The future of ⁸⁹Zr-based immuno-PET is reliant upon the development of new chelators with improved stability compared to the currently used deferoxamine (DFO).

A large number of applications for fibroblast activation protein inhibitors (FAPI)-based PET agents have been evaluated in conditions ranging from cancer to non-malignant diseases such as myocardial infarction. In particular, ⁶⁸Ga-FAPI-46 was reported to have a high specificity and affinity for FAP-expressing cells, a fast and high accumulation in tumor lesions/injuries together with a fast body clearance when investigated in vivo. Due to the increasing interest in the use of the agent both preclinically and clinically, we developed an automated synthesis for the production of ⁶⁸Ga-FAPI-46 on a Trasis AiO platform. The new synthetic procedure, which included the processing of the generator eluate using a strong cation exchange resin and a final purification step through an HLB followed by a QMA cartridge, yielded ⁶⁸Ga-FAPI-46 with high radiochemical purity (>98%) and apparent molar activity (271.1 ± 105.6 MBq/nmol). Additionally, the in vitro and in vivo properties of the product were assessed on glioblastoma cells and mouse model. Although developed for the preparation of ⁶⁸Ga-FAPI-46 for preclinical use, our method can be adapted for clinical production as a reliable alternative to the manual (i.e., cold kit) or modular systems preparations already described in the literature.

The new macrocyclic ligand 15-fluoro-15-methyl-1,4,7,10,13-pentaazacyclohexadecan-14,16-dione (2) was synthesised and its crystal structure determined together with the ones of the known analogues of 2, 15-fluoro-1,4,7,10,13-pentaazacyclohexadecan-14,16-dione (1) and 15,15-difluoro-1,4,7,10,13-pentaazacyclohexadecan-14,16-dione (3). The binding behaviour of all three ligands to copper and zinc was studied in the solid state. They can bind to the metal centre by either triple coordination (N3) with all secondary amines or after double deprotonation of the two amides with all five nitrogen atoms (N5). The N5 coordination mode is favoured by the presence of one or two fluorine substituents at the C(15) position and by a high pH in the case of aqueous solutions. Circular dichroism titrations of poly d(GC) with the metal complexes showed that only 4 and 5, that is the copper complexes of 1 and 2, induced a complete B- to Z-DNA transition. The degree of cooperativity of the transition was found to be 3.4 and 7.3 for 4 and 5 respectively. As a possible hypothesis to explain this difference, the additional methyl group in 5 compared with 4 may be involved in a hydrophobic interaction with the DNA. Ligand 2, the copper complex 6 of the bis fluoro substituted ligand 3, and the zinc complex 7 of ligand 1 did not induce any change in the direction of Z-DNA. In the case of 6, the CD spectrum of the DNA actually showed no change at all, indicating that the complex was even not interacting with the B form of DNA. Therefore it is assumed that the bis fluoro substitution is causing the complex to be in the neutral N5 coordination mode at the experimental conditions of pH 7. The electrostatic contribution together with the shielding effect of the ligand might explain the absence of any interaction with the DNA.

Purpose: Recent studies have highlighted a role of HER3 in HER2-driven cancers (e.g., breast cancer), implicating the upregulation of the receptor in resistance to HER-targeted therapies and Hsp90 inhibitors (e.g., AUY922). Therefore, we have developed an affibody-based PET radioconjugate that quantitatively assesses HER3 changes induced by Hsp90 inhibition in vivo. Experimental Design: Z_HER3:8698 affibody molecules were conjugated via the C-terminus cysteine to DFO-maleimide for ⁸⁹Zr radiolabeling. The probe was characterized in vitro and in vivo in a panel of human breast cell lines and xenograft models with varying HER3 receptor levels. In addition, the radioconjugate was investigated as a tool to monitor the outcome of AUY922, an Hsp90 inhibitor, in an MCF-7 xenograft model. Results: We demonstrated that ⁸⁹Zr-DFO-Z_HER3:8698 can track changes in receptor expression in HER3-positive xenograft models and monitor the outcome of AUY922 treatment. Our in vitro findings showed that MCF-7 cells, which are phenotypically different from BT474, develop resistance to treatment with AUY922 through HER3/IGF-1Rβ–mediated signaling. Of note, the lack of response in vitro due to HER3 recovery was confirmed in vivo using ⁸⁹Zr-DFO-Z_HER3:8698–based imaging. Upon AUY922 treatment, higher radioconjugate uptake was detected in treated MCF-7 xenografts, correlating with an AUY922-induced HER3 upregulation concomitant with an increase in IGF-1Rβ expression. Conclusions: These data underline the potential of HER3-based PET imaging to noninvasively provide information about HER3 expression and to identify patients not responding to targeted therapies due to HER3 recovery. Clin Cancer Res; 24(8); 1853–65. ©2018 AACR.

Fluorescence-guided surgery is set to play a pivotal role in the intraoperative management of pediatric tumors. Shortwave infrared imaging (SWIR) has advantages over conventional near-infrared I (NIR-I) imaging with reduced tissue scattering and autofluorescence. Here, two NIR-I dyes (IRDye800CW and IR12), with long tails emitting in the SWIR range, were conjugated with a clinical-grade anti-GD2 monoclonal antibody (dinutuximab-beta) to compare NIR-I and SWIR imaging for neuroblastoma surgery. A first-of-its-kind multispectral NIR-I/SWIR fluorescence imaging device was constructed to allow an objective comparison between the two imaging windows. Conjugates were first characterized in vitro. Tissue-mimicking phantoms, imaging specimens of known geometric and material composition, were used to assess the sensitivity and depth penetration of the NIR-I/SWIR device, showing a minimum detectable volume of ∼0.9 mm3 and depth penetration up to 3 mm. In vivo, fluorescence imaging using the NIR-I/SWIR device showed a high tumor-to-background ratio (TBR) for both dyes, with anti–GD2-IR800 being significantly brighter than anti–GD2-IR12. Crucially, the system enabled higher TBR at SWIR wavelengths than at NIR-I wavelengths, verifying SWIR imaging enables high-contrast delineation of tumor margins. This work demonstrates that by combining the high specificity of anti-GD2 antibodies with the availability and translatability of existing NIR-I dyes, along with the advantages of SWIR in terms of depth and tumor signal-to-background ratio, GD2-targeted NIR-I/SWIR-guided surgery could improve the treatment of patients with neuroblastoma, warranting investigation in future clinical trials. Significance: Multispectral near-infrared I/shortwave infrared fluorescence imaging is a versatile system enabling high tumor-to-background signal for safer and more complete resection of pediatric tumors during surgery.

There is no established method to assess the PD-L1 expression in brain tumours. Therefore, we investigated the suitability of affibody molecule (Z_PD-L1) radiolabelled with F-18 (Al¹⁸F) and Ga-68 to measure the expression of PD-L1 in xenograft mouse models of GBM. Mice bearing subcutaneous and orthotopic tumours were imaged 1 h post-radioconjugate administration. Ex vivo biodistribution studies and immunohistochemistry (IHC) staining were performed. Tumoural PD-L1 expression and CD4+/CD8+ tumour-infiltrating lymphocytes were evaluated in human GBM specimens. Z_PD-L1 was radiolabelled with radiochemical yields of 32.2 ± 4.4% (F-18) and 73.3 ± 1.8% (Ga-68). The cell-associated radioactivity in vitro was consistent with PD-L1 expression levels assessed with flow cytometry. In vivo imaging demonstrated that ¹⁸F-AlF-NOTA-Z_PD-L1 can distinguish between PD-L1 high-expressing tumours (U87-MGvIII) and PD-L1-negative ones (H292_PD-L1Ko). The radioconjugate was quickly cleared from the blood and normal tissues, allowing for high-contrast images of brain tumours as early as 1 h post-injection. ⁶⁸Ga-NOTA-Z_PD-L1 showed heterogeneous and diffuse accumulation that corresponded to the extensively infiltrating GCGR-E55 tumours involving contiguous lobes of the brain. Lastly, 39% of analysed GBM patient samples showed PD-L1+ staining of tumour cells that was associated with elevated levels of CD4+ and CD8+ lymphocytes. Our results suggest that the investigated radioconjugates are very promising agents with the potential to facilitate the future design of treatment regimens for GBM patients.