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Experimental Therapeutics, Molecular Targets, and Chemical Biology |
vß3
1 The Molecular Imaging Program at Stanford, Department of Radiology and Bio-X Program, Stanford University School of Medicine, Stanford, California and 2 MedImmune, Inc., Gaithersburg, Maryland
Requests for reprints: Xiaoyuan Chen, The Molecular Imaging Program at Stanford, Department of Radiology and Bio-X Program, Stanford University School of Medicine, 1201 Welch Road, P095, Stanford, CA 94305-5484. Phone: 650-725-0950; Fax: 650-736-7925; E-mail: shawchen{at}stanford.edu.
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vß3, is in clinical trials for cancer therapy. In vivo imaging using AbegrinTM-based probes is needed for better treatment monitoring and dose optimization. Here, we conjugated AbegrinTM with macrocyclic chelating agent 1,4,7,10-tetra-azacylododecane N,N',N'',N'''-tetraacetic (DOTA) at five different DOTA/AbegrinTM ratios. The conjugates were labeled with 64Cu (half-life = 12.7 hours) and tested in three human (U87MG, MDA-MB-435, and PC-3) and one mouse (GL-26) tumor models. The in vitro and in vivo effects of these 64Cu-DOTA-AbegrinTM conjugates were evaluated. The number of DOTA per AbegrinTM varied from 1.65 ± 0.32 to 38.53 ± 5.71 and the radiolabeling yield varied from 5.20 ± 3.16% to 88.12 ± 6.98% (based on 2 mCi 64Cu per 50 µg DOTA-AbegrinTM conjugate). No significant difference in radioimmunoreactivity was found among these conjugates (between 59.78 ± 1.33 % and 71.13 ± 2.58 %). Micro-positron emission tomography studies revealed that 64Cu-DOTA-AbegrinTM (1,000:1) had the highest tumor activity accumulation (49.41 ± 4.54% injected dose/g at 71-hour postinjection for U87MG tumor). The receptor specificity of 64Cu-DOTA-Abegrin was confirmed by effective blocking of MDA-MB-435 tumor uptake with coadministration of nonradioactive Abegrin. 64Cu-DOTA-IgG exhibited background level tumor uptake at all time points examined. Integrin vß3-specific tumor imaging using 64Cu-DOTA-AbegrinTM may be translated into the clinic to characterize the pharmacokinetics, tumor targeting efficacy, dose optimization, and dose interval of AbegrinTM and/or Abegrin conjugates. Chemotherapeutics or radiotherapeutics using AbegrinTM as the delivering vehicle may also be effective in treating integrin vß3-positive tumors. (Cancer Res 2006; 66(19): 9673-81) |
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and ß subunits, are involved in a wide range of cell-ECM and cell-cell interactions (4–6). Inhibition of v integrin activity by monoclonal antibodies (mAb), cyclic RGD peptide antagonists, and peptidomimetics has been shown to induce endothelial cell apoptosis, to inhibit angiogenesis, and to increase endothelial monolayer permeability (7, 8). The vß3 integrin, which recruits and activates matrix metalloproteinase-2 and plasmin to degrade the basement membrane and interstitial matrix (9), is significantly up-regulated on endothelium during angiogenesis but not in quiescent endothelium (4, 5, 7). Integrin vß3 is also expressed in melanoma, late-stage glioblastoma, ovarian, breast, and prostate cancer cells (10), where it can potentiate metastasis by facilitating cancer cell invasion and movement across blood vessels.
LM609, a mouse anti-human integrin vß3 mAb, which cross-reacts with vß3 originated from rabbits, chicken, and hamsters but not from mice and rats, was found to be able to immunoprecipitate integrin vß3 from M21 human melanoma cells (11). Recognizing the heterodimer as one entity, LM609 is much more specific and superior to other mAbs, which recognizes either the v or ß3 subunit. However, being a murine mAb, inefficient interactions with human immune effector cells and the short serum half-life (t1/2) of LM609 caused by its immunogenicity in humans severely limits the therapeutic potential of LM609 when chronic administrations are needed (12). For these reasons, LM609 was humanized and later affinity matured as reported previously (13). The humanized version, named VitaxinTM or Vitaxin I (later named MEDI-523) was used in several phase I clinical trials (13–15). Radioimaging of tumor vasculature using this early version of VitaxinTM labeled with 99mTc was unsuccessful due to the instability of the 99mTc labeling in vivo (16). The affinity matured version, now called AbegrinTM (also called VitaxinTM or MEDI-522), is also in clinical trials. In phase I studies of Abegrin, prolonged stable disease was observed in several patients with renal cell carcinoma and an effect on tumor perfusion was correlated with treatment, although no significant toxicity or immune response was noted at the dose levels tested (17). In 2003, MedImmune, Inc. (Gaithersburg, MD), who licensed VitaxinTM and designated it as AbegrinTM for clinical development (18), announced the initiation of phase II clinical trials in prostate cancer and melanoma. Recently, the company announced the initiation of phase III for patients with metastatic melanoma based on promising results from their phase II trial with AbegrinTM.
We and others have reported small molecule, peptide, protein, and antibody-based probes for fluorescence (19, 20), magnetic resonance (21, 22), ultrasound (23), single-photon emission computed tomography (24, 25), and positron emission tomography (PET; refs. 26–37) imaging of integrin vß3 expression in vivo. To date, most of integrin vß3 targeted PET studies have been focused on the radio labeling of arginine-glycine-aspartic acid (RGD) peptide antagonists of integrin vß3 due to their high-binding affinity (31, 33, 37, 38). [18F]Galacto-RGD has been tested in healthy volunteers and cancer patients (33, 35). Very recently, we reported that [18F]FRGD2 can be used to quantify tumor integrin vß3 expression level in vivo with static PET scans in xenograft tumor models (29, 37). Both peptide and antibody-based imaging can help in evaluating integrin vß3 expression level. RGD peptide-based probes are useful in planning and monitoring peptide-based therapeutics due to the similar pharmacokinetics, whereas antibody-based tracer is more relevant to antibody-based therapy. The relatively longer t1/2 of 64Cu [t1/2 = 12.7 hours; ß+ = 655 keV (17%); ß– = 573 keV (39%)] is well suited for the mAb labeling and imaging. To date, no imaging using AbegrinTM has been reported. Here, we report the first 64Cu-labeled AbegrinTM through 1,4,7,10-tetra-azacylododecane N,N',N'',N'''-tetraacetic (DOTA) chelator and the in vitro as well as in vivo characterizations of the resulting 64Cu-DOTA-AbegrinTM.
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Materials and Methods |
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DOTA conjugation and radiolabeling. DOTA was activated by EDC and SNHS at pH 5.5 for 30 minutes with a molar ratio of 10:5:4 (DOTA/EDC/SNHS). Without purification, the DOTA-N-hydroxysulfosuccinimidyl (OSSu) was cooled to 4°C and added to Abegrin at five different molar ratios (DOTA-OSSu/AbegrinTM of 20:1, 50:1, 100:1, 200:1, and 1,000:1). The reaction mixture was adjusted to pH 8.5 with 0.1 N of NaOH and allowed to incubate for overnight at 4°C. The DOTA-Abegrin conjugates were then purified by PD-10 column and concentrated by Centricon filter (Millipore, Bedford, MA), and the final concentration was measured based on UV absorbance at 280 nm using unconjugated AbegrinTM of known concentrations as standard. 64CuCl2 (2 mCi) was diluted in 300 µL of 0.1 mol/L sodium acetate buffer (pH 6.5) and added to the DOTA-AbegrinTM conjugates (25 µg DOTA-AbegrinTM/mCi of 64Cu). The reaction mixture was incubated for 1 hour at 40°C with constant shaking. The 64Cu-DOTA-AbegrinTM conjugates were then purified by PD-10 column using PBS as the mobile phase. The radioactive fractions containing 64Cu-DOTA-AbegrinTM was collected and passed through a 0.2-µm syringe filter for further in vitro and in vivo experiments.
Number of DOTA per AbegrinTM and immunoreactivity. The average number of DOTA chelators per AbegrinTM antibody was determined using a previously reported procedure with slight modifications (39). Briefly, a defined amount of nonradioactive CuCl2 (80-fold excess of DOTA-AbegrinTM) in 40 µL 0.1 N sodium acetate (NaOAc) buffer (pH 6.5) was added to 1.0 mCi 64CuCl2 in 20 µL 0.1 N NaOAc buffer, and 20 µg of each DOTA-AbegrinTM conjugate in 90 µL 0.1 N NaOAc buffer were added to the above carrier-added 64CuCl2 solution. The reaction mixture was incubated with constant shaking at 40°C for 1 hour. The 64Cu-DOTA-AbegrinTM was purified using PD-10 column and the radiolabeling yield was calculated. The number of DOTA per AbegrinTM was calculated using the following equation: number of DOTA per AbegrinTM = moles (Cu2+) x yield/moles (DOTA-AbegrinTM). The results were expressed as mean ± SD (n = 3). An isotype control IgG (against a bacterial antigen with no cross-reaction with human or mouse integrin vß3; supplied by MedImmune) was conjugated with DOTA under the same condition and the number of DOTA per IgG was also determined as described above.
The immunoreactivity of each 64Cu-DOTA-AbegrinTM conjugate (with different number of DOTA per AbegrinTM) was determined by incubating with integrin vß3-positive U87MG cells in suspension culture under conditions of antigen excess (n = 3; ref. 40). The immunoreactivity was calculated as follows: immunoreactivity = bound activity on cells/total added activity x 100%.
Cell lines and cell integrin receptor assay. Four cell lines were used for in vitro and in vivo experiments. U87MG human glioblastoma, MDA-MB-435 human breast cancer carcinoma, and PC-3 human prostate adenocarcinoma cell lines were obtained from American Type Culture Collection (Manassas, VA). The GL-26 mouse glioblastoma cell line was kindly provided by Dr. Victor K. Tse (Department of Neurosurgery, Stanford University, Stanford, CA). All culture media were obtained from Invitrogen Corp. (Carlsbad, CA). U87MG cells were grown in DMEM (low glucose), MDA-MB-435 cells were grown in Leibovitz's L-15 Medium, PC-3 cells were grown in F-12K nutrient mixture (Kaighn's Modification), and GL-26 cells were grown in DMEM (high glucose). All cell lines were cultured in medium supplemented with 10% (v/v) fetal bovine serum at 37°C in a humidified atmosphere with 5% CO2, except for MDA-MB-435, which was cultured without CO2. The procedure of cell integrin receptor assay has been reported earlier (29, 41).
Flow cytometry. Cells were collected and washed with PBS and incubated with AbegrinTM (20 µg/mL) in PBS supplemented with 1% bovine serum albumin (BSA) for 30 minutes at 4°C. After washing with PBS containing 1% BSA, the cells were incubated with FITC donkey anti-human IgG (1:100; Jackson ImmunoResearch Laboratories, Inc., West Grove, CA) for 30 minutes at 4°C. The cells were washed again, resuspended in PBS, and analyzed using a LSR model 1A analyzer (Becton Dickinson, Heidelberg, Germany) and FlowJo analysis software (Tree Star, Inc., Ashland, OR).
Animal models. Animal procedures were done according to a protocol approved by Stanford University Institutional Animal Care and Use Committee. The MDA-MB-435 breast cancer model was established by orthotopic injection of 5 x 106 cells (in 50 µL PBS) into the left mammary fat pad. The U87MG, PC-3, and GL-26 tumor models were obtained by s.c. injection of the corresponding cells (5 x 106 in 50 µL PBS) into the right front leg of the mice (male mice were used for PC-3 tumor). The mice were subjected to microPET imaging and biodistribution studies when the tumor volume reached 200 to 500 mm3 (2 weeks after inoculation for GL-26 tumor model; 3-4 weeks after inoculation for U87MG, MDA-MB-435, and PC-3 tumor models).
MicroPET imaging studies. PET imaging of tumor-bearing mice was done on a microPET R4 rodent model scanner (Concorde Microsystems, Knoxville, TN). The scanner has computer controlled vertical and horizontal bed motion, with an effective axial field of view (FOV) of 7.8 cm and transaxial FOV of 10 cm. The tumor-bearing mice were imaged in prone position in the microPET scanner. The mice were injected with 200 to 300 µCi 64Cu-DOTA-AbegrinTM via the tail vein, anesthetized with 2% isoflurane, and placed near the center of the FOV where the highest image resolution and sensitivity is available. Three- to five-minute static scans were done before 24-hour postinjection, whereas 10- to 20-minute static scans were carried out after 24-hour postinjection For each microPET scan, three-dimensional regions of interests (ROI) were drawn over the tumor, heart, liver, kidneys, and muscle on decay-corrected whole-body coronal images. The average radioactivity concentration (accumulation) within a tumor or an organ was obtained from mean pixel values within the ROI volume, which were converted to counts per milliliter per minute by using a conversion factor. Assuming a tissue density of 1 g/mL, the counts per milliliter per minute were converted to counts per gram per minute and then divided by the injected dose (ID) to obtain an imaging ROI-derived percent injected dose per gram of tissue (% ID/g) of tissue. A mouse bearing a MDA-MB-435 tumor was also imaged using 64Cu-DOTA-AbegrinTM coinjected with 2 mg of unconjugated Abegrin. As a control experiment, 64Cu-DOTA-IgG (200-300 µCi) was injected into U87MG tumor-bearing mice and microPET scans were done as described above.
Biodistribution studies. Female nude mice bearing MDA-MB-435 tumors were injected with about 20 µCi 64Cu-DOTA-AbegrinTM. The mice were sacrificed and dissected at 18-, 44-, and 68-hour postinjection. Blood, tumor, major organs, and tissues were collected and wet weighed. The radioactivity in the tissues was measured using a gamma counter (Packard, Meriden, CT). The results were presented as %ID/g. For each mouse, the radioactivity of the tissue samples was calibrated against a known aliquot of the injectate and normalized to a body weight of 20 g. Values are presented as mean ± SD for a group of three animals.
Radiation dosimetry extrapolation to human. Estimated human dosimetry was calculated from microPET imaging results on Sprague-Dawley female rats (Harlan) injected with 64Cu-DOTA-AbegrinTM. The rats were scanned at two bed positions to cover the whole body and ROI analysis were carried out on major organs. Time-activity curves were generated from the mean values obtained in rats for each organ of interest. We then calculated source organ residence times for the human model by integrating a monoexponential fit to the experimental biodistribution data for major organs (heart, lung, liver, kidneys, and spleen) and the whole body. The source organ residence times obtained forthwith were used with a standard quantitation platform organ level internal dose assessment (Vanderbilt University, Nashville TN; ref. 42).
Immunofluorescence staining. Frozen tumor sections were warmed to room temperature, fixed with ice-cold acetone for 10 minutes, and dried in the air for 30 minutes. The sections were blocked with 10% donkey serum for 1 hour at room temperature. For CD31 and human integrin vß3 double staining, the sections were incubated with rat anti-mouse CD31 (1:100; BD Biosciences, San Jose, CA) and AbegrinTM (100 µg/mL) for 1 hour at room temperature. After incubating with Cy3-conjugated donkey anti-mouse secondary antibody (1:200; Jackson ImmunoResearch Laboratories) and FITC-conjugated donkey anti-human secondary antibody (1:200), the tumor sections were examined under the microscope (Carl Zeiss Axiovert 200M, Carl Zeiss, Thornwood, NY). For CD31 and mouse integrin ß3 double staining, hamster anti-mouse ß3 (1:100) and FITC-conjugated goat anti-hamster secondary antibody (1:400; Jackson ImmunoResearch Laboratories) were used.
Statistical analysis. Quantitative data were expressed as mean ± SD. Means were compared using one-way ANOVA and Student's t test. Ps < 0.05 were considered statistically significant.
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vß3 expression level. Fluorescence-activated cell sorting (FACS) analysis using Abegrin as the primary antibody against integrin vß3 clearly showed that U87MG, PC-3, and MDA-MB-435 cells are integrin vß3 positive (Fig. 2A
). Although GL-26 cells are mouse integrin vß3 positive (where anti-mouse ß3 mAb was used as the primary antibody; Fig. 2A, bottom, right), it is not recognized by Abegrin, which showed that AbegrinTM is human integrin vß3 specific and does not cross-react with mouse vß3. Next, we quantified the vß3 integrin expression level of the four cell lines through competitive cell-binding assay using 125I-echistatin as radioligand. The density of integrin vß3 on the cell surface follows the order U87MG > MDA-MB-435 > GL-26 
PC-3 (Fig. 2B). Note that echistatin binds to integrin vß3 as one entity but not integrin v or integrin ß3 alone and it recognizes both human and mouse integrin vß3.
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vß3 expression level. The localization of 64Cu-DOTA-AbegrinTM in all four tumor models was evaluated by multiple time point microPET imaging. Selected coronal images at different time points postinjection were shown in Fig. 3A
. At 4-hour postinjection, the tumor was already visible. The tumor signal at this early time point may be due to both specific targeting and nonspecific targeting because of the enhanced permeability and retention (EPR) effect, as tumors have abnormal and leaky vasculature and lack lymphatic drainage. High tumor activity accumulation was observed as early as 17-hour postinjection The tumor signal of U87MG-, PC-3-, and MDA-MB-435-bearing mice increased over time (Fig. 3B), indicating specific binding between Abegrin and human integrin vß3. The tumor signal intensity was similar for MDA-MB-435 and PC-3 tumors, whereas the U87MG tumor uptake is much higher (Fig. 1B), agreeing with the cell integrin receptor level. The fact that the GL-26 tumor signal intensity reached a peak at 24-hour postinjection and steadily dropped afterwards is indicative of passive targeting due to the EPR effect, corroborating with the FACS analysis data because Abegrin does not cross-react with mouse integrin vß3. As all tumor models in this study are murine xenografts, the integrin vß3 expressed on the tumor vasculature is of mouse origin and is not recognized by AbegrinTM. The uptake profile in the heart, liver, kidneys, and muscle were similar in all four tumor models.
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vß3, and mouse integrin ß3. Only the images of U87MG and GL-26 tumors were shown. As can be seen from Fig. 4A
, the CD31 and mouse ß3 staining colocalizes very well, indicating that the tumor vasculature expresses mouse integrin vß3. For the GL-26 tumor, both the tumor vasculature and the GL-26 tumor cell express mouse integrin vß3 as evidenced by the staining (anti-mouse ß3 antibody stains both the tumor cells and the tumor vasculature). When using Abegrin as the primary antibody, there is no colocalization with CD31 for the U87MG tumor because only the human tumor cells but not the mouse tumor vasculature are recognized by AbegrinTM (Fig. 4B). For GL-26 tumor, no Abegrin staining was visible as neither the tumor vasculature nor the tumor cells express human integrin vß3. Ex vivo staining experiments again confirmed the human integrin vß3-specific binding of Abegrin.
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vß3-specific binding of 64Cu-DOTA-AbegrinTM. Biodistribution of 64Cu-DOTA-AbegrinTM was also carried out in the MDA-MB-435 tumor model at 18-, 44-, and 68-hour postinjection (Fig. 5B). It can be seen that the major tracer clearance is through the liver and the spleen. All other organs had background level uptake of the tracer, providing good tumor contrast of the tracer. The MDA-MB-435 tumor uptake was 4.54 ± 0.39 %ID/g based on biodistribution studies and 5.63 ± 1.16 %ID/g based on microPET studies at 18-hour postinjection (P = 0.17). At 46-hour postinjection, the MDA-MB-435 tumor uptake was 5.40 ± 0.75 %ID/g based on biodistribution experiments and 7.04 ± 1.47 %ID/g based on microPET studies, respectively (P = 0.16). Comparing the quantification results of the tracer uptake between microPET and biodistribution studies, the uptake in all the other organs follow similar pattern, indicating that the quantification methods used for the noninvasive microPET study is a true reflection of the tracer uptake in vivo.
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Discussion |
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vß3, exhibits high integrin vß3 specificity in vitro and in vivo. As AbegrinTM is currently in phase I/II clinical trials and soon to enter phase III, PET imaging is critical in early and sensitive lesion detection, patient selection for clinical trials based on in vivo integrin expression quantification, better treatment monitoring and dose optimization, and elucidation of the mechanisms of treatment efficacy underlying integrin signaling. We have optimized the DOTA/AbegrinTM ratio for better in vivo targeting and maximized tumor uptake. As the equivalence of DOTA used for conjugation reaction increases, the number of DOTA residues per AbegrinTM, the specific activity of the tracer, as well as the absolute tracer uptake in the tumor all increases. Because the difference in immumoreactivity is minimal between the five tracers, the conjugate with the most DOTA residues per AbegrinTM is more suitable for future clinical translation. The fact that DOTA conjugation of AbegrinTM does not deteriorate the receptor affinity of the antibody indicates that the accessible lysine residues are mostly located away from the antigen-binding site. High specific activity may also play a role here. It has been reported previously that a dendrimer-mAb conjugate with high specific activity exhibited higher tumor uptake (44). Although not tested, we envision that Abegrin coupled to dendrimeric DOTAs may give comparable or potentially even higher tumor uptake. Due to the relatively high molecular weight of the mAb (150 kDa), the tracer exhibited mainly hepatic clearance. Intact IgG is designed to remain in the bloodstream for many weeks, which is of considerable advantage when acting in the natural setting of removing foreign entities. The serum t1/2 of 64Cu-DOTA-AbegrinTM in mouse is about 12 to 24 hours based on microPET studies, much shorter than the t1/2 in humans (5-8 days; ref. 17), presumably due to the much faster metabolic rate of mouse compared with human. In this study, we followed the tracer uptake for about 72 hours (almost six t1/2 of 64Cu). Longer term monitoring may be necessary for future studies and 89Zr with a t1/2 of 79.3 hours can be used. It has been reported that 89Zr imaging might be a good reflection of 90Y distribution (45), which may shed light on future radioimmunotherapy studies using 90Y-AbegrinTM conjugates.
For antiangiogenic treatment using Abegrin alone or in combination with other treatment modalities, the long circulation t1/2 is advantageous because less administration is needed to maintain serum levels of the antibody. The very high tracer uptake in the tumor (up to 49.41 ± 4.54 %ID/g in the U87MG tumor) along with minimal signal in the nontargeting organs warrants future radioimmunotherapy studies using DOTA-AbegrinTM, as therapeutic radioisotopes (67Cu, 177Lu, 90Y, etc.) can be incorporated using a similar strategy. Previous report has shown prominent expression of integrin vß3 by glioma cells and vasculature in cancer patients, especially in the periphery of high-grade gliomas (46). Systematic investigation of vß3 expression on tumor-associated vessels in cancer patients also revealed that a considerable number of colon, pancreas, lung, and breast carcinoma lesions have many vß3-expressing vessels that could be targets for anti-integrin vß3 therapy (47). Clinical translation of 64Cu-DOTA-AbegrinTM will be critical for the maximum benefit of Abegrin-based anticancer agents as imaging can provide a straightforward and convenient way to monitor the biological changes at the molecular level in vivo.
Abegrin is a humanized antibody, which recognizes only human but not murine integrin vß3. Both the microPET imaging studies and the immunofluorescence staining confirmed that AbegrinTM does not bind to mouse tumor vasculature. Therefore, the difference in tumor uptake is a good reflection of the integrin vß3 expression level of different tumor cells. For GL-26 tumor, which only expresses murine integrin vß3, the tracer uptake is mostly nonspecific. As can be seen from Fig. 3B, the tracer uptake in the tumor reached maximum at about 24-hour postinjection and dropped steadily afterwards. This is characteristic of passive targeting due to the EPR effect of the tumor. For all the other tumors, the tracer uptake increased or remained steady over time, which is characteristic of specific targeting. In this study, the passive targeting of GL-26 is quite prominent and, at its peak, the uptake is even higher than integrin vß3-positive tumors MDA-MB-435 and PC-3. PET imaging of other tumors (e.g., C6 rat glioma) that are human/mouse integrin vß3 negative revealed a similar tracer uptake profile as the GL-26 tumor (data not shown), although the time point of maximum tumor uptake varied between these tumors because of the difference in vascular density and leakiness. The tracer uptake, tracer clearance, and the pharmacokinetics of 64Cu-DOTA-AbegrinTM are quite different from our previously reported RGD peptide-based imaging (29, 31, 48). The passive targeting of this antibody may provide a more general use in cancer therapy as most tumors have leaky vasculature, which may result in more uptake of AbegrinTM-based agents and greater efficacy.
We realize that the pharmacokinetics of 64Cu-DOTA-AbegrinTM in the present study may not be a true mimicry of the clinical situation because Abegrin does not bind mouse integrin vß3. This may also be partially responsible for the faster tracer clearance. For future studies, other rodent models, such as hamsters or primates (AbegrinTM cross-reacts with hamster and certain primate integrin vß3), may be used to obtain dosimetry instead of mice and rats. Transgenic mouse models that express both human and mouse integrin vß3 may also be relevant. Due to both the heterogeneity of the tumor itself and the weaker tissue penetration of the mAb-based tracer because of its large molecular weight, the tracer uptake in the tumor is also somewhat heterogeneous. As can be seen from Fig. 1A, the tracer uptake of the U87MG tumor is higher in the periphery region of the tumor and significantly lower in the center of the tumor, although ex vivo examination revealed that the tumor center was not necrotic.
In summary, we describe here the in vitro and in vivo characterization of 64Cu-labeled AbegrinTM, a humanized mAb against human integrin vß3, in various tumor models. No significant difference in immunoreactivity was found among the five 64Cu-DOTA-AbegrinTM conjugates tested. MicroPET imaging of 64Cu-DOTA-Abegrin (1,000:1) showed U87MG tumor uptake as high as 49.41 ± 4.54 %ID/g at 71-hour postinjection. The success of human integrin vß3-specific tumor imaging using 64Cu-DOTA-AbegrinTM may be translated into the clinic to evaluate the pharmacokinetics, tumor targeting efficacy, dose optimization, and dose interval of Abegrin and Abegrin-based cancer therapeutics. A certain level of nonspecific targeting due to the leaky vasculature of the tumors may also have potential applications in radioimmunotherapy in general.
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Acknowledgments |
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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 accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Ms. Pauline Chu and Dr. Xianzhong Zhang for their excellent technical support and the cyclotron teams of Washington University and University of Wisconsin for 64Cu production.
Received 4/24/06. Revised 6/14/06. Accepted 8/ 1/06.
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 A. H. Schmieder, S. D. Caruthers, H. Zhang, T. A. Williams, J. D. Robertson, S. A. Wickline, and G. M. Lanza Three-dimensional MR mapping of angiogenesis with {alpha}5{beta}1({alpha}{nu}{beta}3)-targeted theranostic nanoparticles in the MDA-MB-435 xenograft mouse model FASEB J, December 1, 2008; 22(12): 4179 - 4189. [Abstract] [Full Text] [PDF]
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 A. Veeravagu, Z. Liu, G. Niu, K. Chen, B. Jia, W. Cai, C. Jin, A. R. Hsu, A. J. Connolly, V. Tse, et al. Integrin {alpha}v{beta}3-Targeted Radioimmunotherapy of Glioblastoma Multiforme Clin. Cancer Res., November 15, 2008; 14(22): 7330 - 7339. [Abstract] [Full Text] [PDF]
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Q. Cao, W. Cai, G. Niu, L. He, and X. Chen Multimodality Imaging of IL-18-Binding Protein-Fc Therapy of Experimental Lung Metastasis Clin. Cancer Res., October 1, 2008; 14(19): 6137 - 6145. [Abstract] [Full Text] [PDF]
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P. M. Winter, A. H. Schmieder, S. D. Caruthers, J. L. Keene, H. Zhang, S. A. Wickline, and G. M. Lanza Minute dosages of {alpha}{nu}{beta}3-targeted fumagillin nanoparticles impair Vx-2 tumor angiogenesis and development in rabbits FASEB J, August 1, 2008; 22(8): 2758 - 2767. [Abstract] [Full Text] [PDF]
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P. T. Winnard Jr., A. P. Pathak, S. Dhara, S. Y. Cho, V. Raman, and M. G. Pomper Molecular Imaging of Metastatic Potential J. Nucl. Med., June 1, 2008; 49(Suppl_2): 96S - 112S. [Abstract] [Full Text] [PDF]
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W. Cai and X. Chen Multimodality Molecular Imaging of Tumor Angiogenesis J. Nucl. Med., June 1, 2008; 49(Suppl_2): 113S - 128S. [Abstract] [Full Text] [PDF]
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 H. Wang, K. Chen, W. Cai, Z. Li, L. He, A. Kashefi, and X. Chen Integrin-targeted imaging and therapy with RGD4C-TNF fusion protein Mol. Cancer Ther., May 1, 2008; 7(5): 1044 - 1053. [Abstract] [Full Text] [PDF]
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Z.-B. Li, Z. Wu, K. Chen, E. K. Ryu, and X. Chen 18F-Labeled BBN-RGD Heterodimer for Prostate Cancer Imaging J. Nucl. Med., March 1, 2008; 49(3): 453 - 461. [Abstract] [Full Text] [PDF]
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 G. A.M.S. van Dongen, G. W.M. Visser, M. N. Lub-de Hooge, E. G. de Vries, and L. R. Perk Immuno-PET: A Navigator in Monoclonal Antibody Development and Applications Oncologist, December 1, 2007; 12(12): 1379 - 1389. [Abstract] [Full Text] [PDF]
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W. Cai, K. Chen, Z.-B. Li, S. S. Gambhir, and X. Chen Dual-Function Probe for PET and Near-Infrared Fluorescence Imaging of Tumor Vasculature J. Nucl. Med., November 1, 2007; 48(11): 1862 - 1870. [Abstract] [Full Text] [PDF]
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 S. D. Voss, S. V. Smith, N. DiBartolo, L. J. McIntosh, E. M. Cyr, A. A. Bonab, J. L. J. Dearling, E. A. Carter, A. J. Fischman, S. T. Treves, et al. Positron emission tomography (PET) imaging of neuroblastoma and melanoma with 64Cu-SarAr immunoconjugates PNAS, October 30, 2007; 104(44): 17489 - 17493. [Abstract] [Full Text] [PDF]
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A. R. Hsu, W. Cai, A. Veeravagu, K. A. Mohamedali, K. Chen, S. Kim, H. Vogel, L. C. Hou, V. Tse, M. G. Rosenblum, et al. Multimodality Molecular Imaging of Glioblastoma Growth Inhibition with Vasculature-Targeting Fusion Toxin VEGF121/rGel J. Nucl. Med., March 1, 2007; 48(3): 445 - 454. [Abstract] [Full Text] [PDF]
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K. Mulgrew, K. Kinneer, X.-T. Yao, B. K. Ward, M. M. Damschroder, B. Walsh, S.-Y. Mao, C. Gao, P. A. Kiener, S. Coats, et al. Direct targeting of {alpha}v{beta}3 integrin on tumor cells with a monoclonal antibody, AbegrinTM Mol. Cancer Ther., December 1, 2006; 5(12): 3122 - 3129. [Abstract] [Full Text] [PDF]
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