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.