Noninvasive localization of human atherosclerotic lesions with indium 111-labeled monoclonal Z2D3 antibody specific for proliferating smooth muscle cells.

BACKGROUND: Targeting exclusive antigens in atherosclerotic plaques with antibodies may provide a noninvasive means to detect rapidly proliferative atherosclerotic lesions. 111In-labeled negative charge-modified Z2D3 F(ab')2 (Z2D3) specific for an antigen expressed exclusively by proliferating smooth muscle cells has been shown to accumulate in rabbit atherosclerotic plaques. METHODS: The safety, biodistribution, accumulation, and elimination of Z2D3 were assessed in 11 patients who were candidates for carotid endarterectomy. The presence of atheromas in these patients was confirmed by angiography and Doppler ultrasound. Z2D3 (250 microg) labeled with 5 mCi of 111In was administered by slow intravenous injection. Planar and single photon emission computed tomography (SPECT) images were obtained 4, 24, 48, and 72 hours later. Carotid endarterectomy was performed and the surgical specimens were imaged, weighed, gamma-counted, and analyzed by immunostaining. RESULTS: Uptake of Z2D3 at the site of the carotid plaques was observed in the planar and SPECT views at 4 hours in all subjects. In addition, antibody uptake was noted in the contralateral vessel in 5 subjects. SPECT images identified the atherosclerotic plaques with focal uptake. The antibody uptake corresponded with the angiographic location of the disease. Immunohistochemical studies of the endarterectomy specimens confirmed the localization of Z2D3 into the plaque areas containing smooth muscle cells. Adverse drug reactions were not observed. CONCLUSION: This study demonstrates the feasibility of targeting atherosclerotic lesions with negative charge-modified antibody. It also proposes the possibility of selective identification of various components of atherosclerotic plaque, which may contribute to determining strategies of intervention in future.

Noninvasive detection of atherosclerotic lesions by 99mTc-based immunoscintigraphic targeting of proliferating smooth muscle cells.

BACKGROUND: Mouse/human chimeric antibody Z2D3 identifies an antigen produced exclusively by proliferating smooth muscle cells of human atheroma, and also cross reacts with experimentally induced atherosclerotic lesions in rabbits. Fab' fragments of Z2D3 antibody were labeled with (99m)Tc using glucaric acid as a weak transchelator. The potential role of (99m)Tc-labeled Z2D3 scintigraphy was explored for noninvasive imaging of experimental atherosclerotic lesions. METHODS AND RESULTS: (99m)Tc-Z2D3 Fab' was utilized for noninvasive imaging in four rabbits with experimentally induced atherosclerotic lesions and in one control rabbit. In addition, (99m)Tc-labeled nonspecific 103D2 Fab' was used for comparison in four other rabbits with atherosclerotic lesions. The atherosclerotic lesions were induced by balloon de-endothelialization of the infradiaphragmatic abdominal aorta and 12 weeks of hyperlipidemic diet. An aliquot of 15 mCi (550 mBq) of (99m)Tc pertechnetate was incubated with 6.25 mg of glucaric acid for 30 min followed by incubation of (99m)Tc glucarate with 375 microg of Z2D3 Fab' or 103D2 Fab' for an additional 30 min. Instant thin-layer chromatography demonstrated almost complete radiolabeling. (99m)Tc-Z2D3 was administered IV and gamma imaging was performed at the time of injection, 3, 6, 9, and 12 h, followed by ex vivo imaging of the excised aorta, and biodistribution was performed. Unequivocal visualization of atherosclerotic lesions was possible in all four animals at 9 to 12 h with Z2D3 Fab'. Quantitative uptake, as represented by mean lesion-to-liver count density ratio, was 0.6+/-0.05. Imaging with nonspecific 103D2 Fab' did not show any localization in the abdominal aorta (lesion-to-liver ratio, 0.45+/-0.02, p=0.02). Ex vivo lesion-to-normal aortic segment ratio was 4.3+/-0.9 for Z2D3 and 1.04+/-0.08 for nonspecific 103D2 Fab' (p=0.01). Biodistribution studies demonstrated 0.03+/-0.003% injected Z2D3 dose per gram in the atherosclerotic lesions as compared with 0.01+/-0.003% in the nondenuded thoracic aorta of atherosclerotic rabbits (p=0.008). However, only 0.008+/-0.002% of the mean injected dose per gram was obtained in the atherosclerotic lesions (p=0.001) as compared with 0.005+/-0.003% in the normal aortic segments with 103D2. No Z2D3 uptake in normal rabbits was observed on either the in vivo or ex vivo images. CONCLUSIONS: The present study demonstrates that (99m)Tc-based immunoimaging of the vascular lesions may be feasible by the use of smaller antibody fragments. Earlier visualization is possible at the expense of a lower absolute antibody uptake in the lesions as compared to the use of intact antibody or larger fragments with longer circulating time.

Maximizing radiotracer delivery to experimental atherosclerotic lesions with high-dose, negative charge-modified Z2D3 antibody for immunoscintigraphic targeting.

BACKGROUND: Two factors that directly affect target/background ratio in immunoscintigraphy are the concentration of the antibody bound to the target and the concentration of the antibody in the circulation. High dosages of monoclonal antibody have been reported to be more efficacious in visualization of tumors. Although administration of a higher dosage of antibody increases the absolute target accumulation of the radiotracer, it also increases the background activity, which may offset this advantage. Negative charge-modified antibodies carry high specific radioactivity to the target sites without significantly increasing the background activity. Therefore we investigated whether higher dosages of negative charge-modified antibody can be used to improve imaging of experimental atherosclerotic lesions. METHODS AND RESULTS: Experimental atherosclerotic lesions were produced in 16 New Zealand White rabbits by balloon deendothelialization of the infradiaphragmatic aorta and hyperlipidemic diet for 12 weeks. Negative charge-modified Z2D3 antibody F(ab')2 specific for an antigen on proliferating smooth muscle cells of human atheroma labeled with (111)In was used for imaging experimental atherosclerotic lesions either at high (100 to 125 microg) or low (25 to 50 microg) dosages. A lower dosage of Z2D3 was labeled with 507 +/- 29.5 microCi (25 to 50 microg) (111)In label, compared with 2.9 +/- 0.24 mCi (100 to 125 microg) for the higher dosage. Although noninvasive visualization of atherosclerotic lesions was possible in all animals at 24 hours, high antibody dose allowed unequivocal visualization of the lesion as early as 3 hours after intravenous administration of the antibody. Eight animals were killed at 24 hours and the remaining eight animals at 48 hours. Mean radioactivity dose delivered per gram of lesion with the low-dose protocol at 24 hours was 0.46 +/- 0.09 microCi, which remained essentially unchanged at 48 hours (0.37 +/- 0.09 microCi; p = 0.51). With the high-dosage protocol, the total radioactivity (dose) per gram uptake in the lesion increased by about eightfold (3.49 +/- 0.58 microCi; p = 0.002) at 24 hours and was sixfold higher at 48 hours (2.21 +/- 0.45 microCi; p < 0.02). CONCLUSIONS: The study demonstrated that the increase in the dosage of negatively charge-modified antibody allows a very high delivery of specific radioactivity to the target, which in turn enables early visualization of experimental atherosclerotic lesions.

Gamma imaging of atherosclerotic lesions: the role of antibody affinity in in vivo target localization.

BACKGROUND: Monoclonal antibodies are attractive agents for noninvasive localization of various cardiovascular disorders. Because proliferating intimal smooth muscle cells are important components of atherosclerotic lesions, radiolabeled antibody Z2D3 specific for proliferating smooth muscle cells has been used for immunoscintigraphic localization of experimental atherosclerotic lesions. This study was undertaken to assess the role of antibody affinity in optimization of immunoscintigraphic localization of these lesions. Z2D3 belongs to the immunoglobulin (Ig) M class of antibodies. For immunoscintigraphic studies, attempts were made to prepare F(ab')2 or Fab fragments from the parent cell line. Fragmentation of Z2D3-IgM or its two subclones (B7 and 2B12) was not possible; therefore the parent hybridoma line was subjected to class switching. Cell lines 5C5 and 3E5 secreted antibody of the IgG1 subclass. The Z2D3-IgG1 antibodies were enzymatically digested to provide F(ab')2. Because of a tenfold loss of immunoreactivity of these class-switched antibodies, the parent clone was subsequently genetically engineered to obtain a mouse/human chimeric antibody with human IgG1 constant region. F(ab')2 of Z2D3-73.30 chimeric antibody retained the immunoreactivity relative to the original Z2D3-IgM. Radiolabeled murine and chimeric F(ab')2 fragments were used to assess the role of affinity in gamma scintigraphic visualization of experimental atherosclerotic lesions. METHODS AND RESULTS: Experimental atherosclerotic lesions were induced in 19 rabbits by abdominal aortic balloon deendothelialization followed by a hyperlipidemic diet for 12 weeks. 111In-labeled chimeric high-affinity Z2D3 F(ab')2 fragments (111In-Hi.aff Z2D3) were administered in four animals. Uptake was compared with 111In-labeled F(ab')2 of nonspecific human IgG1 (n = 4), murine low-affinity Z2D3-5C5 (111In-Lo.aff Z2D3; n = 4), nonspecific murine IgG1 monoclonal antibodies (n = 4), and 123I-labeled murine low-affinity Z2D3-3E5 (n = 3). Atherosclerotic lesions were visualized 48 hours after administration of the chimeric Hi.aff-Z2D3 antibody in all animals. Lesions were not visualized in rabbits injected with Lo.aff-Z2D3 or murine or human nonspecific antibodies. Mean percent injected dose per gram in the lesion was significantly higher with the 111In-Hi.aff-Z2D3 (0.112% +/- 0.024%) compared with 111In-Lo.aff-Z2D3 (0.037% +/- 0.005%; p = 0.03), human nonspecific (0.027% +/- 0.004%; p = 0.01), or murine nonspecific antibodies (0.006% +/- 0.001%; p = 0.0004). Nonspecific activity in unballooned thoracic aortic segments (normal) was comparable in the 111In-Hi.aff-Z2D3 (0.019 +/- 0.003) and the 111In-Lo.aff-Z2D3 (0.011% +/- 0.005%; p = 0.3) antibodies. The lesion activities of the Lo.aff-Z2D3 labeled with 111In (0.037 +/- 0.005) or 123I (0.034 +/- 0.007; p = 0.71) were similar regardless of the radioisotopes used for labeling. CONCLUSIONS: Our study demonstrates that the specificity of an antibody for the target antigen in the atheroma is a necessary condition for in vivo targeting. However, high enough affinity of an antibody is an essential component for noninvasive diagnostic visualization of experimental atherosclerotic lesions.

Noninvasive localization of experimental atherosclerotic lesions with mouse/human chimeric Z2D3 F(ab')2 specific for the proliferating smooth muscle cells of human atheroma. Imaging with conventional and negative charge-modified antibody fragments.

BACKGROUND: A murine monoclonal antibody designated Z2D3 (IgM) generated against homogenized human atherosclerotic plaques was demonstrated to be highly specific for proliferating smooth muscle cells. The primary clone subsequently was genetically engineered to provide a mouse/human chimeric antibody with human IgG1 constant region expressed in a rat myeloma cell line. The resulting Z2D3-73.30 chimeric retained the immunoreactivity relative to the parent Z2D3-IgM and was pepsin-digested to yield F(ab')2. 111In-labeled chimeric Z2D3 F(ab')2 was then used for noninvasive imaging of experimental atherosclerotic lesions. To improve the imaging characteristics, we modified chimeric Z2D3 F(ab')2 fragments to carry a high negative charge. Improved visualization of targets with 111In-labeled, negatively charged, polymer-modified antibodies most probably is the result of faster blood clearance and a decrease in nontarget background activity. METHODS AND RESULTS: Experimental atherosclerotic lesions were induced in rabbits by deendothelialization of the infradiaphragmatic aorta followed by a 6% peanut oil-2% cholesterol diet. After 12 weeks, localization of the conventionally labeled 111In-Z2D3 F(ab')2 (24 Mbq [650 microCi]/500 to 750 micrograms) (n = 4) was compared with 111In-labeled, negatively charged, polymer-modified Z2D3 F(ab')2 (24 Mbq [650 microCi]/25 to 50 micrograms) in eight atherosclerotic rabbits. Three control rabbits also received radiolabeled polymer-modified Z2D3. Ten rabbits with atherosclerotic lesions received 111In-labeled nonspecific human IgG1 F(ab')2 with (n = 6) or without (n = 4) negative charge modification. Atherosclerotic lesions were visualized in all rabbits with the conventional Z2D3 F(ab')2 at 48 hours. However, unequivocal lesion visualization was possible at 24 hours only with negatively charged, polymer-modified Z2D3 F(ab')2. Quantitative uptake of F(ab')2 fragments was essentially determined by the presence of atherosclerotic lesions (F1.37 = 69.8; P < .0001) and the specificity of the antibody (F1.37 = 36.6; P < .0001). Uptake of the conventional Z2D3 in atherosclerotic lesions (mean +/- SEM percent injected dose per gram, 0.112 +/- 0.024%) was six times higher than background activity in the normal aortic segments (nondenuded thoracic aorta; mean percent injected dose per gram, 0.019 +/- 0.003%). Uptake of the conventional Z2D3 was also significantly higher than that of nonspecific human IgG1 F(ab')2 (0.027 +/- 0.004%). Specific uptake of the conventional Z2D3 in the lesions was comparable to the charge-modified Z2D3 uptake (0.084 +/- 0.017; P = .20). Uptake of negative charge-modified Z2D3 in the lesions was significantly higher than in the corresponding background activity in normal thoracic aorta (0.021 +/- 0.002). Uptake of negative charge-modified Z2D3 F(ab')2 in the lesions was higher than the uptake of negative charge-modified nonspecific IgG1 F(ab')2 (0.020 +/- 0.002) in the lesions. Uptake of charge-modified Z2D3 in the atherosclerotic lesions was also significantly higher than the corresponding regions of the aorta of the control rabbits (0.017 +/- 0.002; F1.18 = 27.9; P = .0001). There was, however, no difference in the specific lesion uptake of negative charge-modified Z2D3 at 24 hours (0.079 +/- 0.014) and 48 hours (0.084 +/- 0.0017; P = .99) after intravenous administration. Nontarget organ activities were lower with negative charge-modified 111In-labeled Z2D3 F(ab')2 than with the conventional Z2D3 F(ab')2. Mean kidney activity was fourfold less with the modified (0.45 +/- 0.06) than with the conventionally radiolabeled (1.67 +/- 0.264; P = .001) Z2D3 F(ab')2.

Identification of human alcohol dehydrogenase isozymes by disc polyacrylamide gel electrophoresis in 7 M urea.

Polyacrylamide gel electrophoresis in the presence of 7 M urea provides a simple, reproducible method for the identification of cathodic alcohol dehydrogenase (ADH) isozymes. Treatment of native ADH dimers with 7 M urea and 1 mM dithiothreitol results in a complete dissociation of the 40,000 Mr subunits. Electrophoresis of urea-dissociated ADH isozymes yields a single protein band for homodimers and two bands of equal intensity for heterodimers. The ADH subunits pi, alpha, gamma 2, gamma 1, and beta exhibit electrophoretic mobilities of 0.71, 0.79, 0.88, 0.95, and 1.0, respectively. Thus, the identity of any cathodic ADH isozyme can be determined from the electrophoretic mobilities of its component subunits.

Physical and enzymatic properties of a class II alcohol dehydrogenase isozyme of human liver: pi-ADH.

Homogeneous class II alcohol dehydrogenase (pi-ADH) has been isolated from human liver homogenates by chromatography on DE-52 cellulose, 4-[3-[N-(6-amino-caproyl)amino]propyl]pyrazole-Sepharose, SP-Sephadex C-50, and agarose-hexane-AMP, yielding an enzyme that has a significantly higher specific activity and is markedly more stable than that isolated by an earlier procedure. pi-ADH is composed of two identical 40 000-dalton subunits, contains 4 mol of zinc/dimer, and is readily inhibited by metal-chelating agents. The purified enzyme binds two molecules of coenzyme per dimer, exhibits an absorption maximum at 280 nm, epsilon 280 = 57 000, and exhibits an isoelectric point of 8.6. The class II isozyme catalyzes the oxidation of a variety of alcohols with Km values ranging from 7 microM to 560 mM and with kcat values from 32 min-1 to 600 min-1 and demonstrates a preference for hydrophobic substrates. The kcat/Km ratio for ethanol oxidation exhibits a pH maximum at 10.4.