(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.
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.
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Abstract |
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 Top Abstract IntroductionMethods Results Discussion References |
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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
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Introduction |
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TopAbstractIntroductionMethods Results Discussion References |
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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
-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.
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Methods |
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TopAbstractIntroductionMethods Results Discussion References |
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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 (
light chain)
monoclonal antibody. The antibody demonstrated intense immunoperoxidase
staining of frozen sections of atheromatous plaques in
postmortem human coronary artery (Fig 1A
).19 A
nonspecific IgM antibody used for
comparative purpose did not localize in the human coronary
artery atherosclerotic lesion (Fig 1B), 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 1C). 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 1D), and one specific for
smooth
muscle 
-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
1E 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 1C). 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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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 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 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 1H
).
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 1C).
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 2). 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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Radiolabeling of Z2D3
F(ab')2 With
111In
To 1 aliquot of 111In chloride (
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 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 1). 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 1).
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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.
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Results |
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TopAbstractIntroductionMethods Results Discussion References |
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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 3A
). The T/L of the antibody uptake was
0.486±0.047 (Table 2). 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 3B; T/L,
0.341±0.013; P=.01; Table 2). The lack
of radiotracer
uptake with nonspecific IgG1 F(ab')2 was
further confirmed in the ex vivo image of the excised aorta (Fig
3D)
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 3A
and 3C).
The tracer localization was further confirmed by the comparison of the
macroautoradiograph (Fig 3E) with the gross pathological
specimen (Fig 3F).
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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 4A).
Unequivocal delineation of the atheromatous lesions was
feasible 24 hours after intravenous administration of the
negative charge–modified antibody (Fig 4B). The lesion
activity was
reconfirmed in the 48-hour image (Fig 4C; 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 4D) and
macroautoradiography of the excised descending aorta
(Fig 4E), which corresponded to the gross pathological lesions
in the
descending aorta (white regions, Fig 4F).
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The
111In-labeled negative charge–modified
nonspecific human IgG1 F(ab')2 did not localize
in the atheromas (Fig 5). The immediate
post–intravenous tracer administration image showed
primarily blood pool activity (Fig 5A) similar to that seen in
Fig 4A.
However, by 24 hours, there was no activity in the region of the
atheromatous abdominal aorta, except for minimal
residual blood pool activity (Fig 5B, solid arrow), which
cleared
completely by 48 hours (Fig 5C; T/L, 0.178±0.029;
P=.002).
The corresponding ex vivo gamma image of the descending aorta (Fig
5D)
and the macroautoradiograph (Fig 5E) showed minimal nonspecific
radiotracer accumulation, which did not correspond to the severity of
the lesion by gross pathological examination of the aorta (Fig
5F).
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 6A through 6C)
or
ex vivo (Fig 6D) gamma imaging and macroautoradiography
(Fig 6E). On gross inspection the specimen also did not show
presence
of any lesions (Fig 6F).
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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 7). 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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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 =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 8). 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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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
9). 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).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethods Results Discussion References |
|---|
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 1D). Also, smooth muscle -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 1E 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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