Non-tumor applications of radioimmune imaging

0883-2897191 $3.00+ 0.00 Copyright a 1991Pergamon Press plc

Nucl. Med. Biol. Vol. 18, No. I, PP. 127-134,1991 Iat. J. Radiat. Appl. Instrum. Part B Printed in Great Britain. All rights reserved

Non-tumor

H. W. STRAUSS,

Applications of Radioimmune Imaging A. J. FISCHMAN,

B. A. KHAW and R. H. RUBIN

Division of Nuclear Medicine, Department of Radiology, Massachusetts General Hospital, Boston, Massachusetts, U.S.A. Abstract-The application of radioimmune imaging techniques to the evaluation of non-neoplastic disease is largely based on the same principles as tumor imaging; specificity and high affinity of antibodies. Currently the most common non-tumor applications of antibody imaging are the detection of clots (peripheral and pulmonary emboli), myocardial necrosis (myocardial infarction, myocarditis and cardiac transplant rejection) and focal sites of infection/inflammation. In the area of injection imaging, both antigen-specific and non-specific properties of antibodies have been successfully exploited in imaging studies. While the number of non-tumor applications of antibodies are far fewer than the number of tumor studies, in many cases, they appear to be more reliable. The basis for the reliability of antibodies for detecting non-neoplastic lesions is probably related to the availability of abundant antigen, lack of antigen modulation and enhanced permeability at the lesion site. These observations suggest that there will be rapid proliferation of work in this area.

Introduction Non-tumor applications of antibodies were pioneered by Spar in 1965, when he described the use of a polyclonal antibody directed against fibrinogen to identify preformed venous thrombi in dogs (Spar et al., 1965). Within

2 years, this concept

had been al., 1967), and 2 years later was applied to the detection of thrombus in the left atria of patients with atria1 fibrillation (Spar et al., 1969). While antibodies are

tested for the detection of tumors (Spar

et

still advocated for the detection of clot, two other non-tumor areas where antibodies appear to have a clinical role are the detection of myocardial necrosis and localization of infection (Table 1). The non-neoplastic applications of radioimmune imaging are based on the specificity and high affinity of these protein based radiopharmaceuticals for specific binding sites. Clot, myocardial necrosis and infection each offer unique targets that are not usually found in normal tissue, or provide a marked increase in the local abundance of a binding site that is present in normal tissues. As pointed out by Fischman et al. in a recent editorial (Fischman et al., 1989) the combination Table

of large amounts

I. Radioimmune

imaging: applications

of antigen

non-neoplastic

Application

Antigen

Clot detection

Fibrin Platelet Myosin heavy chain Granulocyte surface ? Fc receptors

Myocarditis/infarction Infection/inflammation

and high affinity antibodies is important in defining the maximal theoretical target-to-background ratio that can be achieved in the radioimmune image. An additional factor, the absolute level of antibody accumulation, is equally important for lesion detection. Increasing either the mass of antibody administered or the quantity of antigen tends to improve localization. In small animals, where the lesion is a significant fraction of total body weight, the mass of antigen usually drives the equilibrium reaction to capture a large portion of administered antibody, resulting in marked antibody localization. This could explain the spectacular imaging results obtained with xenografts of human tumors in nude mice [up to 25% injected dose/tumor (Keenan, 1988)] compared to studies of the same tumors in patients (0.01-0.001% injected dose/g tumor). Since antibodies raised against non-tumor antigens generally have affinities of 10s-lO’o L/mol-about the same as that for tumor directed antibodies, the consistent visualization of non-neoplastic lesions must be related to antigen availability or antigen abundance. The number of antigenic sites exposed per gram of necrotic myocardium studied with anti-myosin imaging is many orders of magnitude greater than that available with a surface directed tumor antibody. Thus even the reduction of antibody delivery associated with severe ischemia can be compensated by the enhanced availability of antigen. In addition the diffusion barriers presented by the capillary are also decreased by the presence of edema and necrosis, making the antigen more available for interaction with the antibody. Taken together, these phenomena offer at least a 121

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H. W. STRAUSSet al.

partial explanation for the consistent ability of antimyosin to localize between 0.5 and 2% of the injected dose/infarct (0.025-0.2% dose/g). The wide clinical application of non-neoplastic radioimmune imaging will be enhanced when four major problem areas are addressed: (1) antibody delivery to the target site should be maximized, to increase the concentration of antibody at the target site by an order of magnitude if possible; (2) clearance of the unbound antibody from non-antigen containing background should be enhanced, to permit high contrast imaging; (3) the antigen-antibody complex must be maintained at the site of antigen expression, to allow imaging at late times after administration; and (4) the HAMA response must be nil, since the disease states where the imaging procedure will be applied are serious but not life threatening, and other procedures are available for diagnosis. Thrombosis, myocardial infarction and infection each cause inflammation, resulting in a local increase of perfusion and capillary permeability, enhancing the likelihood of antibody presentation to the lesion. These phenomena reduce the likelihood that the lesion is at an immunologically privileged site, where antibody delivery would be impaired. Since the antigens are rarely normal cell components, the likelihood of internalization and metabolism of the antigen-antibody complex is reduced. This increases the likelihood that the radiopharmaceutical will remain at the site of antigen expression. The HAMA problem is far more difficult to address. Fab fragments of murine monoclonal antibodies (MoAbs) at 0.5 mg doses rarely elicit a HAMA response with a single exposure, and have been used in some patients on multiple occassions without inducing HAMA. This response to murine Fab is different than that with F(ab’), of intact antibody, where the HAMA response increases with increasing doses or repeated administrations. Aside from the potential allergic responses associated with HAMA, the presence of HAMA decreases the efficacy of murine antibody localization at the target site, by accelerating blood clearance. In practice non-neoplastic applications of antibodies have the same difficulties encountered with radioimmune imaging for tumors: (A) the circulating half-times of these proteins or fragments maintain a relatively high blood background, making lesion detection difficult; (B) the concentration in the lesion is relatively low, rarely exceeding 2% of the injected dose, requiring long imaging times to be certain about the presence of a lesion; and (C) the distribution of the proteins in the extravascular space results in non-specific uptake in uninvolved tissue, reducing the contrast between the lesion site and adjacent background. Improvements in any of these parameters can markedly enhance the value of radioimmune imaging for lesion detection. The following is a brief review of the status of radioimmune imaging of clot, inflammation and myocardial necrosis.

Thromhus Detection Identification of clot in vivo can be accomplished either by binding to a feature of the preformed clot or by labeling a substance that will be incorporated in forming clot. Evolving thrombus passes through several stages before the fibrin is crosslinked. marking the presence of mature clot. At that point, endogenous thrombolytic agents begin to work at dissolving the clot. While there is some additional clot formed during the dissolution process, the equilibrium favors lysis and the bulk of thrombus is reduced over hours to days. These observations suggest that an agent that binds to preformed clot is more likely to identify thrombi in vivo than an agent that requires formation of additional thrombus to achieve localization. While there is no unanimity of opinion, two types of antibodies have had the greatest recent successes in localizing clot in vivo (Oster and Som, 1989; Stuttle et al., 1989)--those directed against a portion of cross linked fibrin (Liau et al., 1987) or against the fibrin binding site of activated platelets (Caller ef al.. 1985). In experimental studies in dogs with pulmonary emboli and peripheral thrombi, both anti-platelet and anti-fibrin antibodies failed to detect about 2/3 of the pulmonary emboli, but did image all peripheral lesions. When the pulmonary emboli were harvested and counted, significant activity was found on all clots-but the small size of some lesions ( < 50 mg) suggested that antibody affinity is not yet sufficient to permit detection of small lesions. While preliminary data suggested that antibody imaging has high sensitivity for detection of thrombi in the lower extremities, specificity for detection of thrombi in calf was surprisingly low (Aronen, 1989) perhaps due to imaging the legs flat on the imaging table. Elevation of the legs may reduce the volume of blood in the venous system, and hence avoid some of the zones of increased uptake that were not associated with thrombus. Defaucal et al. (1991) described studies in 44 patients with suspected deep venous thrombosis using an anti-fibrin antibody, and found, unfortunately, that the sensitivity of the procedure decreased with lesions in the thigh. A more difficult application of clot imaging is the detection of thrombi or emboli in the coronary or pulmonary beds. Both of these applications tax the system-due to the small size of the lesions-typically 5-50 mg, and their location-surrounded by blood. The small clot size requires very high uptake on the lesion to render it visible with the limited resolution of current generation instrumentation. Superimposed on this is the difficulty of detecting lesions over residual activity in the blood pool. While these problems seem insurmountable at this stage of development, antibodies with affinities of about lOI that have minimal localization in non-target areas could achieve this daunting task. Overall, clot detection with radioimmune imaging is in evolution. While it appears promising in the

Non-tumor applications of radioimmune imaging periphery, tition from to work in substantial

the technique faces substantial compeultrasound. If the technology can be made the heart and lungs, it promises to play a role in patient management.

Myocardial Necrosis Detection of myocardial necrosis with MoAbs is one of the more successful applications of non-tumor radioimmune imaging with MoAb fragments. Once the myocyte sarcolemma is irreversibly damaged (Khaw et al., 1982) it becomes permeable and soluble substances in the cell, even those of very high molecular weight, leak into the interstitium. Insoluble proteins, such as the heavy chain of cardiac myosin, remain in situ. Antibodies directed against the heavy chain of cardiac myosin will localize in the irreversibly damaged cell. Antibody localization occurs quickly (Khaw et al., 1987). In a study of animals with reperfused infarction, anti-myosin localization occurred within 1 h of i.v. administration. The extent of localization correlated with TTC staining of the extent of necrosis. Co-administration of 99mTCpyrophosphate in these same animals delineated a larger area of localization, suggesting concentration in both irreversibly and severely injured but recoverable tissue. Under circumstances of persistent occlusion, two factors contribute to the rapid delivery of the antibody to the site of necrosis: (1) sufficient residual perfusion (usually about 3-S% of normal flow-insufficient to maintain tissue viability) usually remains to present antibody to the disrupted cell within minutes of tracer administration-while blood levels are very high; (2) edema at the site of necrosis increases local protein content, thereby maintaining a relatively high concentration of protein in the vicinity of the antigen (Khaw et al., 1976). Studies in patients with myocardial infarction confirmed the experimental studies (Khaw et al., 1986; Johnson et al., 1989). The limited solubility of the antigen, maintaining myosin at the site of necrosis, is critical to the success of the imaging procedure. This limited solubility may also maintain some of the antigen in the vicinity of necrosis for an extended interval of time after the acute event-permitting identification of necrosis months after the acute episode. This fact may explain the sensitivity of anti-myosin imaging for the detection of myocarditis (Rezakalla et al., 1989; Obrador et al., 1989) a process where minimal necrosis occurs over an extended interval of time. At any one time, the number of myocytes undergoing necrosis may be small, but the “integral” over months of the disease appears to be sufficient for detection with antimyosin imaging. Similarly, rejection in cardiac transplantation is associated with modest amounts of ongoing necrosis, and may also be visible on antimyosin imaging for the same reason (Frist et al., 1987). The clinical application of anti-myosin imaging for the detection of acute infarction, has not gained wide popularity. The modest photon flux available from an

129

administered dose of ~1.5 mCi (66 MBq) of “‘In labeled material makes it necessary to record images for lO-20min/view. In addition, it usually takes 2448 h after injection for the blood pool to clear and for non-specific antibody concentration in normal myocardium to decrease sufficiently to permit definitive interpretation of the image. Competing laboratory and other imaging procedures have usually provided a clear answer during that time, making anti-myosin imaging a secondary procedure for the identification of acute necrosis. If anti-myosin imaging could be made to work within l-2 h of tracer administration, it may become the procedure of choice for definitive detection of acute infarction. Experiments to make this hypothesis a reality are under way using charge modified antimyosin (Khaw et al., 1989). Khaw reasoned that normal cells maintain a high negative zeta potential, while injured cells lose their potential in proportion to their degree of injury. By enhancing the negative charge on anti-myosin Fab, the non-specific leakage of anti-myosin into the extravascular space could be reduced, by enhanced electrostatic repulsion between the negatively charged antibody and the negative charge of normal healthy cells. The change in charge should not limit uptake at a site of antigen, because the binding affinity is far greater than the electrostatic forces. Preliminary studies in dogs with acute coronary occlusion support this hypothesis-infarction can be visualized in less than 30 min after antibody administration.

Detection of Inflammation/Infection Three approaches have been described radioimmune imaging of infection:

for the

(1) Antibodies directed against an attribute of the infectious organism. If the antibody is directed against a surface determinant, a large number of different agents are necessary, since cell wall antigens are remarkably different on each strain of organism. Since infections, like tumors, have a large number of dead or dying organisms, an internal antigen, such as a portion of the highly immunogenic lipopolysaccharide, endotoxin, that is exposed as the cell dies is a reasonable alternative (similar to the concept of anti-myosin). (2) Antibodies directed against the cells responding to the infection. Several antibodies react with white blood cells. Those that do not inhibit white cell function are capable of labeling the cells in oivo, to permit direct identification of sites of inflammation. (3) Since the site of infection has an increased protein leak as a component of the inflammation induced edema (increased efflux from the capillary and decreased drainage by lymphatics producing and increased protein

130

H. W. STRAUSS et al. space), an alternative approach to inflammation imaging utilizes human polyclonal IgG.

We tested the hypothesis of specific imaging in a rat model of deep soft tissue Pseudomonas aeruginosa infections. First, specific immune imaging of the infectious process using various MoAbs specific for a unique microbial antigen, in this case the Pseudomonas Type I specific polysaccharide, was shown to be possible. The success of this experiment, however, was tempered by the realization that specific immune imaging would require detailed characterization of the causative organism before this approach could be applied to detect focal infection. The inflammatory infiltrate consists of bacteria, dead (partially digested) bacteria, inflammatory cells, and debris. The dead bacteria offered an opportunity to utilize an antibody directed against an internal component of the organism, which, in contrast to the unique antigens on the bacterial surface, the interior of most Gram negative organisms share an antigenitally common core, with the components of endotoxin comprising a major portion of the antigen. Based on the histopathology, we studied an antibody directed against Lipid-A, a component of endotoxin contained in the interior of the bacteria, in the thigh abscess model of infection. In contrast to the polysaccharide antigen, which did not cross react with other types of Pseudomonas, the anti-Lipid-A antibody not only localized in Ps. aeruginosa Type I, but also in Type II, E. coli, and other Gram negative infections. This observation suggested that a generic Gram negative inflammation scan might bc possible. Throughout these experiments, however, we observed localization of control (non-specific) MoAbs at the site of inflammation. The kinetics of the control and specific antibodies at the site of inflammation were substantially different: both antibodies localized rapidly, permitting visualization of the lesion in less than 6 h, but clearance of the specific antibody (either surface or core directed) was slower than that of the control antibody (Rubin et al., 1988). As a result, it was possible to differentiate “specific” from “nonspecific” localization. The consistent localization of the control (non-specific) antibody at the inflammatory site suggested that a second non-specific approach might be possible, using a non-specific IgG (Fig. 1). While the basis for localization of specific antibodies on the surface or core of bacteria is clear, the mechanism of localization of the non-specific IgG requires further investigation. One explanation may be the enhanced expression of Fc receptors on macrophages, polymorphonuclear leukocytes and lymphocytes in the presence of focal inflammation. We tested this hypothesis by administering Fab fragments of the control IgG to animals with infection, and found minimal localization of the fragment at the site of inflammation, while the intact IgG and Fc fragments localized well (Fischman et al., 1989).

Thus, the mechanism of localization of the intact IgG requires the Fc portion of the molecule, and may be due to expression of Fc receptors at the site of infection. In the rat model of deep thigh E. cofi infection, focal localization of radiolabeled immunoglobulin “‘In-1gG was seen as early as 3 h post-injection, with the target-to-background ratio of the image continuing to increase until 24 h after injection. In other experiments, we have demonstrated that treatment of infected animals with anti-inflammatory agents, both steroidal and non-steroidal had not effect on image quality (Rubin et al., 1988). In contrast, infected rats that were made uremic by total nephrectomy showed minima1 lesion localization. To determine that imaging with polyclonal IgG was truly non-specific, additional experiments were performed in the thigh infected rat, using organisms other than E. coli. Similar results were obtained with inflammation due to Pseudomonas aeruginosa, Klebsiella pneumoniae, Bacteriodes fragilis, Staphylococcus aureus, Candida albicans and turpentine. These stud-

ies, coupled with the experiments demonstrating successful imaging with the Fc portion of the IgG molecule but not its Fab portion, suggest that this reagent could for the basis of a “generic” inflammation scan. To determine if non-specific imaging could be employed to detect focal inflammation in human subjects, human polyclonal IgG labeled with “‘In was administered intravenously to subjects with suspected infection (Fischman et al., 1988). Human polyclonal IgG, a commonly used therapeutic agent for the treatment of immunoglobulin deficiency, is readily available from many blood banks as a sterile, pyrogen free, reagent that has been pre-tested and certified against the presence of the human immuno-deficiency virus and hepatitis. When employed for therapy, doses of several grams are typically administered several times/year to each patient and the safety record has been excellent. An initial group of 84 patients with suspected sites of infection had sequential images of the whole body and selected sites (based on symptoms or signs) recorded at intervals from 6 to 72 h following injection of 37-55 MBq of “‘In-1gG. The presence of focal inflammation was determined by surgery, biopsy, x-ray CT and clinical follow-up. The suspected sites of infection were located in the abdomen or pelvis in 38 subjects, on vascular grafts in 25 subjects, in lung in 5 subjects, or in bones or joints in 15. Overall, in 48 of 52 patients with focal lesions determined by other modalities, the “‘In-1gG scan correctly identified the site, while 31 patients without focal inflammation were correctly identified. Four patients had false negative scans and one patient had a false positive result. This small series provides a sensitivity of 92% and a specificity of 95%. In 10 patients, the “‘In-1gG scan was the only imaging modality to correctly localize the site of inflammation. Ten patients were reinjected because of

Fig. 1. Anterior whole body scintillation camera images of 3 rats with focal thigh infection. The animals were infected 48 h prior to imaging. Twenty-four hours after infection “‘InIgG was administered intravenously, followed 24 h later by imaging. Focal tracer concentration can be appreciated in the infected left thigh. Fig. 2. Anterior view of the pelvis 48 h after administration of the descending colon. A focal zone of enhanced tracer quadrant. Fig. 3. Anterior Fig. 4. Anterior

of “‘In-1gG concentration

in a patient is apparent

view of the abdomen of a patient with inflammatory bowel disease. recorded 24 h after iv. administration of “‘In-IgG. image of the pelvis in a patient with osteomyelitis was recorded 24 h after i.v. administration 131

of the left proximal of “‘In-1gG.

with diverticulitis in the left lower The image

was

femur. The image

Non-tumor applications of radioimmune imaging Table 2. Results of “‘In-1gG scans in patients with suspected focal infection False True True False Site + + Vascular Abdomen Lung Sk&al Totals

I1 21 7 12 ?i

0 0 0 0 0

21 30 0 12 63

I 3

I

0 3

[After Rubin er al. (1989)]

In one subject the scan became positive and in the remainder, positive scans became more intense. A recent study in a larger series of patients (Rubin et al., 1989) confirmed these findings (Table 2) and Figs 2-4. Regardless of the mechanism, the IgG scan has proven to be an extremely useful technique for localizing focal sites of inflammation in humans. Infection sites due to numerous types of pathogens at various anatomic sites have been accurately localized. Treatment with antibodies, anti-inflammatory agents or immunosuppressive agents does not appear to affect the diagnostic quality of the images. Similarly, concurrent medical illness such as, diabetes, hepatic disorders or cardiovascular disease do not affect the scan. In contrast, uremia alters the biodistribution of the agent and severely limits sensitivity for lesion detection. In addition to the up-regulation of Fc receptors in focal infection, some tumors evoke an inflammatory response, while others are known to express Fc receptors. Co-incidentally, while seeking focal sites of infection in patients with co-existing tumors, we have observed striking focal localization of human polyclonal IgG in 2 patients with metastatic melanoma and 3 patients with metastatic carcinoma of the uterine cervix. While additional experience is necessary to determine the relative utility of this nonspecific imaging approach compared to that with specific antibodies directed against tumor antigens, this preliminary experience suggests a potential role for IgG imaging in patients with tumors. continued

symptoms.

Summary and Conclusions Radioimmune imaging for the detection of nonneoplastic processes is in its infancy. While only three major applications have emerged to date, the reliability of imaging infarction and infection suggest that these procedures work more reliably than radioimmune imaging of tumors (Fischman et al., 1989). The reliability may be based on the availability of abundant antigen, lack of antigen modulation and enhanced permeability at the lesion site. These observations suggest that non-neoplastic applications of radioimmune imaging will continue to grow. The potential of enhancing the speed of localization suggests that PET imaging, with its enhanced spatial resolution and quantitative abilities will play a role in radioimmune imaging in the near future.

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