In vitro autoradiography of carcinoembryonic antigen in tissue from patients with colorectal cancer using multifunctional antibody TF2 and (67/68Ga)-labeled haptens by pretargeting.

The carcinoembryonic antigen (CEA) was visualized in vitro in tissue from patients with colorectal cancer with trivalent bispecific antibody TF2 and two hapten molecules, [(67/68)Ga]Ga-IMP461 and [(67/68)Ga]Ga-IMP485 by means of pretargeting. Colorectal cancer tissue samples obtained from surgery at Uppsala University Hospital, were frozen fresh and cryosectioned. The two hapten molecules comprising 1,4,7-triazacyclononanetriacetic acid chelate moiety (NOTA) were labeled with (67)Ga or (68)Ga. The autoradiography was conducted by incubating the tissue samples with the bispecific antibody TF2, followed by washing and incubation with one of the radiolabeled hapten molecules. After washing, drying and exposure to phosphor imager plates, the autoradiograms were analyzed and compared to standard histochemistry (hematoxylin-eosin). Pronounced binding was found in the tissue from colorectal cancer using the bispecific antibody TF2 and either of the haptens [(67/68)Ga]Ga-IMP461 and [(67/68)Ga]Ga-IMP485. Distinct binding was also detected in the epithelium of most samples of neighboring tissue, taken at a minimum of 10 cm from the site of the tumor. It is concluded that pretargeting CEA with the bispecific antibody TF2 followed by the addition of (67/68)Ga-labeled hapten is extremely sensitive for visualizing this marker for colorectal cancer. This methodology is therefore a very specific complement to other histochemical techniques in the diagnosis of biopsies or in samples taken from surgery. Use of the pretargeting technique in vivo may also be an advance in diagnosing patients with colorectal cancer, either using (67)Ga and SPECT or (68)Ga and PET.

(68)Ga-Labeling of RGD peptides and biodistribution.

Several peptides comprising Arg-Gly-Asp (RGD) domain and macrocyclic chelator were labeled with (68)Ga for the imaging of angiogenesis. The analogues varied in peptide constitution, linker and chelator type. The labeling efficiency did not vary with the peptide constitution and linker type, but depended on the chelator type. Four of the compounds containing 2,2',2'',2'''-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid (DOTA) chelator were labeled at 90 ± 5°C using conventional or microwave heating reaching 90% of (68)Ga incorporation after 5 and 2 min respectively, when the concentration of the precursor was 2.5 µM. The compound having 2,2',2''-(1,4,7-triazonane-1,4,7-triyl)triacetic acid (NOTA) as the chelator could be labeled at room temperature within 5 min using 2.5 µM peptide precursor. Two of the compounds contained a poly (ethylene glycol) (PEG) linker to the chelator. The biodistribution of the analogues was studied in male rats.

68Ga-autoclabeling of DOTA-TATE and DOTA-NOC.

INTRODUCTION: A new method combining (68)Ga-labeling and steam sterilization, here called autoclabeling, has been evaluated for two somatostatin receptor binding tracers used for positron emission tomography (PET) imaging of neuroendocrine tumors; DOTA-TATE and -NOC. METHODS: The two peptides DOTA-TATE and -NOC were labeled with (68)Ga by heating for 15 min at 121°C in the presence of acetate buffer at pH 4.3. The product solutions were tested for sterility, presence of endotoxins, degradation of peptide and osmolality. RESULTS: Complete incorporation of (68)Ga was obtained after the autoclabeling reaction and no degradation of the peptides was observed. Sterility was verified and the presence of endotoxins was well within Ph. Eur limits (175IU/maximum injected volume). CONCLUSIONS: The autoclabeling method provides a convenient procedure for (68)Ga-labeling by combining the labeling reaction and steam sterilization into one single step.

Preclinical evaluation of a 68Ga-labeled biotin analogue for applications in islet transplantation.

INTRODUCTION: Islet transplantation is a promising treatment for type 1 diabetes mellitus, but the fate of the cells after intraportal infusion is unclear. It is therefore imperative to develop novel techniques for noninvasive imaging and quantification of events following islet transplantation. METHODS: Small islet-like microbeads, avidin-covered agarose resins (AARs), were used as a model system for islet transplantation. Capability for specific [(68)Ga]Ga-DOTA-(PEG)(2)-biotin uptake and retention for either AARs or human islets conjugated with avidin by means of a heparin scaffold was studied in vitro. Biodistribution of the novel positron emission tomography (PET) tracer [(68)Ga]Ga-DOTA-(PEG)(2)-biotin was evaluated in mice treated by intraportal transplantation of AARs by µPET/computed tomography and ex vivo organ distribution and compared with control mice. RESULTS: AARs had high capability to bind [(68)Ga]Ga-DOTA-(PEG)(2)-biotin, close to 50% of administrated tracer/µl in vitro (>0.25 MBq/µl). Avidin-tagged human islets could bind on average 2.2% of administered tracer/µl. Specificity (>90%) and retention (>90% after 1 h) were high for both AARs and avidin-tagged islets. Hepatic tracer uptake and retention were increased in mice transplanted with AARs [standardized uptake value (SUV)=2.6] compared to the untreated group (SUV=1.4). In vivo uptake of tracer to AARs was blocked by preadministration of unlabeled biotin. CONCLUSIONS: Avidin-tagged islet-like objects can be tracked in hepatic volume after intraportal transplantation by using [(68)Ga]Ga-DOTA-(PEG)(2)-biotin and PET.

68Ga-labeling of biotin analogues and their characterization.

Biotin- and (68)Ga-based tracers have been suggested as tools that could be used to monitor the survival of avidin-coated islets of Langerhans isolated from pancreas and used in transplantation, i.e., to liver. Three biotin analogues with various alkyl and poly(ethylene glycol) (PEG) chains coupled to DOTA were synthesized and labeled with (68)Ga. The (68)Ga labeling was studied at room temperature as well as elevated temperature using either conventional or microwave heating. Radioactivity incorporation reached 95% within 5 and 2 min using the, respectively, conventional and microwave heating modes. The specific activity of the tracers was improved by preconcentration and purification of the generator eluate. The binding of the labeled and nonlabeled conjugates to avidin in solution was compared to the binding of native biotin. All compounds maintained good affinity for avidin, though introducing the linkers and chelator, especially the PEG-groups, somewhat decreased the binding affinity. The extent of binding of the labeled compounds to avidin was 54-91% after 5 min. Blocking experiments were performed confirming the specificity of the binding of biotin analogues to avidin. The stability of the three labeled compounds in human serum was studied. The stability of the biotin analogue 8 (65% within 30 min) and avidin-biotin complex (80% within 120 min) might be sufficient for the monitoring of the islets of Langerhans. The tracers will be evaluated in in vitro experiments of avidin-coated islets of Langerhans and in transplantation models in vivo.