DOTAGA-Trastuzumab. A New Antibody Conjugate Targeting

Article pubs.acs.org/bc

DOTAGA-Trastuzumab. A New Antibody Conjugate Targeting HER2/ Neu Antigen for Diagnostic Purposes Mathieu Moreau,† Olivier Raguin,‡ Jean-Marc Vrigneaud,§ Bertrand Collin,§ Claire Bernhard,† Xavier Tizon,‡ Frédéric Boschetti,∥ Olivier Duchamp,‡ François Brunotte,§ and Franck Denat*,† †

Institut de Chimie Moléculaire de l’Université de Bourgogne, UMR CNRS 6302, 21078 Dijon Cedex, France Oncodesign, 21076 Dijon Cedex, France § Service de médecine nucléaire, Centre Georges-François Leclerc, 21000 Dijon, France ∥ Chematech, 21000 Dijon, France ‡

ABSTRACT: Improved bifunctional chelating agents (BFC) are required for indium-111 radiolabeling of monoclonal antibodies (mAbs) under mild conditions to yield stable, target-specific agents. 2,2′,2″-(10-(2,6-Dioxotetrahydro-2H-pyran-3-yl)1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (DOTAGA-anhydride) was evaluated for mAb conjugation and labeling with indium-111. The DOTA analogue was synthesized and conjugated to trastuzumabwhich targets the HER2/neu receptorin mild conditions (PBS pH 7.4, 25 °C, 30 min) and gave a mean degree of conjugation of 2.6 macrocycle per antibody. Labeling of this immunoconjugate with indium-111 was performed in 75% yield after 1 h at 37 °C, and the proportion of 111In-DOTAGA-trastuzumab reached 97% after purification. The affinity of DOTAGA-trastuzumab was 5.5 ± 0.6 nM as evaluated by in vitro saturation assays using HCC1954 breast cancer cell line. SPECT/CT imaging and biodistribution studies were performed in mice bearing breast cancer BT-474 xenografts. BT-474 tumors were clearly visualized on SPECT images at 24, 48, and 72 h postinjection. The tumor uptake of [111In-DOTAGA]-trastuzumab reached 65%ID/g 72 h postinjection. These results show that the DOTAGA BFC appears to be a valuable tool for biologics conjugation.

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INTRODUCTION Monoclonal antibodies (mAb) are attractive molecules in the field of molecular imaging agent development, partly because of the development of personalized medicine.1,2 Indeed, tumorspecific antibodies have been widely used as delivery vectors to transport radiometal ions for cancer therapy.1,3 They have also been used for cancer imaging to determine antigen expression in the whole body, to evaluate therapeutic response, or to classify patients for specific treatments.4 Nevertheless, this field of research is in constant evolution in order to develop and improve labeling technique suitable to antibodies’ half-life, for both preclinical biodistribution studies and further possible applications in diagnosis and treatments. Although the specificity of mAbs makes them attractive targeting vectors, their large size (150 kDa) is sterically hindering: the slow accumulation rate limits their accretion in tumors. Besides, mAbs have a slow blood and hepatic clearance. Consequently, image acquisitions are usually done 24−72 h © 2012 American Chemical Society

postinjection when the optimal target to background ratio is reached (this requires the use of long half-life radioisotopes). To overcome these drawbacks, two options can be considered. The first one is to improve the specific activity of the radiolabeled mAb by increasing the radiolabeling yield. Such an improvement can be obtained by raising the incubation temperature, but this is not always a viable option as it can alter the mAb. Moreover, increasing the specific activity may lead to an increase of radiolysis. The second option is to use mAb fragments or smaller targeting agents such as affibodies or peptides, which are designed to clear more rapidly from blood and allow early image acquisition, but these moieties frequently show lower target accumulation and higher kidney uptake.5,6 Finally, it is still very difficult to determine if a small fragment Received: December 22, 2011 Revised: March 2, 2012 Published: April 23, 2012 1181

dx.doi.org/10.1021/bc200680x | Bioconjugate Chem. 2012, 23, 1181−1188

Bioconjugate Chemistry

Figure 1. Strategy for the construction of

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111

In-modified trastuzumab bioconjugate using DOTAGA-anhydride.

will be more efficient than a “native” mAb for molecular imaging, because each species shows benefits and disadvantages.7 As a consequence, and despite their drawbacks, mAbs are still highly valuable vectors in targeted radiopharmaceutical development, providing that stable complexes with long half-life radiometals are used for their labeling. The humanized mAb trastuzumab (Herceptin) is directed against the human epithelial growth factor receptor 2 (HER2/ neu), which is overexpressed in a variety of epithelial tumors, especially breast cancer.8,9 HER2/neu amplification is normally assessed ex vivo in a primary tumor biopsy by immunohistochemical staining for HER2 protein or by fluorescence in situ hybridization (FISH) to detect increased HER2/neu gene copy number.10 However, this invasive technique has shown discordance in HER2/neu expression between primary and metastatic breast cancer,11,12 and thus, it would be useful to have a more reliable imaging technique to assess HER2/neu phenotype in vivo in breast cancer lesions. Trastuzumab has been radiolabeled for preclinical studies and clinical trials, using a large variety of BFCs to chelate metallic radioisotopes such as indium-111, zirconium-89, or lutetium-177,13−20 whose halflives fit perfectly with the clearance of the antibody. During past years, polyamine carboxylate ligands such as diethylenetriaminepentaacetic acid (DTPA) and 1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-tetraacetic acid (DOTA) have been the BFCs of choice for trivalent radiometals.14,16 Conjugation of these chelates to the biomolecule can be accomplished by introduction of a primary amine targeting grafting function such as N-hydroxysuccinimidyl (NHS), isothiocyanate (NCS), or cyclic anhydride. One should note that the number of chelating agents conjugated to the antibody has to be controlled, since too high a degree of conjugation could induce modifications in the protein (such as chelating agent linked to an amino acid located at the binding site, modification of the structure) that reduce or inhibit its ability to bind its target. The DOTA analogues result generally in more stable radiometal bioconjugates than the DTPA, due to the so-called macrocyclic effect, coupled with the hepta- or octadenticity of these chelates.21−23 Among these BFCs, p-SCN-Bn-DOTA is the most commonly used, because of the high in vivo stability of the resulting radiometal complexes. If the isothiocyanate grafting function shows good stability in water, several hours are needed to get good yields when reacting with proteins. Besides, the synthesis of such molecules remains difficult and costly, and results in low yields.24,25 2-(4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pentanedioic acid (DOTAGA) is a DOTA derivative that leaves four acetate pendant arms intact and can be easily

synthesized in good yield.26,27 This macrocycle has shown good chelation properties with trivalent metallic cations such as Gd3+ or Y3+,28 and has been attached to biologic vectors for therapy and nuclear imaging.29 In this work, we describe the synthesis and the conjugation of the BFC 2,2′,2″-(10-(2,6-dioxotetrahydro-2H-pyran-3-yl)1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (DOTAGA-anhydride), bearing a cyclic anhydride grafting function (Figure 1). This BFC, which has been very recently grafted on gadolinium nanoparticles,30 was conjugated to trastuzumab. The yield of the conjugation reaction was determined by MALDI-TOF mass spectrometry. The DOTAGA-trastuzumab conjugate was then radiolabeled with indium-111, and its biological activity was evaluated by in vitro assays on HER2/ neu-expressing cell lines and in vivo in a model of human breast tumor-bearing mouse model.

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EXPERIMENTAL PROCEDURES Instrument and Methods. Matrix-assisted laser desorption ionization/time of flight (MALDI/TOF) mass spectra were obtained on a Bruker DALTONICS Ultraflex II (Bruker Daltonics, Bremen, Germany) mass spectrometer using sinapinic acid as matrix (Sigma-Aldrich, St. Quentin Falavier, France), at the “Plateforme d’Analyse Chimique et de Synthèse Moléculaire de l’Université de Bourgogne” (PACSMUB, Dijon, France). Radiochromatograms were carried out with a Raytest miniGITA-Star γ radiochromatograph (Raytest, Straubenhardt, Germany) or with a Bioscan AR-2000 radio-TLC Imaging Scanner (Bioscan Inc., Washington, DC). Reagents. Pure water (18.2 MΩ.cm−1, PURELAB Ultra, ELGA) was used throughout. When metal-free conditions were needed, glassware and plasticware were washed with 3 N HCl and thoroughly rinsed with deionized distilled water. Ammonium acetate buffer was prepared under metal-free conditions using Trace select ammonium acetate and acetic acid (Sigma-Aldrich, St. Quentin Falavier, France). Pyridine and acetic anhydride were purchased from Acros Organics (Illkirch, France) and used without further purification. DOTAGA was provided by CheMatech (Dijon, France), and trastuzumab (Herceptin; Roche, Welwyn Garden City, U.K.) was obtained through the “Centre de lutte contre le cancer G.-F. Leclerc” (Dijon, France). [ 111In]indium chloride (111 InCl 3, 370 MBq.mL−1 in 0.05 N HCl) was purchased from Covidien (Petten, The Netherlands) for in vitro studies and the first in vivo biodistribution study. For the second in vivo biodistribution study, [111In]indium chloride (111InCl3, 370 MBq.mL−1 in 0.05 N HCl) was purchased from Perkin-Elmer (Boston, MA, USA). The radiolabeling yield and the absence of free indium-111 in 1182



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Stability Testing of the Radiolabeled Conjugate. A DTPAchallenge test was performed to evaluate the labeling stability of 111 In-DOTAGA-trastuzumab. Two samples were diluted with a 2000-fold molar excess of a DTPA solution in PBS 0.1 M, pH 7.4, and were incubated for several days. Radio-ITLC controls were performed every day for 5 consecutive days. In Vitro Studies. Tumor Cells. HCC1954 and BT-474 cells (ATCC, Rockville, MD) are human breast adenocarcinoma cells31,32 that express the HER2/neu antigen at their surface. These cells were grown as adherent monolayer in RPMI1640 medium (Lonza, Belgium) supplemented with 10% fetal calf serum (Lonza). Tumor cells were harvested just prior to experiments. For in vitro experiments, cells were washed twice with PBS 0.1 M pH 7.4 (Lonza) and resuspended in PBS 0.1 M pH 7.4 containing 0.2% bovine serum albumin (BSA) and 0.02% azide (Sigma, France). For in vivo experiments, cells were washed twice with RPMI1640 medium and resuspended in a mixture of RPMI1640 and matrigel (BD Biosciences) (1/1, v/v). Before all experiments, the viability of the cells was assessed by trypan blue exclusion assay. In all cases, viability was greater than 90%. Quality Control. The fraction of 111In-DOTAGA-trastuzumab able to bind to HER2/neu (i.e., immunoreactivity) was determined by incubating trace amounts of 111In-DOTAGAtrastuzumab (7.0 × 10−9 M) with increasing concentrations of HCC1954 tumor cells (0.4 to 24 × 106 cells.mL−1) in a total volume of 0.2 mL for 1 h at 4 °C. At the end of the incubation period, cells were centrifuged, rinsed twice with ice-cold PBS 0.1 M pH 7.4, and then dissolved in 0.1 N NaOH. The radioactivity associated to cells (bound radioactivity, B) and an aliquot of the supernatant (total radioactivity, T) were counted with a scintillation γ counter (Cobra 4180, Perkin-Elmer Inc.) in order to calculate the bound-to-total ratios (B/T, expressed in %). Nonspecific binding was evaluated in the presence of a >100-fold excess unlabeled trastuzumab. Immunoreactivity was defined as the highest B/T% ratio that could be reached. Determination of DOTAGA-Trastuzumab Binding Affinity. The HER2/neu antigen density and the affinity constant (Kd) of the 111In-DOTAGA-trastuzumab were determined in radioligand binding saturation assays. Approximately 3 × 105 HCC1954 tumor cells were incubated with increasing concentrations of 111In-DOTAGAtrastuzumab (5.8 × 10−11 to 6.0 × 10−8 M, 100 MBq.mg−1) in a total volume of 0.2 mL for 1 h at 4 °C. At the end of the incubation period, cells were centrifuged, rinsed twice with icecold PBS 0.1 M pH 7.4, and then dissolved in 0.1 N NaOH. Radioactivity associated to cells was counted with a scintillation γ-counter. Nonspecific binding was evaluated in the presence of a >100-fold excess unlabeled trastuzumab. The dissociation constant and the number of binding sites were determined from experimental results by curve-fitting using GraphPad Prism v 5.04 for Windows (GraphPad Software, San Diego, California, USA, www.graphpad.com). Biodistribution and Tumor Imaging. All animal experiments were performed in compliance with guidelines governing such work and according to the ethical committee protocols.33 Female Balb/c nu/nu mice (6−8 weeks old, purchased from Charles River, France) were grafted by subcutaneous injection in the flank with 2 × 107 BT-474 human breast carcinoma cells. Three or five weeks after tumor cells injection, BT-474 tumor bearing-mice were given 25 μg 111In-DOTAGAtrastuzumab (13−15 [first experiment] or 3−3.5 MBq [second experiment]) by intravenous injection. In the second experi-

the labeled antibody were determined using ITLC-SG strips from Pall Gelman Sciences (Ann Arbor, MI, USA). Synthesis of DOTAGA-Anhydride. Two milliliters of acetic anhydride under nitrogen were added to a suspension of DOTAGA, 2HCl (1 g, 1.85 mmol) in pyridine (0.85 mL, 0.01 mol, 6 equiv), and the mixture was stirred at 65 °C for 18 h. After cooling down, the suspension was filtered and washed with acetic anhydride (10 mL), acetonitrile (15 mL), and diethyl ether (20 mL). The precipitate was dried under vacuum to afford DOTAGA-anhyride as a gray powder (0.95 g, 96%). The BFC was used without further purification. Mp: 108 ± 2 °C. HRMS (ESI): m/z calculated for C19H30N4O9 + Na+: 481.19050; found 481.18922. Conjugation of DOTAGA-Anhydride to Trastuzumab. Following the instructions of the manufacturer, trastuzumab was reconstituted in water for injection to obtain a 21 mg.mL−1 solution which was purified from other excipients (histidine, polysorbate, and α,α-trehalose) by ultrafiltration (Vivaspin 20 filter 10 kDa, Sartorius; 1 h at 2684 g). Conjugation was performed at a 20:1 DOTAGA-anhydride/ trastuzumab molar ratio. 36.2 μL of a 5 mg.mL−1 suspension of DOTAGA-anhydride (181.6 μg, 0.39 μmol, 20 equiv) in dry chloroform (Carlo Erba, Val de Reuil, France) were pipetted under ultrasonication and transferred to a polypropylene microtube. The chloroform was evaporated under a gentle stream of air. 88.4 μL of a solution of purified trastuzumab (33.92 mg.mL−1, 3 mg, 19.8 nmol, 1 equiv) in PBS 0.1 M, pH 7.4, (Fisher Scientific, Illkirch, France) was subsequently added. The solution was completed to 750 μL with PBS 0.1 M, pH 7.4, and gently mixed at 25 °C for 30 min. Unbound DOTAGA was then removed by ultrafiltration (Nanosep filter 30 kDa, Pall, 12 min at 8100 g, 4 °C). Conjugated trastuzumab was washed twice with 500 μL of PBS 0.1 M, pH 7.4, and the concentrated solution was diluted in 500 μL of ammonium acetate buffer 0.1 M pH 5.7. The purified immunoconjugate DOTAGAtrastuzumab was stored at 4 °C. Concentration of the antibody was determined by UV spectrophotometry at 280 nm. The molar extinction coefficient ε280Trastuzumab (218 500 M−1.cm−1) was calculated using ExPASy ProtParam tool (http://web. expasy.org/protparam). Radiolabeling. Radiolabeling for in Vitro Studies. 6 MBq of 111InCl3 was added to 40 μg of the immunoconjugate in 0.1 M ammonium acetate buffer, pH 5.7, to reach a buffer/HCl (from 111InCl3 solution) ratio of 1.5:1 resulting in a pH 5 solution. The solution was stirred in a thermomixer at 37 °C during 1 h. After incubation, 50 mM EDTA in 0.1 M ammonium acetate was added in order to chelate free indium111. The resulting 111In-EDTA was then removed by ultrafiltration, and the product was diluted in PBS 0.1 M, pH 7.4. Instant thin layer chromatographies (ITLC) were performed before and after ultrafiltration to determine the radiolabeling yield and to assess the absence of free indium-111. 1 μL of each solution was deposited on ITLC-SG strips at 2 cm of the bottom. The solvent (sodium citrate 0.1 M, pH 5) was allowed to rise to 10 cm from the bottom of the strips. Radiolabeled antibody remained at the application point while free indium-111 or 111In-chelates migrated with solvent front. The strips were then analyzed using a γ radiochromatograph. Radiolabeling for in Vivo Studies. The radiolabeling was performed as described for in vitro studies, using 125 MBq of 111 InCl3 and 125 μg of trastuzumab, and the solution was stirred for 3 h at 37 °C. After purification, the radiolabeled antibody was diluted in PBS 0.1 M, pH 7.4, for injection. 1183



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Figure 2. MALDI-TOF MS characterization of unmodified trastuzumab (blue) vs DOTAGA-trastuzumab (red). The difference of mass (δ = 1207) between the molecular peaks gives a degree of conjugation of 2.6 macrocycles per antibody.

ment, to assess the specificity of the targeting in vivo, a group of mice received the 111In-DOTA-trastuzumab in coinjection with excess (2500 μg) trastuzumab. SPECT/CT dual imaging was performed 24, 48, and 72 h after the injection of the radiolabeled conjugate using a NanoSPECT/CT small animal imaging tomographic γ-camera (Bioscan Inc., Washington, DC). Mice were anaesthetized with isoflurane (1.5−3% in air) and positioned in a dedicated cradle. CT and SPECT acquisitions were performed in immediate sequence. CT acquisitions (55 kVp, 34 mAs) were first acquired during 15−20 min, followed by helical SPECT acquisitions with 90−120 s per projection frame resulting in acquisition times of 45−60 min. Both indium-111 photopeaks (171 and 245 keV) were used with 10% wide energy windows. After the last image acquisition, animals were terminated. Blood, tumor, and organs were collected, and radioactivity in these samples was measured with a scintillation γ-counter. Data were then converted to percentage of injected dose and to percentage of injected dose per gram of tissue (the injected doses being corrected for subcutaneously injected material remaining in the animal tail). The CT and SPECT reconstructions were performed using image processing softwares provided by Bioscan Inc. Eventually, the SPECT/CT fusion image was obtained using the InVivoScope software (Bioscan Inc.). Each scan was then visually interpreted, and 3D regions of interest corresponding to the tumor and whole body were manually drawn in order to determine their radioactivity content. In vivo quantification was obtained by accurate calibration of the NanoSPECT/CT γcamera. Radioactivity contents from image analysis were

expressed in Bq, converted to percentage of injected dose, and compared to those determined by ex vivo counting.

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RESULTS AND DISCUSSION The aim of the study was to radiolabel trastuzumab (a humanized mAb) with indium-111 using DOTAGA-anhydride and to characterize this conjugate in vivo and in vitro in human breast cancer models expressing high levels of HER-2/neu antigen. Indium-111 was chosen because of its suitable physical characteristics. Its physical half-life (2.8 days) is highly compatible with the labeling procedures using macrocyclic chelating agent and with in vivo experiments with antibodies known to have long biological half lifes (several days). Besides, indium-111 emits two photons (171 and 245 keV) that are both detected for SPECT imaging. The coupling of DOTAGA-anhydride on trastuzumab was performed at a 20:1 molar excess of chelate to protein, using slight modifications of previous methods.14,34,35 Concentration of the immunoconjugate solution was deduced from absorbance at 280 nm, assuming that conjugation of DOTAGA on trastuzumab did not change significantly ε280Trastuzumab. The number of DOTAGA moieties on trastuzumab was determined by MALDI-TOF mass spectrometry. As shown in Figure 2, a difference of 1207 mass units was observed, which corresponds to an average of 2.6 DOTAGAs per antibody. Conjugation with an equimolar amount of anhydride chelate and antibody was not satisfactory probably due to an excessively fast hydrolysis of the anhydride function, resulting in a poor degree of conjugation. The large excess of macrocycle used for conjugation did not result in major modifications of the 1184



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binding capabilities. Despite the rapid hydrolysis of the anhydride function, cyclic anhydride remains a powerful tool for bioconjugation. Indeed, conjugation of DOTAGA-anhydride is achieved in only 30 min at room temperature and in PBS, while conjugation of molecules containing isothiocyanate or succinimidyl functions needs several hours and/or requires heating to reach a satisfactory yield.36,37 Moreover, the ringopening of the cyclic anhydride leads to a DOTA moiety bearing four acetate arms, which is a key point for transposition to yttrium-90 radioimmunotherapy.38−40 Radiolabeling was performed using two different procedures based on a previous protocol,41 depending on whether the radioimmunoconjugate was used for in vitro or in vivo studies. For in vitro studies, radiolabeling was carried out with a specific activity of 150 MBq.mg−1 of protein. The reaction was achieved in 1 h at 37 °C and gave a radiolabeling yield of 90% (ITLC) before purification. Reaction was quenched with excess EDTA, followed by another radio-ITLC quality control, and radiolabeling yield fell to 75%. This experiment demonstrated the crucial importance of quenching by EDTA to prevent nonspecific chelation of indium-111 on the antibody or incomplete coordination of the metal. Crude product was then purified by ultrafiltration to reach a proportion of 111InDOTAGA-trastuzumab >97% and was used immediately for in vitro experiments after dilution in PBS 0.1 M pH 7.4 containing 0.2% BSA and 0.02% azide. For in vivo studies, radiolabeling was carried out with a specific activity of 1 GBq.mg−1 of protein. The reaction was achieved in 3 h at 37 °C and gave a radiolabeling yield of 80%. In the same manner as for in vitro studies, radiolabeling yield fell down to 66% after quenching by EDTA. After purification by ultracentrifugation, the proportion of 111In-DOTAGAtrastuzumab was >97% and the product was diluted in PBS 0.1 M pH 7.4 and injected to animals within 30 min. The stability of 111In-DOTAGA-trastuzumab was evaluated by competition with 2000-fold molar excess of DTPA over 5 consecutive days as described in a previous study.6 The complex showed good stability with no significant transchelation observed. The proportion of 111In-DOTAGAtrastuzumab in the treated sample was >97% after 24 h and did not vary significantly during the following days, which was comparable to results obtained for p-SCN-Bn-DOTA or DOTA-NHS radiolabeling.23,42 The HCC1954 cell line was used for in vitro assays because it was easier to handle than the BT-474 cell line. For in vivo experiments, we chose the BT-474 model, as the growth of BT474 tumors is more homogeneous than HCC1954 tumors. Besides, the BT-474 tumor model is the gold standard for evaluation of molecules that target HER2/neu. The immunoreactivity was determined from a binding experiment of the 111In-DOTAGA-trastuzumab to increasing concentrations of HCC1954 tumor cells and was found to be 65% (Figure 3). The immunoreactivity of our DOTAGA trastuzumab conjugate appeared to be slightly lower than values reported for DOTA/DTPA trastuzumab conjugates bearing the chelating agent on lysine residues.14,18 The binding affinity constant was 5.5 ± 0.6 nM as determined in a saturation experiment (Figure 4), being similar to values published for DOTA/DTPA trastuzumab conjugates.43,44 The biological characteristics of our DOTAGA-trastuzumab conjugate are suitable for in vivo targeting applications. The ability of the 111In-DOTAGA-trastuzumab to target HER2/neu expressing tumors was evaluated in nude mice

Figure 3. Determination of the immunoreactivity of 111In-DOTAGAtrastuzumab (■, total binding; ▲, nonspecific binding). Trace amounts of 111In-DOTAGA-trastuzumab were incubated with increasing concentration of HCC1954 tumor cells. Nonspecific binding was evaluated with excess unlabeled trastuzumab. The fraction of radioactivity bound to cells (B/T%) was counted. The means of duplicate samples are plotted ± standard deviation (unless smaller than the point as plotted).

Figure 4. Dissociation constant of the 111In-DOTAGA-trastuzumab was determined in in vitro binding saturation assays (■, total binding; ▲, nonspecific binding; solid line, curve-fitting). Briefly, HCC1954 tumor cells were incubated with increasing concentration of the radiolabeled conjugate. Nonspecific binding was evaluated with excess unlabeled trastuzumab. The radioactivity associated to cells (expressed in counts per minute, cpm) was counted. The means of duplicate samples are plotted ± standard deviation (unless smaller than the point as plotted).

bearing subcutaneous human breast tumors. For the first experiment, mice were given 25 μg of the 111In-DOTAGAtrastuzumab (13−15 MBq). Tomographic SPECT/CT images were recorded 24, 48, and 72 h postinjection (Figure 5). Image analysis showed that tumor uptake reached 11.0 ± 5.6, 13.1 ± 7.6, and 14.0 ± 8.1%ID at 24, 48, and 72 h postinjection, respectively, demonstrating that the accretion of the 111InDOTAGA-trastuzumab had reached a plateau within 24 h. The 111 In-DOTAGA-trastuzumab was located mainly in the rim of the tumor, while a lower accumulation was seen inside, which may be due to necrosis in the center of the tumor. The effective half-life of the 111In-DOTAGA-trastuzumab was 53.5 ± 4.0 h from image analysis. The biological half-life was then found to be 268 ± 92 h, which appeared higher than that of a trastuzumab conjugate labeled with yttrium-86 (160 h).13 The observed difference could be related to the higher accretion in tumors of our conjugate (66.9 ± 0.9%ID/g at 72 h vs 7.4 ± 1.2%ID/g at 144 h). Also, the study from Palm et al.13 was conducted in mice bearing ovarian disseminated tumors with DTPA as chelating agent, whereas our own study involved mice 1185



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Table 1. Biodistribution Study of 111In-DOTAGATrastuzumab in balb/c Nude Mice Bearing Subcutaneous BT-474 Breast Human Tumorsa Tissue

%ID/g

Tumor Blood Heart Lungs Liver Spleen Kidneys Ovaries Gastrointestinal tract

66.9 10.0 3.6 5.6 4.3 6.9 6.3 8.0 1.8

± ± ± ± ± ± ± ± ±

0.9 3.5 0.5 0.5 0.5 1.2 0.2 3.7 0.2

tumor-to-organ ratio 7.8 18.8 11.9 15.7 10.0 10.6 9.6 38.1

± ± ± ± ± ± ± ±

4.3 2.6 1.0 1.8 1.9 0.2 3.4 3.5

a

Mice were grafted subcutaneously by injection of BT-474 tumor cells. Mice received a single IV injection of 111In-DOTAGA-trastuzumab and were terminated 72 h later. Blood, tumor, and organs were collected, and the radioactivity in these samples was determined. Tissue radioactivity is expressed as percentage injected dose/g tissue (%ID/g). Data are expressed as mean ± standard deviation (n = 4).

uptake in tumors was reduced from 65.2 ± 2.1 to 17.6 ± 2.4% ID/g, showing the specificity of HER2/neu targeting (Figure 6). Radioactivity persistence of the 111In-DOTAGA-trastuzumab in blood was not affected by the injection of the unlabeled trastuzumab (10.0 ± 2.7 vs 11.9 ± 0.7%ID/g). These results suggest that the coinjection of the excess of trastuzumab may

Figure 5. First in vivo experiment. SPECT/CT reconstructed images of a mouse bearing subcutaneous BT-474 breast human tumors (A, coronal slice; B, transverse slice). Mice received a single IV injection of 111 In-DOTAGA-trastuzumab (25 μg, 13−15 MBq) and images were recorded 24, 48, and 72 h later. All images have the same color scale. Image analysis showed that tumor uptake reached 11.0 ± 5.6%ID, 13.1 ± 7.6%ID, and 14.0 ± 8.1%ID at 24, 48, and 72 h postinjection.

bearing breast human tumors with a macrocyclic chelating agent. To our knowledge, no other publication describes the determination of the pharmacokinetic parameters of a trastuzumab conjugate in rodent obtained from image analysis. Extrapolation of these data to humans was not performed because assessment of organ-absorbed radiation doses was beyond the scope of this study. After the last imaging time point (i.e., 72 h postinjection), tumor, blood, and organs were collected and radioactivity in these samples was determined. Tumor uptake was 13.2 ± 8.2% ID, confirming image analysis (14.0 ± 8.1%ID). Radioactivity uptakes were 66.9 ± 0.9 and 10.0 ± 3.5%ID/g in tumor and blood, respectively, leading to a tumor-to-blood uptake ratio of 7.8 ± 4.3 (Table 1). For the liver and the spleen, involved in the catabolism of antibodies, radioactivity uptakes were 4.3 ± 0.5 and 6.9 ± 1.2%ID/g, respectively, and tumor-to-organ ratios were over 10. Radioactivity in kidneys (6.3 ± 0.2%ID/g) could result from a partial degradation of the 111In-DOTAGAtrastuzumab into small fragments excreted via the urinary pathway. Nevertheless, the fate of the 111In-DOTAGAtrastuzumab after injection in mice remains to be elucidated. Radioactivity uptakes in blood, liver, and kidneys were similar to those of other radiolabeled trastuzumab analogues,14,18,44 while an increased uptake (by a factor 3 to 4) was observed in tumors. This could be related to a high expression of HER2/ neu antigen in vivo in our tumor model or to tumor physiologic parameters such as vascular density or interstitial pressure. The specificity of 111In-DOTAGA-trastuzumab was assessed in the second experiment. Mice were given 25 μg of the 111InDOTAGA-trastuzumab (3−3.5 MBq) alone or coinjected with a 100-fold excess unlabeled trastuzumab. Animals were terminated 72 h postinjection, and blood and tumors were collected for radioactivity uptake determination. Radioactivity

Figure 6. Second in vivo experiment. Biodistribution study of the 111InDOTAGA-trastuzumab in Balb/c nude mice bearing subcutaneous BT-474 breast human tumors. Mice received a single IV injection of 111 In-DOTAGA-trastuzumab (25 μg, 3−3.5 MBq) with (blocking) or without (no blocking) excess unlabeled trastuzumab (2500 μg). Animals were terminated 72 h later just after SPECT/CT dual imaging. Blood and tumor were collected and the radioactivity in these samples was determined. Tissue radioactivity is expressed as percentage injected dose/g tissue (%ID/g). Data are expressed as mean ± standard deviation (n = 3). 1186



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have no influence on the kinetic parameters of the 111InDOTAGA-trastuzumab in blood. Similar data were obtained when the 100-fold excess unlabeled trastuzumab was administered 24 h before the injection of the 111 InDOTAGA-trastuzumab (data not shown).

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CONCLUSION The bifunctional chelating agent DOTAGA-anhydride was successfully linked to a humanized mAb. The DOTAGA conjugation and the labeling procedure with indium-111 did not alter the binding properties of the mAb. A high specific tumor targeting was shown in a model of human breast tumors overexpressing HER2/neu antigen. The DOTAGA-anhydride appears to be a valuable tool for biologic conjugation in the development of new radiolabeled tumor targeting agents suitable for diagnostic and/or therapeutic applications. The scope of use of this BFC is currently being extended to the labeling of peptides and bioengineered molecules such as antibody fragments, for both imaging and therapeutic purposes.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +33 (0)3 80 39 61 15; E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study is a part of Pharm’image® project. Support was provided by OSEO, the CNRS, the ‘‘Université de Bourgogne’’ and the ‘‘Conseil Régional de Bourgogne’’ through the 3MIM integrated project (‘‘Marquage de Molécules par les Métaux pour l’Imagerie Médicale’’).

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DOTAGA-Trastuzumab. A New Antibody Conjugate Targeting

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