title: 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.
abstract: 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.
  
  
  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.
authors.affiliation: Center for Drug Targeting and Analysis, Northeastern University, Boston, Mass., USA.
authors.forename: J
authors.initials: J
authors.lastname: Narula
authors.forename: A
authors.initials: A
authors.lastname: Petrov
authors.forename: C
authors.initials: C
authors.lastname: Bianchi
authors.forename: C C
authors.initials: CC
authors.lastname: Ditlow
authors.forename: B C
authors.initials: BC
authors.lastname: Lister
authors.forename: J
authors.initials: J
authors.lastname: Dilley
authors.forename: I
authors.initials: I
authors.lastname: Pieslak
authors.forename: F W
authors.initials: FW
authors.lastname: Chen
authors.forename: V P
authors.initials: VP
authors.lastname: Torchilin
authors.forename: B A
authors.initials: BA
authors.lastname: Khaw
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification; D001161:Arteriosclerosis / Q000000981:diagnostic imaging* / Q000276:immunology / Q000473:pathology; D002455:Cell Division / Q000276:immunology; D015195:Drug Design; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000276:immunology; D007205:Indium Radioisotopes; D051379:Mice; D009131:Muscle, Smooth, Vascular / Q000276:immunology* / Q000473:pathology; D011108:Polymers; D011817:Rabbits; D011877:Radionuclide Imaging; D011993:Recombinant Fusion Proteins / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D007205:Indium Radioisotopes; D011108:Polymers; D011993:Recombinant Fusion Proteins