Tumor-associated macrophages constitute a major component of the stroma of solid tumors, encompassing distinct subpopulations with different characteristics and functions. We aimed to identify M2-oriented tumor-supporting macrophages within the tumor microenvironment as indicators of cancer progression and prognosis, using PET imaging. This can be realized by designing 18 F-labeled camelid single-domain antibody fragments (sdAbs) specifically targeting the macrophage mannose receptor (MMR), which has been identified as an important biomarker on this cell population. Methods: Crossreactive anti-MMR sdAbs were generated after immunization of an alpaca with the extracellular domains of both human and mouse MMR. The lead binder was chosen on the basis of comparisons of binding affinity and in vivo pharmacokinetics. The PET tracer 18 F-fluorobenzoate (FB)-anti-MMR sdAb was developed using the prosthetic group N-succinimidyl-4-18 F-fluorobenzoate ( 18 F-SFB), and its biodistribution, tumor-targeting potential, and specificity in terms of macrophage and MMR targeting were evaluated in mouse tumor models. Results: Four sdAbs were selected after affinity screening, but only 2 were found to be cross-reactive for human and mouse MMR. The lead anti-MMR 3.49 sdAb, bearing an affinity of 12 and 1.8 nM for mouse and human MMR, respectively, was chosen for its favorable in vivo biodistribution profile and tumor-targeting capacity. 18 F-FB-anti-MMR 3.49 sdAb was synthesized with a 5%-10% radiochemical yield using an automated and optimized protocol. In vivo biodistribution analyses showed fast clearance via the kidneys and retention in MMRexpressing organs and tumor. The kidney retention of the fluorinated sdAb was 20-fold lower than a 99m Tc-labeled counterpart. Compared with MMR-and C-C chemokine receptor 2-deficient mice, significantly higher uptake was observed in tumors grown in wild-type mice, demonstrating the specificity of the 18 F tracer for MMR and macrophages, respectively. Conclusion: Anti-MMR 3.49 was denoted as the lead cross-reactive MMR-targeting sdAb. 18 F radiosynthesis was optimized, providing an optimal probe for PET imaging of the tumor-promoting macrophage subpopulation in the tumor stroma. Dur ing tumor development, myeloid cells are attracted to the tumor stroma. These infiltrating immune cells are versatile, adopting different activation states in response to a changing microenvironment, leading to subsets of tumor-associated macrophages (TAMs) with specialized functions (1,2). Two main morphologically distinct TAM subsets can be distinguished on the basis of major histocompatibility complex (MHC) class II expression levels in multiple mouse tumor models. Tumor promotion has been linked with an accumulation of M2-oriented MHC II low TAMs in lung and breast carcinoma (3,4). Accordingly, MHC II low TAMs were found to reside primarily in less oxygenated zones, express hypoxia-regulated genes, and facilitate the angiogenic switch (5). Interestingly, the macrophage mannose receptor (MMR, CD206), a typical M2...

Positron emission tomography (PET) using radiolabeled biomolecules is a translational molecular imaging technology that is increasingly used in support of drug development. Current methods for radiolabeling biomolecules with fluorine-18 are laborious and require multistep procedures with moderate labeling yields. The Al18F-labeling strategy involves chelation in aqueous medium of aluminum mono[18F]fluoride ({Al18F}2+) by a suitable chelator conjugated to a biomolecule. However, the need for elevated temperatures (100-120 °C) required for the chelation reaction limits its widespread use. Therefore, we designed a new restrained complexing agent (RESCA) for application of the AlF strategy at room temperature.Methods. The new chelator RESCA was conjugated to three relevant biologicals and the constructs were labeled with {Al18F}2+ to evaluate the generic applicability of the one-step Al18F-RESCA-method.Results. We successfully labeled human serum albumin with excellent radiochemical yields in less than 30 minutes and confirmed in vivo stability of the Al18F-labeled protein in rats. In addition, we efficiently labeled nanobodies targeting the Kupffer cell marker CRIg, and performed µPET studies in healthy and CRIg deficient mice to demonstrate that the proposed radiolabeling method does not affect the functional integrity of the protein. Finally, an affibody targeting HER2 (PEP04314) was labeled site-specifically, and the distribution profile of (±)-[18F]AlF(RESCA)-PEP04314 in a rhesus monkey was compared with that of [18F]AlF(NOTA)-PEP04314 using whole-body PET/CT.Conclusion. This generic radiolabeling method has the potential to be a kit-based fluorine-18 labeling strategy, and could have a large impact on PET radiochemical space, potentially enabling the development of many new fluorine-18 labeled protein-based radiotracers.

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This study evaluates the performance of the Bruker PET insert combined with a BioSpec 70/30 USR MRI scanner using the manufacturer acceptance protocol and the NEMA NU 4-2008 for small animal positron emission tomographs (PET). The PET insert is made of 3 rings of 8 monolithic LYSO crystals (50 x 50 x 10 mm 3 ) coupled to silicon photomultipliers (SiPM) arrays, conferring an axial and transaxial FOV of 15 cm and 8 cm. The MRI performance was evaluated with and without the insert for the following radiofrequency noise, magnetic field homogeneity and image quality. For the PET performance, we extended the NEMA protocol featuring system sensitivity, count rates, spatial resolution and image quality to homogeneity and accuracy for quantification using several MRI sequences (RARE, FLASH, EPI and UTE). The PET insert does not show any adverse effect on the MRI performances. The MR field homogeneity is well preserved (Diameter Spherical Volume, for 20 mm of 1.98 ± 4.78 without and -0.96 ± 5.16 Hz with the PET insert). The PET insert has no major effect on the radiofrequency field. The SNR measurements also do not show major differences. Image ghosting is well within the manufacturer specifications (<2.5%) and no RF noise is visible. Maximum sensitivity of the PET insert is 11.0% at the center of the FOV even with simultaneous acquisition of EPI and RARE. PET MLEM resolution is 0.87 mm (FWHM) at 5 mm off-center of the FOV and 0.97 mm at 25 mm radial offset. The peaks for true/noise equivalent count rates are 410/240 and 628/486 kcps for the rat and mouse phantoms, and are reached at 30.34/22.85 and 27.94/22.58 MBq. PET image quality is minimally altered by the different MRI sequences. The Bruker PET insert shows no adverse effect on the MRI performance and demonstrated a high sensitivity, sub-millimeter resolution and good image quality even during simultaneous MRI acquisition.