The preparation of the contrast agent, Gd-DTPA-anti-VEGFR2 antibody conjugate (Fig. 1), was based on a similar method previously described by our laboratory (14) for the preparation of the contrast agent Gd-DTPA-anti-intercellular adhesion molecule 1 (ICAM-1). The VEGFR2 antibody was purified from the culture supernatant of rat hybridoma using protein A/G-coupled affinity chromatography. Purified antibodies were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blot analysis, and bicinchoninic acid (BCA) assay. Diethylenetriaminepentaacetic acid bisanhydride (DTPABA) was dissolved in dimethylformamide and then added to the antibody solution. The anti-VEGFR2 antibody was conjugated to DTPABA in phosphate-buffered saline (PBS) (pH 8.5) for one hour at room temperature. The reaction solution was purified using a PD-10 column (Sephadex G-25M, Amersham Biosciences, Amersham, UK). The concentration of the antibody was measured using the BCA assay. Gd chloride was dissolved in deionized water and then added to the DTPA-antibody conjugate solution. One part of the DTPA-antibody complex was then reacted with 40 parts of Gd chloride in 0.5 M sodium acetate (pH 5.5). The solution was loaded onto a PD-10 column and was eluted from the column with 0.15 M NaCl (pH 5.5). Gd-DTPA antibody conjugates were analyzed using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) (Voyager-DE STR 4349) spectrometry. The Gd content was determined with the aid of inductively coupled plasma mass spectrometry (ICP-MS) (Agilent 7500A, Agilent Technologies, Palo Alto, CA), and the final concentration of the conjugated antibody was measured using BCA analysis. The molar ratio of gadolinium and antibody was then calculated. The control antibody conjugate, Gd-DTPA-anti-rat-IgG, was prepared using a similar method.

A rat hybridoma used for the isolation of anti-VEGFR2 antibody was purchased from the American Type Culture Collection (ATCC, Manassas, VA) and was cultured in Dulbecco's Modified Eagle's Medium (DMEM) with 4 mM L-glutamine, which was adjusted to contain 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, and 10% fetal bovine serum. The murine endothelial cell line (MS-1) and murine colon adenocarcinoma cell line (CT-26) were also obtained from ATCC and the cells were cultured according to the supplier's instructions. Cells were grown as monolayer cultures in DMEM with 10% fetal bovine serum (Invitrogen, Grand Island, NY) supplemented with 1% glutamine and 1% antibiotics. Cells were maintained in a 37℃ incubator in 5% CO₂ humidified air.

For the preparation of the endothelial cell-specific contrast effect, MS-1 cells (5 × 10⁶) were cultured and were incubated with the Gd-DTPA-VEGFR2 antibody complexes (300 nmol Gd) in the presence or absence of anti-VEGFR2 antibody (600 nmol) at 4℃ for four hours. The cells were then washed three times with PBS, harvested by scraping, and pelleted by centrifugation.

In vitro MRI was performed on a 4.7-T animal MRI instrument (Bruker, Ettlingen, Germany). The endothelial cell-specific contrast effect was assessed by determining MRI contrast effects with the endothelial MS-1 cells. An MR image of the cells in the tubes placed in a water-filled chamber was obtained with a spin echo sequence using the following imaging parameters: TR = 300 milliseconds, TE = 10 milliseconds, field of view (FOV) = 25.6 mm × 25.6 mm, slice thickness = 1 mm, pixel resolution = 100 × 100 µm, in the 4.7-T instrument. The signal intensity of T1-weighted imaging (WI) of the cell pellets was normalized against that of the surrounding water. Each experiment was performed in triplicate and the signal intensity was shown as the mean ± standard deviation.

A region of interest (ROI = 0.02 cm²) for cell and water was calculated. The average of the ROIs included areas of maximal and minimal enhancement in each slice. For the in vitro MRI study, we defined the relative signal intensity (SI) as: ([mean of ROI] _cell)/([mean of ROI] _water).

Male Balb/c nude mice (n = 16, aged 6 weeks and each weighing 20-25 g) were purchased from the Central Animal Laboratory (Seoul, South Korea) and used for this study. The Balb/c nude mice were injected subcutaneously in their back with CT-26 cells (1 × 10⁶ cells) suspended in 0.1 mL phosphate-buffered saline. The injected cells were allowed to expand for 10 days until the tumors grew to a size approximately 0.5 cm³.

In vivo MRI was performed on a 4.7-T animal MRI instrument. T1WI was obtained at 10 minutes and at 12, 24, and 48 hours after the injection of the Gd-DTPA-anti-VEGFR2 antibody conjugate (12 µmol of Gd/kg of body weight) in eight mice, followed by the injection of the Gd-DTPA-anti-rat IgG conjugate (12 µmol of Gd/kg of body weight) in another eight mice. All the animals were examined by contrast-enhanced T1-weighted MRI using the following imaging parameters: TR = 300 milliseconds, TE = 10 milliseconds, FOV = 25.6 mm × 25.6 mm, slice thickness =1 mm, pixel resolution = 100 × 100 µm, in the 4.7-T instrument. All of the animal studies were carried out in accordance with the regulations set by the Institutional Review Board of our university.

An ROI (= 0.02 cm²) for the tumor center and muscle was calculated for the mean value. The average of the ROIs included areas of maximal and minimal enhancement in each slice. The SI was calculated using the following equations: SI = ([mean of ROI] _tumor)/([mean of ROI] _muscle). For the in vivo MRI study, we defined the contrast enhancement ratio (CER) as ([SI_post-SI_pre]/SI_pre) × 100%, where SI_pre = signal intensity before administration of contrast medium and SI_post = signal intensity after contrast medium administration.

After analyzing the mice by MRI, tumor tissue was collected and the expression of VEGFR2 was then examined by way of an immunohistochemical analysis. Tissue sections were fixed with 4% paraformaldehyde and sections were reacted with rabbit anti-mouse VEGFR2 antibody for two hours at 37℃. The primary antibody that bound to VEGFR2 and expressed in cells was then visualized with a secondary peroxidase-conjugated antibody and substrate dye. To detect the injected Gd-DTPA-anti-VEGFR2 antibody, an adjacent section was incubated with only the second antibody (goat anti-rabbit IgG antibody) for the Gd-conjugated antibody (rabbit anti-mouse VEGFR2 antibody) and was detected with the substrate dye.

The data were presented as the mean ± standard deviation, and the significance of the data was assessed using the Mann-Whitney test. The statistical analyses were performed using commercial software (SPSS, Chicago, IL).

The purified antibody fraction was identified by SDS-PAGE (separating the 50 kDa and 25 kDa) of the IgG. These antibody fragments were also identified using Western blot analysis, which also detected the 50 kDa and 25 kDa. The intermediate product, DTPA antibody, and the final product, Gd-DTPA antibody, were quantified by BCA assay for their antibody content and were characterized using MALDI-TOF spectrometry analysis for the conjugated product. The molecular weight shift from anti-VEGFR2 to Gd-DTPA-anti-VEGFR2 antibody in MALDI-TOF spectrometry analysis represents the conjugation of the antibody with Gd-DTPA. A quantitative analysis of the Gd content in the Gd-DTPA-antibody was performed using ICP-MS. The molar ratio of Gd to antibody was about 1:20.

To examine whether the Gd-DTPA-anti-VEGFR2 antibody conjugate can bind to cells in a VEGFR2-specific manner, the contrast agent was incubated with MS-1 cells in the presence or absence of a competitive binding inhibitor, the anti-VEGFR2 antibody (Fig. 2). The competitive antibody was added at a three-fold higher concentration than the anti-VEGFR2 antibody content of the Gd-DTPA-anti-VEGFR2 antibody complex. The value of signal intensity showed that binding of the Gd-DTPA-anti-VEGFR2 antibody complex to the VEGFR2-expressing cells was significantly inhibited by competition with the anti-VEGFR2 antibody (Table 1).

Angiogenesis-targeted MRI using the Gd-DTPA-anti-VEGFR2 antibody conjugate was examined in a mouse tumor model. The Gd-DTPA-anti-VEGFR2 antibody conjugate displayed significant signal enhancement and accumulation of tumor tissues until 48 hours after contrast agent injection (Fig. 3A-E). The Gd-DTPA-anti-rat IgG and the Gd-conjugated control antibody containing the same amount of Gd as the Gd-DTPA-anti-VEGFR2 antibody conjugate, showed no significant enhancement in T1 until 48 hours after injection (Fig. 3F-J). The CER in the mouse tumors that received Gd-DTPA-anti-VEGFR2 antibody conjugate increased at 24 hours (29%) and at 48 hours (9%) after injection. Conversely, the CER in the mouse tumors that received Gd-DTPA-anti-rat IgG was increased at 24 hours (11%) and at 48 hours (11%) after injection. The CER value was significantly different for mouse tumors that were administered Gd-DTPA-anti-VEGFR2 antibody conjugate and Gd-DTPA-anti-rat IgG (p < 0.05). The Gd-DTPA-anti-VEGFR2 antibody conjugate injected into tumors showed a peak time at 24 hours. Conversely, Gd-DTPA-anti-rat IgG injected into tumors showed a peak time at 24 hours with a low level of enhancement (Fig. 3).

CT-26 tumor tissue was examined using immunohistochemical staining for VEGFR2 expression to assess whether the angiogenesis-specific imaging, using the Gd-DTPA-anti-VEGFR2 antibody, corresponded to VEGFR2 expression in tumors. The expression of VEGFR2 showed an extremely high level in vessels of CT-26 tumor tissue (Fig. 4).