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Circulation. 1995;92:474-484

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(Circulation. 1995;92:474-484.)
© 1995 American Heart Association, Inc.

Articles

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

Presented in part at 1992 (New Orleans, La, November) and 1993 (Atlanta, Ga, November) Scientific Sessions of the American Heart Association.

Jagat Narula, MD, PhD; Artiom Petrov, PhD; Cesario Bianchi, MD, PhD; Charles C. Ditlow, PhD; Bradford C. Lister, PhD; Jeanette Dilley, BS; Isabella Pieslak, BS; Francis W. Chen, PhD; Vladimir P. Torchilin, DSc; Ban-An Khaw, PhD

From Center for Drug Targeting and Analysis (J.N., A.P., B.C.L., B.-A.K.), Northeastern University, Boston, Mass; Massachusetts General Hospital (J.N., C.B., V.P.T., B.-A.K.), Harvard Medical School, Boston, Mass; and Scotgen Biopharmaceuticals Inc (C.C.D., J.D., I.P., F.W.C.), Menlo Park, Calif.

Correspondence to Ban-An Khaw, PhD, George D. Behrakis Professor of Pharmaceutical Sciences, Northeastern University, Director, Center for Drug Targeting and Analysis, Bouvé College of Pharmacy, 205 Mugar Building, 360 Huntington Avenue, Boston, MA 02115.


*    Abstract
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 *Abstract
down arrowIntroduction
 down arrowMethods
down arrowResults
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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 µCi]/500 to 750 µg) (n=4) was compared with 111In-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 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.

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.


Key Words: atherosclerosis • imaging • angioplasty • antibodies • smooth muscle cells


*    Introduction
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 up arrowAbstract
 *Introduction
down arrowMethods
down arrowResults
down arrowDiscussion
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Arteriography, an invasive procedure, is the standard technique for localization of atherosclerotic lesions. A noninvasive test that identifies metabolically active lesions may provide a window for the atherosclerotic process. Such a test may be important not only for diagnosis but also for the development and monitoring of therapies directed at altering the natural history of these lesions. There are a limited number of reports describing noninvasive visualization of atherosclerotic lesions. These studies have targeted the thrombotic component overlying the atherosclerotic lesion (with radiolabeled fibrinogen),1 2 platelet aggregation at regions of turbulent flow (with labeled platelets or platelet-specific antibodies),3 4 5 or proteins likely to be incorporated into atherosclerotic lesions (with radiolabeled autologous lipoproteins).6 7 8 9 10 11 Nonspecific uptake of human {gamma}-globulin through Fc receptors of macrophages has also been used as the basis for a targeting strategy.12 13

Atherogenesis involves interactions among the injured endothelium and platelets and mononuclear cells, the attendant release of growth factors, and the consequent acquisition by otherwise quiescent medial smooth muscle cells of migratory and proliferative properties.14 15 16 17 18 Although the molecular mechanisms of the atherogenic process are still not clear, some of the events may be amenable to exploratory and diagnostic research. We reasoned that if a diagnostic test could identify an early as well as metabolically active lesion, it would permit therapeutic or dietary interventions to have maximal benefit. Because all active atheromatous lesions contain proliferating smooth muscle cells, these cells provide a logical target.

Therefore, a monoclonal IgM antibody, Z2D3, was initially developed with specificity for an antigen associated with the proliferating smooth muscle cells of human atherosclerotic lesion. Subsequently, a mouse/human chimeric monoclonal antibody, Z2D3, was developed that retained exclusive specificity for the antigen. In the present study, F(ab')2 fragments of the chimeric Z2D3 labeled with 111In were used to demonstrate the feasibility of noninvasive imaging of experimental atherosclerotic lesions in rabbits. Furthermore, to enhance immunotargeting of this antibody, chimeric Z2D3 F(ab')2 was modified to carry a highly negatively charged polymer with multiple diethylenetriaminepenta-acetic acid (DTPA). This enabled the use of Z2D3 F(ab')2 with very high specific radioactivity, better in vivo localization characteristics, and earlier visualization of experimental atherosclerotic lesions than with the conventional 111In-labeled antibody.


*    Methods
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 up arrowAbstract
 up arrowIntroduction
 *Methods
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Z2D3 Antibody: Development, Characterization, Specificity, and Affinity
Development of Z2D3 Antibody
Isolated, homogenized, postmortem human atheromatous plaques were used for the development of murine monoclonal antibodies. Hybridoma cell line Z2D3 (as well as its two subclones, B7 and 2B12) produced an IgM ({kappa} light chain) monoclonal antibody. The antibody demonstrated intense immunoperoxidase staining of frozen sections of atheromatous plaques in postmortem human coronary artery (Fig 1ADown).19 A nonspecific IgM antibody used for comparative purpose did not localize in the human coronary artery atherosclerotic lesion (Fig 1BDown), and Z2D3 antibody did not localize in the control artery section. The antibody also specifically cross-reacted with experimental atherosclerotic lesions in the rabbit aorta (Fig 1CDown). Comparative immunohistological studies of subjacent frozen sections of rabbit aorta with Z2D3 antibody, another standard antibody specific for proliferating cell nuclear antigens (PCNA)20 (Fig 1DDown), and one specific for smooth muscle {alpha}-actin21 demonstrated Z2D3 antibody to stain atherosclerotic lesions containing proliferating smooth muscle cells. The regions of immunohistochemical staining for macrophages were also distinctly different from the regions of Z2D3 staining (Fig 1EDown through 1G). The antigen homologous for Z2D3 antibody has not been completely characterized. Results indicate (unpublished data) that the epitope may be a complex of two or more chemically dissimilar, low-molecular-weight molecules. Without more detailed knowledge, the antigen cannot be quantified. Therefore, relative affinity of the antibody was determined by immunohistochemical studies of frozen sections of rabbit aorta. The relative affinity of the antibody was represented as the concentration of antibody (micrograms per milliliter in phosphate-buffered saline) required for an intensity of immunohistochemical staining score of 3+ (Fig 1CDown). The parent Z2D3 IgM clone had a relative affinity (avidity) of 6.25x10-8 mol/L at 50 µg/mL for 3+ staining.



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  		Figure 1. The specificity of Z2D3 localization as demonstrated by immunoperoxidase staining of frozen sections obtained from atherosclerotic human coronary artery (A, B, E, and F) and from rabbit aorta with experimentally induced atherosclerotic lesions (C, D, and H). A, Staining of the human atherosclerotic lesion with Z2D3 parent IgM antibody; B, no staining in the same region of a serial frozen section with a nonspecific control IgM antibody. C, Cross-reactivity of Z2D3 IgM with experimentally induced atherosclerotic lesion in the rabbit. D, A comparative immunohistochemical study shows that the areas of nuclear staining with an antibody specific for the proliferating cell nuclear antigen correspond to the areas stained by Z2D3 in an experimental atheromatous lesion. Medial smooth muscle cells do not stain with the antibody specific for proliferating cell nuclear antigen. Furthermore, comparison of staining with anti-macrophage antibody (E) and Z2D3 antibody (F) demonstrates that the regions of macrophage/foam cell concentration and Z2D3 concentration are mutually exclusive in a human coronary atherosclerotic lesion. The outline of the anti-macrophage antibody section has been traced to show the area of macrophage presence (large dots and curved arrows) (G). On this outline, the area of Z2D3 staining (hatched areas) has been superimposed (G). The area enclosed by the thick black line represents calcification; the area with tiny dots is the intima; and striped areas represent the medial layer of the artery. The open area above the Z2D3 concentration (G) is the area of the narrowed lumen of the artery. H, Demonstrates the high relative affinity of genetically engineered Z2D3-73.30 antibody for the experimentally induced atherosclerotic lesion in a rabbit aorta. F(ab')2 fragments of this antibody were used for noninvasive gamma imaging studies.

Genetically Engineered Z2D3 Antibody
To optimize the use of Z2D3 for in vivo targeting and visualization of atherosclerotic lesions, mouse/human chimeric Z2D3 was engineered from parent Z2D3-2B12.22 23 24 25 26 For this purpose, total cytoplasmic RNA was isolated from approximately 107 cells in the late logarithmic growth phase. First-strand DNA was prepared from Z2D3 mRNA by reverse transcription with primer sequences based on the 5' ends of the murine IgM and {kappa} constant regions. The VH and VK then were amplified with use of the polymerase chain reaction. The Z2D3 VH and VK fragments were cloned into M13 vectors containing the heavy-chain immunoglobulin promoter, signal sequence, and appropriate splice sites. The VH and VK genes together with their appropriate expression elements then were excised from their respective M13 vector and cloned into pSVgpt and pSVhyg, respectively. pSVgpt contains an immunoglobulin gene enhancer sequence, SV40 origin of replication, the gpt gene (for selection), and genes for replication and selection in Escherichia coli. A human IgG1 constant region24 was also added to pSVgpt. The pSVhyg vector (for expression of the light chain) was essentially the same as pSVgpt, except that the gpt gene was replaced with the hygromyecin-resistant gene and a human {kappa} chain constant region was added.25 Subsequently, YB2/O rat myeloma cells were cotransfected by electroporation with constructed heavy- and light-chain expression vectors. gpt+ transfects from the wells showing the highest concentration of chimeric antibody were expanded, and the antibody was purified by protein A chromatography. The resulting 73.30 chimeric produced IgG1 antibody, which showed immunoreactivity comparable to the parent Z2D3 IgM (Fig 1HUp). The chimeric antibody was fragmented to F(ab')2 by pepsin digestion. Genetically engineered Z2D3-73.30 and its pepsin-digested F(ab')2 consistently required concentrations of less than 50 µg/mL for 3+ staining (relative affinity, 3.33 to 4.55x107 mol/L or 6.6 to 9.1x10-8 mol/L after normalization to the decavalency of Z2D3 IgM) in experimentally induced atherosclerotic lesions, comparable to the staining with parent IgM (Fig 1CUp).

Processing and Radiolabeling of Z2D3-73.30 F(ab')2
F(ab')2 fragments of the chimeric Z2D3 antibody were either covalently linked with DTPA (Sigma Chemical Co) by the bicyclic anhydride method27 (designated as conventional Z2D3 hereafter) or were modified to carry a highly negatively charged polymer28 (designated as modified Z2D3 hereafter) to be used for imaging of experimental atherosclerotic lesions.

Preparation of Conventional DTPA-Z2D3 Antibody
Z2D3-(Fab')2 fragments were covalently coupled with a bifunctional chelating agent—DTPA using bicyclic anhydride of DTPA according to the published procedure.27 In this preparation, the molar ratio of DTPA to Z2D3-F(ab')2 as assessed by mass spectroscopy and gadolinium-labeled magnetic resonance spectroscopy was 2:1.

Preparation of Negatively Charged, Polymer-Modified Z2D3-F(ab')2
A highly negatively charged polymer consisting of starting polymer polylysine (PL) that had been linked with multiple DTPA and succinylated was conjugated to chimeric Z2D3 F(ab')2 or nonspecific human F(ab')2 (Fig 2Down). Briefly, solid bicyclic anhydride of DTPA was added slowly to PL with an average molecular weight of 9800 to a final ratio of 10 mg DTPA/1 mg PL (pH 7.2 to 8.2). Solid succinic anhydride (Sigma Chemical Co) was added slowly with stirring to a final ratio of 10 mg succinic anhydride/1 mg PL. Subsequently, carbodiimide activation of carboxyl groups on DTPA-succinylated PL was performed according to a modification of the method of Khaw et al.28 The activated DTPA-PL fractions were added immediately to Z2D3-F(ab')2 or nonspecific F(ab')2. The conjugate was then purified by ion exchange chromatography on Sephadex-DEAE A25 (Pharmacia). The elution conditions for this step were adjusted (0.35 mol/L sodium chloride in sodium phosphate) so that only conjugates with a 1:1 F(ab')2-to-polymer ratio were eluted in the fraction of interest. Free F(ab')2 fragments were not retained by the column and were eluted in the wash through fraction. Aggregates containing multiple polymer chains were bound tightly to column and were not eluted in 0.35 mol/L sodium chloride–sodium phosphate. Analytical size exclusion high-performance liquid chromatography (Bio-Rad SEC-250) of the purified conjugate exhibited a single symmetrical peak of molecular weight consistent with the predicted weight of a 1:1 conjugation ratio (130 000 D).



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  		Figure 2. Flow chart of the synthesis of negatively charged chelating polymer (DTPA-PL) and its conjugation to chimeric Z2D3 F(ab')2. DTPA ca indicates bicyclic anhydride of DTPA; Su2O, succinic anhydride; CDI, water-soluble carbodiimide; NHSS, N-hydroxy-sulfosuccinimide.

Radiolabeling of Z2D3 F(ab')2 With 111In
To 1 aliquot of 111In chloride ({approx}700 to 800 µCi) an equal volume of 1 mol/L sodium citrate (pH 5.5) was added, followed by 1 aliquot of conventional (500 to 750 µg) or modified (25 to 50 µg) Z2D3-F(ab')2.29 The reaction mixture was allowed to incubate at room temperature for 30 to 45 minutes. Antibody-bound 111In was separated from free 111In by the use of Sephadex G-25 column chromatography. The peak tubes in the void volume containing the radiolabeled antibody were pooled and used within 1 hour of radiolabeling. An average of 95% of the initial antibody concentration was recovered in the peak tubes containing the radiolabeled antibody.

Experimental Model and Protocol
Experimental Atherosclerotic Lesions
Male New Zealand White rabbits weighing 2.5 to 3.0 kg (Charles River Farm) were maintained on a custom-made 2% cholesterol–6% peanut oil diet (ICN Biomedicals) for 3 months. After 1 week of the hyperlipidemic diet, the abdominal aorta was denuded of the endothelium by a modified Baumgartener technique.30 Briefly, each animal was anesthetized with a mixture of ketamine and xylazine (100 mg/mL, 10:1 vol/vol; 1.5 to 2.5 mL sc), and the right femoral artery was isolated. A 4F Fogarty embolectomy catheter (12-040-4F; Edwards Laboratories Inc) was introduced through an arteriotomy and advanced under fluoroscopic guidance to the level of the diaphragm. The catheter was inflated to a pressure of {approx}3 psi above the balloon inflation pressure with radiographic contrast media (Conray, Mallinckrodt). Three passes were made down the abdominal aorta with the inflated catheter. The femoral artery was then ligated, and the wound was closed. The animals were allowed to recover from anesthesia and then returned to their cages. This protocol has been approved by both Northeastern University and Massachusetts General Hospital animal studies committees and is in compliance with National Institutes of Health–approved guidelines.

Classification of the Animals Used
Immunoscintigraphic studies were performed in a total of 25 rabbits (Table 1Down). Twenty-two were rabbits with aortic deendothelialization that had had 12 weeks of the hyperlipidemic diet, and 3 were control rabbits. The conventional 111In-labeled Z2D3 F(ab')2 was used in 4 atherosclerotic rabbits and compared with 4 atherosclerotic rabbits injected with nonspecific conventionally labeled human IgG1 F(ab')2. 111In-labeled negative charge–modified Z2D3 F(ab')2 was administered intravenously to 8 atherosclerotic rabbits. Three control rabbits were injected with negative charge–modified Z2D3 F(ab')2, and 6 atherosclerotic rabbits were injected with negative charge–modified nonspecific human IgG1 F(ab')2. All studies were performed for 48 hours, except for 7 atherosclerotic rabbits that were studied for 24 hours; 4 of these 7 rabbits received modified chimeric Z2D3, and the remaining 3 rabbits received charge-modified nonspecific human IgG1 F(ab')2 (Table 1Down).


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  		Table 1. Classification of Animals Receiving Various Antibodies

Radioimmunoscintigraphy
Approximately 650 µCi (24 Mbq) of 111In-labeled negative charge–modified or conventional specific or nonspecific F(ab')2 was injected into the marginal ear vein of rabbits. Serial blood samples were obtained from the opposite ear during the ensuing 24 to 48 hours for determination of blood clearance of various radiotracer preparations. Serial gamma images in the anterior and lateral decubitus views were recorded at the time of injection and at 24 and 48 hours with a standard field-of-view gamma camera (Series 100, Ohio Nuclear) equipped with a medium-energy, parallel-hole collimator. At each imaging session, 3 mL of blood was drawn into a syringe and placed on the right side of the animal as a reference for blood pool activity in the images. The pulse height analyzers were set at the centerlines of 173 and 247 keV to record both photopeaks of indium with symmetrical 15% windows. Planar images were obtained for 5 minutes (x3) at 0 hours and for 10 minutes (x3) at 24 and 48 hours in a 256x256 matrix. Each image was examined separately. Target-to-liver activity ratios (T/L) were obtained with the use of computer planimetry. The lesion in the area of the descending aorta was planimetered for target activity, and an area over the hepatic silhouette was used to obtain liver activity. In control rabbits and in atherosclerotic rabbits injected with nonspecific antibody, the region of descending abdominal aorta was planimetered. At the end of the final in vivo imaging session, the animals were administered heparin and given a lethal injection of intravenous sodium pentobarbital. The aorta was removed en bloc and washed free of blood with saline. The adventitia were removed completely, and the aorta was opened along the ventral surface. All aortas were examined visually and then photographed or the outline of the aorta and lesions was traced for comparison with the ex vivo studies. The isolated aorta was then imaged ex vivo by laying it flat directly on the gamma camera for 15x2, or 30, minutes in a 256x256 matrix. After imaging, the aorta was divided into three segments: arch (just distal to left subclavian artery), thoracic (nondenuded region), and abdominal (balloon-denuded region). All segments were weighed, and the samples were counted in an automatic well–type gamma counter (model 1282, LKB Compugamma). Counts-per-minute activities were obtained, and the uptake of the antibody in the samples was calculated and represented as percent total injected dose per gram of the tissue. Immediately after being counted, the aortas were reassembled and the opened vessels were covered with a single layer of plastic wrap. The reassembled aorta was placed en face on high-speed x-ray film (Kodak Ortho-Film OH-1) and stored in a cassette (Kodak X-Omatic) at -80°C. All macroautoradiographs were exposed for 1 week, after which the radiographs were developed in a 90-second X-Omat processor. Random samples of the aorta were subjected to histological and histochemical confirmation of atherosclerotic lesions.

Statistical Analysis
All animals used in the present study were of the same age, sex, weight, and size and were obtained from the same source. All were subjected to the same protocol for induction of experimental atherosclerotic lesions. Antibody and radioactivity dosages, imaging protocols, and ex vivo data collection were standardized. The chimeric Z2D3 and nonspecific F(ab')2 (regardless of negative charge modification) were prepared simultaneously, and the animal experiments were randomly performed using one antibody or the other. Animal experiments with negative charge–modified F(ab')2 were performed after completion of experiments with conventionally labeled F(ab')2.

Percent total injected dose per gram uptake of Z2D3 and nonspecific F(ab')2 fragments in tissue samples were represented as mean±SEM and were arcsine-transformed before analysis. The main experimental design was multifactorial with each factor having two levels, ie, a 2k design. Because time of death (24 and 48 hours) was not a significant factor (P=.99), we set k=3 for the ANOVA. The three factors analyzed were aortic site of antibody uptake (lesion versus normal aorta), charge modification of the antibodies (conventional versus negative charge–modified), and antibody specificity (specific versus nonspecific). The experiments comparing antibody uptake in experimental and control rabbits were analyzed with the use of a two-factor ANOVA. The factors were animal model (controls versus experimental rabbits) and uptake site.


*    Results
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 up arrowIntroduction
 up arrowMethods
*Results
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In Vivo and Ex Vivo Image Analysis
The left lateral decubitus gamma images of rabbits with experimental atherosclerosis injected with the conventional 111In-labeled chimeric Z2D3 F(ab')2 showed primarily blood pool activity at the time of tracer administration. At 24 hours, lesion activity could not be distinguished from blood pool activity. However, by 48 hours, unequivocal delineation of the atheromatous lesions was possible (Fig 3ADown). The T/L of the antibody uptake was 0.486±0.047 (Table 2Down). Rabbits with experimental atherosclerotic lesions that had been injected with 111In-labeled nonspecific human IgG1 F(ab')2 showed no localization of the radiotracer in the region of experimental atheromas (Fig 3BDown; T/L, 0.341±0.013; P=.01; Table 2Down). The lack of radiotracer uptake with nonspecific IgG1 F(ab')2 was further confirmed in the ex vivo image of the excised aorta (Fig 3DDown) despite the similarity in the severity of the atheromatous lesions in the nonspecific and specific antibody groups. Ex vivo imaging of the excised atherosclerotic aortas from the animals injected with 111In-labeled conventional Z2D3 F(ab')2 showed tracer localization in regions corresponding to the in vivo radiotracer localization (Fig 3ADown and 3CDown). The tracer localization was further confirmed by the comparison of the macroautoradiograph (Fig 3EDown) with the gross pathological specimen (Fig 3FDown).



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  		Figure 3. Comparison of the left lateral oblique gamma images of rabbits injected with 111In-labeled conventional Z2D3 F(ab')2 (A) and nonspecific F(ab')2 (B) for visualization of experimental atherosclerotic lesions at 48 hours. Focal accumulation of radiolabeled Z2D3 is seen (solid arrows) between the vertebral column activity (open arrow) and the kidney activity (bottom K). No localization of the nonspecific F(ab')2 is seen anterior to the vertebral column (open arrow) or behind the kidney (K) and urinary bladder (U) activities. The in vivo localization of Z2D3 in A corresponds to the regions of tracer accumulation observed in the ex vivo image (C). The nonspecific antibody did not show localization in the lesions in the ex vivo image (D) despite the presence of significant atherosclerosis observed at necropsy. Macroautoradiograph of the aorta of the animal injected with the 111In-labeled conventional Z2D3 F(ab')2 (E) shows localization of the radioactivity in the pathological lesions (F). The ex vivo aortic image (C) is divided into arch (ar), thoracic (th), and abdominal (ab) segments.


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  		Table 2. Target (Atherosclerotic Lesion)-to-Liver Activity Ratio of Z2D3 and Nonspecific F(ab')2 in Experimental and Control Rabbits by Computer Planimetry

In one of the four rabbits that received the conventional Z2D3 F(ab')2, in vivo focal tracer localization was visualized only in the pathologically most severe lesion. In most experimental animals, the arch segments (which were not denuded) almost always developed spontaneous atherosclerotic lesions. Although these lesions were not detectable with the use of in vivo gamma imaging, ex vivo images and macroautoradiographs invariably demonstrated equally intense radiotracer localization.

With the use of 111In-labeled negative charge–modified Z2D3 F(ab')2, the non–target organ radioactivity distribution decreased, and the blood clearance increased. The left lateral oblique gamma image at the time of intravenous administration showed blood pool activity (Fig 4ADown). Unequivocal delineation of the atheromatous lesions was feasible 24 hours after intravenous administration of the negative charge–modified antibody (Fig 4BDown). The lesion activity was reconfirmed in the 48-hour image (Fig 4CDown; T/L, 0.40±0.021). The regions of radiotracer accumulation seen in the in vivo images were confirmed by ex vivo gamma imaging (Fig 4DDown) and macroautoradiography of the excised descending aorta (Fig 4EDown), which corresponded to the gross pathological lesions in the descending aorta (white regions, Fig 4FDown).



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  		Figure 4. Left lateral oblique gamma images of an experimental atherosclerotic rabbit injected with negative charge–modified 111In-labeled chimeric Z2D3-73.30 F(ab')2 immediately after intravenous administration (A) and at 24 (B) and 48 (C) hours. The lesions can be unequivocally visualized in the 24- and 48-hour images (solid arrows) anterior to the vertebral activity (open arrow). The corresponding ex vivo image (D), macroautoradiograph (E), and the photograph (F) of the gross pathological sample of the descending aorta including the arch (ar), thoracic (th), and abdominal (ab) aorta regions show intense tracer localization corresponding to the region of atherosclerotic lesions. In F, the lesions are seen as white areas and normal aorta as clear regions. L indicates liver; K, kidney.

The 111In-labeled negative charge–modified nonspecific human IgG1 F(ab')2 did not localize in the atheromas (Fig 5Down). The immediate post–intravenous tracer administration image showed primarily blood pool activity (Fig 5ADown) similar to that seen in Fig 4AUp. However, by 24 hours, there was no activity in the region of the atheromatous abdominal aorta, except for minimal residual blood pool activity (Fig 5BDown, solid arrow), which cleared completely by 48 hours (Fig 5CDown; T/L, 0.178±0.029; P=.002). The corresponding ex vivo gamma image of the descending aorta (Fig 5DDown) and the macroautoradiograph (Fig 5EDown) showed minimal nonspecific radiotracer accumulation, which did not correspond to the severity of the lesion by gross pathological examination of the aorta (Fig 5FDown). Similarly, when 111In-labeled negative charge–modified Z2D3 F(ab')2 was administered to control rabbits, no radioactivity accumulated in the descending aorta by in vivo (T/L, 0.225±0.026; P=.005; Fig 6ADown through 6C) or ex vivo (Fig 6DDown) gamma imaging and macroautoradiography (Fig 6EDown). On gross inspection the specimen also did not show presence of any lesions (Fig 6FDown).



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  		Figure 5. Left lateral oblique gamma images of an experimental atherosclerotic rabbit injected with negative charge–modified 111In-labeled nonspecific human IgG1 F(ab')2 immediately after intravenous administration (A) and at 24 (B) and 48 (C) hours. The image obtained immediately after radiotracer administration shows predominantly a blood pool image similar to that seen in Fig 4AUp. At 24 (B) and 48 (C) hours later, no aortic lesion activity is observed in the area of the descending aorta anterior to the vertebral column activity (open arrow). Minimal blood pool activity is seen at 24 hours (solid arrow in B), which clears completely by 48 hours (C). The ex vivo gamma image (D) and the macroautoradiograph (E) of the excised aorta do not show radiotracer localization in areas corresponding to the gross pathological lesions (white areas in F). L indicates liver; K, kidney.



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  		Figure 6. Left lateral oblique gamma images of a control rabbit injected with negative charge–modified 111In-labeled chimeric Z2D3-73.30 F(ab')2 immediately after intravenous administration (A) and at 24 (B) and 48 (C) hours. Blood pool image is seen in A. The activity anterior to the ventral aspect of the rabbit represents activity in a syringe of blood obtained just before commencement of data acquisition. No negative charge–modified 111In-labeled chimeric Z2D3-73.30 F(ab')2 localization is observed in the region of the normal descending aorta (C and D) in the in vivo or in the ex vivo images (D) and macroautoradiograph (E). Gross examination shows the aorta to be free of lesions (F). L indicates liver; K, kidney; open arrow, vertebral activity.

Blood Clearance of 111In-Labeled Z2D3 F(ab')2
Blood clearance of the modified Z2D3 was significantly faster compared with the conventional Z2D3. At 24 hours, 20±2% (mean percent injected dose per gram, 0.095±0.011) activity remained in the circulation compared with 11.9±1.1% (mean percent injected dose per gram, 0.053±0.007) at 48 hours for the negative charge–modified Z2D3 F(ab')2 (Fig 7Down). On the other hand, residual mean percent dose per gram activity of the conventional Z2D3 F(ab')2 at 24 hours was 0.294±0.039 (P=.0001). Residual activity at 48 hours was 0.105±0.013 (P=.014), which was equal to the 24-hour residual activity of the negative charge–modified Z2D3. In vitro blood clearance data corroborated the in vivo imaging data where no blood pool was seen in the descending aorta region of control animals with modified Z2D3 by 24 hours and with the conventional Z2D3 by 48 hours.



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  		Figure 7. Plot of blood clearance of 111In-labeled negative charge–modified chimeric Z2D3 F(ab')2 in control rabbits. The temporal blood activities represent biexponential clearance (mean t1/2=757 minutes) with an initial fast component (t1/2 {alpha}=148 minutes) followed by a slower component (t1/2 ß=1472 minutes). The 24- and 48-hour mean blood activity values for the conventional 111In-labeled Z2D3 F(ab')2 are also shown (·I).

Blood clearance of 111In-labeled negative charge–modified Z2D3 F(ab')2 in control rabbits was used for assessment of half-life. The temporal blood activities represented biexponential clearance (mean t1/2=757 minutes) with an initial fast component (t1/2 {alpha}=148 minutes) followed by a slower component (t1/2 ß=1472 minutes).

Antibody Uptake in Atherosclerotic Lesions
Accumulation of the conventional Z2D3 F(ab')2 in atherosclerotic lesions in the balloon-denuded (abdominal) region of the aorta was sixfold greater than that in the nondenuded (thoracic) region (Fig 8Down). The mean±SEM percent injected dose per gram localization of conventional Z2D3 F(ab')2 in the specimens with lesion (0.112±0.024) was significantly higher than the background activity in the normal specimens (0.019±0.003). The conventional 111In Z2D3 F(ab')2 accumulation in the aortic lesions was also significantly higher than that of 111In-labeled nonspecific human IgG1 F(ab')2 (0.027±0.004). The arch segments (which were not denuded) also showed a similar increase in antibody uptake, comparable to that of the abdominal balloon-denuded segments.



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  		Figure 8. Bar graph showing mean percent injected dose per gram in the lesions is significantly higher with the conventional (NoPL-48H) and modified chimeric Z2D3 F(ab')2 (PL-24H, PL-48H) than with the corresponding control nonspecific human IgG1 F(ab')2 fragments with (PL-NS-24H or PL-NS-48H) or without (NoPL-NS-48H) negative charge modification. PL-NOR represents biodistribution of modified Z2D3 in the normal control rabbits. No significant difference is observed in the lesion activity of the modified Z2D3 F(ab')2 at 24 and 48 hours after the administration of the antibody. PL indicates polylysine.

Negative charge–modified chimeric Z2D3 F(ab')2 accumulation in the aortic lesions (0.079±0.014 at 24 hours and 0.084±0.017 at 48 hours) was comparable to the conventional Z2D3 F(ab')2 localization and was significantly greater than the background activity of negative charge–modified antibody in the nondenuded normal segments of the thoracic aorta (0.023±0.003 at 24 hours and 0.021±0.002 at 48 hours, respectively). Uptake of modified Z2D3 F(ab')2 in the lesions was also significantly higher than 111In-labeled negative charge–modified nonspecific human IgG1 F(ab')2 (0.025±0.007 at 24 hours and 0.020±0.002 at 48 hours, respectively). Similar to the conventional antibody, uptake of the modified Z2D3 was also high in spontaneous lesions of the unballooned arch segments. 111In-labeled negative charge–modified Z2D3 administered to three control rabbits did not show tracer localization in the (normal) abdominal segment of the aorta. Mean background tracer activity in the aorta of control rabbits (0.017±0.002) was similar to the background, nonspecific antibody sequestration in nondenuded thoracic segments (0.021±0.002) of atherosclerotic rabbits. No significant difference in the negative charge–modified Z2D3 incorporation in the aortic lesions was seen between 24 (0.079±0.014) and 48 hours (0.084±0.017) after administration of radiolabeled negative charge–modified antibodies.

The results of the three-factor ANOVA indicated that the uptake of the antibody in the aortas of experimental animals was determined only by site (ie, lesion versus normal aorta; F1.37=69.8; P<.0001) and the specificity of the antibody [ie, Z2D3 versus F(ab')2 fragments of nonspecific human IgG1; F1.37=36.6; P<.0001]. The interaction between the aortic site and specificity of the antibody was also significant, indicating that the antibody uptake due to specificity was dependent on where uptake was measured (F1.37=9.2; P=.004). Charge modification of the antibody did not affect the quantitative antibody uptake in the atherosclerotic lesions (F1.37=1.7; P=.20). The two-factorial ANOVA for the comparison of Z2D3 uptake in experimental and control rabbits indicated that rabbit type (experimental versus control; F1.18=27.9; P=.0001), uptake site (lesion versus normal aorta; F1.18=18.2; P=.0005), and the interaction between these two factors (F1.18=16.6; P=.0007) were significant.

Biodistribution in Nontarget Organs
Biodistribution of the conventionally labeled Z2D3 F(ab')2 in nontarget organs showed significant differences compared with that of modified Z2D3 F(ab')2 (Fig 9Down). Mean percent injected dose per gram sequestration of the modified chimeric Z2D3 F(ab')2 in all nontarget organs was lower than that of conventionally labeled antibody. The most significant difference was observed in the kidney cortices where negative charge modification reduced the radiation burden by 3.7-fold (0.045±0.058 versus 1.67±0.264; P=.001). Other organs demonstrated, although to a lesser extent, a decrease in nonspecific sequestration of the antibody, such as in heart (0.026±0.002 versus 0.044±0.010; P=NS), spleen (0.073±0.004 versus 0.138±0.044; P=.07), kidney medulla (0.047±0.01 versus 0.118±0.02; P=.02), lungs (0.023±0.002 versus 0.055±0.002; P=.0001), and liver (0.069±0.016 versus 0.087±0.022; P=NS).



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  		Figure 9. Bar graph of biodistribution data showing modified Z2D3 F(ab')2 localization in nontarget organs to be lower than the conventional F(ab')2. The maximal difference is observed in the kidney cortex, where mean percent injected dose per gram is 3.7-fold lower with modified than with conventional F(ab')2. PL indicates polylysine.


*    Discussion
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*Discussion
down arrowReferences
 
Radionuclide imaging of atherosclerosis uses the pathophysiology of the atherosclerotic process to deliver radiopharmaceuticals to the atherosclerotic lesion.31 The effectiveness of the imaging technique depends on the concentration of labeled element in the lesions relative to that in the surrounding tissues. Based on this concept, a number of constituents of the atheroma, including radiolabeled lipoproteins,9 10 monocytes,32 platelets,3 4 5 and fibrinogen,1 have been explored as potential plaque-imaging agents. Clinical applicability of these agents, however, has been minimal31 for several reasons. To achieve successful imaging with the above reagents, they must be incorporated into the slow pathological process of atherogenesis. Furthermore, because all of these agents are normal constituents of the blood, use of antibodies directed against them also may not be feasible. Antibodies obviously cannot differentiate between the circulating particles and those incorporated into the lesions. Therefore, efforts should be concentrated on selective targeting of the components within the atherosclerotic plaques such as activated platelets33 or macrophages,34 35 induction of endothelial adhesion molecules (intercellular adhesion molecule, vascular cell adhesion molecule) and growth factors (platelet-derived growth factor, endothelin-1, endothelial cell growth factor), or expression of receptors for growth factors.31 The presence of smooth muscle migration in the intima may provide another approach for targeting of metabolically active atherosclerotic lesions. Targeting of proliferating smooth muscle cells, such as with Z2D3, is expected to identify metabolically active atherosclerotic lesions. Such lesions may be more amenable to pharmaceutical or dietary interventions (or gene therapy).36 Local smooth muscle cell proliferation at the site of intimal damage is a frequent consequence of angioplasty and results in a 10% to 56% restenosis rate in the first year.37 An imaging technique that identifies local smooth muscle cell proliferation may also be useful in monitoring such patients and may help identify those who are likely to restenose. Furthermore, such a test may be useful in the noninvasive assessment of the natural history of asymptomatic atherosclerotic lesions.

Immunohistochemical analysis of a mouse monoclonal antibody Z2D3 IgM showed its specificity for an antigen on the proliferating smooth muscle cells of human atherosclerotic lesions. The regions of the normal aorta devoid of such lesions were not stained by Z2D3 antibody. Additional evidence of the specificity of Z2D3 antibody for the atherosclerotic lesion containing proliferating smooth muscle cells was provided by immunohistochemical staining of the subjacent aortic sections for PCNA (Fig 1DUp). Also, smooth muscle {alpha}-actin stained the intimal regions similar to Z2D3. However, the regions of atherosclerotic lesions stained by anti-macrophage antibody were distinctly different from Z2D3 staining (Fig 1EUp through 1G). Once the specificity of the antibody was established in human and experimental atheromatous lesions, it was reasoned that appropriately radiolabeled antibody administered intravenously may be useful for the development of a noninvasive method for localization and visualization of atherosclerotic lesions.

The antibody was observed to cross-react with experimentally induced atherosclerotic lesions in the rabbit aorta, permitting the use of rabbits as an animal model for the evaluation of the feasibility of noninvasive imaging. For the selection of the animal model, preliminary studies were performed in three rabbit models of atherosclerosis (unpublished data): rabbits with only balloon deendothelialization of infradiaphragmatic aorta, Watanabe rabbits, and rabbits with aortic deendothelialization followed by ingestion of a hyperlipidemic diet. Histologically, the arterial deendothelialization model preferentially results in intimal proliferation of the upper medial smooth muscle cells. Although Watanabe rabbits have lesions histologically more similar to human lesions, combined endothelial denudation and hyperlipidemic diet produce lesions that are very similar to advanced human lesions.38 39 Furthermore, the extent and the thickness of the lesions produced by the combined procedure are far greater than those of the lesions expected by summation of the effects of the two procedures used separately.39 Intravenous administration of radiolabeled Z2D3 antibody in Watanabe rabbits did not demonstrate any significant uptake in the atherosclerotic lesions, probably due to the slower rate of smooth muscle cell proliferation (data not shown). In the 4-week-old balloon deendothelialization model, Z2D3 uptake was intense and occurred preferentially in the healing edges of the endothelium. In this model, control antibody demonstrated diffuse uptake in the denuded area with no specificity for the healing edges. Uptake of nonspecific antibody is due to increased endothelial permeability, which is known to heal by 12 weeks after endothelial denudation.6 Therefore, we chose to use the deendothelialization plus hyperlipidemic diet model more than 12 weeks after denudation.

Antibody Z2D3-IgM could not be fragmented to Fab or F(ab')2 for immunoimaging. Therefore, a chimeric antibody (Z2D3-73.30) was genetically engineered with human IgG1 constant region. F(ab')2 fragments of this Z2D3-73.30 that retained the immunoreactivity were used for our in vivo studies. Visualization of the abdominal aortic lesions was possible at 48 hours in all animals that received the conventional 111In-labeled chimeric Z2D3 F(ab')2. The imaging characteristics of Z2D3 F(ab')2 were further improved by imparting a high negative charge to the fragments.

Antibodies are positively charged glycoproteins.40 On the other hand, cell surfaces are negatively charged due to an abundant distribution of acidic residues.41 42 43 This results in a nonimmunological nonspecific electrostatic interaction between the antibodies and the interstitial tissue in the target and nontarget organs as well as macrophages in the reticuloendothelial system. We recently demonstrated that these nonspecific interactions can be minimized if antibodies are modified to carry a high negative charge if the affinity of the antibody was not adversely altered.28 For this purpose, DTPA-succinyl-PL polymer was linked to the antibody. It significantly reduced the isoelectric point of the antibody and imparted a high negative charge to the antibody, which results in a lower background and nontarget organ activities. Furthermore, the use of synthetic polymers provided a method of coupling a large quantity of DTPA, which in turn enabled chelation of a large number of 111In ions providing antibody preparations of high specific radioactivity [40 to 50 mol 111In/1 mol F(ab')2 fragment]. Due to the high specific radioactivity, a substantially lower amount of antibody is needed for in vivo use. The decrease in nonspecific radiation exposure to the limiting nontarget organ such as the kidneys may also permit the use of an increased radioisotopic dose to provide higher photon flux for better image characteristics at the target sites. This has been substantiated in our previous experimental studies using antimyosin Fab for the evaluation of experimental cardiomyocyte necrosis.28 In addition to the advantages of requiring a lower dose of protein, the high specific radioactivity, the lower background activity, and the faster blood clearance of the negative charge–modified antibody enabled visualization of lesions at 24 hours, which was not possible with the conventional 111In-labeled Z2D3 F(ab')2 in the rabbit model of atherosclerosis.

In the present study, unequivocal visualization of experimental atherosclerotic lesions was possible in rabbits where the lesions were only a few cell layers thick and the aorta was only 5 to 7 mm in diameter. The diameter of the rabbit aorta is similar to that of human coronary arteries, and peripheral human arteries are larger. In humans, the lesions are also likely to be thicker and more obliterative. It is therefore expected that a higher antigenic concentration would be encountered within the atherosclerotic plaque, which could result in better accumulation of the antibody. However, use of this technique for visualization of atherosclerotic lesions in the human vascular system will involve overcoming the limitations posed by the blood pool activity and the cardiac motion, as well as by the greater dilution factor of a large blood volume in humans, and by the use of disproportionately less antibody relative to the dose used in rabbits.

Conclusions
Antibody Z2D3 specifically recognizes proliferating smooth muscle cells and cross-reacts with proliferating smooth muscle cells in experimentally induced atherosclerotic lesions in rabbits. The present study demonstrated the feasibility of noninvasive visualization of experimental atherosclerotic lesions with radiolabeled F(ab')2 fragments of Z2D3 antibody. Highly negatively charged polymer was conjugated to the F(ab')2 fragments for the reduction of nonspecific interaction between positively charged (basic) conventional antibody and negatively charged cell surfaces. Negative charge modification of the Z2D3 F(ab')2 resulted in earlier visualization of atherosclerotic lesions with the use of 10 to 15 times less antibody than that required with conventional Z2D3 F(ab')2. It also resulted in a significant reduction in radiation to the nontarget organs.


*    Acknowledgments
 
This work was supported by a grant from Scotgen Biopharmaceuticals Inc, Menlo Park, Calif. We are grateful to Drs Anthony Parker and Shiv Pillai for critical review of the manuscript and Thomas McVarish for editorial assistance.

Received November 14, 1994; revision received January 23, 1995; accepted January 30, 1995.


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