ABSTRACT: 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.
(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.
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.
Chest 07/1997; 111(6):1684-90. · 5.25 Impact Factor
Journal of nuclear biology and medicine (Turin, Italy: 1991) 36(2 Suppl):35-40.
ABSTRACT: 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.
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.
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.
Journal of Nuclear Cardiology 3(3):231-41. · 2.67 Impact Factor
ABSTRACT: 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.
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.
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.
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.
Journal of Nuclear Cardiology 5(6):551-7. · 2.67 Impact Factor