Harald Hauser

90Nb - A potential PET nuclide: Production and labeling of monoclonal antibodies

Abstract Fast progressing immuno-PET gives reasons to develop new potential medium-long and long-lived radioisotopes. One of the promising candidates is 90Nb. It has a half-life of 14.6 h, which allows visualizing and quantifying processes with medium and slow kinetics, such as tumor accumulation of antibodies and antibodies fragments or polymers and other nanoparticles. 90Nb exhibits a high positron branching of 53% and an optimal energy of β+ emission of Emean=0.35 MeV only. Consequently, efficient radionuclide production routes and NbV labeling techniques are required. 90Nb was produced by the 90Zr(p,n) 90Nb nuclear reaction on natural zirconium targets. No-carrier-added (n.c.a.) 90Nb was separated from the zirconium target via a multi-step separation procedure including extraction steps and ion-exchange chromatography. Protein labeling was exemplified using the bifunctional chelator desferrioxamine attached to the monoclonal antibody rituximab. Desferrioxamine was coupled to rituximab via two different routes, by the use of N-succinyl-desferrioxamine (N-suc-Df) and by means of the bifunctional derivative p-isothiocyanatobenzyl-desferrioxamine B (Df-Bz-NCS), respectively. Following antibody modification, labeling with 90Nb was performed in HEPES buffer at pH 7 at room temperature. In vitro stability of the radiolabeled conjugates was tested in saline buffer at room temperature and in fetal calf serum (FCS) at 37 ºC. The selected production route led to a high yield of 145 ± 10 MBq/μA h of 90Nb with high radioisotopic purity of >97%. This yield may allow for large scale production of about 10 GBq 90Nb. The separation procedure resulted in 76–81% yield. The Zr/90Nb decontamination factor reaches 107. Subsequent radiolabeling of the two different conjugates with 90Nb gave high yields; after one hour incubation at room temperature, more than 90% of 90Nb-Df-mAb was formed in both cases. At room temperature in aqueous solution, both 90Nb-Df-mAb constructs were more than 99% stable over a period of 18 d. The developed production and separation strategy provided 90Nb with purity appropriate for radiolabeling applications. Labeling and stability studies proved the applicability of 90Nb as a potential positron emitter for immuno-PET.

Contribution of paramagnetic trace elements to the spin-lattice relaxation time in the liver.

The relaxation times of water protons in rat liver tissue were measured with a NMR spectrometer at 20 MHz. The paramagnetic trace elements Cu, Fe, and Mn were determined by neutron activation analysis. No shortening of T1 could be observed when liver Cu or Fe concentration was increased in the microgram range. T1 was strongly correlated with the liver Mn concentration of untreated animals and animals whose liver Mn concentration was artificially increased or decreased by intravenous injection of manganous acetate or a metal chelating agent with high affinity for hepatobiliary excretion. Deviations from this Mn-T1 correlation were found in the initial phase of liver cirrhosis induced by thioacetamide (elongated T1, normal Mn concentration) and after stimulation of liver growth by phenobarbital (normal T1, decreased Mn concentration). An increased or decreased enhancement factor for Mn may have contributed to the observed deviations during phenobarbital and thioacetamide treatment.

Labeling of monoclonal antibodies with a 67Ga-phenolic aminocarboxylic acid chelate

As a chelating agent for labeling antibodies (Abs) with metallic radionuclides, a propionic acid substituted ethylenediamine N,N′-di-[(o-hydroxyphenyl) acetic acid] (P-EDDHA), which tighly complexes 67Ga, was synthesized. The 67Ga-P-EDDHA chelate was coupled in aqueous solution to IgG at a molar ratio of 1:1 via carbodiimide. The average coupling yield was 15%. A specific activity of 4 mCi/mg IgG could be obtained with commercially supplied 67Ga. In vitro stability was evaluated in human serum at 37° C and showed a half-life of about 120 h for the release of 67Ga from the labeled Ab during the initial phase of incubation. This in vitro halflife is similar to that measured for 111In-DTPA labeled Abs. Because of the high stability of the 67Ga-P-EDDHA chelate, the in vivo formation of radioactive labeled transferrin by transchelation, as described for 111In-DTPA labeled Abs, should, however, be reduced by this labeling technique.

Separation of 90Nb from zirconium target for application in immuno-PET

Abstract Fast progressing immuno-PET asks to explore new radionuclides. One of the promising candidates is 90Nb. It has a half-life of 14.6 h that allows visualizing and quantifying biological processes with medium and slow kinetics, such as tumor accumulation of antibodies and antibodies fragments or drug delivery systems and nanoparticles. 90Nb exhibits a positron branching of 53% and an average kinetic energy of emitted positrons of Emean =0.35 MeV. Currently, radionuclide production routes and NbV labeling techniques are explored to turn this radionuclide into a useful imaging probe. However, efficient separation of 90Nb from irradiated targets remains in challenge. Ion exchange based separation of 90Nb from zirconium targets was investigated in systems AG 1 × 8 – HCl/H2O2 and UTEVA-HCl. 95Nb (t1/2 = 35.0 d), 95Zr (t1/2 = 64.0 d) and 92mNb (t1/2 = 10.15 d) were chosen for studies on distribution coefficients. Separation after AG 1 × 8 anion exchange yields 99% of 90/95Nb. Subsequent use of a solid-phase extraction step on UTEVA resin further decontaminates 90/95Nb from traces of zirconium with yields 95% of 90/95Nb. A semi-automated separation takes one hour to obtain an overall recovery of 90/95Nb of 90%. The amount of Zr was reduced by factor of 108. The selected separation provides rapid preparation (< 1 h) of high purity 90Nb appropriate for the synthesis of 90Nb-radiopharmaceuticals, relevant for purposes of immuno-PET. Applying the radioniobium obtained, 90/95Nb-labeling of a monoclonal antibody (rituximab) modified with desferrioxamine achieved labeling yields of >90% after 1 h incubation at room temperature.

A bifunctional HBED-derivative for labeling of antibodies with 67Ga, 111In and 59Fe. Comparative biodistribution with 111In-DPTA and 131I-labeled antibodies in mice bearing antibody internalizing and non-internalizing tumors

To investigate whether bifunctional ligands containing chelating structures other than EDTA and DTPA and metallic radiotracers other than 111In will reduce the non-specific radioactivity uptake in the liver during immunoscintigraphy, we synthetized an isothiocyanato-substituted phenolic polyaminocarboxylic acid (HBED-CI) for labeling of MAbs with 67Ga, 111In and 59Fe. Biodistribution of HBED-CI-labeled MAbs was compared to that of 131I and 111In-DTPA labeled MAbs in nude mice bearing tumors, which differ with regard to intracellular internalization and catabolism of the corresponding MAb-antigen complex. In the liver a continuous radioactivity excretion for 67Ga-HBED-CI-labeled MAbs was observed with kinetics that parallel 131I clearance after administration of 131I-MAbs, while 111In-HBED-CI-labeling led to a constant 111In liver level quite similar to that of 111In-DTPA-MAbs. In tumors, 67Ga-HBED-CI-MAb uptake again paralleled that of 131I-MAbs, showing continuous accumulation in tumor tissues when internalization of the MAb-antigen complex was not involved. A much lower uptake, which peaked between 24 and 48 h, was found in the case of MAb-antigen internalization. 111In of 111In-HBED-CI- and 111In-DTPA-labeled MAbs continuously accumulated in both types of tumors. Compared with 111In-DTPA-MAbs, an improvement in tumor-to-liver ratios, due to the reduced liver radioactivity associated with 67Ga-HBED-CI-labeled MAbs, could only be obtained with non-internalizing tumors. The time course of radioactivity distribution in the liver and in MAb-internalizing tumors after administration of 67Ga-HBED-CI-, 111In-HBED-CI- and 111In-DTPA-labeled MAbs further indicates a dominating influence of the metallic radiotracer rather than the ligand on retention or excretion of radioactivity in MAb-catabolizing tissues.