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Basic Science Investigations |
Division of Diagnostic Imaging and Department of Surgical Oncology, University of Texas M.D. Anderson Cancer Center, Houston, Texas
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMaterials and MethodsResultsDiscussionConclusionReferences |
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-imaging properties of an antiepidermal growth factor receptor (EGFR) antibody, C225, conjugated with heterofunctional poly(ethylene glycol) (PEG) with 1 terminus of the polymer attached to a radiometal chelator, diethylenetriaminepentaacetic acid (DTPA). Methods: Two preparations of PEG-modified C225, one with 20% and the other with 60% amine substitution, were labeled with 111In. The conjugates, 111In-DTPA-PEG-C225, were injected intravenously into nude mice with EGFR-positive A431 tumors. For comparison, C225 directly labeled with 111In was also injected. In a competitive study, mice with A431 tumors were pretreated intravenously with 100-fold excess of native C225, followed by an injection of 111In-DTPA-PEG-C225 30 min or 20 h later. In addition, 111In-DTPA-PEG-C225 was injected into mice with EGFR-positive MDA-MB-468 tumors and EGFR-negative MDA-MB-435 tumors. Images were acquired at 5 min and at 2, 6, 24, and 48 h after injection of the radiotracers. Regions of interest (ROIs) were drawn on the computer images around the whole body, liver, muscle, and tumor. The counts per pixel in the tumor and normal tissues were calculated. At 48 h, the mice were killed and dissected. Blood, liver, muscle, and tumor samples were removed and the radioactivity of each sample was measured. Results: In A431 tumor xenografts, the tumor uptake of C225 modified with PEG was not significantly different than the uptake of unmodified 111In-DTPA-C225. Uptake in the liver, however, was reduced by 38%–45%, and the reduction increased with increasing degree of PEG substitution. Tumors of A431 and MDA-MB-468 xenografts were clearly visualized with 111In-DTPA-PEG-C225, whereas tumors of the MDA-MB-435 xenograft, which expresses low levels of EGFR, were not as readily visible. The tumor-to-blood ratios of 111In-DTPA-PEG-C225 in A431 and MDA-MB-468 xenografts were about 3 fold higher than in MDA-MB-435 xenografts. Blocking EGFR by pretreatment with native C225 significantly reduced the uptake of 111In-DTPA-PEG-C225 in the liver. The tumor-to-blood ratios in mice with A431 tumors were decreased 2.5–2.7 fold after pretreatment with a large excess of C225. Similar results were obtained with MDA-MB-468 tumor xenografts. In contrast, the tumor-to-blood ratios in mice with MDA-MB-435 tumor xenografts were not significantly different in C225-pretreated mice than in nonpretreated mice. Conclusion: These findings indicate that 111In-DTPA-PEG-C225 selectively localized to the tumors expressing high levels of EGFR. PEG-modification of C225 significantly reduced its liver uptake, resulting in improved visualization of EGFR-positive tumors. Using PEG as a linker between the monoclonal antibody and metal chelator is a useful strategy to optimize the imaging characteristics of antibody-based scintigraphic agents.
Key Words: epidermal growth factor receptor • monoclonal antibody C225 • 111In • imaging
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMaterials and MethodsResultsDiscussionConclusionReferences |
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The effectiveness of a therapeutic intervention targeted to EGFR will depend on its ability to detect and characterize EGFR-expressing lesions throughout the body. To achieve the best results for C225-directed immunotherapy, it is necessary to assess the presence of EGFR before treatment, to confirm in vivo tumor targeting by the antibody, and to evaluate the therapeutic response. The gold standard for the evaluation of EGFR status is immunohistologic examinations. This method, however, is limited by its invasiveness, the heterogeneity of tumors, and the inaccessibility of some tumors, particularly metastases, to biopsy procedures. A noninvasive imaging method using radiopharmaceuticals targeted to EGFR would be useful in characterizing the level of EGFR expression at the tumor sites and appropriately selecting patients for therapy directed against EGFR.
C225 attached to the radiometal chelator diethylenetriaminepentaacetic acid (DTPA) and labeled with 111In has been shown to localize specifically in tumors that overexpress EGFR (12). Phase I studies showed that 111In-DTPA-C225 was able to image squamous cell lung carcinomas expressing high levels of EGFR and metastases >1 cm in diameter. However, considerable hepatic radioactivity was seen as a result of nonspecific uptake (13). In a recent study, another anti-EGFR monoclonal antibody, mAb 528, was labeled with 111In through DTPA (14). Although radiolabeled mAb 528 localized in EGFR-positive MDA-MB-468 human tumor xenografts more efficiently than radiolabeled EGF peptide, both compounds had high uptake in the liver and kidney. The accumulation of radiolabeled anti-EGFR antibodies in a healthy liver and kidney could limit their clinical usefulness for the detection of primary tumors as well as metastases in the chest and abdominal area. To overcome this drawback and optimize the imaging properties of C225, we introduced flexible, linear poly(ethylene glycol) (PEG) molecules into C225 to reduce nonspecific interaction. In a previous study, we developed a simple, 1-step synthesis of DTPA-PEG-conjugated C225 from a key PEG intermediate that has metal chelator DTPA attached to the termini of the polymer chain. We also have shown that DTPA-PEG-C225 and 111In-labeled DTPA-PEG-C225 retained the biologic activity of C225 with reduced nonspecific binding (15). The objective of this study was to investigate the imaging properties of 111In-DTPA-PEG-C225 in EGFR-positive and EGFR-negative human tumor xenografts and to compare the results with those obtained with 111In-DTPA-C225.
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Materials and Methods |
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TOPABSTRACTINTRODUCTIONMaterials and MethodsResultsDiscussionConclusionReferences |
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-maleimidobutyryloxysuccinimide ester; Sigma Chemicals), the protecting group in DTPA-PEG-acetylthioacetate was removed by hydroxylamine (Sigma Chemicals) to give DTPA-PEG-SH. The product was purified by gel filtration chromatography using the Sephacryl S-200 HR column (Amersham Pharmacia Biotech, Piscataway, NJ). Two preparations of DTPA-PEG-C225 were used in the imaging study. In the 1:10 DTPA-PEG-C225 conjugate (where 1:10 denotes the molar ratio of C225 to maleimide), approximately 20% of the lysine amine groups in the antibody were activated. In the 1:30 DTPA-PEG-C225 conjugate, approximately 60% of the amines were derived. The degree of derivation was estimated based on analysis of unreacted amino groups in the modified antibodies using TNBS assay (picrylsulfonic acid; Sigma Chemicals). DTPA-C225 was prepared by reacting C225 directly with DTPA-dianhydride as described previously (16). The conjugate was purified by gel filtration on a PD-10 column (Amersham Pharmacia Biotech).
Radiolabeling
Generally, 40 µg of each antibody conjugate in 100 µL phosphate-buffered saline (PBS) was incubated with 13.0–14.8 MBq (350–400 µCi) 111InCl3 in 20 µL 1 mol/L sodium acetate buffer, pH 5.5 (Dupont–New England Nuclear, Boston, MA), at room temperature for 15 min. The resulting radiopharmaceutical was purified from free 111In by gel filtration on a PD-10 column using PBS as the eluent. Fractions of 0.5 mL were collected. The radioactivity of each fraction was measured by a radioisotope calibrator (CRC-12R; Capintec Instruments, Ramsey, NJ). The protein content in each fraction was determined using a protein assay kit (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer’s instructions. The fractions containing the protein were combined. The radiochemical purities of 1:10 111In-DTPA-PEG-C225, 1:30 111In-DTPA-PEG-C225, and 111In-DTPA-C225, determined by gel permeation chromatography, were 97%, 99%, and 99%, respectively.
Cell Lines
Human breast adenocarcinoma cell lines MDA-MB-468, MDA-MB-435, and human vulvar squamous carcinoma cell line A431 were maintained in 1:1 (v/v) Dulbecco’s modified Eagle’s medium/Ham’s F-12 mixture supplemented with 10% fetal bovine serum (Gibco Laboratories, Grand Island, NY) at 37°C in 5% CO2/95% air. Both MDA-MB-468 and A431 cell lines express high levels of EGFR (12). To determine the EGFR expression in MDA-MB-435 cells, 80 µg total protein from cell lysates of each sample were resolved by 10% sodium dodecylsulfate polyacrylamide gel electrophoresis. The proteins were electroblotted onto Immobilon-NC HAHY nitrocellulose membrane (Millipore Corporation, Bedford, MA). The membrane was blocked in 10% nonfat dry milk for 2 h at room temperature, incubated with monoclonal anti-EGFR antibody (Sigma Chemicals) at 4°C overnight, and treated with horseradish peroxidase-conjugated goat antimouse secondary antibodies (Jackson Immuno Research Laboratories, West Grove, PA) for 1 h at room temperature. A signal was detected using the ECL Western blotting detection system (Amersham Pharmacia Biotech).
Whole-Body Scintigraphy and Dissection Analysis
Female BALB/c mice with a nu/nu background were subcutaneously injected with A431, MDA-MB-468, or MDA-MB-435 cells (1 x 107/site) in the chest and the right hind limb. When the xenografts reached 8–10 mm in diameter, the mice were divided into groups of 3 each. The mice were anesthetized by an intraperitoneal injection of sodium pentobarbital (35 mg/kg), then administered with 10 µg 1:10 111In-DTPA-PEG-C225, 1:30 111In-DTPA-PEG-C225, or 1.85–3.7 MBq (50–100 µCi) 111In-DTPA-C225 through the tail vein. An Orbiter gamma camera (Siemens Gammasonics, Des Plaines, IL), equipped with a medium-energy collimator and Elscint Apex SPX-1 software (Siemens), was used for the -imaging. The mice were placed prone on the camera’s pinhole collimator. The images were acquired in a 128 x 128 matrix for 15 min, immediately after injection and at 2, 6, 24, and 48 h after injection of the radiotracer. Regions of interest (ROIs) were drawn on the computer images around the whole body, liver, muscle, and tumor. The counts per pixel in the tumor and normal tissues were calculated.
At 48 h after injection, the mice were killed and dissected. Blood samples were obtained by cardiac puncture, and samples of the liver, muscle, and tumor were removed from each animal. Radioactivity of each sample was measured with the Cobra Auto-gamma Counter (Packard, Downers Grove, IL). The percentage of the injected dose per gram of tissue (%ID/g tissue) was calculated for each sample.
Competition with Unlabeled C225
In separate experiments, native C225 at a dose of 1 mg per mouse was injected intravenously 30 min or 20 h before injection of 10 µg 1:30 111In-DTPA-PEG-C225 (1.85 MBq [50 µCi]) into mice with A431, MDA-MB-468, or MDA-MB-435 tumors. The mice were scanned at 24 and 48 h after injection of the radiotracer. The mice were killed at 48 h, and tumor, liver, muscle, and blood samples were collected for the quantification of radioactivity by a -counter.
Statistical Analysis
Statistical comparisons were calculated using a Student t test (P < 0.05).
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Results |
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TOPABSTRACTINTRODUCTIONMaterials and MethodsResultsDiscussionConclusionReferences |
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-scintigrams of mice bearing A431 tumors obtained at different intervals after intravenous injection of 111In-DTPA-C225, 1:10 111In-DTPA-PEG-C225, or 1:30 111In-DTPA-PEG-C225 are presented in Figures 1, 2, and 3, respectively. Immediately after injection of each radiotracer, images showed the highest activity in the central location, which is attributable to the cardiac blood pool, the liver, and the spleen. Although activity in the liver of mice injected with 111In-DTPA-C225 dominated the images at 24 and 48 h (Fig. 1), significant reduction of radioactivity in the liver was seen with PEG-modified C225 conjugates, particularly at 24 and 48 h (Figs. 2 and 3). Tumors at both inoculation sites (hind limb and chest) were visualized 24 h after injection with all 3 C225 radiotracers. However, only tumors in mice injected with 1:30 111In-DTPA-PEG-C225 were clearly seen 48 h after injection (Fig. 3).
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-Scintigrams of the mice taken at 6 and 24 h after the radiotracer injection showed markedly reduced liver uptake of 111In-DTPA-PEG-C225 in both groups. Although suppression of tumor activity was seen in mice injected with C225 20 h before radiotracer injection (Fig. 5), this effect was not obvious in scintigrams of mice given C225 30 min before injection of the radiotracer (data not shown).
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Imaging of Tumors as Function of EGFR Expression
To show that 111In-DTPA-PEG-C225 can localize specifically in tumors that overexpress EGFR, we used human tumor xenografts expressing different levels of EGFR in the -imaging study. Western analysis of A431, MDA-MB-468, and MDA-MB-435 confirmed that both A431 and MDA-MB-468 express high levels of EGFR, whereas MDA-MB-435 expresses negligible amounts of EGFR (data not shown). Although the quantification of EGFR by Western blot is not accurate, the assay allowed comparison of the intensities of the bands for the 3 cell lines and estimation of relative receptor expression. -Scintigrams of A431, MDA-MB-468, and MDA-MB-435 xenografts are presented in Figure 6. Both A431 and MDA-MB-468 tumors in the chest and hind limb sites showed substantial activity 24 h after injection of 1:30 111In-DTPA-PEG-C225. In contrast, less tumor activity was visualized in MDA-MB-435 tumors than in the other 2 tumor xenografts.
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Discussion |
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TOPABSTRACTINTRODUCTIONMaterials and MethodsResultsDiscussionConclusionReferences |
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EGFR-positive A431 cancer xenografts were imaged successfully using 1:10 DTPA-PEG-C225 or 1:30 DTPA-PEG-C225 labeled with 111In (Figs. 2 and 3). PEG modification significantly reduced liver uptake of C225, which was clearly visualized at later time points, that is, at 24 and 48 h after radiotracer injection (compare Fig. 1 with Figs. 2 and 3). The extent of reduction in liver activity was a function of the degree of PEG modification; 38% and 45% reductions were observed with 1:10 DTPA-PEG-C225 and 1:30 DTPA-PEG-C225, respectively. Modification with PEG had no effect (for 1:10 DTPA-PEG-C225) or only a mild effect (for 1:30 DTPA-PEG-C225) on tumor uptake of 111In-DTPA-PEG-C225. As a result, tumors were better delineated with 111In-DTPA-PEG-C225 conjugates than with 111In-DTPA-C225. Observations made by visualization of images were supported by quantification from sequential scintigraphic images. Reduction of hepatic uptake of PEG-modified C225 was clearly indicated by the increased values of tumor-to-liver ratio per pixel for both 1:10 and 1:30 DTPA-PEG-C225 derivatives at each time point (Fig. 4). This increase was mainly a function of decreased liver uptake rather than increased tumor uptake, because modification with PEG molecules did not cause significant changes in tumor uptake of C225. These data indicated that, with appropriate control of the degree of substitution, PEG-modified C225 was a more effective tumor-targeting agent than directly labeled C225 for receptor imaging of cancer.
Quantitative ROI analysis showed that the radioactivity in tumors relative to the whole body increased with time and reached the maximum at 24 h for all 3 C225 radiotracers (data not given). The similarity in the kinetics of tumor uptake for PEG-modified C225 and C225 without PEG suggests that the retention of DTPA-PEG-C225 conjugates and DTPA-C225 in tumors is mediated by the same mechanism. To show that the tumor uptake of 111In-DTPA-PEG-C225 is indeed mediated through its binding to EGFR, we pretreated the A431 tumor-bearing mice with 100-fold excess of unlabeled C225 to block the receptors on cell surfaces. Reduced tumor radioactivity was clearly visualized at 6 h and 24 h after radiotracer injection in mice pretreated with C225 20 h before the injection of 111In-DTPA-PEG-C225 (Fig. 5). The observations made with -scintigrams were supported by dissection analysis performed 48 h after the injection of 111In-DTPA-PEG-C225. The tumor-to-blood ratios decreased 2.5–2.7 fold after pretreatment with C225 either 30 min or 20 h before the injection of 111In-DTPA-PEG-C225. Pretreatment with C225 also markedly reduced the liver uptake of 111In-DTPA-PEG-C225, probably as a result of blockage of nonspecific phagocytosis. The higher tumor uptake of 111In-DTPA-PEG-C225 observed when native C225 was injected 30 min before the radiotracer (Table 1) was most likely the result of its higher blood activity caused by the blockage of liver uptake and elimination. The high level of blood activity of the radiotracer conjugate permitted a greater proportion of the injected dose of the radiotracer to diffuse into the interstitial space and bind to receptors on the cancer cells. It is interesting to note that the 20-h blocking group had decreased uptake in the blood, liver, muscle, and tumor when compared with the 30-min blocking group (Table 1), suggesting that C225 preinjection may affect the clearance of PEGylated C225. Clearly, more studies are needed to clarify the role of C225 preinjection on the biodistribution properties of PEGylated C225.
-Imaging studies with human tumor xenografts that express different levels of EGFR provide further support for the specific binding of PEG-modified C225 to the EGFR in tumors. Tumors in A431 and MDA-MB-468 xenografts that express high levels of EGFR were clearly visualized on -images, whereas tumors in MDA-MB-435 xenografts with low levels of EGFR expression were not as readily seen (Fig. 6). In fact, the tumor-to-blood ratios of 111In-DTPA-PEG-C225 in A431 and MDA-MB-468 xenografts were about 3 fold higher than in MDA-MB-435 xenografts. The values were significantly reduced by pretreatment with C225 in mice with A431 and MDA-MB-468 tumors but not in mice with MDA-MB-435 tumors (Fig. 7). These results suggest that PEG-modified C225 retained its receptor-binding affinity in vivo and that its uptake in the tumor is mediated through EGFR.
The ability of 111In-DTPA-PEG-C225 to differentiate the level of EGFR expression in vivo suggests that this radioligand may be useful in characterizing the EGFR status of solid tumors. It is important to note that C225 does not react with mouse EGFR. Therefore, reduction in nonspecific liver uptake may not be sufficient to reduce liver radioactivity of PEGylated C225 in humans, where specific uptake of C225 in the liver by hepatic EGFR is expected. Divgi et al. (13) have shown that with 111In-DTPA-labeled C225, nearly all radioactivity was imaged in the liver if the C225 dose was <40 mg per patient. Improved tumor visualization was achieved only at higher doses of C225 when specific uptake is suppressed by saturating the hepatic EGFR (13). It is, therefore, possible to achieve further reduction in the liver uptake of PEGylated antibody by pretherapy with native antibody. Obviously, these points need to be addressed in a clinical setting.
In this study, C225 was used as a model compound. Whether PEGylation causes changes in potential immunogenicity has not been determined. Thus far, clinical experiences with C225 show little human antimouse antibody response with this antibody. Therefore, PEGylation may not benefit C225 with regard to reduction of immunogenicity. However, PEGylation should have a significant impact on immunogenicity of other proteins or nonhumanized antibodies intended for imaging purposes.
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Conclusion |
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TOPABSTRACTINTRODUCTIONMaterials and MethodsResultsDiscussionConclusionReferences |
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-Imaging and distribution studies with a monoclonal antibody directed against EGFR indicate that the tumor uptake of a PEG-modified antibody was mediated through the receptors. The level of tumor localization in vivo is different in tumors with a different amount of receptors, suggesting that noninvasive characterization of receptor expression is possible. Clinical studies of 111In-DTPA-PEG-C225 are needed to clarify the effect of PEGylation on the specific liver uptake of the C225 antibody.
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ACKNOWLEDGMENTS |
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-scintigraphic studies and Kathryn Hale for editorial assistance. Human breast adenocarcinoma cell lines MDA-MB-468, MDA-MB-435, and human vulvar squamous carcinoma cell line A431 were obtained from Dr. Zhen Fan. This study was supported in part by the John S. Dunn Foundation. |
FOOTNOTES |
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For correspondence or reprints contact: Chun Li, PhD, Division of Diagnostic Imaging, Box 59, University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030.
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TOPABSTRACTINTRODUCTIONMaterials and MethodsResultsDiscussionConclusionReferences |
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