Antibodies as delivery systems for diagnostic functions

Advanced Drug Delivery Reviews 37 (1999) 63–80

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Antibodies as delivery systems for diagnostic functions Ban-An Khaw a ,b , * a

Northeastern University, Bouve´ College of Pharmacy and Health Sciences, Center for Drug Targeting Rm. 205, Mugar Building, Boston MA 02115, USA b Massachusetts General Hospital and Harvard Medical School, Boston MA, USA

Abstract Antibodies are highly specific targeting agents. Therefore, they are invaluable for in vitro and in vivo diagnostic applications. With the advent of monoclonal antibody technology, the utilization of antibodies has increased dramatically in almost every field of biological sciences. The present review describes the utility of monoclonal antibodies primarily in the cardiovascular diseases. Monoclonal antimyosin antibodies have been developed for noninvasive scintigraphic imaging of equivocal acute myocardial infarction. They have been negative charge modified to provide quicker in vivo visualization of the targeted antibody, as well as being applied for diagnosis of other cardiomyopathies that have disruption of myocardial cell membrane as an obligatory component of the disease. The radiolabeling techniques developed initially for myocardial necrosis imaging have also been applied for imaging of intravascular blood clots and atherosclerotic lesions. The applications of antimyosin, antifibrin and anti-atherosclerotic lesion specific monoclonal antibodies have all achieved initial clinical verification of their efficacy to target the respective lesions. However, to date, only antimyosin has been approved by the FDA for commercialization. Others must await additional studies to unequivocally verify the clinical utilities.  1999 Elsevier Science B.V. All rights reserved. Keywords: Monoclonal antibodies; Acute myocardial infarction; Cardiomyopathies; Thrombosis; Athersclerotic lesions; Imaging; Immunoscintigraphy; Charge-modified antibodies; Antimyosin antibody; Antifibrin antibody; Anti-atherosclerotic lesion specific antibody

Contents 1. 2. 3. 4.

Introduction ............................................................................................................................................................................ Antibodies in cancer imaging ................................................................................................................................................... Imaging with antibodies in the cardiovascular system ................................................................................................................ Antimyosin Fab imaging of acute myocardial infarction ............................................................................................................. 4.1. Targeting the necrotic myocardium with negative charge-modified antimyosin Fab............................................................... 4.2. Antimyosin imaging in diagnosis of various cardiomyopathies ............................................................................................ 4.2.1. Myocarditis ........................................................................................................................................................... 5. Dilated cardiomyopathy ........................................................................................................................................................... 6. Heart transplant rejection ......................................................................................................................................................... 7. Other cardiomyopathies ........................................................................................................................................................... 7.1. Antibody imaging diagnosis of vascular disorders .............................................................................................................. *Tel.: 1 1-617-373-4203; fax: 1 1-617-373-3663. E-mail address: [email protected] (B.-A. Khaw) 0169-409X / 99 / $ – see front matter  1999 Elsevier Science B.V. All rights reserved. PII: S0169-409X( 98 )00111-2

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7.1.1. Imaging blood clots................................................................................................................................................ 7.2. Imaging atherosclerotic lesions ......................................................................................................................................... 8. Conclusion ............................................................................................................................................................................. References ..................................................................................................................................................................................

1. Introduction Antibodies are glycoproteins that can be made to specifically target the immunizing antigen. As such, their potential as specific targeting agents for both diagnosis and therapy have been recognized since the discovery of antibodies. At the beginning of the 20 th century, the proposed ‘magic bullet’ of Paul Ehrlich for specific eradication of the spirochete of syphilis pointed to the potential of antibodies for targeted diagnosis and therapy [1]. However, it was not until the seminal work of Pressman and Keighley in 1948 [2] that antibodies were demonstrated to be able to target specific organs. This opened the door for the application of antibodies to tumor targeting [3]. However, the early years of tumor targeting were fraught with equivocal studies since investigators were using impure preparations of antibodies such as the immunoglobulin fractions of various antisera directed to antigens of questionable tumor specificity, as well as the utilization of suboptimal radiolabels. The next major impetus was provided by the revolutionary publication of Kohier and Milstein on the technology of monoclonal antibody production [4]. Since then, the application of antibodies as delivery system for radioisotopes for diagnosis has played a major role in the field of oncology and the cardiovascular identification of pathophysiological conditions. This chapter will not deal in detail on cancer imaging. A brief introduction on cancer imaging will be used to lead into the field of cardiovascular targeting with antibodies since the targets are usually better defined in cardiovascular imaging.

2. Antibodies in cancer imaging As indicated above, Pressman and Keighley demonstrated that the immunoglobulin fraction containing antibodies to normal rat kidneys can be radiolabeled with I-131 and used to specifically target the

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kidneys in vivo [2]. Their work suggested the potential of antibodies for tumor imaging and therapy. However, progress was slow and haphazard due to a lack of specific targets associated with various tumors, lack of pure antibodies for in vivo trials and appropriate radiolabels for tagging the available antibodies. Nevertheless, Jean Pierre Mach and co-workers [5], and Goldenberg and coworkers [6] initially used polyclonal antibodies to carcinoembryonic antigen (CEA) and subsequently used monoclonal antibodies to show that carcinomas can be imaged with radiolabeled anti-CEA antibodies [7,8]. Since those early days of tumor imaging with immunoglobulin fractions, or affinity purified antibody fractions radiolabeled with I-131, monoclonal antibodies and new and improved radiolabeling methods have enabled investigators to image breast [9], colon [10], lung [11], ovarian [12] and prostate cancers [13]. Melanomas [14], T-cell lymphomas [15], pancreatic cancer [16] and indeed every form of cancer have been targeted with polyclonal or monoclonal antibodies in either experimental or clinical trails. Improvements in labeling with radioisotopes such as Tc-99m, In-111 and I-123 to antibodies have also led to better and more efficient gamma imaging. Further improvements in radioimmunoscintigraphy include the use of bispecific monoclonal antibodies [17] and multistep avidin– biotin conjugated antibodies to reduce nontarget organ activities and enhanced target to background ratios [18]. Despite these advances, utilization of monoclonal antibodies for diagnosis of various tumors has been quite slow in recent years. Receptor imaging [19] and metabolic imaging with positron isotope radiolabeled FDG [20] have been out-pacing monoclonal antibody tumor imaging. Whatever the reasons, tumor imaging with monoclonal antibodies has not completely met the high expectations once held for monoclonal antibodies, yet progress towards that expectation is proceeding.

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3. Imaging with antibodies in the cardiovascular system The application of monoclonal antibodies, for that matter all antibodies, in the cardiovascular arena started with the use of antimyosin antibody for diagnosis of acute myocardial infarction in 1976 [21]. Since then, applications of monoclonal antibodies have expended to imaging of myocarditis [22], heart transplant rejection [23], dilated cardiomyopathy [24], alcoholic cardiomyopathy [25], adriamycin cardiotoxicity [26], various other cardiomyopathies [27], vascular clots [28], atherosclerotic lesions [29] and even certain cancers such as soft tissue sarcomas [30]. Yet the best characterized and maximally used antibody for cardiovascular diagnostic imaging is monoclonal antimyosin Fab for its exquisite specificity in the detection of myocardial cell death associated with various forms of cardiomyopathies.

4. Antimyosin Fab imaging of acute myocardial infarction There are approximately 8 million emergency room visits associated with various forms of chest pain in the United States each year [31]. Of these patients, 1.5 million will have chest pain due to acute myocardial infarction. Diagnosis of the run-of-themill acute myocardial infarction (MI) is relatively easy utilizing classical clinical symptoms, electrocardiogram and serum enzyme assessments. Using this triad of diagnostic criteria, only three out of ten myocardial infarct-suspect patients having chest pain and admitted to the coronary care units will have acute myocardial infarction [32]. Furthermore, of the 1.5 million acute myocardial infarct patients, approximately 1 / 3 rd will be misdiagnosed by the existing traditional methods. Therefore, there is a need for a reagent such as radiolabeled antimyosin antibody that is a highly specific and sensitive method for diagnosis of acute and subacute myocardial infarct ion, and for verification of equivocal myocardial infarction. The basis for specifically targeting the damaged myocardium in acute myocardial infarction is predicated on the hypothesis that normal myocardial cells

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will not allow (antimyosin) antibodies to be internalized into the cytosol in any appreciable concentrations, where the homologous antigen, myosin exists as a component of the contractile myofibrils [21,33]. However, following development of sarcolemmal membrane lesions due to an insult, be it ischemic, chemical or physical, the intracellular myosin becomes exposed to the extracellular milieu. If the antimyosin antibody were introduced into the extracellular milieu, the antibody should interact with the once privileged cardiac myosin. If this antimyosin antibody were appropriately labeled, then the regions of cell membrane disruption could be visualized by identification of the accumulation of the labeled anti-myosin antibody [34]. The hypothesis was validated using hypoxic neonatal murine myocytes in primary culture treated with antimyosin antibody attached covalently to 1-micron diameter polystyrene beads [33]. Normal myocytes with intact sarcolemma prevented accumulation of antimyosin beads (Fig. 1a) whereas hypoxic myocytes with sarcolemmal lesions showed specific targeting of the antimyosin beads at the lesions sites (Fig. 1b). Such specific targeting of the necrotic myocardium can also be seen in clinical situations. Fig. 2 shows gamma images of patients with persistently occluded left anterior descending coronary artery (LAD) and another with reperfused LAD injected with In-111-labeled monoclonal antimyosin R11D10 Fab [35]. Radiolabeled antimyosin localized in the region of the hearts corresponding to the areas subtended by the occluded LADs. In patients with no infarction, there was no accumulation of radioactivity in the region of the heart (Fig. 3). Despite the apparent specificity in these clinical studies, the absolute specificity of antimyosin antibody for delineation of the necrotic myocardium remains to be established since radiolabeled antimyosin also accumulated in the myocardium of some patients with unstable angina pectoris [36] and chronically in some infarct patients even up till 9 months after the acute event [37]. To establish without equivocation that antimyosin Fab is highly specific for delineation of the necrotic myocardium, studies utilizing a mixture of I-125labeled antimyosin antibody and I-131-labeled normal IgG administered by intracoronary delivery into dogs with experimental MI were undertaken [38].

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Fig. 1. Scanning electron micrographs of murine neonatal primary myocytes in culture treated with antimyosin-linked 1-mm diameter fluorescent polystyrene beads. (a) A normal myocyte showing the intact cell membrane and a lack of antimyosin bead binding. (b) A necrotic myocyte with a region of sarcolemmal disruption showing antimyosin-bead binding in that region (Ref. [33], with permission).

Uptake of both radiolabeled immunoglobulin species was assessed by gamma scintillation counting [38]. I-125-labeled antimyosin antibody localized in the necrotic myocardium with a target to nontarget ratio of . 32:1 at the infarct center, whereas nonspecific localization was only about 6.5:1 in the same tissue samples [38]. Similar to the clinical images, canine infarcts can be visualized by gamma imaging within a few hours after intravenous administration of In111-labeled antimyosin Fab (Fig. 4, right panel), whereas use of nonspecific antibody Fab also labeled with In-111 showed no infarct localization even at 5 h post intravenous administration of the radiotrace (Fig. 4, left panel) [39]. To further demonstrate the

Fig. 2. Anterior and 458 LAO images of two patients with acute MI at 24 and 26 h after intravenous administration of In-111labeled antimyosin Fab. Images of a patient with persistently occluded LAD (no reperfusion) (top) and those of a patient with successful reperfusion (bottom) (Ref. [35], with permission).

profound specificity of antimyosin for delineation of the necrotic myocardium, two monoclonal antimyosin antibodies, one with high and the other with low affinities were examined in canine acute myocardial infarct model. When the low affinity antimyosin Fab (3H31E6) with an apparent affinity of . 6.5 3 10 6 l / mole was used to visualize acute myocardial infarction, no in vivo infarct activity was seen even 5 h after antibody administration, whereas with the higher apparent affinity antimyosin Fab (R11D10) (1 3 10 9 l / mole), the infarct was visualized unequivocally (Fig. 5) [39]. The gamma images of dogs from this study were also used to quantitate the ratios

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Fig. 3. Negative antimyosin images (anterior and LAO) of a patient showing no myocardial accumulation of the In-111 antimyosin radioactivity.

Fig. 4. Gamma images of two dogs with acute experimental myocardial infarction, injected with 111 In-labeled antimyosin Fab (right panel) and 111 In-labeled nonspecific monoclonal Fab (left panel), at 5 h post intravenous administration of these antibodies. Only the image with antimyosin showed unequivocal infarct uptake, whereas the image with nonantimyosin specific monoclonal Fab showed only blood pool activity. (Ref. [39], with permission).

of activity in the infarct (I) to activity in the blood pool area (B) of the in vivo gamma images by computer planimetry. The mean ratios of I /B for R11D10 was 1.4860.14 (mean6SD), which was significantly greater than that of 3H31E6 Fab

(0.8560.115). The latter was the same as the I /B ratios obtained with nonspecific Fab (0.75–0.771) [39]. Thus these experimental studies showed that not only is antimyosin specific for the delineation of acute myocardial necrosis, its specificity is doubly

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Fig. 5. Left lateral gamma images of two dogs with experimental myocardial infarction injected intravenously with 111 In-labeled 3H3 antimyosin Fab (Ka 5 5 3 10 6 L / M) (right panel) and 111 In-labeled R11D10 antimyosin Fab (Ka 5 0.5–1 3 10 9 L / M) (left panel) obtained at 5 h post injection. The infarcts in both animals were approximately the same size but unequivocal visualization was possible only with R11D10 Fab but not with 3H3 Fab. (Ref. [39], with permission).

enhanced by the need for the antimyosin antibody to have sufficiently high enough affinity for successful in vivo visualization. Despite the ability of antimyosin Fab to specifically delineate acute myocardial infarction, its utility is hampered by the slow blood clearance of the Fab fragments [35]. This causes delayed development of high enough target-to-background ratios for in vivo visualization by gamma scintigraphy, especially in clinical use, of to . 12 h after intravenous administration. Usually, images were acquired 18 to 24 h after IV administration, however small MI may require 48 hours of blood clearance to enable unequivocal diagnosis. If on the other hand, qualitative diagnostic end-point is the desired outcome, irrespective of visual confirmation of the infarct size, then infarcts may be detected rather early after intravenous antibody administration over and above the blood pool activity. These rate-limiting steps may be overcome by using smaller antibody fragments such as sFv [40], CDR [41], and mimetics [42]. However, by increasing the clearance rate, there is a concomitant decrease in the absolute available antibody for target localization. Therefore, we developed a new approach to improve target-to-background ratios in experimental myocardial infarct visualiza-

tion by decreasing the background activity without affecting the target activity at any time point.

4.1. Targeting the necrotic myocardium with negative charge-modified antimyosin Fab Since antibodies are basic glycoproteins, under physiological conditions they are positively charged [43]. Cells and extracellular matrices, on the other hand are negatively charged due to the presence of cell membrane bound acidic residues such as sialic acids [44,45] and heparan sulfate proteoglycans [46] respectively. Therefore, the potential for nonspecific ionic interaction between the positively charged molecules and the negatively charged cell surfaces or extracellular matrices exists. Such nonspecific interaction has been utilized to deliver methotrexate– polylysine conjugates to malignant cells [47], protracted release of basic fibroblast growth factor for the salvage of the infarcted myocardium [46], as well as for the delivery of genetic constructs by lipofection or cationic liposomes [48]. Alternatively, we proposed that if the basic (positively charged) antibodies were to be modified to carry a highly anionic polymer so that the isoelectric point of the modified antibody becomes low (e.g. PI , 5), then this modi-

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fied antibody should have reduced nonspecific ionic interaction with nontarget cells and extracellular matrices [49]. However, the affinity of the antibody at . 1 3 10 9 l / mole can easily overcome the repulsive ionic forces when the charge-modified antibody approaches its homologous antigen. This would permit the same targeting of the charge-modified antibody as with the noncharge modified antibody due to the same antigenic specificity and affinity, but should result in lower nontarget background activity and thereby allow earlier development of high enough target-to-background ratios for visualization of the target [49]. Antimyosin Fab was modified with DTPA-conjugated polylysine that has been made totally anionic by succinylation, by covalent attachment to AM-Fab [50]. Ion-exchange chromatography was used to separate modified AM-Fab from unmodified AMFab. The immunoreactivity of the negatively-chargemodified AM-Fab carrying a polymer of approximate 3.3 kD or 17 kD was assessed by radioimmunoassay or ELISA to be identical to that of the unmodified AM-Fab [50]. When negative charge-modified AMFab with a 3.3- or 17-kD polymer was labeled with In-111, a specific radioactivity of 50–100 mCi per milligram of Ab Fab was obtained. The conventional AM-Fab specific activity was only 2–10 mCi / mg of AM-Fab. Thus less negative charge-modified AMFab was needed to deliver the same radioactivity relative to the conventional AM-Fab dose. Further, when the negative charge-modified AM-Fab was administered into dogs with reperfused experimental MI, myocardial infarcts were visualized within 30 min of intravenous administration of the antibody preparation (Fig. 6), whereas the conventional In111-labeled AM-Fab required 1–2 h of antibody circulation and clearance before infarcts were visualized. This earlier target visualization could be, as stated previously, due to quicker development of high target-to-background ratios for visualization by gamma imaging which requires a ratio of about 10:1. It could also be due to a new concept developed in our laboratory where visualization could be achieved not based on target-to-background ratios but on the difference in activity between target and background if that difference is evidenced as high absolute radiotracer accumulation [51]. Assuming that the same

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Fig. 6. Serial left lateral gamma images of two dogs with acute experimental myocardial infarction injected with negative chargemodified In-111 labeled antimyosin Fab (left panels) and conventionally In-111 labeled antimyosin Fab (right panels); (a) and (b) 5 30-mm images, (c) and (d) 5 1 h, and (e) and (f) 5 3 h post intravenous administration. (Ref. [50], with permission).

amount (500 mg) of charged modified and unmodified antimyosin Fab radiolabeled with In-111, were injected into dogs with experimental MI, and at 5-h post injection there were 0.1718 1 0.0201% ID/ g [49] of negative charge-modified antimyosin Fab in the infarct, since the specific radioactivity of negative charge-modified antimyosin Fab was 100 mCi / mg, the absolute radioactivity in 1 g of infarct would be 85.9 mCi. On the other hand, 500 mg of unmodified antimyosin Fab with a specific radioactivity of 5 mCi / mg with 0.204160.0204% ID/ g would only have 5 mCi / g of the infarcted myocardium. Since normal myocardial activities were 0.005660.0004% ID/ g with charge-modified antimyosin Fab and 0.026360.0037%ID/ g with unmodified antimyosin Fab, the absolute background radioactivities were 2.8 mCi and 0.65 mCi respectively. Therefore, the difference between target and background for charge-modified antimyosin Fab would be 83.1 mCi and the ratio between target to

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background would be 30.7:1. The difference in the absolute activity between target and background on the other hand for unmodified antimyosin Fab would be only 4.34 mCi and the ratio 7.6:1. Therefore, with a differential of 83 mCi / g between target to background, one should be able to visualize the target early, whereas a differential of 4.34 mCi may not permit easy early visualization. To determine whether earlier visualization was due to increased infarct activity or decreased nontarget organ activities, biodistribution data were also compared. Fig. 7 shows that even in the normal myocardium (nontarget tissue), the radioactivity was significantly lower for the 3.3- and 17-kD negative charge-modified polymer – AM-Fab than that of the conventionally labeled AM-Fab (P , O.OO1) [49]. The applicability of negative charge modification was further demonstrated in a rabbit model where an infarct as small as 216 mg was visualized by in vivo imaging within 3 h after intravenous administration (Fig. 8) [52]. The percent injected dose localization in this animal was as high as 1.73% ID/ g providing . 86.3 mCi / g of the target. The mean maximal target-to-nontarget (minimal) ratio from the seven rabbits was 53.9618.4 [52].

Fig. 7. Comparison of normal myocardial background activity of two preparations of negative charge-modified antimyosin Fab and the conventional In-111-labeled antimyosin Fab. The normal myocardial distribution of I-125 labeled antimyosin Fab is also shown for comparison. However, I-125 is subjected to dehalogenation in vivo, yet I-125 antimyosin normal myocardial activity is significantly higher than the negative-charge modified antimyosin Fab (P , 0.001) (Ref. [50], with permission).

Fig. 8. Left lateral gamma image of a rabbit with a 216-mg experimental myocardial infarct visualized with In-111-labeled negative charge-modified antimyosin Fab. Arrow points to the small infarct visualized in vivo (Ref. [52], with permission).

Therefore, negative charge modification of AMFab not only imparted lower nontarget organ activity without affecting the target activity, it also provided antibody preparations with very high specific radioactivity enabling the use of less proteinaceous compounds for in vivo administration. Furthermore, it also showed that when high target activity is achieved, visualization was possible earlier based on the difference between target and background activities, rather than target to background ratios. Whether this process of negative charge modification will ever find clinical application may not be determined by scientific feasibility but more by commercial considerations. Nevertheless, a murine– human chimeric antibody F(ab9) 2 specific for proliferating smooth muscle cells associated with atherosclerotic lesions has been negatively charge modified, labeled with In-111 and shown to localize in the carotid artery atherosclerotic lesions in all nine patients studied to date [53]. Antimyosin is highly specific and sensitive for diagnosis of myocardial necrosis associated with acute myocardial infarction. Despite its requirement of approximately 24 h lag time for unequivocal diagnostic imaging, it is highly useful for diagnosis

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of equivocal myocardial infarction, myocardial necrosis associated with unstable angina, right ventricular infarction [54] and perioperative myocardial infarction in by-pass surgery [55].

4.2. Antimyosin imaging in diagnosis of various cardiomyopathies 4.2.1. Myocarditis Myocarditis is a cardiomyopathy of highly variable clinical manifestations that can in more severe cases, lead to dilated cardiomyopathy and heart failure [56]. It is believed to have a viral origin but the chronic component of the etiology is believed to be autoimmnue in nature. To unify the diagnosis of the disease, the Dallas Criteria were formulated [57]. The criteria mandated the presence of mononuclear cell infiltration and myonecrosis demonstrated in endomyocardial biopsies for unequivocal diagnosis of myocarditis. However, the criteria focused only at a limited phase of myocarditogenesis. Irrespective of the inflammatory obligatory component of the criteria, the presence of myonecrosis led us to propose that antimyosin immunoscintigraphy should be able to target the myonecrotic component of the disease and provide a very sensitive diagnostic indicator for noninvasive diagnosis of myocarditis. In the initial study of 28 patients with histories and clinical findings suggestive of myocarditis, In-111 antimyosin immunoscintigraphy was positive in 17

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patients (61%) (Fig. 9, left panel) and negative in 11 (39%) (Fig. 9, right panel) [22]. All antimyosin negative patients were also negative by endomyocardial biopsy criteria, whereas all biopsy positive patients were also positive by biopsy criteria [22]. A potential complication in the use of In-111 antimyosin Fab for diagnosis of acute myocarditis is the misinterpretation of residual blood pool activity for myocardial uptake. Since myonecrosis associated with myocarditis is generally diffused and not as intense as seen in acute myocardial infarction, uptake of In-111-labeled antimyosin could also be diffused and of low contrast. Therefore, it is recommended that single photon emission tomographic imaging be performed in case of equivocation. The tomographic reconstruction images in the transverse, sagittal and coronal views should show myocardial activities rather than blood pool activities.

5. Dilated cardiomyopathy It has been clinically suspected that active myocarditis is capable of resulting in heart failure and acute dilated cardiomyopathy. Although the exact number of cases of active myocarditis or ongoing inflammation that ultimately resulted in ideopathic cardiomyopathy is not known. Dec et al. [24] studied 74 patients (50 men and 24 women) with dilated cardiomyopathy with global ejection fraction less

Fig. 9. In-111 antimyosin gamma images of two patients suspected of having myocarditis; (a) positive image (left panel) and (b) a negative image (right panel). The arrows point to the myocardial activity.

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than 0.45 with In-111 antimyosin Fab. Thirty-nine patients (53%) were positive by imaging criteria. Of these 39, 11 patients had histologically verified myocarditis whereas 28 showed no evidence of myocarditis on biopsy. Out of the remaining 35 patients with normal antimyosin images, 33 were also negative by endomyocardial biopsy criteria. However, two were false negative since they had biopsy verified myocarditis. Thus the sensitivity of antimyosin imaging was 85% and the predictive value of a normal scan was 94%. However, the specificity was only 54% using the Dallas Criteria as the gold standard. This low specificity is most probably due to the need to use a Gold Standard that is highly insensitive and because myocarditis is a disease of either right or left or both ventricles, and endomyocardial biopsies are primarily obtained from the right ventricles. Irrespective of this low specificity of antimyosin imaging for diagnosis of myocarditis, patients with abnormal antimyosin scan and biopsy results showed significant improvement in the mean ejection fraction form 0.2760.02 to 0.4360.04 within six months follow up examination.

Whereas, patients with normal scans and nondiagnostic biopsy results had only slight improvement in the ejection fraction from 0.1960.02 to 0.2460.03. Furthermore, those patients with positive antimyosin scans and negative biopsy results showed significant improvement in the ejection fraction at follow-up indicating that since spontaneous improvement in cardiac function is a recognized feature of active myocarditis, the subset of patients with a positive antimyosin scan and spontaneous improvement in the cardiac function had myocarditis which biopsy failed to detect. Negative antimyosin imaging can also be used to follow efficacy of therapy. Fig. 10 shows that although the initial antimyosin images were positive, after 6 months of steroid therapy, the images were negative for the myonecrosis component of the Dallas Criteria.

6. Heart transplant rejection Since commencement of immunosuppressive treatments for acute heart transplant rejection is predi-

Fig. 10. In-111 antimyosin gamma images of a patient with biopsy positivity for myocarditis obtained initially when the left ventricular ejection fraction was only 34% (left panels) and after 6 months of steroid therapy (right panels) when the LVEF had normalized to 55%. Planar anterior and LAO images are shown at the top and the tomographic reconstruction images in the transverse, sagittal and coronal reconstructions are shown in the bottom panels.

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cated on the presence of myonecrosis on endomyocardial biopsies, antimyosin imaging should also be applicable for directing therapy. Frist and coworkers [58] showed that antimyosin could be used to detect myonecrosis associated with acute rejection (Fig. 11). However, they also noted that immediately after transplantation, there appeared to be a basal uptake of In-111-labeled antimyosin Fab probably due to myocardial injury associated with transplantation related procedures. Subsequently Ballester and coworkers showed systematically that this initial antimyosin positivity could return to baseline as quickly as 3 months after transplantation [59]. Furthermore, they showed that if antimyosin uptake assessed as heart-to-lung activity ratios remained elevated for greater than 1 year after transplantation, prognosis for the patient was poor and that patient was a prime candidate for retransplantation. Various degrees of intensity of In-111 antimyosin uptake were shown in Fig. 12, ranging form normal antimyosin scan (A), to mild uptake (b), to moderate uptake (c) to significant uptake (D) [60]. Antimyosin imaging appears to be highly sensitive for the detection of acute heart transplant rejection. Relative to the gold standard of endomyocardial

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biopsy, antimyosin has a sensitivity of 95% [61] however, specificity was only 33%. This discrepancy may be due to a lack of specificity of antimyosin or to a low sensitivity of biopsy in the detection of rejection. However, sampling error of endomyocardial biopsy is a distinct possibility that may account for the discrepancy since transplant rejection is histologically a patchy process. Therefore, if antimyosin were taken as the gold standard, then the sensitivity and specificity of endomyocardial biopsy would be 31 and 95% respectively [61]. This is consistent with the low diagnostic yield of endomyocardial biopsy reported for the diagnosis of active myocarditis [62,63].

7. Other cardiomyopathies Due to the mechanism of targeting of antimyosin Fab, it appears that antimyosin could also be used to delineate various cardiomyopathies as long as there is an association of the disease process with irreversible myocardial injury where the integrity of the cell membrane has been compromised. Therefore, antimyosin Fab has been used successfully to demonstrate myocardial injury due to Adriamycin cardiotoxicity (Fig. 13) [64], in rheumatic carditis [65], in Lyme carditis [66], Churg-Strauss Disease [67] and Cardiac Contusion [68].

7.1. Antibody imaging diagnosis of vascular disorders

Fig. 11. Anterior image of a patient with biopsy proven heart transplant rejection. The image was obtained 48 h after iv administration of the antibody (Ref. [58]).

7.1.1. Imaging blood clots Attempts to image blood clots preceded the monoclonal antibody technology. In the mid 1960s, Spar and co-workers used polyclonal antifibrinogen antibodies to detect thrombi in vivo [69]. The antibody used reacted with fibrinogen as well as fibrin thereby lowering the specificity for detection of preformed clots but were effective in the detection of actively forming blood clots. It was not until 1983 that Hui and co-workers [70] developed a monoclonal antibodies (59D8 and 64C5) specific for the b chain of the fibrin molecule. It was made to a seven amino acid N-terminal sequence of the b chain of fibrin. Since the N-terminus of the b chain of fibrin constitutes neoantigen generated when fibrinogen is

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Fig. 12. Anterior gamma images of patients with various degrees of rejection detected by In-111 antimyosin imaging. A 5 normal scan, B 5 mild uptake, C 5 moderate uptake and D 5 significant uptake (from Ref. [60], with permission).

cleaved by thrombin, it did not cross react with fibrinogen. Kudryk et al. [71] also generated a monoclonal antifibrin T2G1S that reacted with the same N-terminal of the b chain of fibrin. Although there are other monoclonal antibodies with varying specificities for fibrin, only 59D8 and T2G1S have seen clinical studies. Fig. 14 (left panel) shows a 24-h image of the lower extremities of a normal subject, whereas Fig. 14 (right panel) shows a set of spot images in a patient with venographically documented DVT. It appears that imaging of DVT was quite easily feasible in experimental and clinical trials. Whether imaging of pulmonary emboli in a clinical situation with monoclonal antifibrin antibody can be success-

ful is not known. Experimentally, Kanke et al. [72] showed that 64C5 monoclonal antifibrin was able to image PE. However, small PE of , 50 mg were not visualized in vivo by gamma imaging despite a correlation between clot size and total antifibrin uptake. Further improvements are needed to make this method of detection of DVT or PE a clinical reality.

7.2. Imaging atherosclerotic lesions Atherosclerosis is another intravascular pathology that appears to be amenable to detection by the use of monoclonal antibodies. It was initially thought that atherosclerotic lesion possessed no specific

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Fig. 13. Anterior gamma images of patients undergoing adriamycin cancer chemotherapy with In-111 antimyosin Fab. (A) In-111 antimyosin image before initiation of chemotherapy. (B) Scan from a patient who had received a cumulative dose of 300 mg / m 2 of doxorubicin. (C) Scan of a patient treated with a doxorubicin cumulative dose of 500 mg / m 2 and (D) scan from patient treated with . 500 mg / m 2 of doxorubicin. Cardiotoxicity can be seen as myocardial damage delineated by antimyosin antibody uptake. (Ref. [64], with permission).

Fig. 14. Anterior gamma images of normal control (left) and patient with DVT (right) with Tc-99m-labeled antifibrin Fab. Radiolabeled antifibrin uptake can be seen in the left thigh and calf regions. (Ref. [28]).

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targets for development of imaging modalities. Although atherosclerotic lesions are composed of oxidized LDL, that had been ingested by macrophages that had become foam cells, and the presence in fibrous lesions of smooth muscles cells, no differentiated antigens were thought to exist in the lesions. Therefore, conventional modes of diagnosis such as arteriography or ultrasonography were believed to suffice for providing information on anatomical narrowing of the affected vessels. Although these methods are effective, they cannot provide pathophysiologic information that may shed light on the stability as well as the pathogenesis of the lesions. To elucidate the potential metabolic component of the pathogenesis of atherosclerotic lesions, several approaches have been taken experimentally. Radiolabeled oxidized LDL [73] as well as to the activated macrophages [74] have been tried. However, noninvasive imaging has not been impressive. Scientists of Scotgen Biopharmaceuticals, developed a monoclonal antibody that is specific for a complex antigen produced only by proliferating smooth muscle cells [75]. The parent monoclonal antibody Z2D3 was an IgM subclass [76]. This antibody was shown to be able to target experimental atherosclerotic lesions in a rabbit model [77]. To enable easier utilization, the IgM subclass of Z2D3 was class switched to IgG as well as genetically engineered to produce a murine–human chimeric IgG Z2D3 antibody. Fig. 15 (top panels) shows a set of gamma images of a rabbit with experimental atherosclerotic lesions imaged with In-111-labeled chimeric Z2D3 F(ab9) 2 . The lesions could be visualized in the region of the descending aorta that was the site of experimentally induced lesions. In this model, the lesions were induced by de-endothelialization of the descending aorta from the region of the diaphragm down to the bifurcation of the femoral arteries [75] and then the rabbits were kept on 6% peanut oil, 2% cholesterol-enriched chow for at least 3 months. This produced lesions that are more akin to human fibrous lesions, unlike the fatty streak lesions. As controls, rabbits with similar lesions were injected with In111-labeled normal human IgG F(ab9) 2 (Fig. 15 lower panels). In this figure, no uptake was seen in the region of the experimental lesions in vivo. Immunohistologically, this antibody also stains the

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B.-A. Khaw / Advanced Drug Delivery Reviews 37 (1999) 63 – 80

Fig. 15. Left lateral oblique images of rabbits with experimental atherosclerotic lesions induced in the descending aorta imaged with In-111 labeled murine–human chimeric Z2D3 F(ab9) 2 (top panels) and In-111 labeled human IgG F(ab9) 2 (bottom panels). The in vivo gamma images are shown in the left panels (k 5 kidney, U 5 urinary bladder activity, solid thin arrows 5 atherosclerotic lesions, and open larger arrow 5 spinal activity), and the ex vivo images of the excised aortas from the aortic arch to the femoral bifurcation are shown in the right panels.

region of smooth muscle cell proliferation in the blood vessels. It did not stain the smooth muscles of the media (Fig. 16). Ultimately, the negative chargemodified chimeric Z2D3 F(ab9) 2 was used to determine whether it could delineate atherosclerotic lesions in carotid lesions in patients [78]. Using planar and SPECT imaging, it was observed that the lesions were better detected with SPECT imaging (Fig. 17) [79]. Whether this antibody Z2D3 will have wide clinical application must await additional trials.

Fig. 16. Immunoperoxidase staining of frozen sections of atherosclerotic human coronary artery (top) and rabbit aorta with experimentally induced atherosclerotic lesions. In the human atherosclerotic lesion, the area stained with Z2D3 was different from the areas stained with antimacrophage antibody. The antibody cross-reacted with rabbit lesions (bottom panel) (Ref. [77]).

8. Conclusion Thus we have shown selective applications of monoclonal antibodies for imaging of various targets. Whether they are in oncologic or cardiovascular disorders, the important parameters for successful application of antibodies as delivery systems for diagnostic functions are the specificity of the antibody for the target antigen, sufficiently high affinity of the chosen antibody and the appropriate radiolabel. Improvements in the in vivo gamma images obtained with radiolabeled antibodies may be obtained by modulation of the size of the antibody, the global charge and the specific radioactivity. Combination of the above should ultimately provide

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Fig. 17. Coronal tomographic image of a patient with carotid atherosclerotic lesions obtained 4 h after iv administration of In-111-labeled negative-charge modified murine–human chimeric Z2D3 F(ab9) 2 (right panel). The arrow denotes the carotid lesion. The carotid angiogram demonstrating a severe right internal carotid artery lesion (arrow) is shown in the left panel).

antibodies with optimal qualities for very early detection of various targets in cancer detection, cardiovascular diseases or for any other noninvasive in vivo diagnosis by gamma imaging.

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