Myocardial kinetics of Tc-99m glucarate in low flow, hypoxia, and aglycemia.

BACKGROUND: Technetium-99m glucarate is a myocardial infarct-avid imaging agent. Recent conflicting and inconclusive reports have suggested that the agent may be taken up by ischemic but viable myocardium. The purposes of this study were (1) to determine conclusively whether there is Tc-99m glucarate uptake in ischemic viable myocardium and (2) to investigate the potential mechanisms for such uptake by studying components of ischemia, namely, low flow, hypoxia, and aglycemia. METHODS AND RESULTS: Rat hearts were isolated and perfused in a modified Langendorff preparation with a crytalloid perfusate. Tc-99m glucarate was studied in control (n = 6), low-flow (n = 5), hypoxic (n = 5), and aglycemic (n = 5) conditions. The experimental protocol consisted of 20-minute baseline (12 mL/min flow) and 20-minute treatment (low flow at 1 mL/min, hypoxia, or aglycemia), followed by tracer uptake (20 minute) and washout (20 minutes). Activity was monitored with a sodium iodide detector. The tracer was delivered continuously over a 20-minute uptake period. The injected dose was 150 micro Ci (5.6 MBq). Hemodynamics were monitored throughout. Triphenyltetrazolium chloride staining was used to assess myocardial viability. There was no evidence of myocardial necrosis. Low flow tended to delay tracer uptake compared with control for the first 10 minutes, but this did not reach statistical significance. Low flow increased end fractional retention significantly compared with control (mean +/- SEM, 59.0% +/- 0.9% peak vs 41.2% +/- 1.4%, respectively; P <.05). Hypoxia resulted in a trend toward increased uptake; however, this was significant only at one early time point during the uptake phase. Retention in the hypoxia group was similar to control. Tc-99m glucarate uptake was significantly increased in aglycemia from 16 minutes to peak compared with control (1.36% +/- 0.71% injected dose per gram vs 0.91% +/- 0.37% injected dose per gram, respectively; P <.05). Aglycemia produced significantly higher end fractional retention compared with control (51.6% +/- 1.8% peak vs 41.2% +/- 1.4%, respectively; P <.05). CONCLUSIONS: Tc-99m glucarate myocardial retention is increased in the setting of ischemia, even in the absence of necrosis. This increased retention is not due to hypoxia. Furthermore, the retention is only partially explained by tissue hypoglycemia. Thus low flow per se appears to have a role in this increased retention, probably as a result of delayed flow-dependent washout.

Detection of acute myocardial infarction by 99mTc-labeled D-glucaric acid imaging in patients with acute chest pain.

UNLABELLED: Definitive diagnosis of acute myocardial infarction early in the process is often difficult. An imaging agent that localized quickly and specifically in areas of acute necrosis could provide this critical diagnostic information. To determine whether imaging with 99mTc-labeled D-glucaric acid (GLA) could provide this information, we imaged a group of patients presenting with symptoms suggestive of acute infarction. METHODS: Twenty-eight patients presenting to the emergency department with symptoms highly suggestive of acute infarction were injected with 99mTC-GLA and imaged about 3 h later. RESULTS: The sensitivity of lesion detection was remarkably time dependent. Fourteen patients with acute infarction injected within 9 h of onset of chest pain had positive scans, even in the presence of persistent occlusion. The remaining 14 patients had negative scans. Nine patients with negative scans had acute infarction but were injected more than 9 h after onset of chest pain. The final diagnosis in the remaining 5 patients was unstable angina (3 injected <9 h and 2 injected >9 h after onset of chest pain). Six patients were reinjected with 99mTc-GLA 4-6 wk after their initial study to determine whether persistent positive scans occurred with this agent. All 6 had negative scans. CONCLUSION: This study suggests that 99mTc-GLA localizes in zones of acute myocardial necrosis when injected within 9 h of onset of infarction.

The relationship between plasma leptin and nutritional status in chronic hemodialysis patients.

Leptin serves an important role in suppressing appetite in mice and is known to be elevated in chronic renal failure (CRF) patients. But clinical significance of leptin as an appetite-reducing uremic toxin, remains to be determined. So we studied the relationship between plasma leptin and nutritional status in 46 chronic hemodialysis (HD) patients. Pre HD leptin was measured and divided by body mass index (BMI) to give adjusted leptin levels. KT/Vurea (K, dialyzer urea clearance; T, duration of HD; V, volume of distribution of urea), C-reactive protein (CRP), plasma insulin and nutritional parameters such as serum albumin, normalized protein catabolic rate (nPCR), subjective global assessment (SGA), BMI and mid-arm muscle circumference (MAMC) were also measured. Mean plasma leptin levels were 8.13+/-2.91 ng/mL (male 3.15+/-0.70; female 14.07+/-6.14, p<0.05). Adjusted leptin levels were positively correlated with nPCR (male r=0.47, p<0.05; female r=0.46, p<0.05), SGA (male r=0.43, p<0.05; female r=0.51, p<0.05) and MAMC (male r=0.60, p<0.005; female r=0.61, p<0.05). They did not correlate with KT/Vurea, serum albumin, hematocrit, bicarbonate, insulin and CRP. Presence of DM and erythropoietin therapy had no effect on leptin levels. These results suggest that leptin is a marker of good nutritional status rather than a cause of protein energy malnutrition in chronic HD patients.

Avidity of technetium 99m glucarate for the necrotic myocardium: in vivo and in vitro assessment.

BACKGROUND: Similar to other 99mTc-based infarct-avid agents, 99mTc-glucarate localizes in myocardial infarcts. Whether severely ischemic viable myocytes sequester 99mTc-glucarate is uncertain. To assess the infarct specificity, in vitro and in vivo studies were performed. METHODS AND RESULTS: H9C2 embryonic rat cardiocytes cultured under normoxia (N) or hypoxia (H) for 24 hours in 7.5 muCi 99mTc-glucarate were compared with necrotic cardiocytes. Mean H/N ratio (3.0 +/- 0.004, mean +/- SD) was significantly less than that of the necrotic/N ratio (39.9 +/- 6.5, p < 0.01). Reperfused myocardial infarction (MI) in 4 dogs confirmed by 201Tl (0.5 to 1.0 mCi) scintigraphy were imaged serially with simultaneously injected mixture of 99mTc-glucarate and 111In-antimyosin Fab. Infarcts were detected scintigraphically within 4 to 10 minutes with 99mTc-glucarate. 111In-antimyosin required more than 1 hour. Myocardial distribution at 5 hours showed a direct correlation between 99mTc-glucarate and 111In-antimyosin uptake (r = 0.99, p < 0.0001). Both 99mTc-glucarate (r = -0.777, p < 0.0001) and 111In-antimyosin (r = -0.775, p < 0.0001) were inversely related to 201Tl distribution. CONCLUSIONS: The near perfect correlation between 99mTc-glucarate and 111In-antimyosin uptake (r = 0.99) in reperfused canine MI and the insignificant glucarate uptake by viable cardiocytes in vitro attest to the avidity of 99mTc-glucarate for the necrotic myocardium and favor its use as a specific early marker of myocyte necrosis in acute MI.

Noninvasive detection of atherosclerotic lesions by 99mTc-based immunoscintigraphic targeting of proliferating smooth muscle cells.

BACKGROUND: Mouse/human chimeric antibody Z2D3 identifies an antigen produced exclusively by proliferating smooth muscle cells of human atheroma, and also cross reacts with experimentally induced atherosclerotic lesions in rabbits. Fab' fragments of Z2D3 antibody were labeled with (99m)Tc using glucaric acid as a weak transchelator. The potential role of (99m)Tc-labeled Z2D3 scintigraphy was explored for noninvasive imaging of experimental atherosclerotic lesions. METHODS AND RESULTS: (99m)Tc-Z2D3 Fab' was utilized for noninvasive imaging in four rabbits with experimentally induced atherosclerotic lesions and in one control rabbit. In addition, (99m)Tc-labeled nonspecific 103D2 Fab' was used for comparison in four other rabbits with atherosclerotic lesions. The atherosclerotic lesions were induced by balloon de-endothelialization of the infradiaphragmatic abdominal aorta and 12 weeks of hyperlipidemic diet. An aliquot of 15 mCi (550 mBq) of (99m)Tc pertechnetate was incubated with 6.25 mg of glucaric acid for 30 min followed by incubation of (99m)Tc glucarate with 375 microg of Z2D3 Fab' or 103D2 Fab' for an additional 30 min. Instant thin-layer chromatography demonstrated almost complete radiolabeling. (99m)Tc-Z2D3 was administered IV and gamma imaging was performed at the time of injection, 3, 6, 9, and 12 h, followed by ex vivo imaging of the excised aorta, and biodistribution was performed. Unequivocal visualization of atherosclerotic lesions was possible in all four animals at 9 to 12 h with Z2D3 Fab'. Quantitative uptake, as represented by mean lesion-to-liver count density ratio, was 0.6+/-0.05. Imaging with nonspecific 103D2 Fab' did not show any localization in the abdominal aorta (lesion-to-liver ratio, 0.45+/-0.02, p=0.02). Ex vivo lesion-to-normal aortic segment ratio was 4.3+/-0.9 for Z2D3 and 1.04+/-0.08 for nonspecific 103D2 Fab' (p=0.01). Biodistribution studies demonstrated 0.03+/-0.003% injected Z2D3 dose per gram in the atherosclerotic lesions as compared with 0.01+/-0.003% in the nondenuded thoracic aorta of atherosclerotic rabbits (p=0.008). However, only 0.008+/-0.002% of the mean injected dose per gram was obtained in the atherosclerotic lesions (p=0.001) as compared with 0.005+/-0.003% in the normal aortic segments with 103D2. No Z2D3 uptake in normal rabbits was observed on either the in vivo or ex vivo images. CONCLUSIONS: The present study demonstrates that (99m)Tc-based immunoimaging of the vascular lesions may be feasible by the use of smaller antibody fragments. Earlier visualization is possible at the expense of a lower absolute antibody uptake in the lesions as compared to the use of intact antibody or larger fragments with longer circulating time.

Maximizing radiotracer delivery to experimental atherosclerotic lesions with high-dose, negative charge-modified Z2D3 antibody for immunoscintigraphic targeting.

BACKGROUND: Two factors that directly affect target/background ratio in immunoscintigraphy are the concentration of the antibody bound to the target and the concentration of the antibody in the circulation. High dosages of monoclonal antibody have been reported to be more efficacious in visualization of tumors. Although administration of a higher dosage of antibody increases the absolute target accumulation of the radiotracer, it also increases the background activity, which may offset this advantage. Negative charge-modified antibodies carry high specific radioactivity to the target sites without significantly increasing the background activity. Therefore we investigated whether higher dosages of negative charge-modified antibody can be used to improve imaging of experimental atherosclerotic lesions. METHODS AND RESULTS: Experimental atherosclerotic lesions were produced in 16 New Zealand White rabbits by balloon deendothelialization of the infradiaphragmatic aorta and hyperlipidemic diet for 12 weeks. Negative charge-modified Z2D3 antibody F(ab')2 specific for an antigen on proliferating smooth muscle cells of human atheroma labeled with (111)In was used for imaging experimental atherosclerotic lesions either at high (100 to 125 microg) or low (25 to 50 microg) dosages. A lower dosage of Z2D3 was labeled with 507 +/- 29.5 microCi (25 to 50 microg) (111)In label, compared with 2.9 +/- 0.24 mCi (100 to 125 microg) for the higher dosage. Although noninvasive visualization of atherosclerotic lesions was possible in all animals at 24 hours, high antibody dose allowed unequivocal visualization of the lesion as early as 3 hours after intravenous administration of the antibody. Eight animals were killed at 24 hours and the remaining eight animals at 48 hours. Mean radioactivity dose delivered per gram of lesion with the low-dose protocol at 24 hours was 0.46 +/- 0.09 microCi, which remained essentially unchanged at 48 hours (0.37 +/- 0.09 microCi; p = 0.51). With the high-dosage protocol, the total radioactivity (dose) per gram uptake in the lesion increased by about eightfold (3.49 +/- 0.58 microCi; p = 0.002) at 24 hours and was sixfold higher at 48 hours (2.21 +/- 0.45 microCi; p < 0.02). CONCLUSIONS: The study demonstrated that the increase in the dosage of negatively charge-modified antibody allows a very high delivery of specific radioactivity to the target, which in turn enables early visualization of experimental atherosclerotic lesions.

Targeting human breast tumour in xeno-grafted SCID mice with 99Tcm-glucarate.

99Tcm-glucarate accumulation in human mammary BT-20 tumours hosted in severe combined immune deficiency (SCID) mice was compared to 111In-monoclonal antibody 103D2-F(ab')2, 99Tcm-methoxyisobutyl isonitrile (99Tcm-MIBI) and 99Tcm-diethylenetriamine pentaacetate (99Tcm-DTPA). The intracellular tumour distribution of 99Tcm-glucarate was also determined. SCID mice injected with a mixture of 99Tcm-glucarate and 111In-103D2-F(ab')2 were imaged serially up until 24 h. Computer planimetered tumour-to-blood activity (in the heart) ratios (T/BH) to 8 h were significantly greater for 99Tcm-glucarate than 111In-103D2. The mean (+/-S.D.) tumour-to-blood ratio (T/B) from biodistribution was 1.21 +/- 0.31 and 0.35 +/- 0.06 (P < 0.0001) at 5 h, and 1.526 +/- 0.29 and 0.75 +/- 0.2 (P < 0.0001) at 8 h, for 99Tcm-glucarate and 111In-103D2 respectively. At 24 h, T/B for 111In-103D2 (1.76 +/- 0.22) exceeded that of 99Tcm-glucarate (1.44 +/- 0.2, P = 0.01). 99Tcm-glucarate uptake in the tumours at 5 h (1.133 +/- 0.25 %ID g-1) and 8 h (1.213 +/- 0.23 %ID g-1) was significantly greater than that of 99Tcm-MIBI (0.340 +/- 0.09, P = 0.0002; 0.220 +/- 0.04, P = 0.0001) and 99Tcm-DTPA (0.091 +/- 0.03, P < 0.0002; 0.016 +/- 0.01, P < 0.0001) respectively. Intracellular tumour distribution of 99Tcm-glucarate was 50.91 +/- 6.55% in the nuclear fraction, 34.34 +/- 2.88% in the cytoplasmic fraction and 14.75 +/- 7.66% in the mitochondrial fraction. Thus glucarate may provide a 99Tcm-based mammary tumour imaging modality for visualization of tumours very quickly after tracer administration with maximal targeting in the nuclei of the tumour cells.

Very early noninvasive detection of acute experimental nonreperfused myocardial infarction with 99mTc-labeled glucarate.

BACKGROUND: 99mTc glucarate has recently been reported to be an infarct-avid agent. The feasibility of imaging with 99mTc glucarate was evaluated for the early diagnosis of nonreperfused and reperfused myocardial infarction and compared with localization of simultaneously administered 111In anti-myosin. METHODS AND RESULTS: Four groups of six rabbits each were studied. The left anterior descending coronary artery (LAD) was kept persistently occluded (n = 6) or reperfused after 40 minutes (n = 6) in rabbits. After confirmation of LAD occlusion by 201Tl scintigraphy, a mixture of 99mTc glucarate (15.7 +/- 1.6 mCi) and 111In anti-myosin (0.53 +/- 0.03 mCi) was administered intravenously. Another group of rabbits (n = 6) with 5 or 15 minutes of LAD occlusion were used to assess the affinity of 99mTc glucarate for the ischemic myocardium. The remaining 6 rabbits with reperfused myocardial infarction were used for the assessment of subcellular localization of 99mTc glucarate. 99mTc glucarate cleared rapidly from circulation (elimination t1/2, 36 minutes). Infarcts were visualized within 10 minutes in reperfused and within 30 minutes in nonreperfused coronary territories after intravenous administration. 111In anti-myosin delineated reperfused infarcts within 1 to 3 hours, but no uptake was seen in persistently occluded rabbits. 99mTc glucarate uptake in reperfused and nonreperfused infarct centers was 28 and 12 times greater, respectively, than that in normal myocardium (P = .0001). A direct correlation between glucarate and anti-myosin localization (r = .60 for nonreperfused; 0.76 for reperfused; P < .0001) was observed. Ischemic hearts showed no glucarate uptake. Subcellularly, 99mTc glucarate localized predominantly in the nuclear fraction of the infarct, with lesser extents in the mitochondrial and cytoplasmic fractions. CONCLUSIONS: Noninvasive imaging of myocardial infarcts with 99mTc glucarate is possible within minutes in persistently occluded or reperfused myocardial infarcts. Early detectability results from the rapid blood clearance and high avidity of glucarate for the acutely necrotic myocardial tissue.

Decreased kidney uptake of technetium-99m-labelled Fab' fragments in ovarian carcinoma bearing nude mice using a cleavable chelator.

A new 99mTc labelling method using a cleavable chelator, RP-1, was developed. In this study Balb/c mice with ovarian carcinoma xenografts received various Fab' fragments labelled with 99mTc either directly or via RP-1. Kidney uptake was significantly lower for the RP-1 linked conjugates. Tumour uptake showed no significant differences between RP-1 conjugates and directly labelled preparations. In conclusion, with the use of the cleavable linker RP-1, kidney uptake can be reduced significantly resulting in a lower radiation dose to the kidneys.

In vivo treatment with a monoclonal chimeric anti-CD4 antibody results in prolonged depletion of circulating CD4+ cells in chimpanzees.

Chimeric M-T412 (cM-T412), an anti-CD4 antibody, was tolerated in chimpanzees at a dosage of 5 mg/kg per day for up to 7 consecutive days, or 5 mg/kg per dose, twice weekly for 4 weeks. All cM-T412-treated chimpanzees showed a prolonged CD4-cell depression. Weak chimpanzee antibody responses to chimeric M-T412 were observed. One of the chimpanzees on the biweekly dosage regimen exhibited a hypersensitivity reaction immediately after receiving its seventh dose. Following supportive treatment, the animal recovered and remained asymptomatic during the non-treatment observation period. The hypersensitivity reaction was not an unexpected response considering the animal received repeated intermittent i.v. administration of a foreign protein. This animal also showed a chimpanzee antibody response to chimeric M-T412 after the seventh dose. Chimeric M-T412 also induced an anti-cM-T412 response in some of the other animals. The level of this response was lower than the anti-mouse responses observed in animals treated with murine anti-CD4. Moreover, the anti-cM-T412 response was mainly directed to idiotypic determinants. The decrease in CD4+ cells observed for all chimeric M-T412-treated chimpanzees is an expected effect of the anti-CD4 antibody. The duration of this CD4+ cell decrease is, however, much longer than observed for other CD4-specific MoAbs described. No selective loss of either memory or naive CD4+ cells was observed after either the single, 7-day or twice-weekly treatments. The CD4+ cell depression was reversible, although individual variation in time to recovery was observed. Therefore, cM-T412 could be a good candidate for clinical use in autoimmune conditions.

Reactivity of the human antiendotoxin immunoglobulin M monoclonal antibody HA-1A with lipopolysaccharides from rough and smooth gram-negative organisms.

Clinical data suggest that the human immunoglobulin M antiendotoxin antibody HA-1A reduced mortality in patients diagnosed with gram-negative bacteremia and bacteremia with shock. Previous studies have demonstrated that HA-1A binds to the lipid A domain of lipopolysaccharide (LPS). The present study evaluated the ability of HA-1A to interact with LPs isolated from various strains of gram-negative bacteria by using liquid-phase rate nephelometry and solid-phase immunoblotting assays. HA-1A formed immune complexes in solution with LPSs isolated from both rough and smooth gram-negative organisms. Western blot (immunoblot) analysis of these LPS preparations revealed that HA-1A bound to LPS isolated from rough gram-negative organisms and to a rough LPS-like component present in smooth LPS. HA-1A also bound to LPS-protein complexes found in certain commercial rough LPS preparations. Preincubation of HA-1A with lipid A completely blocked subsequent binding of HA-1A to LPS in both liquid- and solid-phase assay formats, suggesting that the interaction of HA-1A with LPS is through the lipid A domain. Evidence that the binding of HA-1A to LPS was mediated through the antigen-combining (Fv) region of the antibody was provided by the finding that a murine anti-idiotypic antibody to HA-1A inhibited binding. These findings suggested that the broad antiendotoxin reactivity exhibited by HA-1A appeared to be due to the ability of HA-1A to bind to the conserved lipid A moiety of LPSs derived from both smooth- and rough-phenotype gram-negative bacterial strains.

Effects of isotype and Fc region on in vitro function of a mouse/human chimeric CD4 antibody.

Murine CD4 mAbs have shown potential for the treatment of allograft rejection and autoimmune disorders including rheumatoid arthritis. Clinical usefulness of the murine mAbs has been limited by immunogenicity and a short circulating half-life. Mouse/human chimeric antibodies have been constructed, composed of the variable region of M-T412 (a murine G2a mAb specific for the human CD4 molecule) and human G1 (cM-T412 G1) or G4 (cM-T412 G4) Fc regions. F(ab')2 and F(ab) fragments of the murine G2a and chimeric G1 mAbs were generated by enzymatic digestion. The chimeric mAbs and all fragments retained the avidity and specificity of the murine M-T412 and were evaluated in in vitro assays measuring Ig production by pokeweed mitogen (PWM)-stimulated peripheral blood mononuclear cells (PBMC), sIL-2R produced by phytohemagglutinin-stimulated PBMC, and proliferation in response to tetanus toxoid, CD3 mAb plus IL-2, and mixed lymphocyte response (MLR). When PBMC were stimulated with tetanus toxoid, 10 ng/ml of cM-T412 G1 inhibited proliferation by 90%, while neither the cM-T412 G4, M-T412 G2a, nor any mAb fragment produced > 65% inhibition, even at 1000-fold higher concentrations. A similar pattern of inhibition was observed in MLR assays. In contrast, the F(ab')2 fragment of the cM-T412 G1 was as effective as the whole antibody in inhibiting PWM-stimulated IgM synthesis and PBMC proliferation in response to stimulation by a CD3 mAb plus IL-2.(ABSTRACT TRUNCATED AT 250 WORDS)

Tumour detection and localization using 99Tcm-labelled OV-TL 3 Fab' in patients suspected of ovarian cancer.

Fab' fragments of the monoclonal antibody OV-TL 3, that recognizes an ovarian carcinoma-associated antigen (OA3), were labelled with 99Tcm using D-glucarate as a ligand. Twenty patients suspected of having primary or recurrent ovarian cancer received intravenously 1 mg of the Fab' labelled with 740 MBq 99Tcm. Both planar and single photon emission computed tomographic (SPECT) scintigraphy were performed up to 30 h after intravenous infusion. In 19 out of 20 patients surgical and histopathological evaluation was performed between 2 and 6 days postinfusion. Imaging results were compared with X-ray computed tomography (CT), ultrasonography (US) and CA 125 serum level. Blood clearance was fast with median t1/2 beta of 9.5 h. Thirty-seven per cent of the injected dose (% ID) was excreted in the urine within the first 24 h, whereas 7% ID was excreted in the 24 h faeces. In one patient with an OA3 negative ovarian carcinoma, radioimmunoscintigraphy (RIS) did not visualize the tumour. In two other patients a benign ovarian cyst was found, also showing no elevated uptake. In 13 out of 17 patients ovarian cancer lesions were detected with RIS, whereas CT and US detected lesions in, respectively, 15 and 12 patients. Of 36 surgically defined tumour deposits larger than 1 cm in diameter, 53% were detected and localized with RIS, whereas CT and US detected 61 and 40%, respectively. Radioimmunoscintigraphy with 99Tcm-OV-TL 3 Fab' is less distressing for the patients but the overall imaging performance is not improved when compared with 111In-OV-TL 3 F(ab')2.

High-level expression and characterization of a mouse-human chimeric CD4 antibody with therapeutic potential.

The use of murine anti-CD4 monoclonal antibodies (MAbs) has shown considerable promise for the treatment of allograft rejection and rheumatoid arthritis. We have constructed mouse-human anti-CD4 antibodies with the goal of increasing their clinical potential by decreasing immunogenicity and improving effector functions. The chimeric antibodies were constructed by cloning the heavy and light chain variable regions of M-T412, a murine antibody raised against the human CD4 antigen, and joining them to the human G1, G4, or kappa constant regions in mammalian expression vectors. After transfection into mouse myeloma cells, stable cell lines were isolated that secrete up to 140 micrograms/ml chimeric antibody in static culture. The chimeric antibodies were equivalent to the murine antibody in their binding characteristics and relative affinities. However, the chimeric M-T412 MAbs have enhanced activity when compared to the murine G2a MAb in mediating antibody-dependent cell-mediated cytotoxicity using human CD4+ target and effector cells.

Labeling and stability of radiolabeled antibody fragments by a direct 99mTc-labeling method.

The in vitro labeling and stability of 99mTc-labeled antibody Fab' fragments prepared by a direct labeling technique were evaluated. Eight antibody fragments derived from murine IgG1 (N = 5), IgG2a (N = 2) and IgG3 (N = 1) isotypes were labeled with a preformed 99mTc-D-glucarate complex. No loss of radioactivity incorporation was observed for all the 99mTc-labeled antibody fragments after 24 h incubation at 37 degrees C. The 99mTc-labeled antibody fragments (IgG1, N = 2; IgG2a, N = 2; IgG3, N = 1) were stable upon challenge with DTPA, EDTA or acidic pH. Furthermore, using the affinity chromatography technique, two of the 99mTc-labeled antibody fragments displayed no loss of immunoreactivity after prolonged incubation in phosphate buffer up to 24 h at 37 degrees C. The bonding between 99mTc and antibody fragments was elucidated by challenging with a diamide ditholate (N2S2) compound. The Fab' with IgG2a isotype displayed tighter binding to 99mTc in comparison to the Fab' from IgG1 and IgG3 isotype in N2S2 challenge and incubation with human plasma. The in vivo biodistribution of five 99mTc-labeled fragments were evaluated in normal mice. In conclusion, the direct labeling method allows stable 99mTc labeling of antibody fragments from three of the major murine isotypes.

An instant kit method for labeling antimyosin Fab' with technetium-99m: evaluation in an experimental myocardial infarct model.

An instant kit method for labeling antibody Fab' fragments was developed. The method utilizes a ligand exchange reaction between the intermediate complex 99mTc-D-glucarate and the free sulfhydryl groups on the antibody Fab' fragment. Radiolabeling of the Fab' using generator eluate achieves quantitative 99mTc incorporation in less than 30 min at room temperature. The radiolabel is stable in human plasma for at least 24 hr and stable to incubation with 10 mM diethylene-triaminepentaacetic acid (24 hr) and 1 mM diaminodithiol agent (up to 3 hr). Mouse biodistribution of 99mTc-antimyosin shows faster blood clearance and lower uptake in the lungs, liver, and spleen in comparison to 111In-antimyosin. Technetium-99m-antimyosin and 111In-antimyosin showed equivalent ability to detect myocardial infarct in a canine model.

Evaluation of the 323/A3 monoclonal antibody and the use of technetium-99m-labeled 323/A3 Fab' for the detection of pan adenocarcinoma.

The 323/A3 murine monoclonal antibody, initially described as reactive to breast carcinomas, is found by immunohistological analyses to have broad cross reactivity with adenocarcinomas of diverse histologic origin. The 323/A3 antigen is similar to the tumor-associated 17-1A antigen as revealed by immunoblot and cross-competition cell binding studies. We have investigated the potential use of the 323/A3 monoclonal antibody for tumor imaging as a Fab' molecule labeled with 99mTc. In vitro studies demonstrate that 323/A3 Fab' has high affinity (2-3 x 10(9) M-1) with no significant loss of immunoreactivity compared to the intact IgG. In vivo studies demonstrate that 99mTc 323/A3 Fab' can rapidly detect human breast and colon tumor xenografts growing in athymic nude mice. Distinct breast tumor visualization is observed as early as 1 h post intravenous administration with the 99mTc 323/A3 Fab'. Distinct colon tumor visualization is observed by 3 h (the earliest time point imaged). Tumor-to-blood ratios are higher for 99mTc 323/A3 Fab' than with a 99mTc-labeled nonspecific isotype-matched Fab' antibody. These results suggest that 99mTc 323/A3 Fab' can detect 17-1A antigen and may have potential clinical utility for the rapid diagnostic imaging of adenocarcinomas.

In vivo technetium-99m S12 antibody imaging of platelet alpha-granules in rabbit endothelial neointimal proliferation after angioplasty.

To examine the specificity of technetium-99m monoclonal antibody (S12) imaging for identifying activated platelets at interventional injury sites in atherosclerotic rabbit arteries, subgroups of unheparinized rabbits (n = 39) underwent serial percutaneous transluminal aortic angioplasty (PTA) procedures (with or without intravascular stent placement) followed by in vivo and then ex vivo gamma camera imaging, scanning, and immunoelectron microscopy to determine the intravascular loci of S12 Fab' antibody binding. Despite angiographic vessel patency, image-derived ratios of in vivo S12 binding in injured versus uninjured vascular segments were significantly increased (p less than 0.05) after one PTA (1.3 +/- 0.17, n = 7), PTA twice at 6-week intervals (1.4 +/- 0.22, n = 7), and PTA plus stent placement (1.6 +/- 0.28, n = 7) compared with control experiments (1.1 +/- 0.13, n = 7). Ex vivo imaging of blood-free excised aortas confirmed S12 localization at PTA (2 +/- 0.4, n = 3) and PTA plus stent placement (5 +/- 3.8, n = 7) sites (both p less than 0.05 versus controls). S12 antibody uptake decreased significantly (p less than 0.05) at 1 week after PTA plus stent placement in vivo (1.1 +/- 0.10, n = 4) and ex vivo (1.6 +/- 0.7, n = 3). Electron microscopic studies confirmed dense platelet, fibrin, and red blood cell deposition in regions of acute injury, with endothelial neointimal proliferation at 1 week after PTA. Immunoelectron microscopic studies confirmed specific in vivo S12 binding (22:1 versus nonrelevant IgG) at sites of alpha-granule GMP-140 expression in activated platelets. Therefore, S12 studies may be useful to localize sites of platelet-derived mitogen release at arterial PTA injury sites.

Tc-99m antifibrin Fab' fragments for imaging venous thrombi: evaluation in a canine model.

An antifibrin antibody (T2G1s) Fab' fragment labeled with technetium-99m was tested for its ability to produce images of fresh thrombi in dogs. In gamma camera images, all thrombi were evident by 2-4 hours after injection. Mean thrombus-to-blood and thrombus-to-muscle ratios averaged 4.0 and 69 at four hours after injection and increased to 24 and 270, respectively, by 24 hours after injection. When compared with I-125 fibrinogen injected into the same dogs, Tc-99m-antifibrin Fab' had lower absolute uptake in thrombus but higher thrombus-to-blood ratios due to a faster rate of disappearance from the blood. The primary route of excretion was through the kidneys. Tc-99m-antifibrin Fab' was highly stable in vivo, with an average of 82% of the circulating radioactivity able to bind to fibrin at 4 hours after injection. When compared with an In-111-labeled Fab fragment of antifibrin antibody 59D8, thrombus-to-blood and thrombus-to-muscle ratios were slightly higher for the Tc-99m-labeled antibody, and the blood disappearance rate was slightly faster. The absolute uptake in thrombus, however, was not significantly different, and the thrombus was visualized at about the same time after injection. These studies suggest that Tc-99m T2G1s Fab' is a potential agent for detecting thrombi in a clinical setting.