Quantitative Immuno-Positron Emission Tomography Imaging of HER2Positive Tumor Xenografts with an...

Quantitative Immuno-Positron Emission Tomography Imaging

of HER2-Positive Tumor Xenografts with an Iodine-124

Labeled Anti-HER2 Diabody

Matthew K. Robinson,1Mohan Doss,

1Calvin Shaller,

1Deepa Narayanan,

1James D. Marks,

2

Lee P. Adler,2Dinko E. Gonzalez Trotter,

3and Gregory P. Adams

1

1Fox Chase Cancer Center, Philadelphia, Pennsylvania; 2University of California at San Francisco, San Francisco, California;and 3G.E. Global Research, Niskayuna, New York

Abstract

Positron emission tomography (PET) provides an effectivemeans of both diagnosing/staging several types of cancerand evaluating efficacy of treatment. To date, the only U.S.Food and Drug Administration–approved radiotracer foroncologic PET is 18F-fluoro-deoxyglucose, which measuresglucose accumulation as a surrogate for malignant activity.Engineered antibody fragments have been developed withthe appropriate targeting specificity and systemic elimina-tion properties predicted to allow for effective imaging ofcancer based on expression of tumor associated antigens.We evaluated a small engineered antibody fragment specificfor the HER2 receptor tyrosine kinase (C6.5 diabody) for itsability to function as a PET radiotracer when labeled withiodine-124. Our studies revealed HER2-dependent imaging ofmouse tumor xenografts with a time-dependent increase intumor-to-background signal over the course of the experi-ments. Radioiodination via an indirect method attenuateduptake of radioiodine in tissues that express the Na/Isymporter without affecting the ability to image the tumorxenografts. In addition, we validated a method for using aclinical PET/computed tomography scanner to quantifytumor uptake in small-animal model systems; quantitationof the tumor targeting by PET correlated with traditionalnecropsy-based analysis at all time points analyzed. Thus,diabodies may represent an effective molecular structure fordevelopment of novel PET radiotracers. (Cancer Res 2005;65(4): 1471-8)

Introduction

The antigen-binding specificity displayed by monoclonal anti-bodies (mAb) is increasingly being exploited as a way to deliverradionuclides for diagnosis and treatment of cancer (1, 2). This isbest exemplified by the Food and Drug Administration approval ofthe anti-CD20 mAbs Zevalin (ibritumomab tiuxetan) and Bexxar(tositumomab and 131I-tositumomab) for the radioimmunotherapytreatment of non-Hodgkin’s lymphoma. The requirement foraccurate dosimetry predictions is inherent to the properadministration of radiopharmaceuticals (3). In the case of theseagents, g-camera imaging protocols that use the g emissionsassociated with an 111In-labeled form of ibritumomab tiuxetan,

or those inherent to 131I-tositumomab, are used to generate thenecessary information. Although well suited for predictingdosimetry of the associated therapeutic agent, the effectivenessof these and other mAbs as true diagnostic agents is hampered bytheir slow clearance rate, which leads to high background levels inthe diagnostic scans. Chemical cleavage of mAbs to more rapidlyclearing Fab molecules has led to diagnostic agents such as theanti-carcinoembryonic antigen (CEA) arcitumomab (CEA-Scan)that exhibit higher tumor-to-background ratios than intact mAbs(4). However, these reagents are still suboptimal due in part totheir monovalent association with tumor antigens.Positron emission tomography (PET) is an imaging modality

based on coincidence detection of the two opposing photons thatresult from annihilation of a positron and an electron (5). The useof mAbs as PET radiotracers has been investigated (6, 7) and recentwork has shown promising preclinical results using the U36chimeric mAb labeled with the positron emitting isotope 89Zr togenerate a PET-based dosimetry prediction for radioimmunother-apy with an 90Y-U36 immunoconjugate (8). Despite the technicaladvantages of PET over g-camera imaging, the backgroundassociated with mAb-based PET radiotracers still limits theirusefulness as diagnostic agents.Advances in antibody engineering have facilitated the develop-

ment of novel antibody-based fragments with physical andpharmacokinetic properties consistent with use as dedicateddiagnostic agents (9). In addition, a number of positron-emittingisotopes (e.g., 124I and 64Cu) exist that have physical half-lives thatmatch well with the biological half-lives of smaller engineeredantibody fragments such as diabodies (Fig. 1). We and others havepostulated that when labeled with these PET isotopes, the tumor-targeting properties and systemic clearance rates of engineeredantibody fragments would partner effectively with the imagingadvantages of PET for the radioimmunodetection of cancer. Wu etal. (10) validated these predictions in preclinical imaging studiesdone on a dedicated small animal imager (microPET) with an anti-CEA minibody labeled with 64Cu (64Cu-DOTA-T84.66). The 64Cu-DOTA-T84.66 showed specific uptake into CEA-positive LS174Thuman colon carcinoma tumor xenografts compared with bilateralCEA-negative C6 glioblastoma tumor xenografts and surroundingsoft tissue (average tumor/nontarget ratio was 3-4:1). Subsequentwork with minibody and diabody forms of T84.66 showed the needto consider the biological properties of both the antibody moleculeand the radionuclide when developing a novel radiotracer. The sizeof the T84.66 minibody (80 kDa) resulted in hepatic clearance andsignificant retention of radiometals in the liver (e.g., f26% ID/g at24 hours when labeled with 111In). By comparison, the 111In-T84.66diabody (52 kDa) showed high levels of retention (>180% ID/g at6 hours) in the kidney due to its renal clearance (11). Labeling both

Requests for reprints: Gregory P. Adams, Fox Chase Cancer Center, Departmentof Medical Oncology, Medical Science Division, 333 Cottman Avenue, W364,Philadelphia , PA 19111. Phone: 215-728-3890; 215-728-2741; E-mail :[email protected].

#2005 American Association for Cancer Research.

www.aacrjournals.org 1471 Cancer Res 2005; 65: (4). February 15, 2005

Research Article

of these molecules with radioiodine decreased the nontarget tissueaccumulation and resulted in higher target-to-background ratios inPET images (12).We have previously shown efficient targeting of HER2-positive

tumor xenografts by the anti-HER2 C6.5 diabody. As with theT84.66 diabody, the size of the C6.5 diabody (52 kDa) results in first-pass renal clearance and rapid systemic elimination (C6.5 diabodyT1/2h = 6.4 hours; ref. 13). The uptake and retention of 125I-C6.5diabody in tumor combined with the clearance kinetics of themolecule results in tumor levels (% ID/g) surpassing those in blood(% ID/mL) as early as 4 hours post-injection. These propertiessuggested that the C6.5 diabody could be an effective agent for thedelivery of diagnostic radionuclides for the detection of HER2-positive cancer via PET. In this study, we evaluate the potential of124I-C6.5 diabody to function as a PET radiotracer in a mousetumor xenograft model.

Materials and Methods

Production of C6.5 Diabody. C6.5 diabody (apparent kD= 4 � 10�10)

was expressed in TG1 Escherichia coli and purified by immobilized metalaffinity chromatography followed by high-performance liquid chromatog-

raphy size-exclusion chromatography over a Superdex 75 column

(Amersham Pharmacia, Piscataway, NJ) as previously described (14).

Endotoxin was removed by ion exchange chromatography over PBE94resin (Amersham Pharmacia) and protein was stored at �70jC at 0.5 mg/

mL in PBS containing 10% glycerol until ready to use.

Radioiodination of C6.5 Diabody. The anti-HER2 diabody C6.5 was

radioiodinated with iodine-124 produced by RITVERC Isotope Products (St.Petersburg, Russia) and supplied by Advanced Nuclide Technologies, LLC

(Indianapolis, IN) using either iodogen-coated glass beads (15) or with the

water-soluble form of Bolton-Hunter reagent sulfosuccinimidyl-3-(4-hydrox-

yphenyl) propionate (SHPP, Pierce Biotechnology, Rockford, IL). To radio-iodinate with iodogen-coated glass beads, 0.25 mL of Na124I (1.48 GBq/mL;

2,220 GBq/mg) was neutralized with an equal volume of 2� acidified saline

(300 mmol/L NaCl and 20 mmol/L HCl). Four glass beads and unlabeled

carrier NaI (70 Amol/L) were added to the Na124I and incubated at roomtemperature for 5 minutes with constant mixing. Three milliliters of C6.5

diabody (0.56 mg/mL in PBS, pH 7.2) were added to the reaction and

incubated for 6minuteswith constantmixing ( final concentration of NaI was10 Amol/L, specific activity was 88.8 GBq/mg). Beads were removed to stop

the iodination reaction, percent incorporation was determined by instant

TLC (Biodex, Shirley, NY) using a PBS mobile phase, and unincorporated 124I

was separated from labeled diabody by size exclusion chromatography over aPD10 column (Amersham Pharmacia). To radioiodinate the C6.5 diabody

with SHPP, 0.25 mL of neutralized Na124I (1.48 GBq/mL; 2,220 GBq/mg) was

added to 0.2 mL PBS (pH 7.2) that contained 10 Amol/L NaI. The reaction was

initiated by adding 0.2 mL Chloramine T (5 mg/mL in PBS, pH 7.2) and 20 ALSHPP (3 mmol/L in DMSO). After 1 minute at room temperature with gentle

mixing, the reaction was quenched with 0.2 mL NaS2O5 (12 mg/mL). The

radiolabeled SHPP was then conjugated to 3.3 mL of C6.5 (0.5 mg/mL in 0.5mol/L sodium borate, pH 8.5) by incubating at room temperature for 60

minutes with gentle mixing. The reaction was stopped with the addition of 6

mL of 0.5 mol/L sodium borate (pH 8.5), 0.2 mol/L glycine. Radiolabeled

diabody was then purified over a PD10 column as described above.Radioiodination of the C6.5 diabody with iodogen occurred at efficiencies

between 13.75% and 18.8% and produced productwith a specific activity of 33

to 102 MBq/mg that was >93.5% pure by instant TLC. The SHPP-labeling

protocol worked at efficiencies of 6.5% to 8% and produced labeled diabodywith specific activities between 37 and 48 MBq/mg that was >97% pure by

instant TLC. Immunoreactivity of the labeled diabodywas assayed by live-cell

binding assays with MDA-361/DYT2 cells (gift of Dr. Dajong Yong, GeorgeWashington University) as described previously (16). Preps of iodogen-

labeled diabody were 71% to 73% active and SHPP-labeled diabody were 31%

to 41% active.

Biodistribution. CB.17 Icr severe combined immunodeficient (SCID)

mice, ages 6 to 8 weeks, were obtained from the Fox Chase Cancer Center

Laboratory Animal Facility. SK-OV-3 (3.0� 106), MDA-361/DYT2 (3.0 � 106),

or MDA-MB-468 (6.0 � 106) cells were implanted s.c. in the inguinal regionof mice. At f7 weeks post-implantation, Lugol’s solution (Sigma, St. Louis,

MO) was added to the drinking water at a final concentration of 0.2% to

block thyroid accumulation of radioiodine, and biodistribution studies wereinitiated f5 days later. Cohorts of mice (n z 5 per cohort) received 124I-

C6.5 (labeled by either the iodogen or SHPP methods) via tail-vein injection,

were euthanized at indicated times post-injection, imaged (as appropriate,

see below), dissected and major organs weighed and counted in a g wellcounter (Cobra Quantum, Packard Instruments, Meriden, CT) with a

window of 400 to 650 keV. The retention in tumor and nontarget tissues was

expressed as a percentage of the injected dose localized per gram of tissue

(% ID/g) as determined from decay-corrected counts as previouslydescribed (17). Values are reported as the mean and SE. Animals exhibiting

tumor or organ uptakeF2 SDs from the mean were considered outliers and

removed from the biodistribution analysis. The percentage of protein-bound radioiodine in the blood of animals was determined by trichloro-

acetic acid precipitation as previously described (18).

Statistics. The Excel spreadsheet package was used to perform two-

tailed t tests set up for samples with equal population variances. For eachcomparison, F tests were done using a web-based calculator (http://

nimitz.mcs.kent.edu/~blewis/stat/tTest.html) to determine the validity of

using a t test set up for samples with equal population variances.

PET/CT Imaging. Before necropsy, individual cohorts were imagedsimultaneously on a Discovery LS clinical PET/computed tomography (CT)

scanner (GE Healthcare, Milwaukee, WI) using a custom built acrylic holder

mounted on the patient bed. First, a low dose CT was done, with 140 kV, 50

mA, 0.8 second per rotation, 5-mm slice thickness, and 4.25-mm sliceinterval. Next, a high axial resolution CT was done with 120 kV, 50 mA, 0.63-

mm slice thickness, 0.5 second per rotation, with the same axial limits as

initial CT. Immediately after, a PET emission scan was acquired for 10

minutes using delayed random event subtraction in two-dimensional highsensitivity mode. PET images were reconstructed on a 128 � 128 matrix for

a 30-cm-diameter field of view, with vendor supplied software using

ordered subsets expectation maximization algorithm, using the initial CT

Figure 1. ImmunoPET imaging. A, schematic representation of the structure ofan IgG and a diabody. B, photograph of custom-built acrylic holder and micearranged for PET/CT imaging. C, transaxial view of resulting PET/CT fusion atlevel of tumor xenografts.

Cancer Research

Cancer Res 2005; 65: (4). February 15, 2005 1472 www.aacrjournals.org

for attenuation correction. The PET, CT, and fused images were visualized

with the MIM software package (MIMVista Co., Cleveland, OH).

Quantitation of tumor uptake by PET was done as previously described(19). Briefly, recovery coefficients were determined through use of small-

animal phantoms that were positioned at various distances from the

longitudinal axis of the scanner. Phantoms contained spheres of relevant

volumes (0.0625–1.0 mL), activity concentrations (27 F 2 kBq/mL), andtarget to background ratios (10:1 and 20:1). Tumor uptake (activity

concentration in the lesions) was quantitated from filtered back-projection

(2-mm full-width half-maximum Gaussian filter) images reconstructed on256 � 256 size axial slices (1.0725 � 1.0725 � 4.25 mm3 voxels) with

appropriate recovery coefficients applied to the maximum intensity voxels

associated with each tumor xenograft. Animals that were removed from the

biodistribution analysis, described above, due either to tumor or nontargetorgan uptake that differed by >2 SDs were not removed from this analysis.

Results124I-C6.5 Diabody Efficiently Targets to HER2-Positive

Tumor Xenografts. To test the hypothesis that the C6.5 diabodywould function as an effective PET radiotracer for the detectionof HER2-positive tumors, C6.5 diabody was radioiodinated withthe positron emitting isotope 124I and given via tail-vein injectionto SCID mice bearing established s.c. HER2-positive humanovarian carcinoma (SK-OV-3) xenografts. Tumor xenografts usedin this study ranged in size from 0.11 to 0.86 gram (mean tumorsize = 0.43 g), all of which were detectable by CT scan (seebelow). Cohorts of animals (n = 7 per cohort) were euthanizedat the appropriate time points, positioned within a custom-designed holder (Fig. 1B) and imaged simultaneously using asingle bed position on a dedicated clinical PET/CT scanner(Discovery LS PET/CT, GE Healthcare). A benefit of the clinicalPET/CT scanner is that the resulting PET and CT images wereautomatically registered, allowing for fusion of the resultingimages to confirm the position of the PET signal as emanatingfrom the tumor xenografts. This is exemplified by the transaxial

slice of a PET/CT fusion shown in Fig. 1C . Subsequent toimaging, animals were dissected and biodistribution of theradioiodinated diabody was determined (Table 1). Targetingkinetics of the 124I-C6.5 diabody was consistent with thatpreviously seen with both the 125I-C6.5 diabody and the anti-CEA diabody T84.66 (13, 20). Uptake, as measured as percent ofinjected dose per gram of tissue (% ID/g), peaked by 4 hours post-injection (9.8% ID/g) and cleared f3-fold from the tumor over theremainder of the study (6.3% ID/g at 24 hours and 3% ID/g at 48hours). Coincident with the drop in % ID/g in tumor was a morerapid clearance from nontarget tissue (0.95% ID/mL of blood at24 hours and 0.22% ID/mL at 48 hours), leading to increasedtumor-to-background ratios at later time points (Table 1). Theclearance rate of the C6.5 diabody pairs well with the physicalhalf-life of iodine-124 (4.18 days), allowing for significant retentionof radioactivity in the tumor at 48 hours. This time-dependentincrease in tumor-to-background signal is displayed very dramat-ically in coronal sections through the level of the tumorxenografts (Fig. 2). However, tumor uptake was detectable andquantifiable (see below) by PET for all tumor sizes studied and bythe earliest time point (4 hours) analyzed in this study.Modification of Labeling Strategy. The divalent nature of the

C6.5 diabody increases tumor retention due to avid binding butalso results in cross-linking and internalization of the HER2 targetantigen. A byproduct of internalization is catabolism of thediabody with subsequent release of 124I-tyrosine and free 124I intocirculation (21). This process is evident at later time points in Fig.2 as uptake of 124I by both the stomach and the thyroid, naturalsites of iodine metabolism. Dehalogenation may impair radiotracerfunction due to both durable retention of 124I in these normaltissues and loss of internalized tracer from tumor. Multiple

Table 1. Biodistribution of 124I-labeled C6.5 diabody in

SCID mice bearing s.c. SKOV-3 tumors

Tissue 24 h 48 h

Tumor 6.34 (1) 2.95 (1)Blood 0.95 (6.7) 0.22 (13.4)

Liver 0.65 (9.8) 0.14 (21.1)

Lung 0.74 (8.6) 0.15 (19.7)

Spleen 0.89 (7.1) 0.21 (14)Kidney 0.76 (8.3) 0.18 (16.4)

Heart 0.61 (10.4) 0.11 (26.8)

Stomach 3.01*(2.1) 0.29c(10.2)

Bone 0.28 (22.6) 0.09b(32.8)

Intestine 0.39 (16.3) 0.07 (42)

Muscle 0.21 (30.2) 0.05 (59)

NOTE: Cohorts of mice (n z 5 per cohort) were analyzed at each timepoint. Average tumor and organ uptakes are expressed as percentage

injected dose per gram of tissue (% ID/g).

All standard errors of the means (SE) were <15% of the average unless

otherwise noted. Values in parenthesis are the ratios of the averagetumor to organ uptake.*46% SE.c26% SE.b53% SE.

Figure 2. ImmunoPET imaging with directly labeled C6.5 diabody. Animalsimaged 4, 8, 24, and 48 hours post-injection of directly radioiodinated C6.5diabody showed targeting of the radiotracer to the SK-OV-3 tumor xenografts(arrowheads ). However, dehalogenation of the radiotracer resulted in significantuptake of iodine by the thyroid (chevron ) and stomach (arrow ).

Anti-HER2 ImmunoPET


labeling strategies have been developed to block the dehalogena-tion process (22–24). For our purposes, we selected a water-solubleform of Bolton-Hunter reagent, SHPP, to indirectly label lysineresidues in the C6.5 diabody.To compare the function of 124I-SHPP-labeled C6.5 diabody with

directly labeled diabody, aliquots of C6.5 were labeled simulta-neously with either the iodogen or SHPP methodologies andinjected into tumor bearing mice. Both methodologies resulted inradiolabeled diabody that was >97% radiochemically pure byinstant TLC but the SHPP methodology decreased the immuno-reactive fraction of the diabody f2-fold compared with theiodogen labeling methodology. This decrease in immunoreactivitydid not seem to greatly affect its function as a PET radiotracer; C6.5diabody labeled with either method exhibited tumor targeting thatwas readily visible by PET imaging 48 hours post-injection (Fig. 3).The f40% decrease in the % ID/g of indirectly labeled diabodytargeted to the MDA-361/DYT2 tumors (0.9% ID/g for SHPPcompared with 1.5% ID/g for iodogen at 48 hours, Table 2) is mostlikely explained by the decrease in immunoreactivity resulting fromthis labeling procedure.MDA-361/DYT2 tumor xenografts were selected for these studies

in an effort to enhance any differences between the labelingstrategies by taking advantage of the faster rate of HER2internalization exhibited by MDA-361/DYT2 cells compared withSK-OV-3 cells (data not shown). Despite the partially residualizingnature of the SHPP reagent, necropsy analysis revealed that iodogenand SHPP-labeled C6.5 exhibited similar levels of tumor targeting inthe MDA-361/DYT2 tumors suggesting that internalization did not

significantly impact the outcome of the experiment. It should benoted that the quantity of iodogen-labeled C6.5 diabody retained inthe MDA-361/DYT2 tumors was f2-fold lower than that normallyseen for SK-OV-3 tumors at 48 hours post-injection. This may beexplained by the lower level of HER2 expression on MDA-361/DYT2as compared with SK-OV-3 cells (see below). Consistent with thethyroid and stomach uptake seen in the PET images (Fig. 3), animalsreceiving SHPP-labeled diabody contained lower levels of free io-dine in their blood than animals receiving iodogen-labeled diabody(P < 0.005); 97.9F 1.39% of all iodine in the blood of the SHPP cohort(n = 5) was trichloroacetic acid precipitable at 48 hours post-injection compared with 88.9F 1.33% for the iodogen cohort (n = 5).PET images of animals injected with 124I-SHPP-C6.5 diabody

exhibited an overall increase in background signal compared withscans of animals injected with iodogen-labeled diabody (Fig. 3). Theincreased background in the scans was consistent with biodis-tribution results (Table 2) that showed statistically significantdifferences in retention in nontarget tissues such as muscle (0.12versus 0.06% ID/g, P = 0.005), intestine (0.15 versus 0.09% ID/g, P =0.01), lung (0.31 versus 0.21% ID/g, P = 0.02) and heart (0.6 versus0.11% ID/g, P < 0.0001). The increase in retention of radioiodine inthese tissues, presumably due to the residualizing nature of theSHPP moiety, could account for the broad diffuse increase in PETsignal. In addition, the decrease in the signal to noise ratioobserved in scans of the 124I-SHPP-C6.5–injected animals was mostlikely compounded by the adverse effect of the labeling method onthe immunoreactivity of the radiotracer.Tumor Targeting Is HER2 Dependent. We did PET and

biodistribution studies in mice bearing either HER2-positive (SK-OV-3) or HER2-negative (MDA-MB-468) tumor xenografts toconfirm that targeting of radioiodinated C6.5 diabody to thetumor xenografts was due to antigen-dependent recognition oftumor cells rather than nonspecific trapping of diabody withintumors due to differences in the physiology of tumor versus normaltissue (e.g., lack of draining lymphatics, leaky vasculature, etc.).

Table 2. Effect of labeling method on targeting

Tissue SHPP Iodogen

Tumor 0.9 (1) 1.5 (1)

Blood 0.29 (3.1) 0.25 (6)Liver 0.16 (5.6) 0.19 (7.9)

Lung 0.31 (2.9) 0.21 (7.1)

Spleen 0.14 (6.4) 0.21 (7.1)

Kidney 0.43 (2.1) 0.42 (3.6)Heart 0.6 (1.5) 0.11 (13.6)

Stomach 0.18 (5) 0.23 (6.5)

Bone 0.09 (10) 0.09 (16.6)

Intestine 0.15 (6) 0.09 (16.6)Muscle 0.12 (7.5) 0.06 (25)

NOTE: Cohorts of SCID mice (n = 8 per cohort) bearing s.c. HER2-

positive (MDA-361/DYT2) tumor xenografts were injected with C6.5

diabody labeled with 124I either indirectly (SHPP) or directly (iodogen)and biodistribution was analyzed 48 hours post-injection. Average

tumor and organ uptakes are expressed as percentage injected dose

per gram of tissue (% ID/g). All standard errors of the means (SE) were<15% of the average. Values in parenthesis are the ratios of the average

tumor to organ uptake.

Figure 3. Comparison of direct and indirect labeling methods on radiotracerfunction. C6.5 diabody was radioiodinated with 124I either (A) indirectly withSHPP or (B) directly with iodogen, given to SCID mice bearing MDA-MB-361tumor xenografts, and animals were imaged 48 hours post-injection.

Cancer Research


MDA-MB-468 cells were selected as the HER2-negative tumori-genic cell line based on reports in the literature (25, 26) and wereconfirmed to be HER2-negative by flow cytometry (data notshown). Mice bearing MDA-MB-468 (mean tumor size = 0.21 g) orSK-OV-3 (mean tumor size = 0.51 g) tumor xenografts wereinjected with 124I-SHPP-C6.5 diabody and tumor targeting wasanalyzed 48 hours post-injection. PET imaging showed antigendependent targeting of the C6.5 diabody (Fig. 4). C6.5 diabodyaccumulated in HER-2 positive SK-OV-3 tumors to a significantlyhigher level (P < 0.0001) than that seen in blood at 48 hours post-injection (1.1 F 0.12% ID/g in tumor versus 0.26 F 0.03% ID/mL inblood). In contrast, C6.5 accumulated to 0.32 F 0.02% ID/g in theHER2-negative MDA-MB-468 tumors at 48 hours post-injection, alevel equal to that seen in the blood of those animals (0.33 F 0.01%ID/mL). The 3.4-fold increase in tumor uptake in SK-OV-3 tumorscompared with MDA-MB-468 tumors (P < 0.0001) was the onlytissue analyzed which showed a statistically significant increase intissue uptake in the SK-OV-3 tumor bearing mice compared withthe MDA-MB-468 bearing mice (Table 3). This targeting specificityis indicative of the predicted antigen-dependent uptake of the C6.5diabody into the tumor xenografts.Quantitation of Tumor Targeting by PET. Quantitative PET

imaging can be a powerful component of the development and invivo evaluation of novel radiotracers in murine model systems. Thebore size of clinical PET scanners allow for simultaneous imaging ofmultiple mice (see above) but the large intrinsic resolution (f5 mm

full-width half-maximum) compared with the size of murine tumorxenografts force the use of correction methods to obtainmeaningful radiotracer concentration data from PET images. Inthe case of I-124–labeled antibodies, the resulting blurring is alsosomewhat exacerbated by the long positron range of 124I. We haverecently developed a recovery-coefficient correction method toobtain meaningful tracer concentration data from murine PETimages produced with a clinical scanner (19). To validate thiscorrection method, we applied it to mice imaged in this study. Thetumor uptake (% ID/g) of C6.5 and background signal at the 4, 8, 24,and 48 hour time points represent a large range of the valuesexpected to be encountered during development of novel smallantibody-based radiotracers. Activity concentrations in the tumorxenografts of all 28 mice were determined and converted to % ID/gusing the correction methodology and the tumor weights obtainedduring necropsy. Those values were then compared the % ID/gvalues derived from necropsy-based analysis (Fig. 5). Over the entirerange of % ID/g present in the tumors, the PET-based quantitationmethod showed a strong correlation with the necropsy-basedanalysis (R2= 0.96291). This was also true when comparing theaverage % ID/g for a given time point. At all four time points, the %ID/g tumor derived by PET differed from the necropsy-based valueby less than the experimental error (4 hours = 9.76 F 0.41 versus11.26F 1.41; 8 hours = 9.39F 0.9 versus 10.30F 2.25; 24 hours = 5.71F 0.69 versus 6.07F 2.12; 48 hours = 2.99F 0.14 versus 3.16F 0.59).Although the resolution of the clinical scanner that we employed forthese studies decreases from 5-mm full-width half-maximum at thecenter of the field-of-view to 7-mm full-width half-maximum 10 cmfrom the center, this had no noticeable effect on the quality of theimages we were able to obtain. Additionally, this decrease inresolution was incorporated into the described quantitation method.

Figure 4. Imaging with anti-HER2 diabody is antigen dependent. C6.5diabody was radiolabeled with 124I using SHPP. Radiolabeled diabody was givento SCID mice bearing (A) HER2-positive SK-OV-3 or (B ) HER2-negativeMDA-MB-468 tumor xenografts, and animals were imaged 48 hourspost-injection.

Table 3. Biodistribution of 124I-labeled C6.5 diabody in

SCID mice bearing either HER2-positive or HER2-negative

tumors

Tissue HER2 positive HER2 negative

Tumor 1.1 (1) 0.32 (1)

Blood 0.26 (4.2) 0.33 (1)

Liver 0.26 (4.2) 0.29 (1.1)

Lung 0.22 (5) 0.25 (1.3)Spleen 0.14* (7.9) 0.18 (1.8)

Kidney 0.4 (2.8) 0.44 (0.7)

Heart 0.26 (4.2) 0.27 (1.2)

Stomach 0.09c(12.2) 0.12

b(2.7)

Bone 0.06 (18.3) 0.06 (5.3)

Intestine 0.13 (8.5) 0.12x (2.7)

Muscle 0.05 (22) 0.05 (6.4)

NOTE: Cohorts of mice (n = 5 per cohort) bearing s.c. tumorxenografts derived from either HER2-positive SK-OV-3 or HER2-

negative MDA-MB-468 cells were analyzed at 48 hours post-injection.

Average tumor and organ uptakes are expressed as percentage

injected dose per gram of tissue (% ID/g). All standard errors of themeans (SE) were <15% of the average unless otherwise noted. Values

in parenthesis are the ratios of the average tumor to organ uptake.*19.4% SE.c24.7% SE.b33.4% SE.x23.5% SE.

Anti-HER2 ImmunoPET


Discussion

We have developed a divalent antibody fragment that functionseffectively in our preclinical model as a radiotracer for the imagingof HER2-positive tumors with PET. Using 124I-labeled C6.5 diabody,we successfully imaged and quantitated radiotracer uptake intumors of clinically relevant size (0.11–0.86 g) on a dedicatedclinical scanner. In addition to demonstrating the performance ofthe anti-HER2 C6.5 diabody as a radiotracer for the detection ofHER2-positive disease, these experiments show that a clinical PET/CT scanner can be used during preclinical development to rapidlyquantitate the performance of novel radiotracers in multiple micein a single scan.The high resolution, sensitivity, and quantitative nature of PET

make it an effective method for both detection and staging ofcancerous lesions. The only approved radiotracer for PET-baseddetection of cancer is 18F-fluoro-2-deoxy-glucose (T1/2h = 110minutes). This glucose derivative measures increased glucoseaccumulation as a surrogate for malignant activity and has proveneffective in the diagnosis and staging of a large variety of tumors(e.g., lung and colon; ref. 27). However, there are a number oftumors (e.g., prostate, bronchoalveolar carcinoma, and neuroen-docrine tumors) that do not readily incorporate 18F-fluoro-deoxyglucose (28). Furthermore, 18F-fluoro-deoxyglucose imagingcan be complicated by significant uptake into metabolically activetissues (e.g., brain and heart) and at sites of infection (29).Thus, imaging compounds targeted at tumor-associated antigens,like the 124I-conjugated anti-HER2 diabody described here, mayprovide a means for overcoming the deficiencies associated with18F-fluoro-deoxyglucose PET.In this study, we showed that the C6.5 diabody and the clinical

scanner could be used to effectively image and quantify uptake intumor xenografts at least as small as 0.1 g in size when derived fromcells that express high (SK-OV-3) levels of HER2 and 0.3 g when thetumors expressed moderate levels of HER2 (MDA-361/DYT2). Thetumor targeting we achievedwith the C6.5 diabody against theMDA-

361/DYT2 and SK-OV-3 tumors allowed for comparable tumor:-background ratios to those seen with the anti-CEA diabody andminibody constructs against LS174T tumors in studies with adedicated animal scanner (12). Based on quantitative flow cytometryand Skatchard analysis, the SK-OV-3 and MDA-361/DYT2 cells thatwere used to generate the tumor xenografts for these experimentswere found to express f1 � 106 and 3.75 � 105 copies of HER2 percell, respectively, when cultured in vitro .4 By comparison, LS174Tcells can bind 2.7� 106 copies of the anti-CEA mAb COL-1 implyinga minimum of 2.7 � 106 copies of CEA per cell (30). Our resultssuggest that immunoPET imaging can be effectively used to detecttumors with significantly lower quantity of antigen.A number of additional factors need to be considered in

selecting an antigen target and antibody construct for immunoPETimaging. For example, the presence of shed antigen can decreasethe sensitivity of the imaging system and the size of the antibody-based construct will dictate its rate of systemic clearance and itsability to extravasate and penetrate into tumor masses. WhereasCEA is commonly shed from tumors, HER2 can also be shed atsignificant levels and its impact will need to be addressed. In termsof size of the antibody construct, increased tumor targeting can beachieved with intact mAbs or larger fragments, but it comes at acost of increased time between injections and imaging, increaseddose to patients, and higher background levels. This is evidentwhen our results are compared with those reported by others usingintact mAb. HuA33 is a humanized mAb that binds the A33antigen expressed on >95% of colorectal cancers. A33 is expressedby SW1222 colorectal cancer cells in vitro at f3.7 � 105 copies percell (31). A comparison of the tumor-targeting specificity between124I-HuA33 mAb in BALB/c nude mice bearing SW1222 tumorscompared with the results reported here for C6.5 show thatdirectly labeled C6.5 reaches tumor/blood ratios (>13:1) by 48hours that are not reached until after 96 hours post-injectionwith the mAb. Consistent with the increased blood retention of the124I-HuA33mAb (T1/2h = 38.2 hours) % ID/g in tumor peaks atmuch higher levels of % ID/g than that seen with the C6.5 diabody(50 % ID/g at 96 hours post-injection compared with 10% ID/g at 4hours post-injection for the C6.5 diabody). This increased tumoruptake may come at the expense of increased patient dose; inaddition to positron emission (Eaverage = 819 keV), 124I decays via anumber of high-energy g emissions.Unlike the CEA or A33 antigens mentioned above, the HER2

receptor is readily internalized. Cross-linking of two HER2molecules by the C6.5 diabody is thought to induce internaliza-tion of HER2 into the lysosomal compartment. Once internalized,radioiodinated antibodies are particularly susceptible to deiodi-nation when the iodine is directly conjugated to tyrosine residues(21). In vivo this leads to active retention of radioiodine in tissuesthat express the NA-I symporter (e.g., thyroid, stomach, andsalivary glands). Signal associated with such radioiodine uptakemay obscure signal from tumor-targeted diabody, especially inareas surrounding those sites of normal iodine uptake. To addressthis, we examined the utility of using a modified version of theBolton-Hunter conjugation method that indirectly attaches the124I to lysine residues (32). When radiolabeled, a significantportion of the indirectly labeled diabody was capable of bindingto HER2-positive cells. However, the immunoreactive fraction of

4 Manuscript in preparation.

Figure 5. PET-based quantitation of tumor uptake correlates withnecropsy-based analysis. Tumor targeting (% ID/g) of directly radioiodinatedC6.5 diabody was determined by PET for mice imaged at 4, 8, 24, and 48 hourspost-injection. Results from individual tumors were compared directly with thoseobtained by traditional necropsy analysis.

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the SHPP-conjugated C6.5 diabody was still markedly decreasedcompared with the directly labeled preparation (36% versus 72%)indicating that modifications to this diabody will be necessary totake full advantage of this labeling strategy. For example, apotential conjugation site for SHPP exists within the heavy chainCDR3 of the C6.5 scFv. This lysine residue (100 g, Kabatnomenclature; ref. 33) can be substituted to an alanine residuewithout affecting affinity of the molecule for HER2 (34).Alternatively, an approach similar to the one described byOlafsen et al. (35) could be used to modify the COOH terminiof the diabody with free cysteine residues for site-specificconjugation of the SHPP moiety. Despite this decrease inimmunoreactivity, imaging studies done with the 124I-SHPP-labeled diabody revealed sufficient tumor-targeting specificity toallow for high resolution imaging of the tumor xenografts.Significant decreases in radioiodine accumulation in the thyroidand stomach were also observed. This occurred without increasingrenal retention, a possible side effect due to the renal clearance ofthis molecule combined with the residualizing nature of theradiolabeled SHPP. These results indicate that active uptake bynormal tissues can be moderated by altering the labeling strategy,leading to significant improvements in the specificity of PET/CTimaging.Necropsy-based biodistribution analysis has historically been the

accepted method for determining the tumor-targeting of novelagents during preclinical development. We have recently describeda PET-based quantitation method which we developed usingphantoms to generate recovery coefficients that could be appliedto small animal models for the purpose of determining tumortargeting (19). This method provided an accurate account of tumoruptake in a small pilot study done in tumor bearing mice. A majorgoal of the current study was to validate the results of that pilotstudy in larger cohorts of mice bearing tumors over a range of sizes.In the studies reported here, we obtained close agreement with thetumor uptake we measured by traditional necropsy-based methodsthereby validating this PET-based methodology. Our method of PETquantitation differs from other methodologies (8, 36) in that it doesnot rely on direct measurement of the complete activity in the target(which would require segmentation of the PET target image), but onthe application of recovery coefficients estimated a priori to the

maximum intensity voxel of the PET image of the target. Therecovery coefficient depends on the target mass (obtained atnecropsy), background activity concentration (measured directlyfrom the PET image) and position of the target in the bore of thescanner (measured directly from the PET image). By removing theneed for segmentation, we have eliminated an important potentialsource of uncertainty in the quantitation procedure. Thus, ourmethod may prove more accurate in the estimation of target activityconcentration, especially when the target activity concentration isfairly homogenous and its size is comparable to the resolution full-width half-maximum of the scanner. Our results indicate that theseassumptions are well-founded for tumors of the type and size usedin this study and validates the method for acquiring tumor-targetingdata in the context of an in vivo model system. In addition, themethod may prove useful for monitoring the impact of therapeuticstrategies, particularly those directed at the same target antigen.However, although we achieved close agreement between the twoquantitation methods, the PET-based quantitation methodreported here depended upon tumor volumes determined bynecropsy. Therefore, it will be necessary to develop reliable CT-based tumor-volume estimates before the utility of this method canbe fully realized.As cancer therapeutics become increasingly targeted, an

understanding of the molecular underpinnings driving themetastatic progression of an individual’s disease will be invaluablefor both guiding therapy and determining its effectiveness. This isexemplified by the successful use of the HER2-directed antibody,Trastuzumab, in women with HER2 overexpressing breast cancer(37, 38). Development of diagnostic radiotracers, such as the anti-HER2 diabody described here, may ultimately provide us with theability to predict which patients may benefit from these therapeuticstrategies.

Acknowledgments

Received 6/7/2004; revised 11/9/2004; accepted 11/22/2004.Grant support: ACS-IRG 92-027-09 (M.K. Robinson).The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

We thank the members of the Fox Chase Cancer Center Laboratory Animal Facilityfor their expert technical assistance.

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