RESEARCH ARTICLE

Evaluation of a HER2-targeting affibody moleculecombining an N-terminal HEHEHE-tag with a GGGCchelator for 99mTc-labelling at the C terminus

Hanna Lindberg & Camilla Hofström & Mohamed Altai &Hadis Honorvar & Helena Wållberg & Anna Orlova &

Stefan Ståhl & Torbjörn Gräslund & Vladimir Tolmachev

Received: 10 October 2011 /Accepted: 21 December 2011 /Published online: 17 January 2012# International Society of Oncology and BioMarkers (ISOBM) 2012

Abstract Affibody molecules are a class of small (ca.7 kDa)robust scaffold proteins with high potential as tracers forradionuclide molecular imaging in vivo. Incorporation of acysteine-containing peptide-based chelator at the C terminusprovides an opportunity for stable labelling with the radionu-clide 99mTc. The use of a GGGC chelator at the C terminus hasprovided the lowest renal radioactivity retention of the previ-ously investigated peptide-based chelators. Previously, it hasalso been demonstrated that replacement of the His6-tag withthe negatively charged histidine-glutamate-histidine-gluta-mate-histidine-glutamate (HEHEHE)-tag permits purificationof affibody molecules by immobilized metal ion affinity chro-matography (IMAC) and provides low hepatic accumulationof radioactivity of conjugates site-specifically labelled at the Cterminus using several different nuclides. We hypothesizedthat the combination of a HEHEHE-tag at the N terminus anda GGGC chelator at the C terminus of an affibody moleculewould be a favourable format permitting IMAC purificationand providing low uptake in excretory organs. To investigate

this hypothesis, a (HE)3-ZHER2:342-GGGC affibody moleculewas generated. It could be efficiently purified by IMAC andstably labelled with 99mTc. 99mTc-(HE)3-ZHER2:342-GGGCpreserved specific binding to HER2-expressing cells. InNMRImice, hepatic uptake of 99mTc-(HE)3-ZHER2:342-GGGCwas lower than the uptake of the control affibody molecules,99mTc-ZHER2:2395-VDC and 99mTc-ZHER2:342-GGGC. At 1and 4 h after injection, the renal uptake of 99mTc-(HE)3-ZHER2:342-GGGC was 2–3-fold lower than uptake of 99mTc-ZHER2:2395-VDC, but it was substantially higher than uptakeof 99mTc-ZHER2:342-GGGC. Further investigation indicatedthat a fraction of 99mTc was chelated by the HEHEHE-tagwhich caused a higher accumulation of radioactivity in thekidneys. Thus, a combination of a HEHEHE-tag and theGGGC chelator in targeting scaffold proteins was found tobe undesirable in the case of 99mTc labelling due to a partialloss of site-specificity of nuclide chelation.

Keywords Affibody molecules . Radionuclide molecularimaging . Technetium-99m . HEHEHE-tag .

GGGC chelator . Biodistribution

Introduction

Selective systemic therapy is a promising strategy for treatmentof disseminated cancers using drugs targeting malignancy-associated alterations on the tumour cells, using, e.g. mono-clonal antibodies. An example of successful application of thisstrategy is the targeting of human epidermal growth factorreceptor type 2 (HER2)-expressing breast cancer. HER2 is amember of the transmembrane tyrosine kinase receptor family,which is overexpressed in 25–30% of breast and ovariancarcinomas [1, 2] and in 80% of urinary bladder carcinomas

Hanna Lindberg and Camilla Hofström contributed equally to thisstudy.

H. Lindberg : C. Hofström :H. Wållberg : S. Ståhl : T. GräslundDivision of Molecular Biotechnology, School of Biotechnology,AlbaNova University Center, Royal Institute of Technology,Stockholm, Sweden

M. Altai :H. Honorvar :V. Tolmachev (*)Biomedical Radiation Sciences, Rudbeck Laboratory,Uppsala University,751 85 Uppsala, Swedene-mail: vladimir.tolmachev@bms.uu.se

A. OrlovaDepartment of Medicinal Chemistry, Preclinical PET Platform,Uppsala University,Uppsala, Sweden

Tumor Biol. (2012) 33:641–651DOI 10.1007/s13277-011-0305-z

[3]. HER2 signalling occursmainly by heterodimerisation withother members of the HER family providing a growth advan-tage to the malignant cells [4]. Suppression of HER2 signallingby the monoclonal antibody trastuzumab or the tyrosine kinaseinhibitor lapatinib improves survival of patients withmetastaticHER2-positive breast [5] and gastric [6] cancer.

Apparently, such therapy can be efficiently used only forpatients with tumours expressing HER2. Current biopsy-based methods for testing of HER2 expression in tumoursare invasive and suffer from low accuracy [7]. Radionuclidemolecular imaging of molecular therapeutic targets has astrong potential for selection of patients suitable for target-ing therapy, thus avoiding biopsy-associated pitfalls such assampling errors due to intra- and intertumour heterogeneityof HER2 expression [8]. Recently, a new class of targetingproteins for molecular imaging, affibody molecules, hasbeen developed [9]. Several different approaches for label-ling of affibody molecules with radionuclides suitable forsingle photon computed tomography (SPECT) and positronemission tomography have been evaluated [10]. For radio-nuclide imaging of HER2-expressing tumours, an affibodymolecule ZHER2:342 with affinity of 22 pM to HER2 hasbeen developed [11]. A combination of its small size(7 kDa) and very high affinity (subnanomolar) providedsuperior contrast of HER2 imaging in comparison withother HER2 imaging agents [12].

The radionuclide 99mTc (T1/206h, E0140.5 keV) iswidely used in SPECT imaging due to its photon energy(nearly ideal for SPECT cameras), low cost, excellentavailability and low absorbed dose burden to the patient.Several approaches for labelling of anti-HER2 affibodymolecules with 99mTc have previously been investigated.Initially, labelling of affibody molecules containing ahexahistidine tag at the N terminus with [99mTc(CO)3]

+

was evaluated [13]. The hexahistidine tag was introducedin affibody molecules for purification using immobilizedmetal ion affinity chromatography (IMAC). The labellingwas stable and HER2-specificity of the conjugate waspreserved, but this kind of labelling chemistry was shownto cause high hepatic uptake of radioactivity. Furthermore,it was later shown that the presence of a hexahistidine tagis associated with high liver uptake of affibody moleculeseven if other labelling methods and different nuclideswere utilized [14–16]. Production of affibody moleculeswithout a hexahistidine tag was therefore evaluated. Thisimproved biodistribution [14, 15] but necessitated the useof much more complicated and work-intensive purificationprocedures.

A series of studies demonstrated that the use ofmercaptoacetyl-containing peptide based chelators at the Nterminus of synthetic affibody molecules would permit notonly their stable labelling with 99mTc and 186Re, but alsoenable modification of their biodistribution [17–22]. It

has been shown that it is possible to reduce renal retentionof radioactivity by using chelators that provided non-residualizing properties to 99mTc and 186Re labels [20, 22].It has to be noted that residualizing properties of a label arenot critical for anti-HER2 affibody molecules because theirinternalization by cancer cells is slow [23].

We have shown that incorporation of a cysteine at theC terminus of the ZHER2:342 affibody molecule forms aN3S-type chelator VDC (Fig. 1a), which permits stablegluconate-mediated labelling with 99mTc at 90°C [14]. Theuse of the same labelling condition to a counterpart withoutcysteine gave no labelling, which demonstrated that thelabelling was chelator-specific [24]. This new ZHER2:342

derivative, designated as ZHER2:2395, demonstrated high-contrast imaging of HER2-expressing xenografts in mice[14]. However, a combination of high renal re-absorptionof ZHER2:2395 and strong residualizing properties of 99mTc-VDC resulted in high accumulation of radioactivity inkidneys [14]. A systematic study demonstrated a stronginfluence of composition of cysteine-containing peptide-based chelators at the C terminus on biodistribution of99mTc-labelled affibody molecules [25, 26]. It was shownthat a GGGC chelator (Fig. 1b) provided the lowest renalretention of 99mTc, while 99mTc-ZHER2:342-GGGC (Fig. 2)demonstrated excellent tumour targeting [25]. This ap-proach opened a way for complete recombinant productionof affibody-based tracers, without any necessity of furthercoupling of chelators. Due to absence of a histidine tag,purification of the novel conjugates was performed usingimmobilized anti-idiotypic affibody molecules specific tothe binding site of ZHER2:342 [27]. This is a relatively low-capacity method, and prospects for its industrial up-scalingremain uncertain. Therefore, it would be desirable to developa general high-throughput purification approach for affibodymolecules for use as imaging tracers. An approach to reachthis goal is to modify the hexahistidine tag. Our recent find-ings suggest that it is possible to alleviate the negative influ-ence of histidine-based tags on biodistribution of affibody

Fig. 1 Cysteine-containing peptide-based chelators expressed aspeptide extensions at the C terminus of affibody molecules. a VDCand b GGGC

642 Tumor Biol. (2012) 33:641–651

molecules without compromising the attractive features ofIMAC purification [16, 28]. In these studies we replaced theHis6-tag with the negatively charged histidine-glutamate-histidine-glutamate-histidine-glutamate (HEHEHE)-tag. Thistag permitted purification by IMAC, enabled stable labellingwith [99mTc(CO)3]

+, and provided much lower hepatic accu-mulation of radioactivity than the case of hexahistidine tags[28]. However, the renal retention of 99mTc(CO)3-HEHEHE-ZHER2:342 radioactivity was high. Further studies have shownthat the use of an N-terminal HEHEHE-tag provided lowhepatic uptake when other nuclides or another technetiumcore was conjugated to the C terminus of affibody moleculesusing other labelling chemistries [16].

Based on these data, we hypothesised that a combinationof an N-terminal HEHEHE-tag with a GGGC chelator at theC terminus of an affibody molecule would be a favourableformat. The incorporation of the HEHEHE-tag would allowIMAC purification without causing high liver uptake, whilethe use of a GGGC peptide extension would provide stable99mTc-labelling, but low kidney retention of radioactivity.To test this hypothesis, a (HE)3-ZHER2:342-GGGC affibodymolecule (Fig. 2) was generated and labelled with 99mTc.The biodistribution of 99mTc-(HE)3-ZHER2:342-GGGC wascompared with the biodistribution of 99mTc-ZHER2:2395 and99mTc-ZHER2:342-GGGC in normal mice.

Materials and methods

General

Production, purification and characterisation of ZHER2:2395,

ZHER2:342-GGGC and (HE)3-ZHER2:342 has been describedearlier [14, 27, 28]. High-quality Milli-Q© water (resistancehigher than 18 MΩ/cm) was used for preparing solutions.99mTc was obtained as pertechnetate from an Ultra-TechneKow generator (Covidien) by elution with sterile0.9% NaCl. NAP-5 size exclusion columns were from GEHealthcare, Uppsala, Sweden. Cells used during in vitroexperiments were detached using trypsin-EDTA solution(0.25% trypsin, 0.02%EDTA in buffer, BiochromAG, Berlin,Germany). For in vivo experiments Ketalar (50 mg/ml, Pfizer,NY), Rompun (20 mg/mL, Bayer, Leverkusen, Germany) andheparin (5,000 IE/ml, Leo Pharma, Copenhagen, Denmark)were used. Radioactivity was measured using an automatedgamma-counter with a 3-in. NaI(Tl) detector (1480WIZARD,

Wallac Oy, Turku, Finland). The distribution of radioactivityalong the thin layer chromatography strips and SDS-PAGEgels was measured on a Cyclone™ Storage Phosphor Systemand analysed using the OptiQuant™ image analysis software(PerkinElmer). Purity of non-labelled affibody molecules wasdetermined using reversed phase HPLC. The purity of radio-labeled affibody molecules was determined by radio-instantthin layer chromatography (radio-ITLC) (150-771 DarkGreen, Tec-Control Chromatography strips from BiodexMedical Systems) cross-validated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). The purityof all studied compounds was over 95%. Data on cellularuptake and biodistribution were assessed by an unpaired,two-tailed t test using GraphPad Prism (version 4.00 forWindows GraphPad Software, San Diego CA) in order todetermine any significant differences (p<0.05).

Production, purification and characterisationof the (HE)3-ZHER2:342-GGGC affibody molecule

The production and purification of (HE)3-ZHER2:342-GGGCaffibody molecule was made essentially as previously de-scribed [28]. Briefly, PCR fragment encoding for (HE)3-ZHER2:342-GGGC was sub-cloned into the expression vectorpET21a(+) (Novagen, Darmstadt, Germany) and the correctDNA sequence was verified by DNA-sequencing. The affi-body molecule was expressed in Escherichia coli strainBL21(DE3) as previously described [29]. After cell disrup-tion by sonication followed by cell debris removal by cen-trifugation, the clarified cell lysate was heat-treated for 10min at 60°C. The heat-treated cell lysate was then centri-fuged and filtered through a 0.45 μm filter (Sartorius StedimBiotech, Aubagne Cedex, France). (HE)3-ZHER2:342-GGGCaffibody molecule was recovered by IMAC using a TalonMetal Affinity Resin (BD Bioscience, San Jose, CA).

The (HE)3-ZHER2:342-GGGC affibody molecule was fur-ther purified by reverse phase high-performance liquid chro-matography on an Agilent 1200 HPLC system (AgilentTechnologies, Santa Clara, CA). To reduce potential disul-fide bridges, TCEP-HCl (Sigma-Aldrich, Sweden) wasadded to a final concentration of 50 mM followed by incu-bation at room temperature for 1 h prior to RP-HPLCpurification. The sample was injected onto a C18 columnusing a 20-min gradient of 20–65% B (A—0.1% TFA-H2Oand B—0.1% TFA-CH3CN), with a flow rate of 2.5 mL/min.The final purity of (HE)3-ZHER2:342-GGGC was analysed by

Fig. 2 Amino acid sequences, in single letter code, of the (HE)3-ZHER2:342-GGGC and control proteins, ZHER2:2395, ZHER2:342-GGGC and(HE)3-ZHER2:342

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analytic RP-HPLC as previously described [16]. The puritywas determined by diving the area of the peak correspondingto the affibody molecule with the total area of all peaks in thechromatogram. The protein concentration was determined byamino acid analysis (Amino Acid Analysis Center, UppsalaUniversity).

Mass spectrometry analysis

The molecular mass of (HE)3-ZHER2:342-GGGC wasdetermined on a 6520 Accurate-Mass Q-TOF liquidchromatography–mass spectrometry (LC-MS) instrument(Agilent Technologies, Santa Clara, CA), and the isotopicand charge state information was deconvoluted using AgilentMassHunter B.02.00 software (Agilent Technologies).

Melting point analysis

Variable temperature measurement of (HE)3-ZHER2:342-GGGC was performed using a JASCO J-810 spectropo-larimeter instrument (JASCO, Tokyo, Japan). The samplewas diluted to a concentration of 60 μM in phosphate-buffered saline (PBS), and absorbance was measured at221 nm using a temperature gradient increasing 5°C/minranging from 20°C to 90°C. Circular dichroism spectra werealso recorded from 250 to 195 nm at 20°C before and aftereach variable temperature measurement.

Biosensor analysis

Real-time biospecific interaction analyses on a Biacore 3000instrument (Biacore Life Science, GE Healthcare, Uppsala,Sweden) were performed to determine the affinity betweenHER2 and (HE)3-ZHER2:342-GGGC. Prior to the analysis, theaffibody molecule was reduced with 30 mM DTT (dithio-threitol) followed by cysteine alkylation in the presence of 5×molar excess of N-ethylmaleimide. Recombinant humanHER2-ECD fused to the Fc region of human IgG (R&DSystems, Minneapolis, MN) was immobilized (~1,000 RU)on a flow-cell surface of a CM5 sensor chip (Biacore LifeScience, GE Healthcare, Uppsala, Sweden) by amine cou-pling, according to the manufacturer’s instructions.

A 3-fold dilution series consisting of five different con-centrations were prepared in duplicates. Samples wereinjected with a flow rate of 50 μL/min and regeneration ofthe flow-cell surface was accomplished by injecting 20 μLof 15 mM HCl. The concentrations used were ranging from30 pM to 2.4 nM. Finally, the dissociation equilibriumconstant (KD), the association rate constant (ka) and thedissociation rate constant (kd) were calculated using BIAe-valuation 3.2 software (Biacore Life Science, GE Health-care, Uppsala, Sweden), assuming a one-to-one interactionmodel.

Radiolabelling and in vitro stability

Radiolabelling of the (HE)3-ZHER2:342-GGGC, ZHER2:2395 andZHER2:342-GGGC affibody variants was performed usingthe optimized two-vial kit method described by Ahlgren andco-workers [24]. Briefly, each lyophilized kit contained 5 mgof sodium α-D-gluconate dihydrate, 100 μg of edetate diso-dium (Na2EDTA) and 75 μg of tin(II) chloride dihydrate(SnCl2×2H2O). Labelling was performed by adding the con-tents of the kit, dissolved in 100 μL degassed PBS, to 100 μgof affibody molecule. To the reaction mixture, 100 μL(~200 MBq) of the 99mTc-pertechnetate-containing generatoreluate was added, and the vial was filled with argon gas toprotect the mixture from oxidation. The reaction vial wasthoroughly vortexed and incubated at 95°C for 1 h. Labellingyield and percentage of reduced hydrolyzed technetium col-loids (RHT) was thereafter determined by ITLC after coolingfor 15 min. PBS was used as mobile phase for analysis of thelabelling efficiency and radiochemical purity. In this system,unbound radioactivity migrates with the eluent front andradiolabelled affibody molecule remains at the applicationpoint. A mixture of pyridine/acetic acid/water (5:3:1.5) wasused as eluent for ITLC to estimate the presence of RHT.

Labelling of (HE)3-ZHER2:342 (100 μg) was performedusing the same kit, and the yield was determined using ITLCeluted with PBS. 99mTc-(HE)3-ZHER2:342 was purified using aNAP-5 column pre-equilibrated and eluted with PBS followedby measurement of the radiochemical purity of the 99mTc-(HE)3-ZHER2:342. The cycle was repeated 3 times, until onlya stable 99mTc-(HE)3-ZHER2:342 conjugate remained.

For serum stability studies, freshly 99mTc-labelled affibodymolecule (10 μL) was diluted in a murine serum sample(240 μL) to a concentration similar to the concentration inblood at themoment of injection, incubated for 1 h at 37°C andanalysed by radio-SDS PAGE usingNuPAGE 4–12%Bis-TrisGel (Invitrogen AB, Lidingö, Sweden) in MES buffer (Invi-trogen AB, Lidingö, Sweden) at 200 V constant. A sample of99mTc-pertechnetate was run in a parallel lane as reference.

Binding specificity and cellular retention

The HER2-expressing ovarian cancer cell line SKOV-3,expressing 1.2×106 HER2-receptors per cell [30] (pur-chased from American Type Tissue Culture Collection viaLGC Promochem, Borås, Sweden) was used in the cellstudies. The specificity of radiolabelled affibody moleculesbinding to these cells and cellular retention and internaliza-tion of radioactivity was evaluated as described by Wållbergand Orlova [23].

Briefly, a group of six Petri dishes containing a cellmonolayer (1×106 cells/dish) was used for the bindingspecificity test. Cells in three dishes were pre-saturated with500-fold excess of unlabelled affibody molecules 5 min

644 Tumor Biol. (2012) 33:641–651

before addition of the labelled affibody conjugates. Thecells were incubated with 0.6 nM 99mTc-(HE)3-ZHER2:342-GGGC in a humidified incubator (5% CO2, 37°C) for 1 h.Thereafter, the medium was collected, the cells were washedwith cold serum-free medium and then trypsin-EDTA solu-tion was added and incubated for 10 min. Detached cellswere collected. The radioactivity of cells and media wasmeasured using an automated gamma counter and the percent of cell-bound radioactivity was calculated.

To evaluate cellular retention and processing, SKOV-3cells were incubated with 0.6-nM solution of labelled con-jugate at 4°C. After 1-h incubation, the medium with thelabelled compound was removed and cells were washedwith ice-cold serum-free medium. One millilitre of completemedia was added to each dish and cells were further incu-bated at 37°C in an atmosphere containing 5% CO2. Atdesignated time points (0, 1, 2, 4, 8 and 24 h), a group ofthree dishes was removed from the incubator, the media wascollected and cells were washed with ice cold serum-freemedium. Thereafter cells were treated with 0.5 mL 0.2 Mglycine buffer, pH 2, containing 4 M urea, for 5 min on ice.The acidic solution was collected and cells were additionallywashed with 0.5 mL glycine buffer. The acidic fractionswere pooled. The cells were then incubated with 0.5 mL1 M NaOH at 37°C for 10 min. The cell debris was collected,and the dishes were additionally washed with 0.5 mL ofNaOH solution. The alkaline fractions were pooled. The ra-dioactivity in the acidic solution was considered as membranebound, and in the alkaline fractions as internalized. For

comparison, processing of 99mTc-ZHER2:2395 affibody mole-cule (VDC chelator with known residualizing properties) wasevaluated simultaneously using the same protocol.

Animal studies

The animal experiments were planned and performed inaccordance with national legislation on laboratory animalsprotection. The animal study plans have been approved bythe local Ethics Committee for Animal Research in Uppsala.

Biodistribution studies were performed in female immu-nocompetent Naval Medical Research Institute (NMRI) mice.All mice were acclimatized for 1 week at the RudbeckLaboratory animal facility before any experimental proce-dures. For each conjugate, 12 mice, randomized into groupsof four, were used. Animals were injected intravenously with1μg (40 kBq) conjugate per animal in 100 μL PBS. Onegroup of mice was sacrificed at predetermined time points (1,4 and 24 h after injection) by an intraperitoneal injection ofanesthesia, Ketalar–Rompun solution (20 μL/g body weight;Ketalar, 10 mg\mL; Rompun, 1 mg\mL) with subsequentexsanguination by heart puncture using a 1-mL syringe pre-washed with diluted heparin. Blood, lung, liver, spleen, stom-ach, kidney, salivary glands, muscles and intestines, and theremaining carcass were collected. Organs and tissue sampleswere weighed, and their radioactivity was measured. Thetissue uptake values were calculated as per cent of injecteddose per gram tissue (% ID/g) except for the intestines andthe carcass, which was calculated as %ID per whole sample.

Fig. 3 Analysis of (HE)3-ZHER2:342-GGGC. a SDS-PAGE analysis ofsamples taken during the purification of (HE)3-ZHER2:342-GGGC.Lane 1, sample after cell lysis and initial clarification; lane 2, sample

after heat-treatment; lane 3, sample after IMAC purification; lane 4,sample after RP-HPLC. b HPLC chromatorgram of (HE)3-ZHER2:342-GGGC. c Deconvolution of (HE)3-ZHER2:342-GGGC mass spectrum

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Results and discussion

Production, purification and characterisationof the (HE)3-ZHER2:342-GGGC affibody molecule

The (HE)3-ZHER2:342-GGGC affibody molecule was recom-binantly produced in E. coli. After harvest, disruption bysonication and clarification, the lysate containing (HE)3-ZHER2:342-GGGC was heat-treated for 10 min at 60°C to

precipitate a portion of endogenous E. coli proteins prior toIMAC purification on a Co2+-derived affinity resin. The(HE)3-ZHER2:342-GGGC affibody molecule could success-fully be recovered by IMAC, and was further purified byreversed-phase high performance liquid chromatography(RP-HPLC). Analysis by sodium dodecyl sulfate polyacryl-amide gel electrophoresis of samples taken after IMAC andafter RP-HPLC showed only a single band having a molec-ular weight corresponding to the expected molecular mass

Fig. 4 Circular dichroismanalysis. a Ellipticity measuredin a CD spectrometer at 221 nmas a function of temperature.b An overlay of CD-spectrarecorded before and afterthermal unfolding of(HE)3-ZHER2:342-GGGC

Fig. 5 Biosensor analysis usingsurface plasmon resonance.Sensorgrams obtained afterinjection of (HE)3-ZHER2:342-GGGC over a sensor surfacecontaining amine-coupledHER2-Fc fusion protein. Theconcentrations used of(HE)3-ZHER2:342-GGGC areindicated next to eachsensorgram and eachconcentration was run induplicates. Experimental curveswere fitted to a 1:1 (Langmuir)binding model

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of (HE)3-ZHER2:342-GGGC (Fig. 3). LC-MS analysis con-firmed the molecular weight of the recovered protein (cal-culated molecular weight of (HE)3-ZHER2:342-GGGC is7,599 Da, found molecular weight was 7,600 Da), and RP-HPLC analysis demonstrated an exceptional purity, 99.5%.These experiments confirmed that highly efficient produc-tion of (HE)3-ZHER2:342-GGGC was possible.

Circular dichroism analysis (Fig. 4a) showed that (HE)3-ZHER2:342-GGGC had a melting temperature of 62°C, whichwas similar to the previously reported values of 65°C forZHER2:342-GGGC [25] and 64°C for (HE)3-ZHER2:342 [28].As expected, (HE)3-ZHER2:342-GGGC gave CD-spectrashowing high alpha-helical content (Fig. 4b). Also, thespectra recorded for (HE)3-ZHER2:342-GGGC before andafter heating from 20°C to 90°C were highly similar, indi-cating that the construct was able to completely refold afterthermal denaturation. This is considered an important pre-condition for gluconate-mediated labelling with 99mTc atelevated temperature.

Analysis of (HE)3-ZHER2:342-GGGC binding kinetics toimmobilized HER2-ECD using surface plasmon resonanceis presented in Fig. 5. Fitting the sensorgrams with a 1:1(Langmuir) binding model suggested that the associationrate constant (ka) was 6.3×10

6 M−1 s−1, and the dissociationrate constant (kd) was 1.7×10

−4 s−1. The dissociation equi-librium constant (KD ) was 27 pM, which is comparablewith values for ZHER2:342-GGGC (150 pM) [25] and (HE)3-ZHER2:342 (18 pM) [28].

Radiolabelling and in vitro stabilityof 99mTc-(HE)3-ZHER2:342-GGGC

The use of an optimized two-vial kit method [24] for label-ling of (HE)3-ZHER2:342-GGGC with 99mTc provided a yieldgreater than 98.2±0.2%, and the level of RHT colloids were0.4±0.3%. A specific radioactivity of 3.5 GBq/mmol wasachieved for 99mTc-(HE)3-ZHER2:342-GGGC.

PhosphorImager analysis of the radioactivity distributionon a polyacrylamide gel (Fig. 6a) showed a high stability of99mTc-(HE)3-ZHER2:342-GGGC after incubation at 37°C for1 h in mouse plasma. The majority of the radioactivity wasassociated with 99mTc-(HE)3-ZHER2:342-GGGC (peak 1;Fig. 6a), and only very weak bands (less than 2% totally) wereassociated with release of low molecular 99mTc (peak 3 withthe same migration path as free 99mTc-pertechnetate, peak 2),or transchelation to blood proteins (peaks 4 and 5). Due to thehigh purity, 99mTc-(HE)3-ZHER2:342-GGGC could be used forbiological experiments without additional purification.

Binding specificity and cellular retention

The result of a binding specificity test is presented in Fig. 7.The test demonstrated that the binding of 99mTc-(HE)3-

ZHER2:342-GGGC to living HER2-expressing SKOV-3 cellswas receptor mediated, since saturation of the receptors bypre-incubation with non-labelled ZHER2:342 significantly de-creased the binding of the radiolabelled affibody molecule

1

1

3

3

2

2

4

B

C

5

A 1

3

2

4 5

Fig. 6 SDS-PAGE analysis of the stability of radiolabelled Affibodymolecules. 99mTc-pertechnetate was run on the same gel for compari-son. Distribution of radioactivity along lanes was visualized usingCyclone™ Storage Phosphor system. a Stability of 99mTc-(HE)3-ZHER2:342-GGGC in serum (37°C, 1 h). b Stability of 99mTc-(HE)3-ZHER2:342 in PBS (ambient temperature, 1 h). c Stability of 99mTc-(HE)3-ZHER2:342 in murine serum (37°C, 1 h). Peak 1 corresponds tomigration path of the tested affibody protein, peak 2-99mTc-pertechne-tate. The nature of other indicated peaks in discussed in the text

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(p<0.0001). It has to be noted that this test confirms onlythe specificity of the binding of the radiolabelled proteins toHER2-expressing cells but does not allow comparing theirbinding affinities.

Retention and cellular processing of 99mTc-(HE)3-ZHER2:342-GGGC by HER2-expressing SKOV-3 cells ispresented in Fig. 8a. For comparison, the retention of99mTc-ZHER2:2395 affibody molecule, which was labelledvia the VDC chelator with known residualizing properties,was also evaluated (Fig. 8b). The retention and processingpattern of these two conjugates was clearly different. Pro-cessing of 99mTc-ZHER2:2395 demonstrated, in agreementwith previous data [14], relatively slow internalization anda good overall retention of radioactivity by SKOV-3 cells.Processing of 99mTc-(HE)3-ZHER2:342-GGGC showed a lowerlevel of internalized radioactivity and a lower level of over-all cellular retention of radioactivity, i.e. the residualizingproperties were much less pronounced for this construct.Still, the cellular retention of 99mTc-(HE)3-ZHER2:342-GGGCafter 4 h (a time point relevant for in vivo imaging), wasquite good, 68.4±0.4%. At the same time, low residualizingproperties should provide lower renal retention of 99mTc-

(HE)3-ZHER2:342-GGGC in comparison with99mTc-ZHER2:2395

in vivo, as previous studies have shown rapid internalizationof affibody molecules in the kidney leading to prompt excre-tion of radiocatabolites of non-residualizing labels [20].

Animal studies

The data concerning biodistribution of 99mTc-(HE)3-ZHER2:342-GGGC, 99mTc-ZHER2:2395 and 99mTc-ZHER2:342-GGGC inNMRI mice are presented in Table 1. The data concerningbiodistribution of 99mTc-ZHER2:2395 and 99mTc-ZHER2:342-GGGC were in an excellent agreement with the previouslypublished data [14, 24, 25]. All three conjugates demonstratedgood in vivo stability, which was manifested in low uptake ofradioactivity in the stomach and salivary glands; organs whichaccumulate free pertechnetate by the Na+/I− symporter. Allconjugates showed rapid clearance from blood and non-excretory organs. Low accumulation in liver and intestines(with content) suggested that hepatobiliary clearance played aminor role in the excretion of 99mTc-(HE)3-ZHER2:342-GGGC,99mTc-ZHER2:2395 and

99mTc-ZHER2:342-GGGC. There were ap-parent differences in the biodistribution of the conjugates. Forexample, blood clearance of 99mTc-(HE)3-ZHER2:342-GGGCwas the most rapid, which was manifested in the lowest con-centration of radioactivity in the blood at 1 h after injection.This conjugate provided also the lowest uptake in lung at alltime points. As it was predicted [16], the use of HEHEHE-tag atN terminus provided lower hepatic uptake in comparison with99mTc-ZHER2:2395. There was, however, one puzzling issue. Theuse of the GGGC chelator resulted in appreciable reduction ofrenal retention of radioactivity at 1 and 4 h after injection for99mTc-(HE)3-ZHER2:342-GGGC in comparison with 99mTc-ZHER2:2395 having a VDC chelator. However, the renal retentionwas appreciably lower for 99mTc-ZHER2:342-GGGC than for99mTc-(HE)3-ZHER2:342-GGGC.

The fact that the renal activity after injection of 99mTc-(HE)3-ZHER2:342-GGGC was higher than that of 99mTc-ZHER2:342-GGGC was indeed surprising. A number of pre-vious studies [25, 26, 31] have demonstrated a strong

Fig. 7 Specificity of 99mTc-(HE)3-ZHER2:342-GGGC binding to HER2-expressing SKOV-3 cells. Two groups of culture dishes containingSKOV-3 cells were incubated with 0.6 nM 99mTc-(HE)3-ZHER2:342-GGGC. One group of culture dishes was pretreated with saturatingamounts of nonlabelled ZHER2:342 before incubation with 99mTc-(HE)3-ZHER2:342-GGGC. Cell-associated radioactivity was calculated aspercentage of total added radioactivity

Fig. 8 Retention andinternalization of 99mTc-(HE)3-ZHER2:342-GGGC (a) and99mTc-ZHER2:2395 (b) by theHER2-expressing SKOV-3 cellline. Cells were pre-incubatedwith radiolabelled affibodymolecules on ice, and then theculture medium was changed.Data are presented as anaverage of three samples withstandard deviations; error barsmight be not seen since they, inmost cases, are smaller than thesymbols

648 Tumor Biol. (2012) 33:641–651

influence of peptide-based cysteine-containing chelators onrenal retention of radioactivity of 99mTc-labelled affibodymolecules. Moreover, both overall biodistribution and renaluptake of 99mTc-ZHER2:2395 and 99mTc-ZHER2:342-GGGC inthis study was in an excellent agreement (variation withinexperimental error) with our previous results [14, 24, 25].This observation allowed us to rule out that there was adifference in kidney function between the batches of miceused in the current and in previous studies. Another possi-bility was that the site-specificity of labelling of (HE)3-

ZHER2:342-GGGC was impaired, so that 99mTc was partiallyattached to another chelating sequence, which would resultin a different profile of renal retention of radioactivity com-pared with GGGC. An assumption concerning site-specificity of labelling was based on previous results [24],when the use of current labelling condition for non-cysteine-containing ZHER2:342 resulted in zero yield. However, thosetests were performed using an affibody molecule withoutany kind of histidine tag. This led us to hypothesize that theMHEHEHE-sequence at the N terminus of the affibody

Table 1 Biodistribution of 99mTc-labelled affibody molecules in NMRI mice after intravenous injection

99mTc-(HE)3-ZHER2:342-GGGC99mTc-ZHER2:342-GGGC

99mTc-ZHER2:239599mTc-(HE)3-ZHER2:342

1 h

Blood 0.5±0.1*, **, *** 1.1±0.1 0.9±0.1 1.2±0.1

Lung 0.6±0.1*, **, *** 2.1±0.2 1.4±0.2 0.9±0.1****

Liver 1.2±0.1*, **, *** 2.0±0.1 2.0±0.3 1.5±0.1****

Spleen 0.36±0.02*, **, *** 0.67±0.16 0.60±0.17 0.64±0.07

Stomach 0.6±0.2*** 0.8±0.1 0.8±0.2 1.4±0.2****

Kidney 63±10*, **, *** 24±3 148±13 112±4****

Salivary gland 0.3±0.1**, *** 0.4±0.1 0.5±0.1 0.6±0.1****

Muscle 0.16±0.03*, **, *** 0.36±0.03 0.28±0.02 0.29±0.05

Intestinesa 1.1±0.1*, **, *** 1.9±0.1 2.2±0.4 2.1±0.2

Carcassa 5.6±0.9*, **, *** 6.9±0.3 10.1±1.6 14.1±0.9****

4 h

Blood 0.10±0.01**, *** 0.08±0.02 0.15±0.03 0.47±0.06****

Lung 0.19±0.04*, **, *** 0.5±0.2 0.4±0.1 0.59±0.05

Liver 0.8±0.1**, *** 0.8±0.1 2.0±0.5 1.3±0.2****

Spleen 0.17±0.05**, *** 0.22±0.05 0.4±0.1 0.5±0.1****

Stomach 0.4±0.2*, *** 0.11±0.05 0.34±0.05 1.5±0.4****

Kidney 46±11*, **, *** 5.4±0.9 146±20 88±6****

Salivary gland 0.15±0.07*, *** 0.04±0.02 0.20±0.06 0.4±0.1****

Muscle 0.07±0.03*, *** 0.04±0.01 0.08±0.01 0.18±0.04****

Intestinesa 1.0±0.5**, *** 0.9±0.2 1.9±0.2 3.6±0.8****

Carcassa 1.8±0.4**, *** 1.4±0.2 3.8±0.4 12±1****

24 h

Blood 0.020±0.004**, *** 0.03±0.01 0.08±0.03 0.09±0.03****

Lung 0.09±0.02*, **, *** 0.21±0.08 0.15±0.03 0.25±0.07

Liver 0.50±0.05* 0.39±0.07 0.50±0.09 0.60±0.08****

Spleen 0.11±0.04*** 0.15±0.06 0.17±0.04 0.3±0.1****

Stomach 0.15±0.04**, *** 0.2±0.1 0.3±0.1 0.5±0.2

Kidney 28±4*, *** 1.9±0.3 37±9 44±7****

Salivary gland 0.07±0.04*** 0.05±0.01 0.09±0.02 0.14±0.02****

Muscle 0.03±0.01*, *** 0.016±0.003 0.030±0.006 0.10±0.05****

Intestinesa 0.34±0.23** 1.8±1.6 1.2±0.3 0.6±0.1

Carcassa 1.1±0.1*, **, *** 0.8±0.1 1.7±0.2 8.6±1.3****

The uptake is expressed as per cent ID per gram and presented as an average value from four animals ± standard deviationa Data for intestines with content and carcass are presented as per cent of injected radioactivity per whole sample

*p<0.05, significant difference between 99m Tc-(HE)3-ZHER2:342-GGGC and 99m Tc-ZHER2:342-GGGC; **p<0.05, significant difference between99m Tc-(HE)3-ZHER2:342-GGGC and 99m Tc-ZHER2:2395; ***p<0.05, significant difference between 99m Tc-(HE)3-ZHER2:342-GGGC and99m Tc-(HE)3-ZHER2:342; ****p<0.05, significant difference between 99m Tc-(HE)3-ZHER2:342 and 99m Tc-ZHER2:342-GGGC

Tumor Biol. (2012) 33:641–651 649

molecule could possess also chelating properties for techne-tium species formed after reduction by tin chloride.

To investigate this hypothesis, we performed a series ofexperiments with a (HE)3-ZHER2:342 affibody molecule thatdoes not contain cysteine. The same kit and labelling con-dition were applied as for 99mTc-(HE)3-ZHER2:342-GGGC,99mTc-ZHER2:2395 and

99mTc-ZHER2:342-GGGC. This experi-ment demonstrated coupling of 99mTc to (HE)3-ZHER2:342,although the yield was significantly lower than that forcysteine-containing affibody molecules, only 43±1%. Fur-thermore, the stability of the labelling was appreciably low-er. A SDS PAGE analysis of size-exclusion purified 99mTc-(HE)3-ZHER2:342 showed that the majority of 99mTc wasdissociated (peak 3) from the conjugate after 1 h incubationin PBS (Fig. 6b). In the murine plasma (Fig. 6c), the disso-ciation (peak 3) was accompanied with transchelation toblood proteins (peak 4) and possibly with a proteolyticcleavage of the 99mTc-chelator complex (peak 5). Thus, thestability profile was completely different from the profile of99mTc-(HE)3-ZHER2:342-GGGC, for which very little releaseof radioactivity was observed in plasma (Fig. 6a). However,a small fraction of 99mTc was still associated with (HE)3-ZHER2:342 after incubation in both PBS and plasma. Thissuggests that coupling of 99mTc to (HE)3-ZHER2:342 is medi-ated by several separate chelating sites. Most likely, most ofthese chelating sites are weak and the coupling of 99mTc tothese sites does not occur in the presence of a strong compet-ing cysteine-containing chelator site as in (HE)3-ZHER2:342-GGGC. However, a small fraction of 99mTc is chelated strong-ly enough by the MHEHEHE-tag, and this could also happenin the presence of GGGC. This chelate might be processeddifferently by tubular cells in kidneys, and this could explainhigher renal retention of radioactivity for 99mTc-(HE)3-ZHER2:342-GGGC compared with 99mTc-ZHER2:342-GGGC.To investigate this hypothesis, we labelled (HE)3-ZHER2:342with 99mTc, and performed a series of size-exclusion purifica-tions to ensure that all weakly bound nuclide would dissociateand only firmly bound 99mTc would be coupled with the

conjugate. Thereafter, we injected 99mTc-(HE)3-ZHER2:342 inNMRImice andmeasured biodistribution at 1, 4 and 24 h afterinjection. The results are presented in the Table 1 and Fig. 9.The data suggest that the renal uptake of 99mTc-(HE)3-ZHER2:342 was significantly higher than uptake of both99mTc-(HE)3-ZHER2:342-GGGC and 99mTc-ZHER2:342-GGGC.This indicates that the renal retention profile of 99mTc-(HE)3-ZHER2:342-GGGC is influenced by partial chelation of 99mTcby a HEHEHE-tag of the construct.

It has to be noted that whether this explanation of elevatedrenal uptake of 99mTc-(HE)3-ZHER2:342-GGGC is correct ornot, the use of HEHEHE-tag is associated with impairment ofthe biodistribution of 99mTc-labelled GGGC-containing affi-body molecules. However, the HEHEHE-tag can still besuitable for simplified production of tracers, when site-specificity of labelling is not dependent on peptide-basedchelators, e.g. labelled via maleimido-derivatives polyamino-carboxylate chelators as for 111In, 68Ga or 86Y.

In conclusion, incorporation of a HEHEHE-tag permittedsimple and efficient purification of the (HE)3-ZHER2:342-GGGC affibody molecule by IMAC. The (HE)3-ZHER2:342-GGGC could be labelled with 99mTc providing a conjugatethat was found to be stable in vitro and in vivo. The 99mTc-(HE)3-ZHER2:342-GGGC was furthermore found to bind spe-cifically to HER2-expressing cells in vitro. Cellular process-ing experiments suggested that the residualizing propertiesof 99mTc-(HE)3-ZHER2:342-GGGC were weaker than those of99mTc-ZHER2:2395. In a murine model, the renal retention99mTc-(HE)3-ZHER2:342-GGGC was significantly higher thanthe retention of 99mTc-ZHER2:342-GGGC, lacking theHEHEHE-tag. A likely explanation for this phenomenon isthat the site-specificity of the labelling is lost, and a fractionof 99mTc is chelated by the HEHEHE tag providing a tracerwith a different retention profile in the kidneys.

Acknowledgements This research was financially supported bygrants from Swedish Cancer Society (Cancerfonden) and SwedishResearch Council (Vetenskapsrådet).

Conflicts of interest None.

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