Three-step monoclonal antibody tumor targeting in carcinoembryonic antigen-positive patients.

We describe a method to postlabel, in vivo, biotinylated monoclonal antibodies pretargeted onto tumor deposits when most of the non-tumor-bound antibodies have already been cleared as avidin-bound complexes. The application of this principle to tumor detection by immunoscintigraphy was tested in 20 patients with histologically documented cancer and increased circulating carcinoembryonic antigen levels. One mg of biotinylated anti-carcinoembryonic antigen monoclonal antibody (FO23C5) was administered i.v. (first step). After 3 days, 4-6 mg of cold avidin were injected i.v. (second step), followed 48 h later by 0.2-0.3 mg of a biotin derivative labeled with 111In (2-3 mCi) (third step). No evidence of toxicity was observed. Whole body radioactivity distribution was measured in five patients at various intervals postinjection by the conjugate counting technique. Tumors and metastases were detected in 18 of 19 patients (the remaining patient was a true negative) within 3 h after administration of 111In-biotin by planar or single photon emission tomography imaging. At the time of imaging, tumor/blood pool ratio was 5.5 +/- 3.2, and tumor/liver ratio was 6.7 +/- 3.9. Blood clearance of 111In-biotin was multiexponential, with the fast component having a t1/2 of 5 +/- 3 min. Urinary excretion of radioactivity over 3 h was 63.5 +/- 4.9% of the injected dose. Radioactivity at 3 h was 6.5 +/- 1.8% in blood, 1.6 +/- 0.3% in the kidney, and 2.4 +/- 0.6% in the liver. This approach represents an improvement in immunoscintigraphic techniques for tumor localization. The potential use for radioimmunotherapy is discussed.

Characterisation of biotinylated liposomes for in vivo targeting applications.

Liposomes containing monosialoganglioside (GM1) or polyethylene glycol (PEG) lipid derivatives have prolonged circulation in the blood. This favours liposome extravasation to tumour sites. In this report it is shown that inclusion of GM1, PEG550-DPPE or PEG2000-DPPE in liposomes containing biotin-DPPE significantly diminished the ability of vesicles to bind to streptavidin in vitro. Steric inhibition due to the bulky head group of these lipids was least for biotin-DPPE liposomes containing GM1. Biodistribution studies in C26 tumour-bearing mice showed that GM1-liposomes containing small amounts of biotin-DPPE have long circulation life-times in the blood. Using fluorescent microscopic techniques, liposomes containing both GM1 and biotin-DPPE were detected within extra-vascular spaces in tumours. In addition it was shown that biotin-DPPE in GM1-liposomes bound streptavidin in situ. These results suggest that GM1-liposomes containing biotin-DPPE have potential use as diagnostic or therapeutic reagents in pre-targeting applications dependent on the high-affinity interaction of biotin with streptavidin.

Labeling and evaluation of N-[11C]methylated quinoline-2-carboxamides as potential radioligands for visualization of peripheral benzodiazepine receptors.

The novel quinoline-2-carboxamide derivatives N-[methyl-11C]-3-methyl-4-phenyl-N-(phenylmethyl)quinoline-2-carboxamide ([11C]4), (+/-)-N-[methyl-11C]-3-methyl-N-(1-methylpropyl)-4-phenylquinoline-2-carboxamide ([11C]5), and (+/-)-N-[methyl-11C]-3-methyl-4-(2-fluorophenyl)-N-(1-methylpropyl)quinoline-2-carboxamide ([11C]6) were labeled with carbon-11 (t1/2 = 20.4 min, beta+ = 99.8%) as potential radioligands for the noninvasive assessment of peripheral benzodiazepine type receptors (PBR) in vivo with positron emission tomography (PET). The radiosynthesis consisted of N-methylation of the desmethyl precursors 3-methyl-4-phenyl-N-(phenylmethyl)quinoline-2-carboxamide (4a), (+/-)-3-methyl-N-(1-methylpropyl)-4-phenylquinoline-2-carboxamide (5a), and (+/-)-4-(2-fluorophenyl)-3-methyl-N-(1-methylpropyl)quinoline-2-carboxamide (6a) with either [11C]methyl iodide or [11C]methyl triflate in the presence of tetrabutylammonium hydroxide or potassium hydroxide in dimethylformamide. The radioligands [11C]4, [11C]5, and [11C]6 were synthesized with over 99% radiochemical purity in 30 min, 30 +/- 5% radiochemical yield, calculated at the end of synthesis (EOS) non-decay-corrected, and 2.5 +/- 1.2 Ci/micromol of specific radioactivity. Inhibition studies in rats following intravenous pre-administration of 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide (PK 11195, 1) showed high specific binding to PBR of [11C]4, [11C]5, and [11C]6 in heart, lung, kidney, adrenal gland, spleen, and brain. The biological data suggest that [11C]5, [11C]6, and particularly [11C]4 are promising radioligands for PBR imaging in vivo with PET.

Detection of prosthetic vascular graft infection using avidin/indium-111-biotin scintigraphy.

UNLABELLED: Prosthetic vascular graft infection, though rare, carries high morbidity and mortality rates; therefore, timely diagnosis is important. Patients, however, often present with vague symptoms, and radiological investigations are frequently inconclusive. These factors may lead to prolonged periods of observation and hospitalization, with the resultant increase in costs and complication rates, before reaching a final diagnosis. This prospective study evaluates the use of nonspecific avidin/111In-biotin imaging in diagnosing prosthetic vascular graft infection. METHODS: Twenty-five patients with a total of 29 grafts were investigated. Eighteen patients (19 grafts) had low probability of disease, whereas the remaining 7 patients (10 grafts) warranted surgical exploration based on clinical, laboratory or radiological evidence. Avidin was first injected intravenously and then followed 24 hr later by administration of 111In-biotin. Whole-body images were obtained 10 min and 2 hr postinjection of 111In-labeled biotin. SPECT imaging was performed 1 hr postinjection. Increased uptake along part or the whole length of the graft was considered evidence of graft infection. RESULTS: Avidin/111In-biotin scintigraphy correctly identified all infected grafts, as confirmed by culturing surgical specimens. In contrast, infection was correctly excluded in all but one of the grafts, and long-term follow-up was used to assess the presence of infection in patients who did not undergo surgical intervention. CONCLUSION: Avidin/111In-biotin scintigraphy is a simple and accurate imaging method for the routine diagnosis of vascular graft infection, and it may have a role in identifying the disease process in its initial stages, thus improving prognosis.

Imaging osteomyelitis with streptavidin and indium-111-labeled biotin.

UNLABELLED: Animal studies of infection imaging by a two-step protocol have shown that important improvements in target to nontarget ratios are possible. In this protocol, unlabeled streptavidin is administered and allowed sufficient time to accumulate in the lesion, probably by nonspecific processes, and to clear elsewhere. Thereafter, 111Inbiotin is administered. A fraction of the labeled biotin may be retained in the lesion because of biotin's high affinity for streptavidin while most of the activity is cleared through the kidneys. METHODS: Radioscintigraphy with unlabeled streptavidin followed with 111Inlabeled biotin was performed in 15 patients with chronic osteomyelitis. As controls, each patients received either 111In-labeled biotin without the preadministration of streptavidin or 111In-labeled nonspecific IgG. RESULTS: Regions of focal uptake were identified in all patients receiving streptavidin followed by radiolabeled biotin as early as 10 min postadministration of radioactivity, and retention of label was evident through 24 hr. Coincident regions of abnormal accumulation were apparent with 111In-IgG, but only in delayed images. Moreover, with 111In-biotin alone, without the preadministration of streptavidin, focal accumulations were detected in areas similar to that identified with the two-step protocol. Although, these observations were only in the earliest images. CONCLUSION: The results of this preliminary clinical investigation suggest that a two-step protocol with unlabeled streptavidin and radiolabeled biotin may be an alternative for the detection of infection.

Quantitative comparison of direct antibody labeling and tumor pretargeting in uveal melanoma.

UNLABELLED: SPECT radioimmunoscintigraphy with 99mTc-labeled anti-melanoma monoclonal antibodies (MAbs) 225.28S is being used to detect uveal melanoma. Recently, pretargeting methods have been described to reduce background activity and perform imaging in a shorter time interval. METHODS: We compared the three-step pretargeting method with conventional radioimmunoscintigraphy in 15 patients with a clinical and laboratory diagnosis of uveal lesion. High-resolution SPECT radioimmunoscintigraphy was performed in all patients with directly labeled MAbs and, 1 wk later, with the three-step pretargeting technique. Eleven patients underwent eye enucleation and specimens of uveal melanoma were available for histology, whereas four patients underwent conservative therapy. The percent injected dose (%ID) delivered to the tumor and the tumor-to-background ratio were calculated. RESULTS: In all three-step radioimmunoscintigraphy studies, there was a reduction of nonspecific nasopharyngeal background. The three-step radioimmunoscintigraphy tumor-to-nontumor ratio was 3.1 +/- 1.3 versus 1.5 +/- 0.5 of conventional radioimmunoscintigraphy, while the percent injected dose on the tumor was similar for the two methods (4.4 +/- 3.0 versus 3.8 +/- 2.8) x 10(-3). CONCLUSION: Improved SPECT imaging with the three-step radioimmunoscintigraphy results from reduced background and from higher counting statistics due to reduction of time interval between radiotracer administration and imaging, whereas the absolute amount of tracer delivered to the tumor by the two methods is comparable.

Asymmetric synthesis and preliminary evaluation of (R)- and (S)-[11C]bisoprolol, a putative beta1-selective adrenoceptor radioligand.

(+/-)-1-[4-(2-Isopropoxyethoxymethyl)-phenoxy]-3-isopropylamino-2-propanol (bisoprolol) is a potent, clinically used beta(1)-adrenergic agent. (R)-(+) and (S)-(-) enantiomers of bisoprolol were labelled with carbon-11 (t(1/2)=20.4 min) as putative tracers for the non-invasive assessment of the beta(1)-adrenoceptor subtype in the human heart and brain with positron emission tomography (PET). The radiosynthesis consisted of reductive alkylation of des-iso-propyl precursor with [2-11C]acetone in the presence of sodium cyanoborohydride and acetic acid. The stereo-conservative synthesis of (R)-(+) and (S)-(-)-1-[4-(2-isopropoxyethoxymethyl)-phenoxy]-3-amino-2-propanol to be used as the precursors for the radiosynthesis of [11C]bisoprolol enantiomers was readily accomplished by the use of the corresponding chiral epoxide in three steps starting from the commercially available hydroxybenzyl alcohol. The final labelled product (either (+) or (-)-1-[4-(-isopropoxyethoxymethyl)-phenoxy]-3- [11C]isopropylamino-2-propanol) was obtained in 99% radiochemical purity in 30 min with 15+/-5% (EOS, non-decay corrected) radiochemical yield and 3.5+/-1 Ci/micromol specific radioactivity. Preliminary biological evaluation of the tracer in rats showed that about 30% of heart uptake of [11C](S)-bisoprolol is due to specific binding. The high non-specific uptake in lung might mask the heart uptake, thus precluding the use of [11C](S)-bisoprolol for heart and lung studies by PET. The remarkably high uptake of the tracer in rat brain areas rich of beta-adrenergic receptors such as pituitary (1.8+/-0.3% I.D. at 30 min) was blocked by pre-treatment with the beta-adrenergic antagonists propranolol (45%) and bisoprolol (51%, p<0.05). [11C](S)-bisoprolol deserves further evaluation in other animal models as a putative beta(1) selective radioligand for in vivo investigation of central adrenoceptors.

Biodistribution in tumour-bearing mice of two 90Y-labelled biotins using three-step tumour targeting.

Tumour pretargeting techniques based on a three-step approach with the avidin/biotin system and associated with the high-energy beta-emitter yttrium-90 (90Y) have been applied with encouraging results in cancer therapy. While in-vivo stable binding of 90Y through a chelating agent like DOTA is essential for directly labelled monoclonal antibodies in the labelling of a fast clearing molecule like biotin, this may not be so important, since the time for catabolic processes to occur is drastically reduced. In this study, the biodistribution of 90Y bound to biotin through DTPA or DOTA was evaluated. Tumour-bearing mice received biotinylated antibody on day 1, avidin on day 2 and 90Y-labelled biotin on day 3. The biodistribution was determined 4 and 24 h after the injection of radiolabelled biotin. The majority of the injected dose was recovered in the urine 4 h after injection of both radiolabelled compounds. Higher bone uptake of 90Y was observed in animals administered 90Y-DTPA-biotin. In general, all organs including tumour showed higher accumulation of activity following the injection of 90Y-DAPA-biotin. Despite the lower levels of accumulation in the tumour after injection of 90Y-DOTA-biotin, the tumour-to-liver and tumour-to-bone ratios for the DOTA ligand were higher compared to DTPA. The higher tumour uptake observed after injection of 90Y-DTPA-biotin may be attributed to the presence of two biotin molecules in its dimeric structure. Our results indicate that the ideal 90Y-labelled biotin derivative for therapy studies should be formed from a DOTA core with two non-cleavable spacers, each ending with a biotin molecule.

Radiolocalization of 99mTc-labeled fragments in a melanoma xenografted model.

A xenograft model of human malignant melanoma was used to compare, in terms of tumor localization, the specific antimelanoma monoclonal antibody (MoAb) 225.28S with an irrelevant antibody (4C4). Both specific and non-specific 99mTc-labeled fragments were injected in 12 nude mice bearing subcutaneous tumor. The animals were then sacrificed at 6 and 24 hours post-injection and immediately dissected. Radioactivity of the tumor and normal tissues was measured in a well scintillation counter and autoradiography of tumor, liver and kidneys was also obtained. Tumor localization of 99mTc-labeled MoAb 225.28S fragments was highly specific compared with 99mTc-labeled irrelevant antibody 4C4. With the exception of the kidneys, already at six hours there was a satisfactory tumor-to-normal tissue ratio, which improved at 24 hours. However, the percentage of injected dose per gram of tumor decreased with time, probably due to the weaker bond of radiolabeled Fab' fragments to tumor cells. These results would indicate 99mTc-fragments of the antimelanoma MoAb 225.28S as a suitable radiotracer in clinical nuclear medicine.

Pretargeted immunoscintigraphy in patients with medullary thyroid carcinoma.

To evaluate the use of pretargeted immunoscintigraphy (ISG) in the diagnosis and follow-up of patients with medullary thyroid carcinoma (MTC), we studied 25 patients with histologically proven disease; ISG was repeated after surgery in two patients. The antibody, either an anticarcinoembryonic antigen (CEA) or an antichromogranin A (CgA) biotinylated monoclonal antibody (MAb) or a cocktail of the two biotinylated MAbs was first injected. After 24 h, avidin was administrated i.v., followed by 111In-labelled biotin 24 h later. Fifty-two lesions were visualised. Six primary tumours, diagnosed by increased calcitonin levels, were all correctly diagnosed; 47 recurrences, also suspected by blood tumour markers, were detected and confirmed by cytology or histology. In one case, single photon emission tomography allowed the detection of small lymph nodes with a diameter of 4-7 mm. These lesions, not judged neoplastic by ultrasound, were confirmed to be neoplastic by fine needle aspiration. Pretargeted ISG correctly localises primary tumours and recurrences in MTC patients, when the only marker of relapse is serum elevation of calcitonin. With this three-step pretargeting method, cocktails of potentially useful MAbs can be used, avoiding false-negative studies that may occur when CEA or CgA are not expressed.

Pre-targeted immunodetection in glioma patients: tumour localization and single-photon emission tomography imaging of [99mTc]PnAO-biotin.

The imaging of cerebral gliomas with radiolabelled monoclonal antibodies (MoAbs) has been previously reported. However, previous studies have been hampered by the drawback of a low tumour to non-tumour ratio. In order to overcome this problem we have developed a three-step pre-targeting method using the avidin-biotin system. The rationale of this technique consists in vivo labelling of biotinylated MoAbs targeted onto tumour deposits, when most of the unbound antibodies have been cleared from the bloodstream as avidin-bound complexes. The anti-tenascin MoAb BC2, specific for the majority of gliomas, was biotinylated and 1 mg was administered i.v. in 20 patients with histologically documented cerebral lesions. After 24-36 h, 5 mg avidin was injected i.v. followed 24 h later by a third i.v. injection of 0.2 mg PnAO-biotin labelled with 15-20 mCi technetium-99m. No evidence of toxicity was observed. Whole-body biodistribution was measured at 20 min, 3 h and 5 h post-injection. [99mTc]PnAO-biotin had a fast blood clearance and was primarily excreted through the biliary system. A dedicated single-photon emission tomography system was used to acquire brain tomographic images 1-2 h after the administration of [99mTc]PnAO-biotin. Tumours were detected in 15/18 glioma patients with a tumour to non-tumour ratio of up 14:1. This three-step method, based on the sequential administration of anti-tenascin MoAb BC2, avidin and [99mTc]PnAO-biotin, can support computed tomography or magnetic resonance imaging for the diagnosis and follow-up of patients with glioma. Further studies are required to evaluate the potential of this technique for therapeutic application.