Scintigraphy using a technetium 99m-labelled anti-E-selectin Fab fragment in rheumatoid arthritis
F. Jamar1,
F. A. Houssiau2,
J.-P. Devogelaer2,
P. T. Chapman3,
D. O. Haskard3,
V. Beaujean1,
C. Beckers1,
D.-H. Manicourt1,2 and
A. M. Peters4,
1 Centre of Nuclear Medicine and
2 Department of Rheumatology, University of Louvain Medical School, Brussels, Belgium and the
3 National Heart and Lung Institute and
4 Department of Imaging, Imperial College (Hammersmith Campus), London, UK
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Abstract
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Objective. We previously described a novel radiolabelled monoclonal antibody (1.2B6), which reacts with porcine E-selectin, for targeting activated endothelium as a means of imaging inflammatory disorders, and presented initial clinical work based on 111In-labelled antibody. The aim of the present study was to evaluate a Fab fragment of 1.2B6 labelled with 99mTc in patients with rheumatoid arthritis (RA) by comparison with (i) 111In-labelled 1.2B6 F(ab')2 and (ii) conventional bone scanning.
Methods. 99mTc-1.2B6-Fab (
440 MBq) and 111In-1.2B6-F(ab')2 (
27 MBq) were compared in 10 patients using a double-isotope protocol. Images were obtained 4 and 2024 h after injection. Two normal volunteers were also imaged. In a separate group of 16 patients, 99mTc-1.2B6-Fab and 99mTc-oxidronate (99mTc-HDP) (
740 MBq) were compared on the basis of visual and semi-quantitative analysis of joint uptake (joint/soft tissue ratios) 4 h after injection. The respective biodistributions and blood clearances of the two 1.2B6 fragments were also compared.
Results. Image contrast was slightly better with 99mTc-Fab at 4 h but equal for the two tracers at 24 h. Diagnostic accuracy, taking joint tenderness or swelling as the clinical endpoint, was 76% for both fragments at 24 h. Plasma clearance of 99mTc-Fab was faster than that of 111In-F(ab')2 (t1/2 142 vs 421 min; P<0.0001). 99mTc-Fab appeared somewhat unstable in vivo, as shown by activity in the thyroid gland and bowel. The diagnostic accuracy of 99mTc-Fab was 88%, higher than that of 99mTc-HDP (57%) as a result of the low specificity of the latter in RA. Receiver operating characteristic (ROC) curve analysis using joint/soft tissue ratios as a variable cut-off showed that 99mTc-Fab discriminates better than 99mTc-HDP between actively inflamed and silent joints (Z=4.72; P<0.0001). No uptake of 99mTc-Fab was observed by inactive or normal joints, whereas 99mTc-HDP was taken up by all joints to a variable degree, making the decision as to whether a particular joint is actively involved or chronically damaged very difficult.
Conclusion. 99mTc-anti-E-selectin-Fab scintigraphy can be used successfully to image synovitis with better specificity than 99mTc-HDP bone scanning. The advantages over 111In-1.2B6-F(ab')2 are easier availability of the radionuclide, improved physical properties and optimal imaging 4 h after injection.
KEY WORDS: Rheumatoid arthritis, E-selectin, Endothelium, Monoclonal antibody.
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Introduction
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Objective assessment of arthritis by radionuclide imaging has been a challenge now for 25 yr. To provide an accessible, reliable and objective technique, many radiotracers have been proposed, including radioiodinated albumin, sodium [99mTcO4]pertechnetate, gallium-67 citrate, phosphate compounds, liposomes and labelled leucocytes [16]. Although 99mTc-labelled human non-specific immunoglobulin (HIG) scintigraphy has been suggested more recently as a useful tracer for arthritis [7, 8], it targets inflammation non-specifically, prompting efforts to develop monoclonal antibodies with specific targets.
Taking advantage of recent progress in cell biology, especially in the field of adhesion molecules, we recently proposed scintigraphy using a monoclonal antibody (mAb) (termed 1.2B6) directed against the human and porcine cytokine-inducible endothelial cell-specific adhesion molecule, E-selectin, involved in the recruitment of various leucocyte subsets [9]. Importantly, while there is virtually no expression of E-selectin by endothelial cells of unstimulated tissues, immunohistochemistry has shown its presence in a wide range of diseases of inflammatory, infectious and immunological aetiology [10]. More specifically, the expression of E-selectin has been demonstrated in postcapillary venules in inflamed synovia of patients with rheumatoid arthritis (RA), but not by other cell types or normal synovium [11, 12]. In addition, the direct accessibility of the molecule at the luminal surface of small vessels is an advantage for targeting with radiolabelled macromolecules.
111In-labelled 1.2B6 was first validated in porcine models of acute inflammation [13], including experimental acute arthritis in pigs [14, 15]. In preliminary studies in patients with RA we compared the 111In-labelled F(ab')2 fragment of 1.2B6 with HIG, labelled with either the same radionuclide or with 99mTc [16, 17], and, by showing higher specificity and better image contrast, demonstrated the potential of 1.2B6 for identifying actively inflamed joints. However, for practical reasons (availability of the radionuclide, chemistry, cost) and because of its physical properties (suboptimal spatial resolution, low count rate and relatively high radiation dose to the patient), 111In is not the tracer of choice for wider application of this method.
The aim of this study, therefore, was to evaluate the 99mTc-labelled Fab fragment of 1.2B6 in patients with RA, first by comparison with the 111In-F(ab')2 fragment and secondly by comparison with a widely available tracer for bone and joint disorders, 99mTc-oxidronate (HDP).
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Materials and methods
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Patients
Twenty-six patients with RA fulfilling the 1987 ARA criteria [18] were enrolled prospectively. All but one had a disease duration of >1 yr and a therapeutic regimen which remained unchanged during the 2 weeks before the study and which included non-steroidal anti-inflammatory drugs, low-dose steroids (
7.5 mg equivalent prednisolone) and/or disease-modifying anti-rheumatic drugs.
The patients were divided into two subgroups. In group I, six females and four males [mean age 51.4 (S.D. 13.2) yr] were recruited for a double-isotope comparative study of 99mTc-1.2B6-Fab and 111In-1.2B6-F(ab')2. In addition, two healthy volunteers without any history of a joint disorder were studied; one of them underwent the double-isotope study and the other a 99mTc-1.2B6-Fab study only. In group II, 13 females and three males [mean age 50.9 (14.9) yr] were evaluated for a comparison between 99mTc-1.2B6-Fab and 99mTc-HDP. Most patients were studied on an outpatient basis and, in all cases, treatment remained unchanged during the 2-week interval between the 1.2B6 and HDP studies.
The study, for which patients and volunteers gave written informed consent, was approved by the Ethics Committee of the University of Louvain Medical School, Brussels.
Tracers
The anti-E-selectin 1.2B6 mAb is a mouse IgG1 generated against cytokine-activated human umbilical vein endothelial cells [19]. At each step of the preparation, the immunoreactivity of the mAb fragments, controlled by enzyme-linked immunosorbent assay (ELISA) on cultured tumour necrosis factor
-activated endothelial cells, remained unchanged. Prior to clinical use, the mAb and fragments underwent extensive toxicity and safety testing by independent laboratories [16].
F(ab')2 fragments were generated by pepsin digestion [20] and labelled with 111In following diethyltriaminepentaacetic acid (DTPA) coupling according to the method of Hnatowich et al. [21]. On the day of each study, 35 µg DTPA-F(ab')2 was labelled with approximately 3040 MBq 111In-citrate. After purification by gel chromatography using a PD10 column (Pharmacia, Uppsala, Sweden) and saline as solvent, the radiopharmaceutical purity, assessed by thin-layer chromatography (ITLC-SG; Gelman Sciences, Ann Arbor, MI, USA) in phosphate-buffered saline (PBS), was >97% in all cases. An activity of 26.7 (7.1) MBq of 111In-1.2B6 was injected intravenously (i.v.).
Fab fragments were produced by papain digestion followed by purification using protein A affinity chromatography and dialysis [22]. Radiolabelling was performed by means of the reduction-mediated method using 2-mercaptoethanol (ME) [23]. The Fab was concentrated to
2.2 mg/ml and incubated with a 2000-fold excess ME for 1 h at room temperature. ME was removed by gel chromatography using a PD10 column and PBS as solvent (0.5 ml fractions). To avoid reoxidation in air, the protein peak fractions were immediately pooled, filtered on a Millipore 0.22 µM filter and stored as aliquots of 100200 µg at -20 °C. Labelling of the ME-reduced Fab with 99mTc was achieved by incubating the protein with Sn(II)F2 (obtained from an Amerscan MDP kit; Amersham, Amersham, UK), immediately followed by addition of
925 MBq freshly eluted 99mTcO4-. Preliminary experiments showed that the optimum Sn++/Fab ratio was 20 and that, in these conditions, up to 7.4 GBq 99mTc could be attached to 1 mg Fab with labelling efficiency >85% and <2% TcO2/colloid species, as determined by the method of Thrall et al. [24]. Due to incomplete labelling efficiency, a purification step using gel chromatography (PD10; Pharmacia) was necessary prior to injection. The in vitro stability of the labelled Fab was assessed by dilution in PBS and fresh human serum and by a DTPA challenge with a DTPA/Fab molar excess of 2000. In these conditions, >85% of the bound 99mTc was retained after 6 h. All patients were given Fab i.v. within 30 min of final purification, with a mean activity of 440 (95) MBq. The labelling efficiency, determined from the peak of protein-associated radioactivity of the PD10 purification (Fig. 1
), was 94.9% on average after exclusion of the mean 6.9% of the radioactivity found to stick to the column. The radiopharmaceutical purity, assessed by ITLC, was >95% (range 95.199.4%). In group I patients, the ratio of 99mTc to 111In activity injected was 21 on average; tracers were injected i.v. in separate syringes in saline containing 0.2% human serum albumin (Mérieux, Brussels, Belgium). The amounts of 1.2B6-F(ab')2 and 1.2B6-Fab injected were less than 35 and 100 µg respectively (after losses during purification). The specific activities were 220 (48) GBq/mmol for 99mTc-Fab and 76 (20) GBq/mmol for 111In-F(ab')2. In double-isotope studies, these amounts are minimal and far below the saturating quantities. Therefore, the differentially labelled fragments are not likely to compete with one another for binding to the available antigen. The injected dose was measured precisely by comparison with a standard used later to measure the blood clearance of radiotracer. Using pig lectin as specific ligand and ouabain as negative control, the immunoreactivity of the labelled fragments was assessed qualitatively by means of dot blot in the preclinical phase, during which the labelling methods were initially developed. The immunoreactivity of the fragments was not further assessed during the clinical studies.

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FIG. 1. Examples of elution profiles of the 99mTc-1.2B6-Fab and 111In-1.2B6-F(ab')2 fragments by gel chromatography with a PD10 column and saline as solvent (fractions of 0.5 ml).
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99mTc-HDP (TechneScan HDP; Mallinckrodt Medical, Petten, The Netherlands) was given i.v. (740 MBq).
Imaging protocol
In group I, imaging was performed 4 and 2024 h after tracer injection. Five-minute images were obtained over the chest, abdomen, pelvis (anterior and posterior views) and all peripheral joints (anterior views and posterior views for the feet). Images were recorded simultaneously in two energy windows: 133147 keV for 99mTc and 220270 keV for 111In. The gamma camera (Diacam; Siemens, Hofman Estates, IL, USA) was equipped with a medium-energy collimator. Using these settings, the cross-over of 111In activity into the 99mTc window is 21.3% and the cross-up of 99mTc into the 111In windows is 0.2%. These values were used for cross-over corrections of semi-quantitative data. Venous blood was drawn
1, 2 and 3 h after injection to measure the distribution volume and plasma clearance.
In group II patients, the 99mTc-HDP and 99mTc-1.2B6-Fab studies were performed within 2 weeks of each other but at least 3 days apart. In eight subjects 99mTc-HDP was performed first, and in the remaining patients 99mTc-1.2B6 was completed first. Images were obtained 4 h after injection of either tracer. Five-minute spot views of all joints were obtained with the gamma camera fitted with a low-energy, high-resolution collimator (Diacam; Siemens). In five cases, the bone scan was acquired in whole-body mode (Toshiba GCA-901A) with additional spot views over the feet and hands.
Data analysis
Biodistribution.
Plasma activity data were fitted with a monoexponential function from which the disappearance half-time was calculated. The distribution volume was estimated using the intercept of the exponential curve at time 0 (A0). Because intra-patient comparisons were performed, no normalization for plasma volume was attempted. Activities in liver, spleen, kidneys, urinary bladder, heart and large vessels, thyroid, small bowel, large bowel and bone marrow (pelvis) were assessed visually as present or absent.
Comparison of joint scintigraphy using the two 1.2B6 fragments.
Joint scores were established using a four-point visual scoring system in which 0 represents no uptake and 1, 2 and 3 represent moderate, definite and marked uptake respectively [8]. Twenty-one joints were assessed in each patient: shoulders, elbows, wrists, metacarpophalangeal and proximal interphalangeal joints, hips, knees, ankles, midtarsal and metatarsophalangeal joints (including toes), and the cervical spine. Because of limitations in spatial resolution, especially in the upper 111In window, scores for small joints of the hands and feet were recorded as the mean of five scores. Scintigraphic joint scores obtained for both 1.2B6 fragments were compared with each other and with scores of clinical activity, namely tenderness (or pain at mobilization) [25] and swelling [16]. Although these parameters were assessed initially on a three-point scale, for simplicity of data presentation we considered a particular joint to be active if tenderness and/or swelling was recorded prior to radiotracer injection.
Semi-quantitative analysis was performed for those joints that displayed abnormal activity with both tracers at both scanning times. Regions of interest (ROIs) were drawn over joints displaying abnormal activity and over adjacent soft tissues. Joint/soft tissue ratios (JSRs) and contrast figures were calculated after appropriate physical corrections (ROI size, background and cross-over). Contrast figure (CF) is defined as {[(activity in joint)-(activity in soft tissues)]/[activity in joint]}. Retention of radioactivity in joints and soft tissues was calculated by dividing the activity in ROIs at 2024 h, normalized for physical decay, by the activity in ROIs at 4 h. The same ROIs were used for sets of four images.
Comparison of 99mTc-1.2B6-Fab with bone scanning.
Because 99mTc-HDP is taken up in normal joints, a simplified visual scoring system was used for both agents, in which 0=no uptake, 1=visible uptake and 2=marked uptake. The same joints as listed previously were analysed, except that the metacarpophalangeal and proximal interphalangeal joints were analysed individually, giving 37 joints for each patient. Again, clinical activity of the joint was defined as the presence of either tenderness or swelling. In the 11 patients in whom both tracers were imaged with the same camera, semi-quantification (JSR) was performed on 32 joints [shoulders, elbows, wrists, MCP and PIP joints (individual), knees, ankles and MTP joints] regardless of the scintigraphic appearance.
Statistical analysis
Unless otherwise stated, data are reported as mean (S.D.). Biodistribution data were compared by paired Student t-test (blood clearance) and Fisher's exact test (visual analysis). Results of joint scintigraphy were analysed using non-parametric tests. Concordances and discordances between two scintigrams and between scintigraphic and clinical scores were assessed using the
2 and McNemar tests respectively. The kappa (
) test of Cohen was used to test whether concordance between two tests was the result of chance [26]. Taking tenderness and/or swelling as the clinical endpoint, sensitivity (TP/TP+FN), specificity (TN/TN+FP) and accuracy [(TN+TP)/all patients] were calculated (TP, true positive; TN, true negative; FP, false positive; FN, false negative). Semi-quantitative 99mTc-Fab and 99mTc-HDP image data were compared using discriminant analysis. ROC curves were computed by varying the JSR as the cut-off for positivity. The areas under the curves were compared using the method of Hanley [27, 28] and Z>1.96 was considered significant. Otherwise, P<0.05 was considered significant.
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Results
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Biodistribution of anti-E-selectin preparations
The plasma clearance of both tracers differed significantly: using a mono-exponential fit of plasma activity data after 1 h, the mean t1/2 was 142 (S.D. 23) min for 99mTc-Fab and 421 (107) min for 111In-F(ab')2 (P<0.0001). The distribution volumes of the tracers were not significantly different, with an A0 of 29.4 (9.5) % of injected data/l plasma for 99mTc-Fab and 35.0 (10.1) %ID/l plasma for the 111In-F(ab')2 (P=0.086). These translate to average distribution volumes of 3.40 and 2.86 l respectively, values which are close to plasma volume. The distribution of the activity in various organs is shown in Table 1
. The major differences are the marked bladder activity at 4 h and the almost complete disappearance of a 99mTc signal in the circulation at 24 h, because of the faster blood clearance of this radiotracer. In addition, with 99mTc-Fab, some activity was regularly noted in bowel at 24 h but not 4 h. Thyroid activity was noted in 6/11 subjects at 4 h, in spite of the fact that virtually no free 99mTc was injected, as determined by the preinjection ITLC. Liver, spleen and kidney activities appeared equal with the two tracers, the highest uptake being observed in the kidneys. Although some 111In activity was noted in the urinary bladder, it was only barely visible, whereas with the 99mTc tracer bladder activity was marked.
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TABLE 1. Frequency of visualization (%) of normal organs with 99mTc-1.2B6-Fab and 111In-1.2B6-F(ab')2 at 4 and 2024 h in 11 subjects
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Comparison between 99mTc-Fab and 111In-F(ab')2 joint scintigraphy
No activity was observed in the joints of the two normal volunteers. Overall, both 99mTc-Fab and 111In-F(ab')2 gave the same information. Image quality was clearly better for 99mTc-Fab at 4 h than for 111In-F(ab')2. However, as a result of radioactive decay, the image quality appeared similar for the two tracers at 24 h. Accordingly, interpretation of the early images was easier with 99mTc-Fab (Fig. 2
). For both tracers, vascular activity was noted on the early scans but did not hamper adequate delineation of articular uptake. The pattern of joint activity was similar for the two tracers (Fig. 3
).

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FIG. 2. Images of the knees obtained 4 h (top) and 20 h (bottom) after injection of 99mTc-1.2B6-Fab (left) and 111In-1.2B6-F(ab')2 (right) in a patient with RA, showing deposition of radiotracer in the joints.
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FIG. 3. Images of the hands obtained 4 h (top) and 20 h (bottom) after injection of 99mTc-1.2B6-Fab (left) and 111In-1.2B6-F(ab')2 (right) in a patient with RA.
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Joint scores observed for the two fragments showed a high degree of concordance (Table 2
). However, regardless of the clinical picture, the 99mTc-Fab images allowed the detection of more positive joints than the 111In-F(ab')2. At 4 h, 90 joints (42% of the total), were scored higher for 99mTc-Fab than for 111In-F(ab')2, whereas this number was 47 (i.e. 22%) at 24 h. The opposite was seldom seen, only two joints (1%) being scored higher for 111In-F(ab')2 at 4 h and 12 (6%) at 24 h. Moreover, regardless of the intensity of uptake, 99mTc-Fab showed abnormal activity in 52 and 20 joints that were negative on the 111In images, at 4 and 24 h, respectively. These differences were significant (McNemar test; P<0.0001 and P<0.005 at 4 and 24 h respectively). Both tracers yielded more positive images at 24 h than at 4 h (P<0.0001 for 111In and P<0.05 for 99mTc).
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TABLE 2. Absolute and partial* (in parentheses) concordance between joint scintigraphic scores for 99mTc-1.2B6-Fab and 111In-1.2B6-F(ab')2 at 4 and 24 h
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Semi-quantitative analysis was used to compare the degree of uptake of the two fragments. Only those joints that were visually judged abnormal on all four scans were considered for this analysis. As shown in Fig. 4
, the contrast was slightly better with 99mTc than with 111In at 4 h, whereas no significant difference was noted at 24 h. For both tracers, the contrast factor increased with time, although this was more marked for the 111In fragment. Measurement of the retention of tracers in inflamed joints, using decay correction for actual scanning times and appropriate cross-over correction, showed that the 99mTc activity decreased by a mean of 20% [95% confidence interval (CI) 15.424.7], whereas the activity in soft tissues decreased by a mean of 38.1% (95% CI 33.642.5). Conversely, the 111In activity in joints increased on average by 27.4% (95% CI 18.636.2), whereas the activity over soft tissues decreased by 22.7% (95% CI 17.827.6). All changes were significantly different from unity (P<0.0001).

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FIG. 4. Contrast figures and JSRs for 111In-1.2B6-F(ab')2 and 99mTc-1.2B6-Fab at 4 or 24 h. *P<0.001 between 4 and 24 h for the same tracer; #P<0.05 between 111In-1.2B6-F(ab')2 and 99mTc-1.2B6-Fab at the same time point.
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Sensitivity, specificity and accuracy are compared between the two tracers in Table 3
. The diagnostic performances of 99mTc-Fab at 4 and 24 h and of 111In-F(ab')2 at 24 h were quite similar. In particular, the best accuracies were obtained for the early 99mTc and delayed 111In images. There was a highly significant correlation between the scintigraphic findings and the clinical scores (
2 test; P<0.0001 for all comparisons). This was not the effect of chance, as demonstrated by the
test (P<0.05 for all).
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TABLE 3. Diagnostic performances of 99mTc-1.2B6-Fab and 111In-1.2B6-F(ab')2 at 4 and 24 h in detecting actively inflamed joints taking tenderness and/or swelling as the reference for clinical involvement (%)
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Comparison between 99mTc-Fab and 99mTc-HDP scintigraphy
99mTc-HDP provided high-quality images 34 h after injection without significant residual vascular activity. As expected, uptake was observed over almost all joints, regardless of clinical involvement (Fig. 5
). Contrast between joints, adjacent bone and soft tissues was highly variable. Conversely, image intensity with the 99mTc-Fab appeared slightly less impressive and images were characterized by the persistence of some vascular activity and soft tissue background. Importantly, uptake was highly variable from joint to joint, 46.6% of the joints (276/592) showing no activity that could be delineated from the background. In addition, Fab uptake was restricted to the inflamed synovium, whereas HDP uptake not only involved the joint itself but extended beyond the subchondral bone.

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FIG. 5. Images obtained 4 h after injection of 99mTc-1.2B6-Fab (left) and 99mTc-HDP (right) in two patients with RA. The images on the top correlate well; the bottom images show discordance between the lack of uptake of the mAb fragment and diffuse bony uptake.
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There was a high degree of concordance between clinical scores (i.e. positive or negative) and the scoring of 99mTc-Fab images (P<0.0001), which was not the effect of chance (
=0.76; P=0.035). Taking tenderness or swelling as evidence of clinical activity, 340/592 joints were considered active. Among these, 47 (13.8%) displayed no activity with 99mTc-Fab whereas 129 and 164 received scores of 1 and 2 respectively. Twenty-three of the 252 clinically inactive joints (9.1%) were judged positive with the 1.2B6 scan. The positive and negative predictive values of 99mTc-Fab were thus 92.7 and 83.0% respectively, with an overall diagnostic accuracy of 88.2%. Conversely, as 99mTc-HDP is taken up by normal bone, including juxta-epiphyseal bone, visual discrimination between normal and abnormal uptake is more difficult. If any joint uptake was considered abnormal, the negative and positive predictive values of bone scanning were 42.9 and 57.4% respectively, with an overall accuracy of 57.4%. Only 140 out of the 340 clinically active joints (41.1%) displayed activity that was considered intense (i.e. score 2). Taking marked uptake as indicative of a positive bone scan, the positive and negative predictive values were 72.2 and 49.7% respectively, with an overall accuracy of 57.1%. Using this criterion, although there was a high degree of concordance between the clinical and scintigraphic score (
2=25.7; P<0.0001), this agreement could have been the effect of chance (
=0.18; P=0.062).
In close agreement with the visual findings, taking all joints together, the JSR was higher for 99mTc-HDP than for 99mTc-Fab (Fig. 6
). The mean JSR was 1.13 (0.52, 95% CI 1.081.17) for 99mTc-Fab and 2.11 (1.42, 95% CI 1.962.26) for 99mTc-HDP (P<0.0001). Similar differences were noted if subsets of clinically active or inactive joints were considered. There are, however, three major differences between the radiotracers.

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FIG. 6. JSRs for 99mTc-1.2B6-Fab and 99mTc-HDP in clinically active (hatched bars) and inactive (open bars) joints. Data are mean (S.D.) of all joints or subsets of joints. Note the different scales of the ordinates.
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First, among the 252 clinically inactive joints, the JSR was lower than unity in 75% of the joints with 1.2B6 and in only 19% for HDP. Secondly, the spread of JSR values was clearly more marked for HDP than for 1.2B6. This is illustrated by the coefficients of variation of the JSR values, which were 33 and 37% for 1.2B6 in clinically negative and positive joints respectively compared with 45 and 65% for HDP. Thirdly, as shown in Fig. 7
, discrimination between clinically positive and negative joints was excellent with 1.2B6 (except for the shoulders and ankles) and poor for HDP (except for the small finger joints). This was confirmed by discriminant analysis, taking the clinical result as the dependent variable and JSR for both tracers as independent variables. 99mTc-1.2B6 was a better discriminant than 99mTc-HDP for the identification of inflamed joints in general (P<0.0001). This was true in subsets of large joints and small finger joints (P<0.0001 for both subgroups). This is clearly illustrated by ROC curves computed using JSR as the variable cut-off (Fig. 7). The best sensitivity/specificity pairs were JSR >1.1 for 99mTc-Fab and >1.7 for 99mTc-HDP. The areas under the curves for both tracers were significantly different (Z=4.72; P<0.0001). However, it should be stressed that ROC curve analysis did not yield better accuracy than visual scoring and accordingly it is only reported for comparative purposes.

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FIG. 7. Receiver operating characteristics (ROC) curves obtained for 99mTc-1.2B6-Fab and 99mTc-HDP using JSR as the variable cut-off to discriminate between clinically active and inactive joints. The arrows point to the best cut-offs, namely 1.1 for 99mTc-1.2B6-Fab and 1.7 for 99mTc-HDP. The areas under the curves are significantly different (P<0.0001).
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Discussion
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This study demonstrates that joint inflammation can be imaged successfully by targeting activated endothelial cells with a 99mTc-labelled Fab fragment of an mAb antibody against the inducible endothelial adhesion molecule, E-selectin. After the encouraging experimental results in acute arthritis and in patients with RA obtained with an 111In-labelled F(ab')2 fragment of the 1.2B6 mAb [1517], it was important to develop a tracer labelled with 99mTc. This radionuclide, instead of 111In, is a prerequisite for the wider use of the method. Labelling of the Fab fragment was shown to be possible without evident loss of affinity for E-selectin in vivo. The labelled Fab appears stable enough in vitro for administration up to 23 h after preparation. Radiopharmaceutical purity was satisfactory after gel chromatography, although the need for a purification step is obviously not optimal for routine applications. Nevertheless, the labelling procedure can be optimized and a cold kit formulation could be envisaged in a dedicated radiopharmaceutical environment.
We chose to develop the Fab fragment because of its faster blood clearance compared with F(ab')2. Although blood vessel activity and tissue background were both lower with the monovalent fragment as early as 4 h after injection, an improved target/background ratio was not in fact observed because, in parallel to the decrease in soft tissue activity between 4 and 24 h, joint uptake of 99mTc-Fab also decreased slightly but significantly. This may represent elution of 99mTc from the inflamed joint itself, as has been reported for 99mTc-HIG [29, 30], whereas the increase in 111In signal over inflamed joints is in accordance with previous findings and consistent with internalization of the label after antibodyantigen interaction [31]. The prominent liver and kidney uptake of both fragments will limit the potential value of this mAb in these organs. This is, however, a problem with most, but not all, mAbs [32] as well as with other tracers used for imaging inflammation. Thyroid and bowel activity was not observed with 111In and is thus likely to represent free 99mTc. Although this is not a significant drawback for imaging peripheral joints, it calls for an improvement in the labelling method, perhaps through a bifunctional chelating system, especially if 99mTc-Fab were to be used in other indications, such as inflammatory bowel disease and pyrexia of unknown origin.
Globally, anti-E-selectin scanning with 99mTc-Fab at 4 h appears almost equivalent to scanning with the 111In-F(ab')2 at 24 h. In terms of clinical use, this is a clear advantage for the 99mTc-Fab as it can be used in a one-day protocol, similarly to 99mTc-HIG. By comparing 111In-F(ab')2 with both 111In-HIG and 99mTc-HIG [16, 17], we have demonstrated previously that the contrast achieved with anti-E-selectin is better than that obtained with non-specific antibody, consistent with the notion that specificity provides significant added value to the non-specific uptake common to any macromolecule.
99mTc-anti-E-selectin Fab demonstrated more specific targeting of active joint inflammation compared with 99mTc-HDP, which displays abnormal uptake over both currently inflamed and chronically damaged joints. 99mTc-HDP is very sensitive for the detection of joint and subchondral bone abnormalities [4, 33] but it cannot discriminate accurately between actively inflamed and chronically affected joints [34]. Moreover, the deposition of 99mTc-HDP in normal subchondral and juxta-epiphyseal bone sometimes makes discrimination between a normal joint and recently developed arthritis very difficult. As shown in the present study, neither visual assessment nor semi-quantification of HDP images allowed better diagnostic accuracy than anti-E-selectin. Although the latter radiotracer leads to less impressive JSRs as a result of the background signal, the absence of uptake by normal joints allows the markedly improved detection of active joints.
The new tracer described in this study is an mAb of murine origin, raising the issue of immunogenicity. However, the small amount of mAb administered and the use of the Fab fragment devoid of Fc portions, which are thought to be responsible for immunization, should reduce the likelihood of immunogenicity. Although patients were not tested for a human anti-mouse antibody response, some were imaged again with 99mTc-Fab 24 months later but with no change in the biodistribution. In future, other approaches using humanized or chimeric mAb or smaller fragments, such as sFv, which can be produced by bacterial systems [35], will be considered. The results obtained with a 99mTc-labelled Fab, which is only twice the size of an sFv, suggest that sFv might be large enough for appropriate imaging. Alternatively, the development of other molecules to target E-selectin, such as glycoproteins and peptide ligands, might be the best way for the future [36, 37].
In conclusion, this study has demonstrated that imaging of inflammatory arthritis with a 99mTc-labelled Fab directed against E-selectin is as effective as a F(ab')2 of the same mAb labelled with 111In. The method has proven superior to 99mTc-HDP bone scanning and should now be used in prospective studies to demonstrate its clinical relevance and impact on patient management, not only in arthritis but also in patients with various other inflammatory and/or immune disorders. The expression of E-selectin on the vascular endothelium of many diseased organs, but not of normal tissues, makes this technique an attractive one in a wide range of clinical situations.
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Notes
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Correspondence to: A. M. Peters, Department of Nuclear Medicine, Box 170, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2QQ, UK. 
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Submitted 2 February 2001;
Accepted 6 July 2001