Noninvasive Localization of Experimental Atherosclerotic Lesions With Mouse/Human Chimeric Z2D3 F(ab′)
2
Specific for the Proliferating Smooth Muscle Cells of Human Atheroma
Authors: Narula Jagat , Petrov Artiom , Bianchi Cesario , Ditlow Charles C. , Lister Bradford C. , Dilley Jeanette , Pieslak Isabella , Chen Francis W. , Torchilin Vladimir P. , Khaw Ban-An 
Published: June 2012

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 IgG
1
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
.
111
In-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
111
In-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
111
In-Z2D3 F(ab′)
2
(24 Mbq [650 μCi]/500 to 750 μg) (n=4) was compared with
111
In-labeled, negatively charged, polymer-modified Z2D3 F(ab′)
2
(24 Mbq [650 μCi]/25 to 50 μg) in eight atherosclerotic rabbits. Three control rabbits also received radiolabeled polymer-modified Z2D3. Ten rabbits with atherosclerotic lesions received
111
In-labeled nonspecific human IgG
1
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 (
F
1.37
=69.8;
P
<.0001) and the specificity of the antibody (
F
1.37
=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 IgG
1
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 IgG
1
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;
F
1.18
=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
111
In-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
.

Conclusions
The present study demonstrates the feasibility of noninvasive visualization of experimental atherosclerotic lesions with a mouse/human chimeric antibody Z2D3 F(ab′)
2
directed against the proliferating smooth muscle cells. Furthermore, negative charge modification of the chimeric Z2D3 F(ab′)
2
enabled (1) earlier visualization of the atherosclerotic lesions, (2) use of 10- to 15-fold less antibody than with conventional Z2D3 F(ab′)
2
, and (3) reduction of the radiation burden to nontarget organs.