review article zirconium-89 labeled antibodies: a new tool for...

Review ArticleZirconium-89 Labeled Antibodies A New Tool forMolecular Imaging in Cancer Patients

Floor C J van de Watering1 Mark Rijpkema1 Lars Perk2

Ulrich Brinkmann3 Wim J G Oyen1 and Otto C Boerman1

1 Department of Radiology and Nuclear Medicine Radboud University Medical Center Geert Grooteplein Zuid 106525 GA Nijmegen The Netherlands

2 Radboud Translational Medicine BV Reinier Postlaan 2 6525 GC Nijmegen The Netherlands3 Roche Pharma Research amp Early Development Large Molecule Research Nonnenwald 2 82377 Penzberg Germany

Correspondence should be addressed to Otto C Boerman ottoboermanradboudumcnl

Received 14 March 2014 Accepted 23 April 2014 Published 28 May 2014

Academic Editor Roland Haubner

Copyright copy 2014 Floor C J van de Watering et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Antibody based positron emission tomography (immuno-PET) imaging is of increasing importance to visualize and characterizetumor lesions Additionally it can be used to identify patients who may benefit from a particular therapy and monitor the therapyoutcome In recent years the field is focused on 89Zr a radiometal with near ideal physical and chemical properties for immuno-PETIn this reviewwewill discuss the production of 89Zr the bioconjugation strategies and applications in (pre-)clinical studies of 89Zr-based immuno-PET in oncology To date 89Zr-based PET imaging has been investigated in a wide variety of cancer-related targetsMoreover clinical studies have shown the feasibility for 89Zr-based immuno-PET to predict andmonitor treatment which could beused to tailor treatment for the individual patient Further research should be directed towards the development of standardized androbust conjugation methods and improved chelators to minimize the amount of released Zr4+ from the antibodies Additionallyfurther validation of the imaging method is required The ongoing development of new 89Zr-labeled antibodies directed againstnovel tumor targets is expected to expand applications of 89Zr-labeled immuno-PET to a valuable method in the medical imaging

1 Introduction

Molecular biomarkers can be used to monitor image andmeasure biological processes at molecular or cellular levelDifferent types of biomarkers are known including diagnos-tic prognostic and predictive biomarkers or a combina-tion of these [1] Extensive research has been done on thedevelopment of molecular imaging biomarkers in the field ofcancer This has led to tools that can be used to visualize andcharacterize tumor lesions An advantage of using molecularimaging agents is the noninvasive nature of these procedureswhereas in conventional methods a more invasive procedureis used (eg blood sample or biopsy) Various imagingmoda-lities can be used for tumor visualization such as fluorescentimaging magnetic resonance imaging (MRI) or radionuclideimaging with positron emission tomography (PET) or singlephoton emission computed tomography (SPECT) In most

cases the use of PET is preferred over SPECT since higherspatial resolution images can be obtained and images can beanalyzed quantitatively more accurately with PET Specificuptake of molecular biomarkers can be achieved usingradiolabeled targeting agents such as antibodies directedagainst tumor-associated antigens like epidermal growthfactor receptor (EGFR) [2] human epidermal growth factorreceptor 2 (HER2) andmany othersThe high specificity andaffinity of radiolabeled antibodies make them attractive can-didates as an imaging agent For example 89Zr-labeled anti-HER2 antibodies can be used to differentiate betweenHER2+and HER2minus tumors [3] also appreciating intra- and inter-tumoral heterogeneity An additional application of radiola-beled antibodies is to identify patients who may benefit froma particular therapy and monitor therapy outcome based onthe level of tumor-associated antigen expression [4] How-ever the relative slow pharmacokinetics of intact antibodies

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 203601 13 pageshttpdxdoiorg1011552014203601

2 BioMed Research International

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= 3-4 days) requires the use of radionuclides with longhalf-lives (eg 111In (28 days) for SPECT or 89Zr (33 days)and 124I (42 days) for PET [5]) For antibody based PETimaging (immune-PET) 89Zr has several advantages 89Zr hasa half-life of 784 h which matches the pharmacokinetics ofantibodies and it has a relative low average positron energy of395 keV making it an ideal candidate for high resolution PETimaging of slow-accumulating biomolecules In addition89Zr-based agents are safer to handle and more stable in vivomaking them better candidates than 124I-based agents forclinical applications Due to the numerous advantages of89Zr-based immuno-PET the field is progressing at a rapidand exciting pace In this review the potential of 89Zr-basedimmuno-PET in oncology will be reviewed The productionof 89Zr the bioconjugation strategies and applications in(pre-)clinical studies are discussed

2 Radiochemical Properties of 89Zr89Zr decays (half-life of 784 h) first via positron emission andelectron capture to 89mY (half-life of 157 s) which in turndecays via gamma ray emission (909 keV) to the stable 89YWith its relatively low energy positrons (average energy395 keV) 89Zr provides high resolution PET images In addi-tion the energy disparity between the photons (511 keV) andthe gamma rays (909 keV) prevents the latter from interferingwith the detection of 511 keV photons In contrast its halogencompetitor 124I produces high energy photons of differentenergies (603 keV (630) 1691 keV (109) and 723 keV(104) [6]) which may result in random and scatter coinci-dences and therefore in more background noise as comparedto 89Zr Hence reconstruction of 89Zr-based PET scans ismore straightforward to attain good image quality comparedto 124I Although 89Zr has many advantages over other PETradionuclides some essential shielding requirements duringtransport and handling of 89Zr are needed (half-value layerof 89Zr in lead is roughly 10mm) High energy and highlypenetrating photons (909 keV) are emitted during 89Zr decayin high abundance

3 Production of 89Zr

The first production of 89Zr was done by Link et al [7] bya (pn) nuclear reaction by bombarding 89Y on Y foil with13MeV protons [5]The produced 89Zr needed several purifi-cation steps andwas obtained in 80 yield with radionuclidicpurity exceeding 99 Nowadays many medical centers areable to producemedical isotopes using low-energy cyclotronsthat are capable of bombarding targets with protons of lowenergy (lt20MeV) Therefore the most common route toproduce 89Zr is via the 89Y (p n) 89Zr reaction on commer-cially available 89Y target foilsThe above route will in generalresult in high yields (94-95) and high radionuclidic purities(gt99) Competing nuclear reaction like (p 2n) reactionscan result in small amounts radionuclidic byproducts suchas 88Zr and 88Y [8] Several separation and purification

techniques with variable outcomes are used including anionexchange cation exchange and solvent extraction [9ndash11] Forsynthesizing such radiopharmaceuticals for patients auto-mated units for a clean fast safe and reproducible radionu-clide synthesis according to good manufacturing practice(GMP) are necessary Several groups have designed and builtautomated systems for 89Zr [12 13] For exampleWooten et al[14] reported a custom-made system to safely and routinelyproduce 89Zr with high radionuclidic purity (gt9999) andsatisfactory effective specific activity (5ndash353 mCisdot120583molminus1(001ndash088of theoretical specific activity)) based on previ-ous developments in separation and purification techniques[9ndash11 15]

4 The Need for Efficient Chelators

The release of 89Zr4+ from the antibodies needs to be pre-vented because the free radionuclide can accumulate in themineral bone and can associate with plasma proteins Thisleads to depositing significant doses to the bone marrow [16]Therefore an appropriate chelator system is necessary tomin-imize the disassociation of 89Zr from the antibodies Over theyears several chelators have been used with different suc-cess such as diethylenetriaminepentaacetic acid (DTPA)ethylenediaminetetraacetic acid (EDTA) 14710-tetraaceticacid (DOTA) and desferoxamine (DFO) [17]The stability ofZr-DOTA Zr-DTPA and Zr-EDTA was found to be limitedThe thermodynamic stability of Zr-DTPA is slightly higherthan that of Zr-EDTA most likely because DTPA coordi-natively saturates the Zr4+ while EDTA requires exogenouswater molecules [18] DFO is the most prominent chelatorof Zr4+ DFO is a hexadentate siderophore containing threehydroxamate groups for chelating metals and a primaryamine tail for conjugation to a biomolecule (Figure 1) Besidesa zirconium chelator it is a chelating agent for several othermetal ions [19] It demonstrated good stability releasing lessthan 02 of Zr4+ after 24 h in serum [20] and after seven daysin serum still less than 2 demetallation occurs [21] Severalproof-of-principle preclinical studies have been conductedusing DFO to label antibodies with 89Zr however the in vivostability of this complex remains an issue because free 89Zr isobserved in the bone dependent on the in vivo behavior of theantibody [22] Several studies have attempted to improve thelinkage between DFO and the antibody [9 23] whereas oth-ers have focused on improving the chelate itself [24] Even-tually a ligand that is both octadentate and oxygen-rich isbelieved to be the most stable Zr4+ chelator since it would beable to incorporate all eight coordination sites of zirconium[22] This novel high stability Zr4+ ligand would in theoryminimize the uptake of liberated Zr4+ in the bone and othernontargeted tissues To date the design synthesis and theevaluation of such a Zr4+ chelate requires further research

41 Conjugation of Antibodies withDFO AsDFO is currentlythe most promising chelator for 89Zr4+ conjugation of anti-bodies with DFO will be discussed here in detail (Figure 1)Several methods are available to conjugate DFO based on

BioMed Research International 3

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Figure 1 Schematic overview of 89Zr-labeled antibody using DFO as chelator

the reaction of an activated bifunctional chelator with a lysineor cysteine residue of the antibody The different conjugationtechniques do not only have different conjugation efficienciesbut also affect the biodistribution of the radiolabeled anti-body [22]

The earliest reports on conjugation of DFO to bioactivemolecules is based on the addition of thiols to the aminogroup ofDFO [20] In this approachDFOwasmodified byN-succinimidyl-S-acetylthioacetate (SATA) resulting in an S-acetyl-protected thiol derivatized formof chelator In parallelmaleimide moieties were introduced in the antibody bythe reaction with 4-(N-maleimidomethyl) cyclohexane car-boxylic acid N-hydroxysuccinimide ester (SMCC) Next thetwo formed compounds were combined and in the presenceof hydroxylamine at physiological pH the DFO-antibodyconjugate was formed Following this early work Verel et alintroduced a novel conjugation approach which was basedon an activated 2356-tetrafluorophenol (TFP) chelate esterwhich can form a stable amide bondwith the 120576-amino-groupsof the lysine residues of the monoclonal antibody (mAb) [9]This laborious approach consisted of 5 steps which involve (i)the extension of DFOwith succinyl anhydride (ii) protectionof side-reactions of the hydroxamate groups of the ligandby complexation with Fe3+ (iii) formation of activated TFPester (iv) conjugation of activated DFO-ester to the unmod-ified antibody and (v) removal of Fe3+ from the chelatorNowadays the most widely used method in preclinical 89Zr-based immuno-PET uses the conjugation strategy with N-succinimidyl-DFOwhich addresses 120576-amino groups of lysineside chains [22 25ndash30] Since the Zr4+ field is rapidly growingand becomingmoremainstream in the clinical setting simplemethods for DFO conjugation preferably using commercialavailable starting materials are essential Perk et al intro-duced a simplified method using a commercially availablep-isothiocyanatobenzyl-DFO (DFO-Bz-NCS) chelate whichcan be directly attached to the 120576-aminogroups of the lysineresidues of an antibody by forming a stable thiourea linkage[23] Despite the fact that this method is simpler than theN-succinimidyl-DFO chemistry it requires more expertisemainly because of the limited water solubility of the chelatorprecursor

A limitation of the conjugation of DFO to antibodies iscompromised immunoreactivity because the chelator mayinterfere with the antigen-binding domain of the antibodyespecially if there are lysines in or close to the complemen-tarity determining regions of the antibody To overcome theselimitations site-specific strategies using engineered cysteineresidues can be used in combination with thiol-reactive DFOderivatives such as bromoacetamido-desferrioxamine (DFO-Bac) iodoacetamido-desferrioxamine (DFO-Iac) and male-imidocyclohexyl-desferrioxamine (DFO-CHX-Mal) [31]The radiolabeled antibodies using these thiol-reactive DFOderivatives were stable and showed similar characteristicsas the lysine-linked complexes Remarkably no significantdifference was observed between the immunoreactivity of thesite-specific complex and the lysine-linked complexes

Another novel conjugation approach for effective labelingof 89Zr to antibodies is the use of click chemistry between anacetylene group and an azideThis approachmight not signif-icantly improve the targeting of tumors compared with DFO-based conjugation strategies however with this approach itis possible to fully tailor the constructs Furthermore themodular system can be used for direct comparison of bio-conjugates with different radiometals as the the chelator-modified antibodies are synthesized using identical ligationconditions resulting in similar immunoreactivity and chela-torantibody ratios [32] Several studies have been reportedon bioorthogonal click chemistry [32] Staudinger ligation[33] or catalyst-free click chemistry [34]The click chemistryas specialized conjugation method is expected to expand thescope of 89Zr-based PET

5 Preclinical Studies with 89Zr

Over the last years several 89Zr-labeled antibodies directedagainst different tumor types have been evaluated in preclin-ical studies (eg [9 18 22 35 36] see Table 1) Here thesedevelopments of 89Zr-labeled antibodies in preclinical studieswill be discussed based on their tumor target

51 Targeting CD20 The glycosylated phosphoproteinCD20 is expressed on the surface of B-cell lymphomas hairy


Table 1 Overview of the described preclinical and clinical studies using 89Zr-labeled antibodies

Target Type of tumor Targeting vectorCD147 Pancreas 059-053CD20lowast Non-Hodgkinrsquos lymphoma ibritumomab tiuxetanCD44v6lowast Head and neck squamous cell carcinoma cmAb U36EGFR Multiple CetuximabEGP-1 Prostate hRS7GPC3 Liver 120572GPC3HER1 Colorectal PanitumunmabHER2lowast Breast and ovarian TrastuzumabIGF-1R Triple negative breast cancer R1507MET Head and neck squamous cell carcinoma and gastric DN30MNCA IX Renal cell carcinoma cG250PSMA Prostate 7E11PIGF Liver RO5323441VEGFlowast Breast head and neck squamous cell carcinoma and ovarian BevacizumablowastTargets evaluated in clinical studies

leukemia B-cell chronic lymphocytic leukemia and mela-noma cells 89Zr-labeled antibodies directed against CD20might be useful tomeasure andmonitor the therapeutic effectof non-Hodgkinrsquos lymphoma (NHL) therapy [18 37] The89Zr-Desferrioxamine-rituximab an antibody directedagainst CD20 specifically targeted the human CD20 antigenin a humanized CD20-expressing transgenic mouse model(huCD20TM) 90Y-labeled anti-CD20 mAb ibritumomabtiuxetan (Zevalin) is approved for treatment of patients withrelapsed and refractory NHL In a pilot study 89Zr-labeledibritumomab tiuxetan was shown to have a nearly identicalbiodistribution compared to 90Y-labeled counterpart [18]This indicated that a scout scan with 89Zr-ibritumomabimmuno-PET can be used to assess predict and quantify thebiodistribution of 90Y-ibritumomab tiuxetan

52 Targeting CD44 The cell-surface glycoprotein CD44 isinvolved in many biological processes including adhesion ofcells to extracellular matrix proteins lymphocyte-endothelialcell interactionsmetastasis formationmigration of cells andT cell activationadherence [38] The v6 splice variant ofCD44 is involved in tumorigenesis tumor cell invasion andmetastasis and is expressed preferentially in squamous cellcarcinomas [39] Preclinical studies using 89Zr-labeled anti-CD44v6 chimeric monoclonal antibody cU36 demonstratedthat the tracer was able to detect small tumors in nude micewith HNSCC xenografts [9 40] In addition it was reportedthat 89Zr-cU36 PET imaging was a suitable candidatefor scouting of therapeutic doses of 90Y-cU36 [40 41]Recently evaluation of 89Zr-RG7356 an antibody directedagainst the constant part of CD44 was performed in micebearing tumor xenografts with different levels of CD44expression and RG7356 responsiveness namely MDA-MB-231 (CD44+ responsive) PL45 (CD44+ nonresponsive) andHepG2 (CD44minus nonresponsive) [42] 89Zr-RG7356 selec-tively targeted CD44+ responsive and nonresponsive tumors

in mice 89Zr-RG7356 whole body immuno-PET in healthycynomolgus monkeys revealed antibody uptake in spleensalivary gland and bone marrow which might be related tothe expression of CD44 in these organs The 89Zr-RG7356uptake in the normal organs decreased with increasing doseof unlabeled RG7356 indicating saturable targeting of CD44in these animals

53 Targeting EGFR The epidermal growth factor receptor(EGFR) is a member of the ErbB family It plays a crucial rolein differentiation proliferation and survival of many differ-ent tumor types including breast lung bladder and coloncarcinoma [2]The overexpression of EGFR is associatedwithmore aggressive tumors and poor prognosis due to the resis-tance of treatment [43 44] ManymAbs have been developedto inhibit the EGFR activation [2] A well-known exampleis cetuximab (Erbitux) a chimeric IgG which upon bindingto the ligand-binding domain induces internalization ofEGFR and thereby blocking downstream signalling [45 46]Several studies showed tumor regression upon treatmentwith cetuximab [47ndash50]89Zr-labeled cetuximab was evaluated for scouting the

biodistribution of 90Y- and 177Lu-cetuximab in tumor bear-ing mouse and thus potentially allowing the estimation of theradiation dose delivered to tumors and normal tissues duringradioimmunotherapy with 90Y- and 177Lu-cetuximab [51] Itwas reported that the 89Zr-immuno-PET could be used forin vivo scouting of 90Y- and 177Lu-labeled mAbs Howeveran increased bone uptake of 89Zr-cetuximab compared with90Y- and 177Lu labeled cetuximab was observed indicatingthat 89Zr is more efficiently incorporated in the bone com-pared to the other radiometals (90Y- and 177Lu) Thereforeestimating bone marrow doses based on 89Zr-bone uptake isnot straightforward Another study investigated the relationbetween the in vivo expression of EGFR and the tumoruptake of 89Zr-cetuximab [52] In this study no clear-cutrelationship was found suggesting that apart from antigen


(+ve) (minusve)Her2 Her2

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18F-FDG 18F-FLT 89Zr-trastuzumab

0 4 0 10 0 25( IDg) ( IDg) ( IDg)

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Figure 2 Specificity of 89Zr-trastuzumab for HER2-positive tumors Coronal 89Zr-trastuzumab 18F-FDG and 18F-FLT PET images ofathymic nude mice bearing subcutaneous HER2-positive NCI-N87 (left) and HER2-negative MKN-74 (right) are shown ROIs (IDg) for89Zr-trastuzumab 18F-FDG and 18F-FLT are indicated +ve = positive minusve = negative This research was originally published in [3] copy by theSociety of Nuclear Medicine and Molecular Imaging Inc

expression other parameters determine the tumor uptake of89Zr-cetuximab

Another approved mAb to inhibit the EGFR signallingis panitumumab It was the first recombinant human mon-oclonal antibody (IgG2) approved by the FDA for the treat-ment of patients with EGFR-expressing metastatic colorectalcancer (mCRC) [53] In several studies the use of panitu-mumab for noninvasive in vivo imaging of HER1 expressionin tumors is reported [54ndash58]The use of 89Zr-panitumumabfor immuno-PET of HER1 expression was recently evaluatedin a direct comparison with 111In-panitumumab The organbiodistribution between 111In- and 89Zr-panitumumab wasalmost identical [55] In addition the targeting of 89Zr-panitumumab correlated well with the HER1 expressionRecently a standardized and straightforward stepwise sim5 hproduction method was reported for the production ofclinical-grade 89Zr-panitumumab [59] In this methodclinical-grade panitumumab is conjugated with DFO chelateand subsequently radiolabeled with 89Zr resulting in highyields (gt70) and high radiochemical purity (gt98)

54 Targeting HER2 Human epidermal growth factor recep-tor 2 (HER2) is another member of the ErbB family It isinvolved in angiogenesis differentiation metastasis prolifer-ation and cell survival upon heterodimerization with othermembers of the EGF receptor family [60] HER2 overex-pression is found in many types of tumors including breastand ovarian cancer The FDA approved anti-HER2 mAbtrastuzumab (Herceptin Genentech CAUSA) to be used forthe treatment of HER2 positive breast tumors since it blocksthe HER2 activation [60] The efficacy of the treatment is

dependent on the HER2 expression level The HER2 expres-sion level in a tumor is not static and may vary over time[60] In addition theHER2 expression is found to be differentbetween the primary lesion and the distant metastatic lesionsin the same patient Noninvasive in vivo imaging to visualizeHER2 expressing using radiolabeled trastuzumab has beenextensively investigated [29 30 61] PET imaging using89Zr-trastuzumab has been performed in different murinetumor models and accumulation of the tracer was found tobe HER2 specific [29 30 61] For example the tumor uptakeof 89Zr-trastuzumab in nude mice with a subcutaneoushuman ovarian cancer xenografts (SK-OV-3) was high(sim30 IDg) and the biodistribution was similar to thatof 111In-trastuzumab [29] Recently the specificity of 89Zr-trastuzumab 18F-FDG and 18F-FLT PET for HER2-positivegastric cancer was evaluated ([3] Figure 2) The studyrevealed a high specific uptake of 89Zr-trastuzumab inHER2-positive tumors whereas 18F-FDG and 18F-FLT PET wereunable to differentiate between HER2-positive and HER2-negative tumors In addition 89Zr-trastuzumab was used toquantitatively determine the HER2 expression level aftertreatment For example after treatment with a heat shockprotein 90 (hsp90) inhibitor a significant decrease in HER2expression could bemeasured based on the 89Zr-trastuzumabtumor targeting [30 62] A combination treatment ofhsp90 inhibitor 17AAG and the EGFRHER2 tyrosine kinaseinhibitor lapatinib revealed an even stronger reduction of theHER2 expression levels using 89Zr-Trastuzumab-F(ab1015840)

2

fragment as probe [63] Additionally the biological effect ofafatinib an EGFRHER2HER4 inhibitor in aHER2-positivegastric xenograft models was evaluated [3] In this model theuptake of 18F-FDG did not change after afatinib therapy


whereas a decrease in 89Zr-trastuzumab uptake was observedupon treatment The lower uptake of the 89Zr-trastuzumabcorrelated with the decreased HER2 expression as deter-mined by immunoblots and immunohistochemistry Thus89Zr-trastuzumab PET might be useful for the characteriza-tion treatment planning and treatment monitoring of HER-2 positive cancers

55 Targeting VEGF Vascular endothelial growth factor(VEGF) is a proangiogenic factor in both normal tissues andin tumors The overexpression of VEGF and its receptors(VEGFR) are associated with poor prognosis [64] Thehumanized anti-VEGF mAb bevacizumab (AvastinGenentechHoffmann-La Roche) is capable of blockingangiogenesis by depleting VEGF and thereby preventing itsbinding to the VEGFR This neutralizes VEGF actions (seeeg [65ndash72]) A direct comparison between 89Zr-bevacizumab and an irrelevant 89Zr-labeled IgG revealed asignificantly higher tumor uptake of 89Zr-bevacizumab innude mice with human ovarian SK-OV-3 tumors [73]Besides using 89Zr-bevacizumab as PET tracer for non-invasive in vivo imaging of VEGF expression in the tumormicroenvironment potentially it can also be used to predictormonitor an antiangiogenic response For example hsp90 iscrucial player in VEGF transcription and can be used to treatovarian tumors In nude mice with a subcutaneous humanovarian cancer xenografts (A2780) uptake of 89Zr-bevacizumab in the tumors correlated with the therapeuticeffect of the hsp90 inhibitor NVP-AUY922 [74] In anotherstudy the effect of the mTOR inhibitor everolimus on theVEGF production was evaluated [75] Everolimus treatmentcaused decreased 89Zr-bevacizumab uptake in subcutaneousA2780 human ovarian tumor The results were in linewith the lower VEGF-A protein levels in tumor lysates oftreated versus untreated tumors These results indicate 89Zr-bevacizumab can be used to monitor tumor VEGF-A levelsas an early biomarker of the antiangiogenic effect of mTORinhibitor treatment89Zr-labeled ranibizumab a monoclonal antibody frag-

ment (Fab) derivative of bevacizumab was used to detect andmonitor the early antiangiogenic response to treatment withsunitinib a VEGFR tyrosine kinase inhibitor in nude micebearing a subcutaneous A2780 human ovarian tumor orColo205 human colon cancer xenografts 89Zr-ranibizumabPET matched better with the observed results obtained byhistology immunohistochemistry and tumor proliferationand vascularization assays than 18F-FDGPET and 15O-waterPET Since ranibizumab has a serum half-life of only 2 to 6hours rapid and sequential follow-up PET scans are feasiblewith 89Zr-ranibizumab [76] Therefore in contrast to 89Zr-bevacizumab 89Zr-ranibizumab can be used for imaging ofrapid dynamic alterations in VEGF response in tumors

56 Targeting PIGF The clinical benefits of angiogenesisinhibitors can be compromised by the upregulation ofproangiogenic factors such as the placental growth factor(PIGF) PIGF a VEGF homolog is expressed in low levels innormal tissue and can be overexpressed in tumor cells PIGF

contributes to angiogenesis in pregnancy wound healingischemic conditions and tumor growth [77 78] PIGFinhibitors are able to reduce the angiogenesis and tumor cellmotilityThe antitumor activity of a humanizedmAbdirectedagainst PIGF-1 and PIGF-2 RO5323441 in human tumorxenograft models has been reported [79] To further exploreand validate the use of RO5323441 the tumor and normaltissue uptake of 89Zr-RO5323441 at different time points wasevaluated in mice bearing human PlGF-expressing Huh7hepatocellular cancer xenografts Tumor accumulation of89Zr-RO5323441 was specific and time- and dose-dependent

57 Targeting PSMA Prostate-specific membrane antigen(PSMA) is a transmembrane glycoprotein which is associatedwith increased tumor progression development of castrationresistance andor resistance to hormone-based treatments[80ndash82] PMSA is expressed in a limited range of normaltissues including benign prostatic epithelium renal proximaltubule small bowel and the brain however the expressionlevel is 2 to 3 times lower than in prostate cancer speci-mens [83] 89Zr-labeled anti-PSMA mAb J591 was able todifferentiate between subcutaneous PSMA positive and neg-ative tumors in athymic nude mice [21] making it a potentialtarget for clinical noninvasive identification and quantifica-tion of PSMA-positive tumors

58 Targeting CD147 CD147 a member of the immunoglob-ulin superfamily is involved in many physiological functionsincluding embryo implantation early stage neural networkformation and spermatogenesis [85] Overexpression ofCD147 is found in many types of cancer including pancreaticcancer and induces expression of matrix metalloproteinases(MMPs) and VEGF [86 87] Several (pre-)clinical studieshave been performed using anti-CD147 antibodies to inhibitthe actions of CD147 and revealed a reduction in prolifera-tion invasion andmetastasis of tumors [88ndash90] Almost 90of the pancreatic cancers have high CD147 expression levels[86] Sugyo et al evaluated the CD147 expression in fourpancreatic cancer cell lines (MIA Paca-2 PANC-1 BxPC-3and AsPC-1) using the human 125I- 67Ga- or 89Zr-labeledanti-CD147 mAb (059-053) [84] Additionally the in vivoCD147 expression was evaluated using 125I- or 89Zr-labeled059-053 in mice with sc and orthotopic MIA Paca-2 andA4 (non-CD147-expressing) tumorsThebiodistribution datarevealed significantly higher tumor uptake of 89Zr-059-053in MIA Paca-2 tumors than in the A4 tumors (Figure 3)PETCT imaging demonstrated that orthotopic MIA Paca-2tumors could be visualized with 89Zr-059-053 PET Highexpression of CD147 is not only restricted to pancreaticcancer but is also found in other types of cancer includingbladder breast colorectal cervical liver and ovarian cancer[84ndash86] Therefore 89Zr-059-053 might also be applied inpatients with these cancer types

59 Targeting CAIX Hypoxia in tumors is associated with apoor prognosis inmany tumor types since it is associatedwithresistance to radiotherapy and chemotherapy Inmany tumortypes carbonic anhydrase IX (CAIX) has been validated as


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Figure 3 In vivo biodistribution experiments in nude mice bearing MIA PaCa-2 and A4 xenografts of radiolabeled anti-CD147 antibody059-053 Samples were collected and weighted and radioactivity was measured at days 1 (white bars) 2 (dot bars) 4 (gray bars) and 6 (blackbars) after intravenous injection of 37 kBq each of 89Zr-059-053 (a) and 125I-059-053 (b) Data are expressed as mean plusmn SD (119899 = 5) lowast119875 lt 001versus 89Zr-059-053 tumor uptake at each time point analyzed by ANOVA with the Student-Newman-Keuls method multiple comparisontest This research was originally published in [84]

an intrinsic hypoxia-related cell marker [91] Using antibod-ies directed against CAIX it is possible to select patientsfor hypoxia-targeting or -modifying treatment combinedwith radiotherapy For example it is possible to visualizetumor hypoxia in mice bearing sc SCCNij3 head and necksquamous cell carcinomas using 89Zr-cG250-F(ab1015840)

2 an anti-

CAIX antibody fragment [92] In a direct comparison thetumor uptake of mAb 89Zr-cG250 in mice with CAIX-expressing clear cell renal cell carcinoma (ccRCC) xenografts(NU-12) was significantly higher compared to that of 124I-cG250 [93]This indicates that PET imaging of ccRCC tumorswith 89Zr-cG250 could be more sensitive than 124I-cG250-PET CAIX targeted 89Zr-PET imaging is a candidate forimaging hypoxia in different types of tumors and deservesfurther exploration

510 Targeting IGF-1R The insulin like growth factor 1receptor (IGF-1R) is a transmembrane receptor expressed inmany human cancers including insim35 of all triple-negativebreast carcinomas It is involved in the proliferation apop-tosis angiogenesis and tumor invasion Heskamp et alreported excellent tracer uptake of 111In-R1507 and 89Zr-R1507 a human mAb directed against IGF-1R in mice withsc SUM149 triple-negative breast cancer xenografts [94]This suggests that the use of 89Zr-R1507 in patient selectionof IGF-1R-targeted therapy is possible

511 Targeting Met The expression of hepatocyte growthfactor receptor tyrosine kinase (Met) was measured by PETusing 76Br or 89Zr-labeled-onartuzumab a mAb against Met[95] Both tracers specifically targeted Met however at latertime points a higher tumor uptake was observed with 89Zr-Onartuzumab This suggests that 89Zr-onartuzumab is thepreferred tracer to identify Met expression in cancer patientsand possibly to predict and monitor the treatment with

Met-targeted therapeutics In another study the potentialof immune-PET using 89Zr (residualising radionuclide) or124I-labeled (non-residualising radionuclide) anti-Met mAbDN30 was evaluated in mice with sc GLT-16 (high Metexpression) and FaDu (low Met expression) tumors [96]The biodistribution data revealed significantly higher tumoruptake of 89Zr-DN30 than 124I-DN30 in GTL-16 tumor-bearing mice Similar blood levels were found indicating thatDN30 is internalized 89Zr-DN30 immuno-PET imaging wasable to visualize small tumor lesions with a higher 89Zr tumoruptake in GTL-16 than FaDu tumor-bearing mice Addition-ally the correlation was high for PET-image-derived 89Zrtumor uptake and the ex vivo-assessed 89Zr tumor uptakeThis indicates that 89Zr-labeled immuno-PET is an attractivemethod to evaluate Met-targeted therapeutics

512 Targeting GPC3 The glypican-3 (GPC3) is a hepato-cellular-specific cell surface proteoglycan overexpressed inmost hepatocellular carcinomas (HCC) Sham et al reportedexcellent tracer uptake of 89Zr-120572GPC3 a mAb directedagainst GPC3 in mice with GPC3-expressing HepG2 livertumors [97] This suggests that the use of 89Zr-120572GPC3 toimage HCC in the liver is possible

6 Clinical Translation of 89Zr Immuno-PET

The 89Zr-labeled antibodies against the targets mentionedabove all show promising results for clinical translation Todate several clinical investigations using 89Zr-labeled anti-body constructs have been reported [1 22 98] Here theserecent clinical studies will be discussed

61 89Zr-Labeled cU36 The first clinical trial using the 89Zr-cU36 PET to target CD44 expressing tumors showed that


the tracer was able to detect primary tumors as well asmetastases in the neck region with similar sensitivity as com-puted tomography (CT) and magnetic resonance imaging(MRI) [99]The results are promising although several issuesremain to be addressed In the clinical studymicrometastaseswere missed with 89Zr-cU36 PET so immuno-PET may beless suited as a staging tool but more suitable to characterizetumorsMoreover 2 out of the 20 patients developed antibod-ies against the chimeric cU36 antibody (HACA) which mayhinder repetitive imaging procedures

62 89Zr-Ibritumomab A clinical prospective study was con-ducted to evaluate the biodistribution and radiation dosime-try of CD20-targeting 90Y-ibritumomab tiuxetan using 89Zr-ibritumomab tiuxetan [100] Patients with relapsed or refrac-tory aggressive B-cell (CD20-positive) NHL underwent aPET scan at 1 72 and 144 h after injection of 70MBq 89Zr-ibritumomab tiuxetan and again 2 weeks later after coin-jection of 15MBqkg or 30MBqkg 90Y-ibritumomab tiuxe-tan The results revealed that simultaneous therapy of 90Y-ibritumomab tiuxetan did not affect the biodistribution of89Zr-ibritumomab A second aim of the study was to estimatethe radiation doses during radioimmunotherapy with 90Y-ibritumomab tiuxetan based on 89Zr-ibritumomab PET Thehighest 90Y absorbed dose was observed in liver (32 plusmn18mGyMBq) followed by the spleen (29 plusmn 07mGyMBq)Additionally the correlation was high for standardizeduptake value (SUV) of 89Zr-ibritumomab tiuxetan andabsorbed dose of 90Y-ibritumomab tiuxetan in the liver at 72 hpi and 144 h piThis suggests that in the future a single 89Zr-ibritumomab tiuxetan PET scan is sufficient to optimize theadministered amount of 90Y-ibritumomab tiuxetan RIT forindividual patients

63 89Zr-Trastuzumab In 2010 the first-in-man report of89Zr-trastuzumab for imaging of HER2-positive lesions inpatients with metastatic breast cancer was published [101]14 Patients were included in the study that either received 10(119899 = 2) or 50 (119899 = 5) mg 89Zr-trastuzumab if trastuzumab-naıve and 10mg 89Zr-trastuzumab (119899 = 7) if on trastuzumabtreatment (37MBq 89Zr-trastuzumab) Per patient at leasttwo PET scans were acquired between day 2 and day 5after injection of 89Zr-trastuzumab The trastuzumab-naıvepatients required a 50mg dose for effective imaging whereas10mg was sufficient in the trastuzumab-treated patients Ahigher dose in the trastuzumab-naıve patients was requiredas an increased 89Zr-trastuzumab clearance was observedat lower doses due to presence of extracellular domains ofthe HER2 receptor in the circulation [102] After binding of89Zr-trastuzumab to these extracellular domains theimmune complex was cleared by the liver and excreted inthe intestines In patients treated with trastuzumab at thetime of injection higher doses of 89Zr-trastuzumab did notimprove imaging since complex formation was minimalOverall the uptake of 89Zr-trastuzumab in the tumor lesionswas high The best time to assess tumor uptake was 4 to5 days after injection of 89Zr-trastuzumab (Figure 4) All

the known and even some unknown lesions were detectedwith PET Of interest metastatic brain lesions were detectedin several patients despite the fact that trastuzumab cannotpenetrate the blood-brain barrier This is probably becausethe blood-brain barrier in patients with brain metastasis isdisrupted allowing 89Zr-trastuzumab to pass In this studyHER2 overexpressing lesions could be distinguishedfrom non-HER2 expressing lesions These data indicate thepotential use of 89Zr-trastuzumab to improve the diagnosis ofpatients with HER2-positive breast cancer especially whenlesions are inaccessible for biopsy

64 89Zr-Bevacizumab Recently a clinical study was per-formed to assess the use of 89Zr-bevacizumab for the visu-alization of VEGF-A in primary breast cancer [103] In 23patients 26 tumors were detected by conventional imagingmodalities mammography (119899 = 22) ultrasound (119899 = 25) orMRI (119899 = 1) Prior to surgery and 4 days pi of 37MBq of89Zr-bevacizumab the patients underwent a PETCT scan ofthe breasts and the axillary regions (Figure 5(a)) 25 of the26 breast cancer nodules (961) were detected using 89Zr-bevacizumab Also a correlation between the VEGF-A pro-tein level in the tumors observed as measured by VEGF-A ELISA and the tumor uptake 89Zr-bevacizumab wasfound (Figure 5(b)) This study provides evidence that 89Zr-bevacizumab might be a potential candidate for the classifi-cation of breast tumors and to predict and monitor the effectof VEGF-A targeted therapies

7 Conclusions

Clinical studies revealed that the use of 89Zr-based immuno-PET results in high spatial resolution images with high tumoruptake and a good signal to noise ratio Therefore the use of89Zr-labeled antibodies is very promising for noninvasivevisualization of tumor-associated antigens before duringand after therapy This makes 89Zr-based immuno-PET anexcellent imaging modality to predict and monitor treatmentand to tailor treatment for individual patients However tofully integrate 89Zr-based immuno-PET in the clinic severalhurdles still need to be overcome For example standard-ized and robust methods for stable conjugation of DFO toantibodies should become available to obtain clinical-gradeconjugates In addition research should focus on the devel-opment of improved chelators to minimize the amount ofliberated Zr4+ Although some direct comparison studiesbetween 89Zr-based immuno-PET and immuno-PET usingother PET isotopes have been performed and supplementaryquantitative and comprehensive comparison studies areneeded to evaluate the value of 89Zr-based immuno-PETAdditionally the radiation dose for patients undergoing a89Zr-based immuno-PET (75MBq of 89Zr-cmAb U36) wasfound to result in a mean effective dose of 053 to066mSvMBq [104] which is significantly higher comparedto themean effective dose of clinically used 111In- and 99Tcm-based tracers (111In-IgG (75MBq) was 025mSvMBq and99Tcm-IgG (750MBq) was mu SvMBq) [105] The high


(a) (b) (c)

Figure 4 Examples of 89Zr-trastuzumab uptake 5 days after the injection (a) a patient with liver and bone metastases and ((b) and (c)) twopatients withmultiple bonemetastases A number of lesions have been specifically indicated by arrowsThis research was originally publishedin [101]

1

2

(a)

VEG

F-A

(pg

mg)

89Zr-bevaciuzmab uptake (SUVmax)

0 2 4 6

600

400

200

0

(b)

Figure 5 (a) Axial slices of 89Zr-bevacizumab PET frompatient with primary breast tumor (1) and lymph nodemetastasis (2) (b) Correlationbetween 89Zr-bevacizumab tumor uptake (119909-axis) and tumor VEGF-A (119910-axis) levels as measured by ELISA (Pearson 119903 = 049 119875 = 004)This research was originally published in [103] copy by the Society of Nuclear Medicine and Molecular Imaging Inc

radiation dose for patients will limit repeated application of89Zr-based immuno-PET [104] However introducing newPETCT scanners to allow better-quality immuno-PETimages to be obtained with a lower 89Zr radioactivity(37MBq) dose have reduced the radiation dose [102 103]Furthermore research is focusing on combining 89Zr-basedimmuno-PET with other imaging modalities For examplethe use of 89Zr-immuno-PET in combination with near-infrared fluorescence (NIRF) imaging has been reported byseveral groups [106ndash108] The ongoing development of new89Zr-labeled antibodies directed against novel tumor targetsis believed to rapidly expand applications of 89Zr-labeledimmuno-PET to a valuable method in the medical imaging

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] Y Zhang H Hong and W Cai ldquoPET tracers based on zirc-onium-89rdquo Current Radiopharmaceuticals vol 4 no 2 pp 131ndash139 2011

[2] R S Herbst and D M Shin ldquoMonoclonal antibodies to targetepidermal growth factor receptor-positive tumors a newparadigm for cancer therapyrdquo Cancer vol 94 no 5 pp 1593ndash1611 2002


[3] Y Y Janjigian N Viola-Villegas J P Holland et al ldquoMonitoringafatinib treatment in HER2-positive gastric cancer with 18F-FDG and 89Zr-trastuzumab PETrdquo Journal of Nuclear Medicinevol 54 no 6 pp 936ndash943 2013

[4] C H Muselaers A B Stillebroer I M Desar et al ldquoTyrosinekinase inhibitor sorafenib decreases 111In-girentuximab uptakein patients with clear cell renal cell carcinomardquo Journal ofNuclear Medicine vol 55 no 2 pp 242ndash247 2014

[5] T J Wadas E H Wong G R Weisman and C J AndersonldquoCoordinating radiometals of copper gallium indium yttriumand zirconium for PET and SPECT imaging of diseaserdquo Chem-ical Reviews vol 110 no 5 pp 2858ndash2902 2010

[6] M Lubberink and H Herzog ldquoQuantitative imaging of 124Iand 86Y with PETrdquo European Journal of Nuclear Medicine andMolecular Imaging vol 38 supplement 1 pp S10ndashS18 2011

[7] J M Link K A Krohn J F Eary et al ldquoSixth internationalsymposium on radiopharmaceutical chemistry Abstracts PartIIIrdquo Journal of Labelled Compounds and Radiopharmaceuticalsvol 23 no 10-12 p 1297 1986

[8] A Kasbollah P Eu S Cowell and P Deb ldquoReview on produc-tion of 89Zr in a medical cyclotron for PET radiopharmaceuti-calsrdquo Journal of Nuclear Medicine Technology vol 41 no 1 pp35ndash41 2013

[9] I Verel GWVisser R BoellaardM Stigter-vanWalsumG BSnow and G A van Dongen ldquo89Zr immuno-PET comprehen-sive procedures for the production of 89Zr-labeled monoclonalantibodiesrdquo Journal of Nuclear Medicine vol 44 no 8 pp 1271ndash1281 2003

[10] W E Meijs J D M Herscheid H J Haisma et al ldquoProductionof highly pure no-carrier added 89Zr for the labelling ofantibodies with a positron emitterrdquo Applied Radiation andIsotopes vol 45 no 12 pp 1143ndash1147 1994

[11] J P Holland Y Sheh and J S Lewis ldquoStandardizedmethods forthe production of high specific-activity zirconium-89rdquo NuclearMedicine and Biology vol 36 no 7 pp 729ndash739 2009

[12] A L Wooten G D Schweitzer L A Lawrence E Madrid andS E Lapi ldquoAn automated system for production of 89Zrrdquo inProceedings of the 14th International Workshop on Targetry andTarget Chemistry (WTTC rsquo12) vol 1509 of AIP Conference pp201ndash205 Playa del Carmen Mexico August 2012

[13] J Siikanen M Peterson T A Tran P Roos T Ohlsson and ASandell ldquoA peristaltic pump driven 89Zr separation modulerdquo inProceedings of the 14th International Workshop on Targetry andTarget Chemistry (WTTC rsquo12) vol 1509 of AIP Conference pp206ndash210 Playa del Carmen Mexico August 2012

[14] A L Wooten E Madrid and G D Schweitzer ldquoRoutine pro-duction of 89Zr using an automated modulerdquo Applied Sciencesvol 3 no 3 pp 593ndash613 2013

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[16] D S Abou T Ku and P M Smith-Jones ldquoIn vivo biodistribu-tion and accumulation of 89Zr in micerdquo Nuclear Medicine andBiology vol 38 no 5 pp 675ndash681 2011

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[19] T Kiss and E Farkas ldquoMetal-binding ability of desferrioxamineBrdquo Journal of Inclusion Phenomena and Molecular Recognitionin Chemistry vol 32 no 2-3 pp 385ndash403 1998

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[22] M A Deri BM Zeglis L C Francesconi and J S Lewis ldquoPETimaging with 89Zr from radiochemistry to the clinicrdquo NuclearMedicine and Biology vol 40 no 1 pp 3ndash14 2013

[23] L Perk M W D Vosjan G W Visser et al ldquop-isothiocya-natobenzyl-desferrioxamine a new bifunctional chelate forfacile radiolabeling of monoclonal antibodies with zirconium-89 for immuno-PET imagingrdquo European Journal of NuclearMedicine and Molecular Imaging vol 37 no 2 pp 250ndash2592010

[24] F Guerard Y-S Lee R Tripier L P Szajek J R Deschampsand M W Brechbiel ldquoInvestigation of Zr(IV) and 89Zr(IV)complexation with hydroxamates progress towards designing abetter chelator than desferrioxamine B for immuno-PET imag-ingrdquo Chemical Communications vol 49 no 10 pp 1002ndash10042013

[25] S Bhattacharyya K Kurdziel L Wei et al ldquoZirconium-89labeled panitumumab a potential immuno-PET probe forHER1-expressing carcinomasrdquo Nuclear Medicine and Biologyvol 40 no 4 pp 451ndash457 2013

[26] D J Vugts GW Visser and G A van Dongen ldquo89Zr-PET rad-iochemistry in the development and application of therapeuticmonoclonal antibodies and other biologicalsrdquo Current Topics inMedicinal Chemistry vol 13 no 4 pp 446ndash457 2013

[27] K Leung ldquo89Zr-desferrioxamine b-j591 anti-prostate-specificmembrane antigenmonoclonal antibodyrdquo inMolecular Imagingand Contrast Agent Database (MICAD) National Center forBiotechnology Information Bethesda Md USA 2004

[28] A Chopra ldquo89Zr-labeled p-isothiocyanatobenzyl-desferrioxa-mine b (df-bz-ncs)-conjugated panitumumab a fully humanmonoclonal antibody directed against the extracellular domainIII of the epidermal growth factor receptorrdquo inMolecular Imag-ing and Contrast Agent Database (MICAD) National Center forBiotechnology Information Bethesda Md USA 2004

[29] E C Dijkers J G Kosterink A P Rademaker et al ldquoDevelop-ment and characterization of clinical-grade 89Zr- trastuzumabfor HER2neu immunoPET imagingrdquo Journal of NuclearMedicine vol 50 no 6 pp 974ndash981 2009

[30] T H Oude Munnink M A de Korte W B Nagengast et alldquo89Zr-trastuzumab PET visualises HER2 downregulation by theHSP90 inhibitor NVP-AUY922 in a human tumour xenograftrdquoEuropean Journal of Cancer vol 46 no 3 pp 678ndash684 2010

[31] J N Tinianow H S Gill A Ogasawara et al ldquoSite-specifically89Zr-labeled monoclonal antibodies for ImmunoPETrdquo NuclearMedicine and Biology vol 37 no 3 pp 289ndash297 2010

[32] B M Zeglis P Mohindra G I Weissmann et al ldquoModularstrategy for the construction of radiometalated antibodies forpositron emission tomography based on inverse electron


demand diels-alder click chemistryrdquo Bioconjugate Chemistryvol 22 no 10 pp 2048ndash2059 2011

[33] D J Vugts AVervoortM Stigter-vanWalsumet al ldquoSynthesisof phosphine and antibody-azide probes for in vivo staudingerligation in a pretargeted imaging and therapy approachrdquo Bio-conjugate Chemistry vol 22 no 10 pp 2072ndash2081 2011

[34] B M Zeglis C B Davis R Aggeler et al ldquoEnzyme-mediatedmethodology for the site-specific radiolabeling of antibodiesbased on catalyst-free click chemistryrdquo Bioconjugate Chemistryvol 24 no 6 pp 1057ndash1067 2013

[35] G Fischer U Seibold R Schirrmacher B Wangler and CWangler ldquo89Zr a radiometal nuclide with high potential formolecular imaging with pet chemistry applications and rem-aining challengesrdquoMolecules vol 18 no 6 pp 6469ndash6490 2013

[36] H H Yeh K Ogawa J Balatoni et al ldquoMolecular imaging ofactive mutant L858R EGF receptor (EGFR) kinase-expressingnonsmall cell lung carcinomas using PETCTrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 108 no 4 pp 1603ndash1608 2011

[37] A Natarajan F Habte and S S Gambhir ldquoDevelopment of anovel long-lived immunoPET tracer for monitoring lymphomatherapy in a humanized transgenic mouse modelrdquo BioconjugateChemistry vol 23 no 6 pp 1221ndash1229 2012

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[39] J W Mulder P M Kruyt M Sewnath et al ldquoColorectalcancer prognosis and expression of exon-v6-containing CD44proteinsrdquoThe Lancet vol 344 no 8935 pp 1470ndash1472 1994

[40] I Verel G W Visser R Boellaard et al ldquoQuantitative 89Zrimmuno-PET for in vivo scouting of 90Y-labeled monoclonalantibodies in xenograft-bearing nude micerdquo Journal of NuclearMedicine vol 44 no 10 pp 1663ndash1670 2003

[41] I Verel GW Visser O C Boerman et al ldquoLong-lived positronemitters zirconium-89 and iodine-124 for scouting of therapeu-tic radioimmunoconjugates with PETrdquo Cancer Biotherapy andRadiopharmaceuticals vol 18 no 4 pp 655ndash661 2003

[42] D Vugts D Heuveling M Stigter-vanWalsum et al ldquoPreclini-cal evaluation of 89Zr-labeled anti-CD44 monoclonal antibodyRG7356 in mice and cynomolgus monkeys prelude to phase 1clinical studiesrdquoMAbs vol 6 no 2 pp 567ndash575 2013

[43] F Meric-Bernstam and M-C Hung ldquoAdvances in targetinghuman epidermal growth factor receptor-2 signaling for cancertherapyrdquoClinical Cancer Research vol 12 no 21 pp 6326ndash63302006

[44] H J Burstein ldquoThe distinctive nature of HER2-positive breastcancersrdquoThe New England Journal of Medicine vol 353 no 16pp 1652ndash1654 2005

[45] B A W Hoeben J D M Molkenboer-Kuenen W J G Oyenet al ldquoRadiolabeled cetuximab dose optimization for epider-mal growth factor receptor imaging in a head-and-neck squa-mous cell carcinoma modelrdquo International Journal of Cancervol 129 no 4 pp 870ndash878 2011

[46] L Koi R Bergmann K Bruchner et al ldquoRadiolabeled anti-EGFR-antibody improves local tumor control after externalbeam radiotherapy and offers theragnostic potentialrdquo Radio-therapy and Oncology vol 110 no 2 pp 362ndash369 2014

[47] P Specenier and J B Vermorken ldquoCetuximab its unique placein head and neck cancer treatmentrdquo Biologics vol 7 no 1 pp77ndash90 2013

[48] C Jones M A Taylor and B McWilliams ldquoThe role of cetux-imab as first-line treatment of colorectal liver metastasesrdquoHPBvol 15 no 1 pp 11ndash17 2013

[49] K Boyd S M Shea and J Patterson ldquoCetuximab for treatmentof advanced squamous cell carcinoma in solid organ transplantrecipientsrdquoWiener Medizinische Wochenschrift vol 163 no 15-16 pp 372ndash375 2013

[50] K Unger U Niehammer A Hahn et al ldquoTreatment ofmetastatic colorectal cancer with cetuximab influence on thequality of liferdquo Zeitschrift fur Gastroenterologie vol 51 no 8 pp733ndash739 2013

[51] L R Perk G W Visser M J Vosjan et al ldquo89Zr as a PETsurrogate radioisotope for scouting biodistribution of the ther-apeutic radiometals 90Y and 177Lu in tumor-bearing nude miceafter coupling to the internalizing antibody cetuximabrdquo Journalof Nuclear Medicine vol 46 no 11 pp 1898ndash1906 2005

[52] H J Aerts L Dubois L Perk et al ldquoDisparity between in vivoEGFR expression and 89Zr-labeled cetuximab uptake assessedwith PETrdquo Journal of Nuclear Medicine vol 50 no 1 pp 123ndash131 2009

[53] MWu A Rivkin and T Pham ldquoPanitumumab humanmono-clonal antibody against epidermal growth factor receptors forthe treatment of metastatic colorectal cancerrdquo Clinical Thera-peutics vol 30 no 1 pp 14ndash30 2008

[54] K E Day L Sweeny B Kulbersh K R Zinn and E LRosenthal ldquoPreclinical comparison of near-infrared-labeledcetuximab and panitumumab for optical imaging of head andneck squamous cell carcinomardquoMolecular Imaging and Biologyvol 15 no 6 pp 722ndash729 2013

[55] A J Chang R A de Silva and S E Lapi ldquoDevelopment andcharacterization of 89Zr-labeled panitumumab for immuno-positron emission tomographic imaging of the epidermalgrowth factor receptorrdquoMolecular Imaging vol 12 no 1 pp 17ndash27 2013

[56] L Shan ldquoActivatable alexa fluor680-conjugated panitumumaband indocyanine green-conjugated trastuzumab cocktailrdquo inMolecular Imaging and Contrast Agent Database (MICAD)National Center for Biotechnology Information Bethesda MdUSA 2004

[57] A Chopra ldquo111In-labeled panitumumab a fully human mono-clonal antibody directed against the extracellular domain III ofthe epidermal growth factor receptorrdquo inMolecular Imaging andContrast Agent Database (MICAD) National Center for Bio-technology Information Bethesda Md USA 2004

[58] K J Wong K E Baidoo T K Nayak K Garmestani M WBrechbiel and D E Milenic ldquoIn vitro and in vivo pre-clinicalanalysis of a F(ab1015840)

2fragment of panitumumab for molecular

imaging and therapy of HER1-positive cancersrdquo EJNMMIResearch vol 1 no 1 pp 1ndash15 2011

[59] L Wei J Shi G Afari and S Bhattacharyya ldquoPreparationof clinical-grade 89Zr-panitumumab as a positron emissiontomography biomarker for evaluating epidermal growth factorreceptor-targeted therapyrdquo Journal of Labelled Compounds andRadiopharmaceuticals vol 57 no 1 pp 25ndash35 2014

[60] M E Gross R L Shazer and D B Agus ldquoTargeting the HER-kinase axis in cancerrdquo Seminars in Oncology vol 31 supplement3 no 1 pp 9ndash20 2004

[61] A J Chang R DeSilva S Jain K Lears B Rogers and S Lapildquo89Zr-radiolabeled trastuzumab imaging in orthotopic andmetastatic breast tumorsrdquo Pharmaceuticals vol 5 no 1 pp 79ndash93 2012


[62] J P Holland E Caldas-Lopes V Divilov et al ldquoMeasuring thepharmacodynamic effects of a novel Hsp90 inhibitor onHER2neu expression in mice using 89Zr-DFO-trastuzumabrdquoPLoS ONE vol 5 no 1 Article ID e8859 2010

[63] T H Oude Munnink E G de Vries S R Vedelaar et al ldquoLap-atinib and 17AAG reduce 89Zr-trastuzumab-F(ab1015840)2 uptake inSKBR3 tumor xenograftsrdquo Molecular Pharmaceutics vol 9 no11 pp 2995ndash3002 2012

[64] I S Moreira P A Fernandes and M J Ramos ldquoVascularendothelial growth factor (VEGF) inhibition a critical reviewrdquoAnti-Cancer Agents in Medicinal Chemistry vol 7 no 2 pp223ndash245 2007

[65] J Lin and W K Kelly ldquoTargeting angiogenesis as a promisingmodality for the treatment of prostate cancerrdquo Urologic Clinicsof North America vol 39 no 4 pp 547ndash560 2012

[66] D Maru A P Venook and L M Ellis ldquoPredictive biomarkersfor bevacizumab are we there yetrdquo Clinical Cancer Researchvol 19 no 11 pp 2824ndash2827 2013

[67] M T Schweizer and M A Carducci ldquoFrom bevacizumab totasquinimod angiogenesis as a therapeutic target in prostatecancerrdquo Cancer Journal vol 19 no 1 pp 99ndash106 2013

[68] A Argiris A P Kotsakis T Hoang et al ldquoCetuximab andbevacizumab preclinical data and phase II trial in recurrentor metastatic squamous cell carcinoma of the head and neckrdquoAnnals of Oncology vol 24 no 1 pp 220ndash225 2013

[69] J A Chan K Stuart C C Earle et al ldquoProspective studyof bevacizumab plus temozolomide in patients with advancedneuroendocrine tumorsrdquo Journal of Clinical Oncology vol 30no 24 pp 2963ndash2968 2012

[70] J R Kroep and J W Nortier ldquoThe role of bevacizumab inadvanced epithelial ovarian cancerrdquo Current PharmaceuticalDesign vol 18 no 25 pp 3775ndash3783 2012

[71] PGMorris ldquoBevacizumab is an active agent for recurrent high-grade glioma but do we need randomized controlled trialsrdquoAnti-Cancer Drugs vol 23 no 6 pp 579ndash583 2012

[72] S Sato and H Itamochi ldquoBevacizumab and ovarian cancerrdquoCurrent Opinion in Obstetrics and Gynecology vol 24 no 1 pp8ndash13 2012

[73] W B Nagengast E G de Vries G A Hospers et al ldquoIn vivoVEGF imaging with radiolabeled bevacizumab in a humanovarian tumor xenograftrdquo Journal of Nuclear Medicine vol 48no 8 pp 1313ndash1319 2007

[74] W B Nagengast M A de Korte T H Oude Munnink et alldquo89Zr-bevacizumabPETof early antiangiogenic tumor responseto treatment with HSP90 inhibitor NVP-AUY922rdquo Journal ofNuclear Medicine vol 51 no 5 pp 761ndash767 2010

[75] A R van der Bilt A G T van Scheltinga H Timmer-Bosschaet al ldquoMeasurement of tumor VEGF-A levels with 89Zr-bevacizumab PET as an early biomarker for the antiangiogeniceffect of everolimus treatment in an ovarian cancer xenograftmodelrdquo Clinical Cancer Research vol 18 no 22 pp 6306ndash63142012

[76] W B Nagengast M N Lub-de Hooge S F Oosting et alldquoVEGF-PET imaging is a noninvasive biomarker showingdifferential changes in the tumor during sunitinib treatmentrdquoCancer Research vol 71 no 1 pp 143ndash153 2011

[77] P Carmeliet L Moons A Luttun et al ldquoSynergism betweenvascular endothelial growth factor and placental growth factorcontributes to angiogenesis and plasma extravasation in patho-logical conditionsrdquo Nature Medicine vol 7 no 5 pp 575ndash5832001

[78] JMRakicV Lambert LDevy et al ldquoPlacental growth factor amember of the VEGF family contributes to the development ofchoroidal neovascularizationrdquo Investigative Ophthalmology andVisual Science vol 44 no 7 pp 3186ndash3193 2003

[79] T H Oude Munnink K R Tamas M N Lub-de Hooge et alldquoPlacental growth factor (PlGF)-specific uptake in tumormicroenvironment of 89Zr-labeled PlGF antibody RO5323441rdquoJournal of Nuclear Medicine vol 54 no 6 pp 929ndash935 2013

[80] J R Osborne N H Akhtar S Vallabhajosula A Anand KDeh and S T Tagawa ldquoProstate-specific membrane antigen-based imagingrdquo Urologic Oncology vol 31 no 2 pp 144ndash1542013

[81] Y Zhang Z Guo T Du et al ldquoProstate specific membraneantigen (PSMA) a novel modulator of p38 for proliferationmigration and survival in prostate cancer cellsrdquo Prostate vol73 no 8 pp 835ndash841 2013

[82] A Afshar-Oromieh A Malcher M Eder et al ldquoPET imagingwith a [68Ga]gallium-labelled PSMA ligand for the diagnosis ofprostate cancer biodistribution in humans and first evaluationof tumour lesionsrdquo European Journal of Nuclear Medicine andMolecular Imaging vol 40 no 4 pp 486ndash495 2013

[83] M J Manyak ldquoIndium-111 capromab pendetide in the manage-ment of recurrent prostate cancerrdquo Expert Review of AnticancerTherapy vol 8 no 2 pp 175ndash181 2008

[84] A Sugyo A B Tsuji H Sudo et al ldquoEvaluation of 89Zr-labeledhuman anti-CD147monoclonal antibody as a positron emissiontomography probe in amousemodel of pancreatic cancerrdquoPLoSONE vol 8 no 4 Article ID e61230 2013

[85] U H Weidle W Scheuer D Eggle S Klostermann and HStockinger ldquoCancer-related issues of CD147rdquo Cancer Genomicsand Proteomics vol 7 no 3 pp 157ndash169 2010

[86] S Riethdorf N Reimers V Assmann et al ldquoHigh incidence ofEMMPRIN expression in human tumorsrdquo International Journalof Cancer vol 119 no 8 pp 1800ndash1810 2006

[87] S Zucker M Hymowitz E E Rollo et al ldquoTumorigenic poten-tial of extracellularmatrixmetalloproteinase inducerrdquoTheAme-rican Journal of Pathology vol 158 no 6 pp 1921ndash1928 2001

[88] Z N Chen L Mi J Xu et al ldquoTargeting radioimmunotherapyof hepatocellular carcinoma with iodine (131I) metuximabinjection clinical phase III trialsrdquo International Journal ofRadiation Oncology Biology Physics vol 65 no 2 pp 435ndash4442006

[89] Z Zhang H Bian Q Feng et al ldquoBiodistribution and localiza-tion of iodine-131-labeled Metuximab in patients with hepato-cellular carcinomardquo Cancer Biology and Therapy vol 5 no 3pp 318ndash322 2006

[90] J Xu Z Y Shen X G Chen et al ldquoA randomized controlledtrial of licartin for preventing hepatoma recurrence after livertransplantationrdquo Hepatology vol 45 no 2 pp 269ndash276 2007

[91] P Swietach A Hulikova R D Vaughan-Jones andA L HarrisldquoNew insights into the physiological role of carbonic anhydraseIX in tumour pH regulationrdquo Oncogene vol 29 no 50 pp6509ndash6521 2010

[92] B A W Hoeben J H A M Kaanders G M Franssen et alldquoPET of hypoxia with 89Zr-labeled cG250-F(ab1015840)

2in head and

neck tumorsrdquo Journal of Nuclear Medicine vol 51 no 7 pp1076ndash1083 2010

[93] AM Stillebroer GM Franssen P F AMulders et al ldquoImmu-noPET imaging of renal cell carcinoma with 124I- and 89Zr-Labeled Anti-CAIX monoclonal antibody cG250 in micerdquoCancer Biotherapy amp Radiopharmaceuticals vol 28 no 7 pp510ndash515 2013


[94] S Heskamp H W Van Laarhoven J D Molkenboer-Kuenenet al ldquoImmunoSPECT and immunoPET of IGF-1R expressionwith the radiolabeled antibody R1507 in a triple-negative breastcancer modelrdquo Journal of Nuclear Medicine vol 51 no 10 pp1565ndash1572 2010

[95] EM Jagoda L LangV Bhadrasetty et al ldquoImmuno-PETof thehepatocyte growth factor receptor met using the 1-armed anti-body onartuzumabrdquo Journal of Nuclear Medicine vol 53 no 10pp 1592ndash1600 2012

[96] L R Perk M Stigter-van Walsum G W Visser et al ldquoQuanti-tative PET imaging ofMet-expressing human cancer xenograftswith 89Zr-labelled monoclonal antibody DN30rdquo European Jour-nal of Nuclear Medicine and Molecular Imaging vol 35 no 10pp 1857ndash1867 2008

[97] J G Sham F M Kievit J R Grierson et al ldquoGlypican-3-targeted 89Zr PET imaging of hepatocellular carcinomardquoJournal of Nuclear Medicine vol 55 no 5 pp 799ndash804 2014

[98] J L Seitchik J C Peeler M T Taylor et al ldquoGeneticallyencoded tetrazine amino acid directs rapid site-specific in vivobioorthogonal ligation with trans-cyclooctenesrdquo Journal of theAmerican Chemical Society vol 134 no 6 pp 2898ndash2901 2012

[99] P K Borjesson Y W Jauw R Boellaard et al ldquoPerformance ofimmuno-positron emission tomography with zirconium-89-labeled chimeric monoclonal antibody U36 in the detectionof lymph node metastases in head and neck cancer patientsrdquoClinical Cancer Research vol 12 no 7 part 1 pp 2133ndash21402006

[100] S N Rizvi O J Visser M J W Vosjan et al ldquoBiodistributionradiation dosimetry and scouting of 90Y- ibritumomab tiuxetantherapy in patients with relapsed B-cell non-Hodgkinrsquos lym-phoma using 89Zr-ibritumomab tiuxetan and PETrdquo EuropeanJournal of Nuclear Medicine and Molecular Imaging vol 39 no3 pp 512ndash520 2012

[101] E C Dijkers T H Oude Munnink J G Kosterink et al ldquoBio-distribution of 89Zr-trastuzumab and PET imaging of HER2-positive lesions in patients with metastatic breast cancerrdquo Clin-ical Pharmacology and Therapeutics vol 87 no 5 pp 586ndash5922010

[102] R Bruno C BWashington J Lu G Lieberman L Banken andP Klein ldquoPopulation pharmacokinetics of trastuzumab inpatients with HER2+ metastatic breast cancerrdquo Cancer Chem-otherapy and Pharmacology vol 56 no 4 pp 361ndash369 2005

[103] S B M Gaykema A H Brouwers M N L Hooge et al ldquo89Zr-bevacizumab PET imaging in primary breast cancerrdquo Journal ofNuclear Medicine vol 54 no 7 pp 1014ndash1018 2013

[104] P K Borjesson YW Jauw R de Bree et al ldquoRadiation dosime-try of 89Zr-labeled chimeric monoclonal antibody U36 as usedfor immuno-PET in head and neck cancer patientsrdquo Journal ofNuclear Medicine vol 50 no 11 pp 1828ndash1836 2009

[105] W C Buijs W J Oyen E T Dams et al ldquoDynamic dis-tribution and dosimetric evaluation of human non-specificimmunoglobulin G labelled with 111In or 99Tcmrdquo NuclearMedicine Communications vol 19 no 8 pp 743ndash751 1998

[106] A G T Terwisscha van Scheltinga G M van Dam W BNagengast et al ldquoIntraoperative near-infrared fluorescencetumor imaging with vascular endothelial growth factor andhuman epidermal growth factor receptor 2 targeting antibod-iesrdquo Journal of Nuclear Medicine vol 52 no 11 pp 1778ndash17852011

[107] HHong Y Zhang GW Severin et al ldquoMultimodality imagingof breast cancer experimental lung metastasis with biolumines-cence and a monoclonal antibody dual-labeled with 89Zr and

IRDye 800CWrdquoMolecular Pharmaceutics vol 9 no 8 pp 2339ndash2349 2012

[108] Y Zhang H Hong G W Severin et al ldquoImmunoPET andnear-infrared fluorescence imaging of CD105 expression using amonoclonal antibody dual-labeled with 89Zr and IRDye800CWrdquo American Journal of Translational Research vol 4 no3 pp 333ndash346 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


(11990512

= 3-4 days) requires the use of radionuclides with longhalf-lives (eg 111In (28 days) for SPECT or 89Zr (33 days)and 124I (42 days) for PET [5]) For antibody based PETimaging (immune-PET) 89Zr has several advantages 89Zr hasa half-life of 784 h which matches the pharmacokinetics ofantibodies and it has a relative low average positron energy of395 keV making it an ideal candidate for high resolution PETimaging of slow-accumulating biomolecules In addition89Zr-based agents are safer to handle and more stable in vivomaking them better candidates than 124I-based agents forclinical applications Due to the numerous advantages of89Zr-based immuno-PET the field is progressing at a rapidand exciting pace In this review the potential of 89Zr-basedimmuno-PET in oncology will be reviewed The productionof 89Zr the bioconjugation strategies and applications in(pre-)clinical studies are discussed

2 Radiochemical Properties of 89Zr89Zr decays (half-life of 784 h) first via positron emission andelectron capture to 89mY (half-life of 157 s) which in turndecays via gamma ray emission (909 keV) to the stable 89YWith its relatively low energy positrons (average energy395 keV) 89Zr provides high resolution PET images In addi-tion the energy disparity between the photons (511 keV) andthe gamma rays (909 keV) prevents the latter from interferingwith the detection of 511 keV photons In contrast its halogencompetitor 124I produces high energy photons of differentenergies (603 keV (630) 1691 keV (109) and 723 keV(104) [6]) which may result in random and scatter coinci-dences and therefore in more background noise as comparedto 89Zr Hence reconstruction of 89Zr-based PET scans ismore straightforward to attain good image quality comparedto 124I Although 89Zr has many advantages over other PETradionuclides some essential shielding requirements duringtransport and handling of 89Zr are needed (half-value layerof 89Zr in lead is roughly 10mm) High energy and highlypenetrating photons (909 keV) are emitted during 89Zr decayin high abundance

3 Production of 89Zr

The first production of 89Zr was done by Link et al [7] bya (pn) nuclear reaction by bombarding 89Y on Y foil with13MeV protons [5]The produced 89Zr needed several purifi-cation steps andwas obtained in 80 yield with radionuclidicpurity exceeding 99 Nowadays many medical centers areable to producemedical isotopes using low-energy cyclotronsthat are capable of bombarding targets with protons of lowenergy (lt20MeV) Therefore the most common route toproduce 89Zr is via the 89Y (p n) 89Zr reaction on commer-cially available 89Y target foilsThe above route will in generalresult in high yields (94-95) and high radionuclidic purities(gt99) Competing nuclear reaction like (p 2n) reactionscan result in small amounts radionuclidic byproducts suchas 88Zr and 88Y [8] Several separation and purification

techniques with variable outcomes are used including anionexchange cation exchange and solvent extraction [9ndash11] Forsynthesizing such radiopharmaceuticals for patients auto-mated units for a clean fast safe and reproducible radionu-clide synthesis according to good manufacturing practice(GMP) are necessary Several groups have designed and builtautomated systems for 89Zr [12 13] For exampleWooten et al[14] reported a custom-made system to safely and routinelyproduce 89Zr with high radionuclidic purity (gt9999) andsatisfactory effective specific activity (5ndash353 mCisdot120583molminus1(001ndash088of theoretical specific activity)) based on previ-ous developments in separation and purification techniques[9ndash11 15]

4 The Need for Efficient Chelators

The release of 89Zr4+ from the antibodies needs to be pre-vented because the free radionuclide can accumulate in themineral bone and can associate with plasma proteins Thisleads to depositing significant doses to the bone marrow [16]Therefore an appropriate chelator system is necessary tomin-imize the disassociation of 89Zr from the antibodies Over theyears several chelators have been used with different suc-cess such as diethylenetriaminepentaacetic acid (DTPA)ethylenediaminetetraacetic acid (EDTA) 14710-tetraaceticacid (DOTA) and desferoxamine (DFO) [17]The stability ofZr-DOTA Zr-DTPA and Zr-EDTA was found to be limitedThe thermodynamic stability of Zr-DTPA is slightly higherthan that of Zr-EDTA most likely because DTPA coordi-natively saturates the Zr4+ while EDTA requires exogenouswater molecules [18] DFO is the most prominent chelatorof Zr4+ DFO is a hexadentate siderophore containing threehydroxamate groups for chelating metals and a primaryamine tail for conjugation to a biomolecule (Figure 1) Besidesa zirconium chelator it is a chelating agent for several othermetal ions [19] It demonstrated good stability releasing lessthan 02 of Zr4+ after 24 h in serum [20] and after seven daysin serum still less than 2 demetallation occurs [21] Severalproof-of-principle preclinical studies have been conductedusing DFO to label antibodies with 89Zr however the in vivostability of this complex remains an issue because free 89Zr isobserved in the bone dependent on the in vivo behavior of theantibody [22] Several studies have attempted to improve thelinkage between DFO and the antibody [9 23] whereas oth-ers have focused on improving the chelate itself [24] Even-tually a ligand that is both octadentate and oxygen-rich isbelieved to be the most stable Zr4+ chelator since it would beable to incorporate all eight coordination sites of zirconium[22] This novel high stability Zr4+ ligand would in theoryminimize the uptake of liberated Zr4+ in the bone and othernontargeted tissues To date the design synthesis and theevaluation of such a Zr4+ chelate requires further research

41 Conjugation of Antibodies withDFO AsDFO is currentlythe most promising chelator for 89Zr4+ conjugation of anti-bodies with DFO will be discussed here in detail (Figure 1)Several methods are available to conjugate DFO based on


HN

O

O

O

O O

OO

O

O

ON

N

N

NN

NH

H

H

89Zr4+



















(a)


0 4 0 10 0 25( IDg) ( IDg) ( IDg)

(b)






2












( ID

g)

25

20

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(a)

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25

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(b)



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7 Conclusions



(a) (b) (c)


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2

(a)

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(pg

mg)


0 2 4 6

600

400

200

0

(b)





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2in head and

























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















HN

O

O

O

O O

OO

O

O

ON

N

N

NN

NH

H

H

89Zr4+



















(a)


0 4 0 10 0 25( IDg) ( IDg) ( IDg)

(b)






2












( ID

g)

25

20

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cle

Bone

MIA

pac

a-2

A4


(a)

( ID

g)

25

20

15

10

5

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Lung

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Panc

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Stom

ach

Inte

stine

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cle

Bone

MIA

pac

a-2

A4



(b)



2 an anti-















7 Conclusions



(a) (b) (c)


1

2

(a)

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F-A

(pg

mg)


0 2 4 6

600

400

200

0

(b)





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2in head and

























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























(a)


0 4 0 10 0 25( IDg) ( IDg) ( IDg)

(b)






2












( ID

g)

25

20

15

10

5

0

Bloo

d

Lung

Live

r

Sple

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Panc

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Stom

ach

Inte

stine

Kidn

ey

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cle

Bone

MIA

pac

a-2

A4


(a)

( ID

g)

25

20

15

10

5

0

Bloo

d

Lung

Live

r

Sple

en

Panc

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Stom

ach

Inte

stine

Kidn

ey

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cle

Bone

MIA

pac

a-2

A4



(b)



2 an anti-















7 Conclusions



(a) (b) (c)


1

2

(a)

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F-A

(pg

mg)


0 2 4 6

600

400

200

0

(b)





References




































































































2in head and

























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























( ID

g)

25

20

15

10

5

0

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d

Lung

Live

r

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Panc

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Inte

stine

Kidn

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cle

Bone

MIA

pac

a-2

A4


(a)

( ID

g)

25

20

15

10

5

0

Bloo

d

Lung

Live

r

Sple

en

Panc

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Stom

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Inte

stine

Kidn

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Mus

cle

Bone

MIA

pac

a-2

A4



(b)



2 an anti-















7 Conclusions



(a) (b) (c)


1

2

(a)

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F-A

(pg

mg)


0 2 4 6

600

400

200

0

(b)





References




































































































2in head and

























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















7 Conclusions



(a) (b) (c)


1

2

(a)

VEG

F-A

(pg

mg)


0 2 4 6

600

400

200

0

(b)





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2in head and

























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b) (c)


1

2

(a)

VEG

F-A

(pg

mg)


0 2 4 6

600

400

200

0

(b)





References




































































































2in head and

























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















































































































2in head and

























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















review article zirconium-89 labeled antibodies: a new tool for...

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