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Basic Science Investigation |
1 Center for Cardiovascular Targeting, School of Pharmacy, Bouve College of Health Sciences, Northeastern University, Boston, Massachusetts; and 2 Division of Cardiology, Department of Medicine, Columbia University Medical Center, New York, New York
Correspondence: For correspondence or reprints contact: Ban-An Khaw, PhD, Center for Cardiovascular Targeting, School of Pharmacy, Bouve College of Health Sciences, Northeastern University, Boston, MA 02115 E-mail: b.khaw{at}neu.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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15.0 MBq 99mTc-DTPA–succinyl-polylysine (2 µg; DTPA is diethylenetriaminepentaacetic acid) were injected intravenously. Results: In vivo 
-images showed that lesions were observed unequivocally by 2–3 h. Sham-operated right femoral regions showed no radiotracer accumulation. Ex vivo -scintillation counting corrected for sham-operated nonspecific activity and lesion mass showed that the mean lesion activity was 10.10 ± 6.76 %ID/g (percentage injected dose per gram), whereas nonspecific human IgG bispecific control (NSB control) also corrected similarly was 0.939 ± 0.877 %ID/g (P < 0.03). Atherosclerotic lesions were confirmed by immunohistochemical staining. Computer planimetry of immunohistograms showed the mean lesion size to be 2.64 ± 2.46 mg. Conclusion: Use of bispecific antibody complexes and 99mTc-DTPA–succinyl-polylysine enabled in vivo visualization of very small atherosclerotic lesions in ApoE knockout mice.
Key Words: bispecific antibody • pretargeting • radiolabeled polymer
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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-imaging even with standard -cameras. Narula et al. demonstrated that experimental atherosclerotic lesions in rabbits can be visualized within 3 h if a very high specific radioactivity-labeled high antibody dose of Z2D3 F(ab')2 were used in comparison with a lower specific radioactivity, low antibody dose (5). However, nontarget organ activity in these animals was still unacceptably high. The approach we have taken to improve in vivo molecular imaging is to use a bispecific antibody. The targeting arm consists of chimeric Z2D3 F(ab')2 antibody, which is specific for a lipid antigen complex produced by proliferating smooth muscle cells (SMCs) of atherosclerotic lesions or postangioplasty restenosis, and a murine anti-DTPA monoclonal F(ab')2 antibody (6C31H3; DTPA is diethylenetriaminepentaacetic acid) as the capture arm. A negatively charged, high specific radioactivity 99mTc-DTPA–succinylated polylysine polymer (99mTc-DTPA–Suc-PL14.6 kDa) is used as the radiopharmaceutical that will be captured by the capture arm of the pretargeted bispecific antibody. This 2-step targeting approach not only reduced the nontarget background activities but also enabled enhanced unequivocal visualization of very small femoral artery atherosclerotic lesions in ApoE–/– mice within 2–3 h after intravenous administration of the radiotracer.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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Cross-Linking of F(ab')2 of Z2D3 to F(ab')2 of Anti-DTPA 6C31H3
An aliquot of Z2D3 F(ab')2 (1–2 mg) was reacted with 14 molar excess of N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and the same amount of 6C31H3 F(ab')2 was modified with 300–1,000 times molar excess of iminothiolane (8,9). The immunoreactivities of SPDP-modified or thiolated antibodies were assessed by enzyme-linked immunosorbent assays (ELISAs) (9). Modified F(ab')2 preparations without loss of immunoreactivity were reacted at equimolar concentration (1.75 mg) for 1 h at room temperature and then overnight at 4°C (9). The reaction mixture was then subjected to Ultrogel AcA-22 size-exclusion column (2.5 x 100 cm) chromatography (Fig. 1A). The samples were eluted from the column with 0.1 mol/L PBS. Dimeric bispecific F(ab')2 antibodies were eluted in the first peak and the monomeric F(ab')2 fragments were eluted in the second peak. Antibodies in these 2 peaks were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) (X Cell Sure lock Mini-Cell, Nu PAGE 6% precast gels; Invitrogen Inc.) under nonreducing condition and compared with the molecular weight standards (Fig. 1B). Nonspecific-human IgG F(ab')2 was also cross-linked to 6C31H3 F(ab')2 and used as nonspecific bispecific (NSB) controls.
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Assessment of Bispecific Antibody Immunoreactivity with Surrogate Antigen (7-Dehydrocholesterol [DHC] and Benzyldimethylhexadecylammonium Chloride [BDMHDAC])
The surrogate antigen for Z2D3 was prepared with minimal modification as described in U.S. patents (10,11). Briefly, to prepare antigen-coated polystyrene beads, beads with an average diameter of 3 µm (Sigma Chemical Co.) were washed and resuspended in absolute ethanol. The resulting suspension was separated into aliquots, each containing 4 µg of beads. To each aliquot, 500 µg of DHC (250 µL of a 2 mg/mL solution in ethanol) and 31 µg of BDMHDAC (31 µL of a 1 mg/mL solution in ethanol) were added. After thoroughly mixing, the solvent was allowed to evaporate at room temperature. The coated beads were stored at 4°C until they were used. The control beads were made by drying the ethanol and resuspending the beads in 0.1 mol/L PBS.
Alternatively, surrogate antigen-coated 96-well microtiter plates were also used for the assessment of the immunoreactivity of various Z2D3 antibody preparations as described in U.S. patent 5 811 248 (10). Briefly, aliquots of 500 µg DHC (250 µL ethanol) and 31 µg BDMHDAC (31 µL in ethanol) were mixed and added to each of the 96 wells of the microtiter plates (Beckton Dickinson & Co.). The ethanol was allowed to evaporate, and the wells were washed with PBS/Tween (0.05% Tween) (PBS-T) and then blocked with 1% horse serum, followed by additional washing. To each well, serial dilutions of bispecific or unmodified Z2D3 were added (10–0.0001 µg/mL) and incubated overnight at 4°C. After washing, goat antihuman IgG-conjugated horseradish peroxidase (HRP) (1:1,000 dilution) was added and incubated at 37°C for 1 h. The excess secondary antibody was washed as above, and 100 µL of o-phenyldiamine (OPD) (1 mg/mL OPD in 0.1 mol/L citrate, 0.2 mol/L disodium phosphate, pH 6.0) containing 30 µg/mL H2O2 were added to each well. After 15–30 min of incubation at room temperature in the dark, 25 µL of 2.5 mol/L H2SO4 were added and the optical density at 490 nm was read in an ELISA reader (EL307C; Biotech Instruments).
To determine the in vitro signal enhancement, serial dilutions of bispecific antibody or unmodified Z2D3 F(ab')2 were added to the surrogate antigen-coated microtiter wells, prepared as described. After incubation, the unmodified Z2D3 binding was assessed with a secondary antibody modified with HRP as described. To test the activity of the bispecific antibody for signal enhancement, DTPA-linked PL46 kDa modified with 5.5 or 9 HRP modified DTPA-PL46 kDa was used (9). Incubation with the chromogen, stoppage of the reaction with H2SO4, and determination of the optical density were as described.
Anti-DTPA Antibody Immunoassay
Microtiter plates (96 well) were coated with 100 µL of DTPA/bovine serum albumin (BSA) (1 µg/mL) as described. The DTPA/BSA–coated wells were used to assess anti-DTPA activity by the standard ELISA protocol.
Preparation of 99mTc-Labeled DTPA–Suc-PL14.6 kDa
An aliquot of 50 mg of polylysine (PL) (14.6 kDa; Sigma Chemical Co.) was dissolved in 5 mL of 0.1 mol/L NaHCO3, pH. 8.7. Ten times molar excess (relative to lysine residues) of bicyclic anhydride of DTPA (Sigma Chemical Co.) in 1 mL of dimethyl sulfoxide (DMSO) was added slowly to the above solution while stirring vigorously. The number of lysyl residues modified was assessed by the trinitrobenzylsulfonic acid (TNBS) assay relative to the standard unmodified PL solution (9,12). The reaction mixture was dialyzed against excess (4 L) 0.1 mol/L carbonate, pH 9.6, at 4°C overnight. Then the DTPA–PL solution was succinylated with 100 times molar excess of succinic anhydride to modify any residual lysyl residues. The DTPA–Suc-PL14.6 kDa was dialyzed in 0.1 mol/L Na2CO3, pH 9.6, and stored at 4°C until used.
An approximately 50-µg aliquot of DTPA–PL14.6 kDa in 0.1 mol/L Na2CO3 was reacted with 1,125 MBq of 
in 50 µg of SnCl2 in 100 µL of 0.1N HCl that has been flushed with N2 for 15 min. After 30 min of incubation, the 99mTc-DTPA–Suc-PL14.6 kDa was separated from free 99mTc by Sephadex-G25 (10 mL) column chromatography.
Preparation of Rhodamine-Labeled DTPA–Suc-PL14.6 kDa
Rhodamine-labeled DTPA-PL was prepared by reacting 50 mg of PL14.6 kDa with 3 times molar excess of DTPA anhydride (Sigma Chemical Co.) (9). The DTPA modification was assessed by the TNBS assay as before (12); then 2 mg/mL of DTPA-linked PL were reacted with 24 times molar excess of rhodamine isothiocyanate (Pierce Chemical Co.) in N,N-dimethylformamide (DMF). The preparation was succinylated with 100 times molar excess of succinic anhydride. The sample was dialyzed against 0.1 mol/L PBS, pH 7.4, and stored in the dark at 4°C.
In Vitro Demonstration of Specificity of Bispecific Z2D3–6C31H3 with Rhodamine-Labeled DTPA–PL14.6 kDa
One percent agarose gels in PBS were prepared on microscope slides. Sample wells (2-mm diameter) were made in the gels approximately 5 mm apart. One well was filled with 75 µL of surrogate antigen-coated (DHC/ BDMHDAC) beads, and the other was filled with bispecific antibody at a concentration of 1–10 µg/mL. The antibody was allowed to diffuse out of the wells overnight at 4°C. Controls consisted of unmodified beads in the first well with the same amount of bispecific antibody in the second well or surrogate antigen-coated beads in the first well and NSB control in the second well. The excess antibody or the NSB control was washed from the gels. Then the antibody or NSB control wells were filled with 50 µg/mL of rhodamine-conjugated succinylated DTPA–PL14.6 kDa. The polymers were allowed to diffuse through the gel overnight at 4°C, followed by extensive washing as described. The microscope slides were then viewed with an epifluorescent microscope (Zeiss). Fluorescent and light photomicrographs were then digitally recorded.
In Vivo Imaging Experimental Protocol
C57BL/6 mice, 8–12 wk of age with ApoE–/–, were purchased from The Jackson Laboratory. Eleven ApoE–/– female mice were used to induce accelerated femoral artery atherosclerotic lesions (13) as approved by the Institutional Animal Care and Use Committee of Northeastern University. Mice were fed Western diet (21%, w/w, fat [polyunsaturated/saturated ratio = 0.07]) and 0.15%, w/w, cholesterol (Harlan Teklad) for 2 wk. Each mouse was then anesthetized with injections of intraperitoneal ketamine (90 mg/kg) and xylazine (10 mg/kg). The ventral sides of the hind legs were shaved and swabbed with butadiene and alcohol. An incision was made in the left femoral artery region. Under a surgical microscope, the femoral artery was isolated by placing 2-0 silk sutures 0.5 cm apart, isolating a segment of the femoral artery. A small incision was made with microscissors, and then a 0.25-mm-diameter angioplasty guide wire (Advanced Cardiovascular Systems) was inserted into the femoral artery segment. The guide wire was advanced and pulled back 3 times as described by Roque et al. (13) to induce endothelial injury that resulted in intimal SMC proliferative lesions. The guide wire was withdrawn and bleeding was stopped by applying pressure at the incision site for 5–10 min. The femoral incision was closed with a 3-0 silk suture using interrupted surgical knots. Antibiotic ointment (bacitracin; Denison Pharmaceuticals Inc.) was applied to the site. Similarly, the right femoral artery region underwent femoral incision and closure without endothelial injury. This right femoral artery region was used as the sham-operated control site. After surgery, all mice were returned to the cages and vital signs were monitored until the animals recovered from anesthesia. The wounds and vital signs were monitored daily until the surgical wounds were healed. ApoE–/– mice with femoral endothelial denudation were kept on Western diet for an additional 2 wk. Seven mice were then injected with 30–50 µg bispecific antibody and 4 remaining mice were injected with 50 µg NSB control. The next day (15 h later), approximately 15 MBq of 99mTc–DTPA–Suc-PL14.6 kDa (2 µg) were injected intravenously. Anteroposterior images of the lower torso of mice were acquired using a 3-mm pinhole collimator-equipped -camera (Picker SX300) attached to an Apple computer with a Gamma 600 acquisition program. Ten-minute acquisition serial images at injection or between 30 min and 1 h, 1 and 2 h, and 2 and 3 h after injection of the radiotracer were obtained for each mouse. The photo peak was set at 140 keV with a 15% window. After the last imaging session, mice were euthanized by intraperitoneal injection of pentobarbital (100 mg/kg). Right and left femoral arteries were excised and rinsed in saline. Other organs (blood, heart, lungs, liver, kidneys, spleen, stomach, small and large intestine) were obtained directly for biodistribution.
Assessment of Lesion Size by Computer Planimetry
Because femoral arteries in mice are <1.0 mm in diameter, it was not practical to dissect out nonvessel adventitial tissue and other tissues from the femoral artery segment that was harvested with any efficiency. Furthermore, it was not possible to harvest atherosclerotic lesions exclusively at the time of sacrifice. Therefore, the vessel segments with the surrounding adventitia were excised, weighed, and counted in a -scintillation counter. To enable assessment of the atherosclerotic lesion activity without the normal adherent tissue activities, a method to estimate the percentage lesion of the whole tissue sample was developed. Ten-micron-thick frozen cross-sections of the midregion of the excised vessels and accompanying adventitia were subjected to immunoperoxidase staining using Z2D3. The frozen sections were incubated with Z2D3 overnight, followed by washing. These sections were counterstained with rabbit antihuman IgG antibody conjugated with HRP. After further washing, 3,3'-diaminobenzidine (DAB) substrate solution was added and incubated at room temperature for 5–10 min. The stained slides were washed extensively. The immunoperoxidase-stained slides were counterstained with methyl green, followed by washing as described. Sections were dehydrated in succession in ethanol: 70%, 75%, 95%, and 100% for 2 min each. Slides were mounted with Permount (Fisher Scientific), coverslips were applied, and then slides were viewed under light microscopy.
The immunohistochemical micrographs were photographed digitally and the digital micrographs were used to planimeter the regions of antigen-positive staining as the number of pixels and compared with the total area of the whole tissue cross-section. The percentage of the area of antigen-positive staining was determined relative to the whole tissue cross-section from 4 different left femoral arteries with lesions chosen randomly. The mean percentage of the lesion was then used to estimate the mass of the lesion in each atherosclerotic femoral artery segment.
To obtain specific lesion activity, counts per minute per gram (cpm/g) of the sham vessel (i.e., without lesion) were subtracted from the cpm/g of the lesion vessel. The nonspecific activity–corrected left femoral artery tissue activity was then divided by 0.114 (lesion = 11.4% of the whole vessel segment). Similar corrections were also made for the left femoral arteries from ApoE–/– mice with atherosclerotic lesions injected with NSB control. The following formula was used:
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Statistical Analysis
Data are expressed as mean ± SD. ANOVA (single factor) was used to determine statistical significance using the statistical package of Microsoft Excell XP. A P value of 
0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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210 kDa), whereas the second peak consisted of an 110-kDa protein band consistent with the size of monomeric F(ab')2. ELISA was used to determine the immunoreactivity of the dimeric bispecific (Z2D3-6C31H3 F(ab')2) antibody. The surrogate antigen (Fig. 2A) or DTPA-linked BSA-coated microtiter plates were used (Fig. 2B). Binding of the bispecific antibody was compared with that of Z2D3 F(ab')2 or anti-DTPA 6C31H3 F(ab')2 using HRP-conjugated secondary antihuman or antimouse IgG antibodies, respectively. There was no difference in the binding of the bispecific antibody relative to unmodified Z2D3 or 6C31H3 F(ab')2, indicating that there was no loss of immunoreactivity after formation of the bispecific antibody complexes via the disulfide linkage.
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Immunohistochemical Signal Enhancement
Frozen sections of rabbit aortas with experimental atherosclerotic lesions (6) were used to demonstrate signal enhancement by immunoperoxidase staining (Fig. 3A). The intensity of staining with 150 µg/mL of Z2D3 F(ab')2 (Fig. 3A, panel e) was the same as the staining obtained between 5 and 10 µg/mL of bispecific antibody–5.5 HRP polymer (Fig. 3A, panels b and c). This resulted in 15–30 times enhancement in staining signal intensity.
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Radiolabeling of DTPA–Suc-PL14.6 kDa
Assuming an 80% recovery of the DTPA–Suc-PL14.6 kDa after Sephadex-G25 column chromatography and because only the peak tube was used for in vivo studies, 40 µg of the polymer were assumed to be recovered and only 20 µg were assumed to be in the peak tube labeled with 150 MBq 99mTc. Each mouse was injected intravenously via the tail vein with 15 MBq of 99mTc-DTPA–Suc-PL14.6 kDa (2 µg; 37 MBq is equivalent to 0.38 nmol of 99mTc).
In Vivo Imaging of Atherosclerotic Lesions in ApoE–/– Mice
Figure 4A shows that there was no lesion (red arrow) or sham vessel (yellow arrow) activity in the ApoE–/– mouse injected with NSB control followed by 99mTc-DTPA–Suc-PL14.6 kDa administration. Whereas, in the ApoE–/– mouse injected with bispecific antibody followed by intravenous injection of 99mTc-DTPA–Suc-PL14.6 kDa, the 30-min image shows a slight increase in radioactivity in the region of the atherosclerotic lesion (Fig. 4B, left red arrow, bottom), which by 3 h became unequivocally delineated (right red arrow, bottom). There is no radiotracer accumulation in the contralateral sham-operated right leg region (yellow arrows). Furthermore, the only other organs showing radiotracer activities were the kidneys and the bladder. In the 3-h postinjection image, the lesion activity was greater than that seen in the kidneys. The 2-h postinjection 99mTc-DTPA–succinyl-PL14.6 kDa image in another ApoE–/– mouse with an atherosclerotic lesion is shown in Figure 4C. Although there is more kidney activity, the atherosclerotic lesion is also delineated unequivocally (red arrow) and there was no activity in the contralateral sham-operated right femoral artery region (yellow arrow).
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-imaging. The lesion activity in these mice (range, 3–4 pixels) did not differ from that of the sham-operated sites (range, 2–3). These were significantly lower than the pixel density of the lesions in bispecific antibody–injected mice (P < 0.01). The ratio of lesion to sham pixel density was calculated to be 12:1. Localization of the radiotracer in the femoral arteries determined as percentage injected dose per gram (%ID/g) of total tissue mass containing the lesions is shown in Figure 5A. There was no significant difference between the mean activity in the femoral arteries with lesions (0.668 ± 0.167) and that of the sham-operated legs (0.561 ± 0.142) in mice injected with NSB control (solid bars, left to right, respectively; n = 4, P = not significant). However, uptake of radiotracer in the whole femoral arteries with lesions in bispecific antibody–injected mice (left open bar, 2.003 ± 1.024) was significantly greater than that of the contralateral sham-operated right femoral artery (right open bar, 0.852 ± 0.368; P < 0.02) or the lesion activity (0.668 ± 0.167) of mice injected with NSB control (P < 0.03). The left to right femoral artery activity per gram of total vessel tissue also showed that the mean ratio in ApoE–/– mice with bispecific antibody injection (2.40:1) was significantly greater than that of mice injected with NSB control (1.19:1) (P < 0.007).
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-scintillation counter. The corrected radioactivity of bispecific antibody 99mTc-DTPA–Suc-PL14.6 kDa was 10.10 ± 6.76 %ID/g of lesion and that of the NSB control in the lesion was 0.939 ± 0.877 %ID/g of lesion (P = 0.027) (Fig. 5C, far right open and solid bars, respectively). The mean ratio of the corrected lesion radioactivity to sham vessel radioactivity (0.852 + 0.368) was calculated to be 11.85:1, which was consistent with the mean target-to-nontarget (sham) activity ratio from computer planimetry (12.05:1).
Biodistribution of 99mTc-DTPA–Suc-PL14.6 kDa in all ApoE–/– mice determined at 2–3 h after radiotracer administration in bispecific antibody (n = 7, open bars) or NSB control (n = 4, black bars) injected mice is shown in Figure 5C. The mean radioactivity in atherosclerotic lesions containing left femoral artery of mice injected with bispecific antibody was significantly greater than the mean radioactivity of atherosclerotic lesions in mice injected with NSB control (P = 0.03). The difference between left and right femoral artery activities of mice injected with bispecific antibody or NSB control was also statistically significant (P 0.01). There was no statistical difference in other organs.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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In an attempt to target the SMC proliferation component of atheromas, mice were immunized with homogenized human atherosclerotic plaques and an antibody designated as Z2D3 (IgM class, 
light chain) was produced. Z2D3 reacted specifically with intimal proliferating SMCs in human atheroma (20), experimental rabbit (6), and swine lesions (21). The parent Z2D3 IgM monoclonal antibody was subsequently genetically engineered to produce a murine–human chimera with a human IgG1 constant region. The F(ab')2 of Z2D3 labeled with 111In was successfully used to image rabbit atheromas at 48 h after antibody injection (6).
To improve the counts per pixel and target-to-background ratio in single-photon imaging, we have linked multiple DTPAs with PL that could chelate a large number of trivalent metallic radiolabels to Z2D3 (5,22). This process improved target-to-background activities by simultaneously increasing the amount of radiolabeled antibody delivered to the target and reducing the electrostatic attraction to nontarget cells with overall weak negative charge. Using 111In-labeled PL Z2D3 F(ab')2, we demonstrated that with higher antibody doses experimental lesions could be visualized earlier (5). In a phase I study, Carrio et al. showed focal carotid uptake of 111In-DTPA–succinylated PL-modified Z2D3 F(ab')2 in patients with recent ischemic cerebral events (22).
We have now improved targeting of very small lesions while maintaining in vivo background activity to a minimum. This enabled in vivo scintigraphic visualization of small lesions (2.64 ± 2.46 mg) using a vintage clinical -camera equipped with a pinhole collimator. The only other organs that showed radiotracer activity were the kidneys and the bladder. Such organ activities are unavoidable, as the size of the polymers was chosen to enable elimination by renal excretion. An obstacle that we had encountered previously in the assessment of radiotracer localization was that SMC proliferation in experimental atherosclerotic lesions constitutes a very minor portion of the total vascular segment that was harvested to determine tissue activity. By staining the vascular segments containing lesions with Z2D3, then by planimetering the areas stained relative to the total tissue segment, we were able to normalize the activity to the estimated lesion size. It appears that, of the mean femoral mass of 22.96 ± 21.58 mg that was used for -scintillation counting, only 11.4% ± 2.66% constituted antigen-positive tissue, resulting in 2.64 ± 2.46 mg of mean lesion mass. This process of normalization demonstrated that the ratio of the intensity of the lesions (20.6 ± 12.21 pixels) to sham regions (1.71 ± 0.76) of 12:1 was consistent with the ratio of the ex vivo -counting of 11.86:1 (10.10/0.8516). This also demonstrated that lesions as small as a few milligrams (<1 to 5 mg) can be visualized by in vivo imaging as long as there is sufficient radioactivity at the target sites and the background activity remains low, even though the resolution of the -camera used was not as high as that of the new rodent mini-SPECT -cameras. Even with the best Bioscan NanoSPECT -cameras, the resolution is only 0.6 mm. However, by delivering targeting radiopharmaceutical with high specific radioactivity, the ability to detect small lesions in rodents should be further enhanced.
The limitation of the current study is that the tissue sections used for immunohistochemical staining for calculation of the lesion size were assumed to be representative of the whole lesion vessel. Furthermore, only sections from 4 randomly chosen femoral arteries were used. In reality, the extent of the lesions could be more or less. Because these sections were obtained from the midregions of the lesion vessels, the extent of the lesion should be maximum. The 2 extremities of the lesion vessels would normally have less injury. Therefore, our assumption that the extent of the lesion is consistent throughout the segment of the vessel with lesion may be an overestimation of the lesion size and, therefore, an underestimation of the target activity. The only way to improve on this assumption is to undertake immunohistochemical staining of serial sections of each vessel. Nonetheless, the activity ratio of the target to sham-operated region from computer planimetry of in vivo images was similar to that obtained after correction of lesion mass by -scintillation counting, indicating that both methods of lesion assessment are comparable.
Although the specific radioactivity that is used in these studies has not been optimized, the present study showed the potential of using bispecific antibodies with 99mTc-labeled negatively charged polymers for enhanced in vivo imaging. Radiolabeling at 5.4–140 MBq/µg of polymers or 1–5.5 mol of 99mTc per mol of polymer radiospecific activity has been achieved (data not shown). In the current in vivo studies, 7.5 MBq/µg of polymer were used. Most radiopharmaceuticals labeled with 99mTc used higher concentrations of the pharmaceuticals. In the glucaric acid kits used for imaging acute myocardial infarction (23), 12.5 mg of glucaric acid were labeled with 1,125 MBq 99mTc. Assuming 100% labeling efficiency, the specific activity would be 0.09 MBq/µg of glucaric acid. This is 2.3 x 10–4 mol 99mTc per mol of glucaric acid. Therefore, the polymers were radiolabeled with 99mTc at a relatively high specific activity (83–1,400 times more efficient than 99mTc labeling of glucaric acid). Furthermore, because the polymer is the molecule that is radiolabeled, the problems associated with radiolabeling of antibodies are avoided.
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CONCLUSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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-imaging within 2 h. Substitution of single-photon radioisotopes with other signaling molecules may allow pretargeted imaging with positron- emission radioisotopes or paramagnetic ions for enhanced MRI. Substitution with therapeutic agents on these polymers may result in more efficient therapy similar to the use of bispecific antibodies and radiotherapeutic iodine-labeled dipeptides (24). |
ACKNOWLEDGMENTS |
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