89Zr-Labeled Domain II-Specific scFv-Fc ImmunoPET Probe for Imaging Epidermal Growth Factor Receptor In Vivo.

Epidermal growth factor receptor I (EGFR) is overexpressed in many cancers. The extracellular domain of EGFR has four binding epitopes (domains I- IV). All clinically approved anti-EGFR antibodies bind to domain III. Imaging agents that bind to domains other than domain III of EGFR are needed for accurate quantification of EGFR, patient selection for anti-EGFR therapeutics and monitoring of response to therapies. We recently developed a domain II-specific antibody fragment 8709. In this study, we have evaluated the in vitro and in vivo properties of 89Zr-8709-scFv-Fc (105 kDa). We conjugated 8709-scFv-Fc with the deferoxamine (DFO) chelator and radiolabeled the DFO-8970-scFv with 89Zr. We evaluated the binding of 89Zr-DFO-8709-scFv-Fc in EGFR positive and negative cell lines DLD-1, MDA-MB-231 and MDA-MB-435, respectively, and in mouse xenograft models. Simultaneously, we have compared the binding of 89Zr-8709-scFv-Fc with 111In-nimotuzumab, a domain III anti-EGFR antibody. DFO-8709-scFv-Fc displayed similar cell binding specificity as 8709-scFv-Fc. Saturation cell binding assay and immunoreactive fraction showed that radiolabeling did not alter the binding of 8709-scFv-Fc. Biodistribution and microPET showed good uptake of 89Zr-8709-scFv-Fc in xenografts after 120 h post injection (p.i). and was domain-specific to EGFR domain II. 89Zr-8709-scFv-Fc did not compete for binding in vitro and in vivo with a known domain III binder nimotuzumab. The results show that 89Zr-8709-scFv-Fc is specific to domain II of EGFR making it favorable for quantification of EGFR in vivo, hence, patient selection and monitoring of response to treatment with anti-EGFR antibodies.

Production and Semi-Automated Processing of 89Zr Using a Commercially Available TRASIS MiniAiO Module.

The increased interest in 89Zr-labelled immunoPET imaging probes for use in preclinical and clinical studies has led to a rising demand for the isotope. The highly penetrating 511 and 909 keV photons emitted by 89Zr deliver an undesirably high radiation dose, which makes it difficult to produce large amounts manually. Additionally, there is a growing demand for Good Manufacturing Practices (GMP)-grade radionuclides for clinical applications. In this study, we have adopted the commercially available TRASIS mini AllinOne (miniAiO) automated synthesis unit to achieve efficient and reproducible batches of 89Zr. This automated module is used for the target dissolution and separation of 89Zr from the yttrium target material. The 89Zr is eluted with a very small volume of oxalic acid (1.5 mL) directly over the sterile filter into the final vial. Using this sophisticated automated purification method, we obtained satisfactory amount of 89Zr in high radionuclidic and radiochemical purities in excess of 99.99%. The specific activity of three production batches were calculated and was found to be in the range of 1351-2323 MBq/µmol. ICP-MS analysis of final solutions showed impurity levels always below 1 ppm.

Nitrogen-13: historical review and future perspectives.

Positron emission tomography is an ultra-sensitive, in vivo molecular imaging technique that allows the determination of the spatiotemporal distribution of a positron emitter labeled radiotracer after administration into living organisms. Among all existing positron emitters, (18) F has been by far the most widely used both in clinical diagnosis and in preclinical investigation, while the use of (11) C significantly increased after the 1980s because of the widespread installation of biomedical cyclotrons. The use of other shorter-lived positron emitters such as (13) N (T1/2 = 9.97 min) has been historically more restricted. Paradoxically, its stable isotope ((14) N) is present in many biological active molecules; consequently, the development of strategies for the efficient incorporation of (13) N into radiotracers would represent an interesting alternative to (11) C- and (18) F-labeling. In the current paper, the developments related to (13) N chemistry are reviewed, including different production routes of primary precursors and their applications to the preparation of more complex (13) N-labeled molecules. The current situation and future perspectives are also briefly discussed.

Synthesis and evaluation of (13)N-labelled azo compounds for ß-amyloid imaging in mice.

PURPOSE: The aim of the present study was to develop short half-lived tools for in vitro and in vivo ß-amyloid imaging in mice, for which no suitable PET tracers are available. PROCEDURES: Five (13)N-labelled azo compounds (1-5) were synthesized using a three-step process using cyclotron-produced [(13)N]NO3 (-). Biodistribution studies were performed using positron emission tomography-computed tomography (PET-CT) on 20-month-old healthy, wild-type (WT) mice. In vivo and in vitro binding assays were performed using PET-CT and autoradiography, respectively, on 20-month-old healthy (WT) mice and transgenic (Tg2576) Alzheimer's disease model mice. RESULTS: (13)N-labelled azo compounds were prepared with decay corrected radiochemical yields in the range 27 ± 4 % to 39 ± 4 %. Biodistribution studies showed good blood-brain barrier penetration for compounds 1 and 3-5; good clearance data were also obtained for compounds 1-3 and 5. Compounds 2, 3 and 5 (but not 1) showed a significant uptake in ß-amyloid-rich structures when assayed in in vitro autoradiographic studies. PET studies showed significant uptake of compounds 2 and 3 in the cortex of transgenic animals that exhibit ß-amyloid deposits. CONCLUSIONS: The results underscore the potential of compounds 2 and 3 as in vitro and in vivo markers for ß-amyloid in animal models of Alzheimer's disease.