCA A11 …clincancerres.aacrjournals.org/content/clincanres/20/24/6367.full.pdfwork, we will compare the sensitivity of the A11 minibody to18F-Fluoride

Personalized Medicine and Imaging

Applications of ImmunoPET: Using 124I-Anti-PSCA A11Minibody for Imaging Disease Progression and Response toTherapy in Mouse Xenograft Models of Prostate Cancer

Scott M. Knowles1, Richard Tavar�e1, Kirstin A. Zettlitz1, Matthew M. Rochefort2, Felix B. Salazar1,Ziyue Karen Jiang1,3, Robert E. Reiter3, and Anna M. Wu1

AbstractPurpose: Prostate stem cell antigen (PSCA) is highly expressed in local prostate cancers and prostate

cancer bone metastases and its expression correlates with androgen receptor activation and a poor

prognosis. In this study, we investigate the potential clinical applications of immunoPET with the anti-

PSCA A11 minibody, an antibody fragment optimized for use as an imaging agent. We compare A11

minibody immunoPET to 18F-Fluoride PET bone scans for detecting prostate cancer bone tumors and

evaluate the ability of the A11 minibody to image tumor response to androgen deprivation.

Experimental Design: Osteoblastic, PSCA-expressing, LAPC-9 intratibial xenografts were imaged with

serial 124I-anti-PSCA A11 minibody immunoPET and 18F-Fluoride bone scans. Mice bearing LAPC-9

subcutaneous xenografts were treated with either vehicle or MDV-3100 and imaged with A11 minibody

immunoPET/CT scans pre- and posttreatment. Ex vivo flow cytometry measured the change in PSCA

expression in response to androgen deprivation.

Results: A11 minibody demonstrated improved sensitivity and specificity over 18F-Fluoride bone scans

for detecting LAPC-9 intratibial xenografts at all time points. LAPC-9 subcutaneous xenografts showed

downregulationofPSCAwhen treatedwithMDV-3100whichA11minibody immunoPETwas able todetect

in vivo.

Conclusions: A11 minibody immunoPET has the potential to improve the sensitivity and specificity

of clinical prostate cancer metastasis detection over bone scans, which are the current clinical standard-

of-care. A11 minibody immunoPET additionally has the potential to image the activity of the androgen

signaling axis in vivo which may help evaluate the clinical response to androgen deprivation and the

development of castration resistance. Clin Cancer Res; 20(24); 6367–78. �2014 AACR.

IntroductionThere is a significant clinical need for improving the

detection of prostate cancer metastases and measuring theresponse of tumors to systemic therapy. The most commonmethod of screening for prostate cancer, a prostate-specificantigen (PSA) blood test, is notoriously nonspecific and is

commonly elevated in benign prostate hyperplasia, pro-statitis, and low-risk indolent prostate cancer. Prostatecancer survival is high for the 91% of patients that arediagnosed with local or regional disease and PSA testing isassociated with overdiagnosis and treatment of indolentdisease (1).On the other hand, prostate cancer is the secondmost common cause of cancer-related deaths in men andone of the great challenges in treating it is distinguishing thepatients whodonot need to be treated from those forwhomit is life threatening (2). Metastases, and especially bonemetastases, represent the primary cause of morbidity andmortality and prostate cancer survival rates fall quickly oncethe disease hasmetastasized to distant sites (2).Detectionofmetastases, therefore, stratifies a subset of patients that needto be treated aggressively and systemically from the largemajority of patientswheremore conservative local therapiesor active surveillance is preferred. Following the detectionofdistantmetastases, androgen deprivation becomes the stan-dard-of-care and PSA tests are frequently used to monitorthe effectiveness of androgen deprivation and the develop-ment of castration-resistant prostate cancer (CRPC).

1Crump Institute for Molecular Imaging, Department of Molecular andMedical Pharmacology, David Geffen School of Medicine at the UniversityofCalifornia-Los Angeles, LosAngeles,California. 2Department of Surgery,David Geffen School of Medicine at the University of California-LosAngeles, Los Angeles, California. 3Department of Urology, David GeffenSchool of Medicine at the University of California-Los Angeles, LosAngeles, California.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Anna M. Wu, David Geffen School of Medicine atUCLA,Crump Institute, CNSI 4335, 570WestwoodPlaza, Box 951770, LosAngeles, CA 90095-1770. Phone: 310-794-5088; Fax: 310-206-8975;E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-14-1452

�2014 American Association for Cancer Research.

ClinicalCancer

Research

www.aacrjournals.org 6367

on June 13, 2018. © 2014 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst October 17, 2014; DOI: 10.1158/1078-0432.CCR-14-1452

http://clincancerres.aacrjournals.org/

However, PSA levels can only measure the response of thetumors as awhole and cannot determinewhether there is anandrogen-independent subset of tumors that fails torespond and may necessitate the use of targeted radiationor chemotherapy. There is a need for an imaging modalitythat can improve the early detection and localization ofprostate cancer metastases and image response to androgendeprivation therapy (ADT) to guide therapeutic decisionmaking.

Despite the large influence of bone metastases on patientprognosis and therapy decisions, the current methods ofdetecting them are unsatisfactory. Most current clinicalmonitoring for metastases utilizes 99mTc-Methylene dipho-sphonate (99mTc-MDP) planar or SPECT imaging of boneformation in response to osteoblastic prostate cancer bonemetastases, though other methods are at various stages ofdevelopment (3, 4). Bone scans are clinically recommendedfor symptomatic patients and asymptomatic men withserum PSA >10–20 ng/mL; however, due to relatively lowsensitivity of bone scans and their inability to detect metas-tases to other sites the presence of prostate cancer recurrenceand/or metastasis often must be inferred on the basis ofother risk factors such as a rising PSA in a patient who hasreceived a radical prostatectomy (4).

Recently 18F-Fluoride PET bone scans have shownimproved sensitivity over 99mTc-MDP in initial small clin-ical trials and are currently the subject of a phase III studyto determine whether their use improves metastasis detec-tion (5–8). However, while the sensitivity of bone metas-tasis detection may be improved using 18F-Fluoride bonescans, bone scans are obviously limited to detectingmetas-tases to bone without the ability to image local prostatecancer or metastases to other sites. Bone scans also have

considerable false positives due to any benign process thatincreases bone formation such as trauma and fractures,degenerative diseases (e.g. osteoporosis), Paget disease,and inflammatory processes (e.g., arthritis) which arerelatively common in the elderly patients most likely todevelop prostate cancer (4, 8). Bone scans, additionally,have difficulty measuring response to therapy due to theflare phenomenon where uptake in bone scan lesionsoften increases in response to therapy due to bone healingin response to the shrinking tumor. This flare can last formonths after successful therapy has been initiated andlesions may continue to appear on bone scans long afterthe viable tumor has been eliminated (4).

While the mainstay of metastatic prostate cancer therapyis ADT, patients progress to CRPC a median of 2 to 3 yearsafter initiating treatment (9). MDV-3100 (enzalutamide) isa second-generation antiandrogen that has recently wonFDA approval for the treatment of CRPC (10). MDV-3100has continued efficacy in cell lines resistant to other anti-androgens and results in a larger repression of androgen-dependent genes and shorter time period to maximal effi-cacy than castration in mouse models (11–13). A phase IIIstudyofMDV-3100 showeda�50%PSAdecrease in54%ofpatients who had previously failed both ADT and chemo-therapy regimens and prolonged median survival 4.8months over placebo (14). However, many patients do notrespond to therapy with MDV-3100 and many initialresponders develop resistance rapidly, possibly through thegeneration of constitutively active splice variants that lackthe AR C-terminal ligand binding domain on which MDV-3100 acts (15–17). A method of imaging the activity of theandrogen receptor (AR) signaling axis in vivo could lead toimproved therapeutic strategies and could allow for targetedradiotherapy or a more prompt transition to alternativeandrogen deprivation agents or systemic chemotherapy inthe patients whose tumors reactivate the androgen-signal-ing axis.

Prostate stem cell antigen (PSCA) is highly expressed in83% to 100% of prostate cancers and overexpressed in thegreat majority of prostate cancer bone metastases (87%–100%) and in many metastases to other sites (67% liver,67%–95% lymph node; refs. 18–21). Its expression corre-lates with the Gleason score, tumor invasion, androgenindependence, metastasis, and a poor prognosis (21–26).The PSCA promoter contains an androgen response ele-ment and PSCA expression is regulated by androgens in thenormal mouse prostate (27, 28). Likewise, androgen dep-rivation decreases PSCAmRNA expression in human high-grade prostatic intraepithelial neoplasia and prostate cancer(29, 30). We have previously shown that immunoPET withan affinity matured 124I-labeled A11 anti-PSCA minibody,an antibody fragment with pharmacokinetics optimized forimaging, can be used for specific and quantitative imagingof PSCA expression in vivo (31–33). We therefore hypoth-esize that imaging PSCA expression using theA11minibodymay outperform bone scans for imaging prostate cancerbone metastases and allow for imaging changes in PSCAexpression in response to androgen deprivation. In this

Translational RelevanceClinical detection of prostate cancer metastases and

monitoring of the response to androgen deprivationtherapy remains inadequate. We have previously devel-oped an engineered anti-PSCA antibody fragment withaffinity and pharmacokinetics optimized for imaging,the A11 minibody, and demonstrated high-contrastquantitative immunoPET imaging of PSCA expression.In this work, we investigate the translational applica-tions of A11 minibody immunoPET. We demonstratedetection of bone tumors with higher sensitivity andspecificity than 18F-Fluoride bone scans and that the A11minibody can detect tumor response to androgen dep-rivation earlier than conventional imaging. These resultsmay directly translate into improved clinical metastasisdetection and the ability to noninvasively image theefficacy of androgen deprivation therapy and the devel-opment of castration-resistant prostate cancer. We haverecently begun a clinical trial to compare A11 minibodyimmunoPET to bone scans in men with known meta-static disease to investigate this translational potential.

Knowles et al.

Clin Cancer Res; 20(24) December 15, 2014 Clinical Cancer Research6368




work, we will compare the sensitivity of the A11 minibodyto 18F-Fluoride bone scans for detecting bone tumors usinga naturally PSCA expressing, purely osteoblastic, LAPC-9intratibial xenograftmodel.Wewill also investigate changesin PSCA expression in LAPC-9 subcutaneous xenografts inresponse to androgen deprivation with MDV-3100 anddemonstrate that anti-PSCA A11 minibody immunoPETcan image these changes in vivo.

Materials and MethodsSubcutaneous and intratibial xenograft modelsLAPC-9 subcutaneous xenografts were passaged surgi-

cally in male SCID mice as previously described (34).Single-cell suspensions of LAPC-9 cells were prepared bydigesting freshly excised LAPC-9 subcutaneous xenograftsin 0.1% Pronase (Sigma) in Iscove modified Dulbeccomedium (Gibco) for 18 minutes at room temperature(35, 36). The cells were then passaged through 18 gaugeneedles and strained through a 70-mm cell strainer (BDBiosciences).Viable cells (1�105)wereprepared in10mLof1:1Media:

Matrigel and injected into the tibias of anesthetized maleSCIDmice by drilling a needle through the proximal end ofthe tibial plateau. Once the needle tip entered the intrame-dullary space of the tibial metaphysis, the cells were slowlyinjected and the needle was removed (37). A sham injec-tion, where the needle was inserted using the samemethod,but onlyMedia:Matrigelwas injected,was performedon thecontralateral leg. Injections of PSCA-negative 22rv1 cellsand 22rv1 � PSCA cells transfected with PSCA were used,respectively, as negative and positive controls (see Supple-mentaryMaterials). All animal experimentswere conductedin compliancewith a protocol approved by the InstitutionalAnimal Care and Use Committee of the University ofCalifornia-Los Angeles (Los Angeles, CA).

18F-Fluoride and A11 minibody imaging of intratibialxenograftsIntratibial tumor-bearing mice were serially imaged

with both bone scans and immunoPET at 4, 6, and/or8 weeks postintratibial injection. Intratibial xenograftbearing mice were injected with approximately 100 mCiof 18F-Fluoride in 100 mL of saline via tail vein injection.After an hour of conscious uptake, the mice were anes-thetized, their bladders were manually expressed, and themice were imaged with a 10-minute acquisition on anInveon microPET scanner (Siemens Preclinical Solutions)followed by a microCT scan (MicroCAT II, Siemens Pre-clinical Solutions). MicroPET and microCT were auto-matically coregistered on the basis of empirically deter-mined scanner alignments. All image manipulation andquantification was performed using AMIDE (38). Align-ment was manually verified and adjusted using the bladderas a fiduciary marker. Uptake (%ID/g) was then quantifiedfrom the coregistered microPET/CT images using microCTisocontour region of interests (�200 Hounsfield units)to capture a region encompassing either only the tumor-

bearing tibia or the sham tibia. The mean value of themicroPET scan was converted to %ID/g using the decay-corrected injected dose of 18F-Fluoride and an empiricallydetermined cylinder factor for 18F.

Either immediately following the 18F-Fluoride bone scanor the day following the bone scan the intratibial tumorbearing mice were injected with approximately 3 mg 124I-labeled A11 minibody. Radiolabeling, purification, andimmunoreactivity were performed as previously describedexcept for the much higher specific activity used here (�30-50 mCi/mg; ref. 32). Forty-four hours after A11 minibodyinjection, the mice again received microPET/CT scans. Themice were then either kept for serial imaging at later timepoints or at the last time point sacrificed for biodistributionas previously described (32). The images were analyzedusing the same method as for the 18F-Fluoride bone scans.No partial volume correction was performed on either the18F-Fluoride or 124I-A11 immunoPET as determining thetumor volume by CT would be too arbitrary to be repro-ducible andhence only quantification of the entire tibiawasperformed without attempting to approximate the tumorboundaries. Thismethod provided the additional benefit ofallowing direct comparison between the biodistributionand imaging results.

High-resolution ex vivo microCT and histologyAfter biodistribution, tibias were stored in 10% phos-

phate-buffered formalin until radioactivity had decayed.The tibias were then analyzed ex vivo by 20-mm resolutionmicroCT (mCT40, SANCO Medical). Volume renderingswere generated with OsirX 5.6 (39). The tibia samples werethendecalcified andembedded inparaffin and sectioned forhistologic analysis. Only those mice with intratibial tumorestablishment confirmed by gross and/or histologic analy-sis were included in the analysis.

A11 minibody imaging of response to therapy withMDV-3100

LAPC-9 subcutaneous xenografts were implanted bilat-erally and allowed to grow for 3 weeks. The mice thenreceived a pretreatment A11 minibody immunoPET/CTat 44 hours postinjection as previously described, with theexception that each mouse received a dose of approxi-mately 50 mg of 124I-A11 minibody (32). Immediately,after the pretreatment scan, the mice were randomizedinto treatment groups and received either 40 mg/kg MDV-3100 (ChemScene) or vehicle by daily gavage. The vehicleconsisted of 300 mL of water with 1% carboxymethylcel-lulose (Sigma-Aldrich), 0.1% Tween-80 (Sigma-Aldrich),and 1.6% DMSO. After five days of treatment, one mousefrom each group underwent a microPET/CT scan to con-firm that minimal signal was retained from the firstimaging injection, and then all mice were again injectedwith approximately 50 mg of 124I-labeled A11 minibody.On the seventh day of treatment, 44 hours post-A11minibody injection, the mice received a posttreatmentscan, following which the mice were sacrificed and bio-distribution and microPET image analysis and

Applications of ImmunoPET with 124I-Anti-PSCA Minibody

www.aacrjournals.org Clin Cancer Res; 20(24) December 15, 2014 6369




quantification with partial volume correction were per-formed as previously described (32). The tumors werethen fixed and examined histologically (see Supplemen-tary Materials and Methods).

Quantitative flow cytometryLAPC-9 subcutaneous xenograft-bearing mice treated

identically to the imaging cohorts, except without theinjection of the radioactive tracers, were used for quantita-tive flow cytometry. After 7 days of treatment with MDV-3100 or vehicle, mice were sacrificed and LAPC-9 tumorswere reduced to single-cell suspensions by incubation in1 mg/mL collagenase IV (Sigma) in HBSS for 1 hour at37�C. The cells were then passaged through 18 and24 gauge needles followed by a 70 mm cell strainer (BDBiosciences). A total of 5 � 105 LAPC-9 cells from eachtumor were incubated in 16 mg/mL [approximately 100nmol/L) 1G8 mouse anti-PSCA antibody (produced aspreviously described)] followed by 8 mg/mL (approxi-mately 50 nmol/L) Dylight-649 conjugated anti-mouse-Fc (secondary antibody (Jackson ImmunoResearch;ref. 40). After secondary antibody incubation, sampleswere incubated with Alexa-488 anti-PSMA antibody(FOLH1, BioLegend) and stained with 7-AAD (BD Bios-ciences), as per the manufacturers’ instructions, to allowgating for viable prostate epithelial cells. The final ana-lyzed population was FSC and SSC gated, 7-AADLow,PSMAþ. Acquisition was performed with an LSRII flowcytometer (BD Biosciences) and analysis was performedin FlowJo 9.3.2 (TreeStar). PSCA receptor density wasquantified using the QIFIKIT calibration beads (Dako) asper the manufacturer’s instructions.

Statistical analysisStatistical analysis of both the bone scans and the immu-

noPET scans in the intratibial model were performed usingtwo-way repeated measure ANOVA with intra-time pointsignificance testing and adjustments for multiple compar-isons performed using the Holm–Sidak method. As thevariance of the A11 immunoPET dataset increases signifi-cantly as the uptake increases, a log transformation wasapplied to fulfill the homoscedasticity requirement of bothANOVA and Holm–Sidak before significance testing. Sig-nificance testing of the MDV-3100 response to therapymodel was performed using two-tailed student t tests. The95% confidence level (P < 0.05) was used for all analysis.Except where indicated otherwise, all values are reported asmean � SD. All microPET images are displayed as fullthickness maximum intensity projections.

ResultsRadiolabeling

124I-labeled A11 minibody used in the intratibial xeno-graftmodel had amean specific activity of 23.7�6.5mCi/mg(n¼ 8). After purification by size exclusion, themean purityof the protein injected was 95� 5%with immunoreactivityof 62 � 5%. For the MDV-3100 treatment model, the

specific activity for the pretreatment imaging was 1.23mCi/mg with injected radiochemical purity of 93.8% andimmunoreactivity of 72.7%. The posttreatment imaginghad specific activity of 2.38 mCi/mg, radiochemical purityof 98.6%, and immunoreactivity of 83.3%.

Comparison of 18F-Fluoride ion bone scans and124I-A11 minibody immunoPET in mice bearingintratibial xenografts

18F-Fluoride bone scans of mice bearing LAPC-9 intrati-bial xenografts show a large amount of nonspecific uptakeand a qualitative increase in the positive tibia over thenegative tibia in only 16.7% of mice at 4 weeks (1/6),50% (3/6) at 6 weeks, and 50% (1/2) at 8 weeks (Fig.1A).Quantificationof the 18F-Fluoride bone scans shows anoverall increase in tibial uptake of 18F- Fluoride in thetumor-bearing tibia for all time points in aggregate (P ¼0.01, two-way ANOVA), with no trend in increased uptakein the positive tibia over time (P¼ 0.89). The negative tibiasshow a large amount of non-specific background uptake,especially in the knee, which leads to a large degree ofoverlap between the positive and negative tibias andmakes quantitative determination of a positive signal dueto tumor growth difficult. In fact, the increase in 18F-Fluo-ride uptake in the positive tibia only reaches significancein aggregate across time points and fails to reach signi-ficance for any individual time point on its own (Fig. 1B).

A11 minibody immunoPET imaging shows intratibialtumor targeting which can be appreciated above bloodactivity in 67% of mice (4/6) at 4 weeks, 100% of mice(6/6), at 6 weeks and 100% of mice at 8 weeks (2/2)postxenograft implantation (Fig. 2A). Imaging of two miceat 8 weeks after intratibial tumor implantation shows thatthe tumor has invaded through the bone and into thesurrounding muscle. Quantification of the serial A11immunoPET scans shows anoverall increase in tibial uptakeof the A11 minibody in the tumor-bearing tibia across alltime points (P<0.0001, two-wayANOVA),with an increasein the positive tibia over time (P ¼ 0.001). Because of thehigh specificity of the A11 minibody and universally lowbackground in normal bone, the positive tibia has signif-icantly higher uptake at all time points (Fig. 2B). The highspecificity of A11 minibody, therefore, allows for highlysensitive imaging of bone tumors as any bone uptake aboveblood can be interpreted as a PSCA-expressing tumor.

Biodistribution of the LAPC-9 mice at 6 (n ¼ 4) or8 weeks (n ¼ 2) after tumor implantation confirmsthe results of the immunoPET imaging (Table 1). The 6week after implantation, mice show an average of 1.51 �0.79 %ID/g uptake in the positive tibia and 0.08 � 0.03%ID/g in the negative tibia (P ¼ 0.02). At 8 weeks afterinjection, the uptake is similar with 1.50 � 0.35 %ID/g inthe positive tibia and 0.12 � 0.00 %ID/g in the negativetibia.

A11 minibody immunoPET of mice bearing negativecontrol 22rv1 intratibial tumors (n ¼ 5) showed no distin-guishable intratibial uptake by microPET and showed onlya small increase in tibial uptake (0.36 � 0.19 %ID/g)

Knowles et al.





compared with the contralateral sham tibia (0.08 �0.02 %ID/g) by biodistribution (P ¼ 0.025). The smallincrease in intratibial uptake in the 22rv1 tumor was lessthan the blood activity (0.40� 0.11%ID/g) andwas likely aresult of the enhanced permeability and retention effectuptake due to malformed tumor vasculature. Biodistribu-tion of a mouse bearing a 22rv1 � PSCA intratibial xeno-graft at 6weeks after injection showed uptake of 3.31%ID/gfor the 22rv1� PSCA tibia, 46.9 times higher than the shamtibia, 8.5 times higher than tibias bearing 22rv1 tumors, and10.7 times higher than blood (Supplementary Table S1 andSupplementary Figs. S1–S4).

Ex vivo microCT and histologyGross analysis, ex vivo microCT, and histology confirm

the presence of an osteoblastic intratibial tumor with spi-culated bone formation in each mouse. High-resolution exvivo microCT imaging of the LAPC-9 xenograft-bearingtibias and the sham controls shows spiculated bone forma-tion in the LAPC-9–bearing tibias with sham injected tibiasshowing normal appearing bone (Fig. 3A). Histology ofLAPC-9–bearing tibias shows osteoblastic tumor forma-tion, consistent with previous reports, whereas histologyof sham injected tibias showsnormal bonemarrow (Fig. 3B;refs. 35, 36, 41–46). For mice that grew muscle tumors due

4 Weeks 6 Weeks 8 Weeks0

5

10

15

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-Flu

orid

e up

take

(%ID

/g)

0.15910.15020.1591ns ns ns

Positive tibiaNegative tibia

B

4 weeks

6 weeks

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A

15 %ID/g

15 %ID/g

15 %ID/g

0.5 %ID/g

0.5 %ID/g

0.5 %ID/g

Figure 1. Serial 18F-Fluoride bonescans of mice bearing LAPC-9intratibial xenografts (A) andquantification of the positive andnegative tibias (B). Cleardetermination of increased signalin tumor bearing tibias is difficultdue to the large degree ofnonspecific uptake. Each columndisplays serial imaging of the samemouse and each is matched withthe corresponding A11immunoPET in Fig. 2.






to missed intratibial injections, neither gross analysis norhistology showed the presence of tumor cells in the intra-medullary space and on biodistribution only the ipsilateralmuscle uptake of A11 minibody was elevated due to thetumor presence and these mice were excluded from theanalysis.

Changes in PSCA expression withMDV-3100 treatmentQuantitative flow cytometry on digested LAPC-9

tumors following 7 days of treatment with MDV-3100(40 mg/kg) or vehicle shows that MDV-3100 treatment

downregulates PSCA expression 62.8% � 4.9% (P <0.0001, n ¼ 4) compared with the vehicle-treated control(Fig. 4A). Quantitative flow finds expression of 4.75� 105

PSCA antigens per cell for vehicle-treated mice, whereasmice treated with MDV-3100 express only 1.65 � 105

PSCA antigens per cell.

ImmunoPET imaging of PSCA downregulation inresponse to ADT

Partial volume corrected quantification of the pre-treatment results shows equivalent tumor volumes and

A4 weeks

6 weeks

8 weeks

3.0 %ID/g

5.0 %ID/g

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0.5 %ID/g

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2

4

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min

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y up

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(%

ID/g

)

Positive tibiaNegative tibia

< 0.0001< 0.00010.0003*** **** ****B

Figure 2. Serial 124I-anti-PSCAimmunoPET imaging of micebearing LAPC-9 intratibialxenografts (A) and quantification ofthe positive and negative tibias (B).Increased uptake of A11 minibodycan be easily discerned in themajority of tumor-bearing tibias.Each column displays serialimaging of the same mouse andeach is matched with thecorresponding 18F-Fluoride bonescan in Fig. 1. All mice aredisplayed on the scale indicated onthe right except for those with ascale indicated directly abovethem.

Knowles et al.





uptake in the two groups before treatment is initiated(2.43 � 0.42 %ID/g vehicle, 2.65 � 0.72 %ID/g MDV-3100, P ¼ 0.46; Fig. 4C). After one week of treatmentwith MDV-3100, tumor volumes show no significantdifference as measured by CT (P ¼ 0.54) or by mass(P ¼ 0.53). However, following treatment, the A11minibody uptake in the MDV-3100 treated cohort is29% lower than in the control group by partial volume

corrected microPET (3.70 � 0.20 %ID/g vehicle vs.2.61 � 0.23 %ID/g MDV-3100, P < 0.003; Fig. 4Band C) and 24.0% lower than in the vehicle-treatedcontrols by biodistribution (Table 2; P ¼ 0.03).

Normalizing posttreatment imaging by the pretreatmentimaging similarly shows uptake in the MDV-3100–treatedgroup 32% lower than the vehicle-treated group (P ¼0.0003). However, rather than the MDV-3100–treated

Table 1. Forty-four–hour biodistribution of 124I-A11minibody inmice bearing LAPC-9 intratibial xenograftsat 6 and 8 weeks posttumor implantation

LAPC-9 Intratibial xenograft

6 weeks 8 weeks

n %ID/g � SD n %ID/g � SD

Blood 4 0.57 � 0.15 2 0.45 � 0.11Positive (left) tibia 4 1.51 � 0.79 2 1.50 � 0.35Sham (right) tibia 4 0.08 � 0.03 2 0.12 � 0.00Left calf muscle 4 0.45 � 0.33 2 2.54 � 1.26Right calf muscle 4 0.06 � 0.02 2 0.07 � 0.01Left femur 4 0.10 � 0.03 2 0.14 � 0.03Right femur 4 0.10 � 0.03 2 0.13 � 0.00Liver 4 0.14 � 0.02 2 0.28 � 0.04Kidney 4 0.24 � 0.04 2 0.37 � 0.04Spleen 3 0.18 � 0.08Heart 3 0.21 � 0.04Lung 3 0.32 � 0.07Carcass 4 0.12 � 0.04 2 0.25 � 0.05

Pos Tibia: blood 4 2.85 � 1.49 2 3.55 � 1.68Pos:neg tibia 4 18.98 � 7.10 2 12.50 � 2.98Pos tibia:muscle 4 27.16 � 15.5 2 20.08 � 0.80

LAPC-9 ShamA B×4

×10

LAPC-9 Sham

Figure 3. At 6 weeks posttumorinoculation, tibias bearingLAPC-9intratibial xenografts grossly showbonemarrow displacement by thetumor and high-resolution ex vivomicroCT demonstratesosteoblastic changes in theLAPC-9 injected tibias withnormal appearing sham controls(A). Histology of LAPC-9–bearingtibias shows tumor interspersedwith osteoblastic bone formation,whereas histology of sham tibiasshows normal bone marrow (B).






group decreasing from the baseline scan, we find that theMDV-3100–treated group is not significantly changed frompretreatment (pre:posttreatment ratio 1.10 � 0.16, P ¼

0.90) and rather the difference posttreatment is due to anincrease in the vehicle-treated group between the scans(pre:post treatment ratio 1.61 � 0.26, P ¼ 0.0002).

Vehicle MDV31000

200

400

600

Vol

ume

(mm

3)

P = 0.83 P = 0.54

P = 0.80 P = 0.004

Vehicle MDV31000

200

400

600V

olum

e (m

m3)

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2

4

6

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/gP

VC

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CT

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ume

A11

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y

imm

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ET

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2

4

6

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/gP

VC

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icle

MD

V-3

100

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0.5 %ID/g

Vehicle

MDV-3100

% M

ax

PSCA per Cell

A B

C

100

80

60

40

20

0

103 104 105 106 107

Figure 4. Ex vivo quantitative flowcytometry shows downregulationof PSCA on LAPC-9 tumor cells inresponse to treatment with MDV-3100 (A). A11 minibody imaging ofmice bearing LAPC-9 xenograftstreated with MDV-3100 showsdecreased tumor uptake in vivo ascomparedwith vehicle controls (B).Quantification of the microPET/CTresults shows no difference intumor volumes or minibody uptakepretreatment (C). However,following a week of treatment, A11immunoPET shows significantlydecreased uptake in MDV-3100–treated mice compared withvehicle controls (P ¼ 0.004),whereas CT volume shows nodifference between the groups(P ¼ 0.54).

Table 2. Biodistribution of 124I-A11minibody inmice bearing LAPC-9 xenografts at 44 hours after injectionshow decreased uptake in MDV-3100 (40 mg/kg) treated mice compared with vehicle control

Vehicle MDV-3100

n %ID/g � SD n %ID/g � SD P

LAPC-9 9 3.63 � 0.59 9 2.75 � 0.95 0.03Blood 5 0.65 � 0.11 5 0.61 � 0.14 0.63Liver 5 0.19 � 0.01 5 0.18 � 0.03 0.48Kidney 5 0.26 � 0.03 5 0.26 � 0.08 0.95Heart 5 0.32 � 0.06 5 0.25 � 0.05 0.08Lungs 5 0.54 � 0.08 5 0.62 � 0.19 0.42Spleen 5 0.27 � 0.04 5 0.36 � 0.11 0.15Stomach 5 0.38 � 0.09 5 0.30 � 0.05 0.14Tail 5 0.56 � 0.27 5 0.59 � 0.25 0.86Muscle 5 0.05 � 0.02 5 0.05 � 0.01 0.92Carcass 5 0.13 � 0.02 5 0.12 � 0.02 0.57

Tumor:blood 9 5.58 � 1.07 9 4.51 � 1.32 0.08Tumor:muscle 9 74.0 � 29.1 9 53.6 � 17.7 0.09

Knowles et al.





DiscussionAn ideal imaging modality for prostate cancer would

allow for early detection of metastases that would informtreatment decisions. Imaging of prostate cancer metastaseswith an imaging agent specific to the tumor itself instead ofimaging a downstream process such as bone formationcould yield both higher sensitivity and specificity for detect-ing prostate cancer bone metastases as well as the potentialto image local prostate cancer and metastases to other sites.An ideal imagingmodality could likewise determinewheth-er a chosen therapy is effective in a patient.While PSA bloodtests can measure the response of the tumors in aggregate,they cannot detect whether only a subset of lesions areresistant to therapy. Molecular imaging of AR activity couldyield more detailed information regarding individualtumor response to androgen deprivation therapy and devel-opment of resistance through the reactivation of AR path-ways. ImmunoPET imaging with radiolabeled antibodiestargeted to cell surface biomarkers has shown potential foruse in this role (13, 31, 47). In previous work, we haveshown that 124I-A11 anti-PSCA minibody is capable ofspecifically imaging PSCA-expressing tumors and that therapid clearance of the minibody provides excellent imagingcontrast at 44 hour postinjection (32, 33). In this work,we demonstrate that A11 minibody immunoPET outper-forms the sensitivity and specificity of 18F-Fluoride bonescans for detecting intratibial tumors and can image PSCAdownregulation in response the androgen deprivation withMDV-3100 in vivo.While all LAPC-9 intratibial xenografts in this study show

osteoblastic changes and spiculated bone formation on exvivo microCT and histology, bone scans of LAPC-9 intrati-bial xenografts show qualitative increases over the shamtibia in only 50% (3/6) mice at 6 weeks after tumorinoculation indicating that the bone scans missed a signif-icant number of osteoblastic lesions. Even at 8 weeks, whenthe tumors have eroded through the bone and into thesurroundingmuscle, the 18F-Fluoride bone scans only showa fairly subtle andquestionable increase in uptake in 50%ofmice (1/2). The low sensitivity of the 18F-Fluoride bonescans may have been due to the intratibial tumors’ prox-imity to the nonspecific uptake in the tibial growth plateillustrating the problems caused by nonspecific uptake of18F-Fluoride. Quantification of the LAPC-9 bone scanslikewise revealed that while there was a significant increasein uptake overall, no significant increase was seen for anytime point individually due to the large overlap in uptakebetween the tumor-bearing tibia and the sham control.Comparison of the bone scans with A11 minibody

immunoPET reveals that immunoPET imaging is moresensitive for imaging PSCA-expressing bone tumors with100% (6/6) mice bearing LAPC-9 intratibial xenograftsshowing clear tumor uptake at 6 weeks. In contrast withthe bone scans, A11 minibody immunoPET shows highlyspecific imaging with minimal activity anywhere in themouse other than in the PSCA-expressing tumor. This highspecificity results in easier interpretation of scan results asvirtually any lesion above blood activity can be interpreted

as a tumor with the exception of only the thyroid, stomach,and bladder due to free 124I released from catabolizedantibody. It should be noted that the quantification of thetibial uptake underestimates the actual uptake of A11mini-body in the LAPC-9 tumors by a large margin. Whilequantification of uptake in the entire tibia was the leastbiased method of comparing both A11 immunoPET andbone scans and in vivo and ex vivo measurements using theA11 minibody, the tumor makes up only a fraction of thetibial volume and mass and the %ID/g is decreased by theinclusion of bone and other nontumor tissue in themeasurement.

The results of this work indicate that the A11 minibodyhas the potential to translate into an imaging agent withhigher sensitivity and specificity than bone scans for imag-ing prostate cancer bone metastases in the clinic. In addi-tion, while bone scans are limited to the detection ofrelatively large bone metastases that cause osteoblasticchanges in the surrounding bone, immunoPET imagingcan not only image tumors before changes are seen on bonescan, but also image tumors in locations other than bone.Lymph node metastases, lung metastases, liver metastases,and metastases to other locations have been shown toexpress PSCA and may be able to be imaged with A11minibody immunoPET adding to its diagnostic value as upto 19% of patients have been found to have only visceralmetastases with no bone involvement (18, 19, 48). Inaddition, PSCA expression has been correlated with tumorstage,metastatic potential, and poor outcomes and imagingof PSCA may help stratify patients with high-risk localdisease even in the absence of metastases (21–26).

It has previously been suggested that PSCAmay be down-regulated in prostate cancer in response to androgen dep-rivation (27–30). In this work, we found that treatment ofmice bearing naturally PSCA-expressing LAPC-9 xenograftswith the antiandrogen MDV-3100 causes a significant,nearly 3-fold, downregulation of PSCA in vivo. Imaging ofPSCA expression, in addition to helping aid in the locali-zation of metastases, therefore, holds the potential forimaging the activity of the AR signaling axis. After 1 weekof treatment, LAPC-9 xenografts treated with MDV-3100show no significant differences in volume or mass com-pared with vehicle-treated controls. However, the MDV-3100–treated mice show significantly lower uptake of the124I-A11minibody than vehicle controls by bothmicroPETand biodistribution. These results suggest that quantitativeimaging of PSCA expression holds the potential to measurethe efficacy of ADT in men with prostate cancer beforechanges in tumor volume can be observed. A11 minibodyimmunoPET may also be effective in imaging the reactiva-tion of the AR axis upon development of CRPC.

Modeling work by Thurber andWeissleder has suggestedthat antibody uptake into a tumor will only directly reflectantigen expression when the binding sites are relativelyclose to saturation and that, otherwise, the antibody uptakewill be limited by the rate of antibody extravasation fromthe vasculature and diffusion into the tissue (49). However,large antibody doses and receptor saturation produces a






blocking effect that reduces imaging contrast and this effectneeds to be balanced with the desire for antigen quantita-tion. While, the dose of A11 minibody in the response totherapy experiments was increased, the PSCA antigensremained relatively far from saturation and hence thedecreased uptake of A11 minibody into the tumor (29%)does not directly reflect the degree of PSCA downregulation(63%). The decrease in A11 minibody uptake with MDV-3100 treatment is significant by both microPET/CT andbiodistribution. However, the picture is complicated bycomparison to the pretreatment imaging. Rather than asimple decrease in A11 minibody uptake in the MDV-3100–treated mice compared with the pretreatment imag-ing, we instead see an increase in the vehicle-treated mice.A11 minibody uptake increased by 59.7 � 8.5% (P ¼0.0002) in the vehicle–treated mice compared with thepretreatment imaging, whereas the MDV-3100–treatedmice show no significant difference between pre- and post-treatment scans (P ¼ 0.90). These results cannot beexplained by differences in vehicle and MDV-3100 cohorttumor volumes, masses, or necrosis as no significant differ-ences in these variables were observed (Supplementary Figs.S5–S6). The result was also not due to residual activity fromthe pretreatment scan as residual activity was minimal andequivalent between the groups. Other explanations for theincrease in uptake posttreatment include changes in tumorvascularity and permeability as the tumors of both cohortsgrew in the 7 days between scans. We recently published amethod that uses diffusion-limited kinetic modeling ofdynamic imagingwith the A11minibody tumor tomeasurepermeability, vascularity, and antigen concentration inde-pendently which we intend to utilize to address thesequestions in future works (50). Regardless of the cause ofthe baseline shift between pre- and posttherapy scans,normalizing of posttreatment uptake values by pretreat-ment imaging reduces the intracohort variance and resultsin large effect size compared with the non-normalized data(P¼ 0.0003). While the explanation for the increase in A11minibody uptake in the vehicle-treated group between thepre- and posttreatment scans requires further investigation,there was an unequivocal decrease in uptake in the MDV-3100–treatedmice comparedwith the vehicle control that islikely due to the PSCA downregulation seen by ex vivo flowcytometry.

In summary, the majority of local prostate cancer tumorsand metastases express PSCA and its expression correlateswith tumor grade, stage, invasiveness, andmetastatic poten-tial. The A11 anti-PSCA minibody has shown the ability tospecifically image PSCA-expressing cells in vivo and has

potential diagnostic utility in noninvasive imaging, staging,and risk stratification of prostate cancer (32). In this work,theA11minibody achievedhigher sensitivity and specificitythan 18F-Fluoride bone scans for detecting osteoblasticintratibial xenografts, which may allow for earlier clinicalmetastasis detection and improved patient risk stratifica-tion. Furthermore, we demonstrated that A11 minibodyimmunoPET showed decreased tumor uptake in responseto androgen deprivation in vivo. The A11 minibody, there-fore, has clinical potential for monitoring the response toandrogen deprivation and the development of castrationresistance. As the response to ADT and development ofandrogen independence can be quite heterogeneousbetween different tumors, determination of whether a sub-set of tumors fails to respond to treatment could potentiallyallow for targeted therapies (e.g., external beam radiation)to have a larger role in treating metastatic disease or for amore prompt transition to systemic chemotherapy. Wehave, therefore, begun investigation of 124 I-labeled anti-PSCA A11 minibody in the clinical setting.

Disclosure of Potential Conflicts of InterestA. Wu is an employee of, has ownership interest in, and is a consultant/

advisory board member for ImaginAb, Inc. No potential conflicts of interestwere disclosed by the other authors.

Authors' ContributionsConception and design: S.M. Knowles, R. Tavare, K.A. Zettlitz, R.E. Reiter,A.M. WuDevelopment of methodology: S.M. Knowles, Z.K. JiangAcquisition of data (provided animals, acquired and managed pati-ents, provided facilities, etc.): S.M. Knowles, R. Tavare, K.A. Zettlitz,M.M. Rochefort, F. Salazar, A.M. WuAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): S.M. Knowles, R. Tavare, K.A. Zettlitz,M.M. Rochefort, R.E. Reiter, A.M. WuWriting, review, and/or revision of the manuscript: S.M. KnowlesAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): F. Salazar, A.M. WuStudy supervision: R.E. Reiter

AcknowledgmentsThe authors thank Waldemar Ladno, Darin Williams, Melissa

McCracken, Dr. David Stout, and Dr. John David for technical assistancewith these experiments.

Grant SupportThis work was supported by NIH grants CA092131, CA016042, and

T32GM008042, Department of Defense W81WXH-08-1-0442, Departmentof Energy DE-SC0001220, and NCI F30CA165824.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received June 6, 2014; revised September 14, 2014; accepted September15, 2014; published OnlineFirst October 17, 2014.

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2014;20:6367-6378. Published OnlineFirst October 17, 2014.Clin Cancer Res Scott M. Knowles, Richard Tavaré, Kirstin A. Zettlitz, et al. Mouse Xenograft Models of Prostate Cancerfor Imaging Disease Progression and Response to Therapy in

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Applications of ImmunoPET: Using I-Anti-PSCA A11 …clincancerres.aacrjournals.org/content/clincanres/20/24/6367.full.pdfwork, we will compare the sensitivity of the A11 minibody to18F-Fluoride